JXB Advance Access originally published online on March 19, 2008
Journal of Experimental Botany 2008 59(6):1267-1277; doi:10.1093/jxb/ern037
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues
1Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín (CSIC), C/ Prof. Albareda 1, E-18008-Granada, Spain
2Laboratoire Génome et Développement des Plantes, Université de Perpignan, UMR 5096 CNRS-UP-IRD, F-66860 Perpignan, France
3Laboratoire d'Agrophysiologie, EI Purpan, F-31076 Toulouse cedex 3, France
4Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y CSIC, Avda. Américo Vespucio 49, E-41092-Sevilla, Spain
* Present address and to whom correspondence should be sent: Institut des Sciences du Végétal, UPR2355-CNRS Bt23, Centre National de la Recherche Scientifique, F-91198 Gif/Yvette cedex France. E-mail: jose.traverso{at}isv.cnrs-gif.fr
Received 20 December 2007; Revised 22 January 2008 Accepted 22 January 2008
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Abstract |
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Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.
Key words: Heterologous complementation, oxidative stress, pea, Pisum sativum, thioredoxin, vascular tissue, yeast EMY63
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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Thioredoxins (TRXs) are small proteins (12–14 kDa) with a characteristic folding and a conserved active site (WCG/PPC) located at the surface of the enzyme (Schürmann and Jacquot, 2000). The two Cys residues of the active site are involved in thiol–disulphide interchange and interact with a large number of targets, which show the participation of TRXs in redox control of many cellular processes in any organism from bacteria to mammals or plants (Buchanan and Balmer, 2005).
In contrast to mammals, yeast, and bacteria, which contain a low number of TRXs (Laurente et al., 1964; Gan, 1991; Spyrou et al., 1997), these proteins are encoded by a large gene family in plants (Meyer et al., 2002). TRX genes in Arabidopsis are grouped in different families according to sequence similarities and intron positions (Sahrawy et al., 1996). Of these subgroups, the most complex one encodes h-type TRXs, which is formed by nine members in Arabidopsis (Meyer et al., 2005). Type-h TRXs are very abundant in the vascular tissue (Ishiwatari et al., 1995, 1998; Schobert et al., 1998) and, initially, it was assumed that they are localized in the cytoplasm. However, a TRXh2 is localized in mitochondria (Gelhaye et al., 2004) and accumulation of these TRXs in the nucleus of seed cells undergoing oxidative stress has been shown in both developing and germinating wheat seeds (Serrato et al., 2001; Serrato and Cejudo, 2003).
In addition, plants contain TRXs targeted to organelles. In mitochondria from Arabidopsis, an NADP/thioredoxin system was identified formed by NADPH-dependent thioredoxin reductase (NTR) and o-type thioredoxins (Laloi et al., 2001; Reichheld et al., 2005). The chloroplast is an organelle with a complex set of TRXs. Up to nine plastidial TRXs have been described, which were classified into four subgroups: m, f, x, and y (Buchanan and Balmer, 2005). Whilst m- and f-type TRXs play an important role in the redox regulation of enzymes of the Calvin cycle (Buchanan, 1980), x-type TRX is an efficient electron donor of 2-Cys peroxiredoxins (PRX) (Issakidis-Bourguet et al., 2001; Collin et al, 2003), thus being involved in the mechanism of plant protection against oxidative damage.
The chloroplast is also unusual because it contains a specific system for TRX reduction based on reduced ferredoxin and ferredoxin reductase (FTR) (Schürmann and Jacquot, 2000), whereas cytosolic and mitochondrial TRXs are reduced by NADPH in a reaction catalysed by NTR (Laloi et al., 2001; Serrato et al., 2002; Reichheld et al., 2005). Moreover, the complexity of the chloroplast regarding TRX-dependent processes was completed by the discovery of a new type of NTR, stated NTRC, displaying both NTR and TRX activity (Serrato et al., 2004). This new enzyme is, in fact, able to conjugate both activities to reduce 2-Cys PRX efficiently, using NADPH as the source of electrons (Pérez-Ruiz et al., 2006).
Type f and m TRXs were initially identified, and their names given accordingly, by their preferential ability to reduce and activate enzymes involved in the photosynthetic carbon assimilation; fructose-1,6-bisphosphatase and NADP-malate-dehydrogenase, respectively (Wolosiuk and Buchanan, 1977). However, our knowledge of plastidial TRXs has increased considerably since then. Four m-type and two f-type TRXs have been described in A. thaliana (Meyer et al., 2005), and the number of chloroplast targets so far identified for these TRXs is also very large, suggesting the involvement of TRXs in other processes than carbon assimilation (Motohashi et al., 2001; Balmer et al., 2003). A new type of plastidial TRX was characterized, stated as y-type, of which Arabidopsis contains two genes. Type-y TRXs reduce PRX Q with a higher efficiency than the other plastidial TRXs (Collin et al., 2004). These results suggest that TRX y is also involved in ROS detoxification, thus reinforcing the important role of TRXs in the mechanism of chloroplast protection against oxidative damage.
In previous reports, the expression of TRX f and, to a lower extent, of TRX m, were described in non-photosynthetic tissues in pea plants grown under standard conditions or under light and temperature stress (Pagano et al., 2000; Barajas-López et al., 2007). These studies are based on the analysis of the level of TRX f and m transcripts or the activity of the corresponding promoters fused to GUS in transgenic Arabidopsis plants. To analyse the potential function of these plastidial TRXs in non-photosynthetic tissues further, the spatial pattern of expression of both proteins has been studied by immunocytochemical techniques. To address the possible function of these TRXs, their capability to complement the yeast TRX-deficient mutant EMY63 has been analysed.
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Materials and methods |
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Plant material and growth conditions
Pea (Pisum sativum L. cv. Lincoln) seeds were germinated on vermiculite in plastic trays and grown in a growth chamber for 12–15 d under a 16/8 h photoperiod at a light intensity of 200 µmol m–2 s–1, with a day/night temperature of 25/20 °C. Plants were collected and dissected into different parts and then processed or immediately frozen in liquid nitrogen and kept at –80 °C until use. For stress treatments, roots of 12-d-old pea seedlings were placed in the presence of oxidant conditions (up to 15 mM H2O2) and maintained for 15 h in darkness. Roots and leaves were then washed in distilled water and either analysed or frozen in liquid nitrogen and stored at –80 °C until use.
Immunocytochemical analysis
Freshly cut organs were immediately fixed by incubation in FAE (50% ethanol, 5% acetic acid, 3.7% formaldehyde) with occasional vacuum, dehydrated in a graded series of aqueous ethanol solutions and embedded in Paraplast Plus (Sigma Chemical Co.) as described in González et al. (1998). Sections (10 µm thick) were cut with a Leica RM 2025 microtome and placed on poly-L-lysine coated microscope slides. After deparaffinizing in xylol and rehydrating in decreasing concentrations of ethanol, sections were blocked for 3 h in TBS buffer containing 1% (w/v) BSA. Anti-TRX f and anti-TRX m antibodies (diluted 1:1000 in TBS), or pre-immune serum, were added to the samples and incubated overnight at 4 °C. Unbound primary antibodies were removed by three washes of 10 min in TBS. Tissue sections were then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG for 2 h at 37 °C. The reaction of alkaline phosphatase was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. Western-blot analysis was performed according to Traverso et al. (2007a). Both anti-TRXf and anti-TRXm antibodies employed were previously described (Pagano et al., 2000). For cross-reaction assays, different amounts of each pea recombinant protein were dropped on polyvinylidene difluoride membranes and immunodetected using the anti-PsTRXf and PsTRXm (1:1000) coupled with an ECL western-blotting detection system (Amersham Biosciences). Antibodies and pea recombinant TRXs h1, h2, f, and m were already available and have been described elsewhere (Pagano et al., 2000; Traverso et al., 2007a). PsTRXh3 and PsTRXh4 recombinant proteins were kindly provided by Dr F Montrichard (Angers, France).
Heterologous complementation analysis
Complementation experiments were performed in the Saccharomyces cerevisiae strain EMY63 (Müller, 1991) according to previous reports (Issakidis-Bourget et al., 2001; Traverso et al., 2007a). Plant TRX expression in yeast cells was carried out with either the inducible Ycp2 shuttle vector (Cole et al., 1990) or the constitutive pFL61 vector (Minet et al., 1992). Each plant TRX was amplified from the corresponding full-length cDNA by PCR using specific pairs of oligonucleotides. To amplify and clone PsTRXf cDNA, the pairs of primers MluIfN/PeafC and NotIfN/NotIfC (Table 1) were used for Ycp2 and pFL61 vectors, respectively. The pairs MluImN/PeamC and NotImN/NotImC (Table 1) were used to clone the PsTRXm cDNA in the same vectors. Primers were designed to introduce MluI (5')/BamHI (3') or NotI (5')/NotI (3') sites necessary for cloning into Ycp2 and pFL61 vectors. The yeast TRX1 (YLR043c) and pea PsTRXh1 and PsTRXh2 were cloned in both vectors to be used as controls, as previously described (Traverso et al., 2007a). All constructs were introduced into the yeast EMY63 cells by the lithium acetate method (Ito et al., 1983). Yeast transformants were verified by PCR using Ycp2-5'/Ycp2-3' and FLDR/FLGA pairs of primer for Ycp2 and pFL61 constructs, respectively (data not shown). Efficient production of foreign proteins in EMY63 was checked by western blot analysis. Heterologous complementation was performed according to Traverso et al. (2007a). YNB medium supplemented with glucose or galactose (2%, w/v) was used in the oxidant complementation test, containing either 0.8 mM H2O2 or 0.4 mM TBHP. Assays involving methionine or methionine sulphoxide (both at 0.5 mM final concentration) were performed on B medium (Cherest and Surdinkerjan, 1992). Yeast cells were first grown to a density of 107 cells ml–1 in YNB-Glu medium and 7 µl of serial dilutions (5x105, 5x104, 5x103 cells ml–1) were plated on the appropriate solid medium, and incubated for 72–96 h at 30 °C.
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Real-time quantitative RT-PCR analysis
For gene expression analysis by real-time PCR, samples of total RNA (500 ng) extracted from stressed or control plants using the RNeasy Plant Mini kit (Qiagen) were reverse transcribed with the Multiscribe Reverse Transcriptase and random hexamer primers (Applied Biosystems). For quantification, the SYBR Green technology, an ABI Prism 7700 Sequence detector (Applied Biosystems) and the QuantiTect SYBR Green PCR kit (Qiagen) were used. Specific primers at the 3'-untranslated region of each gene were designed using the Primer Express (Applied Biosystems) software, the corresponding pair of primers were f up/down for PsTRXf, m up/down for PsTRXm, apx up/down for PsAPX, and 18S up/down for rRNA 18S (Table 1). Relative quantification of gene expression was monitored after normalization by 18S rRNA expression as internal control, as fold variation over a calibrator using the 2–
CT method (Livak and Schmittgen, 2001).
Thioredoxin and peroxiredoxin activity assays
Peroxiredoxin activity was assayed as disappearance of hydrogen peroxide determined with the Peroxoquant reagent (Perbio Science) at 560 nm in a reaction mixture containing 100 mM phosphate buffer pH 7.0, 0.1 mM hydrogen peroxide, 0.5 mM DTT, 4 µM type II PRX from pea, and 2 µM TRXm or TRXf. Thioredoxin was determined with the DTT-dependent reduction of insulin assay or oxidation of NADPH as previously reported (Serrato et al., 2002). NADPH-dependent reduction of insulin was performed with purified recombinant NTR and Trxh1 from wheat (Serrato et al., 2001, 2002).
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Results |
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TRXs f and m are localized in vascular tissue of pea plants
Previous reports described the expression in non-photosynthetic organs of two pea nuclear genes PsTRXf and PsTRXm, encoding classical plastidial TRXf and TRXm (Pagano et al., 2000; Barajas-López et al., 2007). To confirm this pattern of expression at the protein level and to gain insight on the possible function of these TRXs in non-photosynthetic organs, their spatial localization was analysed by immunohistochemistry using anti-PsTRXf and anti-PsTRXm antibodies, which did not show cross-reaction with either m- or f-type TRXs and with h-type TRXs localized in heterotrophic tissues (Fig. 1). As expected, PsTRXf was abundant in leaf photosynthetic tissues, being the signal strongest in palisade mesophyll cells (Fig. 2A) and cells of the photosynthetic cortex from stems (Fig. 2B, D). In addition, a clear signal was detected in non-photosynthetic tissues, such as cells close to the central vascular bundle from both the stem (Fig. 2B, C) and the root (Fig. 2E, F). Labelling in non-photosynthetic tissues was not homogeneous, but associated to cells next to the xylem vessels of the stem (Fig. 2C) and to unique cells from the vascular cambium in roots (Fig. 2F).
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Regarding PsTRXm, immunolocalization experiments revealed the presence of this protein in leaf photosynthetic cells (Fig. 3A, B), the signal being more clear in cells next to the edge of the leaf (Fig. 3B) than in photosynthetic cells close to the central vascular bundle (Fig. 3A). PsTRXm was less abundant in cells of the photosynthetic cortex from the stem (Fig. 3C, D). However, the most unexpected result was the high labelling detected in the vascular tissue in all the organs analysed: leaf (Fig. 3A), stem (Fig. 3C, D, E), and root (Fig. 3F, G, H). No signal above background was detected using any of the rabbit pre-immune serums (data not shown).
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Functional in vivo approach to search non-classical functions for TRXs f and m from pea
The above-described results showing the presence of both PsTRXf and PsTRXm in non-photosynthetic tissues suggested additional functions for these TRXs besides its well-established role in redox regulation of carbon assimilation in plants (Ruelland and Miginiac-Maslow, 1999). To search for these functions, their ability to complement the yeast EMY63 mutant (Müller, 1991) devoid of the two TRXs present in yeast cells (trx1
, trx2) was tested. This approach has previously been used to identity the function of the cytosolic counterparts PsTRXh1 and PsTRXh2 from pea (Traverso et al., 2007a) and the Arabidopsis thaliana TRXs (Mouaheb et al., 1998; Issakidis-Bourguet et al., 2001; Vignols et al., 2003). Both PsTRXf and PsTRXm full-length cDNAs, excluding the sequence encoding the putative transit peptide, were cloned into the low-copy centromeric Ycp2 (Mouaheb et al., 1998) and into the high-copy pFL61 (Minet et al., 1992) vectors and separately introduced into EMY63 mutant cells. After verifying the presence of both genes and the expression of the corresponding proteins in the transformed yeast cells at the expected level (Fig. 4A), the function(s) of both proteins were tested by EMY63 complementation assays under different growth conditions, using as positive controls the yeast ScTRX1 and the two pea h-type TRXs previously characterized (Traverso et al., 2007a).
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None of the pea TRXs were able to restore yeast sulphate assimilation, compared to yeast cells expressing its own TRX (ScTRX1) (Fig. 4B, Met-). When methionine sulphoxide was added to the B-medium, EMY63 cells expressing either PsTRXf or PsTRXm from the pFL61 vector were able to restore growth partially, whereas no complementation was observed in cells expressing the pea proteins from the Ycp2 vector (Fig. 4B, SOMet). This result suggests the ability of both heterologous proteins to reduce in vivo the yeast methionine sulphoxide reductase, an enzyme involved in oxidative damage repair and required to reverse the oxidation of methionine residues (Moskovitz et al., 1997).
To analyse the possible involvement of PsTRXf and PsTRXm in the response to oxidative stress further, the growth of EMY63 cells expressing both heterologous m and f type pea TRX was tested in medium supplemented with either hydrogen peroxide or tert-butyl hydroperoxide (TBHP) since it is well known that the TRX-deficient yeast mutant is sensitive to both oxidant agents (Issakidis-Bourget et al., 2001). PsTRXm expression conferred to EMY63 mutant cells the ability to grow in the presence of 0.8 mM H2O2, but at a lower extent than the positive controls PsTRXh1 and ScTRX1 (Fig. 4B, H2O2). When the oxidant agent used was TBHP, PsTRXm conferred a weak growth when expressed at low level, from the Ycp2 vector, but promoted a clearer complementation when expressed from the pFL61 vector (Fig. 4B). EMY63 expressing PsTRXf showed a weak growth in medium supplemented with H2O2, but not with TBHP, when it was expressed from the Ycp2 vector, whereas it promoted complementation in the presence of either H2O2 or TBHP when expressed at a high level from the pFL61 vector (Fig. 4B). Therefore, these results suggest the involvement of TRXm, more clearly than TRXf, in protection against oxidative damage.
Effect of oxidative stress treatments on PsTRXf and PsTRXm expression
To test in planta the possible involvement of both PsTRXf and PsTRXm, as deduced from the yeast complementation assay, in the response to oxidative stress, the effect of oxidant agents on the accumulation of PsTRXf and PsTRXm transcripts in roots of pea plants was analysed. The amount of both transcripts was significantly increased in response to hydrogen peroxide treatment, this increase being higher in the case of the PsTRXm gene (Fig. 5A, B). As a positive control, the expression of ascorbate peroxidase 1 (Apx1, Genbank accession number X62077
[GenBank]
), a gene previously shown to be induced in response to oxidative stress (Mittler and Zilinskas, 1992) was analysed. Apx1 transcript accumulated at a higher level in response to increasing concentrations of H2O2 (Fig. 5C), transcript accumulation being higher than for PsTRXf and PsTRXm genes.
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In vitro analysis of TRXs m and f involvement in peroxide detoxification
Both yeast complementation results and induction of expression in response to oxidative stress suggest the involvement of TRXs f and m in the process of peroxide detoxification. To test this possibility, the activity of TRXs f and m as the reductant of a non-plastidial peroxiredoxin was analysed. To that end, a mitochondrial peroxiredoxin (PRX II) was assayed in vitro using DTT as the electron donor. No activity was detected with TRXm and only a low activity was detected with TRXf (Fig. 6A).
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It is well known that the source of reducing power for heterotrophic TRXs (type h) is NADPH in a reaction catalysed by NTR (Schurmann and Jacquot, 2000). Therefore it was tested whether purified recombinant NTR from wheat (Serrato et al., 2002) was able to reduce TRXs m and f. Both TRXm and TRXf, as well as TRXh1 from wheat used as positive control (Serrato et al., 2001), showed the characteristic thioredoxin activity as determined in vitro with the DTT-dependent reduction of insulin assay (Fig. 6B). However, no activity was observed in the presence of NADPH and NTR as reductant of either TRXf or TRXm (Fig. 6C), whereas NTR was an efficient donor of wheat TRXh1, used as positive control (Fig. 6C), as previously shown (Serrato et al., 2002).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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In a previous work, the presence of PsTRXm and PsTRXf mRNAs in non-photosynthetic tissues of pea plants was reported, although the amount of the corresponding proteins, especially of PsTRXf, was unexpectedly low (Pagano et al., 2000). In the present work, a double approach was used based on immunocytochemistry and functional analysis by heterologous complementation of a yeast TRX-deficient mutant and in vitro activity assays, to analyse the expression and possible function of these plastidial TRXs in non-photosynthetic tissues further.
The immunocytochemical approach showed the presence of both TRXs in their classical localization, the photosynthetic cells from leaves and stems, in accordance with its well-established regulatory functions. PsTRXm was detected in the adaxial layer of leaves, as its counterpart PsTRXf, but showing a different pattern within the leaf structure. This protein was also detected in the photosynthetic cortex from the stem, although at a lower level than PsTRXf. The different spatial pattern of expression of both proteins in leaves may indicate different functions in the chloroplast context. Concerning their detection in non-photosynthetic organs, both TRXs were localized to vascular tissues, but showing remarkable differences. PsTRXf localized to cells next to the xylem vascular tubes from the stem, and some cells from the vascular cambium in roots. However, the most unexpected result was the localization of PsTRXm in all the different elements of the vascular tissues from the whole plant. Therefore, the immunocytochemical approach confirmed the localization of both f and m TRXs associated with vascular tissues, apart from their well-known localization in photosynthetic cells. The antibodies used for the immunocytochemical study, anti-TRXf and anti-TRXm, are highly specific and do not cross-react with other types of TRXs (Fig. 1). Furthermore, the pattern of localization of both TRXs is in agreement with the pattern of expression of the corresponding genes based on in situ hybridization and promoter–GUS fusion analysis (Barajas-Lopez et al., 2007). In A. thaliana, four m and two f-type TRXs have been described; therefore, the possibility cannot be excluded of cross-reaction with other f or m-type TRXs most probably present in pea, but not yet characterized. Four h-type TRXs and both PsTRXf and PsTRXm have previously been characterized in P. sativum (Montrichard et al., 2003; Traverso et al., 2007a, b). In addition, there is a new pea m-type TRX, named m2, described in the database (Accession number: AJ316577 [GenBank] ), which has not been characterized.
Although, to our knowledge, this is the first report of vascular localization of plastidial TRXs, the presence of TRXs of the h-type as an abundant component of the phloem sieve tube is well documented (Ishiwatari et al., 1995, 1998; Schobert et al., 1998). The function of TRXs in vascular tissue was initially related to the differentiation of this tissue during the early stages of plant development (Ishiwatari et al., 2000). However, the description of several TRX-related antioxidant systems in phloem, including glutaredoxins or peroxiredoxins (Szederkenyi et al., 1997; Rouhier et al., 2001; Walz et al., 2002), suggested the involvement of TRXs in the mechanism of ROS detoxification (Reichheld et al., 2002; Traverso et al., 2007a). In addition, it has recently been proposed that TRXs in vascular veins could serve as a long-distance thiol signal between different parts of the plant (Balmer et al., 2006).
To search possible functions of m- and f-type TRXs in non-photosynthetic tissues, in vivo heterologous complementation of the yeast mutant EMY63 (Müller, 1991) was performed, a tool previously used to study the function of plant TRXs (Mouaheb et al., 1998; Issakidis-Bourguet et al., 2001; Vignols et al., 2003; Traverso et al., 2007a). EMY63 yeast cells are unable to grow in medium devoid of an organic-sulphur source because the deficiency of TRXs does not allow PAPS-reductase activity, which provides reduced equivalents for methionine synthesis in yeast (Vignols et al., 2005). Heterologous complementation of the yeast EMY63 mutant showed that none of the pea TRXs were able to restore efficient growth in an organic-sulphur-free medium. This result showed that none of the pea plastidial TRXs were able to reduce the yeast PAPS reductase in vivo, in contrast with yeast ScTRX1 (Fig. 3B) or Arabidopsis AtTRXh2 (Mouaheb et al., 1998).
When MetSO was added as a unique source of organic-sulphur both pea heterologous TRXs were able to confer efficient growth to the yeast mutant expressing these proteins from the pFL61 vector. Methionine residues are readily oxidized by reactive oxygen species to form methionine sulphoxides (MetSO), which is reduced to methionine by methionine sulphoxide reductase (MSR) (Romero et al., 2004). This system acts as an antioxidant, repairing proteins damaged by oxidative stress (Mostkovitz et al., 1997). TRXs have been proposed as biological reductants for MSRs (Brot et al., 1981). Our results with the pFL61 expression system suggest that the f and m-type TRXs can interact in vivo with the yeast methionine sulphoxide reductase in agreement with results showing the interaction between methionine sulphoxide reductase and TRXs in Arabidopsis (Marchand et al., 2004; Vieira Dos Santos et al., 2005). In addition, one A. thaliana m-type TRX has been described as a preferential and efficient donor towards MSRB2, a plastidial methionine sulphoxide reductase (Vieira Dos Santos et al., 2007). Altogether these results suggest that both pea TRXs might act as reductants of pea MSR. However, pea TRXs only complement this phenotype of the yeast EMY63 strain when expressed at a high level (from pFL61 vector), therefore more studies including in vitro analysis of the interaction of these proteins are still needed.
The yeast complementation test suggests the involvement of pea TRXs m and f in the mechanism of peroxide detoxification. The trx1 trx2 double yeast mutant is sensitive to oxidative stress (Müller, 1991), a phenotype due to its inability to reduce a type II-PRX (Verdoucq et al., 1999). Pea m- and f-type TRXs restored growth of the yeast mutant in the presence of both hydrogen peroxide and TBHP when expressed at high level, whereas TRXf showed a lower capacity to complement when expressed at a low level (from the Ycp2 vector) (Fig. 4B). Therefore, pea TRXm seems more active in peroxide detoxification than TRXf. Further support for this proposal is provided by the higher efficiency of pea TRXm, as compared to TRXf, to reduce 2-Cys PRX (Bernier-Villamor et al., 2004). In addition, the antioxidant effect of pea TRXs m and f might be due to their capacity to reduce other types of PRXs, however, both TRXs show poor activity as reductants of the mitochondrial type II PRX (Fig. 6A). So, although more information is still needed about the complexity of the PRX family in pea, the antioxidant function of TRXm is more probably based on its capacity to reduce 2-Cys PRX.
Taking advantage of the Arabidopsis microarray database ACT (Arabidopsis Co-expression Tool; Manfield et al., 2006), the set of genes co-expressed with the Arabidopsis m and f-type TRXs was analysed (see Supplementary Table S1 at JXB online). The PsTRXm gene analysed in this work is more closely related to AtTRXm1, 2 and 4 than with AtTRXm3 (Barajas-Lopez et al., 2007). Within the gene pool co-expressed with AtTRXm1, 2 and 4, several genes involved in the response to oxidative stress were found, most of them showing correlation with AtTRXm1 expression: dehydroascorbate reductase (At5g16710), x-type TRX (At1g50320), 2-Cys PRX (At5g06290), ascorbate peroxidases (At4g09010 and At1g77490), 2-Cys PRX BAS1 (At3g11630), PRX Q (At3g26060), and an MSR domain-containing protein (At4g21860), among others. These genes were not well represented in the pool of genes co-expressed with the Arabidopsis f- and m3-type TRX genes (see Supplementary Table S1 at JXB online), thus lending support to the involvement of TRXm in the response to oxidative stress.
An additional question raised by the finding of TRXs m and f in non-photosynthetic tissues is the source of reducing power for these TRXs. The possibility that the source of reducing power is NADPH in a reaction catalysed by NTR can be clearly ruled out based on in vitro assays (Fig. 6B, C). An additional possibility is that TRXs are reduced by reduced ferredoxin and FTR. This is most probably the case in amyloplasts, which contain the enzymes necessary to produce reduced ferredoxin from NADPH (Balmer et al., 2006). Moreover, the expression of an FTR gene in pea roots has been shown (Barajas-López et al., 2007). Lastly, the finding that an Arabidopsis double NtrA-NtrB mutant is viable has led to the description of GRXs as TRX reductants in vivo (Reichheld et al., 2007). The high number of GRXs isoforms that exist in plants (Meyer et al., 2006) could also account for such role.
Regarding gene expression analysis in response to oxidative stress in pea plants, PsTRXf and PsTRXm transcripts increased in roots of plants subjected to hydrogen peroxide treatment. However, similar to the results shown by Pagano et al. (2000), PsTRXf was hardly detected by western blot in root tissues (data not shown), which is in concordance with the low signal that has been detected in the immunohistochemistry studies. By using anti-PsTRXm, no significant variation in root extracts could be found (data not shown). A similar behaviour was described for a TRXm in the green algae C. reinhardtii, showing no change in its protein level although it displayed an increase of gene expression in response to heavy metals (Lemaire et al., 1999).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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The unexpected localization of m and f-type TRXs in non-photosynthetic tissues opens the possibility of additional roles apart from their classical function in photosynthetic enzyme regulation. The pattern of expression in pea vascular tissue, as well as their differential ability to complement the trx1
trx2 double yeast mutant, suggest that pea m-type TRX is involved in the mechanism of oxidant detoxification, probably through its interaction with PRXs. However, the function of PsTRXf as an antioxidant is uncertain and not supported by our results. |
Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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The following supplementary data are available at JXB on line.
Table S1. Gene co-expressed with the four m and two f-type TRXs from Arabidopsis thaliana using ‘Arabidopsis Co-expression Tool’ (Manfield et al., 2006; http://www.arabidopsis.leeds.ac.uk/).
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Acknowledgements |
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This research was supported by grants PB98-0474, BF12002-00401, and BIO2004-02023 from Dirección General de Investigación Científica y Técnica (Spain), CVI 154 and CVI 182 from Junta de Andalucía (Spain), and CSIC (Acción Integrada HF2001-0136). JAT was supported by a fellowship from the Spanish government (FPI98) and PP by a fellowship from the PhD Program of Sevilla University. We thank Dr Amada Pulido for technical assistance, Dr F Montrichard for the gift of PsTRXh3 and PsTRXh4 and Dr Juan J Lazaro for the pea mitochondrial PRX.
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Abbreviations |
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TRX, thioredoxin; PRX, peroxiredoxin; NTR, NADPH-dependent thioredoxin reductase; FTR, ferredoxin-dependent thioredoxin reductase; MetSo, methionine sulphoxide; MSR, methionine sulphoxide reductase.
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