Solid State NMR for Plant Structural Biology

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Soybean leaves labeled with 13CO2 at sub-ambient concentrations (simulating a water stress) contain high concentrations of glycine from the photorespiratory pathway. The glycine is incorporated into protein within minutes of the start of labeling. This routing may result from the short-term use of photorespiration to sense a water stress and to help regulate the synthesis of protoxylem elements, which are part of the intercellular water-transport network in the leaf. Four specific aims have been formulated to measure the response of soybean leaves to a variety of environmental stresses as a function of the concentration of CO2 in the atmosphere. (1) The first aim is to prove that the observed high-glycine protein resulting from the photorespiratory pathway operating under sub-ambient CO2 concentrations is cell-wall glycine-rich protein (GRP) concentrated in leaf veins. This will require the use of new rotational-echo double-resonance (REDOR) experiments on both intact leaves and cross-linked components of leaves. Correlation of increased GRP synthesis and sub-ambient leaf CO2 levels would be strong evidence for a short-term function for photorespiration related to water stress. (2) The second aim is to label glycine with 17O2 under genuine water-stress conditions in which CO2 uptake has been suppressed. By flooding the leaf with labeled oxygen and then forcing leaf stomata to close completely, the oxygenase activity along the photorespiratory pathway can be measured for minutes to hours in the absence of gas exchange. (3) The third aim is to correlate carbon and nitrogen assimilation in soybean leaves under environmental and chemical stress. Productivity in the field depends on an effective response to changing conditions. REDOR NMR methods to characterize whole-leaf stress response under normal conditions, and under the elevated CO2 concentrations that are expected in the near future, will be developed to search for high-CO2-induced modifications of plant response to stress that may be deleterious to productivity. (4) The fourth aim is to characterize GRPs in the cross-linked cell-wall fraction of cultured soybean cells. The plant may use GRPs (and other cell-wall proteins) in response to a variety of environmental stresses and the results of cell-culture experiments will be used to guide work on intact leaves.If the hypothesis about the short-term function of photorespiration is true, current efforts to produce transgenic crop plants in which photorespiration has been suppressed could ultimately result in plants that are less tolerant to drought and less productive under the increased atomospheric CO2 levels expected within 40 years. This project could guide genetic modifications more in harmony with total plant metabolism. Meeting the demands of anticipated high atmospheric CO2 concentrations (at 550 ppm the highest on Earth for millions of years) will be a goal that engages undergraduates, who will be recruited in the summers for labor-intensive growing, labeling, harvesting, and extracting soybean plants. The effect of the environment on the future on crop plants is a good topic to use in reaching out to inform the community about modern technology. The PI is an active participant in a formal education training program at Washington University for St. Louis K-8 science teachers.