NSF 2010 Background Page

Project Summary

In Arabidopsis, G proteins couple multiple and diverse signals (e.g. light, pathogens, hormones) recognized by receptors to downstream effectors in a cell-specific manner. With the identification and characterization of heterotrimeric Ga and Gß subunit mutants and a set of candidate G-protein-coupled receptors (GPCRs), we are uniquely poised to ask which cells express G-protein pathway components and under what circumstances G proteins become activated in vivo? Our gene set comprises 16 heptahelical membrane proteins (candidate GPCRs), one canonical Ga subunit, three ‘extralarge’ Ga-related subunits (XLGs), one Gß, and two Gg subunits.

The first objective is to assess expression patterns of the gene set at different developmental stages and growth conditions using gene promoter::GUS fusions. Subcellular localization of Ga, Gß, XLGs and selected candidate GPCRs will be accomplished by visualizing translational fusions with yellow fluorescent protein (YFP).

The second objective is an in vivo determination of the regulation of physical interactions between G-protein pathway components. Physical interaction will be visualized in real time and with spatial dimensions using fluorescence energy transfer (FRET) between cyan fluorescent protein (CFP)-tagged and YFP-tagged proteins. To assess G-protein activation, loss of FRET interaction between a Ga-CFP fusion (or XLG-CFP fusions) and YFP-Gß will be assessed during responses to hormonal and abiotic stimuli implicated in G protein signaling. In plants, no GPCR has been unequivocally identified. We will address this issue by assessing FRET interactions between candidate GPCRs (tagged with YFP) expressed in our systems of interest (root and guard cells) and each of the 4 Ga-type proteins (GPA1 + 3 XLGs).

Our research will develop tools for the community enabling direct in vivo tests of hypothesized G-protein involvement in any signal transduction (light, hormone, biotic and abiotic stresses), or biological context (cell growth, division, stomatal physiology, host/pathogen interactions).

About Classical G Protein-Coupled Signaling

The largest gene family in animals encodes heptahelical transmembrane proteins that physically interact with a complex containing a GTPase, called a heterotrimeric G protein. These polytopic membrane proteins, for which there are over a thousand in metazoans, are collectively termed G protein-coupled receptors (GPCRs, colored red in the figure) and their ligands are as diverse as the GPCR family is itself. Small molecules like serotonin, peptides like somatostatin, and even large proteins like thrombin bind to their cognate GPCRs and induce cytoplasmic conformations by such shifts in helix positions that translate into specific loop conformations.

This is the “apical step” in many signal transductions and it works for so well for so many signals because the consequence of receptor occupancy is a very simple output: a specific cytoplasmic protein conformation that can be recognized by a family of heterotrimeric G proteins able to couple this signal to downstream effectors such as adenyl cyclase (AC, green in figure), phospholipases, ion channels (purple in figure),

phosphodiesterases, etc. Heterotrimeric G proteins (henceforth referred to as G proteins or simply “G” here) have an alpha subunit (Ga, blue ribbon structure in figure) with two domains, one is called the ras domain containing a GDP/GTP binding site, GTP hydrolase activity, and a covalently-attached lipid which anchors this subunit to the bilayer. Also, located within the ras domain are backbone loops, called switches which position themselves depending on whether GDP or GTP occupies the nucleotide binding site. When GDP is bound, the switches orient to permit tight association of Ga to the beta subunit (Gß, magenta ribbon structure in figure), but upon GTP binding, these switches re-orient such that the Ga/Gß interaction is disrupted permitting a slightly different interaction at the same interface with a membrane-localized enzyme. These G-protein activated enzymes are collectively called effectors. The nucleotide exchange is catalyzed by interaction of the Ga subunit with the activated conformation of the GPCR loop described above. This interaction between Ga and the cytoplasmic loop of the activated GPCR occurs at Ga’s N- and C-terminal domains which are located distal to the switch regions. On the other hand, the interaction between Ga and its cognate effector occurs along the same interface between Ga and Gß. While Ga undergoes a conformational change upon activation, Gß does not. Whether sequestered by Ga or free, Gß remains tightly bound to the gamma subunit (Gg, gold ribbon structure in figure), which tethers the complex to the bilayer via lipid modification at its C-terminus. Over time, and often assisted by accessory GTPase activating proteins (see RGS in Figure), GTP is hydrolyzed to GDP, permitting reassociation to Gßg and readied for another cycle of activation by its cognate GPCR. Similar interaction cycles are repeated over and over for each of the thousands of signals using the GPCR pathway; obviously, mother nature knew a good thing when she saw it.

Animals have 23 different Ga, 6 Gß, and 12 Gg subunits, potentially assembling over a thousand different G proteins, although given differences in cell expression and the known exclusion of some subunit pairs, we can more conservatively estimate that there are over a hundred heterotrimeric complexes existing in a cell. Ga forms 4 subfamilies, Gs, Gi, Gq, and G12, based upon their sequence. All except one member of the Gi subfamily can be covalently modified by ribosylation on a C-terminal cysteine residue catalyzed by pertusis toxin from Bordetella pertusis, thus Ga ribosylation is a diagnostic of subtype Gi. In general, Gs members stimulate adenyl cyclase (AC) while Gi inhibits AC activity. Gq typically stimulates phospholipase C-b (PLC) activity and G12 operates via another class of GTP-binding proteins, the rho family. The Gßg subunits also can activate effectors such as channels and phospholipase A2 (PLA).

Such enormous multiplicity raises the central question: how can so many signals, each recognized independently by a separate GPCR, specifically couple to only a dozen or less effectors by G proteins? Specificity in signal coupling in metazoans is accomplished by two mechanisms: 1. some G proteins are able to recognize a specific GPCR and a specific effector, 2. promiscuous G proteins are sequestered in signaling rafts containing a specific GPCR, the cognate effector and all other components that operate on a particular pathway.

G Proteins in Arabidopsis

In contrast to animals, Arabidopsis has single canonical Ga (GPA1) and Gß subunits (AGB1) and possibly only two Gg subunits (AGG1 and AGG2). GPA1 is roughly 30% identical to mammalian Ga's and essentially all of this conservation lies in the few critical domains for interacting with receptors and effectors (see "About Classical G protein signaling" for a discussion of the important domains).

Arabidopsis mutants lacking GPA1 (gpa1), have reduced cell division during hypocotyl and leaf formation (see Ullah, et al 2001, Ullah, et al, 2002 in "selected readings"). Expression of GPA1 causes ectopic cell divisions, including massive overproliferation of meristem formation at high GPA1 expression levels suggesting that GPA1 couples a signal that controls cell division. A likely candidate is the plant growth hormone, auxin, but auxin-induced cell division still occurs in mutants lacking either Ga or Gß, thus indicating that auxin can not be directly coupled by a G protein. However, while G-protein mutants respond to auxin they have dramatically altered auxin sensitivity suggesting that some other, as yet unknown, G protein-coupled pathway crosstalks to auxin signaling in a way that controls auxin sensitivity. Unlike for auxin, it can be concluded that an ABA signaling pathway is directly coupled by a G protein. Wang, et al (see Wang, et al. 2001 in selected readings) demonstrated that ABA inhibition of light-induced stomata opening is completely lacking in gpa1 mutants. Consistent with the loss in ABA responsiveness, gpa1 mutants lack ABA inhibition of inward K+ channels and lack activation of pH-independent anion channels. Interestingly, ABA-induced stomatal closure mediated by pH change remains unaffected by the loss of function of GPA1, indicating independent ABA pathways in guard cells.

Not only can a specific cell type contain multiple mechanisms of signaling for one hormone such as ABA, but different cell types can have different mechanisms as well. For example, in contrast to the ABA insensitivity in gpa1 guard cells, gpa1 seeds have wildtype sensitivity to ABA, but are 100-fold less sensitive to gibberellic acid (GA) and completely insensitive to brassinosteroid (BR), two other plant hormones. Seeds overexpressing GPA1 are a million-fold more sensitive to GA but still require GA for germination. One interpretation of the loss and gain-of-function results is that GA signaling of seed germination is not directly coupled by G, but rather that some other G-coupled pathway crosstalks in a way that controls GA sensitivity. This indirect effect on a pathway via control of sensitivity is a re-occurring theme. Because it is known that BR regulates GA sensitivity and that BR application to seeds having reduced GA levels will fully germinate, it is possible that a BR pathway coupled by a G protein is the sought after pathway. Consistent with this, we have shown that BR synthesis and response mutants have the same reduced GA sensitivity as the gpa1 mutants and that BR was completely ineffective at rescuing germination of gpa1 seeds when GA levels were reduced.

What is Upstream of G in Plants?
To date, no plant receptor has been shown to be directly coupled by G. Furthermore, in contrast to the thousand or more heptahelical transmembrane proteins in animals, Arabidopsis has only a few candidates with only one plant protein to date actually confirmed to be heptahelical (see Resources). This protein, called MLO1, confers resistance to powdery mildew when present in its recessive form, but the mechanism of resistance is unknown. Recent evidence indicates that disease resistance conferred by mlo is independent of a G protein, however, the possibility remains that MLO is coupled by a G protein in another signaling pathway because the function of this putative orphan receptor is unknown.

GCR1 is a protein that shares some sequence identity to animal GPCRs of the serotonin family (see Resources). GCR1 has a predicted heptahelical structure but this has not yet been confirmed by direct analyses. Receptor-independent, G-protein signaling occurs in animals. Using a functional screen in yeast, Lanier's group at Univ. South Carolina found three proteins (AGS1-3) capable of activating G-protein signaling in the absence of a cognate receptor (see figure in "About classical G protein signaling"). Perhaps the most interesting of these is AGS3 which has subsequently shown to be a guanine dissociation inhibitor. AGS3, a protein interaction involving a GoLoco motif, binds the GDP-bound form of Ga to release Gßg, which in yeast directly activates a MAP kinase pathway. However, at this time, it is not known if AGS3 homologs or GoLoco-containing proteins are found in plants.

Thus, we are left with only three possible conclusions: 1. in contrast to animals, plants couple only one or a few heptahelical receptors by a G protein to downstream effectors, and/or 2. receptor-independent G protein signaling occurs as the primary mechanism in plants, and/or 3. plants couple nonheptahelical receptors. While the jury is still out, some interesting facts shed light on this problem. First, the C-terminal domain of all plant G proteins is nearly 100% conserved, unlike in animals where this region is poorly conserved due to the diversity in Ga/receptor interactions. By reverse reasoning, complete conservation in sequence among plant Ga C-terminal domains suggests that there is a single or only a few receptors with which Ga can interact. Second, indirect observations are consistent with G coupling to nontraditional receptors. For example, as discussed above, we found that germination of gpa1 seeds is insensitive to BR. They also showed that bri1 seeds have the same BR insensitive phenotype as gpa1 seeds. BRI1 encodes a putative BR receptor-like kinase. John Walker's lab at Univ. Missouri found that a putative protein-null allele of AGB1 shares many fruit phenotypes with the receptor-like kinase mutant, erecta (er). Interestingly, double er, agb1 mutants have complex phenotypes. Depending on the trait, AGB1 is epistatic to ER or vice versa, suggesting either that either there are multiple parallel pathways operating or that both encoded proteins interact. These recent observations raise the exciting possibility that G couples one or more of the over 400 receptor-like kinases in plants.

What is Downstream of G in Plants?
Phospholipase D (PLD), potassium and calcium channels are presently the only three candidate effectors implicated in G-protein signaling in plants. In rice aleurone, GA-induced a-amylase secretion is greatly reduced in a Ga mutant, d1, suggesting that some signal which crosstalks to the GA/ABA pathway is coupled by G. Simon Gilroy at Penn State Univ. (PSU) showed that GTPgS alters PLD activity consistent with PLD residing directly downstream of an activated G protein. Sally Assmann at PSU showed that the gpa1 mutant, which likely has excessive Gßg, lacks ABA inhibition of K+ influx, suggesting either GPA1 is required for channel activation (via PLD?) or that Gßg blocks channel activation in contrast to animals. While another phospholipase activity, namely PLA2, increases activity rapidly after applied hormone, but in this case by auxin, it is still unknown if this occurs via coupling of auxin to PLA2 by G. The lipid by-products of PLA2 have been shown in animals to activate a K+ channel and PLA2 activation there probably occurs by interaction with the released Gßg. Finally, there is evidence that a heterotrimeric G protein may also activate calcium conductance in plant cells because cation influx increases in the presence of recombinant Ga, although it is not known yet if this regulation is direct as shown in animal cells.

Two Mechanisms for Signaling Specificity with a Heterotrimeric Plant G Protein Many signals in plants are coupled by this G protein complex leaving us wondering how only one or two G-protein complexes in Arabidopsis can provide specificity in coupling a particular signal to a particular effect. Obviously, one mechanism is to control the constellation of receptors and effectors expressed in a particular cell type; in other words, by a temporal control of the up and downstream components to G in a cell. A second mechanism is to physically sequester receptors and effectors with G and other signaling components in a type of signaling raft as described in the introduction for b-adrenergic signaling. Not surprisingly, signaling rafts are found in plants, although none yet have been shown to include a heterotrimeric G protein component.

About Arabidopsis G-protein Structure

in brevia… The structure of a composite mammalian G protein complex is known from the crystallographic work of John Sondek’s group at the University of North Carolina- Chapel Hill. With a known structure, it is possible to take another molecule that is suspected to share the same or similar structure and model it. In this case, it is the Arabidopsis G protein complex. The model is just a model but it enables you to test computationally it to determine how plausible it is. In the case of our modeling the Arabidopsis G protein complex, the results of the tests indicate that this plant complex is extremely likely to be similar to the animal complex. The figure here compares the structure of the composite animal G complex (panel A) to the modeled Arabidopsis complex (panel B).

If you want the details of how we did it… Experimentally-determined structures for two different mammalian G protein heterotrimers have been previously reported. The PDB model with accession code 1GOT is a 2.0 Å structure of the heterotrimer Gt-a (bovine)/Gi-a (rat) chimera, Gi-b1 (human), Gt-g1 (bovine) (Lambright et al., 1996) . The model with PDB accession code 1GP2 is a 2.3 Å structure of the Gi-a1 (rat), Gi-b1 (human), Gi-g2 (C68S) (bovine) (Wall et al., 1995; 1998) . The structures of 1GOT and 1GP2 are very similar and superimpose with a root-mean-square deviation of 1.2 Å. The mammalian heterotrimer 1GOT is shown in Figure 1A with the a, b and g subunits colored blue, purple and gold, respectively. The Ga subunit is composed of an N-terminal helix that interacts with Gß, a mixed a-helical/b-strand Ras-like domain with GTPase function and an all a-helical domain. The Gß subunit is a seven-bladed b-propeller structure with an N-terminal helix. The g subunit contains two helices which interact with the N-terminal helix of the subunit and the b-propeller structure itself.

The higher resolution structure 1GOT was used as a template to build a homology model (panel B) of the Arabidopsis G protein heterotrimer Gabg. Models for each of the three subunits were built independently and then superimposed onto the heterotrimer structure of the G protein. The fold-recognition servers bioinbgu (Fischer, 2000) , 3D-PSSM (Kelley et al., 2000) , GenTHREADER (Jones, 1999) and FUGUE (Shi et al., 2001) all identified the Arabidopsis sequence GPA1 as a Ga with greater than 99% confidence and all but 3D-PSSM identified the Arabidopsis sequence AGB1 as a Gß protein with greater than 99% confidence. The 3D-PSSM server selected the 7-bladed b-propeller structure as the most compatible structure with the AGB1 sequence with 80-90% confidence. All of the servers determined the Gg protein to be the most compatible structure with the Arabidopsis AGG1 sequence, although none of the predictions had greater than 99% confidence. The final theoretical model for the Arabidopsis Ga was given a self-compatibility score of 146.9 by the Profiles-3D/Verify module of Insight II. The typical score expected for an experimentally-determined protein structure of 362 residues was 165.0 while a score less than 74.2 was considered to be indicative of an incorrect structure. For comparison, the self-compatibility score for the 338 residue mammalian Ga structural template was 148.9, with a typical score of 154.0 and a minimum score of 69.3. Self-compatibility scores of 167.5 and 165.9 were calculated for the theoretical Arabidopsis Gß structure and the experimentally determined mammalian Gß, respectively. The Arabidopsis Gß model contained 362 residues and the typical self-compatibility score was reported at 165.0 while the mammalian Gß contained 339 residues and had a typical self-compatibility score of 154.4. The 786-residue theoretical Arabidopsis heterotrimer had self-compatibility and typical scores of 336.8 and 360.4, respectively. The self-compatibility score for the mammalian composite heterotrimer was 346.9 with a typical score of 331.3 for a 723 residue protein.

The theoretical models (panel B) of the Arabidopsis G protein heterotrimer monomers based on the mammalian templates (panel A) are “valid” structures overall (model deposited at PDB). The compatibility of the Arabidopsis sequences with the mammalian G-protein structures was predicted by the fold recognition servers bioinbgu, 3D-PSSM, GenTHREADER and FUGUE. The Profiles-3D/Verify self-compatibility scores indicated that the final theoretical structures for Arabidopsis Ga and Gß are nearly as compatible with the Arabidopsis sequences as the experimentally-determined mammalian structures of Ga and Gß are with the mammalian sequences. While the overall structures are valid, there are some minor differences between the Arabidopsis structures and the mammalian structures due to insertions in the Arabidopsis proteins. The insertions are generally small with an average size of 5.0 residues for 5 inserts in the Arabidopsis Ga and an average size of 2.3 residues for 10 inserts in the Arabidopsis Ga subunit. The unpredicted conformations are colored green in panel B.

The functionally important regions of the G protein heterotrimer structure are highly conserved in the Arabidopsis heterotrimer. Guanine nucleotide-binding proteins contain 5 conserved sequence elements that are highlighted in Figure 1C. These elements are: (1) the NKxD motif (orange) that interacts with the nucleotide base and is responsible for guanine specificity; (2) the P-loop (green), a GxxxxGKS motif that interacts with the b, g-phosphates; (3) the DxxG motif (yellow) that is responsible for distinguishing between GTP and GDP; and the two switches: (4) switch I (red) and (5) switch II (dark blue). Panel D of the Arabidopsis heterotrimer, in the same orientation as the mammalian heterotrimer Figure 1C, shows residues highlighted according to degree of conservation among G proteins. Invariant or highly conserved residues are dark blue, dark magenta or dark gold in the Ga, Gß or Gg, respectively.

Conserved residues are correspondingly light blue, light magenta or light gold. Residues which are not conserved are white in the Arabidopsis heterotrimer. This coloring scheme is reproduced in panels E and F where the heterotrimer has been rotated to show the Ga/Gß interactions (panel E) and the Gß/Gg interactions (panel F). The side chains of residues involved in protein/protein interactions are highlighted and are also colored according to conservation in panels E and F. Residues in Ga interacting with Gß highlighted in panel E are Ala7, Ile10, Glu11, Ile14 and Glu17. Residues highlighted in Gg are Val11, Leu14, Glu17, Val27, Ser28, Leu34, Leu48, Leu49 and Trp60 (Arabidopsis numbering). Clearly, the sequence elements of the guanine nucleotide-binding proteins that are responsible for their action as molecular switches are conserved in the Arabidopsis thaliana Ga subunit. The interface residues involved in formation of the heterotrimer are also highly conserved. A cycling view of the conserved interacting between Ga and Gß and Gß and Gg subunits as shown in panel E and F, is provided as a flash video of 5 Å increment rotation. If the movie has already loaded click the reset button and then press play.

About Arabidopsis

The genus Arabidopsis is a member of the Brassicaceae (mustard or crucifer) family in the tribe Sisymbriae that contains several species including the most well-known Arabidopsis thaliana. This particular species has emerged as a model plant for studies in classical and molecular genetics, developmental biology, physiology, biochemistry and functional genomics for a variety of reasons. These include the facts that:

Arabidopsis develops, reproduces and responds to disease and environmental stresses in ways comparable to many crop plants.
Arabidopsis plants are relatively small making it easy and inexpensive to grow them under a variety of research conditions.
Arabidopsis thaliana possesses a relatively small genome marked by a remarkable absence of highly and moderately repetitive sequences. This feature has allowed researchers to adopt saturation mutagenesis strategies and/or map-based cloning strategies not possible in other plants with genomes containing significant proportions of repetitive DNA.
Arabidopsis has a short non-seasonal generation time (5 weeks) and the ability to produce thousands of seeds per plant. These features have facilitated the extensive genetic analyses needed to map individual gene traits.
Arabidopsis is autogamous (self-fertile), which makes the maintenance of homozygous lines straightforward, and capable of being cross-pollinated, which makes genetic crosses and gene mapping possible.
Arabidopsis thaliana is a diploid with a small number of chromosomes (five). This has facilitated the identification of recessive traits and allowed researchers to avoid the complications of varying gene dosages encountered in many other plant species.
Arabidopsis is readily transformed by vacuum infiltration with Agrobacterium tumefaciens containing T-DNA based binary vectors. This has allowed researchers to analyze the phenotypic variations associated with overexpression of endogenous and exogenous proteins in intact plants as well as the visual expression patterns of promoter: reporter genes in intact cells.

Arabidopsis mutants can be readily generated with many different chemical and biological mutagens that randomly alter genomic DNA sequences. This feature has made it possible to generate and screen large mutant collections of progeny for phenotypic changes derived from single nucleotide changes, T-DNA insertions or transposon insertions.

from http://arabidopsis-p450.biotec.uiuc.edu

About FRET

Fluorescence Resonance Energy Transfer (FRET)

Fluorescent Resonance Energy Transfer (FRET) is a phenomena used in microscopy to observe and demonstrate interactions between biological molecules.

The two biological molecules of interest are labeled with different fluorophores. One molecule will act as a donor , the second molecule will act as an acceptor fluorophore. As the acceptor comes close the donor, the donor transfers its energy without emission of a photon (i.e. nonradiatively) to the acceptor. As a result of this energy transfer, the donor fluorescence is quenched and the acceptor fluorescence will be stimulated.

The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor.

Excitation and Emission Spectra for BD Living Colors Fluorescent Proteins

Efficiency of the energy transfer process is dependent on the inverse of the sixth power of the distance separating the donor and acceptor labeled molecules, the FRET process can provide very sensitive measurements on the separation distance of the donor and acceptor labeled molecules. Donor and acceptor molecules must be in close proximity (typically 10–100 Å).

Typically, interaction distances of 10 – 100 Angstroms can be measured using this approach when FRET is used; co-localization of proteins and other molecules can be imaged with spatial resolution.

2010 Project Background

2010 Project: To determine the function of 25,000 genes in Arabidopsis thaliana by the year 2010

INTRODUCTION
The Directorate for Biological Sciences (BIO) of the National Science Foundation (NSF) announces its intention to continue support of research to determine the function of all genes in the model plant Arabidopsis thaliana by the year 2010. This represents a continuation of the Arabidopsis genome research initiative BIO has supported since 1990 and of the 2010 Project begun in FY 2001.

In 1990, plant scientists established an international research effort called "The Multinational Coordinated Arabidopsis thaliana Genome Research Project", by adopting the widespread use of an easily manipulated model plant called Arabidopsis thaliana. (For a long-range plan, see NSF 90-80.) One outcome of this effort is the entire DNA sequence of this plant, that was completed at the end of 2000. For the first time, we know the sequence of approximately 25,000 genes necessary for a flowering plant to function.

As a follow-up to the Arabidopsis genome sequencing efforts, and to take full advantage of the opportunities created by them, the community of plant biologists has proposed an important and revolutionary new initiative: to determine the function of all genes of a reference species within their cellular, organismal and evolutionary contexts by the year 2010 (the 2010 Project). Details can be found at http://www.nsf.gov/cgi-bin/getpub?bio011 and http://www.arabidopsis.org/workshop1.html. In response, BIO has established the 2010 Project as a Directorate-wide activity.

One of the factors that contributed to the success of the Multinational Coordinated Arabidopsis thaliana Genome Research Project was world-wide collaboration among the researchers involved. The Arabidopsis research community has become a model for international research collaboration. It is expected that continued efforts by the international community of scientists will be essential for the success of the 2010 Project. NSF will continue to foster activities to advance international collaboration and coordination of the 2010 Project.

PROGRAM DESCRIPTION
Individual investigators, or groups of investigators, will be supported to conduct creative and innovative research designed to determine, using all available means, the function of a set of genes in Arabidopsis thaliana of the investigator's interest and choice. The number of investigators involved in a single proposal should be determined by the scope and approach used in the proposal. The NSF expects that both large and small laboratories should be able to participate in the 2010 Project by taking advantage of various publicly available whole genome tools and resources.

Investigators are expected to have selected a set of genes as the subject of their research prior to the submission of a proposal. These genes must be identified in the proposal by GenBank accession number or by other identifiers from a publicly accessible database. The size of the selected gene set will depend on a number of factors, such as how the particular set was selected (e.g., based on a specific sequence motif or a set of genes expressed under a specific condition), and the interest and throughput capacity of the applicant. Genes being investigated by FY 2001 2010 Project awardees are publicly posted on a website at The Arabidopsis Information Resources or on an individual project's website whose address can be found in the award abstract available through the FY 2001 2010 award list.

Development of Research Tools and Resources: While the genome sequence and annotation information in public databases, microarray expression data at public websites, and a variety of biological resources at the Arabidopsis Biological Resource Center provide a good start for the community to begin the 2010 Project, NSF recognizes that additional tools and community biological resources are needed to enable individuals or groups of individuals in the scientific community to participate in the 2010 Project. As in FY 2001, building the necessary, critical tools and biological resources will be supported in FY 2002. Especially encouraged are new informatics tools that would allow individual investigators to access, analyze and utilize the massive amounts of Arabidopsis data accumulating rapidly. The goal of new informatics tools would be to enable individual researchers to formulate a query of all the available resources so that they can make full use of the tools and resources that are in place as well as those that will become available in the future.

Integration of Research and Education
One of the principal strategies in support of NSF's goals is to foster integration of research and education through the programs, projects, and activities it supports at academic and research institutions. These institutions provide abundant opportunities where individuals may concurrently assume responsibilities as researchers, educators, and students and where all can engage in joint efforts that infuse education with the excitement of discovery and enrich research through the diversity of learning perspectives.

Integrating Diversity into NSF Programs, Projects, and Activities
Broadening opportunities and enabling the participation of all citizens -- women and men, underrepresented minorities, and persons with disabilities -- is essential to the health and vitality of science and engineering. NSF is committed to this principle of diversity and deems it central to the programs, projects, and activities it considers and supports.

ABOUT THE NATIONAL SCIENCE FOUNDATION
The National Science Foundation (NSF) funds research and education in most fields of science and engineering. NSF welcomes proposals from all qualified scientists, engineers and educators. The Foundation strongly encourages women, minorities and persons with disabilities to compete fully in its programs. In accordance with Federal statutes, regulations and NSF policies, no person on grounds of race, color, age, sex, national origin or disability shall be excluded from participation in, be denied the benefits of, or be subjected to discrimination under any program or activity receiving financial assistance from NSF (unless otherwise specified in the eligibility requirements for a particular program).

Selected Readings

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Ashikari, M., Wu, J., Yano, M., Sasaki, T., and Yoshimura, A. (1999). Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the a -subunit of GTP-binding protein. Proc. Natl. Acad. Sci. USA 96, 10284–10289.[Abstract/Full Text]

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Fujisawa, Y., Kato, T., Ohki, S., Ishikawa, A., Kitano, H., Sasaki, T., Asahi, T., and Iwasaki, Y. (1999). Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice. Proc. Natl. Acad. Sci. USA 96, 7575–7580.[Abstract/Full Text]

Humphrey, T.V., and Botella, J.R. (2001). Re-evaluation of the cytokinin receptor role of the Arabidopsis gene GCR1. J. Plant Physiol. 158, 645–653.[ISI]

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Ishikawa, A., Tsubouchi, H., Iwasaki, Y., and Asahi, T. (1995). Molecular cloning and characterization of a cDNA for the a subunit of a G protein from rice. Plant Cell Physiol. 36, 353–359.[ISI][Medline]

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Kim, W.Y., Cheong, N.E., Lee, D.C., Je., D.Y., Bahk, J.D., Cho, M.J., and Lee, S.Y. (1995). Cloning and sequencing analysis of a full-length cDNA encoding a G protein a subunit, SGA1, from soybean. Plant Physiol. 108, 1315–1316.[CrossRef][ISI][Medline]

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Lee, Y.-R.J., and Assmann, S.M. (1999). Arabidopsis thaliana ‘extra-large GTP-binding protein’ (AtXLG1): A new class of G-protein. Plant Mol. Biol. 40, 55–64.[ISI][Medline]

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