Download making cotton significantly more drought-tolerant

MAKING COTTON SIGNIFICANTLY MORE DROUGHT-TOLERANT
Hong Zhang and Dick Auld, Texas Tech University, Lubbock, TX 79409
Drought is the major limiting factor for cotton production in the Southwest. To increase droughttolerance in cotton, a new approach is proposed. Dr. Eduardo Blumwald of UC Davis has recently
expressed a gene called IPT in tobacco plants and observed a significantly improved droughttolerance phenotype in transgenic tobacco plants. The IPT gene encodes an isopentenyl transferase
that plays a critical role in cytokinin biosynthesis in Arabisopsis. When IPT expression is controlled
by a water-deficit inducible promoter, it would increase cytokinin biosynthesis under water-deficit
conditions, which in turn activates defense mechanisms that protect plants under water-deficit
conditions. We introduced the same gene construct into Arabidopsis and also obtained transgenic
plants that are significantly more drought-tolerant than wild-type plants. These preliminary data
indicate a possbility that cotton may be made significantly more drought-tolerant when IPT is
overexpressed under severe water-deficit conditions.
1
TOWARDS THE GENETIC IMPROVEMENT OF COTTON FIBER QUALITY IN
RESPONSE TO LOW TEMPERATURES
Thea A. Wilkins1, Noureddine Abidi1,2 and Eric Hequet1,2
1
Department of Plant & Soil Science and 2International Textile Center, Texas Tech University,
Lubbock, Texas 79409
Cotton is a vital agricultural commodity to the U.S. economy as an important renewable resource
and number one value-added crop, generating revenues in excess of $100 billion annually. More
than 90% of cotton’s value resides in the production of fiber, yet yield and fiber quality is on the
decline. This downward trend has been attributed to the erosion of genetic diversity in breeding
programs and increasing vulnerability of germplasm to environmental stress. Fiber maturity, a
major component of yield and fiber quality, is deleteriously impacted by the low night temperatures
typical of the southwestern states of the cotton growing belt, costing producers millions of dollars in
lost revenue. Understanding the molecular mechanism by which cold stress affects fiber growth and
development, and hence, impacts yield and fiber quality will facilitate the design and
implementation of strategies aimed towards developing chilling tolerant germplasm using molecular
approaches.
Fiber maturity is dictated by the thickness, composition and structure of the secondary cell wall
(SCW), which is composed of almost pure celluose, and as such, fiber maturity is therefore
governed by the rate and duration of the cellulose synthesis during SCW biogenesis. The optimal
temperature for cellulose biosynthesis in field-grown and fibers from in vitro cultured ovules ranges
between 28-30°C, and temperatures below 28°C not only reduce fiber maturity, but also result in
structural changes to the SCW. It has been hypothesized that suboptimal temperatures suppress the
rate of cellulose synthesis and induce the formation of diagnostic tree-like “growth rings” in the
SCW as a consequence, especially in cold-sensitive varieties. In chilling tolerant species, rapid
induction of the conserved CBF regulon results in changes in the structure, composition and
function of the plasma membrane, and indeed, it has been demonstrated that overexpression of CBF
transcription factors can confer chilling tolerance in otherwise cold-sensitive species.
The main goal of the submitted proposal is to gain novel insight into the molecular mechanisms
regulating fiber maturity in response to low temperatures as a step towards the genetic enhancement
of fiber quality by breeding programs using molecular strategies. A combination of genetic,
genomic, structural, and biochemical approaches as well as the physical sciences will be used to test
the operating premise that i) suboptimal temperatures negatively impact carbon flux and the rate of
cellulose synthesis, ii) the response varies in genotypes that differ in response to suboptimal
temperatures, and iii) overexpression of CBF transcription factors to manipulate the CBF regulon
can confer enhanced chilling tolerance in elite cotton cultivars. The successful completion of these
studies will provide new insight into the regulation of carbon metabolism and cell wall biogenesis in
response to cold stress. The proposed project will develop tools and resources, including unique
germplasm, to address fundamental and applied questions that will enhance our understanding of
fiber maturity, and facilitate generating chilling tolerant cotton germplasm for the southwest.
2
THE USE OF NATURALLY OCCURRING ENDOPHYTES AND MYCORRHIZAE OF
CAPSICUM TO REDUCE DISEASE AND DROUGHT DAMAGE OF CULTIVATED
CHILE
Scott Kroken, University of Arizona, and Soum Sanogo, New Mexico State University
Chile (pepper) is a culturally and economically important crop of the American southwest, and a
significant vegetable and spice crop worldwide. The cultivation of chile is impacted by two
common stresses: diseases and drought. Phytophthora blight, caused by Phytophthora capsici, and
Verticillium wilt, caused by Verticillium dahliae, are two diseases that limit profitable production of
many crops, especially in chile. These pathogens cause root, stem, and vascular dysfunction,
culminating in wilting and plant death. Chemical control is not routinely employed owing to costs,
variable effectiveness, the potential of rapid development of resistance within populations of
pathogens, and concerns for environmental health. Research on chile resistance to wilt diseases has
not yet yielded resistant commercial cultivars. Although crop rotation and water management are
the most recommended control methods, they are not used consistently due to economic constraints.
Given the unavailability of resistant cultivars and the economic impracticability of using chemical
and cultural control methods, there is a need to explore new avenues of control. Drought stress also
reduces the quantity and quality of chiles, and causes an increase their levels of capsaicin, which
gives chiles their heat. Therefore, minimizing water stress will result in higher yields and a more
uniform product.
Both disease and drought damage can be minimized through the use of mutualistic fungi.
Mycorrhizal fungi improve the host plant’s ability to avoid water stress by acting as extensions of
the root system. In addition, under certain conditions mycorrhizal fungi induce resistance in the
host plant to subsequent infections to pathogenic fungi and other microbes. The roles of endophytic
fungi are more newly appreciated. Endophytes, which live entirely inside plant tissues, improve the
host plant’s ability to tolerate drought stress through the accumulation of osmotically active solutes.
And, like mycorrhizal fungi, endophytes are known to induce systemic acquired responses in the
host plant against pathogenic microbes. The positive effects of mycorrhizal fungi on drought
avoidance on chile have been documented. In contrast, no studies have been conducted to look for
naturally occurring endophytic fungi of Capsicum, or to demonstrate positive benefits of inoculating
chile with endophytes as agents of biological control. However, endophytic fungi have a potential
advantage in that they are more likely to establish and survive in their host plant in spite of variance
in the external environment, as they are not subject to external (root zone) conditions, but rather
reside entirely within host plant tissue. Endophytes also have another potential advantage, as they
improve drought tolerance, which may be a complimentary effect to the drought avoidance
provided by mycorrhizal fungi.
In order to be agriculturally useful, mutualistic fungi must both be compatible with cultivated chile,
and be able to establish under field conditions. Therefore, in this proposed study, mutualistic fungi
from New Mexico and Arizona will be collected and evaluated along with fungi that occur in wild
populations of Capsicum spp. in Latin America, the center of genetic diversity. These comparisons
will help determine if it is more important that the mutualistic fungi are from a similar habitat, or if
they have evolved to be compatible with Capsicum. With this approach, we aim to identify
beneficial fungi that, when introduced into chile under agricultural conditions, will improve the
crop’s ability to acclimate to arid land agriculture of the southwestern US, and to improve the crop’s
resistance to soilborne diseases.
3
MYCORRHIZAL EFFECTS ON PATHOGEN RESISTANCE AND WATER RELATIONS IN
ARID AGRO-ECOSYSTEMS
Peter Lammers, Department of Chemistry and Biochemistry and Steven Hanson, Department of
Entomology, Plant Pathology and Weed Science, New Mexico State University
Arbuscular mycorrizal fungi (AMF) are abundant and obligate symbionts of root tissue in most
plants. In natural settings, AMF supply host plants with a significant fraction of their phosphorus
and nitrogen in return for carbohydrate derived from photosynthesis. Some 200 species of AMF
colonize more than 200,000 plants, but the regular use of chemical fertilizers suppresses
colonization, leading to depletion of AMF diversity and biomass1. Yet AMF colonization is well
known to improve drought tolerance2 and suppress plant diseases in many crop species3. The
mechanism(s) of disease suppression by AMF not understood despite a variety of hypotheses4.
Bioprotection of Capsicum annuum against verticillum wilt is known to vary between AMF
species5, yet only a single AMF isolate is produced commercially in North American. So, while
AMF hold tremendous potential for enhancing plant dehydration tolerance and disease resistance,
implementation suffers from a) a century of reliance on chemical fertilizers, b) our ignorance of the
mechanisms of dehydration tolerance and disease suppression, and c) the paucity of studies
identifying superior AMF strains for specific crops. We propose a study of AMF bioprotection of
chile against Phytophthora capsici, which is a particular concern in NM. Thirteen strains of AM
fungi in root organ culture will be analyzed including the model AMF Glomus intraradices. Strains
will be evaluated for the ability to protect chile seedlings from challenge with spores of P. capsici.
Genetic mechanisms of disease suppression and strain differences between fungi will be assessed
using microarrays based techniques.
1.
2.
3.
4.
5.
Treseder, K. & Allen, M. Direct nitrogen and phosphorus limitation of arbuscular
mycorrhizal fungi: a model and field test. New Phytologist 155, 507-515 (2002).
Auge, R. M. et al. Relating foliar dehydration tolerance of mycorrhizal Phaseolus vulgaris to
soil and root colonization by hyphae. J Plant Physiol 160, 1147-56 (2003).
Borowicz, V. Do arbuscular mycorrhizal fungi alter plant-pathogen relations? Ecology 82,
3057-3068 (2001).
Garcia-Garrido, J. M. & Ocampo, J. A. Regulation of the plant defence response in
arbuscular mycorrhizal symbiosis. J Exp Bot 53, 1377-86 (2002).
Garmendia, I., Goicoechea, N. & Aguirreolea, J. Effectiveness of three Glomus species in
protecting pepper (Capsicum annuum L.) against verticillium wilt. Biological Control 31,
296-305 (2004).
4
WATER STRESS ADAPTATION OF SWEET SORGHUM GENOTYPES
FOR ETHANOL PRODUCTION IN THE SOUTHWEST
Michael J. Ottman, Plant Sciences Department, University of Arizona, Tucson, AZ 85721
Donald C. Slack, Dept. Ag. Biosystems Engineering., U. of Ariz., Tucson, AZ 85721
Mark R. Riley, Dept. Ag. Biosystems Engineering., U. of Ariz., Tucson, AZ 85721
Due to recent increases in petroleum prices, and policy declarations from our federal administration,
there is a renewed interest in alternative fuels. Ethanol is increasingly being used as an additive to
unleaded gasoline as MTBE (methyl tertiary butyl ether) is phased out. Ethanol blended fuels must
be handled differently than fuels blended with MTBE. Gasoline/ethanol blends can not be delivered
in pipelines, making shipping and storage more costly. Therefore, local production of ethanol is
advantageous.
The growing demand for ethanol can not be met locally in the southwest at this time. Most of the
ethanol plants in the United States are situated in Midwestern states near the source of the most
commonly used feedstock, corn. In the southwestern US, only 3 ethanol plants are functional
representing less than 1% of the ethanol capacity in the US.
Most of the ethanol produced in the United States is derived from corn. However, ethanol can also
be produced from sugar obtained from sugarcane or from sweet sorghum, as we propose in our
research. Sweet sorghum can be grown efficiently in the Southwest because it requires less
production inputs and fossil fuel than most other crops. Since it is salt tolerant, municipal
wastewater or other saline irrigation water can be used to irrigate the crop. Increasing the sugar
content in sweet sorghum using salt or water stress, as is done in grapes and tomato, may be
possible.
We propose to evaluate sweet sorghum genotype response to water stress through field studies at
the University of Arizona Campus Agricultural Center in Tucson, Arizona. Twelve genotypes will
be grown under three water regimes. The water regimes will be based on soil water depletion and
include 35, 50, and 65% depletion of plant available water. Plant water potential will be measured
before irrigation. Crop water use, growth, and development will be monitored in order to develop a
thermally-based model of water use and development. At harvest, the biomass of the total plant and
stalks, leaves, and heads will be measured. Juice will be pressed from the stalks and the sugars in
the juice will be determined. The juice will be fermented and ethanol production measured. The
feeding quality of the byproducts (head, leaves, and pressed stems) will be evaluated by measuring
acid detergent fiber, neutral detergent fiber, and protein content.
This research is significant since we are attempting to take advantage of arid land agricultural
conditions to meet the increasing demand for alternative fuels.
5
GROWTH, FLOWERING, RUBBER PRODUCTION, AND GENE FLOW OF
TRANSGENIC GUAYULE UNDER ARID AND SEMI-ARID ENVIRONMENTAL
STRESSES
Dennis T. Ray, Division of Horticultural and Crop Sciences, Department of Plant Sciences,
University of Arizona, Tucson, AZ 85721
Normand C. Ellstrand, Biotechnology Impacts Center, Department of Botany and Plant Sciences,
University of California, Riverside, CA 92521
Joceline C. Lega, Department of Mathematics, University of Arizona, Tucson, AZ 85721
Guayule (Parthenium argentatum Gray) has long been a source of natural rubber, but on a minor
scale. However, with the advent of latex allergies to natural rubber from the Brazilian rubber tree
(Hevea brasilensis) guayule has become a commercial rubber-producing crop in the southwestern
United States. There are already commercial acreages in Arizona and California, with
approximately 50,000 new acres being planted in the next five years. Yields per acre have been
increased significantly through traditional plant breeding, because the components of yield that have
to do with biomass production are highly heritable and easily manipulated. However, rubber
content (% rubber), the most important component of yield to the rubber/processing industry, has
not been increased, and in fact lowered in some cases, through traditional plant breeding. The
reasons for this include guayule’s complicated reproductive biology, the low heritability for rubber
production, and the elasticity of the isoprenoid pathway. Because of this lack of progress in raising
the rubber content in new lines, many of the improved guayule lines being developed are
genetically engineered. Because guayule is a perennial crop, there will be previously planted nontransgenic and seed increase fields in the same geographic areas where the new transgenic lines will
be established. In addition, prime guayule growing areas include Arizona, California, New Mexico
and Texas are where related Parthenium species are found; therefore, there is potential for pollenmediated gene transfer from transgenic guayule to not only cultivated plants, but to wild and related
species. Our research will address the following objectives: (1) determine pollen viability and
longevity under environmental conditions encountered throughout the growing season; (2) monitor
flowering and pollen dispersal in the field throughout the flowering period; (3) determine the effect
of different irrigation levels on plant growth, rubber production, and flowering; (4) determine the
level of gene transfer between transgenic guayule and non-transgenic guayule under field
conditions; and (5) develop a mathematical model to predict the probability of pollen mediated gene
transfer, which considers pollen dispersal patterns, and the environmental effects on pollen viability
and longevity so that environmental risk assessment can be made, and containment strategies
developed before the industry is ready to use transgenic plants in their field plantings. This research
is directed toward agriculture in the southwestern United States, and addresses the growth of
guayule under arid and semiarid environmental stresses, and how these environmental stresses
affect growth, flowering, rubber production, and gene flow from transgenic guayule to nontransgenic guayule being grown in the same area, as well as wild Parthenium species.
6
MICROBIAL ARSENIC BIOMETHYLATION: ITS POSSIBLE APPLICATION IN
RHIZOREMEDIATION.
Christopher Rensing (PI)*, Hans VanEtten and Leland S. Pierson
The University of Arizona
Arsenic contamination is a serious world-wide problem with severe consequences for human
health. Arsenic is also prevalent across the Southwest and is present above current EPA standards
in several sources of Southwest water and soils. Elevated As levels in soil and water is a concern
with respect to plant uptake and subsequent entry into wildlife and human food chains. One
mechanism of resistance and reduced uptake of arsenic in plants is achieved by suppression of the
high-affinity phosphate/arsenate uptake system. Fungi and bacteria in the rhizosphere are therefore
likely to have a strong influence on arsenic speciation and uptake as could be demonstrated in
recent studies. We recently characterized an enzyme, ArsM, responsible for arsenite methylation
and subsequent volatilization. This reaction physically removes arsenic from the source. We
propose to engineer well-characterized rhizosphere bacteria to express arsM to aid in arsenic
reduction. At the same time, we will characterize an arsM homolog identified in the rhizosphere
fungus Nectria haematococca. The potential of both organisms to reduce the presence of arsenic
from the rhizosphere and subsequently the plant will be determined.
7
MOLECULAR GENETIC AND BIOCHEMICAL ANALYSES OF HARLEQUIN (HLQ)
AND DC3-OVEREXPRESSED1 (DOR1), PLEIOTROPIC MUTANTS OF ARABIDOPSIS
THAT HAVE ECTOPIC EXPRESSION OF A HORMONE-REGULATED REPORTER
GENE AND CELL WALL DEFECTS
Christopher D. Rock1 and Eugene A. Nothnagel.2
1
Department of Biological Sciences, Texas Tech University, Lubbock TX 79409-3131
2
Department of Botany and Plant Sciences, University of California, Riverside CA 92521
The next few years will mark the epoch of novel gene discovery, after which there will be another
paradigm shift in biological research. The current task of matching data with gene function or with
means of affecting the function is huge. The primary difference between plant and animal cells is
the cell wall. Plant cell walls are complex carbohydrate polymers that comprise the bulk of biomass
on earth and are the ultimate source of fodder and fiber in agriculture, yet knowledge of their
biogenesis and functions in signal transduction of environmental stimuli and plant development is
quite limited, even in model organisms. In some tissues, inhibition of cellulose biosynthesis leads
to hormone-dependent lignin accumulation. Interestingly, several recent reports have established
links between the stress hormone abscisic acid (ABA) and the cell wall. The P. I. has isolated two
mutants of Arabidopsis, harlequin (hlq), and Dc3 overexpression in roots (dor1), that have altered
ABA-, stress- and auxin-inducible late-embryogenesis-abundant Dc3:GUS reporter gene expression
and pleiotropic effects on epidermal cell morphogenesis and the cell wall. Mutants of dor1 have
arrested root elongation with abnormal root hair morphogenesis, and are seedling lethal. The HLQ
gene will be cloned by map-based methods and its subcellular and tissue-specific expression
characterized. The DOR1 gene will be fine-mapped using PCR-based molecular markers (Cleaved
Amplified Polymorphic Sequences [CAPS] and Simple Sequence Length Polymorphisms [SSLPs])
with a view to clone DOR1 in the future. Detailed phenotypic characterization of hlq and dor1 cell
walls by immunocytological, chemical and spectroscopic means will be carried out in reference to
known cell wall mutants (isoxaben resistant2/cellulase synthase 6/procuste 1,
furca2/katanin1/ectopic root hair 3/botero, and radially swollen2/defective cytokinesis/korrigan).
The transcriptome of hlq will be profiled in order to identify the molecular events mediated by the
HLQ gene and to facilitate HLQ gene ontology, especially if the sequence of the HLQ gene is
uninformative. Elucidation of the gene structure and function of HLQ and DOR1 can serve as a
starting point for comparative genomics as a tool to understand plant evolution and diversification.
Characterization and molecular cloning of these genes will lead to new insights into fundamental
cell biological processes involved in ABA-, auxin-, stress perception/responses and cell wall
metabolism affecting morphogenesis in response to environmental and hormonal cues. A major
application of the knowledge gained will be to more fully realize plants' potential for adaptation to
more marginal environments and to genetically improve food and fiber crops for the constraints of
the 21st century– water deficits and high salinity.
8
ROLES OF SALINITY STRESS AND PATHOGEN POPULATION DIVERSITY IN
DEVELOPMENT OF RAPID BLIGHT DISEASE OF TURFGRASS IN THE
SOUTHWESTERN UNITED STATES
Mary W. Olsen, Department of Plant Sciences, Forbes 303, The University of Arizona, Tucson,
AZ 85721.
Francis P. Wong, Department of Plant Pathology, 202 Fawcett Lab, University of California,
Riverside, Riverside, CA 92521.
Greg W. Douhan, Department of Plant Pathology, 238 Fawcett Lab, University of California,
Riverside, CA 92521.
Natalie P. Goldberg, New Mexico State University - Cooperative Extension Service,
Box 30003, MSC 3AE, Las Cruces, NM 88003.
David Kopec, Department of Plant Sciences, Forbes 303, The University of Arizona, Tucson, AZ
85721.
Salinity stress is associated with a new and unique disease of cool season turfgrasses known as
“rapid blight” that has been described in eleven states, including Arizona, California and Texas in
the southwest US. Since its discovery, rapid blight has become a chronic problem on turf irrigated
with high salinity water (usually >3.0 dS/m). In the desert Southwest, it affects cool season turfgrass
varieties used for overseeding bermudagrass such as rough bluegrass (Poa trivialis) and perennial
rye (Lolium perenne) as well as annual bluegrass (Poa annua), creeping bentgrass (Agrostis
paulustris) and colonial bentgrass (Agrostis tenuis). In preliminary laboratory trials for host range
determination, other grasses such as wheat, barley and rice also were susceptible.
The causal agent, first described in 2003, is a new Labyrinthula species, Labyrinthula terrestris. It
is an unusual organism with a confused taxonomy, and other known Labyrinthula species are
associated only with marine plants and algae. A study with ten different isolates of L. terrestris
from turfgrass using r-DNA sequence analysis indicated that these ten isolates were very closely
related to each other but not closely to isolates from marine systems. Nothing is known of the
population structure and genetic diversity of L. terrestris.
This proposal is based on an observed increase in disease in the Southwest in turfgrass irrigated
with high salinity water; the prospect of widespread colonization of warm season turfgrasses with L.
terrestris; a recent discovery of isolates with variations in morphology and pathogenicity; and
decreases in water quality along with increases in salinity of irrigation water as urban water needs
increase. Our objectives are to (1) collect isolates of L. terrestris from sites with different disease
history, quality of irrigation water, soil salinity and turfgrass varieties; (2) use this collection of
isolates to determine ranges in salinity for optimal growth and hosts; and (3) use AFLP analyses to
correlate genotypes with diversities in phenotypes - host range, virulence or salinity tolerance. A
better understanding of the biology of L. terrestris will help in choosing turfgrass species that are
tolerant to the disease, in utilizing the best agronomic practices for these species, in providing
information on how the pathogen is spreading to new locations and its potential to cause disease in
other economic hosts.
9
THE ROLE OF INCREASED SUCROSE PHOSPHATE SYNTHASE (SPS) ACTIVITY ON COLD
TOLERANCE AND SOURCE-SINK RELATIONSHIP IN ALFALFA AND COTTON
Champa Sengupta-Gopalan1 and Scott Holaday2. 1Dept. of Plant & Environ Sci, New Mexico St
Univ, Las Cruces, NM88003. 2Dept of Biological Sci, Texas Tech Univ, Lubbock, TX79409
Frost tolerance is essential for temperate crops while in tropical crops such as cotton, productivity
and quality are affected by even non-freezing low temperature. In alfalfa, non fall-dormancy is a
desired trait because it produces more herbage. However, the major constraint preventing
widespread use of nondormant alfalfa in temperate regions is their poor winter hardiness. Similarly
cold tolerance is a desired trait in cotton since it would help cotton producers to do early planting
without being hampered by low temperatures and early planting would allow peak flowering to
occur during the longest summer days and may set bolls before soil moisture supplies are depleted.
Accumulation of soluble sugars, mostly sucrose, have been documented during the acquisition of
freezing tolerance of many plant species. It is accepted that sucrose synthesis is regulated by the
activity of the enzyme, sucrose phosphate synthase (SPS) which catalyzes the formation of sucrose6-phosphate from uridine 5’-diphosphate (UDP)-glucose and fructose-6-phosphate.
Low
temperature induced increase in SPS activity has been documented in a number of plants.
SPS is found mostly in photosynthetic tissues and the abundance of sucrose in the source leaf is the
determinant of the rate of sucrose export from the leaf. However, SPS is also present in
heterotrophic tissues and it has been proposed that SPS in these tissues could allow re-synthesis of
sucrose after import via apoplastic cleavage or SPS could be involved in a regulatory cycle in which
sucrose is simultaneously degraded and re-synthesized. The root nodules in alfalfa where the
symbiotic bacteria, Rhizobia, fix atmospheric nitrogen acts as a large sink for photosynthetically
produced sucrose. The products of sucrose metabolism is in nodules is to provide a substrate to
provide the ATP and reducing power for the fixation of N2 and to provide carbon skeletons for
ammonia assimilation. Similarly the fiber in cotton bolls also act as a C sink since sucrose is the
required substrate for cellulose synthesis in the cotton fiber.
We have recently isolated two different SPS gene members from alfalfa, SPSA and SPSB. While
the former is expressed in all organs but at much higher levels in the nodules followed by the roots,
the latter is mostly leaf-specific in its expression. High SPS activity in the nodules suggests a
possible role of SPS in the nodules. Cotton plants transformed with a spinach SPS gene driven by
the CaMV 35S promoter exhibited a small but statistically significant increase in fiber wall
thickness and quality under cool temperature conditions.
The objectives of this proposal are to overexpress SPS in a constitutive and leaf-specific manner in
a non-dormant alfalfa cultivar and compare their performance under freezing conditions with
control plants. The same transformants along with those that have the SPS gene driven in a nodulespecific manner will be analyzed for nodule function and overall yield during the growing period.
The results of the alfalfa transformants will be compared with those of cotton transformants
expressing SPS in a leaf-specific or boll-specific manner. Besides the applied aspects, the proposal
is addressing the role of SPS in two kinds of sink tissues, the nodules and the cotton fiber.
10
ROLE OF NITRIC OXIDE SIGNALING IN POLLEN TUBE REPULSION AND
GROWTH
Ravishankar Palanivelu1 and William R. Montfort2. 1Dept. of Plant Sciences, Marley 541E, and
2
Dept. of Biochemistry & Molecular Biophysics, BSW 533, University of Arizona, Tucson, AZ
85721.
After pollen lands on the surface of the pistil, it extends a tube to transport the sperm cells to an
ovule. Based on imaging of fixed flower tissues, it is known that this journey involves a series of
cell-cell interactions that includes adhesion, attraction and repulsion. However, since pollen tube
growth occurs within opaque floral structures, it has been difficult to examine the dynamic nature of
these interactions. To overcome this technical limitation, we developed an in vitro assay to directly
observe pollen tube guidance to the ovules. By using this assay, we have performed detailed
characterization of pollen tube entry into the ovule and subsequent arrest within an embryo sac.
When pistils in this system are coated with excess pollen, multiple tubes can associate with each
ovule. However, pollen tubes turn away sharply from an ovule if it is already been entered by a
pollen tube. These preliminary observations suggest that a short-range, diffusible repellant
mechanism prevents multiple tubes from entering one ovule. Such interactions are reminiscent of
polyspermy blocks in vivo, where only one tube generally migrates up the funiculus and enters an
ovule. Since pollen tube entry and bursting can be easily monitored in this in vitro assay, it will
allow us to collect ovules as they are being targeted and use them to dissect the ovule repellant
signals–an opportunity that has remained so far impossible in vivo.
The immediate goal of our proposal is to use this newly developed in vitro guidance assay to
characterize pollen tube repellant signals in a model plant system (A. thaliana) before they are
tested in a crop species. Specifically, the proposed experiments will:
1) Characterize pollen tube repulsion by a) identifying the temporal and spatial origins of repulsion
signals, b) analyzing the developmental regulation of pollen tube repulsion signals, c) using
mutants to understand interplay between pollen tube repulsion and previously defined pollen
tube guidance events and d) exploring the degree to which this repulsion pathway has diverged
in plants beginning with close relatives of A. thaliana.
2) Investigate role of a candidate repellent nitric oxide (NO) accumulation and release from ovules
in PT repulsion by a) evaluating the effect of NO loss–by chemicals that disrupt NO
biosynthesis and mutants defective in NO accumulation–in ovules on PT repulsion in the in
vitro guidance system, b) quantifying total NO levels in untargeted and targeted ovule extracts,
c) localizing NO accumulation in targeted ovules using NO-sensitive dyes and d) developing
assays to probe NO release from ovule.
Research significance and relevance: Upon completion, this study not only would have
characterized a unique process in plants but also will have impact on crop improvement. The
outcome of this study will help us understand how plants regulate spurious fertilization events.
Insights into this process, could then be used to contain pollen from genetically modified crop
varieties from spreading into native species, a key challenge breeders face in the southwest while
developing improved transgenic crop varieties (for example, cotton) and an area of concern to
public and regulatory agencies. Since this work will be part of a research program aimed at
understanding how seeds are made, it will have a long-term benefit in gaining knowledge to
increase crop yield, overcome inter species hybridization barriers, and generate novel plant hybrids
using wild relatives that are more likely to withstand environmental stresses.
11
TOWARDS DEVELOPMENT OF MARKERS FOR HEAT STRESS
TOLERANCE IN PEANUT.
Kameswara Rao Kottapalli1, Michael Gomez Selvaraj2, Gloria B. Burow3 , Jamie L. Ayers2,4,
John J. Burke3, Naveen Puppala1, And Mark D. Burow2,4. 1Agricultural Sciences Center, New
Mexico State University, Clovis, NM 88101; 2 Department of Plant and Soil Science, Texas Tech
University, Lubbock, TX 79409; 3 USDA-ARS Plant Stress and Germplasm Development Unit,
3810 4th Street, Lubbock, TX 79415; and 4 Texas Agricultural Experiment Station, 1102 East
FM1294, Lubbock TX 79403.
Heat stress is a significant factor affecting peanut production. Genetic improvement of peanut is
hampered by limited genetic variability in the germplasm used commonly by breeding programs,
and by lack of study and markers for selection of improved plant response. In this project, we are
working to identify additional sources of genes for heat stress tolerance, and to identify candidate
genes and markers for use in peanut improvement.
Greater genetic variability is present in the U. S. peanut core and core subset collections than among
peanut varieties, but utilization of these collections could be enhanced by genetic characterization of
these accessions. We report characterization of the U. S. peanut minicore collection using simple
sequence repeat (SSR) markers. Seventy two peanut accessions were genotyped using 73 primer
pairs. Substantial genetic variation was found to exist in the core subset, contrary to previous
reports of little or no molecular variation in the cultivated species. A group of twelve unlinked
markers with known map positions identified less variation among the accessions but was found
sufficient to differentiate both subspecies and market types and gave results similar to those
obtained using the larger number of primer pairs. The genetic variation observed indicate that SSR
markers are highly suitable for development of genetic maps of cultivated tetraploid peanut.
Greenhouse experiments to identify sources of heat stress resistance within the minicore collection
found significant differences among accessions by the enhanced respiratory biodemand assay,
suggesting the potential utility of the minicore as a source of different genes for tolerance to heat
stress. A subset of these accessions has also been characterized to date at seedling and reproductive
stages using acquired thermotolerance and metabolic fitness index (MFI) measurements, counts of
flowers during heat stress and recovery, and number of fertile pollen grains. Both MFI and
acquired thermotolerance varied considerably among the genotypes. There was no strong
correlation between the two measurements, indicating differences in the mechanisms of tolerance
associated with the measurements. Interestingly the number of flowers under stress showed highly
significant positive correlations with both acquired thermotolerance (0.643***) and MFI (0.300**).
The present study indicates that to screen the peanut genotypes at the seedling level for heat
tolerance, different heat stress assays will be required.
Based on the present and previous results, the genotypes ICGS76 and ICGV87157 possessed good
tolerance to heat stress while Tamrun OL02 and Spanco were susceptible. Based on these results,
parents with contrasting heat stress responses and of different market types for greater molecular
polymorphism have been selected. Crosses (ICGV87157  Tamrun OL02, ICGS76  Tamrun
OL02, and ICGS76  Spanco) were made to develop F1 hybrids for expansion of current or
production of additional F2 mapping populations. Microsatellite marker analysis identified primer
pairs useful for identification of true F1 hybrids. Since ICGS76 is very compatible in crossing
studies, identified heat tolerance QTLs will be easily introgressed to susceptible varieties.
12
DEVELOPMENT OF A VALENCIA PEANUT CORE COLLECTION, AND
ASSOCIATION MAPPING TO IDENTIFY MARKERS ASSOCIATED WITH YIELD,
RESISTANCE TO POD ROT COMPLEX DISEASES, AFLATOXIN CONTAMINATION,
AND TOLERANCE TO DROUGHT IN PEANUT
Naveen Puppala1 Mark Burow2 Soum Sanogo3 and Jorge Fonseca4
1
New Mexico State University, Agricultural Science Center at Clovis, 2346 SR 288, NM 88101,
Texas Tech University, Department of Plant and Soil Science, Lubbock, TX 79409, 3New Mexico
State University, Entomology, Plant Pathology and Weed Science, Las Cruces, NM 88003, 4The
University of Arizona, Department of Plant Sciences, Yuma Agricultural Center, Yuma, Arizona
85364.
2
The Valencia peanuts for in-shell market are predominantly grown in eastern New Mexico, and
west Texas. Of recent, water has become a scarce resource in this region. The area under peanut
cultivation has therefore declined thus affecting the monopoly of this region to produce peanuts for
in-shell market types. Development and cultivation of high yielding Valencia peanut cultivars that
mature early, uses less water to produce more (high water use efficiency), tolerant to drought, and
resistant to pod rot and aflatoxin will benefit peanut producers in this part of the US. The overall
goal of this proposal is to develop a core collection specific to Valencia peanut that will be
evaluated for various morpho-agronomic traits, physiological traits related to drought, and
resistance to pod rot and aflatoxin contamination to identify promising germplasm for enhancing the
genetic potential of Valencia peanuts. Association mapping on this core collection will enable us to
identify markers associated with beneficial traits, as discussed above, for use in breeding programs.
Over 500 publicly available SSRs will be initially screened to identify polymorphic markers on a set
of diverse germplasm and then the full core collection will be genotyped using 100 polymorphic
SSRs and high throughput assay (ABI3700) to dissect the genetic structure of this core collection.
The New Mexico Peanut Research Board provides limited funds to support ongoing peanut
breeding program at NMSU; however, additional support from SWC through this proposed project
will help us identify critical germplasm and the marker technology for effective use in development
of high-yielding Valencia peanut cultivars.
13
FUNCTIONAL CHARACTERIZATION OF STRESS-INDUCIBLE
RNA SILENCING FACTORS IN PLANTS
Zhixin Xie1 and Thea Wilkins2
1
( Department of Biological Sciences, and 2Department of Plant and Soil Sciences,
Texas Tech University, Lubbock, TX 79409)
RNA silencing is a remarkably conserved system that negatively regulates gene expression at the
transcriptional or posttranscriptional level in most eukaryotic organisms. Recent genetic and
biochemical studies have revealed a framework for the core RNA silencing machinery. Several
evolutionarily conserved protein families, including DICER (DCR) or DICER-LIKE (DCL),
ARGONAUTE (AGO), and RNA-DEPENDENT RNA POLYMERASE (RDR) have been identified
as the central components of the silencing machinery. A hallmark for RNA silencing is the production
of 21- to 24-nucleotide small RNAs by DCRs or DCLs from RNA precursors containing extensive
double-stranded (ds) structure. In some cases, formation of a dsRNA precursor requires an RDR
activity. The small RNAs, once incorporated into AGO-containing effector complexes, guide target
RNA recognition and downstream gene suppression events in a sequence-specific manner. The small
RNA-mediated gene silencing pathways play important roles in an amazingly diverse set of biological
processes, including development, chromatin modification, genome integrity, antiviral defense, and
stress response.
Plants encode multiple functional DCL, RDR, and AGO proteins. The Arabidopsis genome contains
four expressed DCLs, six putative RDRs, and ten AGOs. Genetic analyses have revealed multiple small
RNA pathways in plants; each requires distinct RNA silencing factors. To date, functions for each of
the four DCLs, two RDRs (RDR2 and RDR6), and at least three AGOs (AGO1, AGO4, and AGO7)
have been associated with distinct endogenous small RNA pathways. Functions for other members of
the RDR and AGO family proteins have yet to be uncovered. Using the publicly accessible
AtGenExpress data set, we have found that several AGO family members, including AGO2 and
AGO3, which are expressed at very low levels under normal growth conditions, are highly expressed
upon certain abiotic stress such heat (AGO2), and high salinity (AGO3). Several other putative RNA
silencing factors including a dsRNA-binding protein (DRB3) and an RNase III-like protein also
exhibit inducible expression patterns under various stress conditions. These observations raised an
interesting possibility that certain RNA silencing factors may play important roles in small RNAdirected gene regulation during stress responses in plants.
This proposal seeks to uncover the role of these previously uncharacterized putative RNA silencing
factors in plant stress responses as a step towards the genetic improvement of crop plants. A
combination of genetic, genomic, and biochemical approaches will be used to dissect the function of
stress-inducible AGO-family members in Arabidopsis. The analyses will be extended to explore the
functional homologs of these Arabidopsis genes in cotton, a major crop in the Southwest of the United
States. The proposed project has three specific aims: (1).Examine the role of stress-inducible AGO
family members in plant stress response; (2) Identify the endogenous small RNAs associated with the
stress-inducible AGO complexes; (3) Identify the targets of the stress-associated small RNAs. More
details on the research strategy that would allow us to achieve these goals will be presented.
Achievements of these research aims would not only reveal novel aspects on the molecular mechanism
of plant stress responses, but also will advance our understanding on the functional diversification of
plant small RNA pathways.
14
A FUTURISTIC VIEW OF
AGRICULTURAL BIOTECHNOLOGY
By Invited Guest Speaker
Lowell B. Catlett, Regents Professor and Dean
College of Agriculture and Home Economics
New Mexico State University
During the next two decades four major trends will converge to permanently alter not only North
America’s food system but most developed nations’. Demographics, economics, biotech and
quantum mechanics will change the food system more during the next twenty years then during the
last 200 years. Everything from surface emitting lasers to bacteriocins will alter not only what we
eat but why and how. This is a journey through the strange but fascinating world where new
technologies blend with society’s changing values.
15
HIGH RESOLUTION GENETIC ANALYSIS OF A QTL FOR ROOT
CHARACTERISTICS ON THE SHORT ARM OF CHROMOSOME 1 OF RYE IN BREAD
WHEAT
J. Giles Waines, Prasanna R Bhat, Bahman Ehdaie, Sundrish Sharma, Timothy Close and Adam J.
Lukaszewski, Department of Botany & Plant Sciences, University of California, Riverside, CA
92521-0124, USA
‘Pavon 76’ (Triticum aestivum, 2n = 6x = 42, BBAADD) is a semidwarf, green- revolution bread
wheat previously grown in Mexico and southwest USA that is susceptible to heat and drought
stress. ‘Pavon 1RS.1BL’ is a rye / wheat translocation line formed from it, homozygous for the
1RSk.1BLp translocated chromosome, markedly less susceptible to water stress, that exhibits a
larger root biomass and more root branches than Pavon 76. The 1RS arm is originally from
‘Kavkaz’ winter wheat, and is originally thought to derive from a Petkus rye (Secale cereale, 2n =
2x = 14, RR). This project aims to map the QTL for root characteristics on the short arm of
chromosome 1 of rye. Homoeologous recombinants of 1RS with 1BS were made by suppression of
the Ph1 locus. As the proximal 70% of the 1RS-1BS arms generally do not undergo recombination,
all breakpoints are located in the terminal 30% of the arms. Sixty of these recombinants are being
used, along with parents Pavon 76 and Pavon 1RS.1BL, to phenotype the translocation lines for root
characteristics in sand tubes; 81 recombinants are used to generate a genetic map of the
translocation break points. The Affymetrix Wheat GeneChip was used to map the translocation
breakpoints to syntenic intervals on rice chromosomes and to identify a total of 193 probe sets from
the wheat genome array corresponding to the 1BS arm of wheat. Overall, a total of 20 polymorphic
DNA and phenotypic markers were used to generate a genetic map of the 1S arms with the average
spacing of 2.5 cM. A method using Quade statistics was developed to score root and shoot
characters of the translocation lines, using 45 day-old seedlings grown in 80cm long sand-culture
tubes. Wheat root growth and development are affected differentially by photoperiod and
temperature regimes at different seasons of the year. A QTL for increased root biomass was
allocated to the terminal 15% of the genetic map of 1RS and we plan to refine this map position in
the upcoming growing season.
16
GENETIC AND MOLECULAR INTERACTION OF SALT TOLERANCE
DETERMINANTS IN ARABIDOPSIS
Huazhong Shi
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409
Plant salt tolerance is a complex trait that is controlled by many genes. The recent identified SOS
signaling pathway crucial for salt tolerance in Arabidopsis has provided insight in how salt stress
signal is perceived, transduced and responded in plants. The SOS signaling pathway includes three
components: the Ca2+ sensor SOS3, the serine/threonine protein kniase SOS2, and the plasma
membrane Na+/H+ antiporter SOS1. Under salt stress, cellular Ca2+ concentration increases, which
leads to Ca2+ binding onto the SOS3 protein. Ca2+-bound SOS3 interacts with the SOS2 and
activates SOS2 kinase activity. SOS3 contains a myristoylation site at its N-terminus and the
myristoylated SOS3 brings the complex onto the plasma membrane, providing the opportunity for
SOS3 / SOS2 to interact with SOS1. SOS2 phosphorylates SOS1, which enhances SOS1 Na+ / H+
exchange activity and promotes Na+ efflux. The plasma membrane-localized SOS1 contains a large
cytosolic C-terminus that could interact with different molecules, which suggests that SOS1 might
also serve as a signaling component to sense and transduce salt stress signal. Genetic screening of
sos3 suppressors identified another important salt tolerance determinant AtHKT1, a Na+ transporter
in the plasma membrane. Single athkt1 mutant exhibits salt sensitivity primarily in the leaves due
to excessive Na+ accumulation, while mutation in AtHKT1 suppresses sos3 salt hypersensitive
phenotype. Ion content measurements of xylem sap, roots and leaves revealed a complex
interaction between SOS3 and AtHKT1 in control of Na+ and K+ uptake. We recently identified a
weak allele of sos3hkt1 containing a T-DNA insertion in the promoter of AtHKT1 that is 3.6 kb
upstream of the coding region, indicating the distal sequence above 3.6 kb is required for AtHKT1
expression. Sequence analysis revealed a tandem repeat structure that is separated from the 3.6 kb
promoter by the T-DNA insertion. This distal repeat sequence in the AtHKT1 promoter could
function as an enhancer element. The role of the tandem repeat in the regulation of AtHKT1
expression will be discussed. Mutation in AtHKT1 also suppresses sos1 salt hypersensitive
phenotype. A genetic screening for sos1 suppressors has identified twenty additional suppressors.
The molecular identities of these suppressors are to be uncovered in the future.
17
DROUGHT RESPONSIVE GENES AND PHYSIOLOGICAL TRAITS AS CANDIDATE
MARKERS FOR DROUGHT TOLERANCE QTL IN ALFALFA
Ian Ray1, Tracy Sterling2, Mary Sledge3, Johnny Maruthavanan2, Lei E1, and Chris Meenach1
1
Dept. of Plant and Environmental Science and 2Dept. of Entomology, Plant Pathology, and Weed
Science, New Mexico State Univ., Las Cruces, NM, 88003. 3Samuel Roberts Noble Foundation,
Ardmore, OK.
The goal of this project is to genetically and physiologically characterize drought response in alfalfa
(Medicago sativa). Two first generation backcross mapping populations (~90 genotypes each) of
tetraploid alfalfa were generated between a high yielding but low water use efficient ‘Chilean’
parent, and a low yielding but high water-use efficient ‘M. falcata’ parent. Half-sib families derived
from each mapping population genotype and both parents were seeded in replicated field plots at
Las Cruces, NM in 2004. Forage biomass and a variety of physiological and biochemical traits
were measured on these plots under water-deficit conditions during the May and June regrowth
periods in 2005 and 2006.
Pre-dawn and solar noon water potential and leaf relative water content of the parents and extreme
high and low yielding families indicated that all plots were under water stress at the time gas
exchange was determined. Net photosynthesis, transpiration, and conductance were measured on
fully-expanded leaves under ambient light; additional leaves were sampled concurrently and
immediately frozen for antioxidant and biochemical marker determination. Under well-watered
conditions, net photosynthesis was ca. 20 μmol m-2 s-1 and was consistent regardless of time of day
when light was not limiting. With water deficit, gas exchange was reduced regardless of the alfalfa
genotype. In general, levels of most antioxidants were unchanged under water deficit except for
beta-carotene, alpha-tocopherol and the xanthophylls, which varied depending on the alfalfa
genotype.
286 markers derived from M. truncatula cDNAs containing SSR motifs were evaluated for their
association with forage yield under water deficit conditions. Based on 2005 data, 29 marker alleles
were associated with biomass production (p<0.01) with some alleles accounting for up to 18% of
the yield variance. Individual allele effects ranged from -10% to +12% of the population mean.
The magnitude each marker allele effect differed between harvests, however, the direction of the
influence for each allele was consistent across harvests. Most marker-trait associations localized to
two regions on linkage group 1 (LG1) and one region on LG8. Marker alleles on LG8 primarily
influenced May forage regrowth, while those on LG1 influenced June forage regrowth. Annotation
results of 18 markers with greatest effects on forage yield indicated that half of them represented
regulatory factors. The next most common functional marker group was associated with ion/sugar
transport. Additional biomass data were collected in 2006 to verify the influence of these loci.
Forty-five additional markers representing drought responsive genes and genes associated with key
physiological responses, have been constructed with another 55 underway. These markers are
currently undergoing segregation analysis to determine their allelic dosage prior to conducting
additional mapping and QTL analysis. The frequency with which drought-responsive genes are
associated with drought tolerance QTL will be compared to that of the random cDNA-SSR markers
to determine if a candidate gene approach provides an enriched source of markers for detecting
drought tolerance QTL.
18
DEVELOPMENT OF CANDIDATE GENE MARKERS FOR ROOT-KNOT NEMATODE
RESISTANCE IN COTTON
Jinfa F. Zhang, Department of Plant and Environmental Sciences, New Mexico State University, Las
Cruces, NM 88003
Philip A. Roberts, Department of Nematology, University of California-Riverside, Riverside, CA
92521
Root-knot nematodes (RKN) cause cotton yield loss of 2.24% ($140 million) nationwide and 0.55% ($30 million) in the southwest states, highest among cotton diseases. Host plant resistance
represents the most cost effective strategy for RKN management. Even though the breeding history
for RKN resistance in cotton can be traced back to the early 1900s, the first major breakthrough was
the development of a highly resistant line, Auburn 623 RNR, by combining moderate resistance
from two sources (Wild Mexico Jack Jones and Clevewilt) in the early 1970s. Auburn 623 RNR is a
highly valuable source of RKN resistance, but its high level of RKN resistance has never been
transferred to any commercial cotton cultivars. Acala NemX is a moderately resistant cultivar, with
its source unknown. The inheritance of RKN resistance in Auburn 623 RNR has been debated for
>30 years due to the lack of reliable resistance phenotyping method. Even though the results still
remain inconclusive, it has become clearer that, for the high level of RKN resistance in the Auburn
source, race stock Wild Mexico Jack Jones contributes resistance, presumably gene Mi1, while
Clevewilt 6 may contribute resistance gene Mi2, which might be the same as or allelic to rkn1 in
Acala NemX. At present, only cultivar ST 5599 BG containing Mi2 gene with moderate RKN
resistance derived from Clevewilt 6 has been grown on only 7% U.S. cotton acreage, and Acala
NemX has been grown on limited acreage in California. However, high resistance requires the
presence of both Mi1 and Mi2/rkn1 genes. Cotton production still depends heavily on the application
of nematicides to suppress RKN.
The identification and mapping of molecular markers linked to nematode resistance genes in cotton
are essential steps in understanding the relationships of resistance genes and incorporating the
resistance into elite cotton cultivars through marker-assisted selection, rather than through
phenotypic selection. Recently two SSR markers amplified by primers BNL 1231 and CIR 316 and
one cleaved amplified polymorphic sequence (CAPS) marker converted from an AFLP marker on
Chromosome A11 have been identified that are tightly linked to the resistance gene rkn1 in Acala
NemX. These rkn1-linked markers, together with two RAPD markers and one STS marker have
been confirmed/identified to be associated with one of the RKN resistance genes (presumably Mi2)
in the Auburn source. However, the chromosomal location of gene Mi1 is unknown and no DNA
markers have been developed for it.
To tackle the RKN problem and provide ultimate solutions for RKN control through cotton
breeding, several issues need to be addressed: (1) Where is the Mi1 gene locus and how does it
interact with rkn1/Mi2 to render the high level of RKN resistance? High resolution mapping of the
RKN R genes using various markers will provide answers to this question. (2) What are the
relationships between the RKN R genes and resistance gene analogs (RGAs)? High resolution
mapping of RGAs and RGA-AFLP markers, their co-localization with the RKN R genes and their
expression levels in relation to the RKN R genes will help address this question. Our hypothesis is
that the two RKN R genes belong to the NBS-LRR R gene family and thus can be tagged by
candidate RGA markers. Therefore, this project proposes separating the two major RKN R genes
(Mi1 and rkn1/Mi2) by using and developing functional/candidate markers. This will result in the
establishment of a highly efficient MAS method for RKN resistance breeding and lay the
foundation for cloning of the R genes.
19
CHARACTERIZATION OF A COTTON MUTANT WITH IMPROVED YARN SPINNING
PERFORMANCE
Dick Auld1, Efrem Bechere1, Eric Hequet2, and Noureddine Abidi2
Plant & Soil Science Dept.; Texas Tech University; Box 42122; Lubbock, TX 79409
2
International Textile Center; Texas Tech University; Box 5019; Lubbock, TX 79409
1
In 2005, 40 M7 lines selected for naked or partially naked seed coats and five check varieties of
cotton were grown in replicated plots at Lubbock, TX. The fibers harvested from these plots were
analyzed at the Texas Tech University – International Textile Center using High Volume
Instruments (HVI), Advanced Fiber Information Systems (AFIS), and standard yarn quality
analyses. One of these mutants (SC 9023-NS-57-13-3) produced yarn that would provide
exceptional performances for ring spinning applications. HVI analyses did not identify this line as
superior, but AFIS analyses showed that this line had very low neps/gram, low short fiber content
(weight), and a very high maturity ratio. This study showed that cotton breeders need to increasing
rely on AFIS analyses in late generation screening for fiber quality. Future studies will be needed
to determine if the enhanced yarn characteristics of SC 9023-NS-57-13-3 are the result of the
partially naked seed coat characteristic or other traits inherent in this line.
20
STUDIES ON TRANSFORMATION AND REGENERATION IN CHILE (CAPSICUM
ANNUUM).
2
2
Charlene Carr , Forest Ross , Samantha Clark2, Suman Bagga2, Champa SenguptaGopalan1,2
Department of Molecular Biology1, Plant and Environmental Sciences2,
New Mexico State University, Las Cruces, New Mexico 88003
Chile pepper is a high value crop in the world. Most chile peppers are susceptible to different
Phytopathogenic fungi, bacteria and viruses. Insects and other pests like the root knot nematodes
can also cause extensive losses in yield and quality of peppers. The abiotic factors can also affect
the performance of chile pepper plants.
Breeding programs are in place to improve the resistance or tolerance against different biotic and
abiotic factors. While traditional breeding techniques have been of great value for chile pepper
genetic improvement, biotechnological techniques involving plant tissue culture and recombinant
DNA technologies could be powerful auxiliary tools to accelerate and achieve this goal.
The successful application of genetic engineering strategies to improve chile pepper depends on
having an efficient and reproducible regeneration and transformation system. Although pepper
regeneration in vitro has been reported by some laboratories, there have been only a few reports on
reliable genetic transformation of pepper, but in all the reported cases the efficiency of
transformation has been low. Preliminary data in our Lab suggests high regeneration potential
using hypocotyls and cotyledon explants from seedlings. Two kinds of shoots were produced on
the callus that grew into plantlets, those that were callus-mediated (indirect shooting) or shoots
growing from the wounded surface (direct shooting). The regeneration response was higher in
hypocotyledons. Protocols are in the works that will optimize conditions for Agrobacterium
tumefaciens mediated transformation using Gus as a reporter gene.
21
INCREASING FREE METHIONINE LEVELS IN ALFALFA BY MODULATING GENES
IN THE METHIONINE BIOSYNTHETIC PATHWAY AND THE
S-METHYLMETHIONINE CYCLE.
Matthew Barrow1, Suman Bagga2, Jagtar Singh2, Champa Sengupta-Gopalan 1,2
Department of Molecular Biology1, Plant and Environmental Sciences2,
New Mexico State University, Las Cruces, New Mexico 88003
Alfalfa is a forage legume used as a principal feed source for livestock throughout the world
because it provides much of the protein required in animals’ diets. Alfalfa, however, is deficient in
the essential amino acid, methionine (Met). One strategy of improving the Met content of alfalfa has
involved introducing the corn genes encoding the Met-rich and rumen stable β- and δ- zein seed
storage proteins. However, preliminary results indicated that the synthesis of the zein proteins in
alfalfa leaves is limited by the availability of free Met. We are interested in increasing Met levels by
genetically modifying the alfalfa Met metabolic pathway. Cystathionine γ-synthase (CγS) is the key
enzyme in Met synthesis. The major route for Met metabolism is in the synthesis of Sadenosylmethionine (SAM), catalyzed by SAM synthase (SAMS) and into S-methylmethionine
(SMM). SMM is formed by the SAM dependent methylation of Met, catalyzed by Met Smethyltransferase (MMT) and can be reconverted to Met in a reaction mediated by homocysteine Smethyltransferase (HMT). Constitutive expression of the Arabidopsis CγS gene in alfalfa did lead to
an increase in Met levels, and only a slight increase in zein protein accumulation; however, there
was a more significant increase in the level of SMM. Our research goal is to understand how the
SMM cycle is regulated in alfalfa and how overexpression of CγS affects this cycle. Towards
accomplishing this goal we have isolated the genes for these three enzymes from alfalfa and are
using them as probes to determine how these genes are regulated. We will also present data on our
efforts to genetically modify Met metabolism in alfalfa by targeting expression of HMT, MMT, and
SAMS.
22
A DISTAL TANDEM REPEAT IN THE PROMOTER IS IMPORTANT FOR AtHKT1
GENE EXPRESSION.
1
Jung-Sung Chung , Prasanth Pathange1, Paul M. Hasegawa2, Huazhong Shi1
1
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409
2
Department of Horticulture and Landscape Architecture, Purdue University, West
Lafayette, IN 47907
Several lines of evidence have shown that AtHKT1 controls Na+ homeostasis under salt stress. First,
electrophysiological studies revealed that AtHKT1 is a transporter highly selective to Na+. Second,
mutations in AtHKT1 resulted in hypersensitive of the mutants to Na+. Third, mutations in AtHKT1
suppress salt hypersensitive phenotype of sos3 mutant. These findings suggest that AtHKT1 plays
important roles in salt tolerance. Recently, we identified several weak alleles of sos3athkt1 carrying
a T-DNA insertion in the AtHKT1 coding region. By using TAIL-PCR, we identified a T-DNA
insertion in the promoter of AtHKT1 gene in one of the sos3athkt1 weak alleles, designated as
sos3athkt1P. This T-DNA insertion is located at 3.6kb upstream of the ATG start codon.
Interestingly, sequence analysis of the AtHKT1 promoter revealed a distal tandem repeat located
4.0kb upstream of the ATG codon. Each repeat is 780bp. The T-DNA insertion separating this
direct tandem repeat from 3.6kb promoter of AtHKT1 gene results in weaker suppression of sos3
salt sensitive phenotype comparing with the T-DNA insertion in AtHKT1 coding region. Expression
of AtHKT1 gene driven by 2.0kb promoter excluding the tandem repeat in the sos3athkt1 mutant
resulted in not only abolishment of the suppression phenotype, but more sensitive to NaCl than sos3
mutants. These results indicate that the distal tandem repeat plays crucial role in AtHKT1 gene
expression. Na+ content measurements revealed that both sos3 and hkt1 mutants accumulated higher
Na+ in the xylem sap and leaves, while sos3athkt1 null suppressors had reduced Na+ content relative
to sos3. The weak allele sos3athkt1P displayed intermediate Na+ accumulation between sos3 and
sos3athkt1. The expression of AtHKT1 was induced by low K+ treatment. How ever, this induced
expression is abolished in both sos3athkt1P and sos3 mutants, which suggest that the tandem
repeat might be required for the induced expression of AtHKT1 by K+ deficiency. The repeat
sequence is able to enhance the GUS gene expression driven by the minimal 35S promoter that
confers only basal gene expression. More importantly, the repeat sequence also confers induced
gene expression by K+ deficiency. These results suggest that the tandem repeat could be a distal
enhancer for AtHKT1 transcription and confer fine regulation of AtHKT1 expression in response to
salt stress and K+ deficiency.
23
SCREENING FOR ANTI-MICROBIAL COMPOUNDS IN DATURA INNOXIA
Jeanne Curry, Thurman Redhouse, Laura Hernandez, F. Omar Holguin and Mary O’Connell
Department of Plant & Environmental Sciences, New Mexico State University,
Las Cruces, NM
Southwestern plants have been used as medicines for thousands of years. Datura innoxia
(Solanaceae), containing the abundant tropane alkaloids, is under investigation for additional novel
anti-cancer bioactivities. Preliminary extractions identified a methanol soluble fraction with
activity inhibiting the growth of breast cancer cells. To determine the chemical structure of this
bioactive compound more material is needed. The goal of this project is to isolate sufficient
quantities of the bioactive anti- cancer compound (~20 mg) for NMR analyses so that a chemical
class for this compound could be predicted. An additional goal is to screen the other fractions in the
extraction process for additional bioactivities. Leaves (~860 g, dry weight) were collected from
plants in the wild and sequentially extracted (soxhlet) with hexane, chloroform and methanol. The
methanol soluble fraction was further fractionated by extraction with butanol and finally
precipitated with diethyl ether. This last fraction is dissolved and then separated by HPLC and a
single peak containing the bioactivity is recovered. All extracts and fractions were tested for
several bioactivities: anti-bacterial, anti-fungal and anti-cancer using cell growth assays. Microbial
cultures to be screened include: Bacillus cereus, Bacillus megaterium, E. coli (JM109),
Enterobacter cloacae, Pseudomonas fluorescens, Candida kefyr, Geotrichum candidum, Salmonella
enteritidis , Shigella flexneri, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus
pyogenes, and eleven Saccharomyces cerevisiae mutant yeast strains: Bub3, Mec2, Mlh1, Mgt1,
Sgs1/Mgt1, Sgs1, Rad14, Rad18, Rad50/Epp+, Rad50, Rad52, plus wild type. Chemical
composition of the crude fractions will be determined using gas chromatography/mass
spectrometry.
This work was supported in part by NIH grants R25 GM48998, MBRS RISE GM61222 and NCI
MICCP 5U56CA09286.
24
CHEMICAL CONTROL OF ROOT CAP DEVELOPMENT:
IMPLICATIONS FOR ROOT DEVELOPMENT AND FUNCTION
J.J. Ebolo, H.H. Woo, F. Wen, M.C. Hawes. Division of Plant Pathology and Microbiology,
Department of Plant Sciences, University of Arizona, Tucson AZ 85721
Plants find water and other nutrients required for survival through the sensory-response capabilities
of the root cap, a small organ at the root apex consisting of layers of morphologically differentiated
cells.
Charles Darwin noted that when the root cap is removed surgically or destroyed by
mechanical or chemical injury, the root continues to grow at a normal rate but the capacity to move
toward or away from distinct stimuli is lost. After removal of the cap, the root meristem is induced
to produce a new one and once it has been replaced, sensing of water and other stimuli is restored
along with the capacity for directional movement. The mechanism(s) linking sensing of stimuli
with control of directional movement are not well defined but potentially offer an economically
feasible means to control plant responses to diverse environmental conditions. Root cap
development is controlled by unknown signals released into the extracellular environment from
border cells, specialized cells programmed to detach from the cap periphery into the soil. When
border cells are removed from the cap periphery, renewed cap development is induced within
minutes. Metabolites can be screened for their ability to influence cap function by exposing the root
cap to specific chemicals during the first 5-10 minutes after cap development is induced, and then
measuring the impact on growth, development, and directional movement in response to external
stimuli. A chemical profiling approach is being used to identify products that may prove useful in
influencing the ability of roots to sense and respond to soilborne signals by controlling directional
movement.
25
EXTRACTION OF OLEORESIN USING SUPER CRITICAL FLUID TECHNOLOGY
Laura Hernandez and Mary A. O’Connell
Department of Plant and Environmental Sciences, New Mexico State University
Las Cruces, NM, 88003
Oleoresin is the extracted pigment obtained from red chile. It is traditionally extracted from nonpungent peppers and uses large amounts of hexane. The hexane is then driven off, leaving a thick
oil. This oil is used by the pharmaceutical, cosmetic, and food industries as a coloring agent. Value
is based on several factors, such as the amount of red pigment, and the level of contaminants, such
as plant material and capsaicin. The use of Supercritical Fluid Technology was investigated as a
“green” method of extracting oleoresin. Using supercritical CO2 as an extraction solvent and 95%
ethanol as a capture solvent allows for the production of high quality oleoresin from pungent chile
peppers without the use of toxic chemicals.
26
POPULATION ANALYSIS OF ANEMOPSIS CALIFORNICA IN NEW MEXICO
Andrea Holguin, Sandra Micheletto, F. Omar Holguin and Mary O’Connell
New Mexico State University, Department of Plant and Environmental Sciences, Las Cruces, NM
88003
Anemopsis californica is a medicinal plant native to the Southwestern United States and Northern
Mexico. For generations, people living in these areas have relied on Anemopsis to treat a variety of
ailments. Root tissue from seventeen populations of Anemopsis collected in New Mexico were
extracted by Super Critical Fluid Extraction (SFE) and analyzed by Gas Chromatography/Mass
Spectrometry (GC/MS). This work was performed to determine environmental and inter-population
differences in support of cultivating Anemopsis as a high value crop.
An early objective was to examine the root essential oil of Anemopsis growing in New Mexico and
determine chemical variability of specific, abundant compounds: elemicin, methyleugenol, thymol,
and piperitone between populations. Variables such as temperature, annual rainfall, and cultivation
have been examined for their effects on the relative abundances of these compounds. Analysis
revealed remarkable chemical diversity between Anemopsis populations that are growing and being
consumed as medicine in New Mexico. The climate in much of New Mexico is favorable for oil
accumulation in Anemopsis root tissue. Optimal environmental conditions for essential oil
production in Anemopsis are 11.67 to 15.56 °C and less than 25.4 cm of annual rainfall.
Herbalists often argue that medicinal plants are not as potent when grown in a cultivated setting
compared with their wild counterparts. To examine these issues, this analysis included a
comparison of plants growing under cultivated conditions to plants growing at the original
collection site. Growing under cultivated conditions appears to have little effect on the chemistry of
wild New Mexico Anemopsis. Analysis of tissue recollected at selected sites after three years
reveals the stability of chemical profiles within a population, as well as the retention of unique
chemical profiles between populations. Based on these studies we can expect genetic influences to
have a significant effect on variability in New Mexico Anemopsis populations. High oil yielding
populations such as Mesilla Valley Anemopsis, which retain their chemical profiles through
cultivation and changing environmental conditions, may be reliable seed sources for farmers
seeking a high value crop.
In addition the roots of seventeen New Mexico Anemopsis populations were analyzed and fourteen
compounds were quantified (phenylpropanoids: eugenol, methyleugnol, isoeugenol and elemicin;
monocyclic monterpenes: cymene, limonene, piperitone, thymol and carvacrol; and bicyclic
monterpenes: alpha-pinene, carene, 1,8-cineole, camphor and myrtenol). The abundances of the
different chemicals within and between chemical classes will be used to suggest biochemical
pathways and genetic controls on secondary metabolite accumulations. These analyses are
underway using clustering algorithms.
This work was supported in part by the NM Agricultural Experiment Station and NIH grants
GM6122 and U56 CA96286, and NSF NM-AGEP.
27
TRANSCRIPTIONAL REGULATION OF CAPSAICINOID BIOSYNTHESIS
Neda Keyhaninejed and Mary O’Connell
Department of Plant and Environmental Sciences,
New Mexico State University, Las Cruces, NM
Chile (Capsicum spp.) is an important vegetable and spice crop grown in the Southwest. The quality
trait in chile fruit is pungency, due to the accumulation of the alkaloid capsaicin and its analogs
(capsaicinoids) in vesicles or blisters on the epidermis of the chile placenta. Therefore, the spicy and
hot taste of chile depends on how much capsaicinoid is produced. In addition to its spice value,
capsaicinoids are responsible for the analgesic use of this plant in many cultures, its antimicrobial
uses, and recently investigations for cancer therapies. Considering the economic and agricultural
importance of these compounds, little is known about the regulation of this pathway and the
enzymes involved in capsaicin biosynthesis are not well characterized. This pathway also appears to
be environmentally sensitive, with different pungency levels accumulating in cultivars grown in
different locations.
Biosynthesis of capsaicin results from the acylation of an aromatic moiety, vanillylamine, with a
branched-chain fatty acid. The current model for the accumulation of these intermediates starts with
the amino acids phenylalanine and valine respectively. Our lab has cloned and characterized many
of the structural genes for the capsaicinoid pathway and observed that the mRNA abundances for
many of these genes appears to be coordinately regulated. One goal of our research is to find
transcription factors, which are responsible for coordinating the transcription of the capsaicinoid
biosynthetic genes.
Candidate clones for a wide range of transcription factors were identified following EST sequencing
of several cDNA libraries in the lab; C. annuum CM334 root cDNA library, Phaseolus acutifolius
root and leaf cDNA libraries. Among the forty clones first identified, fourteen were confirmed as
transcription factor following high quality re-sequencing. These 14 clones were then used as probes
against northern blots containing total RNA from pungent and non-pungent chile lines, isolated
from leaf, root, fruit wall, flower and placenta. In the case of placenta, RNA samples were also
collected from two different fruit maturity stages. Among the 14 clones, three hybridized with
patterns expected for transcription factors associated with capsaicinoid biosynthesis. These
expression patterns will be presented.
The DNA sequence of these three clones predicts them to encode transcription factors like Jerf1,
Jerf3 and Scarecrow. The data supporting the annotation of these clones will be presented. To
isolate the placental cDNA forms of these genes, a placental cDNA library of habanero transcripts
will be screened using primers based on the DNA sequences of the Jerf1, Jerf 3 and Scarecrow
clones. Those clones that are identified with the primers to conserved regions of the transcription
factors will then be isolated and hopefully the full-length clone and DNA sequence will be
determined.
This work was supported in part by the NM Agricultural Experiment Station and NIH grant S06
GM08136.
28
MYB TRANSCRIPTION FACTOR IN THE PLACENTA OF PUNGENT CHILE
Yingzhi Lu, Jeanne Curry, Mary O’Connell
Department of Plant and Environmental Sciences, New Mexico State University
Las Cruces, NM 88003
MYB proteins are a major group of transcription factors in plant-specific processes including
secondary metabolism. A PCR approach was used to isolate genes for MYBs from the pungent
chile, habanero. Degenerate primers were designed using available tomato and chile MYB cDNA
sequences in the highly conserved R2 and R3 regions of the DNA binding domain. PCR
amplifications were obtained from chile genomic DNA as well as tomato genomic DNA. DNA
fragments were cloned and sequenced thereafter. A chile clone of 791 bp was found to be partial
MYB gene containing 519 bp of intron, which was shorter than that of tomato clone. A cluster
analysis indicated the chile clone was closely grouped to another known chile MYB protein
involved in pigment metabolism. This clone will be used to probe RNAs extracted from chiles of
different pungency classes, different tissues and organs, and different development stages of fruits.
A placental-specific myb would be a candidate transcription factor to regulate expression of the
capsaicinoid pathway.
This work was funded in part by the NM Agricultural Experiment Station and by NIH grant S06
GM 08136.
29
PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF FIELD GROWN ALFALFA
TO WATER DEFICIT
Janakiraman Maruthavanan1, Ian M. Ray2 and Tracy M. Sterling1
1
Department of Entomology, Plant Pathology and Weed Science, 2Department of Agronomy and
Horticulture, New Mexico State University, Las Cruces, NM 88003.
Water deficit is a major environmental factor limiting crop production. The effect of water deficit on
alfalfa (Medicago sativa L. var. ‘Peruvian’) grown at the Leyendecker Plant Science Research Center
near Las Cruces, NM was characterized by withholding irrigation. Physiology and biochemical
measurements were taken overtime at 10 A.M. everyday 7 to 20 (Experiment 1) and 7 to 30
(Experiment 2) days after irrigation (DAI). Diurnal measurements were sampled hourly from 5: 30
A.M. through 5: 30 P.M. at 10 (baseline) and 20 (drought) DAI for both experiments. Trifoliate leaves
were harvested from the top of the canopy and frozen immediately to quantify antioxidants and
pigments using HPLC. Plant RWC decreased in the time course and diurnal studies with exposure to
water deficit and corresponded to increased ambient temperature. The time course study (Experiment 2)
showed that prolonged drought stress (> 20 DAI) induced antioxidants like α-tocopherol. In the diurnal
studies, conversion of violaxanthin to zeaxanthin through antheraxanthin increased gradually during the
morning, reaching a maximum around solar noon and declining in the later part of the day. An inverse
relationship between chlorophyll a:b ratio and xanthophyll cycle conversion was also observed in the
diurnal studies. Results from this preliminary study characterizing the ecophysiological and
biochemical responses of alfalfa to water deficit was important to targeting optimal sampling times for
studies comparing multiple progeny of alfalfa genotypes which differ in their water stress tolerance.
30
IDENTIFICATION OF DROUGHT RESPONSIVE TRANSCRIPTS IN ROOTS OF
PHASEOLUS VULGARIS AND P. ACUTIFOLIUS USING A MICROARRAY APPROACH
Sandra Micheletto, Laura Rodriguez-Uribe, Ricardo Hernandez, Jeanne Curry, Richard
Richins and Mary A. O’Connell
Plant and Environmental Sciences, New Mexico State University, MSC 3Q, P.O. Box 30003, Las
Cruces, New Mexico 88003 USA
We conducted microarray analysis on drought-treated Phaseolus acutifolius (drought tolerant) and
P. vulgaris (non-tolerant) to compare their roots response to water deficit. Customs arrays were
printed with 2300 clones from a cDNA library from water stressed P. acutifolius (tepary) roots, 943
clones from a drought-treated tepary leaf cDNA library and 1962 clones from a cDNA library from
P. vulgaris var. Negro Jamapa. Hybridizations were performed using roots samples from tepary
under two different leaf water potentials, -2.20 and -2.52 MPa and compared with tepary well
watered plants (controls). Another analysis was conducted in P. vulgaris under -2.56 MPa and -2.53
MPa leaf water potential versus controls. Based on the microarrays analysis, 702 cDNA clones were
identified as differentially regulated by drought. These clones were selected for sequencing and
submitted to Gene Bank. Several known drought responsive genes such as LEA and
hydroxyproline-rich proteins were induced in both species. Novel genes with no sequence
similarities to any known cDNA sequences in existing public databases were also abundant in both
species. Several pathogenesis-related proteins, a senescence-associated protein, glutathione Stransferase, metallothionein were all highly induced in P. acutifolius compared with P.vulgaris
suggesting a role in the tolerance to water deficit.
31
CLUSTERING COMPARISONS USING MICROARRAY DATA FROM CAPSICUM
SPECIES INFECTED WITH PHYTOPHTHORA CAPSICI
Sandra Micheletto, Richard D. Richins and Mary A. O’Connell
Plant and Environmental Sciences, New Mexico State University, MSC 3Q, P.O. Box 30003, Las
Cruces, New Mexico 88003 USA
A wide range of statistical approaches is available to analyze the data generated by microarrays,
including dimension reduction tools such as Principal Component Analysis (PCA), and
unsupervised clustering tools like Hierarchical Clustering (HC) or K-means. In this study, the
results of different data analysis tools are compared on data acquired from an interaction between
Capsicum and Phytophthora. Gene transcription time course patterns were characterized following
root infection by Phytophthora capsici in two Capsicum annuum varieties, the resistant Criollo de
Morelos-334 and the susceptible New Mexico 6-4. Different data processing steps were applied and
the microarray results were analyzed using PCA, HC, and K-means. PCA allowed us to interpret
global elements of the gene expression changes in the resistant and susceptible lines. K-means
allowed us to identify the sub-sets of transcription patterns as a function of time and indicated
which genes were members of each set. HC established the relationships between genes classified
by K-means and displayed them in a dendrogram. These tools allowed us to infer functional
relationships even among genes without good annotation, based on other members of the set or
cluster. PCA, K-means and HC are widely available in a broad variety of statistical packages and
therefore should be applied together on filtered datasets.
32
THE 3’ UNTRANSLATED REGION OF THE MESSENGER RNA FOR GLUTAMINE
SYNTHETASE AFFECTS TRANSCRIPT STABILITY AND TRANSLATION RATES IN
TRANSGENIC PLANTS.
Jose L. Ortega1,2 and Champa Sengupta-Gopalan 1,2
Department of Molecular Biology1, Plant and Environmental Sciences2,
New Mexico State University, Las Cruces, New Mexico 88003
Glutamine synthetase (GS) is a key enzyme in the metabolism of higher plants. It converts
inorganic into organic nitrogen by incorporating ammonia into glutamate to form glutamine. The
GS occurs as two isoforms in plants, the chloroplastic form, GS2 and the cytosolic form, GS1. The
assimilation of nitrogen consumes energy and carbon skeletons derived from photosynthesis. This
makes the ammonia assimilation by GS a highly regulated process, subject to regulation by different
mechanisms at different levels.
We have found that the 3’ untranslated region (UTR) of a soybean GS gene (Gmglnb1) affects the
stability of its transcript and also represses the translation of this transcript. To identify the
mechanisms that determine transcript stability and translatability, we also transformed tobacco
plants with a gene construct consisting of a reporter gene (udiA) driven by the constitutive CaMV
35S promoter and the Nopaline Synthase (NOS) 3’ UTR and a similar gene construct in which the
NOS terminator was replaced by the 3’ UTR of the soybean Gmglnb1 gene.
The expression of the Gmglnb1 and the reporter gene constructs in transformed tobacco and alfalfa
plants show that the 3’ UTR or the Gmglnb1 3’ has a role in regulating the expression of the GS1
by affecting the mRNA stability and translation.
33
CHARACTERIZATION OF AN ABIOTIC STRESS RESPONSIVE
SULFOTRANSFERASE GENE FROM ARABIDOPSIS
Prasanth Pathange1, Jung Sung Chung1 and Huazhong Shi1
1
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409
Plants possess a remarkable ability to cope with abiotic stresses. Such ability of plants is
accomplished by changes in gene expression which are fundamental for stress adaptation. Based on
microarray data, we identified an Arabidopsis gene, At2g03760 that is highly induced by salt stress.
Sequence analysis indicated that this gene encodes a putative steroid sulfotransferase showing high
similarity with a brassinosteroid sulfotransferase in Brassica napus. Northern blotting confirmed
that the expression of At2g03760 gene is indeed highly induced by salt stress. Other treatments
including iso-osmotic mannitol, ABA, H2O2, and jasmonic acid had no effects on the gene
regulation of At2g03760. These results indicate that the up-regulation of At2g03760 gene is saltspecific and not due to osmotic stress and is ABA-independent. According to Arabidopsis database,
this gene includes a 207 bp promoter and a 212 bp 5’UTR. To elucidate the cis-element responsible
for salt-specific up-regulation, deletion analysis of the promoter and 5’ UTR was carried out. We
analyzed transgenic plants carrying various deletions of promoter and 5’ UTR fused with luciferase
reporter gene. Upon salt stress treatment, transgenic plants harboring promoter deletions displayed
highly induced luciferase activities. Northern analysis confirmed that the induced luciferase activity
is due to the increased mRNA transcript levels of the transgenic plants. However, deletions of
5’UTR nearly abolished the expression of luciferase. Furthermore, we analyzed the transgenic
plants harboring promoter and 5’UTR fused with GUS reporter gene. Consistently, induced
expression of GUS reporter gene was observed only in transgenic plants harboring promoter–
5’UTR–GUS fusion but not in the transgenic plants carrying only promoter–GUS fusion (without
5’UTR). These results suggest that the regulation of this stress-responsive gene is likely due to the
modulation of mRNA stability, which will be the focus of our future research. In addition,
functional characterization involving biochemical and genetic analysis of this protein will be carried
out.
34
HOST SPECIFICITY AND STRESS RESPONSE OF THE PVY IRES
Jennifer J. Randall1, Mercy S. Thokala1, Swati Mukherjee2, Rio A. Stamler1,
Jenna M. Painter1, Kathryn A. Hanley2, and Stephen F. Hanson1
EPPWS1, Department of Biology2, New Mexico State University
Eukaryotic messenger RNAs (mRNAs) characteristically include a methylated G7 cap located at
their 5’ end and a polyadenylated tail located at their 3’ end. Eukaryotic translation of mRNAs
occurs with recruitment of the ribosome via an interaction of the initiation complex with the
methylated 5’ cap. In the 1980’s it was demonstrated that cap-dependant translation is temporarily
shut down in eukaryotic cells infected with Polio virus (Etchison et al, 1982). However, this RNA
virus is successfully translated and subsequently replicated within the cell. The virus RNA lacks a
cap but does have a secondary structural element known as an internal ribosome initiation site
(IRES) which allows for the direct recruitment of the ribosome and viral translation (Pelletier and
Sonenberg, 1988).
Since the 1980’s, several IRES elements have been identified in the Picornavirus family (including
polio, ECMV, and potyviruses). IRES elements differ in their structure and their functionality in
different hosts. The Potyvirdiae family is the largest plant viral family and contains several hundred
members (Adams et al., 2005). Potato virus Y (PVY), a potyvirus, is transmitted by aphids; and
upon infection, this virus is translated into a polyprotein prior to its replication within plant cells. In
our lab, a novel IRES element from PVY has recently been identified and characterized in tobacco
(Thokala and Hanson, manuscript submitted). In order to understand the host specificity of IRES
functionality we are characterizing the PVY IRES in a “cross-kingdom” approach. We are currently
testing the PVY IRES using a reporter gene (GUS) behind both cap-dependant promoters
(polymerase II) and cap-independent promoters (polymerase I) in Arabidopsis thaliana as well as
yeast, insect, and mammalian cells.
Besides characterizing the PVY IRES in other kingdoms, we are also testing the PVY IRES
functionality under specific stress responses (heat shock, cold shock, and starvation). In Eukaryotic
cells, cap-dependent translation is impaired during heat shock, cold shock, apoptosis, and cell
division. Recently, putative IRES elements were described in the 5’ untranslated regions (UTR) of
mRNAs for maize heat shock protein 101(Hsp101), Nicotiana tabacum heat-shock factor 1
(NtHSF-1), human binding luminal protein (BiP), and drosophila BiP. Translation of these mRNAs
is specifically activated during heat shock. Therefore, characterizing the stress response of the
PVY IRES will increase our understanding of both viral replication in the host during times of
stress and the conservation of IRES function.
35
MODULATION OF CYTOSOLIC GLUTAMINE SYNTHETASE IN MEDICAGO SATIVA
(ALFALFA): MESOPHYLL-SPECIFIC VS CONSTITUTIVE OVEREXPRESSION
Mark Seger1, Jose Ortega1,2, Martha Martinez1, Champa Sengupta-Gopalan 1,2
Department of Molecular Biology1, Plant and Environmental Sciences2,
New Mexico State University, Las Cruces, New Mexico 88003
Glutamine synthetase (GS) is a key enzyme in plant nitrogen metabolism and catalyzes the ATP
dependent condensation of NH+4 and glutamate to yield glutamine. In plants, GS is an octameric
enzyme that is found as 2 major isoforms: cytosolic GS (GS1) and chloroplastic GS (GS2). Reports
in the literature have shown improved performance of plants engineered to overexpress GS1 in a
constitutive manner. Authors believe improved plant performance seen in these transformants may
have been attributed to enhanced reassimilation of photorespiratory NH+4. To address this, we have
produced alfalfa transformants that are engineered for constitutive or mesophyll-specific
overexpression of the soybean GS1 (Gmglnβ1). The transformants were analyzed for transgene
transcript, polypeptide, holoprotein, and enzyme activity. Transformants constitutively
overexpressing GS1 exhibited increased GS activity, but showed poor performance when grown
under N-sufficient conditions. Transformants overexpressing Gmglnβ1 in a photosynthetic cellspecific manner, however, showed much superior performance with respect to total protein content,
photosynthetic rates, and overall growth rates compared to control plants and plants constitutively
over-expressing Gmglnβ1. We propose that continuous overexpression of Gmglnβ1 takes away from
the C and ATP reserves that are made during photosynthesis. We will present data to test our
hypothesis. Experiments are in progress monitoring GS activity and amino acid free pools during a
day/night cycle in the transformants and control plants grown under different light and nitrogen
conditions.
36
REGULATION OF CYTOSOLIC GLUTAMINE SYNTHETASE IN SENESCENCE
Fernando Solorzano1, Esther Paul2, Olivia Wilson1, & Champa Sengupta-Gopalan1
Molecular Biology Program1, New Mexico State University2, Las Cruces, NM 88003
Glutamine synthetase (GS) is the key enzyme in nitrogen assimilation, catalyzing the ATPdependent biosynthesis of glutamine from ammonia and glutamate. Plant GS exists in various
isoforms, located either in the cytosol (GS1) or chloroplastid/plastid (GS2). GS2 is the major form
found in the mesophyllic cells of young and mature photosynthetic leaves. Here, it assimilates
ammonia reduced from nitrate and reassimilates ammonia produced by photorespiration. GS1 is
found in the phloem generating glutamine for nitrogen transport. Leaf senescence is a
developmentally programmed degenerative process making up the final step of leaf development.
In senescent leaves, GS1 predominates as it plays a key role in the reassimilation and remobilization
of nitrogen released from the degeneration of cellular structures. Previous work in our lab has
shown that besides being regulated at the transcriptional level, GS1 is also regulated at the level of
transcript stability and translation mediated by its 3’ untranslated region (UTR) and at the level of
holoprotein turnover. This work is focused on resolving if GS1 is subject to posttranscriptional
regulation during senescence. Tobacco plants have been transformed with a soybean GS1 gene
(Gmglnβ1) with and without its 3’ UTR driven by the constitutive CaMV35S promoter. Leaves
from the transformants and control were analyzed at different developmental stages for polypeptide
and holoenzyme levels corresponding to the transgene and the endogenous GS1 gene. Our initial
results show that age mediated regulation of GS1 and GS2 may be species specific. Also, the
Gmglnβ1 gene is not regulated at the 3’ UTR-mediated transcript turnover level during leaf
senescence.
37
DEVELOPING A TRANSIENT ASSAY SYSTEM TO STUDY POSTTRANSCRIPTIONAL
REGULATION OF GLUTAMINE SYNTHETASE IN PLANTS
Olivia Wilson1, Fernando Solorzano1, Dr. Champa Sengupta-Gopalan 1,2
Department of Molecular Biology1, Plant and Environmental Sciences2,
New Mexico State University, Las Cruces, New Mexico 88003
Glutamine synthetase (GS) plays a central role in nitrogen metabolism in all plants. GS catalyzes
the ATP dependent condensation of ammonia with glutamate to yield glutamine. Plant GS occurs
as a number of isoenzyme forms and these GS isoforms are located either in the cytosol (GS1) or
plastid (GS2). While there is ample evidence in the literature supporting the concept that GS is
regulated transcriptionally, recent evidence from our laboratory has shown that a soybean GS1 gene
is also regulated posttranscriptionally at the level of transcript turnover and translational initiation
(Ortega et al., 2006). Furthermore, it was also shown that the posttranscriptional regulation is
mediated via the gene’s 3’UTR. The objective of this study is to determine if other GS1 genes are
also subject to the 3’UTR mediated posttranscriptional regulation. Towards the objective, we have
made alfalfa GS1 gene constructs with and without its 3’UTR driven by a constitutive promoter.
These constructs have been introduced into Agrobacterium tumefaciens and the A. tumefaciens with
the gene construct have been introduced into tobacco and alfalfa via transient Agro infiltration and
by whole plant transformation. We will present our data on transgene expression in the transient
assay.
38