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Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of -7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants.The major source of nitrogen for developing leaves and ears in mature rice plants is the nitrogen released from older senescing leaves. Our previous study with 'sN showed that remobilized nitrogen accounted for 64% of the total nitrogen in the youngest leaf blades (12). Rubisco, the major soluble protein in leaves, decreases with senescence and is the principal source of transported nitrogen (8,12 Glutamate is a major free amino acid in mature leaf blades of rice. Recently, Hayashi and Chino (7) showed that glutamine and asparagine accounted for 42 and 12%, respectively, of the total amino acids in phloem sap of rice plants. These amides are derived from amino acids and ammonia released by the hydrolysis of Rubisco, other leaf proteins, and Chl.GS2 is a candidate for the conversion of glutamate and NH4' to glutamine in senescing leaves and the resultant glutamine could be the precursor for asparagine (18,19).However, GS activity is known to decrease rapidly during either natural senescence of wheat leaves (2, 21) or dark induced senescence of detached Lolium temulentum leaves (24) and radish cotyledons (10). In many plants, there are two isoforms of GS in leaves (17): one located in the cytosol (GS 1) and the other in the chloroplast stroma (GS2). Because the physiological function of GS2 is considered to be the reassimilation of NH4' released during photorespiration (26,27) and because the rate of photosynth...

The amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), total chlorophyll (Chi), and total leaf nitrogen were measured in fully expanded, young leaves of wheat (Triticum aestivum L.), rice (Oryza sativa L.), spinach (Spinacia oleracea L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.). In addition, the activities of whole-chain electron transport and carbonic anhydrase were measured. All plants were grown hydroponically at different nitrogen concentrations. Although a greater than proportional increase in Rubisco content relative to leaf nitrogen content and Chi was found with increasing nitrogen supply for rice, spinach, bean, and pea, the ratio of Rubisco to total leaf nitrogen or Chi in wheat was essentially independent of nitrogen treatment. In addition, the ratio of Rubisco to electron transport activities remained constant only in wheat. Nevertheless, gas-exchange analysis showed that the in vivo balance between the capacities of Rubisco and electron transport in wheat, rice, and spinach remained almost constant, irrespective of nitrogen treatment. The in vitro carbonic anhydrase activity in wheat was very low and strongly responsive to increasing nitrogen content. Such a response was not found for the other C3 plants examined, which had 10-to 30-fold higher carbonic anhydrase activity than wheat at any leafnitrogen content. These distinctive responses of carbonic anhydrase activity in wheat were discussed in relation to CO2-transfer resistance and the in vivo balance between the capacities of Rubisco and electron transport. ach, clarified the relation between nitrogen nutrition and nitrogen partitioning into the various photosynthetic components and activities. They found that although nitrogen supply increased the ratio of Rubisco activity to electron transport activity, ATPase, Chl, or total leaf nitrogen, the balance between the in vivo activities of Rubisco and electron transport remained constant. They concluded that this difference was compensated for by the presence of a C02-transfer resistance between intercellular air spaces and the carboxylation sites. As a result of this resistance, the in vivo Rubisco specific activity was reduced progressively with increasing amount of enzyme because the partial pressure of CO2 at the carboxylation sites was reduced and kept in a constant balance with electron transport activity. The increase in the ratio of Rubisco to total leaf nitrogen or Chl with nitrogen supply is frequently found for other C3 species, such as tobacco (1), cotton (32), Solanum (11), bean (26), and pea (18).However, in spite of the existence of significant C02-transfer resistance in wheat (5,8,23, 30), the ratio of Rubisco to total leaf nitrogen or Chl in fully expanded young leaves seems to be independent of nitrogen nutrition (5,17,18 Plant Physiol. Vol. 100, 1992 CO2 diffusion to the carboxylation sites remains uncertain. In addition, the response of CA activity to changing nitrogen content is not known.In this study, we used fully expanded, youn...

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...

Abstract.Tissue and cellular localization of NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) in the unexpanced leaf blades and young grains of rice (Oryza sativa L.) was investigated using tissueprint immunoblot and immunocytological methods with an affinity-purified anti-NADH-GOGAT immunoglobulin G. Tissue-print immunoblots showed that the NADH-GOGAT protein was mostly located in large and small vascular bundles of the unexpanded blades. When the cross-sections (10 Ixm in thickness) prepared from the paraffin-embedded blades were stained with the antibody, the NADH-GOGAT protein was detected in vascular-parenchyma cells and mestome-sheath cells. In developing grains, the NADH-GOGAT protein was detected in both phloem-and xylem-parenchyma cells of dorsal and lateral vascular bundles, and in the nucellar projection, nucellar epidermis, and aleurone cells. On the other hand, ferredoxin (Fd)-dependent GOGAT (EC 1.4.7.1) was located mainly in mesophyll cells of the leaf blade and in chloroplast-containing cross-cells of the pericarp of the grains. The spatial expression of these GOGAT proteins indicates distinct and non-overlapping roles in rice plants. In the leaf blades and young grains, NADH-GOGAT could be involved in the synthesis of glutamate from the glutamine that is transported through the vascular system from roots and senescing tissues.