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

Overexpression of AtCpNifS enhances selenium tolerance and accumulation in Arabidopsis

Selenium (Se) is an essential element for many organisms but is toxic at higher levels. CpNifS is a chloroplastic NifS-like protein in Arabidopsis (Arabidopsis thaliana) that can catalyze the conversion of cysteine into alanine and elemental sulfur (S 0 ) and of selenocysteine into alanine and elemental Se (Se 0 ). We overexpressed CpNifS to investigate the effects on Se metabolism in plants. CpNifS overexpression significantly enhanced selenate tolerance (1.9-fold) and Se accumulation (2.2-fold). CpNifS overexpressors showed significantly reduced Se incorporation into protein, which may explain their higher Se tolerance. Also, sulfur accumulation was enhanced by approximately 30% in CpNifS overexpressors, both on media with and without selenate. Root transcriptome changes in response to selenate mimicked the effects observed under sulfur starvation. There were only a few transcriptome differences between CpNifS-overexpressing plants and wild type, besides the 25-to 40-fold increase in CpNifS levels. Judged from x-ray analysis of near edge spectrum, both CpNifS overexpressors and wild type accumulated mostly selenate (Se VI ). In conclusion, overexpression of this plant NifS-like protein had a pronounced effect on plant Se metabolism. The observed enhanced Se accumulation and tolerance of CpNifS overexpressors show promise for use in phytoremediation

Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2

Abstract Background Our previous transcriptomic analysis revealed that downregulation of nitrogen and carbon metabolism in the basal portions of the shoots inhibited cytosolic glutamine synthetase1;2 (GS1;2), which severely reduced rice tiller number. In the present study, we used rice mutants lacking GS1;2 (gs1;2 mutants) to determine the contribution of carbon metabolism to tiller growth. Results Metabolomic analysis indicated the effects of carbon metabolism disorder such as reductions in the levels of sugar metabolites (e.g., sucrose and glucose 6-phosphate) in the shoot basal portions of the gs1;2 mutant seedlings. Decrease in sucrose caused by the lack of GS1;2 was successfully restored to the wild-type levels by introducing OsGS1;2 cDNA into the mutants. In the basal portions of the shoots, the lack of GS1;2 caused low expression of cytosolic fructose 1,6-bisphosphatase2 (OscFBP2), which is a key cytosolic sucrose synthesis enzyme; it is especially important in the phloem companion cells of the nodal vascular anastomoses. NH4 + supply upregulated OscFBP2 expression in the shoot basal portions of the wild type but not in those of the gs1;2 mutants. Rice mutants lacking cFBPase2 presented with ~ 30% reduction in total cFBPase activity in the basal portions of their shoots. These mutants displayed reductions in sucrose levels of the basal portions of their shoots but not in their leaf blades. They also had relatively lower tiller numbers at the early growth stage. Conclusions Metabolomic analysis revealed that the lack of GS1;2 reduced sucrose metabolism in the basal portions of the shoots. Our results indicated that sucrose reduction was caused by the downregulation of OscFBP2 expression in the basal portions of the gs1;2 mutant shoots. The reduction in sucrose content caused by the lack of cFBPase2 resulted in lower tiller number at the early growth stage. Therefore, adequate sucrose supply via cFBPase2 may be necessary for tiller growth in the basal portions of rice shoots