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和利用的关键酶。本研究采用免疫金电镜技术，对该酶蛋白在水稻根中的胞内定位进行了研究。作为第一个实例，我们确定NADH-GOGAT蛋白定位于根的表皮、外皮和中央圆柱体的质体中。我们还成功地从大麦根中纯化了该酶，进一步分离了水稻NADH-GOGAT的基因组克隆和cDNA克隆，并对其基因结构进行了深入分析。将核苷酸序列登记到基因库中。该基因的表达已被研究在响应水稻幼苗根中的氮供应。在GOGAT基因和谷氨酰胺合成酶基因中，NADH-GOGAT基因在NH_4^+供应后3小时内被特异性诱导。通过比较不同类型水稻（粳稻、籼稻和爪哇稻）叶片中NADH-GOGAT蛋白的含量，发现籼稻叶片中NADH-GOGAT蛋白的含量低于粳稻叶片中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.