2011 Annual Report
1a.Objectives (from AD-416)
The objective of this proposal is to develop the technology for improving the efficiency of Rubisco, particularly under the warmer temperatures predicted from climate change. To resolve some of the basic scientific questions that are impeding wide-spread implementation of the technology, we propose to move beyond Arabidopsis by using the oilseed plant, Camelina sativa, to refine the technology and then evaluate the results of the refinements under more natural conditions. The research will allow us to evaluate several mechanistic questions, while also improving camelina germplasm.
1b.Approach (from AD-416)
To test the feasibility of improving photosynthetic thermotolerance, we would like to launch an extensive transgenic project with the oilseed crop, camelina. In this project we would express both regulated and non-regulated forms of modified activase behind activase and heat-inducible promoters in a background of wild type activase. Transgenic camelina plants harboring the various constructs will be evaluated under natural conditions of light under control and elevated temperatures in the greenhouse and plants with superior performance will be tested in the field. Successful completion of the three objectives with camelina and a fourth objective with tobacco will resolve outstanding questions about the feasibility of improving plant thermotolerance by introducing an improved activase and answer several basic mechanistic questions about the regulation of Rubisco in plants.
To characterize the thermotolerance of photosynthesis in camelina, Rubisco activation was measured both in vivo and by assaying Rubisco activase activity using a new method developed for leaf extracts. The results were consistent with the idea that the thermal properties of Rubisco activase determine the thermotolerance of photosynthesis. In growth chamber experiments, a moderate heat stress of 35°C reduced seed weight and seed number in camelina by more than 50%. That photosynthetic performance and yield in camelina are acutely sensitive to inhibition by moderate heat stress indicates that this plant is an excellent choice for the planned experiments to improve thermotolerance. A manuscript detailing the responses of photosynthesis and yield to temperature in camelina is in preparation and one describing the new method for measuring Rubisco activase activity in leaf extracts has been accepted for publication.
A chimeric Rubisco activase have been constructed using the sensor-2 retention method to improve the thermotolerance of the enzyme. Recombinant proteins have been purified for testing of thermotolerance and the ability to activate camelina Rubisco. If greater thermotolerance and Rubisco activation are observed, camelina plants will be transformed with these activases via Agrobacterium-mediated transformation using vector designed to produce high level expression of modified activases. The plasmid is being used in both its original form containing kanamycin resistance and in a re-engineered form that replaces kanamycin resistance with the gene for dsRed monomer, a fluorescent protein.
To understand how photosynthesis is regulated by activase, experiments are in progress to modify the mechanism for regulation of activase in tobacco. Genes have been synthesized for expressing tobacco activase with a redox-regulated C-terminal extension from creosote activase. Recombinant protein has been produced from the cDNAs and is being characterized. These experiments require thioredoxin-f, the native reducing agent for activase-A. Thus far, we have successfully produced recombinant thioredoxin-f and have purified the recombinant protein for use in these experiments. In addition, tobacco plants have been transformed with the modified enzyme and we are currently screening for transformants.