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Microarray analysis of gene expression in rice lacking cytosolic glutamine synthetase 1;1 and 1;2 grown under the ammonium sufficient condition

ABSTRACT: Nitrogen (N) is a critical limiting element in the provision of important components, e.g., chlorophylls, to plants. Organic N synthesized by glutamine synthetase (GS) may help regulate metabolic networks for plant growth and development. However, how multiple isoforms of cytosolic GSs act for metabolic regulation remain unclear. Using the knockout mutants, Osgs1;1 and Osgs1;2, we compared the effects of two cytosolic GSs on metabolism and cell differentiation in rice roots using reverse genetic-, metabolite- and transcript profiling- and microscopic analyzes together with integrated metabolite-transcript network analysis. We used microarrays to detail the global programme of gene expression in rice Osgs1;1 and Osgs1;2 roots.

ORGANISM(S): Oryza sativa Oryza sativa Japonica Group

PROVIDER: GSE49789 | GEO | 2020/04/20

REPOSITORIES: GEO

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Project description:Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in Cys biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidposis thaliana. Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favour of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to Cd. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under non-stress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. To further elucidate the specific function(s) of the OAS-A1 isoform in the adaptation response to cadmium we extended the trasncriptome experiment to the wild type and oas-a1.1 mutant plants exposed to Cd. The comparison of transcriptomic profiles showed a higher proportion of genes with altered expression in the mutant than in the wild type, highlighting up-regulated genes identified as of the general oxidative stress response rather than metal-responsive genes. Wild type and oas-a1.1 mutant plants were grown hydroponically and, after a two-week acclimation period, the roots and shoots were harvested separately. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. We made two different comparisons to classify the differently expressed genes in the mutant plant: oas-a1.1 roots versus wild-type roots and oas-a1.1 shoots versus wild-type shoots. Hydroponically-grown wild type and oas-a1.1 mutant plants were further treated with 50µM CdCl2 and 18h-treated-roots and 24h-treated-shoots were harvested. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. Different comparisons were performed as follows: 18h Cd-treated wild type roots versus untreated wild type roots; 24h Cd-treated wild type shoots versus untreated wild type shoots; 18h Cd-treated oas-a1.1 roots versus untreated oas-a1.1 roots; 24h Cd-treated oas-a1.1 shoots versus untreated oas-a1.1 shoots; 18h Cd-treated oas-a1.1 roots versus 18h Cd-treated wild type roots; 24h Cd-treated oas-a1.1 shoots versus 24h Cd-treated wild type shoots

Project description:Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1M-NM-^T acs2M-NM-^T S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.21 h-1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While, for the first time, demonstrating that yeast ACS can be fully replaced by alternative reactions, this study demonstrates that further modifications are needed to achieve optimal in vivo efficiencies of the supply of acetyl-CoA as product precursor. To investigate the impact of introduced changes in native pathway of cytosolic acetyl-CoA formation in S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic or anaerobic, glucose-limited chemostat cultures.

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Microarray analysis of gene expression in rice lacking cytosolic glutamine synthetase 1;1 and 1;2 grown under the ammonium sufficient condition

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