Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Depleting cytosolic cysteine compromises the antioxidant capacity of the cytosol in Arabidopsis thaliana

ABSTRACT: 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

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Cecilia Gotor

PROVIDER: E-GEOD-19245 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Knocking out cytosolic cysteine synthesis compromises the antioxidant capacity of the cytosol to maintain discrete concentrations of hydrogen peroxide in Arabidopsis.

López-Martín M Carmen MC Becana Manuel M Romero Luis C LC Gotor Cecilia C

Plant physiology 20080425 2

Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in cysteine (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 Arabidopsis (Arabidopsis thaliana). Total intr ...[more]

PMID: 18441224

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Project description:Purpose: To investigate the expression profiles of genes regulated by bHLH6, we performed RNA-seq analysis using bHLH6 OV, spx4, and wild type treated with high and low P Methods: Roots and shoots mRNA profiles of 7-day-old bHLH6 OV, spx4, and wild-type (WT) rice seedlings treated with 200 μM or 10 μM Pi for 2 weeks three biological replicates were generated by deep sequencing using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA). The mapped reads of each sample were assembled by Hisat2 (v2.0.5). qRT–PCR validation was performed using SYBR Green assays Results: RNA-seq data confirmed that in shoots, 4,694 genes were upregulated and 4,126 were downregulated in bHLH6 OV lines compared with wild type under high-P conditions.Among the differentially expressed genes, 1,805 (38.5%) were upregulated in wild type under low-P conditions, whereas 1,598 (38.7%) of the downregulated genes were downregulated in wild type under low-P conditions, suggesting that bHLH6 overexpression upregulates a subset of PSI genes. In total, 971 genes were upregulated and 853 genes were downregulated in spx4 compared to wild type in high-P conditions. Among these upregulated genes, 371 (38.2%) were also upregulated in shoots of bHLH6 OV plants compared to in wild type; 456 (53.4%) of the downregulated genes were also downregulated in shoots of bHLH6 OV compared with wild type. In roots, 311 (10.5%) out of the 2,969 upregulated genes in bHLH6 OV lines under high-P conditions were also upregulated in wild type under low-P conditions; 501 (19.1%) out of the 2,628 downregulated genes in bHLH6 OV were also downregulated in wild type under low-P conditions. However, 313 (60.2%) out of the 520 genes upregulated in spx4 in roots were also upregulated in bHLH6 OV lines compared to in wild type, whereas 497 (54.6%) out of 910 the downregulated genes were also downregulated in bHLH6 OV compared to in wild type. To validate the RNA-seq results, we selected the upregulated PT and PAP (PURPLE ACID PHOSPHATASE) genes for expression analysis using RT-qPCR. The expression levels of PT2, PT10, PAP10a, and PAP21b were considerably higher in roots of bHLH6 OV plants than in those of wild type and bhlh6 mutants, whereas the expression of SPX2 was much lower in bHLH6 OV plants than in wild type Conclusion: our study is the first to analyze in detail the bHLH6 transcriptome produced by rna-seq technology and perform biological replication. Our results suggest that bHLH6 mediates the regulation of Pi homeostasis by SPX4. Our results advance the current understanding of the Pi-signaling regulatory network and will potentially contribute to the genetic improvement of crop P efficiency.

Dataset Information

Depleting cytosolic cysteine compromises the antioxidant capacity of the cytosol in Arabidopsis thaliana

Publications

Knocking out cytosolic cysteine synthesis compromises the antioxidant capacity of the cytosol to maintain discrete concentrations of hydrogen peroxide in Arabidopsis.

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