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.
A reverse genetic approach combined with metabolic profiling showed that three cytosolic glutamine synthetases and two NADH-glutamate synthases have distinct functions in primary ammonium assimilation and nitrogen-remobilization processes in rice.
Nitrogen (N), as an essential element in amino acids, nucleotides, and proteins, is a key factor in plant growth and development. Omics approaches such as metabolomics and transcriptomics have become ...a promising way to inspect complex network interactions in N metabolism and can be used for monitoring the uptake and regulation, translocation, and remobilization of N. In this review, the authors highlight recent progress in omics approaches, including transcript profiling using microarrays and deep sequencing, and show recent technical developments in metabolite profiling for N studies. Further, network analysis studies including network inference methods with correlations, information-theoretic measures, and a network concept to examine gene expression clusters in relation to N regulatory systems in plants are introduced, and integrating network inference methods and integrated networks using multiple omics data are discussed. Finally, this review summarizes recent omics application examples using metabolite and/or transcript profiling analysis to elucidate the regulation of N metabolism and signalling and the coordination of N and carbon metabolism in model plants (Arabidopsis and rice), crops (tomato, maize, and legumes), and trees (Populus).
This review presents recent progress and applications of transcript and metabolite profiling for nitrogen studies. Time-series “omics” datasets are useful for constructing a stoichiometric metabolic model in future studies.
Although postmortem aging of pork improves its umami taste when roasted, the relationship between roasted aroma and taste has not been investigated. Therefore, in this study, we investigated the role ...of taste-related metabolites in the aroma composition of roasted meat during postmortem aging. Comprehensive metabolomic analyses of taste-related metabolites and volatile organic compounds in aged pork were conducted by gas chromatography-time-of-flight mass spectrometry. Taste-related metabolites, including amino acids, sugars, fatty acids, and nucleic acids, accumulated with age. Among the detected aroma compounds, benzeneacetaldehyde and 2,5-dimethyl-3-(3-methylbutyl)-pyrazine levels increased with age; these compounds potentially contribute to roasty and meaty aromas. Furthermore, there was a positive correlation between aroma compounds and their precursors in raw meat; some amino acids contributed to the generation of benzeneacetaldehyde, 3-methylbutanal, 3-ethyl-2,5-dimethyl-pyrazine, and 2,5-dimethyl-3-(3-methylbutyl)-pyrazine via the Maillard reaction. These results confirm that the changes in taste-related metabolites in raw meat affect the specific aroma compositions of roasted meat.
•Taste-related metabolites in aged pork were analyzed by metabolomic profiling.•Benzeneacetaldehyde and 2,5-dimethyl-3-(3-methylbutyl)-pyrazine accumulate with age.•Roasty and meaty aroma compounds are generated via the Maillard reaction.•Taste-related metabolites in aged raw meat alter the aroma profile of roasted pork.•Trace volatile organic compounds also modulate the aroma profile of aged pork.
Sugar content is one of the most important quality traits of tomato. Cell wall invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source ...leaves and fruits, while invertase inhibitor (INVINH) regulates this process. In this study, knock-out of cell wall INVINH in tomato (SlINVINH1) was performed by genome editing using, CRISPR/Cas9 and Target-AID technologies. Most of the genome-edited lines set higher soluble solid content (SSC) fruit than the original cultivar 'Suzukoma', while fruit weight was different among the genome-edited lines. From these genome-edited lines, three lines (193-3, 199-2, and 247-2), whose SSC was significantly higher than 'Suzukoma' and fruit weight were almost the same as the original cultivar, were selected. The fruit weight and overall plant growth of the two lines were comparable to those of the original cultivar. In contrast, the fructose and glucose contents in the mature fruits of the two lines were significantly higher than those of the original cultivar. The mature fruits of genome edited line 193-3 showed the highest sugar content, and the fructose and glucose contents were 29% and 36% higher than that of the original cultivar, respectively. Whole genome sequence data showed no off-target mutations in the genome-edited lines. Non-target metabolome analysis of mature fruits revealed that fructose was the highest loading factor in principal component analysis (PCA) between the genome-edited line and the original cultivar, and no unexpected metabolites appeared in the genome-edited line. In this study, we succeeded in producing tomato lines with high sugar content without a decrease in fruit weight and deterioration of plant growth by knock-out of SlINVINH1 using genome editing technology. This study showed that functional disruption of SlINVINH1 is an effective approach to produce tomato cultivars with high sugar content.
Alternative promoter usage is a proteome-expanding mechanism that allows multiple pre-mRNAs to be transcribed from a single gene. The impact of this mechanism on the proteome and whether it is ...positively exploited in normal organismal responses remain unclear. We found that the plant photoreceptor phytochrome induces genome-wide changes in alternative promoter selection in Arabidopsis thaliana. Through this mechanism, protein isoforms with different N termini are produced that display light-dependent differences in localization. For instance, shade-grown plants accumulate a cytoplasmic isoform of glycerate kinase (GLYK), an essential photorespiration enzyme that was previously thought to localize exclusively to the chloroplast. Cytoplasmic GLYK constitutes a photorespiratory bypass that alleviates fluctuating light-induced photoinhibition. Therefore, phytochrome controls alternative promoter selection to modulate protein localization in response to changing light conditions. This study suggests that alternative promoter usage represents another ubiquitous layer of gene expression regulation in eukaryotes that contributes to diversification of the proteome.
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•Phytochrome induces genome-wide changes in alternative promoter selection•Phytochrome-mediated alternative promoter selection modulates protein localization•This mechanism produces a cytoplasmic isoform of glycerate kinase (cytGLYK)•cytGLYK accumulates in shade to alleviate fluctuating light-induced photoinhibition
Light signaling through phytochrome receptors changes protein localization through alternative promoter selection, allowing plants to metabolically respond to changing light conditions.
Key message
By using the organelle glue technique, we artificially manipulated organelle interactions and controlled the plant metabolome at the pathway level.
Plant cell metabolic activity changes ...with fluctuating environmental conditions, in part via adjustments in the arrangement and interaction of organelles. This hints at the potential for designing plants with desirable metabolic activities for food and pharmaceutical industries by artificially controlling the interaction of organelles through genetic modification. We previously developed a method called the organelle glue technique, in which chloroplast–chloroplast adhesion is induced in plant cells using the multimerization properties of split fluorescent proteins. Here, we generated transgenic Arabidopsis (
Arabidopsis thaliana
) plants in which chloroplasts adhere to each other and performed metabolome analysis to examine the metabolic changes in these lines. In plant cells expressing a construct encoding the red fluorescent protein mCherry targeted to the chloroplast outer envelope by fusion with a signal sequence (cTP-mCherry), chloroplasts adhered to each other and formed chloroplast aggregations. Mitochondria and peroxisomes were embedded in the aggregates, suggesting that normal interactions between chloroplasts and these organelles were also affected. Metabolome analysis of the
cTP-mCherry
-expressing Arabidopsis shoots revealed significantly higher levels of glycine, serine, and glycerate compared to control plants. Notably, these are photorespiratory metabolites that are normally transported between chloroplasts, mitochondria, and peroxisomes. Together, our data indicate that chloroplast–chloroplast adhesion alters organellar interactions with mitochondria and peroxisomes and disrupts photorespiratory metabolite transport. These results highlight the possibility of controlling plant metabolism at the pathway level by manipulating organelle interactions.
Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great ...challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non‐transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.
Both inorganic fertilizer inputs and crop yields have increased globally, with the concurrent increase in the pollution of water bodies due to nitrogen leaching from soils. Designing agroecosystems ...that are environmentally friendly is urgently required. Since agroecosystems are highly complex and consist of entangled webs of interactions between plants, microbes, and soils, identifying critical components in crop production remain elusive. To understand the network structure in agroecosystems engineered by several farming methods, including environmentally friendly soil solarization, we utilized a multiomics approach on a field planted with Brassica rapa. We found that the soil solarization increased plant shoot biomass irrespective of the type of fertilizer applied. Our multiomics and integrated informatics revealed complex interactions in the agroecosystem showing multiple network modules represented by plant traits heterogeneously associated with soil metabolites, minerals, and microbes. Unexpectedly, we identified soil organic nitrogen induced by soil solarization as one of the key components to increase crop yield. A germ-free plant in vitro assay and a pot experiment using arable soils confirmed that specific organic nitrogen, namely alanine and choline, directly increased plant biomass by acting as a nitrogen source and a biologically active compound. Thus, our study provides evidence at the agroecosystem level that organic nitrogen plays a key role in plant growth.
Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more ...than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an ammo-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology.
We have developed a comprehensive method combining analytical techniques of one-dimensional (1D) and two-dimensional (GC
×
GC) gas chromatography-time-of-flight (TOF)–mass spectrometry. This method ...was applied to the metabolic phenotyping of natural variants in rice for the 68 world rice core collection (WRC) and two other varieties. Ten metabolites were selected as metabolite representatives, and the selected ion current of each metabolite peak obtained from both techniques were statistically compared. Our method of combining 1D- and GC
×
GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs.
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