A. Low Temperature Regulatory Circuits and Gene Regulons in Arabidopsis
1. Paint a detailed picture of the low temperature transcriptome in Arabidopsis
2. Define regulons of cold-responsive genes
3. Determine regulons involved in cold tolerance
4. Low temperature signaling genes

B. Conservation of the CBF Cold-Response Pathway
1. Determine the structure and expression of CBF loci in plants that differ in freezing tolerance
2. Role of CBF genes in barley winter hardiness
3. Characterization of CBF regulons in plants that differ in freezing tolerance

C. Comparative Cold-Gene-Network Studies with Plants that Differ in Cold Tolerance

References

A. Low Temperature Regulatory Circuits and Gene Regulons in Arabidopsis
We will provide key components of the diagram including determining the composition of the low temperature transcriptome (i.e., the array of gene transcripts that change in levels in response to low temperature); the organization of cold-regulated genes into regulons; an identification of loci that control expression of the regulons; and a determination of the regulons have important roles in cold tolerance.
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1. Paint a detailed picture of the low temperature transcriptome in Arabidopsis
  • determine how the transcriptome is altered when plants experience a rapid downshift in temperature and how it changes over time of exposure to low temperature
  • determine whether the rate of temperature change has a significant effect on changes in gene expression
  • determine what happens to global gene expression when plants freeze, a condition that plants are routinely subjected to in nature and causes an increase in freezing tolerance in wheat and other plants

    The general form of these experiments will be to grow Arabidopsis plants to the seedling stage at 20C, transfer them to 4C for various lengths of time, isolate the RNA and determine the transcriptome. In addition, cold acclimated plants will be frozen at -5C and the effects on the transcriptome determined after various times. To determine whether certain of the changes in gene expression are a consequence of "cold-shock" as opposed to low temperature per se, will grow plants to the seedling stage at 20C (constant light) and decrease the temperature 2C per hour for 8 h and then hold the plants at 4C. RNA will be isolated from the plants after each temperature shift and followed for days 4C. In these experiments, we will harvest all "above soil" plant material (which will be primarily leaf rosettes). The ultimate low temperature wiring diagram would include informational networking for all cold responsive genes. Toward this end, our intent is monitor gene expression using "full-genome" Affymetrix-type microarrays to optimize our changes of detecting cold-responsive genes. Once specific cold-regulated genes of interest have been identified, their expression can be monitored at higher resolution (more time points) by printing microarrays (slides) or transferring to filters and monitoring expression. Until the full-genome chips are available, we will conduct "baseline" exploratory experiments using the currently available Affymetrix chips which includes about 8,200 genes (the chips include the three CBF genes, but not the highly cold-induced COR15a gene). The experiments will be carried out at Michigan State University (MSU) in the Genomics Facility which includes instrumentation required for microarray printing and analysis, including both slide-based and Affymetrix chips. MSU is part of the Arabidopsis Functional Genomics Consortium (AFGC) and is responsible for conducting the microarray experiments.
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    2. Define regulons of cold-responsive genes
    By carefully analyzing the expression patterns of the cold-responsive genes over time, we anticipate that it will be possible to assign genes to groups that are potentially coordinately regulated by the same transcription factor(s). By using clustering programs (Eisen et al., 1998) and GeneSpring software package, we will assign genes to putative regulons. In addition, we search for short sequence elements that are conserved in promoters of a gene cluster and thus, represent potential cold-regulatory. To prove that a given transcription factor has a role in controlling a putative regulon of genes, we will overexpress the transcription factor (using constitutive and inducible promoters) and monitor expression of the putative target genes.
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    3. Determine regulons involved in cold tolerance
    To determine whether any of the cold-regulated regulons that we identify have discernable roles in cold tolerance, transgenic plants that either overexpress or under-express a given regulatory gene will be tested for both chilling tolerance (growth and development at low temperature; photosynthetic capacity; etc.) and freezing tolerance (electrolyte leakage analysis and whole plant survival) as previously described (Gilmour et al., 2000; Jaglo-Ottosen et al., 1998). Given the connection between freezing and dehydration tolerance and the cross-protection observed with overexpression of the CBF genes, we will also test for water-stress and high salinity tolerance as described by Shinozaki and colleagues (Kasuga et al., 1999; Liu et al., 1998).
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    4. Low temperature signaling genes
    Not all transcription factors that control expression of cold-regulated genes will themselves be cold-induced and thus, would not be directly implicated as being part of the low temperature regulatory circuits in the expression profiling experiments. Such genes, however, are key to the cold-networks and thus, need to be identified. In addition, we are interested in identifying more "upstream" genes/proteins that are potentially part of low temperature signaling. As part of another project, we are currently trying to isolate mutants that are affected in cold induction of the CBF genes with the intent of identifying genes involved in cold sensing. The approach we are using is the one that Zhu and colleagues (Ishitani et al., 1997) have used so successfully to identify genes involved in induction of the RD29A (COR78), in particular, HOS1 and HOS2 (Lee et al., 1999, 2000) that appear to encode negative regulators of low temperature signal transduction. The strategy is to fuse the promoter of the gene of interest to the firefly luciferase reporter gene, to mutagenize the plants (either chemical mutagenesis or insertional mutagenesis), and to screen for altered promoter activity by determining light production using a CCD camera.
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    B. Conservation of the CBF Cold-Response Pathway
    1. Determine the structure and expression of CBF loci in plants that differ in freezing tolerance
    We will characterize CBF loci in regard to their structure (genomic sequence) and expression (transcription) in plants that have evolved in different climes and differ in freezing tolerance. In this project, we will focus on cereals, solanaceous and leguminous plants. If the results point to differences in locus composition or expression being casually related to differences in low temperature tolerance, we would expand our analysis (in a future project) to include a broader range of plants.
    Comparative analysis of cereals. The cereals differ dramatically in freezing tolerance. The question we have is whether all of these plants have CBF cold-response pathways and whether differences in the structure of the CBF loci, alleles of the CBF genes, or regulation of the CBF genes might contribute to differences in cold tolerance. To address these issues, we will isolate the CBF loci from rye, barley and rice, determine the nucleotide sequences of the regions, and test for expression of the CBF genes.
    Comparative analysis of solanaceous plants. Solanaceous plants also differ dramatically in freezing tolerance. Solanum commersonii is a tuber bearing-plant that can cold acclimate and attain a moderate level of freezing tolerance (killed at about -10C) whereas its close tuber-bearing relative S. tuberosum, the common potato, does not cold acclimate and is freezing sensitive. Tomato, Lycopersicon esculentum, also does not cold acclimate and moreover, is prone to chilling injury. We will perform that same general analysis with these plants as we described above for the cereals.
    Comparative analysis of leguminous plants. Both soybean and M. truncatula harbor sequence homologs of the Arabidopsis CBF proteins and one might predict that the encoded proteins would bind to CRT/DRE sequences and activate a CBF regulon if activated. To isolate the CBF loci from these species we will screen genomic phage libraries using these ESTs as probes. As with the analyses cereals and the solanaceous plants we will complete the sequencing of the CBF genes and determine the expression of the individual genes by northern analysis.
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    2. Role of CBF genes in barley winter hardiness
    Winter hardiness is a complex trait with three principal components: low temperature tolerance, vernalization, and photoperiod. The phenotypic complexity of these traits has necessitated a Quantitative Trait Locus (QTL) strategy based on identifying associations between marker genotypes and winter hardiness trait phenotypes (see review by Hayes et al., 1998). Because of the known role of CBF genes in low temperature tolerance in Arabidopsis, and linkage map data showing a linkage relationship of a CBF ortholog with a winter hardiness QTL cluster in barley, we are interested in determining the role of CBF genes in winter hardiness in the Triticeae, using barley as a model system. We will map all members of the CBF gene family in barley to determine their genome locations on linkage maps and relationships to winter hardiness-related QTLs. We will also determine the relationships of CBF genes to winter hardiness-related QTL in barley, as well as the allelic variation at CBF genes in an array of barley germplasm.
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    3. Characterization of CBF regulons in plants that differ in freezing tolerance Does the CBF regulon of freezing tolerant barley differ from the CBF regulon of freezing sensitive rice? Do they differ in freezing tolerant S. commersonii and freezing sensitive S. tuberosum? The general experimental design to address this issue will be to overexpress CBF genes from a given plant in that plant and determine what genes are induced. We will begin by using constitutive promoters and assess whether we should extend the experiments to include the use of an inducible promoter (this would be in a future project). The specific plants that we will transform and compare are the cereals barley (freezing tolerant) and rice (freezing sensitive); the solanaceous plants tomato (freezing sensitive), potato (freezing sensitive) and S. commersonii (freezing tolerant) and the legume soybean (freezing sensitive). What we will do is identify CBF-responsive genes by screening for cDNAs that are up-regulated in CBF-expressing plants. This will be done by making "suppressive subtraction" cDNA libraries and doing high throughput sequencing to identify the genes.
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    C. Comparative Cold-Gene-Network Studies with Plants that Differ in Cold Tolerance Is the low temperature transcriptome of Arabidopsis typical of plants that cold acclimate? How do the low temperature gene networks of various plants, which differ so greatly in their response to low temperature, compare to each other? We will address this issue by comparing the complements of low temperature-responsive genes in wheat, barley, maize, tomato, potato, S. commersonii and soybean. We are coming from a comparative biology perspective with the intent of better understanding the array of responses that plants have evolved (or potentially have NOT evolved in the case of tropical plants) to adjust to cold temperature. Conducting these experiments would be easy if full-genome microarrays were available for these plants, but they are not. So, instead, we will conduct two lines of investigation. First, as microarrays become available for the cereals, solanaceous plants and legumes, we will use them to test for low temperature responsive gene expression. We anticipate that we will be able to determine the identify of many cold-responsive genes, including most of those that are down-regulated in response to low temperature; they should be present among the available ESTs. However, we will probably not identify a significant number of cold-induced genes as most of the EST projects have not specifically included efforts to include cold-responsive genes. Thus, our second line of investigation is to test this supposition. In particular, we will construct cDNA libraries from barley, rice, tomato, potato, S. commersonii and soybean that are enriched for inserts representing cold-responsive genes. RNA will be isolated from plants treated at low temperature for various lengths of time and "subtracted" cDNA libraries constructed. The libraries will then be screened by differential hybridization for clones representing putative cold-responsive genes and sequenced.
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    References
  • Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA. 95, 14863-14868.
  • Gilmour, S. J., Sebolt, A. M., Salazar, M. P., Everard, J. D., and Thomashow, M. F. (2000). Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124, 1854-1865.
  • Hayes, P.M., F.Q. Chen, A. Corey, A. Pan, T.H.H. Chen, E. Baird, W. Powell, W. Thomas, R. Waugh, Z. Bedo, I. Karsai, T. Blake, and L. Oberthur (1998). The Dicktoo x Morex population: a model for dissecting components of winterhardiness in barley. In: P. H. Li and T. H. Chen (ed.). Plant Cold Hardiness. Plenum Press, New York, USA
  • Ishitani, M., Xiong, L., Stevenson, B., and Zhu, J. K. (1997). Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid- independent pathways. Plant Cell 9, 1935-1949.
  • Jaglo-Ottosen, K. R., Gilmour, S. J., Zarka, D. G., Schabenberger, O., and Thomashow, M. F. (1998). Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, 104-106.
  • Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999). Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat. Biotechnol. 17, 287-291.
  • Lee, H., Xiong, L., Ishitani, M., Stevenson, B., and Zhu, J. K. (1999). Cold-regulated gene expression and freezing tolerance in an Arabidopsis thaliana mutant. Plant J. 17, 301-308.
  • Lee, H., Xiong, L., Ishitani, M., Stevenson, B., and Zhu, J. K. (2000) 11 th International Conference on Arabidopsis Research, Madison, WI, Abst 107, 54
  • Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998). Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10, 1391-1406.
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