Location: Plant Physiology and Genetics Research2013 Annual Report
1a. Objectives (from AD-416):
Objective 1: Discover and characterize biochemical and molecular factors that limit photosynthetic capacity of plants at elevated temperatures and under water-limited conditions that can be exploited to develop molecular targets for generating stress-tolerant crops. Objective 2: Identify genes and biochemical pathways involved in production of the protective, waxy layer of plant surfaces, and determine their relationships with tolerance to abiotic stress. Subobjective 2.1: Develop high-throughput screening strategies to identify new genes and alleles controlling production of plant cuticle lipids related to drought stress tolerance. Subobjective 2.2: Elucidate the cellular and drought-stress-related physiological functions of newly identified cuticle genes. Objective 3: Identify genes and/or molecular processes that regulate seed oil concentration and composition in oilseed crops, including under abiotic stress conditions, and determine molecular mechanisms that regulate oil production pathways in plant vegetative biomass. Subobjective 3.1: Identify and characterize genes involved in determining oil content and composition in B. napus, including under heat and drought conditions. Subobjective 3.2: Elucidate the role of the CGI-58 gene in regulating triacylglycerol content and lipid signaling pathways in plant leaves and seeds.
1b. Approach (from AD-416):
The research program includes a variety of experimental approaches including statistical genetics and genomics as well as molecular and biochemical studies of model plants such as Arabidopsis and camelina, as well as field-based studies of cotton and Brassica napus. To uncover natural mechanisms for heat and drought tolerance in cotton, we will examine the genetic variability for these traits using a high-throughput phenotyping platform to determine canopy temperature in combination with measurements of lint yield and carbon isotope discrimination. We will further develop a high-throughput assay for Rubisco activity as a tool in our high-throughput phenotyping platform to uncover natural mechanisms that might improve or stabilize Rubisco activase in response to heat stress. To identify genes associated with cuticle and oil production, we will use both statistical genetics and candidate gene-based approaches to identify highly active allese in a diverse B. napus germplasm that are associated with altered cuticle or oil properties. We will also employ mutagenic screens of Arabidopsis to isolate a unique class of putative cuticle stress responsive genes. The molecular and biochemical function of oil and cuticle related genes and their encoded enzymes will be examined using a variety of model systems including Arabidopsis, yeast cells, and bacteria. Finally, production of oil in plant leaves will be studied by examining the function of a gene called CGI-58 gene, which plays a key role in regulating lipid metabolism, oil content, and stress-response signaling in plant leaves. The end-goal is to develop the knowledge base and molecular tools required for dramatically increasing the oil (energy) content of a rapidly growing biomass crop such as switchgrass or Miscanthus.
3. Progress Report:
This is the first report for this new project and represents a continuation and expansion of work described in the previous project, 5347-21000-011-00D, entitled "Physiological and Genetic Basis of Cotton Acclimation to Abiotic Stress". A collection of approximately 500 different varieties of Brassica napus, representing worldwide diversity, was planted and grown in the warm, arid climate in Maricopa, Arizona. Visual scores revealed tremendous variation in leaf wax deposition on leaves, and analytical measurements revealed extensive differences in wax content and composition. Analysis of wax content of all plant lines is ongoing. The resulting dataset will be used for linking phenotypic differences to specific molecular markers determined by genome sequencing and genome-wide association studies. A collaborating partner, KeyGene, has determined the genome sequences for a portion of the Brassica varieties, and a post-doctoral fellow has been hired to initiate statistical genetics analyses. Seeds of each Brassica variety were collected for oil analysis, which will also be used as a dataset for statistical genetics analysis, but seed yield was very poor due to the hot temperatures during seed development and harvest. As a contingency, the plant lines are now being grown in a greenhouse to obtain seeds for oil analysis. Studies on the molecular mechanisms of wax and oil production have included the expression of an Arabidopsis transcription factor called MYB96 in camelina, with the end goal of increasing wax production on plant leaves and a potential increase in drought tolerance. A second study focused on the role of a protein called CGI-58 in regulating oil content in plant leaves, which revealed a key association with a second protein called PXA1, and that the two proteins collaboratively regulate both oil content in leaves as well as stress response in plants, in general. Other studies revealed that CGI-58 was also involved in regulating seed germination during stress response, specifically, by regulating the rupture of the seed coat. Lastly, CGI-58 has also been unexpectedly found to regulate the production of polyamines in plants, which are known to play essential roles in a variety of plant stress response pathways including drought, heat, and cold tolerance. Investigation of the underlying molecular mechanisms is ongoing. Research on heat stress in cotton focused on testing and validating phenotyping platforms for field analysis of various traits and on the development of a high-throughput assay from Rubisco and Rubisco activase. The assay is now fully develop and is being used as a screening tool to assess photosynthetic responses to heat and drought stress.