Regulatory Mechanism by Nitrogen of Gene Expression of Enzymes Involved in Photosynthesis and Nitrogen-Assimilation Pathways

本课题取得的主要成果如下： 1．通过在接近最佳光照和温度条件下向缺氮植物幼苗补充氮的过程中，通过检查氮在过渡状态下分配给 rubisCO、PEP 羧化酶和丙酮酸、Pi 二激酶，研究了玉米叶片中碳同化酶的氮对基因表达的调节。在完全发育的最年轻的叶子中，观察到这三种酶之间蛋白质和 mRNA 的积累存在差异。根据数据，PEP 羧化酶被判断为玉米叶光合作用的最限制性酶。还提供了证据表明氮是这三种酶的光刺激mRNA和蛋白质积累所必需的调节因子。2．天冬氨酸转氨酶是从 C_4 植物的 PEP 羧激酶型黍的叶肉细胞中纯化得到的。对该酶进行了部分表征，该酶似乎涉及光合碳同化，并制备了其特异性多克隆抗体。使用该抗体，从源自牛筋草(C_4植物的一种NAD-苹果酸酶类型)的mRNA的DNA文库制备该酶的互补cDNA。并制备了抗Gln合成酶和GOGAT的抗体。 3．研究了玉米叶PEP羧化酶的光/暗调节，并通过对暗失活和光激活形式的分析，提出了其可能的机制。

The results obtained in this project were summarized as follows.1. The regulation of gene expression by nitrogen of carbon assimilating enzymes in maize leaf was studied by examining partitioning of nitrogen into rubisCO, PEP carboxylase, and pyruvate, Pi dikinase in transitional state during supplementing nitrogen to nitrogen deficient plant seedlings grown under near optimal light and temperature conditions. In a fully developed youngest leaf a differential accumulation of protein and mRNA was observed among these three enzymes. Based upon the data PEP carboxylase was judged as a most limiting enzyme to photosynthesis in maize leaf. An evidence was also provided to suggest that nitrogen is a regulatory factor essential for the light-stimulation of accumulation of mRNA and protein for these three enzymes.2. Aspartate aminotransferase was purified from the mesophyll cells of Panicum maximum trichoglume , a PEP carboxykinase type of C_4 plant. Partial characterization of the enzyme which appears to involve photosynthetic carbon assimilation was achieved and its specific polyclonal antibody was prepared. Using this antibody the complementary cDNA of this enzyme was prepared from DNA library of mRNAs derived from Eleusine coracana, a NAD- malic enzyme type of C_4 plant. Also, antibodies against Gln synthetase and GOGAT were prepared.3. Light/dark regulation of maize leaf PEP carboxylase was studies and its possible mechanism was proposed based upon analysis of dark-inactivated and light-activated forms.