Solid State NMR for Plant Structural Biology

在亚环境浓度(模拟水分胁迫)下，标记有13CO2的大豆叶片含有来自光呼吸途径的高浓度甘氨酸。甘氨酸在标记开始的几分钟内就被结合到蛋白质中。这种途径可能是由于短期使用光呼吸来感受水分胁迫并帮助调节原生木质部元素的合成，原生木质部元素是叶片细胞间水分运输网络的一部分。已经制定了四个具体目标来测量大豆叶片对各种环境胁迫的反应作为大气中二氧化碳浓度的函数。(1)第一个目的是证明在亚环境CO2浓度下光呼吸途径产生的高甘氨酸蛋白是集中在叶脉中的富含甘氨酸的细胞壁蛋白(GRP)。这将需要使用新的旋转-回声双共振(REDOR)实验，对完整的叶片和叶片的交联组分进行实验。GRP合成增加和亚环境叶片二氧化碳水平的相关性将是与水分胁迫相关的光呼吸短期功能的有力证据。(2)第二个目标是在真正的水分胁迫条件下用17O2标记甘氨酸，在这种条件下CO2吸收已经被抑制。通过向叶片注入标记的氧气，然后迫使叶片气孔完全关闭，在没有气体交换的情况下，可以测量沿着光呼吸途径的加氧酶活性几分钟到几个小时。(3)研究环境胁迫和化学胁迫下大豆叶片碳氮同化作用的相关性。该领域的生产力取决于对不断变化的条件的有效反应。REDOR核磁共振方法将被用来表征在正常条件下和在不久的将来预期的二氧化碳浓度升高下的全叶胁迫反应，以寻找可能对生产力有害的高二氧化碳诱导的植物对胁迫反应的修饰。(4)第四个目的是研究培养的大豆细胞交联壁部分的GRPs。植物可能会利用GRPs(和其他细胞壁蛋白质)来应对各种环境压力，细胞培养实验的结果将被用来指导对完整叶片的工作。如果关于光呼吸短期功能的假设是正确的，目前培育光呼吸被抑制的转基因作物的努力最终可能导致植物更不耐旱，在预计40年内大气二氧化碳水平增加的情况下产量更低。该项目可以指导基因修改更符合植物的整体代谢。满足预期的高大气二氧化碳浓度(550ppm，是地球上数百万年来最高的浓度)的要求将是本科生的目标，他们将在夏季被招募从事劳动密集型的大豆种植、标记、收获和提取。环境对未来农作物的影响是一个很好的话题，可以用来向社区宣传现代技术。PI是华盛顿大学为圣路易斯K-8科学教师举办的正式教育培训计划的积极参与者。

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.