Localization of nitrogen-related enzymes in higher plants

NADH依赖的谷氨酸合成酶(GOGAT)是高等植物氮素同化利用的关键酶。然而，关于该酶在细胞内的定位和分子性质还知之甚少。在这项联合研究中，该酶蛋白在水稻根中通过免疫金电子显微镜方法被证明是在细胞内定位的。作为第一个例子，我们确定NADH-GOGAT蛋白定位于根的表皮、外胚层和中央圆柱体的质体中。此外，我们还成功地从大麦根中纯化了NADH-GOGAT酶，并对水稻NADH-GOGAT的基因组克隆和cDNA克隆进行了分离和测序，并对其基因结构进行了深入的分析。核苷酸序列在基因库中注册。该基因的表达已被研究对水稻幼苗根中氮素供应的响应。在另一个GOGAT基因和谷氨酰胺合成酶基因中，NADH-GOGAT基因在提供NH4+后3小时内被特异性地诱导。通过对不同类型水稻叶片中NADH-GOGAT蛋白含量的比较发现，不同类型水稻叶片中NADH-GOGAT蛋白的含量均低于不同类型水稻品种的叶片中NADH-GOGAT蛋白的含量。

NADH-dependent glutamate synthase (GOGAT) is a key enzyme in nitrogen assimilation and utilization in higher plants. However, little is known on the intracellular localization and molecular properties of this enzyme.In this joint research, intracellular localization of this enzyme protein has been demonstrated by the methods of immuno-gold electron microscopy in rice roots. As the first instance, we determine that the NADH-GOGAT protein locates in plastids of epidermis, exodermis, and central cylinder of the roots. We also succeeded to purify the enzyme from barley roots.Further, genomic clones, as well as cDNA clones, for rice NADH-GOGAT have been isolated and sequenced and the gene structure was thoroughly analyzed. The nucleotide sequences were registered into a Gene-Bank. Expression of this gene has been studied in response to the nitrogen supply in the roots of rice seedlings. The NADH-GOGAT gene is specifically induced within 3 hr after the supply of NH_4^+ among another GOGAT gene and glutamine synthetase genes. When the content of NADH-GOGAT protein in developing leaves were compared among various types of rice, i.e.japonica, indica, and javanica, the leaves in indica-type rice contained less amounts of the protein than those in japonica-type.From those studies, molecular and physiological properties of NADH-GOGAT are getting clear, at least in rice plants.