Functional Genomics of Plant Stress Tolerance
A. Goals and Objectives.

How crop plants respond to stresses in their physical environment is crucial to productivity. Of all parameters, abiotic environmental stresses contribute most significantly to the reduction in potential yield (USDA Statistical Yearbooks; Flowers & Yeo, 1995). We have chosen for our studies the sensing of osmotic and ion imbalance and the functional basis of responses to osmotic and ionic stresses. We will compare higher plants and models with different capacities for the expression of biochemical and physiological defense mechanisms that counter stress.

Our results will provide the number and nature of all genes that are implicated in stress tolerance and their response to alterations in osmotic and ionic conditions. To achieve this, we will identify how many and which genes provide stress responses, which genes make essential contributions to stress tolerance, and how many and which mechanisms serve in stress protection. As the major result of our integrative approach, we envision characterizing the genes that constitute the "osmome" or "xerome" - that portion of the genome with specific functions under osmotic challenge or water-limiting conditions. Such knowledge can eventually be provided as a diagnostic set of microarrayed sequences. Our objectives are:

One important point of this project is that the data are not only useful to those working on salinity and osmotic stress. Stresses targeted in our project show considerable overlap with responses of plants to cold stress, drought, and insect, fungal, bacterial and viral stresses. The tools and resources which we will assemble will be available and can be used for a comparative profile for those other stresses in addition.

The results from our program will provide important knowledge for future plant breeding. Salinity and drought stresses constitute a permanent and increasing agronomical problem in the arid midwest/western growing areas. On irrigated land, with a productivity more than three times that of rain-fed areas, long-term irrigation inevitably increases salinity. Plant breeding has not produced varieties suitable for use in stressful environments (Flowers & Yeo, 1995). Our work will provide the genes, their putative nature by homology and function, comparative analysis of different stress-adaptable organisms, and a description of gene expression over the life-time of plants in their unstressed and stressed states.

B. Complexity and Multigenicity of Stress Responses.

1. Variations on common physiological Themes. The responses of plants to osmotic and ionic stress have been reviewed by PIs on this proposal and others (McCue & Hanson, 1990; Bartels & Nelson, 1994; Niu et al., 1995; Bohnert et al., 1995; Serrano, 1996; Bray, 1997; Zhu et al., 1997; Shinozaki & Yamaguchi-Shinozaki, 1997; Nelson et al., 1998; Bohnert et al., 1998; Noctor & Foyer, 1998), often with an emphasis on transgenic stress tolerance engineering (Bohnert & Jensen, 1996; Jain & Selvaraj, 1997; Holmberg & Bülow, 1998).

Flowers & Yeo (1995) reviewed the complexity of salinity stress responses in an article assigning the failure of current breeding strategies to a lack of mechanistic understanding. Indeed, such breeding amounts to merging two multigenic, quantitative traits: tolerance and productivity. Our limited knowledge about the molecular genetic basis of this complexity has been derived largely from analysis of single to few genes and the expression of corresponding transcripts and proteins in a wide variety of different species. although the functional mechanisms served by different gene products may be the same. For example, accumulation of mannitol in celery, of pinitol in the ice plant, glycerol in yeast/ Dunaliella and ectoine in halobacteria all seem to assure "osmotic adjustment". Even though the importance of osmotic adjustment is questionable, the "compatible solute" concept appears to hold up. The accumulating solutes seem to act in specific functions such as protein stabilization (ectoine, glycinebetaine), and uncharged solutes (mannitol, pinitol) seem to act as scavengers of reactive oxygen species (ROS). The latter has recently been documented by a different strategy - over-expression of the Fe-binding protein ferritin resulted in increased tolerance, attributable to the removal of free iron from the cells (D. Dudits, personal communication). Protection by the removal of iron, which participates in a "Fenton-reaction" through which hydroxyl radicals are produced, has already been shown (Shen et al., 1997). Many reports suggest ROS scavenging as most essential for protection (Allen, 1995; Roxas et al., 1997; Noctor & Foyer, 1998).

There are other biochemical mechanisms contributing to tolerance, such as reactions that lower the NADH/NAD+-ratio thereby decreasing reducing power. The balance, also, between nitrogen assimilation and carbon provision requires adjustments. Uptake, transport and compartmentation of sodium ions - while maintaining uptake of the essential potassium - requires several genes and pathways. Cellular integrity is maintained by changes in membrane fluidity and protein composition of membranes. Accumulation of stress-specific proteins, such as the late embryogenesis abundant (LEA) proteins to high concentrations within specific tissues is correlated with or causally related to improved tolerance under drought, cold, or salinity stress conditions (Moons et al., 1995; Imai et al., 1996; Close, 1996; Xu et al., 1996), although the mechanistic basis of their effects remains elusive. Apparently, different species have solved the problems posed by salinity and osmotic stress by strengthening unique biochemical pathways, but there seems to be a limited number of ways in which this can be achieved.

2. Evolutionary Conservation of Stress Responses. Recently, substantial data from genetic transformation experiments have established that functionally analogous tolerance determinants exist in both unicellular organisms and plants. Largely conserved are stress sensing, transduction of the signal and the regulation and functioning of the downstream gene induction and repression machinery (Frommer & Ninnemann, 1995; Serrano, 1995; Shinozaki & Yamaguchi-Shinozaki, 1997: Zhu et al., 1997; Ishitani et al., 1997; Nelson et al., 1998). This justifies a large-scale genetics approach aimed at finding all genes involved in stress responses in selected model organisms: Synechocystis sp. PCC6803, yeast and Aspergillus nidulans. The three models have been chosen to demonstrate and exploit the significant evolutionary conservation of homologous adaptive strategies. Differences exist, even among higher plants, in how signal transduction pathways and gene expression patterns are connected via a finite set of cis- and trans-acting elements and transcriptional activators.. For this reason, we include halophytic models, Dunaliella and Mesembryanthemum, to compare and contrast the overlapping and unique sets of stress-related transcripts from Arabidopsis and rice. From a technological standpoint, determining the common and unique portions of the genome which dictate all stress responses will be an important prerequisite for understanding and modifying preexisting adaptations and targeting and exploiting new adaptive processes in the post-genomic era of biology.

C. Experimental Plan.

The nine PIs from the University of Arizona, Purdue University and Oklahoma State University forming the "Plant Stress Consortium" contribute strategies and technologies to a project that could not be carried out by any individual laboratory. Our expertise covers the entire field of study in model organisms, genome analysis - including reverse genetics, EST and microarray analyses - and functional studies in plant cell biology and physiology. The Consortium project integrates our individual approaches to understanding the genomic basis of plant stress responses in a comparative, comprehensive way. We will use models for various overlapping aspects of stress tolerance including cellular traits (Saccharomyces), photosynthetic traits in a simple prokaryote (Synechocystis) and more complex adaptive strategies in a multicellular fungus (Aspergillus). All of these organisms are halotolerant and have evolved related, yet unique aspects of adaptation to ionic and osmotic stresses. For all of these organisms facile insertional mutagenesis and complementation systems have been worked out which will allow for the rapid functional identification of endogenous adaptive genes as well as the isolation of gene homologs from higher plant species.

These organisms also offer the advantage of rapid, targeted gene disruption through homologous recombination. The use of saturation mutagenesis in Arabidopsis by T-DNA insertions provides a powerful tool for identifying mutations in a particular gene of interest (Krysan et al., 1996). Targeted gene disruption is now also feasible in the moss Physcomitrella patens (Schaefer and Zyrd, 1997; Puchta, 1998) and in Arabidopsis using the vacuum infiltration method (Miao & Lam, 1995; Kempin et al., 1997). We plan to exploit each of these approaches to isolate and functionally characterize knockout mutants. In addition to functional assays, we plan to focus strongly on direct gene discovery strategies (EST collections and databases) with corresponding developments of specialized bioinformatics resources. This strategy will bring us much closer to comprehensive characterization of the osmome (xerome) - the set of transcripts that is stress specific - in higher plant models where genomic sequencing remains incomplete. Another advantage of this approach is the ability of using EST collections to identify stress-induced genes of known or unknown function solely on the basis of their expression profiles using microarray hybridization technology. This will have the added advantage of providing the tools for assessing gene family (functional) redundancy and complexity which will facilitate targeted gene disruption strategies in Arabidopsis and other higher plant models. Possibly the most interesting outcome of EST microarrays will be the ability to monitor and compare stress-induced gene expression profiles among different related stresses and in specific mutants. This will allow us to capture individual "transcriptome profiles" or stress fingerprints and discern interrelationships or connections relating stresses or mutants not immediately obvious through the study of one or a few genes. Also available are our unique methods for rapidly identifying genes co-transcribed with the stress genes of interest. Applying whole genome analysis technologies will revolutionize stress biology in short order. Because the field of genomics is moving so rapidly, largely driven by technological developments, we will inevitably adjust our procedures to find those best suited for collecting pertinent data in the most efficient way possible. This consideration also underscores the necessity for the Consortium to provide training for pre- and post-graduate students for working within the context of integrative, large-scale and collaborative projects which likely will dominate future biological research. Therefore, we propose to develop an extensive education and training network and formal workshops and conferences.

1. Setting up Model Systems for Analysis.

a. Models for cellular aspects of stress tolerance.

(i) Saccharomyces cerevisiae.

The genome of brewer’s yeast, 15 Mb, is completely sequenced (Goffeau et al., 1996). Among the more than 5,800 potential proteins, about 70 are known to be differentially expressed under salinity stress, but we expect more than 200 genes that have a functional relationship to salt stress tolerance (see: Nelson et al., 1998). According to Dr. Goffeau, who is involved in the yeast international project, a collection of knock-out strains will not be available for the next three years, although individual strains may be forthcoming. As yet there is no coordinated effort towards the isolation of stress-related functions either for yeast itself or in a plant context. We will generate strains with deletions in genes necessary for our work, based on work that is ongoing in the labs of Drs. Bressan, Hasegawa & Pardo (Purdue) and, to a smaller degree, in the Bohnert lab (UA).

In this sub-project we will identify plant osmotic stress tolerance determinants as (i) suppressors of stress-sensitive yeast mutants, (ii) functional plant homologs of yeast osmotic stress tolerance based on sequence homology and gain of function analysis, and (iii) interacting proteins with either plant or yeast osmotic stress tolerance. The objectives are:

Isolation of osmotic stress responsive mutants by insertional mutagenesis. We will isolate yeast mutants with increased or decreased sensitivity to stress; and mutants in stress signal transduction pathways using osmotically regulated promoter-reporter fusion traps (Purdue).

Identification of osmotically regulated transcripts by genome array analysis. We will establish gain or loss of function by gene deletion or overexpression (Purdue, UA, OSU).

Identification of plant suppressors of stress responsive mutants. This will be accomplished by direct genetic suppression of the yeast mutant phenotype by complementation and determination of their sequence or functional homology with yeast osmotic stress tolerance determinants. (Purdue, UA, OSU).

Protein/protein interaction determinants. We will isolate plant proteins that interact with the previously isolated gene products. (Purdue, UA).


Part 1. Stress Responsive Yeast Mutants by Insertional Mutagenesis. An existing Tn3 insertion library will be used for transposon tagging mutagenesis in yeast (Burns et al., 1994). A yeast genomic library constructed in an E. coli plasmid is mutagenized with a Tn3 derivative containing lacZ, yeast LEU2 and E. coli b -lactamase. NotI digestion releases the vector. Linear DNA is transformed into yeast and recombinations are selected that replace homologous genomic sequences flanking the transposon. The ORF in one of the transposon-derived termini extends into the adjacent lacZ coding region. Therefore, some of the Tn3 insertions in yeast DNA will generate in-frame fusions with lacZ which result in identification of osmotically induced genes. The large size of this library, ~20 genome equivalents, and the ease with which the tagged mutant allele can be rescued make this system useful for identification of osmo-sensitive mutants (Burns et al., 1994). After establishing linkage between LEU2 and the mutation, the gene fusion will be isolated by plasmid rescue (Burns et al., 1994) and the interrupted gene identified by sequence analysis using the yeast genome database.

> Mutants with altered sensitivity to osmotic/salt stress. To isolate osmotically sensitive mutants, yeast cells transformed with the Tn3 library will be screened for growth on medium with NaCl (in varying amount) representing a moderate salt shock (Mendoza et al., 1994). Systematic comparisons will be made on medium supplemented with NaCl, KCl, LiCl or polyethylene glycol/ mannitol to distinguish between salt and osmotic sensitivity for the isolation of mutants with altered stress signaling capacity or effector determinants. Besides cnb, we have isolated several independent osmotic stress sensitive strains and are presently identifying wild type alleles (Pardo et al., unpublished).

> Mutants in stress signal transduction pathways using osmotically regulated promoter-reporter screening. Osmotic stress signal transduction pathways will be dissected by utilizing screens involving promoter-reporter fusions. Stress-responsive promoters will be used for gene fusions of tolerance determinants such as GPD1 (glycerol-3-P DH) or ENA1/PMR2A (Na+-ATPase) regulated by sensing or salt stress signaling pathways. GPD1 is regulated by the HOG pathway and is responsible for osmotic adjustment by glycerol (Varela & Mager, 1996). The ENA1/PMR2A ATPase is primarily responsible for Na+ efflux across the plasma membrane. The gene is regulated by NaCl through calcineurin-dependent processes (Mendoza et al., 1994, 1996; Matheos et al., 1997; Stathopoulos & Cyert, 1997). We have used ENA1 (-1384 to +40)-lacZ translation fusion to monitor calcineurin-dependent activation (Mendoza et al., 1994; 1996). Since numerous components of the HOG pathway have been identified (Valera & Mager, 1996; Posas & Saito, 1997), a focus will be further characterization of the salt stress signal pathway. A promoter-reporter construct is integrated into the genome, and the strain will be transformed with the Tn3 library to isolate mutants that are responsive to the stress, or are constitutively expressing the promoter.

Part 2. Identification of osmotically regulated Transcripts by Genome Arrays. Microarray analysis will be used to identify genes differentially expressed in response to stress (details below). We are currently using an array from Research Genetics [] which contains 6,000 yeast ORF (DeRisi et al., 1997; Wodicka et al., 1997). Gene expression differences are monitored in wild type in a time-dependent adaptation to salt-shock (minutes to one generation time) by using 33P-labelled RT-products. Signals are measured by phosphorimaging. Similar analyses will be carried out with mutant strains, but we expect to switch to non-radioactive detection of inexpensive yeast microarray s soon. Mutant analysis will facilitate further categorization of genes that are differentially expressed in response to stress. For example, genes expressed after salt treatment in hog or pbs2 will not include those regulated by the HOG pathway. To date, about 70 yeast genes are known to be differentially expressed in response to salt stress (Nelson et al., 1998).

The functional involvement in stress tolerance of differentially expressed genes will be determined through gain or loss of function experiments. Genes will be constructed into expression plasmids and transformed into wild type or stress sensitive (e.g. cna1D cna2D or cnbD ) mutant strains (Mendoza et al., 1994; 1996). Stress tolerance sufficiency of the wild type or suppression of the mutant phenotype will be indicative of stress tolerance determinants. Negative regulators of stress tolerance may mediate a stress sensitive phenotype of the wild type. Gene inactivation mutants will be generated by replacement with alleles disrupted with a selectable marker. These will be evaluated for stress sensitivity (loss of function).

Part 3. Identify Plant Suppressors of Stress-responsive Mutants: Arabidopsis, Dunaliella and Mesembryanthemum. Plant osmotic stress tolerance determinants will be identified based on direct functional selection as suppressors of osmotic stress sensitive yeast mutants. Alternatively, yeast stress tolerance will be used to identify plant genes by sequence homology. Functionality of the plant gene will be assessed by complementation of disrupted mutants or suppression of a stress sensitive signal pathway mutant. We will use cDNA libraries from stress-adapted plants which are enriched for functional determinants of tolerance.

> Direct selection of plant suppressors of osmotic stress sensitive mutants. To date, calcineurin null mutants have been used to isolate putative plant transcription factors STZ, STO and TOB175 (Lippuner et al., 1996; this laboratory, unpublished). The functions of STZ and TOB175 are dependent on ENA1and current efforts are directed at determining if the plant transcription factors can activate ENA1. Maize Opaque-2 can transactivate the yeast GCN4 promoter (Mauri et al., 1993). Arabidopsis SAL1 (encoding: 3’(2’),5’-bisphosphate nucleotidase/ inositol polyphosphate 1-phosphatase) was isolated by complementation of a ena1-4 mutant (Quintero et al., 1996). Osmotically adapted Arabidopsis thaliana, Mesembryanthemum crystallinum and Dunaliella salina cDNA libraries will be constructed (directional cloning) into yeast/ E. coli l-expression vectors containing the cre/lox recombination system for excision, e.g., lYES (Invitrogen), lPG15, lAD5 (Brunelli & Pall, 1993; Mumberg et al., 1995). Plasmid DNA will be used to transform osmo-sensitive strains. Presently, we are screening a 428mM NaCl-adapted Chlamydomonas reinhardtii in pYES for cDNAs that can suppress the Na+/Li+ sensitive phenotype of W303 (ura3 cna1::TRP1 cna2::HIS3 ena2-4::LEU2).

> Plant homologs of yeast tolerance determinants. Sequence comparisons will be made of the yeast osmotic stress tolerance determinants with the available plant sequence databases to identify plant homologs. Confirmation of functionality of the plant determinants will be based on complementing the phenotype of the corresponding yeast mutant or suppression of a stress-sensitive signal pathway mutant.

Part 4. Isolation of Plant Proteins interacting with Yeast or Plant Stress Tolerance Determinants. Future development of the workplan includes isolation of plant genes based on biochemical interactions of the encoded proteins with yeast/ plant stress tolerance determinants using the yeast two-hybrid system (Chien et al., 1991; Fields & Sternglanz, 1994). The yeast/plant homologs will be used as bait to screen cDNA libraries derived from Arabidopsis, Mesembryanthemum and Dunaliella. Functional significance of interacting proteins will be confirmed by genetic suppression of the appropriate yeast mutant.

This strategy is feasible, because many plant genes function in yeast, for example STO, STZ, SAL1, HKT, AtKUP1, HAK1, CAX1, phosphate or sulfate transporters (Lippuner et al., 1996; Rubio et al., 1995; Hirschi et al., 1996; Liang et al., 1997; Santa-Maria et al., 1997; Fu & Luan, 1998; Kim et al., 1998). Similarly, HKT from rice and Mesembryanthemum: (Golldack et al., 1997; 1998) and the Mesembryanthemum ITR1 homolog of the yeast proton/inositol symporter (Nelson & Bohnert, 1998) function in yeast. Also, the yeast CNA/B (calcineurin) genes affect plant salinity tolerance (Cunningham & Fink, 1996; K Hirschi, personal communication; Pardo et al., 1998). Recently, it has been determined that vacuolar compartmentation of Na+ is a component of ion homeostasis in yeast (Nass et al., 1997) as it is in plants (Niu et al., 1995).

(ii) Synechocystis sp. PCC6803. Synechocystis represents an ideal model for stress responses in a simple autotroph - photosynthesis, ROS scavenging, respiration and chloroplast functions. The organism can be transformed with very high efficiency relying on homologous recombination. This cyanobacterial genome, 3.6 Mb, is completely sequenced , and the small, non-redundant genome allows precise DNA engineering by PCR-mediated, not plasmid-based, manipulations .

Among the more salient findings of the sequencing project is the detection of over 80 ORFs identified as members of two-component signal transducers and homologs of genes involved in stress responses in higher plants. Synechocystis sp. PCC6803 also represents an excellent model for osmoregulation since it tolerates up to 1.2M NaCl. Tolerance is effected through the accumulation the osmolyte glucosylglycerol, accumulating by de novo synthesis. Following salt-shock, maximal levels of glucosylglycerol are achieved within 3 hours. Mutant analysis has provided insight regarding the role of two genes expressed in response to salt-shock. StpA encodes glucosylglycerol-P phosphatase involved in the salt-induced accumulation of glucosylglycerol . GgtA encodes a subunit of an ABC-type transporter which permits the maintenance of high levels of solutes in the cytoplasm by recovering leaked glucosylglycerol . Paralleling osmolyte accumulation, changes in the expression of photosynthesis- and respiration-related enzymes have been reported (Jeanjean et al., 1993; Joset et al., 1996).

Using Synechocystis sp. PCC6803, options for generating mutants include random insertion of a resistance marker and screening for salt-sensitivity and precise knockout mutagenesis. We chose the latter method, different from that used for yeast, because of the efficiency of insertional mutagenesis and the low complexity of the Synechocystis genome. The objectives are three-fold:

Generating knock-out strains for known and suggested functions. We will target 300 to 400 functions (~100/year) that are known or suspected elements of the stress response (OSU).

Analyzing Synechocystis microarrays. We will utilize existing arrays and generate arrays for stress-related ORFs which will be used for monitoring gene expression under stress in wild type, knock-out mutants and complemented mutants (OSU, UA).

Utilizing plant cDNA libraries for complementation. Knock-out strains will be complemented by cloned transcripts expressed from the higher plants (OSU, UA, Purdue).


Part 1. Stress-responsive Mutants by targeted Knockouts. Identified in the Synechocystis genome, mostly by homology, are 80 two-component regulatory genes (e.g., ethylene response sensor-like), kinases and phosphatases, GTP-binding proteins, transcriptional repressors, and approximately 100 other genes possibly involved in stress-related functions. These are components of photosystems (e.g., psbH), enzymes (inositol monophosphatase-like), ROS scavenging (SOD, gluthathione peroxidase) or they seem to be homologs of ion/ water/ and metabolite transporters (e.g., mipE [a Mesembryanthemum homolog], ferritin, mscL).

Targeted genes will be identified, primers synthesized and the genes will be disrupted by a spectinomycin resistance cassette using a two-step PCR method. After disruption is verified, we will use, depending on which function is eliminated, a set of informative assays to characterize the phenotype. Test reagents include NaCl, H2O2, H2O2 and iron, methylviologen and glucose. Synechocystis provides an additional advantage because defects that affect photosynthesis can be monitored by fluorescence spectroscopy that highlight individual colonies. This is routinely used in the Burnap laboratory (OSU). The strains will be available for complementation, either with cDNA libraries from the higher plant/ algal models, or for functional analysis through the expression of full-length cDNAs from the plants.

Part 2. Microarray Analysis. Building on an existing collaboration, the Burnap laboratory will receive microarrays on filters for the approximately 6,000 indexed clones from the laboratory of Dr. Tabata (Kazuza, Japan). Wild type and knockout mutants will be subjected to stress regimes (e.g., NaCl, high light, oxidative stress) and the resulting changes in gene expression will be monitored. We will establish a clone collection for stress-related genes at OSU for printing arrays. RNA will be isolated, depleted of rRNA and tRNA species and labelled as documented by the Burnap lab for unstable Fe-stress transcripts (Al-Khaldi et al., 1998). Signals will be detected by phosphorimaging.

This strategy will identify new stress-regulated genes (gene discovery), and will permit the identification of stress-regulated genes. The results can be compared with those from the higher plant EST sub-project. In addition, the information will help feed the Synechocystis sp. PCC6803 knock-out group with new targets, and ultimately new phenotypes.

Part 3. Genetic Complementation. Initial targets for complementation are the full-length cDNAs which are already available from the Cushman/Bohnert collections from Mesembryanthemum, the Bressan/ Hasegawa Chlamydomonas/ Dunaliella transcripts, and the Zhu collection of Arabidopsis transcripts.

Foreign genes and libraries from Dunaliella, Mesembryanthemum and Arabidopsis are brought into the disruption mutant using neutral site shuttle vector. The neutral recombination site is at a unique, separate location on the chromosome which is distinct from the locus of the spectinomycin cassette-disrupted gene, does not confer a phenotype when disrupted in the wild-type, and serves a generalized recombination platform to facilitate recombination of the test gene(s). The recombination of test genes will be forced to coincide with the acquisition of a second antibiotic resistance (kanamycin). Therefore, the first selection will be for conversion of the Spr to the Spr/Kmr phenotype. Transformants (or populations of transformants in case of an introduced library) will then be screened for re-acquisition of the character lost by the targeted disruption. For example, complementation of a disrupted salt-tolerance gene would be selected on high salt plates on which the mutant cannot grow or shows impaired photosynthesis by fluorescence criteria. Because different organisms have evolved unique solutions to common biochemical problems, thus we expect that the complementing gene will encode for a non-homologous protein performing an analogous cellular function.

(iii) Dunaliella salina. Dunaliella species are unicellular algae that include some of the most halotolerant plants with some species capable of growth in salt of up to 5.5 M (Avron, 1986). The alga adjusts to osmotic perturbations by volume regulation through a signal transduction pathway that involves phospholipids and small G proteins (Bental et al., 1990; Memon et al., 1993). Volume regulation is followed by osmotic adjustment resulting mainly from glycerol synthesis and accumulation (Sadka et al., 1989). Also, Dunaliella has great capacity for the maintenance of ion homeostasis in saline environments (Pick, 1992) through the plasma membrane H+-ATPase and Na+/H+ antiporter, and other P-type ATPases (Katz et al., 1991; Weiss & Pick, 1996). Additionally, 60 and 150 kD plasma membrane proteins have been implicated in the control of ion homeostasis for the growth of D. salina in high salt concentrations (Sadka et al., 1991; Fisher et al., 1994). Currently, the Purdue group is constructing libraries in the yeast l vectors (Brunelli & Pall, 1993; Mumberg et al., 1995) using the GAL1 promoter with RNA from 2 M NaCl adapted D. salina cells. The pYES2 library will be transformed into the salt sensitive yeast strain W303 cna1-2D ena2-4D and screened for galactose-dependent complementation of salt sensitivity. Alternatively, subtracted cDNA libraries will be generated for complementation of salt sensitive mutants.

cDNA libraries from salt-stressed Dunaliella will be used for EST analysis (OSU), for complementation analysis (OSU, Purdue) and for microarrays (UA).

(iv) Aspergillus nidulans, a simple multicellular eukaryote includes ~8,000 genes (Kupfer et al., 1997) in a 31 Mb genome (Brody et al., 1991), a 28% increase in gene complexity over the unicellular yeast (Dujon, 1996). A. nidulans is renowned for its metabolic diversity (Brown et al., 1996), able to metabolize numerous complex food sources S. cerevisiae is unable to utilize. Thus, this gain in genetic complexity is likely to reflect the biological differences between these organisms, especially with respect to genes required for colonizing habitats in which osmotic potentials vary greatly. A. nidulans is xero- and osmo-tolerant due to the accumulation of glycerol/erithritol as osmoregulatory solutes (Griffin, 1981; Beever & Laracy, 1986). In the A. nidulans EST collection (release 1/1998), 9% of the cDNAs with known biological function encode domains related to various stress response pathways, including those from plants (Prade & Ayoubi, unpublished). For example, EST entries for the heat shock and ubiquitination pathways (8.6% of the EST-set), for ROS scavenging proteins (catalase, peroxidase, Cu,Zn-SOD), for uptake, transport and compartmentalization of ions (ATPases/ pumps), and for compatible solute synthesis (glycerol, trehalose, proline, betaine, mannitol and sorbitol) were found. Homologs for regulatory proteins are also represented (HAL2, HOG1 and calcineurin) (da-Silva et al., 1994; Kim et al., 1996; Lippuner et al., 1996; Quintero et al., 1996; Parrou et al., 1997). Finally, 75% of the non-redundant stress-EST-subset were found to be more homologous to genes identified in eukaryotic organisms other than S. cerevisiae (Prade & Ayoubi, unpublished).

The Prade lab (OSU) is interested in a comparative genomics approach towards stress biology. Recognizing the large number of genes involved in stress adaptation in eukaryotic organisms, the lab is interested in generating a homology-based network between a cyanobacterium, a unicellular and a multicellular tractable model eukaryote, and the higher plant models that will render the complete gene complement for the salinity stress response. The specific objectives are:

Identification of plant stress genes as A. nidulans homologs. We will generate and examine inactivation mutants in A. nidulans for higher plant genes involved in salinity stress (OSU, UA).

Examination of A. nidulans salinity stress responsive genes. We will generate A. nidulans random insertional and overexpression mutants and select strains with altered sensitivity to osmolytes for in-depth analysis. Novel genes will provide the model to identify plant homologs (OSU, Purdue, UA).

Expression profile of salinity stressed A. nidulans. We will use DNA arrays to determine stress induced gene expression profiles in wild-type and mutant A. nidulans (OSU, Purdue, UA).


Part 1. Identification of Plant Stress Genes as A. nidulans Homologs.

Plant - fungus homologies. Fungal homologs will be identified by one of three ways: a) direct amino acid sequence comparisons between A. nidulans and higher plant EST databases; b) DNA/DNA hybridization (Prade et al., 1997) using probes derived from Dunaliella and higher plant models and, c) functional complementation (bi-directional gene transfer) of A. nidulans salinity stress mutants with plant genes or libraries (Borgia et al., 1994; Lippuner et al., 1996).

Inactivation of plant stress homologs in A. nidulans. Null strains will be constructed via gene replacements (Prade & Timberlake, 1993; Prade & Timberlake, 1994) using pRP100B (argB) as the homologous recombination driver and RMS11 (yA2, pabaA1, argB::trpC801, trpC801)as the recipient strain. This simple gene replacement system is robust and efficient for large-scale experiments.

The phenotype of salinity stress response inactivation mutants. A. nidulans mutants will be subjected to a standardized set of osmotolerance tests, revealing loss or gain of osmotolerance or indicating altered regulatory properties. In addition, we will determine the effect of various mutations on radial growth and sporulation. Alterations of radial growth or changes in conidiation are easily detected and are criteria for functional abnormalities in multicellular processes associated with nuclear movement (Beckwith et al., 1995), septation (Kaminskyj & Hamer, 1998), wide-domain gene regulation and compartmentation and metabolite traffic. The results are likely to highlight sets of plant genes with functional equivalence in fungi.

Part 2. Examination of A. nidulans Salinity Stress Responsive Genes. The experiments for this section are designed to reveal new A. nidulans stress responsive genes that are not yet known as determinants in the plant stress response.

Isolation of salinity stress responsive genes using RIM. We will utilize the recently developed RIM (Recombination Induced Mutagenesis) technique to create random collections of insertional mutants (Prade & Ayoubi, unpublished). RIM has been designed for large-scale functional genetics experiments and is based on optimized frequencies for disruptive and homologous random recombination events. RIM is also useful for creating random, in frame reporter fusions. RIM-mutants are automatically tagged and incorporate a series of features useful in physical and genetic mapping, generation of STSs, molecular cloning and gene characterization. Salinity stress responsive loss-of-function mutants will be recognized as colonies with altered radial growth rates or other phenotypic markers in response to "high" (tolerant) or "low" (sensitive) Na+ ion concentrations.

Isolation of stress responsive genes by overexpression. We will utilize a nutritionally inducible (threonine) overexpression DNA library, constructed by the Adams group (Marhoul and Adams, 1995), to identify genes that cause changes in salinity response when overexpressed.

Cloning of stress responsive genes. Osmo-/halo-tolerant or -sensitive mutants, obtained through both mutagenesis methods, will be subjected to tagged-rescue, STS sequencing, gene cloning and characterization following standard techniques. Recovery of one-step STSs is a standard feature of our RIM libraries which allows rapid physical and genetic mapping.

The main focus of this section is to outline the biological functions necessary and sufficient to mediate the salinity stress response in A. nidulans. It is anticipated that we will find genes not yet known in higher plants, yeast or other model systems. We will use these genes (or equivalent null mutants) to discover homologs (or functional complements) in the other models and in higher plants. In addition, we expect to find novel stress-related genes, not obtainable from unicellular models such as, for example, the ones involved in cell-cell interactions.

Part 3. Expression Profile of Salinity Stressed A. nidulans. We will initially construct a partial but informative A. nidulans DNA-based array that contains three well defined clusters: (a) a partial, but comprehensive, collection of ESTs representing major cellular and metabolic functions; (b) the complete set of all stress related STS-tags from A. nidulans ; and (c) all A. nidulans genes with homologies to plant stress genes. These arrays will be utilized to produce expression profiles of wild type A. nidulans and mutant strains under a variety of conditions. The results will be integrated into an expression network with other model- and plant-profiles, priming the backbone on which comprehensive gene complements will be determined.

In summary, we hypothesize that these simple prokaryotic and eukaryotic models exemplify, in large part, the basic adaptive responses to salinity and osmotic stress that have evolved early in evolution. Therefore, comparative EST analyses among these organisms (see below) will provide a strong foundation for identifying a common set of stress-relevant genes in higher plants. Undoubtedly, there will be unique adaptations and signaling pathways found in other organisms not represented in this set of models. For future comparative studies in other organisms, the proposed work will provide an important, definitive reference resource.

b. Higher Plant Models.

We have chosen Arabidopsis thaliana and Oryza sativa as models for salt- and osmotic stress-sensitive species. We propose to exploit the Arabidopsis genetic system and to extend the analysis of rice and Arabidopsis EST accessions (Newman et al., 1994; Sasaki et al., 1994; Cook et al., 1996; Rounsley et al., 1996) to include salinity and osmotically challenged plants. This work will complement functional analysis of genes using T-DNA mutagenesis strategies carried out simultaneously in Arabidopsis. Most ESTs have been obtained from plants or cells that had not been subjected to any stress treatment, although several Arabidopsis groups (e.g., Michigan State Univ. and Gyeongsang National Univ.) are now including EST-sequencing with clones from libraries of specifically treated plants. We know that cDNA libraries from unstressed plants are missing a set of ionic and osmotic stress-related transcripts (see ice plant example, below), which we estimate comprises approximately 20% of all transcripts. For Arabidopsis, this would amount to approximately 4,000 stress-related transcripts, assuming approximately 21,000 genes in total (Bevan et al., 1998). We include Mesembryanthemum in our research plan because it is naturally salinity- and osmotic-stress-tolerant and is known to contain genes that are absent or are not expressed in a stress context in crop plants. Examples of such genes are documented (Vernon & Bohnert, 1992; see: Nelson et al., 1998; Bohnert et al., 1998). Mesembryanthemum libraries from UA and OSU will include tissue-, cell- and developmental state-specific cDNA libraries. In addition, Dunaliella libraries from Purdue will be used as halophytic sources of novel genes for (1) complementation experiments in unicellular models and Arabidopsis, (2) EST analysis for "digital" expression profiles, and (3) microarray and functional analyses of stress-related transcripts.

Arabidopsis thaliana. Arabidopsis as the most popular model for higher plants has been used successfully for studies of plant development, phytohormones, light signaling and disease responses. The establishment of Arabidopsis as a model organism to study salt and drought stress tolerance or signaling has been recent, but is proving to be extremely powerful (Wu et al., 1996; Zhu et al., 1997; Ishitani et al., 1997). Already (Zhu lab, UA) more than 40 salt-overly-sensitive (sos) Arabidopsis mutants (Wu et al., 1996; Liu & Zhu, 1997) have been identified. One of these loci, SOS3, has now been positionally cloned and encodes a key signaling component with homology to the B subunit of calcineurin (Liu & Zhu, 1998). cDNA libraries will be generated from ecotype Columbia for the EST and microarray s (UA).

Oryza sativa, rice, is our model for a crop plant for several reasons, in particular because of a small genome size and because of availability of a large number of EST sequences, genomic sequences, high transformation efficiency, and information about cell biology, biochemistry and physiology (Matsuo et al., 1993; 1995). In addition, among the more than 2,000 breeding lines several salt-tolerant indica-lines are included to which we have access (Drs. G. Kush, D. Senadhira, J. Bennett, IRRI, Manila). The Bohnert lab (UA) is working on salt-tolerant (Pokkali) and -sensitive (IR29/ IR64) rice and has established cDNA libraries for salt-stressed (150mM NaCl, 24 h) rice Pokkali. Specific projects in the lab include the analysis of Pokkali/IR29 potassium transporters, analysis of inositol biosynthesis under stress and transfer of multigene vectors into rice (Golldack et al., 1997, 1998; Zhu et al., 1998). cDNA libraries for complementation will be provided to Purdue and OSU .

Mesembryanthemum crystallinum. The halophytic ice plant with a genome size twice that of Arabidopsis (DeRocher et al., 1990; Meyer et al., 1990; Adams et al., 1998) provides an excellent model for inducible responses to salinity and drought which allow the plant to colonize extreme habitats. The plant has been a rich source of novel genes for salinity-adaptive responses such as the synthesis of osmoprotectants (Vernon and Bohnert, 1992; Ishitani et al., 1996), facilitated water uptake (Yamada et al., 1995) or ion sequestration (Tsiantis et al., 1996; Löw et al., 1996; Su et al., 1997; 1998). The long-term adaptation to water stress by a switch from C3 to Crassulacean acid metabolism (CAM) is a paradigm for metabolic adaptations that improve water use efficiency (Cushman and Bohnert, 1997). A large-scale EST sequencing project is in progress for leaf tissues of adult plants (OSU) and epidermal bladder cells (UA), a solute and ion storage organ, making this the best studied higher plant halophyte available. Large EMS- and gamma irradiation-induced mutant populations have now been established (Adams et al., 1998; Cushman et al., 1998). An efficient somatic embryogenesis-based regeneration system that is now available for the ice plant (Cushman et al., unpublished) is expected to streamline transformation studies now in progress.

2. Collecting T-DNA tagged Arabidopsis Mutants.

a. Tagged Lines. We will generate Arabidopsis tagged lines defective in stress tolerance and/or signaling. Gene knock-out in Arabidopsis via T-DNA insertions provides direct and definitive functional assessment of the gene product. One important aspect of our genome-wide analysis of stress tolerance is to identify and characterize all possible Arabidopsis mutations that would lead to alterations of plant stress tolerance and/or stress gene regulation. The Zhu lab (UA) has developed a high throughput genetic screen for Arabidopsis mutants defective in stress gene regulation (Ishitani et al., 1997). The screen utilizes transgenic Arabidopsis plants expressing the firefly luciferase marker (LUC) under control of a salt-, drought-, cold- and ABA-responsive promoter (RD29A) and low-light video imaging technology. Stress-induced gene expression in hundreds of plants can be quantitatively detected by luminescence imaging in a matter of several minutes. A Petri dish containing up to 1,000 seedlings can be subjected to multiple stress and hormonal treatments and imaged repeatedly. From screening 300,000 EMS-mutagenized M2 Arabidopsis seedlings containing RD29A-LUC, the Zhu lab has recovered several hundred mutants defective in various components of the stress response pathways (Ishitani et al., 1997). Preliminary genetic analysis of 100 of the mutants exhibiting strong luminescence phenotypes indicated that the screen is still far from being saturated because most of the loci are defined by single alleles.

We propose to expand the genetic screen for stress tolerance/signaling mutants and to screen for T-DNA alleles. This calls for a large scale T-DNA mutagenesis of the plants expressing the RD29A-LUC transgene. Since the RD29A-LUC plants already contain a NPTII gene for kanamycin resistance, we are using a construct with the BAR gene for T-DNA insertional mutagenesis. With the very efficient Agrobacterium-infiltration method for Arabidopsis transformation, we expect to generate 150,000 independent transformed lines within the next two years. Our initial transformation efficiency has ranged from 2% to 5%. Assuming an average 1.5 inserts/per plant line, the 150,000 lines would contain more than 200,000 inserts, which would nearly saturate the Arabidopsis genome, i.e., one insertion per approximately 1,000 bp. We are fully aware of the large number of T-DNA lines that have been generated in various labs around the world. Although most of these lines have not yet been made available to the public, our intention is not to duplicate their effort. Rather, we will generate lines appropriate for our goal, because the mutagenesis has to be in the RD29A-LUC transgene background in order for us to screen for stress-related mutants. We pledge to distribute these T-DNA lines and the mutants freely to others as they become available. Also, lines will be made available to others for mapping purposes.

The screen of the T-DNA populations will begin as soon as we have T2 seeds from 5,000 or more independently transformed lines. Therefore, most of our screening and mutant characterization work will be carried out in parallel with mutagenesis and selection of more transformed lines. Mutants will be characterized initially by examining endogenous RD29A and other stress gene expression via Northern analysis and, later microarray analysis with the stress-responsive set of transcripts. Mutants exhibiting altered tolerance to salt and/or drought stresses will be chosen for T-DNA co-segregation analysis to determine whether the genes are tagged. While it is not realistic to expect complete detailed characterization of every mutant we isolate, we do expect to complete within the tenure of this project the mutagenesis, mutant screening, and tagging analysis of at least the most interesting mutants.

b. Expansions of the Concept. In addition to the RD29A-LUC system, we have constructed Arabidopsis plants expressing luciferase under control of other stress-activated promoters, the osmotin promoter (in collaboration with Bressan lab, Purdue) and a stress-regulated water channel promoter (Bohnert lab, UA). These other stress responsive promoters contain cis- elements different from those found in the RD29A promoter. The use of these promoters will enable us to recover mutants that define other stress response pathways. EMS mutagenesis, mutant screening and characterization will be according to the RD29A-LUC system (Ishitani et al., 1997).

These new EMS mutations, together with the more than 100 strong-EMS mutants already identified through RD29A-LUC will be genetically mapped. Mapping relies on inter-ecotype DNA polymorphism. We have extensive experience using CAPS and SSLP markers (Wu et al., 1996; Liu & Zhu, 1997). Our genetic analysis of the mutants from the RD29A-LUC system has revealed at least 30 complementation groups (Zhu et al., unpublished). Therefore, with new mutants from the osmotin-LUC (UA, Purdue) and water channel-LUC systems (UA), we will likely be looking at over a hundred genetic loci to map with. We will initially rely on SSLP and CAPS markers but will use SNPs (single nucleotide polymorphisms) markers that can be of extremely high density and automated, once the methodology is better defined (F Ausubel, personal communication). With a large number of genetic loci placed on specific regions of the chromosomes, we will be able to best take advantage of the genome sequence of Arabidopsis, because candidate genes can be identified and confirmed through functional complementation of our mutants.

3. Generating Libraries of stress-adaptation Transcripts.

Estimating 20% or 4,000 transcripts to be stress-relevant in Arabidopsis (and a slightly higher, but similar number in Mesembryanthemum and rice), we propose to sequence at least 10,000 ESTs from these species to ensure good representation of stress-regulated transcripts in the EST databases. At OSU (and UA), the M. crystallinum EST database and clone collection for the ice plant now includes 1,600 accessions representing randomly selected cDNAs from libraries constructed in l -Uni-Zap-XR (Stratagene) using mRNA isolated from shoots of six-week old, hydroponically grown plants either well-watered or osmotically stressed (30 h; 0.4 M NaCl). Approximately 15% of the EST represent genes characterized previously in the ice plant. On average, more than 42% of the clones show no significant homology to the non-redundant databases and presumably represent unidentified or novel genes. Importantly, 31% of the clones isolated from well-watered plants are unknown, whereas 54% of the clones from salinity-stressed plants represent unidentified transcripts, with greater than 50% of these transcripts having no significant homology to EST accessions derived from other higher plants (e.g., rice, Arabidopsis). The high proportion of unidentified and novel genes in stressed plants underscores how little we know about gene function and plant gene expression patterns under environmental stress conditions. Characterization of expression patterns of unknown genes using microarrays will facilitate targeted functional and comparative analyses of these genes. Dramatic changes in expression patterns are apparent when comparing unstressed and salinity stressed plants. For example, transcripts for photosynthetic functions (RUBISCO, photosystem and light harvesting complexes) decrease 5-7 -fold in abundance, whereas transcripts encoding ion transport and compartmentation, osmotic adaptation, and CAM (H+-translocating vacuolar ATPase, glycolytic/gluconeogenic, malate synthesis and decarboxylation, inositol biosynthesis) increase 5-6-fold in abundance. ESTs representing proteolytic enzymes (ubiquitin hydrolase, proteosome components, cysteine proteases) increase 12-fold in abundance (Cushman et al., unpublished). We propose to expand the scope of our current EST sequencing efforts to include the following additional libraries:

(i) From Arabidopsis (ecotype Columbia) one library each from stressed (100mM NaCl) seedlings and mature early-bolting plants (generated at UA).

(ii) From rice (Pokkali) one library for plants approximately three weeks of age (three to four leaf stage) stressed for 12 h by the addition of 125 mM NaCl (generated at UA).

(iii) From Dunaliella one library from an upshift experiment (e.g., 300mM to 2M NaCl) within less than one hour after the upshift (generated at Purdue).

(iv) From Mesembryanthemum we will utilize four types of libraries. One library will be for seedlings (250 mM NaCl) and one for plants between the juvenile and reproductive phase (5 weeks old, 500 mM NaCl) (Adams et al., 1998, for a description of growth phases). The third library will be for epidermal bladder cells, a salt-storage organ (Adams et al., 1998), after approximately 24 hours of stress when these cells begin to develop (UA). A fourth library of salt-stressed roots (6 weeks old, 400 mM NaCl) will be included (provided by OSU) to define adaptive changes occurring at the primary site of ionic contact. At present all cDNA libraries, after mass excision, are available as double-stranded plasmids. For the complementation projects at OSU and Purdue, vectors compatible with the models will be used. At UA, we will make new libraries for this purpose, while using existing libraries for the EST sequencing, generating full-length cDNAs by 5’ RACE (see below) and microarrays at present. To reduce the probability of redundant sequencing of abundant stress-related transcripts, libraries will be prescreened by colony hybridization. We will also limit the number of non-stress-related transcripts by using subtracted libraries. Current estimates are based on manual preparation of templates and sequencing reactions. However, we expect projected cost to be lower in the future as we implement operation of robotic workstations requested for UA and OSU. With these capabilities, we will be able to accelerate the rate of finding new transcripts. Although limited EST sequencing will be done at UA, most ESTs information will be generated at OSU which will also serve as the project-hub for bioinformatics, including sequence data processing, polishing, annotation, submission, and analysis.

4. Constructing Diagnostic Micro-arrays for Stress Parameters.

a. Microarray Analysis of Wildtype Plants under Stress Conditions and of Mutants with Defects in Different Stress Signaling Components. Microarray technologies have greatly facilitated the analysis of perturbations of gene expression (DeRisi et al. 1996, 1997; Heller et al., 1997; Schena, 1996; Schena et al., 1995, 1996; Shalon et al., 1996). Microarrays are available in two different forms: oligonucleotides photolithographically synthesized in situ on "chips" (Affymetrix, InCyte), or DNAs mechanically deposited onto microscope slides (Synteni). Information is provided using mRNA from the tissue under control and test conditions. cDNAs are then synthesized in the presence of two fluorescent NTPs, typically Cy3 and Cy5-dUTP, mixed and hybridized to the arrays, and the intensities of the two fluorescent signals recorded. We will not discuss photolithographic production of oligonucleotide chips, because of the presently high cost, although this technology has advantages which could be exploited in our project. Deposition of DNA for microarrays is done using robotic spotters. They typically employ a quill-type tool for sampling/depositing the DNA; other approaches are under development. Element densities are determined by drop size and positional accuracy of the robot. A typical array spacing of 500 µm results in 2x103 elements on a 22 x 22 mm microscope slide; forthcoming instruments aim at a spacing of 200 µm, resulting in 12 x 103 elements/slide. Following hybridization, arrays are read using fluorescent scanners, which are either custom fabricated or, recently, have become commercially available. Software to quantitate, compare, and store hybridization signals is available in both the public and private sectors.

Technical Details. The Galbraith lab (UA) has adapted a Biomek 2000® Laboratory Automation Workstation for first-generation robotic cDNA spotting (Macas et al. 1998). The resultant arrays have cDNA elements of ~125 m m diameter, and a spacing of 500 m m. We wrote software in Tcl to control the robot, and have made this software freely available. Arraying involves dipping the pins into the DNA samples, followed by consecutive spotting of the samples onto 28 slides. Pins are cleaned by two washing steps and one drying step before reloading. Using four pins, the Biomek will spot the samples of a 96-well microtiter plate onto 28 slides in 1 h (~one-half the speed described for the NIH robot (http://www.; spotting 104 elements on 28 slides using 16 pins would take about 24 h.

As part of this proposal, the Galbraith lab wishes to continue development of arraying technologies by constructing a second-generation arraying instrument with an increased array density (center-to-center spacing of 200 µm), permitting arrays of 12,100 DNAs within a 22 x 22 mm area. It should also have an increased print speed to permit arraying of elements within a shorter time. Unpublished and web-based data of other groups suggest these increases are possible. Short of purchasing an arrayer, should instruments become available, there are two ways that a second-generation instrument can be developed for use in this project. They involve custom fabrication of the instrument either in house, or by a third party, using available or modified design specifications. A final decision on which direction to take will depend on the developments over the next few months in this extremely rapidly-changing field.

Mechanical deposition typically employs cDNA samples that have been PCR amplified from library clones. We employ 5’ amino-modified primers, which produce amplified DNA that can be attached to silylated slides using aldehyde chemistry. For array scanning, the Galbraith lab (UA) has obtained a General Scanning ScanArray® 3000 DNA array hybridization scanner. With a pixel size of 10x10 µm the instrument provides spatially accurate and sensitive analyses of the hybridization patterns of the DNA arrays for analyzing first and second generation arrays.

For microarray-based analysis of stress-specific transcription, our experimental approach involves first placing wild type DNA from EST/clones of the higher plant models, derived mostly from "stressed libraries", in the array analysis. Arrays from an increasing number of elements will be probed using the mixed fluorescent cDNAs prepared from these plants. In the second set of experiments, comparisons will be made between different mutants and wild type, under control and stressed conditions. Array capacity is sufficient to gradually accumulate samples from the unicellular models and their mutants. Arraying is established at the UA where we have begun to analyze ESTs (see the dBEST database) from Mesembryanthemum (provided by OSU and UA). All participant laboratories will be contributing to array analyses, mailing 96-well plates to UA; a second arrayer/reader will be set up at OSU.

b. Direct Identification of Stress-affected Transcripts. At UA, the Galbraith lab has recently developed techniques that directly characterize changes in nuclear transcripts that occur in response to alterations to a stimulus applied in vivo (Macas et al., 1998b). Termed Nuclear Expressed Sequence Tag (NEST) analysis, this technique starts with control and test plants that are subjected to the stimulus of interest. Next, tissue homogenates are prepared and nuclei rapidly isolated through flow sorting, after staining with a DNA-specific fluorochrome. RNA is extracted from the sorted nuclei, and is captured on magnetic beads carrying oligo-dT. cDNA is synthesized, and is restricted using an enzyme that cuts frequently (NlaI). Linkers are then ligated onto the 5’ ends of the bead-linked cDNA tags, and PCR is employed for amplification. Tags are then displayed on large, diagnostic polyacrylamide gels. Differences in transcription produce altered profiles in the different lanes, which are visualized using silver staining. Individual bands are excised, reamplified and directly sequenced.

NEST analysis is conceptually similar to differential display, but has important advantages. First, since a single linker is ligated onto the cDNA tags, PCR amplification is done under stringent conditions, which eliminates low stringency artifacts (c.f. differential display). Second, NEST analysis is highly sensitive, employing few nuclei and correspondingly requiring small amounts of RNA. Finally, this approach samples the nuclear RNA transcriptional spectrum, rather than the steady-state cytoplasmic mRNA pool. It therefore is not biased against transcripts having short cytoplasmic half-lives and, therefore, represents faithfully transcriptional activities of cells.

c. Analysis of Gene Expression Coordinated with Promoters of Interest. For the various stress regulated transcripts that are identified, a critical next step is to define those genes that are coordinately up- and down-regulated. This is done as follows: first, the promoter of interest is placed upstream from a chimeric coding region comprising an effective nuclear localization signal, GFP, and b -glucuronidase (GUS). We have previously described the characteristics of this recombinant construction (Grebenok et al., 1997a), and the properties of transgenic plants constitutively expressing the chimeric protein (Grebenok et al., 1997b). Briefly, transgenic plants exhibit green-fluorescent nuclei within all the cells that are expressing NLS-GFP-GUS. These nuclei can be isolated from homogenates and flow-sorted based on green fluorescence. For transgenic plants expressing NLS-GFP-GUS under the control of tissue specific promoters, only these particular tissues have green nuclei; therefore, flow cytometry can be used to purify them away from the remaining, non-fluorescent nuclei. The second part of the strategy involves NEST analysis as described above (UA, with sub-sets of cDNAs provided by Purdue, OSU).

d. Subcellular localization of the protein products of stress-induced genes. A large proportion of the genes identified as being stress-induced will bear no homology to known genes. For this reason alone, systematic approaches are needed to provide information about the potential roles of the encoded protein products. We propose construction of translational gene fusions between these proteins and GFP. The chimeric coding sequence is then placed under the control of a suitable promoter, and the subcellular distributions of the chimeric protein product examined in transfected protoplasts or transgenic plants using confocal microscopy, techniques for which considerable experience and equipment is available at UA (Sheen et al., 1995; Yamada et al., 1995; Galbraith et al., 1995; Grebenok et al., 1997a; 1997b). For GFP fusions, variants are available that improve protein expression and enhance fluorescence emission through altered fluorescence yield, photostability, and emission/excitation spectra (Haseloff et al., 1997; reviewed by Galbraith et al., 1998). For construction of translational fusions, Clontech provides several vectors (pEGFP-N & pEGFP-C series) whose coding regions are identical to ours (Grebenok et al., 1997a, 1997b).

An additional advantage of a transgenic approach will be that subcellular distributions of chimeric proteins can be examined as a function of the imposition of stress. Stress-induced alterations in subcellular location, e.g. membrane cycling of water channels, will provide information about functions. We propose this as a systematic approach in the sense that all genes that are identified as being stress-relevant will be candidates for analysis, but considering the high number of expected transcripts, only a subset of these will be selected for study as translational GFP fusions. The subset will be chosen based on whether the predicted coding sequence contains oligopeptide motifs or structural domains of interest, such as putative transmembrane domains, nuclear targeting signals, or transcription-relevant motifs (zinc-fingers, activation domains), or motifs that imply protein-protein interactions (zinc-fingers, leucine zippers). The approach is highly sensitive and measurements can be made with a high degree of spatial resolution in vivo. We anticipate that some gene products will be recalcitrant to over-expression in plants as fusion proteins. Thus, for all proteins displaying specific subcellular patterns of targeting, these patterns will be confirmed through independent methods (biochemical fractionation; western blotting; immunolocalization). GFP spectral variants also provide the possibility of co-localization experiments. Considering the finite number of different intracellular locations to which proteins can be targeted, it will be possible to categorize the targeting patterns of the different proteins.

5. Defining the Master Set of Stress-relevant Genes.

Generating stress-responsive EST collections represents the first step in our complete characterization of genes involved in stress adaptation. We propose to generate and sequence a collection of full-length cDNA clones representing the master set of stress-responsive genes in Arabidopsis, Rice, Mesembryanthemum and Dunaliella which will serve as an adjunct to chromosome sequencing projects and molecular marker development in, for example, corn and Arabidopsis. This master set of stress genes will be extremely useful in guiding the choice of future, directed mutagenesis strategies and in identifying open reading frames in genomic DNA sequences of genes identified by random insertional mutagenesis. Full-length cDNAs will also be needed for systematic, functional analysis of the potential roles of unknown protein products using gene fusions between these proteins and GFP (see previous section). cDNAs will provide essential reagents for systematic microarray analysis for comparing expression patterns in response to different stresses and in specific mutants analogous to the commercially available yeast ORF collections. Complete cDNA sequences will also be important for correct categorization and selection of clones based on predicted structural motifs and domains, as well as comparative informatics studies across phyla. Selection criteria for inclusion in this set of genes will not be limited to stress-inducible expression patterns discerned from microarray analyses. Since not all genes encoding stress-relevant functions will be inducible, we will include additional genes defined by genetic and reverse genetic approaches. In yeast, for example, constitutively expressed components of the HOG signaling pathway are known (Posas & Saito, 1997). This fact justifies the emphasis we place on transcripts present in libraries from long-term stressed plants, as well as the emphasis on three different unicellular models for complementation and the mutant screens using T-DNA tagged Arabidopsis lines.

Technical Aspects. Standard cDNA libraries contain truncated versions of the original mRNA. Rather than repeating all analyses with the new cDNA libraries, we will capitalize on what is already available in the current EST collections in Arabidopsis and Mesembryanthemum and conduct 5' RACE for the master set of ESTs. Similarly, all new stress-responsive ESTs will be converted into full-length cDNAs for the functional analyses (UA, Purdue, OSU). To favor production of full-length cDNAs, double stranded cDNA will be made from different tissue types (e.g., leaves and roots) using "lock-docking" oligo(dT) primers to eliminate 3' heterogeneity during first strand synthesis reactions and Marathon cDNA adaptors which block 3' end extension of ligated adaptor primers during double-stranded cDNA synthesis (Chenchik et al., 1996). 5' RACE reactions will be primed with gene-specific primers designed from EST information. 5' RACE amplifications will be performed using KlenTaq polymerase mix (Clontech, Inc.) to ensure recovery of large cDNAs. RACE products will be cloned and sequenced. Using the 5' sequence information, full-length cDNAs will be recovered by "end-to-end" PCR. Rapid sequencing of longer cDNA inserts will be accomplished at the OSU sequencing facility which currently houses an AB 373 automated DNA sequencing systems with the stretch liner and 64 well upgrades (Perkin-Elmer). Recent technological improvements in automated sequencing systems including more sensitive fluorescent dyes (e.g., BigDye™, Perkin-Elmer), more accurate base-calling algorithms, and higher resolution long (48 cm) gels will allow relatively good coverage of cDNA less than 2 kb by just two overlapping reads on opposite DNA strands (OSU will be the lead institution, also UA). A second sequencer (AB 377) is requested which will be housed at OSU for EST and full-length cDNA clone sequencing. Contig assembly of ESTs and full-length cDNA sequences will be conducted using bioinformatics resources at OSU. All sequence information generated by this project will be curated at the proposed Stress Site Database (SsDb) (see Appendix 2).

6. Testing of identified Halotolerance Genes.

a. Transgenic Plant Analysis. Genes which are identified in various screens (complementation, microarray, etc.) with the greatest promise for affecting salinity tolerance in plants will be tested for sufficiency in model plant transformations using Arabidopsis and tobacco. Either overexpression or permanent activation (if a mechanism such as the presence of an inhibitor domain is known) will be achieved using constitutive promoter expression systems. In addition, we will use our T-DNA mutation library of Arabidopsis to identify by reverse genetics knockouts of identified halotolerance genes and subsequently test salinity tolerance. Labs at the UA and Purdue have considerable experience transforming and testing in these two species for salinity tolerance. Recently a new $7.8 million environmental-controlled growth facility has been added to the Horticulture Dept at Purdue which will be available. Purdue will be our hub for transgenics.

b. Reverse Genetics. Based on the preliminary statistics from the ice plant ESTs, and extension to Arabidopsis would mean that there may be 4,000 salt/osmotic stress-regulated genes. The functionality of some of these genes will become clear if they correspond to those identified through yeast-, Synechocystis- or Aspergillus-complementation, or those defined by Arabidopsis stress tolerance/signaling mutants. For others, we will establish their role in stress responses by two approaches: (1) identifying knock-out mutants with T-DNA or transposon insertions in the genes of interest. This can be done by PCR screening of DNA from T-DNA populations (Krysan et al., 1996), or more efficiently by searching databases for sequences which flank T-DNA or transposon insertions, which are being built in various labs (Klimyuk et al., 1997); and (2) targeted gene disruption via homologous recombination (Kempin et al., 1997). These powerful reverse genetics approaches are presently in their infancy, but are expected to mature during the next several years, and will lead to the disruption and functional evaluation of every one of the stress-relevant genes we find (UA leading, OSU, Purdue).

D. Undergraduate/Graduate Training/ Outreach Programs.

OSU (enrollment 26,000) provides valuable research opportunities to undergraduate students in a variety of science and engineering disciplines (currently >50% are from underrepresented groups). OSU consortium faculty will recruit students for research training through several different outreach programs such as the Oklahoma Partners for Biological Sciences (OPBS) program (HHMI), the Native Americans in Biological Sciences (NABS) program, and the Oklahoma Alliance for Minority Participation in Science, Mathematics, Engineering and Technology (OKAMP-SMET) both NSF-funded programs. The OPBS program enhances training and research opportunities for undergraduates as freshman or upperclassmen transferring to OSU from regional two-year colleges. The NABS and OKAMP-SMET programs target minority and native American student training, recruitment and retention in scientific disciplines. NABS and OKAMP-SMET, comprising 27 colleges, offer summer research internship programs to over 2,000 minority students making the transition from high school-to-college, 2-year to 4-year college, or undergraduate-to-graduate study (~150 students).

Programs at UA (enrollment 38,000) are similar in breath to those at OSU for underrepresented groups, including NABS. The UA has a nationally recognized Undergraduate Biology Research Program (funding: NSF, HHMI, NIH, and Am. Soc. Pharmaceut. & Exptl. Therapeutics) providing research experience to students working in biology. The program has served more than 750 students (84% graduating with bachelors degrees) and now supports 140 students annually. More than 200 students have published their work. Our alumni are in medical school or practicing medicine (33%), in biology graduate programs (21%), working in university/ governmental/ private sector research (16%), and 15% are either teachers, working in regulatory agencies, or went to law school. Also funded by NSF and HHMI is a program in which (presently 37) high school teachers work towards a M.S. degree in Biochem. & Mol. Cell. Biol.

The Purdue University (enrollment 36,000) campus includes nearly 6,000 students in graduate programs of 57 departments. A large number of ethnic minorities have received Ph.D. degrees in science at Purdue. Competitive fellowships (30 to 35/year) are offered to Ph.D. candidates from underrepresented groups. Purdue participates in the federal Minority Access to Research Centers (MARC) program which provides funds for student participation in the sciences. Undergraduate participants in this project may receive academic credit through a Special Assignments or a Honors Thesis Research course. Graduate students will have access to the Horticulture, Plant Biology or Genetics interdisciplinary graduate programs.

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    Appendix 1.

    Relationship between the proposed Activity and current Research.

    Hans Bohnert (Dept. of Biochemistry, The University of Arizona, Tucson, AZ)

    The main activity in the laboratory is closely related to this proposal - working on (1) stress responses in the ice plant (Mesembryanthemum crystallinum) for ten years [1-5, 8]; (2) gene transfer into tobacco/ rice to study mechanisms of ionic and osmotic stress tolerance [many of the concepts have come from work with the ice plant] [2, 6-7, 11]; and (3) modifications of enzymes of carbohydrate metabolism [3, 9-10].

    The halophytic ice plant switches during water/ion stress from C3 to Crassulacean acid metabolism, CAM. While Dr. Cushman, OSU, is focusing on the molecular induction of CAM, which began in my lab, continued collaboration includes signaling and signal transduction under stress and CAM switching [3].

    I consider all we do as one project, different portions of which are funded by different agencies. NSF (IBN or, maybe, MCB) funds tracing inositol biosynthesis and its relationship to stress tolerance in the ice plant (1-2, 4-5). Funding by NSF-INT is for analyzing ion transport proteins expressed in insect cells and oocytes (with Dr. Pantoja, Mexico). Funding by DOE is for analysis of transgenic tobacco expressing proteins that may provide enhanced stress tolerance (2, 6-7, 11). USDA-NRI has just begun funding work on water channel protein turnover during stress. Additionally, people with their own funding by DFG (Germany), NEDO/RITE (Japan), the Rockefeller Fndtn, and JSPS (Japan) have worked or are presently in the lab.

    Our work is inspired by physiological and biochemical approaches to study mechanisms of osmotic and ionic stress tolerance. The relationship to the proposed project is that we bring the accumulated knowledge of the laboratory on stress responses in halophyte models to the center. I expect that the characterization of the core set of stress-related transcripts will provide a better understanding of the mechanisms of plant stress tolerance and shed light on the reasons for the lack of tolerance in crop plants. We have already begun working on multi-gene vectors, up to nine genes at present, but I imagine that we will make mini-chromosomes in the near future for the transfer of an even larger number of genes into rice.

    Robert Burnap (Dept. Microbiology & Molecular Genetics, Oklahoma State University, Stillwater, OK)

    Research in this laboratory aims at an understanding the molecular basis of oxygenic photosynthesis. The PI investigates two major aspects of the oxygenic photosynthetic mechanism: 1.) assembly and function of the PSII H2O oxidation complex and 2.) adaptation of the photosynthetic apparatus to environmental stress. The scientific approach involves isolation and directed modification of photosynthetic genes combined with biochemical and biophysical characterization. The goal is to deduce mechanism by analyzing the phenotypic consequences of defined genetic changes. Targeted deletion strains are constructed in the experimental model, the transformable, genomically determined cyanobacterium Synechocystis sp. PCC6803, by replacing target genes with antibiotic resistance cassettes. Site-directed and random mutations are introduced into the gene in vitro and the modified gene returned to the cognate deletion strain, such that the transformed strains only express the mutant forms of the gene. The proposed project builds on the P.I.’s expertise in engineering specific mutations into the cyanobacterial genome , but represents a distinct and new line of experimentation. The PI. studied the function and expression of Fe-responsive genes that are involved in a major reorganization of the photosynthetic and respiratory complexes during episodes of Fe-limitation . The most recent contributions from the P.I.’s lab concern the photoassembly and catalytic properties of PSII complex and studies on the interaction of the respiratory and photosynthetic pathways . Studies of the H2O oxidation complex have led to an investigation into the source and activity of reactive oxygen species (ROS) that appear to increase as a consequence of environmental or mutational perturbation of the H2O oxidation complex. These studies are connected with the recent characterization of a novel heme-containing component of the cyanobacterial H2O oxidation enzyme, that is not found in higher plant PSII . This protein is hypothesized to cope with oxidative stress. As part of this effort, the laboratory is currently developing bacterial luciferase constructs in Synechocystis to monitor changes in the expression of genes induced to cope with these stress conditions and to deconvolute the regulatory pathways involved.

    John C. Cushman (Dept. of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, OK)

    Current research in the Cushman laboratory is directed towards understanding the genetic and molecular genetic basis of Crassulacean acid metabolism (CAM) in the facultative CAM plant M. crystallinum [1]. We maintain an active and continuing collaboration with the Bohnert lab on various aspects of ice plant biology. Major projects include 1) large-scale EST sequencing of ice plant leaf cDNAs 2) establishing fast neutron and EMS mutagenized plant populations to isolate and characterize mutants defective in CAM, 3) characterization of the stress-inducible enzymatic machinery of the CAM pathway [2, 3, 4], 4) understanding the mechanisms of transcriptional activation of CAM-specific enzymes [5], and 5) elucidation of the signal transduction pathways leading to CAM induction and other adaptive responses brought about by environmental stress [6, 7]. The EST and mutant projects are currently funded by NSF (Integrative Plant Biology) whereas the transcriptional activation and signal transduction aspects of CAM are funded by USDA-NRI (Plant Responses to the Environment).

    The proposed EST sequencing and microarray characterization of a master set of stress-inducible genes represent a natural extension of ongoing research in the Cushman laboratory. To date, over 1600 cDNA randomly selected cDNA clones derived from mRNA isolated from leaves well-watered or NaCl stressed plants. The long-term goal of this project is to sequence all of the expressed genes in ice plant leaves to fully understand the molecular aspects of CAM and to lay the foundation for physical mapping and sequencing of the ice plant genome. Once established, mutant populations can also be screened to identify mutants defective in stress tolerance. The current proposal would allow us to expand our current EST project to include cDNA clones derived from other tissues (e.g., roots, floral tissues, epidermal bladder cells, etc.) of the ice plant and from other halophytic and glycophytic species under study within the consortium. My laboratory is adjacent to the OSU Recombinant DNA/Protein Resource Facility where I oversee the automated DNA synthesis and sequencing services. With an additional automated DNA sequencing system and technical support, we would be able to meet the DNA sequencing needs of the entire stress consortium. My lab, in collaboration with other OSU researchers, has received funding from the Oklahoma Agricultural Experiment Station to build and operate a microarrayer within the Recombinant DNA/Protein Resource Facility to monitor global gene expression patterns. Analysis of gene expression patterns using microarray technology will allow us to rapidly identify a master set of salt-responsive genes implicated in stress-adaptation responses including CAM.

    David W. Galbraith (Dept. of Plant Sciences, The University of Arizona, Tucson, AZ)

    My research program has historically combined interests in the development of biological instrumentation and reagents with the investigation of specific biological questions concerning plant cell, molecular, and developmental biology [1-3]. The specific aims of our funded research projects are: (I) Flow cytometry: digital processing of molecular information (NSF Instrument Development Program). This involves the development of hardware and software for the analysis of flow cytometric pulse waveforms through digital signal processing, and the real-time sorting of cells based on waveform shapes [4,6,7]. This aims at providing new ways to examine cell structure and function. (II). Novel techniques for gene characterization in higher plants (USDA-NRICGP, Plant Genome).  This involves developing flow sorting methods for the identification of genes expressed within specific cell types, and is achieved via cell-specific expression of GFP, targeting of this molecule to the nucleus, recovery of labeled nuclei through flow sorting, and analysis of the nuclear transcripts [5,8,9]. We are also developing microarray techniques for analyzing the expression of these transcripts [10]. (III). Systematic analysis of the P450 superfamily in Arabidopsis (USDA-NRICGP, Plant Genome). This project involves PCR-based reverse genetics, using pooled, T-DNA tagged Arabidopsis lines, and primers based on P450 and T-DNA gene sequences, to identify mutants carrying specific P450 gene insertions. We are analyzing the mutant phenotypes to gain an understanding of the enzymatic activities encoded by the different P450s. We also employ microarray technologies to systematically analyze global P450 gene expression, as a function of development and in response to applied stimuli [10]. (IV). Flow karyotyping and sorting of translocations in maize (NSF, International Programs). This project involves use of flow cytometry for the production of chromosome-specific genomic libraries. (V). Interdisciplinary research training group on plant-insect interactions (NSF, Biological Infrastructure). This program supports postdoctoral research associates and graduate students in various laboratories within three UA Departments.

    My research activities as a whole emphasize technology development and implementation. My contribution to this proposal involves the development and application of novel technologies to examine stress responses at the molecular level.

    Paul M. Hasegawa/Ray A. Bressan (Dept. of Horticulture and Landscape Architecture, Purdue University, W. Lafayette, IN)

    Our laboratories are organized for everyone in both groups to work together in completely unhindered collaboration. We currently focus on 3 (surprisingly related) areas: 1) Insect resistance, 2) fungal disease resistance, and 3) osmotic stress resistance. All projects have basic science components seeking to elucidate molecular mechanisms of tolerance. In addition, we conduct work aimed at the more practical application of genomics technology mainly by the production of transgenic plants with altered expression of "tolerance" genes. Our recent collaboration with José Pardo's group in Spain has led to the extensive use of yeast as a model system to dissect the functional genomics of both resistance to osmotic stress and plant antifungal toxins. We are in a particularly unique position with these collaborations to unify the powerful molecular genetics of yeast with plant molecular biology and gene manipulation approaches toward salinity tolerance. For this project, our experience and expertise with the yeast model system and with plant molecular biology allows us to interface extremely well with the even more developed projects at the University of Arizona designed to allow multi-gene transferes to plants.

    José M. Pardo (Department of Horticulture and Landscape Architecture, and Instituto de Recursos Naturales y Agrobiologia in Seville, Spain.

    Dr. Pardo has an active lab at the Instituto de Recursos Naturales y Agrobiologia in Seville, Spain. Working with colleagues on a European Union Sponsored project, he has developed an extensive set of salinity sensitive mutants of yeast. By exploiting the many important advantages of the yeast model (genomic sequence completed saturation transposon tagged mutagenesis/gene knockout replacement/haploid genome with sexual cycle/efficient transformation/etc.). The Pardo lab has been able to characterize some of these mutants extensively and isolate the genes responsible for altered salinity tolerance. Dr. Pardo has spent three of the last five years as a visiting professor at Purdue University, where his collaboration with the Purdue group has led to the isolation of plant genes that complement salt sensitive yeast mutants and the transformation of plants with some of these genes. He will continue to spend part of his time at Purdue. His participation on this project will greatly facilitate the exhaustive isolation of plant genes involved in salinity tolerance using yeast as a model for mutant complementation and micro-assay analysis.

    Rolf A. Prade (Dept. Microbiology & Molecular Genetics, Oklahoma State University, Stillwater, OK) (In collaboration with Dr. P. Ayoubi).

    Current research in the Prade laboratory is focused towards the understanding of fungal molecular sensing mechanisms involved in environmental (e.g., stress and fungal plant infections) and developmental [1, 2] adaptations. Particularly, we are interested in the genetics of how and why fungi, such as Cochliobolus sativus (plant pathogen) and Aspergillus nidulans (model organism), sense and respond to the presence of complex carbon sources (e.g., polysaccharides). In addition, numerous experimental clues strongly point to a direct relationship between various stress response pathways and carbon sensing. Our current research activities related to carbon sensing are funded by grants from the USDA-NRI (Plant Pathology) and the Oklahoma Agricultural Experiment Station (in 1997).

    Several of the large-scale genetics resources that will be extensively utilized during this project have been developed in the last few years in collaboration with several groups. Some of these resources include: the A. nidulans physical map (Prade RA, Arnold JA ) and the A. nidulans EST database (Prade RA, Roe BA ). Both resources are publicly available ( and many of the related technical aspects have also been published [3-5]. In addition, construction of A. nidulans recombinant strains, mycological techniques, classical Mendelian and bacterial mobilization genetics, manipulation of proteins, eukaryotic and prokaryotic nucleic acids are routinely employed in our laboratory [1-4, 6-9].

    Our current limitation in providing clear answers to key questions on how, when and why living cells need to sense and respond to specific carbon driven cues are twofold: i) we realize that carbon sensing affects the expression of a large number of genes. Thus, the answers to basic questions require the analysis of many genes (genomics), and ii) carbon sensing is not only directly linked to development and the cell-cycle but is also strongly associated with various stress conditions as well. We recently determined that 9.2% of all genes expressed in A. nidulans during asexual development are associated with known stress responses encompassing; heat- and cold-shock proteins (7.1% and 0.1% of all ESTs, respectively), ubiquitination (1.5% ESTs), ROS scavenging (catalase, peroxidase, Cu-, Zn-superoxide dismutase), uptake, transport and compartmentalization of sodium and other compounds (8 ATPase proton efflux pumps), compatible solutes (0.4% ESTs and comprising, glycerol-, trehalose-, proline-, betaine-, mannitol- and sorbitol-synthesis genes) and various regulatory homologs such as, HAL2, HOG1, and calcineurin (R.A. Prade and P. Ayoubi, unpublished results).

    Jian-Kang Zhu (Dept. of Plant Sciences, The University of Arizona, Tucson, AZ)

    Efforts in the laboratory are focused on the genetic analysis of salt tolerance and osmotic stress signal transduction using Arabidopsis thaliana as the model organism. Our goal is to identify genes that are essential for plant salt tolerance and osmotic stress signaling, no matter they are induced by stresses or not. To this end, we have been working to isolate and characterize Arabidopsis mutants defective in NaCl tolerance (1-5) or stress gene regulation (6).

    Work on the isolation and characterization of salt-hypersensitive Arabidopsis mutants, and positional cloning of the salt tolerance loci is supported by USDA-NRI. Funding by Southwest Consortium on Plant Genetics and Water Resources is for genetic analysis of osmotic and cold stress signal transduction. We also work on cell surface proteins that mediate plasma membrane-cell wall interaction, which is funded by NSF (IBN).

    Our current work firmly establishes Arabidopsis as a powerful model to study salt stress tolerance and signaling. The proposed work builds on our expertise and knowledge of T-DNA mutagenesis of Arabidopsis (5), mutant screening (1, 2, 5), genetic mapping and positional cloning (7) to achieve genome-wide genetic analysis of salt stress responses.