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S-Nitrosylated proteins in pea (Pisum sativum L.) leaf peroxisomes: changes under abiotic stress.

Ortega-Galisteo AP, Rodríguez-Serrano M, Pazmiño DM, Gupta DK, Sandalio LM, Romero-Puertas MC - J. Exp. Bot. (2012)

Bottom Line: NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D).Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes.However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Granada, Spain.

ABSTRACT
Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, β-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.

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Detection of S-nitrosylated proteins in pea-leaf (Pisum sativum L.) extracts under abiotic stress. Protein extracts (150 μg) from pea leaves were not treated (control, C) or treated with 50 μM cadmium or 22.6 mM 2,4-dichlorophenoxy acetic acid (2,4-D) and were subjected to the biotin-switch assay, separated by SDS-PAGE, and immunoblotted with an anti-biotin antibody. Protein loading was verified by Ponceau staining. B, Histogram showing relative quantification of Western blot showed in (A). The intensity of bands was quantified as described in the legend for Fig. 3. The results are representative of four different Western blots assayed.
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fig4: Detection of S-nitrosylated proteins in pea-leaf (Pisum sativum L.) extracts under abiotic stress. Protein extracts (150 μg) from pea leaves were not treated (control, C) or treated with 50 μM cadmium or 22.6 mM 2,4-dichlorophenoxy acetic acid (2,4-D) and were subjected to the biotin-switch assay, separated by SDS-PAGE, and immunoblotted with an anti-biotin antibody. Protein loading was verified by Ponceau staining. B, Histogram showing relative quantification of Western blot showed in (A). The intensity of bands was quantified as described in the legend for Fig. 3. The results are representative of four different Western blots assayed.

Mentions: The biotin-switch assay (Jaffrey et al., 2001) that converts S-nitrosylated cysteine into biotinylated cysteine was first evaluated in pea-leaf extracts, for which no previous description is available. Pea-leaf extracts were incubated with the NO donors SNAP or GSNO to induce S-nitrosylation, with GSSG as the glutathionylating agent, or with the reducing agent TCEP as a negative control, subjected to the biotin-switch method, separated by SDS-PAGE, and immunoblotted into a PVDF membrane. Some endogenous S-nitrosylated proteins can be detected in control (non-treated) extracts. Meanwhile, the signals corresponding to S-nitrosylated proteins increased in both extracts treated with a NO donor, SNAP or GSNO, although the signal was higher with the trans-nitrosylating agent GSNO. A similar signal was detected in non-treated extracts, in proteins pre-incubated with glutathionylating agent GSSG, showing that the biotin-switch method does not detect glutathionylation (Fig. 3). Incubation with TCEP, which reduces all cysteine, eliminated the signal, demonstrating the specificity of the method. Based on these results, this method was considered suitable for studying S-nitrosylation in the pea and for analysing the S-nitrosylation pattern during abiotic stress. S-Nitrosylation in pea leaves was studied under cadmium (50 μM) and 2,4-D (22.6 mM) treatments. Both treatments altered NO metabolism in pea plants, although no data on S-nitrosylation has been previously provided. No differences in S-nitrosylation were found in pea-leaf extracts from cadmium-treated plants with respect to control (Fig. 4A), whereas an increase in this post-translational modification was found in plants after 2,4-D treatment (Fig. 4B).


S-Nitrosylated proteins in pea (Pisum sativum L.) leaf peroxisomes: changes under abiotic stress.

Ortega-Galisteo AP, Rodríguez-Serrano M, Pazmiño DM, Gupta DK, Sandalio LM, Romero-Puertas MC - J. Exp. Bot. (2012)

Detection of S-nitrosylated proteins in pea-leaf (Pisum sativum L.) extracts under abiotic stress. Protein extracts (150 μg) from pea leaves were not treated (control, C) or treated with 50 μM cadmium or 22.6 mM 2,4-dichlorophenoxy acetic acid (2,4-D) and were subjected to the biotin-switch assay, separated by SDS-PAGE, and immunoblotted with an anti-biotin antibody. Protein loading was verified by Ponceau staining. B, Histogram showing relative quantification of Western blot showed in (A). The intensity of bands was quantified as described in the legend for Fig. 3. The results are representative of four different Western blots assayed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3295397&req=5

fig4: Detection of S-nitrosylated proteins in pea-leaf (Pisum sativum L.) extracts under abiotic stress. Protein extracts (150 μg) from pea leaves were not treated (control, C) or treated with 50 μM cadmium or 22.6 mM 2,4-dichlorophenoxy acetic acid (2,4-D) and were subjected to the biotin-switch assay, separated by SDS-PAGE, and immunoblotted with an anti-biotin antibody. Protein loading was verified by Ponceau staining. B, Histogram showing relative quantification of Western blot showed in (A). The intensity of bands was quantified as described in the legend for Fig. 3. The results are representative of four different Western blots assayed.
Mentions: The biotin-switch assay (Jaffrey et al., 2001) that converts S-nitrosylated cysteine into biotinylated cysteine was first evaluated in pea-leaf extracts, for which no previous description is available. Pea-leaf extracts were incubated with the NO donors SNAP or GSNO to induce S-nitrosylation, with GSSG as the glutathionylating agent, or with the reducing agent TCEP as a negative control, subjected to the biotin-switch method, separated by SDS-PAGE, and immunoblotted into a PVDF membrane. Some endogenous S-nitrosylated proteins can be detected in control (non-treated) extracts. Meanwhile, the signals corresponding to S-nitrosylated proteins increased in both extracts treated with a NO donor, SNAP or GSNO, although the signal was higher with the trans-nitrosylating agent GSNO. A similar signal was detected in non-treated extracts, in proteins pre-incubated with glutathionylating agent GSSG, showing that the biotin-switch method does not detect glutathionylation (Fig. 3). Incubation with TCEP, which reduces all cysteine, eliminated the signal, demonstrating the specificity of the method. Based on these results, this method was considered suitable for studying S-nitrosylation in the pea and for analysing the S-nitrosylation pattern during abiotic stress. S-Nitrosylation in pea leaves was studied under cadmium (50 μM) and 2,4-D (22.6 mM) treatments. Both treatments altered NO metabolism in pea plants, although no data on S-nitrosylation has been previously provided. No differences in S-nitrosylation were found in pea-leaf extracts from cadmium-treated plants with respect to control (Fig. 4A), whereas an increase in this post-translational modification was found in plants after 2,4-D treatment (Fig. 4B).

Bottom Line: NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D).Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes.However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Granada, Spain.

ABSTRACT
Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, β-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.

Show MeSH