Regulation and Assembly of Pyruvate Dehydrogenase Complexes

呼吸作用是活细胞利用能量做功。生长和繁殖都受到呼吸作用的影响，必须仔细控制呼吸，以避免生长减慢，就植物而言，还会降低农业生产率。尽管进行了大量研究，但关于植物细胞中呼吸作用是如何控制的细节仍然是一个谜。丙酮酸脱氢酶是一种多组分的酶复合体，位于植物细胞的特定亚细胞室内。它处在呼吸的多个组成部分之间相互作用的十字路口位置。这个综合体的位置非常理想，可以在呼吸的总体控制中发挥重要作用。此外，综合体的多组件架构允许来自几个不同机制的输入。调控机制的一个元素可能是丙酮酸脱氢酶复合体的内在成分。对这一成分的详细生化和分子分析将为推测的控制机制提供关键的洞察。此外，还将采用一种使用整个植物的遗传策略。将使用一种小型模型植物--老鼠耳水芹。分子遗传学实验将允许完全消除丙酮酸脱氢酶复合体的拟议控制成分。如果控制假说是正确的，不受控制的呼吸将导致小的、产量较低的植物。这些工厂将通过更换控制部件来拯救。将使用在实验室中修改过的本地控制组件和版本。这将使我们能够具体了解控制机制。与微生物或动物不同，植物细胞含有几种不同版本的丙酮酸脱氢酶复合体的另一种成分。初步研究表明，情况并不是在不同的时间或不同的地点有不同的版本那么简单。同样，老鼠耳朵的水芹植物将被操纵，以消除该组件的三个版本中的两个。这些实验将是迭代和组合的。这意味着将生成只包含组分1、2、3、1和2、1和3、1和3以及2和3的植物。这些操作将允许更好地了解每个组分对整个复合体的贡献。丙酮酸脱氢酶复合体的第三个成分一直是个谜。有相当多的证据表明它的存在，但到目前为止，它并不是孤立的。通过分子遗传学分离这种成分的尝试并不成功。将采取更经典的生化分离策略。然而，这将是一个基于其他植物和动物实验系统的结果的更具体的策略。分离这第三个组分将允许随后分离基因。一旦这一点完成，那么上述生物化学和分子策略将被应用。从这些实验中获得的信息将增进对植物生长发育的基本了解。此外，这一结果有可能使研究人员通过改变植物细胞呼吸的控制来提高农业生产率。最后，研究结果将为通过经典育种或生物技术设计更高效的农作物提供参考。

Respiration is the use of energy by living cells to do work. Both growth and reproduction are affected by respiration and it must be carefully controlled to avoid decreased growth and, in the case of plants, reduced agricultural productivity. Despite considerable research, details of how respiration is controlled in plant cells remain a puzzle. Pyruvate dehydrogenase is a multi-component enzyme complex located within a specific sub-cellular compartment of plant cells. It occupies a cross-roads position where there is interaction among multiple components of respiration. This complex is ideally situated to play a major role in the overall control of respiration. Furthermore, the multi-component architecture of the complex allows input from several different mechanisms. One element of the regulatory scheme may be an intrinsic component of the pyruvate dehydrogenase complex. Detailed biochemical and molecular analyses of this component will provide critical insight into the putative mechanism of control. In addition, a genetic strategy that uses whole plants will be employed. A small model plant, mouse-eared cress, will be used. Molecular genetic experiments will allow the complete elimination of the proposed control component of the pyruvate dehydrogenase complex. If the control hypothesis is correct, uncontrolled respiration will result in small, less-productive plants. These plants will be rescued by replacing the control component. Both the native control component and versions that have been modified in the laboratory will be used. This will allow specific understanding of the mechanism of control. In contrast to microbes or animals, plant cells contain several different versions of another component of the pyruvate dehydrogenase complex. Preliminary studies have shown that the situation is not so simple as having different versions functioning at different times or in different places. Again, the mouse-eared cress plant will be manipulated so as to eliminate two of the three versions of this component. These experiments will be iterative and combinatorial. This means that plants will be generated that contain only component 1, only 2, only 3, 1 and 2, 1 and 3, and 2 and 3. These manipulations will allow a better understanding of the contribution that each component makes to the whole complex. A third component of the pyruvate dehydrogenase complex has been an enigma. There is considerable evidence for its existence, but it has thus far not been isolated. Attempts to isolate this component by molecular genetics have not been successful. The return to a more classical biochemical isolation strategy will be undertaken. However it will be a more specific strategy based upon results from other plant and animal experimental systems. Isolation of this third component will allow subsequent isolation of the gene. Once this is accomplished, then the biochemical and molecular strategies described above will be applied. The information gained from these experiments will improve basic understanding of plant growth and development. Furthermore, there is the potential that the results will allow researchers to increase agricultural productivity by altering the control of plant cell respiration. Finally, the results will inform the design of more efficient crop plants through classical breeding or biotechnology.