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LECTURES
Silver Medal Lectures
L101 FROM GIBBERELLINS TO PLANT HORMONOME
Kamiya Yuji RIKEN Plant Science Center, Yokohama, 230-0045 Japan
[email protected]
Almost all the enzyme genes involved in gibberellin (GA) biosynthesis have been cloned from Arabidopsis. There are
12 steps from geranylgeranyl diphosphate (GGDP) to GA4, which is a physiologically active GA in Arabidopsis. GGDP
is synthesized in plastids by a methyl erythritol phosphate (MEP) pathway, which is different from mevalonate pathway
in cytosol. Many of the enzymes involved GA biosynthesis are multi-functional and some of them catalyze several steps.
In addition to known pathways, we have found two new deactivation pathways in Arabidopsis and rice.
During light-induced seed germination of Arabidopsis, GA and ABA biosyntheses interact each other. Therefore it is
important to analyze all the hormones from the same plant materials to understand the net-work regulation of hormone
biosynthesis. We have recently introduced a LC-ESI-MS/MS system, which does not require derivatization of hormones.
Using both GC-MS and LC-MS we have performed comprehensive hormone analysis. We could identify 16 different
GAs by a single LC-MS run from Arabidopsis. ABA and PA, DPA and ABA-GE were also analyzed. In a special case,
we could quantify ABA even from a single seed of Arabidopsis.
L102 MAPPING GENOTYPE INTO PHENOTYPE: NATURAL, INDUCED AND EPIGENETIC VARIATION IN ARABIDOPSIS GENOMES
Ecker, Joseph R. et al.
[email protected]
A precise and comprehensive understanding of DNA sequence and epigenetic variation in natural populations of a species will be essential for elucidating the basis of phenotypic variation. We have applied oligonucleotide tiling microarrays
and next-generation ultra-high throughput DNA sequencing methods to identify DNA sequence and epigenetic variation
in wild strains (accessions) of Arabidopsis thaliana that were chosen for maximal genetic diversity. The entire Arabidopsis Genome Initiative (AGI) reference genome sequence of A. thaliana Col-0 was tiled on high-density tiling microarrays
at single base resolution using over a billion 25 base oligonucleotides. The arrays were hybridized with genomic DNA
from 19 accessions, isothermally amplified DNA to minimize ascertainment biases. In addition, whole genome sequencing using a Solexa/Illumina Genetic Analyzer allowed more complete sequence coverage of the genomes of several
strains (Col-0, Ler-1, Cvi-0, etc.), allowing independent assessment of the quality of the AGI Sanger and Perlegen arraybased genome sequences. The degree of polymorphism and types of genes that harbor major effect polymorphisms in
natural populations will be described. We are also using next-generation sequencing technology to develop methods for
creating large populations of T-DNA insertion mutants in diverse genetic backgrounds as well as for determining the extent of epigenetic diversity in wild accessions. A proposal for cost effective re-sequencing of 1,001 or more Arabidopsis
genomes will be outlined. When coupled with community-wide phenotypic screens for traits of interest such as responses
to all growth regulators, this database would allow realization of whole genome association mapping studies and provide
an unprecedented resource for understanding the genetic/epigenetic basis of phenotypic variation in plants.
IPGSA 19th Annual Meeting - Abstracts
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EMBO Plenary Lecture
L301 PHOSPHATE STARVATION SIGNALING IN PLANTS
PHOSPHATE STARVATION SIGNALING IN PLANTS Bustos Regla, Franco Zorrilla José Manuel, Catarecha Pablo,
Castrillo Gabriel, Mateos Isabel, Puga Mabel, Rubio Vicente, Leyva Antonio, Paz-Ares Javier Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, 28049-Madrid, Spain; [email protected]
[email protected]
Plants are endowed with nutrient sensing mechanisms that allow them to respond and adapt their growth to conditions
of limited nutrient supply. These sensing mechanims and responses are greatly nutrient specific an involve biochemical
and developmental changes that improve acquisition and recycling of the limiting nutrient, and protect against the nutrient starvation stress. In consonance with the importante of phosphorus nutrition, which is usually absorbed as phosphate
(Pi) from the soil, the control of Pi starvation responses involves a highly elaborated regulatory mechanism, whose details are just beginning to emerge. In addition to the signalling role of Pi, this regulatory system is responsive to positive
modulators, such as sugars, and to repressing signals, such as cytokinins and other unknown signal(s) mediating long
distance systemic repression. Moreover, transcription of Pi starvation-induced genes is repressed by arsenate, potentially
reflecting the existence of a savage system to protect plants from arsenate, particularly in Pi poor soils. Transcription factor PHR1 is a master regulator of Pi starvation responses, that recognises an imperfect palindromic motif, P1BS (GNATATNC). In line with the key role of PHR1, P1BS motifs are over-represented in the promoter region of Pi starvation
induced genes. We have shown that a minimal promoter containing a multimerised P1BS motif is specifically induced
by Pi starvation and is responsive to the stimulatory effect of sugars and the inhibitory effects of cytokinins, long distance repression signals and arsenate. Recent work in other laboratories has shown the key role of starvation responsive
miRNAs (miR399) in the control of Pi homeostasis. In addition the existence of other noncoding RNAs highly specifically responsive,encoded by the Mt4/TPS1 gene family, has been known for some time. These noncoding RNAs display
sequence complementarity with miR399 wich is impaired at the predicted cleavage site. As a result, we have shown that
these Mt4/TPS1 RNAs interact miR399, but this interaction does not result in miR399 guided degradation of Mt4/TPS1
RNAs but rather on miR399 sequestration. We use the term target mimicry to define this riboregulatory mechanism of
miRNA activity.
Plenary
Hormone biosynthesis and metabolism
P101 GENETICS OF AUXIN REGULATION IN ARABIDOPSIS
Bonnie Bartel
[email protected]
Indole-3-butyric acid (IBA) and amide-linked conjugates of indole-3-acetic acid (IAA) both can act as auxin storage
forms. We have exploited the observation that IBA and certain IAA-amino acid conjugates mimic IAA in bioassays to
isolate Arabidopsis mutants defective in IBA or conjugate responses. Some IBA response mutants are generally impaired
in peroxisomal fatty acid chain shortening, whereas others appear to have defects specific to IBA metabolism. These
results imply that the conversion of IBA to IAA is disrupted in the mutants, and that IBA acts, at least in Arabidopsis,
through its conversion to IAA. Genes defective in the IAA-conjugate response mutants encode IAA-amino acid conjugate hydrolases and proteins implicated in metal homeostasis in subcellular compartments. Our results suggest that the
IAA released from IBA and IAA-amino acids contributes to lateral root formation.
P102 REGULATION OF CYTOKININ METABOLISM: A NEW INSIGHT INTO THE HORMONE FUNCTION
Sakakibara, Hitoshi RIKEN Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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Cytokinin (CK) plays a crucial role in various phases of plant growth and development and the concentration is finely
controlled by internal and external environmental factors such as phytohormones and nitrogen sources. CK in plants is
first synthesized as isopentenyladenine (iP) nucleoside phosphate by adenosine phosphate-isopentenyltransferase (IPT)
and then hydroxylated to trans-zeatin (tZ) nucleoside phosphate by a cytochrome P450 monooxygenase, CYP735A. The
expression of a subset of IPT genes is tightly regulated by nitrogen sources, such as nitrate, andn that of CYP735A is
induced by cytokinin and repressed by auxin. Our recent study with a rice mutant, log, an activation step of CK is catalyzed by LOG, a cytokinin-specific phosphoribohydrolase, which converts the CK-nucleotide to the biologically active
free-base form. Spatial expression patterns of the genes indicate that CK is locally synthesized and activated at various
sites where it is needed. We will outline the recent progress of study on metabolic pathway of CK and discuss the regulatory system in plants.
P103 GIBBERELLIN BISYNTHESIS AND DEACTIVATION
Shinjiro Yamaguchi RIKEN Plant Science Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, 230-0045, Japan
[email protected]
Among numerous gibberellins (GAs) found in plants, only a limited number of them are able to activate the GA signaling pathway as hormones. The concentrations of such biologically active GAs in plant tissues are determined by the rates
of their formation and deactivation. GA 2-oxidation has been well characterized as a mechanism for GA deactivation in
a number of plant species, and this reaction is catalyzed by a class of 2-oxoglutarate-dependent dioxygenases. However,
our recent work on the recessive ʻtall riceʼ mutant, elongated uppermost internode (eui), revealed that a cytochrome P450
monooxygenase plays a major role in deactivating GAs in rice internodes at the heading stage. We found that the eui mutant accumulated a large amount of bioactive GAs due to a defect in GA 16,17-epoxidation. More recently, methylation
of the carboxyl group of GAs by SAM-dependent methyltransferases has been shown to act as another GA deactivation
reaction. Our data demonstrate that GAs are deactivated through multiple pathways in plants, and suggest that some
GA deactivation genes are tightly regulated by developmental and/ or environmental signals. Therefore, the presence
of multiple deactivation pathways may play an important role in the integration of a number of signals to the levels of
bioactive GAs.
P104 HORMONE REGULATION BY AMINO ACID CONJUGATION: JASMONATES, AUXINS AND MORE
Staswick, Paul Dept. of Agronomy and Horticulture, University of Nebraska, Lincoln NE, 68583 USA
[email protected]
Plants use a variety of hormones to regulate their growth and development and to respond to biotic and abiotic assaults.
Amide-linked amino acid conjugates of some plant hormones are known to help regulate the level and activity of these
hormones. However, our knowledge of how conjugation controls signaling has been limited because the conjugating enzymes were unknown. We have discovered an enzyme family that conjugates amino acids to a several plant signaling molecules. The jasmonic acid (JA)-amido synthetase encoded by the Arabidopsis thaliana JAR1 locus is required for several
jasmonate responses. A marked and specific increase in the amount of JA-isoleucine occurs in response to leaf wounding
and the jar1 mutant is defective in the induction of some genes associated with plant defense in response to wounding.
Application of exogenous JA-Ile also functionally complements defects seen in jar1, establishing that this conjugate is
required for signaling. JAR1 belongs to a 19-member family of proteins called the GH3s, named for the auxin-induced
soybean GH3 gene that has been studied for over two decades. Eight other GH3s conjugate indole-3-acetic acid (IAA). In
contrast with JA conjugation, the activity of these enzymes helps to maintain auxin homeostasis by removing excess free
IAA from the active hormone pool. Transcriptional induction of several of these genes by exogenous IAA is consistent
with this inactivating role. Over expression of several of these genes in transgenic plants results in severely compromised
plant growth and development and lowered amounts of free IAA. Of the 10 other GH3 enzymes, at least one conjugates
indole-3-butyric acid (IBA), but not IAA Some other Arabidopsis GH3s prefer various phenolic acids as substrates. The
long term goal of this research is to understand the biochemical function of the GH3 family of conjugating enzymes and
to establish how they help to regulate and coordinate these important signaling pathways in plants.
IPGSA 19th Annual Meeting - Abstracts
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P105 ABA BIOSYNTHESIS AND COMMUNICATION OF WATER STRESS
Christmann, Alexander, Grill, Erwin Lehrstuhl für Botanik, Technische Universität München, Am Hochanger 4, D85354 Weihenstephan, Germany
[email protected]
Plants experiencing a limiting water supply due to drying soil respond by inducing ABA biosynthesis and adjusting
leaf transpiration. An attractive hypothesis for root to shoot communication of water stress conditions favours ABA as an
endogenous signal. According to this hypothesis, ABA is generated in roots upon drought stress and is transported via the
transpiration stream to the shoot to initiate stomatal closure. Our study does not support ABA as being the long-distance
signal. Rather, we show that soil water deficit elicits ABA responses of the shoot in the absence of root-generated ABA
and that shoot-derived ABA is necessary and sufficient for stomatal closure. Water stress resulted in a hydraulic response
in the shoot, which preceded ABA signalling and stomatal closure. Attenuation of the hydraulic response prevented longdistance signalling of water stress. Thus, the analysis revealed root to shoot communication of plant water status by a
hydraulic signal.
Long range signaling and horomone transport
P201 LONG DISTANCE SIGNALLING IN THE CONTROL OF SHOOT BRANCHING
Leyser, Ottoline
[email protected]
A unique feature of plant development is the ability to alter body plan in response to environmental conditions. The
primary body axis of plants is laid down during embryogenesis with the establishment of the shoot apical meristem at
one end and a root apical meristem at the other. Post-embryonically, the meristems elaborate this basic axis, but in addition secondary meristems arise in both the root and shoot, which give rise to new axes of growth- lateral branches. It is
this ability to produce lateral branches that gives plants their spectacular plasticity of form. As a model for understanding
the role of plant hormones in plant developmental plasticity, we are investigating the hormonal control of shoot branching. We are focusing on two hormones that inhibit branching-auxin, which has been known to regulate branching for 80
years; and a novel hormone, which has not yet been chemically defined. In Arabidopsis, the novel hormone came to light
through the analysis of mutants at 4 loci, called MAX1-MAX4, with increased shoot branching. These genes define an
additional pathway that interacts with auxin to mediate branch inhibition. Grafting studies have demonstrated that three
of these loci are involved in the production of a long-range graft transmissible signal that inhibits bud growth, while the
third acts locally in the transduction of this signal, and this is consistent with the molecular identities of these genes.
The pathway appears to act by modulating auxin transport capacity in the main stem, suggesting an interesting indirect
mechanism for apical dominance and its modulation.
P202 PIN-DEPENDENT POLAR AUXIN TRANSPORT
Ji_í Friml Department of Plant Cell Biology, University of Göttingen, Germany
[email protected]
Auxin is a prominent intercellular signal in plants. Directional, active, cell-to-cell auxin transport mediates local auxin
gradients, which are required for various patterning processes including apical-basal axis formation, organogenesis and
tropisms. The chemiosmotic hypothesis postulates that auxin transport is accomplished by the action of auxin influx and
efflux carriers, which are localized at the plasma membrane of transporting cells. The strictly controlled directionality
of auxin flow is supposed to be determined by the asymmetric cellular localisation of auxin efflux carrier. Genetic approaches in Arabidopsis thaliana identified candidate genes coding for regulators of auxin efflux including homologues of
human multiple drug resistance/P-glycoproteins (PGP1 and PGP19) and plant-specific plasma membrane PIN proteins.
PIN proteins show asymmetric subcellular localisation, which well correlates with known directions of auxin flow. Plants
defective in PIN function show decreased auxin transport, altered auxin distribution patterns, and diverse developmental
defects, all of which can be phenocopied by the chemical inhibition of auxin efflux. All these results provide strong evidence that PIN proteins are essential for auxin transport but offer only little insight into the molecular basis of function
IPGSA 19th Annual Meeting - Abstracts
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of PIN proteins. Here we show, by assessing the impact of PIN and PGP activity in plant, yeast and HeLa cultured cells,
that PIN proteins are rate-limiting catalysts of auxin efflux, which can act independently of PGP proteins. PIN action is
specific to auxins, sensitive to known inhibitors and causes phenotypes indicative of auxin starvation. Moreover, interfering with PIN sequence-embedded polarity signals directly demonstrates that polarity of PIN localisation is a necessary
and sufficient factor determining direction of local auxin flow. Our results suggest that PINs are catalytic components of
auxin efflux carriers and that their polarity determines the direction of vectorial auxin movement.
P203 AUXIN AND PHYLLOTAXIS
Kuhlemeier, Cris 1, Guyomarcʼh, Soazig 1, Bainbridge, Kath 1, Bayer, Emmanuelle 1, Mandel, Therese 1, Reinhardt,
Didier 1, Smith, Richard S. 2, Prusinkiewicz, Przemyslaw 2 1 Institute of Plant Sciences, University of Berne, Altenbergrain 21, CH-3013 Berne 2 Dept of Computer Science, University of Calgary
[email protected]
Phyllotaxis, the regular arrangement of leaves or flowers around a plant stem, is an example of developmental pattern formation and organogenesis. Phyllotaxis is characterized by the divergence angles between the organs, the most
common angle being 137.5°, the golden angle. Recently, we showed that the central regulator of phyllotaxis is the plant
hormone auxin. A key characteristic of the system is the redistribution of auxin within the shoot apical meristem through
active transport. I will describe the relevant experiments and how they form the basis for a new generation of experimentbased computer simulations. Such models can be tested by experiment and, conversely, guide further experiments.
P204 MOLECULAR CONTROL OF SECONDARY GROWTH INITIATION IN ARABIDOPSIS
Sehr, Eva Maria; Lichtenberger, Raffael; Nehlin, Lilian; Kerzendorfer, Claudia; Greb, Thomas Gregor Mendel Institute
of Molecular Plant Biology, Dr. Bohr-Gasse 3, 1030 Vienna, Austria Correspondence: [email protected],
phone: +43-1-79044-9871, fax: +43-1-79044-23-9870
[email protected]
In all vascular plants, the initial establishment of the shoot by the shoot apical meristem (SAM) is characterised by
longitudinal growth and the establishment of the procambial strands, which give rise to the primary pattern of discrete
vascular bundles. Subsequently to primary growth, many vascular plants, including Arabidopsis, undergo secondary
growth leading to an increase in shoot diameter. For this process, a cylindrical meristem, the vascular cambium, is established at the periphery of the stem which produces secondary vascular tissue, resulting in a continuous cylinder of
vascular tissue along the shoot axis.
The initiation of secondary growth with the formation of a closed cambial cylinder is anatomically well described. However, the molecular control of secondary growth initiation and the establishment of the vascular cambium is far from
being understood. Based on previous results, we hypothesised that post-embryonic establishment of meristematic activity depends on the function of members of the GRAS family of transcriptional regulators. Indeed, three closely related
members of this family, SCL6, SCL22, and SCL27, are expressed in the shoot in a cylindrical domain where cambial
activity is induced. We suggest that GRAS gene expression mediates a meristematic potential in interfascicular regions of
the shoot and that cells located in the SCL6/22/27 expression domain are especially susceptible to auxin redirected from
the basipetal auxin flow conducted through the stem. Our observation that auxin accumulates in interfascicular regions
during the onset of cell division supports the concept that lateral auxin transport from primary vascular bundles is an
initial step in the initiation of secondary growth.
P205 L-SYSTEM MODELS OF APICAL DOMINANCE
Prusinkiewicz, Przemyslaw 1, Smith, Richard S. 1, Leyser, Ottoline 2 1 Department of Computer Science, University of
Calgary; 2 Department of Biology, University of York.
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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An important aspect of plant development is the order in which lateral buds are activated to produce branches. This
order may manifest itself, for example, in the gradient of branch lengths, or in the flowering sequence. Basipetal activation progression is likely related to apical dominance. The hypothesis is that the shoot apical meristem in the vegetative
state has a strong inhibitory influence on the lateral buds below, which is lifted upon the transition of the apex to the
flowering state. This information propagates down the stem, causing gradual activation of the lateral buds. The inhibitory
signal may be auxin, produced by the shoot apex and actively transported down the plant. To better understand the dynamics of the activation, we implemented and examined several simulation models of this process. The models confirm
the plausibility of postulated molecular-level mechanisms: mediation of auxin action by cytokinin, and competition for
the limited auxin-carrying capacity of the stem. From a methodological perspective, the models illustrate the usefulness
of the formalism of Lindenmayer systems in the simulation of long-distance signaling and hormone transport in plants.
Hormone signaling
P301 GENETIC AND BIOCHEMICAL ANALYSIS OF THE AUXIN RECEPTOR REVEALS A NOVEL MECHANISM OF
HORMONE SIGNALING
Calderon-Villalobos, Luz Irina; Greenham, Katie; Lavy, Meirav; Mockaitis, Keithanne; Mooney, Sutton; Parry, Geraint;
Prigge, Mike; Santner, Aaron; Estelle, Mark Department of Biology, Indiana University, Bloomington IN 47405, USA
[email protected]
The auxin class of plant hormones, including the endogenous auxin IAA, are a relatively heterogeneous collection
of molecules. These compounds regulate diverse aspects of plant growth and development by promoting the degradation of transcriptional regulators called Aux/IAA proteins through the action of the ubiquitin protein ligase SCFTIR1. In
recent work the F-box protein subunit of SCFTIR1, a protein called TIR1, was shown to function as an auxin receptor.
Auxin binds directly to TIR1 to promote binding of the Aux/IAA proteins. Structural studies of the ASK1-TIR1 complex
indicate that auxin binding does not produce a conformational change in TIR1. Rather, auxin appears to function as a
ʻmolecular glue” to stabilize a weak interaction between TIR1 and the Aux/IAA substrates. These studies also suggest
how structurally diverse compounds function as auxins. In addition, genetic analysis of TIR1 and other members of the
TIR1/AFB auxin receptor family, provide new insight into the complexity of auxin signaling.
P302 BRASSINOSTEROID SIGNALING: THE FIRST DECADE
Chory, Joanne, Geldner, Niko, Savaldi-Goldstein, Sigal, Wang, Xuelu Plant Biology Laboratory, The Salk Institute for
Biological Studies and The Howard Hughes Medical Institute, La Jolla, CA 92037 USA
[email protected]
Brassinosteroids (BRs) are a class of hormones that are essential for normal plant growth and development. In the
absence of BR production or perception, plants exhibit an extreme dwarf stature, which has been attributed to defects
in cell expansion and division. During the past decade, the BR receptor has been characterized and key players in both
the BR signaling and biosynthesis pathways have been identified. In this talk, I will summarize the state of the field and
discuss our most recent results in dissecting the activation mechanism of the BR receptor. In addition, I will present data
that shows that BRs act locally to promote cell expansion, with the major target tissue being the epidermis.
P303 CYTOKININ SIGNALLING: TWO-COMPONENTS AND MORE
To, Jennifer1, Rashotte, Aaron1, Hutchison, Claire1, Ferreira, Fernando1, Schaller, Eric2, Kieber, Joseph1 1The Department of Biology, Univeristy of North Carolina, Chapel Hill, NC 27599; 2 Dept. of Biological Sciences, Dartmouth College, Hanover, NH 03755 [email protected]; Ph:(919) 962-2144
[email protected]
Cytokinins have been implicated a wide variety of plant growth and development processes and have been shown to
interact with various other signals. Recent studies have demonstrated that cytokinin signal transduction occurs through
a classic bacterial two-component signaling system, in which signal propagation relies on the transfer of phosphates between alternating histidine and aspartic acid residues. Genes encoding proteins corresponding to each of these two-com-
IPGSA 19th Annual Meeting - Abstracts
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ponent elements have been identified in Arabidopsis. Using molecular, genetic and biochemical approaches, the role of
the Arabidopsis two-component genes in plant growth and development has been defined. There is extensive functional
redundancy in these gene families. Analysis of lines harboring multiple disruptions in multiple two-component genes has
indicated that these elements play roles in various signaling pathways. In related studies, we have defined the response
of the transcriptome to cytokinin. One set of cytokinin-regulated genes encode transcription factors, and we have begun
to analyze the role of these in the response to cytokinin. We have found a group of AP2-related transcription factors that
translocate to the nucleus rapidly in response to cytokinin, and analysis of hypomorphic mutations indicates that these
CRFs mediate a subset of the transcriptional response to cytokinin. A model incorporating these and other results will be
presented.
P304 IDENTIFICATION OF NOVEL ABA SIGNALING COMPONENTS AND TRANSDUCTION MECHANISMS IN
GUARD CELLS.
Julian I. Schroeder, Tae-Houn Kim, Izumi Mori, Maik Böhmer, Yingzhen Yang, Yoshiyuki Murata1, Jaakko Kangasjarvi2 , Hannes Kollist2 , Erwin Grill3, Yong-Fei Wang Cell and Developmental Biology Section, Division of Biological
Sciences, University of California, San Diego, La Jolla, CA 92093-0116, USA. 1Dept. of Agriculture Okayama University, Okayama 700, Japan. 2 Dept of Biol and Environ Sciences, University of Helsinki, Finland. 3Botany Institute, T.U.
Munich, Germany.
[email protected]
Guard cells have been developed as a model system for dissecting ion channel functions and early signal transduction
mechanisms. Genetic loss-of-function mutants in Ca2+ sensors that disrupt Ca2+- and abscisic acid-regulated stomatal
movements have been lacking. We have recently identified two calcium-dependent protein kinases (CDPKs) that function in abscisic acid (ABA) and Ca2+ regulation of guard cell ion channels and stomatal closing (I. Mori et al., 2006
PLoS Biol.). Furthermore, several independent signal transduction analyses suggest a new model for how plant cells can
achieve specificity in calcium signaling through “priming” and “de-priming” of Ca2+ sensitive mechanisms (J. Young
et al., 2006 PNAS). Further evidence that correlates with this “Ca2+ sensor priming” hypothesis will be presented. Evidence for parallel pathways that function in the ABA signaling network will also be presented. An central target of ABA
and cytosolic Ca2+ signaling is the activation of S-type anion channels in guard cells. Identification of a new membrane
protein-encoding gene that is essential for mediating this response will be presented.
Genetic, genomic and signal transduction analyses in several laboratories indicate that genetic redundancies and robustness exist within the abscisic acid signal transduction network. To address this complexity we have pursued gain-of-function screens and genomic approaches (e.g. N. Leonhardt et al., 2004 Pl Cell; Mori et al., 2006 PLoS Biol.). More recently
we have developed a chemical genetics approach that allows high-throughput screening for molecules and mutants that
affect ABA signal transduction. Progress at isolating a small molecule that blocks ABA responses and isolation and characterization of mutants in ABA signaling that are insensitive to this compound will be presented.
P305 GIBBERELLIN PERCEPTION AND SIGNAL TRANSDUCTION IN RICE
Ueguchi-Tanaka, Miyako1, Nakajima, Masatoshi2, Ashikari, Motoyuki1, Matsuoka, Makoto1 1Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan; 2Department of Applied Biological Chemistry, The
University of Tokyo, Tokyo 113-8657, Japan;
[email protected]
Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. Recently, we
isolated a GA insensitive dwarf mutant of rice, gid1. GID1 encodes a soluble GA receptor that is mainly located in nuclei
and shares sequence similarity with a hormone sensitive lipase (HSL). Recombinant GID1 (rGID1) protein had a high
affinity only for bioactive GAs, and the mutated rGID1 did not. The Kd value of rGID1 for GA4 was estimated to be
around 10-7M, enough to account for the GA dependency on shoot elongation. Moreover, GID1 bound to SLR1, a rice
DELLA protein, in a GA dependent manner in yeast cells. GID1 overexpression gave a GA-hypersensitive phenotype.
For further study on the GID1 function, we performed the domain analysis using 94 GID1 proteins, which were independently mutated in the manner of alanine scanning. Using these mutated proteins, we analyzed the in vitro GA binding
activity and SLR1 interacting activity.
IPGSA 19th Annual Meeting - Abstracts
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We also examined the interaction between GID1 and SLR1 protein in vivo. GID1-GFP was over-expressed in rice callus and the callus was treated with or without GA4. SLR1 was co-immnoprecipitated with GID1-GFP protein only from
GA4-treated callus, which indicated that GID1 also interacts with SLR1 in vivo in GA dependent manner.
Hormone interaction
P401 CHEMICAL BIOLOGY AS A METHOD OF DISSECTING HORMONE ACTION IN ARABIDOPSIS
McCourt Peter 1, Tsuchiya, Yuichiro 1,2, Delmas, Frederic 1, Petal, Jignasha 1 1 Department of Cell & Systems Biology, University of Toronto, Toronto, Canada M5S 3B2 2 RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku,
Yokohama, JAPAN 230-0045
[email protected]
The scrutiny of the genetic eye has been the extremely successful for understanding how plant hormones are perceived
and transduced their signals. This success mostly stems from the ability to design simple forward genetic screen that
identify mutants that altered the sensitivity of the plant to a particular hormone. We are now, however, entering a new
phase of genetic analysis that requires more sophisticated screening methods to uncover new genes that contribute to a
particular hormone pathway but whose contribution may be subtle. By definition, such genetic screens must be exquisitely sensitive to signaling fluxes and at the same time not dramatically reduce the viability of the organism. The use
of sensitizing screens in which a known hormone response mutant is enhanced or suppressed by a second site mutation
are useful but the nuances of genetic analysis of suppressor and enhance mutations can often make the experimental manipulation of such lines difficult. Recently with the advent of chemical genetics it is possible to screen large libraries of
annotated chemicals for phenotypes that enhance or suppress known genetic mutations. The combination of controlled
application of the chemical with its specificity for a particular genotype, in principle, can identify new components in a
particular signaling pathway. Furthermore, because the chemical can be reduced or withdrawn at any time detrimental
effects can be minimized. In this presentation, I will address some of the issues of adapting chemical genetics to furthering our understanding of hormone signaling in the system of seed development. In particular, I will focus on examples
related to the ABA response pathway.
P402 ROLE OF THE POLARIS PEPTIDE IN ETHYLENE AND AUXIN CROSS-TALK
Lindsey, Keith, Chilley, Paul, Casson, Stuart, Mehdi, Saher
[email protected]
The root is an excellent model system in which to study hormonal interactions. It represents a system in which tight
control of auxin distribution and responses play a central role in patterning cells by the spatial regulation of gene expression. It is the site of synthesis of cytokinin, which can antagonise auxin responses. Root growth and development is also
influenced by ethylene through the effects of this hormone on cell division, expansion and differentiation.
The mechanistic links between these signalling pathways are not clear. To investigate this question, we screened for mutants defective in root growth and development. One gene, identified in a promoter trap screen of Arabidopsis, encodes
a 36 amino acids peptide, and is designated POLARIS (PLS) on the basis of its expression pattern in the embryo and
seedling root. PLS is required for root cell division and elongation, and for lateral root formation. The mutant is defective
in auxin transport and responses, though genetic and physiological studies suggest the PLS peptide acts primarily as a
component of the ethylene signal transduction pathway. We will discuss evidence for a role for PLS and ethylene signalling in modulating auxin responses in the context of root development.
P403 TO GROW OR NOT TO GROW
Achard, Patrick1,2, Hedden, Peter3, Moritz, Thomas4, Van Der Straeten, Dominique5, Genschik, Pascal2, Peng, JinRong6, Fu, Xiangdong7, Harberd, P. Nicholas1 1Department of Cell and Developmental Biology, John Innes Centre,
Norwich NR4 7UJ, United Kingdom; 2Institut de Biologie Moléculaire des Plantes, CNRS UPR2357, Conventionné
avec lʼUniversité Louis Pasteur, 67084 Strasbourg, France; 3Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ,
IPGSA 19th Annual Meeting - Abstracts
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United Kingdom; 4Umeå Plant Science Center, Department of Forest and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden; 5Unit Plant Hormone Signaling and Bio-Imaging, Department of Molecular Genetics, Ghent University, Ledeganckstraat 35, B-9000 Gent, Belgium; 6Institute of Molecular and Cell Biology,
61 Biopolis Drive, Proteos 138673, Singapore; 7The State Key Laboratory of Plant Cell and Chromosome Engineering,
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, P.R. China.
[email protected]
Plants live in fixed locations, and survive adversity by integrating growth responses to diverse environmental signals.
For example, an excess of salt in the soil delays plant life-cycle and decreases yields. Therefore, understanding mechanisms regulating plant growth in adverse conditions is of key strategic importance. Recent reports have highlighted the
importance of growth-repressing DELLA proteins (DELLAs). DELLAs (GAI, RGA, RGL1, RGL2 and RGL3 in Arabidopsis) are nuclear proteins that restrain the cell expansion that drives plant growth. The phytohormone gibberellin (GA)
stimulates growth by promoting the destruction of DELLAs by the proteasome via a specific SCF E3 ubiquitin ligase involving the SLEEPY F-box proteins (SLY1 and SLY2). Mutant GA-deficient plants (ga1-3) are dwarfed and late flowering, whilst treating these plants with GA restores normal growth. Moreover, the stress response phytohormones ethylene
and abscisic acid also control DELLA restraint. For example, ethylene decreases GA levels and thus enhances DELLAmediated plant growth repression. Furthermore, DELLA-accumulation and quantitative modulation of hypocotyl growth
is proportional to light energy dose (the product of exposure duration and fluence rate). Hence DELLAs inhibit hypocotyl
growth during the light phase of the day-night cycle, via a mechanism that is quantitatively responsive to natural light
variability. Finally, the growth restraint conferred by DELLAs is beneficial to plants because it promotes survival under
adverse conditions. Lack of DELLAs GAI, RGA, RGL1 and RGL2 suppressed the salt tolerance conferred by ga1-3.
Therefore we propose that DELLAs permit flexible and appropriate growth in response to environmental changes.
P404 O-GLCNAC MODIFICATION OF PROTEINS ACTS IN MULTIPLE SIGNALING PATHWAYS
Hartweck, Lynn, Gopalraj, Manjula, Kim, Yung-Chun, Oldenhof, Harriëtte, Scott, Cheryl, Matsumoto, Peter, Wu Qiuxia
and Olszewski, Neil Dept. Plant Biology, Univ. of Minnesota, St. Paul, MN 55108 USA
[email protected]
Genetic studies with Arabidopsis thaliana indicate that posttranslation modification of nuclear and cytoplasmic proteins with O-linked N-acetylglucosamine (O-GlcNAc) is involved in gibberellin, cytokinin, light and circadian regulation. This modification is required for embryo development and thus is likely to be involved in additional response pathways. While two O-GlcNAc transferase (OGT) genes, SECRET AGENT and SPINDLY, have been identified, lack of
information about the identities of the modified proteins is a constraint to understanding how this modification functions
in plants. We are using two approaches to identify the O-GlcNAc-modified proteins of arabidopsis. An E. coli-based coexpression system was used to identify OGT substrates and map the modifications. Several of the substrates were previously identified as potential substrates through genetic studies or yeast two-hybrid assays. A number of the substrates are
transcription factors and some are known to participate in gibberellin or light signaling pathways. We have also initiated
the use of mass spectrometry-based approaches to identify and map modifications on proteins isolated from plants. Experiments assessing the role of this modification by characterizing the functionality of non-modifiable mutant proteins in
transgenic plants are in progress.
This work is supported by grants from the NSF (MCB-0516690), DOE (DE-FG02-02ER15353) and BARD (US-389606).
P405 LIPOCHITIN-OLIGOSACCHARIDES AND CYTOKININ GIVE PLANTS THE NOD
Szczyglowski Krzysztof, Jeremy Murray1, Karas Bogumil, Held Mark, Amyot Lisa, and Kosuta Sonia. Agriculture and
Agri-Food Canada, SCPFRC, London, ON N5V 4T3 Canada; [email protected] 1Current address: Plant Biology
Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401 USA
[email protected]
IPGSA 19th Annual Meeting - Abstracts
10
Biologically active oligosaccharides (oligosaccharins) are best known as elicitors of non-host resistance but their
involvement in modifying plant growth and development has also been documented. Legumes have evolved a specific
response to Rhizobium-encoded lipochito-oligosacharides known as nodulation factors (NFs). Instead of evoking plant
defences, NF signalling leads to the establishment of a symbiotic relationship. In addition to playing an essential role in
the mechanism of selective recognition which allows the compatible bacteria to enter the host root, the NF-dependent response pathway acts to stimulate the organogenesis of symbiotic organs, root nodules. Recent results provide unequivocal support for the existence of cross-talk between NF and cytokinin during initiation of nodule primordia organogenesis
1-3. How perception and early plant responses to NFs may lead to cytokinin signalling, nodule organogenesis and establishment of functional nitrogen fixing nodules will be discussed.
1. S. Gonzalez-Rizo, M. Crespi, F. Frugier, Plant Cell, 18, 2680 (2006).
2. J. Murray et al., Science 315, 101 (2007).
3. L. Tirichine et al., Science 315, 104, (2007).
Minisymposia
Fruit and Seed Development
MS101 ETHYLENE RECEPTOR TURNOVER AND THE CONTROL OF FRUIT RIPENING
Klee, Harry, Kevany, Brian, Tieman, Denise
hjklee@ufl.edu
Fruit ripening in tomato requires integration of developmental cues with the phytohormone ethylene. Although we
know much about the role of ethylene in initiating ripening in climacteric fruits, much less is known about the role of
ethylene in fruit development prior to ripening. For example, there is strong evidence that fruits measure cumulative
exposure to ethylene throughout development and use this measurement as a clock to determine when to initiate the ripening program. How the fruit measures ethylene exposure is not known.
The multigene ethylene receptor family negatively regulates ethylene signal transduction by suppressing ethylene responses in the absence of the hormone. Tomato has seven receptor genes. Our results indicate that a reduction in the
levels of either of two receptors, LeETR4 or LeETR6, causes an early ripening phenotype. Gene expression measurements indicate that there is an approximately ten-fold increase in overall receptor mRNA levels at the onset of ripening.
This result is puzzling since the receptors are negative regulators of ethylene responses and fruit ripening is dependent
upon ethylene action. However, analysis of receptor proteins during ripening revealed that there is rapid degradation of
receptor proteins in the presence of ethylene and that degradation likely occurs through the 26S proteasome-dependent
pathway. Ethylene exposure of immature fruits depletes receptor proteins and causes earlier ripening. The results are
consistent with a model in which receptor levels modulate timing of the onset of fruit ripening by measuring cumulative
ethylene exposure.
MS102 REPRESSION OF THE LEC1/B3 NETWORK FOR REGULATION OF EMBRYO DEVELOPMENT BY THE VAL B3
DOMAIN TRANSCRIPTION FACTORS.
Donald R. McCarty1 and Masaharu Suzuki1. 1Plant Molecular and Cellular Biology, University of Florida, Gainesville,
FL 32611.
drm@ufl.edu
The B3 domain proteins are a diverse family of plant specific DNA binding factors that have key roles in hormone signaling and plant development. We have focused on the role of the VP1/ABI3 family of B3 factors in seed development.
In Arabidopsis, the VP1/ABI3 family of B3 factors includes ABI3, FUS3, LEC2 and three VP1-ABI3-Like (VAL) genes.
ABI3, FUS3 and LEC2 B3 proteins function with the LEC1 and L1L HAP3-related factors in a regulatory network controlling embryogenesis and maturation. The VAL B3 proteins form a sister clade to the ABI3/FUS3/LEC2 sub-family and
are uniquely equipped with PHD-like and CW domains often associated with chromatin factors. Our findings highlight a
IPGSA 19th Annual Meeting - Abstracts
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striking functional symmetry between two clades within the VP1/ABI3 B3 group that control the fundamental transition
between embryo and plant development. Analysis of double and triple mutant genotypes indicates that the VAL genes are
essential for repression of the LEC1/B3 network during early seedling development. An embryonic seedling phenotype
of the val1 val2 double mutant is associated with de-repression of the LEC1, L1L, ABI3 and FUS3 genes and massive upregulation of embryogenesis related gene expression. Key features of this phenotype resemble the conditional phenotype
of the PICKLE (PKL) mutant induced by treatments that block GA synthesis. PKL encodes a SNF/SWI2 chromatin factor implicated in chromatin remodeling. Consistent with the hypothesis that VAL and PKL genes function in a common
pathway, we show that GA synthesis inhibitors also induce embryonic phenotypes in val1 single mutant seedlings.
MS103 USE OF MUTANTS AND GENOMICS TO IDENTIFY GENES REGULATING ETHYLENE RESPONSE DURING
TOMATO FRUIT RIPENING
James Giovannoni, Boyce Thompson Institute for Plant Research and USDA-ARS, Tower Road, Ithaca, NY 14853.
[email protected]
Considerable knowledge regarding general mechanisms of ethylene signal perception and transduction has been
developed through analysis of Arabidopsis thaliana. Identification and characterization of homologous genes in tomato
has provided insight into both evolutionary conservation of ethylene signal transduction machinery and how some constituents have evolved to play more specific roles in fruit development and ripening. Little is known regarding ethylene
signaling activities that are specific to fruit development and ripening. Characterization of the Green-ripe (Gr) locus revealed a novel gene which, when over-expressed, specifically represses ethylene response in a subset of plant tissues with
the most extreme response in fruits. Both mutant Gr fruit that carry a mutation resulting in GR over-expression, and transgenic lines expressing GR under the direction of the CaMV35s promoter, yield fruit which are ethylene insensitive and
fail to ripen. GR is a member of a small multi-gene family in tomato and is the only member of this family that can confer
the ethylene insensitive phenotype in transgenic fruit. The homologous RTE1 gene of Arabadopsis does not confer ethylene insensitivity in transgenic tomato fruit suggesting that GR has a novel function relative to fruit ethylene response.
Progress in characterization of GR function will be presented in addition to functional analysis of ripening-related genes
associated with ethylene response and identified via transcriptome analysis of fruit development and ripening.
MS104 ABA SIGNALING NETWORKS IN SEEDS AND SEEDLINGS
Finkelstein, Ruth, Lynch, Tim, Reeves, Wendy, Garcia, Emily, Molecular, Cellular and Developmental Biology Department, UC Santa Barbara, Santa Barbara, CA 93117, USA
fi[email protected]
Seed maturation and subsequent germination and seedling growth are regulated in part by abscisic acid (ABA) and
several ABA-insensitive (ABI) loci, some of which encode transcription factors. Studies of this signaling network have
revealed extensive cross-regulation among the ABI and related factors, and identified a novel plant-specific family of
proteins that interact with ABI5 and related bZIP factors to modify ABA and abiotic stress sensitivity.
Over-expression of these ABI factors is sufficient to confer hypersensitivity to ABA and other stresses even though the
ABI factors directly regulate less than 10% of all ABA-regulated genes, suggesting that their targets are central components of the stress response. These targets include presumed desiccation protectants and additional regulators. Surprisingly, mutations in many ABI-regulated or –interacting regulatory factors result in ABA hypersensitivity, suggesting that
the networks include both positive and negative feedback loops.
MS105 HORMONAL REGULATION OF FRUIT-SET AND PISTIL SENESCENCE IN ARABIDOPSIS THALIANA
Perez-Amador, Miguel A, Dorcey, Eavan, Carbonell, Pablo, Urbez, Cristina, Granell, Antonio, Blázquez, Miguel Angel,
Carbonell, Juan. Instituto de Biologia Molecular y Celular de Plantas (CSIC). Avda de los Naranjos s/n, 46022 Valencia,
Spain; [email protected]
In Arabidopsis, fruit-set can be initiated by hormones generated after fertilization but their hierarchy and signalling is
unknown. Our data indicates that fruit-set is initiated by auxin accumulation in ovules, which triggers auxin signalling,
GA biosynthesis and signalling. GA application bypasses auxin accumulation in ovules, while 2,4-D and NPA treatment
mimics pollination-related signalling events. If no fruit development is initiated, the pistil becomes senescent, with the
IPGSA 19th Annual Meeting - Abstracts
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style and ovules being degraded as soon as 2 days post-anthesis. Ethylene signalling controls ovule senescence, which
parallels loss in response of the pistil to external GA application. This data suggest that a functional ovule is needed to
initiate fruit development.
MS106 AN AUXIN RESPONSE FACTOR AS A NEGATIVE REGULATOR OF FRUIT DEVELOPMENT IN TOMATO
de Jong, Maaike; Feron, Richard; Wolters-Arts, Mieke; Mariani, Celestina; Vriezen, Wim H. Dept. of Plant Cell Biology,
Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, the Netherlands
[email protected]
It is well established that phytohormones as auxin, gibberellin, cytokinin, abscisic acid and ethylene are required
for the normal fruit developmental program. However, the hormonal regulatory mechanisms that regulate fruit set are
largely unknown. To unravel these mechanisms, we performed a transcriptome analysis of tomato ovaries (Solanum
lycopersicum cv. Moneymaker). One of the identified genes was down regulated in the ovules within 48 hours after pollination, suggesting that it might have a regulatory role in fruit set. The deduced protein sequence of this gene contains
all conserved domains typical for Auxin Response Factor (ARF) protein family members and shares high homology with
Arabidopsis NPH4/ARF7. ARFs are encoded by a gene family of transcription factors that control specific auxin dependent growth- and developmental processes. Transgenic plants in which the tomato ARF is silenced by means of RNAi
approach produce seedless (parthenocarpic) fruits. This phenotype, together with expression analysis of the tomato ARF
gene suggest that the tomato ARF inhibits fruit set in unfertilized ovules and that it functions as a negative regulator of
fruit set until pollination and fertilization have taken place.
Shoot Development
MS201 GNOM AND BODENLOS - AUXIN MEDIATORS IN ARABIDOPSIS EMBRYOGENESIS
Gerd Jürgens ZMBP, Entwicklungsgenetik, Univ. Tübingen, Auf der Morgenstelle 3, 72076 Tübingen, Germany
[email protected]
Plant embryogenesis establishes a basic body organisation, including an apical-basal axis with the two primary meristems of shoot and root at opposite ends. The GNOM gene encodes a brefeldin A (BFA)-sensitive guanine-nucleotide
exchange factor for an ARF GTPase (ARF-GEF) that mediates recycling of the putative auxin efflux carrier PIN1 to the
basal plasma membrane, which is required for both embryo axis formation and lateral root initiation (Geldner et al., 2003,
Cell 112, 219; Friml et al., 2003, Nature 426, 147; Geldner et al., 2004, Development 131, 389). A major focus of our
current research is to identify mechanisms of ARF-GEF action in PIN1 recycling. The BODENLOS (BDL) gene encodes
auxin-response regulator IAA12, which is thought to interfere with the activation of auxin-inducible target genes by the
auxin-response factor ARF5 encoded by the MONOPTEROS (MP) gene.
Although both genes are involved in the specification of the hypophysis, which initiates the formation of the primary
root meristem, they are expressed in the adjacent proembryo cells rather than the hypophysis itself, suggesting that
auxin-dependent cell-cell communication plays a role in root meristem initiation (Hamann et al., 2002, Genes & Dev.
16, 1610; Weijers et al., 2006, Dev. Cell 10, 265). We are currently investigating mechanisms underlying BDL action in
early embryogenesis.
MS202 AUXIN-MEDIATED LOCALIZED GROWTH UNDERPINS DIVERGENCE OF LEAF FORMS OF C. HIRSUTA AND
A. THALIANA.
Michalis Barkoulas, Angela Hay, Evagelia Kougioumoutzi and Miltos Tsiantis
[email protected]
Plant leaves show a considerable degree of morphological diversity and hence present attractive opportunities to investigate evolution of form. The developmental basis for generation of different leaf forms remains largely unclear. Here we
investigate this problem by studying the processes that distinguish development of two related species A.thaliana which
has simple undivided leaves and C. hirsuta that elaborates a subdived leaf blade comprising individual leaflets.
IPGSA 19th Annual Meeting - Abstracts
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We report that in C.hirsuta leaflet formation depends on de novo action of leaflet initial cells that are recruited in response
to maxima of auxin, a small hormone also known to facilitate leaf initiation. Action of KNOX proteins known to promote
leaflet initation is contigent on the ability to organise such auxin maxima. Thus, evolutionary tinkering with the activity
of a mechanism facilitating leaf initiation contributes to diversification of leaf form by facilitating establishiment of novel
growth centers in developing leaf primordia.
MS203 AUXIN TRANSPORT PATTERNING IN THE SHOOT APICAL MERISTEM
Heisler, Marcus1, Ohno, Carolyn1, Jönsson, Henrik2, Mjolsness, Eric3 and Meyerowitz, Elliot M.1 1California Institute
of Technology, USA; 2Lund University, Sweden; 3University of California, Irvine USA.
[email protected]
The plant apical meristem is able to maintain its complex structure and periodically generate lateral organs despite
the continuous generation and exodus of cells from its flanks. In order to understand how this is achieved, detailed
knowledge of its gene expression and growth patterns is required. We have developed new imaging and computational
techniques for analysing and obtaining this data using in vivo confocal microscopy. By simultaneously imaging multiple
fluorescent marker proteins we have discovered novel spatial and temporal relationships that give insight into auxin related patterning processes taking place during primordium development. Causal investigation into these relationships is
underway and our latest results will be presented.
MS204 TOWARDS A VIRTUAL MERISTEM
J.Traas1, S. Stoma2, J. Chopard2, O. Hamant 1, C. Godin2 1Laboratoire de Reproduction et Développement des Plantes,
UMR INRA/CNRS/ENS, 46 allée dʼItalie, 69364 Lyon Cedex 7, France 2 Team Virtual Plants UMR DAP CIRAD, TA
A-96/02, 34398 Montpellier Cedex 5, France
[email protected]
During the last decade an impressive body of knowledge on meristem function has been generated. This concerns
information on the genes involved, their expression patterns, cell differentiation, cell division patterns, etc. The complexity of these data is such, that an integrated view of our current knowledge on meristem function is no longer possible.
Therefore, we are currently developing adapted mathematical and informatics approaches to integrate the knowledge and
to advance the level of understanding in the field. Our efforts are oriented in two directions. First we are trying to link
the gene regulatory and signalling networks to specific morphogenetic events. Although much is known on the overall
composition of these networks, almost nothing is known on the way in which they interact with the structural elements
of the cells and tissues to create particular shapes. It is therefore important to link specific gene activities to precisely
quantified growth characteristics of the cells and tissues in which they are expressed.
In parallel to this largely descriptive effort, we are also developing modelling tools.
To formulate and test hypotheses on spatial aspects such as flows of signalling molecules, strain within tissues, and the
role of gene products in the spatial control of cell proliferation, we are creating a model in the form of a virtual meristem.
This model will integrate as much spatial, dynamic and quantitative information as possible.
MS205 THE ROLE OF CYTOKININ RESPONSE FACTORS IN PLANT DEVELOPMENT
Aaron M. Rashotte 1* and Joseph J. Kieber 1 1 University of North Carolina at Chapel Hill, Biology Department, Chapel
Hill, NC 27599 * Current address Auburn University, Department of Biological Sciences, Auburn, AL 36849
[email protected]
Cytokinin is an essential plant hormone involved in numerous aspects of plant growth and development. Recently we
have shown that six closely related Arabidopsis AP2/ERF transcription factor genes lacking any ascribed function are
novel components of the cytokinin signaling pathway. These Cytokinin Response Factor (CRF) proteins show a rapid
nuclear localization (5-10min) after the addition of cytokinin that requires the AHK cytokinin receptors.
Exactly how the CRF function in the regulation of an initial cytokinin response is under investigation. We will present
work on several phenotypic analyses of single, double and multiple knockout CRF mutants that reveals that CRF genes
have overlapping function and that they are involved in cotyledon, leaf and embryo development. Additionally, we examine the link between CRFs and cytokinin regulated developmental processes.
IPGSA 19th Annual Meeting - Abstracts
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MS206 DIRECT CONTROL OF SHOOT MERISTEM ACTIVITY BY A CYTOKININ ACTIVATING ENZYME
Junko Kyozuka1, Takashi Kurakawa1, Nanae Ueda2, Erika Sasaki1, Kaoru Kobayashi1, Masahiko Maekawa3, Mikiko
Kojima2, Yasuo Nagato1, Hitoshi Sakakibara2 1Graduate School of Agriculture and Life Sciences, University of Tokyo,
Yayoi, Bunkyo, Tokyo 113-0032, Japan; 2RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045, Japan; 3Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan
[email protected]
The growth of plants depends on the continuous function of the meristems. Shoot meristems are responsible for all the
post-embryonic aerial organs, such as leaves, stems, and flowers. It has been assumed that the phytohormone cytokinin
plays a positive role in shoot meristem function. Here, we report a novel regulation of meristem activity, which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice is required to maintain
meristem activity and its loss of function causes a severe reduction of meristem size and premature termination of the
shoot meristem. LOG encodes a novel cytokinin activating enzyme that works in the final step of bioactive cytokinin
synthesis. LOG mRNA is specifically localized in shoot meristem tips, indicating the activation of cytokinins in a specific
developmental domain. We propose the fine-tuning of concentrations and the spatial distribution of bioactive cytokinins
by a cytokinin activating enzyme as a novel regulation of meristem activity.
MS207 THE EPIDERMIS BOTH DRIVES AND RESTRICTS GROWTH – A LESSON FROM BRASSINOSTEROIDS
Sigal Savaldi-Goldstein1 and Joanne Chory 1,2 1 The Salk Institute, La Jolla, 92037, CA, USA 2 Howard Hughes Medical Institute
[email protected]
The growth process involves coordination of cell division and expansion, which largely depends on developmental
programs governed by plant hormones. Brassinosteroids (BRs) are essential for plant development. In the absence of BR
production or perception, plants exhibit extreme dwarf stature which is mainly attributed to defects in cell expansion.
Thus far, key players in the BR signaling and biosynthesis pathway have been cloned, but their sites of action responsible
for the control of plant growth remains unknown. We therefore dissected the interaction and contribution of the outer and
inner layers to BR mediated growth responses. To this end we used targeted expression of BR signaling, biosynthesis and
catabolic components in their corresponding mutant and wild-type background. Our data suggest that the epidermis, but
not the vasculature, is sufficient to drive shoot growth and that shoot growth depends on available active BRs or active
BR signaling in the epidermis. Our current working model will be presented and discussed.
Senescence
MS301 ATMYB2 REGULATES WHOLE PLANT SENESCENCE BY SUPPRESSING CYTOKININ ANABOLISM
Susheng Gan, Yongfeng Guo Department of Horticulture, Cornell University, Ithaca, NY 14853-5904, USA
[email protected]
In order for a monocarpic plant to complete its life history, axillary buds need to be suppressed permanently. The
buds are initially inhibited by shoot apical meristem-produced auxin, a mechanism known as apical dominance. How the
buds are suppressed after the shoot apical meristem becomes arrested is unknown. Here we report an AtMYB2-regulated
post-apical dominance mechanism by which Arabidopsis inhibits outgrowth of axillary buds. When the main shoot apical
meristem is arrested, AtMYB2 is expressed in the compressed basal internode region of Arabidopsis to suppress genes
encoding cytokinin-synthesizing isopentenyltransferases. Cytokinins are required for the axillary bud outgrowth at late
stages of plant development. atmyb2 knockout lines contain higher levels of cytokinins and display a bushy phenotype.
AtMYB2 promoter-directed cytokinin oxidase 1 in the knockouts reduces the endogenous cytokinin levels and restores
the bushy phenotype to wild type.
MS302 MOLECULAR GENETIC UNDERSTANDING OF THE ROLE OF PLANT HORMONES IN REGULATING LEAF
SENESCENCE
Hyo Jung Kim1, Ja Choon Koo2, Jin Hee Kim1, In Chul Lee1, Seung Hee Choi1, Hong Gil Nam1, 1Division of Molecular Life Sciences and the I-BIO graduate school, Pohang University of Science & Technology, Pohang, Kyungbuk
790-784, Republic of Korea; 2Department of Biological Education, Chonbuk National Univ. Chonju, Chonbuk, Korea
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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Leaf development ends with senescence, a genetically programmed degeneration process that ultimately leads to leaf
death. Although occurring in an age-dependent manner, leaf senescence is greatly affected by various endogenous and
environmental signals to attune the life span of leaves to optimized fitness of plants. Among the endogenous developmental signals, plant hormones appear to play important roles during leaf senescence, including the initiation, progression,
and terminal phases of leaf senescence. Through forward and reverse genetic approaches, we are trying to unveil the roles
of plant hormones in regulating leaf senescence.
Using forward genetic approach, we initially found that ore12-1 mutant has increased leaf longevity due to a missense
mutation in AHK3, one of the three-cytokinin receptors in Arabidopsis. A loss-of-function mutation in AHK3 but not
of the other cytokinin receptors conferred a reduced sensitivity to cytokinin in cytokinin-dependent delay of leaf senescence, and abolished cytokinin-dependent phosphorylation of ARR2. Furthermore, ARR2 underwent gradual degradation through a proteasome-dependent pathway. We, thus, suggest that a specific phosphorelay cascade from AHK3 to
ARR2 positively controls cytokinin-mediated leaf longevity along with a proteasome-dependent desensitization mechanism. The screening for suppressors of ore12-1 mutant is underway for further understanding of the role of cytokinin
signaling in leaf senescence.
Using a PCR-based subtractive hybridization, we found that RPK1 encoding an ABA-inducible receptor-like kinase is
highly induced during natural leaf senescence. The rpk1 knock-out mutants exhibited the extended leaf longevity with
significant delays in cell death process during natural leaf senescence. In particular, ABA-induced artificial senescence
and cell death was dramatically impaired in the rpk1 mutants. Microarray analysis of RPK1-inducible transgenic plant
indicated RPK1 functions at the upstream of stress-related and senescence-associated signaling pathway. These results
may provide a clue to an integration mechanism of leaf senescence and environmental stress.
MS303 THE IMPACT OF TEMPORAL AND SPATIAL REGULATION OF THE IPT GENE EXPRESSION BY VARIOUS
PROMOTERS ON LEAF SENESCENCE AND WATER STRESS TOLERANCE
Shimon Gepstein1 Banner Idit1 Eduardo Blumwald2 and Amira Gepstein1 1Faculty of Biology, Technion-Israel Institute
of Technology, Haifa, Israel;2Department of Plant Sciences University of California Davis, CA 95616
[email protected]
A most dramatic transgenic intervention leading to a delay senescence phenotype concerns the manipulation of levels
of endogenous cytokinins. An effective current approach is the autoregulation of cytokinins synthesis, based on a specific
promoter of a senescence gene - SAG12. Surprisingly, this autoregulatory system did not show delay of leaf senescence
in Arabidopsis. Two promising strategies for delaying senescence in tobacco and Arabidopsis plants are presented. The
autoregulated biosynthesis of cytokinins, mediated by the expression of a isopentyltransferase (IPT) gene driven by an
inducible early-senescence/late maturation promoter (SARK, senescence-associated receptor kinase), resulted in the
remarkable delay of senescence of tobacco plants. Additional approach is the estrogen-inducible XVE system of the IPT
gene for the production cytokinins in Arabidopsis transgenic plants. These transgenic plants displayed altered seedlings
development and delayed leaf senescence.
Most surprisingly, the tobacco transgenic plants also displayed water stress tolerance as reflected by vigorous growth
after a severe drought (18 days without watering). The transgenic plants retained photosynthetic activity during drought
and did not show chlorophyll degradation. After rewatering, the plants recovered photosynthetic activity and active
growth. Our results showed that plants expressing SARK::IPT, cytokinins were synthesized in the whole plant and transported to the bottom.
MS304 SENESCENCE-INDUCIBLE EXPRESSION OF ISOPENTENYL TRANSFERASE EXTENDS LEAF LIFE AND INCREASES DROUGHT STRESS RESISTANCE IN CASSAVA
Peng Zhang1,2, Wenquan Wang3, Genliang Zhang3 and Wilhelm Gruissem1,2 1 Institute of Plant Sciences, ETH Zurich,
Universitätstrasse 2, 8092 Zurich, Switzerland 2 Shanghai Center for Cassava Biotechnology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Rd, 200032
Shanghai, China 3 Institute of Tropical Biosciences and Biotechnology, CATAS, Haikou 571101, China
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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Cassava (Manihot esculent Crantz) can withstand prolonged abiotic stress and survives by shedding its leaves. We
have developed cassava lines that express the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene, which encodes a key enzyme for cytokinin biosynthesis, under control of the senescence-inducible Arabidopsis SAG12 promoter.
With these lines we want to test if cassava leaf senescence can be delayed via an autoregulatory senescence inhibition
mechanism. Insertion of the pSAG12::ipt cassette was confirmed in seven cassava plant lines using PCR and Southern
analyses. In five lines we could detect low levels of ipt expression in mature leaves. Detached mature leaves from in vitro
and greenhouse-grown pSAG12::ipt lines 529-28 and 529-48 retained high levels of chlorophyll compared to wild type
leaves after dark-induced senescence. These lines also showed significant delays in leaf senescence after drought treatment . Expression of ipt was increased in older leaves of drought-stressed 529-28 plants. During development leaves of
pSAG12::ipt lines retained higher levels of chlorophyll, total protein and Rubisco. The pSAG12::ipt lines also showed
early storage root bulking in comparison with wild-type plants.
Lines 529-28 and 529-48, together with wild-type TMS60444 plants, were further evaluated by growing in vitro shoot
cultures in an experimental field in Haikou, China during the 2006 growth season. During the 10-month field experiment, leaf senescence was significantly delayed line 529-28 plants compared to wild-type and 529-48 plants. Although
there was no significant difference in storage root numbers and yield among wild-type and transgenic lines, storage roots
of 529-28 plants showed a strong reduction in post-harvest physiological deterioration. Further investigation of agronomical traits under different field conditions is currently underway using stem cuttings harvested from first-phase field
experiment.
MS305 ANTIOXIDANT RESPONSE OF (DACTYLIS GLOMERATA L.) TO THE CYTOKININ BAP DURING GROWTH
AND SENESCENCE
Wilson-García Claudia Y. (1), Zavaleta-Mancera Hilda A. (1), López-Delgado Humberto A.(2), Hernández-Garay Alfonso (1), Delgado-Alvarado Adriana (1). (1) Colegio de Postgraduados Km. 36.5 Carr. México-Texcoco, Montecillo,
Edo. Mex. 56230 México ([email protected]) (2) Programa Nacional de Papa, INIFAP, Sedagro, Metepec, 52140,
Edo. Mex. México.
[email protected]
Leaf senescence promotes the production of reactive oxygen species ROS (1O2, O-2, OH- and H2O2), which damage the cell. In forage grasses, senescence reduces it nutritional quality. The senescence delaying effect of the cytokinin
6-bencylaminopurine (BAP) is well documented in several species but its effect on forage grasses is poorly understood.
Dactylis glomerata plants were weekly (seven weeks) sprayed with a solution of BAP. Foliar growth and senescence
were monitored during 16 weeks, measuring the green length of the leaf. The levels of hydrogen peroxide and ascorbic
acid (Vitamin C) and the activity of ascorbate peroxidase (APX) were determined on young, mature and senescent tissue. Growth and delay of senescence was significantly increased on BAP treated plants with respect to the controls. The
APX and vitamin C levels were increased on BAP treated plants as an antioxidant response to the growth regulator. The
delaying senescence effect was accompanied by high contents of soluble protein. The role of BAP on the promotion of
antioxidants during leaf development is discussed.
The present research was supported by CONACYT- 43866-Z granted to Zavaleta-Mancera
MS306 CHARACTERIZATION OF A NON-YELLOWING MUTANT AND ISOLATION OF ATNYE1 ENCODING A NOVEL
PROTEIN PRIMARILY MODULATING CHLOROPHYLL DEGRADATION DURING SENESCENCE IN ARABIDOPSIS
Benke,Kuai, Guodong,Ren, Kun,An, Yang,Liao, Xiao,Zhou, Yajun,Cao, Xiaochun,Ge, State Key Lab.Genet.Engineering, School of Life Sci. Fudan University, Shanghai 200433, P.R.China
[email protected]
A dramatic increase of chlorophyll (Chl) degradation occurs during senescence of vegetative plant organs and fruit
ripening. Although the biochemical pathway of Chl degradation has long been proposed, little is known about its regulatory mechanism. Identification of Chl degradation-disturbed mutants and subsequently isolation of responsible genes
would greatly facilitate the elucidation of the regulation of Chl degradation. Here, we describe a non-yellowing mutant
of Arabidopsis, nye1-1, in which 50% Chls was retained, compared to less than 10% in the wild-type, at the end of a six-
IPGSA 19th Annual Meeting - Abstracts
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day dark incubation. Nevertheless, neither photosynthesis- nor senescence-associated process was significantly affected
in nye1-1. Characteristically, no detectable accumulations of either chlorophyllide a or pheophorbide a were observed,
and no obvious changes in the transcript levels of AtCLHs (A.thaliana chlorophyll-chlorophyllido hydrolases) and PaO
(pheophorbide a oxygenase) in the senescing leaves of nye1-1. However, a significant reduction in PaO activity was
detected in nye1-1. Reciprocal crossings revealed that the mutant phenotype was caused by a monogenic semi-dominant
nuclear mutation. We have identified AtNYE1 by positional cloning. However, no clue about its biochemical role is revealed from its DNA and amino acid sequences. Dozens of its putative orthologs, predominantly appeared in higher plant
species, were identified from the Database, some of which have been indicatively associated with Chl degradation in a
few crop species. Real-time RT-PCR analysis showed that AtNYE1 was drastically induced by senescence signals, however, its constitutive over-expression, driven by 35S promoter, could result in albino seedlings. These results collectively
indicate that NYE1 may normally accelerate Chl degradation during senescence by maintaining sufficient PaO activity.
microRNAs in Hormone Signaling
MS401 AUXIN RESPONSE FACTORS AND A MICRO-RNA THAT REGULATE FLOWER DEVELOPMENT
Jason W. Reed, Miin-Feng Wu, Paul H. Reeves, and Punita Nagpal
[email protected]
In flowering plants, male and female development must be coordinated with each other for effective pollination to
occur. ARF6 and ARF8, two closely related transcription activators of the Auxin Response Factor (ARF) family in the
model plant Arabidopsis, regulate the transition from immature buds to mature fertile flowers and coordinate male and
female reproductive development. In arf6 arf8 double mutants, flower buds arrest and are both male- and female-sterile.
The male sterility arises from loss of jasmonic acid production leading to failure of anther dehiscence. Female sterility is
at least in part due to poor pollen germination or pollen tube growth in mutant gynoecia. arf6 and arf8 single mutants are
less fecund than wild-type plants because they have delayed stamen development and consequently self-pollinate inefficiently. We are studying regulatory pathways both upstream and downstream of ARF6 and ARF8.
Several factors regulate ARF6 and ARF8 abundance or activity. The microRNA miR167 targets both ARF6 and ARF8
transcripts for turnover. We have determined the function of miR167 by making transgenic plants with the miR167 target
site mutated in either ARF6 or ARF8. These mARF6 and mARF8 plants are both male- and female-sterile. Anthers grow
larger than normal and fail to dehisce. Ovules lack outer (and sometimes also inner) integument growth. These phenotypes correlate with expansion of the ARF6 or ARF8 expression domain, indicating that miR167 functions to pattern
gene expression in anthers and ovules. We are currently studying whether auxin, light, or the circadian rhythm regulates
ARF6 and ARF8 activity.
To dissect the transcriptional network regulated by ARF6 and ARF8, we are analyzing gene expression in mutant flowers, and we are screening for additional mutants with flower maturation phenotypes. In a screen for enhancers of an arf6
single mutant, we isolated mutations in MYB21, which cause a spectrum of phenotypes similar to those of arf6 and
arf8 mutations, including decreased stamen filament elongation, anther dehiscence and volatile emission. Expression of
MYB21 and three other closely related MYB genes depends on ARF6 and ARF8, and higher-order myb mutants have
stronger phenotypes than myb21 single mutants, indicating that these MYB proteins are downstream effectors of ARF6
and ARF8 action.
MS402 ROLE OF AUXIN AND MIRNA IN REGULATING ROOT CAP DEVELOPMENT AND GRAVITROPISM
Wen-Juan Cai, Jiang-Wei Wang, Nan Yu, Xiao-Ya Chen National Key Laboratory of Plant Molecular Genetics, Institute
of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200032, China
[email protected]
A number of microRNA target genes encode proteins of auxin signaling pathways, linking the microRNAs to hormone
network. In previous research we showed that the Arabidopsis thaliana auxin response factors ARF10 and 16, targeted
by microRNA160 (miR160), control root cap cell differentiation. Results obtained from the 35S::MIR160 and the arf103arf16-2 double mutant plants demonstrated that ARF10 and 16 are indispensable for balancing the cell division and
IPGSA 19th Annual Meeting - Abstracts
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differentiation in root cap region, and auxin cannot bypass the two ARFs to promote columella cell differentiation. In
root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. Furthermore, we found that regulation of ARF10 and ARF16 by miR160 is essential for
maintaining normal development and growth of Arabidopsis plants. Having identified ARF10 and ARF16 as the key controller of root cap development, it becomes interesting to isolate factors that act down-stream of the two auxin response
factors in the root cap pathway.
We have isolated a mutant with EMS mutagenesis in the background of 35S::miR160c, which shows recovered gravitropism with abnormally arranged starch granules; however, the root cap still displays deviant cell division and cell
morphology, suggesting that the function of ARF10 and ARF16 are only partially resumed. It gives us a clue that there is
a factor that may negatively regulate the formation of starch granules. We¡¯ll provide data showing that this factor functions positively in auxin response. Further research will be focused on identifying the candidate gene(s) and dissecting
the signaling pathway which acts downstream of miR160 and its target genes.
MS403 THE REGULATION OF VEGETATIVE PHASE CHANGE BY MIRNAS AND TA-SIRNAS
R. Scott Poethig
[email protected]
Many organisms undergo programmed changes in their morphology and physiology during development. In plants,
this occurs most obviously during the transition from vegetative to reproductive growth, when the shoot stops making
leaves and begins to manufacture flowers or other types of reproductive structures. This event is preceded by the transition from a juvenile to an adult phase of vegetative development (vegetative phase change), which is marked by changes
in leaf anatomy and morphology and by an increase in reproductive competency. Genetic analysis of vegetative phase
change in maize and Arabidopsis has revealed that this phenomenon is regulated by several endogenous small (21 nt)
RNAs.
These include the trans-acting siRNA, tasi-ARF, and two miRNAs—miR156 and miR172. tasi-ARF represses the expression of the auxin-related transcription factors ETTIN/ARF3 and ARF4. miR156 and miR172 have complementary
temporal expression patterns and target different families of transcription factors. miR156 targets members of the SBP/
SPL family whereas miR172 targets AP2-like transcription factors. These genes operate in a pathway that regulates epidermal identity and flowering time. ETT/ARF3 and ARF4 regulate the amplitude of SPL gene expression, but are not part
of the switch mechanism. The structure and developmental functions of the pathways in which these genes are involved
will be discussed.
MS404 THE TOMATO WIRY SYNDROME RESULTS FROM IMPAIRED SILENCING OF SLARF3/4
Eshed, Yuval, Pekker, Irena, Peled, Dror, Amsellem, Ziva, Saban, Moti, Alvarez, John, Yifhar, Tamar Dep. Plant Sciences, Weizmann Institute of Science, Rehovot, Israel
[email protected]
The auxin-response factor ARF3/ETT and its closest homolog, ARF4, are essential components of the KAN signaling
pathway, which specifies abaxial fate early in organ development. Recently, ARF3 and ARF4 transcripts were shown to
be co-negatively regulated by trans-acting siRNAs (ta-siRNA) derived from the TAS3 gene. To evaluate the in planta
consequences of disrupting ta-siRNA regulation, we constructed ta-siRNA-resistant versions of tomato ARF3 and ARF4.
While misexpression of the sensitive ARF3 and ARF4 versions resulted in a nearly wild type-looking plants, siRNA
resistant versions gave rise to plants with filamentous organs, a phenotype strikingly similar to the known wiry tomato
mutants.
We show that three non-allelic loci in tomato, wiry, wiry1 and wiry4 encode for the RDR6, ARGONAUTE7, and SGS3
homologs, respectively, all of which are components of the TAS3 ta-siRNA biogenesis pathway in Arabidopsis. We
demonstrate that in the tomato wiry mutants, the ta-siRNAs derived from the TAS3 gene are absent leading to the accumulation of target transcripts. In agreement, simultaneous down-regulation of ARF3/4 using a synthetic microRNA
could rescue the wiry phenotype. Thus, in tomato, posttranscriptional regulation of ARF3/4 is crucial for organ asymmetry patterning.
IPGSA 19th Annual Meeting - Abstracts
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MS405 MICRO-RNAS FROM PHASEOLUS VULGARIS IN RESPONSE TO ABA AND DROUGHT
Reyes, Jose Luis, Arenas, Catalina, Pérez, Beatriz, Contreras, Karina, Covarrubias Alejandra. Instituto de Biotecnologia,
UNAM, Cuernavaca Mor. 62250, Mexico.
[email protected]
In plants, drought stress produces several physiological and cellular changes, with Abscisic Acid (ABA) signaling
playing a central role in mediating the corresponding responses. Phaseolus vulgaris (common bean) represents an important crop in Mexico, and it provides a unique model for the analysis of different adaptation strategies. We are exploring
the participation of microRNAs in the regulation of processes related to perception/adaptation to drought stress. To address this question, we have constructed cDNA libraries to identify small RNAs present under optimal conditions, water
stress conditions or after ABA treatment. To date, we have found microRNAs conserved in Arabidopsis and other species as well as sequences representing potential microRNAs. We will present the characterization of novel microRNAs
we have found as specific to the legume lineage and their relationship to stress responses. In addition, we will show the
development of experimental tools to study microRNAs in this and other model legumes such as Medicago truncatula
or Lotus japonicus.
MS406 REGULATING THE EXPRESSION OF THE TIR1/AFB FAMILY OF AUXIN RECEPTORS
Parry, Geraint, Estelle, Mark
[email protected]
The TIR1/AFB family of F-box proteins function as auxin receptors. Their interaction with auxin causes the degradation
of negative regulators of the auxin response called Aux/IAA proteins. This process is of fundamental importance to plant
development as plants that lack auxin receptor function exhibit early seedling lethality.
We have created transgenic plants containing TIR1/AFB proteins fused to GUS expressed by their native promoters.
These have enabled us to discover that TIR1, AFB2 and AFB3 undergo significant post-transcriptional regulation but that
AFB1 does not. We are investigating the nature of the differences between these lines, particularly focusing on regulation
of TIR1/AFB transcripts by the micro-RNA mi393.
Furthermore we have used these translational GUS-fusion lines to investigate the role of plant hormones on the control of
auxin receptor function. Our initial findings suggest that both ABA and MeJA are able to reduce GUS expression in these
lines whereas brassinosteroid appears to increase GUS expression. We are continuing to investigate how auxin receptor
proteins mediate the crosstalk between different hormone signaling pathways.
In order to investigate the individual function of each of these genes we attempted to rescue the tir1 mutant phenotype by
driving expression of AFB1:GUS or AFB2:GUS proteins using the native TIR1 promoter. Interestingly these transgenes
are unable to rescue auxin resistance of tir1 roots suggesting that AFB1 and AFB2 may have cellular functions distinct
from those of TIR1.
Peptide Hormones
MS501 FUNCTION OF DODECA-CLE PEPTIDES IN PLANT STEM CELLS
Hiroo Fukuda1, Ikuko Nakanomyo1, Asuka Inoue1, Atsuko Kinoshita1, Shinichiro Sawa1 1Department of Biological
Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Tokyo 113-0033, JAPAN
[email protected]
In plants as well as animals, small peptide ligands have been suggested to play crucial roles in development as signal
molecules of cell-cell communication. However, their chemical properties that allow them to function as signaling ligands in plants remain little understood. We identified TDIF (Tracheary element Differentiation Inhibitory Factor) by using a single cell transdifferentation system of Zinnia as a bioassay. TDIF was a dodeca-peptide with two hydroxyproline
IPGSA 19th Annual Meeting - Abstracts
20
residues, which was the functional product of a gene from the CLE family containing CLAVATA3 ( CLV3). TDIF at 30
nM, can suppress xylem cell development and promotes cell division, suggesting a new function for the CLE peptides.
Microarray analysis suggested that TDIF suppresses the differentiation from procambium-like cells into tracheary elements. In contrast, dodeca-CLV3 peptide rather promoted tracheary element differentiation. Therefore there may be the
two counteracting pathways in the CLE signaling, one promotive and the other inhibitory in differentiation from stem
cells. The application to plants of 26 different synthetic dodeca-peptides corresponding to all of the Arabidopsis CLE
genes, revealed diverse signaling pathways involved in cell fate of stem cells.
MS502 INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) AND IDA-LIKE (IDL) PEPTIDES AND THEIR ROLES IN
PLANT DEVELOPMENT
Stenvik, Grethe-Elisabeth,Tandstad, Nora M., Holmgren, Asbjørn, Kristiansen, Wenche, Butenko, Melinka A.,Aalen,
Reidunn B.
[email protected]
Small peptides may act as signaling molecules that coordinate development, growth and differentiation. We have
identified the gene INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), encoding a protein of 77 amino acids (aa),
that is absolutely required for floral organ abscission in Arabidopsis (1).
When IDA is overexpressed, floral organ abscission takes place earlier and ectopic abscission is induced at the base of
pedicels and cauline leaves and at inflorescence branch points, indicating that IDA induce cell separation in preformed
abscission zones (AZ) (2). We have suggested that IDA and IDA-LIKE (IDL) proteins, identified based on their similarities to IDA, represent a novel group of putative ligands in plants. These proteins are all small, with a predicted N-terminal
signal sequence, and a conserved 12 aa motiv (PIP) at the C-terminus. Histochemical analyses of promoter-GUS constructs for the five IDL genes in transgenic Arabidopsis have been performed. Since IDA is involved in the cell separation
process that results in the shedding of the floral organs, it was interesting to find that the GUS expression directed by IDL
promoters in many cases was associated with sites of cell separation, such as AZs, valves of developing carpels, the base
of pedicels, and root tips. GFP-fusions to IDA/IDL signal peptides suggest that these proteins are exported out of the cells
(3). Localization in the extracellular space is consistent with the hypothesis that these proteins are ligands. Overexpression of the IDL genes resulted in similar phenotypes as overexpression of IDA, while overexpression of IDA/IDL lacking the signal peptide, were not phenotypically affected, suggesting that the extracellular localization of these proteins is
necessary for function. These data also suggest some degree of functional redundancy between IDA and the IDL proteins,
and that this redundancy is made possible due to the conserved PIP motif. We have therefore made a number of constructs
to test whether this motif is sufficient for IDA function, and whether part of the IDA and IDL proteins can be swapped
and still function in floral organ abscission.
1. Butenko, M.A., Patterson, S.E., Grini, P.E., Stenvik, G.E., Amundsen, S.S., Mandal, A. and Aalen, R.B. (2003) Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative
ligands in plants. Plant Cell, 15, 2296-2307.
2. Stenvik, G.-E., Butenko, M.A., Urbanowicz, B.R., Rose, J.K. and Aalen, R.B. (2006) Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial abscission zones in Arabidopsis. Plant Cell,
18, 1467-1476
3. Aalen, R.B., Butenko, M.A., Stenvik, G.-E., Tandstad, N.M. and Patterson, S.E. (2006) In Silva, J. T. d. (ed.), Floriculture, Ornamental and Plant Biotechnology: advances and topical issues Global Science Books Ltd. , London, pp.
101-108.
MS503 ROLES OF TWO STRUCTURALLY DISTINCT SULFATED PEPTIDES, PSK AND PSY1, IN ARABIDOPSIS
GROWTH AND DEVELOPMENT
Yoshikatsu Matsubayashi, Yukari Amano, Mari Ogawa, Hidefumi Shinohara, Hiroko Tsubouchi and Youji Sakagami:
Graduate School of Bio-Agricultural Sciences, Nagoya University Chikusa, Nagoya 464-8601, JAPAN
[email protected]
Phytosulfokine (PSK) is a 5-amino-acid secreted peptide that has been identified in the medium of plant cell cultures,
based on the results of assays of growth-promoting activity of cultured cells. PSK contains two sulfated tyrosine residues.
IPGSA 19th Annual Meeting - Abstracts
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Genes encoding PSK precursors are expressed at considerable level in a variety of tissues throughout plant life cycle,
suggesting a role in plant development. The PSK receptor PSKR1 was purified from microsomal fractions of carrot cells
by ligand-based affinity chromatography and identified as a member of leucine-rich repeat receptor kinase (LRR-RK).
Disruption and overexpression of Arabidopsis ortholog of PSKR1 (AtPSKR1) affects cellular longevity and potential for
growth during plant development. We further identified two LRR-RKs with high sequence identity to AtPSKR1 from the
Arabidopsis genome,. We report the results of a series of analysis including complementation tests, expression patterns,
PSK-binding activities and phenotypes of multiple loss-of-function mutants of these LRR-RKs. PSY1 is a novel 18amino-acid sulfated peptide that has been identified in the medium of Arabidopsis cell cultures by the exhaustive analysis
of sulfated peptides using LC-MS. When applied to Arabidopsis seedlings, PSY1 peptide stimulates seedling growth by
promoting cellular enlargement. A gene encoding PSY1 precursor is expressed at considerable level in a variety of tissues. We discuss possible roles of PSY1 peptide in Arabidopsis growth.
MS504 PEPTIDE DEFENSE SIGNALS IN ARABIDOPSIS AMPLIFY THE INNATE IMMUNE RESPONSE
Huffaker, Alisa, Pearce, Gregory, Yamaguchi, Yube & Ryan, Clarence A.
[email protected]
AtPep1, a 23 amino acid peptide from Arabidopsis leaves, activates expression of the defense genes PDF1.2 (defensin) and PR-1 through the JA/Et and SA signaling pathways, mediated by a cell surface receptor, PEPR1. PROPEP1
is a member of a small, six membered gene family, and overexpression of two of the genes, PROPEP1 and PROPEP2
enhance resistance of Arabidopsis plants against the pathogen Pythium irregulare. The six Arabidopsis PROPEP gene
family members are differentially expressed in leaves of intact plants infected with pathogens or sprayed with MeJA
and MeSA, or in excised leaves supplied through the cut petioles with homologs of AtPep1 peptides derived from the
C-terminus of each of the proteins encoded by PROPEP genes. The peptides also differentially induce the expression of
PDF1.2 and PR-1. The induction of defense genes by the peptides is blocked in mutant plants deficient in the JA/Et and
SA pathways and by treatment of wild-type plants with DPI, an inhibitor of hydrogen peroxide production. PROPEP
genes appear to regulate the expression of defense genes in response to PAMPs to produce a feed back loop amplifying
defense signaling initiated by pathogen attack.
MS505 LONG DISTANCE TRANSPORT OF PEPTIDE HORMONE ACTIVITY IN ROOT XYLEM SAP
Peter M. Neumann,Environmental, Water and Agricultural Eng., Rabin Building, Technion IIT, Haifa 32000, Israel
[email protected]
The means by which plant signal peptide activity may be transported over long distances remain unclear. This report
investigates the possible occurrence and root to shoot transport of signal peptide activity in the xylem. Suspension cultured cells of wild tomato Lycopersicon peruvanium L. were used in an established bioassay for detecting nanomolar
concentrations of signal peptides via the induction of alkalinizing activity. Xylem sap naturally exuded from the cut and
washed stem-surfaces of de-topped tomato plants (Lycopersicon esculantum L. cv Castlemart ) was collected, partially
purified, concentrated and shown by the bioassay to consistently contain significant alkalinizing activity. Plant salinity
treatment induced further small increases in activity. Subsidiary experiments indicated that the alkalinizing activity found
in the xylem-sap had properties similar to those of known plant signal peptides and was root derived. Thus, it was:- a)
Detectable within minutes. b) Eluted similarly during HPLC chromatography. c) Destroyed by incubation with proteases
and stable in the presence of protease inhibitor cocktail. d) Not found in bioassays of simulated xylem sap placed on the
cut stem-surfaces of non-exuding roots in order to detect any significant release of wound peptides. Further investigations
of the signal peptide activity found in root xylem sap could provide new insights into its identity, genes, receptors, origins
and possible hormonal roles in regulating shoot growth and development.
MS506 IDENTIFICATION OF THE FUNCTIONAL PEPTIDE OF CLV3 BY IN SITU MALDI-TOF MS ANALYSIS.
Tatsuhiko Kondo1, Satoko Mizuno1, Shinichiro Sawa2, Hiroo Fukuda2, and Youji, Sakagami1 1Grad. Sch. Bio-Agricultural Sci., Nagoya Univ., Chikusa, Nagoya, 464-8601, Japan 2Grad. Sch. Sci. Univ. of Tokyo, 7-3-1 Hongo, Tokyo,
113-0033, Japan
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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The Arabidopsis CLAVATA3 (CLV3) gene encodes a stem cell specific protein presumed to be a precursor of a secreted peptide hormone that controls the size of the shoot apical meristem. Matrix-assisted laser desorption/ionization
(MALDI) time-of-flight mass spectrometry (TOF-MS) applied to in situ Arabidopsis tissues to determine the structure of
functional peptide derived from CLV3 protein.
We generated transgenic plants constitutively overexpressing CLV3 under the control of the CaMV35S promoter
(CLV3OX), and performed in situ MALDI TOF-MS experiments with frozen slices of CLV3OX calli to detect typical
ions. We observed a CLV3OX-specific ion and following MS/MS analysis revealed the structure of a modified 12-amino
acid peptide (MCLV3), which was derived from a conserved motif in the CLV3/ESR related (CLE) sequences and had
two hydroxy proline residues. Synthetic MCLV3 induced shoot and root meristem consumption as cells differentiate into
other organs; the typical phenotype of transgenic plants overexpressing CLV3.
These results suggest that the functional peptide of CLV3 is MCLV3. We developed a highly sensitive method of detection and quantification of MCLV3 using a related peptide containing hydroxy proline residues at different positions as an
internal standard. The relationship between MCLV3 produciton and hormone conditions will be discussed.
Reference
Kondo T., Sawa S., Kinoshita A., Mizuno S., Kakimoto T., Fukuda H., Sakagami Y. 2006. The plant peptide encoded by
CLV3 idntified by in situ MALDI-TOF MS analysis. Science 313, 845-848.
Hormones in Agriculture and Horticulture, and Synthetic Plant Growth Regulators
MS601 REGULATING SHOOT GROWTH AND MORE IN POME FRUIT TREES
Rademacher, Wilhelm BASF Agricultural Center, 67114 Limburgerhof, Germany
[email protected]
Prohexadione-calcium (ProCa) is a structural mimick of 2-oxoglutarate and ascorbate. It blocks distinct dioxygenases
involved in the biosynthesis of GAs, ethylene and flavonoids, which require these compounds as co-substrates. As a
result of lowered levels of growth-active GAs, annual shoot growth in apple and pear trees is reduced and significantly
less labor is required for dormant and summer pruning. Treated trees have a more open canopy, which enables better
light penetration into central parts and a more efficient crop protection. Due to lowered ethylene formation, fruit set and,
hence, tree productivity can often be improved. ProCa induces the formation of 3-deoxyflavonoids with phytoalexin-like
activities. This leads to clear reductions in the incidence of diseases. Attack by several insect pests is also diminished.
Favorable toxicological and ecotoxicological features and a relatively rapid disintegration into naturally occurring metabolites add further to the value of ProCa-based products for bioreglation in pome fruit trees.
MS602 POTENTIALS AND LIMITS OF ETHYLENE CONTROL IN HORTICULTURAL CROPS
Prange, Robert; Schotsmans, Wendy; Daniels-Lake, Barbara; DeLong, John Agriculture and Agri-Food Canada, Atlantic
Food and Horticulture Research Centre, 32 Main St., Kentville, NS B4N 1J5, Canada;
[email protected]
Research on ethylene over the last 30 years has clearly shown that it is a dominant plant growth substance. Although
our scientific knowledge of its role in plants has increased greatly during the past few decades, ethylene continues to be
one of the most studied plant growth substances. This presentation will summarise what has been learned in recent years
about ethylene metabolism and action through the use of inhibitors, especially AVG and 1-MCP. From this work, much
more is known about ethylene binding and ethylene production but also that ethylene metabolism and its effects are not
universal in all fruits and vegetables. Recent research has also resulted in new uses of ethylene in horticultural crops
alone or in combination with inhibitors such as 1-MCP. For example, in our research we have demonstrated how ethylene
controls sprouting of potato tubers, leading to its commercial application as a sprout inhibitor in a number of countries.
This has led to a better understanding of the interaction between ethylene and polyamine metabolism; the interaction of
CO2 and ethylene in tubers and how 1-MCP can modify the ethylene responses.
IPGSA 19th Annual Meeting - Abstracts
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MS603 POTENTIAL USE OF ABSCISIC ACID IN HORTICULTURE AND AGRICULTURE
Petracek, Peter; Woolard, Derek; Liu, Xiaozhong; Campbell, Craig; Hansen, James; Venburg, Greg Valent BioSciences
Corporation, 6131 Oakwood Road, Long Grove, IL 60047 USA
[email protected]
S-Abscisic acid (S-ABA) is a naturally-occurring plant hormone that plays a role in many key physiological processes
including dormancy, germination, bud break, flowering, fruit set, general growth and development, stress tolerance, fruit
ripening, abscission, and senescence. For example, S-ABA application reduces transpiration and water use by closing
the stomata, and thus reduces water stress in many plants. ABA analogs also can reduce transpiration, but at lower rates
than S-ABA. Valent BioSciences Corporation is currently evaluating the use of S-ABA and ABA analogs to reduce water
stress symptoms. Research to date has shown that spray or drench applications of S-ABA to ornamental crops such as
impatiens, petunia, and chrysanthemum, and vegetable crops such as pepper and tomato significantly delays wilting of
plants receiving insufficient water. This effect may be commercially useful for reducing losses of ornamental crops at the
retail level and improving plant growth in row-crop fields under water stress conditions.
MS604 INHIBITORS OF HORMONE BIOSYNTHESIS AS CHEMICAL PROBES
Asami, Tadao1,2, Kitahata, Nobutaka2, Nakano, Takeshi2, Tsujimoto Masafumi2 1Dep. Appl. Biol. Chem., Univ. Tokyo,
Tokyo, Japan; 2DRI RIKEN, Saitama, Japan
[email protected]
Development of chemicals that induce phenotypes of interest is now emerging as a useful way to study biological
systems in plants. This way of plant research would be a complement to classical biochemical and genetic methods.
Brassinosteroid (BR) biosynthesis inhibitors allowed the investigation of functions of BRs in a variety of plant species.
Application of BR biosynthesis inhibitors to a standard genetic screen to identify mutants that confer resistance to these
inhibitors allowed the identification of new components working in BR signal transduction, such as BIL1/BZR1. With
the results achieved by brassinosteroid biosynthesis inhibitors, next, we started the screening of inhibitors targeting the
biosynthesis or catabolism of chemicals derived from carotenoids, such as ABA, strigols, or MAX factor. Some of the
inhibitors that targeting carotenoid cleaving enzyme (dioxygenase) existing in the upstream of ABA or MAX factor were
found to be new biosynthesis inhibitor. They will be useful for the future research.
MS605 PREHARVEST MANIPULATION OF CHLORO-CHROMOPLAST TRANSFORMATION OF CITRUS FRUIT FOLLOWING THE APPLICATION OF A GIBBERELLIN BIOSYNTHESIS INHIBITOR
Barry, Graham H. 1, le Roux, Smit 2 1 Citrus Research International, Department of Horticultural Science, Stellenbosch
University, P. Bag X01, Matieland 7602, Stellenbosch, South Africa; 2 Department of Horticultural Science, Stellenbosch University, P. Bag X01, Matieland 7602, Stellenbosch, South Africa
[email protected]
Rind colour is one of the main cosmetic preferences for the marketing of fresh citrus fruit. Acceptable rind colour is
obtained when an adequate amount of carotenoids are synthesised together with chlorophyll degradation. Tree vegetative vigour, as well as high gibberellin and cytokinin levels, are thought to adversely affect rind colour. Thus, methods
to increase preharvest rind colour by manipulating vegetative vigour were investigated. Prohexadione-calcium (ProCa;
Regalis®) was applied to ʻNules Clementineʼ mandarin (Citrus reticulata Blanco), ʻNavelina Navelʼ and ʻPalmer Navelʼ
oranges [C. sinensis (L.) Osbeck], and ʻEurekaʼ lemon [C. limon (L.) Burm.f.] during the 2005 and 2006 seasons at 200
and 400 mg_L-1 active ingredient. Rind colour rating, colorimeter measurements and pigment analysis were done after
harvest, after ethylene degreening, and 3 weeks after cold-storage. During the 2005 season, ProCa significantly increased
rind colour by increasing carotenoid and decreasing chlorophyll concentrations in flavedo of fruit before and after ethylene degreening for all Citrus spp. tested, except ʻEurekaʼ lemon. However, after cold-storage, rind colour was not
significantly different among treatments. During the 2006 season, rind colour was significantly increased after harvest
and chlorophyll degradation plus carotenoid biosynthesis were stimulated by the late 400 mg_L-1 ProCa application on
all Citrus spp. tested. Foliar spray application of ProCa at a concentration of 400 mg_L-1 applied 6 plus 3 weeks before
anticipated harvest has the potential to increase preharvest rind colour of early-maturing citrus cultivars and these results
support the hypothesis that there may be a relationship between vegetative vigour and rind colour development of citrus
fruit.
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MS606 MANIPULATING GIBBERELLINS TO MODIFY PLANT STATURE
Bhattacharya, Anjanabha1, Davey, Michael1, Power, Brian1, Ward, Dennis2, Phillips, Andy2, Hedden, Peter2 1University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK 2Rothamsted Research, Harpenden AL5
2JQ, UK
[email protected]
Gibberellins (GAs) are endogenous plant hormones that control key aspects of growth and development viz., seed
germination, shoot growth, flower induction and development, and fruit expansion.
Two species, Solanum nigrum and Nicotiana sylvestris, have been employed as targets for Agrobacterium-mediated
gene delivery of GA biosynthetic genes to evaluate their role in controlling stature. The constructs used in this study
contained the CaMV 35S promoter driving GA-biosynthetic genes, which include a GA 20-oxidase from Arabidopsis thaliana(AtGA20ox1) and two functionally different GA 3-oxidases from Marah macrocarpus (MmGA3ox1 and
MmGA3ox2). Both GA 3-oxidases produce bioactive GAs, the latter forming products that are not inactivated by GA
2-oxidases. In addition, plants were transformed with a GA 2-oxidase from Phaseolus coccineus (PcGA2ox1). A GA4
desaturase (des) gene from the fungus Gibberella fujikuroi is also being studied. Multiple transformations of both species with 35S::MmGA3ox1 + 35S::MmGA3ox2, 35S::MmGA3ox1 + 35S::MmGA3ox2 + 35S::AtGA20ox1, 35S::
MmGA3ox1 + 35S::AtGA20ox1 and 35S::PcGA2ox1 + 35S::des genes are also being carried out in order to evaluate
the combined effect of genes in modifying GA metabolism. The plants are currently undergoing detailed phenotypic and
molecular analysis. Changes in shoot architecture, including increases in stature were observed in plants transformed
with 35S::MmGA3ox1, 35S::MmGA3ox2 and AtGA20ox1 genes, and particularly in S. nigrum transformed with 35S::
MmGA3ox1 + 35S::MmGA3ox2. Plants carrying PcGA2ox1 were dwarfed. Modification of plant stature by this approach may have commercial applications in horticulture.
Dormancy and Germination
MS701 REGULATION OF DORMANCY AND GERMINATION IN BARLEY AND ARABIDOPSIS
Barrero, José María1, Millar, Anthony1, Jacobsen, John1, Waterhouse, Peter1, Czechowski, Tomasz2, Scheible, WolfRüdiger2, Udvardi, Michael2, Gubler, Frank1. 1CSIRO Plant Industry, Canberra, Australia 2Max Planck Institute of
Molecular Plant Physiology, Golm, Germany
[email protected]
Recent work in our lab has focused on regulation of ABA metabolism in dormant seeds from two model plants, barley
and Arabidopsis. We have used transcription factor profiling to discover novel regulators of dormancy in Arabidopsis
seeds. Over 30 differentially expressed transcription factors were identified in the screen of dormant and nondormant
seeds. T-DNA insertions in 4 of the transcription factors exhibited either a loss or gain of dormancy phenotype compared
to wild type Columbia seeds. One of the genes which encodes a RING finger domain has been shown to regulate NCED
expression in developing and imbibed mature seeds. In addition we have been analyzing the role of light in regulating
dormancy and ABA metabolism in barley grains. Light has been shown to strongly promote dormancy in cereal grains
while dark has the opposite effect. Analysis of NCED and ABA 8ʼ-hydroxylase gene expression in imbibing barley
grains, shows that light promotes HvNCED1 expression and represses HvABA 8ʼ-hydroxylase-1 expression correlating
with increased ABA content and the pattern is reversed when the grains are imbibed in the dark. RNAi experiments in
transgenic barley plants support the role of HvABA 8ʼ-hydroxylase-1 in light regulation of dormancy.
MS702 MOLECULAR MECHANISMS OF SEED DORMANCY IN ARABIDOPSIS
Wim Soppe, Yongxiu Liu, Melanie Schwab, Matteo Sugliani, Regina Geyer, Kazumi Nakabayashi and Maarten Koornneef Max Planck Institute for Plant Breeding Research, Carl von Linné Weg 10, 50829 Cologne, Germany
[email protected]
Seed dormancy is defined as the failure of a viable seed to germinate under favorable conditions. Besides having an
adaptive role in nature by optimizing germination to the best suitable time, dormancy control is important in crop plants.
Plant hormones have a central role in the regulation of dormancy. ABA is required to induce dormancy during seed maturation and GA is necessary for seed germination. Many aspects of the molecular mechanisms underlying the regulation
of dormancy are still unknown. We have taken a molecular genetic approach to study seed dormancy in Arabidopsis, with
IPGSA 19th Annual Meeting - Abstracts
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the aim to unravel the molecular pathways that lead to establishment and release of dormancy. Analysis of natural variation for seed dormancy has led to the identification of several QTLs. In addition, several mutants have been identified in
mutagenesis screens for altered dormancy levels. The cloning and characterization in our lab of several of the underlying
genes revealed a role for alternative splicing, transcript elongation and histone H2B monoubiquitination. This suggests
that transcript processing and chromatin remodeling are essential components of the seed dormancy mechanism. The
relation between these novel components and the hormones ABA and GA is still not clear, but will be the focus of our
future work.
MS703 SITES OF SYNTHESIS AND ROLES OF ABSCISIC ACID IN SEEDS
Marion-Poll, Annie1, Frey, Anne1, Clément, Sabine1, Effroy, Delphine1, Seo, Mitsunori1, Botran, Lucy1, Sotta, Bruno2,
North, Helen M.1 1Institut Jean-Pierre Bourgin, Laboratoire de Biologie des Semences, UMR204 INRA, AgroParisTech,
F-78026 Versailles Cedex, France; 2Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, FRE 2846, CNRS,
Université Pierre et Marie Curie, Le Raphaël, 3 rue Galilée, F-94200 Ivry-sur-Seine, France
[email protected]
Abscisic acid (ABA) is a key regulator of seed development and germination. A major increase in ABA levels occurs
during the maturation phase, that positively regulates seed reserve accumulation and induces desiccation tolerance and
seed dormancy. In order to gain a better understanding of its role in these different processes it is important to understand
how ABA biosynthesis is regulated and exactly which tissues are involved. ABA biosynthesis starts by the generation of
oxygenated carotenoid C40 precursors termed xanthophylls. Xanthophyll conversions involving epoxidation and isomerisation occur prior to cleavage to form a C15 compound, xanthoxin, which is then converted to ABA in the cytoplasm.
To date a number of genes coding enzymes responsible for most of the steps of the ABA biosynthesis pathway have
been identified in Arabidopsis. Our group has studied the regulation of genes involved in the conversion of xanthophylls
up until the cleavage step, specifically zeaxanthin epoxidase and 9–cis-epoxycarotenoid dioxygenase, furthermore we
have identified a new ABA-deficient mutant, Ataba4, affected in neoxanthin synthesis. The ABA4 gene encodes a small
protein containing putative transmembrane domains and a predicted chloroplast peptide signal. The precise function of
ABA4 protein in neoxanthin synthesis is currently investigated. The phenotypes of deficient mutants and the tissue specific expression patterns of biosynthesis genes have been analyzed in relation to the role of ABA in seed development
and dormancy.
MS704 THE ROLE OF DELLA PROTEINS IN THE INTEGRATION OF GA AND ABA CONTROL OF SEED DORMANCY
Penfield, Steven1, Josse, Eve-Marie2, Gan, Yinbo1, Gilday, Alison1, Halliday, Karen2 and Graham, Ian1. 1Centre for
Novel Agricultural Products, Department of Biology, University of York, PO BOX 373, York YO10 5YW, UK. 2School
of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, UK.
[email protected]
Seed dormancy is a key adaptive trait in plants responsible for the soil seed bank. Germination terminates dispersal
and therefore regulates the time and place of plant growth. The long established hormone balance theory describes the antagonistic roles of the dormancy promoting plant hormone abscisic acid (ABA), and the germination promoting hormone
gibberellin (GA) in dormancy control, yet little is known about how these signals are integrated in the seed. Furthermore,
although many genetic loci mediate the hormonal regulation of seed dormancy, the way in which these genes influence
germination potential has remained elusive. Following on from our demonstration that the control of Arabidopsis seed
dormancy by cold and light is mediated through the SPT and PIL5 bHLH transcription factors, at least in part through
regulation of GA biosynthesis (Penfield et al., 2005 Current Biology 15:1998-200), we have found that the DELLA protein regulators of the GA response are required for seed dormancy control. DELLA proteins RGL2, RGA and GAI are
required for dormancy and the ABA inhibition of seed germination (Penfield et al., 2006 Current Biology 16:2366-70).
This is the first demonstration that DELLA proteins integrate ABA and GA signals during seed dormancy and germination. We have also found that cotyledon expansion precedes radicle emergence during Arabidopsis seed germination, and
that seedling cotyledon size correlates strongly with dormancy in the DELLA mutants. GA, ABA and mutants affected in
hormonal regulation of seed dormancy also control cotyledon size in a manner consistent with their effect on germination
potential. We propose that light, temperature and hormonally responsive cotyledon expansion prior to radicle emergence
overcomes dormancy imposed by the seed coat and underlies seed dormancy control in Arabidopsis.
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MS705 RGL2 PROTEIN DOES NOT DISAPPEAR DURING SLY1 MUTANT SEED GERMINATION
Tohru Ariizumi 2, Camille M. Steber 1,2 1 USDA-ARS and 2 Dept. of Crop and Soil Science, Washington State University, Pullman WA 99164-6420 USA, [email protected] (509)335-2887
[email protected]
The SLEEPY1 (SLY1) and RGA-like2 (RGL2) genes play an important role in the regulation of seed germination by
GA in Arabidopsis. The control of seed dormancy and germination is critical for plant survival and important for proper
stand establishment in crop species. The plant hormone gibberellin (GA) stimulates seed germination in many plant species and is required for seed germination in Arabidopsis. The Arabidopsis GA biosynthesis mutant ga1-3 is unable to germinate unless GA is exogenously applied. The failure of seed germination in ga1-3 is suppressed by mutations in RGL2,
and GA stimulation of germination in ga1-3 is associated with rapid disappearance of RGL2 protein. This suggests
that the RGL2 DELLA protein is an important repressor of seed germination. The current model is that GA stimulates
seed germination by causing the SLY1 SCF complex to trigger the ubiquitination and destruction of the RGL2 protein.
Unlike ga1-3, the GA-insensitive sly1 mutant shows a variable seed dormancy phenotype. Those seed lots showing
high seed dormancy eventually afterripen. Stronger alleles showed a stronger seed germination phenotype and required
more time to afterripen. It was expected that if RGL2 is the major repressor of seed germination, sly1 mutant seeds that
germinate well should accumulate lower levels of RGL2 protein than those that fail to germinate. Surprisingly, RGL2
protein accumulated at high levels even in after-ripened sly1 mutant seeds capable of 100% seed germination. RGL2
protein accumulation actually appeared to be higher in sly1-10 which shows a milder seed germination phenotype than
the sly1-2 allele. This suggests that SCFSLY1 is the main E3 ubiquitin ligase controlling RGL2 protein accumulation,
and that RGL2 disappearance is not a prerequisite for seed germination in the sly1 mutant background. In the absence of
GA, several GA-induced genes show increased accumulation in sly1 mutant seeds compared to ga1-3. It is possible, that
the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds or that a parallel pathway
controls seed dormancy and germination in the sly1 mutant background.
MS706 THE CELL CYCLE DURING MAIZE GERMINATION AND THE EFFECT OF PHYTOHORMONES
Vázquez-Ramos, Jorge. Facultad de Química, UNAM. Av. Universidad y Copilco, México DF, 04510, México.
[email protected], tel and fax 5255 56225284.
[email protected]
A successful seed germination should run in parallel with the activation of the cell proliferation mechanisms that
would allow seedling establishment. During maize seed germination, cell cycle events can be perceived since the early
hours and onset of S phase clearly precedes radicle protrusion, not so the first mitotic figures, that are concomitant with
radicle emergence. The timing of cell cycle events can be modified if maize seeds are imbibed in the presence of plant
hormones like abscisic acid (ABA), cytokinins (CK) and auxins (AUX). Whereas ABA stops germination prior to the
start of S phase, CK and AUX accelerate entry into S phase. Thus, these phytoregulators seem to exert their action during the G1 phase. We have cloned several genes whose products participate in G1 and the G1-S transition: Four D-type
cyclins, Cdk-A, PCNA, KRP, and their expression both at the transcriptional and protein accumulation levels has been
studied in control and hormone-treated embryo axes. Also, the effect of phytoregulators on kinase activity in cyclin-Cdk
complexes has been followed. The four D cyclins show distinctive mRNA expression patterns during germination, and
these are strongly modified by CK and AUX; however, at the protein level, there seems to be no distinction. Nonetheless,
the timing of appearance of the associated kinase activity appears to be regulated by phytoregulators, particularly AUX.
PCNA responds to CK only at the protein level, whereas Cdk-A and KRP transcripts or proteins respond to none. These
results will be discussed in terms of their relevance to the germination process.
MS707 In vitro germination and somatic embryogenesis induction in Cyrtochilum loxense an endemic vulnerable orchid from Ecuador.
Augusta Cueva A. 1 Yadira González R.1
1
Unidad de Ecofisiología Vegetal, Universidad Técnica Particular de Loja, Ecuador, CP 11-01-608
IPGSA 19th Annual Meeting - Abstracts
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Cyrtochilum loxense (Lindl.) Kraenzl, is an endemic orchid species distributed in the southern Ecuador (Loja province).
Considering the rapid loss of habitats this species is critically endangered. The main goal of this study was to evaluate
the effects of medium type and plant growth regulators in the somatic embryogenesis induction. Somatic embryogenesis
induction was proposed as an alternative method for C. loxense conservation. Seeds of C. loxense were germinated in
MS culture media (Murashige & Skoog, 1962), Knudson C and an alternative medium (MA) in base of a leaf fertilizer
(Leaf Nitrofoska ®). One month old protocorms (approximate height 0.5 cm) were cultivated in: MS media, Knudson C
and MA without plant growth regulators and MS medium with twelve combination levels of 2,4-dichlorophenoxyacetic
acid (2,4-D) and thidiazuron (TDZ). The highest rate of germination (90%) was obtained in MS media. Not significantly
differences in somatic embryogenesis induction were observed between the different culture media, although the highest rate of somatic embryogenesis induction was achieved in MS medium (37%) and Knudson C (37%). The embryo
number by callus was evaluated too. The highest average number of embryo per callus (14) was found on MS medium. 2,
4-D y TDZ promotes lower percentages of embryo formation (10-22%). The embryogenic callus induction was achieved
only in presence of quantities lowers than 0.1 mg/l of 2, 4-D in combination with the same rate of TDZ or in the absence
of it.
Key words: Embryogenesis, 2, 4-D, TDZ.
Novel Hormones/Hormones in Other Systems
MS801 GIBBERELLIN BIOSYNTHESIS AND ITS EVOLUTION IN FUNGI
Hedden, Peter1, Rojas, Cecilia2, Bömke, Christiane3, Tudzynski, Bettina3 1Rothamsted Research, Harpenden, Herts
AL5 2JQ, UK; 2Dept. Quimica, Univ. de Chile, Casilla 653, Santiago, Chile; 3Institüt für Botanik, Univ. Münster,
Schlossgarten 3, D-48149 Münster, Germany
[email protected]
Gibberellins (GAs), as well as being ubiquitous in higher plants, are present also in some bacterial and fungal species.
Indeed, they were first discovered in the fungus Gibberella fujikuroi from which they obtained their name. Comparison of
the biochemistry and genetics of GA biosynthesis in fungi and higher plants has revealed profound differences indicating
that GA production was acquired independently in these kingdoms. G. fujikuroi consists of a complex of at least nine different sexually fertile species (mating populations), which differ in their plant hosts and types of secondary metabolites
produced. Gibberellins are produced mainly by the rice pathogen Fusarium fujikuroi (MP C), which contains a cluster
of seven GA-biosynthesis genes. Although most of the other species of the complex contain the complete gene cluster,
most of them do not produce GAs due to mutations in the promoter and coding regions of the biosynthetic genes. While
more distantly-related Fusarium species lack the GA biosynthesis cluster, some unrelated species, such as Sphaceloma
manihoticola and Phaeosphaeria species, produce GAs using enzymes related to those in F. fujikuroi. Thus it appears that
GA biosynthesis in fungi has a common origin and may have been propagated by horizontal gene transfer.
MS802 COMPARATIVE ‘HORMONOMICS’: GENES AND SIGNALLING PATHWAYS IN A MOSS, PHYSCOMITRELLA
PATENS
Reski, Ralf Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Germany [email protected]
[email protected]
Mosses are characterised by a simple body plan and distinct cellular differentiation responses to ABA, auxin and cytokinin. Effects of brassinosteroids, GAs, or JA on moss development have not been described so far.
The last common ancestor of mosses and seed plants lived about 500 million years ago, a time when plants conquered
the land. Thus mosses occupy a phylogenetic key position in land plant development.
Recently, the Physcomitrella genome was completely sequenced as the first genome of a non-seed plant.
This talk will review our current knowledge of genes for phytohormone metabolism, receptors and signalling molecules
encoded by the Physcomitrella genome. It may thus provide detailed insights into the evolution of the hormonal system
in plants.
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MS803 STRIGOLACTONES, PLANT DERIVED SIGNALS FOR SYMBIOSIS AND PARASITISM
Yoneyama Koichi, Xie Xiaonan, Yoneyama Kaori, Dai Kusumoto, Takeuchi Yasutomo Weed Sci. Ctr., Utsunomiya
Univ., 350 Mine-machi, Utsunomiya 321-8505, Japan
[email protected]
Strigolactones are host-recognition signals for both arbuscular mycorrhizal (AM) fungi symbionts and root parasitic
plants Orobanche and Striga spp. Even non-mycotrophic plant species produce strigolactones, indicating that strigolactones may also have other important functions in plants and/or in rhizosphere communities. Therefore, chemical structures of major strigolactones and their distribution in the plant kingdom should be determined to promote symbiosis and
prevent parasitism through the manipulation of production and/or exudation of strigolactones.
Structures of novel strigolactones from tobacco, sorghum, flax, and other plant species will be presented and discussed.
MS804 INVESTIGATION OF GERMINATION-PROMOTING COMPOUNDS IN SMOKE
Flematti, Gavin1, Goddard-Borger, Ethan1, Heath, Charles1, Merritt, David2,3, Ghisalberti, Emilio1, Dixon, Kingsley2,3. 1School of Biomedical, Biomolecular and Chemical Science, The University of Western Australia, Crawley, WA
6009, Australia. 2School of Plant Biology, The University of Western Australia, Crawley, WA 6009, Australia. 3Kings
Park and Botanic Garden, West Perth, WA 6005, Australia.
gfl[email protected]
The newly discovered butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one (1) is a potent germination stimulant present
in smoke that shows biological activity at remarkably low concentrations (< 1 nM or 1 ppb) with a range of plant species
from Australia, South Africa, Europe and North America. To reflect the origin of this and related compounds also found
in smoke, we have named them ʻkarrikinsʼ from the Nyungar Aboriginal word for smoke (ʻkarrikʼ). The parent butenolide is thus named karrikinolide. Since discovering karrikinolide, our recent investigations have been concerned with
improving its synthesis and the preparation of synthetic analogues for structure-activity studies. A number of analogues
have now been prepared in our laboratory and have been assessed for germination activity. Preliminary structure activity
studies show that removal of the methyl group at C-3 or addition of a methyl at C-7 results in a considerable reduction in
germination promoting activity. In contrast, modification at the C-5 position with a methyl does not affect the activity of
the molecule. This indicates that modification at C-5 is better tolerated and should provide a site for further elaboration
with labelling strategies in mind. Further substitution of the parent molecule has revealed that polar hydroxyl or ester
functionalities at C-5 severely reduce the bioactivity of the molecule. On the other hand, alkyl or benzyl ethers show
strong bioactivity and indicate that lipophilic substituents are preferred. In addition to structure activity studies, we have
also investigated the interaction of karrikinolide with phytohormones, such as GA and ABA. Seeds of the model plant
species Stylidium maritimum are promoted to germinate with lower concentrations of GA when in the presence of karrikinolide. We have attempted to measure the changes in endogenous levels of GA and ABA to gain some insight into the
mode of action of karrikinolide. A summary of this work as well as our recent synthetic endeavours will be presented.
MS805 AMINO COMPOUND-CONTAINING LIPIDS: A NOVEL CLASS OF SIGNALS INVOLVED IN PLANT GROWTH
AND DEVELOPMENT
López-Bucio, José, Morquecho-Contreras, Alina, Pelagio-Flores, Ramón, Millán-Godínez, Mayra, Méndez-Bravo, Alfonso. Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo. Edificio
B3, Ciudad Universitaria, C.P. 58030 Morelia, Michoacán, México.
[email protected]
Most organisms are known to contain in their inner and outer membranes amphipatic lipids based on one or two amino
acids linked to a fatty acid through an amide bond. Thus, they have a structural similarity to ceramides. Lipids containing serine include N-acyl homoserine lactones (acyl HSLs), a group of compounds used by gram-negative bacteria for
cell-to-cell communication and for controlling gene expression in response to population density. N-acylethanolamines
(NAEs) represent compounds with aminoalcohol linked as an amide to the fatty acid. They were first reported as constituents of soy lecithin and peanut meal and as anti-inflammatory agents, but now they have been identified in a variety
of plant tissues. Anandamide (NAE 20:4), the main ligand of the cannabinoid receptor in mammals, regulates a variety of
IPGSA 19th Annual Meeting - Abstracts
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cellular and developmental responses. It appears that NAE signaling has important biological functions in plants, such as
defense responses, but research is still at a relatively early stage. Previously, we showed that alkamides, a class of plantproduced amino compound-containing lipids, structurally related to NAEs and acyl-HSLs, modified root system architecture. In this work, we present evidence that alkamides regulate important aspects of shoot development. A. thaliana
seedlings treated with low micromolar concentrations of alkamides develop ectopic blades on petioles and disorganized
tumorous tissue from the leaf lamina. Ectopic organ formation by alkamide treatment was related to altered expression
of ARR5:uidA, and CycD3:uidA, two markers of cytokinin signaling. The involvement of cytokinins in mediated the
biological activity of alkamides was tested using cytokinin-signaling mutants lacking one, two or three of the putative
cytokinin receptors CRE1, AHK2, AHK3, and RPN12 a gene involved in cytokinin responses. The cytokinin-signaling
mutants showed normal primary root growth inhibition by alkamides, but were resistant to the cell proliferative effects
caused by these compounds in shoots.
We went further to characterize the plant genetic responses to alkamides by isolating alkamide-response mutants of A.
thaliana. Alkamide-oversensitive mutants showed dramatic neoplasic growth in shoots that increase with alkamide treatment. On the contrary, alkamide resistant mutants showed absence of ectopic organs in leaves and continued root growth
under elevated levels of alkamides. Interestingly, alkamide-signaling mutants showed resistance to acyl-HSLs in root
growth assays, indicating that a bacterial quorum sensing-response pathway likely operates in plants. Taken together, our
results suggest that alkamides and other amino compound-containing lipids represent a new class of plant growth regulating substances that interact with cytokinins to control cell proliferation responses during plant development.
MS806 AN OXYLIPIN, (12Z,15Z)-9-HYDROXY-10-OXO-OCTADECADIENOIC ACID [KODA] SHOWS UNIQUE SIGNALING ON FLORAL DEVELOPMENT OF HIGHER PLANTS.
Madoka Ayano1, Hideki Goda1, Kazuo Shinozaki1, Naoharu Watanabe3, Mineyuki Yokoyama2, Shigeo Yoshida1 1.
Plant Science Center, RIKEN, Yokohama 230-0045, Japan 2. H&BC Research Center, Shiseido Co. Ltd., Yokohama
224-8558, Japan 3. Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan
[email protected]
KODA, (12Z,15Z)-9-hydroxy-10-oxo-octadecadienoic acid was originally found as an oxylipin relating with the floral
signal in Lemna paucicostata after exposure to stress. Interestingly exogenous application of KODA promotes flowering
at various plants so that we analyzed endogenous levels of KODA in the flower-induction process of Pharbitis nil, Citrus
unshu and Arabidopsis thaliana by means of LC-SIM and LC-MS/MS. KODA was clearly detected in seedlings of P. nil
and in shoots of C. unshu grown under flower-inductive conditions. The KODA concentration sharply increased in seedlings of P. nil during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. Similar fluctuation trend of KODA was observed in C. unshu shoots under flowering condition. In the case
of A. thaliana KODA indicated no floral promotion, but it showed obvious enlargement of leaves and increasing number
of roots. This effect of KODA was analysed by use of GeneChip showing no similarity in the pattern of gene expressions
between KODA and Jasmonate, nevertheless both belongs a class of oxydative products of ƒ¿-linolenic acid.
Root Development
MS1001 THE ROLE OF CYTOKININS IN CONTROLLING VASCULAR MORPHOGENESIS IN ROOT
Helariutta, Ykä , Lindgren, Ove , Bishopp, Anthony , Decourteix, Mélanie , Campilho, Ana , Dettmer, Jan, Lehesranta,
Satu , Nieminen, Kaisa , Honkanen, Anne , Immanen, Juha , Zeng, Jun
yrjo.helariutta@helsinki.fi
The cell lineages of the root vascular cylinder that form the transporting tissues (xylem and phloem) and the intervening procambial tissue originate from stem cells near the root tip. We and others have taken a combination of genetic and
genomic approaches to understand how the specification of vascular cell lineages is determined at a molecular level. We
have recently demonstrated that in Arabidopsis, cytokinin phytohormones negatively regulate protoxylem specification,
a “default” identity. AHP6, an inhibitory pseudphosphotransfer protein, counteracts cytokinin signaling in a spatially spe-
IPGSA 19th Annual Meeting - Abstracts
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cific manner, allowing protoxylem formation in this domain. On the other hand, we have shown that cytokinin signaling
stimulates proliferation of cell files within the vascular cylinder, indicating that cytokinins have a dual role in controlling
vascular morphogenesis. Recent progress in understanding the molecular nature of cytokinin responses and their interaction with other regulatory pathways in controlling cell specification and cell proliferation will be presented.
MS1002 THE INTRINSIC STEM CELL FACTOR, FEZ AND ITS PARTNER SOMBRERO, CONTROL AUXIN-SENSITIVE
ORIENTED STEM CELL DIVISIONS IN THE ROOT.
Viola Willemsen, Marion Bauch, Ana Campilho, Harald Wolkenfelt, Jian Xu, Ben Scheres. Dpt. of Molecular Genetics,
Utrecht University, Padualaan 8 Utrecht, 3584 CH The Netherlands
[email protected]
Stem cells divide to self-renew and to create daughter cells that differentiate. Stem cell function is controlled by extracellular cues from the stem cell niche that modulate intrinsic factors within the stem cell.
In the Arabidopsis root, stem cells surround the quiescent centre and produce al the tissues of the main root. The identity,
activity and division plane orientation of root stem cells correlates with accumulation of auxin (Sabatini et al., 1999). The
orientation of cell division in the root stem cell niche is tightly controlled and highly regular. Two novel genes, FEZ and
SOMBRERO (SMB), encode for NAC domain transcription factors. FEZ and SMB are specifically expressed in the Col
and LRC stem- and daughter cells and regulate the orientation and extent of cell division in these stem cells. These characteristics make FEZ and SMB candidate intrinsic stem cell factors acting at a different level than stem cell patterning
factors reported so far. Loss of FEZ leads to reduction LRC and columella layers while loss of SMB leads to ectopic stem
cell divisions suggesting that SMB promotes stem cell divisions but represses stem cell-like divisions in their daughter
cells. Inhibition of polar auxin transport results in reorientation of the cell division plane. It will be discussed whether
auxin plays a role in the function and regulation of FEZ and SMB.
MS1003 NATURAL GENETIC VARIATION AND ROOT DEVELOPMENT
Christian S. Hardtke Dept. Plant Mol. Biol., University of Lausanne, Switzerland
[email protected]
Arabidopsis wild accessions are an increasingly popular resource to identify allelic variants and even novel genes of
interest for a given developmental process. In our lab, we exploit this natural genetic variation to identify factors that
modulate growth rate and thereby contribute to morphological differences between accessions. Growth is most easily
monitored in the root, where growth measurements are essentially one-dimensional. In a survey of primary root growth
rate in a sample of 44 accessions, we identified one line, Uk-1, in which root growth is markedly reduced. Molecular
genetic analysis of this line revealed that this phenotype is largely due to loss-of-function of one locus, named BREVIS
RADIX (BRX). Gene expression analyses and physiological experiments suggest that BRX plays a role in the interaction
of the auxin and brassinosteroid pathways. Both hormones have been implicated in vascular differentiation, and therefore
it was not surprising that vascular growth is decreased in Uk-1 plants, both in the hypocotyl and root, when compared
to transgenic Uk-1 plants complemented by a 35S:BRX transgene. These transgenic lines enable an overall more comprehensive analysis of the brx phenotype, because they eliminate the influence of background-specific modifiers in the
previously analyzed brx introgression lines. Details about genetic interactions of BRX with other loci will be reported.
MS1004 DEVELOPMENTAL AND ENVIRONMENTAL REGULATION OF LATERAL ROOT FORMATION
Karen Deak, Dana Macgregor, Paul Ingram, Peter Roycewitz, Jocelyn Malamy
[email protected]
One important checkpoint in lateral root formation is the development of the root from the lateral root primordia. We
developed an in vitro lab assay to assess the environmental and developmental mechanisms regulating this process. We
found that under our assay conditions the fate of a lateral root primordium is determined by signals originating at the root
tip and shoot, and that the primordium alone is not able to coordinate its development with environmental cues. In depth
analysis of the signaling events from the shoot demonstrate the importance of physical contact of the cotyledons with the
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growth media and the permeability of the leaf cuticle. Indeed, mutants with increased lateral root formation, including
ABA deficient mutants, show increased cuticle permeability, which appears to be sufficient for the root system phenotype. The promotive factor in the media and its mode of action will be discussed, as well as the implications of these in
vitro findings for root system development in the soil.
MS1005 HYDROTROPISM: ROOT GROWTH RESPONSES TO WATER REGULATES ROOT SYSTEM ARCHITECTURE
IN ARABIDOPSIS.
Cassab Gladys, Saucedo Manuel, Ponce Georgina, Razgado Fátima, Campos María Eugenia, Romero Luis. Dep. Biol.
Mol. de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Mor. 62250, México.
[email protected]
The sessile plant requires a morphological and developmental pattern that enables exploitation of water, local minerals, and light if proper growth is to be achieved. Roots are capable to construct a three-dimensional perspective of their
local space by sensing and responding to a variety of stimuli. By doing this, plants actively forage resources from their
environment. Roots have positive hydrotropic response and modify their growth direction in search of water overcoming
their positive gravitropic response. Even though the lack of sufficient water is the single-most important factor affecting
world agriculture, there are few studies on hydrotropism. Recent genetic analysis of hydrotropism in Arabidopsis has
provided new insights about the mechanisms that the root cap uses to perceive and respond simultaneously to moisture
and gravity signals. Using a screening system with a water potential gradient, we isolated a no hydrotropic response
(nhr1) semi-dominant mutant of Arabidopsis that continued to grow downwardly into the medium with the lowest water
potential contrary to the positive hydrotropic and negative gravitropic response seen in wild type-roots. The root gravitropic response of nhr1 seedlings was significantly faster in comparison with those of wild type. The enhanced gravitropic of nhr1 roots might suggest that the reduction in the strength of hydrotropism increases the net effectiveness of the
gravitropic response. Expression of the nhr1 phenotype by heterozygous nhr1 roots exhibited intermediate penetrance,
typically 50% depending on the batch of seed. Addition of 10mM abscisic acid (ABA) increased penetrance of the nhr1
phenotype up to 100%. Furthermore, ABA induces gravitropism and inhibits hydrotropism in wild-type roots growing
in the screening system with a water potential gradient. These results indicate that ABA might play an important role in
hydrotropism. We have also developed a screening system for the isolation of putative super hydrotropic response (suh)
mutants in Arabidopsis. suh1 mutant roots continuously grew under water deficit for 10 days and reach the moderate
water potential conditions present in the lower section of the Petri dish in contrast with wild type roots. suh1 mutant roots
also modify their root system architecture according to the position of water availability in the medium and the presence
of ABA. These analyses indicate that the application of new genetic screens for dissecting complex signaling phenomena
such as hydrotropism is promising.
Supported by CONACyT: 46022Q and DeGAPA, UNAM: IN220807.
MS1006 LATERAL ROOT INITIATION: CELL COMPETENCE AND AUXINS
Dubrovsky, Joseph G.1, Sauer, Michael2, Napsucialy-Mendivil, Selene1, Friml, Jirí2, Shishkova, Svetlana1, Ivanchenko, Maria3, Celenza, John4 & Benková, Eva2 1Inst. de Biotecnología, Universidad Nacional Autónoma de México, A.P.
510-3, 62250, Cuernavaca, Mor., México; 2Center for Plant Molecular Biology (ZMBP), Tübingen University, D-72076
Tübingen, Germany; 3Dep. Botany & Plant Pathology, 2082 Cordley Hall, Oregon State Univ., Corvallis, OR 97331,
USA; 4Dep. of Biology, Boston University, Boston, MA 02125, USA
[email protected]
Lateral roots in Arabidopsis thaliana occur in an acropetal pattern along the primary root axis and their initiation takes
place during a relatively narrow developmental window (Dubrovsky et al., 2006, Ann Bot, 97: 903-915). We analysed
how this developmental window is established and what triggers certain pericycle cells to initiate lateral root formation.
As the phytohormone auxin is known to be important for lateral root formation, we analyzed changes in auxin response
along the primary root. Full spatial reconstruction of DR5rev::GFP activity along the root demonstrated that it is practically absent in all tissues from about one mm from the root tip up to the lateral root initiation (LRI) site. Starting from the
IPGSA 19th Annual Meeting - Abstracts
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LRI site, DR5 activity appears again and gradually increases reaching a maximum at the level of lateral root emergence.
The data suggest that the established auxin-response gradient is instructive for determination of a LRI site and is independent of the ALF4 gene. We will discuss our new data, which indicate that auxins but not brassinolides are required
for triggering of a LRI event in Arabidopsis. We will also present expression analysis of DR5::GUS in tomato roots to
demonstrate that the DR5 reporter can be used to monitor auxin response in plant species other than Arabidopsis. We
thank DGAPA-PAPIIT, UNAM (project IN225906) for financial support.
MS1007 AUXIN RESPONSE NETWORKS IN EMBRYONIC ROOT FORMATION
Schlereth, Alexandra1,2, Rademacher, Eike2, Moller, Barbara2, Biernat, Magdalena2, van Haperen, Anja2, Juergens,
Gerd1, Weijers, Dolf2 1 Center for Plant Molecular Biology (ZMBP), Tuebingen University, Auf der Morgenstelle 3,
D72076 Tuebingen, Germany. 2 Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands
[email protected]
The root meristem is first initiated in the developing embryo, and requires specification of an extra-embryonic suspensor cell as hypophysis, the quiescent centre precursor cell. Specification of the hypophysis is controlled by the Auxin Response transcription Factor (ARF) MONOPTEROS (MP), and its inhibitor IAA12/BODENLOS. We have found previously that MP acts in a small group of cells adjacent to the future hypophysis, which implicates cell-cell communication
through secondary signals in hypophysis specification. Auxin itself is such a signal, since MP controls PIN1-dependent
auxin transport from the embryo to the upper suspensor cell, where it elicits a second auxin response. However, auxin
accumulation alone seems insufficient for hypophysis specification. In order to identify additional signalling pathways,
we have used microarrays to isolate a number of transcription factor genes that are immediate targets of MP. We will present functional analysis of these target genes in root initiation. Furthermore, we have used a reverse genetics approach to
identify additional ARF and AUX/IAA proteins in hypophysis specification and found that in addition to hypophysis cell
fate, many cell fates in the early embryo are controlled by ARF transcription factors. We will also present our progress
on the systematic dissection of auxin responses in embryo development.
Hormone Signaling
MS1101 THE ETHYLENE SIGNALING PATHWAY OF ARABIDOPSIS: ACTION AND INTERACTION
Schaller, G. Eric, Chen, Yi-Feng, Gao, Zhiyong, Shakeel, Samina, Hall, Brenda P., Qu, Xiang Dartmouth College, Dept.
of Biological Sciences, Hanover, NH 03755
[email protected]
The gaseous hormone ethylene is perceived by a five-member receptor family in Arabidopsis. Here we report on three
aspects of ethylene receptor signaling. First, we address the differing roles for the receptors in signal output based on
the analysis of single and higher order loss-of-function mutations. Second, we address the importance of protein-protein
interactions for signaling, including physical interactions among the receptors themselves. Our recent data indicate that
receptors may exist in plants as clusters in a manner potentially analogous to that found with the histidine kinase-linked
chemoreceptors of bacteria. Clustering of receptors may explain the broad range of ethylene responsiveness found in
plants as well as the dominant nature of some ethylene-insensitive receptor mutations. Third, we address how post-transcriptional regulation modulates ethylene receptor protein levels. Ethylene, in addition to stimulating expression of some
receptors at the transcriptional level, can also induce their degradation at the post-transcriptional level. Thus, in a similar
fashion to what has been found for animal growth factor receptors, output from ethylene receptors may be regulated by
ligand-induced degradation. By incorporating a family of five different receptors with varying expression levels, differing roles in signal output, and the ability to interact with and modulate the activity of each other, a tremendous range of
signaling capacity can potentially be achieved.
MS1102 A NOVEL REGULATOR OF ETHYLENE RECEPTOR SIGNALING
Resnick, Josephine, Dong, Chunhai, Rivarola, Maximo and Chang, Caren
[email protected]
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The gaseous hormone ethylene plays an important role in plant growth and development and responses to environmental stresses. We have uncovered a negative regulator of ethylene responses that acts by positively regulating ETR1
ethylene receptor function in Arabidopsis. Arabidopsis has five ethylene receptors, which exhibit functional redundancy
and negatively regulate ethylene responses. In a genetic screen for suppressors of the dominant ethylene-insensitive
receptor mutant etr1-2, we identified REVERSION-TO-ETHYLENE–SENSITIVITY1 (RTE1), which encodes a novel
integral membrane protein found in plants, animals and protists. The rte1 null mutant displays ethylene hypersensitivity,
which appears to be caused by reduced ETR1 function, whereas over-expression of RTE1 confers ethylene insensitivity
that is largely dependent on ETR1.
Interestingly, loss of rte1 function can suppress only certain etr1 dominant alleles; 7 alleles were suppressed out of 13
etr1 alleles tested, suggesting that RTE1 and ETR1 interact at the protein level. The other four ethylene receptors do not
appear to be affected by rte1. A functional version of RFP-tagged RTE1, expressed under the native RTE1 promoter,
localizes to the ER and Golgi, and shows partial co-localization with CFP-tagged ETR1. Preliminary biochemical data
suggests that RTE1 can interact with ETR1 in living plant cells.
MS1103 DOWNSTREAM EVENTS IN BRASSINOSTEROID SIGNAL TRANSDUCTION
Wenqiang Tang1, Yu Sun1, Srinivas S. Gampala1, Zhiping Deng1, Joshua M. Gendron1, Tae-Wuk Kim1, Juan A. OsesPrieto2,, Shenheng Guan2, Alma L. Burlingame2, Zhi-Yong Wang1 1Department of Plant Biology, Carnegie Institution,
Stanford, CA 94305, USA 2Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco CA 94143, USA
[email protected]
Brassinosteroids (BRs) play important roles in a wide range of developmental processes. BR deficiency or insensitivity causes multiple growth defects, including dwarfism, male sterility, delayed flowering, and light-grown morphology in
the dark. How BRs regulate specific cellular and developmental processes remain largely unknown. Molecular genetic
studies in Arabidopsis have established a BR signal transduction pathway, in which perception of BR by BRI1 at the
cell surface leads to dephosphorylation of the BZR1 and BZR2/BES1 transcription factors, possibly by inhibiting the
BIN2 kinase or activating the BSU1 phosphatase. BIN2 phosphorylation of BZR1 and BZR2/BES1 inhibits their DNA
binding and promotes their binding to the 14-3-3 proteins. Mutation of a BIN2-phosphorylation site in BZR1 abolished
14-3-3 binding and increased nuclear localization and in vivo activity of BZR1, indicating that 14-3-3 protein binding is
essential for efficient inhibition of BZR1 by BIN2-phosphorylaiton. When activated by BR signaling, BZR1 and BZR2/
BES1 bind to the promoters of target genes to either activate or repress transcription. A large number of BZR1-target
genes, identified by chromatin immunoprecipitation, establish links to specific growth responses. Proteomic studies of
BR-responsive proteins have identified components that mediate BR signal transduction as well as downstream cellular
responses. A complete signaling pathway from BR perception to cellular and developmental responses is emerging.
MS1104 HETEROTRIMERIC G PROTEINS AND ABA SIGNALING IN ARABIDOPSIS
Xigang Liu, Wei Li, Fangming Wu, and Ligeng Ma National Institute of Biological Sciences, Beijing 102206, CHINA
[email protected]
Abscisic acid (ABA) is an important hormone that mediates many aspects of plant growth and development, particularly in response to the environmental stresses. Several components involved in ABA response were identified, however,
the ABA signaling pathways is not well defined. Recent reports revealed that the nuclear RNA-binding protein FCA and
the chloroplast protein Mg-chelatase H subunit are ABA receptors, suggesting the existence of multiple ABA receptors
in Arabidopsis. In addition, previous report verified that the only canonical heterotrimeric G protein _Á subunit, GPA1,
is involved in ABA-mediated seed germination, stomata opening, and inward K+ channel in guard cell, suggesting that
GPA1 is involved in ABA signaling pathway. Heterotrimeric G proteins coupled with a plasma membrane localized receptor to transduce the excellular signaling. In the present work, we characterized a putative G protein-coupled receptor.
We found that this receptor interacts with both GPA1 and AGB1 (Arabidopsis G_Â subunit), and the interaction between
this receptor and GPA1 is dependent on the intrinsic GTPase activity of GPA1. We also observed that mutation in this
receptor leads to the defects in ABA responses from seed germination, stomata clousure and opening to gene expression.
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This receptor specific binds to ABA, and the binding between the receptor and ABA follows receptor kinetics. Thus, our
results revealed that this receptor is the plasma membrane receptor for ABA, and it mediates all major ABA response via
heterotrimeric G proteins in Arabidopsis.
MS1105 EARLY GIBBERELLIN SIGNAL TRANSDUCTION AND DELLA-PROTEIN TARGETS IN ARABIDOPSIS
Zentella Rodolfo, Zhang Zhonglin and Sun Tai-ping. Dept. of Biology, Duke University, Durham, NC 27708, U.S.A.
[email protected]
Genetic and molecular studies have identified DELLA proteins as negative regulators of the gibberellin (GA) signaling
pathway. GA promotes rapid degradation of the DELLA proteins, which leads to activation of the signaling pathway. In
Arabidopsis there are five DELLA homologs with partially redundant roles. DELLA proteins are localized to the nucleus
and are believed to act as transcriptional regulators. This work aimed to elucidate the early events of GA signaling and
to identify direct DELLA target genes using microarrays. Most of the early GA-responsive genes that we identified are
down-regulated after 1h of GA treatment and only very few are weakly induced. To identify early DELLA-responsive
genes, we generated a chemically inducible transgenic line that expresses the mutant protein rga-delta17. This mutant
form of the DELLA protein RGA, lacks a 17 amino acid motif that is indispensable for its GA-mediated degradation and
renders a constitutively active repressor of GA signaling. Induction of rga-delta17 caused up-regulation of a battery of
genes, including those that are down-regulated by GA. Quantitative RT-PCR and chromatin immunoprecipitation experiments verified that some of the genes identified by this process are RGA direct targets. Moreover, transient expression
studies supported the notion that, although DELLA proteins are GA-signaling repressors, such repression may be caused
by activation of downstream repressors.
MS1106 NITRIC OXIDE FUNCTIONS IN ABA- AND AUXIN-REGULATED SIGNALLING PATHWAYS
Lamattina, Lorenzo Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, CC 1245 (7600) Mar
del Plata, Argentina. E-mail: [email protected]
[email protected]
Plants display a series of mechanisms for perception and transduction of environmental and endogenous stimuli. Phytohormones play key roles for the integration and synchronization of physiological responses once stimuli are perceived.
Then, signal transduction processes construct the cell responses to a stimulus. As well as it was demonstrated in animal
systems that nitric oxide (NO), cGMP and Ca2+ are interconnected to play key roles in vascular cell physiology, the same
evidence are forthcoming from plant systems. It has been demonstrated that abscisic acid (ABA) triggers a complex sequence of signaling events to drive stomatal closure. Results indicate that NO is a prerequisite for ABA-regulated signal
transduction in guard cells leading to stomatal closure. NO regulates Ca2+-sensitive ion channel through the promotion
of Ca2+ release from intracellular stores. Besides, it is known that auxin is involved in the regulation of most aspects
of root growth and development. Recent results demonstrate that NO is a critical molecule in auxin-triggered signaling
pathways that lead to adventitious root formation, lateral root and root hair development. Again, Ca2+ and cGMP play
central roles in NO-regulated mechanisms that determine root architecture. Supported by ANPCyT, CONICET, UNMdP,
Argentina. L.L. is a J.S. Guggenheim fellow.
Light Responses
MS1201 A NUCLEAR PROTEIN INTERACTION CASCADE MEDIATING COORDINATED REGULATION OF ARABIDOPSIS DEVELOPMENT BY PLANT HORMONE GIBBERELLINS AND LIGHT SIGNAL
Suhua Feng1, Cristina Martinez1, Yu Wang2, Liying Chen3, Giuliana Gusmaroli1, Juan M. Iglesias-Pedraz4, Feng
Wang3, Lu Yu3, Xiangdong Fu5, Liu-Min Fan2, & Xing Wang Deng1 1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104, USA. 2Peking-Yale Joint Center for Plant
Molecular Genetics and Agrobiotechnology, and National Laboratory for Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China. 3National Institute of Biological Sciences,
IPGSA 19th Annual Meeting - Abstracts
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Zhongguancun Life Science Park, Beijing 102206, China. 4Departamento Genetica Molecular de Plantas, Centro Nacional de Biotecnologia-CSIC, Campus Universidad Autonoma de Madrid, 28049 Madrid, Spain. 5Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China.
[email protected]
Gibberellins (GAs) are a family of plant hormones that mediate many essential plant growth and developmental
processes, many of which are also regulated by ambient light signal. DELLA proteins function as repressors of GA
responses and inhibit plant growth, largely through their effect on gene expression. The stability of DELLA proteins is
inversely controlled by GA via ubiquitin/proteasome-mediated proteolysis. However, the precise mechanism of DELLA
proteins¡_ function in coordinating GA signalling and gene expression remains unknown. Here we characterize a nuclear
protein-protein interaction cascade mediating transduction of GA signals to the activity regulation of a light-responsive
transcription factor in Arabidopsis. In the absence of GA, nuclear localized DELLA proteins accumulate to higher level,
interact with phytochrome-interacting factor 3 (PIF3, a bHLH-type transcription factor), and prevent PIF3 from binding
to its target promoters, therefore abrogating PIF3-mediated light control of hypocotyl elongation of Arabidopsis seedlings. In the presence of GA, three largely redundant GA receptors (GID1 proteins) elevate their direct interaction with
DELLA proteins in the nucleus, trigger DELLA proteins¡_ ubiquitination and proteasome-mediated degradation, and
thus release the negative effect of DELLA proteins on PIF3. Collectively, our data suggests that two main sets of GAmodulated antagonistic protein-protein interactions within the nucleus, between GA receptors and DELLA proteins, and
between DELLA proteins and light-responsive transcription factors, are likely responsible for coordinated regulation of
gene expression and seedling development by GA and light.
MS1202 SIGNAL INTEGRATION DURING SEEDLING PHOTOMORPHOGENESIS
Jennifer Nemhauser
[email protected]
Our studies focus on two interlocking questions: how does light shape seedling development and how do different
hormone response pathways coordinately regulate growth. We have uncovered a striking interconnection between two
hormones--brassinosteroids (BRs) and auxin that act in opposition to the light response. We are investigating the mechanism by which auxin and BR signals together modulate gene expression and growth. We are also taking a genomics
approach to investigate hormone response networks. Our analysis of publicly available global transcriptome data suggests that the relationship between auxin and BRs may be unusual. This analysis suggests that different plant hormones
regulate growth through largely nonoverlapping transcriptional responses.
MS1203 FUNCTIONAL CROSS TALK BETWEEN TWO-COMPONENT AND PHYTOCHROME B SIGNAL TRANSDUCTION IN ARABIDOPSIS
Mira-Rodado, Virtudes, Harter, Klaus Center for Plant Molecular Biology, Department of Plant Physiology, University
of Tuebingen, Auf der Morgenstelle 1, D-72076 Tuebingen, Germany
[email protected]
The A-type response regulator ARR4 is an element in the two-component signalling network of Arabidopsis. ARR4
interacts with the N-terminus of the red/far-red light photoreceptor phytochrome B (phyB) and functions as a modulator
of photomorphogenesis. In concert with other A-type response regulators, ARR4 also participates in the modulation of
the cytokinin response pathway. We present evidence that ARR4 directly modulates the activity state of phyB in planta,
not only under inductive but also under extended irradiation with red light. Mutation of the phosphorylatable aspartate
to asparagine within the receiver domain creates an ARR4 version that negatively affects photomorphogenesis. Additional evidence suggests that ARR4 activity is regulated by a phosphorelay mechanism that depends on the AHK family
of cytokinin receptors. Accordingly, the ability of ARR4 to function on phyB is modified by exogenous application of
cytokinin. These results implicate a cross talk between cytokinin and light signalling mediated by ARR4. This cross talk
enables the plant to adjust light reponsiveness to endogenous requirements in growth and development.
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MS1204 THE CIRCADIAN CLOCK REGULATES AUXIN SIGNAL TRANSDUCTION
Covington, Michael F., Harmer, Stacey L. Section of Plant Biology, University of California, Davis CA 95616, USA
[email protected]
The circadian clock plays a pervasive role in the temporal regulation of plant physiology, environmental responsiveness, and development. In contrast, the phytohormone auxin plays a similarly far-reaching role in the spatial regulation
of plant growth and development. Seventy years ago, Went and Thimann noted that plant sensitivity to auxin varied according to the time of day, an observation which they could not explain. We have found an explanation for this observation: the clock modulates transcriptional and growth responses to auxin. We are currently investigating the mechanism
by which the clock modulates auxin signal transduction and the physiological consequences for the plant.
MS1205 PIL5 REGULATES GIBBERELLIN RESPONSIVENESS BY DIRECTLY BINDING TO THE GAI AND RGA PROMOTERS IN ARABIDOPSIS SEEDS
Eunkyoo Oh1, Shinjiro Yamaguchi2, Jianhong Hu3, Jikumaru Yusuke2, Inyup Paik1, Tai-ping Sun3, Yuji Kamiya2, and
Giltsu Choi1 1Department of Biological Sciences, KAIST, Daejeon 305-701, Korea 2RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan 3Department of Biology, Duke University, Durham, NC27708-1000, USA
[email protected]
Gibberellin (GA) plays a key role in mediating light-regulation of Arabidopsis seed germination. It was shown that
light increases GA biosynthesis by inducing expression of two GA biosynthetic genes (GA3ox1 and GA3ox2). Previous
reports also indicated that PIL5, a phytochrome-interacting light-liable bHLH protein, inhibits seed germination by repressing GA3ox1 and GA3ox2, and activating a GA catabolic gene (GA2ox2). However, the persistent light-dependent
and PIL5-inhibited germination behavior in the absence of both de novo GA biosynthesis and deactivation by GA2ox2
suggested that PIL5 regulates not only GA metabolism but also GA responsiveness. Gene expression analyses showed
that PIL5 increases the expression of two GA repressor (DELLA) genes, GAI and RGA, in darkness. The significant
role of this transcriptional regulation in seed germination was shown by the hypersensitivity of the gai-t6 rga-28 double
mutant to red light for seed germination and the suppression of germination defects of a PIL5-overexpression line by the
rga-28 mutation. Beyond regulation of GA-related genes, PIL5 increases abscisic acid (ABA) levels by activating ABA
biosynthetic genes and repressing an ABA catabolic gene. Chromatin immunoprecipitation analyses indicated that PIL5
directly binds to GAI and RGA promoters, but not to GA and ABA metabolic gene promoters. Taken together, our results
show that light signals perceived by phytochromes cause a reduction in the PIL5 protein level, which in turn regulates
the transcription of two DELLA genes directly and that of GA and ABA metabolic genes indirectly.
MS1206 THE NATURE OF LONG DAY SIGNALS IN ARABIDOPSIS FLOWERING: ROLES FOR FLOWERING LOCUS T
(FT) AND GIBBERELLIN.
Tamotsu Hisamatsu1, and Rod W King2 1 National Institute of Floricultural Science (NIFS),Tsukuba, 305-8519, Japan.2
CSIRO Plant Industry GPO Box 1600 Canberra ACT 2600 Australia
[email protected]
Floral signals are sourced in the leaf and transported to the shoot apex. Their nature is unresolved but, in the long
day (LD)-responsive grass, Lolium temulentum, gibberellins (GA) are a floral signal (King and Evans ARPB 54:307-28,
2003) and in Arabidopsis (Ecotype Columbia), mRNA or protein of the gene FT is a likely LD signal (Huang et al., Science 309:1694-6, 2005). Here, using the GA biosynthesis mutant, ga1-3 in Columbia, we show its flowering can be GA
dependent but FT-independent. GA rapidly reversed (<1 d) the extreme dwarfing of ga1-3 and caused flowering in short
days (SD). FT expression was not increased sufficiently to contribute to this response. Also, in LD ga1-3 did not flower
unless GA was applied and, then, FT expression increased 1500% in <16 h. FT was essential for this GA/LD response as
flowering was delayed in the ft-1 mutant. Contradictorily, FT alone is also sufficient for flowering as ga1-3 flowers when
FT is overexpressed (Blazquez et al. Plant Physiol130:1770-5, 2002) and, in Columbia, GA application only increases
LD-regulated FT expression by 35% (cf. 1500% in ga1-3). Thus, in the LD flowering of Columbia, because endogenous
GA levels are near-to saturating, GA acts permissively and FT is the dominant signal.
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Abiotic Stress
MS1301 REGULATORY GENE NETWORK IN DROUGHT AND ABA RESPONSES
Kazuo Shinozaki1 and Kazuko Yamaguchi-Shinozaki 2, 3 1RIKEN Plant Science Center, Suehiro-cho, Tsurumi-ku,
Yokohama, Japan, 2Laboratory of Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, 3Biological Resources Division, Japan International Research Center for
Agricultural Sciences (JIRCAS), Tsukuba, Japan [email protected]
[email protected]
Drought stress induces a variety of genes of which products function in drought stress tolerance and response in plants.
Many stress-inducible genes have been used to improve stress tolerance by gene transfer. In this meeting, we present our
recent studies on regulatory networks in drought and ABA responses. We have identified complex regulatory systems
in stress-responsive gene expression: ABA-dependent and ABA-independent systems. In one of the ABA-independent
pathways, a cis-acting element (DRE/CRT) and its binding proteins, DREB2s, are important cis- and trans-acting elements in drought-responsive gene expression, respectively. DREB2 is also involved in heat stress response. In the ABAdependent pathways, bZIP transcription factors (AREB/ABF) are involved in the major process. Protein phosphorylation
is important for the activation of AREB proteins. The MYB/MYC and NAC transcription factors are involved in ABAresponsive gene expression. In the ABA-dependent pathway, stress-inducible NCED3 is mainly involved in the ABA
biosynthesis during drought stress. We analyzed metabolic profiles regulated by ABA using T-DNA tagged mutant and
with GC-MS and LC-MS. We analyzed the function of CYP707A3 in the regulation of ABA metabolism during stress
responses. We also report the functions of SnRK2 protein kinases in drought and ABA responses using mutants and transgenic overexpressors. In addition, we discuss a novel MAP kinase cascade, MKK3-MPK6 in stress response.
Umezawa et al. Curr Opin Biotech 17:113-122 (2006)
Yamaguchi-Shinozaki and Shinozaki. Ann Rev Plant Bio 57:781-803 (2006)
MS1302 JASMONATE-INSENSITIVE 3 IS A NUCLEAR TARGET OF SCFCOI1 NEGATIVELY REGULATING JA SIGNALLING
Solano, Roberto. Departamento de Genética Molecular de Plantas. Centro Nacional de Biotecnología-CSIC. Campus
Cantoblanco Universidad Autónoma, 28049 Madrid, Spain. E-mail [email protected]; Tel. 0034 91 5855429; fax
0034 91 5854506
[email protected]
Jasmonates (JAs) are essential phytohormones structurally similar to metazoan prostaglandins and potent anti-cancer
agents in humans. In spite of their importance for plant development and survival the molecular details of their signalling pathway remain largely unknown. The identification of COI1 as an F-box protein almost a decade ago suggested
the existence of a repressor of JA responses targeted by SCFCOI1 for degradation by the proteasome in response to JA.
We have identified JASMONATE-INSENSITIVE 3 (JAI3) and show that it belongs to a novel family of JA-regulated
nuclear targets of SCFCOI1, named JAIL (JAI3-Like). JAI3 and other JAIL proteins physically interact with COI1, and
JA treatment induces their SCFCOI1-dependent, proteasome degradation. The jai3-1 allele encodes a mutant protein
resistant to degradation that also inhibits degradation of the wild-type JAI3 and other JAILs, explaining its dominant
JA-insensitive phenotype. In addition, JAI3 and other JAILs physically interact with the key transcriptional activator of
JA-regulated gene expression, AtMYC2, which suggests a model of JAI3/JAILs action as repressors of AtMYC2. Our results demonstrate that JAILs are direct targets of the E3 ligase SCFCOI1, linking ubiquitin-mediated protein degradation
to transcriptional activation of JA-responses. Moreover, our results show the existence of a negative regulatory feed-back
loop involving AtMYC2 and JAILs that provides a mechanistic explanation for the pulsed response to the hormone and
the subsequent desensitization of the cell.
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MS1303 STRESS-REGULATED SIRNAS AND MIRNAS
Zhu, Jian-Kang Department of Botany and Plant Sciences, University of California, Riverside
[email protected]
It is often presumed that RNA interference (RNAi) evolved as a cellular surveillance mechanism to silence “foreign”
or invasive double stranded RNAs (dsRNAs) so that cells can defend against viral infection or transposons. However,
recent work suggests a widespread occurrence of dsRNAs unrelated to viral replication or transposons. A significant
portion of cellular dsRNAs can be formed by natural antisense transcripts (NATs). Approximately 10-20% of genes in
plant and animal genomes encode cis-NATs, i.e. these genes overlap but are on the opposite strands of DNA. In Arabidopsis, dsRNAs generated from cis-NATs can be processed by Dicer-like enzymes into 21-24 nt nat-siRNAs which then
direct the cleavage of complementary mRNAs. Evidence indicates that many cis-NAT genes are regulated by biotic and
abiotic stresses, and they generate nat-siRNAs only under specific stress conditions. The stress-induced nat-siRNAs are
important components in the regulatory circuits leading to stress acclimation. These results suggest that RNAi is not only
critical for cellular surveillance but it is also an important cellular gene regulatory mechanism. In this presentation, I will
talk about abiotic stress-regulated nat-siRNAs and other endogenous siRNAs in Arabidopsis. In addition, I will discuss
the role of miRNAs in plant responses to abiotic stresses.
MS1304 LIPID SIGNALING IN PLANT HORMONAL AND STRESS RESPONSES
Xuemin Wang, Yueyun Hong, Sungchul Bahn, Xiangqing Pan, Yanyan Zhang, Wenhua Zhang, Girish Mishra Dept. of
Biology, University of Missouri; Danforth Plant Science Center, St. Louis, MO 63121, USA
[email protected]
Cell membranes are the initial and focal points of stimulus perception and signaling messenger production. In addition, signal transduction, vesicular trafficking, and many other critical cellular functions are initiated by the assembly
of cytosolic protein complexes to specific sites in cellular membranes. Binding to specific lipid ligands are required for
the recruitment and/or regulation of the signaling and meatbolic complexes. In recent years, phosphatidic acid (PA), the
simplest membrane phospholipid and also a central intermediate of glycerolipid metabolism, has emerged as a class of
pivotal lipid mediators in various cellular processes. The effects of PA have been linked to signaling and production
of phytohormones and to plant growth, development, and responses to abiotic and biotic stresses. Signaling PA can be
produced by multiple enzymes, and the activation of specific enzymes regulates the timing, location, and molecular species of PA. The modes of PA action are multifaceted and include membrane tethering, direct modulation of enzymatic
activity, and effects on membrane structures and metabolism. Phospholipase D (PLD) is one major family of enzymes
that produce PA. Recent results have provided insights into the molecular mechanism by which the PLD family and PA
molecular species mediate hormonal and stress responses.
MS1305 THE CONTRIBUTION OF OXOPHYTODIENOATE REDUCTASES TO OXIDATIVE STRESS TOLERANCE IN
ARABIDOPSIS THALIANA
Abu Romman Saeid, Schaller Andreas, Stintzi Annick Institute of Plant Physiology and Biotechnology. University of
Hohenheim. D-70593 Stuttgart, Germany.
[email protected]
A small family of six flavin-dependent oxidoreductases exists in Arabidopsis thaliana designated as oxophytodienoate
reductases (OPRs). Despite their name, OPR3 is the only family member catalyzing the reduction of oxophytodienoic
acid, a key step in the jasmonic acid biosynthetic pathway. Our characterization of Arabidopsis opr1, 2, and 4 loss-offunction mutants indicates a role for these enzymes in the alleviation of (photo)-oxidative stress by detoxification of
lipid peroxidation products. Biochemical and structural data indicate that á,â-unsaturated carbonyls are likely to be the
physiological substrates of Arabidopsis OPRs 1, 2, 4, and 5/6. Potential substrates that meet the criteria deduced from the
biochemical and structural data are generated abundantly under conditions of abiotic and biotic stress.
IPGSA 19th Annual Meeting - Abstracts
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They include reactive aldehydes that are derived from lipid peroxidation products formed non-enzymatically as a consequence of oxidative stress. The characteristic á,â-unsaturated carbonyl motif is also present in many potential signaling
molecules of the oxylipin family. Results will be presented on the function and physiological substrates of the different
OPR isozymes, and the stress-associated phenotypes of opr mutants.
MS1306 THE IMPACT OF HEAT SHOCK AND/OR DROUGHT STRESS ON PLANT HORMONE AND POLYAMINE LEVELS IN TOBACCO PLANTS
Marie Havlova1, Milena Cvikrova1, Jiri Malbeck1, Vaclav Motyka1, Petre Dobrev1, Alena Gaudinova1, Jozef Gubis2,
Radomira Vankova1 1Institute of Experimental Botany AS CR, Rozvojova 263, 165 02 Prague 6, Czech Republic; 2Research Institute of Plant Production, Bratislavska cesta 122, 921 68 Piestany, Slovak Republic
[email protected]
Plants have evolved considerable capacity to cope with adverse environmental conditions, including elevated temperatures and water deficit. Heat shock (HS) and drought stress had differential impact on polyamine content in tobacco
plants. While spermidine and spermine were increased after both treatments, putrescine decreased during HS, being
elevated after prolonged water deficit. Transgenic plants over-expressing &Delta1-pyrroline-5-carboxylate synthetase
gene (P5CS), which codes for the key enzyme of proline biosynthesis, had higher total polyamine content under both
control and stress conditions. Potential link, which might exist between proline and polyamine metabolism due to sharing
one of the biosynthetic pathways, will be discussed.
Both stresses, which are unfavourable for plant growth, were connected with the decrease of bioactive CK levels (observed already after 3-h HS). Nevertheless, within 6-h HS bioactive CK gradient in favor of the upper leaves was established, indicating their preferential protection. Severe water deficit (RWC decrease by ca 30%) led to much more
profound decrease of bioactive CK levels in leaves than HS. Transgenic plants, which were more drought stress tolerant
(exhibiting prolonged chlorophyll retention), had relatively higher levels of bioactive CKs. Differences in CK metabolism, including CK glucosylation and degradation by cytokinin oxidase/dehydrogenase, in leaves in dependence on their
position and stress strength will be described. Drought caused high elevation of abscisic acid levels as well as re-distribution of auxin in favor of the lower leaves and roots. These changes were not observed after HS. The results suggest strict
regulation of plant hormone levels during the stress progression.
Interactions Between Hormonal Pathways: Biosynthesis/Signaling
MS1401 REGULATIONS OF ABA METABOLISM ON ITS ACTION AND REACTIONS
Eiji Nambara
[email protected]
The endogenous ABA level is regulated by the rate of its biosynthesis and catabolism. Among the multiple pathways,
nine-cis-epoxycarotenoid dioxygenase (NCED) and ABA 8ʼ-hydroxylase (CYP707A) are the rate-limiting enzymes of
ABA biosynthesis and catabolism, respectively. Therefore, these genes often act as the node of the multiple regulatory
networks in the homeostatic controls as well as the interactions of multiple plant hormone actions. I will discuss about
the spatial and temporal regulation of Arabidopsis NCED and CYP707A genes in response to external and internal (hormonal) cues.
MS1402 HORMONE INTERACTIONS DURING DEVELOPMENTAL PHASE TRANSITIONS
Sonia Gazzarrini 1, Mimmie Lu 1, Lumba Shelley 2, Yuichiro Tsuchiya 3 McCourt Peter 2. 1Department of Life Sciences, University of Toronto, 1265 Military Trail, Toronto, ON, M5S 3B2, Canada 2Department of Cell and Systems
Biology, University of Toronto, 25 Willcocks St. Toronto, ON, M5S 3B2 Canada 3Plant Science Center, RIKEN, 1-7-22
Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
[email protected]
The incredible variation observed in plant morphology often derives from changes in the timing of expression of
certain developmental programs. During their lifetime plants undergo major developmental phase transitions, such as
embryonic-to-vegetative (germination) and vegetative-to-reproductive (flowering) phase changes. Mutational analysis
has identified genes that accelerate or delay the timing of transition from the embryonic to the vegetative phase of de-
IPGSA 19th Annual Meeting - Abstracts
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velopment. The B3 domain transcription factor FUS3, which plays an important role in the establishment of late embryo
functions, controls developmental phase transitions and foliar organ identity by regulating the levels of the hormones
ABA and GA. These two hormones in turn regulate the stability of the FUS3 protein. Data will be presented on possible
mechanisms that regulate the stability and localization of the FUS3 protein during embryonic and vegetative development as well as the FUS3-dependent control of hormones levels.
MS1403 CHANGING VIEWS ON THE ROLE OF ABA IN ROOT AND SHOOT GROWTH RESPONSES TO WATER DEFICITS
Sharp, Robert E. Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, USA
[email protected]
Root and shoot growth are differentially sensitive to water deficits. Interest in the involvement of hormones in regulating these responses has focused on abscisic acid (ABA) because it accumulates under water-limited conditions, and because it usually inhibits growth when applied to well-watered plants. However, studies utilizing ABA-deficient mutants
and inhibitors of ABA synthesis to decrease endogenous ABA levels, and experimental strategies to circumvent variation
in plant water status with ABA deficiency, are changing the view of the role of ABA from the traditional idea that the
hormone is generally involved in growth inhibition. In particular, studies of several species indicate that endogenous
ABA limits ethylene production, and that as a result of this interaction ABA can function to maintain rather than inhibit
growth. The involvement of this interaction in the regulation of root and shoot growth responses to water deficits will
be evaluated. In addition, we are investigating the hypothesis that ABA functions in regulating the antioxidant system to
maintain reactive oxygen species (ROS) at non-damaging levels. In the ABA-deficient maize mutant vp14, ROS levels
increase dramatically in the growth zone of water-stressed roots, causing loss of plasma membrane and cell wall integrity
in association with inhibition of cell expansion. Broader implications for the role of ABA in stressed plants will also be
discussed.
MS1404 SHADE AND SUBMERGENCE AVOIDANCE REQUIRES A CONCERTED ACTION OF ETHYLENE, ABA, GA
AND AUXIN
Peeters, Ton, van Zanten, Martijn, Millenaar, Frank F., Voesenek, Laurentius A.C.J. Plant Ecophysiology, Institute of
Environmental Biology, Utrecht University, Sorbonnelaan 16, 3584CA Utrecht, The Netherlands E: A.J.M.Peeters@uu.
nl, T: +31302536850, F: +31302518366
[email protected]
Due to their sessile lifestyle plants are continuously threatened by all kinds of biotic attackers and abiotic disturbances. Since they can not escape their environment they have to adjust to the changes in the environment appropriately
to improve their chances for survival and to limit the use of resources. One of the abiotic stresses that plants encounter
is flooding. It has a dramatic effect on growth and yield of many plants since most plant species are very intolerant to
flooding. Species originating from semi-aquatic environments, however, have the capacity to cope with flooding stress.
More specifically, Rumex palustris (Rp), the marsh dock, is capable to redirect growth when submerged to bring leaf tips
above the water thus restoring gas exchange. Phenotypically, this has a striking resemblance with the shade avoidance
syndrome observed in shaded plants and when plants are subjected to a higher temperature (38ºC). Experimentally, we
have a system using Rp and Arabidopsis where we can use three different stimuli to evoke ultimately a similar response.
Experiments using this system have shown that ethylene, ABA and GA and to a lesser extend auxin are crucial components of the submergence avoidance system in Rumex. Preliminary data from Arabidopsis show an involvement of
ethylene and ABA in the submergence response, auxin and possibly GA during shade avoidance and auxin and ethylene
in the high temperature response.
MS1405 CHARACTERIZATION OF THE ROLE OF THE APETALA2 FAMILY MEMBER, ABI4, DURING SUGAR SIGNALING IN ARABIDOPSIS THALIANA
Flavia Bossi, Carolina San Román, Elizabeth Cordoba, Patricia Dupreé, and Patricia León1 1Departamento de Biología
Molecular de Plantas Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo Postal 510-3 Cuernavaca, Morelos 62250 México.
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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Glucose in addition to their essential role as structural component and energy provider acts as an evolutionary conserved hormone-like signal used by all living cells, to control their growth and development. Plants have evolved a
complex sugar-detection system that includes several signaling pathways, though which the expression of an important
number of genes involved in diverse processes are up- or down-regulated. Still today the sugar signal transduction
mechanism and the factors involved remain poorly defined.
The characterization of mutants altered in sugar signaling and perception (gin) has been valuable to identify some components required for sugar responses in plants. Analysis of some of these gin mutants permit to define the participation of
the hormone abscisic acid (ABA) as an essential component of the glucose response during early seedling development
and to uncover a complex network that links sugar and plant hormone signaling. The characterization of the gin6 mutant
demonstrated the participation of ABI4 as key factor in sugar signaling, in addition to its critical role in the ABA signaling pathway for seed development and germination. However, the mechanism by which this transcriptional factor acts
during sugar signaling is still unclear. To advance in the understanding of the role(s) of ABI4 during sugar responses, in
this study we demonstrate that ABI4 functions both as a positive and as a negative regulator for the expression of glucoseregulated genes. We demonstrate in vitro the capacity of ABI4 to directly interact to specific target genes, including its
own gene. In vivo using a protoplast system, we also demonstrate that this interaction affect the expression of these genes
in a sugar-dependent manner. Finally, in planta we evaluate results in that ABI4 is directly required for transcriptional activation during glucose responses. Thus, a fusion of the ABI4 promoter to the GUS reporter gene was introduced into the
abi4 mutant background and the expression of this transgene was followed. Quantitative analysis of the GUS levels corroborated that the absence of ABI4 dramatically decreases the glucose ABI4 promoter induction. These in planta results
confirm that ABI4 is required for glucose responses over this gene, and also uncover factor still present the participation
of an additional factor that participates in this glucose induction. Thus this works allowed to advance in our understanding of the role of ABI4 for glucose and also for stress responses.
MS1406 REGULATION OF ABA AND GA ACTION BY HIGH TEMPERATURE IN ARABIDOPSIS SEEDS
Toh, Shigeo1, Imamura, Akane1, Okamoto, Masanori2, Hanada, Atsushi2, Yamaguchi, Shinjiro2, Kamiya, Yuji2, Nambara, Eiji2, Kawakami, Naoto1 1Dep. Life Sci., Meiji University, Higashimita 1-1-1, Tama-ku, Kawasaki 214-8571,
Japan; 2Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
[email protected]
Suppression of seed germination by environmental high temperature (thermoinhibition) during summer is crucial for
Arabidopsis to establish vegetative growth in autumn. Alleviation of thermoinhibition by an ABA biosynthesis inhibitor,
fluridone, and by exogenous GA3 indicated that stimulation of ABA synthesis and suppression of GA action are critical for germination inhibition. GC/MS analyses showed that high temperature elevated ABA level and suppressed the
increase of bioactive GA level in the imbibed seeds. Quantitative real time-PCR analyses revealed that expressions of
ABA biosynthesis genes, ZEP, NCED2, NCED5 and NCED9 are up-regulated, and GA biosynthesis genes GA20ox1,
GA20ox2, GA20ox3, GA3ox1 and GA3ox2 are down-regulated by high temperature. The seeds of loss-of-function
mutants of GA negative regulators, SPY and RGL2, showed thermoinhibition tolerance, and expression of SPY was upregulated significantly by high temperature. These results demonstrate that high temperature stimulates ABA action by
enhancing expressions of ABA biosynthesis genes and represses GA action not only by suppressing expressions of GA
synthesis genes but also by stimulating GA negative regulator gene in Arabidopsis seeds.
MS1407 THE EMBRYONIC REGULATOR FUSCA3 ACTS THROUGH ETHYLENE IN VEGETATIVE LEAF DEVELOPMENT
Lumba, Shelley1, Tsuchiya, Yuichiro 2, Gazzarrini, Sonia 1, Provart, Nick 1, Hezky, Jodi, McCourt, Peter 1 1Department
of Cell and Systems Biology, University of Toronto, 25 Willcocks St. Toronto, ON, M5S 3B2 CANADA, 2RIKEN Plant
Science Center
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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In Arabidopsis thaliana the FUSCA3 (FUS3) transcription factor is thought to be both necessary and sufficient for correct embryonic leaf development. Loss-of-function mutations result in replacement of embryonic leaves (cotyledons) with
vegetative leaves and constitutive misexpression causes the production of cotyledons in place of vegetative leaves. Using
controlled activation, however, we show that FUS3 can only instruct cotyledon development outside of embryogenesis if
it is misexpressed during germination but not after seedling establishment. Transcriptome analysis does indicate that later
activation of FUS3 results in changes in vegetative gene expression but these changes are not related to classic embryonic
functions. These results suggested FUS3 may have roles unrelated to cotyledon identity and a phenotypic reexamination
of loss-of-function fus3 mutants post germination does show that these plants have a reduced number of vegetative juvenile leaves. Consistent with this, a short activation of FUS3 during germination can rescue this alteration in leaf phase
variation. Interestingly, ectopic activation of FUS3 preferentially dampens normal temporal changes in the expression of
a group of genes involved in ethylene action. Conversely, loss of FUS3 function stabilizes the ethylene dependent transcription factor EIN3 and results in misexpression of ethylene response genes during germination. Finally, mutations that
confer an ethylene insensitive phenotype have the opposite juvenile leaf phenotypes observed in fus3 loss-of-function
plantlets and inhibition of ethylene signaling suppresses fus3 juvenile leaf defects. Together, these results suggest ethylene plays a key downstream role in FUS3-dependent vegetative leaf phase transitions. Based on these results, models are
discussed to reconcile how FUS3 can have roles in both cotyledon identity and juvenile leaf transitions.
Hormones in Cellular Growth
MS1501 HORMONE INTERACTIONS IN ARABIDOPSIS: THE JIGSAW OF SIGNALING PATHWAYS
Van Der Straeten Dominique1, Decat Jan1, De Grauwe Liesbeth1, Dugardeyn Jasper1, Hagenbeek Dik1, Vandenbussche
Filip1, Achard Patrick2, Harberd Nicholas2, Cheng Hui3, Peng Jingrong3, Rieu Ivo4, Phillips Andy4, Hedden Peter4,
Moritz Thomas5, Tietz Olaf6, Palme Klaus6 1Unit Hormone Signalling and Bio-imaging, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent; 2John Innes Centre, Norwich, UK; 3Institute of Cell and Molecular Biology, National
University of Singapore; 4Rothamsted Research, Harpenden, Herts, UK; 5Umeå Plant Science Centre, SE-901 83 Umeå,
Sweden; 6Institut für Biologie II-Botanik, Universität Freiburg, Germany
dominique.vanderstraeten@ugent
Plants perceive a plethora of external signals and continuously adapt to changes in their environment. This phenotypic
plasticity is obtained by integration of external signals with hormonal signals that regulate developmental alterations.
Much progress has been made in understanding how these pathways interact, and which proteins serve as signal integrators, leading to the biologically most adequate output. DELLA proteins – a family of nuclear proteins that restrain cell
proliferation and expansion, and control gibberellin response- have been shown to act as integrators of multiple growth
regulatory inputs. Ethylene, abscissic acid, and auxin signals have all been demonstrated to influence DELLA stability.
It can be hypothesized that also brassinolide, jasmonate, and external signals as light co-interact with these pathways,
in order to determine a final developmental output. Examples of bipartite and tripartite signal interactions in seedling
growth in skoto- and photomorphogenesis will be illustrated.
MS1502 DYNAMICS OF AUXIN TRANSPORT ACROSS THE PLASMA MEMBRANE - PINS, PGPS, AUX/LAXS (AND
OTHERS ?)
Zazimalova Eva Institute of Experimental Botany, Rozvojova 263, 16502 Prague 6, Czech Republic
[email protected]
Cell-to-cell polar auxin transport plays a key role in the regulation of spatial and temporal aspects of plant growth
and development. The activity of auxin carriers is crucial for both quantity and direction of cell-to-cell polar auxin flow.
At present in Arabidopsis there are two main candidates for auxin efflux carriers - plant specific PIN-FORMED proteins
(PINs) and multidrug-resistance-like P-glycoprotein transporters (MDR/PGPs), and one main candidate family for auxin
influx proteins – permease-like AUX1/LAX proteins. We have transformed BY-2 tobacco cells with various genes of
PIN-, MDR/PGP- and AUX1-type. After their overexpression, we have characterized concomitant changes in cell morphology and development together with the biochemical characteristics of intracellular auxin accumulation. Possible
IPGSA 19th Annual Meeting - Abstracts
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functions of relevant proteins in cellular growth have been suggested.
This work was supported by the Ministry of Education of the Czech Republic, project LC06034.
MS1503 INTEGRATING GROWTH, FROM HORMONES TO IONS
Simon Gilroy, Gabriele Monshausen, Tatiana Bibikova, Peter Dowd, Sarah Swanson
Biology Department, PennState University, 208 Mueller Lab, University Park, PA 16802, USA
[email protected]
Roots must sense and respond to a variety of stimuli such as the direction of gravity, availability of nutrients and water
and mechanical signals from objects they touch and integrate these to an appropriate growth response. We are interested
in defining the regulatory processes that underlie this integration of stimuli and how this system translates to tropic
growth control and development of root system architecture. We have found that both tropic and vertical root growth are
associated with highly complex and dynamic oscillatory changes in surface pH and ROS centered over the proximal and
distal elongation zones. Tropic growth is associated with a coalescence and stabilization of surface alkalinization and
ROS production to the non-growing side of the root. Equivalent changes can be induced by mechanical stress of the same
regions, suggesting a possible link between the mechanical forces inherent in growth and changes in wall properties that
mediate local growth control. Auxin also elicits a rapid (within seconds) transient alkalinization over the surface of the
root. This auxin-dependent dynamic change in surface pH is still observed in tir-1 and a range of auxin transport mutants
such as the pins and aux-1, suggesting it does not simply reflect auxin transport phenomena and is not triggered by the
TIR-1-dependent receptor system. Current work aims at defining the molecular mechanism behind auxin-dependent
apoplastic alkalinization and ROS production and its relationship to dynamic growth control of the root as a whole. This
work is supported by NSF.
MS1504 ETHYLENE UP REGULATES AUXIN BIOSYNTHESIS IN ARABIDOPSIS SEEDLINGS TO ENHANCE INHIBITION OF ROOT CELL ELONGATION
Swarup, Ranjan1, Perry, Paula1, 2, Hagenbeek, Dik3, Van Der Straeten, Dominique3, Beemster, Gerrit2, 4, Sandberg,
Goran5, Bhalerao, Rishikesh6, Ljung, Karin6 and Bennett, Malcolm1
[email protected]
Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why
auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin
biosynthesis in the root apex. We subsequently demonstrated that ethylene regulated root growth is dependent on (A) the
transport of auxin from the root apex via the lateral root cap and (B) auxin responses occurring in multiple elongation
zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor ACC to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by up regulating auxin biosynthesis ethylene
facilitates its ability to inhibit root cell expansion.
MS1505 STEROL FUNCTION IN POLAR PIN2 AUXIN EFFLUX CARRIER POSITIONING DURING ARABIDOPSIS
ROOT GRAVITROPISM
Shuzhen Men, Yohann Boutté, Thomas Moritz and Markus Grebe Umeå Plant Science Centre, Department of Forest
Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden. E-mail: markus.
[email protected]; Phone: +46 90 786 8237; Fax: +46 90 786 8165
[email protected]
Plant membrane sterols are crucial for diverse cellular processes during plant growth and development including cell
polarity [1, 2]. The directional growth of plant roots along the gravity vector requires correct membrane sterol composition [2] and is mediated by polar auxin transport which involves the Arabidopsis PIN2 auxin efflux carrier [3-6]. Previous studies suggested that polar membrane localisation of the auxin efflux carrier protein PIN1 in root tip cells requires
correct sterol biosynthesis [2]. and that PIN2 co-traffics with sterols along a common transport route [7]. However, a
mechanistic link between sterol and PIN protein in a tropic growth process has not been provided. We have extended our
IPGSA 19th Annual Meeting - Abstracts
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previous studies and functionally analysed the dependence of polar PIN2 protein positioning on sterol composition, employing a combination of pharmacological and new genetic tools for modulation of sterol composition. We provide subcellular localization data for one component of the sterol biosynthesis machinery and employ cell biological approaches
to demonstrate that sterol composition in meristematic cells just after cytokinesis is required for establishment of PIN2
polarity. Our results suggest that sterol biosynthesis in dividing cells and post-cytokinetic cells is critical for polar PIN2
partitioning and root gravitropism. We further report more detailed results that post-cytokinetic polarity acquisition of
PIN2 requires sterol-dependent endocytosis, providing a mechanism for sterol action on auxin efflux carrier positioning
during root gravitropism.
1. Fischer, Men, and Grebe (2004). Curr. Opin. Plant Biol. 7, 670-676.
2. Willemsen et al. (2003). Plant Cell 15, 612-625.
3. Luschnig et al. (1998). Genes Dev. 12, 2175-2187.
4. Müller et al. (1998). EMBO J. 17, 6903-6911.
5. Chen et al. (1998). Proc. Natl. Acad. Sci. USA. 95, 15112-15117.
6. Utsuno et al. (1998). Plant Cell Physiol. 39, 1111-1118.
7. Grebe et al. (2003). Curr. Biol. 13, 1378-1387.
MS1506 A ROLE FOR SPERMINE IN XYLEM DEVELOPMENT
Minguet Eugenio G2, Muñiz Luis1, Pesquet Edouard1, Vera Francisco2, Carbonell Juan2, Blazquez Miguel A2, Tuominen Hannele1 1Dep Plant Physiol, Umea University, S-90187 Umea, Sweden; 2Inst Biol Mol Cel Plantas (UPV-CSIC)
Avda Naranjos s/n, 46022-Valencia, Spain [email protected]
[email protected]
Mutation in the spermine synthase ACL5 results in altered xylem development in Arabidopsis thaliana. The xylem
vessels of the acl5 mutant are smaller and more simple in structure than the wild type, and the xylem fibers are completely
missing. The expression of ACL5 in the early developing vessels, as detected by in situ hybridisation and reporter gene
analyses, suggests a cell-autonomous function of spermine during xylem development. Based on the observation that exogenous spermine prolongs vessel differentiation and stimulates vessel cell expansion and cell wall elaboration in Zinnia
elegans cell cultures, spermine is hypothesized to prevent premature death of the developing vessels to allow complete
differentiation. This model is supported by transgenic Arabidopsis plants expressing the DT-A toxin gene under the control of the ACL5 promoter, which display alterations in xylem development similar to the acl5 mutant.
Hormone Metabolism
MS1601 BETA-GLUCOSIDASE HOMOLOGS IN HOMEOSTASIS OF ABA LEVELS IN ARABIDOPSIS
Hwang, Inhwan, Lee, Kwanghee, Song, Kyungyong, Jang, Mee hee Division of Molecular and Life Sciences, Pohang
University of Science and Technology, Pohang, 790-784, Korea
[email protected]
The phytohormone, abscisic acid (ABA), is critical for plant growth and development, as well as for adaptive responses. To elicit proper physiological responses, ABA levels have to be constantly adjusted to changing physiological
and environmental conditions. To date, the mechanisms for fine-tuning ABA levels remain elusive. In this presentation,
I will present evidence that AtBG1 and AtBG2, two beta-glucosidase homologs localized to the ER and the vacuole, respectively, hydrolyze glucose-conjugated ABA (ABA-GE) to produce ABA. Loss-of-function mutants, atbg1 and atbg2,
displayed early germination, abiotic stress-sensitive phenotypes, and lower ABA levels, whereas transgenic plants overexpressing AtBG1 or AtBG2 accumulated higher ABA levels and displayed enhanced tolerance to abiotic stress. Under
dehydration stress, AtBG1 undergoes rapid polymerization into a high molecular weight forms, resulting in a 4-fold
increase in its enzymatic activity and AtBG2 was protected from degradation, resulting in higher protein levels. Furthermore, diurnal increases in ABA levels were attributable to polymerization-mediated AtBG1 activation. We propose
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that plants accomplish rapid increases in ABA levels in response to environmental cues by hydrolysis of ABA-GE using
organelle-specific AtBG isoforms whose activity is modulated by mechanisms involving transcriptional and post-translational modulation.
MS1602 AUXIN BIOSYNTHESIS IN ARABIDOPSIS
Youfa Cheng, Xinhua Dai, Yunde Zhao
[email protected]
Auxin is essential for many aspects of plant growth and development. Here we analyze the roles of the YUC family of
flavin monooxygenases in auxin biosynthesis and plant development in Arabidopsis. There are eleven YUC genes in the
Arabidopsis genome. The YUC genes mainly express in young developing primordia and each YUC gene has a unique
expression pattern that overlaps with other YUC gene expression domains. Systematic analyses of plants with double,
triple, quadruple, or higher order of loss-of-function yuc mutations indicate that the YUC genes play an essential role
in embryogenesis, seedling development, vascular development, and flower development. Interactions between auxin
biosynthesis and polar transport will also be discussed.
MS1603 GIBBERELLIN BIOSYNTHESIS IN THE MOSS PHYSCOMITRELLA PATENS
Kawaide, Hiroshi Div. Biosci. & Agrisci. Tokyo Univ. Agric. Technol.
[email protected]
The moss Physcomitrella patens is a model organism for the studies on genetics and development in early land plants.
It has been demonstrated that some plant hormones, such as auxin, abscisic acid and cytokinin, are biosynthesized and
act as plant hormones in P. patens. In contrast, there is no clear evidence for the presence and biosynthesis of endogenous
gibberellins (GAs) in the moss. We demonstrated the structure and function of the gene encoding the ent-kaurene biosynthetic enzyme from the moss. ent-Kaurene is the key intermidiate in GA biosyntehsis in plants and fungi. In higher
plants, ent-kaurene is synthesized by two independet diterpene cyclases, ent-copalyl diphosphate synthase (CPS) and
ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). We identified ent-kaurene synthase (PpCPSKS)
as a bifunctional diterpene cylcase from P. patens. The recombinant protein produced in vitro catalyzed the cyclization
reaction of GGDP to ent-kaurene and ent-16alpha-hydroxykaurene. Site-directed mutagenesis showed that two Asp-rich
motifs were independently involved in the CPS and KS activities. This function is similar to that of fungal CPSKS, which
catalyzes the bifunctional cyclization reaction with the single polypeptide. Truncations of the C-terminus of PpCPSKS
showed neither of two activities indicating that C-terminal region of PpCPSKS requiered not only KS activity but also
CPS activity.
MS1604 THE CAMPESTANOL-INDEPENDENT PATHWAY OF BRASSINOSTEROID BIOSYNTHESIS
Masaharu Mizutani1, Toshiyuki Ohnishi1, Takao Yokota2, Miklos Szekeres3 1Institute for Chemical Research, Kyoto
University, Uji, Kyoto 611-0011, Japan; 2Department of Biosciences, Teikyo University, Utsunomiya, 320-8551, Japan;
3 Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, POB 521, H-6701
Szeged, Hungary
[email protected]
Brassinosteroids (BRs) are plant steroid hormones that are essential for normal growth and development in plants.
Cytochrome P450 monooxygenases (P450s) play crucial roles in BR biosynthesis from campesterol to brassinolide, in
which many oxygenations at steroidal skeleton and side-chain structure occur. Recent molecular genetic studies for BRdeficient mutants of Arabidopsis, rice, tomato and garden pea have identified several P450 genes (CYP85A, 90A, 90B,
90C, 90D, and 724B) so far. However, the catalytic functions of them except for CYP85A remain ambiguous due to lack
of biochemical study. Recently, we have succeeded in functional expression of these P450s in a baculovirus-insect cell
system, and their catalytic activities were determined in an in vitro assay reconstituted with NADPH-P450 reductase. We
found that CYP90B and CYP724B are redundant C-22 hydroxylases. Campesterol is a predominant substrate of CYP90B
and CYP724B, suggesting the importance of the early C-22 hydroxylation route in the BR biosynthetic pathway. Next,
we found that CYP90C and CYP90D are redundant C-23 hydroxylases. Their substrate specificity revealed a novel
IPGSA 19th Annual Meeting - Abstracts
46
shortcut in BR biosynthesis, which allows direct conversion of early 22-hydroxylated intermediates to 6-deoxo3DT and
6-deoxoTY via C-23 hydroxylation. Furthermore, we have found that CYP90A is a C-3 oxidase/isomerase. CYP90A
catalyzes C-3 oxidation and isomerization of 22-hydroxycampesterol and 22,23-dihydroxycampesterol to produce their
corresponding 4-en-3-one. In contrast, campesterol is not metabolized by CYP90A at all. Taken together, we propose
the campestanol-independent pathway of BR biosynthesis, which predominantly converts campesterol to 22-hydroxycampesterol and (22S,24R)-22-hydroxyergost-4-en-3-one to form bioactive BRs, without going through campestanol.
MS1605 JASMONATE METABOLISM OCCURRING UPON WOUNDING AND IN SEED DEVELOPMENT OF TOMATO
Claus Wasternack Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale), Germany
[email protected]
Jasmonic acid (JA) and its metabolites such as the methyl ester and amino acid conjugates are lipid-derived compounds active in plant responses to biotic and abiotic stress as well as development (Wasternack, 2006). JA is synthesized in chloroplasts and peroxisomes via the intermediate 12-oxophytodienoic acid (OPDA). OPDA is formed by an
essential enzyme in JA biosynthesis, the allene oxide cyclase (AOC) (Ziegler et al., 2000). In tomato leaves, this enzyme
is exclusively located in the chloroplast of companion cells and even in plastid-like structures of sieve elements of vascular bundles, where also preceding enzymes in JA biosynthesis are located (Hause et al., 2003). Transgenic approaches
and mutant analyses showed amplification in wound signaling by AOC and JA and a putative role of JA in systemic
responses, which was documented by grafting experiments in G. Howeʼs lab. Here, we show that hydroxylation at C-12
of the pentenyl side chain of JA occurs upon wounding of tomato leaves leading to a switch off in JA signaling during
wound-responsive gene expression.
Moreover, 12-OH-JA is an ubiquitously occurring metabolite of JA which can also accumulate abundantly in seed tissues up to 2-3 orders of magnitude higher levels than JA. Other metabolites of JA are 12-OH-JA sulfate formed by a
sulfotransferase (LeST2a) and the 12-OH-JA glucose ester. Transgenic approaches with overexpression and repression
of LeAOC and LeST2a revealed the role of JA and 12-OH-JA in the wound response and flower development. Furthermore, specific role of OPDA, which accumulates abundantly in the seed coat during embryo development, was shown by
analyzing wild-type and mutant plants being deficient in JA or in JA and OPDA accumulation. Functional diversity of JA
given by its metabolic fate is discussed.
References
Hause B, Hause G, Kutter C, Miersch O, Wasternack C. 2003. Enzymes of jasmonate biosynthesis occur in tomato sieve
elements. Plant & -648.-Cell Physiology 44, 643
Wasternack C. 2006. Oxilipins: biosynthesis, signal transduction and action. In: Plant Hormone Signaling. (Hedden, P.
Thomas, S., eds.) Annual Plant Reviews, Blackwell, Oxford, UK. 185-228
Ziegler J, Stenzel I, Hause B, Maucher H, Miersch O, Hamberg M, Grimm R, Ganal M, Wasternack C. 2000. Molecular
cloning of allene oxide cyclase: The enzyme establishing the stereochemistry of octadecanoids and 19138.-jasmonates.
Journal of Biological Chemistry 275: 19132-19138.
MS1606 THE ROLES OF GIBBERELLIN 20-OXIDASE AND 2-OXIDASE GENES IN ARABIDOPSIS GROWTH AND DEVELOPMENT
Rieu, Ivo1, , Ruiz-Rivero, Omar1 , Fernandez-Garcia, Nieves1, Griffiths, Jayne1, Powers, Stephen1, Linhartova, Terezie1, Eriksson, Sven2, Nilsson, Ove2, Thomas, Stephen1, Phillips, Andrew1, Hedden, Peter1 1Rothamsted Research,
Harpenden, Herts AL5 2JQ, UK; 2Dept Forest Genet. Plant Physiol., SLU, S-90183 Umeå, Sweden
[email protected]
The gibberellin (GA) biosynthetic enzymes GA 20-oxidase (GA20ox) and 3-oxidase (GA3ox), and the inactivating 2oxidases (GA2ox) are encoded by gene families, whose members differ in their expression profiles. We are using reverse
genetics to determine the physiological roles of the GA20ox and GA2ox paralogues in Arabidopsis. Analysis of ga20ox1,
ga20ox2 and the ga20ox1 ga20ox2 double mutant reveals distinct, but overlapping roles for these genes in stem elongation, flower development and silique elongation. For example, while GA20ox1 makes a major contribution to the length
of vegetative internodes, GA20ox2 expression determines the numbers of internodes that elongate. Both genes contribute
IPGSA 19th Annual Meeting - Abstracts
47
to male fertility and silique elongation, with the major contributions to these processes from GA20ox1 and GA20ox2,
respectively. Mutants lacking all five C19-GA 2-oxidases resemble, in part, plants treated with GA, with individual genes
making major contributions to particular processes. For example, GA2ox4, whose expression is restricted to the base of
the shoot apical meristem, makes the largest contribution to flowering time, while GA2ox2 has a major role in preventing
parthenocarpic fruit growth in the absence of pollination.
Plant Growth Regulation Society of America Industry UPdate
Jeff Dobbs (OHP)
Gary Stutte (Dynamac)
Jeff Norrie (Acadian Seaplants)
Tom Stopyra (Chemical Dynamics)
Jim Hansen (Valent)
John Immaraju (AMVAC)
Jerry Stoller (Stoller Enterprises)
Gary Custis (PBI Gordon)
Poster Sessions
Fruit and Seed Development
PS0101 SLR, A SELECTION USEFUL IN THE PEACH FRUIT GROWTH AND RIPENING RESEARCH.
Bonghi Claudio 1 , Begheldo Maura 1, Ziliotto Fiorenza 1, Rasori Angela 1, Varotto Serena 1, Tonutti Pietro 2 and Ramina Angelo 1 1Department Environmental Agronomy & Crop Science, University of Padova; Viale dellʼUniversità, 16;
Legnaro (PD); I-35020-Italy,22 Scuola Superiore SantʼAnna di Studi Universitari e di Perfezionamento Piazza Martiri
della Libertà, 33 Pisa I-56127 Italy
[email protected]
The availability of natural mutants or those obtained by antisense technology, co-suppression and RNA-interference
is essential for the elucidation of complex physiological processes as fruit growth and ripening.
In peach a series of genotypes with altered fruit development and ripening as stony hard (hd) and slow-ripening (slr) are
available. slr, originated from an open pollination of the cv Fantasia, a yellow-fleshed nectarine, is bearing fruits characterized by a reduction in size, ethylene biosynthesis, pulp softening and mesocarp pigmentation. However the exposure
to exogenous propylene at advances stages of development (90 days after full bloom, advanced S3 phase) enhanced
ethylene biosynthesis, although the progression of the ripening syndrome is still blocked.
These data would indicate that the transductive pathway of the ethylene autocatalytic signal is separated from that of
ripening,
Differences in transcrpitome profiling at late stage of fruit development between the wild type (Fantasia) and slr were
investigate in air and propylene enriched atmosphere (500 ppm for 48 h) using the µPEACH 1.0, a microarray containing
4806 oligos corresponding to unigenes isolated mainly from a ripe fruit .
This approach allowed the identification of 322 uniges differentially expressed among which 195 and 127 were up-regulated in Fantasia and in slr, respectively. In the Fantasia fruit up-regulated genes encoding proteins related to ripening as
ACC oxidase, ethylene receptor of ERS and ETR2 types, cell wall hydrolases (endopolygalacturonases) and transcription
factors (TF) related to ethylene (EREBP) and auxin (AUX/IAA) action. On the basis of previous histologycal information indicating that in slr mesocarp small cell clusters with an altered cell wall were present, the attention was addressed
to clones putatively controlling cell wall biosynthesis and cell cycle. Among the first category a clone showing similarity
to LIM of Nicotiana tabacum, a TF controlling phenylpropanoid biosynthesis. Among the second, a tomato-like D3,2 cy-
IPGSA 19th Annual Meeting - Abstracts
48
clin involved in G1/S transition was found. Expression analysis and in situ hybridization reconfirmed that both mRNAs
were more abundant in slr mesocarp and that cyclin transcripts were preferentially localized in the small cell clusters.
Propylene treatment induced a differential expression of 151 and 101 unigenes in Fantasia and slr fruit, respectively.
Among the up-regulated genes ( 65 in Fantasia and 49 in slr) only five were shared by the two genotypes indicating that
ethylene is acting in a different genetic background.
PS0102 DEVLOPING A SUITE OF FRUIT-SPECIFIC PROMOTERS: TARGETING ALTERED GENE EXPRESSION TO
PRECISE STAGES OF TOMATO FRUIT DEVELOPMENT.
Nigel Gapper, Mi-Young Chung, Julia Vrebelov, James Giovannoni. Boyce Thompson Institute for Plant Research,
Tower Road, Cornell University, Ithaca, NY, 14850, USA.
[email protected]
Until now, attempts to manipulate the expression of target genes during tomato fruit ripening have produced varying results. Constitutive expression of target genes, can cause unwanted plieotropic effects on other aspects of plant
development, as a consequence, analyses of the transgene effect in the fruit is often difficult. Even in cases where fruit
development can be monitored, secondary effects on horticultural characteristics leave resulting transgenics of little
practical value. More accurate phenotypic determination of fruit function and utility for assessment of other production
or quality influences will be achieved through use of appropriate tissue and development-specific promoters. While several fruit-specific promoters are available, most are restricted by IP and even they represent a narrow range of expression
options.
PS0103 HORMONAL INTERACTIONS IN FRUIT DEVELOPMENT
Ozga, Jocelyn A., Ayele, Belay T., Reinecke, Dennis M. Department of Agricultural, Food and Nutritional Science,
University of Alberta Edmonton, Alberta, T6G 2P5, Canada ([email protected], 780-492-2653; Fax: 780-4924265)
[email protected]
Fruit development involves a complex interplay of cell division, differentiation and expansion of sporophytic and
gametophytic tissues that is carefully coordinated over time. Plant hormones are signals that regulate many processes of
plant development including fruit development leading to mature fruit and viable mature seed. Auxins and gibberellins
(GAs) have been implicated at various stages of fruit development. In this study, the role of endogenous auxins on GA
deactivation during pea fruit development was investigated using physiological, biochemical, and molecular approaches.
Using quantitative RT-PCR, we found that the growth-active auxin, 4-Cl-IAA, represses PsGA2ox1 (mainly responsible
for conversion of GA20 to biologically inactive GA29) and stimulates PsGA2ox2 (mainly responsible for conversion of
growth-active GA1 to inactive GA8) mRNA levels. The observed 4-Cl-IAA-induced expression pattern for PsGA2ox
genes, along with the previously reported 4-Cl-IAA stimulation of PsGA20ox and PsGA3ox gene expression (Ozga and
Reinecke, J Plant Growth Regul 22: 73-81, 2003), is accompanied by production of GA1 as demonstrated by [14C]GA1
synthesis from [14C]GA12 by 4-Cl-IAA-treated pericarps. These data suggest that a pulse of bioactive GA is produced
by the pollinated ovary in response to auxin, which leads to fruit set and development.
This research was supported by a grant from NSERC to JAO.
PS0104 RELATIONSHIP BETWEEN ENDOGENOUS HORMONAL LEVELS AND ZYGOTIC EMBRYOGENESIS OF
ACCA SELLOWIANA BERG. MYTACEAE
Pescador1, Rosete; Kerbauy2, Gilberto Barbante; Ferreira3 ,Wagner de Melo , Purgatto4 , Eduardo and Suzuki5,Rogério
Mamoru. 1Departamento de Ciências Naturais, Fundação Universidade Regional de Blumenau, Caixa Postal 1507,
89010-971 Blumenau, Santa Catarina, Brazil. 2Universidade Federal do Tocantins, NEAMB), Caixa Postal 111, 77500000 Porto Nacional, Tocantins, Brazil.3Departamento de Botânica, Universidade de São Paulo, Caixa Postal 11461,
05422-970 São Paulo, Brazil.4Department of Food and Experimental Nutrition, FCF, University of São Paulo.5Seção de
Orquidário, Instituto de Botânica de São Paulo. Av. Miguel Stéfano 3687, 04301-902 São Paulo, Brazil. rosetep@furb.
br
[email protected]
IPGSA 19th Annual Meeting - Abstracts
49
The effects of some endogenous hormones on the control of zygotic embryo development of Acca sellowiana Berg
were analyzed. Ovules and seeds containing embryos were collected at distinct stages of development. Endogenous levels
of indole-3-acetic acid (IAA) (GC-MS-SIM) and the cytokinins: isopentenyladenine (iP), isopentenyladenine 9-riboside
(iPR), zeatin (Z) and zeatin riboside (ZR) were quantified through high performance liquid chromatography (HPLC) and
enzyme-linked immunosorbent assay (ELISA). The amounts of these hormones, IAA and cytokinins revealed conspicuous differences during the embryonic development. A sharp and intense increase in the levels of IAA occurred between
the 21th and 24th day after fertilization. On the other hand, a consistent decrease in the levels of the auxin took place
along the globular, cordiform, torpedo and cotyledon phases. The levels of total cytokinins were always higher than IAA.
Zeatin was the most prominent cytokinin, an intense increase in the 18th after pollination as well in globular, cordiform
and torpedo embryo stages, with a decrease in the cotyledon stage.
PS0105 IAA, ABA, POLYAMINES AND AMINO ACID METABOLISM DURING OCOTEA CATHARINENSIS SEED GERMINATION
Santa-Catarina, Claudete; Dias, Leonardo L.C.; Pieruzzi, Fernanda P.; Silveira, Vanildo; Handro, Walter; Floh, Eny I.S.
Plant Cell Biology Laboratory, Department of Botany, IB-University of São Paulo, CP 11461, 05422-970. São Paulo,
Brazil. Email: enyfl[email protected].
enyfl[email protected]
The aim of this work was to study biochemical variations at the endogenous levels of IAA (indol acetic acid), ABA
(abscisic acid), polyamines and amino acids, during seed germination in O. catharinensis. The seeds were germinated
in vermiculite substrate with a photoperiod of 16 h light, at 27 °C, samples being collected after 15, 30 and 60 days of
germination. Total amino acid levels decreased during the first 15 days, followed by an increment at the end of germination (60 days). Among the amino acids, asparagine was observed in larger concentration, this being the predominant
amino acid during the whole germination period. On the other hand, the content of total polyamines (free + conjugated)
increased during the first 15 days, followed by a decrease and stabilization between 30 and 60 days after germination.
Among the polyamines, free putrescine levels increased during the first 15 days, followed by a decrease and stabilization
until 60 days of germination, while the contents of spermidine and spermine decreased during the whole germination
period. Only spermine was detected in the conjugated form, in increasing concentrations starting from 30 days after
the germination. IAA levels increased during the first 15 days, followed by a decrease and stabilization until the end of
germination (60 days), while the endogenous levels of ABA decreased during the first 15 days, presenting similar values
until the end of germination.
These results, besides being important for comparative studies on zigotyc and somatic embryogenesis, can be used for the
improvement and monitoring of the germination process in somatic embryos of O. catharinensis (supported by Fapesp
and CNPq).
PS0106 COMPARATIVE PROTEOMIC DURING SEED DEVELOPMENT IN ARAUCARIA ANGUSTIFOLIA
Balbuena, Tiago S.; Silveira, Vanildo; Dias, Leonardo L.C.; Santa-Catarina, Claudete; Handro, Walter; Floh, Eny I.S.
Plant Cell Biology Laboratory, Department of Botany, IB-University of São Paulo, CP 11461, 05422-970. São Paulo,
Brazil. Email: enyfl[email protected].
enyfl[email protected]
The aim of this work was to study the differential expression of proteins, through the analysis of comparative proteomics, during seed development of A. angustifolia. The protein content increased significantly until the cotyledonary
stage followed by a decrease in the mature stage, with embryonic tissues presenting a larger protein content when compared with to megagametophyte tissues. Throughout bidimensional electrophoresis (2-DE) 1108 distinct polypeptides
were detected during all the analyzed stages, 31 of them being common in all the tissues and stages. Specifically, in the
initial stages 426±42 polypeptides at the proembryogenic, 425±81 polypeptides at the globular and 353±124 polypeptides
at the torpedo stages, were detected. The analysis of the 2-DE maps of the seeds at the pre-cotyledonary, stage showed
IPGSA 19th Annual Meeting - Abstracts
50
an increase in the number of expressed proteins, presenting 955±21 polypeptides in embryonic and 545±74 polypeptides
in megagametophyte tissues. After the cotyledonary stage, a constant reduction in the number of expressed proteins on
2-DE gels was observed. A significant variation was observed in relation to the molecular weight of the expressed polypeptides during seed development of zigótica of A. angustifolia. In the cotyledonary and mature stages, an increase in the
number of peptides with low molecular weight in both embryonic and megagametophytes tissues, was observed. These
low molecular weight polypeptides are probably related to the synthesis of specific proteins such as late embryogenesis
abundant (LEAs) and storage proteins, frequent at the end of seed development (Supported by FAPESP and CNPq).
PS0107 GIBBERELLINS IN FLOWERS AND FRUIT OF THE CHRISTMAS ROSE (HELLEBORUS NIGER L.)
Magnus, Volker1, Ayele, Belay T.2, Mihaljevic, Snjezana1, Reinecke, Dennis M.2, Ozga, Jocelyn A.2, Salopek-Sondi,
Branka1
1Dept. Mol. Biol., Rudjer Boskovic Institute, P.O.Box 180, 10002 Zagreb, Croatia; 2Agric./Forestry Center, University
of Alberta, Edmonton, Alberta T6G 2P5, Canada
[email protected]
The perianth of the Christmas rose, which is white or pink at anthesis, attains photosynthetic capacity during seed
development. Surgical experiments, combined with hormone treatments, indicated a role for cytokinins (Tarkowski et
al., JXB 57, 2237, 2006) and gibberellins in this process. GC-MS-SIM analysis of fruit extracts suggested the presence
of GA1 and GA4, supplemented by minor amounts of GA3 and GA7. GA1 peaked twice: at late anthesis (17 ng/g FW)
and shortly before seed ripening (18 ng/g FW), GA4 levels decreased from early anthesis (40 ng/g FW) toward seed
ripening (25 ng/g FW), with a minor secondary peak (35 ng/g FW) when seed development was inititiated. In the perianth, only GA1 (0.2 ng/g FW at fully established photosynthetic capacity) was so far detected. These data demonstrate
that the developing fruit have the capacity to supply GAs to the perianth, as one of the signals that induce and maintain
photosynthetic activity. The high overall gibberellin levels in fruit shortly before seed ripening may also be correlated
to embryo development which only proceeds to an early cotyledonary stage while the seeds are attached to the mother
plant. Supported by grants no. 138166 (National Sciences and Engineering and Research Council of Canada) to J.A.O.
and 098-0982913-2829 (Croatian Ministry of Science, Education and Sports) to B.S.
PS0108 GIBBERELLIN-AUXIN INTERACTION DURING TOMATO FRUIT-SET
Serrani, Juan C, Sanjuán, Rafael, Ruiz-Rivero, Omar, Fos, Mariano, García-Martínez, José L Inst Biol Mol Cel Plant
(IBMCP), Univ Polit Valencia-CSIC, Avda Los Naranjos s/n, 46022-Valencia, Spain
[email protected]
Gibberellins (GAs) and auxins (IAA, NAA, 2,4-D) induce fruit-set and growth in tomato, although their effect at histological level is different. Application of diverse GAs and inhibitors of GA biosynthesis (paclobutrazol and LAB 198999),
and quantitative analyses show that fruit-set depends on GAs, and that the active form is GA1. Expression analysis of
GA metabolism genes, by semiquantitative RT-PCR, shows that the increase of GA content upon pollination is mainly
the result of upregulation of genes encoding CPS and GA 20-oxidases (GA20ox1, -2 and -3), but not of those encoding
GA3ox1 and -2. Five tomato genes encoding GA2ox (1 to 5) have been isolated and characterized by determining the activity of expressed proteins and phylogenetic analysis. It is shown that the expression of SlGA2ox was not affected early
after pollination. Induction of fruit-set by auxins and by inhibitors of auxin transport (NPA) was negated by inhibitors of
GA biosynthesis, suggesting that auxin effect was mediated by GA. Auxin (2,4-D) application increased GA content in
unpollinated ovaries associated with upregulation of GA20ox and GA3ox, and downregulation of GA2ox genes. Therefore, it is concluded that auxins induce fruit-set by increasing active GA levels in the ovary.
PS0109 MODULATION OF AUXIN SIGNALING DURING SEED DEVELOPMENT IN ARABIDOPSIS
Yasuaki Kagaya1_CAkiko Yamamoto2, Michiko Kagaya1, Kumiko Tsutsumida1, Keita Taniguchi1_CKazuhiko Sakai1,
Masashi Suzuki1 CMikiko Kojima3, Hitoshi Sakakibara3, Tsukaho Hattori2 1Life Sci. Ctr, Mie Univ., Tsu 514-8506,
Japan; 2Biosci. & Biotech. Ctr., Nagoya Univ., Nagoya 8601, Japan; 3RIKEN Plant Sci.Ctr., Yokohama 230-0045, Japan
[email protected]
[email protected]
IPGSA 19th Annual Meeting - Abstracts
51
Auxin plays vital role in embryonic patterning. However, the function of auxin during the maturation phase of embryogenesis has been relatively unexplored. We demonstrate the evidence that the seed maturation program modulates
auxin signaling, which appears to contribute to the developmental arrest of the embryo during the maturation phase.
FUS3 is the one of the four master regulators of seed maturation. The embryos of the fus3 mutant do not arrest its growth
and show precocious expression of postembryonic characters during seed development. Analysis with an auxin signaling
reporter gene demonstrated that auxin signaling was ectopically activated in the radicle region in the fus3 embryo without
an increase in the IAA level. Consistently with this observation, ectopic expression of FUS3 caused the repression of the
auxin reporter during the early seedling development. Microarray analysis of the fus3 mutant seed and transgenic plants
ectopically expressing FUS3 revealed that some of IAA/AUX protein genes including IAA31, the negative regulators of
auxin induced transcription, are positively regulated by FUS3. Unlike the canonical members of the family, these IAA/
AUX proteins wholly or partially lack the domain II and the conserved Lys residue, which are required for destabilization
of the protein. With these and additional data, we propose a model for the developmental control of embryo maturation,
which involves a modulation of auxin signaling.
PS0110 STY1 ACTS AS A TRANSCRIPTIONAL ACTIVATOR THAT AFFECTS AUXIN REGULATED GYNOECIUM MORPHOGENESIS IN THE SAME PATHWAY AS LUG
Ståldal, Veronika, Eklund, Magnus, Sohlberg, Joel and Sundberg, Eva Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, S-750 07 Uppsala, Sweden [email protected];
[email protected]; [email protected]
[email protected]
The SHI gene family consists of nine active members in Arabidopsis (STY1, STY2, SHI, LRP1, SRS3, SRS4, SRS5,
SRS6, SRS7). We have previously reported that mutations in STY1, and related genes, affect style morphogenesis and
apical-basal patterning of the gynoecium, that STY1 activates transcription of the flavin monooxygenase-encoding gene
THREAD/YUCCA4, involved in auxin biosynthesis and that changes in expression of STY1 and related genes, leads
to altered auxin homeostasis. Now we can show that the style phenotype, which is gradually enhanced in SHI-family
double, triple, quadruple and quintuple mutants, can be restored by exogenous application of auxin in the apical end of
the gynoecia and that STY1 physically interacts with the YUCCA4 promoter as a homodimer. Using a polar auxin transport assay we can also show that loss of SHI-family function does not affect the auxin transport capacity. This suggests
that reduced auxin levels are causing the phenotype and that all effects are biosynthesis related.
We have previously shown that the transcriptional co repressor LUG appears to act upstream of SHI-family members and
this is supported by the finding that STY1 transcription is down regulated in lug-1 mutants. SEUSS, another transcriptional co repressor, interacts with LUG and we could show that both lug and seuss mutants respond to inhibited PAT in
a similar way as sty1-1 sty2-1. The apical-basal patterning is hypersensitive to NPA and the apical tissue defects can be
restored by NPA treatment, suggesting that auxin homeostasis or responses are affected in the mutant gynoecia. Taken
together these results suggest that STY1 affects gynoecium morphogenesis in the same pathway as LUG, and possibly
also SEUSS, through regulation of auxin homeostasis.
PS0111 SEED SET AND FRUIT GROWTH IN ARABIDOPSIS
Steve Swain, Mikihiro Ogawa, Pippa Kay, Davinder P. Singh, Michael Groszmann
[email protected]
In the majority of Angiosperm species, the formation of seeds is directly linked to fruit development. Seed set influences fruit initiation and, depending on the species examined, fruit abscission. With the exception of completely parthenocarpic genotypes, seeds also affect final fruit size, and a positive relationship exists between total seed number and
weight, and fruit size. These observations suggest that signals exist, possibly generated by developing seeds, that promote
fruit initiation and growth. Likely candidates for these signals are plant hormones such as auxin and gibberellin (GA),
both of which are present in developing seeds and other fruit tissues. In addition, application of these hormones can often
IPGSA 19th Annual Meeting - Abstracts
52
induce parthenocarpy, in which fruit growth takes place without seed development. To investigate the Arabidopsis seed/
fruit (silique) relationship in more detail, we are characterizing a range of auxin and GA mutants with defects in biosynthesis and response. Detailed examination of plants with reduced seed set caused by partial GA-deficiency reveals that
seeds act in an essentially localized manner to promote fruit growth and each seed contributes to final fruit size in a near
additive manner. In addition, GA production in both the embryo/endosperm and maternal tissues is required for normal
fruit growth suggesting that seeds may change GA levels or response in surrounding maternal tissues.
PS0112 INDEHISCENT DIRECTS CELL FATE IN THE ARABIDOPSIS FRUIT BY REGULATING AUXIN DYNAMICS
Sorefan, Karim1, Ljung, Karin2, Yanofsky Martin F.3, and Østergaard, Lars1
1 Crop Genetics Department, John Innes Centre, Norwich, NR4 7UH, United Kingdom; 2 Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, S-901 83 Umeå, Sweden; 3 Section of Cell and Developmental
Biology, University of California at San Diego, La Jolla, California 92093-0116, USA; [email protected]; +44
1603 450572; +44 1603 450027
[email protected]
Auxin plays important roles in organogenesis and tissue patterning. Members of the PINFORMED (PIN) family of
auxin efflux carriers are required for directional flow of auxin from cell to cell, and their asymmetrical localisation in the
plasma membrane is regulated by the protein kinase PINOID (PID). The polar auxin transport process ensures regulation
of local auxin concentrations, which mediate specification of organ outgrowths, polarity within organs or formation of
specific cell types. Although significant leaps have been made in the understanding of how auxin mediates its function
on a molecular level, not much is known about the upstream events that are likely to be specific to individual tissues.
Here we show that tight regulation of auxin levels and distribution is essential for proper Arabidopsis fruit development
and seed dispersal. We found that the valve margin identity factor INDEHISCENT (IND) negatively regulates PIN1 localisation at the plasma membrane, and loss-of-function analyses indicate this occurs at least partially by repressing PID
expression. Furthermore we found that IND blocks auxin-induced gene expression. Consistent with these data, localised
ectopic production of auxin in valve margin cells resulted in loss of valve margin specification. Our results provide a
detailed description of auxin concentrations and flow during Arabidopsis fruit development and firmly embed auxin dynamics within the established regulatory pathway of Arabidopsis fruit patterning.
PS0113 THE WPK1 GENE REGULATES EMBRYO AND ALEURONE DEVELOPMENT IN MAIZE, POSSIBLY THROUGH
REGULATION OF LEC1 AND B3 DOMAIN TRANSCRIPTION FACTORS
Suzuki, Masaharu 1, Latshaw, Susan 1, Sato, Yutaka 2, Settles, A. Mark 1, Koch, Karen E. 1, Hannah, L. Curtis 1, Kojima, Mikiko 3, Sakakibara, Hitoshi 3, McCarty, Donald R. 1 1 PMCB program, Horticultural Sciences Dept., University
of Florida, Gaineville FL32611, USA 2 Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi
464_E601, Japan 3 Institute of Physical and Chemical Research Plant Science Center, Yokohama, Kanagawa 230_E045,
Japan
masaharu@ufl.edu
Two classes of DNA binding proteins, LEC1 and B3 domain transcription factors, are known to play central roles
during plant seed development. We have identified a maize mutant that alters expression of these factors in developing
seeds. This mutant, designated as widowʼs peak 1 (wpk1), perturbs embryo development at late stage of embryogenesis
and modulates polarity of aleurone differentiation of maize kernels. In the developing seeds of wpk1 mutant, expression
of a subset of LEC1 and B3 genes is reduced. In addition, endogenous ABA level is significantly decreased in the mutant
embryos, together with a reduction of Vp14 gene expression. We isolated three independent Mutator transposon tagged
wpk1 alleles from UniformMu W22 inbred mutagenic population. We subsequently cloned Wpk1 gene by using Mutator adapted TAIL PCR method. Further genetic analysis revealed that the wpk1 is allelic to viviparous8 (vp8) mutant.
Unlike the wpk1 mutants, vp8-R in its original background develops a viviparous yet viable embryo. Upon introgression
into the W22 inbred background, vp8-R expression was transformed to a defective embryo phenotype indistinguishable
from the three UniformMu wpk1 alleles. Further genetic tests implied that the Stock Center vp8-R stock contains a single
dominant suppressor of the wpk1/vp8 mutant.
IPGSA 19th Annual Meeting - Abstracts
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PS0114 THE ROLE OF GIBBERELLIN IN THE REGULATION OF WHEAT GRAIN DEVELOPMENT
Vaughan, Simon., Wilkinson, Mark., Evans, Dan., Wan, Yongfang., Li, Keixa., Sparkes, Caroline., Jones, Huw., Huttly,
Alison., Lenton, John., Phillips, Andy., Hedden, Peter
[email protected]
Gibberellin (GA) produced by the germinating wheat embryo induces the production of hydrolases, including _ amylase, in the aleurone. Under certain environmental conditions _ amylase is produced prematurely in developing grain resulting in flour with poor bread-making quality. To investigate the role of GA in grain development and to establish a link
between GA levels and premature _ amylase we utilised transgenic wheat (cv. Cadenza) over-producing either of two GA
metabolic genes: the GA inactivating 2 oxidase gene from bean (PcGA2ox1), introduced with the aim of decreasing GA
levels, and the GA biosynthetic enzyme, 20 oxidase, gene from Arabidopsis (AtGA20ox1), introduced with the aim of increasing GA levels. Expression of both transgenes was targeted to the endosperm under control of the endosperm-specific
High Molecular Weight Glutenin (HMWG) promoter. Significant effects were observed in _ amylase levels in the mature
grain and on grain morphology. In the HMWG::GA20ox lines _ amylase levels at harvest were found to be over three
times higher than those observed in control lines. Immunoblotting indicated that the _ amylase was present mainly in the
aleurone. Furthermore, increases in grain weight of between 10% - 40% were observed in the primary transgenic lines
and of between 19% - 32% maintained into the T2 generation. However, sections through the grain revealed the presence
of large air spaces which were absent in control lines. This may be the result of the high _ amylase in the developing grain
and/or the endosperm volume being increased beyond the capacity of the source tissues in the GA20ox lines. In contrast,
grain size and weight was found to be unaffected by over-expression of the HMWG::GA2ox gene. However _ amylase
levels in the GA2ox lines at harvest were found to be lower than those observed in control lines. Additional studies to
elucidate the effect of increasing or suppressing GA expression in specific tissues and at specific developmental stages in
the developing wheat seed have been initiated.
PS0115 Genetic interactions of LEC2 and GA
Mónica Santos Mendoza, Pierre Bechereau, Bertrand Dubreucq, Loic Lepiniec
Institut Jean-Pierre Bourgin, Laboratoire de Biologie des Semences, UMR204 INRA, AgroParisTech, F-78026 Versailles
Cedex, France.
[email protected]
It is well known that GA is a hormone involved in many cellular processes (Swain and Singh, 2005). The high GA
level is important for germination and subsequent differentiation of several cell types. Mutants of the chromatin-remodeling factor PICKLE (pkl) maintain embryonic traits after germination specifically in the primary roots. This phenotype
is enhanced with the use of GA inhibitors or by crossing pkl with a GA biosynthetic mutant (ga) (Ogas et al, 1999).
Furthermore, the addition of GA during pkl seedling development inhibits embryonic traits. The embryonic roots of pkl
accumulats transcripts for three important transcription factors (TF) specific of embryogenesis, the leafy cotyledons:
Leafy cotyledon 1 (LEC1), LEC2 and Fusca3 (FUS3). These factors modulate the expression of a variety of genes
generating a complex regulatory network. It is known that FUS3 negatively regulates the biosynthesis of GA by inhibiting the transcription of the AtGA3ox1, which codes for an enzyme that promotes the synthesis of active GA (Curaba,
2004). Complementary, the Agamous-like 15 TF (AGL15) favors the production of inactive GA through the induction of
AtGA2ox3; interestingly, FUS3 and AGL15 are positively regulated by LEC2. Thus, LEC2 negatively regulates active
GA in an indirect manner. Transgenic plantlets that ectopically produce LEC2 under the control of the glucocorticoid
receptor (LEC2:GR) have shown the production of seed specific fatty acids in leaves treated with the synthetic hormone
dexametasone (DEX). Trying to shed some light about the action of GA on LEC2, these plants were crossed with a ga1
mutant. The results of the molecular and biochemical analyses of these plants will be presented.
Curaba, J., Moritz, T., Blervaque, R., Parcy, F., Raz, V., Herzog, M., and Vachon, G. (2004). Plant Physiol 136, 36603669.
Ogas, J., Kaufmann, S., Henderson, J., and Somerville, C. (1999). Proc Natl Acad Sci U S A 96, 13839-13844.
Swan SM, Shing DO. (2005). Trends Plant Sci, 10, 123-129
IPGSA 19th Annual Meeting - Abstracts
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Shoot Development
PS0201 SHOOTING CONTROL IN THE MARUBAKAIDO APPLE-ROOTSTOCK: EFFECT OF BRASSINAZOLE ON
SHOOT FORMATION, ELONGATION AND ENDOGENOUS LEVELS OF BRASSINOSTEROIDS
Pereira-Netto, Adaucto B.1; Fujioka, Shozo2; Asami, Tadao2; Yoshida, Shigeo2 & Clouse, Steve3 1Dept. of BotanySCB, Paraná Federal University, Centro Politecnico, C.P. 19031, 81531-970 Curitiba-Paraná, Brazil; 2The Institute of
Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; 3Dept. of Horticultural Science, North Carolina State University, Campus Box 7609, Raleigh, North Carolina 27695-7609, USA; apereira@ufpr.
br
[email protected]
The use of specific biosynthesis inhibitors is an alternative to the use of mutants for the determination of physiological
functions of brassinosteroids (BRs), especially for tree species since no BR-deficient mutant has been identified for trees
so far. Here, we report on the use of brassinazoles (Brzs), inhibitors of BR biosynthesis known to selectively and directly
binds to the DWF4 protein, to help to unravel the role of BRs in shooting control in the marubakaido apple rootsock
(Malus prunifolia). Progressive inhibition of main shoot elongation was found for shoots treated with increasing doses
of Brz220. Somewhat surprisingly, a slight increase in elongation of primary lateral shoots was found for Brz220-treated
shoots, except when Brz220 was used at the highest dose (5mg.shoot-1), when no change in shoot elongation was found.
Treatment of shoots with 0.2 mg.shoot-1 Brz220 resulted in an increase of 16 and 67 % in the formation of main and
primary lateral shoots, respectively. Progressive enhancement of shoot fresh weight was also related to increased doses
of Brz, up to 1 mg.shoot-1. Further enhancement of the Brz dose (5 mg.shoot-1) reduced fresh weight accumulation. For
shoots treated with 1 µg.shoot-1 Brz 22012, a decline of 14 and 25% in shoot elongation was found respectively for main
and primary lateral shoots. In addition, a decline of 15 and 60% was found, respectively, for the number of main and
primary lateral shoots formed after the 1 µg.shoot-1 Brz 22012 treatment. Treatment with increased doses of Brz220 led
to a progressive increase in the endogenous levels of the sterols 24-MC and CR. Progressive decline in the endogenous
levels of 6-DeoxoCT, 6-DeoxoTY and CS was also related to treatment with increased doses of Brz220. However, shoots
treated with 5 µg.shoot-1 Brz220 presented unchanged or increased endogenous levels of 6-DeoxoTE, 6-Deoxo3DT,
6-deoxoCS, TE and TY. Further increase in the Brz220 dose (10 µg.shoot-1) led to a decrease in the endogenous levels
of all of the detected BRs to a level below the ones found for untreated shoots. These results give support to previous
reports showing that brassinazole treatments diminishes the levels of BRs whose side chains possess hydroxy groups.
Neither brassinolide nor cathasterone were identified as endogenous BRs in marubakaido shoots. Taken together, data
in this report, along with previously reported data on the promotive effect of BRs on shoot formation and elongation
(Pereira-Netto et al., 2006), indicate that shooting in marubakaido is under control of changes in the endogenous pool of
bioactive BRs.
PS0202 IN VITRO SHOOT REGENERATION OF POTATO FROM CELL SUSPENSION
Ali Akbar Ehsanpour, Firouzeh Torabi, and Ahmad Majd
[email protected]
Potato (Solanum tuberosum L.) is one of the most important and widely grown food crops after wheat, rice and maize.
Plant regeneration from cell and tissue culture is an important and essential component of biotechnology that is required
for the genetic manipulation of plant. Regeneration of adventitious shoot can be produced both directly from organ culture and in directly via a callus phase or cell suspension.
In direct method stem and leaf segments of potato cultivar White Desiree were cultured on four different regeneration
medium with different combination of BAP, IAA, IBA, NAA, GA3.
In indirect method leaf and stem explants were initially produced callus in MS medium with 2,4-D , NAA and KIN. Then
from callus cell suspension supplied in liquid medium , and cell suspension were transferred to solid medium for callus
recovery, finally callus were transferred to regeneration medium.
Results showed the highest percentage of shoot formation via direct organogenesis on medium with BAP and IBA.
IPGSA 19th Annual Meeting - Abstracts
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Medium containing BAP and NAA resulted in root formation. In other experiment stem and leaf explants initially were
cultured on medium with BAP and NAA, after two weeks they were transferred to medium supplemented with BAP and
GA3 and finally after seven weeks several shoots were initiated.
When callus segments were cultured to the medium with BAP and GA3, they became green and started shoot bud initiation. In other regeneration medium callus became green only.
However, plant regeneration from cell suspension is in progress.
PS0203 OPPOSING EFFECTS OF BRASSINOSTEROIDS AND GIBBERELLINS ON SHOOT BRANCHING
Ross, John J1, Reid, James B1, Bishop, Gerard J2, Foo, Eloise1 1School of Plant Science, University of Tasmania,
Private Bag 55, Hobart, Tasmania, Australia; 2Faculty of Life Sciences, Imperial College London, Wye Campus, Kent,
TN25 5AH, UK
[email protected]
In pea, recombining the brassinosteroid-deficient dwarf mutant lkb with the increased-branching mutant rms2 results
in plants in which basal branching is inhibited, compared with the single mutant rms2. This novel phenotype of lkb rms2
plants indicates that brassinosteroids function as promoters of shoot branching. In contrast, gibberellin-deficient dwarf
mutants tend to branch more than comparable wild-type plants, implicating gibberellin as an inhibitor of branching. To
further explore these roles, we monitored the expression of a key branching gene, RMS1, in brassinosteroid-related mutants. The RMS1 gene is thought to encode a mobile branching inhibitor. However, RMS1 mRNA levels were reduced
in the lkb mutant, even though lkb branches less than the wild type. This supports the existence of a feedback system
in which the branching process influences the expression of RMS1 (Foo et al 2005; The Plant Cell, 17: 464-474). We
will also report on the relationships between gibberellin, RMS1, and DELLA proteins (key signalling molecules), with
respect to the regulation of shoot branching.
PS0204 THE EFFECTS OF LIGHT AND DARK ON ENDOGENOUS HORMONE LEVELS AND THEIR RELATIONSHIP
WITH THE RESTART OF MERISTEMATIC ACTIVITY IN APICAL SHOOTS OF CATASETUM FIMBRIATUM (ORCHIDACEAE)
Rogério M. Suzuki1, Gilberto B. Kerbauy2, Wagner M. Ferreira3, Eduardo Purgatto4 and Rosete Pescador5 1Instituto de
Botânica de São Paulo, Seção de Orquidário do Estado, Av. Miguel Stéfano 3687, 04301-902 São Paulo, Brazil. 2Departamento de Botânica, Universidade de São Paulo, São Paulo, Brazil. 3Universidade Federal do Tocantins, Núcleo de Estudos Ambientais (NEAMB), Porto Nacional, Brazil. 4Department of Food and Experimental Nutrition, FCF, University
of São Paulo, São Paulo, Brazil. 5Departamento de Ciências Naturais, Fundação Universidade Regional de Blumenau,
Blumenau, Brazil – [email protected]
[email protected]
Light plays an important modulatory role on Catasetum fimbriatum development. This plant shows both interruption
of shoot apex growth and pseudobulb formation around 150 days under light incubation. However, their transference to
dark condition rapidly promotes the restart of apical shoot meristem activity as well lateral bud development. Contrarily,
if they are kept under light, the inhibition is maintained. In this study we analyze the variations of endogenous levels of
four cytokinins (IP, IPR, Z, ZR) and IAA in the apex shoot and pseudobulb development in the light, and after transference to the dark. Quantifications were performed by HPLC-ELISA (cytokinins) and GC-MS-SIM (IAA).
The reduced cytokinins/IAA ratio in the apex of plants incubated in the light, due to the presence of high IAA levels, could
be involved in the apical shoot development inhibition. The pseudobulb formation would be related to the photosynthetic
products accumulation in the still short shoot, to the detriment of the apex region, thus contributing for its inhibition. Possibly, the resumption of shoot apex development just after dark transference was induced by a significant raise of cytokinins/IAA ratio in that region. The enhanced cytokinin levels in pseudobulbs verified 20 d after dark transference could
be related to the stimulation of the lateral buds development. The results reveal that light can strongly modulates the C.
fimbriatum growth by promoting marked variations in endogenous levels of cytokinins and IAA, inhibiting or stimulating the apical shoot development, lateral bud and pseudobulb formation. Supported by FAPESP Proc. No 00/02301-8
IPGSA 19th Annual Meeting - Abstracts
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PS0205 NITRIC OXIDE AND HYDROGEN PEROXIDE REGULATE THE EXPRESSION OF THE ZINNIA ELEGANS BASIC PEROXIDASE ISOENZYME, A KEY ENZYME INVOLVED IN LIGNIN BIOSYNTHESIS
Ros Barceló, Alfonso1, Gabaldón, Carlos1, Gómez-Ros, Laura1, Esteban-Carrasco, Alberto2, Zapata, Jose Miguel2,
Gayoso, Carmen3, Merino, Fuencisla3 1Department of Plant Biology, University of Murcia, E-30100, Murcia, Spain
2Department of Plant Biology, University of Alcalá, E-28871, Alcalá de Henares, Spain 3Department of Plant Biology,
University of La Coruña, E-15071, La Coruña, Spain
[email protected]
The Zinnia elegans basic peroxidase (ZePrx) is encoded by at least four putative paralogous genes, which belong to
the class a peroxidase gene subgroup, already described in Oryza and Arabidopsis. These ZePrx paralogous genes arise
from the duplication of a previously duplicated ancestral gene by recombination within introns, as has been described for
two horseradish basic peroxidase genes coded in tandem. Nitric oxide and hydrogen peroxide are two key endogenous
signals whose levels greatly increase during xylem differentiation. In this report, we study i) the stability of the secondary
structure of the four mRNAs codifying this protein, and ii) the effect of nitric oxide and hydrogen peroxide on the expression of these mRNAs by real-time PCR. The results showed that the four mARNs are, theoretically, indiscriminately
translated into active proteins, and that both nitric oxide and hydrogen peroxide regulate the expression of ZePrxs. The
possible cross-talks between these signals are outlined.
This work was supported by grants from the Fundación Séneca (project # 00545/PI/04) and MEC(BFU2006-11577/BFI)FEDER.
PS0206 HORMONAL REGULATION OF LIGNIFICATION DURING THE FIRST STAGES OF DEVELOPMENT IN ZINNIA
ELEGANS SEEDLINGS
López Núñez-Flores, María Josefa1, Gómez-Ros, Laura1, Cuello, Juan1, Ros Barceló, Alfonso1 Department of Plant
Biology, University of Murcia, E-30100, Murcia, Spain [email protected]
[email protected]
Xylem differentiation in plants is under strict hormonal regulation. Auxins and cytokinins, together with gibberellins
and ethylene, appears to be the main hormones controlling vascular differentiation. In this report, we study the effect of
these hormones on xylem differentiation during the first stages of development of Zinnia elegans seedlings. Pleiotropic
hormonal effects on diploid seedlings were compared with phenotypic characters (degree of vascularization, lignification) of tetraploid seedlings, and possible cross-talks between hormones were established. Studies were completed at
protein level by studying, during xylem differentiation, the hormonal regulation of both the protein fingerprint and the
basic peroxidase of Z. elegans, a key enzyme involved in lignin biosynthesis.
This work was supported by grants from the Fundación Séneca (project # 00545/PI/04) and MEC (BFU2006-11577/
BFI)-FEDER. LVGR holds a fellowship (FPI) from the MCYT.
PS0207 HORMONAL REGULATION OF LIGNIFICATION DURING THE FIRST STAGES OF DEVELOPMENT IN ZINNIA
ELEGANSSEEDLINGS
López Núñez-Flores, María Josefa, Gómez-Ros, Laura, Cuello, Juan, Ros Barceló, Alfonso Department of Plant Biology,
University of Murcia, E-30100, Murcia, Spain
[email protected]
Xylem differentiation in plants is under strict hormonal regulation. Auxins and cytokinins, together with gibberellins
and ethylene, appears to be the main hormones controlling vascular differentiation. In this report, we study the effect of
these hormones on xylem differentiation during the first stages of development of Zinnia elegans seedlings. Pleiotropic
hormonal effects on diploid seedlings were compared with phenotypic characters (degree of vascularization, lignification) of tetraploid seedlings, and possible cross-talks between hormones were established. Studies were completed at
protein level by studying, during xylem differentiation, the hormonal regulation of both the protein fingerprint and the
basic peroxidase of Z. elegans, a key enzyme involved in lignin biosynthesis.
IPGSA 19th Annual Meeting - Abstracts
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This work was supported by grants from the Fundación Séneca (project # 00545/PI/04) and MEC (BFU2006-11577/
BFI)-FEDER. LVGR holds a fellowship (FPI) from the MCYT.
PS0208 BRANCHING OUT: CONTROL OF AXILLARY MERISTEM GROWTH IN PETUNIA
Revel S.M. Drummond, Joanne L. Simons, Brian Quinn, Bart J. Janssen, Kimberley C Snowden.
[email protected]
A mutational approach has been used to study the genetic control of axillary branch development in Petunia hybrida.
Mutations at three DAD (Decreased apical dominance) loci act primarily to increase the growth of axillary meristems
into branches during vegetative development. This leads to a highly branched phenotype for dad1 and dad2 mutants and
a moderately branched phenotype for dad3.
Graft analyses of the dad mutants with wild-type plants suggest that there is a branch suppressing factor produced by
wild-type roots which is transported to the stems, but additionally that at least one other transported factor that promotes
branching is produced in dad1 and dad3 mutant roots. Results showing the combined action of dad and wild-type rootstocks on a single plant will be presented.
We have cloned the genes responsible for the dad1 and dad3 mutant phenotypes. These are PhCCD8 and PhCCD7,
respectively, which encode putative carotenoid cleavage dioxygenases. Both genes are expressed in root and shoot tissue, consistent with the observation that grafts of wild-type root, or small pieces of wild-type shoot, tissue are sufficient
to revert dad mutants to near wild-type branching. Data showing the expression of the PhCCD7 and PhCCD8 genes in
mutant and grafted plants will also be presented.
PS0209 PEAPOD GENE FUNCTION IS CONSERVED IN WHITE CLOVER
Alvarez, Nena de Guzman and White, Derek W.R. Grasslands Research Centre, AgResearch, Private Bag 11008, Palmerston North, New Zealand
[email protected]
In Arabidopsis the PEAPOD genes (PPD1 and PPD2) control the arrest of cell proliferation during the later stages of
organ lamina development (1). Loss-of- PPD1/PPD2 function resulted in prolonged dispersed meristematic cell proliferation during lamina growth and abnormally large leaves with positive curvature. Conversely, the duration of dispersed
meristematic cell proliferation was reduced in transgenic plants over expressing either PPD1 or PPD2, and lateral organs
were smaller. PPD proteins have a central putative DNA binding motif, in combination with a highly conserved and
unique N terminal PPD domain (1). Database analysis indicates the presence of PPD-like genes in a wide range of Eudicot plants and their ancestors, but the genes appear to be absent from the grasses. We have initiated a study to determine
if the function of PPD genes is conserved in the Eudicots. Here we report the isolation and characterisation of a white
clover orthologue of Arabidopsis PPD.
Full-length cDNA and genomic DNA ORF clones of a PPD-like gene from white clover, fused to AtPPD1 regulatory
sequences, were able to rescue PPD gene function when transformed into an Arabidopsis ppd deletion mutant. Partial
silencing of the white clover PPD-like gene, using RNAi, resulted in transgenic white clover plants with enlarged, positive curvature leaves. Furthermore, in situ hybridisation analysis demonstrated that the white clover PPD-like gene was
expressed in developing leaves, in a pattern similar to expression of the PPD1 gene in Arabidopsis. We conclude that PPD
gene function is likely to be conserved throughout the Eudicots.
(1.) White, D.W.R. (2006). PEAPOD regulates lamina size and curvature in Arabidopsis. PNAS 103: 13238-13243.
PS0210 AUXIN EXPORT FROM PEA AXILLARY BUDS
Jozef Balla, Petr Kalousek, Zdenka Tesarikova, Vilem Reinohl, Stanislav Prochazka Dept. of Plant Biology, Mendel
University of Agriculture and Forestry, Zemedelska 1, 613 00 Brno, Czech Republic
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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The changes linked to the release of axillary buds from apical dominance were studied on two-week-old plants of
pea (Pisum sativum L.) cv. Vladan decapitated above the 2nd or 3rd axillary bud. The export of auxin from axillary buds
was traced by the use of radioactively labeled [14C]-IAA and the establishment of polar auxin transport was visualized
by immunolocalization of PIN1 protein. Also the expression of PsDRM1, a gene closely related to bud dormancy maintenance, was followed. The results of our experiments show the correlation among the decrease of PsDRM1 expression,
establishment of polar export of auxin from axillary buds into the stem vascular bundles and visible bud outgrowth. Also
the changes of auxin transport and PsDRM1 expression in axillary buds were studied during growth to dormancy cycle.
This work was supported by a grant of the Czech Ministry of Education LC 06034.
PS0211 UNDERSTANDING THE ROLE OF GREEN REVOLUTION GENES IN WHEAT
Pearce, Stephen 1, Thomas, Steve 1, Hedden, Peter 1, Phillips, Andy 1 and Edwards, Keith 2 1 Plant Science Department,
Rothamsted Research, Harpenden, AL5 2JQ. 2 University of Bristol, BS8 1TH [email protected], 01582
763133
[email protected]
During the 1960s, shorter varieties of the worldʼs most important crops were bred that contributed to the Green Revolution, increasing harvest index and reducing yield losses due to lodging. In wheat, the dwarfing gene responsible has
been identified as the GA response modulator gene, RHT. Its product is a DELLA protein, which represses growth by an
unknown mechanism. When bioactive GA binds its soluble receptor, GID1, the GID1-GA complex associates with DELLAs allowing them to be polyubiquitinated by the SCF E3 ligase and thereby targeted for degradation. We are seeking to
understand GA regulation of stem extension in wheat by analyzing the expression of key genes in stem tissues. In order
to prepare promoter::GUS reporters for genes involved in GA signaling, the promoters of the GID1 and RHT genes, as
well as the biosynthetic genes TaGA20ox1 and TaGA3ox2 have been cloned. RHT regulation is achieved mainly at the
protein level. The stability of the RHT protein can be affected by mutations, such as those in the agriculturally important
Rht-B1b and Rht-D1b dwarf mutants in which a STOP codon has been introduced within the GA-binding DELLA domain; this is assumed to result in a truncated product lacking the DELLA domain produced by downstream re-initiation.
With RHT antibodies, it will be possible to test this process and determine how these and other mutations in RHT affect
the proteinʼs stability. In particular, the severe Rht-B1c and the milder Rht-B1e mutations have been characterized at the
DNA level and the effects of these mutations on protein stability are currently being determined.
Senescence
PS0301 ROLES OF CYTOKININS IN THE CONTROL OF NITROGEN ECONOMY, LEAF SENESCENCE AND GRAIN
FORMATION OF WHEAT PLANTS
S_korová Blanka1, Jandová Gabriela2, Hoyerová Klára1, Trcková Marie2, Daskalova Sasha3, Motyka Václav1, Scott
Nigel3, Elliott Malcolm3, Kamínek Miroslav1
[email protected]
Cytokinins control a number of processes related to plant productivity including signaling of the nitrogen status of
the root to the shoot, delay of leaf senescence and strengthening the reproductive sinks. We used transgenic wheat plants
expressing isopentenyltransferase (IPT), a cytokinin biosynthetic gene, under the control of the senescence-inducible
PSAG12 promoter (Gan and Amasino, 1995), to determine the effect of cytokinin levels on the grain yield-related parameters specified above. The extension of the period of active nitrate uptake by the PSAG12-IPT plants was correlated with
the delay of leaf senescence and was more pronounced in plants grown under limited N supply. The delayed senescence
of the flag leaves of the transgenic plants was also related to the accumulation of total as well as currently uptaken [15N].
The ears and developing grains of control plants exhibited a much higher sink for assimilated [15N]. It was demonstrated
that the very sharp and transient peak of cytokinins that appears shortly after anthesis is associated with the late phase of
free nuclear divisions and with the beginning of endosperm cellularization. The strength of the reproductive sinks was
also increased after application of exogenous cytokinin to plants at anthesis. The results will be discussed in relation to
the different reproductive strategies of monocarpic plants.
Reference:
Gan S & Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270, 19861988.
IPGSA 19th Annual Meeting - Abstracts
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PS0302 THE ROLE OF JASMONIC ACID IN MONOCARPIC SENESCENCE IN SOYBEAN
Takada, Noboru1, Matsuno, Junko2, Abe, Mihoko1, Hashimoto, Masaru1, Koda, Yasunori2
1Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, 036-8561, Japan; 2Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan
[email protected]
Senescence in monocarpic plants, such as legume and wheat, is dramatic because the entire resources of the plant
are mobilized and redirected to support seed development. We found that the level of senescence promoting activity in
extract of soybean pods changed greatly during the plant growth. Abscisic acid was first identified as senescence promoting factor. However, Soybean pod did not contain enough amounts of abscisic acid. We found that jasmonic acid played
a role as a synergist against abscisic acid. Furthermore, we found that the level of jasmonic acid in pods increased greatly
just before beginning of monocapic senescence. These results suggested the possibility that jasmonic acid regulates the
commencement of monocarpic senescence of soybean.
PS0303 CYTOKININ AND GIBBERELLIN INFLUENCE RE-GREENING OF SPATHE TISSUE IN HYBRID ZANTEDESCHIA
Jianyu Chen1, Keith A. Funnell, David J. Woolley, David H. Lewis2, Jocelyn R. Eason2 1 Institute of Natural Resources,
Massey University, Palmerston North, New Zealand 2 New Zealand Institute for Crop & Food Research, Palmerston
North, New Zealand; [email protected]
The leaf-like structure (spathe) of Zantedeschia re-greens after it has fully opened and reached reproductive maturity.
Previous studies with Z. aethiopica have found that the spathe undergoes re-greening only when fructification occurs, or
when the inflorescence (spadix) is substituted by the cytokinins isolated from its fruit. Little is known about the re-greening in hybrids of the summer flowering species of Zantedeschia.
This report describes the influence of cytokinins (BAP, Zeatin and CPPU) and gibberellin (GA3) on the spathe re-greening of the hybrid ʻBest Goldʼ. Discs, excised from the spathe prior to pollen release, were floated in solutions of either
BAP, Zeatin, CPPU, or GA3 at 10-4, 10-6, and 10-8M, respectively. Colour variation of both abaxial and adaxial surfaces
of the discs was quantified using reflectance spectrophotometry (L*, C* and H) and pigment analysis. The abaxial surface
re-greened regardless of; removal of the spadix, absence of fruit, or the application of hormones. In contrast the adaxial
surface re-greened only when treated by the cytokinins. Hue Angle (H) increased during re-greening, which described the
colour change from yellow to green and was correlated with the increase in chlorophyll concentration. During re-greening, GA3 (10-4 and 10-6M) altered the rate of change in L*, C* and H on both surfaces, while BAP, Zeatin and CPPU
(10-4M) only affected the adaxial surface. In addition, all hormonal treatments delayed the onset of the subsequent senescence-phase of the re-greened tissue. We conclude therefore that there is a clear role for plant hormones in influencing
the re-greening of flower spathe tissue of both ʻBest Goldʼ and Z. aethiopica. However the exact mechanisms controlling
re-greening within each appears to differ.
PS0304 INDUCTION OF LEAF SENESCENCE BY DARKNESS OR HYDROGEN PEROXIDE IS DELAYED IN RICE TILLERING DWARF MUTANT, DWARF3
Haifang Yan1, Hiroaki Saika1, Masahiko Maekawa2, Itsuro Takamure3, Nobuhiro Tsutsumi1, Junko Kyozuka1, Mikio
Nakazono1 1,Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan; 2,Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama,
710-0046 Japan; 3,Faculty of Agriculture, Hokkaido University, Sapporo, 060 Japan
[email protected]
Leaf senescence in plants is known to be inducible by darkness or H2O2. When the Arabidopsis gene MAX2/ORE9 is
disrupted, leaf senescence in response to the above stimuli is delayed. Because the rice gene D3 is orthologous to MAX2/
ORE9, we wished to know whether disruption of D3 also results in delayed leaf senescence. We found that darknessand H2O2-induced leaf senescence (as measured by chlorophyll degradation, membrane ion leakage and expression of
senescence-associated genes (SAGs)) in a d3 rice mutant was delayed by 1-3 d compared to that in wild-type plants.
IPGSA 19th Annual Meeting - Abstracts
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Moreover, the mRNA levels of D3, HTD1 and OsCCD8, which are orthologs of Arabidopsis MAX2/ORE9, MAX3 and
MAX4, respectively, increased during leaf senescence. These results suggest that D3 protein in rice, like MAX2/ORE9
in Arabidopsis, is involved in leaf senescence.
PS0305 GIBBERELLIC ACID INHIBITS DARK-INDUCED SENESCENCE BY MULTIPLE PATHWAYS
Friedman, Haya, Rosenvasser, Shilo, Rot, Ilona and Harush, Tali Dept. of Postharvest Science of Fresh Produce, ARO,
The Volcani Center, Bet Dagan 50250, Israel,
[email protected]
Dark-induced senescence is characterized by chlorophyll breakdown, an increase in reactive oxygen species (ROS)
levels and induction of senescence-associated gene (SAG) transcripts. Gibberellic acid (GA3) applied to dark-treated
leaves delayed senescence either via or independent of ROS inhibition. GA3 applied to Pelargonium cuttings before the
dark treatment prevented chlorophyll breakdown and also prevented an increase in ROS. This was followed by inhibition of SAGs expression. The pattern of GA3 suppression of ROS levels positively correlated with its inhibitory effect
on chlorophyll breakdown. This may suggest GA3 inhibits senescence via inhibition of ROS. Nevertheless, application
of GA3after ROS accumulation in Pelargonium and in Arabidopsis before the dark treatment hardly affected chlorophyll
breakdown, but had a major effect on inhibiting SAGs expression. This suggests that GA3 acts to inhibit leaf senescence
by interfering with senescence regulation, independent of chlorophyll breakdown, through an as yet unknown mechanism.
microRNAs in Hormone Signaling
Peptide Hormones
PS0501 LIGAND BINDING SITE OF PHYTOSULFOKINE RECEPTOR IS 15-AMINO ACID REGION IN THE ISLAND
DOMAIN
Hidefumi Shinohara, Mari Ogawa, Youji Sakagami, Yoshikatsu Matsubayashi Grad. Sch. Bio-agricultural Sci., Nagoya
Univ., Chikusa, Nagoya, 464-8601, Japan
[email protected]
Phytosulfokine (PSK), an endogenous 5-amino-acid secreted peptide in plants, affects cellular potential for growth
via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with carrot
PSKR1 in a highly specific manner, with a dissociation constant of 4.2 nM. However, it is not known which residues in
the PSKR1 extracellular domain constitute the ligand-binding pocket. Here, we identified the PSK-binding domain of
carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK analog with DcPSKR1
using UV irradiation, and mapped the cross-linked region using chemical and enzymatic fragmentation. We also established a novel _gon-column photoaffinity labeling_h methodology that allows repeated incorporation of the photoaffinity
label to increase the efficiency of the photoaffinity cross-linking reactions. We purified a labeled DcPSKR1 tryptic fragment using anti-PSK antibodies, and identified a peptide fragment that corresponds to the 15-amino-acid Glu503-Lys517
region of DcPSKR1 by MALDI-TOF MS. Deletion of Glu503-Lys517 completely abolishes the ligand-binding activity
of DcPSKR1. This region is in the island domain flanked by extracellular LRRs, indicating that this domain forms a
ligand-binding pocket that directly interacts with PSK.
Reference
Shinohara, H., Ogawa, M., Sakagami, Y. and Matsubayashi, Y. (2007) Identification of ligand binding site of phytosulfokine receptor by on-column photoaffinity labeling. J. Biol. Chem. 282 124-131
IPGSA 19th Annual Meeting - Abstracts
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Hormones in Agriculture and Horticulture, and Synthetic Plant Growth Regulators
PS0601 DIVERSITY IN SALICYLIC ACID EFFECTS ON GROWTH CRITERIA AND DIFFERENT INDOLE ACETIC ACID
FORMS AMONG FABA BEAN AND MAIZE
El-Mergawi , Ragab and Abdel-Wahed , Mohamed. Botany Dept., National Research Centre, Dokki, Egypt
[email protected]
Salicylic acid is a phenolic compound and natural constituent of plant .Phenolics participate in some way on auxin
metabolism by regulating IAA degradation or by control the formation of IAA conjugates .The present study was conducted to investigate the association between diverse effects of SA (1-5 mM) on faba bean and maize growth and IAA
metabolism. All SA doses tended to induce increases on faba bean growth criteria. There was a progressive increase in
the fresh and dry weights of stem, leaves and whole plant as well as number and fresh weight of kernels by increasing SA
level. These characters achieved more than 40% increases by spraying faba bean plants with 5mM of SA. An enhancement effect was obtained by maize plants as affected by 1 or 2mM of SA, but application 3-5mM resulted in a remarkable
reduction in plant height, fresh and dry weights of stem and leaves, maximized by using 4mM after 30 days from SA
spraying. Chlorophylls and total phenolics in the leaves of faba bean and maize did not affect significantly by SA treatments. Free, ester, amide, bound and total IAA forms were determined by using HPLC. At vegetative growth stage, free
IAA in leaves of faba bean and maize plants reached about 31 and 14%, respectively of the total IAA .Whereas, amide
IAA form constituted about 43% and 70%, corresponded by about 26% and 17% for ester form, respectively. Spraying
faba bean plants with SA tended to cause increases in free IAA and decreases in ester IAA, after 3 days from treatment.
Values of free and ester IAA reached 113% and 44%, respectively of control by using SA at 4mM. As for maize, all SA
doses caused decreases in free IAA and increases in ester IAA. Maximum effects (30% decreases in free IAA and 70%
increases in ester IAA) were achieved by 5mM after 6 days from treatment.
In general SA did not induce a great consistent effect on amide-IAA and total IAA, while changes in ester form tended to
be in opposite manner to changes in free IAA, meaning that SA might interfere with IAA-conjugation.
Key words: Salicylic acid, (SA), indole acetic acid forms, phenolics, faba bean,maize.
PS0602 PROHEXADIONE-CA INCREASES LYCOPENE CONTENT AND ENZYMATIC ACTIVITY DURING
FRUIT RIPENING IN TOMATO
Ramirez, Homero, Herrera, Bruno, Benavides, Adalberto, Mendoza, Rosalinda,Rancaño, Hugo,Villareal, Jose Angel
Dep. Hort. Universidad Aut. Agr. Antonio Narro Buenavista, Saltillo, Coah., 25315, Mexico
[email protected]
This study was conducted with the purpose of learning the effects of prohexadione-ca (P-Ca)on the antioxidant lycopene content and the enzymatic activity during fruit ripening in tomato var. Floradade. The growth retardant was sprayed
to experimental plants in one or/and in two occasions.The first application was done when plants had reached 50% blossom; whereas, the second one was performed two weeks after.The concentration dosages of P-Ca were: 0 , 125, 175 and
200 mg L-1.
The results showed that any treatment with P-Ca increased the total antioxidant capacity in evaluated fruits at different
stages of fruit ripening. The content of lycopene showed a remarkable increment in ripen fruits harvested from plants
which have received the P-Ca applications. The activity of catalaze and peroxidase also increased in these tomato fruits
treated with the growth retardant.
PS0603 CYCLANILIDE PROMOTES LATERAL BRANCHING OF CRAPE MYRTLE AND INKBERRY HOLLY
Banko, Thomas1, and Stefani, Marcia1 1Virginia Tech, Hampton Roads AREC, 1444 Diamond Springs Rd., Virginia
Beach, VA 23455, USA
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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During nursery production of landscape trees and shrubs it is desirable to promote the development of well-branched
plants to achieve a uniform, full appearance. Branching may be promoted with multiple prunings but this is labor-intensive and expensive. In this study, spray applications of the bioregulator, cyclanilide (Bayer Environmental Science) were
evaluated for promotion of lateral branching on container-grown crape myrtle (Lagerstroemia _indica ʻMuskogeeʼ) and
inkberry holly (Ilex glabra ʻCompactaʼ). Treatments were applied in the spring as shoots were elongating. Cyclanilide
spray concentrations of 0, 54, 106, and 212 mg/L resulted in a linear increase in new lateral shoots for crape myrtle and
a quadratic increase for inkberry holly. Treatments also caused a delay in flowering, and a small but significant reduction
in height of crape myrtle.
PS0604 PACLOBUTRAZOL TREE GROWTH REGULATOR AFFECTS PHYSIOLOGY, MORPHOLOGY AND GROWTH
OF SWEETGUM AND CHERRYBARK OAK
Qi, Yadong1, Chaney, William R.2, Hilton, Tanya K3 1Urban Forestry Program, Box 11288, Southern University, Baton
Rouge, LA 70813, USA; 2Department of Forestry & Natural Resources, Purdue University, West Lafayette, IN 47907,
USA; 3USDA Forest Service, Southern Region, 1720 Peachtree Road NW, Atlanta, GA 30309, USA
[email protected]
Paclobutrazol (PBZ) treatment was applied by soil drench one time to six years old sweetgum (Liquidamber styraciflua L.) and cherrybark oak (Quercus falcata var. pagodaefolia Ell.) located at Southern University Horticultural Farm,
Baton Rouge, LA in spring 1997. Physiological and morphological study in the third year (2000) after the treatment indicated that PBZ significantly reduced leaf surface area, increased leaf thickness, and enhanced leaf chlorophyll content
in both species. PBZ also reduced leaf petiole length and enhanced the gas exchange rates per unit of leaf area including
net photosynthesis, stomatal conductance and transpiration in sweetgum but not in cherrybark oak. Scanning electron
micrographs taken in the fourth year (2001) showed that PBZ suppressed the radial growth of the epidermal and palisade
cells resulting in cell compaction; and in turn, stimulated the transverse growth causing thickening of the epidermal and
palisade layers in both species. PBZ also caused leaf veinlets to be more deeply imbedded within the leaf surfaces of
sweetgum and induced higher leaf trichome density in cherrybark oak.
The greater density of trichomes could be very beneficial to leaves as a physical barrier to biotic invasion and a means
by which the boundary layer resistance is increased to reduce moisture loss under dry weather conditions. Measurement
of growth at the end of the fifth year (2002) of the treatment indicated that PBZ reduced tree height by 60% in sweetgum
and 50% in cherrybark oak, and decreased DBH by 65% in sweetgum and 58% in cherrybark oak. Since PBZ treatment
provided sweetgum and cherrybark oak trees with specific structural and physiological advantages, it could be used as a
tree growth retardant for utility and urban forestry to effectively maintain healthier and slower-growing trees underneath
power lines, along the streets, and other landscape situations.
PS0605 IMMUNOMODULATIONAL STUDY OF GIBBERELLINS IN PLANTS
Suzuki, Yoshihito1, Mizuno, Toru1, Yamaguchi, Isomaro2, Asami, Tadao1 1Dept. Appl. Biol. Chem., The Univ. of Tokyo, Tokyo, Japan; 2Faculty of Technol., Maebashi Inst. Technol., Gunma, Japan
[email protected]
A single-chain Fv (scFv) is the minimum recombinant form of antibody with antigen-binding activity. ScFv gene
libraries with random mutations were constructed from anti-bioactive gibberellin (GA) monoclonal antibody lines from
which functional scFv clones were screened by their affinity to GA4. These scFv genes were used for immunomodulational study of GAs in plants, i.e., the scFvs were produced in tobacco and arabidopsis to disturb normal function of GAs.
The expression vectors were designed to localize scFvs in various subcellular compartments, which were confirmed by
fluorescence of GFP fused to scFvs.
The production of scFv-GFP was confirmed by ELISA, western and GFP fluorescence analysis for all the constructs.
There was a tendency that scFv-GFPs were more stably accumulated in endoplasmic reticulum (ER) than other compartments. The ER lines showed significantly shorter plant height than wild-type plants in both plant species. The roots
were also shorter in transgenic plants. The endogenous GA4 level was much higher in transgenic plants than in wild-type
plants, strongly suggesting that scFvs inhibited the action of the bioactive GAs by capturing them.
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PS0606 MANIPULATION OF FRUIT DROP AND CROP LOAD IN PECAN WITH AMINOETHOXYVINYLGLYCINE,
FORCHLORFENURON, AND GIBBERELLIN-A3
Wood, Bruce1 1USDA, ARS, Fruit and Tree Nut Research Laboratory, Byron, Georgia 31008, USA Bruce.Wood@ars.
usda.gov
[email protected]
Excessive fruit-drop is a major problem in many tree-fruit and tree-nut crops. A substantial percentage of fruit drop 4-6
weeks post-pollination (i.e., June Drop). This drop is especially severe for certain pecan (Carya illinoinensis) cultivars,
such as ʻDesirableʼcv., with as much as 90% of the potential crop aborting. Field studies found aminoethoxyvinylglycine
(AVG/ReTain) and forchlorfenuron (CPPU/Prestige) to substantially reduce fruit-drop by pecan trees in orchards. AVG is
efficacious when applied as a foliar spray to canopies just after the conclusion of pollination at 66 – 132 mg a.i. per L and
CPPU is efficacious at 15 – 30 mg a.i. per L. Gibberellin-A3 (ProGibb) at 100 mg a.i. per L reduced drop by about 14% in
heavy crop trees, whereas Gibberellin A4+7 & 6-Benzyladenine (Promalin) at 50 mg a.i. per L was ineffective. Activity
by three distinctly different classes of plant growth regulators affecting three different classes of endogenous regulators
appears to indicate that either two or more mechanism likely regulate fruit drop or that a single mechanism is subject to
influenced at different points by different classes of growth regulators. AVG or CPPU increased fruit retention by > 30%
in trees supporting relatively heavy crop loads, but did not retain fruit in trees with light crops. AVG and CPPU are potentially useful for increasing crop yields in moderate to heavy crop load trees. Unfortunately, enhanced fruit retention leads
to reduced flowering the following growing season via pecanʼs innate alternate bearing physiological characteristic.
PS0607 VASE LIFE AND FLOWER SENESCENCE OF EXCISED INFLORESCENCES OF POLIOMINTHA LONGIFOLIA
Mackay, Wayne, Narendra Sankhla Texas A&M University, TAES-Dallas, 17360 Coit Rd., Dallas, TX 75252-6599
[email protected]
Novel cut flower crops continue to derive consumer interest. Members of Novel cut flower crops continue to derive
consumer interest. Members of Lamiaceae< are extensively used as source of essential oils, secondary metabolite production and attractive garden plants. Poliomintha longifolia (Mexican Oregano) is one of the ʻdeer-resistantʼ plants of
Hill country landscapes which blooms from spring through fall and produces striking white- purple tubular flowers. This
report describes our initial results on the effect of ethephon (CEPA), gibberellic acid (GA) and sucrose (SUC) on flower
senescence and vase life of cut inflorescences of this plant with potential as a cut flower crop. Freshly cut excised inflorescences were placed in glass vases containing either 400 ml water or the test solution containing CEPA (20-100 µM),
GA (30 µM) and SUC (30 mM) solutions at 22± 2ºC in light. Presence of CEPA hastened the senescence of flowers which
was accompanied by an increase in the development of purple color. GA, on the other hand, inhibited the development
of flower color and delayed flower senescence. Incorporation of SUC in the vase solution dramatically increased the
opening of new flower buds, improved the overall quality, display life and longevity of cut inflorescences. These results
indicate that it is possible to extend the vase life of Poliomintha cut inflorescences with suitable post harvest treatments.
Lamiaceae are extensively used as source of essential oils, secondary metabolite production and attractive garden plants.
Poliomintha longifolia (Mexican Oregano) is one of the ʻdeer-resistantʼ plants of Hill country landscapes which blooms
from spring through fall and produces striking white- purple tubular flowers. This report describes our initial results on
the effect of ethephon (CEPA), gibberellic acid (GA) and sucrose (SUC) on flower senescence and vase life of cut inflorescences of this plant with potential as a cut flower crop. Freshly cut excised inflorescences were placed in glass vases
containing either 400 ml water or the test solution containing CEPA (20-100 µM), GA (30 µM) and SUC (30 mM) solutions at 22± 2ºC in light. Presence of CEPA hastened the senescence of flowers which was accompanied by an increase
in the development of purple color. GA, on the other hand, inhibited the development of flower color and delayed flower
senescence. Incorporation of SUC in the vase solution dramatically increased the opening of new flower buds, improved
the overall quality, display life and longevity of cut inflorescences. These results indicate that it is possible to extend the
vase life of Poliomintha cut inflorescences with suitable post harvest treatments.
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PS0608 NEW INSIGHTS ON CONTROL OF FRUIT DEVELOPMENT AND QUALITY BY PLANT GROWTH REGULATORS
Bonghi Claudio (1), Bregoli Anna Maria (2), Costa Guglielmo (2), Ramina Angelo(1), Torrigiani Patrizia(3), Tonutti Pietro(1), Ziosi Vanina(2) 1) Dipartimento di Colture Arboree, Bologna University, Italy 2) Department of Environmental
Agronomy and Crop Sciences, Padua University, Italy 3) Dipartimento di Biologia evoluzionistica sperimentale, Bologna University, Italy
[email protected]
PGRs can be used to promote, inhibit, or modify plant growth and developmental processes. Among these, fruit development and ripening represent the most reliable from an economic point of view. PGRs may be applied in fruit production for controlling several phases of development, such as the immature and pre-harvest fruit drop, and for improving
the final fruit quality.
The chemical thinning is usually performed by means of molecules such as Dormex (hydrogen cyanamide), Dinitroorthocresol (DNOC), Armothin (Alkoxylated fatty alkylamine polymer), ammonium thiosulphate (ATS), NAA and its
amide NAD, BA, Carbaryl and CEPA, that induce the drop of flower buds or fruitlets. Fruit size and quality might be
controlled by exogenous application of PGRs as auxins, BA, CEPA, Jasmonates and polyamine. The whole fruit quality
is largely dependent upon the control of ripening. In climacteric fruit this syndrome is efficiently regulated by ethylene.
In fact, chemicals blocking ethylene biosynthesis (aminoethoxyvinylglycin, AVG) or action (1-methylcyclopropene, 1MCP) are currently used in pre- and post-harvest phases, respectively, to delay ripening. For the same purpose, auxin,
PAs and JAs might also be used.
In this presentation the effects of PGRs on fruit growth and development will be analyzed taking into account both the
usual crop parameters and their effects on specific metabolic pathways related to fruit development and ripening. This
will contribute to a further elucidation of the role of plant hormone in controlling these processes and allow the setting of
novel tools to efficiently evaluate the ripening dynamics and the final whole fruit quality.
PS0609 PRELIMINARY STUDY OF THE L-PROLINE EFFECTS ON APPLE FRUIT QUALITY
Bregoli Anna Maria, Fabbroni Cristina, Raimondi Valerius, Andreotti Carlo, Ravaglia Daniela, Costa Guglielmo Dipartimento di Colture Arboree, Bologna University, # 46 Fanin blv., 40127 Bologna, Italy
[email protected]
Foliar application of different compound is become a common technique in agriculture to increase crop productivity
and fruit quality. Many data are available on the application of N fertilizers, while few data are reported on the application
of amino acids and nucleic acids. The present study was performed to investigate the effect of the amino acid L-proline
on the yield, fruit quality and nutraceuticals content of apple (cv ʻGalaxyʼ). Recently, nutraceuticals in fruits and vegetables have attracted a great deal of attention thanks to their role in preventing diseases caused as a result of oxidative
stress. Among them, phenolic compounds may be considered as one factor contributing to the so called “inner quality”
of fruits, as well as enhancing their “outer quality”, in particular the peel red colour.
Trial was carried out at the experimental farm of the Bologna University during the 2005 and 2006 years. Pure L-proline,
and an experimental product containing 50% in weight of this amino acid (Ajinomoto Co., Inc.), were applied at different doses and application times. Fortnightly during the growing season, the fruit diameter, fresh and dry weight were
recorded to measure the L-proline effect on fruit growth. At harvest the main productivity and quality parameters were
also recorded. Mesocarp and epicarp tissues were separately sampled and freezed at -80°C for GC and HPLC determination of sugars, organic acids and phenolic compounds.
L-proline application, even at the highest concentration, has no phytotoxic effect on the vegetation and on the fruit. Results obtained in the first year showed an increase in the fruit fresh weight and in the red skin colour. L-proline seems
also to modify fruit quality: treated fruit, in fact, had higher solid soluble content and fewer starch content than control
ones. In the second year, L-proline application showed no effect on the productivity parameters while the effects on fruit
quality, specially on the red peel colour, were confirmed. The data obtained from the biochemical analysis showed a general increase in the phenolic content in the epicarp tissue. In particular, two classes of phenols were particularly affected,
anthocyanidins and flavonols. No differences were present at the pulp level between untreated and L-proline treated fruit.
The practical importance of the enhancement of anthocyanidins and flavonols of the fruit skin caused by L-proline is
confirmed by the fact that the red skin fruit have a high commercial value on the market.
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PS0610 BENZOIC ACID ON FLORAL QUALITY OF LILIUM CV. DREAMLAND
Benavides, Adalberto, Ramirez, Homero, Rojas, Alfonso Dept. Horticultura Universidad Autonoma Agraria Antonio
Narro Buenavista, Saltillo, Coah., 25315, Mexico
[email protected]
This study was carried on to learning the effects of benzoic acid (benzene carboxylic acid)on the floral quality and
floral abortion rate of Lilium growed in greenhouse.
The results showed that the foliar spraying of benzoic acid (1 X 10-4 M and 1 X 10-3 M) applied before and during
appearance of floral buds did not change the number, abortion rate or morphology of such buds. On the other hand, the
application after appearance of floral buds effectively changed the height and diameter of buds and open flowers.
PS0611 FUNCTION ANALYSIS OF OXYLIPIN, 9-HYDROXY-10-OXO-12(Z), 15(Z)-OCTADECADIENOIC ACID (KODA)
IN ARABIDOPSIS THALIANA.
Madoka Ayano1, Hideki Goda1, Mineyuki Yokoyama2, Kazuo Shinozaki1, Shigeo Yoshida1. 1Plant Science Center,
RIKEN, Yokohama 230-0045, Japan. 2Research Center, Shiseido Co. Ltd., Yokohama 224-8558, Japan.
[email protected]
Oxylipin, 9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic acid (KODA) is a signal compound found in Lemuna
paucicostata after exposure to stress, such as drought, heat or osmotic stress. According to the subsequent research, it has
reported that the flower-inducing activities were found in Lemuna paucicostataand Pharbitis nil (violet). To understand
function of KODA in Arabidopsis thaliana (L.) Coumbia ecotypes, we performed phylogenetic analysis.
First, we measured endogenous KODA levels in Arabisopsis to confirm their existences. The145pg/g-F.W KODA was
certanly detected. Next, to detect KODAʼs function, we sprayed KODA to seedlings. But under the continuous light
condition, none of the KODAʼs influence was detected. However some morphological changes were induced when the
10µM KODA were treated under stress condition. Under the short day condition (7h light-17h dark), both Rosette leaf
enlargement and acceleration of lateral buds formation were detected from the late-vegetative to early-reproductive
stages. Under continuous light condition with high-Nitrogen fertilizer treatment, not only leaf enlargement but also
vigorous inflorescences development was detected. These results suggested that KODA have a potential to recover from
stress condition in A. thaliana.
PS0612 SOME NECESSARY ASPECTS DURING MASS-IN VITRO MULTIPLICATION TO PRODUCE ARTIFICIAL
SEEDS IN BANANA AND SORGHUM
Hassanein Ahmed 1, Hamad Ahmed 2, Abed-Elmaksood Ibrahiem 3, Galal Abed-Elnaser 1, Soltan Dia El-Deen 1, Salem Jehan 1 1 Central Laboratory of Genetic Engineering, Faculty of Science, Sohag University, 82524 Sohag, Egypt. 2
Botany Department, Faculty of Science, Assuit University, 71516 Assuit, Egypt.. 3 Genetic Engineering La., Sadat City,
Monofia University, Monofia, Egypt
[email protected]
Fast multiplication was induced when seedling shoot tips of sorghum were cultured on MS medium supplanted with
2 mg/l BAP and 0.5 mg/l 2,4-D. Suckers shoot tips were used for multiplication of banana on MS medium supplemented
with 5 mg/l BAP. Six subcultures of in vitro obtained shoot tips resulted in the formation of huge number of plant materials to produce of artificial seeds [371293 and 18720 shoot tips/explant in case of sorghum (in 3 months) and banana
(in 6 months), respectively]. Size of in vitro obtained shoot tips of banana and sorghum was big enough to simplify the
encapsulation processes. Consequently, manual mass production of artificial seeds were obtained through encapsulation of in vitro obtained shoot tips of both plants in calcium alginate beads. Storage of encapsulated shoot tips in liquid
multiplication medium under low temperature (4 oC) was important for viability and conservation of the obtained seeds.
Conversion was fulfilled on basal MS medium and the resulted plantlets were transferred to the garden soil. Although
somatic embryos were obtained, production of artificial seeds using in vitro obtained shoot tips were recommended to
decrease the somaclonal variation. Also, to minimise the appearance of somaclonal variation, any condition stimulated
the formation of callus or somatic embryogenesis were avoided, eg. application of relatively high concentration of 2,4-
IPGSA 19th Annual Meeting - Abstracts
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D (1-5 mg/l) and/or incubation under dark condition. In addition, genetic stability sufficient for production of artificial
seeds was obtained when long term culture was avoided, subculture number should be not more than seven in banana
and ten in sorghum.
PS0613 PISTILLATE FLOWER ABORTION AND POLLINATION-INDUCED ETHYLENE PRODUCTION IN ENGLISH
WALNUT
Johnson, Holly, Polito, Vito Department of Plant Sciences, University of California, Davis, Davis, California 95616,
USA; [email protected]
[email protected]
Walnut pistillate flower abortion (PFA) is the loss of female flowers approximately 2 weeks after bloom. Ovaries in
PFA type flowers stop enlarging at approximately 3-4 mm in size, and abscise 10 to 14 days later. PFA in walnut is due to
the presence of excess pollen on the stigma. When it lands on the stigma, pollen induces ethylene production in flowers.
Previous lab and field studies indicate that ethylene is involved in PFA. The cultivar Serr is the most susceptible to PFA;
and PFA in Serr can exceed 90%, but other cultivars are susceptible as well. ReTain™ (active ingredient amino-ethoxyvinyl-glycine (AVG)) has shown to reduce PFA and increase yields on the cultivar Serr in a given year.
The effects of multiple year ReTain™ treatments on Serr yields were evaluated. In year two of treatment, there appeared
to be a slight, but non-significant decrease in yield, in year three, this was not evident. This may have been due to overall
low yields in year three. ReTain™ applications on the variety Chandler do not appear to be effective. Treatments of a
liquid formulation of 1-Methylcyclopropene (1-MCP, AgroFreshInc) were also tested on whole, individual trees, and
showed a significant decrease in PFA associated with treatment. Non-significant increases in yield were also seen. In-vitro measurements of ethylene production by walnut flowers showed that ReTain™ decreases ethylene production, while
1-MCP increases ethylene production.
PS0614 INCREASED WATER-USE EFFICIENCY (WUE) THROUGH MANIPULATION OF ABSCISIC ACID (ABA) BIOSYNTHESIS IN TOMATO AND ARABIDOPSIS
Thompson, Andrew 1, Andrews, John 1, Hilton, Howard 1, Deswarte Jean-Charles 1, Farquhar, Graham 2, Smeeton, Rachel 3, Black, Colin 3, Swee Ang Tung 3, Taylor, Ian 3 1 Warwick HRI, Univ. Warwick, Wellesbourne, Warwick, CV35
9EF, UK; 2 Environmental Biology Group, Research School of Biological Sciences, Australian National Univ., GPO Box
475, Canberra ACT 2601, Australia; 3 Plant Sciences Division, School of Biosciences, Univ. Nottingham, Loughborough, Leic., LE12 5RD, UK. [email protected]; Tel +44(24)76575090; FAX +44(24)76574500
[email protected]
ABA is a key modulator of drought response. Our aim is to improve crop productivity in water-limited environments
through the genetic manipulation of ABA biosynthesis. Over-expression of 9-cis-epoxycarotenoid dioxygenase (NCED)
resulted in increased ABA biosynthesis in the shoot and, to a lesser extent, in root tissues. An rbcS::NCED construct led
to large increases in ABA accumulation in photosynthetic tissues of tomato, together with greatly improved WUE, but
with detrimental effects on growth. In Arabidopsis the same construct also greatly improved WUE but with no significant
effect on biomass. Similarly, tomato lines derived from a constitutive promoter that provided lower expression levels produced the same biomass as WT plants but with nearly double the WUE. Moderately increased ABA levels caused stomatal closure but also increased leaf water status, leaf expansion and root hydraulic conductivity. The combined pleiotropic
effects of elevated ABA are considered in relation to crop productivity and WUE. In roots, we show that steps upstream
of NCED, including â-carotene hydroxylase, are important in regulating ABA biosynthesis and the potential for enhancement of root-to-shoot ABA signaling, by manipulating multiple enzymatic steps in root tissue will be addressed.
PS0615 1-MCP SIGNIFICANTLY AFFECTS THE TRANSCRIPTION PROFILES OF PEACH FRUIT
Bonghi Claudio1, Ziliotto Fiorenza1, Rasori Angela1, Begheldo Maura1, Tonutti Pietro2, and Ramina Angelo1 1Department Environmental Agronomy & Crop Science, University of Padova, Viale dellʼUniversità, 16; Legnaro (PD); i-35020
Italy; 2Scuola Superiore SantʼAnna di Studi Universitari e di Perfezionamento, Piazza Martiri della Libertà, 33 Pisa,
I-56127 Italy
[email protected]
IPGSA 19th Annual Meeting - Abstracts
67
The control of climacteric fruit ripening relies upon the possibility of controlling ethylene biosynthesis and action.
This can be achieved through genetic engineering, control of environmental parameters (temperature, CA) during storage
or using specific inhibitors of ethylene biosynthesis or action,
The µPEACH1.0, a microarray containing 4806 oligos corresponding to unigenes isolated mainly from a ripe fruit, was
used to evaluate changes in trascriptome profile of peach fruits induced by 1-methylcyclopropane (1-MCP), an antagonist
of ethylene for the binding sites.
Peaches (cv Super Crimson Gold) were picked at commercial ripening and incubated at 20° for 24 h with 1-MCP (1µL/L)
and then transferred in air for 48h. The control fruits were maintained in air (A) throughout the experimental period. The
sampling was performed at the beginning of the experiment (T0), at 24 (24-1MCP and 24A) and 72 h (72 1-MCP and
72A). At the end of the treatment with 1-MCP and 48 h later fruit ethylene evolution and firmness were measured. Microarray analysis was carried out according to a simple-loop experiment design taking into account the following comparisons 24 1-MCPvsT0; 24Avs T0; 24 1-MCPvs24A; 72 1-MCPvsT0; 72vsT0 and 72 1-MCPvs72A. Within the selected
genes using the SAM algorithm, a threshold of confidence more than 2 and less than 0.5 for up- and down-regulation,
respectively, was assumed. It has been confirmed that 1-MCP slightly reduced the fruit softening: in fact, 1-MCP-treated
fruit showed an higher firmness than the control ones for a period no longer than three days, after that the fruits started to
soft concurrently with the onset of ethylene evolution.
Microarray analysis revealed that in 24AvsT0 and 24 1MCPvsTO the unigenes differentially expressed (up and down)
were 103 and 9, respectively. These results pointed out that after 24h in air a large set of genes are showing changes in
the transcription activity and many of these are blocked by 1-MCP. In fact, the ethylene positively regulated genes as
Pp-ACO1 and those encoding cell wall hydrolases (as endoPG, Pp-EXP1, PME) were among the down-regulated in 24
1-MCPvs24A, the up-regulated in 24AvsT0, and the unchanged in 1MCPvsTO. The analysis of transcript profiling after
an additional period of 48 h in air (72 1MCP/72A) pointed out that the genes blocked by 1-MCP at the end of the treatment can be grouped in three clusters. In the first, including endoPG, PME, PpEXP1, an EIN3-like factor and an ethylene
responsive element binding proteins (AP2/EREBPs), the expression level was similar to that found in fruits maintained
in air indicating a full recovery. In the second clusters, in which Pp-ACO1, an AUX/IAA-like gene and an ETR2-like
ethylene receptor are present, the block was still maintained, although the transcripts accumulation was increasing. In the
last set, in which genes down-regulated as a glucose acyltransferase and a catalase after 24h in air are present, the positive
effect of 1-MCP on transcription is progressively lost. In conclusion, 1-MCP efficiently impaired the ethylene action, but
its short lasting effect in the delaying the peach fruit ripening might be imputed to a fast recovery of the transcription rate
of some transcription factors controlling the ethylene signaling as EIN3-like or AP2/EREBPs.
PS0616 EFFECTS OF NAA, AVG, AND 1-MCP ON ETHYLENE BIOSYNTHESIS, PREHARVEST FRUIT DROP, FRUIT
MATURITY AND QUALITY OF GOLDEN SUPREME AND GOLDEN DELICIOUS APPLES
Yuan , Rongcai, Carbaugh, David Virginia Polytechnic Institute and State University, Alson H. Smith, Jr. Agricultural
Research and Extension Center, 595 Laurel Grove Road, Winchester, VA 22602
[email protected]
Effects of naphthaleneacetic acid (NAA), aminoethoxyvinylglycine (AVG), and 1- methylcyclopropene (1-MCP)
alone or in combination, on fruit ethylene production, preharvest fruit drop, fruit quality, and fruit maturation were examined in ¡®Golden Supreme¡¯ and ¡®Golden Delicious¡¯ apples (Malus x domestica Borkh.). In ¡®Golden Supreme¡¯
apples, the combination of two applications of AVG and one application of NAA 3 and 1 weeks before the anticipated
optimum harvest date synergistically inhibited fruit ethylene production, and delayed fruit drop and ripening. Compared
with one or two applications of AVG, the combination of one application of AVG and two applications of NAA had much
lower preharvest fruit drop even though there was no significant difference in fruit ethylene production among these
treatments. In ¡®Golden Delicious¡¯ apples, 1-MCP at 396 mg.L-1 had a better effect in delaying fruit drop than did AVG
at 125 mg.L-1 or NAA at 20 mg.L-1 when they were applied a week before the optimum harvest date. The combination
of NAA and 1-MCP or AVG was more effective in delaying fruit drop than were NAA, 1-MCP, or AVG alone. Fruit
ethylene production was inhibited by 1-MCP and AVG but not by NAA. 1-MCP and AVG delayed fruit ripening whereas
NAA increased fruit ripening as determined by fruit firmness and starch.
IPGSA 19th Annual Meeting - Abstracts
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PS0617 CHEMICAL REGULATION OF PLANT STRESS TOLERANCE
Armstrong, Joshua I., Schwarz, Yuka H. Mendel Biotechnology, Inc. 21375 Cabot Blvd., Hayward, CA 94545, USA
[email protected]
The genomics era has yielded a wealth of knowledge surrounding signaling pathways controlling plant health and
development. We have employed both forward and reverse genetic approaches to identify and characterize transcription
factors that regulate abiotic and biotic stress tolerance. This information has guided the generation of fluorescence-based
whole plant reporter lines, enabling straightforward detection of pathway activation. We have used these reporter lines in
high-throughput chemical screens to identify structurally distinct compound classes that up-regulate Arabidopsis drought
tolerance and disease resistance in both plate and soil-based assays. Our strategy and insights into the mechanism of a
select compound will be presented.
PS0618 THE EFFECTS OF PLANT GROWTH RETARDANTS OF RICE STARCH
The effect of gibberellin biosynthetic inhibitors on enzyme activity and physicochemical properties of rice starch Sang
Kuk Kim, Bong Ho Lee Institute for Bioresources Research, Gyeongbuk Provincial Agricultural Technology Administration, Andong 760891, Korea Corresponding author: [email protected]
[email protected]
Application of gibberellic acid and kinetin(6-furfuryl amino purine) improve development and grain yield on all
branches of rice plants (Patel and Mohapatra, 1992). From our previous reports, gibberellin-treated rice plants showed
that endogenous GA 4 content in both superior and inferior part was more promoted in GA 4 -treated rice grain than in
the control. GA 1 content of an inferior part was not detected in the control and GA 4 -treated rice otherwise GA 4 was
detected in all grain parts. Amylopectin from GA 4 -treated grain contained more very short chains with degree of polymerization (DP) between 4 and 8 than amylopectin from the control plant. When gibberellin biosynthetic inhibitors is
applied to rice plants, several enzyme activity related starch synthesis and its physicochemical properties of rice starch
is discussed.
PS0619 EFFECT OF DAY LENGTH ON BULB OF GARLIC
Influence of day length on plant hormones in leaf blades and sheath of garlic (Allium sativum L.) Eun Y. Sohn, Byung
S. Kim, I. J. Lee Division of Plant Biosciences, Kyungpook National University, Daegu 702701, Korea Corresponding
author (E-mail: [email protected])
[email protected]
Endogenous gibberellins during bulb growth were investigated from two garlic tissue parts, leaf sheaths and leaf
blades as affected by different day length conditions. When garlic was grown in the long day condition, the concentrations of total gibberellins in the leaf sheaths began to increase at bulb differentiation times and kept to increase about 15
days before they began to enlarge, and reached a maximum shortly at early bulb enlargement times and declined slightly.
On the contrary, the concentrations of total gibberellins in the leaf sheaths of garlic grown in short day conditions were
increased steadily till early bulb enlargement times and reached a maximum shortly at only full bulb enlargement times.
In addition, non C-13 hydroxylation route was always dominant during bulb growth both long and short day lengths.
Furthermore, GA 4 was also more abundant than GA 1, however its changes were differed from day length with increased
bulb growth.
PS0620 EFFECT OF PUTRESCINE AND SPERMIDINE ON GROWTH AND PRODUCTION OF TWO WHEAT CULTIVARS DERIVED FROM FIELD EXPERIMENTS AND IN VITRO
Abdel-Hady M.S.S* and M.A. Ahmed** * Botany Dept. and ** Filed Crop Research Dept National Research Center,
Dokki, Giza , Egypt. E-mail : [email protected]
[email protected]
Abstract : Two field experiments were carried out in the Agricultural Experimental Station of National Research Center, Shalakan, Qalubeya Governorate, Egypt to study effect of putrescine and spermidine on growth and productivity of
two wheat cultivars drived from field experiments and in vitro. The main results were:
IPGSA 19th Annual Meeting - Abstracts
69
1- There was highly significant response of the two genotypes to putrescine (put) and spermidine (spd) and (put + spd).
The results showed that MS8 medium containing (100 mg/l put + 100 mg/l spd) gave the best embryogenic callus induction and plant regeneration for the two wheat genotypes tested. The highest callus induction, callus growth rate and plant
regeneration were obtained in Gemmiza-9 on MS8 medium.
2- Gemmiza-9 cultivar significantly exceeded Giza-168 cultivar in Chl.a and carotenoids per blades, total carbohydrate
percentage and protein percentage in grains, platn height, No. of tillers/plant, main spike length, main spike weight,
spikes weight/plant, grain index, grain weight/main spike, grain yield/plant and per fed.
3- Foliar application with 100 mg/l put + 100 mg/l spd produced wheat plants characterized by high values of Chl.a and
carotenoids in blades, total carbohydrate % in grians, protein % in grains, plant height, main spike length, main spike
weight, spikes weight/plant, grain index, grain weight/main spike and grain yield/plant and/fed. On the other hand the
highest value of Chl.b obtained by spraying wheat plants with 100 mg/l Put + 50 mg/l Spd., whereas, spraying with 50
mg/l Put + 100 mg/l Spd harvested the greatest value of number of tillers/plant.
4- As for the interaction between wheat cultivars and foliar spraying with putrescine and spermidine, the effect was significant on photosynthetic pigments content/blades, total carbohydrates %, protein % per grains, and yield and its components. Generally, the most favoruable treatment of Chl.a, carotenoids, carbohydrate %, protein % in grains, plant height,
number of tillers/plant, main spike length, main spike weight, spikes weight/plant, grain index, grain weight/main spike,
and grain yield/plant and/fed was spraying Gemmiza-9 cultivar with 100 mg/l Put + 100 mg/l Spd., meanwhile spraying
the same cultivar with 100 mg/l Put + 50 mg/l Spd. gave the height significant value from Chl.b.
Key words: Wheat, putrescine, spermidine, growth and tissue culture.
PS0621 RESULTS OF THE USE OF 3,5,6-TPA AGAINST PREHARVEST FRUIT DROP IN ‘CONFERENCE’ PEAR
Basak, Alina1, Buczek Maria2 1 Research Institute of Pomology and Flor., Pomologiczna 18, 96-100 Skierniewice, Poland; 2 Fruit Experiment Station of Research Institute of Pomology and Flor., Brzezna, 33-386 Podegrodzie, Poland
[email protected]
The preparation TOPS, which contains 3% of 3,5,6-TPA, is the latest product designed to control preharvest fruit drop
in apple, pear and citrus orchards. In 2005 and 2006, the preparation was used at the Research Institute of Pomology
& Flor., in Skierniewice, Poland, on pear trees cv. _Conference_ growing in two orchards under different climatic and
agrotechnical conditions. The preparation TOPS was used in doses of 10 and 15 tablets/ha by spraying the trees 5 weeks
before the anticipated harvest date. The tablets, which contain 1 g of the active ingredient per tablet, are easily soluble in
water. The dose of the preparation was calculated per 1000 litres of water per ha, but the amount of the preparation per
each individual tree depended on the number of trees per ha. The Polish preparation Pomonit Super 050 SL (containing
5% NAA in a salt with triethanolamine) was used in the same way as in commercial orchards (40 ml/100 L at 10 days
before harvest). The untreated trees served as the control for both preparations. The effects on the dynamics of fruit drop,
fruit quality at harvest and after storage, as well as on shoot growth and subsequent blooming were observed. It was
found that 3,5,6-TPA efficiently retarded preharvest fruit drop in pear trees cv. _Conference_ in 3 experiments, but in 1
experiment on trees with parthenocarpic fruits the compound was found to be ineffective.
PS0622 EFFECT OF BA IN APPLE THINNING DEPENDING ON SOME FACTORS (FORMULATION, ADDITIVES,
BLOOM INTENSITY, PHENOLOGICAL STAGE OF FRUITLETS)
Basak, Alina Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland
[email protected]
BA (benzyladenine) is quite a new thinning agent and research must continue to determine its efficiency more precisely. The results of two experiments conducted in 2005 on 9-year-old ʻGala Mustʼ/M26 apple trees, and in 2006 on
10-year-old ʻGolden Deliciousʼ/M26 apple trees will be presented. In the first year, the efficiencies of two preparations
IPGSA 19th Annual Meeting - Abstracts
70
containing BA: Paturyl 10 WC (10% BA), a product of the Hungarian firm Reanal, and VBC 30001 (1.9% BA), a product of Valent Company, USA, were compared. Both forms of the compound were applied at two doses: 50 and 100 mg/l
when fruitlets on old wood were 10-12 mm in diameter. The next year, the preparation VBC 30001 was evaluated. The
preparation was used at one concentration only (150 mg/l BA), but at different development stages of fruitlets on old
wood, i.e. with fruitlet diameter 7-8 mm, 10-11 mm and 13-14 mm, both on abundantly blooming trees and on those with
a weak bloom intensity. Moreover, in three treatments the different doses of VBC 30001 were combined with different
doses of NAA with triethanolamine salt (in the Polish preparation Pomonit Super) and used as mixtures on fruitlets 1012 mm in diameter. For comparison, the product of the Italian firm l.Gobbi, containing a mixture of 41 mg/l BA and 4.1
mg/l NAA, was applied on fruitlets at the same time. The effect on fruit set, average fruit size, the distribution of fruits
in size group and some traits of apple quality were evaluated. It was found that the effects of thinning by means of VBC
30001 and Paturyl 10 WSC were comparable and were related to the concentration of BA applied, but not to the formulation. When VBC 30001 was tested on its own, the best thinning effect was noticed in the trees treated when fruitlets were
10-11 mm in diameter, but when it was applied later, on fruitlets 13-14 mm in diameter, it thinned efficiently, too. Earlier
applications of VBC 30001 at the 7-8 mm stage thinned inadequately. In the case of poorly blooming trees (302 flower
clusters/tree), an increase in fruit set was noticed instead of thinning while the same dose of the preparation applied on
abundantly blooming trees (540 flower clusters/tree) produced a significant thinning effect. The joint use of VBC 30001
at 100-150 mg/l in a mixture with NAA at 10-15 mg/l resulted in overthinning. However, when VBC 30001 was used at
50 mg/l BA in a mixture with NAA at 10 mg/l, it produced the best effect comparable with the results of hand thinning. At
the same time, the Italian preparation LG 183, a mixture of 41% BA and 4.1% NAA, thinned too, but less efficiently.
PS0623 REDUCED SPRING BUDBREAK IN ALTERNATE BEARING CITRUS: THE ROLE OF FRUIT-PRODUCED HORMONES
Arbona, Vicent, Verreynne, Stephan, Zheng, Yusheng, Lovatt, Carol
Dep. Botany and Plant Sci., University of California, Riverside, CA 92521-0124, USA [email protected], phone 951827-4663, fax 951-827-4437
[email protected]
Alternate bearing is a significant problem for citrus growers. For on-crop mandarin (Citrus reticulata) trees, correlative inhibition reduces budbreak of summer and fall vegetative shoots and return bloom. Treatments that stimulated
summer-fall shoot growth of on-crop trees failed to increase return bloom due to inhibition of spring budbreak. Spring
inflorescence number was related to bud starch (r2 = 0.54), isopentyladenosine (IPA) (r2 = 0.41) and abscisic acid (ABA)
(r2 = 0.37) concentrations and ABA:IPA ratio (r2 = 0.57) just before bloom (P ¡Ü 0.004). Yield influenced inflorescence
number (r2 = 0.56) and bud starch (r2 = 0.62), ABA (r2 = 0.57) and IPA (r2 = 0.52) (P ¡Ü 0.006). Yield did not influence
bud glucose or IAA concentrations and neither was related to return bloom.
PS0624 EFFECT OF PLANT GROWTH REGULATIVE SUBSTANCES ON HEIGHT OF ALFALFA
deSoto, Rachel E. Department of Plant Science, University of California, Davis, Davis, CA 95616, USA. Putnam, Daniel
H. University of California Cooperative Extention, Department of Plant Science, University of California, Davis, Davis,
CA 95616, USA.
[email protected]
The purpose of this experiment was to observe the effects of plant growth regulative (PGR) substances on the height
and growth pattern of alfalfa, (Medicago sativa), particularly to determine if an induced summer dormancy state could be
achieved as a water preservation technique. The experiment was conducted using fourteen PGR substances as treatments
in a randomized complete block design with four replications under well-watered conditions and repeated under drought
conditions in the same field. The repetitions were analyzed as two separate trials.
Plant height measurements were taken periodically during the month of July, 2006, between the third and fourth cuttings
to determine the effect of the PGR treatments on alfalfa plant height. Measurements were also taken following the fourth
cutting to determine residual effects. Initial findings show four substances of interest showing consistently significant
IPGSA 19th Annual Meeting - Abstracts
71
growth pattern effects, including Apogee, Atrimmec, B-Nine, and Florel. Both quantitative and qualitative observational
data show little effect toward inducing summer dormancy when the alfalfa had access to water. Under drought conditions, alfalfa plant heights were significantly reduced in general, and the same showed significantly different effects on
plant height than the control. Further research is needed in order to determine if this technique will be an effective management technique for California alfalfa growers under water transfer conditions in the near future.
PS0625 VEGETATIVE GROWTH RESPONSES OF CITRUS NURSERY TREES TO VARIOUS GROWTH RETARDANTS
Barry, Graham H. 1, le Roux, Smit 2 1 Citrus Research International, Department of Horticultural Science, Stellenbosch
University, P. Bag X01, Matieland 7602, Stellenbosch, South Africa 2 Department of Horticultural Science, Stellenbosch
University, P. Bag X01, Matieland 7602, Stellenbosch, South Africa
[email protected]
As part of a larger study to improve rind colour of citrus fruit, an initial study was conducted to determine the concentration of various gibberellin biosynthesis inhibitors required to get a biological response in citrus trees, as measured by
vegetative growth. Repeated foliar applications of ProGibb® (4% v/v GA3) increased growth of ʻEurekaʼ lemon [Citrus
limon (L.) Burm. f.] shoots by 63%, with no significant effect on rootstock and scion diameters. Repeated applications
of Regalis® (10% v/v Prohexadione-calcium) at various concentrations (1, 2, 4 and 8 g_L-1) as well as Sunny® (5% v/v
uniconazole) (at 10 and 20 mL_L-1) and Cultar® (25% v/v paclobutrazol) (at 10 mL_L-1) had no effect on the rootstock
or scion diameters 8 months after the first application. Both the 4 and 8 g_L-1 Regalis® treatments, both Sunny® treatments and the Cultar® treatment significantly reduced shoot growth. Sunny® at 20 mL_L-1 resulted in the most growth
retardation which resulted in 34% shorter shoot length than the control. Although the number of nodes on the longest
shoot did not differ from the untreated control, internode length differed significantly among treatments. Regalis® at 4
and 8 g_L-1, Sunny® at 20 mL_L-1 and Cultar® at 10 mL_L-1 reduced internode length relative to the control by 31%,
56%, 50% and 28%, respectively. Vegetative growth of ʻEurekaʼ lemon nursery trees was retarded following the repeated
(x4) application of gibberellin biosynthesis inhibitors. Regalis® at 4 to 8 g_L-1 and Sunny® at 10 to 20 mL_L-1 are
potential candidates for further field studies to test their effects on rind colour enhancement of citrus fruit.
PS0626 DEVELOPING CONTROL OF PISTILLATE FLOWER ABORTION IN WALNUT TREES AS A NOVEL USAGE OF
AVG, AN ETHYLENE BIOSYNTHESIS INHIBITOR.
Lemus, Gamalier 1; González, Cristián 1; Retamales, Julio 2, 3 1 INIA-Chile, La Platina
Exp. Station; 2 Valent BioSciences Corporation, Field R&D; 3 Universidad de Chile
[email protected]
Pistillate flower abortion (PFA) in walnut trees (Juglans regia) has been shown as resulting from excessive pollen
load on female flowers, leading eventually to their abscission. Ethylene production has been measured following pollination in walnut trees linking excessive ethylene synthesis to rapid abscission under such circumstances. Evaluations
in California and Chile have shown a particularly high incidence of this problem in ʻSerrʼ walnut trees, compromising
fruit set and, subsequently, significantly reducing yield in such variety, extensively planted as derived from its high nut
quality. A whole program of trials during 3 years has being carried out in Chile to ascertain whether applications of AVG
(aminoethoxyvinylglycine), an ethylene biosynthesis inhibitor long used in basic studies and now commercially available, can effectively control PFA in ʻSerrʼ and further walnut varieties. Moreover, trials have allowed to determine most
appropriate application times and concentrations of AVG when applied by spraying whole trees in commercial orchards.
Results from these studies are showing that AVG applications can be effective in order to overcome PFA and subsequent
yield losses in ʻSerrʼ walnut orchards under Chilean Central Valley conditions, enabling to obtain yield levels higher than
previously being considered as normal for this variety.
This research work led to the available AVG formulation (ReTain®) getting registration for this use in both Chile and
USA. Adoption of this practice has been quite rapid and extended among walnut growers in both countries. In our opinion this constitutes a good example of developing further usages for currently available PGRs. Reasons underlying such
development will be analyzed.
IPGSA 19th Annual Meeting - Abstracts
72
PS0627 EFFECT OF PLANT GROWTH REGULATIVE SUBSTANCES ON QUALITY OF ALFALFA
Grande, Rachel. Department of Plant Science, University of California, Davis, Davis, CA 95616, USA. Putnam, Daniel
H. University of California Cooperative Extension, Department of Plant Science, University of California, Davis, Davis,
CA 95616, USA.
[email protected]
The purpose of this experiment was to observe the effects of plant growth regulative (PGR) substances on the quality
ratings of alfalfa, (Medicago sativa). The experiment was conducted using fourteen different PGR substances as treatments in a randomized complete block design with four replications under well-watered conditions and repeated under
drought conditions in the same field. The repetitions were analyzed as two separate trials. Quality analysis was performed
on the third and fourth cuttings to determine the effect of the PGR treatments on alfalfa quality as crude protein, acid
detergent fiber, and neutral detergent fiber. Initial findings show no significant differences in quality under well-watered
conditions.
PS0628 EXPRESSION OF AHNCED1 GENE IN PEANUT ORGANS SUBJECTED TO DEHYDRATION AND DETERMINATION OF AHNCED1 PROTEIN
XiaoHui Liu1,Bo Hu1; LiXia Yang1; Ming Qin1; Xiaorong Wan1,2; Ling LI1,*1 1.Guangdong Provincial Key Lab of
Biotechnology for Plant Development£¬College of life Science, South China Normal University, Guangzhou, China,
510631 2. College of Life Sciences, Zhongkai University of Agriculture and Technology, Guangzhou 510225, China£¨*
requests for offprints£©
[email protected]
A rate-limiting step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). We cloned AhNCED1 gene from dehydrated peanut (Arachis hygogaea L.) leaves that encodes a putative
NCED. The result of duplex RT-PCR reveals that the expression of AhNCED1 is up-regulated by dehydration and that
rehydration represses its expression. To study a variation of AhNCED1 gene expression in different peanut organs of
three-leaves stage or legume stage, since peanut of three-leaves stage and legume stage were hypersensitivity to soil
drought.
Northern blotting analysis indicated that AhNCED1 gene were significantly up-regulated by drought stresses in all organs
of peanut during three-leaves stage. The gene dominantly expressed in both leaf and root. Meantime, the endogenous
ABA accumulate predominantly in both leaf and root in response to dehydration. Under a drought condition, the ABA
content was obviously higher in leaf and legume. The AhNCED1 gene dominant expression was more higher in leaf and
legume. The gene expression and ABA level in stems and roots was less. A good correlation has resulted between the
expression of the AhNCED1 genes described in this work and the data of ABA production.
Construction prokaryotic expression vector of AhNCED1 (AhNCED1-pHT) transformed to competent cell DH5a and
the gene expression was identified by RT-PCR. With the induction of sense strain by IPTG, the expression AhNCED1
protein 65 KD was observed on SDS-PAGE. The protein was a insolubility protein. It showed that the recombinant (His)
6 fusion protein were purified by Ni-NTA purification system and the purification single protein were obtained.
PS0629 STRUCTURE-ACTIVITY STUDIES OF UNICONAZOLE DERIVATIVES AS STRONG INHIBITORS AGAINST
ABA 8’-HYDROXYLASE
Todoroki, Yasushi 1, Yoneyama, Hidetaka 1, Kobayashi, Kyotaro 1, Jin Mei-hong 1, Hirai, Nobuhiro 2, Mizutani, Masaharu 3
1 Dep. Appl. Biol. Chem., Facul. Agric. Shizuoka Univ., Shizuoka 422-8529, Japan; 2 Int. Innov. Center, Kyoto Univ.,
Kyoto 606-8501, Japan; 3 Inst. Chem. Res., Kyoto Univ., Uji, Kyoto 611-0011, Japan
[email protected]
[email protected]
Specific inhibitors of ABA catabolism are potentially very useful tools for cellular and molecular investigations in the
field of plant physiology, and for potential agricultural chemicals. ABA 8ʼ-hydroxylase, a key enzyme of ABA catabo-
IPGSA 19th Annual Meeting - Abstracts
73
lism, is a cytochrome P450 monooxygenase. Many P450 inhibitors are found within the group of azole-containing compounds. The azole-type P450 inhibitors tend to be substantially non-specific, owing to this intrinsic effect of the nitrogen.
Uniconazole-P, a well-known azole-containing P450 inhibitor of GA biosynthesis, acts as a very strong inhibitor against
ABA 8ʼ-hydroxylase. In this study, we designed and synthesized uniconazole derivatives whose conformation is similar
or not similar to uniconazole, for probing 3D-structure-activity relationships for inhibition of ABA 8ʼ-hydroxylase and
developing the specific azole-type inhibitor. Our results suggested that the control of the azole ring rotation may be a key
for developing the isoform specific azole-type inhibitor against plant P450.
PS0630 ABA SPRAYED TO THE LEAVES INCREASES YIELD IN FIELD-CULTURED GRAPE (VITIS VINIFERA L. CV.
CABERNET SAUVIGNON) BY AUGMENTING BERRY SET WITHOUT AFFECTING QUALITY FOR WINEMAKING
Quiroga, Mariela; Cavagnaro, Bruno; Bottini, Rubén Facultad de Ciencias Agrarias-CONICET, Universidad Nacional de
Cuyo, Alte. Brown 500, M5528AHB Chacras de Coria, Argentina; [email protected]
[email protected]
In grapes for red-winemaking periods of water restraint is a tool employed to get higher quality although with lower
yield. The rationale is that ABA produced by the water-stressed plant controls water losses by regulating stomata, and
therefore photosynthesis with decreases in growth and yield, but at the same time either ABA stimulates polyphenol synthesis and/or smaller berries concentrate in the skin more secondary metabolites. We had previously found however that
ABA increases yield in other field-cultured species, either via alleviation of the stress (Ilex paraguariensis) or favoring
C assimilation-translocation (wheat). In an attempt to give the stress signal but without their undesirable effects, a field
experiment was performed during three years with no soil water restriction. During the 2004-5 season ABA 250 mg.L-1
was sprayed weekly to leaves since 15 days after sprouting; in 2005-6 the treatment was done on the same plants of the
year before, or on to a new set; in 2006-7 the spraying was performed on to plants of two, one or zero years of previous
applications. In the mid-term ABA did not significantly influence leaf Øw, stomatal conductance, photosynthesis, or leaf
area. However, ABA consistently increased yield during the three years, ranging between 21 and 102%, without affecting
polyphenol total index or anthocyan content either in berriesʼ skin or in the wine obtained. Yield increases correlated with
an enhanced number of berries per bunch.
PS0631 INDUCTION OF POTATO TUBERS UNDER HIGH TEMPERATURE CONDITIONS BY THEOBROXIDE
Kyong-Hee Nam 1, Kensuke Nabeta 1 and, Teruhiko Yoshihara 1,2 1 Graduate School of Agriculture, Hokkaido University, Sapporo, Japan 2 Asahikawa University, Women_fs Junior College, Asahikawa, Japan
[email protected]
Temperature is one of the major environmental factors affecting potato tuberization. In our previous report, cool
temperatures (15 _‹C) were very favorable for tuber induction, while no tubers were induced at high temperatures (30
_‹C). At low temperature, lipoxygenase (LOX) activity increased strongly and endogenous levels of jasmonic acid (JA),
tuberonic acid (TA) and tuberonic acid glucoside (TAG) rose, indicating their crucial role in tuber induction. On the other
hand, theobroxide, an exogenous potato tuber inducing compound, produced tubers under non-inductive photoperiod
conditions and stimulated the activity of LOX and the synthesis of JA and TA. In this study, to examine the effect of
theobroxide on the induction of potato tubers, different concentration (0, 1, 5, and 10 mM) of theobroxide was spayed
onto the leaves of potato grown at high temperature of 30 _‹C. Theobroxide induced potato tuber at high temperature of
30 _‹C. The plants producing tubers were 20, 60, and 60 % for concentration of 1, 5, and 10 mM, respectively. The maximum number of tubers was observed at a concentration of 5 mM and the maximum fresh weight of tubers was showed
at a concentration of 10 mM. Regardless of concentration, the average diameter of tubers was almost same as 0.70, 0.64,
and 0.65 cm for concentration of 1, 5, and 10 mM, respectively.
PS0632 EFFECT OF BIO-REGULATOR ON VEGETATIVE PROPAGATION OF M. HUPEHENSIS IN TISSUE CULTURE
Xiao-jiao HAN£¬Hong-qiang YANG*£¬ Shu-zhen YOU (College of Horticulture Science and Engineering, Shandong
Agricultural University, Taiʼan, 271000) *Author for Correspondence: Hongqiang Yang, E-mail: [email protected]
[email protected]
The Malus hupehensis is one kind of ornamental plants, commonly used as the rootstock of apple and Chinese flowering crabapple, and used for to manufacture the tea drink which may reduce the animal blood sugar. Malus hupehensis
IPGSA 19th Annual Meeting - Abstracts
74
is typical allotriploid (2n = 3x = 51), mainly reproduces the descendant by apogamy way and propagates by seed. The
vegetative propagation of M. hupehensis is difficult. In this experiment, stems of seedlings of M. hupehensis Rehd as
explants were vaccinated on MS medium for tissue culture and rapid propagation. The results showed that stems of
seedlings were used for suitable explants. The medium MS+ BA0.5mg•L-1+ZT0.2mg•L-1+NAA0.1 mg•L-1 was most
suitable for plantlet propagation with proliferation coefficient of 15.6.The medium MS+ZT0.2 mg•L-1 +NAA0.1 mg•L1+7.0g•L-1agar +40 g•L-1 sucrose changed from vitrification to normal effectively with rate of change to normal of
84.2%. The medium 1/2MS+IBA0.5mg•L-1+NAA0.3 mg•L-1 was most suitable for rooting, rooting rate reached 85.6%
and mean number of roots was 15.5.
PS0633 INTERACTION BETWEEN GIBBERELLIC ACID AND ETHEPHON IN GROWTH OF RICE SEEDLING
Hajime Watanabe, Shu Hase, and Masahiko Saigusa Grad. School of Agric. Sci., Tohoku Univ., Miyagi 989-6711, Japan.
[email protected]
Enhancement of early seedling growth is a major constraint in direct seeding rice cultivations. We investigated the
effects of two plant growth regulators (PGRs), gibberellic acid (GA) and ethephon (ET), on the seedling growth under
growth chamber and field conditions. Seedling growth was increased by the single treatment of GA or ET over that of
the control. However, effects of the combined applications of GA and ET were more pronounced than that of GA or
ET alone. The growth of different organs of rice seedling, such as coleoptiles, first leaves and second leaves were also
increased by PGRs treatments. In conclusion, enhancement of early seedling growth in direct seeding cultivation is possible by using the proper combinations of PGRs. Effects of GA and ET on the expression of expansin and pathogenesisrelated genes will be also assessed.
PS0634 REGULATION OF DROUGHT-RELATED GENES IN ARABIDOPSIS BY BIOFORGE.
Salzman, Ron A1,2 , Liptay, Albert1, Stoller, Jerry H1, Beem, Lance W1 1 Stoller Enterprises Inc. Houston, TX 77043,
USA 2 Department of Entomology, Texas A&M University, College Station, TX 77843, USA
[email protected]
In addition to documented yield increases, improvements in crop water status in the field have been anecdotally observed in plants treated with Stollerʼs Bioforge (diformyl urea). Research into this phenomenon using Arabidopsis thaliana indicated that treatment with Bioforge previous to the imposition of drought increased survival of water-deprived
plants by an average of 44% compared to water-only controls. In subsequent experiments on Petri dishes, a phenotype
of deeper green leaf color, known to be associated with increased ABA content, was observed under Bioforge treatment.
Molecular investigation of these effects by DNA microarray and quantitative real-time PCR analyses revealed upregulated expression of the transcription factor Dreb1A, as well as other genes involved in several aspects of drought and
oxidative stress tolerance, ABA production, photosynthesis, and genes of unknown function. Taken together, our experiments suggest that Bioforge can activate several gene-expression-based drought tolerance mechanisms in plants, which
could account for the increased drought survival observed.
PS0635 POTENTIAL USES OF PROHEXADIONE-CA IN WINE GRAPE PRODUCTION: YIELD RESTRICTION AND
CONTROL OF BUNCH AND SOUR ROT
Doerr, Siegfried, Mann, Wolfram, Kopf, Alexander, Kuehn, Annett, Reimann, Stephan, Marr, Jens, Rademacher, Wilhelm BASF Agricultural Center, 67114 Limburgerhof, Germany
[email protected]
Treating wine grapes at flowering with 125 to 175 g/ha of prohexadione-calcium reduces berry set by approximately
30%. As a result, yield is restriceted by approximately 20%, which is paralleled by increases in must quality. Another
advantage is that the incidence of bunch rot and sour rot at harvest is significantly diminished. So far, the best results
have been obtained with Pinot varieties. Reducing GA formation in the early stages of fruit development is seen as the
underlying reason for reduced berry formation. Having fewer berries, the clusters of grapes are less dense and less susceptible to fungal infections. This and induced resistance against infections with Botrytis cinerea at flowering are seen as
the underlying reasons for reduced incidence of bunch and sour rot at vintage.
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PS0636 METHYL JASMONATE DELAYS THE OXIDATION-MEDIATED CAROTENOID DEGRADATION IN PETALS OF
YELLOW-PIGMENTED CUT ROSES, AND INHIBITS GENE EXPRESSION OF A CAROTENOID CLEAVGE DIOXYGENASE (CCD)
Glick, Alon 1, Philosoph-Hadas, Sonia 1, Vainstein, Alexander 2, Meir, Ayala 3, Tadmor, Yaakov 3, Meir, Shimon 1 1
Dep. of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet-Dagan 50250, Israel; 2 The Robert H.
Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Faculty of Agriculture, Food and Environmental Quality Sciences, Rehovot 76100, Israel; 3 Dep. of Cucurbits, ARO, Newe Yaʼar Research
Center, Ramat Yishay 30095, Israel
[email protected]
The color intensity and the carotenoid content in petals of the yellow-pigmented rose cultivars ʻFriscoʼ and ʻGolden
Gateʼ increased during the first days of vase life and gradually decreased thereafter. Application of methyl jasmonate (MJ)
to the cut roses following harvest increased their petal color intensity and their carotenoid content throughout vase life.
This MJ effect could result from either induction of carotenoid biosynthesis and/or reduced carotenoid degradation in the
petals. However, simultaneous application of the carotenoid biosynthesis inhibitor, Norflurazon (NF) with MJ showed
that MJ delays mainly carotenoid degradation between days 2-6. Using a subtraction library, constructed by MJ-treated
vs. non-treated flowers, we have found a variety of MJ-affected genes related to different metabolic pathways including
oxidative processes which may be involved in carotenoid breakdown, but no genes related to carotenoid biosynthesis.
Incubating the cut flowers in a low oxygen (2%) atmosphere inhibited the loss of petal color and carotenoids, indicating
that oxidative processes are involved in carotenoid degradation, possibly via the carotenoid cleavage dioxygenase (CCD)
enzyme. Therefore, we have cloned segments of two CCD genes, CCD1 and CCD4, whose expression in rose petals
corresponded to the kinetics of carotenoid degradation during vase life. Our results show that at the molecular level, MJ
treatment reduced CCD1 expression during six days of vase life, but it did not affect the expression of CCD4, which was
recently shown to contribute to whitening of Chrysanthemum petals. These results suggest that MJ may delay the oxidation-mediated carotenoid degradation in rose petals by inhibiting the expression of the CCD1 gene but not the expression
of CCD4. However, MJ may still affect CCD4 at the protein level.
PS0637 UNDERSTANDING THE DIFFERENCES BETWEEN 2,4-D AND NAA IN DELAYING FLORET ABSCISSION IN
RED CESTRUM CUT FLOWERS
Abebie, Bekele 1,2, Lers, Amnon 1, Philosoph-Hadas, Sonia 1, Goren, Raphael 2, Huberman, Moshe 2, Riov, Joseph 2
, Meir, Shimon 1
[email protected]
Floret abscission in red cestrum (Cestrum elegans) cut flowers was delayed by the synthetic auxin 2,4-diclorophenoxyacetic acid (2,4-D) after pulse treatment but not by a-naphthaleneacetic acid (NAA). A significant amount of 2,4-D
moved acropetally, sufficient to reduce floret abscission, while NAA failed to do so. In florets and leaves, more 2,4-D
remained in its active free form, while NAA was quickly conjugated.
We hypothesized that these differences in transport and metabolism of the two auxins might result in differential activation of early auxin responsive genes (Aux/IAA), which were used as molecular markers of auxin activity. Northern
hybridization and quantitative RT-PCR results of six different Aux/IAA cDNAs, cloned from the floret abscission zone
(AZ), showed temporal and spatial differences in the expression level of their encoding genes, with 2,4-D inducing
higher expression levels than NAA. The expression level of all six genes decreased after three days from harvest, and
was inversely related to the increased floret abscission during this period. Taken together, our results suggest that due
to the differences in transport and metabolism of the two synthetic auxins, more free auxin accumulates in the floret AZ
following 2,4-D treatment. This results in turn in activation of the Aux/IAA genes for a longer period than NAA, and in
reduction of floret abscission.
PS0638 CROP PLANT PERFORMANCE: VISUAL ESTIMATION OF PLANT HORMONE SIGNALING
Liptay, Albert, Stoller, Jerry H., Salzman, Ron A, Beem Lance W.
[email protected]
Crop yields exhibit about 25% of a plantʼs genetic potential; we are focusing on how to improve this crop performance,
by visualizing crop development in terms of plant hormone signaling. Our commercial, field and lab experience gives
IPGSA 19th Annual Meeting - Abstracts
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an indication that plant hormone signaling can have a greater effect on crop productivity than fertility alone (N,P,K).
Moreover, micronutrients also have a crucial and substantial effect on the activity of the plant growth hormones. The
unexpected aspect of our work was that when crop yield was improved then generally crop quality was greatly enhanced.
Serendipitously we have also found that with improved yields, pest tolerance was also increased (nematodes, viruses,
diseases, insects). We are vigorously in pursuit of enhancing crop genetic expression through field, lab and gene regulation studies, focusing on understanding plant hormone signaling to enhance crop productivity. We will present a summary of how we have visualized hormone balance in a plant to maximize genetic expression, and have thus affected crop
productivity, crop quality and enhanced pest tolerance.
PS0639 POST FLOWER ABORTION IN NUT TREES: NUT YIELD AS AFFECTED BY HORMONE REGULATION
Beem, Lance W, Liptrap Robert, Stoller Jerry H., Liptay Albert, Salzman Ron A.
[email protected]
Flower development in nut trees often occurs during less-than-ideal weather conditions. Weather can affect the hormone balance during flowering and post flowering periods. Excessive ethylene production can result in flower abortion
on certain biotypes of nut trees. The plant growth hormones balance during flowering and post-flowering is critical for
retention of nuts. The use of calcium, known as a second messenger, will affect or move auxin in various plant parts.
This movement is dependent on application method. Endogenous tryptophan plus zinc applications will increase auxin
production, in certain plant tissues. Elements such as boron, cobalt, nickel & silver have been documented to effectively
counter or inhibit ethylene production. Field research conducted over three consecutive years in California, on almond
and walnut tree crops have shown improvement of nut retention with several elements. Applications of Ca, Co, Cu, Mg,
Mn, Mo, and Zn, increased nut retention up to 50% statistically compared to untreated check in replicated plot experiments. This research suggests certain elements have a direct impact on hormone regulation during development of the
nut.
PS0640 MECHANISM OF GIBBERELLINS IN REDUCING RUSSET IN ‘GOLDEN DELICIOUS’ APPLE
Curry, Eric United States Department of Agriculture, Agriculture Research Service, Tree Fruit Research Laboratory,
1104 North Western Avenue, Wenatchee, WA 98801, USA
[email protected]
MECHANISM OF GIBBERELLINS IN REDUCING RUSSET IN ʻGOLDEN DELICIOUSʼ APPLE
Curry, Eric
United States Department of Agriculture, Agriculture Research Service, Tree Fruit Research Laboratory, 1104 North
Western Avenue, Wenatchee, WA 98801, USA;
[email protected]
Although the apple strives to maintain cuticle integrity against desiccation stress-induced suberin production (russet),
certain environmental conditions predispose fruit to so respond. Gibberellins A4+7 have been used for many years to
reduce the severity of such physiological russetting with sequential foliar applications during the first 30 days after anthesis. Both untreated fruitlets and those treated with GA4+7 were sampled after each of 4 sequential applications about
7 days apart. Epidermal cell size was evaluated by SEM examination of fixed or freeze-dried receptacle cross sections
as well as of enzymatically removed cuticle. Treatment-induced reduction in epidermal cell size is likely the result of
enhanced cell division.
PS0641 SPRAYED ABA ENHANCES PHYSIOLOGICAL PARAMETERS AND YIELD COMPONENTS IN FIELD-CULTURED SOYBEAN
Travaglia, Claudia 1; Reinoso, Herminda 1; Bottini, Rubén 2 1 Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Campus Universitario, X5800ARS Río Cuarto; 2 Facultad de Ciencias Agrarias-CONICET, Universidad Nacional de Cuyo, Alte. Brown 500, M5528AHB Chacras de Coria, Argentina; rbottini@fca.
uncu.edu.ar
[email protected]
IPGSA 19th Annual Meeting - Abstracts
77
This work evaluated the effect of exogenous ABA on to the physiology and yield performance of field-cultured soybean during three years. The hypotheses were that ABA alleviates temporary water stress hence promoting growth, and
it upholds carbohydrate re-mobilization towards seeds. ABA 300 mg L-1 was sprayed at the 7 leaves stage and repeated
in anthesis. The results indicate that ABA increased stomatal conductance and transpiration rate in the mid term, with a
lower leaf temperature. ABA also augmented the content of leaf pigments. By consequence aerial mass increased because
of broad shoots and higher leaf area (although only significant in the upper part of canopy), and shoot soluble carbohydrates were enhanced. The main root of ABA-treated plants was shorter, but with more lateral roots and, importantly, the
number and weight of effective nodules were not affected by the hormone. The number of pods per plant was also improved, and the average weight of grains was significantly enhanced. The seeds had higher oil content, without variation
in proteins. The results suggest that ABA favored the photosynthetic activity by both, increases in leaf area and pigments.
It also confirms results with other species (wheat) in incrementing assimilate export towards storage organs. Such effects
were reflected in yield increases, without affecting the seed quality.
PS0642 SCREENING FRUIT LOOSENING AGENTS FOR TABLE OLIVES
Jacqueline K. Burns1, Louise Ferguson2, Kitren Glozer2, William H. Krueger3, and Richard C. Rosecrance4 1Horticultural Sciences Department, Citrus Research and Education Center, University of Florida,700 Experiment Station Road,
Lake Alfred Florida, Lake Alfred, FL 33850-2299; 2Department of Plant Sciences, University of California, Davis;
3University of California Cooperative Extension Glenn County, Orland, CA; 4California State University, Chico, CA
[email protected]
The California olive industry relies almost exclusively on the table olive (Olea europaea L.). Increased harvesting costs
have intensified industry interest in identifying an abscission agent that can be used with developing mechanical harvesting technologies to increase removal rates. Table olives are harvested immature green at horticultural maturity but before
physiological maturity is reached. The ideal abscission agent would be one that selectively loosens target fruit sufficiently
to allow removal with mechanical harvesters, but minimizes drop to the ground before harvesting is scheduled. The goal
of this research was to reevaluate the potential of ethylene-releasing compounds (ERCs) as olive loosening agents, and
to screen additional candidates previously shown to accelerate immature and mature citrus fruit abscission. Fourteen
compounds were screened in Fresno and Tehama counties in California in September and October 2006. Compounds
were applied at various concentrations alone or in combination to run-off. Fruit detachment force (FDF) and % fruit drop
were measured and leaf loss assessed. Of the compounds evaluated, ERCs ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC) were the most efficacious. In whole tree applications, concentrations above 1000 mg L-1 reduced FDF
greater than 50% of the untreated control within 14 days, but leaf drop increased with increasing concentrations.
Combination applications of 1-methylcyclopropene (1-MCP) with high ethephon concentrations reduced efficacy of
ethephon and delayed leaf drop. Monopotassium phosphate + ethephon (4% and 1000 mg L-1) reduced FDF but leaf loss
was as high as the ethephon alone treatment. Compounds such as methyl jasmonate, coronatine, dikegulac, and 5-chloro3-methyl-4-nitro-1H-pyrazole (CMNP) were not efficacious.
PS0643 SCREENING OF CAROTENOID CLEAVAGE DIOXYGENASE INHIBITORS
Nobutaka Kitahata1, Natsumi Noji1, Shigeo Yoshida1, Masafumi Tsujimoto1, Tadao Asami2 1 RIKEN,Saitama, Japan;
2 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
[email protected]
Carotenoid cleavage dioxygenases play important roles in vitamin A biosynthesis in animals and abscisic acid biosynthesis in plants. Recently, it has been reported that carotenoid cleavage dioxygenases play also important role in
regulation of shoot branching in plants. To examine the function of molecules that are synthesized by carotenoid cleavage
enzyme, we screened our chemical libraries for new inhibitors of carotenoid cleavage dioxygenases.
IPGSA 19th Annual Meeting - Abstracts
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PS0644 PROMOTIVE EFFECTS OF PENTAKEEP-V CONTAINING 5-AMINOLEVULINIC ACID AND TRACE ELEMENTS
ON CROPS
Toru Tanaka1, Keitaro Watanabe1, Shigeyuki Watanabe1, Tohru Tanaka1, Kazuya Iwai2, Yasutomo Takeuchi3 1Cosmo
oil Co., Ltd., Tokyo, Japan; 2Cosmo seiwa agriculture Co., Ltd., Tokyo, Japan; 3Utsunomiya University, Utsunomiya,
Tochigi, Japan
[email protected]
We found promotive effects of 5-aminolevulinic acid(ALA) on the growth and yield of several crops at lower concentrations. These effects include increasing photosynthesis, improving and/or maintaining green color of leaves and
increasing resistance to environmental stresses.
In addition, ALA when mixed with trace elements promoted growth of several crops including radish, eggplant, rice and
barley. We have developed the new functional fertilizer _hPENTAKEEP-V_h which contains magnesium nitrate, FeDTPA and other trace elements. In the poster presentation, we introduce excellent performance of _gPENTAKEEP-V_h
in agricultural fields.
For exmple, _gPENTAKEEP-V_h at 0.1 L.ha-1 to 0.5 L.ha-1, increased the yield of green pepper(Capsicum annum)
fruits increased 15.4% to 25.3% which represents the yield increase 6.4 t.ha-1 to 10.6 t.ha-1, and it also decreased the
content of NO3-N of total N in the fruits 17.2% to 21.4%.
PS0645 INFLUENCE OF FOLIAR SPRAY WITH PACLOBUTRAZOL OR GLUTATHIONE ON GROWTH , FLOWERING
AND CHEMICAL COMPOSITION OF CALENDULA OFFICINALIS L. PLANT
Nahed Abd El Aziz, Mona Mahgoub and Abd Elghany Youssef Department of Ornamental Plants and Woody Trees—
Department of Cultivation and Production of Medicinal and Aromatic Plants ,National Research Centre , Egypt. Prof.
[email protected]
[email protected]
Influence of foliar spray with paclobutrazol or glutathione on growth , flowering and chemical composition of Calendula officinalis L. plant.
Nahed Abd El Aziz, Mona Mahgoub and Abd Elghany Youssef
Department of Ornamental Plants and Woody Trees—Department of Cultivation and Production of Medicinal and Aromatic Plants ,National Research Centre , Egypt.
[email protected]
The objective of this study was to evaluate the effect of using paclobutrazol or glutathione with levels of 0, 50, 100 and
150 mg/l on plant growth , flowering and some chemical composition of Calendula officinalis L. plant grown at the green
house of National Research Center , Giza, during two successive seasons of 2003-2004 and 2004-2005. The obtained
data indicated that foliar application of most paclobutrazol treatments significantly decreased plant height compared with
control treatment while number of branches, fresh weight and dry weight of leaves per plant were increased. All growth
parameters were significantly promoted by foliar application of glutathione. In most collections foliar application of glutathione at 150 mg/l gave the highest significant increase in number of flowers/ plant , fresh weight of flowers (g/plant)
and dry weight of flowers (g/plant) followed by paclobutrazol 100 mg/l compared with untreated plants. Foliar application of glutathione at 150 mg/l recorded the highest values of chlorophyll a and chlorophyll b . However, paclobutrazol and glutathione showed no significant increase in carotenoids contents. The maximal value of total carbohydrates
(37.64%) was obtained when 150 mg/l glutathione was applied followed by paclobutrazol (31.42%) at level of 100 mg/l
and total nitrogen percentage had the same trend.
PS0646 EVALUATION OF THE EFFECTS OF OVER-PRODUCTION OF ABSCISIC ACID (ABA) ON WHOLE PLANT WATER USE, GROWTH AND PRODUCTIVITY
Smeeton, Rachel1, Thompson, Andrew 2, Black, Colin1, Taylor, Ian1 1Plant Sciences Division, School of Biosciences,
University of Nottingham, Loughborough, Leic., LE12 5RD, UK; 2Warwick HRI, University of Warwick, Wellesbourne,
Warwick, CV35 9EF, UK. [email protected]; Tel +44 (0)115 9516377; FAX +44 (0)115 9516334
[email protected]
IPGSA 19th Annual Meeting - Abstracts
79
The phytohormone abscisic acid (ABA) is a fundamental component of plant responses to water stress. ABA is synthesised via the oxidative cleavage of C40 epoxycarotenoid precursors, a reaction catalysed by the water stress inducible key enzyme 9-cis-epoxycaroteniod dioxygenase (NCED) encoded by the gene LeNCED1 in tomato. We previously
transformed tomato plants with a construct constitutively over-expressing the LeNCED1 gene and identified two transgenic lines for further study, sp5 and sp12, which have an elevated ABA content. The additional ABA reduced stomatal
conductance under optimal growing conditions and this conserved soil water during periods when wild type plants were
profligate in its use; under well-watered glasshouse conditions whole plant transpiration efficiency was 27 and 79%
greater in sp12 and sp5 than in WT. This water conserving ability was also evident under water-limited conditions; when
sp12 and WT were grown with sub-optimal irrigation, sp12 plants exhibited greater biomass accumulation. Indeed sp12
plants grew at a similar rate under sub-optimal irrigation as WT plants receiving double the irrigation. Implications of
engineering high ABA for the physiology, water use and productivity of plants are being evaluated.
PS0647 DORMANT APPLICATION OF ETHEPHON WITH VEGETOIL® THINS PEACH FLOWER BUDS
Reighard, Gregory, Ouellette, David, and Brock, Kathy. Department of Horticulture, Clemson University, Clemson, SC
29634-0319, USA [email protected]; phone:864-656-4962; FAX:864-656-4960
[email protected]
Early removal of flower buds, flowers or small fruitlets increases peach fruit size and value at harvest. Due to the
scarcity of labeled or consistent thinning chemicals, peach growers generally wait ~30 days after full bloom before handthinning fruit, which limits potential fruit size. Experiments were conducted near Clemson, South Carolina to determine
the efficacy of combining Vegetoil® (VO), an emulsified soybean oil adjuvant (93% soybean oil), with Ethrel® (a.i.
21.7% ethephon) for pre-bloom flower bud thinning of peach cultivars. Contender in February 2005, 2006 and 2007, and
Cresthaven and Rubyprince in January 2006 and 2007 were sprayed with 10% VO except for Rubyprince in 2006 (8%
VO). Ethrel® concentrations used (varied by year) were 25, 50, 75, 100, or 150 ppm. Dormant oil at 3% and VO at 10%
were the control treatments in 2006 and 2007, respectively. VO (10%) plus Ethrel® at 100 and/or 150 ppm significantly
delayed bloom in Contender and Cresthaven in 2006 and in Cresthaven and Rubyprince in 2007. In 2006, VO (10%) significantly reduced the number of flower buds alive at bloom for Contender and Cresthaven, and the addition of Ethrel®
to the VO spray significantly increased flower bud mortality when compared to the 10% VO application. There were no
differences observed among the Rubyprince treatments in 2006, but the addition of 75 and 100 ppm ethephon slightly
increased flower bud death in 2007. Basal flower buds on marked shoots had significantly lower survival than the terminal flower buds in the VO treatments in both years and all cultivars except Cresthaven in 2006. Generally, Rubyprince is
easier to flower bud thin with soybean oil than Cresthaven, but the opposite was observed in this study when using the
Vegetoil® soybean oil product.
PS0648 POTENTIAL FOR 1-METHYLCYCLOPROPENE ENHANCEMENT OF LINT YIELD IN COTTON
Cothren, J. Tom, Bynum, Josh B, Scheiner, Justin J., da Costa, Vladimir A. Soil and Crop Sciences Department Texas
A&M University Texas Agricultural Experiment Station College Station, Texas 77843-2474, USA
[email protected]
The ethylene action inhibitor 1-methylcyclopropene (1-MCP) has shown numerous beneficial physiological effects
in countering senescence and stress responses in various species of plants. Studies were conducted with 1-MCP to determine its potential for alleviating stress responses and improving yield and fiber quality of upland cotton (Gossypium
hirsutum). The studies investigated different rates of 1-MCP and times of application for their impact on canopy health,
fruit retention and development, and ultimately yield and yield parameters. Lint yield was significantly increased in two
of three field studies, however none of the parameters investigated explained the observed yield response. Relative chlorophyll content and electrolytic leakage were among the parameters tested and were used as indicators of canopy senescence. Relative chlorophyll content was not affected by 1-MCP treatment at the times it was determined, but electrolytic
leakage was increased by a 250 g ha-1 rate of 1-MCP.
Presenter: [email protected], 979-845-0360-phone, 979-845-0456-fax
IPGSA 19th Annual Meeting - Abstracts
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PS0649 ARTEMISININ IN ARTEMISIA ANNUA PLANTS: POSSIBLE EFFECTS OF GIBBERELLINS ON ITS SYNTHESIS
OR ACCUMULATION
Sponsel, Valerie1, Sanusi, Olabisi1, Olmos, David1, Mullens, Conor2, Bach, Stephan2. Biology1 and Chemistry2 Depts,
UTSA, San Antonio, UTSA.
[email protected]
Artemisinin, a sesquiterpenoid lactone endoperoxide, is produced in flowering inflorescences of Artemisinin annua
(sweet wormwood) plants. Artemisinin derivatives are essential components of Artemisinin-based Combination Therapy
(ACT), recommended by the World Health Organization (WHO) for the treatment of malaria that is resistant to conventional monotherapies. It is estimated that at least 500 million clinical cases of malaria occur annually, with over a million
deaths. A recent report (Zhang et al 2005) shows that treatment of vegetative Artemisia plants with gibberellic acid can
increase the yield of artemisinin approximately four-fold. We are re-examining the effect of GAs, and of daylength, on
the yield of artemisinin in Artemisia, an obligate short day plant. Artmisinin, which accumulates in glandular hairs on
leaves, stems and sepals, is measured in chloroform extracts by LC-MS. Since 2001 56 countries have adopted a WHOrecommended ACT, underscoring the urgent need for artemisinin.
PS0650 IN VITRO CALLUS PRODUCTION OF GINGKO BILOBA FOR THE GROWING AREA OF NUTRACEUTICALS
Marihelen Glass and Monica Haddix Department of Natural Resources and Environmental Design North Carolina A&T
State University, Greensboro, NC 27411
[email protected]
Ginkgo biloba is an important tree for the landscape. It is known as a living fossil because it dates back 270 million
years ago in Permian Period. Due to geological cataclysms only two species were left. In 1691,Ginkgo was survived
in China. In Chinese Medicine the leaves of the Ginkgo are used for benefiting the brain, as an astringent to the lungs,
asthma and cough.
Today, Ginkgo leaf extracts are manufactured and used for poor circulation, heart disease, eye disease, tinnitus, dementia
and many other conditions.
Ginkgo leaves are the most extensive studied botanical used today as Nutraceuticals.
The pharmaceutical properties called ginkgolides, known as essential oil. This research was initiated, to compare the
amount of ginkgolides produced by callus grown from excised embryos to the amount of the essential oil produced by
the leaves.
The Embryos were disinfested and cultures on a Murashige and Skoog minimal organics medium with various combinations of two Auxins, 2,4-dichlorophenoxyacetis acid (2,4-D) and naphthaleneacetic acid (NAA), to enhance callus
growth. The two Cytokinins used were benzyladenine (BA) promotes callus growth and 2iP to promote cell division.
Ginkgolides A and B were both found in the callus tissue.
PS0651 EVALUATION OF ANTIOXIDANTS TO REDUCE PHYSICAL DAMAGE IN MECHANICALLY-HARVESTED
‘MANZANILLO’ OLIVES
Glozer, Kitren 1, Ferguson, Louise 1, Krueger, William 2, Rosecrance, Richard 3, Burns, Jacqueline 4 1 Dept. Plant Sci.,
Univ. Calif., Davis, CA 95616, USA, 2 Univ. Calif. Coop. Ext., Glenn County, Orland, CA, 95963, USA, 3 Coll. Agric.,
Calif. State Univ., Chico, CA 95928, USA, 4 Citrus Res. Educ. Center, Univ. Flor., Lake Alfred, Florida, 33850, USA.
[email protected]
Several antioxidants were tested for reduction of physical damage induced by simulated mechanical harvest of ʻManzanilloʼ olives in 2006. Different methods of inducing mechanical damage were tested to reproduce as closely as possible the type of damage observed in mechanical harvest trials. Antioxidants tested included glutathione, ascorbic acid, a
proprietary lactic/acetic acid solution quercetin and luteolin. Compounds were applied as sprays on single limb replicates
prior to induced damage, or as immersions overnight after mechanical damage to simulate drenches. Ascorbic acid ap-
IPGSA 19th Annual Meeting - Abstracts
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plied prior to hand harvest and mechanical damage improved the appearance of fruit after overnight storage by reducing
the severity of bruising; luteolin alone and in combination with ascorbic acid also decreased apparent damage.
Keywords: antioxidant, olive, mechanical harvest, mechanical damage, postharvest
PS0652 EFFECT OF DA-6 ON THE COLD-RESISTANCE OF CUCUMBER SEEDLINGS
Guang-jian Liang; Cai-cheng Huang (Department of Biology, Zhaoqing College, CHINA 526061)
[email protected]
LIANG Guang-jian HUANG Cai-cheng
(Department of Biology, Zhaoqing College, Guangdong CHINA 526061)
The cucumber (Cucumis sativus L. cv.Aoshou No.6) seedlings were treated with different concentrations of DA-6,
which chemical name is hexanoic acid 2-diethylamino ethyl ester). 20 mg.L-1 of DA-6 had the best activities of SOD
and fewest MDA content of the seedlings than those of the other treated and untreated plants under 5¢J cold stress for 3d
and following room temperature recovery, did not increase POD activities under cold stress, however, very significantly
increased POD under following recovery. Therefore, the seedlings treated with 20mg/L of DA-6 maintained green under
cold stress and following recovery, but the untreated plants became wilting and yellow. The result showed that DA-6
could outstandingly increase cold resistance of cucumber seedlings
Key words: cucumber seedlings; DA-6; cold-resistance
Dormancy and Germination
PS0701 REGULATION OF STRIGOLACTONE EXUDATION BY PLANT NUTRIENTS
Yoneyama Kaori 1, Sekimoto Hitoshi 2, Takeuchi Yasutomo 1, Yoneyama Koichi 1 1 Weed Sci. Ctr; 2 Fac. Agric., Utsunomiya Univ., 350 Mine-machi, Utsunomiya 321-8505, Japan
[email protected]
Strigolactones are plant-derived host recognition signals for symbiotic arbuscular mycorrhizal (AM) fungi and root
parasitic plants. We already demonstrated that in leguminous plants, reduced supply of P but not the other mineral nutrients significantly promoted the exudation of strigolactones. Here, we show that in non-leguminous plants, N as well
as P deficiency increases the strigolactone exudation. By contrast, in non-hosts of AM fungi, neither N nor P deficiency
affects exudation. These results indicate that the regulation of strigolactone exudation may vary with the nutrient acquisition strategy of plants.
PS0702 REGULATION OF ENDOGENOUS LEVELS OF ABSCISIC ACID IN PHOTOBLASTIC LETTUCE SEEDS
Yoshiaki Sawada1, Miki Aoki1, Kentaro Nakaminami1, Wataru Mitsuhashi1, Kiyoshi Tatematsu2, Yusuke Jikumaru2,
Tetsuo Kushiro2, Yuji Kamiya2, Yasunori Inoue3, Eiji Nambara2, Tomonobu Toyomasu1, 1Yamagata Univ., Tsuruoka,
Yamagata, 2RIKEN, PSC., Yokohama, Kanagawa, 3Tokyo University of Sciece, Noda, Tokyo, Japan.
[email protected]
Germination of lettuce seed is regulated by phytochrome. We have shown that endogenous abscisic acid (ABA) levels
decreased after red light irradiation during germination of seeds. To reveal the molecular mechanism on regulation of
endogenous ABA levels, we isolated 8 cDNAs encoding 9-cis-epoxycarotenoid dioxygenase (NCED; biosynthesis) and
ABA 8ʼ-hydroxylase (ABA8ox; catabolism): LsNCED1-4, and LsABA8ox1-4. The expression analysis of these genes
during germination and quantitative analysis of endogenous levels of ABA and its catabolites suggested that LsNCED4
played an important role in regulation of ABA levels for lettuce seed germination.
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PS0703 POTENTIAL CROSSTALK BETWEEN AUXIN AND ABA DURING SEED GERMINATION AND POSTGERMINATION
Nonogaki, Hiro 1, Liu, Po-Pu 1, Hewitt, Jessica, R. 1, Homrichhausen, Tanja, M. 1, Martin, Ruth, C. 1, Montgomery,
Taiowa, A. 2, 3, Fahlgren, Noah 2, 3, Kasschau, Kristin, D. 2, 3, Carrington, James, C. 2, 3, González-García, Mary-Paz
4, Lorenzo, Oscar 4 1Dept. of Horticulture; 2Center for Genome Research and Biocomputing; 3Dept. of Botany and
Plant Pathology, Oregon State Univ., Corvallis, Oregon 97331, USA; 4Dept. de Fisiología Vegetal, Centro Hispano-Luso
de Investigaciones Agrarias, Facultad de Biología, Universidad de Salamanca, Plaza de los Doctores de la Reina s/n,
Salamanca 37007, Spain
[email protected]
Seed germination sensu stricto that is defined as the physiological events before radicle emergence is regulated by
the balance between ABA and GA. Recent studies using microarrays and hormone response mutants have suggested the
potential involvement of auxin in seed germination. Our studies using two separate approaches, enhance-trap and microRNA (miRNA) de-regulation experiments also indicated the involvement of auxin, especially the crosstalk between
auxin and ABA in sensu stricto germination and post-germinative events. Seeds of loss-of-function mutants for a PWWP
transcription factor exhibited reduced sensitivity to ABA. GUS expression in the PWWP enhancer-trap lines indicated
that this gene is expressed in the cotyledon tips of embryo enclosed by the testa. The postgerminative expression of
PWWP was detected in the hydathodes of rosette leaves, which is similar to the GUS expression driven by DR5, an auxin
responsive promoter. The pwwp mutant seedlings exhibited hypersensitivity to auxin. In miRNA de-regulation experiments, transgenic seeds expressing miR160 resistant form of AUXIN RESPONSE FACTOR10 (ARF10) exhibited ABA
hypersensitivity suggesting that overaccumulation of de-regulated ARF10 affected ABA signal transduction in seeds.
The hypersensitivity to ABA was more apparent during the post-germinative events than in sensu stricto germination.
These results support the idea of potential crosstalk between auxin and ABA during seed germination and post-germination stages.
PS0704 HOW CROSS-TALK BETWEEN NITRIC OXIDE AND HEMOGLOBIN INFLUENCE SEED GERMINATION IN
WHEAT
Shukdeb Sen Biology Department, School of Science, Mathematics and Engineering, Bethune-Cookman College, Daytona Beach, FL 32114-3099, USA
[email protected]
Seed germination is a complex process involving various physical and biochemical cues such as water, light, heat,
and other chemical agents. The question arises how do these seeds germinate? To answer this question a few years ago,
this author proposed a hypothesis that nitric oxide in conjunction with phytochromes play a role in the process of seed
germination (Sen and Cheema, 1995). Recent reports confirmed that plants release nitric oxide and it influences seed
germination (Beligni, et al., 2002). Ubiquitous presence of hemoglobin in plants (Appleby, 1992) and the reports indicating that hemoglobin can deliver nitric oxide to the mammalian tissue by S-nitrosylation of conserved cysteine residues
(Gow and Stamler, 1998; Stamler et. al. 1997; Gross and Lane 1999) present an attractive idea to suggest that plant hemoglobin might be able to deliver NO to the tissue where a low oxygen tension exist due to hypoxic condition such as
seed tissue. Class 1 is stress-related hemoglobin that is found in seeds, roots and stem. Hypoxic stress activates class1
hemoglobin synthesis (Dordas et al. 2003). Database search and ClustalW (Thompson et al. 1994) analysis revealed that
cysteine is conserved in all non-symbiotic hemoglobin. This finding provided support to test if hemoglobin can interact
with NO during different time periods of germination. Data obtained through immunoblotting experiments, showed a
burst of class 1 hemoglobin synthesis during the early stages of seed germination. After 24 hours of water imbibitions,
the emergence of embryonic axes occurred in germinating wheat seed. At this juncture, extensive synthesis of hemoglobin occurred in the tissue that lasted for a short period of time. The levels of hemoglobin decreased after 48-72 hours
of imbibitions. Similarly, the levels of NO measured by Griess reaction indicated a copious amount of nitric oxide is
produced in the germinating seeds after 24 hours of water imbibition. The levels of NO drops as embryonic axes grow.
The NO donor sodium nitroprusside (SNP) increased the NO levels in treated seeds. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)treatment reduced the level of NO drastically. Seeds treated
with 10mM SNP showed a 2-fold increase in the levels of NO. Similarly, seeds treated with 1 mM c-PTIO alone have
IPGSA 19th Annual Meeting - Abstracts
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lowest level of NO. This observation suggests that NO is necessary for seed germination. To identify the S-nitrosated
protein products, anti S-nitrosocysteine antibody was utilized to see if any proteins could be detected by western blot
technique. At least three proteins have been recognized that gave positive immuno-reactive bands. The molecular mass
of those proteins were 75, 41 and 13 kD respectively. All these three proteins were present in the embryonic axes during
the 12-24 hours, but later the levels of these proteins reduced during 48 hours. This finding supports the idea that various
proteins are nitrosated during the seed germination. This parallel activation of NO and hemoglobin in the early stages of
seed germination suggest that hemoglobin play important roles in the NO metabolism. It can scavenge NO as well as it
can bind with cysteine to form S-nitrosothiol-hemoglobin (SNO-Hb), which is stored in the tissue and can deliver NO
at the time of need. This data strengthen our hypothesis that during seed germination hemoglobin and NO interact with
each other and regulate NO levels that control the process of seed germination.
References
Appleby, C.A. 1992. The origin and functions of hemoglobin in plants. Science in Progress 76: 365-398.
Beligni, M.V., A. Fath, P.C. Bethke, L. Lamattina and R.L. Jones 2002. Nitric oxide acts as an antioxidant and delays
programmed cell death in barley aleurone layers. Plant Physiol 129: 1642-1650.
Dordas, C., R. Jean and R.D. Hill 2003. Plant haemoglobins, nitric oxide and hypoxic stress. Annals of Botany 91: 173178.
Gow, A.J. and J.S. Stamler 1998. Reactions between nitric oxide and haemoglobin under physiological conditions. Nature (London) 391: 169-173.
Graziano, M., M.V. Beligni, and L. Lamattina 2002. Nitric oxide improves internal iron availability in plants. Plant
Physiol 130: 1852-1859.
Gross, S.S. and P. Lane 1999. Physiological reactions of nitric oxide and hemoglobin: A radical rethink. Proc. Natl. Acad.
Sci. USA 96: 9967-9969.
Sen, S and I. Cheema 1995. Nitric oxide synthase and calmodulin immunoreactivity in plant embryonic tissue. Biochemical Archives 11: 221-227.
Stamler, J.S., L. Jia, J.P. Eu, T.J. McMahon, I.T. Demchenko, J. Bonaventura, K. Gernert and C.A. Piantadosi 1997.
Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science 276: 2034-2037.
Thompson, J.D., D.G. Higgins, and T.J. Gibson (1994). Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids
Res. 22:4673-80.
PS0705 REGULATION OF EMBRYONIC ABSCISIC ACID CONTENT DURING IMBIBITION OF BARLEY: THE COMPARISON OF COMMERCIAL CULTIVARS WITH HIGH VERSUS LOW LEVEL OF DORMANCY
Makiko Chono1, Ichiro Honda2, Naoyuki Kawada3, Shigenobu Kaneko1, Yoshiaki Watanabe4 1National Institute of
Crop Science, NARO, Tsukuba 305-8518, Japan; 2National Institute of Vegetable and Tea Science, NARO, Ano 5142392, Japan; 3National Agricultural Research Center for Kyushu Okinawa Region, NARO, Chikugo 833-0041, Japan;
4National Agricultural Research Center, NARO, Tsukuba 305-8660, Japan.
[email protected]
We have investigated the correlation between dormancy and the regulation of embryonic abscisic acid (ABA) content
in two commercial barley (Hordeum vulgare L.) cultivars, _gIshukushirazu (IS)_h and _gNishinochikara (NI)_h, which
have, respectively, a high and low dormancy level at harvest. During grain development, IS contained a slightly higher
ABA content than NI. Expression analysis for key ABA-biosynthetic and ABA-catabolic genes [9-cis-epoxycarotenoid
dioxygenase genes (HvNCED1 and HvNCED2) and ABA 8_f-hydroxylase genes (HvABA8_fOH-1, HvABA8_fOH-2),
respectively] revealed that the expression patterns of these genes during grain development were similar between IS and
NI; however, the expression patterns of these genes in response to imbibition were different at harvest. When the freshly
harvested grains were imbibed, the increase in HvABA8_fOH-1 expression in NI was followed by rapid ABA decrease
and a high germination percentage after the start of imbibition. Conversely, the increase in HvNCED1 expression in IS
was followed by the lesser extent of ABA decrease and the lower germination percentage than in NI. The change of ABA
content via the varying expression patterns of HvNCED1 and HvABA8_fOH-1 during imbibition might be reflected, in
part, in dormancy level.
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PS0706 HYDROGEN CYANAMIDE INCREASES BUD BREAK OF PISTACHIO UNDER INSUFFICIENT CHILL HOURS
Arreola, Jesus. Universidad Autonoma Chapingo-URUZA. Apdo. Postal 8. Bermejillo, Dgo. C.P. 35230
[email protected]
[email protected]
This experiment was carried out with the objective to evaluate the effects of hydrogen cyanamide on budbreak of pistachio (Pistacia vera L.) grown in a region with insufficient chill hours (7.2oC). dormant buds on branches of 1 to 5 years
old were treated on February 20th, 2005 and 2006 with 0, 25.5 or 51.0 g.L-1 of H2CN2 (hereafter referred to as 0%, 2.5%
or 5.0% (v/v) H2CN2). Hydrogen cyanamide advanced and increased vegetative budbreak in branches of 1 to 5 years
old. The highest budbreak was observed when H2CN2 at 5.0% was applied without causing phytotoxicity. Hydrogen
cyanamide applied al 5.0% ¡Ö 30 days before expected budbreak may increase budbreak, and substitute for chilling when
pistachio is grown in mild environments.
PS0707 EVOLUTION OF GIBBERELLIN BIOSYNTHESIS AND FUNCTION IN PLANTS
Anterola, Aldwin 1, Shanle, Erin 1, Mansouri, Katayoun 1, Schuette, Scott 1, Vidal-Russell, Romina 1, Coates, Robert
2, Von Schwartzenberg, Klaus 3, Renzaglia, Karen 1 1 Dept. of Plant Biology, Southern Illinois University, Carbondale,
IL 62901, USA 2 Dept. of Chemistry, University of Illinois, Urbana, IL 61801, USA 3 Biozentrum Klein Flottbek und
Botanischer Garten, University of Hamburg, Hamburg, Germany
[email protected]
Gibberellins are essential phytohormones in angiosperms and gymnosperms, as they are required for seed germination, flower development and vegetative growth. In ferns, gibberellins are involved in spore germination, and serve (in
some species) as antheridiogens that induce the formation of male gametangia (antheridium). Inspection of the recently
sequenced genome of the moss Physcomitrella patens revealed the presence of gibberellin biosynthetic genes, though it
appears that this moss uses fewer enzymes than angiosperms do to make gibberellins. An example is the bifunctional diterpene cyclase that has both copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities. To determine
the potential roles of gibberellins in P. patens, the gene encoding the bifunctional diterpene cyclase was heterologously
expressed in E. coli, and then tested for its sensitivity to AMO-1618, which is a known CPS and KS inhibitor in higher
plants. After confirming that AMO-1618 inhibited the moss bifunctional enzyme, the effects of AMO-1618 on antheridia
formation and spore germination of P. patens were tested. Our results show that while antheridia formation in P. patens
was not affected by AMO-1618, the germination of its spores was suppressed, suggesting that an ancient role of gibberellins may be in spore germination, rather than in antheridum formation. Hence, in the evolution of land plants, we see a
gradual expansion in the biosynthetic pathway leading to gibberellins, as well as in its role in plant development.
PS0708 NOVEL SMALL COMPOUND ACTIVATORS OF PLASTID-TO-NUCLEUS SIGNALING PATHWAYS
Yuichiro Tsuchiya12, Yuji Kamiya2, Sean R. Cutler1, Eiji Nambara2 and Peter McCourt1 1Dormancy and Adaptation
Research Team, RIKEN Plant Science Center, Yokohama, Japan; 2Dep. Cell and Systems Biol., Univ. of Toronto, Toronto, Canada
[email protected]
Although genetic analysis has identified genes involved in seed germination many components still remain to be
discovered. We have investigated seed germination of Arabidopsis using chemical genetics. As with mutational analysis, the _gphenotype_h caused by small molecule are used as a starting point to identify genes in a particular biological
process.
From 10,000 non-annotated chemical library, 5 compounds (E compounds) were identified which inhibit cotyledon
greening and expansion. Interestingly ABA or GA signaling mutants showed altered response to E compounds. Consistent with this, genetic screening for E compounds resistant mutants identified new alleles of ABA and GA response
mutants. Surprisingly, suppressor screens also identified new alleles of elongated hypocotyl1 locus (hy1). Although hy1
is known photomorphogenesis mutant it is also allele of genome uncoupled2 locus which is involved in plastid retrograde
signal. In this presentation, we will discuss possible involvement of E compounds on hormone and plastid retrograde
signaling pathways.
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PS0709 DEFENSE-RELATED SIGNALING BY INTERACTION OF ARABINOGALACTAN-PROTEINS AND _-GLUCOSYL YARIV REAGENT INHIBITS GIBBERELLIN SIGNALING IN BARLEY ALEURONE CELLS
Kiyoshi Mashiguchi1, Eriko Urakami1, Morifumi Hasegawa2, Kazutsuka Sanmiya1, Ichiro Matsumoto1, Isomaro
Yamaguchi3, Tadao Asami1_Yoshihito Suzuki1 1 Department of Applied Biological Chemistry, University of Tokyo,
Bunkyo-ku, Tokyo, Japan 2 College of Agriculture, Ibaraki University, Ami, Ibaraki, Japan 3 Faculty of Technology,
Maebashi Institute of Technology, Gunma, Japan
[email protected]
Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at plasma membrane and extracellular space. A synthetic chemical reagent called _-glucosyl Yariv reagent (_-GlcY) binds specifically with AGPs. We have
reported that gibberellin (GA) signaling was specifically inhibited by _-GlcY treatment in barley aleurone protoplasts.
In this study, we examined the specificity of inhibitory effect of _-GlcY on GA signaling using microarray analysis and
found that _-GlcY was largely effective in repressing both the GA-induced- and repressed-gene expressions. In addition,
quite a number of genes were up-regulated by _-GlcY in a GA-independent manner, and many of them were categorized
to defense-related genes including two WRKYs and Esi47, which were recently shown to repress GA signaling. Plant
defense signaling triggered by jasmonic acid or chitin elicitor could inhibit GA-inducible events such as _-amylase secretion, programmed cell death, and gene induction of some GA-inducible genes in aleurone cells. These results indicate
that GA signaling in aleurone cells is under regulation of defense-related signaling, which is presumably mediated by
WRKYs and Esi47 at least in part. It is also probable that AGPs are involved in the perception of stimuli causing defense
responses.
PS0710 EXPRESSION ANALYSIS OF ATNCED6 AND ATNCED9 IN ARABIDOPSIS DEVELOPING SEEDS
Seo, Mitsunori, Effroy, Delphine, Frey, Anne, Clement, Sabine, North, Helen M and Marion-Poll, Annie Laboratoire de
Biologie des Semences, UMR204 AgroParisTech, Institut Jean-Pierre Bourgin, 78026 Versailles Cedex, France
[email protected]
Abscisic acid (ABA) is a plant hormone regulating seed dormancy and germination. We have shown that AtNCED6
and AtNCED9 play key role in ABA biosynthesis in developing and germinating seeds. Mutants defective in these genes
contain reduced ABA levels in seeds and can germinate in the presence of a gibberellin biosynthesis inhibitor, paclobutrazol. Promoter-reporter analysis, in combination with in situ hybridization and RT-PCR, indicated that AtNCED6 was
expressed in endosperm during middle stage of seed development whereas AtNCED9 expression was observed in seed
coat during middle stage and in embryo during late stage of seed development. These data indicates that ABA biosynthesis is regulated in tissue specific manners depending on developmental stages. To clarify the roles of ABA synthesized
in different tissues and elucidate the mechanisms by which ABA biosynthesis is regulated, we are conducting promoter
analysis of AtNCED6 and AtNCED9. Identification of the factors regulating AtNCED6 and AtNCED9 expression will
provide us with new insights into regulation of seed dormancy and germination.
PS0711 UNEQUALLY REDUNDANT FUNCTIONS OF NCED9, NCED5 AND NCED2 GENES IN THERMOINHIBITION
OF ARABIDOPSIS SEED GERMINATION
Shigeo Toh1, Akane Imamura1, Masanori Okamoto2, Yusuke Jikumaru2, Yuji Kamiya2, Eiji Nambara2 and Naoto
Kawakami1 1Meiji Univ., Dep. Life Sci., Higashimita, Tama-ku, Kawasaki, Japan, 2RIKEN Plant Science Center, Suehiro-cho, Tsurumi-ku, Yokohama, Japan,
[email protected]
Like other winter annual species, germination of Arabidopsis seed is inhibited by high temperature (thermoinhibition). We have found that de novo ABA biosynthesis during imbibition is responsible for thermoinhibition of Arabidopsis
seeds. We also found that three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5 and NCED9, were up-regulated by supraoptimal temperatures. To reveal contribution of each NCED gene on thermoinhibition, we observed germination of their T-DNA insertion mutants. The seeds of nced9 mutant showed considerable tolerance to thermoinhibition,
but those of nced2 and nced5 showed no apparent phenotypes. The seeds of nced5nced9 and nced2nced9 double mutants
showed higher thermoinhibition tolerance than nced9, but nced2nced5 double mutants showed similar germination to
their parents. The seeds of nced2nced5nced9 triple mutant showed higher germination tolerance than those of the double
mutants. These results suggest that NCED9, NCED5 and NCED2 genes are unequally redundant and their up-regulation
by high temperature is critical for thermoinhibition of Arabidopsis seeds.
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PS0712 GIBBERELLIN LEVELS IN EMBRYOS OF GERMINATING SEEDS OF TWO SORGHUM LINES WITH CONTRASTING DORMANCY
Piccoli, Patricia 1; Rodríguez, Verónica 2; Benech-Arnold, Roberto 2; Bottini, Rubén 1 1 Facultad de Ciencias AgrariasCONICET, Universidad Nacional de Cuyo, Alte. Brown 500, M5528AHB Chacras de Coria, Argentina; rbottini@fca.
uncu.edu.ar 2 Facultad de Agronomía-CONICET, Universidad de Buenos Aires, San Martín 4453, C1417DSE Buenos
Aires
[email protected]
Caryopse sprouting in planta is an undesirable trait in sorghum. We have previously assessed by functional genomics
that embryos of germinating seeds of a sprouting-resistant line (IS) show an increased expression of one GA2ox as compared to a non-resistant sprouting line (RB9). In order to unravel the mechanism involved in the control of dormancy of
sorghumʼs caryopses, the GA profile of embryos detached from caryopses of both lines along germination was assessed
by GC-EIMS with stable isotopes as standards. GAs from both, the non-hydroxylative and the early-hydroxylative pathways, were searched. From the 2-hydroxy-GAs only GA51 levels were substantially higher in IS embryos after 4 days
of incubation in correlation with the GA2ox expression. Nor GA29, GA8 or GA34 were detectable from the extracts.
Both GA1 and GA4 levels were higher in RB9 in the first 2-3 days. The rest of the GAs assessed, i.e. GA19, GA17, GA3,
GA24, GA25 and GA9 did not show appreciable differences between lines, with the exception of GA20 that increased
after 4 days in RB9. Interestingly, the levels of GA1 and GA3 had values of two orders of magnitude higher respect to the
other GAs. This finding suggests that in the embryos of the germinating seeds there are two different sources of GAs, one
produced by the growing embryo that correlates with the gene expression previously determined, and the other coming
from the endosperm and connecting tissues. It is possible that GA1/3 may be sequestered by some substances, like the
polyphenolic net in which sorghum caryopses are particularly rich, and compartmentation of these GAs may account for
the different sprouting behaviour of the two sorghum lines studied.
PS0713 REGULATION BY ABA OF A GROUP 6 LEA GENE DURING DORMANCY AND SEEDLING ESTABLISHMENT
Jiménez, Ericka, Battaglia, Marina, Arrieta Ma. Paz, Covarrubias, Alejandra A. Dep. Biología Molecular de Plantas,
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Mor. 62250
México.
[email protected]
Jiménez, Ericka, Battaglia, Marina, Arrieta, Ma. Paz, Covarrubias, Alejandra
Dep. Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo.
Postal 510-3, Cuernavaca, Mor. 62250 México.
[email protected]
Abscisic acid (ABA) is a phytohormone that, to some extent, regulates most aspects of plant growth and development,
partly due to interactions with other phytohormones. Some of the processes regulated by ABA include the synthesis of
seed storage proteins and lipids, the promotion of seed desiccation tolerance and dormancy, among others, as well as
some aspects of physiological responses to environmental stresses such as water deficit.
PvLEA-18 is a group 6 LEA gene, whose protein and transcript are highly accumulated in desiccated common bean
seeds, and in vegetative tissues in response to water deficit. Even though PvLEA-18 transcript and protein also accumulate in response to ABA treatments, protein accumulation in response to water deficit is mostly ABA independent, rather
regulated at the translational level through the 3ʼUTR of its transcript. To better characterized the role of ABA in the
expression of the PvLEA-18 gene, in this work we have followed the expression of this gene in Arabidopsis plants using
chimeric constructs containing the PvLEA-18 promoter fused to the GUS gene and to either the 3ʼUTR of the NOS or
of the PvLEA-18 gene. This analysis was carried out during seed development and seedling establishment, using Arabidopsis plants affected in ABI1, ABI3 and ABI5 genes, encoding regulatory proteins known to mediate the ABA response.
Our results show that the 3ʼUTR also favored the expression of the reporter protein during the developmental stages
examined, in an ABA independent mode. Also, our data indicate that ABI1, ABI3 and ABI5 mediate the expression of
the PvLEA-18 gene from its promoter during seed development but not during seedling establishment in well-watered
seedlings.
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PS0714 PROMOTION OF SEED GERMINATION IN ARABIDOPSIS THALIANA BY KARRIKINS, A NEW CLASS OF
NATURALLY OCCURRING PLANT GROWTH REGULATORS
Nelson, David C., Chiwocha, Sheila D.S., Flematti, Gavin R., Ghisalberti, Emilio L., Dixon, Kingsley W., Smith, Steven
M.
[email protected]
The primary active compound in smoke that promotes germination of highly smoke-responsive species has been
identified as a butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one). We have adopted the family name karrikin (from ʻkarrikʼ, the first recorded word for ʻsmokeʼ in Aboriginal Nyungar), to reflect the original source of such compounds, and
karrikinolide (KAR1) to denote the parent butenolide molecule. It has been established that KAR1 can also promote
germination and seedling vigor in species that do not normally encounter smoke, raising the possibility that karrikins
represent a new class of plant growth-promoting substances of wide significance. To uncover the molecular mechanisms
of karrikin action, we have characterized its effects on Arabidopsis thaliana. KAR1 enhances the germination rate of
conditionally dormant seeds. While it does not replace a gibberellin requirement, KAR1 stimulates germination of GAdeficient arabidopsis seed. KAR1 can also alleviate or enhance, respectively, the effects of exogenous ABA or GA on
seed germination. To help dissect the karrikin response pathway, mutants defective in ABA, GA, and ethylene signaling
pathways have been tested for KAR1 effects on rates of germination. Microarray analysis of KAR1-treated tissues was
performed to generate a karrikin response profile. Candidate genes with a strong transcriptional response to KAR1 were
selected to set up a reporter-based forward genetic screen for karrikin response mutants.
Novel Hormones/Hormones in Other Systems
PS0801 PROGESTERONE: ITS OCCURRENCE IN PLANTS AND INVOLVEMENT IN PLANT GROWTH
Mayumi Iino1, Takahito Nomura2, Yuji Tamaki1, Yumiko Yamada2, Koichi Yoneyama1, Yasutomo Takeuchi1, Masaki
Mori3, Tadao Asami4, Takeshi Nakano4, Takao Yokota2 1Center for Research on Wild Plants, Utsunomiya University,
Utsunomiya 321-8505, Japan; 2Department of Biosciences, Teikyo University, Utsunomiya 320-8551, Japan; 3Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 Japan;4RIKEN,
Wako, Saitama 351-0198, Japan
[email protected]
We identified and quantified progesterone, a mammalian gonadal hormone, in a range of higher plants by using GCMS and revealed the effects of progesterone on the vegetative growth of plants. The growth of Arabidopsis seedlings was
promoted by progesterone at low concentrations but suppressed at higher concentrations in both light and darkness. The
growth of the gibberellin-deficient mutant lh of pea was also promoted by progesterone. An earlier study demonstrated
that progesterone binds to MSBP1 protein (Membrane Steroid Binding Protein 1) of Arabidopsis. In this work we cloned
the homologous genes of Arabidopsis, MSBP2 and SBP (soluble Steroid Binding Protein), as well as of rice, OsMSBP1,
OsMSBP2 and OsSBP and examined their expressions in plant tissues. All of these genes, except OsMSBP1, were expressed abundantly in plant tissues. Roles of progesterone in plant growth will be discussed.
PS0802 REGULATION OF PLANT DEVELOPMENT BY A CLASS OF BACTERIAL QUORUM SENSING SIGNALS
Ortíz-Castro, Randy, Morquecho-Contreras, Alina, Pelagio-Flores, Ramón, Martínez-Trujillo, Miguel, López-Bucio,
José. Instituto de Investigaciones Químico-Biológicas. Universidad Michoacana de San Nicolás de Hidalgo. Edificio B3,
Ciudad Universitaria, C.P. 58030 Morelia, Michoacán, México.
[email protected]
Many bacterial species use small molecule signaling to communicate each other and to coordinate their activities, a
process commonly referred to as quorum sensing. Diverse Gram-negative bacteria employ the acyl-homoserine lactone
(acyl-HSL) system. This type of bacterial cell-to-cell communication was discovered in the context of microbial ecology,
but it is now evident that acyl-HSL signaling is important in plant and animal communication with bacteria.
Acyl-HSLs are members of a class of compounds that contain a conserved homoserine lactone (HSL) ring and an amide
(N)-linked acyl side chain. The acyl groups of naturally occurring AHLs range from 4 to 18 carbons in length; they may
be saturated or insaturated, and with or without a C-3 substituent.
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These chemical signals are produced by specific enzymes, and they are detected by specific receptors. Recent work
demonstrated that the presence of HSL-producing bacteria in the rhizosphere of tomato induced the salicylic acid and
ethylene-dependent defense responses. In addition, certain Rhizobium mutants that fail to produce or sense acyl-HSLs
were unable to nodulate legume plants, suggesting that acyl-HSLs might participate in bacterial-plant interactions.
To test the hypothesis that acyl-HSLs could play a role in plant development, we evaluated the A. thaliana growth responses to a variety of acyl-HSL compounds, with acyl chain ranging from 4 to 14 carbons in length. In particular, we
focused in the root response to acyl-HSLs as a means to determine the biological activity of these signals in plants. We
found that acyl-HSL signals differentially regulate root system architecture in a dose-dependent way. Developmental
targets of acyl-HSL signals include root growth, root hair development and lateral root initiation. Alterations in these processes were related to the effects of acyl-HSLs in cell division and differentiation. Interestingly, the finding that alkamide
resistant mutants of A. thaliana show reduced responses to acyl-HSL signals, indicates that plants might sense alkamides
and acyl-HSLs by a common genetic mechanism, and that plants are able to adjust their growth and development in response to bacterial quorum sensing signals.
PS0803 AN UNUSUAL CHIMERA DITERPENE SYNTHASE IS RESPONSIBLE FOR FUSICOCCIN BIOSYNTHESIS
Tomonobu, Toyomasu1, Mai, Tsukahara1, Akane, Kaneko1, Rie, Niida1, Chiho, Ikeda2, Wataru, Mitsuhashi1, Tohru,
Dairi2, Nobuo, Kato3, Takeshi, Sassa1 1Dept. of Bioresource Engineering, Yamagata Univ., Yamagata 997-8555, Japan;
2Biotechnology Research Center, Toyama Prefectural Univ., Toyoma 939-0398, Japan; 3The Institute of Scientific and
Industrial Research, Osaka Univ., Osaka 567-0047, Japan
[email protected]
Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As
modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H+-ATPase in plants. From
the mycelia, we have successfully isolated a cDNA encoding (+)-fusicocca-2,10(14)-diene (a tricyclic hydrocarbon precursor for fusicoccins) synthase (PaFS) which possesses not only cyclase activity but also prenyltransferase activity. The
functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains:
a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus.
PS0804 SYNTHESIS AND PLANT GROWTH REGULATION PROPERTIES OF INDOLDICARBOXYLIC ACIDS DERIVED
FROM PIPERONAL
Pescador1, Rosete; Maiochi1, Antunes Riceli; Rosa2 , Flávia Aparecida Fernandes da; Labes2, Ricardo;, Weidlich2 ,
Rafael; Rebelo2 , Ricardo Andrade. 1Departamento de Ciências Naturais, Universidade Regional de Blumenau, CaixaPostal 1507, 89010-971 Blumenau, Santa Catarina, Brazil 2Departamento de Química, Universidade Regional de Blumenau, CaixaPostal 1507, 89010-971 Blumenau, Santa Catarina, Brazil.l. [email protected]
[email protected]
Continuing our effort towards the synthesis of new plant growth regulators presently is investigated the preparation of
5,6-methylenedioxyindoledicarboxylic acids (1) from piperonal, a derivative of the abundant natural product safrole. The
presence of carboxylic group has been accepted as a structural requirement for auxin candidates. Therefore, we decided
to investigate the influence of two carboxylic groups, which may provide a two binding points to the plant receptors.
The synthetic strategy adopted requires the intermediates 5,6-methylendioxyindol-2-yl-formic acid (2) and 5,6-methylenedioxyindole (3). These were prepared according to literature procedure1 via nitrene insertion reaction and reductive
cyclization, respectively. Compound 2 should be submitted to classical Mannich reaction followed by cianation and hydrolysis to give 1 with acetic and formic acid chains. Compound 1 with a double acetic acid chains, should be accessed
from 3 by means of a double Mannich reaction, cianation and hydrolysis. Plant cell tissue and rooting promotion are the
selected bioassays for the compounds synthesized.
1Rosa, F.A.F. da; Rebelo, R.A.; Nascimento, M.G. J. Braz. Chem. Soc., 14(1), 11-15, 2003.
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PS0805 COORDINATE EXPRESSION OF GENES CLUSTERED ON RICE CHROMOSOME 4 FOR THE BIOSYNTHESIS
OF MOMILACTONE
Okada, Kazunori1, Shimura, Kazuhiro1, Okada, Atushi1, Ko, Kwang-Wook1, Toyomasu, Tomonobu2, Hasegawa,
Morifumi3, Shibuya, Naoto4, Nojiri, Hideaki1, Yamane, Hisakazu1 1Biotech. Res. Center, The Univ. of Tokyo, Tokyo
113-8657, Japan; 2Dept. of Biores. Eng., Yamagata Univ., Yamagata 997-8555, Japan; 3College of Agri., Ibaraki Univ.,
Ibaraki 300-0393, Japan; 4Dept. of Life Sciences, Meiji Univ., Kanagawa 214-8571, Japan
[email protected]
In rice fourteen diterpenoid phytoalexins derived from geranylgeranyl diphosphate are produced in response to exogenously applied elicitors or UV irradiation. Six diterpene cyclase genes for the biosynthesis of these phytoalexins have
been functionally characterized and shown to have inducible expressions upon the elicitation. However, genes involved
in the downstream oxidation of the diterpene hydrocarbons leading to bioactive phytoalexins have not been identified,
although participations of cytochrome P450 monooxygenases (P450s) and a dehydrogenase in the biosynthetic pathways
have so far been suggested. From the information of rice genomic sequences and results of microarray analysis, we found
that the genes encoding diterpene cyclases, P450s, and a dehydrogenase, all of which are closely organized on chromosome 4 as a gene cluster, were coordinately up-regulated by elicitor prior to the accumulation of the phytoalexins. Biochemical and molecular genetic approaches to show the function of these enzymes revealed that the P450s (CYP99A2
and/or CYP99A3) and the dehydrogenase (OsMAS) were responsible for the momilactone biosynthesis. Thus, it appears
that the expressions of these genes in the cluster are controlled by coordinate regulating system to exert efficient and
transient production of the phytoalexins when plants are infected by pathogen. To our knowledge, the presence of a gene
cluster consisting of several genes that are involved in a biosynthetic pathway has never been demonstrated in higher
plants, whereas the gene cluster has been reported for the biosynthesis of gibberellins in the fungus Gibberella fujikuroi.
PS0806 SEARCH FOR NARINGENIN 7-O-METHYLTRANSFERASE, A BIOSYNTHETIC ENZYME OF A RICE FLAVONOID PHYTOALEXIN
Fengqiu Lin1, Kazunori Okada1, Atsushi Okada1, Morifumi Hasegawa2, Hiroyuki Anzai3, Osamu Kodama2, Hideaki
Nojiri1, Hisakazu Yamane1, 1Biotechnology Research Center, The University of Tokyo, 2College of Agriculture, Ibaraki
University, 3Gene Research Center, Ibaraki University, Japan
[email protected]
Sakuranetin (4_f,5-dihydroxy-7-methoxyflavanone) is known as a major phytoalexin among 15 phytoalexins isolated
from rice plants. Naringenin 7-O-methyltransferase (NOMT) is the enzyme that catalyzes the methylation of naringenin,
the final step leading to sakuranetin, and has not been isolated from rice yet. In order to identify the rice NOMT, we have
searched for candidate genes encoding NOMT on the base of sequence homology to HVOMT, which is a pathogen-induced flavonoid 7-O-methyltransferase from Hordeum vulgare. Eight ESTs were screened as the result, and 3 (AK069721,
AK069308 and AK072740) of them were cloned from UV-irradiated rice leaves. On the other hands, sakuranetin and
NOMT were found to be accumulated only in stressed leaves but not in healthy leaves. Therefore, it is highly possible
that the expression of NOMT mRNA is induced by stress. The microarray analysis using suspension-cultured rice cell
showed that the expression of AK069960 mRNA, which is a putative O-methyltransferase, was increased 27-fold after
treatment with chitin elicitor treatment for 8 hours when compared to the control (0 hour). The semi-quantitative RT-PCR
also revealed that the expression of AK069960 mRNA in rice leaves was greatly induced by copper chloride, jasmonic
acid and UV irradiation. Thus, all of the 4 cDNAs were considered as remarkable candidates for the NOMT gene. The
enzyme assay using the recombinant proteins as enzymes, 11 flavonoids including naringenin and 3 non-flavonoid compounds as substrates is under progress.
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PS0807 EFFECTS OF THE BILE ACID ELICITOR, CHOLIC ACID, ON THE BIOSYNTHESIS OF DITERPENOID PHYTOALEXINS IN SUSPENSION-CULTURED RICE CELLS
Shimizu, Takafumi1, Jikumaru, Yusuke1, Okada, Atsushi1, Okada, Kazunori1, Koga, Jinichiro2, Umemura, Kenji3,
Shibuya, Naoto4, Hasegawa, Morifumi5, Kodama, Osamu5, Nojiri, Hideaki1, Yamane, Hisakazu1 1 Biotechnology
Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. 2 Food and Health R&D
Laboratories, Meiji Seika Kaisha, Ltd., Sakado, Saitama 350-0289, Japan. 3 Agricultural and Veterinary Research Laboratories, Meiji Seika Kaisha, Ltd., Kohoku-ku,Yokohama 222-8567, Japan. 4 Department of Life Sciences, School of
Agriculture, Meiji University, Kawasaki, Kanagawa, 214-8571 Japan. 5 College of Agriculture, Ibaraki University, 3-211 Chuo, Ami, Ibaraki 300-0393, Japan
[email protected]
An elicitor of rice defense responses was recently isolated from human feces and was identified as cholic acid (CA).
Whereas pathogen infection in rice leaves induces phytocassanes and momilactones, both of which are major diterpenoid
phytoalexins in rice, CA mainly induces phytocassanes. We established a high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry protocol for the rapid and accurate quantification of phytocassanes and
momilactones. Using this method, we showed that CA also preferentially induced phytocassanes in suspension-cultured
rice cells while a fungal chitin elicitor induced both phytocassanes and momilactones. We further investigated the effects
of CA on the expression of diterpene cyclase genes involved in phytoalexin biosynthesis. CA induced the transcription
of the genes OsCyc2 and OsDTC1, which are involved in phytocassane biosynthesis, to a greater extent than the genes
OsCyc1 and OsKS4 that are involved in momilactone biosynthesis. OsCyc2 was particularly strongly induced, suggesting that it is one of the main mechanisms by which CA induces high levels of phytocassanes.
PS0808 INVOLVEMENT OF THE ELICITOR - INDUCED WRKY GENE OSWRKY53 IN DEFENSE RESPONSES IN RICE
Tetsuya Chujo1, Eiichi Minami2, Naoto Shibuya3, Hideo Nakashita4, Kazunori Okada1, Hideaki Nojiri1, Hisakazu
Yamane1 1Biotech. Res. Ctr., The Univ. of Tokyo, Tokyo, Japan; 2NIAS, Tsukuba, Japan; 3Dep. of Life Science, Meiji
Univ., Kawasaki, Japan; 4RIKEN, Wako, Japan
[email protected]
Plants recognize pathogens by detecting elicitors, such as chitin and sphingolipid elicitors, via specific receptors that
activate defense genes through intracellular signaling cascades. Transcriptional regulation of defense-gene expression is
central to induced disease resistance in higher plants. WRKY proteins form a large family of plant-specific transcription
factors that appear to play a regulatory role in a variety of stress responses. Using microarray analysis, we previously
identified OsWRKY53 as a chitin elicitor-induced gene from suspension-cultured rice cells. OsWRKY53 was induced
in suspension-cultured rice cells also by a sphingolipid elicitor or jasmonic acid, and in rice plants by infection with the
blast fungus Magnaporthe grisea. A fusion of OsWRKY53 with green fluorescent protein was detected exclusively in the
nuclei of onion epidermal cells, and OsWRKY53 protein specifically bound to W-box elements. A transient assay using
the particle bombardment method showed that OsWRKY53 is a transcriptional activator. A microarray analysis revealed
that several defense-related genes, including pathogenesis-related protein genes such as PBZ1, were upregulated in rice
cells overexpressing OsWRKY53. Finally, overexpression of OsWRKY53 in rice plants resulted in enhanced resistance
to M. grisea. These results strongly suggest that OsWRKY53 is a transcription factor that plays important roles in elicitor-induced defense signaling pathways in rice.
PS0809 FLAVONOIDS ARE SELECTIVELY TAKEN UP AND TRANSPORTED LONG DISTANCES IN ARABIDOPSIS
Charles S. Buer 1, Gloria K. Muday 2, Michael A. Djordjevic 1 1 Australian Research Council Centre of Excellence for
Integrative Legume Research, Research School of Biological Sciences, GPO Box 475, The Australian National University Canberra, ACT 2601, Australia; 2 Biology Department, Wake Forest University, Winston-Salem, NC 27109, USA.
[email protected]
Flavonoids are synthesised in response to developmental and environmental signals and have diverse functions. Darkgrown Arabidopsis thaliana roots have no flavonoid accumulation since flavonoid synthesis is light-dependent. However,
flavonoids were detected in wild-type root tips of light-grown seedlings with light-shielded roots, suggesting shoot-to-
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root movement. To further explore this observation, we used fluorescence microscopy to track flavonoid movement. This
was possible using a selective flavonoid stain and localised aglycone application to transparent testa4 A. thaliana mutants
which possess no flavonoids and no flavonoid-dependent fluorescence. The results showed that flavonoids entered tt4
plants and accumulated in tissues distal to the application site. This was confirmed by time-course fluorescence experiments and by HPLC analysis of tissues distal to application sites. Flavonoids applied to root tips showed basipetal movement in epidermal layers. Flavonoids applied mid-root showed unidirectional acropetal movement especially in vascular
tissue. Flavonoids applied at cotyledons moved towards the roots in vascular tissue and accumulated in root tips within
24 h. Naringenin, dihydrokaempferol, and dihydroquercetin were taken up at the root tip, mid-root, and cotyledons and
travelled via cell-to-cell movement (determined by confocal microscopy) to distal tissues followed by conversion to
quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at root tips and did not move to
shoots. Using ATP-binding cassette (ABC) transporter and H+-ATPase inhibitors showed that multidrug resistance-associated ABC proteins facilitated flavonoid movement away from the application site without pH dependency. The results
suggest that flavonoid uptake is selective and movement is an active process.
PS0810 AZOSPIRILLUM BRASILENSE PRODUCES ABA IN CHEMICALLY-DEFINED CULTURE MEDIA
Cohen, Ana; Bottini, Rubén; Piccoli Patricia Facultad de Ciencias Agrarias-CONICET, Universidad Nacional de Cuyo,
Alte. Brown 500, M5528AHB Chacras de Coria, Argentina; [email protected]
[email protected]
Azospirillum sp. are plant growth promoting bacteria (PGPB) that increase grain yield in cereals and other species
via growth promotion and/or stress alleviation. The PGPB beneficial effects have been partially attributed to bacterial
production of plant hormones, especially “growth promoters” like auxins, gibberellins and cytokinins. This work reports
the characterization of the stress-like plant hormone abscisic acid (ABA) by GC-EIMS in cultures of A. brasilense after
120 h of incubation in chemically-defined media. The addition of 100 mM NaCl in the culture media reduced bacterial
growth assessed as OD540 (total biomass) and CFU mL-1 (cells alive) by 1/3. However, under such stressful conditions
Azospirillum improved three-folds the ABA production (73 versus 235 ng.mL-1 of culture medium). Since saline soils
are one of the most problematic aspects in nowadays world agriculture, PGPB with improved capacity of ABA production may help in plant-stress alleviation.
PS0811 AZOSPIRILLUM BRASILENSE AND ABA IMPROVE GROWTH IN ARABIDOPSIS THALIANA
Cohen, Ana; Pontin, Mariela; Bottini, Rubén; Piccoli, Patricia. Facultad de Ciencias Agrarias-CONICET, Universidad
Nacional de Cuyo, Alte. Brown 500, M5528AHB Chacras de Coria, Argentina; [email protected]
[email protected]
Azospirillum sp. are plant growth promoting bacteria (PGPB) that increase grain yield in cereals and general plant
growth in different species. The beneficial effects of PGPB have been partially attributed to bacterial production of plant
hormones, especially “growth promoters” like auxins, gibberellins cytokinins and, more recently, ABA (see Cohen et al.
this meeting). In this work 7 days-old A. thaliana plants inoculated with 10 uL 106 CFU.mL-1 of A. brasilense or sprayed
with 100 uM ABA, both with and without 10 uM fluridone (inhibitor of carotene synthesis), improved root length. However ABA did not increased root hair density and emergence of lateral roots as Azospirillum did. Leaf area was increased
by two-fold in inoculated plants. Interestingly, inoculation with Azospirillum enhanced trichome development. These
preliminary results suggest that Azospirillum beneficial effects may be mediated by different phytohormones, including
ABA.
PS0812 A SIMPLE BENCH TOP METHOD FOR MEASURING NO PRODUCTION
Jan Vitecek1, Vilem Reinohl1, Russell Jones2 1Dept. of Plant Biology, Mendel University of Agriculture and Forestry,
Zemedelska 1, 613 00 Brno, Czech Republic; 2Dept. of Plant and Microbial Biology, 111 Koshland Hall, University of
California, Berkeley, CA 94720-3102 , USA
[email protected]
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A simple flow through detector capable of quantitating NO has been constructed using readily accessible chemicals
and equipment. This system may be also utilized for parallel determination of HNO2 emitted from plant tissue. The apparatus was connected to a computer-operated flow through system (Libourel et al., Planta, 223: 813-816, 2006) but it
also has been proven to work with a simple compressor. Using this setup the limit of detection for NO of 0.2 nmol was
obtained when gas was sampled for one hour at a flow rate of 40 ml min-1. The detector is selective for NO among several volatile nitrogen-containing compounds although a positive signal can be elicited by high levels (~ 1000 ppm) of
nitrous oxide. The functionality of the flow-through detector was verified by two well defined NO producing systems:
nitrate infiltrated leaves (Harper, Plant Physiol., 1488-1493, 1981; Klepper, Plant Physiol., 85: 96-99, 1987) and nitrite
treated extracellular medium of barley aleurone layers (Bethke et al., Plant Cell, 16: 332-341, 2004). The flow-through
detector was also used to monitoring NO emission from leaves of Arabidopsis mutants lacking or over expressing NOS.
Null mutations produced no measurable NO whereas over expressing plants produced twice the amount of NO than wild
type. This method was also used to detect NO production from suspension cultured tobacco treated with fungal elicitor
cryptogein where elevated production of NO was found after elicitor treatment. The data obtained with this simple apparatus confirm measurements made using the NO-sensitive fluorescent probe diaminofluorescein.
This work was supported by grants of the Czech Ministry of Education No. 1P05ME757 to VR and JV and from the US
National Science Foundation to RJ.
PS0813 SHI-GENES AND REGULATION OF AUXIN BIOSYNTHESIS IN THE MOSS PHYSCOMITRELLA PATENS
Magnus Eklund1, Mattias Thelander1, Veronika Ståldal1, Anders Nilsson2, Hans Ronne2 and Eva Sundberg1. 1Dept. of
Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, S-750 07 Uppsala, Sweden;
2Dept. of Medical Biochemistry and Microbiology, Uppsala University, Box 582, S-751 23 Uppsala, Sweden
[email protected]
The SHORT INTERNODES (SHI) gene family consists of nine transcribed members, including the STYLISH1
(STY1) and STY2 genes, in Arabidopsis thaliana. They are unique to plants and have highly redundant functions in the
development of the gynoecia, stamens and leaves, and severe developmental defects can be observed in multiple mutant
lines.
Using Microarray and qRT-PCR we have identified several putative direct targets of STY1 action, one of which is YUCCA4, a flavin monooxygenease, rate limiting it the biosynthesis of Indole-3 acetic acid.
In the moss Physcomitrella patens the orthologs of the Arabidopsis SHI genes are PpSHI1 and PpSHI2. They are highly
similar to each other with an 88% sequence identity at the nucleotide level and differ at the amino acid level only in regions outside of the conserved ZnFn and IGGH domains.
We have produced single knock-outs of PpSHI1 and PpSHI2, as well as lines over-expressing PpSHI1 (PpSHI1ox).
PpSHI1ox lines show an increased caulonemal radial growth and a reduction of chloronemal filaments, and also hyper
pigmentation in the older, central, protonemal tissue and in the older parts of the gametophores. The PpSHI1 and PpSHI2
knock-outs are indistinguishable and show a decreased radial filamentous growth and shorter gametophores having significantly larger leafs. The knock-outs also have more rhizoids.
Auxin has been suggested to induce rhizoid formation, to be responsible for gametophore elongation and for chloronema
to caulonema transition.
Future studies will focus on quantitative and qualitative measurements of auxin in knock-outs and constitutive expressorlines as well as identifying down stream targets of SHI/STY genes in moss.
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Floral Transition
PS0901 GIBBERELLIN 2-OXIDASES AND A 16,17-EPOXIDASE DEACTIVATE GIBBERELLINS INVOLVED IN FLOWERING OF LOLIUM.
King Rod1, Mander Lew2, Asp Torben3, Blundell Cherryl and Evans Lloyd1 1 CSIRO, Plant Industry, Canberra, Australia: 2 RSC, Australian National University, Canberra, Australia: 3 Dept. of Genetics and Biotechnology, University of
Aarhus, 4200 Slagelse, Denmark RWK Ph 61262465235 Fax 61262465000 e-mail [email protected]
rod.king @csiro.au
The gibberellins (GA) cause dramatic increases in plant height and earlier flowering. Mendelʼs findings on dwarfing
in peas highlighted a restriction on GA synthesis. However, dwarfing also results from enzymic inactivation of GAs.
We document in the grass species, Lolium: i) the pattern and timing of expression of GA catabolic genes during floral
induction; ii) effects of an inhibitor or of structural changes which protect GAs from catabolism and; iii) metabolism of
substrates of GAs by catabolic enzymes. Three catabolic genes, two 2â -oxidases and a C16-17 epoxidase, express highly
at or just below (2-3mm) the shoot apex. Together they block flowering by restricting GA access to the shoot apex but
have a lesser effect on stem elongation. Chemically protecting GA4 at the point of attack of 2â-oxidases increased its
florigenicity ~10,000-fold. Similarly, TNE (Trinexapac Ethyl), a 2-oxidase inhibitor, made GA4 florigenic as did addition of a 2á-hydroxyl to sterically hinder C2â hydroxylation. A role for C16-17 epoxidases was implied by the increased
florigenicity of GA4 if protected by conversion of a C16-17 double bond to single bond, or by adding a hydroxyl at the
nearby C13. Importantly, GA5 a highly florigenic GA, was not catabolised in vivo. Overall, our structural and molecular
studies indicate that catabolism of GAs is important for their florigenicity in Lolium. Adaptively, ability to restrict GA
access to the shoot apex may be important for survival of plants such as the grasses which are subject to heavy grazing.
PS0902 REGULATION OF PHOTOPERIODIC FLOWERING-RELATED GENE EXPRESSION BY DNA METHYLATION
Kondo, Hiroshi1, Okazaki, Keiichi2, Takeno, Kiyotoshi1 1Grad. Sch. Sci. Tech., Niigata
Univ.; 2Fac. Agr., Niigata Univ., Ikarashi 2, Niigata 950-2181, Japan
[email protected]
We found that the treatment with a DNA demethylating reagent, 5-azacytidine (azaC) induced flowering in Perilla
frutescens, suggesting that the flowering of P. frutescens was epigenetically regulated. The progeny of the plants flowered
by azaC treatment did not flower under non-inductive photoperiod. The flowering-related genes activated by the azaC
treatment may be deactivated by remethylation during the generation change. Thus, we hypothesized that the expression
of flowering-related genes is regulated by DNA methylation. To examine this idea, we compared the genomic DNA extracted from photoperiodically induced P. frutescens and that from non-induced control plants by MS-AFLP technique,
and detected some fragments specific to the DNA sample of the photo-induced plants. This suggests that the inductive
photoperiod changed DNA methylation, supporting the above hypothesis.
PS0903 INVOLVEMENT OF PHENYLALANINE AMMONIA-LYASE IN THE STRESS-RESPONDING FLOWERING.
Wada, Kaede C., Takeno, Kiyotoshi Grad. Sch. Sci. Tech., Niigata Univ., Niigata 950-2181, Japan
[email protected]
Pharbitis nil, a short-day plant (SDP), can be induced to flower by responding to poor nutrition stress under long days
(LD). This flowering was inhibited by a phenylalanine ammonia-lyase (PAL) inhibitor, aminooxyacetic acid, and this
inhibition was overcome by salicylic acid (SA). Exogenous SA alone did not induce to flower under non-stress condition
indicating that some factors other than SA must be also necessary to induce flowering. We also found that Perilla frutescens, a SDP, can be induced to flower by low-light intensity stress under LD. This flowering was also inhibited by a PAL
inhibitor, aminooxy-phenylpropionic acid. Thus, the metabolic pathway regulated by PAL, possibly that related to SA,
may be involved in the regulation of stress-responding flowering in the both species.
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PS0904 GIBBERELLINS AND FLOWERING IN TOBACCO PLANTS
Gallego-Giraldo Lina1, García-Martínez Jose-Luis 1 Moritz Thomas2 and López-Díaz Isabel1
[email protected]
Flowering of day neutral Nicotiana tabacum cv Xhanti depends on gibberellins (GAs) because GA deficient plants,
due to over-expression of a GA 2-oxidase gene (35S:NoGA2ox3; Ubeda-Tomás et al., 2006) or to treatment with the GA
biosynthesis inhibitor Paclobutrazol, flowered later than wild type. Active GA1 and GA4 contents in apical shoots of tobacco decreased progressively during vegetative growth, reaching very low levels at floral transition and beyond, correlating with the pattern of stem elongation but not with flowering promotion. Moreover, increased GA4 level as a result of
over-expression of a GA 20-oxidase gene (35S:CcGA20ox1; Vidal et al.2001), which promoted shoot elongation, did not
alter flowering, suggesting that this GA is not involved in tobacco flowering. In contrast, application of GA3 or silencing
of GA 2-oxidase genes (using a “hairpin” RNAi construct designed to silence the expression of several GA2ox, which
should increase endogenous GAs) resulted in flowering delay. Tobacco lines with silenced GA2ox displayed a range of
phenotypes including, in addition to flowering delay, increased height and developmental alteration of reproductive organs. (Ubeda-Tomás et al., 2006, J Plant Growth Regul. 25:52–68; Vidal et al. 2001,Physiol. Plant. 112(2):251-260)
PS0905 DEVELOPMENTAL GENETICS OF INFLORESCENCE ARCHITECTURE IN PEA
Maki, Sonja, Mullen, Heidi, and Singer, Susan Biology Department, Carleton College, 1 North College Sreet, Northfiel,
MN 55057, USA
[email protected]
Inflorescence development is intriguing from both agricultural and evolutionary perspectives, affecting reproductive
yield in natural and agricultural settings. Pea has a compound raceme with flowers formed by third order rather than
second order meristems. Indeterminate first order meristems (I1) can be distinguished from second order meristems (I2)
which terminate in a hairy stub and have suppressed bracts rather than the compound leaves produced by I1 meristems.
We are investigating both determinate and indeterminate lines which produce an increased number of flowers due to either extended I2 activity or increased flowers at each node of an I2 as well as crosses between the two multiflowering typologies. One plant resulting from these crosses produced both an inflorescence (I2) and an axillary branch at each node
beginning at the node of flower initiation. In addition, relative quantification of PsAP1, PsCEN, and PsLFY transcript
levels is being used to test and modify our model for the regulation of pattern in pea inflorescences.
Root Development
PS1001 THE ROLE OF PHYTOHORMONES AND GENOTYPE IN ROOT REGENERATION FROM CALLUS OF CACTACEAE WITH DETERMINATE PRIMARY ROOT
Shishkova, Svetlana 1, Castillo-Díaz 1, Vicente 1, Moreno, Norma E. 1, Arellano, Jesús 2 and Dubrovsky, Joseph G. 1 1
Dep. Biol. Mol de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3,
Cuernavaca, Mor., CP 62250, México; 2 Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México,
Cuernavaca, Mor., CP 62250, México; [email protected]
[email protected]
In some Sonoran Desert Cactaceae, the primary root has a determinate root growth: cells of root apical meristem
undergo only a few cell division cycles and then differentiate. To evaluate a possibility for quick analysis of the role of
certain Cactaceae genes in the determinate pattern of root growth, we have established a protocol of root regeneration
from calli of two species with determinate primary root, Stenocereus gummosus and Ferocactus peninsulae. The auxin
1-Naphthaleneacetic acid (NAA) and the cytokinin 6-Benzylaminopurine (BA) were successfully used for callus induction and root regeneration. Both plant genotype and concentrations of phytohormones in culture media seemed to be important for the callus induction: more efficient callus induction on hypocotyl and cotyledon explants was found on media
with higher concentrations of NAA and BA, although high plant-to-plant variation was observed.
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Moreover, when in callus induction media the auxin-to-cytokinin ratio was equal to 1, 0.2 or 5, no differences in the
efficiency of subsequent root regeneration from callus (one-way ANOVA) were found. Media with high concentrations
of NAA and without or with very low concentration of BA were used for root regeneration, and the greater effect of
genotype than that of (a) auxin concentrations, (b) BA presence in the root regeneration media, and (c) the explant type
was found. Regenerated roots had a determinate growth pattern similar to that of primary roots of these species. As all
primary and regenerated roots of the studied Cactaceae are determinate, they represent an excellent model system for the
investigation of the mechanisms of root meristem exhaustion and maintenance.
We thank S. Napsucialy-Mendivil for excellent technical help and DGAPA-PAPIIT, UNAM (project IN227206) for
financial support.
PS1002 RESPONSES OF TRANSFORMED CATHARANTHUS ROSEUS ROOTS TO FEMTOMOLAR CONCENTRATIONS OF SALICYLIC ACID
Echevarría-Machado, Ileana1, Escobedo -G. M. Rosa Maria1, Larqué-Saavedra2, Alfonso. 1UBBMP. 2URN. Centro de
Investigación Científica de Yucatán, A. C. Calle 43 No 130 Col. Chuburná de Hidalgo, CP 97200, Mérida, Yuc. México.
[email protected]., Phone: (999) 9 813914, Fax: (999) 9 81 39 00
[email protected]
RESPONSES OF TRANSFORMED Catharanthus roseus ROOTS TO FEMTOMOLAR CONCENTRATIONS OF
SALICYLIC ACID. Echevarría-Machado, Ileana1, Escobedo -G. M. Rosa Maria1, Larqué-Saavedra2, Alfonso.
1UBBMP. 2URN. Centro de Investigación Científica de Yucatán, A. C. Calle 43 No 130 Col. Chuburná de Hidalgo, CP
97200, Mérida, Yuc. México. [email protected]., Phone: (999) 9 813914, Fax: (999) 9 81 39 00.
C. roseus transformed roots were cultured in the presence of salicylic acid (SA) at concentrations between 0.1 fM and
100 pM and its effect on root growth was evaluated. At the second day of exposure to the SA treatment, significant
stimulation in the development of lateral roots was recorded at femtomolar concentration. This is one of the few reports
that demonstrate that plant tissue can respond to very low quantities of chemical response. SA increased the root cap size
and originated appearance of lateral roots closer to the root tip. The sensitivity of the bioassay used in the present work
allows testing low concentrations of other growth regulators that may have effect on the morphology and physiology of
the roots. We propose a tentative model for the regulation of root growth by SA.
PS1003 INTERACTIONS BETWEEN AUXIN, GIBBERELLIN AND DELLA PROTEINS REGULATE ROOT GROWTH IN
PEA
Weston, Diana E, Lester, Diane R, Elliott, Robert C, Reid, James B, Ross, John J School of Plant Science, University of
Tasmania, Private Bag 55, Hobart, Tasmania, 7001 Australia
[email protected]
Accumulating evidence suggests that interactions between auxin and gibberellin (GA) play an important role in plant
growth regulation. In pea internodes, auxin promotes the biosynthesis of the bioactive GA1 (Ross et al., 2000; Plant Journal 21: 547-552). However, in Arabidopsis roots, auxin is reported to enhance GA signalling, by facilitating GA-induced
degradation of the growth-inhibitory DELLA proteins (Fu and Harberd, 2003; Nature 421: 740-743). DELLA proteins
are known to also promote the expression of GA synthesis genes, and we show here for the first time that this is true for
roots. If the Fu and Harberd theory is the dominant interaction in pea roots, the application of an auxin action inhibitor
should increase the level of DELLA proteins, thereby promoting GA synthesis. However, this result was not found: the
auxin action inhibitor p-chlorophenoxyisobutyric acid (PCIB) inhibited GA biosynthesis, as indicated by GA quantification by GC-SIM and by monitoring GA synthesis gene expression by Real-Time PCR. PCIB also inhibited primary root
elongation. This indicates that in pea roots, as in pea shoots, the main auxin/gibberellin interaction is a promotion of GA
biosynthesis by auxin, a process that appears to be required for normal root growth.
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PS1004 THE ROLE OF AUXIN IN ROOT-HAIR DEVELOPMENT
Jones, Angharad 1, Kramer, Eric 2, Swarup, Ranjan 3, Bennett, Malcolm 3, Leyser, Ottoline 4, Lazarus, Colin 1, Grierson, Claire 1 1Schl. Biol. Sci., University of Bristol, Bristol, BS8 1UG, UK; 2Phys. Dept., Simonʼs Rock College, 84
Alford Road, Great Barrington, MA 01230, USA; 3Schl. Biosci., University of Nottingham, Nottingam, LE12 5RD, UK;
4Dept. Biol., University of York, York, YO10 5YW, UK.
[email protected]
In Arabidopsis thaliana the production of hair and non-hair cells within the epidermal layer of the root is an accessible
example of position-dependent differentiation. Hair cells are found overlying the anticlinal junctions between adjacent
cortical cells, whilst non-hair cells are found in positions allowing contact with single cortical cells. In addition, the
distance of the cell from the root tip may control its progression through the various stages of differentiation. The major
determinant of the fate of epidermal cells upon leaving the meristem is the result of the activity of a network of mobile
transcription factors. However, the involvement of auxin in the processes of root-hair initiation and growth suggests that
this hormone contributes to the final pattern observed. Auxin is actively transported into and out of individual plant cells,
and the tissue wide co-ordination of such cellular transport affords the possibility of its dynamic distribution within the
plant. The creation of spatial and temporal highs and lows of auxin concentration in this way is known to provide essential developmental co-ordinates. We are studying the expression of auxin influx- and efflux carriers in the root epidermis,
and have observed that the auxin influx carrier AUXIN RESISTANT 1 (AUX1) is detectable only in non-hair cells. We
are investigating how this pattern is affected in plants with mutant root hairs, as well as in plants subjected to treatments
(such as altered ethylene levels) that are known to affect the normal patterning and growth of hair- and non-hair cells. Our
hope is to understand the importance of the delivery of an auxin signal in establishing wild-type root-hair growth.
PS1005 THE ROLE OF CYTOKININ IN REGULATION OF EARLY ROOT DEVELOPMENT AND ORGANOGENESIS IN
ARABIDOPSIS
Alena Kuderova1, Ivana Urbankova1, Martina Valkova1, Jiri Malbeck2, Danka Nemethova3, 4 and Jan Hejatko1 1Dept.
of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37, Brno, Czech
Republic. 2Institute of Experimental Botany, The Academy of Sciences of the Czech Republic, Rozvojová 135, 165 02
Prague, Czech Republic. 3Institute of Biostatistics and Analyses, Masaryk University, Kamenice 126/3, 625 00 Brno,
Czech Republic. 4Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice
126/3, 625 00 Brno, Czech Republic.
[email protected]
Cytokinin (CK) has been known to inhibit primary root elongation and suggested to act as an auxin antagonist in the
regulation of lateral root (LR) formation. While auxin role in root development has been thoroughly studied, the detailed
and overall description of CK effects on root system morphology, particularly that of developing lateral root primordia
(LRPs), and hence its role in organogenesis has been missing. Here we examine the effects of conditional CK overproduction on root architecture during the early development of Arabidopsis thaliana. We employed the pOp/LhGR system
to induce ectopic IPT overexpression at designated developmental points. CaMV 35S>GR>ipt transactivation greatly
enhanced levels of biologically active CKs of zeatin type and identified a distinct developmental interval during which
primary root elongation is susceptible to increases in endogenous CK production. CK overproduction inhibited primary
root elongation by reducing quantitative parameters of primary root meristems, disturbed a characteristic graded distribution pattern of auxin response in LRPs and impaired their development. Our findings suggest a negative role of CKs
in the regulation of asymmetric auxin distribution during LRP development and imply that there is crosstalk between
the auxin and cytokinin pathways during de novo organogenesis in A. thaliana. Supported by the Ministry of Education,
Youth and Sports of the Czech Republic, LC06034 and MSM0021622415.
PS1006 SEQUENTIAL ADDITIONS OF PLANT GROWTH REGULATORS IN THE FORCING SOLUTION ENHANCED
PROPAGATION OF WOODY PLANTS
Yang, Guochen1 and Read, Paul E.2 1Department of Natural Resources & Environmental Design, North Carolina A&T
State University, Greensboro, NC 27411; 2Department of Agronomy and Horticulture, University of Nebraska, Lincoln,
NE 68583-0724.
[email protected]
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The research goal was to expedite propagation of woody plants, such as privet. Either IAA or NAA included in the
forcing solution enhanced rooting of forced privet softwood cuttings by increasing number of roots per cutting and root
length. A model forcing solution system was established by sequentially including plant growth regulators (PGR) in the
forcing solution for propagation of woody plants as follows: GA3 was initially included in the basic solution for 1 day to
enhance bud break and shoot elongation, then GA3 was replaced by IBA or other auxins to fresh aliquots of basic forcing
solution to “prime” softwood cuttings for rooting. Addition of IBA to fresh solution following the initial use of GA3 in
the solution counteracted the undesirable effects of GA3 on rooting and stimulated rooting after the favorable effects of
GA3 on bud break and shoot elongation.
PS1007 THE ARABIDOPSIS BEATNIK MUTATION ENHANCES AUXIN GRADIENT-DIRECTED PLANAR POLARITY
OF ROOT HAIR POSITIONING
Ikeda, Yoshihisa, Ljung, Karin, Grebe, Markus Umea_‹ Plant Science Centre, Department of Forest Genetics and Plant
Physiology, Swedish University of Agricultural Sciences 90183 Umea_‹, Sweden E-mail: [email protected].
se.
[email protected]
The polarity of single cells is commonly coordinated within the plane of a single tissue layer. In animals, this phenomenon is referred to as planar polarity and has been highly successfully investigated, for example, during Drosophila
wing hair positioning. We study the plant-specific planar polarity of root hair positioning in the Arabidopsis root epidermis where hairs are strictly formed close to the basal ends of hair-forming cells [1-3]. Hair position along the trichoblast
is directed toward the maximum concentration of an auxin gradient in the root tip [2, 4]. We recently demonstrated
that shaping this gradient requires combined function of the auxin influx carrier AUX1, ETHYLENE INSENSITIVE 2
(EIN2) , and GNOM genes which converges prior to polar Rho-of-plant (ROP) protein recruitment to the hair initiation
site. Moreover, local auxin gradients can direct coordinated planar root hair positioning [2, 5]. In genetic screens for
mutants with altered root hair placement, we have identified the recessive beatnik (btk) mutation. btk causes root-specific
phenotypes, including long hair and hyperpolarized hair initiation at basal-most ends of trichoblasts. We show that in btk,
F-actin prematurely accumulates in the hair bulge and ROPs localizes to the hair initiation site at basal-most ends of cells
with enhanced polarity early during cell elongation. The btk phenotype can be partially to fully suppressed in the act2,
gnom eb, aux1, and ein2 mutant backgrounds, indicating that BTK acts as a repressor of coordinated polarity upstream
of these genes. Intriguingly, the auxin concentration gradient and auxin biosynthesis rates are clearly enhanced in btk
mutant root tips, suggesting that BTK acts on planar polarity as a repressor of auxin biosynthesis and gradient function.
We further report on the molecular identity of btk, expression of the protein, and progress with btk loss-of-function mosaics to study its local effect on planar polarity.
1. Masucci and Schiefelbein, 1994. Plant Physiol. 106, 1335-1346.
2. Grebe M. et al. 2002. Curr. Biol. 12, 329-334.
3. Grebe 2004. Bioessays 26, 719-729.
4. Sabatini et al. 1999. Cell 99, 463-472.
5. Fischer et al. 2006. Curr. Biol. 16, 2143-2149.
PS1008 THE ROLE OF NITRIC OXIDE ON ROOT FORMATION INDUCED BY IBA IN MALUS HUPEHENSIS RHED.
Huajun Gao, Hongqiang Yang*, and Wei Zhang (College of Horticulture Science and Engineering, Shandong Agricultural University, Taian, Shandong 271018, China) *Author for Correspondence: Hongqiang Yang, E-mail: labft@sdau.
edu.cn
[email protected]
As an important regulator of plant growth, nitric oxide is involved in root development in plants. As lateral root primordia (LRP) increased significantly after 96 h of 100 µmol/L IBA treatment in Malus hupehensis Rhed. seedling, effect
of IBA on root nitric oxide (NO) concentration as well as exogenous NO on root architecture were further investigated. A
transient increase of endogenous NO in roots was detected in early (15~30 min) and late (48~96 h) stages after 100 µmol/
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L IBA treatment. However, no increase was observed when 100 µmol/L Quercetin (a protein kinase inhibitor) was also
presented in the solution, suggesting that NO acts as an important messenger in IBA-induced rooting process and protein
kinase is upstream of its generation. The transient increase of endogenous NO in early and late stages of IBA treatment
is very likely to be associated with the initiation and emergence of LRP, respectively. Exogenous NO affects root formation in a dose-dependent manner: SNP concentrations of 10 µmol/L and 50 µmol/L are most effective in increasing root
length and root number, respectively.
PS1009 SALICYLIC ACID SPRAYED TO SHOOTS OF TOMATO PLANTS AFFECTS ROOT GROWTH
R. Martín-Mex1, M. Gutiérrez-Rendón2, J. Barroso-Castro2, A. Nexticapan-Garcés1 and A. Larqué-Saavedra1 1Centro de Investigación Científica de Yucatán. A.C. Calle 43 No. 130. Chuburná de Hidalgo. C.P. 97200. Mérida, Yucatán,
México., 2Instituto Tecnológico de Conkal. Km 16.3 Ant. Carr Mérida,-Motul, Conkal, Yucatán, México.
[email protected]
Shoots of nine days olds tomato seedlings (Lycopersicon esculentum) were sprayed twice with aqueous solutions of
salicylic acid (SA) at different concentrations. Six SA concentrations were tested and compared with a water control. The
seedlings were cultivated in standard trays filled with compost in greenhouse conditions, until harvest, when the soil of
the roots was removed with water. Seven days after the last SA treatment, the plants were harvested and the fresh weight
(FW) of the root was measured. Results showed that a significant increase effect on the growth of the roots and that the
pattern of the effect gave a regression coefficient of 0.809. Thereafter three of the treatments (1.0; 0.01 and 0.0001 µM)
were selected for further measurements of the biomass of the plants. It was recorded that all the concentrations tested
affected the root area, root perimeter and the total root length. Similarly was recorded increase in the values of shoot
fresh and dry weight, leaf area and shoot length. The data are consistent with previous work published from these laboratories.
PS1010 MOLECULAR ANALYSIS OF ROOT INDUCTION IN MODEL LEGUME MEDICAGO TRUNCATULA
Imin, Nijat 1, Holmes, Peta 1, Dawson, Chris 1, Nizamidin, Mahira 1, Goffard, Nicolas 1, Rose, Ray 2, Rolfe, Barry
1 ARC Centre of Excellence for Integrated Legume Research. 1Genomic Interactions Group, Research School of Biological Sciences, Australian National University, Australia, 2School of Environmental and Life Sciences, University of
Newcastle, Australia
[email protected]
How plant cells committed to become roots? In model legume Medicago truncatula . we have the perfect system to
answer this question. Leaf explants supplied exogenously with the synthetic auxin will produce roots under controlled
tissue culture conditions. We have used transcriptomics (Affymetrix GeneChip) and proteomics (2-dimensional gel electrophoresis and mass spectrometry), real-time RT-PCR, promoter reporter analysis and other biochemical approaches
to study global patterns of gene expression during the processes in-vitro root induction. We compared the gene expression of root meristemic tissues such as root tips and week-old root forming tissue cultures, with control tissues such as
leaves, non-root forming tissue cultures and non-meristemic roots to find a group of genes that are closely linked with the
establishment of the root meristem. We have identified hundreds of differentially expressed genes/proteins involved in
transcription regulation, signal transduction, cell proliferation, stress and defence. Some of these genes have previously
reported while many others indicate novel roles in root meristem formation.
We have chosen eight transcription factors for functional analysis. These transcription factors include two auxin-responsive IAA genes, a LATERAL ORGAN BOUNDARIES gene, an unknown helix-loop-helix DNA binding protein,
a GROWTH-REGULATING FACTOR 1 and two AP2 domain containing transcription factors (BABY BOOM and
PLETHORA2). We are examining endogenous expression of these genes using a promoter-GFP fusion and by creating
plants with knockdown phenotypes using Agrobacterium rhizogenes induced hairy-roots. The role of these transcription
factors in the initiation of root nodules are also examined which provided clues about the evolutionary link between root
formation and nodulation. We also have shown that phytohormones such as auxin, cytokinin, ethylene and glutathione
play important roles in the determination of root stem cell niche. We also have identified several MicroRNAs that plays
key role in root formation. The results showed stem cell niche formation during root organogenesis is a complex process
that involves hormone signalling, specific gene regulation, redox homeostasis and stress response.
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PS1011 CHARACTERISATION OF SUPERROOT2-1 SUPPRESSORS
Gutierrez, Laurent 1, Bussell, John 1, Schwambach, Joséli 1, Kowalczyk, Mariusz 1, Castelain, Mathieu 1, Pons, Sébastien 1, Bellini, Catherine 1,2 1 Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish
University of Agricultural Sciences (SLU), 901 83 Umeå, Sweden; 2 Laboratoire de Biologie Cellulaire, Institut National
de la Recherche Agronomique, 78026 Versailles Cedex, France
[email protected]
Adventitious root formation allows clonal propagation and rapid fixation of superior genotypes prior to their introduction into production or breeding programs. This strategy is often used for long-lived woody species. Nevertheless the inability to initiate adventitious roots remains an obstacle for elite genotypes of many crop and woody species and the regulatory mechanisms are still not well understood. The hormone auxin is one of the main endogenous factors controlling
adventitious root formation and the superroot (sur1 and 2) mutants over-produce auxin and spontaneously make adventitious roots. In an attempt to identify new genes involved in the regulation of adventitious rooting we performed a screen
for suppressors of the sur2 mutation. 1800 independent M2 families derived from sur2-1 homozygote seeds mutagenized
with EMS were screened for the suppression of the adventitious root phenotype. Twelve suppressors were confirmed in
the M3 progeny. Complementation tests indicate that they represent independent group of complementation. A mapping
population after crossing with atr4-1, a sur2 allele in Col-0 background, has been produced for 4 of these suppressors.
Their position on the chromosomes has been established and the map based cloning for 2 of the genes is in progress. The
free and conjugated IAA levels were quantified in entire seedlings grown in the light. In suppressors 420, 494 and 677
the free IAA level is similar as in sur2-1 but the content of conjugated auxin is decreased in 420 and 494. In suppressor
266 the total auxin content is back to WT level. These preliminary data show that these mutants will allow in the future
to identify new genes that not only regulate adventitious root formation but most likely also auxin homeostasis.
PS1012 CLONING OF RUM1, A GENE INVOLVED IN LATERAL AND SEMINAL ROOTS DEVELOPMENT IN MAIZE
Komatsu, Mai 1; Hochholdinger, Frank 2; Multani, Dilbag 3; Sakai, Hajime 1; Taramino, Graziana 1 1 DuPont Crop
Genetics, DuPont Experimental Station, Wilmington, DE 19880, USA; 2 Center for Plant Molecular Biology (ZMBP),
Dep. of General Genetics, University of Tuebingen, 72076 Tuebingen, Germany; 3 Pioneer Hi-Bred International, Inc.,
Johnston, IA 50131, USA
[email protected]
The maize root stock comprises a series of root types with distinct shares of contribution in the development of the
plant. The embryonic primary and seminal roots together with their post-embryonic lateral roots form the root stock
of seedlings during early development. The shoot-borne crown and brace roots and their extensive lateral root system
form the main root stock of the plant during the juvenile and mature stages. Among the root types, lateral roots provide
the major surface water and nutrient uptake, shaping the root system architecture according to the plant_fs efficiency in
explore soil resources.
A forward genetics approach is being taken to identify genes involved in the molecular pathways that drive maize root
development. Several monogenic root mutants have been selected for map-based cloning.
Among them, the recently characterized rum1 mutant is defective in the initiation of lateral roots from the primary root,
and in the initiation of seminal roots. Furthermore, rum1 shows reduced auxin transport in the primary root, reduced primary root elongation, delayed gravitropic responses and no lateral root induction by exogenous auxin application. Here
we present results obtained so far in the map-based cloning of the RUM1 gene and the determination of its function in
maize root development.
PS1013 IDENTIFICATION OF NOVEL AUXIN RESPONSES IN THE PLANT EMBRYO
Eike H. Rademacher1, Annemarie Lokerse1, Alexandra Schlereth2, Gerd Jürgens2 & Dolf Weijers1 1Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands; 2Center for Plant Molecular
Biology (ZMBP), Tuebingen University, Auf der Morgenstelle 3, 72076 Tuebingen, Germany.
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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Auxin controls numerous developmental processes in plants by regulating gene expression. These transcriptional
responses are mediated by ARF transcription factors and their Aux/IAA inhibitors. In the embryo, MP/ARF5 and BDL/
IAA12 control root meristem initiation by specifying an extraembryonic suspensor cell as hypophysis, the root meristem precursor. However they do so in a non-cell autonomous way in part by promoting auxin transport to the adjacent
cell. The auxin response machinery within this cell is not known, and we have undertaken a reverse genetic approach to
identify ARFs and Aux/IAAs involved. During these investigations we have found that in addition to hypophysis specification, ARF-mediated auxin response is also critical for maintenance of suspensor cell fate. We have identified strong
candidates for the ARFs and Aux/IAAs involved in both hypophysis specification and suspensor development and will
present our latest results on their characterization.
PS1014 ARABIDOPSIS THALIANA MUTANT WITH ALTERATIONS IN LATERAL ROOT DEVELOPMENT AND DETERMINATE ROOT GROWTH
Hernández-Barrera A., Shishkova S., Lira Ruan V., Dong G., Dubrovsky J. Dep. Biol. Mol. de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Mor., 62250, México.
[email protected]
Root system supports the plant, synthesizes hormones like auxins (1) and cytokinins (2), acquires water and minerals and is the site of interaction with soil and microorganisms. Root architecture is determined by the quantity and
distribution of lateral roots, among other factors. To further reveal the molecular basis of lateral root development, a
new Arabidopsis recessive mutant preliminarily called sr “short roots with determinate growth” was isolated (through
the screening of EMS-mutagenized seeds) and studied. The sr mutant develops very short primary root and short lateral
roots. Primary roots of 3-d old seedlings show disorganized root apical meristem. 13 days after germination, the primary
root meristem is exhausted and no cell proliferation in the meristem is taking place. Pericycle cells adjacent to the protoxylem are shorter than in the wt. Lateral root initiation and primordium morphogenesis are severely affected and the
data will be presented in details. Auxin application did not rescue this phenotype. It promoted lateral root initiation and
induced formation of a multi-layered xylem–adjacent pericycle. This suggests that the mutant responds to auxin. Data
about sr mutant responses to other exogenous hormones will be presented. The SR locus was mapped to the lower arm
of chromosome 4. We thank S. Napsucialy-Mendivil for excellent technical help and DGAPA-PAPIIT, UNAM (project
IN225906) for financial support.
(1) Ljung et al., 2005. Plant Cell 17: 1090–1104.
(2) Kieber, J.J. 2002. Cytokinins. The Arabidopsis book. Rockville, M.D: ASPB.
PS1015 ANALYZING THE ROLE OF NITRIC OXIDE IN ARABIDOPSIS THALIANA LATERAL ROOT INITIATION
Lira-Ruan, Verónica1,2; Dubrovsky, Joseph G. 1. 1 Dep. Biol. Mol. de Plantas, Instituto de Biotecnología, Universidad
Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Mor., 62250 México. 2Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Av. Universidad 2001, Col Chamilpa, Cuernavaca, Mor., 62250 México. Tel (777)
3297020 Fax (777) 3136600 [email protected]
[email protected]
During the last 15 years Nitric Oxide (NO) has been revealed as an important regulator of plant physiology and development. Different roles of NO in root development have been demonstrated, for example, NO increases maize root
cell elongation; in cucumber, NO promotes the adventitious root formation. In tomato seedlings, the addition of NO
decreases the primary root length and favors lateral root development, these responses are similar to auxin addition effect, suggesting that NO participates in the auxin mediated lateral root initiation process (1). Despite these important
results, no detailed research was done neither on the role of NO in Arabidopsis thaliana root development, nor in lateral
root initiation process. The latter issue is of our particular interest, because the mechanisms involved in the lateral root
initiation process are not fully understood, and the possible participation of NO in this process constitutes a new factor
to understand it. Thus in this work the analysis of Arabidopsis root response to NO, specifically the effects on lateral root
primordium initiation will be presented.
1 Correa-Aragunde N. et al. 2004. Planta 218:900-905.
We thank DGAPA-PAPIIT, UNAM (project IN225906) for financial support.
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PS1016 GENETIC ANALYSIS OF VASCULAR PATTERNING OF THE ARABIDOPSIS ROOT
Anne Honkanen 1, Satu Lehesranta 1, Jan Dettmer 1, Ove Lindgren 1, Annelie Carlsbecker 2 & Ykä Helariutta 1 1.
Institute of Biotechnology, Viikinkaari 1, FI-00014 University of Helsinki, Finland 2. Dept. of Physiological Botany,
Evolutionary Biology Centre, Uppsala University, Villav. 6, SE-752 36 Uppsala, Sweden
Anne.Honkanen@Helsinki.fi
Plant vasculature is of great importance for plant growth and development as it connects all parts of the plant and
allows the transport of water, nutrients, and signalling molecules. Currently, our knowledge regarding how the development of plant vasculature is regulated is relatively limited. The influence of various hormones have been emphasized by
several studies, but few regulatory factors have been identified, and only one, APL, has been shown to determine phloem
identity (Bonke et al., 2003, Nature 426:181-186). APL is a transcription factor necessary, but not sufficient for phloem
differentiation, which implies that it represents an intermediate hierarchical level in the transcriptional network determining phloem development. We aim to characterize this network in detail and, furthermore, to identify the targets of APL
using a functional genomics approach.
In order to to identify novel mutants defective in the phloem development, we have performed a genetic screen using
EMS-mutagenized plants expressing the AtSUC2::GFP phloem marker (Imlau A et al., 1999, Plant Cell 11:309-322).
This resulted in the identification of a set of novel mutants with patterning defects specific to the stele which were named
distorted root vascular pattern1-7 (dva1-7). These mutants all have short primary root, lack AtSUC2::GFP expression at
the root tip and are accompanied by delayed and distorted phloem development. Interestingly, in dva1 and dva2 mutants
xylem develop ectopically in the pericycle layer adjacent to xylem axis. Expression of APL and cytokinin signalling
inhibitor, AHP6 ( Mähönen et al. 2006, Science 311:94-98) is reduced. This suggests that dva1 and dva2 act upstream of
the phloem identity determining gene APL and they may interact with cytokinin signalling pathway.
Dva1 and dva2 are both necessary for normal development of vascular tissues in the root. Mapping and characterization
of these genes and their putative roles in vascular development will be discussed.
PS1017 THE ROOT PHENOTYPE OF ARABIDOPSIS THALIANA IS ALTERED AFTER FLAVONOID ACCUMULATION
IS INDUCED BY AN OLIGOGALACTURONIDE.
Hernandez-Mata, G., Richards, A., Beltran-Pena, E., Mellado-Rojas, M.E., Soriano-Bello, E. Instituto de Investigaciones
Quimico-Biologicas. Universidad Michoacana de San Nicolas de Hidalgo. Morelia, Mexico.
[email protected]
The oligogalacturonide (OGA10) has been reported as a stimulator of defense in legumes (Hernández Mata et al.,
2006). The treatment of Arabidopsis seedling with 10-8 M of OGA 10 caused inhibition of cellular elongation in the main
root, but conversely caused an increase in both lateral roots development, and root hairs elongation.
Root system development in Arabidopsis is strongly influenced by auxin metabolism, chiefly by the hormones distribution via polar transport.
Likewise, auxin transport is regulated negatively by the accumulation of flavonoids (Taylor and Grotewold, 2005; Bresseau et al., 2007). We tried to relate flavonoids production and the Arabidopsis response to the defense elicitor. We found
that 10-8 M concentration of this compound stimulates flavonoids accumulation, showing the implication of these metabolites in Arabidopsis root growth inhibition under OGA 10 treatment.
References
Hernández Mata G., Sepúlveda B., Richards A. and Soriano E. (2006). Braz. J. Plant Physiol., 18(2): 351-355.
Taylor L. P. and Grotewold E. (2005). Curr. Opin. Plant Biol. 8: 317-323.
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PS1018 ENDOGENOUS CYTOKININS AND MORPHOLOGICAL VARIATIONS DURING COMPETENCE ACQUISITION
FOR THE ROOT APICAL MERISTEM OF CATASETUM FIMBRIATUM CONVERSION INTO BUDS.
Rodrigues, Maria Aurineide1; Freschi, Luciano1; Ceccantini, Gregório Cardoso Tápias1; Kerbauy, Gilberto Barbante1.
1Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
[email protected]
The conversion of root apical meristems (RAMs) into buds is a rare process of organogenesis that occurs in root tips
isolated from the epiphytic orchid Catasetum fimbriatum. The root-to-shoot conversion is strongly promoted by cytokinins (Cks), and the competence acquisition for this event has been recently related to plant ageing. Based on this, the
aim of this study was to investigate the endogenous levels of four Cks (Z, ZR, iP and iPR) and the morphological variations in the RAM of C. fimbriatum during the competence acquisition for conversion into buds. These parameters were
analyzed during the ageing of RAMs still linked to the plants and, also, over the first 24 hours of RAM isolation. The
results showed a strict correlation among ageing, competence acquisition, decrease in the Ck pool, and alteration in the
RAM pattern. However, both young and old RAMs showed predominantly iP-type Cks, when still linked to the plant.
The isolation of RAMs for two hours was sufficient to cause a sharp decrease in the levels of both iP-type and Z-type Cks.
During the subsequent 10 hours of RAM isolation, the endogenous levels of iP-type Cks continued to decrease; however,
the opposite tendency was observed in the content of Z-type Cks, which started increasing since the forth hour of RAM
detachment. As a result, a clear reversion in the predominant type of Cks was observed during the following period of
RAM isolation, with Z-type being the dominant form of Ck. Simultaneously, a more advanced degree of alteration in
the original architecture of detached RAMs was observed. These results indicate that isolation of root tips from plants of
C. fimbriatum, when compared with the ageing process, intensifies the competence for root-to-shot conversion through
faster structural modifications in RAMs, sharper decrease in Ck content, and, possibly, through an initial predominance
of the Z-type Cks. Supported by FAPESP.
PS1019 INTERACTION OF ETHYLENE AND CYTOKININS ON COMPETENCE ACQUISITION FOR THE CONVERSION
OF ROOT APICAL MERISTEM OF CATASETUM FIMBRIATUM INTO BUDS
Rodrigues, Maria Aurineide1; Freschi, Luciano1; Kerbauy, Gilberto Barbante1. 1Departamento de Botânica, Instituto de
Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
[email protected]
Root apices of the epiphytic orchid Catasetum fimbriatum represent an interesting model to study the hormonal signs
that control root apical meristem (RAM) development, since this tissue loses its typical architecture and shows competence to convert into buds when the determinate root growth is reached. These changes in the RAM of C. fimbriatum
have been shown to be ageing-dependent. Actually, young roots do not display the aforementioned competence; instead,
they keep growing in length and maintain a closed RAM organization even when they are detached from the original
plants. In this study, the role and possible interaction of ethylene and cytokinins (Cks) during C. fimbriatum root growth
and RAM competence acquisition to convert into buds were investigated. The results showed a strong decrease in root
length of young plants treated during 30 days with 102, 103 and 104 ppm of ethylene, with 100% of RAMs converted
into buds. Treatments with 10 ìM of zeatin (Z), zeatin ribozide (ZR), isopentenyladenine (iP), and isopentenyladenosine
(iPR) caused a sharp decrease in root tip growth. However, only Z and ZR induced RAM competence for conversion into
buds, Z being the most effective between the two. In addition, the Ck endogenous content (Z+ZR+iP+iPR) was two times
higher in ethylene-treated-RAMs than in the control, and Z was found to be the Ck with the most increased levels. The
present results indicate that both root growth inhibition and competence acquisition of C. fimbriatum RAMs to convert
into buds are promoted by increased levels of ethylene and Z-type Cks, and the inductive effects of ethylene in root-toshoot conversion are mediated by an enhancement in Cks endogenous levels, especially Z, in the RAM. Supported by
FAPESP.
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PS1020 STUDY OF NITRATE EFFECT ON CAPSICUM CHINENSE JACQ. ROOT SYSTEM AND THE PUTATIVE HIGH
AFFINITY TRANSPORTER
Celis-Arámburo, Teresita, Murillo-Esquivel Kenny, Medina-Lara, Fátima, Martínez-Estévez, Manuel, Echevarría-Machado, Ileana. UBBMP. Centro de Investigación Científica de Yucatán, A. C. Calle 43 No 130 Col. Chuburná de Hidalgo,
CP 97200, Mérida, Yuc. México. [email protected]., Phone: (999) 9 813914, Fax: (999) 9 81 39 00.
[email protected]
Capsicum chinense jaq. grows in extremely adverse conditions in Yucatán soils (rocky and very low level of nutriments, specially in nitrogen and phosphorus). We have investigated how nitrate availability and distribution can affects
the root system of this plant. When habanero pepper roots were exposed to a local supply of nitrate, a dose-depend reduction in primary root growth was observed between the fourth and fifth day of treatment. An increase in lateral root
numbers within nitrate-rich zone was also observed. Lateral root elongation in this zone increased more than of 4 folds
in the presence of nitrate. We present the cDNA sequence of a putative high affinity nitrate transporter for C. chinense
which have a high homology with NRT2;1 and NRT2;2 of Lycopersicum esculentum and discussed their possible role
in this species.
Hormone Signaling
PS1101 ROLES OF PHOSPHOLIPASE D&ZETA2 IN STIMULATING AUXIN RESPONSES THROUGH REGULATING
VESICLE TRAFFICKING
Hong-Wei Xue, Gang Li National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, 200032 Shanghai,
P.R.China [email protected]
[email protected]
Phospholipase D (PLD) is crucial in numerous cellular processes including stress responses, hormone effects, membrane vesicle trafficking and cytoskeleton rearrangements. Genome analysis reveals that there are 12 members of PLD
presented in Arabidopsis, which are classified as different subfamilies and play multiple roles in plant growth and development, including root (hair) development, responses to phosphate starvation and so on. To further elucidate the physiological functions of PLD, an cDNA encoding Arabidopsis thaliana PLD&zeta2 was isolated and functionally characterized. PLD&zeta2 encodes a member of PXPH-PLD and its defective mutant, pld&zeta2, and transgenic plants deficient
of PLD&zeta2 are less sensitive to auxin, have reduced root gravitropism and suppressed auxin-dependent hypocotyl
elongation under high temperature. On the contrast, transgenic seedlings overexpressing PLD&zeta2 showed opposite
responses, indicating that PLD&zeta2 positively mediates auxin signaling. Further analysis and observation revealed the
suppressed vesicle trafficking under deficiency of PLD&zeta2 or by treatment with 1-butanol, a PLD specific inhibitor,
while enhanced vesicle trafficking was detected under treatment with PA or under PLD&zeta2 overexpression. Deficiency of PLD&zeta2 does not affect the localization of PIN proteins (auxin efflux carriers) but but blocked the inhibition
of brefeldin A on PINs cycling. These suggest that PLD&zeta2, through its product PA, stimulates auxin responses via
accelerating vesicle trafficking.
PS1102 IBR5, A PUTATIVE DUAL-SPECIFICITY PROTEIN PHOSPHATASE, AFFECTS AUXIN RESPONSE INDEPENDENTLY OF TIR1
Lucia C. Strader, Melanie Monroe-Augustus, Bonnie Bartel Department of Biochemistry & Cell Biology, Rice University, Houston, TX 77005, USA
[email protected]
The phytohormone auxin affects many aspects of plant growth and development, influencing cell division, elongation, and differentiation at the cellular level and affecting apical dominance, lateral root formation, and tropisms at the
whole-plant level. Mutations in IBR5 (INDOLE 3-BUTYRIC ACID RESPONSE5), encoding a putative dual-specificity
protein phosphatase, confer decreased sensitivity to auxins and auxinic compounds (Monroe-Augustus et al. , 2003, Plant
IPGSA 19th Annual Meeting - Abstracts
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Cell 15:2979). In an effort to understand how IBR5 modulates auxin signaling, we evaluated effects of the ibr5 mutation
on molecular auxin responses. Levels of the auxin-inducible transcripts IAA1 and IAA2 were reduced in ibr5 relative to
wild type, suggesting that IBR5 acts upstream of the transcriptional regulation of these genes. Repression of IAA1 and
IAA2 can be relieved by the TIR1-mediated 26S-proteasomal degradation of AUX/IAA repressor proteins. However, we
found that the AUX/IAA protein AXR3, which is stabilized in several other auxin response mutants, was not stabilized in
ibr5. This result suggests that IBR5 affects auxin-responsive transcription by a method other than promoting AUX/IAA
degradation. We used double mutant analysis to determine how other auxin response players genetically interact with
ibr5. The double mutants ibr5 tir1, ibr5 aux1, and ibr5 axr1 all exhibit additive auxin resistance, suggesting that IBR5 acts
in a separate pathway from TIR1, AUX1, or AXR1. Overall, our results suggest that IBR5 modulates auxin responsive
transcription by an unknown mechanism. We are conducting an ibr5 modifier screen to further elucidate this process.
(This research is supported by the NIH.)
PS1103 NOVEL HORMONE REGULATES CELL GROWTH AND PROLIFERATION IN MAIZE (ZEA MAYS L.). FEATURES OF THE SIGNALING PATHWAY
Rodríguez César, Agredano Lourdes, Sotelo Ricardo, Jiménez Sara, Aguilar Raúl and Sánchez de Jiménez Estela. Depto.
Bioquímica. Facultad de Química, UNAM. México, D. F. 04510 [email protected] Phone 56 22 52 78 Fax. 56
22 53 29
[email protected]
A family of hormones named IGFs (Insulin-like Growth Factors) regulate cell growth and/or proliferation in non-plant
eukaryotes. They stimulate a pathway that activates TOR (target of rapamycin) kinase, central controller that diverts the
signal to several cellular targets. A novel plant growth factor, isolated from maize tissues (ZmIGF), seems to regulate cell
growth through this pathway. The main objetive of this work is to further characterize this effector and the downstream
events it induces. ZmIGF was found to be a small highly hidrophobic peptide, that resembles insulin structure recognized
by insulin antibody.
It stimulates protein synthesis and ribosome biogenesis through ZmTOR (maize ortholog, kinase) activation. Either
embryonic axes or in vitro maize cell cultures stimulated by ZmIGF, showed increase on DNA synthesis and on proteins
related to the cell cycle. All this resulted on fast seedling growth and in vitro cell culture proliferation. A signal pathway
for ZmIGF is proposed.
Supported by DGAPA IN207903 and Conacyt 43371Q
PS1104 CONSTITUTIVE CKI1 ACTIVITY MODULATES CYTOKININ SIGNALING AND REGULATES VASCULAR TISSUE DEVELOPMENT IN ARABIDOPSIS
Jan Hejatko1,2,3, Hojin Ryu4, Gyung-Tae Kim5,6, Romana Dobesova1, Sang Mi Choi4, Premysl Soucek1,2, Petra
Borkovcova1,2, Klaus Palme3,7, Bretislav Brzobohaty1,2 and Ildoo Hwang 4 1Dept. of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Kotlarska 2, CZ-61137, Brno, Czech Republic. 2Institute of Biophysics
AS CR, Kralovopolska 135, CZ-61265, Brno, Czech Republic. 3Max-Delbrück_Laboratorium in der Max-Planck-Gesselschaft, Carl-von-Linne Weg 10, D-508 29, Köln, Germany. 4Department of Life Sciences and Functional Genomics
Center, Pohang University of Science and Technology, Pohang 790-784, Korea. 5Department of Molecular Biotechnology, Dong-A University, Busan 604-714, Korea. 6Environmental Biotechnology National Core Research Center,
Gyeongsang National University, Jinju 660-701, Korea. 7Institut für Biologie II, Universität Freiburg, Schänzlestr. 1,
D-79104 Freiburg, Germany.
[email protected]
Cytokinins play essential roles in cell division and vascular tissue development. While cytokinin signals are perceived
by the histidine kinase receptors AHK2, AHK3, and AHK4/CRE1/WOL, recent genetic analyses indicate the existence of
additional cytokinin signaling pathway in Arabidopsis. Here we show that histidine kinase CKI1 constitutively activates
the cytokinin signaling pathway. CKI1 induces expression of cytokinin-responsive type-A ARRs and ARR2 phosphory-
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lation in cytokinin-deficient and wild-type protoplasts and dominant negative CKI1 allele suppresses AHKʼs-dependent
activation of ARR6. Hormone-regulated CKI1 expression was found in vascular tissues, root tip and shoot apex. Ectopic
expression of CKI1 causes abnormal cell division and differentiation in the vascular bundles of inflorescence stems and
in axillary meristems and CKI1 RNA interference and dominant negative CKI1 plants reveal cytokinin-dependent aberrations in the inflorescence vascular tissue development. These results suggest that the hormone-dependent transcriptional regulation of constitutive CKI1 activity modulates cytokinin signaling and regulates vascular tissue development
in Arabidopsis. Supported by Plant Diversity Research Center of the MOST, R08-2003-000-10819-0 and the Ministry of
Education, Youth and Sports of the Czech Republic, LC06034, MSM0021622415, the BMBF, FCI and DAAD.
PS1105 MOLECULAR INTERACTIONS OF A SOLUBLE GIBBERELLIN RECEPTOR, GID1, WITH A RICE DELLA PROTEIN, SLR1, AND GIBBERELLIN
Ueguchi-Tanaka, Miyako 1, Nakajima, Masatoshi 2, Katoh, Etsuko 3, Ohmiya, Hiroko 1, Asano, Kenji 1, Saji, Shoko 3,
Hongyu, Xiang 3, Ashikari, Motoyuki 1, Kitano, Hidemi 1, Yamaguchi, Isomaro 2 and Matsuoka, Makoto 1 1Bioscience
and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan; 2Department of Applied Biological Chemistry,
The University of Tokyo, Tokyo 113-8657, Japan; 3Department of Biochemistry, National Institute of Agrobiological
Sciences, Tsukuba 305-8602, Japan
[email protected]
GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble GA receptor that shares sequence similarity
with a hormone sensitive lipase (HSL). Previously, a yeast two hybrid (Y2H) assay revealed that the GID1-GA complex
directly interacts with SLR1, a DELLA repressor protein in GA signaling. Here, we demonstrated, by a pull-down and
Bimolecular Fluorescence Complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also
occurs in planta. GA4 was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of
gibberellin in planta. Domain analysis of SLR1 by Y2H assay and gel filtration analysis revealed that the DELLA and
TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction.
To identify the important regions of GID1 for GA- and SLR1-interactions, we used many different mutant versions of
GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with
conserved amino acids replaced with alanine. The amino acid residues important for SLR1-interaction completely overlapped the residues required for GA-binding, that were scattered throughout the GID1 molecule. When we plotted these
residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions
corresponding to the substrate binding pocket and lid. Further, the GA-GID1 interaction was stabilized by SLR1. Based
on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.
PS1106 THE SLENDER PEA MUTANT RESULTS FROM MUTATIONS IN THE DELLA GENES LA AND CRY
Ross, John J1, Elliott, Robert C1, Rameau, Catherine2, Murfet, Ian C1, Reid, James B1 1School of Plant Science, University of Tasmania, Private Bag 55, Hobart, Tasmania, 7001, Australia; 2INRA, Institut Jean-Pierre Bourgin, Station
Genetique et dʼAmelioration des Plantes, 78026 Versailles Cedex, France
[email protected]
The growth-inhibitory DELLA proteins play a key role in gibberellin (GA) signal transduction. In pea, the classical
constitutive GA response mutant, “slender”, known since 1927, has played a pivotal role in thinking on GA action. Here
we show that the mutations responsible for the slender phenotype, la and crys, are mutations in DELLA-encoding genes.
Prior to our current knowledge of DELLA function, Brian (1957) and Potts et al (1985) postulated that GAs “inhibit an
inhibitor” of stem elongation, rather than positively promoting that process. We now know that GAs do indeed inhibit
(destabilise) DELLA proteins, which can be viewed as inhibitors of growth (Fu and Harberd 2003; Nature, 421:740-743).
The la and crys mutations result in proteins that are unable to inhibit growth, explaining why the slender mutant behaves
like a plant with a saturated GA response. Another mutation, cryc, retains some growth-inhibiting capacity, but in the homozygous state appears to interfere with the growth inhibition caused by LA, whereas crys does not. This indicates that
the LA and CRY DELLA proteins might compete for the same protein binding site. We will also discuss other important
implications of cloning DELLA-encoding genes from pea, including insights into the regulation of root elongation.
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PS1107 QUANTITATIVE AND CONTEXTUAL MODELLING OF TRANSCRIPTIONAL RESPONSES TO AUXIN
Kepinski, Stefan1 and King, John2 1Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK; 2Division of
Theoretical Mechanics, School of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK
[email protected]
A key question in plant biology is how auxin controls such a diversity of developmental events. Central to this control
is auxinʼs ability to prompt specific changes in programs of gene expression. Work in several labs has defined a system
of physically interacting components sufficient to provide the basic mechanistic link between auxin perception and transcriptional regulation. The system is essentially a simple derepression mechanism in which auxin causes the degradation of members of the Aux/IAA family of transcriptional repressors which then allows the expression of genes targeted
by certain members of the Auxin Response Factors (ARF) family of DNA-binding transcription factors. Although this
simplified qualitative model provides a conceptual framework for understanding how auxin can turn genes on and by
extension, turn others off, it is unclear how specific information is carried in the system and thus how auxin pulses of
differing length and amplitude can be translated into quantitatively different genomic outputs in various developmental
contexts. To address these questions, we are working to obtain quantitative genomic and biochemical data describing the
essential properties of the system, referenced to defined developmental contexts, to parameterize a mathematical model
and establish rules of auxin signalling that may be applied throughout development. Progress of both the experimental
and theoretical aspects of this work will be presented.
PS1108 EFFCT OF ELECTROPHILICITY OF ABSCISIC ACID ON THE BIOLOGICAL ACTIVITY
Hirai, Nobuhiro1, Iwami, Kumiko2, Horiuchi, Mari2, Kano, Kenji2, Todoroki, Yasushi3, and Ohigashi, Hajime2 1 IIC, c/
o Grad. Sch. Agric., Kyoto University, Kyoto 606-8502, Japan; 2 Grad. Sch. Agric., Kyoto University, Kyoto 606-8502,
Japan; 3 Fac. Agric., Shizuoka University, Shizuoka 422-8529, Japan; [email protected]
[email protected]
A plant hormone, abscisic acid (ABA) has electrophilicity derived from the conjugated dienecarboxyl group. This
electrophilicity may be important for the biological activity of ABA. We have precisely investigated on the electrochemical property of ABA, and identified new products from the cathodic reaction of ABA. Further, new ABA analogues with
the partially saturated side chain were synthesized using the cathodic reaction. The analogues showed low inhibitory
activity in rice seedling growth and lettuce seed germination tests. This finding suggested that the electrophilic side chain
of ABA was necessary for the biological activity.
PS1109 GAMYB PLAYS THE IMPORTANT ROLE IN POLLEN DEVELOPMENT AS A MEMBER OF GA SIGNALING
Aya, Koichiro1, Ueguchi-Tanaka, Miyako1, Kondo, Maki2, Tsuji, Hiroyuki1, Ashikari, Motoyuki1, Nishimura, Mikio2,
Matsuoka, Makoto1 1Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan; 2Department
of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
[email protected]
GAMYB is known as a positive transcriptional regulator of gibberellin (GA)-dependent ƒ¿-amylase expression in
cereal aleurone cells. However, the function of GAMYB in other tissues is still unknown. Recently, Tos-17 insertional
mutants for OsGAMYB were isolated. gamyb mutant showed the defective anther, suggesting that GAMYB functions
as a GA signaling factor in anther as well as in aleurone cells. We examined the anther development of other GA-related
mutants; GA deficient mutant, oscps and GA insensitive mutants, gid1 and gid2, and compared them with gamyb. All
these mutants showed white and contracted anthers, possessing swollen tapetum cells and destroyed pollens. We also
performed microarray analysis using anthers from these mutants. The results demonstrate that the genes under the control
of GAMYB are also regulated by CPS, GID1 and GID2. Taken together, GAMYB may play an important role in pollen
development as a transcription factor involved in GA signaling.
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PS1110 FINDING OF A NOVEL HORMONE-RELATED COMPOUND BY USING ATGENEXPRESS MICROARRAY
DATA.
Hideki Goda, Shigeo Yoshida, Kazuo Shinozaki,Yukihisa Shimada, Plant Science Center, RIKEN, Yokohama 230-0045,
Japan
[email protected]
AtGenExpress is a multinational project to collect and distribute transcriptome data of Arabidopsis thalinana. using
a highly standardized platform, Affymetrix GeneChip, ATH1, which covers most Arabidopsis genes in the genome.
The consortium collected data of developmental series, organ specificity series, light response series, stress response
series, pathogen response series, hormone response series, nutrition response series. We have identified plant hormone
inducible genes by using hormone response series microarray data. In order to find a novel hormone-related compound,
hormone-inducible-gene expressions were compared between plant hormone treatments and several chemical-compound
treatments. These comparison analyses found several novel compounds that correlates to phytohormone actions in gene
expression.
PS1111 HOW SYNTHESIS OF ABA IS INITIATED IN CELLS AND WHERE IT IS STORED
Milborrow, Barry School of Biotechnology and Biomolecular Sciences University of New South Wales Sydney Australia
[email protected]
(+)-Deuterio ABA from gel blocks passes down mung seedling segments into gel blocks. This ABA was entirely deuteriated; no endogenous, unlabelled ABA was detected. The material from “donor” blocks had passed through the cells but
did not mix with the endogenous ABA. Centrifugation of a Commelina leaf homogenate gives a pellet containing most
of the mesophyllʼs ABA. This ABA is released by hyperosmotic Sorbitol, by wilting the leaf beforehand, or by treatment
of the brei with proteinase K. Gel electrophosesis of the pellet gives a band containing ABA (extractable with ethanol or
acetone) closer to the origin than free [14C]ABA added to the gel. This band “Fettered ABA” disappeared after treatment
with proteinase K.
If wilting releases free ABA from the “fettered” fraction, it could be the cause of stomatal closure which has been found
to occur before leaf ABA increases. If ABA released into the cytosol “switched on” the synthesis of the 9-cis neoxanthin dioxygenase then this could account for the rise in stress ABA which occurs 2-3 hours after wilting. Hexadeuterio
(+)-ABA concentrations were vacuum infiltrated into sliced barley leaves and the amounts of unlabelled (+)-ABA and
hexadeuterio ABA were measured by GC/MS after 3 hours. The added ABA increased production of natural (+)-ABA
so it is suggested that wilting releases free ABA from the chloroplasts to initiate synthesis of the enzymes in the cytosol
which then biosynthesise “stress ABA” in the chloroplasts.
PS1112 ANALYSIS OF A PROTEIN KINASE REQUIRED FOR LATERAL ROOT INITIATION
Schwechheimer, Claus 1, Zourelidou, Melina 1, Müller, Isabel 1, Li, Hanbing 1, Maier, Andreas 1, Nill, Carola 1 1 Department of Developmental Genetics, Centre for Plant Molecular Biology, Tuebingen University, Auf der Morgenstelle
5, 72076 Tuebingen, Germany
[email protected]
Throughout plant development, the phytohormone auxin controls many differentiation and growth processes. Both,
proper auxin transport and auxin responses are required for the initiation of lateral root formation. We have characterized
a serine-/threonine protein kinase, which we designated D6 PROTEIN KINASE (D6PK). d6pk mutants are strongly impaired in the initiation of lateral roots, but growth of the primary root is not affected in these mutants. Conversely, overexpression of D6PK induces lateral root formation in the wild type. This indicates that D6PK is required and sufficient
for lateral root initiation. In d6pk mutants, we occassionally identify seedlings with a single or with fused cotyledons.
Both phenotypes, the lateral root phenotype as well as the cotyledon phenotype, are indicative for a role of D6PK in auxin
response or auxin transport. We examined auxin transport and response using the DR5:GUS reporter and by gene expression analysis. In the context of other data, we will discuss the role of D6PK in auxin transport and auxin response.
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PS1113 A NEW AMPEROMETRIC SENSOR FOR INDOLE-3-ACETIC ACID BASED ON PLATINUM NANOWIRES AND
CARBON NANOTUBES
Ruo-Zhong Wang 1, Lang-Tao Xiao 1, Ming-Hui Yang 2, Jun-Hui Ding 1, Yu-Wei Li 1, Feng-Li Qu 2, Guo-Li Shen 2 1
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural University, Changsha 410028 2 Chemistry and Chemical Engineering College, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082
[email protected]
According to the molecule structure of Indole-3-acetic acid (IAA), under proper conditions, the nitrogen atom at
the pyrrole ring can be oxidized. The electrochemical responses of indole-3-acetic acid were examined. As a result, we
developed a novel amperometric sensor for the determination of IAA based on multiwall carbon nanotubes film coated
glassy carbon (GC) electrode. Platinum nanowire (PtNW) can be grown by electrodeposition in polycarbonate membrane, with the average diameter of the nanowires about 250 nm. The PtNW and multiwalled carbon nanotubes (CNT)
are then dispersed into chitosan (CHIT) solution. The resulting PtNW-CNT-CHIT material brings new capabilities for
electrochemical devices by using the synergistic action of the electrocatalytic activity of PtNW and CNT. By dropping
the PtNW-CNT-CHIT film onto the glassy carbon electrode surface, and after evaporation an amperometric sensor for
the determination of indole-3-acetic acid (IAA) was developed.
The oxidation current of IAA increases significantly at the PtNW-CNT-CHIT film coated GC electrode, in contrast to
that at the CNT-CHIT modified GC, and a potential of 0.9V was selected for amperometric determination of IAA. The
electrochemical behaviors of IAA at the PtNW-CNT-CHIT film coated GC electrode have been examined by cyclic
voltammetry. The oxidation peak current of the sensor is linearly from 50 ng/ml to 50 ƒ_g/ml with a detection limit of
25 ng/mL.
PS1114 A DIRECT IMMUNOSENSOR FOR ABSCISIC ACID BASED ON ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY
Hui Chu, Langtao Xiao, Yuwei Li, Ruozhong Wang, Wei Li, Qian Li Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural University, Changsha 410028
[email protected]
The phytohormone abscisic acid (ABA) is the major player in mediating the adaptation of the plant to stresses. Traditionally, phytohormonal biosensors usually employ indirect detection of the products of conjugated oxidase reactions.
We proposed a reagentless electrochemical impedance immunosensor for abscisic acid detection that is based on the antiABA antibody adsorbed directly on a porous nano-gold film. The film was produced electrochemically on a glassy carbon
electrode in 0.008 M hydrogen tetrachloroautate solution containing 0.004 M lead acetate with applied potential of -0.5
V (versus Ag/AgCl) for 50s. The anti-ABA antibody was immobilized onto the porous nano-gold through electrostatic
adsorption and covalently conjugation. The electrochemical impedance spectroscopy (EIS) was used to characterized
the successful construction of the porous nano-gold film and the stepwise modifacation of the glassy-carbon electrode.
The concentration increase of the antigen brought the decrease of the interfacial electron transfer, which also means
the increase of the impedance signal. Experimental parameters as pH, temperature, antibody incubation time, antibody
concentration were optimized. The results showed good linearity with the content of ABA in the range of 5 ng/mL-0.05
mg/mL with a detection limit of 0.5 ng/mL.
PS1115 CYTOKININ ANTAGONISTS – UPDATE AND NEW INSIGHTS
Spíchal, Luká_, Strnad, Miroslav Laboratory of Growth Regulators, IEB AS CR & UP Olomouc, _lechtitelù 11, 78371
Olomouc, Czech Republic; [email protected]
[email protected]
Cytokinins are a class of plant hormones that regulate diverse developmental and physiological processes. Several anticytokinins were identified in past decades. We have recently shown that representative anticytokinins are not competitive inhibitors of Arabidopsis cytokinin receptors, but inhibit cell cycle progression and cause cellular changes consistent
with responses to known CDK inhibitors. Providing new insight we present real anticytokinin PI-55 that competitively
inhibits binding of trans-zeatin to the cytokinin receptors and affects cytokinin activation of the primary response gene
IPGSA 19th Annual Meeting - Abstracts
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ARR5 in Arabidopsis seedlings. Different in vitro cytokinin bioassays were employed to prove the anticytokinin-like effect of PI-55. Arabidopsis seedlings grown on PI-55 had increased number and length of lateral roots, what confirms the
involvement of PI-55 in inhibition of cytokinin perception.
PS1116 SUPPRESSORS OF THE PEA BRANCHING RAMOSUS
Braun Nils1, David Karine1, Pillot Jean Paul1, Delbarre Alain2, Beveridge Christine3, Rameau Catherine1. 1Stat. Genet.
INRA 78026 Versailles cedex, FR. 2ISV CNRS Gif/Yvette FR. 3 Uni. Queensland AU.
[email protected]
In order to characterise new genes controlling branching in the model plant Pisum sativum, a second chemical mutagenesis has been carried out on 2 ramosus (rms) branching lines. Three independent non branched mutants have been obtained and called dormant (dor) as the axillary buds are present but remain dormant. Two of the dor mutants present some
very strong pleiotropic characters as an altered growth and modification of stipules shape. Molecular characterisation
of axillary buds of the dor mutants indicate that dormant related genes (DRM1)are more expressed whereas cell cycle
related genes are down regulated confirming the dormant phenotype at the molecular level. As most of the phenotypes
appear to be auxin related, auxin transport, local auxin levels and auxin response are being investigated at the plant and
molecular levels.
PS1117 THE DOMINANT TRANSCRIPTIONAL SILENCER ARR1-SRDX SUPPRESSES PLEIOTROPIC CYTOKININ ACTIVITIES IN ARABIDOPSIS THALIANA
Heyl, Alexander1, Ramireddy, Eswar1, Brenner, Wolfram 1 and Schmülling, Thomas1
[email protected]
In plants the cytokinin signal transduction is mediated via a multi-step phosphorelay system. In the current model the
cytokinin signal is perceived at the plasma membrane by sensor histidine kinases and transported via phosphotransmitter
proteins to the nucleus. Here they activate the B-type response regulators, which act as transcription factors. The Arabidopsis genome encodes eleven B-type response regulators and genetic analyses have indicated functional redundancy. In
order to study the extent of the contribution of B-type ARRs to cytokinin activities, we generated a dominant repressor
version of Arabidopsis response regulator ARR1 (ARR1-SRDX) using chimeric repressor silencing technology (CREST) and studied the consequences on short-term and long-term responses on the molecular and the morphological level
in transgenic tissues. 35S:ARR1-SRDX Arabidopsis plants showed phenotypic changes reminiscent of plants with a
reduced cytokinin status, such as a strongly reduced leaf size, an enhanced root system and larger seeds. Results of this
study will be presented in detail.
PS1118 THE ROLE OF ETHYLENE ON ENDOGENOUS LEVELS OF IAA, CYTOKININS AND ABA AND DEVELOPMENT OF CATASETUM FIMBRIATUM (ORCHIDACEAE).
Rogério M. Suzuki1, Gilberto B. Kerbauy2 1Instituto de Botânica de São Paulo, Seção de Orquidário do Estado, Av.
Miguel Stéfano 3687, 04301-902 São Paulo, Brazil. 2Departamento de Botânica, Universidade de São Paulo, São Paulo,
Brazil. [email protected]
[email protected]
This study attempted to clarify the effects of ethylene, light and dark on plant growth and endogenous levels of indole-3acetic acid (IAA), cytokinins and abscisic acid (ABA) in Catasetum fimbriatum. Dark-incubation fully inhibited root and
pseudobulb formation as well as leaf growth, but favored shoot elongation. The results of continuous and active growth
in dark-incubated shoots (stolons) are induced by strong apical meristem sink activity and by the significantly increased
of cytokinins in shoots. In fact, shoot length, cytokinin and IAA levels in dark-incubated shoots were about twice as much
as those grown under light conditions. Moreover, total cytokinin level in shoots of C. fimbriatum under light conditions
without ethylene was significantly higher than that found in roots. High levels of cytokinins in dark-grown stolons may
be closely related to the absence of roots in C. fimbriatum. Under light conditions, the increased IAA level in shoots
is mediated by ethylene. However, ethylene caused a significant increase of cytokinins in roots of light-treated plants,
IPGSA 19th Annual Meeting - Abstracts
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which may be involved in the retardation of root growth. Since the difference of cytokinins in shoots between ethylenetreated and non-treated plants under light conditions is small, it is concluded that the marked inhibition of leaf growth
in ethylene-treated plants was attributed to ethylene. Zeatin (Z) and zeatin riboside (ZR) are the major cytokinins in C.
fimbriatum regardless of the light conditions, ethylene treatment and organ types. Supported by CAPES and CNPq.
PS1119 FUNCTIONAL ANALYSIS OF AUXIN AND AUX/IAA BINDING SITES IN THE AUXIN RECEPTOR TRANSPORT
INHIBITOR RESPONSE1 FROM ARABIDOPSIS THALIANA
Calderon-Villalobos, Luz Irina A. 1, Tan, Xu 2, Zheng, Ning 2, Estelle, Mark 1. 1 Department of Biology, Indiana University, Bloomington, Indiana 47405, USA. 2 Department of Pharmacology, University of Washington, School of Medicine, Box 357280, Seattle, Washington 98195, USA.
[email protected]
Auxin is a central regulator of most developmental processes in plants. In Arabidopsis, Transport Inhibitor Response
1 (TIR1) and its three closest homologues, Auxin signalling F-Box Protein 1-3 (AFB1 to 3),have been shown to be auxin
receptors. TIR1/AFB1-3 also function as the substrate receptors of SCF-E3 type ubiquitin ligases, which promote the
degradation of the Aux/IAA transcriptional repressors in response to auxin.
Recently, the crystal structure of TIR1-ASK1 in complex with auxin and an Aux/IAA peptide has been solved. This is
the first demonstration that an SCF complex is regulated through the direct binding of a small ligand, in this case auxin.
The structure allowed us propose a mechanism for how auxin stimulates binding of TIR1 to its targets. According to our
hypothesis, TIR1 has intrinsic binding sites for auxin and the Aux/IAA proteins, which are adjacent to each other. After
binding to TIR1, auxin enhances the affinity between TIR1 and the Aux/IAAs, thereby acting as “molecular glue”. In
order to evaluate the functional significance of each of the predicted auxin and Aux/IAA binding sites in vivo, we have
generated tir1 as well as tir1afb2 transgenic plants carrying mutated versions of TIR1. Furthermore, through in vitro pulldown analysis, we are assessing which predicted binding motifs are essential for auxin-dependent substrate recruitment
by TIR1. Another highlight of the TIR1-ASK1-auxin-Aux/IAA structure has been the presence of hexakisphosphate
(IP6) as a cofactor for TIR1. In this context we are analysing the effect of IP6 on auxin dependent TIR1-Aux/IAA interaction.
PS1120 CO(I)-ORDINATION OF LOCALLY INDUCED RESISTANCE TO HERBIVORES AND DIFFERENTIAL REGULATION OF JASMONATE BIOSYNTHESIS BY COI1 IN NICOTIANA ATTENUATA
Anja Paschold1, Rayko Halitschke2 and Ian T. Baldwin1 1Max-Planck-Institute for Chemical Ecology, Hans-KnoellStrasse 8, 07745 Jena, Germany; 2Department of Ecology and Evolutionary Biology, Cornell University, E443 Corson
Hall, Ithaca, NY 14853, USA
[email protected]
CORONATINE INSENSITIVE 1 (COI1) is an F-box protein that mediates jasmonate (JA) responses. COI1-deficient
Arabidopsis and tomato mutants are more susceptible to herbivores in laboratory trials but the exact mechanisms of
COI1-mediated resistance are not known. We silenced COI1 by transformation with an inverted repeat construct (ir-coi1)
in Nicotiana attenuata, a plant whose direct and indirect defenses against various herbivores have been well studied. Ircoi1 plants are male sterile and impaired in JA-elicited direct (nicotine, caffeoylputrescine, and trypsin proteinase inhibitor [TPI] activity) and indirect (cis-_-bergamotene emission) defense responses; responses not elicited by JA treatment
(ethylene production, flower TPI activity) are unaffected. Larvae of Manduca sexta, a common herbivore of N. attenuata,
gained three times more mass feeding on ir-coi1 than on WT plants in glasshouse experiments. By regularly moving
caterpillars to unattacked leaves of the same plant, we demonstrate that larvae on WT plants can grow and consume
leaves as fast as those on ir-coi1 plants, a result which underscores COI1ʼs role in mediating locally induced resistance
in attacked leaves and the importance of herbivore movement in avoiding a plantʼs induced defenses. When transplanted
into native habitats in the Great Basin Desert, ir-coi1 plants suffer greatly from damage by the local herbivore community, which includes herbivores not commonly found on N. attenuata WT plants. Phytohormone analyses revealed that
JA as well as JA-Ile amounts in herbivore-induced ir-coi1 plants differ from that of WT indicating a regulatory function
of COI1 in the octadecanoid pathway. In addition, we demonstrate that COI1 regulates the expression of some JA biosynthetis genes, while others are COI-independent. We conclude that NaCOI1 is essential for induced resistance in N.
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attenuata, and that ir-coi1 plants highlight the benefits of herbivore movement for avoiding induced defenses. NaCOI1
not only is involved in the perception of jasmonates and/or the transduction of subsequent signals, it also functions as
a regulator of jasmonate biosynthesis. The changed oxylipin profile of ir-coi1 plants might account for the attraction of
herbivores that do not feed on WT plants.
PS1121 A NOVEL STRATEGY TO DISSECT THE ROLE OF HORMONES IN THE REGULATION OF CELL SEPARATION
IN ARABIDOPSIS THALIANA
Basu Manojit, Carranza-Gonzalez Zinnia, Azam-Ali Sayed and Roberts Jeremy School of Biosciences, University of
Nottingham, College Road, Sutton Bonington, UK
[email protected]
Cell separation is an important process in the life cycle of plants, regulating abscission, dehiscence, root elongation
and lateral root emergence. During abscission and lateral root development cell separation is brought about by the action
of several hydrolytic enzymes; one of these has been identified as polygalacturonase (PG).
In this study we have used two genes which are able to modify IAA biosynthesis and have been fused to the promoter of
an Arabidopsis PG gene (At2g41850) for tissue specific expression in the floral AZ, the root pericycle and cortical cells.
A second strategy has been used to study the effect of At2g41850-driven silencing of IAA response during abscission and
lateral root emergence using the AXR3 gene. Interaction between ethylene, ABA, and GA has been investigated during
cell separation. Manipulation of hormone action during development is currently being investigated using the GAL4 enhancer trap lines in early and late abscission zone on the process of organ shedding. Delayed abscission and higher tissue
break strength has been recorded in plants transformed with genes up-regulating IAA biosynthesis. The expression of the
IAA manipulating genes under the control of the At2g41850 promoter has altered the root architecture and the impact of
this on plant abiotic stress tolerance has been evaluated by measuring WUE and root growth under various levels of soil
compaction.
PS1122 REGULATION OF THE ARABIDOPSIS PROTEIN KINASE CTR1 IN ETHYLENE SIGNALING
Reading, Mandy1, Shockey, Jason2, Chang, Caren1 1Dept. of Cell Biology & Molecular Genetics, University of Maryland, College Park, MD 20742 USA; 2Dept. of Biological Sciences, The George Washington University, Washington
D.C. 20052, USA
[email protected]
In Arabidopsis, ethylene is perceived by five receptors that share sequence similarity to prokaryotic two-component
systems. The next-known downstream component from the receptors is CTR1, which has similarity to Raf-like protein
kinases. CTR1 is comprised of a novel N-terminal domain and a C-terminal kinase domain (KD). The N-terminus of
CTR1 interacts with at least two ethylene receptors, ETR1 and ERS1, in the yeast-two-hybrid and in vitro pull-down
assays. Furthermore, in planta evidence support this interaction. Little is known about the mechanism of signaling from
the receptors to CTR1, which raises the question of how CTR1 is regulated. We have shown that the CTR1 N-terminal
domain interacts with the CTR1 KD in vitro and in planta evidence suggests that this interaction is required for regulation of KD activity. Overexpression of the CTR1 KD alone in Arabidopsis does not result in a constitutively active CTR1
kinase, since no phenotypic changes are observed in Arabidopsis seedlings. However, by fusing the CTR1 KD to the Nterminus (transmembrane-containing-region) of the ETR1 receptor, (which we speculate will target the CTR1 KD to its
site of activity based on the aforementioned direct interaction with the ETR1 receptor), we observe ethylene insensitivity
consistent with KD activity. Additionally we are using this fusion-based system to further investigate the nature of the
ctr1-8 mutation (which disrupts the direct interaction between ETR1/ERS1 and CTR1 in yeast).
PS1123 THE _-DIOXYGENASE-MEDIATED INTERACTIONS DURING MANDUCA SEXTA ATTACK ON NICOTIANA
ATTENUATA
Gaquerel, Emmanuel1 , Steppuhn, Anke1 and Baldwin Ian T.1 1Max-Planck-Institute for Chemical Ecology, Hans-Knoell-Strasse 8, 07745 Jena, Germany [email protected]
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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Fatty acid oxygenated derivatives (oxylipins) are central components modelling the expression of genes in most of
plantsʼ responses against insects. Besides the lipoxygenase pathway leading to jasmonic acid (JA) and its derivatives,
additional enzymatic systems catalyze the synthesis of bioactive oxylipins. One of these, the _-dioxygenase (_DOX),
discovered in Nicotiana tabacum and first named PIOX (for pathogen-induced oxygenase), is wound- and JA-inducible. Plantʼs _DOXs are involved in the _-oxidation pathway responsible for the chain-shortening of fatty acids (FA) by
catalyzing among others the _-hydroperoxidation of linolenic acid (C18:3) that may either be reduced to the corresponding alcohol (2-hydroxylinolenic acid, 2-HOT) or spontaneously decarboxylated to the C17 aldehyde (heptadecatrienal,
HDT) which leads after dehydrogenation to heptadecatrienoic acid (C17:3).
The wild tobacco plant, Nicotiana attenuata, has been extensively explored in glasshouse conditions and its natural environment as an excellent model to study defense reprogramming during herbivory. Thus, we silenced the homologue of N.
tabacumʼs _DOX in N. attenuata by transformation with an inverted repeat construct (ir-_DOX). Strongly silenced plants
were stunted, whereas mildly silenced _DOX (ir-_DOX-M) transgenic plants had normal growth. Larvae of Manduca
sexta, a specialist herbivore, gained more mass feeding on ir-_DOX-M than on WT plants.
Moreover, JA biosynthesis and signalling appeared strongly altered in ir-_DOX-M, results which underscore new fundamental interplays between these two signalling pathways. Investigating the fate of _DOX products in both plant and
insectʼs compartments during the feeding process, we demonstrated that M. sextaʼs regurgitant, midgut and feces contain
high amounts of 2-HOT strictly after rearing on WT plants. On the other hand, the synthesis of long chain aldehydes and
chain-shortened FA strikingly prevailed in planta and was reduced in ir-_DOX-M plants. Results from a set of microarray
hybridizations of N. attenuataʼs transcriptome aiming at searching for the targets of _DOX products and at deciphering
their interactions with the JA signalling pathway will also be discussed.
PS1124 PHOSPHORYLATION–DEPENDENT NUCLEOCYTOPLASMIC SHUTTLING OF BZR1 IS ESSENTIAL IN ARABIDOPSIS BRASSINOSTEROID SIGNALING
Hojin Ryu1, Kangmin Kim1, Hyunwoo Cho1, Joonghyuk Park1, Sunghwa Choe2 and Ildoo Hwang1 1Department of
Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea; 2Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea
[email protected]
Phosphorylation–dependent Nucleocytoplasmic Shuttling of BZR1 is Essential in Arabidopsis Brassinosteroid Signaling
Hojin Ryu1, Kangmin Kim1, Hyunwoo Cho1, Joonghyuk Park1, Sunghwa Choe2 and Ildoo Hwang1
[email protected]
1Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea; 2Department of
Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea
Phytohormone brassinosteriods (BRs) play critical roles in plant growth and development. BR exerts its action by modulating the phosphorylation status of two key transcriptional factors, BES1 and BZR1, upon perception of BR by BRI1/
BAK1 receptors at the plasma membrane. However, a regulatory mechanism mediating BR signals from the plasma
membrane-associated BR perception to expression of BR target genes in the nucleus remains unknown. We here show
that phosphorylation by BIN2 directly induces the nuclear export of BZR1; in the cytoplasm, BR-activated BSU1 mediates their dephosphorylation and subsequent nuclear translocation. Two distinct potential phosphorylation domains in
BZR1 were required for the nuclear export induced by BIN2. Transgenic Arabidopsis plants overexpressing BZR1 with
mutations in these phosphorylation sites, displays constitutive BR response phenotypes. Taken together, we propose that
the phosphorylation-dependent spatial redistribution of BZR1 is essential for proper BR signaling.
PS1125 CROSSTALK BETWEEN HORMONES AND THE HOMEOBOX GENE BREVIPEDICELLUS IN ARABIDOPSIS
THALIANA
Soucek Premysl 1,2, Rekova Alena 1,2, Brzobohaty Bretislav 1,2
1Institute of Biophysics AS CR, CZ-61265 Brno, Czech Republic; 2 Department of Molecular Biology and Radiobiology, Mendel University of Agriculture and Forestry, CZ-61300 Brno, Czech Republic
[email protected]
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Developmental and physiological processes in plants are orchestrated by hormones and a cascade of regulatory genes.
The BREVIPEDICELLUS (BP) gene encodes a homeodomain transcription factor belonging to the KNOX I (knotted1like homeobox gene) family. BP predominantly regulates internode patterning and could be involved in other developmental processes including root growth. Hormonal regulation of BP expression and responsiveness of the bp mutant
and overexpressor to hormonal treatment were investigated. Auxin, gibberellin, ethylene and cytokinin treatments did
not result in ectopic BP expression, whereas abscisic acid (ABA) repressed BP in both shoots and roots dramatically.
Any alteration in hormone sensitivity was not observed in the bp mutant. CaMV35S-BP seedlings had fewer and shorter
lateral roots and altered sensitivity to cytokinins, ethylene and ABA. The main root growth of CaMV35S-BP was more
sensitive to ethylene and cytokinin compared to wild type plants. On the contrary, transgenic plants were more resistant
to the inhibitory effect of ABA on lateral root length.
Supported by grants LC06034, IAA600380507, IAA600040612 and AVOZ50040507.
PS1126 AHP GENE EXPRESSION PROFILING: CYTOKININ ACTION AND ORGAN SPECIFICITY
Hradilova, Jana 1,2Malbeck Jiri 3, Brzobohaty, Bretislav 1,2 1Institute of Biophysics AS CR, Kralovopolska 135, CZ61265 Brno, Czech Republic; 2Department of Molecular Biology and Radiobiology, Mendel University of Agriculture
and Forestry, Zemedelska 1, CZ-61300 Brno, Czech Republic; 3Institute of Experimental Botany AS CR, Rozvojova
135, CZ-16500 Prague, Czech Republic [email protected], Tel: +420 545 133 297
[email protected]
In higher plants, two-component systems of signal transduction are involved in hormone, stress and light signaling.
Histidine-containing phosphotransmitters (AHPs) transfer a phosphoryl group from membrane-localized receptors to
nuclear-localized effectors. Five AHPs have been identified in Arabidopsis thaliana. Real-time RT-PCR was employed to
quantify steady state levels of individual AHP transcripts in different Arabidopsis organs. Transcripts of all AHPs were
detected in every organ examined, although at different levels. We also analyzed and compared the effects on steady state
levels of AHP transcripts of a short-term treatment with an aromatic cytokinin (CK) and increase in endogenous isoprenoid CK levels following activation of the Agrobacterial isopentenyltransferase gene (ipt), in Arabidopsis seedlings. Following ipt activation, a rapid and highly preferential increase in trans-zeatin-type CKs was observed. Transient increases
in steady-state levels of AHP1-4 transcripts in response to aromatic CK treatment and the increase in endogenous transzeatin-type CK levels were observed. On the contrary, increase in steady-state levels of AHP1-4 transcripts observed
in seedlings cultivated continuously in presence of exogenous aromatic cytokinin were not paralleled in seedlings with
constitutively increased endogenous trans-zeatin-type cytokinins.
Supported by grants 1M06030, LC06034, AVOZ50040507, IAA600380507 and IAA600040612.
PS1127 GIBBERELLIN RECEPTORS IN WOOD FORMATION
Mauriat, Mélanie and Moritz, Thomas Umeå Plant Science Centre (UPSC), Department of Forest Genetics and Plant
Physiology, Swedish University of Agricultural Sciences, SE-901 87 Umeå, Sweden Tel. 0046 (0)90 786 85 45
[email protected]
Gibberellins (GAs) are plant growth hormones required for numerous of developmental processes, such as shoot
elongation, seed germination and flowering. Particularly, GAs function as a regulator of wood formation is an important
question to address in highlight of current functional and genetic tools. GAs have been shown to stimulate xylem fiber
elongation and are presumed to regulate early stages of xylem differentiation in the wood formation model: hybrid aspen
(Populus tremula x P. tremuloides). However, molecular keys of this mechanism remain widely unknown. Recently,
the first GA receptor (GID1) has been identified in rice and orthologs to GID1 have been found in Arabidopsis thaliana.
We are currently isolating GID1 orthologs in aspen and identifying their function in wood formation by using different
strategies such as phylogenic analyses of GID1 and its aspen and A. thaliana orthologs, RT-qPCR expression analyses in
different organs of the whole plant and in different cells layers of the wood-forming tissues, functional studies of GID1
orthologs by an overexpression strategy and optic microscopic analyses of transgenic plants obtained. Our work will
permit a better understanding of the role of GA signal transduction in wood formation.
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PS1128 A COMPARATIVE STUDY OF TWO F-BOX PROTEINS, SLEEPY1 AND SNEEZY IN GA SIGNALING
Tohru Ariizumi 1, Camille M. Steber 1,2 1Dept. of Crop and Soil Science, Washington State University 2USDA-ARS
[email protected]
SNEEZY (SNE) is a homolog of SLEEPY1 (SLY1), encoding an F-box protein subunit of an SCF E3 ubiquitin ligase
complex in Arabidopsis. SLY1 plays a central role in destruction of DELLA negative family proteins via 26S proteasome pathway in GA signaling pathway. DELLA proteins consist of five members including RGA, GAI, RGL1, RGL2
and RGL3. It is known that SCFSLY1 recognizes these DELLA proteins in response to GA, and trigger DELLA protein
destruction, which releases DELLA inhibitory effects on GA responses. The sly1 mutant shows severe dwarfism, reduced
fertility and seed dormancy apparently due to high accumulation of DELLA proteins. In this study, the effect of SLY1 and
SNE over-accumulation on DELLA protein accumulation was determined.
We found that all sly1 mutant phenotypes were rescued by SLY1 over-expression (SLY1-OE), while these phenotypes
were partly restored by over expression of SNE (SNE-OE). The level of protein accumulation in RGA was decreased
in the SNE-OE lines compared to sly1 mutant, suggesting that SNE could partially compensate GA signaling instead
of SLY1. On the other hand, the level of RGL2 protein accumulation was not altered in the SNE-OE lines, suggesting
that SNE was not able to recognize RGL2 as well as SLY1. sly1 sne double mutants were also constructed in this study.
Characterization of sne single and the double mutants are reported in this study.
PS1129 ABA-HYPERSENSITIVE GERMINATION1 ENCODES A PROTEIN PHOSPHATASE 2C, AN ESSENTIAL COMPONENT OF ABSCISIC ACID SIGNALING IN ARABIDOPSIS SEED
Noriyuki Nishimura 1,2,3,5, Tomo Yoshida 2,3, Nobutaka Kitahata 4, Tadao Asami 4, Kazuo Shinozaki 3and Takashi
Hirayama 1,2 1 Laboratory of Environmental Molecular Biology, RIKEN Wako Institute, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan; 2 Integrated Graduate School of Art and Science, Yokohama City University, 1-7-29 Suehiro, Tsurumi,
Yokohama 230-0045, Japan; 3 Gene Discovery Research Group, RIKEN Plant Science Center, 1-7-22 Tsurumi, Yokohama 230-0045, Japan; 4 Laboratory of Cellular Biochemistry, RIKEN Wako Institute, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan; 5 Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive La Jolla,
CA 92093-0116, USA
[email protected]
The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses in
plants. To reveal the mechanism of response to ABA, we isolated several novel ABA-hypersensitive Arabidopsis thaliana
mutants, named ahg (ABA-hypersensitive germination). ahg1-1 showed hypersensitivity to ABA, NaCl, KCl, mannitol,
glucose, and sucrose in germination and post-germination growth but did not display any significant phenotypes in adult
plants. ahg1-1 seeds accumulated slightly more ABA before stratification and showed increased seed dormancy. Mapbased cloning of AHG1 revealed that ahg1-1 has a nonsense mutation in a gene encoding a novel protein phosphatase
2C (PP2C). We previously showed that ahg3-1 has a point mutation in the AtPP2CA gene, which encodes another PP2C
that has a major role in ABA response in seed (Yoshida et al., 2006). The levels of AHG1 mRNA were higher in dry seed
and increased during late seed maturation_\an expression pattern similar to that of ABI5. Transcriptome analysis revealed
that, in ABA-treated germinating seeds, many seed-specific genes and ABA-inducible genes were highly expressed in
ahg1-1 and ahg3-1 compared with in the wild type. Detailed analysis suggested differences between the functions of
AHG1 and AHG3. Dozens of genes were expressed more strongly in ahg1-1 than in ahg3-1. Promoter_|GUS analyses
demonstrated both overlapping and distinct expression patterns in seed. In addition, the ahg1-1 ahg3-1 double mutant
was more hypersensitive than either monogenic mutant. These results suggest that AHG1 has specific functions in seed
development and germination, shared partly with AHG3.
PS1130 THE ARABIDOPSIS MAPK PHOSPHATASE, MKP5, REGULATES ABA-SIGNALING AND SEEDLING ESTABLISHMENT.
Montserrat Pages and Victoria Lumbreras
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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The phytohormone abscisic acid (ABA) plays an essential role in adaptative stress responses. Although genetic analyses have identified numerous ABA-response mutants, the regulators of ABA signal transduction remain to be fully elucidated. Here, we report the identification and characterization of a novel MAPK phosphatase (MKP5) from Arabidopsis
involved in developmental and stress responses. The mkp5 gene is expressed at very low levels in vegetative tissues, but
is markedly induced during seedling establishment. Seeds expressing an MKP5-GFP fusion protein show phenotypic
alterations, including ABA and salt hypersensitivity and a severe growth arrest after germination. This developmental
arrest is a result from inefficient lipid reserve mobilization and is rescued by providing a source of exogenous sugar in
the growth medium. This phenotype is not observed after overexpression of the normal MKP5 protein, suggesting that it
arises from dominant negative effects of the MKP5-GFP protein, confirmed by a hypomorphic mkp5 allel. We also find
that the constitutive MKP5 overexpression results in ABA and salt hypersensitivity and slight enhanced drought tolerance. Molecular analyses reveal upregulation of ABA signaling genes in those plants.
The spatial expression pattern and protein MKP5 accumulation is consistent with their functional role. MKP5-GFP
shows specific accumulation in tissues involved in water homeostasis, hydathodes and guard cells. Taken together, these
data suggest that MKP5 is a regulator of ABA signaling pathway and present new evidence implicating a link by MAPK
activities in the control of plant development and survival under stress conditions.
PS1131 FUNCTIONAL SIGNIFICANCE OF THE AUX/IAA- ARF INTERACTIONS
Armitage, Lynne, Readshaw, Anne and Leyser, Ottoline
[email protected]
The plant hormone auxin is involved in a diverse range of growth and developmental processes in plants, yet the
mechanisms of how specific auxin responses are generated is not fully understood. Q-rich ARFs are transcriptional activators of auxin regulated genes. Aux/IAA proteins form dimers with the ARF proteins causing transcriptional repression. In the presence of auxin Aux/IAA proteins are degraded, releasing this repression. With 23 ARF and 29 Aux/IAA
genes in Arabidopsis, there are potentially many combinations of homo and heterodimers. Similar mutations in different
Aux/IAA genes can cause different phenotypes, suggesting functional diversity. It is thought that the Aux/IAA and ARF
proteins have preferential binding partners, which, along with differential gene expression, could explain how specific
auxin responses are produced in the plant. The interactions of these proteins is being investigated using dual bait yeast
two-hybrid analysis and in planta using Fluorescence Resonance Energy Transfer (FRET). In a complementary approach
we are testing the phenotype effects of Aux/IAA promoter and domain swap experiments in transgenic plants. Initial
results show both promoter and protein effects on phenotype, with the promoter being the predominant influence. The
protein effects appear at least partially to be associated with the dimerization domains.
PS1132 CHARACTERIZATION OF KNOCKOUT MUTANTS FOR GIBBERELLIN RECEPTOR ATGID1S IN ARABIDOPSIS THALIANA.
Iuchi, Satoshi 1, Suzuki, Hiroyuki 2, Kim, Young-Cheon2, Iuchi, Atsuko1, Kuromori, Takashi3, Ueguchi-Tanaka, Miyako4, Asami Tadao2, Yamaguchi, Isomaro5, Matsuoka, Makoto4, Nakajima Masatoshi2, Kobayashi, Masatomo1 1BioResource Center, RIKEN, Tsukuba, Ibaraki 305-0074, Japan, 2Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, 3Plant Science Center, RIKEN, Yokohama, Kanagawa 230-0045,
Japan, 4Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan, 5Maebashi Institute
of Technology, Maebashi, Gunma 371-0816, Japan
[email protected]
Three receptor genes (AtGID1a, AtGID1b, and AtGID1c) for a phytohormone gibberellin (GA) exist in Arabidopsis
genome. The AtGID1s proteins are able to bind with GA, and the over-expression of each AtGID1s gene could complement dwarf phenotype of rice GA-receptor mutant gid1-1. In this study, we elucidate the function of AtGID1s using TDNA and Ds insertion mutants obtained from RIKEN GSC and ABRC. We could not detect obvious phenotypic change
in the homozygous single mutants (atgid1a, atgid1b, atgid1c) suggesting that the function of AtGID1s is highly redundant. Thus we established double mutants (atgid1ab, atgid1ac, and atgid1bc) to detect any functional difference. The
phenotypes of double mutants including germination, stem elongation, flowering and seed development are examined.
The result of crossbreeding experiment to establish triple mutant (atgidabc) will be also reported.
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PS1133 CYTOKININ SIGNALING IN SECONDARY VASCULAR DEVELOPMENT
Nieminen, Kaisa 1, Immanen, Juha 1, Decourteix, Mélanie 1, Helariutta, Ykä 1,2 1 Department of Biological and Environmental Sciences/Institute of Biotechnology, FI-00014 University of Helsinki, Finland; 2 Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden
kaisa.nieminen@helsinki.fi
The secondary phase of plant development is initiated through activation of a lateral meristem, the vascular cambium.
Secondary vascular tissues are produced via cell divisions taking place in the cambium. We have shown that cytokinin signaling is required for the proliferation and persistence of procambial cell files between xylem and phloem during the primary
phase of Arabidopsis root development (Mähönen et al. 2000, Genes & Dev. 14:2938-2943; Mähönen et al. 2006, Science
311:94-98). These cell files will form the vascular cambium upon initiation of secondary development.
To understand the role of cytokinin signaling in secondary development we have identified orthologs for the three Arabidopsis CRE family cytokinin receptor genes (CRE1/WOL/AHK4, AHK2, AHK3) from two tree species; Betula pendula
(BpCRE1-3) and Populus trichocarpa (PtCRE1-5). We have shown that these genes are active during the cambial development, and that the putative birch ortholog of CRE1, BpCRE1, is functional as a cytokinin receptor in Arabidopsis. To
demonstrate the role of cytokinin signaling during secondary vascular development we are currently studying transgenic
trees, in which cytokinin signaling is downregulated in the cambial zone.
In addition of the receptor genes, we have also identified and annotated all genes coding for cytokinin signal transduction components in the recently sequenced P. trichocarpa genome (Tuskan et al. 2006, Science 313:1596-1604). A few
of the cytokinin signaling related gene families display a particularly high degree of diversification since the speciation
of poplar and Arabidopsis. Our future aim is to explore if the poplar specific members of these gene families manifest
some specific, yet currently unknown, gene functions required for the extensive secondary development characteristic
of tree species.
PS1134 CHARACTERIZATION OF ARABIDOPSIS GIBBERELLIN RECEPTORS
Nakajima, Masatoshi1, Shimada, Asako2, Takashi, Yoshiyuki1, Kim, Young-Cheon1, Park, Seung-Hyun1, UeguchiTanaka, Miyako2, Suzuki, Hiroyuki1, Katoh, Etsuko3, Iuchi, Satoshi4, Kobayashi, Masatomo4, Maeda, Tatsuya5, Matsuoka, Makoto2, Yamaguchi, Isomaro6 1Dept. Appl. Biol. Chem., Univ. of Tokyo, Tokyo, Japan; 2Biosci. Biotech.
Center, Nagoya Univ., Aichi, Japan; 3Dept. Biochem., Natl. Inst. Agrobiol. Sci., Ibaraki, Japan; 4RIKEN-BRC, Ibaraki,
Japan; 5Inst. Mol. Cellular Biosci., Univ. of Tokyo, Tokyo, Japan; 6Maebashi Inst. Tech., Gunma, Japan
[email protected]
Three receptor genes for gibberellin were identified from Arabidopsis, and the characteristics of their recombinant
proteins were examined. The GA-binding activities of them were confirmed by an in vitro assay. Biochemical analyses
revealed similar ligand selectivity among them, and all recombinants showed higher affinity to gibberellin A4 than to
other GAs. AtGID1b showed GA-binding in a narrow pH range with higher affinity than AtGID1a and 1c. A Y2H system showed in vivo interaction_@between each AtGID1 and the DELLA proteins in the presence of GA. The DELLA
boosted the GA-binding activity of AtGID1s in vitro, which suggests the formation of a complex DELLA-AtGID1 leads
to the more tight binding of GA to AtGID1. The expression of each AtGID1 gene in rice gid1-1 mutant rescued its phenotype to the normal. These results demonstrate AtGID1s functioned as GA receptors.
PS1135 PARTICIPATION OF AUXIN AND BRASSINOSTEROID DURING THE THIGMOTROPIC RESPONSE
García-Ponce Berenice1, Lanza Mónica2, Sánchez Federico3, Alvarez-Buylla Elena1 y Leyva Antonio2. 1Instituto de
Ecología, Universidad Nacional Autónoma de México (UNAM), 04510 México, D. F.; 2Centro Nacional de Biotecnología, campus Cantoblanco 28049 Madrid, España; 3Instituto de Biotecnología, UNAM, 62250 Cuernavaca, México.
[email protected]
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When wild type Arabidopsis seedlings are grown on a hard vertical plate a natural root oscillating behaviour known
as circumnutation is observed. However, when these plates are shifted to 45o from the vertical position, such oscillatory
growth is pronounced as a response to gravity and touch (thigmotropic) stimuli (1). In the past, a few mutants with altered wavy root growth have been isolated in angled agar surface screenings (2), but the molecular basis and signals that
control this phenomenon are still largely unknown.
In this study we have isolated an EMS mutant that has a constitutive waving response when grown on vertical plates and
the number of coils per unit of root length increased proportional to the angle of the plate (from 90o to 45o). In contrast,
wt roots coils reach a plateau at 65o angle. Our mutant also shows curly cauline leaves and wrinkled siliques, suggesting
that the gene underlying the mutation regulates no only root thigmotropic responses. In order to further characterize the
possible signal transduction participants implicated in the thigmotropic response, we used several phosphatase inhibitors
and we found that only orthovanadate complements the root oscillatory phenotype.
Orthovanadate and PAO (phenylarsine oxide) have been used to inhibit tyr-phosphatases and the latter has an inhibitory
effect in the bending response of Mimosa pudica (3). Some auxin deficient mutants that are agravitropic, also show a
wavy root phenotype. We analyzed the gravitropic response in our mutant and we found that it is capable of responding
to the gravitropic axis. Further characterization of the auxin response reporter line, DR5::GUS, in the mutant background
will be show. Our data suggest that the protein affected in our mutant participates in the same transduction pathway as the
oscillatory response induced by brassinolide and phosphorylation signal transduction is also implicated in this process.
1 Buer C.S., Masle J. and Wasteneys G. (2000). 42: 1164-1170
2 Okada K. and Y. Shimura (1990). Science 250: 274-276
3 Kameyama et al (2000). Nature 407: 37
PS1136 SBI1 REGULATES BRI1 IN BRASSINOSTEROID SIGNALING
Guang Wu123,Yuji Kamiya1 and Joanne Chory2 1 RIKEN Plant Science Center,1-7-22 Suehiro-cho,Tsurumiku,Yokohama,230-0045,Japan 2 Plant Biology, The Salk Institute for Biological Studies,La Jolla, CA 92078, USA 3
College of Life Science, Shangdong Agricultural University, Taian, Shangdong 271018, China
[email protected]
Brassinosteroids (BRs), the steroid hormones in plants, play important roles in plant growth and development. Over
the past decade, studies have been focusing on finding new components in on BR signaling pathway. As such, the several
key components in BR signaling pathway have been identified, including receptor BRI1 and co-receptor BAK1, and
downstream components BKI1, BIN2, BES1 and BZR1. BRs are perceived by BRI1, which inactivates a negative regulator, BIN2, a GSK3-like kinase, which then dephosphorylates and activates a family of plant-specific transcription factors (such as BES1 and BZR1) to regulate the expression of many BR responsive genes. The loss of function bri1 mutant
is similar to the strongest BR biosynthetic mutant, indicating that BRI1 is the master switch for BR function. However,
we know little about how this master switch, BRI1, is regulated.
We used a genetic approach to identify regulators of BRI1 using an unstable bri1 mutant, a weak bri1 mutant allele that
accumulates less bri1 protein. We screened for suppressors of this bri1 mutant and found a BR signaling specific mutant (sbi1, suppressor of bri1) that accumulates a high level of BRI1 protein. sbi1 does not suppress other bri1 alleles in
which the protein level is not altered. Nevertheless, the sbi1 single mutant has an elevated BRI1 protein level and has
an enhanced response to BL and reduced sensitivity to BR inhibitors. The increased accumulation of BRI1 protein may
explain the basis for its suppression of bri1 and the enhanced growth in sbi1 single mutant. Interestingly, the expression
of BRI1::bri1 in the bri1 mutant had similar phenotype as sbi1/bri1 double mutant, suggesting that the BRI1/bri1 protein
accumulation in sbi1 single mutant and sbi1/bri1 double mutant is responsible for their respective enhanced response to
BL compared to wild type and bri1 respectively. Furthermore, the treatment of bri1 mutant with a protease inhibitor leads
to a similar accumulation of BRI1 protein as in the sbi1/bri1 double mutant. Therefore, SBI1 may play an important role
in early BR signaling events by regulating BRI1 levels.
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PS1137 OSTGA1, A BZIP TRANSCRIPTIONAL FACTOR, REGULATES EXPRESSION OF THE DITERPENE CYCLASE
GENE OSKS4 INVOLVED IN PRODUCTION OF PHYTOALEXINS IN RICE
Atsushi Okada1, Kazunori Okada1, Yoshiaki Nagamura2, Tomonobu Toyomasu3, Jinichiro Koga4, Naoto Shibuya5,
Hideaki Nojiri1, Hisakazu Yamane1 1Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyoku, Tokyo 113-8657, Japan 2Rice Genome Resource Center, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan 3Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan 4Department of Life
Sciences, Faculty of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
5Food and Health R&D Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1, Chiyoda, Sakado-shi, Saitama 350-0289, Japan
[email protected]
Rice diterpenoid phytoalexins are produced in suspension-cultured rice cells treated with a chitin oligosaccharide
elicitor. We have previously reported that the functional identification of 6 diterpene cyclase genes (OsCyc1, OsCyc2,
OsDTC1, OsDTC2, OsKS4, and OsKS10) involved in biosynthesis of diterpenoid phytoalexins in rice and the expression of these genes are activated in suspension-cultured cells upon elicitor treatment.
As part of studies to investigate regulatory mechanisms inducing expression of the diterpene cyclase genes, we first
attempted to identify a transcription factor regulating expression of OsKS4 encoding 9_H-pimara-7,15-diene synthase
involved in momilactones production. We identified a TGACG-sequence as an elicitor-responsive cis-element in the promoter of OsKS4 using reporter gene assays, suggesting that a bZIP transcription factor regulates expression of OsKS4.
Search for the elicitor-responsive bZIP transcriptional factor gene using microarray followed by analysis of OsKS4
expression and momilactones production in the corresponding transposon insertion mutant resulted in identification of a
gene named OsTGA1 as a bZIP transcription factor regulating OsKS4 expression. The subsequent analysis of biochemical properties of OsTGA1 strongly suggests that OsTGA1 regulates OsKS4 expression as a transcriptional activator.
PS1138 PHYSIOLOGICAL AND GENETIC CHARACTERIZATION OF ENHANCERS OF THE AUXIN-RESISTANT4 MUTATION OF ARABIDOPSIS
Lawrence Hobbie, Ogechukwu Eze, Pauline Gould, Michele Adler, Rosaria Anselmo, Stephanie Arredondo, Juan Pablo
Garcia, Michael Hammond, Katherine Herrera, Allison Mead, Velizar Petrov, Alex Sanchez, Pranay Sinha, and Chantelle
Thompson Dept. of Biology, Adelphi University Garden City, NY 11530 USA
[email protected]
The AUXIN-RESISTANT4 protein is required for proper cellular localization and function of the transmembrane
auxin import protein AUX1, which imports the hormone auxin into plant cells. In an attempt to determine the biochemical mechanism of action of AXR4 and to identify other genes involved in the same pathway, a large-scale screen for
genetic modifiers of the axr4 mutant phenotype was carried out. Characterization of over 100 enhancers identified in this
screen is being pursued with genetic, physiological, and molecular approaches. We have shown that most of the enhancer
mutations are auxin-sensitive when not in the presence of the axr4 mutation. 48 recessive auxin-sensitive enhancers and
26 dominant auxin-sensitive enhancers have been identified. In quantitative tests, a number of the enhancer mutations,
when in the presence of axr4, show significantly more resistance to auxin than axr4 alone. Currently we are focusing on
molecular mapping of the mutations, which will enable us both to determine how many genes are defined by our collection of mutants and eventually to isolate the genes.
PS1139 SUPPRESSORS OF IPT161 REVEAL NOVEL CYTOKININ RESPONSE LOCI
Yan Li, Jasmina Kurepa, Jan Smalle University of Kentucky, Plant and Soil Sciences, KTRDC, Lexington, KY 40546,
USA, [email protected] (859) 257-3677
[email protected]
The cytokinin signal transduction pathway includes a two-component system. Binding of cytokinin by histidine
kinase receptors leads to the activation of Type-B response regulators (ARRs), transcriptional activators that promote
cytokinin responses. Type-B ARR activation is mediated by histidine-phosphotransfer proteins and also leads to the
induction of Type-A ARR genes that encode repressors of cytokinin responses. To identify new loci that control the cytokinin response pathway, we screened for mutations that suppress the rosette phenotype of the cytokinin overproducing
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transgenic line ipt161. Eleven suppressor of ipt161 (soi) mutant lines were isolated. The soi1 and soi2 mutants have a
strong suppression phenotype and define separate loci. When introduced in the wild-type background, both mutations
caused decreased cytokinin sensitivity in growth response assays, indicating that the corresponding gene products control
cytokinin responses rather than biosynthesis or metabolism. In addition, the cytokinin induction of Type-A ARR genes
was suppressed in both mutants, suggesting defects in early signaling. Both mutations map to regions of the Arabidopsis
genome that do not contain currently known cytokinin reponse loci. We propose that SOI1 and 2 are novel regulators of
the primary cytokinin response pathway.
PS1140 26S PROTEASOME-DEPENDENT CONTROL OF CYTOKININ SIGNALING
Jasmina Kurepa, Yan Li and Jan Smalle University of Kentucky, Plant and Soil Sciences, KTRDC, Lexington, KY
40546, USA, [email protected] (859) 257-3677
[email protected]
The main goal of this project is to identify the 26S proteasome-dependent control points in the cytokinin response
pathway. Early cytokinin signaling involves the activation of Type-B Arabidopsis response regulators (ARRs) that promote cytokinin responses. Type-B ARRs also promote the upregulation of Type-A ARR transcripts that encode repressors
of cytokinin responses, thus providing feedback-inhibition control. The 26S proteasome mutant rpn12a-1 has reduced
cytokinin sensitivity in various growth response assays suggesting the stabilization of a repressor of cytokinin action.
Indeed, we show that ARR5 (a Type-A ARR) is a target for 26S proteasome-dependent proteolysis. Since ARR5 inhibits
cytokinin responses including the expression of Type-A ARR genes, it was expected that ARR5 stabilization affects the
cytokinin induction of Type-A ARR genes in the rpn12a-1 mutant. More specifically, the feedback inhibition should be
more potent, resulting in a faster decline in Type-A ARR expression level after the initial induction peak. A time course
analysis confirmed this, but surprisingly also revealed a faster increase in Type-A ARR transcript levels in the early stages
of the induction. This suggested that in addition to ARR5 stabilization, loss of proteasome function in rpn12a-1 also leads
to the stabilization of an activator of the early cytokinin response. This was confirmed by the identification of ARR1, a
Type-B ARR, as a proteasome target. Contrary to ARR5, the ARR1 protein was destabilized by cytokinin treatments, a
feature common to the activation of many transcriptional activators. Taken together, our data suggest that the targeted
proteolysis of both an activator (ARR1) and an inhibitor (ARR5) of cytokinin responses allows cells to rapidly readjust
to changes in cytokinin concentrations.
PS1141 MUTATION OF E1-CONJUGATING ENZYME-RELATED1 DECREASES RELATED TO UBIQUITIN CONJUGATION AND ALTERS AUXIN RESPONSE AND DEVELOPMENT
Andrew W. Woodward, Sarah E. Ratzel, Erin E. Woodward, and Bonnie Bartel
[email protected]
The ubiquitin-like protein RUB is conjugated to CULLIN proteins to modulate the activity of SCF ubiquitylation
complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including
Arabidopsis thaliana. Here, we report the isolation and characterization of ecr1-1, an Arabidopsis mutant compromised
in RUB activation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic
site cysteine that normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1
relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function.
The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild
type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are
grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response
mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene
response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals
that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.
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PS1142 THE EFFECT OF PHYTOHORMONES ON THE EXPRESSION OF PHOSPHATIDYLCHOLINE - SPECIFIC PHOSPHOLIPASE C GENES
Veselkova, Stepanka1,2, Pejchar, Premysl1, Potocky, Martin1,2, Novotna, Zuzana2, Valentova, Olga 2, Martinec, Jan1
1Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic 2Department
of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, Czech Republic
[email protected]
Phospholipases are key enzymes involved in signal transduction during responses of plants to the wide range of
environmental stimuli. Phosphatidylcholine-specific phospholipase C (PC-PLC) catalyses hydrolysis of lipid phosphatidycholine into phosphocholine and diacylglycerol, both molecules are potential second messengers. Expression of this
enzyme was recently detected in Arabidopsis thaliana during phosphate starvation. We have revealed in silico four PCPLC homologs in maize genome and we studied the effect of phytohormones on their expression.
Increased expression of genes ZmPC-PLC2, 4 and 6 after auxin, cytokinin and ABA treatment was found by qPCR.
Currently, the effect of these phytohormones on PC-PLC activity in vitro and in situ is investigated. The results obtained
indicate possible role of PC-PLC in phytohormone related signal transduction events.
This work was supported by the grant no. LC06034 of the MSMT and by grants no. 522/05/0340 and 522/07/1614 of
the CSF.
PS1143 ECTOPIC EXPRESSION OF PRE1-INTERACTING PROTEIN1 CONFERS GA-INSENSITIVE DWARFISM IN
ARABIDOPSIS THALINA
Sumin Lee, Moon-Soo Soh Dept. Molecular Biology, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747,
Korea
[email protected]
Previously, we reported that PRE1, a helix-loop-helix nuclear protein, is involved in gibberellin (GA) signaling as
a positive regulator. Here we report that PRE1-interacting protein1 (PRI1) is involved in GA-response in Arabidopsis.
PRI1 was identified as PRE1-interacting protein via yeast-two hybrid screening. The closest homolog of PRI1 also interacted with PRE1, thus we named it as PRE1-interacting protein2 (PRI2). PRI1 formed homodimer or heterodimer with
PRI2. To investigate the function of PRI1 in GA signaling, we overexpressed PRI1 under 35S promoter. The overexpression of PRI1 induced severe dwarfism, dark green leaves, and delayed flowering. While the phenotype is very similar to
that of GA-deficient mutant, GA-treatment did not rescue the phenotype.
PRI1 gene was induced by GA treatment, both in seed and seedlings suggesting possible involvement of PRI1 in GAsignaling. And the PRI1- overexpressing seeds showed hypersensitivity to PAC, a GA inhibitor during germination.
Together with gene expression pattern in PRI1 overexpressing plant, we discuss possible roles of PRI1 in GA response
of Arabidopsis thaliana .
PS1144 CHARACTERIZATION OF COTTON GIBBERELLIN RECEPTORS
Aleman-Sarinana, Lorenzo1, Kitamura Jun2, Nakajima, Masatoshi2, Ueguchi-Tanaka, Miyako3 , Matsuoka, Makoto3,
and Allen, Randy4 1Deptartment of Plant and Soil Sciences, Texas Tech University, Lubbock, TX 79409, USA; 2Department of Applied Biological Chemistry, The University of Tokyo, Tokyo 113-8657, Japan; 31Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan; 4Department of Biological Sciences, Texas Tech University,
Lubbock, TX 79409, USA
[email protected]
Two genes (GhGID1a and GhGID1b) that encode receptors for gibberellin were identified from cotton (Gossypium
hirsutum). These GhGID1 genes along with two cotton DELLA genes (GhSLR1-1 and GhSLR1-5) were functionally
characterized. A yeast two-hybrid system showed GA-dependent in vivo interaction the GhGID1s and the GhSLRs. Similarly, GhGID1s showed interaction with the rice OsSLR1. Recombinant GhGID1 proteins showed binding activity to
GA4 in vitro. The cotton GhGIDs showed an increase in GA-binding with the addition of either, GhSLR1-1, GhSLR1-5,
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or OsSLR1, similar to the effect the Arabidopsis DELLAs had on the binding activity of the Arabidopsis receptor proteins
(AtGID1s)(Nakajima et al., 2006), The expression of the GhGID1a gene in the rice gid1-3 mutant rescued its phenotype
demonstrating that it is a functional GA receptor.
PS1145 A GAIN-OF-FUNCTION MUTATION OF ELONGATED-D SUPPRESS THE DW0RF PHENOTYPES OF BOTH
BRASSINOSTEROIDS BIOSYNTHETIC AND SIGNALING MUTANTS
Yuhee Chung, Mi Kwon, Ok Sun Lee, Woo Suk Cho, and Sunghwa Choe* School of biological sciences, College of
natural sciences, Seoul National University, Seoul 151-747, Korea
[email protected]
Brassinosteroids (BRs) stimulate plant growth through a receptor kinase-mediated signaling pathway. Contrast to
the conventional BR dwarf phenotypes, tall plant stature can be induced due to a gain-of-funtion mutation in a gene for
positive regulator or a loss-of-function mutation in a negative regulator in BR signaling pathways. We obtained a list of
long hypocotyl mutants from Arabidopsis Biological Resources Center (ABRC), and made double mutants with a BR
biosynthetic mutant dwf7-1. Among the mutants tested, elongated-D (elg-D) caused a significant suppression of a dwarf
phenotype seen in dwf7-1. Thus, we hypothesized that elongated-D has a dominant mutation in one of the BR signaling
components. Using a map-based cloning method, we identified that ELG encodes for BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1). The elg-D mutation caused a substitution of Asp at the 123th amino acid
residue for Ile, which is located in an extracellular Leucine-rich repeat domain of BAK1. To further understand the effect of this mutation, we introduced the elg-D mutation into BR signaling mutants including bri1-5, bin2-1D/dwf12-1D,
bzr1-D, and bes1-D through genetic crossing. elg-D mutation suppressed the dwarfism of bri1-5 more than three folds
in the whole plant height. However, the level of suppression of the bin2-1D/dwf12-1D mutant was about 30% that of a
single mutant, confirming that the bin2-1D/dwf12-1D mutation is epistatic to the elg-D. Currently, we hypothesize that
the elg-D mutation constitutively transmits signal even in an absence of brassinolide possibly due to structural change in
the LRR domain into active form, which is normally shaped by brassinolide binding.
PS1146 AUXIN TRANSCRIPTIONAL SIGNATURE OF ARABIDOPSIS
Claude Becker1, Francesco Pinosa1, Ivan A. Paponov1, Martina Paponov1, William Teale1, Margit Menges2, Sohini
Chakrabortee2, Jim A.H. Murray2, Klaus Palme1 1Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104
Freiburg, Germany 2Institute of Biotechnology, University of Cambridge, CB2 1QT Cambridge, United Kingdom
[email protected]
The hormone auxin shapes gene expression to regulate growth and development. Despite the detailed characterization of auxin-inducible genes, a comprehensive overview of the temporal and spatial dynamics of auxin-regulated gene
expression is lacking. We have combined transcriptional data from many experiments and assessed tissue-specific gene
expression in response to auxin concentration and exposure time and in relation to a network of other plant growth regulators. The primary response to auxin comprised almost exclusively the up-regulation of genes. We present a framework
for tissue specific auxin responses and demonstrate context- and sequence-specific patterns of expression. Our analysis
provides a roadmap for auxin-dependent processes underpinning the concept of an ʻauxin codeʼ: a tissue-specific fingerprint of gene expression that initiates specific developmental processes.
Light Responses
PS1201 THE ROLE OF DAYLENGTH AND GIBBERELLIN IN THE REGULATION OF AXILLARY BUD DIFFERENTIATION IN STRAWBERRY
Hytonen Timo 1, Moritz Thomas 3, Elomaa Paula 1, Junttila Olavi 2 1 Dept Applied Biol, P.O.Box 27, FIN-00014 Univ
Helsinki, Finland; 2 Dept Biol, Univ Tromsø, Dramsveien 201, N-9037 Tromsø, Norway; 3 Swedish Univ Agr Sci, Umea
Plant Sci Ctr, Dept Forest Genet & Plant Physiol, SE-90187 Umea, Sweden
timo.hytonen@helsinki.fi
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Differentiation of long and short shoots is an important developmental trait in several species of Rosaceae family, since
flowers are typically formed on short shoots in these species. However, the physiological mechanisms controlling this differentiation are largely unknown. We have studied the role of daylength and gibberellin (GA) in the regulation of shoot differentiation in strawberry (Fragaria x ananassa Duch.). In strawberry, differentiation of axillary buds to runners (long shoot)
and to crown branches (short shoot) is promoted by long-day (LD) and short-day (SD) conditions, respectively.
We found that active GA, produced in the axillary bud or in the vicinity of the bud, is required for runner growth. We
didnʼt find differences in the levels of bioactive GA in SD and LD grown buds just before the determination of bud developmental fate, suggesting that the GA level may not be a primary factor regulating the axillary bud differentiation. Four
days later, however, two-fold increase in GA1 level in LD buds compared to SD was associated with observed runner
growth. We also tested if bud fate could be changed by GA3 treatment. GA3 or transfer from SD to LD alone was virtually ineffective, but the combination of GA3 and transfer from SD to LD restored the runner growth, indicating that the
daylength affects the bud responsiveness to GA. The effect of daylength on the expression of several GA biosynthetic,
signalling and target genes, and on GA sensitivity of strawberry axillary buds will be discussed.
PS1202 ISOLATION AND CHARACTERIZATION OF A NEW PHOTOGERMINATION MUTANT
Dong Hwan Kim, Soohwan Lim, and Giltsu Choi
[email protected]
Seed germination determines the habitat and growth season of a plant. Since these are critical for the survival of
a plant, a seed integrates various environmental signals including temperature, water availability, and light condition.
Phytochromes in the seed are known to integrate light signals to promote seed germination. The activation of seed germination by phytochromes, however, is less well understood phytochrome-mediated light responses. We have previously
identified that PIL5, a phytochrome-interacting bHLH transcription factor, is one of key components that mediate light
signaling during seed germination. The detailed analysis of the pil5 mutant suggested that seed germination is regulated
not only by PIL5 but also by other components. Through a mutant screening, we have isolated a new Arabidopsis mutant
named as somnus1 (som1-1) that germinates irrespective of light condition. The som1-1 mutation was caused by a TDNA insertion into a novel gene encoding a zinc finger protein. A new mutant allele (som1-2) obtained from the SALK
lines also caused the same light-independent seed germination, supporting that the mutation was caused by the disruption of the identified gene. Consistent with mutant phenotypes, the overexpression of SOM1 inhibited both PHYA- and
PHYB-mediated promotion of seed germination. Taken together, our results indicate that SOM1 is a new player in photogermination. We are currently investigating how SOM1 regulate seed germination at the molecular level.
PS1203 THE EFFECTS OF LIGHTS AND GIBBERELLIN ON HOOK OPENING AND COILING RESPONSE OF SEEDLINGS OF CUSCUTA AUSTRALIS
Li Dongxiao 1 Zhang Huihuang 1 Zhang Guoguang 1 Tian Huiqiao1 Chen Liang 1 (corresponding author) 1. School
of life sciences, Shengwu 2 Building, Xiamen University, Xiamen, Fujian 361005, China. [email protected];
[email protected]; [email protected]; [email protected]; [email protected]
[email protected]
Cuscuta spp is a holoparasitic higher plant which can invade a large range of species. This ability indicates that they
must have highly adaptable mechanisms for host attachment, characterized by entwining the stem or leaves of hosts and
developing haustoria to obtain nutrition from the hosts after hook opening of the seedlings. Unfortunately, the nature of
the mechanism(s) is unknown. Furthermore, little is known about the effect of gibberellins (GAs) on responses of hook
opening and coiling of seedlings of dodders, which regulate many aspects of plant development such as germination, leaf
expansion, stem elongation, flowering time and fertility. In the present study, using light-emitting diode (LED) as light
sources, the responses of hook opening and stem twining of seedlings of Cuscuta australis were studied by different light
treatments or by using GA3 and GAs biosynthesis inhibitor paclobutrazol. The results showed that blue light displayed
different effects on hook opening and stem twining in a continuous process: a synergistic interaction with phytochromes
(Pfr) during hook opening and subsequently an antagonistic interaction with Pfr during stem twining process.
IPGSA 19th Annual Meeting - Abstracts
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And there may be also existed dark conversion of Pfr to Pr. We also confirmed definitely that the induction of twining
and haustoria development is triggered by light signals and does not require any chemical signal from the host plants.
Furthermore, the results also showed that GA3 have no distinct effect on hook opening response, but is involved in the
twining response of the seedlings.
PS1204 PIL5 REGULATES GIBBERELLIN RESPONSIVENESS BY DIRECTLY BINDING TO THE GAI AND RGA PROMOTERS IN ARABIDOPSIS SEEDS
Eunkyoo Oh1, Shinjiro Yamaguchi2, Jianhong Hu3, Jikumaru Yusuke2, Inyup Paik1, Tai-ping Sun3, Yuji Kamiya2, and
Giltsu Choi1 1Department of Biological Sciences, KAIST, Daejeon 305-701, Korea 2RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan 3Department of Biology, Duke University, Durham, NC27708-1000, USA
[email protected]
Gibberellin (GA) plays a key role in mediating light-regulation of Arabidopsis seed germination. It was shown that
light increases GA biosynthesis by inducing expression of two GA biosynthetic genes (GA3ox1 and GA3ox2). Previous
reports also indicated that PIL5, a phytochrome-interacting light-liable bHLH protein, inhibits seed germination by repressing GA3ox1 and GA3ox2, and activating a GA catabolic gene (GA2ox2). However, the persistent light-dependent
and PIL5-inhibited germination behavior in the absence of both de novo GA biosynthesis and deactivation by GA2ox2
suggested that PIL5 regulates not only GA metabolism but also GA responsiveness. Gene expression analyses showed
that PIL5 increases the expression of two GA repressor (DELLA) genes, GAI and RGA, in darkness. The significant
role of this transcriptional regulation in seed germination was shown by the hypersensitivity of the gai-t6 rga-28 double
mutant to red light for seed germination and the suppression of germination defects of a PIL5-overexpression line by the
rga-28 mutation. Beyond regulation of GA-related genes, PIL5 increases abscisic acid (ABA) levels by activating ABA
biosynthetic genes and repressing an ABA catabolic gene. Chromatin immunoprecipitation analyses indicated that PIL5
directly binds to GAI and RGA promoters, but not to GA and ABA metabolic gene promoters. Taken together, our results
show that light signals perceived by phytochromes cause a reduction in the PIL5 protein level, which in turn regulates
the transcription of two DELLA genes directly and that of GA and ABA metabolic genes indirectly.
PS1205 ABA MEDIATES THE RESPONSE OF GRAPE LEAF TISSUES TO UV-B RADIATION
Berli, Federico1; Moreno, Daniela1; Hespanhol Viana, Leandro2; Bressan Smith, Ricardo2; Bottini, Rubén2 1 Facultad
de Ciencias Agrarias-CONICET, Universidad Nacional de Cuyo, Alte. Brown 500, M5528AHB Chacras de Coria, Argentina; 2 Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro,
Av. Alberto Lamego 2000, 28013-620 Campos dos Goytacazes, RJ, Brasil; [email protected]
[email protected]
The UV-B radiation affects growth and development in plants, especially by a well documented effect on nucleic acids.
In grapes, it is assumed that UV-B promotes metabolic changes that lead to an increased amount of substances related
with the flavor of the wine. Our hypothesis is that ABA is involved in the grape plant responses to UV-B. An experiment
was performed in which UV-B, ABA and tungstate (W, ABA aldehyde oxidase inhibitor) treatments were combined in a
factorial design. ABA stimulated leaf pigments (chlorophylls a and b, and carotenes) respect to W, but only with significant differences under an UV-B filtered solar radiation. UV-B showed determinant for higher levels of flavonoids, and
both flavonoids and anthocyanin levels were enhanced by ABA as compared to W, but only under enriched (4.5 kJ m-2
d-1) solar radiation and not when the UV-B was filtered. UV-B did not show effects per se on the activity of 4 enzymes
involved in antioxidant response, namely CAT, APX, SOD and GPX. CAT and GPX activities were pharmacologically
enhanced either under enriched- and filtered-UV-B solar radiation, but the response was elicitated by both ABA and W.
APX activity increased only with ABA in UV-B enriched solar radiation. Conversely, SOD activity was inferior in ABAand W-treated plants under enriched UV-B solar radiation. These preliminary results suggest that ABA intermediates the
plant-cell responses to UV-B.
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PS1206 LIGHT QUALITY MODULATES CYTOKININS LEVELS AND ROOT-TO-SHOOT CONVERSION IN CATASETUM FIMBRIATUM.
Rodrigues, Maria Aurineide1; Freschi, Luciano1; Kerbauy, Gilberto Barbante1. 1Departamento de Botânica, Universidade de São Paulo, São Paulo, SP, Brazil.
[email protected]
Cytokinins (Cks) have been shown to mediate a wide range of photomorphogenic events regulated by red and blue
light through the action of phytochromes and cryptochromes, respectively. Both Cks and dark were previously shown as
important signs that regulate the conversion of isolated root tips of Catasetum fimbriatum into buds. In this study we investigated the effects of dark and different light qualities (far-red, white and blue) on the frequency of root-to-shoot conversion and, also, on the predominance of specific forms of Cks (Z, ZR, iP and iPR) over the first 12 hours after the root
tip isolation and incubation under the light treatments. The results showed that the dark was the most effective treatment
in promoting the conversion of root tips into buds, and far-red was the second most inductive. The treatment using white
light, when compared to dark and far-red, caused a slight delay in completing the event, while blue light was strongly
inhibitory to the sequence of changes involved in the root-to-shoot conversion. Before the root tip isolation and light
treatments, iP was found to be the predominant form of Ck. However, after only two hours of root detachment, the levels
of iP dropped and Z started being the most representative form of free Ck in root tips treated with dark, far-red, and white
light. On the other hand, the material incubated in blue light did not show this tendency, and levels of both free Cks were
practically the same during the period of analysis. Furthermore, the treatments that enhanced the conversion frequency
(dark and far-red) showed iPR peaks during the period of incubation. These results suggested that specific organogenetic
responses of isolated root tips of C. fimbriatum are closely associated with the specific action of light quality on Ck
forms: dark and far-red enhanced root-to-shoot conversion and stimulated the predominance of Z as free Ck, whereas
blue light strongly inhibited both events over the first 12 hours of isolation. The possible influence of phytochrome and
cryptochrome on these responses will also be discussed. Supported by FAPESP.
PS1207 LIGHT DEPENDENT TRANSCRIPTIONAL PROFILE OF RAPHANUSANIN-INDUCED GENES IN RADISH HYPOCOTYLS
Moe Hninsi 1, Kosumi Yamada 1, Tsuyoshi Hasegawa 1, Hideyuki Shigemori 1, Takuya Maoka 2, Toshihiko Otomatsu 2,
Katsutoshi Hirose 2, Koji Hasegawa 1,2 1 Graduate School of Life and Environmental Sciences, University of Tsukuba,
Ibaraki, Japan; 2 KNC Laboratories Co., Ltd., Hyogo, Japan
[email protected];[email protected]
The bending of a plant toward the direction of intense light is called phototropism. This directional growth response
is caused by the plant growth regulating substances. In this aspect, Bruinsma-Hasegawa hypothesis (1990) stated that the
gradient of growth-inhibiting substances (designated as phototropism-regulating substances) in the illuminated side is
a key factor of bending during phototropic curvature. Although some phototropism-regulating substances were isolated
from several plant species, the molecular mechanism underlying this phenomenon is still largely remained. The symbolic
phototropism-regulating substances, cis- and trans-raphanusanins have been isolated from radish hypocotyls. These compounds were dramatically accumulated on the side of blue light illumination. To understand the role of raphanusanins
in phototropism and the responsible genes for the growth inhibition, DD-PCR was performed between the raphanusanin
applied and control hypocotyls. We could trace some candidate genes indicating a close linkage between raphanusanins
and phototropic curvature. The detailed roles and functional impacts of these genes for the growth inhibition in response
to phototropic stimulation will be presented.
Abiotic Stress
PS1301 FUNCTION OF PYRROLOQUINOLINE QUINONE (PQQ) IN PLANTS
Natsumi Noji‚P2, Nobutaka Kitahata2, Takeshi Nakano2, Hideki Goda2, Shigeo Yoshida 2, Masafumi Tsujimoto2,
Tamizi Sugiyama‚P, Tadao Asami2 3 (1Graduate School of Agriculture, Meiji University, Kanagawa, Japan ‚QRIKEN,
Saitama, Japan, 3Department of Applied Biological Chemistry, The University of Tokyo, Tokyo, Japan) [email protected]
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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Pyrroloquinoline quinone (PQQ), an element first discovered as a novel cofactor in bacterial dehydrogenase, has
been identified as an important player in improving reproductive performance in mammals and now recognized as a new
vitamin.
Recently, we demonstrated the existence of PQQ in model plant Arabidopsis thaliana by LC/MS/MS analysis. This result
suggests that PQQ should be biosynthesized and might function in plants. In this context, we started the elucidation of
PQQ functions in plants, Treatment of Arabidopsis with PQQ had no effect on germination and growth, but conferred distinct stress resistance to Arabidopsis grown under various abiotic stress. This effect of PQQ treatment was also observed
in rice or bird_fs foot trefoil. Now, we have started the clarification of the mechanism of the stress resistance conferred
by PQQ treatment
PS1302 LOCALIZATION, CROSS-LINKING AND QUANTIFICATION OF TWO CELL WALL PROTEINS ON BEAN
PLANTS DURING WATER-TEMPERATURE STRESS (PHASEOLUS VULGARIS L.)
Mergold Claudia*, Cassab Gladys. Dep. Biol. Mol. de Plantas, Instituto de Biotecnología, Universidad, Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Mor., 62250 México. *[email protected] Tel.: +52(55)5622 7658
Fax: +52(55)313 6600
[email protected]
THE TIGHTLY REGULATED EXPRESSION PATTERNS OF STRUCTURAL CELL WALL PROTEINS IN SEVERAL PLANT SPECIES INDICATE THAT THEY PLAY A CRUCIAL ROLE IN DETERMINING THE FINAL STRUCTURE FOR SPECIFIC CELL TYPES. HERE, WE SHOWED THAT PRP´S, A PROLINE-RICH CELL WALL PROTEINS IN BEAN, ARE HIGHLY SOLUBLE IN THE ELONGATING REGION OF HIPOCOTYLS WHEN PLANTS
ARE WATER-TEMPERATURE STRESSED. THESE RESULTS INDICATED THAT PRP´S ARE REGULATED BY
HIGH TEMPERATURE AND LOW WATER STATUS DURING GROWTH AND DEVELOPMENTAL PATHWAYS.
PS1303 WHEAT GENOTYPIC VARIATION AND PROTEIN MARKERS IN RELATION WITH IN VITRO SELECTION FOR
DROUGHT TOLERANCE
M.S. Abdel-Hady Botany Dept., National Research Center, Dokki, GIZA ,12622 Egypt. E-mail: msabdelhady@hotmail.
com
[email protected]
Abstract: Embryogenic calli isolated from immature embryos of five durum wheat genotypes included one local
cultivar (Benysweif-2), as well as four introduced lines (MSWD-2, MSWD-9, MSWD-19 and NRCWD-16) were subjected in vitro selection for drought tolerance. The effect of polyethylene glycol (PEG) on the embryogenic cultures
and plant regeneration had been investigated. The results indicated that the relative growth weight of callus was highly
significant influenced by differences in durum wheat genotypes and PEG concentrations. The relative growth of callus
decreased rapidly as the concentrations of PEG increased. At the concentration of 5% PEG, all cultures of the five genotypes studied could tolerate that level of drought. At the concentration of 10% PEG, MSWD-2 and MSWD-9 gave the
highest percentage on callus growth rate, while Benysweif-2 the lowest one. At the highest concentration of 20% PEG,
plant regeneration percentage has been succeeded in the genotypes (MSWD-2, MSWD-9 and MSWD-19) with minimal
reduction, while (NRCWD-16 and Benysweif-2) scored highly reduction at that concentration. The plant regeneration
is a good parameter to evaluate the effect of drought. The analysis of water soluble, non-soluble and alcoholic soluble
(gliadin) proteins by SDS PAGE was detected. Some newly protein markers for drought tolerance were induced under
different PEG concentrations, such as water soluble protein bands in the tolerant genotypes MSWD-2 and MSWD-19,
respectively. Other water soluble proteins were expressed only at the higher drought stresses in MSWD-2 and MSWD-9.
Moreover, the gliadin proteins were induced in MSWD-9 and MSWD-19, respectively. While three protein bands were
induced under the higher concentrations of PEG 10 and 20% in MSWD-2. Mean while, some protein bands disappeared
under the highest drought stress in NRCWD-16 and Benysweif-2. The newly induced protein bands could be used as
markers for indirect selection to the drought tolerance, and this could support the development of drought tolerant genotypes of durum wheat.
Keywords:Drought tolerant, durum wheat, in vitro, polyethylene glycol, protein markers.
IPGSA 19th Annual Meeting - Abstracts
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PS1304 WATER RELATIONS, GAS EXCHANGE, NODULATION, NITROGEN FIXATION AND GROWTH OF FABA BEAN
(VICIA FABA L.) IN RELATION TO THE DRYING OF SURFACE ROOTS
Abdelhamid, Magdi A.B; Siddique, K.H.M.C, Turner, NeilB; Palta, JairoD, Veneklaas, ErikE and Atkins, CraigE ANational Research Centre, Botany Department, Al-Behooth Street, Dokki, Cairo, Egypt, Fax: 00202 3370931, E-mail:
mtawfi[email protected] or [email protected] BCentre for Legumes in Mediterranean Agriculture (CLIMA), The University of Western Australia, Faculty of Natural & Agricultural Sciences, 35 Stirling Highway, Crawley,
Western Australia 6009, Australia CInstitute of Agriculture, The University of Western Australia, Faculty of Natural &
Agricultural Sciences, 35 Stirling Highway, Crawley, Western Australia 6009, Australia DCSIRO, Plant Indutry, Private
Bag No. 5 Wembley, WA 6913, Australia ESchool of Plant Biology, The University of Western Australia, Faculty of
Natural & Agricultural Sciences, 35 Stirling Highway, Crawley, Western Australia 6009, Australia EPresent address:
mtawfi[email protected]
The segmented soil column system in which the root system divided into two layers, each 24 cm in diameter and 33
cm high used to test the hypothesis that leaf gas exchange, nodulation, nitrogen fixation and growth of faba bean (Vicia
faba L.) plants were/were not reduced when the surface soil is dried. The experiment was conducted, in a glasshouse
at the University of Western Australia, Perth (31.93 S, 115.83 E), from June to August 2006. Faba bean (Vicia faba L.
var. minor) breeding line SP98002 was subjected to 2 watering regimes. The watering regimes were: (i) well-watered,
and (ii) water withheld at flowering, from 61 to 81 days after sowing (DAS). The treatments imposed as, (i) all two layers adequately watered (control), and (ii) the upper layer un-watered with the remaining layer kept adequately watered
(droughted). The treatments have been applied at flowering (i.e. where 50% of the plants had one open flower), exactly
at 61 days after sowing. Measurements were done at 1, 4, 7, 11, 14, 17 & 21 days after drying (DAD).
Withholding water resulted in significant increase in LWP in all days of measurements compared to control except of
measurement at 1 DAD, control leaves scored higher values than stressed ones. Osmotic potential (OP) did not differ
significantly between treatments, control and drying surface roots. Relative water content (RWC) mean values over time
of measurements were 89.8 and 86.6 % for well-watered and droughted treatments, respectively and the difference was
significant. Drying surface roots significantly resulted in reduction in net photosynthetic rate in all measurements except
of measurements at 7 DAD. Withholding water significantly (pƒ¬ 0.05) reduced stomatal conductance over time of measurements compared to control and mean values were 568.2 and 448.5 mmol/m2/s, respectively. Drying surface roots
significantly (p ƒ¬ 0.05) resulted in reduction in SPAD values over time compared to control and SPAD mean values
were 34.9 & 39.4, respectively. Plant height, branch number/plant, leaf number/plant, green area and shoot biomass at
61 and 81 DAS, respectively were not significantly affected by drying surface roots. Relative growth rate (RGR), leaf
area ratio (LAR), and net assimilation rate (NAR) were not significantly (p ƒ¬ 0.05) reduced due to drying surface roots.
Drying surface roots significantly reduced leaflet width over time compared to control with mean values 18.3 & 20.1 mm,
respectively. Leaflet length over time of droughted plants was significantly reduced due to drying surface roots compared
to control with mean values 49.4 & 54.3 mm, respectively. Drying surface roots resulted in significant reduction in nodule number/plant and nodule biomass (mg/plant) on top layer and total pot. N2 fixation (%) was significantly reduced in
droughted plants and the reduction was representing 20.1% compared to well-watered plants.
PS1305 CLONING AND CHARACTERIZATION OF A VERSATILE C-REPEAT BINDING FACTOR FROM BRASSICA
JUNCEA
Gopal K. Chowdhary, Akhilesh K. Yadav, Parul Saxena and Renu Deswal Plant Molecular Physiology and Biochemistry
Laboratory, Department of Botany, University of Delhi, Delhi-110007, India.
[email protected]
C-repeat binding factor (CBF) in the most well worked out transcriptional activator in low temperature (LT) signaling
network. CBF has been cloned from LT sensitive (tomato, rice etc.) and LT tolerant (Arabidopsis, Brassica napus) plants.
Mostly, it is induced by LT only but in some plants it is induced by salt (Eucalyptus CBF, barley CBF1), drought (Capsicum CBF, barley CBF1, Vitis vinifera CBF 2 & 3) and heat (Capsicum CBF) also in addition to LT. Here, we report the
cloning of a CBF from Brassica juncea which is induced by all the abiotic stresses (LT, drought, salt and heat), although
induction time, peaking and transcript maintenance time varies.
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CBF mediated signaling is principally an ABA independent pathway, but BJCBF (Brassica juncea CBF) is induced by
ABA. In addition to BJCBF only Eucalyptus and Vitis vinifera CBF 2 are induced by ABA. The induction time of BJCBF
with respect to LT and ABA varies but maximum expression is same, 2 hrs for both. BJCBF is the earliest induced CBF
by cold. Moreover, for the first time its induction by calcium is shown. Interestingly, calcium can replace cold to induce
CBF expression.
PS1306 ENHANCED TOLERANCE OF TRANSGENIC SWEET POTATO PLANTS EXPRESSING BOTH CUZNSOD AND
APX IN CHLOROPLASTS TO METHYL VIOLOGEN-MEDIATED OXIDATIVE STRESS AND CHILLING
Soon Lim, Yun-Hee Kim, Sun-Hyung Kim, Haeng-Soon Lee, Sang-Soo Kwak Environmental Biotechnology Research
Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
[email protected]
Oxidative stress is one of the major factors causing injury to plants exposed to environmental stress. To develop
transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants with enhanced tolerance to multiple environmental
stresses, genes of both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of
an oxidative stress-inducible SWPA2 promoter were expressed in chloroplasts of sweetpotato plants (referred to as SSA
plants). SSA plants were successfully generated by the particle bombardment method and were confirmed by PCR analysis. When leaf discs of SSA plants were subjected to 5 uM methyl viologen (MV), they showed approximately 45% less
damage than non-transformed (NT) plants. When 200 uM MV was sprayed onto the whole plants, SSA plants showed a
significant reduction in visible damage compared to leaves of NT plants that were almost destroyed. The expression of
introduced CuZnSOD and APX genes in leaves of SSA plants after MV treatment was significantly induced in well reflecting increased levels of SOD and APX in chloroplasts. The APX activity in chloroplast fractions from SSA plants was
approximately 15 times higher that that from NT plants. In addition, when treated with chilling stress at 4oC for 24 hr,
SSA plants exhibited less decrease of the photosynthetic activity (Fv/Fm) compared to NT plants. Furthermore, after 12
h of recovery following chilling, the Fv/Fm of SSA plants almost fully recovered to the initial levels, whereas NT plants
remained at lower Fv/Fm activity. These results suggest that SSA plants would be useful for commercial cultivation in
unfavorable growth conditions. In addition, the manipulation of the antioxidative mechanism in chloroplasts can be applied in the development of various other transgenic crops with increased tolerance to multiple environmental stresses.
PS1307 DO BRASSINOSTEROIDS MEDIATE THE PLANT’S RESPONSE TO WATER STRESS?
Jager, Corinne E, Symons, Gregory M, Ross, John J, Reid, James B School Plant Sci., Univ. of Tasmania, Private Bag
55, Hobart, TAS, 7001, AUS [email protected], Ph: 61 3 62262605, Fax: 61 3 62262698
[email protected]
The application of brassinosteroids (BRs) has been suggested to increase a plantʼs resistance to a variety of stresses,
including water stress. However, it is not known whether endogenous BRs are involved in mediating the plantʼs response
to these stresses. We have utilised BR mutants in pea (Pisum sativum L.) to determine whether endogenous BRs are involved in water stress and whether altered BR levels help the plant cope better with drought. We show that drought causes
a significant increase in ABA levels, but does not result in altered BR levels in either apical, internode or leaf tissue.
Furthermore, the plantʼs ability to increase ABA levels in response to drought is not affected by BR deficiency, as there
is no significant difference in ABA levels between wild-type and lkb (a BR-deficient mutant) plants before or 11-days
after drought. Therefore, it appears that the plantʼs response to drought is not mediated through changes in endogenous
BR levels.
PS1308 IDENTIFICATION OF ARABIDOPSIS DROUGHT TOLERANCE GENES VIA INFRARED THERMOGRAPHY
North, Helen M.1, Plessis, Anne1, Cournol, Raphaël1, Kraepiel, Yvan2, Botran, Lucy1, Delagrange, Aurore1, Larget,
Sidonie1, De Almeida, Aurélie1, Frey, Anne1, Giraudat, Jérôme3, Leung, Jeffrey3, Marion-Poll, Annie1 1Laboratoire
de Biologie des Semences, UMR204, INRA, AgroParisTech, Institut Jean-Pierre Bourgin, F-78026 Versailles Cedex,
France; 2Laboratoire des Interactions Plantes-Pathogènes, AgroParisTech, F-75251 Paris Cedex, France; 3Institut des
Sciences du Végétal, CNRS UPR 2355, F-91190 Gif-sur-Yvette, France
[email protected]
IPGSA 19th Annual Meeting - Abstracts
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The short-term response of plants to drought stress is stomatal closure mediated by ABA-dependent and ABA-independent mechanisms. Our goal is to identify new elements involved in stomatal closure and drought responses. Using the
technique of infrared thermography it is possible to visualize the temperature difference between plants that have open
stomata whose leaves are cooled by evapotranspiration and those whose stomata are closed which appear hot. A mutant
population, generated by ã-ray mutagenesis of the ABA-deficient mutant aba3, has been screened for plants with altered
leaf temperature. Seven suppressor mutants were identified that were more tolerant that the original mutant to progressive drought stress. Four of the mutants showed a clear recessive inheritance and chromosome walking, combined with
microarray analysis of RNA expression levels in leaves, is currently being used to identify the genes affected in three of
these. Characterization of the response of the suppressor mutants to ABA, abiotic and biotic stress will be presented.
PS1309 RSOSPR10 IS ABUNDANTLY EXPRESSED IN CORTEX CELLS SURROUNDING CENTRAL CYLINDER IN RICE
ROOTS AFTER SALT STRESS
Hatakeyama, Atsushi1, Ishii, Noriko1, Nick, Peter2, Furukawa, Toshiko1, Koshiba, Tomokazu1 1Dept Biol Sci, Tokyo
Metropolitan Univ, Tokyo, 192-0397, Japan; 2Bot Inst, Karlsruhe Univ, 76131 Karlsruhe, Germany E-mail; [email protected]
[email protected]
RSOsPR10 was originally found as the root specific protein induced by drought and salt treatment in rice. The mRNA
was induced specifically in roots by drought, salt, probenazole, rice blast fungus and jasmonic acid (JA), but not by low
temperature, abscisic acid and salicylic acid (SA) (Hashimoto et al., PCP, 2004). In this study, we confirmed that the
expression of RSOsPR10 was upregulated by salt via JA pathway by using JA deficient mutant, hebiba, and the induction was strongly suppressed by SA. Immunohistochemical localization of RSOsPR10 in roots after salt treatment was
investigated using the specific anti-RSOsPR10 antibody. RSOsPR10 protein was detected in the cortex cells that surrounded central cylinder in the roots. The expression was almost completely suppressed by SA treatment. These results
suggested the expressed and accumulated RSOsPR10 protein in the cortex cells functions to protect rice plants from
abiotic stresses.
PS1310 IS ABA RESPONSIBLE FOR THE ENHANCED TRANSPIRATION OF THE TOMATO PHYTOCHROME ENHANCED RESPONSE MUTANT HIGH PIGMENT1?
Carvalho, Rogério Falleiros1, Dodd, Ian2, Peres, Lázaro Eustáquio Pereira1 1Departamento de Ciências Biológicas,
“Escola Superior de Agricultura Luiz de Queiroz”, Universidade de São Paulo, Av. Pádua Dias, 11, Postal 9, Piracicaba,
SP, Brasil; 2 Lancaster University, LA1 4YQ Lancaster, UK. [email protected]
[email protected]
Stomatal aperture (and thus whole plant transpiration) is regulated by many endogenous (eg. plant hormones) and environmental (eg. light, temperature, CO2) factors and there is increasing interest in how these factors interact to regulate
plant water use (Dodd 2003). Our observations that the tomato (Lycopersicon esculentum Mill. Syn Solanum lycopersicum L.) mutant high pigment1 (hp1), which exhibits an exaggerated response to phytochrome (Kendrick et al. 1997),
presented a wilty phenotype typical of ABA-deficient mutants when grown in the glasshouse prompted us to examine
the interaction between photoreceptors and the plant hormone abscisic acid (ABA). When leaves were detached from the
plant and allowed to dry on the bench, both hp1 and the notabilis (not) ABA-deficient tomato mutants showed a greater
rate of water loss then the wild-type (WT) cv. Micro-Tom.
When leaves were detached and placed in vials containing a range of ABA concentrations, transpiration rates of hp1 and
not showed a similar sensitivity to ABA as WT leaves, indicating that neither mutant was ABA-insensitive. Evapotranspiration of whole plants in the greenhouse of hp1 and not mutants was less than WT plants since the mutants had a lower
leaf area. However, transpiration rate (per unit leaf area) was greater in hp1 and not than WT plants. Further experiments
will examine the effect of genetic background (hp1 is present in both dwarf cv. Micro-Tom and tall cv. Ailsa Craig) on
these water relations phenotypes, measure stomatal density and in vivo ABA concentrations of these mutants and construct a hp1not double mutant.
IPGSA 19th Annual Meeting - Abstracts
129
References:
DODD, I. (2003) Journal of Plant Growth Regulation 22, 32-46.
KENDRICK, R.E.; KERCKHOFFS, L.H.; VAN TUINEN, A.; KOORNEEF, M. (1997) Plant, Cell and Environment 20,
746-751.
PS1311 ETHYLENE PROMOTES SUBMERGENCE-INDUCED EXPRESSION OF OSABA8OX1, A GENE THAT ENCODES ABA 8’-HYDROXYLASE IN RICE.
Hiroaki Saika1, Masanori Okamoto2,3, Kentaro Miyoshi1, Tetsuo Kushiro2, Shoko Shinoda2, Yusuke Jikumaru2, Masaru Fujimoto1, Taku Arikawa1, Hirokazu Takahashi1, Miho Ando1, Shin-ichi Arimura1, Akio Miyao4, Hirohiko Hirochika4, Yuji Kamiya2, Nobuhiro Tsutsumi1, Eiji Nambara2 and Mikio Nakazono1 1Graduate School of Agricultural
and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; 2RIKEN Plant Science
Center, 1-7-22 Suehiro-cho, Yokohama, Kanagawa 230-0045, Japan; 3Tokyo Metropolitan University, Hachioji, Tokyo
192-0397, Japan; 4National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan
[email protected]
A rapid decrease of the plant hormone abscisic acid (ABA) under submergence is thought be a prerequisite for the
enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA),
an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is
catalyzed by ABA 8_L-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA
8_L-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-GFP fusion
protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA
levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA
levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment
of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the
expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes.
Moreover, pretreatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed
induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with
OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA
levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.
PS1312 INTERACTION BETWEEN NITRIC OXIDE AND ABSCISIC ACID MODULATES CRASSULACEAN ACID METABOLISM EXPRESSION IN PINEAPPLE PLANTS
Freschi, Luciano1; Rodrigues, Maria Aurineide1; Kerbauy, Gilberto Barbante1; Magalhães, José Ronaldo2; Mercier,
Helenice1. 1Departamento de Botânica, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil; 2EMBRAPA/
CNPGL, Juiz de Fora, MG 36038-330, Brazil
[email protected]
Nitric oxide (NO) has been shown to interact with abscisic acid (ABA) in the induction of important plant responses to
water stress, including stomatal closure and acquisition of drought tolerance. However, several ABA-mediated responses
to drought remain unanalyzed in relation to the possible involvement of NO in their signaling cascade.
Since both water stress and ABA stimulate the expression of Crassulacean acid metabolism (CAM) in C3-CAM facultative plants, this study attempted to investigate a possible interaction between NO and ABA during the C3 to CAM transition in young pineapple plants (Ananas comosus). To achieve this, plants were subjected to CAM induction by drought,
and their leaves were analyzed over 30 days for endogenous levels of ABA, relative intracellular NO content and degree
of CAM expression. Additionally, the effects of exogenous treatments with ABA, NO gas or NO scavenger (cPTIO)
were analyzed on CAM expression in pineapple. In all cases, the degree of CAM expression was evaluated by measuring
the nocturnal malate accumulation and activity of the CAM enzymes phosphoenolpyruvate carboxylase (PEPCase) and
malate dehydrogenase (MDH). The results showed that the endogenous levels of ABA and NO in drought-stressed plants
IPGSA 19th Annual Meeting - Abstracts
130
were positively correlated with the degree of CAM expression. Substantial increases in ABA and NO content, which
were detected after five days of drought, preceded the C3 to CAM transition by one day. Also, the highest levels of ABA,
NO, malate accumulation and activities of CAM enzymes were simultaneously achieved after 15 days of water stress.
Consistent with these results, the treatment with ABA or NO increased the pineapple CAM expression in a dose-dependent manner. Additionally, it was observed that the endogenous ABA content in NO-treated plants increased dramatically,
suggesting the possible influence of NO on ABA synthesis. In agreement with this hypothesis, the removal of NO from
the tissues of drought-stressed plants by treatments with cPTIO partially blocked the ABA accumulation. Altogether,
these results suggest that NO plays an important role in the regulation of CAM expression in pineapple plants, possibly
by acting as an upstream signal of drought-induced ABA accumulation. Supported by FAPESP Proc.05/54515-5
PS1313 THE ROLE OF PXPH-PHOSPHOLIPASE D IN ALUMINUM RESPONSE
Novotna, Zuzana1, Schwarzerova, Katerina2, Krepelova, Anna1, Pejchar, Premysl3, Martinec, Jan3, Valentová Olga1
1Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, Czech Republic
2Department of Plant Physiology, Charles University in Prague, Prague, Czech Republic 3Institute of Experimental
Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic
[email protected]
Aluminum (Al3+), which limits the root growth, has been recognized as the main toxic factor limiting crop productivity on acid tropical and subtropical soils. The inhibition of auxin transport by Al3+ ions has been identified as one of
possible targets of Al toxicity. Recent results suggest that phosphatidic acid (PA), a product of phospholipase D (PLD)
enzymatic activity, is required for the normal cycling of PIN2 containing vesicles as well as for auxin transport and distribution. It has been also shown that the overexpression of PLD_2 partially ameliorated the brefeldin A-induced inhibition
of PIN2 cycling. In our work we show that PLD_2 expression is enhanced by Al3+ treatment in Arabidopsis roots and
that the metabolic activity of PLD_2 and the level of PA are affected by Al3+ treatment in Arabidopsis cell suspension.
These results indicate that PLD_2 could play a role in early stages of Al3+ toxicity. We also hypothesize that Al3+-induced changes in PLD_2 activity are related to auxin transport inhibition in plants during Al3+ stress.
This work was supported by grant of Czech Science Foundation, grant no 522/05/0340 and MSM LC06034.
PS1314 DETERMINING OF TRAFFIC-RELATED HEAVY METAL BY USING TAGETES PATULA L.
J. Caselles1 and P. Zornoza2 1Departamento de Química Aplicada a la Ingeniería, E.T.S. de Ingenieros Industriales,
UNED, Ciudad Universitaria, 28040 Madrid, Spain.2Departamento de Química Agrícola,Geología y Geoquímica, Facultad de Ciencias, Universidad Autónoma, 28049 Madrid, Spain.
[email protected]
In order to evaluate the deposition and accumulation throughout the time of pollutants from vehicle emissions, the
concentration of Fe, Mn, Cu, Zn, Al, Ni and Pb were analysed in washed and unwashed tissues of marigold plants (Tagetes patula L). The plants were cultivated on soil collected from the main street of Madrid (Spain) in an urban and in
a suburban area. Substantial amounts of elements were removed simply by washing with demineralised water, which
removed almost 40% of Fe and at least 50% of Al and Pb in urban areas, where the aerial deposition took place. Washing
decrease also between 8% and 16% of Mn, Zn and Cu.
Throughout the growing season, the concentrations of Fe, Al, Ni and Pb increased and were significantly higher in
washed leaves collected from the urban area than those from the suburban area, indicating that this ornamental plant is
able to absorb Fe, Al, Ni and Pb and reflect the environmental changes in a short time (9 weeks). Periodic assessment of
the accumulation of trace elements in ornamentals plants grown in parks and gardens used as playgrounds for children in
urban areas with intense traffic is important in order to evaluate the rate of environmental pollution of these recreational
spaces.
IPGSA 19th Annual Meeting - Abstracts
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PS1315 nhr1 mutant of Arabidopsis thaliana is tolerant to drought
Salazar-Blas, Marco A.; Campos, María Eugenia, Cassab, Gladys I. and Quiroz-Figueroa, Francisco R.
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México,
Apdo. Postal 510-3, Cuernavaca, Morelos, México. c.p. 62210
[email protected]
Abiotic stress for example drought, salinity, low temperature is an ambient condition that affects plant growth and productivity by decreasing availability of water in cells. The survival of terrestrial plants depends upon the capacity of roots
to obtain water and nutrients from the soil. The aim of this work is to know whether nhr1 or suh1 mutant plants could tolerate severe drought inasmuch as both mutants were isolated under low water available system, and also to characterize
them phenotypically after germination. Seeds of wt, suh1 (superhydrotropic mutant, M. Saucedo and G. I. Cassab, data
unpublished) and nhr1 were germinated in water potential stressed system. Seedlings obtained after 11 days from stressed
condition were transferred into MS medium during 7 days for recovery. After seedlings were plated into pots with metromix at 22 ±1 °C (16/8 h day/night cycle) until being 35 days old, during that time the plants were saturated with water.
To water stressed them; plants were de-hydrated (without irrigation) over 70% of loss water in soil. After this treatment,
plants were watered to recovery. We compared wt, suh1 (homozygous and heterozygous) and nhr1 (heterozygous, homozygous nhr1 is lethal) in each experiment. When we compared the in vitro growth among wt, nhr1 and suh1, wt seeds
germinated and grew faster than both nhr1 and suh1 mutants; however suh1 growth was similar to wt. nhr1 plants showed
a very slow growth as compared with wt and suh1. When we water stressed the plants to know whether both nhr1 and/or
suh1 mutants could resist drought, only nhr1 supported low level of humidity, hnr1 tolerated up to 73±3 percent of water
loss and It had recovered its growth whereas neither wt nor suh1 survived. These results propose that NHR1 gene could
be involved in resistance to drought by regulate water loss through partial close of stomata; this physiological response
is controlled by the plant hormone abscisic acid (ABA). Cross talking between the delay in growth (cell cycle) and tolerance to drought would be mediated by ABA.
Supported by a grant from DeGAPA #
Interactions Between Hormonal Pathways: Biosynthesis/Signaling
PS1401 ABA IS ACTIVELY MODULATED IN FRUIT SET
Nitsch, Lisette 1, Feron, Richard 1, Rieu, Ivo 2, Ma, Jerry 2, Hedden, Peter 2, Mariani, Titti 1, Vriezen, Wim 1 1 Dep.
Plant Cell biology, Radboud university Nijmegen, toernooiveld 1, 6525ED Nijmegen, Netherlands 2 Rothamsted research insititute, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom
[email protected]
The first step of tomato (Solanum lycopersicon) fruit development is fruit set. Cytokinines, gibberellins and auxins are
known to be involved in fruit set and application of these hormones to the ovary induces fruit growth without fertilization.
We show evidence for a role for another plant hormone in this process, abscisic acid (ABA). ABA levels significantly
decrease in the ovary after pollination. Gene expression profiles show that both production and catabolism of ABA are
actively modulated. Especially, CYP707A-homologuous genes (which are catabolic enzymes) show intriguing expression profiles. Both, GA and auxin, influence expression of ABA production, and catabolic genes indicating that cross-talk
occurs between these hormones. ABA keeps seeds and auxillary buds in a dormant state and acts antagonistically to gibberellin and auxin in these tissues. A similar hormone balance appears also to be present in unpollinated ovaries and ABA
might prevent fruit development before fertilization takes place.
PS1402 IDENTIFICATION OF ETHYLENE AND AUXIN-DEPENDENT GENES ASSOCIATED WITH REGULATION AND
EXECUTION OF FLOWER ABSCISSION
Meir, Shimon 1, Lers, Amnon 1, Philosoph-Hadas, Sonia 1, Reid, S. Michael 2, Jiang, Ca-Zhong 2, Halaly, Vita 1,
Kochanek, Bettina 1, Burd, Shaul 1, Volpin, Hanne 3 and Kushnirski, Tzvika 3 1 Department of Postharvest Science
of Fresh Produce, ARO, The Volcani Center, Bet-Dagan 50250,Israel; 2 Department of Plant Sciences, University of
California, Davis, CA 95616 USA; 3 Department of Genomics & Bioinformatics, ARO, The Volcani Center, Bet-Dagan
50250, Israel
[email protected]
IPGSA 19th Annual Meeting - Abstracts
132
It is a generally accepted model that auxin flux through the abscission zone (AZ) prevents organ abscission by rendering the AZ insensitive to ethylene. We have analyzed the molecular changes associated with this sensitivity transition in
response to auxin depletion, which may occur also in other ethylene-dependent processes such as senescence and ripening. For this purpose we have used the newly released Affymetrix Tomato GeneChip to measure global gene expression
in pedicel AZ and near by non-AZ tissues of tomato (Lycopersicon esculentum) at various time points after removal of
the flower which serves as the auxin source. These included the time range of 0-8 h, when increased sensitivity to ethylene was acquired prior to abscission, and the time point of 14 h, when pedicel abscission actually occurred. In addition,
gene expression was measured at the same time points in AZ which was pre-treated with the ethylene action inhibitor,
1-methylcyclopropene (1-MCP) before flower removal, to block any direct effects of ethylene including abscission. We
have identified potential abscission regulatory genes and many new AZ-specific genes, the expression of which was
modified after flower removal. These include: members of the Aux/IAA gene family, ethylene-related genes involved
in its signal transduction and biosynthesis pathways, early and late expressed transcription factors and genes which encode post-translational regulators whose expression was modified specifically in the AZ. We have also identified novel
AZ-specific genes that might function in the later mechanistic stages of the abscission process, encoding for cell wall
degrading enzymes, involved in cell separation, generation of a protective layer and defense. This microarray analysis
allowed us to select an initial set of target genes for further functional analysis, using a useful system of virus-induced
gene silencing (VIGS) in tomato. With this VIGS-based study we could confirm that TAPG, a polygalacturonase specifically associated with the tomato AZ, is a key enzyme in the abscission process. This study represents the first microarray
analysis of the abscission process, which may shed light on the molecular changes associated with acquisition of ethylene
sensitivity in various tissues.
PS1403 SPY, A NEGATIVE REGULATOR OF GIBBERELLIN SIGNALING, PROMOTES CYTOKININ RESPONSES
Weiss David, Mimon Inbar and Greenboim-Wainberg Yaarit Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
[email protected]
We have previously shown that the serine/threonine-O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT)
SPINDLY (SPY), a negative regulator of GA (gibberellin) signaling, promotes cytokinin responses. GA also represses
the effects of cytokinin, suggesting that there is cross-talk between the two hormone-response pathways, which requires
SPY function. The DELLA proteins play a key role in the regulation of GA-signal transduction and in the interactions
between GA and other hormones. The current model suggests that SPY-mediated O-GlcNAc modification of DELLA
proteins is required for their activity. We tested if DELLA proteins are involved in cytokinin action, and whether spy
inhibits cytokinin responses by DELLA inactivation. Our results show that cytokinin has no effect on RGA stability or
on GA-induced RGA degradation. Moreover, while cytokinin responses were strongly suppressed by spy they were unaffected in the quadruple DELLA mutant gai,rga,rgl1,rgl2. Similar to animal OGTs, SPY, is present in both the nucleus and
the cytosol of Arabidopsis cells. To assess whether SPYʼs cytosolic vs. nuclear localization is required for the regulation
of cytokinin responses, we generated transgenic spy plants expressing a GFP-SPY-glucocorticoid receptor (GR) fusion or
GFP-SPY fused to a nuclear export signal (NES). Transgenic spy lines expressing GFP-SPY-NES or GFP-SPY-GR without dexamethasone (DEX) treatment, exhibited wild type phenotypes with normal sensitivity to cytokinin. Moreover,
DEX treatment to SPY-GR lines displayed partial spy phenotypes, including increased resistance to cytokinin. Together,
these results suggest that SPY acts in the cytosol to regulate cytokinin responses, independently of the DELLA-regulated
nuclear GA signaling cascade.
PS1404 MOLECULAR CLONING THE FULL-LENGTH CDNA OF ALDEHYDE OXIDASE
Lixia Yang, Jianhua Liang, Ling Li* Guangdong Provincial Key Lab of Biotechnology for Plant Development£¬College
of life science, South China Normal University, Guangzhou, China, 510631£¨£¨* requests for offprints£©
[email protected]
Aldehyde oxidase is an important enzyme involved in abscisic acid (ABA) biosynthesis in response to drought stress.
Since the enzyme catalyzes the last step of the pathway, ABA production sites may be determined by the presence of Aldehyde oxidase in Arabidopsis (AAO3) and Tomato (sit). In this study, a full-length cDNA corresponding to an aldyhyde
oxidase is isolated from leaves of peanut with RT-PCR strategy. The full-length cDNA, designated AhAO1, consisting of
IPGSA 19th Annual Meeting - Abstracts
133
a 326 bp 5¡¯ untranslated region and a 128 bp 3¡¯ untranslated region including a poly (A) tail of 21 nucleotides, and an
open reading frame of 4131 bp. The open reading frame encodes a polypeptide of 1377 amino acids with a calculated molecular weight of 150.427kDa and an isoelectric point(pI) of 6.889. Sequence analysis shows that the deduced amino acid
sequence of AhAO1 shared 61%, 59% and 55% identity with the AO from tomato, Arabidopsis and maize, respectively.
There are three domains for binding cofactors or prosthetic groups on the polypeptide chain: an N-terminal iron-sulfur
center domain, followed by the FAD binding and molybdopterin binding domains, as already pointed out in plant and
animal AOs.The AhAO1 cDNA was expressed in E.Coli and the purified recombinant AhAO1 protein was found. The
characteristic and expression analysis of AhAO1in peanut plants under drought stress are studying.
PS1405 LOCALIZATION OF ABA BIOSYNTHETIC ENZYMES UNDER DROUGHT STRESS CONDITION IN ARABIDOPSIS
Akira Endo1, Masanori Okamoto1, 2, Hanae Koiwai3, Yusuke Jikumaru1, Yuji Kamiya1, Tomokazu Koshiba2, Eiji
Nambara1 1RIKEN Plant Science Center, 2 Dept. of Biol. Sci. Tokyo Met. Univ., 3National Institute of Aglobiological
Sciences
[email protected]
Regulation of ABA biosynthesis is crucial for plant_fs adaptive responses to osmotic stresses. Arabidopsis 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3) is known to be the rate-limiting enzyme in ABA biosynthesis under drought
stress conditions. However, regulatory mechanisms by which drought signal induces the AtNCED3 expression are still
unknown. In the present study, we investigated the expression and localization of AtNCED3, AtABA2 and AAO3 in
turgid and dehydrated plants using specific antibodies against each enzyme. Western blotting showed that AtABA2 and
AAO3 were expressed in turgid plants, whereas AtNCED3 was undetectable. Induction of AtNCED3 was observed after
30 min of dehydration. The expression patterns of ABA biosynthetic enzymes were investigated by an immunohistochemical technique. In dehydrated leaves, the immunofluorescent signal for AtNCED3 was observed in vascular bundles.
In addition, AtABA2 and AAO3 were also localized in the identical tissues. These results indicate that drought induced
ABA production occurs in vascular systems.
PS1406 CHO1, A (-)-ABA-INSENSITIVE GENETIC LOCUS, IS INVOLVED IN THE REGULATION OF HORMONE METABOLISM GENE EXPRESSION DURING SEED IMBIBITION IN ARABIDOPSIS
Yano, Ryoichi1, Kanno, Yuri1, Jikumaru, Yusuke1, Kamiya, Yuji1, Nambara, Eiji1
1RIKEN Plant Science Center
[email protected]
Abscisic acid (ABA) plays essential roles in seed dormancy and germination. Increasing evidence has demonstrated
that regulation of endogenous ABA levels is important for the regulation of seed dormancy in response to changes in
temperature, light, and nutrition. The ability to integrate these environmental signals with ABA metabolism thus enables
seed to germinate under desirable growth conditions, ensuring successful seedling growth and plant reproduction. We
have previously identified and characterized Arabidopsis mutants that show (-)-ABA-insensitive seed germination. One
of the mutants, named chotto1 (cho1), carries a mutation in a gene encoding a seed-specific transcription factor. We found
that the transcript levels of genes for ABA catabolism and gibberellin biosynthesis were significantly higher in the cho1
mutant than wild type under certain seed imbibition conditions. Possible involvement of CHO1 in the regulation of the
hormone metabolism during seed imbibition will be discussed in this presentation.
PS1407 MECHANISTIC STUDIES EXPLORING THE EFFECT OF ABSCISIC ACID ON GIBBERELLIN CONTENTS
Kumala,Dewi1 and Chandler,Peter2 1 Faculty of Biology, Gadjah Mada University, Jl. Teknika Selatan, Yogyakarta
55281, INDONESIA 2 CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, AUSTRALIA
[email protected]
The effects of ABA on GA content, and the expression levels of mRNAs for GA biosynthesis have been evaluated.
Application of ABA (2 ìM or 5 ìM) or an accumulation of endogenous ABA imposed through dehydration reduced GA1
content. Some GA1 precursors, namely GA44 and GA20, were also present at lower contents following ABA or dehy-
IPGSA 19th Annual Meeting - Abstracts
134
dration treatment. There was no consistent effect of ABA on GA19 content. The content of GA29, a catabolic product of
GA20, decreased in parallel with the reduction in GA20 content. However, the content of GA53 increased about 1.5 to
2.5-fold in both ABA-treated grains and dehydrated seedlings. These results showed that accumulation of endogenous
ABA through dehydration gave similar effects to exogenous ABA on the profile of GAs.
The results of Q-PCR experiments showed that ABA caused reductions to about 20% and 40% in the relative expression levels of Hv20ox1 and Hv3ox2 mRNAs respectively, and it repressed the expression of Hv2ox3 mRNA. When
[14C]GA53 was fed to germinated grains with or without ABA being present, considerable metabolism to [14C]GA44
and [14C]GA19 was observed.
There was no consistent difference between control and ABA-treated grains in metabolism to GA44 and GA19, but there
was a reduction in metabolism to GA20, suggesting that the conversion of GA19 to GA20 was affected by ABA. Feeding
with [14C]GA19 again showed that the conversion to [14C]GA20 was less in ABA-treated grains than in controls. The
conversion of [14C]GA20 to [14C]GA1 was also reduced slightly in germinated grains treated with 5µM ABA compared
to control. These results suggest that the decline in endogenous GA1 content in ABA-treated grains is mainly due to a
reduction in the activity of the GA 20-oxidase which catalyses the conversion of GA19 to GA20.
PS1408 AUXINS STIMULATE DWF4::GUS EXPRESSION IN THE ROOTS VIA AUXIAA AND BES1/BZR1-MEDIATED
REGULATION OF THE DWF4 PROMOTER
Ok Sun Lee, Su Youn Jang, and Sunghwa Choe
School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of
Korea
[email protected]
DWARF4 catalyzes a flux-determining step in the Brassinosteroid biosynthetic pathways and its expression is subjected to a feedback-downregulation by BR treatment. However the molecular mechanisms for this regulation have remained unclear. To elucidate, DWF4-driven GUS expression was studied after various genetic and hormone treatments.
DWF4::GUS expression was up-regulated in the BR-deficient dwarf mutants, whereas the change was not noticeable or
even weaker in axr2 and axr6 background. In addition, the DWF4::GUS expression was down-regulated in the brassinazole-resistant mutant bzr1-D. In contrast, DWF4::GUS expression was significantly up-regulated by 2,4-D treatment,
especially in the bes1-D mutant background, suggesting that auxins may have provided additional room for BES1 action.
Interestingly, the auxin-mediated up-regulation of the DWF4::GUS expression was independent of both BRI1 and BIN2
functions, suggesting that auxin directly acts on the DWF4 promoter. Chromatin Immunoprecipitation (ChIP) analysis
revealed that BES1 directly binds to the DWF4 promoter regardless of BL and 2,4,-D treatments. However, BZR1 interaction to DWF4 promoter was abolished by auxin, suggesting that auxin-mediated increase of DWF4 be due to de-repression mechanisms. Collectively, it is obvious that DWF4 promoter serves as a focal point in a cross talk between BR
and auxin, and this regulation is mediated by transcriptional activator BES1.
PS1409 CYTOKININ ACTION IS LINKED TO OXIDATIVE STRESS AS SHOWN BY PROTEOME ANALYSIS IN ARABIDOPSIS
Vanova, Pavlina 1,2, Chevalier, Francois 3, Rofidal, Valerie 3, Rossignol, Michel 3, Malbeck, Jiri 4, Novak, Ondrej 5,
Brzobohaty, Bretislav 1,2 1 Department of Molecular Biology and Radiobiology, Mendel University of Agriculture and
Forestry, CZ-61300 Brno, Czech Republic; 2 Institute of Biophysics AS CR, CZ-61265 Brno, Czech Republic; 3 Unité
de Protéomique, INRA Montpellier, FR-34060 Montpellier, France; 4 Institute of Experimental Botany AS CR, CZ16502 Prague, Czech Republic; 5 Laboratory of Growth Regulators, Palacky University and Institute of Experimental
Botany AS CR, CZ-78371 Olomouc, Czech Republic
[email protected]
Cytokinins (CKs) play an essential role in plant development, acting in concert, among others, with environmental
factors and other plant hormones. Several global genome expression profiling experiments studying the complexity of
CK action have been reported while corresponding proteomic data remain scarce. Here we report a 2-D electrophoresisbased proteomic study that reveals novel information on CK action in Arabidopsis seedlings.
We used a binary system of transgene activation where the expression of the bacterial CK biosynthesis gene ipt is induced
by dexamethasone (DEX). This system allowed us to compare proteomes of seedlings with large differences in endog-
IPGSA 19th Annual Meeting - Abstracts
135
enous CK levels. To exclude the potential effects of DEX on protein composition, wild type Columbia-0 was included in
the experiment as a control. Both wild-type and transgenic lines were grown for 15 days under long-day conditions on
MS media, in parallel with and without 2.5 _M DEX. The activated transgenic seedlings were strongly affected in both
stem and root growth.
Image analysis of two-dimensional gels in triplicates and subsequent statistical evaluation revealed important changes in
protein expression between seedlings with elevated cytokinin levels and controls. No significant effects of DEX on wildtype protein maps were observed. A total of 58 differentially expressed spots were characterized by MALDI-TOF MS
and classified into functional categories. The majority of them were related to oxidative stress and defense mechanisms
including jasmonic acid (JA) inducible proteins.
Subsequent analyses revealed increased levels of hydrogen peroxide and JA in seedlings with elevated CKs, as well as
changes in activities of several antioxidant enzymes such as ascorbate peroxidase, dehydroascorbate reductase and catalase. Application of thiol scavengers partially reversed the phenotype caused by DEX application. Taken together, the
data demonstrate, for the first time, a link between CKs, JA and oxidative stress at the proteome level.
Supported by grants Nos. 1M06030, IAA600040701, IAA600040612, MSM143100008 and AVOZ50040507.
PS1410 ETHYLENE MODULATES ROOT GROWTH BY REGULATING LOCAL AUXIN BIOSYNTHESIS AND DISTRIBUTION
R__i_ka, Kamil1,2, Podhorská, Radka4, Ljung, Karin3, Friml, Ji_í 2,4, Benková Eva1
1 Center for Plant Molecular Biology (ZMBP), Auf der Morgenstelle 3, University Tübingen, D-72076 Tuebingen, Germany; 2 Department of Plant Cell Biology, Untere Karspüle 2, Göttingen, D-37073, Germany; 3 Umeå Plant Science
Center, Department of Forest Genetics and Plant Physiology, SLU; SE-901 83, Umeå, Sweden; 4 Masaryk University,
Dept. of Functional Genomics and Proteomics, Laboratory of Molecular Plant Physiology, Kamenice 5, CZ-625 00 Brno,
Czech Republic
[email protected]
Numerous physiological and genetic studies demonstrate that in plants, complex interactions between different hormonal pathways are common and of great importance for the final developmental output of hormonal regulation. Recent
genetic and biochemical studies yielded a detailed molecular characterization of most single hormonal pathways, including those of ethylene and auxin. However, common effects of these two classical hormones on regulation of hypocotyl
elongation, root hairs growth and differentiation, apical hook formation and root growth suggest interactions of these
two signaling pathways at the molecular level. Here, we will present a detailed study on auxin-ethylene interaction in
regulation of root growth. The ethylene effect on root growth is largely mediated by regulation of auxin levels and polar
auxin transport-dependent local auxin distribution. Ethylene stimulates auxin biosynthesis and auxin is then basipetally
transported to the elongation zone where it activates a local auxin response leading to inhibition of cell elongation. Consistently, in mutants affected in auxin response and specifically in basipetal auxin delivery to this region, ethylene fails
to activate auxin response and cannot influence root growth. In addition, ethylene modulates transcription of several
components of the auxin transport machinery. Thus, ethylene by both stimulating auxin biosynthesis and modulating the
auxin transport machinery can achieve a local activation of auxin signaling pathway and thus implement large part of its
effects on root growth.
PS1411 THE PPR PROTEIN SLO1 MAY REVEAL A NEW REGULATORY MECHANISM IN HORMONE CROSSTALK
Dugardeyn Jasper, Hagenbeek Dik, Zhang Chun-Yi, Smalle Jan and Van Der Straeten Dominique Unit Plant Hormone
Signaling and Bio-imaging, Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35, B-9000
Gent, Belgium; [email protected]
[email protected]
Previous research proved that appearance of the first leaf pair of seedlings is enhanced by ethylene (Smalle et al. 1997).
Hence, a delay in emergence of the first leaf pair can be used as a screen for ethylene mutants. Screening of 5000 T-DNA
insertion lines for this trait, enabled us to identify a loss of function mutant, that shows a delay in emergence of the first
leaf pair, and is retarded in subsequent developmental transitions, hence named slo(w). As seen for other ethylene-insensitive mutants, the slo1 mutant is hypersensitive to sucrose. This is shown by a delay in germination and seedling growth
arrest on high sucrose. The hypersensitivity of slo1 for sugar is at least in part conferred by enhanced ABA signalling.
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The expression of the SLO1 gene in the presence or absence of both sucrose and ACC was analyzed by a promoter-GUS
fusion. The results suggest that SLO1 can play a role in the crosstalk between these different signals.
The SLO1 gene codes for a PPR protein, which lacks an organel-localisation signal. Therefore, SLO1 is the first example
of a PPR protein that is most probably not localised in chloroplasts, mitochondria or nuclei and is functionally related
to hormone signaling. Although the PPR family in plants consists of about 450 members, the mechanism by which they
function remains unknown. Based on their structure they are predicted to possess RNA binding properties. This has recently been proven in vivo for two PPR proteins (CRP1 and PPR4) by using RIP-CHIP (Schmitz-Linneweber et al. 2005,
2006). Future research will confirm whether this is also the case for SLO1.
References:
Schmitz-Linneweber C. et al. Plant Cell 17: 2791-2804
Schmitz-Linneweber C. et al. Plant Cell 18: 2650-2663
Smalle J. et al. Proc. Natl. Acad. Sci. USA94: 2756-2761
PS1412 APICAL DOMINANCE IS CONTROLLED BY INTERACTION BETWEEN CYTOKININ BIOSYNTHESIS/DEGRADATION AND AUXIN IN STEM
Mori, Hitoshi1,Tanaka, Mina1, Sato-Shimizu,Sae1,Sakakibara, Hitoshi2
[email protected]
Apical dominance is a phenomenon in which a terminal bud inhibits the outgrowth of axillary buds. Although involvement of auxin, which represses axillary bud outgrowth, and cytokinin (CK), which promotes axillary bud outgrowth, has
been proposed, little is known about the underlying molecular mechanisms. Firstly, we demonstrated that auxin negatively regulates local CK synthesis in the nodal stem by controlling the expression level of the gene pea adenosine phosphate–isopentenyltransferase (PsIPT), which encodes a key enzyme in CK biosynthesis. Before decapitation, PsIPT1
and PsIPT2 transcripts were undetectable; after decapitation, they were markedly induced in the nodal stem along with
CK accumulation. PsIPT expression was repressed by the application of indole-3-acetic acid (IAA). In excised nodal
stem, PsIPT expression and CK levels also increased under IAA-free conditions. Furthermore, de novo-synthesized IAA
derived from a new shoot apex, which had previously been a dormant axillary bud, not only flowed to the stem 10 h after
decapitation and again repressed PsIPT expression, but also induced gene expression of CK oxidase, which degrades
CKs in the stem . As the result, CK levels in the stem were low again. Our results indicate that, in apical dominance, one
role of auxin is to control local CK level in the nodal stem.
Hormones in Cellular Growth
PS1501 CHANGE OF EXOGENOUSLY SUPPLEMENTED PLANT GROWTH REGULATOR CAUSES RECOVERY OF
PHYTOPLASMA-INFECTED PERIWINKLES
Æurkoviæ Perica, Mirna 1, Lepedu_, Hrvoje 2, _eruga Musiæ, Martina 1 1Univ. of Zagreb, Fac. of Science, Dept. of
Biology, Maruliæev trg 9a, HR-10000 Zagreb, Croatia; 2 Univ. of Josip Juraj Strossmayer in Osijek, Dept. of Biology,
Trg Ljudevita Gaja 6, HR-31000 Osijek, Croatia; [email protected]; Tel/Fax: **38514843851
[email protected]
Phytoplasmas (genus ʻCandidatus Phytoplasmaʼ) from the class Mollicutes, are wall-less, non-helical, endocellular
prokaryotes that inhabit plant phloem and insects. These bacteria with reduced genome cause numerous economically important diseases infecting several hundred plant species worldwide. Since phytoplasmas cannot be cultivated on artificial
media, they are usually maintained in Catharanthus roseus shoots grown in vitro on MS medium supplemented with benzylaminopurine (BA). In such a system, phytoplasmas are present in a high titer.The aim of our research was to examine
the influence of indole-3-butyric acid (IBA) on C. roseus shoots infected with three different phytoplasma strains. A change
from BA to IBA supplemented in the medium supported plant growth, photosynthesis and remission of symptoms in all
phytoplasma-infected shoots, but had no effect on the presence of two phytoplasma strains in tested tissues. However, after
the 4th subculture, HYDB phytoplasma strain was undetectable in more than 60% of the tested shoots.
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After one year of IBA treatment, HYDB-infected periwinkle shoots were retransferred to the medium supplemented
with BA. Some of the shoots showing remission of symptoms during the IBA treatment permanently recovered from the
infection and remained negative when tested for phytoplasma presence. This is the first report on differential influence of
plant growth regulators on phytoplasma-infected plants.
PS1502 WHEAT GENOTYPIC VARIATION AND PROTEIN MARKERS IN RELATION WITH IN VITRO SELECTION FOR
DROUGHT TOLERANCE
Abdel-Hady, Mohamed Sarwat Botany Dept., National Research Center, Dokki, Giza, Egypt
[email protected]
Embryogenic calli isolated from immature embryos of five durum wheat genotypes included one local cultivar (Benysweif-2), as well as four introduced lines (MSWD-2, MSWD-9, MSWD-19 and NRCWD-16) were subjected in vitro
selection for drought tolerance. The effect of polyethylene glycol (PEG) on the embryogenic cultures and plant regeneration had been investigated. The results indicated that the relative growth weight of callus was highly significant influenced by differences in durum wheat genotypes and PEG concentrations. The relative growth of callus decreased rapidly
as the concentrations of PEG increased. At the concentration of 5% PEG, all cultures of the five genotypes studied could
tolerate that level of drought. At the concentration of 10% PEG, MSWD-2 and MSWD-9 gave the highest percentage
on callus growth rate, while Benysweif-2 the lowest one. At the highest concentration of 20% PEG, plant regeneration
percentage has been succeeded in the genotypes (MSWD-2, MSWD-9 and MSWD-19) with minimal reduction, while
(NRCWD-16 and Benysweif-2) scored highly reduction at that concentration. The plant regeneration is a good parameter to evaluate the effect of drought. The analysis of water soluble, non-soluble and alcoholic soluble (gliadin) proteins
by SDS PAGE was detected. Some newly protein markers for drought tolerance were induced under different PEG
concentrations, such as water soluble protein bands in the tolerant genotypes MSWD-2 and MSWD-19, respectively.
Other water soluble proteins were expressed only at the higher drought stresses in MSWD-2 and MSWD-9. Moreover,
the gliadin proteins were induced in MSWD-9 and MSWD-19, respectively. While three protein bands were induced
under the higher concentrations of PEG 10 and 20% in MSWD-2. Mean while, some protein bands disappeared under
the highest drought stress in NRCWD-16 and Benysweif-2. The newly induced protein bands could be used as markers
for indirect selection to the drought tolerance, and this could support the development of drought tolerant genotypes of
durum wheat.
Keywords: Drought tolerant, durum wheat, in vitro, polyethylene glycol, protein markers.
PS1503 PINS REVEAL DIFFERENTIAL AUXIN EFFLUX ACTIVITIES IN ARABIDOPSIS ROOT HAIR CELLS
Sang Ho Lee, Misuk Cho, and Hyung-Taeg Cho
[email protected]
Auxin is the unique phytohormone that moves cell-to-cell with polarity. Intercellular transport of auxin is mediated
by influx and efflux carriers in the plasma membrane. We have previously demonstrated that the protein kinase PINOID
(PID) and an auxin efflux carrier PIN3 positively regulate auxin efflux using the auxin-sensitive Arabidopsis root hair
cell system (Plant Cell 18: 1604-1616). Here, we have adopted the root hair system to compare the auxin efflux activities among different Arabidopsis PINs. Arabidopsis has eight PIN members whose distinctive primary structures imply
diversity in spatial structures and thus molecular activities. Our assay result of the PIN activities demonstrated that different PINs have differential activities in facilitating auxin efflux in Arabidopsis root hair cells. We will be presenting
the data for changes in root parameters and subcelluar PIN-GFP localizations. Pharmacological approaches, regarding
auxin-transporting and protein kinase activities, also revealed distinctive responses of PINs to the chemicals. Our study
suggests that the root hair cell system provides a quantitative tool to study auxin carrier protein molecules.
This work was supported by grants from the MOST/KOSEF Environmental Biotechnology National Core Research
Center (R15-2003-012-02003-0), and the Korea Research Foundation (KRF-2004-041-C00366).
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PS1504 CHARACTERIZATION OF PEA PLANTS TRANSFORMED WITH A GIBBERELLIN 3_-HYDROXYLASE GENE
Wickramarathna, Aruna D., Ozga, Jocelyn A., Reinecke, Dennis M. Department of Agricultural, Food and Nutritional
Science, University of Alberta, Edmonton, Alberta, T6G 2P5 Canada ([email protected]; 780-492-3293; Fax: 780492-4265)
[email protected]
Gibberellins (GAs) regulate many aspects of plant growth and development. To expand our understanding of the importance of gibberellin biosynthesis in reproductive and vegetative growth, pea (Pisum sativum L. cv. Carneval) plants
have been transformed to over-express the GA 3â-hydroxylase gene, PsGA3ox1, under the control of 35S CaMV promoter. PsGA3ox1 codes for the enzyme that converts GA20 to bioactive GA1. A homozygous transgenic line carrying one
copy of PsGA3ox1 (screened T2 generation with kanamycin), a transformation control line (transgene segregated out),
and a wild-type line (non-cultured) are being characterized. Other independent PsGA3ox1-transformed lines with a similar tall phenotype have been identified by our group for analysis. The characterized transgenic PsGA3ox1 line produced
significantly longer internodes (18% greater than the controls, larger stipule leaves and longer tendrils (both approx. 12%
greater than the controls). The total number of internodes was minimally affected in the PsGA3ox1-transformed plants
compared to controls. These data suggest that the PsGA3ox1-transformed line produced increased PsGA3ox1 transcript
levels which resulted in increased production of GA1, and thus modified the phenotype of the plant. Initial qRT-PCR
data has shown that the PsGA3ox1 gene is expressed higher in young plant tissues of transgenics as in early developing
tendrils. To confirm the segregation studies for the number of PsGA3ox1 genes inserted into the genome, Southern blot
analyses are in progress. GA levels will also be quantified in selected tissues to clarify how regulation of GA levels affects
vegetative and reproductive growth in pea. These results support that pea plant architecture is modified in several organs
by the introduction of a transgene that codes for a GA 3_-hydroxylase.
This research is supported by grants from AARI to JAO and DMR, NSERC to JAO, and a Commonwealth fellowship
to ADW.
PS1505 TRANSCYTOSIS MECHANISM FOR DYNAMIC POLAR DELIVERY OF PIN AUXIN CARRIERS IN ARABIDOPSIS
Jürgen Kleine-Vehn1, Pankaj Dhonukshe1, Michael Sauer1, Philip B. Brewer1, Justyna Wiœniewska1, Tomasz Paciorek1, Eva Benková1 and Jiøí Friml1 1Centre for Plant Molecular Biology, Auf der Morgenstelle 3, University of Tübingen, D-72076 Tübingen, Germany
[email protected]
Mechanisms for generating and maintaining cell polarity belong to central themes of developmental and cell biology
and have been extensively studied in different model systems, prominent among them animal epithelial cells. In these
cells, a transcytosis mechanism has been identified as important for polar cargo delivery, and involves the retargeting of
proteins between basolateral to apical polar domains via internalization and subsequent sorted recycling.
In no other kingdom is the relationship between the polarity of individual cells and macroscopic patterning as prominent
as in plants, but comparable knowledge on cell polarity is still lacking. Classical models and current molecular studies
have identified the plant hormone auxin as a key signal whose asymmetric distribution provides positional and directional
information during major developmental decisions, including the establishment of the apical-basal axis, organogenesis,
tissue regeneration, and tropic growth responses. Auxin distribution strictly depends on the activity of PIN-FORMED
(PIN) proteins that have emerged from genetic studies in Arabidopsis thaliana as key regulators of a plethora of auxinmediated developmental processes. PINs act as mediators of auxin efflux from cells and show distinct polar subcellular
localizations at the plasma membrane (PM) of different cell types, determining the direction of intercellular auxin flow.
The polarity of PIN localization can be rapidly rearranged in response to environmental or developmental cues, rerouting
local auxin flow and consequently triggering various developmental processes.
Here we show that the polar targeting and constitutive endocytic recycling of PIN proteins are functionally linked and
utilized in PIN translocation between different polar plasma membrane domains. We visualize polar PIN transcytosis
between the apical and basal sides of cells in vivo and demonstrate the specific involvement of ARF-GEF vesicle trafficking regulators in apical and basal targeting pathways.
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This ARF-GEF-dependent transcytosis mechanism establishes basal PIN localization in polarized cells and mediates
rapid PIN polarity switches during embryonic axis formation, postembryonic organogenesis and tropistic growth. Our
results reveal that a transcytosis mechanism is operational in plants and underlies developmentally important polarization
events in response to developmental and environmental cues.
PS1506 EARLY DYNAMIC CHANGES IN AUXIN LEVELS AND AUXIN-RESPONSIVE GENES MEDIATE THE GRAVITROPIC BENDING OF SNAPDRAGON FLOWERING SHOOTS
Friedman, Haya 1, Berkovitz-Simantov, Revital 1, Meir, Shimon 1, Rosenberger, Ida 1, Halevy, Abraham 2*, PhilosophHadas, Sonia 1 1 Dept. of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet-Dagan 50250, Israel;
2 The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem,
Faculty of Agriculture, Food and Environmental Quality Sciences, Rehovot 76100, Israel; *Deceased.
[email protected]
The process of the negative gravitropic response of cut flower stalks was characterized in the bending zone of snapdragon (Antirrhinum majus L.) spikes with regard to stimulus transduction mediated through differential changes in
auxin level, transport, action and sensitivity. Asymmetrical distribution of free IAA, differential over-expression of two
early auxin responsive genes (ARGs), SAUR and Aux/IAA, and the involvement of a second messenger, inositol (1,4,5)trisphosphate (IP3) in graviperception leading to auxin redistribution, were studied. Gravistimulation of snapdragon
shoots induced a temporary increase in free IAA levels, which peaked in the upper and lower stem flanks at 15 or 30
min, respectively. This IAA gradient across the stem dissipated following 90 min, before any apparent bending occurred.
Application of the auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), completely abolished the IAA gradient
and nullified stem curvature, indicating the primary role of IAA gradient at early stages of the gravitropic response. The
differential IAA distribution did not result from increased IAA synthesis, but rather from preferential IAA transport from
the apex towards the lower stem flank and/or from differential conjugation. The gravistimulation-induced changes in IAA
content were correlated with similar changes in the expression of two ARGs, Am-Aux/IAA1 and Am-SAUR1, indicating
that higher endogenous IAA levels can activate these genes. Responsiveness to exogenous auxin, monitored by AmAux/IAA1 expression levels, showed that a higher sensitivity to auxin in the lower flank was detected only following 4
h of gravistimulation, when bending commenced. Increased IP3 levels, detected both in the upper and lower flanks as
early as 2 min following horizontal placement, lasted for about 30 min and were followed, until 6 h, by a sustained IP3
gradient in favor of the lower flank. Based on these data, a possible sequence of events in the gravity signaling chain
of snapdragon shoots was suggested: Early asymmetric changes in IP3 induce the differential redistribution of auxin,
obtained via lateral auxin transport and differential auxin conjugation. This auxin gradient across the stem induces in
turn differential expression of ARGs (Am-Aux/IAA1 and Am-SAUR1), leading to differential auxin responsiveness and
asymmetric stem growth. Our results support the Cholodny-Went theory that auxin is the main factor operating in the
gravitropic response, but also demonstrate the complexity of the process in flowering shoots, which was not predicted
by the original model.
PS1507 IMMUNOLOCALIZATION OF PIN PROTEIN IN MAIZE COLEOPTILE TIPS
Takeshi Nishimura, Chiharu Niwa, Tmokazu Koshiba Dept Biol Sci, Tokyo Metropolitan Univ, Tokyo, 192-0397, Japan
[email protected]
[email protected]
Indole-3-acetic acid (IAA) is a plant hormone that plays an important role in many physiological events. Recent studies have suggested an important function of IAA transport by regulating its direction and amount, by which all processes,
such as plant development, differentiation, morphogenesis and growth, are accurately controlled. However, regulation
of IAA biosynthesis has been little investigated. To elucidate the IAA function, it is the most important that the site(s) of
IAA biosynthesis itself is determined. We have reported that maize coleoptile tip is a main site of IAA biosynthesis from
Trp and that the produced IAA immediately moves to the lower parts.
To investigate IAA synthetic site and its flow rout from the site, we studied the localization of PIN(s) in maize coleoptile
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tip by using immunohistochemical method. The purified anti-AtPIN rabbit antibody against a highly conserved peptide
sequence of AtPIN1 could recognize one major band of approximately 69 kDa on western blots of whole protein extracts
and also of membrane proteins of maize coleoptile. In the coleoptile tip about 1 mm, the immunofluorescence was detected in the almost all cells but not in epidermis. The signals were observed in whole cell membranes. In the lower region
(about 3-5 mm from the top) of coleoptile, the antibody recognized a polar signal in a few cell layers located along the
inner epidermis. The signal was polarized in the basal membrane of the cells, indicating that IAA was transported toward
the coleoptile base through the inner a few cell layers of coleoptile.
PS1508 ANALYSIS OF HORMONE-DEPENDENT DE NOVO ORGANOGENESIS IN ARABIDOPSIS THALIANA
Pernisová, Markéta, Hejátko, Jan, Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Masaryk University, Kotlarska 2, CZ-61137 Brno, Czech Republic
[email protected]
Phytohormones play a key role in plant development and morphogenesis. Mutual interactions of the two principal
plant growth regulators, cytokinins and auxins, were identified as a major morphogenetic factor in plants and the ratio of
the auxin and cytokinin concentrations determines the identity of newly formed organs. However, the molecular mechanisms of the phenomenon are still largely unknown.
Taking into account the advantage of the lower complexity and higher sensitivity of the experimental system, we have used
the so called hypocotyl test to study the interactions of cytokinins and auxins during de novo organogenesis in Arabidopsis.
Hypocotyls derived from ethiolated seedlings were affected by various concentration ratios of exogenous auxins and cytokinins. We used detailed microscopy analysis to identify the role of both phytohormones to affect the ability of hypocotyl
explants to form calli, SAM- and/or RAM-like structures and their morphogenesis. We applied two auxins, NAA and 2,4D, differing in the mechanism of their intracellular transport, and auxin transport inhibitor NPA in combination with both
of them. The identified differences in the resulting morphogenetic effect will be discussed. WUS::NLS-GUS and CLV3::
NLS-GUS marker lines were used to recognize the identity of newly formed lateral organs and the role of both phytohormones in the regulation of the cytokinin-to-auxin ratio-dependent developmental switch. Changes in the endogenous
hormone maxima and/or responsibility, respectively were studied using DR5rev::GFP and ARR5::GUS marker lines, and
CycB1::GUS to analyze position-specific, hormone-dependent mitotic cell activity. The results of the above mentioned
marker lines and selected transgenic and mutant lines analysis together with suggested model of the cytokinin and auxin
interactions during de novo organogenesis in Arabidopsis thaliana will be presented.
Supported by the Ministry of Education, Youth and Sports of the Czech Republic, LC06034 and MSM0021622415.
PS1509 PINOID AND PID-LIKE KINASES: A COMPASS ORIENTING AUXIN-REGULATED PLANT DEVELOPMENT?
Galvan Ampudia, Carlos S., Huang, Fang, Maraschin, Felipe, Quint, Ab, Robert, Helene, Zago, Marcelo K., Offringa,
Remko Dep. of Molecular & Developmental Genetics, Institute of Biology, Leiden University, Wassenaarseweg 64, 2333
AL Leiden, The Netherlands.
[email protected]
A major determinant in plant development is the polar transport of the plant hormone auxin, which generates vectorial
gradients that direct growth and provide positional cues for patterning, organogenesis and vascular differentiation. The
directional auxin flow depends on the activity and polar sub-cellular localisation of the PIN auxin efflux carriers. We have
identified the PINOID (PID) protein kinase as regulator of the apico-basal polar localization of PIN proteins 1,2. PID is a
membrane-associated kinase that belongs to the plant-specific AGCVIII family of protein kinases. Recently, we obtained
evidence that PID may influence PIN polarity by phosphorylating the PIN central hydrophilic loop. In Arabidopsis, three
PID-like kinases cluster to the same clade as PID, and we are currently testing the hypothesis that PID and PID-like kinases
provide plants with a compass that integrates both external (e.g. gravity) and internal (e.g. calcium) signals, and directs
auxin transport to determine the proper growth orientation or lateral organ position.
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References
1. Benjamins,R., Quint,A., Weijers,D., Hooykaas,P., and Offringa,R. (2001). The PINOID protein kinase regulates organ
development in Arabidopsis by enhancing polar auxin transport. Development 128, 4057-4067.
2. Friml,J., Yang,X., Michniewicz,M., Weijers,D., Quint,A., Tietz,O., Benjamins,R., Ouwerkerk,P.B., Ljung,K.,
Sandberg,G., Hooykaas,P.J., Palme,K., and Offringa,R. (2004). A PINOID-dependent binary switch in apical-basal PIN
polar targeting directs auxin efflux. Science 306, 862-865.
PS1510 RESPONSE OF DIFFERENT GENOTYPE FOR CALLUS INDUCTION AND PLANT REGENERATION IN CHILI
(CAPSICUM ANNUM L)
Muhammad Shafiq, Muhammad Arshad, Shahid Mansoor, Yusuf Zafar and Shaheen Aftab Plant Biotechnology Division, National Institute for Biotechnology and Genetic Engineering [NIBGE], P.O.Box-577, Jhang Road, Faisalabad,
Pakistan
[email protected]
Five different chili genotypes were evaluated for their callusing response and regeneration potential. Chili hypocotyls
explants obtained from 2 week old seedlings were cultured on solid Murashige and Skoog (MS) medium supplemented with
1% (w/v) sucrose, 0.1mg/l NAA (Naphthalene acetic acid), or 0.5mg/l IAA (Indole acetic acid) or in combination with 2mg/
l Zeatin or 2mg/l BA (Benzyl adenine) to produce callus. After 2 weeks of culturing, callus was visible at the cut edges of
hypocotyls explants. Callused explant were transferred to regeneration medium containing Murashige and Skoog (MS) medium supplemented with 1% (w/v) sucrose, 0.05mg/l NAA (Naphthalene acetic acid), or 0.1mg/l IAA (Indole acetic acid) in
combination with 1mg/ml Zeatin or 1mg/ml BA (Benzyle Adenine) soon after induction. Of the evaluated genotypes, Lungi
and silky red were superior in their response for callusing and regeneration potential. It was found that prolonged period
for callusing leads to green hard/compacted callus losing embryogenic potential and proliferation capability. Of the tested
growth regulators, Zeatin was optimal for the induction of embryogenic callus followed by BA. This protocol demonstrates
the genotype dependent response for somatic embryogenesis and plant regeneration. This type of study will help to suggest
particular chili genotype for transformation and regeneration of transgenic chili plants.
Hormone Metabolism
PS1601 12-OH-JA, 12-HSO4-JA AND 12-O-GLUCOSYL-JA – ABUNDANTLY OCCURRING METABOLITES OF JA
Otto Miersch, Jana Neumerkel, Claus Wasternack Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle
(Saale), Germany; [email protected]
[email protected]
In Solanacean species 12-hydroxyjasmonic acid (12-OH-JA) is a tuber inducing compound, called tuberonic acid (Koda,
1992). Here we show by a screening that 12-OH-JA is an abundantly occurring constituent of numerous plant species exhibiting organ specific differences. Sulfation and glucosylation of 12-OH-JA was shown by detection of 12-HSO4-JA and
12-O-Glc-JA. In immature soybean seeds the level of 12-OH-JA exceeds that of JA 275-fold and the level of 12-HSO4-JA
134-fold, respectively. As shown with JA-deficient tomato mutants and transgenic lines, 12-OH-JA is formed in a JA-dependent manner. In wounded tomato leaves these compounds accumulate in the sequence JA -> 12-OH-JA -> 12-HSO4-JA ->
12-O-Glc-JA. Expression of JA-inducible genes does not occur upon treatment of tomato leaves with 12-OH-JA suggesting
that JA signaling is at least partially switched off by its hydroxylation. Sulfonation of 12-OH-JA seems to be a complete
inactivation step, since 12-HSO4-JA was not cleaved by tomato leaves treated with the labeled compound.
Further insights into distinct roles of JA, 12-OH-JA and 12-HSO4-JA were found with tomato plants expressing constitutively a cDNA in sense and antisense orientation for 12-OH-JA-sulfotransferase.
References:
Koda, Y. (1992) The role of jasmonic acid related compounds in the regulation of plant development. Int. Rev. Cytol.
135, 155-199.
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PS1602 SPECIFIC ROOTING ACTIVITY 0F 4-(3-INDOLYL)-4-BUTANOLIDE FORMED IN EARLY STAGE OF BUPLEURUM FALCATUM L. ROOT CULTURE WITH INDOLE-3-BUTYRIC ACID
Yokoyama, Mineyuki1, Yamaguchi, Shoko1, Yoshida, Seiichi1, Ayano, Madoka2, Nakamura, Kentaro3, Goda, Hideki2, Iida, Toshii1, Yanaki, toshio1, Yoshida, Shigeo2 1Research Center, Shiseido Co. Ltd., Yokohama, 224-8558 Japan;
2Plant Science Center, RIKEN, Yokohama, 230-0045 JapanTsukuba; 3Research Institute, Sumitomo Forestry Co., Ltd.,
Tsukuba, 300-2646 Japan
[email protected]
Indole-3-butyric acid (IBA) induced vigorous rooting in Saiko (Bupleurum falcatum L.)-cultured roots after approximately 2 weeks. We searched stress-induced substances involved in the rooting by the same concept as the discovery of
KODA in flower-induction,1 and found a novel IBA derivative, 4-(3-indolyl)-4-butanolide (IBL) in medium at the first
day of the culture.
Synthetic IBL induced rooting on both cultured roots of B. falcatum and cuttings of Vigna radiata L. Wilczek. Furthermore, it showed much better root-inducing effects than IBA on the cottages of Paraserianthes falcataria. We histologically compared the effects of IBL to IBA in the cultured roots of B. falcatum. IBA induced considerable thickening of the
original root before generating new roots; remarkable cell-expansion and certain degree of cell-division in the cortical
tissue. On the other hand, IBL showed no cell expansion at the same concentration, but induced cell division as strongly
as IBA, resulting a rooting effect without cell-expansion. The results suggest that IBL may play a specific role in the
rooting process induced by IBA.
1Yokoyama et al., (2000) Stress-induced factor involved in flower formation of Lemna is an _-ketol derivative of linolenic acid, Plant Cell Physiol. 41:110-113.
PS1603 NOVEL PATHWAY FOR IAA BIOSYNTHESIS IN MAIZE ENDOSPERM
Angela Hendrickson Culler Jerry D. Cohen
[email protected]
How plants make the hormone indole-3-acetic acid (IAA) has yet to be fully solved. We have approached this question using maize endosperm where we are able to isolate and partially purify tryptophan-dependent IAA biosynthesis in
vitro. Using [2H5] and [13C1115N2] tryptophan along with [13C6]IAA as an internal standard, we were able to quantify
the amount of tryptophan converted to IAA, which occurred at a rate of 140 pmol/min. We then synthesized a series of
intermediates in IAA biosynthesis using stable-labeled precursors that represented all major proposed pathways, and
tested their involvement in IAA biosynthesis by asking three questions: 1) is the intermediate converted to IAA, 2) can
the intermediate interfere in tryptophan conversion to IAA, and 3) is the intermediate endogenous to endosperm? We
were able to measure and distinguish both IAA produced from precursors and IAA from tryptophan on the GC-MS. None
of the proposed precursors proved to be good candidates for intermediates in IAA biosynthesis in this system. Indole-3acetonitrile (IAN) completely inhibited IAA biosynthesis, but we did not find any endogenous IAN in the endosperm.
[3H1] and [14C1] tryptophan supplied to the endosperm enzyme system allowed us to isolate a small membrane protein
that covalently bound tryptophan and released IAA. Tryptophan was linked via a thioester and when hydrolyzed, released
racemic tryptophan. The tryptophan-protein complex was an effective substrate for IAA biosynthesis. The small size of
the protein (between 8-16 kDa) is quite unique from other systems studied, and sequence data should answer more questions about the mechanism of this system for IAA biosynthesis.
PS1604 AUXIN CONJUGATE HYDROLYSIS DURING THE INTERACTION OF TWO SYMBIONTS WITH MEDICAGO
TRUNCATULA
Ludwig-Müller, Jutta1, Campanella, James J.2 1Institut für Botanik, Technische Universität, 01062 Dresden, Germany;
2Montclair State University, Montclair, NJ, USA
[email protected]
The literature suggests a relationship between auxin and symbiosis in plants. We have characterized the regulation of
auxin conjugate hydrolysis in Medicago truncatula during development and interaction with two symbionts. In Medicago
truncatula five putative auxin amidohydrolase genes (MtIAR31, MtIAR32, MtIAR33, MtIAR34, MtIAR36) were identi-
IPGSA 19th Annual Meeting - Abstracts
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fied, homologous to the AtIAR3 gene from Arabidopsis thaliana. The MtIAR32 transcript is the most abundant transcript
in whole seedlings after germination, while MtIAR33 is the least abundant. MtIAR32 is also the most abundant transcript
in roots, stems, and basal-leaves. MtIAR31 is most highly expressed in flowers and MtIAR36 in terminal leaves. Sinorhizobium meliloti-infected seedlings primarily up-regulated MtIAR33 and -34 transcripts. Glomus intraradices-infected
seedlings up-regulated MtIAR33 and -34. MtIAR31, -32, -33, and -34 have hydrolytic activity against IAA-Aspartate
and IBA-Alanine. MtIAR33, -34, and -36 hydrolyze the ester bonds of IAA-glucose. MtIAR36 solely possesses activity
against IAA-Glycine, -Alanine, and -Isoleucine. IBA was increased in Glomus intraradices inoculated roots of Medicago
compared to controls, while IAA was not. Our results suggest an intricate control system that regulates free and conjugated auxins in Medicago.
PS1605 A COMPOUND-BASED APPROACH TO UNRAVELLING AUXIN BIOSYNTHESIS IN PEA
Quittenden, Laura J, Davies, Noel W, Smith, Jason, Symons, Gregory M, Ross, John J School of Plant Science, Private
Bag 55, University of Tasmania, Hobart 7001, Tasmania, Australia
[email protected]
The topic of auxin biosynthesis has puzzled researchers for over five decades. A number of interlinked pathways to
indole-3-acetic acid (IAA) have been proposed; however, it is still unclear as to which pathway(s) predominates. We have
used a compound-based approach to gain insight into the IAA biosynthetic pathway in pea (Pisum sativum). Our main
objective has been to identify putative endogenous precursors, and produce deuterium-labelled forms of these (some
of which are unavailable commercially), for quantification and metabolism purposes. Using these novel labelled compounds, we have identified and quantified endogenous tryptophan, tryptamine, indole-3-acetaldehyde (IAAld), indole3-ethanol (IEt) and IAA from pea tissue. In the bushy mutant, IAA levels are low, while those of tryptamine, IAAld and
IEt are high, with tryptophan levels similar in the mutant and wild type. These results indicate that in the bushy mutant
there may be a disruption in the auxin biosynthesis pathway between IAAld (the putative immediate precursor to IAA),
and IAA itself. This will be further investigated.
Metabolism studies to date have shown that deuterated tryptamine is metabolised to deuterated IAA in sterile wild type
pea roots. A high degree of deuterium labelling was also detected in the putative IAAld storage product IEt. This research
will lead to further elucidation of the auxin biosynthesis pathway in pea.
PS1606 ARABIDOPSIS CYP85A2 CATALYZES THE SYNTHESIS OF 2-DEOXY-7-OXALACTONE TYPE BRASSINOSTEROIDS
Katsumata Takumi, Hasegawa Akiko, Fujiwara Tatsuya, Komatsu Tomoyuki, Natsume Masahiro, Abe Hiroshi, Kawaide
Hiroshi Div. Agric. Sci., Tokyo Univ. Agric. Technol., Fuchu, Tokyo 183-8509, Japan
[email protected]
Occurrence of a new pathway for Brassinosteroids (BRs) synthesis that lactonizes BRs at an earlier step was demonstrated. Endogenous 7-oxateasterone (7-OXTE) and 7-oxatyphasterol (7-OXTY) in Arabidopsis were determined by
GC-MS. Both BRs showed hypocotyls elongation activity using Arabidopsis det2-1 mutant. The activity of 7-OXTY
was as same as that of castasterone, the precursor of brassinolide. To identify the biosynthetic origin of these 2-deoxy-7oxalactone BRs, recombinant CYP85A2 was produced by heterologous expression in Pichia pastoris. The transformants
having both CYP85A2 and Arabidopsis P450 reductase genes were able to convert TE and TY to 7-OXTE and 7-OXTY,
respectively.
PS1607 ENZYMATIC SYNTHESIS AND 2D CARBON-13 NMR OF FULLY 13C-LABELED PRENYL DIPHOSPHATES
Yoshinori Sugai, Isamu Yumoto, Masahiro Natsume, Hiroshi Abe, Hiroshi Kawaide. Div. Agric. Sci., Tokyo Univ. Agric.
Technol., Fuchu, Tokyo 183-8509, Japan 042-367-5698.
[email protected]
All isoprenoids are derived from dimethylallyl diphosphate and isopentenyl diphosphate in organisms. Stable isotopelabeled intermediates in terpenoid biosynthesis will be powerful probes for biosynthetic studies of natural terpenoids. For
example, 13C-labeled prenyl diphosphates will be valuable compounds to study functional analysis of terpenoid biosyn-
IPGSA 19th Annual Meeting - Abstracts
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thetic genes. To synthesize fully 13C-labeled farnesyl and geranylgeranyl diphosphates in vitro, [U-13C6]mevalonate
was prepared. First, [U-13C6]mevalonate was produced by fermentation of Saccharomycopsis fibligera. The yield was
350mg from [U-13C6]glucose as a carbon source. Then, five recombinant proteins responsible for metabolism from
mevalonate to farnesyl diphosphate (FPP) cloned from Neurospora crassa were produced in E. coli. We designed enzyme
cocktails, in which five enzymes, ATP, and cofactors were mixed with several concentration. These cocktails converted
[U-13C6]mevalonate to [U-13C15]FPP in vitro. The overall yield of production of FPP estimated by GC-MS after dephospholization was 80% yield. The structural analysis of [U-13C15]FPP was performed by LC-ESI-MS and two dimensional 13C NMR spectra. These spectral analysis showed the data consistent with structure of 13C-labeled FPP.
PS1608 DITERPENE CYCLASE IN THE LIVERWORT AND MOSS
Kawanabe Ryo1, Hayashi Kenichiro2, Minoda Hiromi1, Notomi Miho1, Nozaki Hiroshi2, Kawaide Hiroshi1 1Div. Agric. Sci., Tokyo Univ. Agric. Technol., Fuchu, Tokyo 183-8509, Japan; 2Dep. Biochem., Okayama Univ. Sci., Okayama
700-0005, Japan.
[email protected]
Structure and function of diterpene cyclases in lower land plants were studied. The moss Physcomitrella patens has
bifunctional ent-kaurene synthase(PpCPS/KS) catalyzing cyclization of geranylgeranyl diphosphate to ent-kaurene and
ent-16alpha-hydroxykaurene. The bifunctional ent-kaurene synthase from the GA-producing fungus Phaeosphaeria sp.
L487 retained CPS activity when the C-terminal region was truncated. In contrast, the C-terminal truncated mutants
of PpCPS/KS lacked both CPS and KS activity. A cDNA encoding diterpene cyclase(DTC) was also cloned from the
liverwort, Jungermannia subulata. The similarity of amino acid sequence of JsDTC showed 58% of similarity against
PpCPS/KS. The analysis of function and structure-activity relationship of JsDTC is in progress.
PS1609 PROPOSALS FOR THE SYNTHESIS AND RELEASE OF ABA PRECURSORS FROM PHOTOSYNTHETIC AND
ROOT CORTICAL CELLS
Netting, Andrew School of BABS, University of New South Wales Sydney, N.S.W. 2052, AUSTRALIA.
[email protected]
PROPOSALS FOR THE SYNTHESIS AND RELEASE OF ABA PRECURSORS FROM PHOTOSYNTHETIC
AND ROOT CORTICAL CELLS.
Netting, Andrew.
School of BABS, University of New South Wales, Sydney, N.S.W. 2052, AUSTRALIA.
[email protected] 61 2 9665 0563.
It is widely accepted that ABA biosynthesis is initiated by the oxidative cleavage of 9-cis-neoxanthin to give xanthoxal.
The ring secondary alcohol of this aldehyde is then oxidised to a ketone to give ABA aldehyde which is then oxidised to
ABA. However, this scheme offers no comment on how ABA-synthesising cells defend themselves against the deleterious effects of these aldehydes and how ABA biosynthesis is integrated into a plantʼs response to stress. When Adduct I,
the first of two ABA precursors, is released from photosynthetic cells (or root cortical cells) it carries two O2 H-bonded
to histidyl residues in both of Adduct Iʼs substituent peptides. Oxidation of Adduct I to Adduct II reduces one O2 to H2O2
which opens a Ca2+ channel. Adduct II moves in the xylem/apoplast until a stress-induced pH increase catalyses its oxygenation so that it releases ABA. These and associated changes will be illustrated with metabolic schema.
PS1610 PROPOSALS FOR THE TRANSPORT AND RELEASE OF ABA IN THE XYLEM
Netting, Andrew School of BABS, University of New South Wales, Sydney, 2052, AUSTRALIA.
[email protected]
PROPOSALS FOR THE TRANSPORT AND RELEASE OF ABA IN THE XYLEM.
Netting, Andrew.
School of BABS, University of New South Wales, Sydney, N.S.W. 2052, AUSTRALIA.
[email protected] 61 2 9665 0563.
IPGSA 19th Annual Meeting - Abstracts
145
The previous abstract outlines a widely accepted scheme for ABA biosynthesis that, as pointed out there, has limitations.
Further, it does not explain how plants stressed in 2H2O incorporate 2H into ABA and its precursors, Adduct I and Adduct
II, while the xanthophylls remain unlabelled. It is proposed that cells within the stele near to the endodermis synthesise
and release Adduct I into the water column at the onset of stress. This release is thought to be initiated by the cessation of
the passage of water through the endodermis, which generates tension on the water column, on receipt of an ABA signal
from the root cortical cells. As this implies a rapid synthesis of Adduct I in the absence of chloroplasts, it is thought that
this synthesis may utilise a ʻdirect pathwayʼ that allows 2H-exchange and the release of Adduct I without H-bonded O2ʼs.
Increasing tension on the water column may ʻsuckʼ O2 in through the cuticle and, together with pH changes, initiate the
release of ABA. Illustrative schema will be presented.
PS1611 ASYMMETRIC LIGAND BINDING OF ABA 8’-HYDROXYLASE
Ueno, Kotomi1, Mizutani, Masaharu2, Hirai, Nobuhiro3, Todoroki, Yasushi4 1 Unit. Grad. School Agric. Sci., Gifu
Univ., Gifu 501-1193, Japan; 2 Inst. Chem. Res., Kyoto Univ., Uji, Kyoto 611-0011, Japan; 3 Int. Innov. Center, Kyoto
Univ., Kyoto 606-8501, Japan; 4 Dep. Appl. Biol. Chem., Facul. Agric., Shizuoka Univ., Shizuoka 422-8529, Japan
[email protected]
ABA 8ʼ-hydroxylase (recombinant CYP707A3) active site specifically binds (S)-(+)-ABA, naturally occurring ABA,
but not its enantiomer (R)-(-)-ABA. In contrast, both enantiomers of a non-azole inhibitor of ABA 8ʼ-hydroxylase AHI1
inhibit the CYP707A3 reaction. By comparing the structures between ABA and AHI1, we found that the 4ʼ-oxo moiety
and 2ʼ,3ʼ-unsaturated system of (-)-ABA prevented binding to the active site of CYP707A3. The elimination of the enone
in the ring of (-)-ABA allows to bind effectively to the active site, whereas the lack of the enone of (+)-ABA decreases
the affinity for the active site. These results suggest that binding interaction of the enone-lacking (-)-ABA analogs with
the active site is different from that of (+)-ABA.
PS1612 CHARACTERIZATION OF TRANSPORT INHIBITOR RESPONSE2 (TIR2) MUTANT IN ARABIDOPSIS
Masashi Yamada1, Mark Estelle1 1Department of Biology, Indiana University, Bloomington, IN 47405, USA
[email protected]
Auxin controls many aspects of plant development. However, it is still unclear how auxin regulates plant development. To identify genes that are required for various aspects of auxin biology we have previously screened for Arabidopsis mutants that are resistant to auxin transport inhibitors. Genes identified in this screen function in auxin signaling
(TIR1), auxin transport (TIR3), and auxin biosynthesis (TIR7). My work concerns the tir2 mutant. This mutant displays
resistance to auxin transport inhibitors, as well as a variety of growth defects including reduced lateral root formation,
decreased hypocotyl elongation, and reduced root gravitropism. Moreover, double mutants of tir2 with mutants related to
auxin synthesis, auxin transport, and auxin signaling show very severe root defects. We have mapped the TIR2 gene to
a small region on chromosome 1 and have identified a candidate gene. Our analysis of this gene, together with a model
describing itʼs function in auxin biology, will be presented.
PS1613 FUNCTIONAL CHARACTERIZATION OF ATLOGS GENES ENCODING CYTOKININ ACTIVATING ENZYMES
IN ARABIDOPSIS
Takeshi Kuroha*, Hitoshi Sakakibara RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045, Japan *tkuroha@psc.
riken.jp, Tel: +81-45-503-9576, FAX: +81-45-503-9609
[email protected]
Cytokinins play a crucial role in various aspects of plant growth and development. The spatial and temporal distribution of bioactive cytokinin levels is strictly controlled in plant development events. Recently, LONELY GUY (LOG)
was identified from rice as a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. In Arabidopsis genome, nine genes (AtLOG1 - AtLOG9) are predicted as homologs of rice LOG. To elucidate
the functions of Arabidopsis LOG family genes, we characterized the activities of AtLOGs proteins and the expression
patterns of AtLOGs genes. The eight cDNAs, AtLOG1 - AtLOG8, with an expected length were obtained by RT-PCR.
The AtLOG6 seems a pseudogene because of the existence of a premature stop codon in AtLOG6 cDNA. The AtLOG1,
IPGSA 19th Annual Meeting - Abstracts
146
2, 3, 4, 5, 7, 8 cDNAs were expressed in Escherichia coli and purified by Ni-NTA agarose. The cytokinin-specific phosphoribohydrolase activities were detected from all of seven proteins as well as rice LOG. AtLOGs proteins displayed
different substrate specificities for cytokinin nucleotides. Analysis of AtLOGs::GUS fusion genes revealed that each AtLOGs genes showed the different expression patterns. Several expression patterns of AtLOGs genes were different from
those of AtIPTs (adenosine phosphate-isopentenyltransferase) genes, whose products catalyze the initial step of cytokinin
synthesis. Possible role of the AtLOGs genes and the regulation of cytokinin activation in plant growth and development
will be discussed._@
PS1614 COORDINATED CONTROL OF BRASSINOSTEROID BIOSYNTHESIS AND PERCEPTION
Szekeres, Miklos 1, Szatmari, Anna-Maria 1, Nagy, Ferenc 1, Yokota, Takao 2, Bishop, Gerard J. 3 1 Inst. Plant Biol.,
BRC HAS, Szeged, Hungary; Dept. Biosciences, 2 Teikyo University, Utsunomiya, Japan; 3 Dept. Agricult. Sci., Imperial College London, Wye, U.K.
[email protected]
Brassinosteroids (BRs) act locally, and their physiological effects depend on their actual level and the local sensitivity to the hormone. To elucidate the importance of these conditions in the activation of BR signaling, we followed the
in vivo expression of the genes that encode key BR-biosynthetic enzymes (CPD and CYP85A2) and the BRI1 receptor
in transgenic Arabidopsis lines carrying promoter-luciferase reporter fusions. Local changes in the amounts of bioactive
BRs were monitored using BR-specific indicator transgenes. Our data suggest that BR synthesis and perception are subtly coordinated processes, which are determined by both developmental and environmental cues.
PS1615 ANALYSIS OF THE GENES ENCODING CYTOKININ HYDROXYLASE IN ARABIDOPSIS
Kentaro Takei, Mikiko Kojima, Hitoshi Sakakibara RIKEN, Plant Science Center, 1-7-22, Suehiro-cho, Tsurumi-ku,
Yokohama, Kanagawa, 230-0045, JAPAN.
[email protected]
In Arabidopsis, CYP735A1 and CYP735A2 catalyze the hydroxylation of isopentenyladenine-type cytokinins
(CKs) to trans-Zeatin (tZ)-type CKs. To elucidate the physiological roles of the tZ-type CKs, we analyzed insertional mutants of the CK hydroxylase genes. cyp735a1 and cyp735a2 did not show any visible phenotype but double mutant, cyp735a1cyp735a2, displayed phenotypes such as reduced shoot size and increased rosette branches. In
cyp735a1cyp735a2, amount of tZ-type CKs was lower than 3 % of that in WT and amount of iP-type CKs was twice of
that in WT. Partial complementation of the mutant phenotypes by the application of tZ indicated that mutant phenotype
was caused by the decreased amount of tZ-type CKs. We will also show the results from gene expression analysis of the
double mutant using DNA microarray chips.
PS1616 THE C-TERMINAL REGION OF ACC SYNTHASE IS INVOLVED IN ITS TURNOVER
Yusuke Kamiyoshihara1, Takayuki Suzuki1, Masayasu Nagata2, Hitoshi Mori1; 1Graduate School of Bioagricultural
Sciences, Nagoya University, Nagoya 464-8601, Japan;2National Institute of Vegetable and Tea Science, National Agriculture and Food Research Organization, Mie 514-2392, Japan
[email protected]
1-Aminocyclopropane-1-carboxylic acid synthase (ACS), a rate-limiting enzyme in the ethylene biosynthesis pathway,
is regulated post-translationally as well as transcriptionally. We previously reported that LeACS2, a wound-inducible
ACS in tomato, was immediately phosphorylated after translation at Ser-460, and then acts in the phosphorylated form in
the cell. Treatments with protein kinase and phosphatase inhibitors led to the finding that phosphorylation regulates ACS
turnover. Furthermore, ETO1 protein binds to the C-terminal region of ACS, including the phosphorylation site, leading to the degradation of ACS via the 26S ubiquitin-proteasome pathway. To confirm the importance of the C-terminal
region on ACS turnover, we examined the turnover of LeACS4 lacking phosphorylation site and ETO1 binding region.
The pulse-chase experiments using transgenic tomato plants expressing LeACS4 under the control of the wound-inducible LeACS2 promoter revealed that the half-life of LeACS4 (240 min) was longer than that of LeACS2 (70 min). This
result suggests that C-terminal region of ACSs that have phosphorylation site is involved in their rapid turnover. Most
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ACSs are induced by biotic and abiotic stresses and have phosphorylation site, whereas LeACS4 expression is developmentally regulated in tomato fruit tissue and does not have it, suggesting that ACS isozymes that are expressed under the
developmental program such as LeACS4 are processed through a distinct pathway from stimuli-induced ACS isozymes
such as LeACS2.
PS1617 HYDROLYTIC ENZYMES ACTING ON CYTOKININS
Pospí_ilová Hana1, Sedláøová Iva1, Kostlánová Nikola2, Frébort Ivo1; 1Department of Biochemistry, Palack_ University, _lechtitelù 11, CZ-78371 Olomouc, Czech Republic; 2National Centre for Biomolecular Research, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic
[email protected]
According to older reports, some yeast and mammalian enzymes can cleave a cytokinin to hypoxanthine and an amine
derived from the N6 side-chain. Of this class of enzymes, adenine deaminase (ADE, EC 3.5.4.2; producing hypoxanthine
and ammonia from adenine) found in yeast and fungi, shows high degree of protein sequence similarity with mammalian
adenosine deaminase (ADA, EC 3.5.4.4). Although genome databases of Arabidopsis thaliana and other plants show
presence of putative ADA-encoding genes, the corresponding proteins have not been obtained so far nor the activity
towards adenosine demonstrated.
We are searching for enzymes involved in possible hydrolytic degradation of cytokinins in plants. Genes encoding yeast
adenine deaminases SPBC1198.02 (Schizosaccharomyces pombe) and AAH1 (Saccharomyces cerevisiae) and a putative
adenosine deaminase from Arabidopsis (At4g04880) were cloned and expressed in Escherichia coli under IPTG-inducible promoter. Homogenous recombinant proteins were then obtained by affinity purification using His-tag. For both
yeast enzymes, significant activity towards adenine was found, but cytokinin degradation was observed only for the S.
pombe enzyme. The activity of the enzyme from Arabidopsis towards adenosine was much lower that those of the yeast
enzymes. All three enzymes were obtained in crystalline form and the conditions for crystal growth are being further
optimized for X-ray structural analysis. Overexpression of the genes in Arabidopsis under 35S promoter that is underway
shall provide more information on the real function of adenine/adenosine deaminase in plants.
PS1618 SUBSTRATE SPECIFICITY AND BIOCHEMICAL CHARACTERIZATION OF CYTOKININ OXIDASES/DEHYDROGENASES FROM ARABIDOPSIS THALIANA
Galuszka Petr1, Marta Kowalska1, Maria _mehilová1, Toma_ Werner2; 1Department of Biochemistry, Palack_ University, _lechtitelù 11, 783 71 Olomouc, Czech Republic; 2Institute of Biology/Applied Genetics, Free University of Berlin,
Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany
[email protected]
Cytokinins are selectively and irreversibly deactivated by enzyme activity of cytokinin oxidase/dehydrogenase (CKX)
encoded by small gene families in plant genomes. In Arabidopsis thaliana, seven distinct genes were found and their
function in planta was proved by constitutive overexpression in tobacco. Partially purified extracts from these plants were
tested for CKX activity with varying pH, electron acceptors, and a range of cytokinin-like substrates. Further all seven
recombinant CKX proteins were prepared in heterologous expression system of Pichia pastoris and as well characterized
in detail. Differences in substrate preferences, reaction mechanism and pH optima among whole CKX family will be
presented within the context of their subcellular localization.
PS1619 CLONING AND CHARACTERIZATION OF A GLUCOSYLTRANSFERASE WHICH METABOLIZES SOME PHYTOHORMONES MULTIPLY
Hiroyuki Suzuki1, Hiroki Hayase1, Masatoshi Nakajima1, Isomaro Yamaguchi2 and Tadao Asami1 1 Department of Applied Biological Chemistry, University of Tokyo, 2 Department of Biotechnology, Maebashi Institute of Technology
[email protected]
_We identified a glucosyltransferase gene InGTase1 from morning glory (Ipomoea nil), which product has a broad
selectivity for phytohormones as substrates. After degenerate RT-PCRs and RACEs for total RNA from the immature
IPGSA 19th Annual Meeting - Abstracts
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seeds (3-33 day-after-flowering), we obtained its full-length sequence, prepared the GST-fused recombinant protein by
using E. coli expression system, and evaluated the enzymatic activity by the reaction with 14C-UDP-glucose. Then we
detected its enzymatic activity to jasmonate, salicylate, abscisic acid and indoleacetic acid. The optimum pH dependency
for the enzymatic reaction was at around pH 6, while the activity was hardly detected at below pH 5. By GC-MS and
1H-NMR, The metabolites of IAA and SA were identified as the glucosyl esters of them. More detail of its substrateselectivity will also be presented.
PS1620 INSIGHTS INTO AUXIN METABOLISM FROM THE ANALYSIS OF ARABIDOPSIS IBA-RESPONSE MUTANTS
Naxhiely Martinez1, Bethany K. Zolman2, Arthur Millius1, Bonnie Bartel1 1Department of Biochemistry & Cell Biology, Rice University, Houston TX, 77005, USA; 2Department of Biology, University of Missouri–St. Louis, St. Louis,
MO, 63121, USA
[email protected]
Indole-3-butyric acid (IBA) is converted to the auxin indole-3-acetic acid (IAA) in the model plant Arabidopsis thaliana. It has been suggested that this conversion is similar to fatty acid _-oxidation (Fawcett et al., 1960), and extensive
genetic evidence supports this suggestion. We are isolating and characterizing IBA-response mutants (ibr) with the aim
of identifying the enzymes that convert IBA to IAA. ibr mutants display enhanced root elongation on IBA, but respond
normally to IAA (Zolman et al., 2000). An unresolved question about IBA _-oxidation to IAA is whether the enzymes
used to convert IBA to IAA are the same as those acting in fatty acid _-oxidation. One possibility is that the enzymes
used in fatty acid metabolism also act directly on IBA-CoA. Another possibility is that enzymes exist that act solely on
IBA-CoA and are independent of fatty acid metabolism. Evidence to support the latter hypothesis is provided by the
analysis of three IBA-response mutants, ibr3, ibr10, and ibr1 that are deficient in IBA responses but lack defects in fatty
acid _-oxidation. These ibr mutants are defective in proteins similar to three fatty acid _-oxidation proteins: an acyl-CoA
oxidase/dehydrogenase (Zolman et al., 2007), an enoyl-CoA hydratase, and a hydroxyacyl-CoA dehydrogenase, respectively. The IBR3, IBR10, and IBR1 proteins contain peroxisomal targeting signals and are likely candidates for catalyzing the various steps of IBA _-oxidation. Analysis of the ibr mutants is providing insight into the roles of IAA that derives
from IBA _-oxidation. (This work is supported by the NSF and the Robert A. Welch Foundation.)
PS1621 SPATIAL AND TEMPORAL EXPRESSION OF CYTOKININ METABOLIC GENES IN WHEAT
Jiancheng Song, Paula Jameson School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
[email protected]
The cytokinins are intimately involved in plant growth and development from the earliest stages of cell division
and organ formation to the final stages of seed development and senescence. At the whole plant level, cytokinin homeostasis may be achieved through the coordinated regulation of several multi-gene families, including those coding
for isopentenyl transferase (IPT) for CK biosynthesis, cytokinin oxidase/dehydrogenase (CKX) for degradation, zeatin
glucosyltransferase (ZOG) for reversible inactivation, and _-glucosidase (GLU) for reactivation. Despite advances in
cytokinin perception and signal transduction, the molecular mechanism(s) that controls cytokinin homeostasis remains
to be elucidated.
We have isolated fragments for most members of the multi-gene families of IPT, CKX, ZOG and GLU homologues in
wheat using specific or degenerate RT-PCR primers based on the publicly available sequence data, including unigenes,
clones, and ESTs. Spatial and temporal expression of each of these genes were quantified using real-time PCR. Significant expression was successfully detected for all tested members of the four gene families, with some 1500- to 2000-fold
expression differences at spatial and temporal levels. The systematic and coordinated regulation of cytokinin homeostasis
in wheat during leaf and seed development will be discussed.
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PS1622 IAR4 REGULATES AUXIN HOMEOSTASIS
Quint, Marcel1,2, Barkawi, Lana S.3, Cohen, Jerry D. 3, and Gray, William M.2 1 Leibniz-Institute for Plant Biochemistry, Department of Stress and Developmental Biology, 06120 Halle, Germany 2 Department of Plant Biology, University of Minnesota,St. Paul, MN 55108, USA 3 Department of Horticultural Science and Microbial and Plant Genomics
Institute,University of Minnesota, St. Paul, MN 55108, USA
[email protected]
In a screen for enhancers of tir1-1 auxin resistance, we identified two novel alleles of the putative mt pyruvate dehydrogenase E1_ subunit, IAR4. The iar4-3 mutation interacts synergistically with tir1-1 in root growth inhibition and
lateral root development assays. Additionally, iar4 single mutants exhibit numerous auxin-related phenotypes including
auxin-resistant root growth and reduced lateral root development, as well as severe defects in primary root growth, root
hair initiation and root hair elongation. To examine the effects of iar4 mutations on SCFTIR1 activity, the HS:AXR3NTGUS reporter was introduced into iar4-3 to examine Aux/IAA protein stability. While the basal level and stability of the
AXR3NT-GUS fusion protein are significantly increased in iar4-3 compared to wild-type, AXR3NT-GUS degradation
in auxin-supplemented media is comparable to wild-type. However, unlike known auxin response components, IAR4:
IAR4:GFP protein localizes to the mitochondria. Remarkably, all of the described iar4 mutant defects are rescued when
the seedlings are grown at high temperature (28°C). Since auxin biosynthesis in planta is increased at high temperature,
the iar4-3 phenotypes may be the result of a defect in auxin homeostasis. In support of this hypothesis, the activationtagged allele of YUCCA, previously shown to confer elevated levels of free IAA, also rescues most of the iar4-3 mutant
phenotypes. IAA measurements detected no significant difference between iar4-3 and wild-type for free IAA, but a
significantly higher level of IAA-amino acid conjugates was observed in the iar4-3 mutant. Furthermore, the tryptophanindependent IAA biosynthesis pathway was upregulated in the mutant. We therefore suggest that iar4 mutations affect
auxin homeostasis with secondary effects on basal auxin responses, perhaps due to localized or transient IAA dynamics.
PS1623 FUNCTIONAL ANALYSIS OF AGLYCONE-BINDING SITE OF THE MAIZE BETA GLUCOSIDASE ZM-P60.1
Radka Fohlerová 1,2, Pavel Mazura 1,2,3, Lubomír Janda 2,3, Radka Chaloupková 4, Petr Jeøábek 4, Jiøí Damborsk_ 4
and Bøetislav Brzobohat_ 1,3 [email protected] 1 Institute of Biophysics AS CR, CZ-61265, Brno, Czech Republic;
2 Department of Functional Genomics and Proteomics, Masaryk University, CZ-61137 Brno, Czech Republic; 3 Department of Molecular Biology and Radiobiology, Mendel University of Agriculture and Forestry, CZ-61300 Brno, Czech
Republic; 4 Loschmidt Laboratories, Masaryk University, CZ-61137 Brno, Czech Republic
[email protected]
Zeatin-O-glucoside was found as a natural substrate for beta-glucosidases Zm-p60.1 (maize) and Bgl4:1 (B. napus).
These enzymes share overall 44 % sequence identity, but amino acids in their active site differ dramatically. To analyze
molecular evolution in the active sites, we generated single-point (F193A, F200K, W373K and F461L) and multiplepoint (F193A+F200K+W373K+F461L) mutant of Zm-p60.1 reproducing the active site of Bgl4:1. All mutant proteins
were expressed as soluble proteins in E. coli. The impact of the mutations on the enzymatic activity and protein structure
was tested. 4-Nitrophenyl beta-D-glucopyranoside (PNPG) and 4-Methylumbelliferyl beta-D-glucopyranoside (MUG)
were used as the substrates for activity measurements. Structural changes were determined by circular dichroism. Compared to the Zm-p60.1, a dramatic alteration in structure accompanied with a loss of activity was observed for the
multiple mutant. Small structural changes were determined also for mutants F200K and F461L. F461L exerted a little
positive and F200K significantly negative effect on enzyme kinetics. Identical structures with Zm-p60.1 were found
in mutants W373K and F193A. Loss of stacking interaction between W373 and substrate aglycone caused the most
dramatic decrease of the enzyme activity. Compared to Zm-p60.1 wild type, F193A mutant showed a 16- and 1.3-fold
decrease in Km, and a 160- and 15-fold decrease in kcat against PNPG and MUG, respectively. Molecular modelling
suggests energetically favourable non-productive binding of PNPG and MUG to the F193A active site as the main cause
of the changed kinetic parameters.
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This project was supported by the Ministry of Education, Youth and Sports of the Czech Republic, LC06034 and
MSM0021622415, the Grant Agency of the Czech Republic (GACR203/02/0865) and the Academy of Sciences of the
Czech Republic (AVOZ50040507).
PS1624 REGULATION OF ZEATIN GLUCOSYLTRANSFERASE GENE EXPRESSION IN ARABIDOPSIS
Albert Pineda Rodo, Sara C. Haines, Jaleh M. Olson, David W.S. Mok and Machteld C. Mok. Dept. of Horticulture and
CGRB, Oregon State Univ., Corvallis, OR 97331, USA
[email protected]
Cytokinins can be conjugated to N- and O-glucosides. While N-glucosylation generally leads to irretrievable loss of
activity, O-glucosides may play an important role in cytokinin storage and homeostasis since they can be hydrolyzed to
the corresponding aglycones. The first zeatin O-glycosyltransferase genes isolated were ZOG1 and ZOX1 from beans
(Martin et al., 1999a,b), and more recently several zeatin O- and N-glucosyltransferase genes were identified in Arabidopsis (Hou et al., 2005). In order to understand the dynamics of zeatin glucosylation, we studied the expression of the
three major Arabidopsis glucosyltransferases: an O-glucosyltransferase (At1g22400) and two N-glucosyltransferases
(At5g05860 and At5g05870). GUS staining of plants transformed with promoter-GUS fusions showed some overlapping functions, particularly in the root tips, but also distinct temporal and tissue-specific expression patterns for each
gene. RT-PCR indicated differential gene expression in response to stress and hormone treatments. Most interestingly,
At1g22400 was consistently expressed in guard cells and induced by drought stress. Preliminary studies with insertional
mutants involving the At1g22400 locus suggest a possible role of zeatin O-glycosylation in stomatal closing. Arabidopsis
transformants harboring ORF:GFP fusion constructs under the control of the CaMV 35S promoter showed cytosolic and
nuclear fluorescence, comparable to that found for 35S::GFP expression. Our findings indicate that cytokinin glucosyltransferases are functionally significant by rapidly changing active cytokinin levels in specific cell types.
PS1625 AN ALTERNATIVE PATHWAY TO PRODUCE CASTASTERONE IN ARABIDOPSIS THALIANA
Se-Hwan, Joo, Jung-Yun, Hwang, Chan Ho, Park, Hojin, Namgung, A-Ram, Kim and Seong-Ki, Kim* *Department of
Life Science, Chung-Ang University, Seoul, 156-756, Korea
[email protected]
In vitro conversion studies using a cell-free enzyme solution prepared from A. thaliana demonstrated that two parallel
biosynthetic pathways to generate 28-norcastasterone, namely the early and late C-6 oxidation for C27-brassinosteroids
(BRs), are operative in the plants. In the presence of S-adenosylmethionine, the enzyme solution successfully mediated conversion of 28-norcastasterone to castasterone, indicating that the C27-BRs biosynthesis is an alternative route
to synthesize biologically active C28-BRs, castasterone and brassinolide, in the plant. In the presentation, biosynthetic
connections between C28-BRs and C27-BRs biosynthesis as well as between the early and late C-6 oxidation pathway
for C27-BRs biosynthesis will be explained. In addition, enzymes (genes) involved in the C27-BRs biosynthesis will be
discussed.
PS1626 ISOLATION AND IDENTIFICATION OF PHENOLIC PRECURSOR OF ELECTRON ACCEPTOR FOR MAIZE CYTOKININ DEHYDROGENASE
Frébortová Jitka 1, Novák Ondøej 1, Jorda Radek 2 1 Laboratory of Growth Regulators, Palack_ University/IEB AS CR;
2 Department of Biochemistry, Palack_ University; Slechtitelù 11, 78371 Olomouc, Czech Republic
[email protected]
Cytokinin dehydrogenase (CKX) catalyzes an oxidative cleavage of a cytokinin side chain. A quinone-type electron
acceptor is used for reoxidation of reduced flavin cofactor by the enzyme in vitro. Co-localization of CKX and polyphenol oxidase (PPO) laccase in maize tissues together with detection of a phenolic precursor enhancing CKX activity
after conversion by PPO in maize phloem sap suggest that CKX may in vivo use oxidation products of plant phenolics
as electron acceptors.
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To identify these phenolic precursors, phloem sap from maize seedlings was collected, fractionated using HPLC and the
presence of phenolic precursors in isolated fractions tested. Active compounds were further examined using HPLC with
Q-TOF detector and were identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 6-methoxybenzoxazolin-2-one (MBOA). Quinone products generated by reaction of DIMBOA with tyrosinase and peroxidase were
studied spectrophotometrically as well as using HPLC and their stability was confirmed by discontinuous CKX activity
assay. Possible involvement of DIMBOA in cytokinin metabolism is an opened question and will be further investigated.
PS1627 THE ROLE OF GA-SENSITIVE DWARF1-1D (GSD1-1D) MUTATION IN GIBBERELLIN METABOLISM
Ayele, Belay T.1, Sumin Lee2, Hiroshi Magome1, Yuji Kamiya1, Shinjiro Yamaguchi1, Moon-Soo Soh2 1 Cellular
Growth and Development Research Team, RIKEN Plant Science Center, Yokohama, Japan; 2 Department of Molecular
Biology, Sejong University, 98, Gunja-Dong, Gwangjin-Gu, Seoul, Republic of Korea 143-747
[email protected]
A new dominant gibberellin (GA)-sensitive semi-dwarf mutant, GA-sensitive dwarf1-1D (gsd1-1D), has recently been
isolated by genetic screening from the pools of Arabidopsis transposon-tagged mutants. Although this mutant was found
in a transposon-tagged pool, genetic analysis revealed that the semi-dwarf phenotype did not co-segregate with the transposon insertion. Phenotypes of this mutant are similar to those displayed by GA deficient mutants, and can be restored by
treatment with exogenous GAs. With the aim to understand the possible role of gsd1-1D in GA metabolism, endogenous
GA (precursors, bioactive GAs and their catabolites) levels were analyzed in 18-day-old wild-type and gsd1-1D mutant
plants (when difference in growth was evident). Consistent to the mutant phenotype, the gsd1-1D mutation reduced bioactive GA4 to a level below the detection limit of our LC/MS/MS system. The result suggests that the gsd1-1D mutation
affects GA metabolism. Further studies are in progress to elucidate the regulatory mechanism.
PS1628 ESTABLISHMENT OF METHOD FOR HIGH SENSITIVE PLANT HORMONE ANALYSIS BY USING LC-ESIMS/MS
Yusuke Jikumaru, Shinjiro Yamaguchi, Eiji Nambara, Yuji Kamiya RIKEN Plant Science Center
[email protected]
Plant hormones have been known as signal compounds which regulate various responses at a low concentration in
plants. To reveal the system, endogenous level of them had been quantified mainly by using GC-EI-MS. Recently, microanalysis by using LC-ESI-MS/MS have been carried out frequently. In this method, samples do not require complicated purification and derivatization that GC-EI-MS requires. However, because ionization efficiency in LC-ESI-MS/MS
method is susceptible to impurities, it requires suitable purification.
Our objective is that establishment of method for comprehensive analysis of all bioactive plant hormones and their precursor/metabolite. Here we will report the method for comprehensive analysis of bioactive hormones which are GA1,
GA4, ABA, IAA, JA, CKs, SA, ACC and castasterone, one of BRs in Arabidopsis and rice. In our method, required
amount of plant material is 1/1000 to 1/10 to that of GC-EI-MS analysis.
PS1629 POTENTIALS FOR CELL SPECIFIC ANALYSIS OF HORMONE SIGNALLING AND METABOLISM
Sara V. Petersson1, Mariusz Kowalczyk1, Göran Sandberg2, Karin Ljung1 1Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden 2Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE-901 87 Umeå, Sweden sara.
[email protected]
[email protected]
The root apex of an Arabidopsis thaliana seedling is an excellent model system for studying IAA metabolism and signalling. We have taken advantage of the large number of available Arabidopsis lines with cell type specific GFP expression and used them in our studies. Protoplasts isolated from these lines and sorted by Fluorescent Activated Cell Sorting
(FACS) in combination with very sensitive analytical instruments such as GC-MS-MS and LC-MS-MS are powerful
tools in order to understand the mechanisms that are regulating hormone metabolism and signalling. We have now started
to unravel some of these mechanisms at a tissue and cellular level. We have recently developed methods to quantify IAA
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and resolve IAA synthesis rates at cellular resolution, making it possible to construct the first map of IAA distribution
in the Arabidopsis root apex. We have also developed methods to describe metabolic profiles of specific cell types and
we are now working on methods to monitor differences in protein concentration in the same cell types. The analyses of
hormone metabolism and metabolite and protein concentrations can then be correlated to differences in gene expression
within the same cells. The ability to isolate and analyse specific cell types from the Arabidopsis root apex and also from
other plant tissues opens up a vast range of possibilities. These methods will hopefully help us to better understand hormone metabolism and signalling and the roles of these substances in the regulation of plant growth and development.
PS1630 NEWLY SYNTHESIZED IAA AT THE TIP REGION IS IMMEDIATELY TRANSPORTED LOWER PARTS IN MAIZE
COLEOPTILES
Hitomi Nakano, Takeshi Nishimura, Tomokazu Koshiba Department of Biological Sciences, Tokyo Metropolitan Univ.,
Tokyo, 192-0397, Japan; [email protected]
[email protected]
Indole-3-acetic acid (IAA) is an important hormone in almost all aspects of plant growth and development. From
our previous work using maize coleoptiles, it has been indicated that the tip region of coleoptiles is the site of IAA biosynthesis from tryptophan (Trp) and the IAA synthesized in the tips is transported to the lower regions. However these
studied was performed with sectioned maize coleoptiles. We have developed an intact experimental system to investigate
Trp-dependent IAA biosynthesis and movement of synthesized IAA, and here we present the results about the high efficient incorporation of stable isotope label from [13C1115N2]-Trp into IAA molecules. Moreover, the labeled IAA was
immediately transported to the lower parts at the rate of approximately 7 mm/hr. These results suggest that IAA in the
tips is continuously synthesized from Trp at the tip region and is just transported to the lower region. Because, even the
concentration of [13C1115N2]-Trp was changed, the amount of IAA in the coleoptile was not varied, this system might
be reflected in vivo IAA biosynthetic situations.
PS1631 SYNTHESIS OF 9,11,11,12,12-DEUTERIUM LABELLED C21- AND C19-STEROIDS
Hideharu Seto1, Kei Yamamoto1,2, Shozo Fujioka1, Takao Saito2, Shogo Matsumoto1 1RIKEN, Discovery Res. Inst.,
Wako-shi, Saitama 351-0198, Japan; 2Tokyo Univ. Sci., Fac. Sci., Dept. Chem., Shinjuku-Ku, Tokyo 162-8601, Japan;
[email protected]
[email protected]
C21-, C19- and C18-steroids including mammalian sex hormones, e.g., progesterone (pregn-4-ene-3,20-dione), testosterone (17ƒÀ-hydroxy-androst-4-en-3-one) and estrone, have been isolated or identified from plants on a number of
occasions, so far. However, their distribution and metabolism as well as physiological roles in plant kingdom remain
almost obscure. Our interests in these themes prompted us to construct a compound library consisting of a variety of
polydeuterium labelled C21-, C19- and C18-steroids. These compounds should be useful as the internal standards in
GC-MS/LC-MS quantitative analysis and substrates in metabolic experiments. In this paper, we describe the synthesis
of [9,11,11,12,12-2H5]progesterone (1) and [9,11,11,12,12-2H5]androstenedione (androst-4-ene-3,17-dione) (2) from
11ƒ¿-hydroxyprogesterone and adrenosterone (androst-4-ene-3,11,17-trione). The deuterium atoms incorporated were
chemically stable and the deuterium content (2H0:2H1:2H2:2H3:2H4:2H5=0:0:0:3:23:73) was sufficient for our purpose.
Since the easily-available starting materials and good overall yields allowed the gram-scale preparation, we now synthesis a variety of 9,11,11,12,12-deuterium labelled C21- and C19-steroids including [9,11,11,12,12-2H5]pregnenolone
(3ƒÀ-hydroxypregn-5-en-20-one) from 1 and 2.
PS1632 ANALYSIS OF THE INTERMEDIATES IN THE IAA BIOSYNTHETIC PATHWAY BY LC-MS/MS
Hiroyuki Kasahara1, Yusuke Jikumaru1, Shojiro Hishiyama2, Satoko Sugawara1 and Yuji Kamiya1 1RIKEN PSC, Yokohama 230-0045, Japan, 2FFPRI, Tsukuba 305-8687, Japan
[email protected]
The biosynthetic pathway for indole-3-acetic acid (IAA) in plants is still largely unknown. To identify intermediates
in the IAA biosynthetic pathway, we have developed a new method for the analysis of IAA intermediates by LC-TOF-
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MS/MS. We synthesized 15N or 2H-labeled tryptamine (TAM), N-hydroxy-tryptamine (HTAM), indole-3-acetaldoxime
(IAOx), indole-3-acetonitrile (IAN) and indole-3-butylic acid (IBA) as internal standards. Using our analytical method,
we detected IAOx as a key intermediate in the IAA biosynthesis from Arabidopsis seedlings. Importantly, we also detected IAOx from cyp79b2cyp79b3 double knockout mutant, in which IAOx production for indole glucosinolate is deficient.
This result suggests that IAOx may be also involved in other IAA biosynthetic pathway, e.g. the YUCCA pathway, in
plants. We will also present the results of IAA intermediate analysis for other IAA biosynthesis-related mutants.
PS1633 EFFECTS OF OVEREXPRESSED CYTOKININ OXIDASE/DEHYDROGENASE ON THE CELL DIVISION, CELL
ELONGATION, POLARITY AND AUXIN RESPONSE OF TOBACCO BY-2 CELLS
Hartig, Katja Dep. Plant Physiol., University of Bayreuth, 95440 Bayreuth, Germany
[email protected]
Cytokinins are together with auxin the essential hormones in respect to the initiation and maintaining of the plant
cell division, however the understanding of their role in the cell cycle is being still fragmentary. One way to learn more
about functions of cytokinins is the manipulation of the cytokinin metabolism. By the overexpression of the cytokinin
degrading cytokinin oxidase/dehydrogenase (AtCKX4) it was able to enhance the natural CKX-activity of tobacco BY-2
cells three-fold. As a result the concentrations of the free and the storage forms of the t-zeatin group of cytokinins are
significantly lower. The cell cycle of the transgenic cell line is shortened by a decreasing length of the G1 and the G2
phases – consequently the grow rate of this cell line is enhanced in comparison with the wild type. Moreover the CKX4overexpressing cells are smaller and their elongation is suppressed, whereas the polarity of cells is stronger pronounced.
Surprisingly the CKX-overexpressing cells show a modified response to applied auxins (2,4-D; NAA and IAA) – the
optimal concentration range for the cell cycle initiation is lower in this cells. The results which are presented here demonstrate ones: That cytokinin degradation represents a time-limiting process during the cell cycle progression at least of
meristematic root cells and secondly: That cytokinin metabolism affects auxin metabolism or response.
PS1634 PROFILING OF ABA METABOLITES IN PLANT TISSUES BY UPLC-MS-MS
Veronika Hradecká, Ondøej Novák, Miroslav Strnad Laboratory of Growth Regulators, Palack_ University & Institute
of Experimental Botany ASCR, _lechtitelù 11, CZ-783 71, Olomouc, Czech Republic
[email protected]
The monocyclic sesquiterpene S-(+)-abscisic acid (ABA) is a key signaling substance in all higher plants. ABA plays
an important role in a number of physiological processes such as seed maturation, seed dormancy, and adaptive responses
to abiotic stress [1]. The development of improved profiling methods is critically important for understanding the role of
hormone-induced signalling networks in controlling specific developmental pathways or physiological responses. Using
the UPLC combined with tandem mass spectrometry (MS/MS) analysis, a 10-fold increase of throughput and sensitively
was obtained compared to the traditional LC/MS system. Furthermore, the UPLC can reduce total time of the analytical
procedure, from sample handover to determination of phytohormone content, only a few days or hours, respectively.
We describe here a simple, reliable and rapid method of extracting, purifying and determining the phytohormone ABA
and its metabolites in different plant tissues by ultra performance liquid chromatography separation combined with tandem mass spectrometry (UPLC-MS-MS).
[1] Nambara, E., Marion-Poll, A.: Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant Biol. 56, 165-185, 2005.
PS1635 THE EFFECTS OF AUXIN TRANSPORT INHIBITORS (ATIS) ON EXPRESSION OF GA 20-OXIDASE ARE DUE
TO AUXIN LEVELS AND FEEDBACK REGULATION
Desgagne-Penix, Isabel, Sponsel, Valerie Biology Dept., UTSA, San Antonio, TX 78249, USA
[email protected]
Auxins have been shown by others to regulate expression of certain genes encoding enzymes in the GA biosynthetic
pathway. We have examined the effects of auxins and ATIs on the expression of GA20oxidase1 in Arabidopsis seedlings.
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The enhanced expression of GA20ox1 in ATI-treated seedlings is several orders of magnitude more than that induced
by auxins. We propose two reasons for this. First, using quantitative reporter gene assays, we observe more auxin accumulation in shoots after ATI treatment than after auxin treatment. Second, it appears that ATIs deplete endogenous GAs,
leading to an additional effect on GA20ox1 related to feedback regulation. Overall the expression of GA20ox1 can be
manipulated by auxins and ATIs but only in shoots, not roots, and only at certain developmental stages. Both organ and
developmental regulation of GA20ox1 expression appear to override regulation by either feedback or auxin.
Supported by MBRS-RISE GM60655 and NSF.
PS1636 DETERMINATION OF BRASSINOSTEROIDS BY IMMUNOAFFINITY CHROMATOGRAPHY AND UPLC/MS
Swaczynová, Jana 1, Novák, Ondrej 1, Kohout,Ladislav 2, and Strnad, Miroslav 1, 1 Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palack_ University, _lechtitelu 11, 78371 Olomouc, Czech Republic,
+420585634859, [email protected] 2 Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo námìstí
2, 16610 Praha 6, Czech Republic.
[email protected]
Brassinosteroids (BRs) are polyhydroxylated steroid plant hormones showing essential effects on growth and development of plants. They induce cell elongation and cell division, increase DNA and RNA polymerase activity; interact synergistically with auxins; stimulate ethylene production, increase tolerance to stress due to temperature, water or salinity.
The detection and quantification of brassinosteroids is quite difficult because its amount in plant tissues is extremely low.
We have established sensitive and specific method for brassinosteroid determination based on combined immunoaffinity
chromatography and UPLC/MS.
We have developed polyclonal antibodies against a brassinosteroid analogue, (20S)-2£\,3£\-dihydroxy-7-oxa-7£\-homo5£\-pregnane-6one-20 carboxylic acid (4812). Antiserum against this substance was raised in rabbits immunized using
4812 - bovine-serum albumin (BSA) conjugate. Polyclonal antibodies were cleaned up by affinity purification on protein
A. The antibodies were coupled to Affi-Gel„¥ 10 and immunoaffinity columns were prepared. The obtained antibodies
were tested in enzyme-linked immunosorbent assay (ELISA) using (20S)-2£\,3£\-dihydroxy-7-oxa-7£\-homo-5£\-pregnane-6one-20 carboxylic acid ¡V horse-radish peroxidase conjugate. The cross-reactivities of 11 compounds including
the natural and synthetic BRs were investigated, revealing the broad specificities of the antibodies.
The tissues of young bean (Phaseolus vulgaris L., cv. Pinto), Arabidopsis thaliana L. Heynh. seedlings and rape pollen
(Brassica napus L were used for the analysis of endogenous brassinosteroids. Purified samples were analyzed by Acquity
Ultra Performance Liquid Chromatography (UPLC) linked to a Quattro micro API mass spectrometer equipped with an
electrospray interface (Waters).
This work was supported by the grant MSM 6198959216 of the Ministry of Education of Czech Republic.
PS1637 METABOLIC PROFILING OF CYTOKININS IN PLANT TISSUES.
Novak, Ondrej, Hauserova, Eva, Amakorova, Petra, Dolezal, Karel, Strnad, Miroslav Laboratory of Growth Regulators,
Palacky University and Institute of Experimental Botany ASCR, Slechtitelu 11, CZ-78371 Olomouc, Czech Republic.
[email protected]
We have developed a simple and efficient purification procedure of plant tissues by batch immunoextraction (IAE),
compatible with a new chromatographic technique, the ultra-performance liquid chromatography (UPLC), for estimation
of wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides). The UPLC was
coupled to tandem (quadrupole) mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg)
amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity step and completed by
fast chromatographic separation of 32 cytokinin derivatives. In multiple reaction monitoring (MRM) mode, the detection
limit for most of cytokinins analysed was close to 1 fmol and the linear range was at least over the five orders of magnitude. The method parameters were tested on various types of plant material. The efficiency of extraction and purification
procedure was determined using samples of the poplar leaves (Populus x canadensis Moench, cv Robusta). The accuracy
of the developed method was further validated using extracts of 10-day-old Arabidopsis thaliana plants spiked with 1 and
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10 pmol of cytokinin derivatives which also showed high reproducibility. Our results will show usefulness of suggested
protocols combined SPE with IAE for cytokinin analyses and UPLC-ESI(+)-MS/MS technology. This approach can be
used for fast and sensitive quantitative and qualitative analysis of broad spectra of natural cytokinins in minute amounts
of plant tissues with high reproducibility and accuracy.
PS1638 FUNCTIONAL ANALYSIS OF RICE CYP85
Bo Kyung Kim, Shozo Fujioka2, Suguru Takatsuto3, Masafumi Tsujimoto2, and Sunghwa Choe1 1 Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea [email protected] 2
RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, Japan 3 Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943-8512, Japan
[email protected]
Brassinosteroids (BRs) collectively refer to steroidal plant hormones that are essential for proper growth and development of plants. It has been proposed that BRs are synthesized via two parallel pathways, the early and the late C-6 oxidation pathways according to the order of C-6 oxidation status. One is the early C-6 oxidation pathway, in which oxidation
at C-6 occurs before the introduction of vicinal hydroxyls at C-22 and C-23 of the side chain. The other is the late C-6
oxidation pathway in which C-6 is oxidized after the introduction of hydroxyls on the side chain. The C-6 oxidation of
BR intermediates is catalyzed by the enzymes encoded by Cytochrome P450 85 (CYP85) genes. Arabidopsis CYP85
enzymes have been shown to catalyze C-6 oxidation of 6-deoxo intermediates. Interestingly, Arabidopsis CYP85A2
was also found to mediate the ultimate step which is Baeyer-Villiger type oxidation of C-6. However, the functions of
rice CYP85 protein as a BL synthase are still unknown. Therefore, we aimed to understand the function through feeding
experiments with a yeast strain that is heterologously expressing the rice CYP85 gene. Feeding tests following GC-MS
based analyses revealed that both Arabidopsis CYP85A2 and rice CYP85 metabolize 6-deoxo-BRs into Castasterone.
However, unlike Arabidopsis CYP85A2, rice CYP85 does not metabolize Castasterone into brassinolide. This result and
previously reported profiles of endogenous brassinosteroids in rice plants suggests that rice has Castasterone as its end
product due to lack of brassinolide synthase activity in the rice CYP85 enzyme.
PS1639 ROLES FOR MULTIPLE AUXIN BIOSYNTHETIC PATHWAYS
Barkawi, Lana S., Gardner, Gary, Cohen, Jerry D.
[email protected]
Biochemical and genetic studies of higher plants have shown that the normal pathway for the production of indole-3acetic acid (IAA) in seedlings is direct from indole by a tryptophan-independent pathway (TI). A tryptophan-dependent
pathway (TD) has been shown to be operative, however, in the first hours after a wounding stress. It is unknown which
TD pathway is activated by wounding or if the IAA produced following wounding has a discrete role from the IAA produced under normal growth conditions. We are testing whether the TD-IAA that is used for local growth at the site of a
wound comes from the same metabolic source as the IAA that is transported by polar auxin transport (PAT) for normal
growth and development. To examine these ideas, PAT studies have been done with maize coleoptile sections using agar
donor (containing 3H-IAA) and receiver blocks. To minimize the effects of wounding, PAT experiments were also done
on intact seedlings by microinjection at the apex. In these experiments, PAT was inhibited by 1-naphthylphthalamic acid
(NPA). Stable isotope labeled tryptophan and anthranilate, precursors whose relative incorporation into IAA reveals
which pathway is being favored, have been used in the donor blocks and at the apex of intact seedlings, and we have
seen evidence of polar transport of both that is partially inhibited by NPA. We are using mass spectrometry in order to
determine the labeling of IAA from each precursor to establish which pathway is being favored. We have found that in
coleoptile sections PAT capacity drops steadily over time after wounding. It may be that callus cells that form at the cut
end of the section do not transport IAA basipetally, perhaps as a mechanism to maintain high levels of IAA at the wound
site.
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