Michael F. Thomashow - US grants

oncogenes; gene expression;

oncogenes; gene expression;

Extremes in environmental conditions such as low and high temperature, drought, and high salinity, severely limit the geographical distribution of plants and account in large part for the shortfalls between potential and actual crop yields. Many plants, however, have evolved mechanisms that enable them to acclimate to and survive to varying degrees, different forms of environmental "stress." The long term research interest of the Principal Investigator is to understand at a molecular and cellular level the adaptive responses that plants make to survive cold and freezing temperatures. He has recently established that Arabidopsis, like many other plants, becomes more frost tolerant when exposed to low nonfreezing temperatures and that changes in gene expression occur during this cold acclimation process. His goal now is to determine the functions of the cold-regulated ("cor") genes. His specific aims include: the isolation and sequencing of cor genes; determining whether COR polypeptides have cryoprotective effects; the construction and analysis of Adrabidopsis cor mutants; determining the cellular and subcellular location of the COR gene products; and determining whether Arabidopsis cor genes are highly conserved among plants. At a minimum, these studies should provide additional insight into the molecular and physiological changes that occur in plants at low temperature. They also have the potential of yielding genes that could be used to improve the cold and freezing tolerance of agronomically important plants.

9728462 Thomashow Many plants increase in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. Recent evidence indicates that cold acclimation involves the induction of cold-regulated genes. A primary research objective is to determine how plants sense low temperature and process the "cold signal" to induce the expression of freezing tolerance genes. Toward this end, an Arabidopsis cDNA has been isolated that encodes a transcriptional activator that binds to the CRT/DRE sequence, a cold- and drought-responsive DNA regulatory element. The focus of this proposal is to determine how this factor, CBF I, activates transcription. The approach that will be taken is based on two important observations: 1)the transcriptional activity of CBF1 in yeast requires the action of at least three transcriptional adaptor proteins, ADA2, ADA3, and GCN5; and 2) Arabidopsis encodes a homolog of the yeast ADA2 protein. Thus, plants may have a transcriptional adaptor system related to the ADA complex of yeast and this system may be involved in cold- and drought-regulated gene expression. These hypotheses will be tested by: 1) defining the boundaries and key residues of the CBF1 activation domain; 2) determining whether the CBF1 activation domain interacts directly with the Arabidopsis ADA2 protein and if so, whether the interaction is affected by temperature or dehydration stress; 3) determining whether the Arabidopsis ADA2 protein interacts with homologs of the ADA3 and GCN5 proteins or with novel plant adaptor proteins; and 4) assessing the extent to which the Arabidopsis ADA2 protein is involved in plant growth and development and response to various environmental stresses. The results of this project will provide fundamental new information on the nature of the proteins with which plant transcriptional activators interact to stimulate transcription, an area about which virtually nothing is known in plants. In addition, they will provide significant new informat ion about cold-regulated gene expression and will open the door to a more globally integrated understanding of plant gene regulation. Plants respond to cold stress by synthesizing specific proteins. This regulatory response is caused by changes in gene expression at the level of transcription, the process by which DNA is copied into RNA. These transcriptional changes are caused by interactions between proteins, called transcription factors, and DNA. This project will study proteins that interact with transcription factors, called transcriptional adaptors, during cold stress. Analogous proteins have been discovered in yeast, but this is the first indication that they exist in plants. This project will determine whether these proteins play a similiar role in plants and define the parts of the transcriptional adaptor molecule that are critical for interacting with the cold-activated transcription factor. This understanding may contribute to our understanding of how plants adapt to cold.

Plants vary greatly in their responses to cold temperatures. At one extreme are plants from tropical and subtropical regions such as soybean and rice, which suffer injury when exposed to chilling temperatures between 0 and 12 degrees Celcius. In sharp contrast, plants from temperate regions are not only chilling tolerant, but many, such as Arabidopsis and wheat, can survive freezing after exposure to low nonfreezing temperatures, a phenomenon known as "cold acclimation."
The overall goal of this project is to understand the "genomic" basis of cold tolerance. The specific focus will be to develop a more detailed understanding of how plants respond to low temperature in terms of altering gene expression. This emphasis is motivated by the recent demonstration in Arabidopsis that cold acclimation involves the action of cold-regulated genes including the CBF regulon, a group of genes that imparts freezing tolerance and is coordinately regulated by the CBF transcriptional activators.
This project will comprise three related lines of investigation. In the first, a low temperature "wiring diagram" will be constructed that includes a definition of low temperature regulatory circuits and the gene regulons that they control. The second line of investigation will be to determine the similarities and differences of low temperature-regulated genes in plants that differ in freezing tolerance. The third line of investigation will be to determine whether the Arabidopsis CBF cold-response pathway is highly conserved in other plants and whether differences in plant cold tolerance can be traced to differences in CBF cold-response pathways. Comparative and functional genomics approaches will be used to address these issues including gene expression profiling using DNA microarrays.
In sum, these lines of investigation will provide a deeper understanding of the "genomic mechanisms" that plants have evolved to cope with low temperature and have the potential to provide "genetic tools" to improve the cold tolerance of plants, traits of considerable agronomic and economic importance. In as much as the CBF regulon not only imparts freezing tolerance, but dehydration tolerance as well, the results of this project also have the potential to provide genetic tools to improve the drought and salinity tolerance of agriculturally important plants.

Deliverables:

Microarray data and other information about the project can be found at:
http://aztec.stanford.edu/cold.

Transcriptional adaptor or coactivator proteins play central integrating roles in regulating eukaryotic gene expression. These proteins function in part to overcome the barrier to transcription imposed by the packaging of eukaryotic genomes in the form of chromatin. The ADA2 and GCN5 proteins are two components of multiprotein coactivator complexes. The biological roles of these coactivator proteins are largely unknown in plants, but the available evidence suggests significant differences from the corresponding mechanisms in yeast and animals. Therefore, the long-term goal of this research project is to define the roles of transcriptional adaptor proteins in regulating gene expression in plants. The mustard cress Arabidopsis is used extensively as a model organism for genetic and molecular biological studies in plants. The genome of Arabidopsis encodes two ADA2 proteins and one GCN5 protein, each with some features in common with yeast and animal homologs and other features unique to plants. Mutations in the ADA2b and GCN5 genes have similar and also distinct effects on plant growth and development, suggesting that the corresponding proteins have both common and distinct biological functions. This project will characterize plant-specific features of these adaptor proteins using genetic and molecular genetic approaches. To identify features of GCN5 or ADA2 proteins that are required for biological activity, specific mutations will be tested for the ability to complement existing gcn5 or ada2 T-DNA disruption mutations. The distinct biological roles of the two ADA2 genes (ADA2a and ADA2b) will be defined by identifying tissue- or cell-specific expression patterns, and by domain-swap experiments. Similar genes from important crop and experimental plants will be characterized to lay the foundation for future genetic and biochemical investigations. This project will fill an important gap in the understanding of the diversity of genetic regulatory mechanisms and may be valuable in future agricultural or biotechnological applications. In addition, this project provides training opportunities specifically tailored for undergraduate, graduate and postdoctoral scientists.

PI: M. Thomashow (Michigan State University)
CoPIs: C. Chan (Michigan State University) and T. Chen (Oregon State University; subawardee)
Collaborator: S.-H. Shiu (Michigan State University)

The long range goals of the project are to gain a systems level understanding of plant responses to abiotic stress and to use the first principles gained to develop novel strategies to improve the stress tolerance of agriculturally important crops. These goals are important as abiotic stresses limit the geographical locations where crops can be grown and account for the majority of losses in yield on an annual basis. The overall objective is to identify the low temperature transcriptional networks that plants have evolved to survive freezing. As there is a direct link between freezing and dehydration injury, the results should also provide further insights into the nature of gene modules that impart tolerance to drought and other dehydration stresses. The specific aims of the project are two-fold. The first is to develop a detailed understanding of the low temperature transcriptional network of Arabidopsis and determine which components contribute to freezing tolerance. This will be accomplished by identifying transcription factors and other regulatory proteins that have key roles in configuring the low temperature transcriptome; identifying the cis-acting DNA regulatory elements through which these regulatory proteins function; and identifying the regulatory programs and gene modules that contribute to freezing tolerance (and potentially tolerance to drought and other abiotic stresses). The second aim of this project is to determine whether the low temperature transcriptional networks and gene modules that impart freezing tolerance in Arabidopsis are conserved in plants that cold acclimate (i.e., increase in freezing tolerance in response to low non-freezing temperatures) and whether ""deficiencies"" in these networks and modules contribute to the freezing sensitivity of those plants that do not cold acclimate. This aim will be accomplished through comparative genomic analysis of three closely related Solanum species which differ in cold tolerance: S. commersonii, potato and tomato. Together, the proposed studies incorporate the determination of gene expression at a genome level, a comparative genomic analysis of model and crop species, and the integration of computational analysis and empirical testing to reconstruct and model transcriptional regulatory networks that are fundamental to plant life and have importance in agriculture.


Broader Impacts

Improving the abiotic stress tolerance of crops is crucial to meeting future demands for food and fiber. In addition, it is a key component of an emerging national vision to produce sufficient biomass per year in the U.S. to replace significant percentages of petroleum-based transportation fuels with biofuels produced from renewable resources. The studies proposed here directly relate to these important areas as they will provide a deeper understanding of the genomic mechanisms that plants have evolved to cope with abiotic stress and have the potential to provide genetic tools to improve the abiotic stress tolerance of plants. Microarray data generated in the course of these studies will be available through a project website (http://aztec.stanford.edu/cold/index.html) and through GEO and ArrayExpress.

The project will also contribute to the training of a next generation of scientists who are experienced in bringing genomic, bioinformatic and computational approaches to bear on fundamental questions in biology. Finally, the project includes a summer undergraduate education and research training program which will target inclusion of underrepresented groups of our society. The goal is to provide the students with opportunities to learn more about different areas of genomic research and to discuss broadly and informally issues that relate to pursuing careers in science.

This research investigates the ecological and genetic mechanisms that allow the plant Arabidopsis thaliana to adapt (grow, survive and reproduce) to different natural environments. Using wild populations from the extremes of a natural gradient in temperature and length of daylight, the investigators will identify the traits and genes that govern plant performance in nature. Traits of particular interest include flowering time, freezing and drought tolerance, and the capacity of plants to photosynthesize (make energy from sunlight) under conditions of physiological stress. The investigators will identify the genes that contribute to adaptation in nature and the magnitude of the effect of individual genes. A major goal of this work is to investigate the importance of genes that have positive effects in one environment but negative effects in other environments. Such genes are poorly understood but are thought to play a key role in adaptation.

The study will provide a deeper understanding of the mechanisms that allow plants to cope with different environments and has the potential to provide genetic tools for producing new varieties of plants/crops. In addition, the study system is well suited to investigate the possible consequences of global climate change and the potential of plants to adapt to rapidly changing conditions.

Understanding how organisms adapt to the many challenges they experience in nature is a central goal in biology. Knowledge of the genetic mechanisms underlying adaptation in nature is also useful for increasing crop yields and for the conservation of wild species. One such challenge faced by organisms at high latitudes is freezing temperatures. This research investigates the genetics of freezing tolerance in the mouse-eared cress, a relative of many crop species that has become a "model" organism for which many genetic tools have been developed. In earlier work, the researchers collected seeds of this species from Italy and Sweden and planted them in experimental gardens located in each country. They found that freezing tolerance is required to survive the long, cold winters in Sweden, whereas freezing tolerance reduces performance in Italy. This result exemplifies the concept of biological "trade-offs"; adaptation to one environment often reduces performance elsewhere. This simple principle can explain why there are so many different species on earth, in that each species is adapted to a particular environment. The research investigates the genetic control of freezing tolerance and explores the mechanisms that contribute to the tradeoff in performance across environments. The genetic mechanisms identified in these studies may be useful tools for producing new crop varieties with increased yield in cold climates. In addition, the research team will perform outreach activities that advance scientific literacy and promote careers in science. Michigan State University scientists will contribute to a partnership between K-12 teachers and the W.K. Kellogg Biological Station to develop lesson plans that support Next Generation Science Standards (NGSS) for inquiry based learning.

The research employs cutting edge genetic technologies to address the mechanisms and adaptive value of freezing tolerance using three complementary approaches. First, the genetic basis of freezing tolerance will be further examined in the two original populations in Sweden and Italy using new lines manipulating freezing tolerance genes in otherwise homogeneous parental backgrounds. These lines will then be grown in controlled environment chambers and in the field to estimate the importance of freezing tolerance genes for genetic trade-offs in performance across environments. Second, the genetic basis of freezing tolerance will be investigated in
four new populations from Scandinavia and Spain with dramatic differences in freezing tolerance. Third, this research will be expanded to an even broader scale, asking if the same genes for freezing tolerance found in these focal populations are found in other geographic regions. The techniques employed in part three are similar to those used to study the genetics of many human diseases. Taken together, these studies examine the genetic basis of freezing tolerance in a broad diversity of natural populations and assess the mechanisms of performance trade-offs for this important adaptive trait.

Plant diseases routinely cause significant losses in crop productivity. Thus, a fundamental goal of plant biology research is to identify genes that impart immunity against plant pathogens and to determine how they are regulated in response to environmental stimuli. Much has been learned about the nature of immunity genes and how they are regulated in response to pathogen attack; i.e., in response to biotic stress. However, recent studies have established that immunity genes can also be induced in response to abiotic stress, namely cold temperatures. In this proposal, the investigators will study this temperature-immunity nexus: how plant immunity is regulated by temperature. This phenomenon has received relatively little attention, but has been observed in crop and other plant species indicating that it may be an underappreciated highly conserved “preemptive” survival strategy; i.e., that plant immunity is activated in response to an abiotic stress that otherwise has the potential to increase the susceptibility of the plant to infection by pathogens. Given the ever-increasing concerns about climate change and the possible negative effects that these changes could have on crop productivity, it is of great importance to understand the connectivity between abiotic and biotic regulation of immunity genes as such information has the potential to be used to develop novel approaches to enhance plant resilience to environmental stresses.


The Arabidopsis Calmodulin-binding Transcription Activator (CAMTA) transcription factors are master regulators of salicylic acid (SA)-mediated immunity. In healthy plants grown at moderate temperature (ca. 22C), the CAMTA proteins repress expression of SA-immunity genes. However, when plants are exposed to pathogens—a biotic stress—or, as has recently been demonstrated by this research team, low temperature (ca. 4C)—an abiotic stress—the CAMTA “brake” is “disabled” resulting in the induction of defense genes. A fundamental goal of this project is to determine how the repression activity of the Arabidopsis CAMTA3 protein is regulated by temperature. In particular, a combination of biochemical, proteomic, genetic and genomic approaches will be used to determine: 1) how the CAMTA3 protein represses expression of target genes at moderate temperature; 2) how low temperature suppresses CAMTA3 repression activity; and 3) whether abiotic factors other than low temperature can inactivate the CAMTA3 brake. An additional integral component of this project is to extend the studies on CAMTA regulation of immunity genes in Arabidopsis to a range of important crop species and to determine the extent to which regulation of immunity in response to low temperature and other abiotic stresses is conserved in plants. Taken together, the results from these lines of investigation promise to expand our knowledge of the basic Rules of Life that govern host defense mechanisms in plants.

This award was co-funded by the Plant Genome Research Program and Plant Biotic Interactions Program.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.