Vascular Bundle-Specific Localization of Cytosolic Glutamine Synthetase in Rice Leaves

Abstract

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 GSI was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissuespecific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of1itrogen exported from the senescing leaf in rice plants. GS2 catalyzes the first step in the assimilation of NH33 in higher plants (12,13). In many plants, there are two isoforms of GS in leaves: one located in the cytosol (GS1) and the other in the chloroplast stroma (GS2) (11,15). GOGAT is a second enzyme active in the transfer of the amide nitrogen of Gln to 2-oxoglutarate and hence in the generation of two Glu molecules (12,13,15 senescing leaves (10). Glu is a major free amino acid in rice (Oryza sativa L.) leaf blades (8), whereas Gln is a major form of the total amino acids in phloem sap of rice plants (6). Therefore, Glu in the blades is probably converted into Gln during the remobilization process. GS is a candidate for this conversion. Because the barley mutant lacking GS2 was able to grow normally under nonphotorespiratory conditions (22), GS1 in leaves could also be important in the synthesis of Gln for normal growth and development. Recently, we showed that the content of GS1 polypeptide remained constant during the natural senescence process in rice leaf blades, whereas that of the GS2 polypeptide declined (8).Although the intracellular distribution of GS isoforms is well established in leaves, there is little information with respect to their tissue localization. If GS1 were truly responsible for export of leaf nitrogen, it would be expected to be localized in close proximity to the phloem in leaf tissues. From molecular-genetic analyses, Edwards et al. (2) recently showed that the promoter for GS1 of pea nodules was expressed within the phloem elements, whereas that for GS2 was expressed within photosynthetic cell types in transgenic tobacco plants. However, direct evidence for tissue localization of GS isoforms in leaves has not yet been described. The same situation is also true for Fd-GO...

Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco

“…Moreover, the close location of the CC-SE complexes to a small number of xylem elements appears to be a prerequisite for a direct xylem-phloem transfer of amino acids translocated from roots (Atkins, 2000). The enzymes of ammonium metabolism, including cytosolic GS1 Kamachi et al, 1992;Pereira et al, 1992), Asn synthetase (Nakano et al, 2000), and GDH (Tercé-Laforgue et al, 2004), are colocalized in the companion cells as well. Although there is no molecular evidence showing that amino acid trafficking takes place by broad affinity amino acid transporters in minor veins (Fischer et al, 1998;van Bel, 2003), phloem loading of Gln, Asn, and Glu from CC-SE complexes likely regulates nitrogen translocation in the sieve streams where these amino acids are maintained at the constant levels.…”

Different location in dark‐adapted leaves of Phaseolus vulgaris of ribulose‐1,5‐bisphosphate carboxylase/oxygenase and 2‐carboxyarabinitol 1‐phosphate

“…However, the whole leaves were ground in liquid N2 or an isotonic medium. It is hard to distinguish the subcellular fractions from the tiny vein network from those from the mesophyll cells of the leaves in these methods, as has been claimed in the case of the subcellular distribution of glutamine synthetase I in rice leaves [28].…”

“…The xylem is a water-conducting tissue and the phloem, composed of sieve elements and companion cells, is a food-conducting tissue. Phloem tissues are the site of glutamine synthetase I in rice leaves [28]. Carboxyarabinitol accumulates in the vacuoles in mesophyll cells [29].…”

Impact of gaseous nitrogen deposition on plant functioning

“…It is located in the vascular bundle of root and leaves, where it might play a role in the synthesis of glutamine for transport (Schjøerring et al, 1993) and in recycling of the NH $ released in production of the monomers for lignin biosynthesis (Raven, Wollenweber & Handley, 1992). Expression of GS " was not found in leaf mesophyll cells of Nicotiana tabacum (Carvalho et al, 1992) and Oryza sativa (Kamachi et al, 1992). Migge et al (1996) found that cytosolic GS activity in cotyledons of Lycopersicon esculentum appeared to be encoded by at least two sets of genes.…”