Ferredoxin-dependent Glutamate Synthase Research Articles

Optimal phosphorus proportion increases cotton yields in low nitrogen environments by enhancing mid-upper canopy biomass and nitrogen utilization

To elucidate the physiological mechanism of optimal phosphorus (P) proportion under reduced nitrogen (N) environments on cotton yield, dry matter (DM) spatiotemporal distribution, and N utilization. A three-year experiment was conducted with reduced N (299 kg hm−2, N3), four proportions of P fertilizer (25:75 % at squaring and bloom-bolling stages (P1), 50 %:50 % (P2), 75 %:25 % (P3), 100 %:0 % (P4)), and a traditional N application (402.5 kg hm−2, N2) with P2 as control. Under the N3 treatment, the seed yield of P2 was 6.7–24.5 % higher than other P, and the yield of N3P2 was 1.6–2.6 % lower than N2P2. During the whole growth period, P2 demonstrated 12.4–46.9 % higher leaf and reproductive organs dry matter (LDM and RoDM) compared to other P, and primarily due to more mid-upper DM. Throughout the growth period, N3P2 maintained dominant LDM and exhibited RoDM 2.5 % higher at late-full bolling stage (LFB) compared to N2P2. Trends in leaf N content and reproductive organ N content were similar to DM, with N3P2 consistently maintaining higher levels. Regarding N metabolism activities, N3P2 displayed 8.5–16.1 % higher activity of nitrate reductase, 9.5–21.9 % higher activity of glutamine synthetase, and 7.5 %-15.6 % higher activity of glutamate synthase compared to other P across all growth stages, with no significant differences observed with N2P2. Moreover, N3P2 and N2P2 exhibited higher relative expression of NR, GS1, and ferredoxin-dependent glutamate synthase compared to other treatments. In Conclusion, at 25 % reduced N (N3), optimizing the proportion of P fertilizer (P2, 50 %:50 %) could lead to higher yields due to enhanced N re-utilization capabilities in the later growth stage and increased distribution of DM in leaves and reproductive organs, particularly in the middle and upper layers (especially the fruiting branches). The study findings hold significant theoretical implications for achieving sustainable and efficient production in drip-irrigated cotton fields under reduced N management.

FERONIA regulates salt tolerance in Arabidopsis by controlling photorespiratory flux.

Photorespiration is an energetically costly metabolic pathway in plants that responds to environmental stresses. The molecular basis of the regulation of the photorespiratory cycle under stress conditions remains unclear. Here, we discovered that FERONIA (FER) regulates photorespiratory flow under salt stress in Arabidopsis (Arabidopsis thaliana). FER mutation results in hypersensitivity to salt stress, but disruption of ferredoxin-dependent glutamate synthase 1 (GLU1), an enzyme that participates in the photorespiratory pathway by producing glutamate, greatly suppresses fer-4 hypersensitivity to salt stress primarily due to reduced glycine yield. In contrast, disrupting mitochondrial serine hydroxymethyltransferase1 (SHM1), which is supposed to increase glycine levels by hampering the conversion of glycine to serine in the photorespiratory cycle, aggravates fer-4 hypersensitivity to salt stress. Biochemical data show that FER interacts with and phosphorylates SHM1, and this phosphorylation modulates SHM1 stability. Additionally, the production of proline and its intermediate △1-pyrroline-5-carboxylate (P5C), which are both synthesized from glutamate, also contributes to fer-4 hypersensitivity to salt stress. In conclusion, this study elucidates the functional mechanism of FER in regulating salt tolerance by modulating photorespiratory flux, which greatly broadens our understanding of how plants adapt to high salinity.

Deciphering proteomic mechanisms explaining the role of glutathione as an aid in improving plant fitness and tolerance against cadmium-toxicity in Brassica napus L.

Cadmium (Cd) hazard is a serious limitation to plants, soils and environments. Cd-toxicity causes stunted growth, chlorosis, necrosis, and plant yield loss. Thus, ecofriendly strategies with understanding of molecular mechanisms of Cd-tolerance in plants is highly demandable. The Cd-toxicity caused plant growth retardation, leaf chlorosis and cellular damages, where the glutathione (GSH) enhanced plant fitness and Cd-toxicity in Brassica through Cd accumulation and antioxidant defense. A high-throughput proteome approach screened 4947 proteins, wherein 370 were differently abundant, 164 were upregulated and 206 were downregulated. These proteins involved in energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense response, heavy metal detoxification, cytoskeleton and cell wall structure, and plant development in Brassica. Interestingly, several key proteins including glutathione S-transferase F9 (A0A078GBY1), ATP sulfurylase 2 (A0A078GW82), cystine lyase CORI3 (A0A078FC13), ferredoxin-dependent glutamate synthase 1 (A0A078HXC0), glutaredoxin-C5 (A0A078ILU9), glutaredoxin-C2 (A0A078HHH4) actively involved in antioxidant defense and sulfur assimilation-mediated Cd detoxification process confirmed by their interactome analyses. These candidate proteins shared common gene networks associated with plant fitness, Cd-detoxification and tolerance in Brassica. The proteome insights may encourage breeders for enhancing multi-omics assisted Cd-tolerance in Brassica, and GSH-mediated hazard free oil seed crop production for global food security.

Glycine-Induced Phosphorylation Plays a Pivotal Role in Energy Metabolism in Roots and Amino Acid Metabolism in Leaves of Tea Plant.

Phosphorylation is the most extensive post-translational modification of proteins and thus regulates plant growth. However, the regulatory mechanism of phosphorylation modification on the growth of tea plants caused by organic nitrogen is still unclear. In order to explore the phosphorylation modification mechanism of tea plants in response to organic nitrogen, we used glycine as the only nitrogen source and determined and analyzed the phosphorylated proteins in tea plants by phosphoproteomic analysis. The results showed that the phosphorylation modification induced by glycine-supply played important roles in the regulation of energy metabolism in tea roots and amino acid metabolism in tea leaves. In roots, glycine-supply induced dephosphorylation of proteins, such as fructose-bisphosphate aldolase cytoplasmic isozyme, glyceraldehyde-3-phosphate dehydrogenase, and phosphoenolpyruvate carboxylase, resulted in increased intensity of glycolysis and decreased intensity of tricarboxylic acid cycle. In leaves, the glycine-supply changed the phosphorylation levels of glycine dehydrogenase, aminomethyltransferase, glutamine synthetase, and ferredoxin-dependent glutamate synthase, which accelerated the decomposition of glycine and enhanced the ability of ammonia assimilation. In addition, glycine-supply could improve the tea quality by increasing the intensity of amino acids, such as theanine and alanine. This research clarified the important regulatory mechanism of amino acid nitrogen on tea plant growth and development through protein phosphorylation.

RNA-seq analysis and transcriptome assembly of Salicornia neei reveals a powerful system for ammonium detoxification

BackgroundSalicornia neei is a halophyte plant that has been proposed for use in the phytoremediation of the saline wastewater generated by land-based aquaculture. To identify the molecular mechanisms related to ammonium response, we analyzed the transcriptome of S. neei in response to growth in saline water containing 3 mM ammonium. ResultsThe RNA sequencing generated a total of 14,680,108 paired-end reads from the control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,020 transcripts and a reference transcriptome with an N50 of 683 base pair.A total of 45,327 genes were annotated, representing 51.2% of the contig predicted from de novo assembly. As regards differentially expressed genes, a total of 9,140 genes were differentially expressed in response to ammonium in saline water; of these, 7,396 could be annotated against functional databases. The upregulated genes were mainly involved in cell wall biosynthesis, transmembrane transport and antiporter activities, including biological Kyoto Encyclopedia of Genes and Genomes, pathways linked to the biosynthesis of secondary metabolites, plant hormone signal transduction, autophagy, and nitrogen metabolism. In addition, a set of 72 genes was directly involved in ammonium metabolism, including glutamine synthetase 1, glutamate synthase 1, and ferredoxin-dependent glutamate synthase chloroplastic. ConclusionsOur results support the hypothesis that an ammonium detoxification system mediated by glutamine and glutamate synthase was activated in S. neei when exposed to ammonium and saline water. The present transcriptome profiling method could be useful when investigating the response of halophyte plants to saline wastewater from land-based aquaculture.This manuscript includes an interactive 360 degree video, supplementary to the materials and methods section. To view the video correctly, it is necessary to scroll through the screen to navigate across the laboratory where you will find 6 interactive points. For an immersive experience a head-mounted display can be used. Please, visit this URL:http://ejbiotechnology.info/public/360view/2022/VTPGALLARDO_1v3/index.htm.How to cite: Díaz-Silva M, Maldonado J, Veloso P, et al. RNA-seq analysis and transcriptome assembly of Salicornia neei reveals a powerful system for ammonium detoxification. Electron J Biotechnol 2022;58. https://doi.org/10.1016/j.ejbt.2022.05.003

PEPC of sugarcane regulated glutathione S-transferase and altered carbon\u2013nitrogen metabolism under different N source concentrations in Oryza sativa

BackgroundPhosphoenolpyruvate carboxylase (PEPC) plays an important role in the primary metabolism of higher plants. Several studies have revealed the critical importance of PEPC in the interaction of carbon and nitrogen metabolism. However, the function mechanism of PEPC in nitrogen metabolism is unclear and needs further investigation.ResultsThis study indicates that transgenic rice expressing the sugarcane C4-PEPC gene displayed shorter primary roots and fewer crown roots at the seedling stage. However, total nitrogen content was significantly higher in transgenic rice than in wild type (WT) plants. Proteomic analysis revealed that there were more differentially expressed proteins (DEPs) responding to nitrogen changes in transgenic rice. In particular, the most enriched pathway “glutathione (GSH) metabolism”, which mainly contains GSH S-transferase (GST), was identified in transgenic rice. The expression of endogenous PEPC, GST and several genes involved in the TCA cycle, glycolysis and nitrogen assimilation changed in transgenic rice. Correspondingly, the activity of enzymes including GST, citrate synthase, 6-phosphofructokinase, pyruvate kinase and ferredoxin-dependent glutamate synthase significantly changed. In addition, the levels of organic acids in the TCA cycle and carbohydrates including sucrose, starch and soluble sugar altered in transgenic rice under different nitrogen source concentrations. GSH that the substrate of GST and its components including glutamic acid, cysteine and glycine accumulated in transgenic rice. Moreover, the levels of phytohormones including indoleacetic acid (IAA), zeatin (ZT) and isopentenyladenosine (2ip) were lower in the roots of transgenic rice under total nutrients. Taken together, the phenotype, physiological and biochemical characteristics of transgenic rice expressing C4-PEPC were different from WT under different nitrogen levels.ConclusionsOur results revealed the possibility that PEPC affects nitrogen metabolism through regulating GST, which provide a new direction and concepts for the further study of the PEPC functional mechanism in nitrogen metabolism.

Transit Peptides From Photosynthesis-Related Proteins Mediate Import of a Marker Protein Into Different Plastid Types and Within Different Species.

Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.

Glutamate synthase 1 is involved in iron-deficiency response and long-distance transportation in Arabidopsis.

Iron is an essential microelement for plant growth. After uptake from the soil, iron is chelated by ligands and translocated from roots to shoots for subsequent utilization. However, the number of ligands involved in iron chelation is unclear. In this study, we identified and demonstrated that GLU1, which encodes a ferredoxin-dependent glutamate synthase, was involved in iron homeostasis. First, the expression of GLU1 was strongly induced by iron deficiency condition. Second, lesion of GLU1 results in reduced transcription of many iron-deficiency-responsive genes in roots and shoots. The mutant plants revealed a decreased iron concentration in the shoots, and displayed severe leaf chlorosis under the condition of Fe limitation, compared to wild-type. Third, the product of GLU1, glutamate, could chelate iron in vivo and promote iron transportation. Last, we also found that supplementation of glutamate in the medium can alleviate cadmium toxicity in plants. Overall, our results provide evidence that GLU1 is involved in iron homeostasis through affecting glutamate synthesis under iron deficiency conditions in Arabidopsis.

Redox Conformation-Specific Protein-Protein Interactions of the 2-Cysteine Peroxiredoxin in Arabidopsis.

2-Cysteine peroxiredoxins (2-CysPRX) are highly abundant thiol peroxidases in chloroplasts and play key roles in reactive oxygen species (ROS) defense and redox signaling. Peroxide-dependent oxidation of cysteines induces conformational changes that alter the ability for protein–protein interactions. For regeneration, 2-CysPRXs withdraw electrons from thioredoxins (TRXs) and participate in redox-dependent regulation by affecting the redox state of TRX-dependent targets, for example, in chloroplast metabolism. This work explores the redox conformation-specific 2-CysPRX interactome using an affinity-based pull down with recombinant variants arrested in specific quaternary conformations. This allowed us to address a critical and poorly explored aspect of the redox-regulatory network and showed that the interaction of TRXs, their interaction partners, and 2-CysPRX occur under contrasting redox conditions. A set of 178 chloroplast proteins were identified from leaf proteins and included proteins with functions in photosynthesis, carbohydrate, fatty acid and amino acid metabolism, and defense. These processes are known to be deregulated in plants devoid of 2-CysPRX. Selected enzymes like LIPOXYGENASE 2, CHLOROPLAST PROTEIN 12-1, CHORISMATE SYNTHASE, ß-CARBONIC ANHYDRASE, and FERREDOXIN-dependent GLUTAMATE SYNTHASE 1 were subjected to far Western, isothermal titration calorimetry, and enzyme assays for validation. The pull down fractions frequently contained TRXs as well as their target proteins, for example, FRUCTOSE-1,6-BISPHOSPHATASE and MALATE DEHYDROGENASE. The difference between TRX-dependent indirect interactions of TRX targets and 2-CysPRX and direct 2-CysPRX binding is hypothesized to be related to quaternary structure formation, where 2-CysPRX oligomers function as scaffold for complex formation, whereas TRX oxidase activity of 2-CysPRX controls the redox state of TRX-related enzyme activity.

High-throughput sequencing reveals genes associated with high-temperature stress tolerance in sugarcane.

Sugarcane (Saccharumspp.) is a major sugar crop grown in tropical and sub-tropical areas throughout the world which is vulnerable to high temperature stress due to climate change. In this present study, we have generated a transcriptome profile of sugarcane variety Co 99004 exposed to high-temperature stress (47°C). The Illumina Nexseq2500 platform yielded a total of 39.28 and 13.44 million reads, corresponding to 3.9 and 1.3 gigabase pair (Gb) of the processed reads for control and high-temperature-stressed samples, respectively. Initially, the reads were de novo assembled into 118,017 unigenes with an average length of 780bp. The longest sequence in the assembly was 21kb. Further, these transcripts were BLASTed against GO, KEGG and COG databases to identify the novel genes/transcripts expressed due to elevated temperature conditions. The different expression analysis showed 1137 transcripts which were up-regulated during heat temperature stress when compared to control conditions. Analysis of relative gene expression showed phytepsin, ferredoxin-dependent glutamate synthase, and stress protein DDR-48 threefold increased expression during heat stress. These findings reveal novel targets for subsequent research on the genomics genetic manipulation and molecular mechanism of elevated stress tolerance in sugarcane.

(Invited) Engineering of Enzymatic Bioelectrochemical Interface for In Vivo Analysis

Bioelectrocatalysts have enabled a rapid development of sensing methods and devices for in vivo quantification of chemical species, which is crucial for understanding the molecular basis of life. However, these sophisticated functional units face a set of challenges when operated in vivo. One inevitable issue is the kinetic barrier for heterogeneous electron transfer at the electrode-enzyme interface, especially when direct electron transfer (DET) is preferred as toxic and unstable mediators are not suitable for in vivo applications. Different strategies have been explored to improve DET efficiency, and lately, a new method for regulating the molecular orientation of laccase on single-walled carbon nanotubes was developed based on the principle of surface wetting. Electrochemical and vibrational spectroscopic investigation drew a mechanistic picture of how enzymes behave in the presence of wetting reagents (such as ethanol), offering a new guidance for the design of bioelectrochemical interfaces. Another issue associated with in vivo biosensing rises from interference of intrinsically existing substrates (other than the analytes) for enzymes, e.g., oxygen and NAD+ that function as electron acceptors for commonly employed oxidases and dehydrogenases. In our recent studies, we have explored a new enzyme candidate, ferredoxin-dependent glutamate synthase (Fd-GltS), as a bioelectrocatalyst. Containing multiple redox centers, Fd-GltS can be fined tuned with various mediators to enable either bioelectrosynthsis of glutamate or bioelectrochemical oxidation of glutamate. By this means, we can construct a new bioelectrochemical interface devoid of oxygen and cofactor interference, thus providing a new option for the development of biosensing platform for glutamate-mediated neurotransmission.

Exploring Ferredoxin-Dependent Glutamate Synthase as an Enzymatic Bioelectrocatalyst.

Ferredoxin-dependent glutamate synthase (Fd-GltS) is reported as an enzymatic bioelectrocatalyst for the first time. By configuring mediated electrochemical interfaces with mediators of different redox potentials, we realize bioelectrosynthesis or bioelectrooxidation of glutamate with recombinant Fd-GltS from cyanobacteria. Particularly, bioelectrocatalytic oxidation of glutamate by Fd-GltS is demonstrated to be oxygen independent. This study reinforces a new catalytic option for developing enzymatic bioelectronic devices for powering, sensing or synthesis.

Protein Hydrolysate Stimulates Growth in Tomato Coupled With N-Dependent Gene Expression Involved in N Assimilation.

Plant-derived protein hydrolysates (PHs) have received increased attention in the last decade because of their potential to improve yield, nutritional quality as well as tolerance to abiotic stressors. The current study investigated the effects and the molecular mechanisms of a legume-derived PH under optimal and sub-optimal nitrogen (N) concentrations (112 and 7 mg L−1, respectively) in tomato (Solanum lycopersicum L.). Growth and mineral composition of tomato plants treated with PHs by foliar spray or substrate drench were compared to untreated plants. In addition, the expression was determined of genes encoding ammonium and nitrate transporters and seven enzymes involved in N metabolism: nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase 1 (GS1), glutamine synthetase 2 (GS2), ferredoxin-dependent glutamate synthase (GLT), NADH-dependent glutamate synthase (GLS), and glutamate dehydrogenase (GDH). The root and total plant dry weight, SPAD index and leaf nitrogen content were higher by 21, 17, 7, and 6%, respectively, in plants treated by a substrate drench in comparison to untreated tomato plants, whereas foliar application of PH gave intermediate values. PH-treated plants grown with lower N availability showed reduced expression of NR and NiR as well as of nitrate and ammonium transporter transcripts in both leaf and root tissues in comparison with untreated plants; this was especially pronounced after application of PH by substrate drench. Conversely, the transcript level of an amino acid transporter gene was up-regulated in comparison with untreated plants. At high N regime, the transcript levels of the ammonium and amino acid transporters and also NR, NiR, and GLT were significantly up-regulated in root after PH foliar and substrate drench applications compared with untreated plants. An up-regulation was also observed for GS1, GS2, and GDH transcripts in leaf after substrate drench. These results highlighted the potential benefits of using legume PH in vegetable production systems to increase growth and N-nutritional status of plants.

Nitrogen metabolism-related enzymes in Mesembryanthemum crystallinum after Botrytis cinerea infection

We compared C3 and CAM (crassulacean acid metabolism) states in Mesembryanthemum crystallinum, a facultative CAM species, with respect to the involvement of phosphoenolpyruvate carboxylase (PEPC) and nitrogen metabolismrelated enzymes in plant response to Botrytis cinerea infection. The enzyme activities were monitored both in pathogeninoculated 2nd leaf pair and non-inoculated 3rd leaf pair. The control activities of most studied enzymes were dependent on the mode of photosynthesis. Compared to C3 plants, those performing CAM exhibited higher PEPC, nitrate reductase (NR), and deaminating glutamate dehydrogenase (NAD-GDH) activities but lower glutamine synthetase (GS) and alanine aminotransferase (ALT) activities. Regardless of the mode of photosynthetic carbon assimilation, the plants responded to infection with enhancement of PEPC and inhibition of NR activities in the inoculated leaves. Whereas the activity of GS remained unaffected, those of all glutamate-yielding enzymes, namely ferredoxin-dependent glutamate synthase (Fd-GOGAT), aspartate aminotransferase (AST), ALT, and aminating glutamate dehydrogenase (NADHGDH) were altered after infection. However, the time-course and extent of the observed changes differed in C3 and CAM plants. In general, CAM plants responded to infection with an earlier increase in PEPC and Fd-GOGAT activities as well as later inhibition of NR activity. Contrary to C3 plants, in those performing CAM the activities of PEPC, Fd-GOGAT, NADH-GDH, and AST in the non-inoculated 3rd leaf pair were similarly influenced by infection as in leaves directly inoculated with the pathogen. This implies that the local infection induced an alteration of carbon/nitrogen status in healthy upper leaves. This reprogramming resulting from changes in PEPC and nitrogen metabolism-related enzymes was C3- and CAM-specific.

Expression Analysis of Nitrogen Metabolism-Related Genes Reveals Differences in Adaptation to Low-Nitrogen Stress between Two Different Barley Cultivars at Seedling Stage.

The excess use of nitrogen fertilizers causes many problems, including higher costs of crop production, lower nitrogen use efficiency, and environmental damage. Crop breeding for low-nitrogen tolerance, especially molecular breeding, has become the major route to solving these issues. Therefore, in crops such as barley (Hordeum vulgare L.), it is crucial to understand the mechanisms of low-nitrogen tolerance at the molecule level. In the present study, two barley cultivars, BI-04 (tolerant to low nitrogen) and BI-45 (sensitive to low nitrogen), were used for gene expression analysis under low-nitrogen stress, including 10 genes related to primary nitrogen metabolism. The results showed that the expressions of HvNIA2 (nitrite reductase), HvGS2 (chloroplastic glutamine synthetase), and HvGLU2 (ferredoxin-dependent glutamate synthase) were only induced in shoots of BI-04 under low-nitrogen stress, HvGLU2 was also only induced in roots of BI-04, and HvGS2 showed a rapid response to low-nitrogen stress in the roots of BI-04. The expression of HvASN1 (asparagine synthetase) was reduced in both cultivars, but it showed a lower reduction in the shoots of BI-04. In addition, gene expression and regulation differences in the shoots and roots were also compared between the barley cultivars. Taken together, the results indicated that the four above-mentioned genes might play important roles in low-nitrogen tolerance in barley.

Identification of the ferredoxin interaction sites on ferredoxin-dependent glutamate synthase from Synechocystis sp. PCC 6803.

Based on in silico docking methods, five amino acids in glutamate synthase (Gln-467, His-1144, Asn-1147, Arg-1162, and Trp-676) likely constitute key binding residues in the interface of a glutamate synthase:ferredoxin complex. Although all interfacial mutants studied showed the ability to form a complex under low ionic strength, these docking mutations showed significantly less ferredoxin-dependent activities, while still retaining enzymatic activity. Furthermore, isothermal titration calorimetry showed a possible 1:2 molar ratio between the wild-type glutamate synthase and ferredoxin. However, each of our interfacial mutants showed only a 1:1 complex with ferredoxin, suggesting that the mutations directly affect the glutamate synthase:ferredoxin heterodimer interface.

ES7, encoding a ferredoxin-dependent glutamate synthase, functions in nitrogen metabolism and impacts leaf senescence in rice

Glutamate synthase (GOGAT) is a key enzyme for nitrogen metabolism and ammonium assimilation in plants. In this study, an early senescence 7 (es7) mutant was identified and characterized. The leaves of the es7 mutant begin to senesce at the tillering stage about 60day after sowing, and become increasingly senescent as the plants develop at the heading stage. When es7 plants are grown under photorespiration-suppressed conditions (high CO2), the senescence phenotype and chlorophyll content are rescued. qRT-PCR analysis showed that senescence-associated genes were up-regulated significantly in es7. A map-based cloning strategy was used to identify ES7, which encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). ES7 was expressed constitutively, and the ES7 protein was localized in chloroplast. qRT-PCR analysis indicated that several genes related to nitrogen metabolism were differentially expressed in es7. Further, we also demonstrated that chlorophyll synthesis-associated genes were significantly down-regulated in es7. In addition, when seedlings are grown under increasing nitrogen concentrations (NH4NO3) for 15days, the contents of chlorophyll a, chlorophyll b and total chlorophyll were significantly lower in es7. Our results demonstrated that ES7 is involved in nitrogen metabolism, effects chlorophyll synthesis, and may also associated with photorespiration, impacting leaf senescence in rice.

Isolation and characterization of a spotted leaf 32 mutant with early leaf senescence and enhanced defense response in rice

Leaf senescence is a complex biological process and defense responses play vital role for rice development, their molecular mechanisms, however, remain elusive in rice. We herein reported a rice mutant spotted leaf 32 (spl32) derived from a rice cultivar 9311 by radiation. The spl32 plants displayed early leaf senescence, identified by disintegration of chloroplasts as cellular evidence, dramatically decreased contents of chlorophyll, up-regulation of superoxide dismutase enzyme activity and malondialdehyde, as physiological characteristic, and both up-regulation of senescence-induced STAY GREEN gene and senescence-associated transcription factors, and down-regulation of photosynthesis-associated genes, as molecular indicators. Positional cloning revealed that SPL32 encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). Compared to wild type, enzyme activity of GOGAT was significantly decreased, and free amino acid contents, particularly for glutamate and glutamine, were altered in spl32 leaves. Moreover, the mutant was subjected to uncontrolled oxidative stress due to over-produced reactive oxygen species and damaged scavenging pathways, in accordance with decreased photorespiration rate. Besides, the mutant showed higher resistance to Xanthomonas oryzae pv. Oryzae than its wild type, coupled with up-regulation of four pathogenesis-related marker genes. Taken together, our results highlight Fd-GOGAT is associated with the regulation of leaf senescence and defense responses in rice.

The ferredoxin-dependent glutamate synthase (OsFd-GOGAT) participates in leaf senescence and the nitrogen remobilization in rice.

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) plays major roles in photorespiration and primary nitrogen assimilation. However, due to no mutant or knockdown lines of OsFd-GOGAT have been reported in rice (Oryza sativa L.), the contribution of OsFd-GOGAT to rice foliar nitrogen metabolism remains little up-to-date. Here, we isolated a rice premature leaf senescence mutant named gogat1, which was reduced in 67% of the total GOGAT enzyme activity in leaves. The gogat1 mutant exhibited chlorosis under natural condition, while showed less extent premature leaf senescence under low light treatment. The gogat1 locus was mapped to a 54.1kb region on chromosome 7, and the sequencing of OsFd-GOGAT showed one substitution (A to T) at the 3017th nucleotide of the open reading frame, leading to the amino-acid substitution of leucine changed to histidine. The gogat1 mutant showed reduced seed setting rate, while the grain protein content in gogat1 mutant was significantly higher than that in wild type. Meanwhile, during the grain-filling stage, total amino acids in the up three leaves and the upmost internode were increased dramatically. The results in this study suggested that OsFd-GOGAT might participate in nitrogen remobilization during leaf senescence, which provides a potential way to improve nitrogen use efficiency in rice.

Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen–Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies

Plants assimilate inorganic nitrogen absorbed from soil into organic forms as Gln and Glu through the glutamine synthetase/glutamine:2-oxoglutarate amidotransferase (GS/GOGAT) cycle. Whereas GS catalyzes the formation of Gln from Glu and ammonia, GOGAT catalyzes the transfer of an amide group from Gln to 2-oxoglutarate to produce two molecules of Glu. However, the regulatory role of the GS/GOGAT cycle in the carbon–nitrogen balance is not well understood. Here, we report the functional characterization of rice ABNORMAL CYTOKININ RESPONSE 1 (ABC1) gene that encodes a ferredoxin-dependent (Fd)-GOGAT. The weak mutant allele abc1-1 mutant shows a typical nitrogen-deficient syndrome, whereas the T-DNA insertional mutant abc1-2 is seedling lethal. Metabolomics analysis revealed the accumulation of an excessive amount of amino acids with high N/C ratio (Gln and Asn) and several intermediates in the tricarboxylic acid cycle in abc1-1, suggesting that ABC1 plays a critical role in nitrogen assimilation and carbon–nitrogen balance. Five non-synonymous single-nucleotide polymorphisms were identified in the ABC1 coding region and characterized as three distinct haplotypes, which have been highly and specifically differentiated between japonica and indica subspecies. Collectively, these results suggest that ABC1/OsFd-GOGAT is essential for plant growth and development by modulating nitrogen assimilation and the carbon–nitrogen balance.