JXB Advance Access originally published online on April 4, 2006
Journal of Experimental Botany 2006 57(8):1785-1793; doi:10.1093/jxb/erj175
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Localization of S-nitrosoglutathione and expression of S-nitrosoglutathione reductase in pea plants under cadmium stress*
1Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (Estación Experimental del Zaidín), Área de Bioquímica y Biología Molecular, Universidad de Jaén, E- 23071 Jaén, Spain
2Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain
To whom correspondence should be addressed. E-mail: javier.corpas{at}eez.csic.es
Received 13 December 2005; Accepted 1 March 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
S-nitrosoglutathione (GSNO) is considered a natural nitric oxide (NO·) reservoir and a reactive nitrogen intermediate in animal cells, but little is known about this molecule and its metabolism in plant systems. In this work, using pea plants as a model system, the presence of GSNO in collenchyma cells was demonstrated by an immunohistochemical method. When pea plants were grown with a toxic Cd concentration (50 µM) the content of GSNO in collenchyma cells was drastically reduced. Determination of the nitric oxide (NO·) and gluthathione contents in leaves by confocal laser scanning microscopy and HPLC, respectively, showed a marked decrease of both compounds in plants treated with cadmium. The analysis of the S-nitrosoglutathione reductase (GSNOR) activity and its transcript expression in leaves showed a reduction of 31% by cadmium. These results indicate that GSNO is associated with a specific plant cell type, and this metabolite and its related catabolic activity, GSNOR, are both down-regulated under Cd stress.
Key words: Abiotic stress, collenchyma, formaldehyde dehydrogenase, nitric oxide, reactive nitrogen species, RNS, S-nitrosoglutathione, S-nitrosoglutathione reductase, signalling
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In animal cells, it is well established that nitric oxide (NO) is generated from L-arginine by a family of enzymes called nitric oxide synthases (NOSs) (Alderton et al., 2001), and NO plays a wide range of important functions, such as vasodilatation, relaxation of muscles, neurotransmission, neuromediation, and host defence reactions (Moncada et al., 1991; Ignarro, 2002). In plants, research on NO has gained considerable attention in recent years due to the discovery of its function in plant growth and development and as a key signalling molecule in different intracellular processes (Lamattina et al., 2003, Neill et al., 2003; del Río et al., 2004; Lamotte et al., 2005).
However, in plants, there is little information on the enzymatic source of endogenous NO during normal development of plants and on the loci where NO is generated. Nitric oxide can be produced in plants by non-enzymatic and enzymatic systems (for a review, see del Río et al., 2004). The enzyme nitrate reductase is a well-established generator of NO (Dean and Harper, 1988; Yamasaki et al., 1999; Rockel et al., 2002) although this enzyme does not produce NO from L-arginine and, therefore, it cannot be considered as a characteristic NOS activity. In the past decade, many plant biologists searched diligently for an NO-generating enzyme similar to the nitric oxide synthases (NOSs) identified in mammalian systems (Wendehenne et al., 2001; Corpas et al., 2004a), and there has been an increasing number of reports showing the presence of nitric oxide synthase-like activities in plants (for a review, see del Río et al., 2004). Recently, the presence of a constitutive arginine-dependent NOS activity in roots, stems, and leaves of pea plants was demonstrated (Corpas et al., 2006). At a subcellular level, a NOS activity sensitive to characteristic inhibitors of animal NOS and with the same cofactor requirements (NADPH, BH4, FAD, FMN, calcium, and calmodulin) has been identified in peroxisomes from pea leaves (Barroso et al., 1999; Corpas et al., 2004b). On the other hand, a gene of a plant protein, AtNOS1, which produces NO from L-arginine, has been identified in Arabidopsis, although this NOS activity does not depend on BH4, FAD, and FMN as cofactors (Guo et al., 2003; Guo and Crawford, 2005). To the best of our knowledge, peroxisomes and mitochondria, are the only plant cell organelles where the arginine-dependent NO generation has been demonstrated (Corpas et al., 2004b; Guo and Crawford, 2005).
In animal cells, knowledge on the physiological and metabolic functions of NO and other reactive nitrogen species (RNS) has experienced a significant advance in recent years. As a result, researchers in plant biology have started to pay attention to the interactions reported in animal cells between NO and molecules like thiols, amino acids, and proteins (Alvarez and Radi, 2003). Some important physiological functions of nitric oxide (NO) are performed through the so-called S-nitrosothiols (SNOs) which are formed by reaction of NO with protein or non-protein sulphydryl-containing compounds (Hogg, 2000; Foster et al., 2003). S-nitrosothiols perform important biological reactions, including nitric oxide release, transnitrosation, S-thiolation, as well as direct actions (Hogg, 2000; Stamler et al., 2001). There is considerable evidence in animal cells indicating that S-nitrosylation of cysteine thiols of proteins is an important redox-based post-translational modification (Stamler et al., 2001; Foster et al., 2003).
If little is known in plant systems on the enzymatic source(s) of NO, much less information is available on S-nitrosothiols and their metabolism. The thiol tripeptide, 
-glutamyl cysteinyl glycine (glutathione, GSH) is one of the major low-molecular-weight soluble antioxidants of plant cells and is involved in the antioxidative ascorbate–glutathione cycle (Noctor and Foyer, 1998; Foyer, 2001). But GSH also has important functions apart from the antioxidative system, including the detoxification of xenobiotics and heavy metals (Steffens, 1990) and involvement in signal transduction processes (Puppo et al., 2005). The S-nitrosylation reaction of NO· with GSH to form S-nitrosoglutathione (GSNO) has a significant physiological relevance in plants since GSNO is thought to function as a mobile reservoir of NO bioactivity (Durner et al., 1999; Díaz et al., 2003), as reported in animals (Stamler et al., 2001). Recently, total S-nitrosothiol (SNO) levels were measured in Arabidopsis plants that were altered in GSNOR activity (Feechan et al., 2005) and, as far as is known, no more experimental evidence has been obtained on the presence of GSNO in plant cells.
In recent years, the glutathione-dependent enzyme formaldehyde dehydrogenase (FALDH; EC 1.2.1.1 [EC] ) has been demonstrated to have GSNO reductase activity in bacteria, yeast, and mammals (Liu et al., 2001) and to be involved in the mechanism of protein S-nitrosation in mammalian cells (Haqqani et al., 2003). The enzyme GSNO reductase (GSNOR) catalyses the NADH-dependent reduction of GSNO to GSSG and NH3 (Liu et al., 2001; Lamotte et al., 2005). In plants, glutathione-dependent formaldehyde dehydrogenase has been found very active in the reduction of S-nitrosoglutathione (GSNO) in Arabidopsis (Sakamoto et al., 2002; Achkor et al., 2003) and tobacco (Díaz et al., 2003). In tobacco plants, the gene coding for this enzyme is modulated in response to wounding, jasmonic acid, and salicylic acid (Díaz et al., 2003). The glutathione-dependent formaldehyde dehydrogenase, also denominated class III alcohol dehydrogenase, has been purified and characterized in pea seeds (Uotila and Koivusalo, 1979; Shafqat et al., 1996) and Arabidopsis plants (Martínez et al., 1996).
Cadmium is a toxic trace pollutant for humans, animals, and plants (Wagner, 1993; He et al., 2005). Different metabolic processes such as photosynthesis and respiration are affected by Cd (Romero-Puertas et al., 2004). In pea plants grown with 50 µM CdCl2 this metal produced a significant inhibition of growth, as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content, and disturbances in the nutrient status of pea plants (Sandalio et al., 2001). Moreover, Cd produced disturbances in plant antioxidant defences and was demonstrated to induce oxidative stress in pea plants (Romero-Puertas et al., 2004).
In this work, using pea plants as a model system and employing biochemical, molecular, and cellular approaches, the presence and tissue distribution of GSNO in leaves was studied, and this S-nitrosothiol and its catabolic activity, GSNO reductase, were found to be down-regulated under Cd stress conditions.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and growth conditions
Pea (Pisum sativum L., cv Lincoln) seeds were obtained from Royal Sluis (Enkhuizen, Holland). Plants were grown in the greenhouse in aerated full-nutrient media under optimum conditions for 14 d (Sandalio et al., 2001). The media were then either unsupplemented (control plants) or were supplemented with 50 µM CdCl2, and plants were grown for a further 14 d.
Preparation of leaf extracts
All operations were carried out at 0–4 °C. Leaves were ground using a mortar and pestle in a solution containing 0.1 M TRIS–HCl pH 7.5, 2 mM DTT, 0.1 mM EDTA, 0.2% (v/v) Triton-X-100, and 10% (v/v) glycerol. Homogenates were centrifuged at 27 000 g for 25 min. Then, the supernatants were passed through Sephadex G-25 gel filtration columns (NAP-10 from Amersham) which were equilibrated and eluted with the same buffer before the enzymatic assays.
Enzyme assay and protein determination
GSNOR activity was assayed spectrophotometrically at 25 °C by monitoring the oxidation of NADH at 340 nm as described by Sakamoto et al. (2002). The leaf extracts were incubated in an assay mixture containing 20 mM TRIS–HCl (pH 8.0), 0.2 mM NADH, and 0.5 mM EDTA, and the reaction was started by adding GSNO (Calbiochem) to the mixture at a final concentration of 400 µM. The activity was expressed as nmol NADH consumed min–1 mg–1 protein (
340=6.22 mM–1 cm–1). Protein concentration was assayed according to Bradford (1976) using bovine serum albumin as standard.
Native polyacrylamide gel electrophoresis was performed using 6% acrylamide gels in TRIS-boric-EDTA buffer, pH 8, as described by Laemmli (1970). Staining for GSNOR activity was performed using a modification of the method reported by Seymour and Lazarus (1989). Gels were soaked in 0.1 M sodium phosphate, pH 7.4, containing 2 mM NADH, for 15 min, in an ice-bath. Excess buffer was drained and gels were covered with filter paper strips soaked in freshly prepared 3 mM GSNO. After 10 min, the filter paper was removed and gels were exposed to ultraviolet light and analysed for the disappearance of the NADH fluorescence, indicating GSNOR activity (Fernández et al., 2003).
Quantification of glutathione
The concentration of reduced glutathione was determined according to Jiménez et al. (1997) in leaf extracts obtained in 6% (v/v) perchloric acid, by measuring the reaction of iodoacetic acid with SH-groups to produce S-carboxymethyl derivatives. Amine groups were then derivatized with the Sanger reagent to produce 2,4-dinitrophenyl derivatives which were detected by HPLC using a 3-aminopropyl column.
RNA isolation and cloning of a pea GSNOR cDNA
Total RNA was isolated from pea leaves with the Trizol Reagent Kit (Gibco BRL) as described in the manufacturer's manual. RNA was quantified spectrophotometrically. One µg of total RNA was used as a template for the reverse transcriptase (RT) reaction. It was added to a mixture containing 5 mM MgCl2, 1 mM dNTPs, 0.5 µg polydT23 primer, 1x RT-Buffer, 20 U Rnasin ribonuclease inhibitor, and 20 U AMV reverse transcriptase (FIZZYMES). The reaction was carried out at 42 °C for 40 min, followed by a 5 min step at 98 °C, and then by cooling to 4 °C. Using the protein sequence of the pea glutathione-dependent formaldehyde dehydrogenase (accession number P80572), oligonucleotides were designed for conserved regions (Table 1) and by RT-PCR a partial cDNA of 257 bp was obtained (accession no. DQ084382).
|
The polymerase chain reaction (PCR) was as follows: 1 µl of the produced cDNA diluted 1/20 was added to 250 µM dNTPs, 1.5 mM MgCl2, 1x PCR buffer, 1.25 U of Hot Start Taq polymerase (Eppendorf) and 0.5 µM of each primer (Table 1) in a final volume of 20 µl. Reactions were carried out in a Master Cycler (Eppendorf). A first step of 2 min at 94 °C was followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 65 °C with a final extension of 10 min at 65 °C. Amplified PCR products were detected after electrophoresis in 1% agarose gels stained with ethidium bromide. The PCR reaction was loaded on a 1% agarose gel and the visualized bands were cut and extracted from the gel (Qiaex II gel extraction kit, Qiagen). The purified fragments were cloned into the pGEM-T easy vector (Promega) and sequenced.
Real-time quantitative PCR
The real-time quantitative PCR was performed in 20 µl of reaction mixture, composed of 1 µl of different cDNAs and master mix IQTM SYBR® Green Supermix with a final concentration of 0.5 U of hot-start iTaqTM DNA Polymerase (Bio-Rad), 20 mM KCl, 16 mM TRIS–HCl, pH 8.4, 0.16 mM each dNTPs, 2.4 mM MgCl2, 0.5 µM gene-specific primers (Table 1), and SYBR Green I, 8 nM fluorescein, using a iCycler iQ system (Bio-Rad). Amplifications were performed under the following conditions: initial polymerase activation: 95 °C, 4 min; then 30 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C.
The specific primers for the pea GSNO reductase (Table 1) were designed to anneal at different exons at distances large enough to avoid the appearance of false positive bands caused by co-amplification of contaminating DNA based on the partial cDNA previously obtained, using the RNA 18S as an internal control.
NO· detection by confocal laser scanning microscopy
Pea leaf segments of approximately 25 mm2 were incubated for 1 h at 25 °C, in darkness, with 10 µM 4,5-diaminoflorescein diacetate (DAF-2 DA, Calbiochem) prepared in 10 mM TRIS–HCl (pH 7.4) (Corpas et al., 2004b), this probe being highly specific for NO (Nakatsubo et al., 1998). They were then washed twice in the same buffer for 15 min each. After washing, leaf sections were embedded in a mixture of 15% acrylamide-bisacrylamide stock solution as described elsewhere (Peinado et al., 2000), and 100 µm-thick sections, as indicated by the vibratome scale, were cut under 10 mM phosphate-buffered saline (PBS). Sections were then soaked in glycerol:PBS (containing azide) (1:1; v:v) and mounted in the same medium for examination with a confocal laser scanning microscope system (Leica TCS SL), using standard filters and collection modalities for DAF-2 green fluorescence (excitation 495 nm; emission 515 nm) and chlorophyll autofluorescence (chlorophyll a and b, excitation 429 and 450 nm, respectively; emission 650 nm and 670 nm, respectively) as orange. Background staining, routinely negligible, was controlled with leaf sections unstained.
Cellular localization of GSNO by immunofluorescence microscopy
Pea leaves were cut into 4–5 mm pieces and fixed in 4% (w/v) p-formaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), for 3 h at room temperature. They were then cryoprotected by immersion in 30% (w/v) sucrose in PB overnight at 4 °C. Serial sections, 60 µm thick, were obtained by means of a cryostat (2800 Frigocut E, Reichert-Jung, Vienna, Austria). Free floating sections were incubated at room temperature overnight with a commercial rat antibody against S-nitrosoglutathione (Calbiochem, Cat. no. 487932) diluted 1:2500 in 5 mM TRIS-buffer, pH 7.2, containing 0.9% (w/v) NaCl, 0.05% (w/v) sodium azide, 0.1% (w/v) bovine serum albumin, and 0.1% (v/v) Triton X-100 (TBSA-BSAT). After several washes with TBSA-BSAT, sections were incubated with biotinylated goat anti-rat IgG (Amersham, Buckinghamshire, UK), diluted 1:1000 in TBSA-BSAT, for 1 h at room temperature. Sections were then washed again and incubated with Cy2-streptavidin (Amersham) and diluted 1:1000 in TBSA-BSAT at room temperature for 1.5 h. The sections were mounted on glass slides with PBS-glycerol (v:v; 1:1), covered with a cover slip, and observed using either a fluorescence microscope or a confocal laser scanning microscope with standard filters for Cy2 (excitation 495 nm; emission, 515 nm). Controls for background staining, usually negligible, were performed by replacing the corresponding primary antiserum by preimmune serum in adjacent sections or without primary antiserum.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The cellular localization of GSNO in pea leaf sections was analysed by immunofluorescence microscopy, using a commercial antibody against this molecule (Fig. 1). A strong green immunofluorescence attributable to GSNO was detected mainly in collenchyma cells (Co) of pea leaf sections from control plants (Fig. 1A), but the green fluorescence was considerably reduced in pea leaves of plants grown with cadmium (Fig. 1B). In both cases, the autofluorescence appeared as a yellowish colour in xylem (X), parenchymal (Pa), and epidermal cuticle (Ec) cells of both control (Fig. 1A) and Cd-treated plants (Fig. 1B), with only a change in Ec intensity in Cd-treated plants. Similar results were obtained when the leaf sections were observed by confocal laser scanning microscopy (data not shown). When the antibody against GSNO was omitted in the localization experiments, no immunofluorescence was detected (Fig. 1E). On the other hand, the effect of cadmium on the NO· production in pea leaves was evaluated by confocal laser scanning microscopy (CLSM) using the fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). An intense green fluorescence, characteristic of NO·, was observed in vascular tissues and was less intense in cells of the epidermis, and the palisade and spongy mesophyll (Fig. 1C). When the leaf sections were incubated without the fluorescencent probe, no fluorescence was detected (Fig. 1F). However, in leaves from plants grown with cadmium, the green fluorescence was considerably reduced (Fig. 1D).
|
The GSH content of pea leaves was also analysed and was found to be 49.3±4.2 nmol ml–1 and 22.0±2.0 nmol ml–1 in control and Cd-treated plants, respectively. This indicated that, in leaves from plants grown with 50 µM Cd2+, a reduction of the GSH level of more that 50% was produced.
It has recently been described that Arabidopsis glutathione-dependent formaldehyde dehydrogenase has also S-nitrosoglutahione reductase (GSNOR) activity (Sakamoto et al., 2002). On the basis of this result, the possible existence of this GSNOR activity in leaves from pea plants was investigated. The specificity of the GSNOR activity was assayed by monitoring the oxidation of NADH which took place after the addition of the substrate GSNO (Fig. 2). In these conditions, a GSNOR activity of 2.67±0.28 nmol NADH min–1 mg–1 protein was determined in crude extracts of pea leaves, and when plants were grown with cadmium this activity was reduced by 31% (Fig. 3A). On the other hand, analysis of leaf crude extracts by native polyacrylamide gel electrophoresis and staining for GSNOR activity showed a single band in control plants and this activity was also reduced in Cd-treated plants (Fig. 4).
|
|
|
To analyse the mRNA expression of the pea GSNOR by quantitative real-time PCR, it was necessary to obtain a partial cDNA of the pea GSNOR (accession number DQ084382). A cDNA was obtained which coded for a protein fragment of 85 amino acids. This cDNA showed a 92% identity with the glutathione-dependent formaldehyde dehydrogenase (which has been demonstrated to have GSNOR activity) of Oryza sativa (BAD21676 [GenBank] ), Lycopersicon peruvianum (AAX44241 [GenBank] ), and Zea mays (CAA71913 [GenBank] ), and a 90% identity with the enzyme of Arabidopsis thaliana (Q96533 [GenBank] ). With this sequence information it was posible to design specific primers (Table 1) to study the mRNA expression of GSNOR by quantitative real-time PCR. The results obtained showed that the transcripts of GSNOR were down-regulated (32%) in leaves of pea plants treated with cadmium (Fig. 3B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nitric oxide has been recognized as an important molecule in many plant physiological processes and, depending on the NO· level in the cell, this free radical can act either as a signal or a toxic molecule (Lamattina et al., 2003; Neill et al., 2003; del Río et al., 2004). However, in plant cells there is little information available on the presence and modulation of molecules derived from the NO· metabolism. In animal cells, GSNO is being intensively studied since this molecule is considered a natural reservoir of NO· (Padgett and Whorton, 1995; Steffen et al., 2001; He et al., 2004; Zhang and Hogg, 2004) and one of the most relevant compounds to perform nitrosation reactions under physiological conditions (Steffen et al., 2001).
Nevertheless, the presence of GSNO in plants has only been hypothesized as a molecule involved in the nitric oxide pathway (Durner and Klessig, 1999; Durner et al., 1999; del Río et al., 2003; Díaz et al., 2003; Lamotte et al., 2005; Lindermayr et al., 2005) but thus far there is very little experimental evidence for the presence and distribution of GSNO in plant cells. This situation contrasts with the abundant information available on the NO content in plants (Foissner et al., 2000; Pedroso et al., 2000; Gould et al., 2003; Lamattina et al., 2003; Neill et al., 2003; Corpas et al., 2004b; Gabaldón et al., 2005). The main goal of this work was to study the metabolic relationships of GSNO using as a model pea plants grown with cadmium which has previously been shown to induce oxidative stress in this plant species (Sandalio et al., 2001). In order to get a deeper knowledge into NO and its metabolism in plant cells, biochemical, molecular, and cellular approaches have been used and the content of nitric oxide (NO), glutathione (GSH), S-nitrosoglutathione (GSNO), and the activity and mRNA expression of GSNOR were analysed.
Pea plants have proved to be a good experimental model under normal and stress conditions. Pioneer studies on nitric oxide were carried out with this leguminous plant (Leshem and Haramaty, 1996), even when the enzymatic source(s) of nitric oxide had still to be identified (Corpas et al., 2004a). In previous studies in pea plants, it was demonstrated that Cd induces oxidative stress symptoms characterized by an increase in the generation of ROS, alterations in the antioxidative systems, lipid peroxidation, and oxidative modification of proteins (Sandalio et al., 2001; Romero-Puertas et al., 2002, 2004). These results, together with the ultrastructural data, pointed to an induction of leaf senescence by cadmium (McCarthy et al., 2001; Sandalio et al., 2001).
Results reported in this work showed that under the Cd-induced oxidative stress conditions, the content of soluble GSH in pea leaves was reduced more than 50% and the cellular analysis of NO by CLSM also revealed a significant reduction of this molecule in the vascular tissues. The ratio of reduced to oxidized glutathione has been proposed as a redox sensor in different signal transduction processes (Foyer and Noctor, 2003). Results recently obtained in pea plants treated with Cd have shown that the ratio GSH/GSSG in leaves was significantly decreased by metal treatment (MC Romero-Puertas et al., unpublished results). Therefore, the results reported in this work on the Cd-induced decrease of GSH content of leaves are consistent with the reduction in the GSNO level determined in the same tissue by immunofluorescence histochemistry. On the other hand, during the natural senescence of pea leaves a decrease of the NO· level was reported (Corpas et al., 2004b) which could contribute to the senescence symptoms induced by Cd in leaves of pea plants (McCarthy et al., 2001; Sandalio et al., 2001).
The GSNOR activity has been described to be associated with the enzyme glutathione-dependent formaldehyde dehydrogenase (Sakamoto et al., 2002). Very recently it has been reported that mutations of AtGSNOR1, an Arabidopsis thaliana GSNOR, modulate the extent of cellular S-nitrosothiol formation and turnover, which seems to regulate multiple modes of plant disease resistance (Feechan et al., 2005). In this work to study the GSNO modulation by Cd in pea leaves, the GSNOR activity (total and isozymic) and mRNA expression were analysed. Under cadmium stress, a down-regulation in both activity and transcripts was found in pea leaves (Fig. 3), and these results were well correlated with the observed decrease of the NO and GSH contents. This suggests that Cd stress down-regulates both activity and expression of GSNOR, probably because, under these conditions, the formation of GSNO, the enzyme's substrate, is also depressed as checked by immunofluorescence histochemistry. The plant GSNOR, as proposed in microbes and mammals (Liu et al., 2001), might have a dual role in turning off GSNO-derived NO signalling and in the cellular protection against nitrosative stress by controlling excess S-nitrosylation (Liu et al., 2001; Sakamoto et al., 2002; Lamotte et al., 2005). Under the conditions used in this work, Cd is known to induce oxidative stress in pea plants (McCarthy et al., 2001; Sandalio et al., 2001; Romero-Puertas et al., 2002, 2004), and the results obtained indicate that it is very unlikely that Cd can cause nitrosative stress in pea plants. This suggests that oxidative and nitrosative stress do not necessarily go hand in hand.
In Arabidopsis the expression of the GS-FDH/GSNOR gene is down-regulated by mechanical wounding and activated by salicylic acid (Díaz et al., 2003). However, in preliminary results obtained in our laboratory with pea plants under different environmental stresses, an increase in the GSNOR activity was found by low and high temperature, continuous light, darkness, and mechanical wounding (FJ Corpas et al., unpublished results). These results contrast with those reported in this work for Cd and suggests under that abiotic stress conditions, the GSNOR activity is differentially regulated depending on the stress type.
An important question to elucidate is the physiological function of GSNO in the collenchyma cells and its down-regulation by cadmium stress. In pea leaves, it has previously been observed that NO· was localized mainly in the xylem of vascular tissue but not in collenchyma cells (Corpas et al., 2004b), and similar results were reported in the xylem of Zinnia elegans (Gabaldón et al., 2005). The collenchyma is a typical supporting tissue of the primary plant body and growing plant organs. Often either phloem or xylem of the vascular bundles is associated with collenchyma cells. The localization of H2O2 and peroxidase in collenchyma tissues of Zinnia elegans stems has been reported (Ferrer and Ros-Barceló, 1999) which could indicate that they are involved in the cross-linking of pectins and hemicelluloses that predominate in the walls of these cells. In addition, a glutathione peroxidase-like protein was localized in the collenchyma of Solanum lycopersicum, and this protein was induced by mechanical stimulation, suggesting that it could regulate apoplastic ROS accumulation (Herbette et al., 2004). Considering that NO· can inhibit the H2O2-metabolizing enzymes catalase and ascorbate peroxidase (Clark et al., 2000), the presence in the collenchyma of the NO· donor GSNO could allow the persistence of hydrogen peroxide, a metabolite involved in the lignification process in vascular tissues (Ros-Barceló, 1997, 2005; Gabaldón et al., 2005). Thus the NO· level regulated by GSNOR might contribute to the gradual lignification of the adjacent cells of collenchyma during the cell proliferation and elongation processes.
In summary, in this work the presence of GSNO and its tissue localization was demonstrated in pea plants. The presence of S-nitrosoglutathione reductase activity was verified in leaves from pea plants and this activity and GSNO were found to be decreased under Cd stress conditions. If it has been demonstrated in Arabidopsis that a decrease in GSNOR activity causes an accumulation of SNO (Feechan et al., 2005), results reported in this work suggest an alternative possibility: that the GSNOR activity could be induced by its substrate GSNO. Further research is necessary to demonstrate this hypothesis, and to localize and characterize the GSNO metabolic pathways in plant cells. This would open new avenues in the research of the physiological function of NO· in higher plants.
|
Acknowledgements |
|---|
MR-S acknowledges a PhD fellowship from the Ministry of Education and Science. This work was supported by the Ministry of Science and Technology (projects AGL2003-05524 and BFI2002-04440-CO2-01) and the Junta de Andalucía (Research Groups CVI 0192 and CVI 0286). The authors are grateful to Dr Francisco Luque, University of Jaén, for his helpful advice in real-time quantitative PCR and Dr Manuel Gómez for his valuable help in the growth of pea plants. The confocal laser scanning microscopy analyses were carried out at the Technical Services of the University of Jaén, and special thanks are given to Miss Nieves de la Casa-Adán for her technical assistance.
|
Footnotes |
|---|
* Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number DQ084382.
|
Abbreviations |
|---|
CLSM, confocal laser scanning microscopy; NO, nitric oxide; ROS, reactive oxygen species; RNS, reactive nitrogen species; DAF-2 DA, 4,5-diaminofluorescein diacetate; GSNO, S-nitrosoglutathione.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Achkor H, Díaz M, Fernández MR, Biosca JA, Parés X, Martínez MC. (2003) Enhanced formaldehyde detoxification by overexpression of glutathione-dependent formaldehyde dehydrogenase from Arabidopsis. Plant Physiology 132:2248–2255.
Alderton WK, Cooper CE, Knowles RG. (2001) Nitric oxide synthases: structure, function and inhibition. Biochemical Journal 357:593–615.[CrossRef][Web of Science][Medline]
Alvarez B and Radi R. (2003) Peroxynitrite reactivity with amino acids and proteins. Amino Acids 25:295–311.[CrossRef][Web of Science][Medline]
Barroso JB, Corpas FJ, Carreras A, Sandalio LM, Valderrama R, Palma JM, Lupiáñez JA, del Río LA. (1999) Localization of nitric oxide synthase in plant peroxisomes. Journal of Biological Chemistry 27:36729–36733.
Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quatities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254.[CrossRef][Web of Science][Medline]
Clark D, Durner J, Navarre DA, Klessig DF. (2000) Nitric oxide inhibition of tobacco catalase and ascorbate peroxidase. Molecular Plant–Microbe Interactions 13:1380–1384.
Corpas FJ, Barroso JB, del Río LA. (2004a) Enzymatic sources of nitric oxide in plant cells: beyond one protein–one function. New Phytologist 162:246–248.[CrossRef]
Corpas FJ, Barroso JB, Carreras A, et al. (2004b) Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants. Plant Physiology 136:2722–2733.
Corpas FJ, Barroso JB, Carreras A, Valderrama R, Palma JM, León AM, Sandalio LM, del Río LA. (2006) Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development. Planta 223: DOI 10.1007/s00425-005-0205-9.
Dean JV and Harper JE. (1988) The conversion of nitrite to nitrogen oxide(s) by the constitutive NAD(P)H-nitrate reductase enzyme from soybean. Plant Physioogy 88:389–395.
del Río LA, Corpas FJ, Barroso JB. (2004) Nitric oxide and nitric oxide synthase activity in plants. Phytochemistry 65:783–792.[CrossRef][Web of Science][Medline]
del Río LA, Corpas FJ, Sandalio LM, Palma JM, Barroso JB. (2003) Plant peroxisomes, reactive oxygen metabolism and nitric oxide. IUBMB Life 55:71–81.[Web of Science][Medline]
Díaz M, Achkor H, Titarenko E, Martínez MC. (2003) The gene encoding glutathione-dependent formaldehyde dehydrogenase/GSNO reductase is responsive to wounding, jasmonic acid and salicylic acid. FEBS Letters 543:136–139.[CrossRef][Web of Science][Medline]
Durner J, Gow AJ, Stamler JS, Glazebrook J. (1999) Ancient origins of nitric oxide signaling in biological systems. Proceedings of the National Academy of Sciences, USA 96:14206–14207.
Durner J and Klessig DF. (1999) Nitric oxide as a signal in plants. Current Opinion in Plant Biology 2:369–374.[CrossRef][Web of Science][Medline]
Feechan A, Kwon E, Yun BW, Wang Y, Pallas JA, Loake GJ. (2005) A central role for S-nitrosothiols in plant disease resistance. Proceedings of the National Academy of Sciences, USA 102:8054–8059.
Fernández MR, Biosca JA, Parés X. (2003) S-nitrosoglutathione reductase activity of human and yeast glutathione-dependent formaldehyde dehydrogenase and its nuclear and cytoplasmic localization. Cellular and Molecular Life Sciences 60:1013–1018.[CrossRef][Medline]
Ferrer MA and Ros Barceló A. (1999) Differential effects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans. Plant, Cell and Environment 22:891–897.[CrossRef]
Foissner I, Wendehenne D, Langebartels C, Durner J. (2000) In vivo imaging of an elicitor-induced nitric oxide burst in tobacco. The Plant Journal 23:817–824.[CrossRef][Web of Science][Medline]
Foster MW, McMahon TJ, Stamler JS. (2003) S-nitrosylation in health and disease. Trends in Molecular Medicine 9:160–168.[CrossRef][Web of Science][Medline]
Foyer CH. (2001) Prospects for enhancement of the soluble antioxidants, ascorbate and glutathione. Biofactors 15:75–78.[Web of Science][Medline]
Foyer CH and Noctor G. (2003) Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiologia Plantarum 119:355–364.[CrossRef]
Gabaldón C, Gómez-Ros LV, Pedreño MA, Ros-Barceló R. (2005) Nitric oxide production by the differentiating xylem of Zinnia elegans. New Phytologist 165:121–130.[CrossRef][Web of Science][Medline]
Gould KS, Lamotte O, Klinguer A, Pugin A, Wendehenne D. (2003) Nitric oxide production in tobacco leaf cells: a generalized stress response? . Plant, Cell and Environment 26:1851–1862.[CrossRef]
Guo FQ and Crawford NM. (2005) Arabidopsis nitric oxide synthase1 is targeted to mitochondria and protects against oxidative damage and dark-induced senescence. The Plant Cell 17:3436–3450.
Guo FQ, Okamoto M, Crawford NM. (2003) Identification of a plant nitric oxide synthase gene involved in hormonal signaling. Science 302:100–103.
Haqqani AS, Do SK, Birnboim HC. (2003) The role of a formaldehyde dehydrogenase-glutathione pathway in protein S-nitrosation in mammalian cells. Nitric Oxide 9:172–181.[CrossRef][Web of Science][Medline]
He J, Kang H, Yan F, Chen C. (2004) The endoplasmic reticulum-related events in S-nitrosoglutathione-induced neurotoxicity in cerebellar granule cells. Brain Research 1015:25–33.[CrossRef][Web of Science][Medline]
He ZL, Yang XE, Stoffella PJ. (2005) Trace elements in agroecosystems and impacts on the environment. Journal of Trace Elements in Medical Biology 19:125–140.
Herbette S, Brunel N, Prensier G, Julien JL, Drevet JR, Roeckel-Drevet P. (2004) Immunolocalization of a plant glutathione peroxidase-like protein. Planta 219:784–789.[CrossRef][Web of Science][Medline]
Hogg N. (2000) Biological chemistry and clinical potential of S-nitrosothiols. Free Radical Biology and Medicine 28:1478–1486.[CrossRef][Web of Science][Medline]
Ignarro LI. (2002) Nitric oxide as a unique signaling molecule in the vascular system: a historial overview. Journal of Physiology and Pharmacology 53:503–514.[Web of Science][Medline]
Jiménez A, Hernández JA, del Río LA, Sevilla F. (1997) Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiology 114:275–284.[Abstract]
Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.[CrossRef][Medline]
Lamattina L, García-Mata C, Graziano M, Pagnussat G. (2003) Nitric oxide: the versatility of an extensive signal molecule. Annual Review of Plant Biology 54:109–136.[CrossRef][Medline]
Lamotte O, Courtois C, Barnavon L, Pugin A, Wendehenne D. (2005) Nitric oxide in plants: the biosynthesis and cell signalling properties of a fascinating molecule. Planta 221:1–4.[CrossRef][Web of Science][Medline]
Leshem YY and Haramaty E. (1996) The characterization and contrasting effects of the nitric oxide free radical in vegetative stress and senescence of Pisum sativum Linn. Journal of Plant Physiology 148:258–263.[Web of Science]
Lindermayr C, Saalbach G, Durner J. (2005) Proteomic identification of S-nitrosylated proteins in Arabidopsis. Plant Physiology 137:921–930.
Liu L, Hausladen A, Zeng M, Que L, Heitman J, Stamler JS. (2001) A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature 410:490–494.[CrossRef][Medline]
Martínez MC, Achkor H, Persson B, Fernández MR, Shafqat J, Farrés J, Jörnvall H, Parés X. (1996) Arabidopsis formaldehyde dehydrogenase. Molecular properties of plant class III alcohol dehydrogenase provide further insights into the origins, structure and function of plant class P and liver class I dehydrogenases. European Journal of Biochemistry 241:849–857.[Web of Science][Medline]
McCarthy I, Romero-Puertas MC, Palma JM, Sandalio LM, Corpas FJ, Gómez M, del Río LA. (2001) Cadmium induces senescence symptoms in leaf peroxisomes of pea plants. Plant, Cell and Environment 24:1065–1073.[CrossRef]
Moncada S, Palmer RMJ, Higgs EA. (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacology Review 43:109–142.
Neill SJ, Desikan R, Hancock JT. (2003) Nitric oxide signalling in plants. New Phytologist 159:11–35.[CrossRef][Web of Science]
Noctor G and Foyer CH. (1998) Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49:249–279.[CrossRef][Web of Science]
Padgett CM and Whorton AR. (1995) S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation. American Journal of Physiology 269:C739–C749.
Pedroso MC, Magalhaes JR, Durzan D. (2000) A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues. Journal of Experimental Botany 51:1027–1036.
Peinado MA, Torres MI, Thompson RP, Esteban FJ. (2000) Immunolocalization of the HNK-1 epitope in the autonomic innervation to the liver and upper digestive tract of the developing rat embryo. Histochemical Journal 32:439–446.[CrossRef][Web of Science][Medline]
Puppo A, Groten K, Bastian F, Carzaniga R, Soussi M, Lucas MM, De Felipe MR, Harrison J, Vanacker H, Foyer CH. (2005) Legume nodule senescence: roles for redox and hormonal signalling in the orchestration of the natural aging process. New Phytologist 165:683–701.[CrossRef][Web of Science][Medline]
Rockel P, Strube F, Rockel A, Wildt J, Kaiser WM. (2002) Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro. Journal of Experimental Botany 53:103–110.
Romero-Puertas MC, Palma JM, Gómez M, del Río LA, Sandalio LM. (2002) Cadmium causes the oxidative modification of proteins in pea plants. Plant, Cell and Environment 25:677–686.[CrossRef]
Romero-Puertas MC, Rodríguez-Serrano M, Corpas FJ, Gómez M, del Río LA, Sandalio LM. (2004) Cadmium-induced subcellular accumulation of 
and H2O2 in pea leaves. Plant, Cell and Environment 27:1122–1134.[CrossRef]
Ros-Barceló A. (1997) Lignification in plant cell walls. International Review of Cytology 176:87–132.[Web of Science][Medline]
Ros-Barceló A. (2005) Xylem parenchyma cells deliver the H2O2 necessary for lignification in differentiating xylem vessels. Planta 220:747–756.[CrossRef][Web of Science][Medline]
Sakamoto A, Ueda M, Morikawa H. (2002) Arabidopsis glutathione-dependent formaldehyde dehydrogenase is an S-nitrosoglutathione reductase. FEBS Letters 515:20–24.[CrossRef][Web of Science][Medline]
Sandalio LM, Dalurzo HC, Gómez M, Romero-Puertas MC, del Río LA. (2001) Cadmium-induced changes in the growth and oxidative metabolism of pea plants. Journal of Experimental Botany 52:2115–2126.
Shafqat J, El-Ahmad M, Danielsson O, Martínez MC, Persson B, Parés X, Jörnvall H. (1996) Pea formaldehyde-active class III alcohol dehydrogenase: common derivation of the plant and animal forms but not of the corresponding ethanol-active forms (classes I and P). Proceedings of the National Academy of Sciences, USA 93:5595–5599.
Seymour JL and Lazarus RA. (1989) Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases. Analytical Biochemistry 178:243–247.[CrossRef][Web of Science][Medline]
Stamler JS, Lamas S, Fang FC. (2001) Nitrosylation: the prototypic redox-based signaling mechanism. Cell 106:675–683.[CrossRef][Web of Science][Medline]
Steffens JC. (1990) The heavy metal-binding peptide of plants. Annual Review of Plant Physiology and Plant Molecular Biology 41:553–575.[Web of Science]
Steffen M, Sarkela TM, Gybina AA, Steele TW, Trasseth NJ, Kuehl D, Giulivi C. (2001) Metabolism of S-nitrosoglutathione in intact mitochondria. Biochemical Journal 356:395–402.[CrossRef][Web of Science][Medline]
Uotila L and Koivusalo M. (1979) Purification of formaldehyde and formate dehydrogenases from pea seeds by affinity chromatography and S-formylglutathione as the intermediate of formaldehyde metabolism. Archives of Biochemistry and Biophysics 196:33–45.[CrossRef][Web of Science][Medline]
Wagner GJ. (1993) Accumulation of cadmium in crop plants and its consequences to human health. Advances in Agronomy 51:173–212.
Wendehenne D, Pugin A, Klessig DF, Durner J. (2001) Nitric oxide: comparative synthesis and signaling in animal and plant cells. Trends Plant Science 6:177–183.[CrossRef][Web of Science][Medline]
Yamasaki H, Sakihama Y, Takahashi S. (1999) An alternative pathway for nitric oxide production in plants: new features of an old enzyme. Trends Plant Science 4:128–129.[CrossRef][Web of Science][Medline]
Zhang Y and Hogg N. (2005) S-Nitrosothiols: cellular formation and transport. Free Radical Biology and Medicine 38:831–838.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Rodriguez-Serrano, M. C. Romero-Puertas, D. M. Pazmino, P. S. Testillano, M. C. Risueno, L. A. del Rio, and L. M. Sandalio Cellular Response of Pea Plants to Cadmium Toxicity: Cross Talk between Reactive Oxygen Species, Nitric Oxide, and Calcium Plant Physiology, May 1, 2009; 150(1): 229 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Chaki, A. M. Fernandez-Ocana, R. Valderrama, A. Carreras, F. J. Esteban, F. Luque, M. V. Gomez-Rodriguez, J. C. Begara-Morales, F. J. Corpas, and J. B. Barroso Involvement of Reactive Nitrogen and Oxygen Species (RNS and ROS) in Sunflower-Mildew Interaction Plant Cell Physiol., March 1, 2009; 50(3): 665 - 679. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chaki, A. M. Fernandez-Ocana, R. Valderrama, A. Carreras, F. J. Esteban, F. Luque, M. V. Gomez-Rodriguez, J. C. Begara-Morales, F. J. Corpas, and J. B. Barroso Involvement of Reactive Nitrogen and Oxygen Species (RNS and ROS) in Sunflower-Mildew Interaction Plant Cell Physiol., February 1, 2009; 50(2): 265 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Corpas, M. Chaki, A. Fernandez-Ocana, R. Valderrama, J. M. Palma, A. Carreras, J. C. Begara-Morales, M. Airaki, L. A del Rio, and J. B. Barroso Metabolism of Reactive Nitrogen Species in Pea Plants Under Abiotic Stress Conditions Plant Cell Physiol., November 1, 2008; 49(11): 1711 - 1722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Lee, C. Wie, B. O. Fernandez, M. Feelisch, and E. Vierling Modulation of Nitrosative Stress by S-Nitrosoglutathione Reductase Is Critical for Thermotolerance and Plant Growth in Arabidopsis PLANT CELL, March 1, 2008; 20(3): 786 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Neill, J. Bright, R. Desikan, J. Hancock, J. Harrison, and I. Wilson Nitric oxide evolution and perception J. Exp. Bot., January 1, 2008; 59(1): 25 - 35. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jasid, M. Simontacchi, C. G. Bartoli, and S. Puntarulo Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins Plant Physiology, November 1, 2006; 142(3): 1246 - 1255. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||