Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves

Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue-print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross-reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd-GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue-specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants.