Tissue and Cellular Localization of NADH-Dependent Glutamate Synthase Protein in Leaves of Spinach

Tissue and cellular localization of NADH-dependent glutamate synthase (NADH-GOGAT, EC1.4. 1.14) in young leaves of dicotyledonous spinach (Spinacia oleacea) was investigated using the immunocytological method with an affinity-purified anti-NADH-GOGAT immunoglobulin G. Immunoblotting analysis showed this antibody specifically cross-reacted with NADH-GOGAT protein in crude soluble proteins from young leaf blades of spinach. When transverse sections (10μm in thickness) prepared from the paraffin-embedded young leaf blades of spinach were stained with the anti-NADH-GOGAT antibody, strong signals for NADH-GOGAT protein were detected in companion cells of large vascular bundles. Weak signals for the NADH-GOGAT protein were also detected in vascular parenchyma cells and mesophyll cells of young leaves. Ferredoxin (Fd)-GOGAT (EC 1.4.7.1) protein was mainly located in mesophyll cells and signals for the protein were also detected in companion cells and xylemparenchyma cells of large vascular bundles. By the way, in young leaf blades of monocotyledonous rice, NADH-GOGAT protein specifically located in vascular cells and Fd-GOGAT protein was abundant in mesophyll cells (Hayakawa et al., 1994). The differences of functions in young leaves for NADH-GOGAT proteins between monocotyledonous rice and dicotyledonous spinach are discussed

Molecular Basis on Nitrogen Utilization in Rice(Recent Topics of the Agricultunal Biological Science in Tohoku University)

Rice (Oryza sativa L.) is the major provision for half of the world population and is the important model crop in terms of synteny. Nitrogen is a massive prerequisite element for rice during its life span. During evolutionary processes, rice has acquired strategic systems of nitrogen metabolism for the survival, i.e., the highly efficient ammonium assimilation in roots and nitrogen remobilization (nitrogen recycling). In our laboratory, research is underway to elucidate molecular mechanisms, cellular functions and the communication mechanisms in nitrogen metabolisms, especially ammonium assimilation in roots and nitrogen recycling, in rice. In this article, aim and overview of our research projects, and some recent research topics are shown

Disruption of a Novel NADH-Glutamate Synthase2 Gene Caused Marked Reduction in Spikelet Number of Rice

Inorganic ammonium ions are assimilated by a coupled reaction of glutamine synthetase and glutamate synthase (GOGAT). In rice, three genes encoding either ferredoxin (Fd)-GOGAT, NADH-GOGAT1, or NADH-GOGAT2, have been identified. OsNADH-GOGAT2, a newly identified gene, was expressed mainly in fully expanded leaf blades and leaf sheaths. Although the distinct expression profile to OsNADH-GOGAT1, which is mainly detected in root tips, developing leaf blades, and grains, was shown in our previous studies, physiological role of NADH-GOGAT2 is not yet known. Here, we isolated retrotransposon mediated-knockout mutants lacking OsNADH-GOGAT2. In rice grown under paddy field conditions, disruption of the OsNADH-GOGAT2 gene caused a remarkable decrease in spikelet number per panicle associated with a reductions in yield and whole plant biomass, when compared with wild-type (WT) plants. The total nitrogen contents in the senescing leaf blade of the mutants were approximately a half of the WT plants. Expression of this gene was mainly detected in phloem companion cells and phloem parenchyma cells associated with large vascular bundles in fully expanded leaf blades, when the promoter region fused with a β-glucuronidase gene was introduced into the WT rice. These results suggest that the NADH-GOGAT2 is important in the process of glutamine generation in senescing leaves for the remobilization of leaf nitrogen through phloem to the panicle during natural senescence. These results also indicate that other GOGATs, i.e., NADH-GOGAT1 and ferredoxin-GOGAT are not able to compensate the function of NADH-GOGAT2