Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (K m ؍ 2450 ؎ 150 M) and glutamate (K m ؍ 3.8 ؎ 0.2 mM). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (K m for ammonium < 10 M; K m for glutamate ؍ 1.1 ؎ 0.4 mM) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.Glutamine synthetase (GS 1 ; EC 6.3.1.2) is responsible for the primary assimilation of ammonium in higher plants (1-4). Ammonium is assimilated into glutamine and glutamate through a consecutive reaction of GS and glutamate synthase (GOGAT), the so-called GS/GOGAT cycle. Plants have two types of GS isoenzymes that localize in different compartments: one located in the cytosol (GS1) and the other in the plastid/chloroplasts (GS2) (1-4). GS1 is the major form of GS in plant roots, and the ammonium taken up from the soil is directly converted to Gln by its reaction. Molecular biological studies have identified a number of genes encoding GS1 from various plant species (5-9). The presence of multiple GS1 isoenzymes complicates the overall understanding of their physiological functions. The isoenzymes of GS1 show organ-and cell-specific patterns of expression and are developmentally regulated (10 -17). In addition, the expression of GS1 is metabolically regulated by the availability of nitrogen and carbon sources (18 -22, 24).In the roots of legumes, GS1 is regulated by ammonium supplied from the environment or the symbiotic nitrogen fixation (18 -21). Transgenic studies with the soybean GS1 promoter suggested that ammonium-dependent regulation is specific for nitrogen assimilation in leguminous plants; the soybean-derived GS1...
Immunocytological studies in this laboratory have suggested that NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in developing organs of rice (Oryza sativa L. cv. Sasanishiki) is involved in the utilization of glutamine remobilized from senescing organs through the phloem. Because most of the indica cultivars contained less NADH-GOGAT in their sink organs than japonica cultivars, over-expression of NADH-GOGAT gene from japonica rice was investigated using Kasalath, an indica cultivar. Several T0 transgenic Kasalath lines over-producing NADH-GOGAT under the control of a NADH-GOGAT promoter of Sasanishiki, a japonica rice, showed an increase in grain weight (80% as a maximum), indicating that NADH-GOGAT is indeed a key step for nitrogen utilization and grain filling in rice. A genetic approach using 98 backcross-inbred lines (BC(1)F(6)) developed between Nipponbare (a japonica rice) and Kasalath were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), which is probably involved in the export of nitrogen from senescing organs and those of NADH-GOGAT. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these BC(1)F(6). Seven chromosomal QTL regions were detected for GS1 protein content and six for NADH-GOGAT. Some of these QTLs were located in QTL regions for various biochemical and agronomic traits affected by nitrogen recycling. The relationships between the genetic variability of complex agronomic traits and traits for these two enzymes are discussed.
Results and discussionBiomass production, yield and NUE. In the experimental conditions tested when more than 10.0 g N m −2 N fertilizer was supplied, the dry matter production and brown rice yield in RBCS-sense plants were increased by up to 23% (Fisher's test, P = 0.000-0.006) and up to 28% (Fisher's test, P = 0.000-0.016), respectively, compared to wild-type plants (Tables 1 and 2). The greatest dry matter production and brown rice yield from the RBCS-sense plants were observed in the 15.0 g N m −2 plot in 2019, at 1,657 g m −2 and 706 g m −2 , respectively, and the highest ratios of increase for both parameters were found in the 14.1 g N m −2 plot in 2018. In the plot with no N application from 2017 to 2019, no difference in dry matter production (Fisher's test, P = 0.506 in 2017; P = 0.208 in 2018; P = 0.208 in 2019) or brown rice yield (Fisher's test, P = 0.842 in 2017; P = 0.335 in 2018; P = 0.911 in 2019) was observed between RBCS-sense and wild-type plants, and brown rice yield of RBCSsense plants in the 7.1 g N m −2 plot in 2018 was lower than that of wild-type plants (Fisher's test, P = 0.000) (Tables 1 and 2). In RBCSantisense plants, the brown rice yield was 14-34% lower than in wild-type plants (Fisher's test, P = 0.000-0.030)-this was regardless of N application except in the plot with no N application in 2019 (Fisher's test, P = 0.934) (Tables 1 and 2). The lack of difference in 2019 might have been the effect of 2 yr without N fertilization. In the 17.0 g N m −2 plot in 2017, all plants were blown down by two typhoons and then almost lodged during their ripening stagestheir dry matter production and brown rice yield were, therefore, The green revolution's breeding of semi-dwarf rice cultivars in the 1960s improved crop yields, with large increases in the use of nitrogen (N) fertilizer. However, excess N application has caused serious environmental problems, including acid rain and the eutrophication of rivers and oceans. To use N to improve crop yields, while minimizing the associated environmental costs, there is a need to produce crops with higher N-use efficiency and higher yield components. Here we show that transgenic rice overproducing ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco)-the key enzyme of photosynthesis-exhibits increased yields with improved N-use efficiency for increasing biomass production when receiving sufficient N fertilization in an experimental paddy field. This field experiment demonstrates an improvement in photosynthesis linked to yield increase due to a higher N-use efficiency in a major crop.
Rice plants grown in anaerobic paddy soil prefer to use ammonium ion as an inorganic nitrogen source for their growth. The ammonium ions are assimilated by the coupled reaction of glutamine synthetase (GS) and glutamate synthase (GOGAT). In rice, there is a small gene family for GOGAT: there are two NADH-dependent types and one ferredoxin (Fd)-dependent type. Fd-GOGAT is important in the re-assimilation of photorespiratorily generated ammonium ions in chloroplasts. Although cell-type and age-dependent expression of two NADH-GOGAT genes has been well characterized, metabolic function of individual gene product is not fully understood. Reverse genetics approach is a direct way to characterize functions of isoenzymes. We have isolated a knockout rice mutant lacking NADH-dependent glutamate synthase1 (NADH-GOGAT1) and our studies show that this isoenzyme is important for primary ammonium assimilation in roots at the seedling stage. NADH-GOGAT1 is also important in the development of active tiller number, when the mutant was grown in paddy field until the harvest. Expression of NADH-GOGAT2 and Fd-GOGAT in the mutant was identical with that in wild-type, suggesting that these GOGATs are not able to compensate for NADH-GOGAT1 function.
Ninety-eight backcross inbred lines (BC1F6) developed between Nipponbare, a japonica rice, and Kasalath, an indica rice were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) and NADH-glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in leaves. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these backcross inbred lines. Seven chromosomal QTL regions for GS1 protein content and six for NADH-GOGAT protein content were detected. Some of these QTLs were located in QTL regions for various biochemical and physiological traits affected by nitrogen recycling. These findings suggested that the variation in GS1 and NADH-GOGAT protein contents in this population is related to the changes in the rate of nitrogen recycling from senescing organs to developing organs, leading to changes in these physiological traits. Furthermore, a structural gene for GS1 was mapped between two RFLP markers, C560 and C1408, on chromosome 2 and co-located in the QTL region for one-spikelet weight. A QTL region for NADH-GOGAT protein content was detected at the position mapped for the NADH-GOGAT structural gene on chromosome 1. A QTL region for soluble protein content in developing leaves was also detected in this region. Although fine mapping is required to identify individual genes in the future, QTL analysis could be a useful post-genomic tool to study the gene functions for regulation of nitrogen recycling in rice.
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