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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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Subcellular localization of S-nitrosoglutathione (GSNO) and glutathione (GSH) in pea leaves. (A) Pre-immune serum control. (B) Pea-leaf cells with anti-GSNO 1:250. (C) Pea-leaf cells with anti-GSH 1:250. Immunogold labelling of GSH and GSNO are indicated by arrowheads. CL, chloroplast; M, mitochondrion; P, peroxisome; PC, cell wall. Bars, 1 μm.
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fig5: Subcellular localization of S-nitrosoglutathione (GSNO) and glutathione (GSH) in pea leaves. (A) Pre-immune serum control. (B) Pea-leaf cells with anti-GSNO 1:250. (C) Pea-leaf cells with anti-GSH 1:250. Immunogold labelling of GSH and GSNO are indicated by arrowheads. CL, chloroplast; M, mitochondrion; P, peroxisome; PC, cell wall. Bars, 1 μm.

Mentions: NO has been shown to be produced in pea peroxisomes and these organelles have been involved in stress/signalling related to oxygen and nitrogen species in response to abiotic stress (del Río, 2011), although no information about S-nitrosylation is available to date. First, by immunodetection, electron microscopy was used to check the presence of GSH in peroxisomes that, together with NO, could give rise to GSNO, a well-known S-nitrosylating agent. GSH has been previously described in peroxisomes by biochemical techniques (Jimenez et al., 1997) and by electron microscopy (Zechmann et al., 2008). In addition to GSH, the current study detected GSNO in peroxisomes under physiological conditions (Fig. 5B,C). The presence of GSNO suggests that S-nitrosylation could occur in peroxisomes. Also, GSNO labelling was observed to be located in the chloroplast (Fig. 5), but also located mainly in the collenchyma cell wall and xylem (data not shown).


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)

Subcellular localization of S-nitrosoglutathione (GSNO) and glutathione (GSH) in pea leaves. (A) Pre-immune serum control. (B) Pea-leaf cells with anti-GSNO 1:250. (C) Pea-leaf cells with anti-GSH 1:250. Immunogold labelling of GSH and GSNO are indicated by arrowheads. CL, chloroplast; M, mitochondrion; P, peroxisome; PC, cell wall. Bars, 1 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig5: Subcellular localization of S-nitrosoglutathione (GSNO) and glutathione (GSH) in pea leaves. (A) Pre-immune serum control. (B) Pea-leaf cells with anti-GSNO 1:250. (C) Pea-leaf cells with anti-GSH 1:250. Immunogold labelling of GSH and GSNO are indicated by arrowheads. CL, chloroplast; M, mitochondrion; P, peroxisome; PC, cell wall. Bars, 1 μm.
Mentions: NO has been shown to be produced in pea peroxisomes and these organelles have been involved in stress/signalling related to oxygen and nitrogen species in response to abiotic stress (del Río, 2011), although no information about S-nitrosylation is available to date. First, by immunodetection, electron microscopy was used to check the presence of GSH in peroxisomes that, together with NO, could give rise to GSNO, a well-known S-nitrosylating agent. GSH has been previously described in peroxisomes by biochemical techniques (Jimenez et al., 1997) and by electron microscopy (Zechmann et al., 2008). In addition to GSH, the current study detected GSNO in peroxisomes under physiological conditions (Fig. 5B,C). The presence of GSNO suggests that S-nitrosylation could occur in peroxisomes. Also, GSNO labelling was observed to be located in the chloroplast (Fig. 5), but also located mainly in the collenchyma cell wall and xylem (data not shown).

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