Cytosolic Glutamine Synthetase GS1;3 Is Involved in Rice Grain Ripening and Germination.

Ammonium is combined with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase (GS or GLN). Plants harbor several isoforms of cytosolic GS (GS1). Rice GS1;3 is highly expressed in seeds during grain filling and germination, suggesting a unique role in these processes. This study aimed to investigate the role of GS1;3 for rice growth and yield. Tos17 insertion lines for GS1;3 were isolated, and the nitrogen (N), amino acid, and ammonium contents of GS1;3 mutant grains were compared to wild-type grains. The spatiotemporal expression of GS1;3 and the growth and yield of rice plants were evaluated in hydroponic culture and the paddy field. Additionally, the stable isotope of N was used to trace the foliar N flux during grain filling. Results showed that the loss of GS1;3 retarded seed germination. Seeds of GS1;3 mutants accumulated glutamate but did not show a marked change in the level of phytohormones. The expression of GS1;3 was detected at the beginning of germination, with limited promoter activity in seeds. GS1;3 mutants showed a considerably decreased ripening ratio and decreased N efflux in the 12th leaf blade under N deficient conditions. The ß-glucuronidase gene expression under control of the GS1;3 promoter was detected in the vascular tissue and aleurone cell layer of developing grains. These data suggest unique physiological roles of GS1;3 in the early stage of seed germination and grain filling under N deficient conditions in rice.

Cytosolic GLUTAMINE SYNTHETASE1;1 Modulates Metabolism and Chloroplast Development in Roots.

Nitrogen (N) is an essential macronutrient, and the final form of endogenous inorganic N is ammonium, which is assimilated by Gln synthetase (GS) into Gln. However, how the multiple isoforms of cytosolic GSs contribute to metabolic systems via the regulation of ammonium assimilation remains unclear. In this study, we compared the effects of two rice (Oryza sativa) cytosolic GSs, namely OsGS1;1 and OsGS1;2, on central metabolism in roots using reverse genetics, metabolomic and transcriptomic profiling, and network analyses. We observed (1) abnormal sugar and organic N accumulation and (2) significant up-regulation of genes associated with photosynthesis and chlorophyll biosynthesis in the roots of Osgs1;1 but not Osgs1;2 knockout mutants. Network analysis of the Osgs1;1 mutant suggested that metabolism of Gln was coordinated with the metabolic modules of sugar metabolism, tricarboxylic acid cycle, and carbon fixation. Transcript profiling of Osgs1;1 mutant roots revealed that expression of the rice sigma-factor (OsSIG) genes in the mutants were transiently upregulated. GOLDEN2-LIKE transcription factor-encoding genes, which are involved in chloroplast biogenesis in rice, could not compensate for the lack of OsSIGs in the Osgs1;1 mutant. Microscopic analysis revealed mature chloroplast development in Osgs1;1 roots but not in the roots of Osgs1;2, Osgs1;2-complemented lines, or the wild type. Thus, organic N assimilated by OsGS1;1 affects a broad range of metabolites and transcripts involved in maintaining metabolic homeostasis and plastid development in rice roots, whereas OsGS1;2 has a more specific role, affecting mainly amino acid homeostasis but not carbon metabolism.

The urea transporter DUR3 contributes to rice production under nitrogen-deficient and field conditions.

Nitrogen is one of the most important elements for plant growth, and urea is one of the most frequently used nitrogen fertilizers worldwide. Besides the exogenously-supplied urea to the soil, urea is endogenously synthesized during secondary nitrogen metabolism. Here, we investigated the contribution of a urea transporter, DUR3, to rice production using a reverse genetic approach combined with localization studies. Tos17 insertion lines for DUR3 showed a 50% yield reduction in hydroponic culture, and a 26.2% yield reduction in a paddy field, because of decreased grain filling. Because shoot biomass production and shoot total N was not reduced, insertion lines were disordered not only in nitrogen acquisition but also in nitrogen allocation. During seed development, DUR3 insertion lines accumulated nitrogen in leaves and could not sufficiently develop their panicles, although shoot and root dry weights were not significantly different from the wild-type. The urea concentration in old leaf harvested from DUR3 insertion lines was lower than that in wild-type. DUR3 promoter-dependent ß-glucuronidase (GUS) activity was localized in vascular tissue and the midribs of old leaves. These results indicate that DUR3 contributes to nitrogen translocation and rice yield under nitrogen-deficient and field conditions.

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

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.

Outgrowth of Rice Tillers Requires Availability of Glutamine in the Basal Portions of Shoots.

BACKGROUND: Our previous studies concluded that metabolic disorder in the basal portions of rice shoots caused by a lack of cytosolic glutamine synthetase1;2 (GS1;2) resulted in a severe reduction in the outgrowth of tillers. Rice mutants lacking GS1;2 (gs1;2 mutants) showed a remarkable reduction in the contents of both glutamine and asparagine in the basal portions of shoots. In the current study, we attempted to reveal the mechanisms for this decrease in asparagine content using rice mutants lacking either GS1;2 or asparagine synthetase 1 (AS1). The contributions of the availability of glutamine and asparagine to the outgrowth of rice tillers were investigated. RESULTS: Rice has two AS genes, and the enzymes catalyse asparagine synthesis from glutamine. In the basal portions of rice shoots, expression of OsAS1, the major species in this tissue, was reduced in gs1;2 mutants, whereas OsAS2 expression was relatively constant. OsAS1 was expressed in phloem companion cells of the nodal vascular anastomoses connected to the axillary bud vasculatures in the basal portions of wild-type shoots, whereas cell-specific expression was markedly reduced in gs1;2 mutants. OsAS1 was up-regulated significantly by NH4+ supply in the wild type but not in gs1;2 mutants. When GS reactions were inhibited by methionine sulfoximine, OsAS1 was up-regulated by glutamine but not by NH4+. The rice mutants lacking AS1 (as1 mutants) showed a decrease in asparagine content in the basal portions of shoots. However, glutamine content and tiller number were less affected by the lack of AS1. CONCLUSION: These results indicate that in phloem companion cells of the nodal vascular anastomoses, asparagine synthesis is largely dependent on glutamine or its related metabolite-responsive AS1. Thus, the decrease in glutamine content caused by a lack of GS1;2 is suggested to result in low expression of OsAS1, decreasing asparagine content. However, the availability of asparagine generated from AS1 reactions is apparently less effective for the outgrowth of tillers. With respect to the tiller number and the contents of glutamine and asparagine in gs1;2 and as1 mutants, the availability of glutamine rather than asparagine in basal portions of rice shoots may be required for the outgrowth of rice tillers.

Lack of ACTPK1, an STY kinase, enhances ammonium uptake and use, and promotes growth of rice seedlings under sufficient external ammonium.

Ammonium influx into plant roots via the high-affinity transport system (HATS) is down-modulated under elevated external ammonium, preventing ammonium toxicity. In ammonium-fed Arabidopsis, ammonium transporter 1 (AMT1) trimers responsible for HATS activity are allosterically inactivated in a dose-dependent manner via phosphorylation of the conserved threonine at the carboxyl-tail by the calcineurin B-like protein 1-calcineurin B-like protein-interacting protein kinase 23 complex and other yet unidentified protein kinases. Using transcriptome and reverse genetics in ammonium-preferring rice, we revealed the role of the serine/threonine/tyrosine protein kinase gene OsACTPK1 in down-modulation of HATS under sufficient ammonium. In wild-type roots, ACTPK1 mRNA and protein accumulated dose-dependently under sufficient ammonium. To determine the function of ACTPK1, two independent mutants lacking ACTPK1 were produced by retrotransposon Tos17 insertion. Compared with segregants lacking insertions, the two mutants showed decreased root growth and increased shoot growth under 1 mm ammonium due to enhanced ammonium acquisition, via aberrantly high HATS activity, and use. Furthermore, introduction of OsACTPK1 cDNA fused to the synthetic green fluorescence protein under its own promoter complemented growth and the HATS influx, and suggested plasma membrane localization. Root cellular expression of OsACTPK1 also overlapped with that of ammonium-induced OsAMT1;1 and OsAMT1;2. Meanwhile, threonine-phosphorylated AMT1 levels were substantially decreased in roots of ACTPK1-deficient mutants grown under sufficient ammonium. Bimolecular fluorescence complementation assay further confirmed interaction between ACTPK1 and AMT1;2 at the cell plasma membrane. Overall, these findings suggest that ACTPK1 directly phosphorylates and inactivates AMT1;2 in rice seedling roots under sufficient ammonium.

Cytosolic Glutamine Synthetase Isozymes Play Redundant Roles in Ammonium Assimilation Under Low-Ammonium Conditions in Roots of Arabidopsis thaliana.

Ammonium is a major nitrogen source for plants; it is assimilated into glutamine via a reaction catalyzed by glutamine synthetase (GLN). Arabidopsis expresses four cytosolic GLN genes, GLN1; 1, GLN1; 2, GLN1; 3 and GLN1; 4, in roots. However, the function and organization of these GLN1 isozymes in ammonium assimilation in roots remain unclear. In this study, we aimed to characterize the four GLN1 isozymes. The levels of growth of the wild type and gln1 single and multiple knockout lines were compared in a hydroponic culture at ammonium concentrations of 0.1 and 3 mM. Under the low-ammonium concentration, in single mutants for each GLN1 gene, there was little effect on growth, whereas the triple mutant for GLN1; 1, GLN1; 2 and GLN1; 3 grew slowly and accumulated ammonium. Under the high-ammonium concentration, the single mutant for GLN1; 2 showed 50% decreases in fresh weight and glutamine, whereas the other gln1 single mutants did not show notable changes in the phenotype. The double mutant for GLN1; 1 and GLN1; 2 showed less growth and a lower glutamine concentration than the single mutant for GLN1; 2. Promoter analysis indicated an overlapping expression of GLN1; 1 with GLN1; 2 in the surface layers of the roots. We thus concluded that: (i) at a low concentration, ammonium was assimilated by GLN1; 1, GLN1; 2 and GLN1; 3, and they were redundant; (ii) low-affinity GLN1; 2 could contribute to ammonium assimilation at concentrations ranging from 0.1 to 3 mM; and (iii) GLN1; 1 supported GLN1; 2 within the outer cell layers of the root.

Nitrate Supply-Dependent Shifts in Communities of Root-Associated Bacteria in Arabidopsis.

Root-associated bacterial communities are necessary for healthy plant growth. Nitrate is a signal molecule as well as a major nitrogen source for plant growth. In this study, nitrate-dependent alterations in root-associated bacterial communities and the relationship between nitrate signaling and root-associated bacteria in Arabidopsis were examined. The bacterial community was analyzed by a ribosomal RNA intergenic spacer analysis (RISA) and 16S rRNA amplicon sequencing. The Arabidopsis root-associated bacterial community shifted depending on the nitrate amount and timing of nitrate application. The relative abundance of operational taxonomic units of 25.8% was significantly changed by the amount of nitrate supplied. Moreover, at the family level, the relative abundance of several major root-associated bacteria including Burkholderiaceae, Paenibacillaceae, Bradyrhizobiaceae, and Rhizobiaceae markedly fluctuated with the application of nitrate. These results suggest that the application of nitrate strongly affects root-associated bacterial ecosystems in Arabidopsis. Bulk soil bacterial communities were also affected by the application of nitrate; however, these changes were markedly different from those in root-associated bacteria. These results also suggest that nitrate-dependent alterations in root-associated bacterial communities are mainly affected by plant-derived factors in Arabidopsis. T-DNA insertion plant lines of the genes for two transcription factors involved in nitrate signaling in Arabidopsis roots, NLP7 and TCP20, showed similar nitrate-dependent shifts in root-associated bacterial communities from the wild-type, whereas minor differences were observed in root-associated bacteria. Thus, these results indicate that NLP7 and TCP20 are not major regulators of nitrate-dependent bacterial communities in Arabidopsis roots.

Lack of Cytosolic Glutamine Synthetase1;2 Activity Reduces Nitrogen-Dependent Biosynthesis of Cytokinin Required for Axillary Bud Outgrowth in Rice Seedlings.

A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in phloem companion cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.

A temporal and spatial contribution of asparaginase to asparagine catabolism during development of rice grains.

BACKGROUND: Asparagine is one of the most dominant organic nitrogen compounds in phloem and xylem sap in a wide range of plant species. Asparaginase (ASNase; EC, 3.5.1.1) catabolizes asparagine into aspartate and ammonium; therefore, it is suggested to play a key role in asparagine metabolism within legume sink organs. However, the metabolic fate of asparagine in source and sink organs during rice seed production remains to be elucidated. Therefore, the main objective of this study is to investigate the asparagine metabolism in a temporal and spatial manner during rice seed production. RESULTS: For this purpose, the expression of genes involved in asparagine catabolism, such as asparaginase1 (OsASNase1) and 2 (OsASNase2), were quantitatively measured, and contents of asparagine, aspartate and ammonium ions were determined in sink and source organs during spikelet ripening. Quantitative real-time PCR and in situ localization studies determined that OsASNase2 is expressed in the dorsal vascular bundles and nucellar projection of developing grains, as well as in mesophyll and phloem companion cells of senescent flag leaves. Amino acid measurements revealed that the aspartate concentration is higher than asparagine in both source and sink organs. CONCLUSION: This work suggests that asparaginase dependent asparagine catabolism occurred not only in sink but also in source organs.

Contributions of two cytosolic glutamine synthetase isozymes to ammonium assimilation in Arabidopsis roots.

Glutamine synthetase (GS) catalyzes a reaction that incorporates ammonium into glutamate and yields glutamine in the cytosol and chloroplasts. Although the enzymatic characteristics of the GS1 isozymes are well known, their physiological functions in ammonium assimilation and regulation in roots remain unclear. In this study we show evidence that two cytosolic GS1 isozymes (GLN1;2 and GLN1;3) contribute to ammonium assimilation in Arabidopsis roots. Arabidopsis T-DNA insertion lines for GLN1;2 and GLN1;3 (i.e. gln1;2 and gln1;3 single-mutants), the gln1;2:gln1;3 double-mutant, and the wild-type accession (Col-0) were grown in hydroponic culture with variable concentrations of ammonium to compare their growth, and their content of nitrogen, carbon, ammonium, and amino acids. GLN1;2 and GLN1;3 promoter-dependent green fluorescent protein was observed under conditions with or without ammonium supply. Loss of GLN1;2 caused significant suppression of plant growth and glutamine biosynthesis under ammonium-replete conditions. In contrast, loss of GLN1;3 caused slight defects in growth and Gln biosynthesis that were only visible based on a comparison of the gln1;2 single- and gln1;2:gln1;3 double-mutants. GLN1;2, being the most abundantly expressed GS1 isozyme, markedly increased following ammonium supply and its promoter activity was localized at the cortex and epidermis, while GLN1;3 showed only low expression at the pericycle, suggesting their different physiological contributions to ammonium assimilation in roots. The GLN1;2 promoter-deletion analysis identified regulatory sequences required for controlling ammonium-responsive gene expression of GLN1;2 in Arabidopsis roots. These results shed light on GLN1 isozyme-specific regulatory mechanisms in Arabidopsis that allow adaptation to an ammonium-replete environment.

Sulfur-Responsive Elements in the 3'-Nontranscribed Intergenic Region Are Essential for the Induction of SULFATE TRANSPORTER 2;1 Gene Expression in Arabidopsis Roots under Sulfur Deficiency.

Under sulfur deficiency (-S), plants induce expression of the sulfate transport systems in roots to increase uptake and root-to-shoot transport of sulfate. The low-affinity sulfate transporter SULTR2;1 is predominantly expressed in xylem parenchyma and pericycle cells in Arabidopsis thaliana roots under -S. The mechanisms underlying -S-inducible expression of SULTR2;1 in roots have remained unclear, despite the possible significance of SULTR2;1 for acclimation to low-sulfur conditions. In this investigation, examination of deletions and base substitutions in the 3'-intergenic region of SULTR2;1 revealed novel sulfur-responsive elements, SURE21A (5'-CAATGTATC-3') and SURE21B (5'-CTAGTAC-3'), located downstream of the SULTR2;1 3'-untranslated region. SURE21A and SULTR21B effectively induced reporter gene expression from fusion constructs under -S in combination with minimal promoters or promoters not inducible by -S, suggesting their versatility in controlling transcription. T-DNA insertions near SURE21A and SULTR21B abolished -S-inducible expression of SULTR2;1 in roots and reduced the uptake and root-to-shoot transport of sulfate. In addition, these mutations partially suppressed SULTR2;1 expression in shoots, without changing its -S-responsive expression. These findings indicate that SULTR2;1 contributes to the increase in uptake and internal translocation of sulfate driven by gene expression induced under the control of sulfur-responsive elements in the 3'-nontranscribed intergenic region of SULTR2;1.

Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice.

Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins, including Rubisco, are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes, and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis (Arabidopsis thaliana). Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared with the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. (15)N pulse chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves.

Asparagine synthetase1, but not asparagine synthetase2, is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots.

Asparagine is synthesized from glutamine by the reaction of asparagine synthetase (AS) and is the major nitrogen form in both xylem and phloem sap in rice (Oryza sativa L.). There are two genes encoding AS, OsAS1 and OsAS2, in rice, but the functions of individual AS isoenzymes are largely unknown. Cell type- and NH4(+)-inducible expression of OsAS1 as well as analyses of knockout mutants were carried out in this study to characterize AS1. OsAS1 was mainly expressed in the roots, with in situ hybridization showing that the corresponding mRNA was specifically accumulated in the three cell layers of the root surface (epidermis, exodermis and sclerenchyma) in an NH4(+)-dependent manner. Conversely, OsAS2 mRNA was abundant in leaf blades and sheathes of rice. Although OsAS2 mRNA was detectable in the roots, its content decreased when NH4(+) was supplied. Retrotransposon-mediated knockout mutants lacking AS1 showed slight stimulation of shoot length and slight reduction in root length at the seedling stage. On the other hand, the mutation caused an approximately 80-90% reduction in free asparagine content in both roots and xylem sap. These results suggest that AS1 is responsible for the synthesis of asparagine in rice roots following the supply of NH4(+). Characteristics of the NH4(+)-dependent increase and the root surface cell-specific expression of OsAS1 gene are very similar to our previous results on cytosolic glutamine synthetase1;2 and NADH-glutamate synthase1 in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4(+) in rice roots.

Lack of cytosolic glutamine synthetase1;2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings.

The development and elongation of active tillers in rice was severely reduced by a lack of cytosolic glutamine synthetase1;2 (GS1;2), and, to a lesser extent, lack of NADH-glutamate synthase1 in knockout mutants. In situ hybridization using the basal part of wild-type seedlings clearly showed that expression of OsGS1;2 was detected in the phloem companion cells of the nodal vascular anastomoses and large vascular bundles of axillary buds. Accumulation of lignin, visualized using phloroglucin HCl, was also observed in these tissues. The lack of GS1;2 resulted in reduced accumulation of lignin. Re-introduction into the mutants of OsGS1;2 cDNA under the control of its own promoter successfully restored the outgrowth of tillers and lignin deposition to wild-type levels. Transcriptomic analysis using a 5 mm basal region of rice shoots showed that the GS1;2 mutants accumulated reduced amounts of mRNAs for carbon and nitrogen metabolism, including C1 unit transfer in lignin synthesis. Although a high content of strigolactone in rice roots is known to reduce active tiller number, the reduction of outgrowth of axillary buds observed in the GS1;2 mutants was independent of the level of strigolactone. Thus metabolic disorder caused by the lack of GS1;2 resulted in a severe reduction in the outgrowth of axillary buds and lignin deposition.

A mathematical model of phloem sucrose transport as a new tool for designing rice panicle structure for high grain yield.

Rice (Oryza sativa) is one of the most important food crops in the world. Numerous quantitative trait loci or genes controlling panicle architecture have been identified to increase grain yield. Yet grain yield, defined as the product of the number of well-ripened grains and their weight, is a complex trait that is determined by multiple factors such as source, sink and translocation capacity. Mechanistic modelling capturing capacities of source, sink and transport will help in the theoretical design of crop ideotypes that guarantee high grain yield. Here we present a mathematical model simulating sucrose transport and grain growth within a complex phloem network. The model predicts that the optimal panicle structure for high yield shows a simple grain arrangement with few higher order branches. In addition, numerical analyses revealed that inefficient delivery of carbon to panicles with higher order branches prevails regardless of source capacity, indicating the importance of designing grain arrangement and phloem structure. Our model highlights the previously unexplored effect of grain arrangement on the yield, and provides numerical solutions for optimal panicle structure under various source and sink capacities.

NADH-dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root.

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2-oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin-dependent glutamate synthase (Fd-GOGAT) and nicotinamide adenine dinucleotide (NADH)-GOGAT. While Fd-GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH-GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH-GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real-time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH-GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH-GOGAT and Fd-GOGAT did not fully overlap. Promoter-ß-glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH-GOGAT was highly accumulated in non-green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH-GOGAT transfer DNA (T-DNA) insertion lines under normal CO2 condition. The data emphasize the importance of NADH-GOGAT in the ammonium assimilation in Arabidopsis roots.

Evidence supporting distinct functions of three cytosolic glutamine synthetases and two NADH-glutamate synthases in rice.

The functions of the three isoenzymes of cytosolic glutamine synthetase (GS1;1, GS1;2, and GS1;3) and two NADH-glutamate synthases (NADH-GOGAT1 and NADH-GOGAT2) in rice (Oryza sativa L.) were characterized using a reverse genetics approach and spatial expression of the corresponding genes. OsGS1;2 and OsNADH-GOGAT1 were mainly expressed in surface cells of rice roots in an NH4 (+)-dependent manner. Disruption of either gene by the insertion of endogenous retrotransposon Tos17 caused reduction in active tiller number and hence panicle number at harvest. Re-introduction of OsGS1;2 cDNA under the control of its own promoter into the knockout mutants successfully restored panicle number to wild-type levels. These results indicate that GS1;2 and NADH-GOGAT1 are important in the primary assimilation of NH4 (+) taken up by rice roots. OsGS1;1 and OsNADH-GOGAT2 were mainly expressed in vascular tissues of mature leaf blades. OsGS1;1 mutants showed severe reduction in growth rate and grain filling, whereas OsNADH-GOGAT2 mutants had marked reduction in spikelet number per panicle. Complementation of phenotypes seen in the OsGS1;1 mutant was successfully observed when OsGS1;1 was re-introduced. Thus, these two enzymes could be important in remobilization of nitrogen during natural senescence. Metabolite profiling data showed a crucial role of GS1;1 in coordinating metabolic balance in rice. Expression of OsGS1:3 was spikelet-specific, indicating that it is probably important in grain ripening and/or germination. Thus, these isoenzymes seem to possess distinct and non-overlapping functions and none was able to compensate for the individual function of another.

NADH-dependent glutamate synthase participated in ammonium assimilation in Arabidopsis root.

Higher plants have 2 GOGAT species, Fd-GOGAT and NADH-GOGAT. While Fd-GOGAT mainly assimilates ammonium in leaves, which is derived from photorespiration, the function of NADH-GOGAT, which is highly expressed in roots, (1) needs to be elucidated. The aim of this study was to clarify the role of NADH-GOGAT in Arabidopsis roots. The supply of ammonium to the roots caused an accumulation of NADH-GOGAT, while Fd-GOGAT 1 and Fd-GOGAT 2 showed no response. A promoter-GUS fusion analysis and immunohistochemistry showed that NADH-GOGAT was located in non-green tissues like vascular bundles, shoot apical meristem, pollen, stigma, and roots. The localization of NADH-GOGAT and Fd-GOGAT was not overlapped. NADH-GOGAT T-DNA insertion lines showed a reduction of glutamate and biomass under normal CO2 conditions. These data emphasizes the importance of NADH-GOGAT in the ammonium assimilation of Arabidopsis roots.

Cytosolic glutamine synthetase1;2 is responsible for the primary assimilation of ammonium in rice roots.

Among three genes for cytosolic glutamine synthetase (OsGS1;1, OsGS1;2 and OsGS1;3) in rice (Oryza sativa L.) plants, the OsGS1;2 gene is known to be mainly expressed in surface cells of roots, but its function was not clearly understood. We characterized knock-out mutants caused by the insertion of an endogenous retrotransposon Tos17 into exon 2 of OsGS1;2. Homozygously inserted mutants showed severe reduction in active tiller number and hence panicle number at harvest. Other yield components, such as spikelet number per panicle, 1,000-spikelet weight and proportion of well ripened grains, were nearly identical between the mutants and wild-type plants. When the contents of free amino acids in roots were compared between the mutants and the wild type, there were marked reductions in contents of glutamine, glutamate, asparagine and aspartate, but a remarkable increase in free ammonium ions in the mutants. Concentrations of amino acids and ammonium ions in xylem sap behaved in a similar fashion. Re-introduction of OsGS1;2 cDNA under the control of its own promoter into the knock-out mutants successfully restored yield components to wild-type levels as well as ammonium concentration in xylem sap. The results indicate that GS1;2 is important in the primary assimilation of ammonium ions taken up by rice roots, with GS1;1 in the roots unable to compensate for GS1;2 functions.