| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online November 10, 2006; 10.1104/pp.106.089524 Plant Physiology 143:300-311 (2007) © 2007 American Society of Plant Biologists PsTRXh1 and PsTRXh2 Are Both Pea h-Type Thioredoxins with Antagonistic Behavior in Redox Imbalances1,2Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, 18008 Granada, Spain (J.A.T., A.P., M.S., A.C.); Laboratoire Génome et Développement des Plantes, Université de Perpignan, Unité Mixte de Recherche 5096, Centre National de la Recherche Scientifique, 66860 Perpignan, France (F.V., Y.M.); Laboratoire d'Agrophysiologie, Unité Mixte de Recherche 1054 Institut National de la Recherche Agronomique, Ecole Supérieure d'Agriculture de Purpan, 31076 Toulouse cedex 3, France (R.C.); and Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, 41092 Seville, Spain (F.J.C.)
Thioredoxins (TRXs) are small ubiquitous oxidoreductases involved in disulfide bond reduction of a large panel of target proteins. The most complex cluster in the family of plant TRXs is formed by h-type TRXs. In Arabidopsis (Arabidopsis thaliana), nine members of this subgroup were described, which are less well known than their plastidial counterparts. The functional study of type-h TRXs is difficult because of the high number of isoforms and their similar biochemical characteristics, thus raising the question whether they have specific or redundant functions. Type-h TRXs are involved in seed germination and self incompatibility in pollen-pistil interaction. Their function as antioxidants has recently been proposed, but further work is needed to clarify this function in plants. In this study, we describe two new h-type TRXs from pea (Pisum sativum; stated PsTRXh1 and PsTRXh2). By functional complementation of a yeast (Saccharomyces cerevisiae) trx1  trx2 double mutant, we demonstrate that PsTRXh1 is involved in the redox-imbalance control, possibly through its interaction with peroxiredoxins. In contrast, PsTRXh2 provokes a phenotype of hypersensitivity to hydrogen peroxide in the yeast mutant. Furthermore, we show differential gene expression and protein accumulation of the two isoforms, PsTRXh1 protein being abundantly detected in vascular tissue and flowers, whereas PsTRXh2 gene expression was hardly detectable. By comparison with previous data of additional PsTRXh isoforms, our results indicate specific functions for the pea h-type TRXs so far described.
Thioredoxins (TRXs) are small proteins (12–14 kD) with a characteristic folding and a conserved redox-active site WCG/PPC. The thiols of the Cys residues at the active site act as powerful reducing agents, able to disrupt disulphide bridges of target proteins. The large number of putative TRX targets identified during the last years indicates that TRXs are involved in an increasing number of processes in plants (Buchanan and Balmer, 2005  ).
TRXs are present in all types of organisms from bacteria to mammals, but plants exhibit the most complex TRX multigenic family, as shown by the analysis performed in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genomes. The Arabidopsis genome encodes more than 25 different TRXs and other TRX-like proteins with multiple TRXs or non-TRX extra domains, being nuclear encoded and grouped by sequence similarity and intron positions (Meyer et al., 2002
In view of such diverse data, a great effort is still needed to clarify their role in the cell, the identification of multiple h-type TRX genes raising questions related to the specificity of their physiological roles. The first attempt to address these questions was the heterologous complementation of the yeast (Saccharomyces cerevisiae) mutant EMY63 (Müller, 1991
In pea (Pisum sativum), five isoforms of the TRX family have been described. Three of them, f, m1, and m2, are chloroplastic (Jaramillo et al., 1997
PsTRXh1 and PsTRXh2 Are TRX Genes Belonging to the h-Type Group
At the time we started this study, only chloroplastic TRXs had been described in pea, none of the cytosolic isoforms being discovered yet. Using an approach based on reverse transcription (RT)-PCR and RACE, we amplified from leaf total RNA two full-length cDNAs, termed PsTRXh1 (GenBank accession no. AJ310990) and PsTRXh2 (GenBank accession no. AJ319808). Both cDNAs contain a unique open reading frame (ORF) that encodes for polypeptides of 120 (PsTRXh1) and 118 residues (PsTRXh2; Fig. 1A
). The deduced proteins exhibit similar molecular mass and present 60% identity at the amino acid level as well as the structural and functional amino acid features found in other h-type TRXs, including the canonical active site WCGPC (Fig. 1A). In addition, three-dimensional models for both deduced sequences, based on resolved h-type TRXs (Coudevylle et al., 2005
The genomic sequences of PsTRXh1 and PsTRXh2 genes were 1,689 bp and 897 bp long, each containing two introns of 1,236 and 90 bp in the PsTRXh1 gene and of 321 and 219 bp in the PsTRXh2 gene, respectively (Fig. 1C). Both intron positions were conserved within the h-type TRX cluster (Sahrawy et al., 1996 ). We could not find any significant similarity between either PsTRXh1 and PsTRXh2 intronic sequences or in 5'- and 3'-untranslated regions. According to both number and position of introns, both genes encode most probably h-type TRXs.
To determine whether PsTRXh1 and PsTRXh2 isoforms possess TRX activity in vitro, their corresponding ORFs were expressed in Escherichia coli to produce recombinant proteins. Both proteins accumulated at a high level in the soluble fraction and were purified to homogeneity (Fig. 2A
). Interestingly, both proteins presented different thermo stability degrees, PsTRXh1 being resistant to higher temperatures (up to 75°C) than PsTRXh2 (45°C). In addition, PsTRXh1 was highly soluble during the purification process, while PsTRXh2 tended to form homodimeric structures (data not shown). TRX activity was determined in vitro by measuring the capacity of the recombinant proteins to reduce disulfide-bridged insulin, using as controls the well-characterized chloroplastic PsTRXf and PsTRXm1 (Pagano et al., 2000
PsTRXh1 and PsTRXh2 Complement Some Aspects of the trx1 trx2 Yeast Mutant in Vivo
To know whether PsTRXh1 and PsTRXh2 are also functional TRXs in vivo, we tested their ability to complement the trx1
Because trx1 trx2 yeast cells are also sensitive to oxidative stress, we analyzed the growth of trx1 trx2 mutant cells expressing heterologous PsTRXs on medium containing oxidant molecules. We found that PsTRXh2 conferred to trx1 trx2 cells the ability to grow in the presence of 0.4 mM terc-butyl-hydroperoxide (TBHP) at a lower extent than the positive controls AtTRXh3 and ScTRX1 but at a higher extent than AtTRXh2 (Fig. 3B, below right). Mutant cells expressing PsTRXh1 grew poorly. On the other hand, trx1 trx2 cells producing PsTRXh1 presented a rapid growth, similar to mutant cells producing ScTRX1 or AtTRXh3, on a medium containing 0.8 mM H2O2 (Fig. 3B, below left), whereas cells expressing PsTRXh2 did not grow in such oxidant growth conditions.
Interestingly, while confirming these results by halo-inhibition disc assays, we noticed that yeast cells expressing PsTRXh2 presented a higher sensitivity toward H2O2 than those transformed by the empty vector Ycp2, showing an excess of sensitivity (20%) in comparison to the negative control (Fig. 3C). To verify this hypersensitive effect of PsTRXh2, we repeated yeast mutant complementation assays to measure sensitivity to 0.4 mM H2O2 using pFL61 as shuttle vector. Figure 3D shows that all yeast mutant cells expressing both plant and yeast TRX from the phosphoglycerate kinase promoter, including those carrying the empty pFL61 vector, could grow in the presence of the oxidant molecules except those expressing PsTRXh2. This result confirmed that PsTRXh2 confers hypersensitivity to H2O2 to yeast cells in vivo. Taken together, our results demonstrate that both PsTRXh1 and PsTRXh2 are functional TRXs in vivo but confer different behavior to trx1
The above results suggest that in addition to a true functional TRX activity in vivo, PsTRXh1 and PsTRXh2 may display a divergent function in pea tissues in light of their different behavior in yeast cells under oxidative stress. To characterize the pattern of expression of PsTRXh1 and PsTRXh2 genes, we first performed a relative quantification of their corresponding mRNAs using real-time PCR on different tissues excised from 100-d-old pea plants. PsTRXh1 transcripts were detected in all the organs tested, showing a higher accumulation in leaves and flowers (Fig. 4A
). In contrast, PsTRXh2 mRNA was almost undetectable, presenting a much weaker accumulation than PsTRXh1 mRNA, with mean values lower than 1% as compared with the PsTRXh1 level of mRNA (data not shown). A higher level of PsTRXh2 gene expression could be detected only in plantlets younger than 12 d but always remained at a maximum expression level of 18% and 16% in respect to the values of PsTRXh1 transcripts in leaves and roots, respectively (data not shown). These results were confirmed in transgenic Arabidopsis plants expressing the
We next analyzed the accumulation of PsTRXh1 and PsTRXh2 proteins in pea tissues and whether their accumulation correlated their corresponding gene expression. For this purpose, recombinant PsTRXh1 and PsTRXh2 proteins were used to obtain antibodies that were subsequently checked for their specificity. Figure 4D shows that the anti-PsTRXh1 antibody is specific to its antigen, recognizing only the PsTRXh1 isoform. No recognition signal was obtained with other PsTRXh proteins despite long-time exposure or when preimmune serum was used. This information allowed us to search for the presence of the PsTRXh1 isoform in pea tissues. Western-blot analysis of protein extracts from different tissues probed with the anti-PsTRXh1 antibody detected a band, close to 13 kD, which was more abundant in leaves of 12-d-old plantlets (Fig. 4E). As the plant became mature (100-d-old plants), the level of PsTRXh1 decreased in leaves while increasing in stem. A high accumulation of PsTRXh1 protein was detected in flowers and dry seeds (Fig. 4E). A more detailed analysis of PsTRXh1 localization using immunohistochemistry revealed that PsTRXh1 accumulates in vascular tissues of the whole plant, as shown in leaves (Fig. 4F). A high signal was also detected in floral tissues, the stigmatic papillae (Fig. 4G), and pollen grains (Fig. 4H). These results corroborate the data obtained in transgenic plants carrying the PromPsTRXh1::UidA construct (Fig. 4, B and C).
Regarding PsTRXh2, the antibody raised against this isoform recognized its antigen but also detected all the h-type isoforms tested (Fig. 4D). Such cross detections were obtained regardless of shortened time exposure and adapted antibody concentrations. When western blots were probed with this antibody, a strong signal was detected in roots from both 12-d-old plantlets and mature plants and also in flowers and seeds (Fig. 4E). This pattern of protein accumulation was not consistent with PsTRXh2 mRNA accumulation. It is likely that these levels mostly correspond to cross recognition of other abundant isoforms such as PsTRXh3, for which similar patterns were previously reported (Montrichard et al., 2003
Heterologous complementation of the yeast EMY63 mutant with pea TRXs h1 and h2 isoforms suggested that they could be specifically involved in different detoxification pathways toward oxidant molecules. To test whether PsTRXh1 and PsTRXh2 accumulation patterns in planta were also influenced by oxidative stress, we performed a series of experiments in which 12-d-old pea plantlets were treated with H2O2. The expression of PsTRXh1 and PsTRXh2 genes was analyzed by real-time PCR on leaves and roots collected from control or stressed plants, and, when possible, the patterns of protein accumulation were also analyzed. When pea plantlets were grown in the presence of increasing concentrations of H2O2, the amount of PsTRXh1 transcripts remained unchanged in leaves (Fig. 5A ), whereas it strongly increased in roots (Fig. 5B) as compared to untreated plants. This effect on the amount of transcripts correlated with the protein level as shown by western-blot analysis performed on the same plantlets using the anti-PsTRXh1 antibody, which showed the higher accumulation of PsTRXh1 in roots from plantlets treated with H2O2, whereas no significant effect was observed on leaves (Fig. 5C).
Regarding PsTRXh2 gene, its messengers were scarcely detectable, as previously mentioned (Fig. 5A, black bars). As it was observed in previous analyses (Fig. 4D), the cross detection of other PsTRX isoforms by anti-PsTRXh2 antibody did not allow the further analysis of PsTRXh2 accumulation in stressed tissues.
In plants, h-type TRXs constitute a complex group of TRXs. Whereas nine TRXs of this type have been described in Arabidopsis among more than 40 TRX-related sequences (Meyer et al., 2005 ), five members have been reported in Populus sp. (Gelhaye et al., 2004b) and three in wheat (Triticum aestivum; Cazalis et al., 2006). In pea, a legume of agronomical interest, only two isoforms, PsTRXh3 and PsTRXh4, have been characterized, both being related to seed development and germination (Montrichard et al., 2003). The first task of this work was to extend our knowledge of the TRX family in this plant. We describe here two new h-type isoforms, PsTRXh1 and PsTRXh2, and show their different pattern of expression. The second aim of this work was to unravel the biological function of h1 and h2 isoforms by comparison with the two previously described PsTRXh3 and PsTRXh4 isoforms. Based on physiological and functional approaches, we provide evidence that h1 and h2 isoforms have different function in the redox imbalances.
The phylogenic analysis confirmed that both PsTRXh1 and PsTRXh2 isoforms belong to the h-type subgroup 1 of TRXs, as their counterpart PsTRXh3 (Montrichard et al., 2003
Though PsTRXh1 and PsTRXh2 are grouped with PsTRXh3 in the same phylogenic subgroup, they differ from the other pea cytosolic TRX characterized up to now by their pattern of accumulation in plant tissues characterized up to now. PsTRXh3 and PsTRXh4 have been described as abundant seed proteins (Montrichard et al., 2003
To analyze further the function of h-type TRXs in pea, we tested the ability of PsTRXh1 and PsTRXh2 to complement the trx1
The second contribution of this study was to reveal a potential function of PsTRXh1 and h2 isoforms in physiological responses to oxidative stress. We provide evidence that both h1 and h2 pea TRXs are able to restore the inability of trx1
Finally, our study shows that the function of h1 and h2 isoforms is not restricted to an antioxidant response through the reduction of MRS enzymes but may be extended to the reduction of other antioxidant enzymes such as peroxiredoxins (PRXs). PsTRXh1 and PsTRXh2 conferred differential response of trx1
Several data suggest that at least the PsTRXh1 isoform could be involved in reactive oxygen species (ROS) detoxification in pea tissues through PRX reduction. Plant PRXs act as a common antioxidant (Dietz et al., 2002
Additionally, we show that PsTRXh1 is present in pollen and stigma papillae. In a recent work, relatively large amounts of ROS, principally H2O2, have been detected in stigma papillae from several species across a range of different angiosperm groups. This effect has been related to possible functions such as pollen-pistil interaction, redox signaling, or resistance to pathogen (McInnis et al., 2006
Plant Materials and Growth Conditions Pea (Pisum sativum) L. cv Lincoln seeds were germinated on vermiculite in plastic trays and grown in a growth chamber for 10 to 15 d under 16-/8-h photoperiod at a light intensity of 200 µmol m–2 s–1, with a day/night temperature of 20°C/25°C. After 3 weeks, plants were transplanted into a soil:vermiculite (3:1, w/w) medium. At different times thereafter, plants were collected and dissected into different parts (flowers, stems, roots, leaves, and seeds) and then processed immediately or frozen until use. For stress treatments, roots of 12-d-old pea seedlings were placed in the oxidant solutions (0, 5, 10, 15, 20, and 25 mM of either H2O2 or TBHP) and maintained for 15 h in darkness. Roots and leaves were then washed in distilled water and either analyzed or frozen in liquid nitrogen and stored at –80°C until use.
DNA was extracted and purified from pea leaves using the Plant DNAzol Reagent kit (Invitrogen). The mRNA was obtained from 12-d-old green leaves using the mRNA Isolation kit (Roche). RT-PCR was performed using the Access RT-PCR system (Promega) according to the manufacturer's instructions. PCR-Walking was performed on a pea genomic DNA library as template following the methods described by Devic et al. (1997)
All primers used for cloning are described in Table I
. Th2N/2C (for h1isoform) and ThN/2C (for h2 isoform) pairs of primers were first used in a RT-PCR on RNA extracted from pea leaf to amplify h1 and h2 partial cDNAs. Based on these sequences, new primers were designed to amplify full-length cDNAs by RACE. 3'-RACE experiments were performed using Th3F and Th4F primers for h1 and h2, respectively, whereas DNA-Walk experiments (Devic et al., 1997
Gene Expression Analysis by Real-Time PCR
Samples of total RNA (500 ng) extracted from stressed plants using the RNeasy Plant Mini kit (Qiagen) were reverse transcribed using 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 were designed using the Primer Express (Applied Biosystems) software (H1up/down for PsTRXh1, H2up/down for PsTRXh2, and 18Sup/down for rRNA 18S; Table I). Relative quantification of gene expression was monitored after normalization by the 18S rRNA expression as internal control, as fold variation over a calibrator using the 2–
To produce recombinant PsTRXh1 and PsTRXh2 in Escherichia coli, the corresponding ORFs were cloned into the expression plasmid pET-3d (Novagen) via NcoI and BamHI restriction sites. Induction of expression of recombinant proteins was performed according to Wangensteen et al. (2001)
The insulin-disulphide reduction assay was performed (Holmgren, 1979
Complementation experiments were performed with the yeast (Saccharomyces cerevisiae) strain EMY63 (Mata ade2-1 ade3-100 his3-11 leu2-3 lys2-801 trp1-1 ura3-1 trx1::TRP1 trx2::LEU2; Müller, 1991
Plant tissues were homogenized with liquid nitrogen and ground in ice-cold mortar with extraction buffer (50 mM Tris-HCl, pH 7.9, 0.2 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, and 5 mM 2-mercaptoethanol). The homogenates were centrifuged, and the supernatant was processed or stored at –80°C. For western-blot analysis, protein samples were separated on SDS-PAGE 16.5% (w/v) polyacrylamide (Schagger and Vonjagow, 1987
Small pieces of plant tissues were dissected and fixed by incubation in FAE (50% ethanol, 5% acetic acid, 3.7% formaldehyde) with occasional vacuum, dehydrated in a graded series of aqueous ethanol solution, and embedded in Paraffin M.P. (Panreac) as described in González et al. (1998)
PsTRXh1 and PsTRXh2 promoter regions were isolated from genomic DNA by PCR walking (Devic et al., 1997 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers PsTRXf (CAA45098) and PsTRXm1 (CAA53900). The accession numbers for the rest of the sequences are in the Figure 1 legend.
We thank Dr. F. Montrichard for the gift of PsTRXh3 and PsTRXh4 recombinant proteins and Dr. R. Cooke for revising the manuscript. Received September 6, 2006; accepted November 6, 2006; published November 10, 2006.
1 This work was supported by the Dirección General de Investigación Científica y Técnica, Spain (grant nos. PB98–0474 and BF12002–00401), by the Junta de Andalucía, Spain (grant no. CVI 154), by CSIC (Acción Integrada grant no. HF2001–0136), and by the Spanish government (fellowship FPI98 to J.A.T.). 
2 This paper is dedicated to the loved and esteemed memory of Professor Julio López Gorgé, who died on June 7, 2004, at the age of 69.
3 Present address: Institut des Sciences du Végétal, UPR2355-CNRS Bt23, Centre National de la Recherche Scientifique, F–91198 Gif/Yvette cedex, France. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: José A. Traverso (jose.traverso{at}isv.cnrs-gif.fr). www.plantphysiol.org/cgi/doi/10.1104/pp.106.089524 * Corresponding author; e-mail jose.traverso{at}isv.cnrs-gif.fr; fax 33–1–69–82–36–07.
Arner ESJ, Holmgren A (2000) Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 267: 6102–6109[ISI][Medline] Balmer Y, Vensel WH, Cai N, Manieri W, Schürmann P, Hurkman WJ, Buchanan BB (2006) A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts. Proc Natl Acad Sci USA 103: 2988–2993 Besse I, Wong JH, Kobrehel K, Buchanan BB (1996) Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium. Proc Natl Acad Sci USA 93: 3169–3175 Bower MS, Matias DD, Fernandes-Carvalho E, Mazzurco M, Gu T, Rothstein SJ, Goring DR (1996) Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase. Plant Cell 8: 1641–1650[Abstract] Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][ISI][Medline] Bréhélin C, Meyer EH, de Souris JP, Bonnard G, Meyer Y (2003) Resemblance and dissemblance of Arabidopsis type II peroxiredoxins: similar sequences for divergent gene expression, protein localization, and activity. Plant Physiol 132: 2045–2057 Bréhélin C, Mouaheb N, Verdoucq L, Lancelin JM, Meyer Y (2000) Characterization of determinants for the specificity of Arabidopsis thioredoxins h in yeast complementation. J Biol Chem 275: 31641–31647 Buchanan BB, Balmer Y (2005) Redox regulation: a broadening horizon. Annu Rev Plant Biol 56: 187–220[CrossRef][Medline] Cabrillac D, Cock JM, Dumas C, Gaude T (2001) The S locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins. Nature 410: 220–223[CrossRef][Medline] Cazalis R, Pulido P, Aussenac T, Pérez-Ruiz JM, Cejudo FJ (2006) Cloning and characterization of three thioredoxin h isoforms from wheat showing differential expression in seeds. J Exp Bot 57: 2165–2172 Chae HZ, Chung SJ, Rhee SG (1994) Thioredoxin-dependent peroxide reductase from yeast. J Biol Chem 269: 27670–27678 Cherest H, Surdin-Kerjan Y (1992) Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. Genetics 130: 51–58[Abstract] Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of A. thaliana. Plant J 16: 735–743[CrossRef][ISI][Medline] Cole GM, Stone DE, Reed SI (1990) Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway. Mol Cell Biol 10: 510–517 Coudevylle N, Thureau A, Hemmerlin C, Gelhaye E, Jacquot JP, Cung MT (2005) Solution structure of a natural CPPC active site variant, the reduced form of thioredoxin h1 from poplar. Biochemistry 44: 2001–2008[CrossRef][Medline] Devic M, Albert S, Delseny M, Roscoe TJ (1997) Efficient PCR walking on plant genomic DNA. Plant Physiol Biochem 35: 331–339[ISI] Dietz KJ (2003) Plant peroxiredoxins. Annu Rev Plant Biol 54: 93–107[CrossRef][Medline] Dietz KJ, Horling F, Konig J, Baier M (2002) The function of the chloroplast 2-cysteine peroxiredoxin in peroxide detoxification and its regulation. J Exp Bot 53: 1321–1329 Draculic T, Dawes IW, Grant CM (2000) A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae. Mol Microbiol 36: 1167–1174[CrossRef][ISI][Medline] Florencio FJ, Yee BC, Johnson TC, Buchanan BB (1988) An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach. Arch Biochem Biophys 266: 496–507[CrossRef][ISI][Medline] Gelhaye E, Rouhier N, Gerard J, Jolivet Y, Gualberto J, Navrot N, Ohlsson PI, Wingsle G, Hirasawa M, Knaff DB, et al (2004a) A specific form of thioredoxin h occurs in plant mitochondria and regulates the alternative oxidase. Proc Natl Acad Sci USA 101: 14545–14550 Gelhaye E, Rouhier N, Jacquot JP (2004b) The thioredoxin h system of higher plants. Plant Physiol Biochem 42: 265–271[CrossRef][ISI][Medline] González MC, Bevia O, Echevarria C, Vidal J, Cejudo FJ (1998) Expression and localization of phosphoenolpyruvate carboxylase in developing and germinating wheat grains. Plant Physiol 116: 1249–1258 Holmgren A (1979) Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem 254: 9627–9632 Ishiwatari Y, Fujiwara T, McFarland KC, Nemoto K, Hayashi H, Chino M, Lucas WJ (1998) Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata. Planta 205: 12–22[CrossRef][ISI][Medline] Ishiwatari Y, Honda C, Kawashima I, Nakamura S, Hirano H, Mori S, Fujiwara T, Hayashi H, Chino M (1995) Thioredoxin h is one of the major proteins in rice phloem sap. Planta 195: 456–463[ISI][Medline] Issakidis-Bourguet E, Mouaheb N, Meyer Y, Miginiac-Maslow M (2001) Heterologous complementation of yeast reveals a new putative function for chloroplast m-type thioredoxin. Plant J 25: 127–135[CrossRef][ISI][Medline] Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153: 163–168 Jaramillo JL, Chueca A, Jacquot JP, Hermoso R, Lazaro JJ, Sahrawy M, Gorge JL (1997) High-yield expression of pea thioredoxin m and assessment of its efficiency in chloroplast fructose-1,6-bisphosphatase activation. Plant Physiol 114: 1169–1175[Abstract] Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: Jeong JS, Kwon SJ, Kang SW, Rhee SG, Kim K (1999) Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae. Biochemistry 38: 776–783[CrossRef][Medline] Kobrehel K, Wong JH, Balogh A, Kiss F, Yee BC, Buchanan BB (1992) Specific reduction of wheat storage proteins by thioredoxin h. Plant Physiol 99: 919–924 Kwon SJ, Park JW, Choi WK, Kim IH, Kim K (1994) Inhibition of metal-catalyzed oxidation systems by a yeast protector protein in the presence of thioredoxin. Biochem Biophys Res Commun 201: 8–15[CrossRef][ISI][Medline] Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline] Laloi C, Mestres-Ortega D, Marco Y, Meyer Y, Reichheld JP (2004) The Arabidopsis cytosolic thioredoxin |
TOP
ABSTRACT
RESULTS
), PsTRXh2 (
), PsTRXm1 (horizontal line), and PsTRXf (x). The negative control (no TRX) is represented by a line without a symbol.
-glucuronidase (GUS; UidA) reporter gene under the control of the promoter of either PsTRXh1 or PsTRXh2 genes. In such plants, we found that PsTRXh1 promoter produced strong UidA expression in vascular veins of the whole plant, except in the root, with expression being also strong in hydathodes (
