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Plant Physiol, June 2002, Vol. 129, pp. 394-437 Summaries of National Science Foundation-Sponsored Arabidopsis 2010 Projects and National Science Foundation-Sponsored Plant Genome Projects That Are Generating Arabidopsis Resources for the CommunityEdited by
Deciphering the functions of the
approximate 25,000 genes encoded in the Arabidopsis genome is an
extraordinarily complex, challenging, and expensive undertaking. In the
United States, federal funding for Arabidopsis genomics research is
coordinated by an interagency governmental program called the National
Plant Genome Initiative (NPGI). Established in 1988, NPGI has been
instrumental in the establishment of two relatively large programs at
the National Science Foundation (NSF), the Plant Genome Program and the
Arabidopsis 2010 Program. NPGI strongly supported the Arabidopsis
Genome Initiative in its goal to obtain the first complete sequence of
a plant genome. The Arabidopsis sequence, published in December 2000 (Arabidopsis Genome Initiative, 2000 The editors of this Plant Physiology special issue devoted to Arabidopsis-related research thought that it would be useful for the community to compile summaries of the Arabidopsis 2010 projects and the Plant Genome projects that are producing Arabidopsis resources. We hope that the following project summaries and/or progress reports will be a valuable and relatively concise source of valuable information that will catalyze the widespread dissemination of the huge body of data being generated about the Arabidopsis genome. Most of the projects have an associated Web site, the URL of which is indicated at the top of each summary.
Private Investigator (PI): David Bird, North Carolina State University, david_bird{at}ncsu.edu; Co-PI: Sandra Clifton, Washington University School of Medicine, sclifton{at}watson.wustl.edu; Co-PI: Thomas Kepler, Santa Fe Institute, kepler{at}santafe.edu; Co-PI: Joseph Kieber, University of North Carolina, Chapel Hill, jkieber{at}biomass.bio.unc.edu; Co-PI: Charles Opperman, North Carolina State University, warthog{at}unity.ncsu.edu; Co-PI: Jeffrey Thorne, North Carolina State University, thorne{at}brooks.statgen.ncsu.edu NSF Plant Genome Project No. 0077503; http://www.nematode.net Plants inhabit a complex environment in the
rhizosphere. Understanding the many interactions they experience with
other organisms (including parasites) is crucial to truly appreciate
plant development and function. Nematodes, which are ubiquitous soil
animals, are the most successful and cosmopolitan plant parasites (Bird
and Koltai, 2000 Nematode Gene Discovery Based on our best understanding of the phylogenetic relationships
within the genus Meloidogyne and the biological differences between species (host range, susceptibility of host R loci,
etc.), we have initiated expressed sequence tag (EST) sequencing
of six species (McCarter et al., 2000 In addition to immediate public submission of all EST data to GenBank, we established a Web site with tools to provide easier access to parasitic nematode sequence data. This site (http://www.nematode.net) allows BLAST and text searches of subsets of available ESTs (by species, library, clade, etc.) and NemaGene clusters. We also provide ftp access to all EST sequences and a viewer to inspect raw sequence trace data. Gene Expression Profiling In a compatible interaction between plants and
Meloidogyne spp., developmental changes ensue in the roots
and local changes in cytokinin and auxin levels are implicated. Thus,
examining the patterns of gene expression in Arabidopsis plants in
response to cytokinins and auxins is likely to be informative about the nematode-plant interaction; conversely, examining transcriptional changes during nematode infection may shed light on hormone regulation during other plant processes. Our preliminary gene expression studies
using Arabidopsis Affymetrix GeneChips (Affymetrix, Santa Clara,
CA) suggest that a set of diverse genes is induced after exogenous application of hormones, which exhibit a range of
induction kinetics. Analysis of these data has been done with a new,
nonparametric method of array normalization that we developed. Our
experiments using Arabidopsis are complemented with array experiments
using spotted tomato (Lycopersicon esculentum) and
nematode ESTs and in situ PCR of nematode-infected tissue (Koltai and
Bird, 2000
PI: Hans J. Bohnert, University of Illinois, bohnerth{at}life.uiuc.edu; Co-PI: Ray A. Bressan, Purdue University, bressan{at}hort.purdue.edu; Co-PI: Robert Burnap, Oklahoma State University, burnap{at}okstate.edu; Co-PI: John C. Cushman, University of Nevada, Reno, jcushman{at}unr.edu; Co-PI: David W. Galbraith, University of Arizona, galbraith{at}arizona.edu; Co-PI: Paul M. Hasegawa, Purdue University, paul.m.hasegawa.1{at}purdue.edu; Co-PI: Rolf A. Prade, Oklahoma State University, prade{at}okstate.edu; Co-PI: Jian-Kang Zhu, University of Arizona, jkzhu{at}ag.arizona.edu NSF Plant Genome Project No. 9813360; http://www.stress-genomics.org; http://www.OSMID.org How plants respond to stress in the environment is crucial to their productivity and survival. Among the yield-reducing factors, abiotic stresses play a significant role. Based on the prevailing view of stress resistance and sensitivity as multigenic traits, we initiated a genome-wide, phylogenetic analysis involving transcriptional profiling analysis of wild-type and stress-related mutants with a focus on sensing and response pathways that constitute the functional basis of osmotic and ionic stress tolerance. Our goal is to determine the number and functional complexity of essential, important, or ancillary genes that prepare plants to cope with stress. The comparative evolutionary approach includes a survey of cellular and organismal stress tolerance response pathways in halophytes and glycophytes alike by carrying out transcript expression analysis in a variety of model species including yeast (Saccharomyces cerevisiae), Aspergillus nidulans, Dunaliella salina, Mesembryanthemum crystallinum, rice (Oryza sativa), and Arabidopsis. Benefiting from Arabidopsis genome sequence and resources, a mutant generation and characterization pipeline has by now provided more than 200,000 T-DNA tagged lines in various genetic backgrounds, many of which affect the stress response phenotype and facilitate detection of mutations in specific stress signaling pathways. The sequenced cyanobacterium Synechocystis sp. PCC6803, which is easily manipulated with gene replacement by homologous recombination, serves as a model for studying the effects of fundamental stress responses on photosynthesis, ion homeostasis, reactive oxygen species scavenging, and respiration. A full-genome Synechocystis sp. DNA microarray has been generated and PCR-based deletion mutagenesis is used to assess the functions of genes identified by microarray analysis. This approach, for example, is being used to determine the functions of five paralogous Na+/H+ antiporter genes. The results to date indicate that redundancy corresponds, in part, to a functional mosaic; that is, specific paralogs seem to be dedicated to specific aspects of different physical parameters (e.g. pH and salinity) or carbon uptake that impacts pH homeostasis. In addition, microarrays let us explore the regulatory cascades involved in the multiphasic cell growth patterns and physiological activity that is observed after salt shock of this simple autotroph. Saprophytes, such as yeast and A. nidulans, must
rapidly adapt metabolism and constantly monitor their changing
environment. In the multicellular fungal salt tolerance model, A. nidulans vegetative growth requires positive turgor pressure.
Deletion of hogA, the nonredundant MAP kinase of the
high-osmotic glycerol pathway, partially reduces the
ability of the fungus to grow on high salt but severely affects
cell wall biogenesis and disrupts cell and nuclear division synchrony.
Transcriptome (microarrays by "aspergillus-genomics.org") analyses
of wild type and As part of our gene discovery efforts, we used repetitive rounds of
differential subtraction screening to identify 84 salt-regulated genes
in Arabidopsis, the majority of which were not previously known to be
salt responsive. Additional mutants, whose characterization is ongoing,
will increase this number. Six of these were implicated in playing
pivotal roles in the SOS signal pathway to mediate ion
homeostasis and salt tolerance. In addition, we have identified a set
of transcripts that comprise a common salinity stress response pathway for cell-specific functions involved in restructuring of the
proteome (RNA and protein turnover and new synthesis). These
latter genes are involved in pathways that preserve cell integrity, protein chaperoning, ion, water and metabolic homeostasis, and radical scavenging and detoxification. Overall, approximately 5%
to 10% of all transcripts are altered in the model organisms for which
salt stress-related microarray datasets have been generated (yeast,
Synechocystis sp., M. crystallinum, rice,
and Arabidopsis). One set of changes is indicative of the "salt
stress emergency response," which has a species-specific threshold.
Such changes are transient unless stress overwhelms the
defense capacity of the species. Responses to salinity stress in the
multicellular models include these cell-specific response categories,
but additionally encompass functions in longer term
adaptation and long-distance integration We have generated and provide to the community Arabidopsis T-DNA-tagged mutants defective in stress tolerance and/or stress signaling. Our screen also identifies lines that have defects in metabolism, transcription and translation machinery, and in protein targeting within cells. Moreover, we are able to provide a large set of tagged Arabidopsis mutant lines useful for different screens. We have established, annotated, and provide ESTs for the core set of stress-related transcripts from the glycophytic Arabidopsis and rice, based on the finding that stressed plants include a significant population of transcripts that are not expressed in the unstressed state. We have provided stress-related ESTs from naturally tolerant
species We have assembled DNA microarrays for the core set of stress-related
transcripts and ESTs for expression analysis in the three higher plant
models Results from this program provide knowledge for future plant improvement; for example, by providing transcripts and microarray data that can be incorporated into genetic engineering of elite germplasm and marker-assisted breeding programs. Several collaborations have started that use salt- and drought-induced transcripts as markers and initial results have correlated such transcripts with quantitative trait locus (QTL) regions. Salinity and drought stresses constitute a permanent and increasing agronomic problem in many areas of the world. Long-term irrigation agriculture, for example, which is about three times more productive than rain-fed agriculture, inevitably will continue to suffer production losses due to increased soil salinity. Plant breeding has not yet produced varieties suitable for use in such environments. Our work provides genes and (Arabidopsis) mutants, putative functions by homology and analysis, evolutionary comparisons, and a description of gene expression changes during stress in comparison with the unstressed state over the lifetime of several model species. The work has resulted in a number of publications, including: Bressan
et al. (2001)
PI: Nick Carpita, Purdue University, carpita{at}btny.purdue.edu; Co-PI: Sara Patterson, University of Wisconsin, spatters{at}facstaff.wisc.edu; Co-PI: Tony Bleecker, University of Wisconsin, bleecker{at}facstaff.wisc.edu; Cooperator: Maureen McCann, John Innes Centre, UK, maureen.mccann{at}bbsrc.ac.uk NSF Plant Genome Project No. 0077719; http://www.btny.purdue.edu/cellwalls Plant cell walls are composed of independent but
interacting networks of carbohydrates, proteins, and aromatic
substances (McCann and Roberts, 1991 We have developed Fourier transform infrared (FTIR) microspectroscopy
as a powerful and selective screening technique to identify broad
classes of cell wall biogenesis-related genes (Chen et al., 1998 Having established the screening and selection protocols and the throughput necessary to accomplish the logistic goals, a comprehensive team has been assembled to identify the genes of potential mutants and to determine their function in a biochemical and cellular context. In addition to the FTIR screen, we have strengthened the overall program to include other spectroscopic approaches. Steve Thomas (Colorado State University, Fort Collins) uses near-infrared spectroscopy as an ultrahigh-throughput means to identify maize secondary wall mutants in plants in the field, and June Medford (Colorado State University) has developed optical coherence microscopy to nondestructively characterize morphological mutants whose defects may arise from changes in wall architecture. "Reverse genetics" will be used to uncover mutant phenotypes resulting from insertions of transposon and T-DNA in genes that are already known to be wall biogenesis-related in maize (Don McCarty and Karen Koch) and in Arabidopsis (Sara Patterson and Tony Bleecker, University of Wisconsin, Madison). Efficient systematic protocols employing biochemical, spectroscopic, and cytological approaches were developed in parallel to deduce specific defects in wall metabolism that result in the infrared phenotypes revealed by our screens. Wolf-Dieter Reiter (University of Connecticut, Storrs), Brad Reuhs (Purdue University, West Lafayette, IN), and Nick Carpita (Purdue University) will develop high-throughput biochemical and spectroscopic technologies to determine linkage structure, polysaccharide unit sequence structures, and wall architecture. Chris Staiger (Purdue University), Maureen McCann (John Innes Centre, Norwich, UK), and June Medford are developing cytological approaches to identify cellular bases of defects that affect the wall structure. Wilfred Vermerris (Purdue University) and Steve Thomas (Colorado State University) are coordinating studies of mutations that affect the polyphenolic structures of maize cell walls. As the heritability of the mutations is confirmed, the plant biology community will be informed of them through a Web site that will be created as a repository for all cell wall-related genomics, and a system will be devised to dispense them. A major practical goal is to generate plants with genetically defined variation in composition and architecture to permit assessment of modifications on wall properties and plant development. Because cell walls are an enormously important source of raw material, we anticipate that several of the genes we identify and characterize, as well as several of the plants with genetically defined alterations, will be of economic importance. Examples include the modification of pectin cross linking or cell-cell adhesion to increase shelf life of fruits and vegetables, the enhancement of dietary fiber contents of cereals, the improvement of yield and quality of fibers, and the relative allocation of carbon to wall biomass for biofuels. The expertise required to fulfill the goals of this project is interdisciplinary, and as part of the effort we will assemble postdoctoral teams to broadly overlap these disciplines and establish an interdisciplinary doctoral student training program in genetics and molecular biology of the plant cell wall.
PI: Gloria Coruzzi, New York University, gloria.coruzzi{at}nyu.edu; Co-PI: Nigel Crawford, University of California, San Diego, ncrawford{at}ucsd.edu; Co-PI: Dan Bush, University of Illinois, U.S. Department of Agriculture-Agricultural Research Service/Plant Biology, Urbana, IL, dbush{at}uiuc.edu; Co-PI: Bud Mishra, New York University, Courant Institute of Math and Computer Sciences, mishra{at}cs.nyu.edu; Collaborator: Dennis Shasha, New York University, Courant Institute of Math and Computer Sciences, shasha{at}cs.nyu.edu NSF Arabidopsis 2010 Project No. 0115586; http://www.nyu.edu/fas/biology/n2010 The goals of our Arabidopsis 2010 genome project entitled: "N2010: Nitrogen Networks in Plants" are to identify networks of genes regulated by nitrogen (N) levels, and to further identify the regulatory genes and cis-acting DNA elements involved in this regulation. These results should substantially advance our understanding of the regulation of N metabolism in the context of plant growth and development, as well as provide new insights into our understanding of complex regulatory metabolic gene networks in plants. Given the central role of N availability and metabolism in crop productivity, these results should also have broad agricultural impacts. The Arabidopsis genome project has uncovered a large set of genes
involved in the uptake, metabolism, and allocation of N (600+).
Expression studies on a small subset of genes encoding N-metabolic/transport proteins have shown that N levels regulate their
transcription. Proposed N signals include nitrate, ammonium, Glu, Gln,
and C to N balance (Coruzzi and Bush, 2001 In our ongoing studies of N regulation of gene expression, a complex
picture has been emerging. N regulation of gene expression appears to
be dependent on multiple variables including starvation, light, and
carbon status, to name a few (Coruzzi and Zhou, 2001 To our knowledge, a first set of genome chip experiments
related to nitrate signaling was conducted by the Crawford lab (Wang et
al., 2000 A computer cluster will store the large amounts of data generated in this project provided via a publicly accessible Web page (http://www.nyu.edu/FAS/biology/N2010/). This Web site will include microarray expression datasets, gene identification information, and all software developed in this project. The new software will include new clustering algorithms, cis-search algorithms, as well as the bioinformatic tool "PathExplore," which can used to query expression datasets to search for coregulated genes in pathways, as described above. These new resources will be linked to the major plant databases for the widest possible distribution of information.
PI: Jeffrey L. Dangl, University of North Carolina, Chapel Hill, dangl{at}emailunc.edu NSF Arabidopsis 2010 Project No. 0114795; http://www.bio.unc.edu/faculty/dangl/lab/superpage.html Plants deploy an innate immune response after infection,
in addition to passive protection afforded by waxy cuticular layers and
preformed antimicrobials. Plant-pathogen interactions, particularly those involving biotrophic parasites, are governed by specific interactions between pathogen avr (avirulence) gene loci and
an allele of the corresponding plant disease resistance (R)
locus. When these are present in both host and
pathogen, the result is disease resistance. If either is inactive or
absent, disease results. R products recognize, directly
or indirectly, avr-dependent signals and trigger the chain
of signal transduction events culminating in a halt of pathogen growth.
Specific R-mediated immunity is layered atop one or more
basal response pathways. Basal defenses stop pathogen spread after
disease onset, protecting the organism at the cost of some tissue
destruction. Genetic overlap between specific and basal resistance
responses suggests that one function of R-mediated signaling
is to more rapidly and effectively deploy shared effector functions
(Dangl and Jones, 2001 Genetic screens, almost exclusively in Arabidopsis, defined loci
required for R gene action (Feys and Parker, 2000 We identified, mapped, and cloned the Arabidopsis RPM1 gene,
which conditions resistance to Pseudomonas syringae strains
carrying the avrRpm1 gene (Grant et al., 1995 We aim to understand "the function of a network of genes," a stated
2010 Project goal (see Table I). The
network begins with RPM1 and the genes required for its
function. However, loci so defined will overlap with loci required for
other R gene functions. Some of the genes to be studied were
identified by forward genetics; thus, we know they are relevant to this
signaling network. Some were isolated in yeast two-hybrid screens and
subsequent reverse genetic analyses confirmed their role in
RPM1-mediated or disease resistance-related
processes. Finally, some are molecular relatives of genes found via the
first two approaches, and we want to test the notion that they function
in similar disease resistance pathways. We cover two small multigene
families. We intend to make all the mutants and reagents generated
available. The first publications funded by the 2010 Project, and the
NSF grant that preceded it, are recently published or currently in
press (Tornero and Dangl, 2001
PI: Deborah Delmer, University of California, Davis, dpdelmer{at}ucdavis.edu; Co-PI: Candace Haigler, Texas Tech University, candace.haigler{at}ttu.edu; Co-PI: Allan Zipf, Alabama A&M University, aamzip01{at}aamu.edu; Co-PI: Andrew Spicer, Co-PI, Texas A&M, Houston, aspicer{at}ibt.tamu.edu; Unfunded Collaborator: Kanwarpal Dhugga, Pioneer HiBred, kanwarpal.dhugga{at}pioneer.com NSF Plant Genome Project No. 0110173; http://www-plb.ucdavis.edu/labs/Delmer/ Cellulose (1, 4-glucan) represents a major sink for carbon
in plants where it exists as a key cell wall polymer. The pattern and
extent of cellulose microfibril deposition contribute to patterns of
morphogenesis, to the unique characteristics of specialized cell types,
and to the strength and flexibility of plant stems. Cellulose is used
extensively as fuel, timber, fiber, forage, and chemical cellulose.
Manipulation of the patterns and extent of cellulose deposition, the
dimensions and crystallinity of the microfibrils, or the ratio of
cellulose to other sinks such as lignin or starch, can be expected to
improve the quality of many economically important plants. This project
seeks to continue work initiated in a previous NSF Plant Genome Grant
to study the functional genomics of the CesA gene family
proposed to encode the catalytic subunits of the multicomponent
cellulose synthase enzyme complex. The new project also extends these
objectives to include discovery and characterization of other genes
that are critical for the process. Research focuses on plants of
economic importance where modifications of this process could yield
most benefit Ongoing work includes: (a) studies of expression patterns of all 10 of the Arabidopsis CesA genes and their related ancestors, the CslD genes. We are defining developmental patterns of expression for all of these genes and also identifying potential pairs or triplets of CesA that are required as functional units within a single cell type, examining affects of carbon status and light on gene expression, and testing the hypothesis that the related CslD genes are the cellulose synthases of tip-growing cells; (b) with respect to maize, these studies will identify expression patterns for four key ZmCesA genes and relate these to any phenotypes generated in the four different selected Mu insertion lines that are mutated in these respective genes; (c) further testing of the hypothesis that at least two distinct CesA proteins and the Korrigan cellulase protein are all required for cellulose synthase complex formation and function; this is being done by co-expressing and analyzing complex formation and the ability to make cellulose when combinations of these genes are expressed in yeast and tobacco (Nicotiana tabacum) Bright-Yellow 2 cells; (d) completion of characterization of the first identified CesA gene from an alga; (e) determination of the comparative topology of a plant CesA protein in the plasma membrane with its related ancestor in animals, hyaluronan synthase, to relate structure of the proteins to their functions in the synthesis of the glucan chains of cellulose; (f) a description of the evolution, diversity, and map locations of CesA genes in cotton, studies that should shed light on the evolution of tetraploid cotton and also identify polymorphisms in these genes to contribute to the genome maps of diploid and tetraploid cottons; and (g) microarray experiments to study global expression patterns of large numbers of genes in Arabidopsis, maize, and cotton under conditions in which we know CesA gene expression is affected, with the goal of identifying other genes that are important for cellulose synthesis in plants. The project will also make available useful tools for the scientific
community such as seeds of transgenic Arabidopsis expressing the
reporter gene
PI: Xinnian Dong, Duke University, xdong{at}acpub.duke.edu; Co-PI: Frederick M. Ausubel, Massachusetts General Hospital, ausubel{at}molbio.mgh.harvard.edu; Co-PI: Shauna Somerville, Carnegie Institute, Stanford University, shauna{at}andrew.stanford.edu NSF Arabidopsis 2010 Project No. 0114783; http://genetics.mgh.harvard.edu/ausubelweb/nsf2010/nsf2010.htm Plants respond to pathogen attack through a variety of
signaling pathways consisting of a large number of regulatory as well as effector genes. During the past several years, many defense-related genes have been identified through genetic analysis conducted in
Arabidopsis. Importantly, Arabidopsis exhibits all of the major kinds
of defense responses present in other plants (Glazebrook, 1999 |
TOP
INTRODUCTION
Genomic Dissection of a...
10% of the gene complement. We found
that 76% of identified transcripts have significant database matches
in other organisms, including five genes with matches only to
rhizosphere bacteria. To investigate the possibility of horizontal
transfer from bacteria to nematodes, we are querying our growing EST
data sets using a Markov chain Monte Carlo technique developed for
studying cospeciation, and we are developing phylogenetic methods that
incorporate information regarding the absence of genes in
genomes due to gene loss as well as the informational content of the sequence itself. The idea is that the topology and
branch lengths of an evolutionary tree affect the number of times that
gene loss must be postulated as well as the probabilities of these gene
loss events.
hogA mutants showed that HogA
only partially regulates genes that respond to high salt. The majority
of salt stress-responsive genes in A. nidulans are
controlled by yet unknown regulatory networks.
through serial engagement of
ion transporters, alterations in cell wall structure, metabolic
readjustment, and signaling through hormones. The balance between
signaling that leads to the known emergency responses and signaling
that leads to the cessation or maintenance of cell cycle, cell
division, and elongation seems to be the defining element that
distinguishes glycophytic from halophytic (and xerophytic) behavior. A
large number of functionally unknown or novel stress-induced
transcripts that remain uncharacterized may contribute to the stress
tolerance phenotype. For example, comparisons of EST sequences from the
halophytic M. crystallinum with Arabidopsis genomic
sequences reveals that between 2.5% and 6% of these genes are not
present in the Arabidopsis genome. Many of these genes have predicted
functional roles in stress tolerance. More detailed analysis of
halophytic species with the experimental advantages of Arabidopsis,
such as Thellungiella halophila, might be helpful in further
identifying both structural and regulatory gene products that
comprise the "osmome." Our work not only sets the stage for
more detailed qualitative analyses that compare different types of
stresses, but also facilitates future quantitative studies of the
responses to different magnitudes of a particular abiotic stress.
Mutants in yeast, A. nidulans,
Synechocystis sp., and Arabidopsis with defects in stress
signaling are being used to determine the linkage between the
constituents of several signaling networks and the relative impact of
different signaling pathways. The results are beginning to provide sets
of sequences for inclusion into diagnostic microarray slides. Examples
of our work can be seen at 
-glucuronidase (GUS) under the control of
each of the individual promoters for CesA and/or
CslD genes, several lines impaired in cellulose synthesis,
and other constructs useful for studying the mechanism of synthesis of
cellulose in plants.