Location: Plant Physiology and Genetics Research2012 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 will launch an extensive transgenic project with the oilseed crop, camelina. In this project we will 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.
3. Progress Report:
This Reimbursable Agreement is in support of objective 1 of the parent project- Improve crop stress tolerance by determining, and developing technology to ameliorate, metabolic limitations by biological processes most sensitive to abiotic stress factors common in arid southwestern U.S. cropping systems, Sub-objective 1a - Improve crop tolerance to heat stress by devising approaches to improve the ability of Rubisco activase to activate Rubisco at leaf temperatures above the optimum for photosynthesis. These studies were conducted with camelina, a model plant species whose photosynthesis is acutely sensitive to considerable heat stress. To characterize the heat stress response in camelina, measurements of Rubisco activase and cpn60 protein levels were conducted. These measurements showed that cpn60 levels increased when plants were grown with a mid-day interval of moderate heat stress (i.e., 35°C). Uexpectedly, the higher levels of cpn60 in heat stressed plants did not acclimate photosynthesis to the higher temperature. Rubisco activase levels were similar under control and heat stress condition, although high molecular mass species of Rubisco activase were more abundant in heat stressed leaves. A manuscript detailing these responses to temperature is in press. Preliminary experiments with the recombinant enzyme showed that creosote Rubisco activase was inefficient in activating camelina Rubisco, whereas cotton Rubisco activase was an effective activator. Consequently, a revised strategy for improving the thermotolerance of camelina photosynthesis has been adopted involving insertion of the alpha- and beta-isoforms of the more temperature tolerant Rubisco activase from cotton. Based on the temperature response of enzyme activity, Rubisco activase from cotton would increase the thermal stability of camelina photosynthesis by about 5°C. To increase the probability of success, camelina was also transformed with the alpha- and beta-isoforms of Rubisco activase from a wild rice species. This species of Oryza is native to hot, arid regions of northern Australia. Together with Macquarie University in Sydney, the temperature response of photosynthesis and Rubisco activation in this species exhibited considerable heat tolerance. Thus, the Rubisco activase from this species is another potential candidate for improving the thermal tolerance of camelina photosynthesis. Considerable progress has been made in addressing aspects of the project related to Rubisco activase regulation. Significant differences in the properties of the non-redox regulated forms of Rubisco activase were found among species. The data showed that the beta-isoform of Rubisco activase from Arabidopsis was insensitive to inhibition by physiological ratios of ADP/ATP when not paired with its alpha-isoform, whereas the beta-isoform from tobacco, a species that does not express an alpha-isoform, was acutely sensitive to inhibition by ADP. To understand the role of regulating Rubisco activase in photosynthesis, photosynthetic induction was measured. In plants like tobacco, that contain a Rubisco activase that is inhibited by physiological ratios of ADP/ATP, the rate of photosynthetic induction was dependent on the duration of low light prior to induction. In contrast, plants that contain a Rubisco activase enzyme that is insensitive to inhibition by ADP exhibited no lag in the induction of CO2 fixation upon transition from low to high light. These data indicate that the sensitivity of Rubisco activase to inhibition by ADP influences the rate of photosynthetic induction. This influence of Rubisco activase is particularly important for carbon gain in environments with highly fluctuating light regimes. Progress performance on this agreement has been monitored through annual reports to DOE, email correspondence with the Project Manager and semi-annual PI meetings. The research on improving plant thermotolerance using camelina relates directly to Component 4 of the Action Plan for NP301: Plant Biological and Molecular Processes and specifically to Problem Statement 4B: Biological Processes that Improve Crop Productivity and Quality