2012 Annual Report 1a.Objectives (from AD-416): Many plant species produce high amounts of industrially important fatty acids (e.g., hydroxy fatty acids) in their seeds, but the plants generally lack agronomic traits that permit these oils to be produced on a large scale for industrial utilization. The objective of this research program is to discover the genetic basis for the production of these high-value oils and use these genes for production of the oils in robust, non-food crop platforms. Cotton is exceptionally well positioned to serve as a platform for production of high value oils because the main economic driver for this crop is cotton fiber. The seed is generally viewed as a lower valued by-product, and raising the value of the seed through production of industrially important oils would increase farm gate value of the crop and provide society with renewable, sustainable sources of oil that are otherwise obtained from non-renewable petroleum. In addition, high-risk/high-payoff research will be conducted to determine if industrial oils can be produced in vegetative (e.g., leaves and stems) parts of plants. The rationale is that vegetative plant biomass is significantly greater than seed tissues, and by developing methods to produce oil ectopically in leaves and stems, the amount of oil obtained per hectare of land can be substantially increased. 1b.Approach (from AD-416): Various species and hybrids of Lesquerella will be analyzed to identify genes involved in the production of hydroxy fatty acids. Enzyme functionality will be investigated by expression in yeast cells, and cellular properties will be evaluated through expression in plant cell culture. Candidate enzymes will be expressed in transgenic Arabidopsis, including both developing seed and leaf tissues. Enzyme stability and regulation (primarily by temperature) will be investigated using western blotting and half-life studies. Select genes will be expressed in transgenic cottonseed to evaluate this crop as a platform for production of industrially important oils. Production of hydroxy fatty acids in non-seed tissues will be evaluated in various wild-type and mutant Arabidopsis plants. 3.Progress Report: This subordinate project is directly related to objective 1 of the parent project: Develop enhanced germplasm and cultivars for low input, high yielding, cost-competitive oilseed, latex, and biomass crops as bio-fuels and bio-based products; Sub-objective 1.B. Manipulate biosynthetic pathways to improve yield traits. Work conducted during this year focused primarily on understanding the underlying molecular mechanisms involved in the production of oils in the leaves of plants. The end-goal is to develop novel strategies for producing and delivering high amounts of industrially important oils and biofuels in plants, which is similar to the overall objectives of the parent CRIS project. In collaboration with scientists at the University of North Texas and the University of Guelph, we have been studying the function of a gene called CGI-58 that, when disrupted in plants, causes a significant increase in the amount of oil present in plant leaves. Research conducted in the prior reporting period identified a number of proteins that physically interacted with the CGI-58 protein, and during the current year additional studies were performed to elucidate the functional relationships with a specific protein called PXA1. Results indicated that these two proteins act together to regulate the total amount of lipid in plant leaves, and that they also co-regulate a variety of lipid signaling pathways involved in plant stress response and plant growth and development. These results provide new insight to the molecular mechanisms involved in regulating oil content in plant leaves, and provide new targets for rationally manipulating the energy content of biomass/biofuel crops. The lead scientist monitored progress on this agreement by directly supervising the research activities of a post doctoral fellow who is vising his lab, and has regular teleconferences and email exchanges with scientists at the University of North Texas and Guelph to monitor overall progress of the research program.