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Statement of Research Interests for Dr. Aaron M. Rashotte
Leaf development and expansion are essential plant processes for which our
understanding is incomplete. Plant hormones are known to be implicated in these processes, but
only the initial connections necessary for this understanding have been made. I am interested in
adding to this area of essential knowledge by studying how the plant hormone cytokinin is
involved in controlling leaf development and expansion.
Both cytokinin mode of action and leaf development are important areas that are poised
to be experimentally explored. Plant hormones are vital compounds that regulate a broad range
of essential functions throughout the lifecycle of a plant. Cytokinin has been implicated as
regulating many leaf related processes such as shoot organogenesis, vascular development,
senescence, sink/source relationships and chloroplast differentiation. Although there has been
much work recently in understanding the primary components of the cytokinin signaling
pathway, little is known as to how this signal acts to regulates cytokinin processes functioning in
the leaf. Leaves are vital plant organs involved in numerous essential aspects of normal plant
growth, including the essential processes of photosynthesis and respiration, yet there is still much
to learn leaf development. Expansion of a leaf is an essential developmental process for normal
plant growth and a better understanding of this process could lead to plants with more desirable
economic traits, such as larger or more photosynthetic efficient leaves. Recently, much of the
study of leaves has focused on understanding their polarity and formation from the meristem,
with less attention paid to the general process of expansion. Many of the tools designed to
understand hormone function in other plant tissues and those methods used to examine other
aspects of leaf function are ideal to be applied together to understand how hormones control leaf
expansion and development. I will take these tools and the knowledge that I have learned in my
postdoctoral work from examining two different plant hormones and apply these to the problem
of understanding leaf development.
My plan to better understand cytokinin regulated control of leaf development is an
extension of goals outlined in my NIH postdoctoral fellowship. I started to examine how
cytokinin regulates growth and developmental processes by conducting microarray experiments
to examine which genes in the plant are regulated by cytokinin. These experiments identified
over 40 genes induced by cytokinin, including three highly related AP2/ERF-like transcription
factors that I named cytokinin regulated transcription factors or CRFs (Rashotte et al., 2003,
Rashotte et al., 2006). I decided to study CRFs in order to reveal potential modes of cytokinin
action that were regulated through the targets of these transcription factors. I have and will
continue to take three distinct approaches in order to attack this problem from different angles
and to achieve a better understanding of the role that cytokinin plays in leaf development: (1) a
basic genetics, mutational, and physiological approach, (2) a molecular, cellular, and signaling
approach, and (3) a microarray and protein-interaction based approach to identify transcription
factor targets. I have already made progress toward this goal in each of these approaches.
I have been able to link cytokinin action to leaf and cotyledon expansion using a genetic
approach in which knockout mutants are identified and physiologically characterized both
generally and in a number of bioassays to examine their response to cytokinin. The three
cytokinin induced genes from the microarray analysis that I designated CRFs, are part of a
subclade of six Arabidopsis genes AP2/ERF-like family of transcription factors of unknown
function, that I have designated CRF1 to CRF6 (Gutterson and Reuber, 2004, Rashotte et al.,
2006). I have identified and characterized cotyledon, leaf and root phenotypes for knockout
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mutants in each of these six genes in the presence and
absence of cytokinin. I have done the same for double and
triple mutants as well as transgenic lines overexpressing
these genes that I have generated. This genetic approach
has revealed some functional redundancy between CRF
genes with one specific CRF gene pair necessary for
normal embryo development. These genes appear to be
primarily involved in the normal development and
expansion of cotyledons and juvenile leaves. This can be
seen in both single and multiple CRF mutants and in
overexpression lines (Figure 1). Alterations in leaf or
cotyledon shape are often seen as areas of tissue that do not
expand properly as in Figure 1. Additionally, inducible
overexpression of CRF5 can result in extreme alterations in
leaf shape, as seen in Figure1I, J.
Cytokinin is involved in this physiological process
as CRF mutants show increased expansion of leaf and
cotyledon area (125-175% of wild-type levels) and a CRF5
overexpression line shows reduced expansion (60% of
wild-type levels) in the presence of cytokinin (data not
shown). These results were obtained using a procedure to
measure radish cotyledon expansion that I modified for use
in Arabidopsis (Letham, 1971). Together, results from this
Figure1. Cotyledons and Leaves Are Altered in
CRF Gene Mutants and Overexpression.
bioassay suggest that CRFs are negative regulators of
Shown are the seedling phenotypes, left, and a
cytokinin based cotyledon and leaf expansion, since the
close up of the affected tissue, right. (A, B)
Wildtype (C, D) crf5 single mutant (E, F)
absence of these genes results in increased expansion and
crf2,5 double mutant (G, H) crf1,2,5 triple
the overabundance of one of these genes results in reduced
mutant and (I, J) Severe Overexpression
phenotype of CRF5. Scale bar is 0.5mm.
expansion.
Using a molecular based approach to study the role
that cytokinin plays in leaf development I have focused on examining how CRF genes and
proteins are involved in this process. My initial molecular studies confirmed the transcriptional
regulation of all six CRFs by cytokinin using both Northern and Real-Time PCR analyses as it
was seen in microarray experiments. Further work from a molecular approach has concentrated
on the generation and subsequent analyses of transgenic constructs to be used as tools to examine
these genes and proteins in transgenic plants in a variety of ways. The constructs I have
generated for the CRFs include constitutive and inducible overexpression, GFP and GUS
reporter genes, protein tags and RNAi. I have begun to examine a number of these constructs in
transformed into both wild-type and mutant plant backgrounds. The continued analyses of these
transgenic plants with a specific focus on the CRF native and cytokinin regulated expression
patterns should help to further determine the role cytokinin is playing in leaf development.
Recent work studying CRF localization at the cellular level led to an examination of how CRFs
function in relation to the cytokinin signaling pathway. I have been able to show that cytokinin
causes the localization of these proteins to the nucleus (Figure 2A and Rashotte et al., 2006).
GFP tagged CRF proteins appear to be found ubiquitously throughout cells in both transgenic
seedlings and protoplasts. Upon the addition of cytokinin CRF proteins localize to the nucleus
rapidly (5-7 min), even in the presence of cycloheximide, suggesting that cytokinin is involved in
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Figure 2. CRF Protein Nuclear Localization is Dependent on Cytokinin Signaling (A) CRF2:GFP nuclear localization by cytokinin was
examined in protoplasts of wild-type WT and multiple mutants of the cytokinin signaling pathway (receptors AHKs, phosphotransferases
AHPs, and both type-Bs and type-A response regulators ARRs) (B) A model of cytokinin signaling including the CRFs indicates that CRF
nuclear localization by cytokinin is dependent on both AHKs and AHPs, but not ARRs. Microarray data from Rashotte et al., 2006 shows
that many cytokinin regulated gene targets of type-B ARRs overlap with the CRFs.
the regulation of this protein movement. As transcription factors act by regulating other genes
within the nucleus, it seems likely that the cytokinin regulated nuclear localization of CRFs
controls the action of these proteins on their target genes. Interestingly, all six CRFs are localized
to the nucleus by cytokinin, independent of the affect of cytokinin on their transcriptional level. I
examined CRF nuclear localization by cytokinin in multiple mutant knockout backgrounds that
our lab has generated in each step of the cytokinin signaling pathway in order to determine where
CRFs fit in this signaling cascade. This work was done in both a transient protoplast system as
shown above in Figure 2A and also in stable transgenic plants (Rashotte et al., 2006). The results
of this study showed that CRFs require both the AHK cytokinin receptors and the AHP
phosphotransferases for nuclear localization, but neither the Type-A or Type-B response
regulators (Figure 2). Additionally, a comparative microarray analysis was conducted to identify
cytokinin regulated targets of CRF genes, using CRF mutants. Genes that were induced or
repressed by cytokinin in the wild-type that did not show the same pattern of regulation by
cytokinin in two different CRF triple mutants, were identified as cytokinin regulated targets of
CRF genes. Interestingly, about two-thirds of the cytokinin regulated CRF target genes were also
identified in a comparative microarray analysis as common cytokinin regulated targets of Type-B
ARRs (Rashotte et al., 2006). Together these examinations of CRF targets and CRF nuclear
localization in signaling mutant backgrounds has allow CRFs to be added into the model of
cytokinin signaling (Figure 2B).
I have also been conducting a detailed examination of the AHP phosphotransferase
nuclear localization by cytokinin in a manner similar to that done for the CRFs. Interestingly
there are striking differences the kinetics and some of the cytokinin signaling components
necessary for nuclear localization of AHPs and CRFs. This research that is in preparation for
publication further redefines the model of how cytokinin signaling is occurring in plants.
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In the first phase of my research as an independent investigator I will focus on further
determining the role that the plant hormone cytokinin is playing in leaf and cotyledon
development by examining how CRFs function. A major area of concentration in my focus on
understanding CRF function is a detailed characterization of CRF mutants and transgenic plants.
An early goal in this research direction is to thoroughly characterize the way that these
transcription factors affect leaf and cotyledon expansion, including what specific tissues or cell
types are involved and whether CRFs act through cell expansion or control of cell division or
both. One approach to achieving this is through a detailed tissue and cellular level examination
of the leaves and cotyledons of CRF mutants during development. In a parallel approach I will
make use of transgenic CRF promoter:reporter and overexpression plants, that I have already
generated, to examine the location of CRF expression and effects of CRF overexpression. Both
mutants and transgenic plants will be examined under a variety of developmental stages and
biotic and abiotic responses with special attention to how cytokinin affects these plants.
Additionally work to determine CRF function will make use of other mutants and reporter gene
constructs that are involved in leaf polarity, meristems, cytokinin responsiveness, and cell
division in CRF mutant backgrounds and under various hormone treatments.
A second key area of concentration I will focus on to better understand the function of
CRFs is the identification and analysis of CRF targets. A detailed characterization of CRF targets
should help to reveal the mode of action for cytokinin regulation of leaf and cotyledon expansion
and development. I plan to identify CRF targets and interactors using several different methods
including yeast two-hybrid and split-YFP analyses to identify interactions at the protein level and
Gel Shift assays to examine the DNA sequence targets of CRF proteins. Another method to
identify CRF targets, specifically interacting gene targets is by using comparative microarray
analyses. I have already begun to use this method to identify cytokinin dependent targets by
comparing expression levels of genes in crf triple mutants to the wild-type in the presence of
cytokinin and have confirmed several of these targets using Real-Time PCR (Rashotte et al.,
2006). Further analyses of promising cytokinin dependent targets involved in leaf development
and expansion will be pursued in a multipronged approach as I used to first examine the CRFs.
Interestingly, many cytokinin regulated CRF gene targets are also type-B ARR gene targets so I
also plan to examine any overlapping regulation between these two different cytokinin related
transcription factors that may be linked to cytokinin and leaf development. Additionally, the
generation of inducible CRF overexpression transgenic plants is in progress such that
experiments can be conducted to identify the immediate downstream targets of the different
CRFs. In all CRF target identification experiments my primary interest is on cytokinin regulated
targets involved in leaf and cotyledon development. These initial approaches to study CRF
function should provide a solid base of knowledge which can be built upon for future study of
cytokinin regulation and leaf expansion and development.
References:
Letham, DS (1971) Regulators of Cell Division in Plant Tissues XII. A Cytokinin Bioassay Using Excised Radish Cotyledons. Physiol. Plant. 25:
391-396.
Gutterson, N and Reuber TL (2004) Regulation of Disease Resistance Pathways by AP2/ERF Transcription Factors. Curr. Op. Plant Bio. 7: 465471.
Rashotte AM, Carson DBS, To JPC, Kieber JJ (2003) Expression profiling of cytokinin action in Arabidopsis. Plant Physiol. 132: 1998-2011.
Rashotte AM, Mason MM, Hutchison CE, Ferreria FJ, Schaller GE, Kieber JJ. (2006) A subset of Arabidopsis AP2 transcription factors mediate
cytokinin responses in concert with a two-component pathway: PNAS 103: 11081-11085.