Dataset Information

Proteomic analysis in Arabidopsis tissues reveals hydrogen cyanide regulation of intracellular processes through posttranslational modification of proteins by S-cyanylation

ABSTRACT: Hydrogen cyanide (HCN) is co-produced with ethylene in plant cell and enzymatically detoxified mainly by the mitochondrial ß-cyanoalanine synthase (CAS-C1). Permanent or transient depletion of CAS-C1 activity in Arabidopsis results in physiological alteration in the plant that point to the function of HCN as a gasotransmitter molecule. Label-free quantitative proteomic analysis of enriched mitochondrial samples isolated from wild type and cas-c1 mutant reveled significant changes in protein content, identifying 451 proteins that are absent or less abundant in cas-c1 and 353 proteins only present or more abundant in the mutant background. Gene onthology classification of these proteins highlights proteomic changes that explains the root hairless phenotype and the altered inmune response observed in cas-c1 mutant. The mechanism of action of cyanide as signaling molecule has been addressed by two proteomic approaches focused on identifying S-cyanylation of cysteine as a posttranslational modification of proteins. Both the 2-imino-thiazolidine chemical method and direct untargeted analysis of proteins by LC-MS/MS identified a set of 163 proteins susceptibles to be S-cyanylated that include sedoheptulose 1, 7 biphosphatase (SBPase), the peptidyl-prolyl cis-trans isomerase (CYP20-3) and enolase 2 (ENO2). In vitro analysis of these proteins identified this modification in SBPase Cys74, CYP20-3 Cys259 and ENO2 Cys346 residues that affected the enzymatic activity of the enzymes. GO classification and protein-protein interaction cluster analysis revealed the function of S-cyanylation in the regulation of primary metabolic pathways as glycolysis, and the Calvin and S-adenosylmethionine cicles.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Root

SUBMITTER: Luis C. Romero

LAB HEAD: Luis C Romero

PROVIDER: PXD009812 | Pride | 2019-01-09

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20160327_03_LRomero_R-_1.group	Other
	20160327_03_LRomero_R-_1.mgf	Mgf
	20160327_03_LRomero_R-_1.wiff	Wiff
	20160327_05_LRomero_R_1.group	Other
	20160327_05_LRomero_R_1.mgf	Mgf

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HCN Regulates Cellular Processes through Posttranslational Modification of Proteins by S-cyanylation.

García Irene I Arenas-Alfonseca Lucía L Moreno Inmaculada I Gotor Cecilia C Romero Luis C LC

Plant physiology 20181030 1

Hydrogen cyanide (HCN) is coproduced with ethylene in plant cells and is primarily enzymatically detoxified by the mitochondrial β-CYANOALANINE SYNTHASE (CAS-C1). Permanent or transient depletion of CAS-C1 activity in Arabidopsis (Arabidopsis thaliana) results in physiological alterations in the plant that suggest that HCN acts as a gasotransmitter molecule. Label-free quantitative proteomic analysis of mitochondrially enriched samples isolated from the wild type and cas-c1 mutant ...[more]

PMID: 30377236

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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:MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHO2 is crucial for phosphate (Pi) acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ-based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis roots by mass spectrometry, 35.2 % of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, 5 were significantly differentially expressed between the wild-type and pho2 mutant under 2 growth conditions. Using immunoblot analysis, we validated the upregulation of several PHT1 Pi transporters and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests their roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the post-endoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research. Spectra Processing: The MS/MS raw spectra generated from the Waters Q-TOF Premier (the first replicate) and the AB SCIEX TripleTOF 5600 (the second and third replicates) instruments were processed to detect MS/MS peaks using UniQua (Chang et al., 2013). For the UniQua processing, the spectra were converted into mzXML format using massWolf (version 4.3.1) and ProteoWizard (version 3.0.4623) for the raw data generated from the Waters and AB SCIEX instruments, respectively. The UniQua parameters for Waters Q-TOF Premier were: smoothing = 9, centroiding high = 80%, maximum resolution = 16,000, baseline cutoff = 1.5 counts, reporter m/z range = 114-117, and reporter dynamic range = 5-1,000. The UniQua parameters for AB SCIEX TripleTOF 5600 were: smoothing = 11, centroiding high = 80%, maximum resolution = 25,000, baseline cutoff = 4 counts, reporter m/z range= 114-117 and reporter dynamic range = 5-100,000. The processed MS/MS spectra were then converted into Mascot generic format (.mgf) using mzXML2Search in Trans Proteomics Pipeline (TPP)1 (Deutsch et al., 2010) version 4.4 rev. 1. To obtain better quantification results, the MS/MS spectra with peak number less than 10, average intensity of the top 10 abundant peaks less than 15 counts and iTRAQ average intensity less than 30 counts were further removed from the peaklist file. Protein Qualification and Quantitation: The processed spectra were searched against the TAIR10 database (December 2011, 27,416 sequences) using the Mascot version 2.3 (Matrix Science, London, UK). For the MASCOT search, the mass tolerance for peptide precursor and MS/MS fragments was 0.1 Da. Only one missed cleavage was allowed for tryptic peptides. The methylthiolation (C), iTRAQ4plex (N-term) and iTRAQ4plex (K) were set as fixed modification. The Oxidation (M) and iTRAQ4plex (Y) were set as variable modification. The quantitation option was enabled and set to the iTRAQ4-plex approach. The peptide was considered to be identified if the MASCOT ion score greater than 27 (P less than 0.05). In addition, the protein was considered to be identified if the MASCOT protein score was greater than or equal to 27 with at least one unique and two different peptides identified. For the protein quantitation, only unique peptide was quantified in MASCOT and used to determine the protein ratio in MASCOT. To minimize the systematic ratio error due to sample preparation, the protein ratios were normalized by the mode of the overall protein ratios. For the data acquired by the Q-TOF Premier, 72,817 peptides and 3107 proteins were quantified. For the data acquired by the TripleTOF 5600, 41,885 peptides and 2893 proteins were quantified in the first replicate; 82,397 peptides and 4939 proteins were quantified in the second replicate.

Dataset Information

Proteomic analysis in Arabidopsis tissues reveals hydrogen cyanide regulation of intracellular processes through posttranslational modification of proteins by S-cyanylation

Dataset's files

Publications

HCN Regulates Cellular Processes through Posttranslational Modification of Proteins by <i>S-</i>cyanylation.

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