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Plant, Cell and Environment (2014)

doi: 10.1111/pce.12276

Original Article

A comprehensive study of thiol reduction gene expression under stress conditions in Arabidopsis thaliana C. Belin1,2, T. Bashandy1,2*, J. Cela1,2, V. Delorme-Hinoux1,2, C. Riondet1,2 & J. P. Reichheld1,2 1

Laboratoire Génome et Développement des Plantes, Université Perpignan Via Domitia, F-66860 Perpignan, France and Laboratoire Génome et Développement des Plantes, CNRS, F-66860 Perpignan, France

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ABSTRACT Thiol reduction proteins are key regulators of the redox state of the cell, managing development and stress response programs. In plants, thiol reduction proteins, namely thioredoxin (TRX), glutaredoxin (GRX), and their respective reducers glutathione reductase (GR) and thioredoxin reductase (TR), are organized in complex multigene families. In order to decipher the function of the different proteins, it is necessary to have a clear picture of their respective expression profiles. By collecting information from gene expression databases, we have performed a comprehensive in silico study of the expression of all members of different classes of thiol reduction genes (TRX, GRX) in Arabidopsis thaliana. Tissue expression profiles and response to many biotic and abiotic stress conditions have been studied systematically. Altogether, the significance of our data is discussed with respect to published biochemical and genetic studies. Key-words: abiotic stress; biotic stress; development; hormone; redoxins; transcriptome

INTRODUCTION Plants are continuously subjected to a large panel of stress depending on the characteristics of their environment. These stresses are known to affect plant growth and development. Plants cope with the deleterious effects of these stress conditions by modulating their gene expression programs and various metabolic functions. Oxidative stress signalling, notably relayed by reactive oxygen species (ROS), is an important part of plant responses to numerous environmental conditions (Apel & Hirt 2004; Rentel et al. 2004; Wagner et al. 2004; Møller et al. 2007; Foyer & Noctor 2009; Mittler et al. 2011). A key candidate in transmitting oxidative signals is modified protein thiol/disulfide status, which is regulated by thioredoxin (TRX) and glutaredoxin (GRX) systems (Nordberg & Arnér 2001; Rouhier et al. 2008; Meyer et al. 2009, 2012). TRX and GRX are well-known dithiol reduction enzymes serving as regulators of protein conformation and electron donor for many enzymes (Holmgren Correspondence: C. Belin. e-mail: [email protected] *Present address: Genetics Department, Agriculture Faculty of the New Valley Branch, Assiut University, Egypt. © 2014 John Wiley & Sons Ltd

1989). Proteomic approaches have been developed that identified the putative TRX and GRX target proteins which are implicated in all aspects of plant growth, including basal metabolism, iron/sulphur cluster formation, development, adaptation to the environment and stress responses (Motohashi et al. 2001; Balmer et al. 2004; Lemaire 2004; Montrichard et al. 2009). In plants, TRX functions in chloroplastic carbon metabolism have been extensively studied. However, in other cellular compartments like the cytosol and mitochondria, their functions are less well understood, in spite of the large number of potential target proteins identified by proteomic studies. Moreover, genetic studies aiming to identify functions of TRX and GRX have turned to be less fruitful, likely due to extensive redundancies between gene functions (Reichheld et al. 2010). Indeed, a characteristic of plants is that TRX and GRX are encoded by large multigenic families of more than 40 members (Meyer et al. 2012). The signification of such a genomic complexity, compared with other organisms that generally harbour a limited number of genes, is not known. One of the reason is that TRX and GRX are localized in distinct cellular compartments (cytosol, chloroplast, mitochondria, etc.) in which the different isoforms might perform specific functions. For example, in the chloroplast, analyses of the biochemical characteristics of specific TRX point to a strong specificity towards some target enzymes (Lemaire et al. 2007).Another explanation for such a complex genomic organization is that the different isoforms are differentially expressed in plant organs and/or environmental stress conditions, which may assign them distinct functions. Recent genetic evidence has assigned specific functions to distinct members of TRX or GRX (for a recent review, see Meyer et al. 2012). For example, the cytosolic TRXh5 has been shown to play a central role in the pathogenic pathway caused by the necrotrophic fungus Cochliobolus victoriae (Sweat & Wolpert 2007; Lorang et al. 2012) and in response to the pathogenic bacteria Pseudomonas syringae infection (Laloi et al. 2004).A type-III GRX is known to play a specific role in petal development in Arabidopsis (Xing et al. 2005). Here, we performed a systematic study of the transcriptome of TRX and GRX gene families in Arabidopsis thaliana. We first give an update of the localization of selected TRX and GRX isoforms, taking into account recent data from the literature. In order to have a clear picture of the expression of all TRX and GRX isoforms, transcriptomic data from the literature have been collected and the respective expression pattern 1

2 C. Belin et al. of each gene in plant tissues/organs has been studied in silico. Gene expression in plants submitted to various abiotic and biotic stresses and hormonal treatment has also been collected.We also validate some of these in silico data by studying the expression of selected genes by promoter-GUS fusion and GFP-fusion protein analyses. These data were confronted with data from the literature, when available, both at the expression level and for the biological function.

MATERIALS AND METHODS Subcellular localization studies In order to get the best prediction of TRX and GRX subcellular localization, we used the predictions from the SUBA3 database (http://suba.plantenergy.uwa.edu.au/; Tanz et al. 2013), together with the experimental data centralized in SUBA3 and the Plant Proteome Database (http:// ppdb.tc.cornell.edu/; Sun et al. 2009). We also carefully confronted these results to the published literature on every TRX and GRX genes.

Transcriptomic data mining In order to get the absolute values from different transcriptomic sets, we chose to use the Expression Browser tool from the BAR (http://bar.utoronto.ca/welcome.htm; Toufighi et al. 2005) and to import all tables. Using Excel, we performed averaging of values from the same tissues or the same conditions each time it was possible, and chose to eliminate certain conditions for which there were no significant data for GRX and TRX genes, in order to make a clear and concise report of expression data. We double-checked that our averaging strategy did not eliminate any critical data (e.g. transient expression).

Sequence analysis and tree constructions Phylogenetic trees were constructed using ClustalW or Mafft (neighbour-joining method), with a weight matrix of Gonnet or Blosum62 type, and visualized using NJplot unrooted software (Perrière & Gouy 1996).

Pro35S:TRXh3-GUS-GFP plasmid construction and transformation The open reading frame of TRXh3 was cloned upstream from a sequence coding for the GUS-GFP fusion protein in the pCAMBIA1303 binary vector. The resulting plasmid was introduced into Agrobacterium tumefaciens LBA4404. Tobacco BY-2 cells were transformed and transformed cali were further selected on 30 mg mL−1 hygromycin. Fluorescence was observed using an Axioplan microscope (Carl Zeiss S.A.S., Le Pecq, France).

GUS expression analysis GRXC1, C2, C3 and C4 promoter regions were isolated from genomic DNA (ecotype Columbia). Fragments of 1 kb of the

regions upstream from the ATG codons were isolated by direct PCR on genomic DNA using primers to introduce unique XhoI and NcoI sites for GRXC2 and GRXC3, and HindIII and NcoI for GRXC1 and GRXC4. The DNA fragments were then digested by the corresponding enzymes and cloned into compatible sites of pGPTV-HYG binary vector. ProTRXh3-GUS and ProTRXh5-GUS plants were described earlier (Reichheld et al. 2002). The resulting plasmids were introduced into A. tumefaciens C18CIRifR. Arabidopsis plants (Col-0) were transformed with agrobacteria by the floral dip method. T1, T2 and T3 seedlings were selected in vitro on MS/2 medium supplemented with 30 mg mL−1 hygromycin. For the GUS assay, seeds and plants were either cultivated in vitro under the same conditions or grown in soil mixed with vermiculite in a greenhouse under continuous light at 22 °C. Flg22 treatments were performed as described in Laloi et al. (2004). GUS histochemical staining was performed according to Reichheld et al. (2002).

Accession numbers Accession numbers for TRX and GRX genes are available in Supporting Information Table S1 and Supporting Information Table S2. AGI numbers for reductases and glutathione biosynthesis genes are: NTRA (At2g17420), NTRB (At4g35460), NTRC (At2G41680), FTRcat (At2g04700), FTRvar1 (At5g23440), FTRvar2 (At5g08410), GR1 (At3g24170), GR2 (At3g54660), GSH1 (At4g23100) and GSH2 (At5g27380).

RESULTS An update on TRX subfamilies and their subcellular localization Arabidopsis genome contains 39 genes encoding putativeTRX, whose biochemical properties have already been extensively reported (Meyer et al. 2009, 2012). About half of these genes encode plastidial proteins, as illustrated in the Arabidopsis TRX phylogenetic tree (Fig. 1a). The predictions for plastidial localization of TRX f, m, x, y, z, CDSP32 and HCF164 have been confirmed by not only an extensive data mining of chloroplastic proteomic studies but also via other experimental approaches in most cases, as reviewed in the Supporting Information Table S1. Moreover, the critical role of several of these proteins for regulating chloroplastic functions has already been reported (Collin et al. 2003, 2004; Motohashi & Hisabori 2006; Arsova et al. 2010; Courteille et al. 2013; Laugier et al. 2013; Thormählen et al. 2013). Among these families, only the TRXm3 has not been identified in any chloroplastic proteome but was rather localized into non-green plastids in Arabidopsis transgenic lines expressing a ProTRXm3:TRXm3-YFP fusion transgene (Benitez-Alfonso et al. 2009). Moreover, the precursor of the chloroplastic TRXm2 was shown to be retained in the cytosol (Meyer et al. 2011). The two WCRKC proteins are also clearly predicted to be chloroplastic, and this is supported by some in vitro import assays into chloroplasts. A family of five atypical Cys His-rich © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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Figure 1. Thioredoxin (TRX) protein family and global pattern of TRX gene expression. (a) Phylogenetic tree of Arabidopsis thaliana TRX. Members of the different subgroups of TRX are clustered, and the cellular localization is represented by different background colours. The green colour corresponds to the chloroplast, blue to the cytosol, white to the mitochondrion, yellow to the nucleus, and red to membranes. A degraded colour circle indicates a double localization. A faded colour circle represents a putative localization. The AGI numbers corresponding to each protein are presented in Supporting Information Table S1. (b) Tissue-specific expression of TRX genes. The table displays the averages of absolute values from the BAR expression browser. The colour scale is given on the left. SAM, shoot apical meristem. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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TRX (ACHT) has been recently reported (Dangoor et al. 2009) in Arabidopsis with a clear localization into the chloroplast. Here we report a new member of this family, ACHT6 (encoded by At1g07700), whose predicted plastidial localization is supported by its presence in three chloroplast proteomes (Supporting Information Table S1). TRXo subfamily groups the mitochondrial protein TRXo1, whose localization has clearly been established, and its very close relative, TRXo2, for which there is no evidence of specific subcellular localization. It is predicted as cytosolic and the only experimental clue reported so far is the incapacity of TRXo2 to enter into mitochondria during in vitro import assays (Laloi et al. 2001). According to Traverso et al. (2013),TRXh family is divided in four subfamilies. TRXhI contains four members in Arabidopsis: TRXh1, TRXh3, TRXh4 and TRXh5. TRXh3 has been identified in some proteomic studies of nearly all cellular compartments (data not shown). We might envisage that TRXh3, abundantly expressed in most Arabidopsis tissues, is a quite frequent contaminant in proteomic studies. Indeed, tobacco transgenic cell lines expressing a TRXh3GUS-GFP fusion protein display a cytoplasmic fluorescence (Supporting Information Fig. S1). However, it is important to notice that, although TRXhI members are not predicted to be N-myristoylated or palmitoylated, TRXh3 has been isolated in most of the plasma membrane (PM) proteomes (Supporting Information Table S1). Interestingly, TRXh5 has clearly been localized in both cytosol and PM in a recent work (Lorang et al. 2012). Its presence in peroxisomal compartment has also been retrieved in proteomic studies (Supporting Information Table S1). Altogether, we should envisage that TRXhI members are not only cytosolic but also associated to the PM or other subcellular compartments. TRXhII is represented in Arabidopsis by TRXh2, TRXh7 and TRXh8. These proteins share a glycine residue as the second amino acid, which is predicted to be myristoylated, and all of them localize to both cytosol and endomembranes [endoplasmic reticulum (ER) and Golgi] when expressed in onion cells (Traverso et al. 2013). Moreover, previous works have reported a mitochondrial localization of TRXh2-GFP when stably overexpressed in Arabidopsis (Meng et al. 2010). TRXhIII group is represented in Arabidopsis by TRXh9, CXXS2 and CXXC2 (previously reported as TRXh10). TRXh9, CXXS2 and CXXC2 harbour both a glycine and a cysteine residue in their N-terminal end, making them prone to be both myristoylated and palmitoylated. In agreement with such modifications, the PM localization of these proteins has been shown in particular for TRXh9 (Supporting Information Table S1). In contrast, the lack of N-terminal maturation of CXXS1 supports a cytosolic localization. TDX, NRX1 and NRX2 are putative cytosolic and nuclear TRX, but we still have no obvious evidence for this double localization. The subcellular localization of the poplar clot homolog was recently established to be cytosolic (Chibani et al. 2012). However, we do not have many clues for the localization of WCGVC (predicted as cytosolic but identified in one chloroplastic proteome) and TARWCGPC, which are predicted to localize in the secretory pathway.

Analysis of TRX gene expression during Arabidopsis development We performed a general investigation of TRX gene expression in publicly available data through the Botany Array Resource expression browser tool (Toufighi et al. 2005). The absolute values of different experiments available for the same tissue were averaged and represented in Fig. 1b. Among chloroplastic TRX-encoding genes, as we could expect, most are highly expressed in green tissues. It is particularly visible for TRXf and TRXm (except m3) families, as well as TRXx, TRXy2, CDSP32, HCF164, WCRKC1, ACHT4, ACHT2 and ACHT6. TRXm3 is ubiquitously but moderately expressed in all organs, like WCRKC2 and TRXz (with a peak in ovary), while TRXy1 expression is particularly low in green tissues and reaches its maximum in dry seeds, as recently reported (Bohrer et al. 2012). ACHT5 and ACHT1 display a general very weak expression, while ACHT3 seems highly specific to male gametophytic tissues, with a very strong expression in mature and germinating pollen. We then checked the expression of these chloroplastic TRX-encoding genes in roots by using the high-resolution spatiotemporal map available (Brady et al. 2007). As expected, we found a general weak expression (Fig. 2), except for TRXm2 that displays an ubiquitous moderate to high expression level in all root tissues. Interestingly, ACHT4 also shows a very specific high expression in the columella. Genes encoding the mitochondrial TRXo1, and its close relative TRXo2, display a mild ubiquitous expression in all Arabidopsis tissues. The TRXh family displays more specific expression patterns, with the notable exception of TRXh3, which is ubiquitously and very highly expressed (Figs 1b & 2). TRXh5 is also strongly expressed but we can notice its very low expression level in meristematic tissues, both in shoots and roots. The other members of the TRXhI subfamily, TRXh1 and TRXh4, display a milder expression in all tissues but are specifically high in the stele and mature pollen, and in unicellular pollen and dry seed, respectively. In the TRXhII subgroup, TRXh2 is ubiquitously expressed, while TRXh7 seems specifically present in the root vasculature (procambium, phloem), and TRXh8 only detected in the ovary. In the TRXhIII subfamily, there are no data available for CXXC2/TRXh10. TRXh9 displays a general and weak expression, slightly more important in root elongation and maturation zone, whereas CXXS2 is only detected in pollen. In the last TRXhIV subfamily, CXXS1 expression concentrates in photosynthetic parts. The putative nuclear TRX-encoding genes, TDX, NRX1 and NRX2, together with WCGVC, Clot and TARWCGPC, display a general very mild expression, even close to the detection level for TARWCGPC, with a moderate peak in meristematic tissues for WCGVC (Figs 1b & 2).

Analysis of TRX gene expression response to stresses and hormones In the second part, we were interested in the modulation of TRX gene expression by environmental constraints (abiotic © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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Figure 2. Details of TRX gene expression in Arabidopsis root. The table displays the averages of absolute values from the BAR expression browser. The colour scale is given on the right. RC, root cap; LRP, lateral root primordia.

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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or biotic stresses) and phytohormones. Figure 3a reports the expression ratios (stress/control) in response to abiotic and biotic stresses. We can notice that core chloroplastic TRX (TRXf, TRXm – except m3, TRXy1, TRXx, TRXz, HCF164, WCRKC1) displays a slight tendency to a decreased expression in response to almost all stresses, with a still mild but slightly stronger effect in response to pathogens or bacterial (HrpZ and Flg22) and oomycete-derived (NPP1) elicitors. Interestingly, CDSP32, which has originally been described as a chloroplastic TRX induced by severe abiotic stresses (Rey et al. 1998), shows a general slight increase in response to abiotic stresses, but only in root tissues, with only a clear expression rise in heat stress conditions. TRXm3 and WCRKC2 display a very slight tendency to be induced by pathogens (Peronospora), osmotic and salt stresses, while only WCRKC2 tends to be repressed by cold stress. TRXy1 expression does not vary significantly in response to stresses (values of log2-transformed ratios between −0.3 and +0.3). ACHT genes do not respond to abiotic or biotic stresses, except ACHT4 and ACHT5, which are mildly up- and downregulated, respectively, in response to cold, osmotic and salt stresses. In TRXhI subfamily, TRXh5 displays a strong increase in response to various abiotic (osmotic, salt, drought, UV-B and wounding) and biotic stresses in shoots (Laloi et al. 2004; Supporting Information Fig. S2), while other members (TRXh1, TRXh3, TRXh4) are quite unaffected by all tested conditions. In TRXhII subgroup, TRXh2 expression is unaffected in most of the conditions, except for Peronospora parasitica where it is induced. TRXh7 is repressed not only in several biotic stress conditions but also in response to osmotic stress in roots, whereas TRXh8 expression is strongly induced in response to wounding and to diverse pathogens and elicitors. In the TRXhIII subgroup, TRXh9 shows a mild tendency to be induced by different stress conditions, in contrast to CXXS2, which expressions tend to be slightly reduced in response to various stresses, as it happens for CXXS1. TRXo genes, together with Clot, TDX and TARWCGPC, do not display any significant change in gene expression in response to stresses. NRX1 expression displays a mild increase in response to pathogens, while NRX2 is slightly induced by osmotic and heat stresses in root. Finally, very few TRX genes respond to hormonal treatments (Fig. 3b). WCRKC1 and ACHT4 are slightly repressed by cytokinins (zeatin) and methyl jasmonate (MeJa) while ACHT4 is induced by abscisic acid (ABA) in seedlings. Interestingly, ACHT4 is also mildly induced in response to ABA and repressed in response to gibberellins (GA) in seeds. Surprisingly, ACHT3 displays a strong but very transient increase in response to all hormonal treatments, but its expression is finally repressed in response to long-term treatments. Among TRXh, TRXh7 is mildly repressed by ABA both in seedlings and seeds, whereas its expression is slightly enhanced in response to ethylene (ACC) or MeJa. CXXS2 is the only TRXh to display strong regulation in response to hormonal treatments, with a rapid and transient increase in response to MeJa, auxin (IAA) and brassinolid (BrsnI), and

a slow induction in response to ACC, cytokinins (zeatin) and ABA. Finally, WCGVC expression is repressed by almost all hormones after 1 or 3 h of treatment, and other TRX genes do not display any strong hormonal regulation. We can notice that GA application on seeds decreases the expression of numerous TRX genes, with the notable exception of TRXy2, which is strongly induced in the same conditions.

An update on GRX subfamilies and their subcellular localization Arabidopsis genome contains 50 genes encoding putative GRX, whose biochemical properties have already been extensively reported (Meyer et al. 2009, 2012; Fig. 4a).The subgroup I of C[P/G/S]Y[C/S] GRX contains six proteins: GRXC1, GRXC2, GRXC3, GRXC4, GRXC5 and GRXS12. The predicted chloroplastic localization of C5 and S12 is strongly supported by experimental data (Supporting Information Table S2). We previously reported that GRXC1 and GRXC2 were found in the cytosol and nucleus when transiently overexpressed in onion epidermal cells (Riondet et al. 2012). However, GRXC1 harbours a myristoylation site and was identified in PM proteomic studies. In addition, GRXC2 was retrieved in numerous proteomic studies (Supporting Information Table S2).Altogether, this might suggest that GRXC1 and GRXC2 are not only cytosolic but could also associate to other subcellular compartments, including the membranes. Finally, GRXC3 and GRXC4 are predicted to localize in the secretory pathway, which is supported by their identification in some secretory pathway compartments via proteomic approaches (ER, Golgi – Supporting Information Table S2). The second group of CGFS GRX is composed of four proteins in Arabidopsis: GRXS14, GRXS15, GRXS16 and GRXS17. Their subcellular localization is quite clear because they were identified in many proteomes, and data from reporter fluorescent lines are available for each of them (Supporting Information Table S2). GRXS14 and GRXS16 localize into the chloroplasts, GRXS15 in the mitochondria, while GRXS17 has a dual targeting in the cytosol and nucleus. In contrast, the subcellular localization of most of the ROXY proteins, which constitute the main family of GRX with 21 members, is still unknown. Not only ROXY1, ROXY2 and ROXY19 have been reported to be localized in the nucleus, where they interact with TGA factors (Ndamukong et al. 2007; Li et al. 2009; Murmu et al. 2010), but also in the cytosol for ROXY1 and ROXY2. The interaction between ROXY18 (also called GRXS13) and TGA2 suggests a nuclear localization for ROXY18 (Laporte et al. 2012), despite only a cytosolic localization has been shown for this GRX (Couturier et al. 2011). All other ROXY members are predicted to be cytosolic, except ROXY3, for which a mitochondrial localization is also predicted. A family of four CXXC GRX-like proteins has been described in plants. Even though they do not present any clear prediction, three of them (4CXXC2, 4CXXC4 and 4CXXC11) have been identified in several PM proteomes. Interestingly, these proteins are predicted to be both © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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Figure 3. Expression of TRX genes in response to stresses and hormonal treatments. (a) Response to abiotic and biotic stresses. (b) Response to hormonal treatments. Values are represented in log2-transformed ratios (treated/control). The colour scale is given on the right. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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Figure 4. Glutaredoxin (GRX) protein family and global pattern of GRX gene expression. (a) Phylogenetic tree of Arabidopsis thaliana GRX. Members of the different subgroups of GRX were clustered in five different subgroups. The AGI numbers corresponding to each protein are presented in Supporting Information Table S2. (b) Tissue-specific expression of GRX genes. The table displays the averages of absolute values from the BAR expression browser. The colour scale is given on the left. SAM, shoot apical meristem. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

Arabidopsis redoxins gene expression myristoylated and palmitoylated, in agreement with such PM targeting. The PM localization of 4CXXC2 has been further validated by reporter fusion lines, and it has also been shown to be associated with the actin microfilaments in some specific light conditions (Whippo et al. 2011). 4CXXC1, 4CXXC6 and 4CXXC8 are also prone to be myristoylated and palmitoylated, whereas 4CXXC3 and 4CXXC5 only harbour myristoylation site. These proteins might localize, at least partially, to the PM or the endomembranes. Only a few 4CXXCs are predicted to localize in specific organelles (4CXXC1 and 4CXXC10 to the plastid, 4CXXC13 to the mitochondrion). Finally, the last subgroup contains six proteins that display both a GRX domain and a glutathione S-transferase (GST) domain. CPFC1 and CPFC3 are targeted to the chloroplast and the mitochondrion, respectively, according to the proteomic studies (Supporting Information Table S2). CPFA4, predicted to be cytosolic, has been identified in proteomes from cytosol and PM. Finally, CPFA5 has clearly been shown to be localized in peroxisomes (Dixon et al. 2009), while its predicted chloroplastic localization is supported by numerous proteomic studies, suggesting a dual targeting.

Analysis of GRX gene expression during Arabidopsis development As for TRX genes, GRX gene expression was investigated through the BAR expression browser tool (Toufighi et al. 2005). The absolute values of different experiments available for the same tissue were averaged and represented in Fig. 4b. Unfortunately, the data are missing for 10 GRX genes (GRXC5, ROXY3, ROXY5, ROXY14, 4CXXC6, 4CXXC7, 4CXXC9, 4CXXC11, 4CXXC13, CPFC2). We can first observe that GRX from the first and second subgroups (GRXC1 to GRXC4 and GRXS12 to GRXS17) are quite ubiquitously expressed except in the male gametophyte where only GRXC2 and GRXS16 are recovered. However, the level of expression differs between these genes with a general strong expression for GRXC2 and GRXC4 (and GRXS15 in roots), compared with the low expression of GRXS12 and GRXS17. GRXC1 displays a very specific expression in roots because it is only significantly expressed in the columella, as we could confirm by using a ProGRXC1:GUS reporter line (Supporting Information Fig. S3). Using the same strategy, we support the ubiquitous pattern of GRXC2 expression by showing its general expression in siliques and embryos (Supporting Information Fig. S4). Finally, GRXC3 displays a strong expression in vascular tissues, as we could show in siliques and embryo (Supporting Information Fig. S4), which is consistent with its strong expression in phloem in roots (Fig. 5). Interestingly, GRXS12 and GRXS17 are strongly expressed in the root meristematic zone (Fig. 5). The chloroplastic GRXS14 displays a strong expression in green parts (leaves, stem, sepals, ovary, young siliques) whereas the other chloroplastic gene, GRXS16, displays its strongest expression in mature pollen and dry seeds. GRXC1 is also very strongly expressed in dry seeds, and in the root columella. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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In contrast to these families, the ROXY genes display rather specific expression profiles, and we can notice that none of them is clearly expressed in the (germinating) pollen and the dry seeds (absolute values inferior to 15), while only ROXY1, ROXY20 and ROXY21 are slightly expressed in the quiescent centre (Figs 4b & 5). ROXY1 expression is limited to the shoot apical meristem and the procambium in roots. ROXY2 to ROXY16 genes are mainly expressed in the green aerial parts, with general low level of expression, except for ROXY8, ROXY9 and ROXY10. However, the expression of some of these genes is detected specifically in root procambium (ROXY6, and ROXY8 to ROXY16) and phloem (ROXY2, ROXY4, ROXY7, ROXY8 and ROXY12–15). Although they are detected in aerial tissues, ROXY17, ROXY18 and ROXY19 are mainly expressed in the root (Fig. 5). ROXY17 and ROXY18 are quite specific to the procambium and the vasculature (phloem and xylem, respectively), whereas ROXY19 is expressed in all root tissues except the procambium (and the quiescent centre). Finally, ROXY20 shows a weak expression with maxima in the hypocotyl, the stem and the root procambium, and ROXY21 is strongly expressed in cauline and senescent leaves, in all flower organs, and in the columella. 4CXXC family also presents a very heterogeneous pattern of expression, with general low levels of expression. 4CXXC1 is nearly undetected in all plant tissues whereas the most expressed gene, 4CXXC10, is quite ubiquitous. 4CXXC3, 4CXXC5 and 4CXXC12 display a general low level of expression, while 4CXXC2 is restricted to the green tissues. Despite its ubiquitous expression, 4CXXC4 is very highly expressed in pollen, while its expression in roots concentrates in the columella, the pericycle and the lateral root primordium. Finally, 4CXXC8 is restricted to shoot apical meristem, root quiescent centre and female reproductive organs (carpels and ovary). In the last family of bifunctional GST-GRX genes (CPFCs and CPFAs), CPFC1, CPFA4 and CPFA5 are predominantly expressed in green tissues. CPFC3 is ubiquitously expressed, whereas CPFA3 is very specifically restricted to the root (lateral root cap and procambium), the senescent leaves and the mature pollen.

Analysis of GRX gene expression response to stresses and hormones As for TRX genes, we also were interested in the modulation of GRX gene expression by environmental constraints (abiotic or biotic stresses) and phytohormones (Fig. 6). We can notice that subgroups I and II of GRX genes are not strongly modulated. GRXC4, GRXS12, GRXS14 and GRXS17 display a slight tendency to be repressed by both abiotic and biotic stresses, whereas GRXC1, GRXC2 and GRXC3 are moderately up-regulated, in particular in response to biotic stresses and in shoot responses to osmotic perturbations and UV-B. In contrast, ROXY genes are particularly responsive to environmental constraints. However, we have to take into account the very low expression levels of some ROXY genes

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Figure 5. Details of GRX gene expression in Arabidopsis root. The table displays the averages of absolute values from the BAR expression browser. The colour scale is given on the right. RC, root cap; LRP, lateral root primordia.

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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Figure 6. Expression of GRX genes in response to stresses and hormonal treatments. (a) Response to abiotic and biotic stresses. (b) Response to hormonal treatments. Values are represented in log2-transformed ratios (treated/control). The colour scale is given on the right. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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when analysing the ratio of expression under stressed versus control conditions. For example, ROXY11 expression in roots is increased in response to many abiotic stresses (Fig. 6a) but we already noted above that ROXY11 is not clearly expressed in roots. ROXY2 to ROXY16 genes, preferentially expressed in aerial parts, are repressed in shoots in response to osmotic and salt stresses, as well as in response to pathogens or elicitors. On the other hand, ROXY11 to ROXY15 genes are induced in shoots by UV-B and wounding (and cold for ROXY11 and ROXY13). ROXY17 displays a general decrease in expression in response to both abiotic and biotic stresses. ROXY18 is strongly induced by pathogens and elicitors (Flg22, HrpZ and NPP1) while ROXY19 is induced by osmotic, salt (in root) and drought stresses, UV-B and wounding (in shoots), and some pathogens. Finally, ROXY20 shows a slight decrease in response to various stresses, whereas ROXY21 is induced by osmotic and salt stresses, more efficiently in shoots. 4CXXC2 and 4CXXC3 genes tend to be repressed by biotic stresses while 4CXXC4 expression is slightly induced in the same conditions, as well as in response to many abiotic stresses. 4CXXC1 displays a weak tendency to be induced in response to any abiotic stress specifically in shoots. 4CXXC2 is induced in both shoots and roots in response to cold and UV-B. Finally, 4CXXC8 is moderately repressed in response to abiotic stresses, and 4CXXC12 also tends to be repressed in response to UV-B, wounding and heat stress. In bifunctional GST-GRX gene family, CPFC1, CPFA4 and CPFA5 are slightly to moderately down-regulated in response to biotic stresses, whereas CPFC3 expression displays a moderate increase not only in response to pathogens and elicitors, but also to UV-B and heat stress. Responses for CPFA3, which seems to be highly induced in response not only to pathogens but also to osmotic stress and UV-B, should be moderated by the fact that CPFA3 expression is extremely low and restricted to very specific tissues (see above). GRX gene expression regulation by phytohormones (Fig. 6) is comparable to their regulation by stresses. Indeed, only ROXY and 4CXXC genes are strongly modulated by phytohormones, whereas subgroup I and II GRX, and bifunctional GST-GRX members are only very slightly modulated. We can notice that GRXC1, GRXC2, GRXC3, GRXS14 and GRXS15 tend to be down-regulated by GA in seeds whereas GRXC4, GRXS12 and GRXS16 tend to be up-regulated in the same conditions. Moreover, GRXC1, GRXC2 and GRXC3 tend to be slightly up-regulated by MeJa and ABA (C1 and C3). Most of the ROXY genes are strongly regulated in response to various, if not all, hormones, but we should emphasize that many of these genes display very low and not significant expression levels. However, we can see that ROXY17 and ROXY18 are repressed in response to MeJa, IAA and ABA (in both seedlings and seeds), whereas ROXY19 is highly induced in response to MeJa. Moreover, ROXY18 is strongly induced by GA in seeds. Among 4CXXC genes, the most significant hormonedependent regulations are the induction of 4CXXC4 expression in response to IAA, of 4CXXC1 in response to ABA in

seeds, and of 4CXXC5 in response to ABA in seedlings, together with the repression of 4CXXC2 in response to MeJa and ABA, of 4CXXC8 and 4CXXC10 in response to ABA in seeds, and of 4CXXC10 in response to GA in seeds.

Analysis of reductases and glutathione biosynthesis gene expression Finally, we report the pattern of expression of the thioredoxin and glutathione reductases (FTR, NTR and GR) in Supporting Information Fig. S5. Firstly, we notice that NTRA and NTRB are not distinguishable in transcriptomic analyses and we report a global pattern of expression for both NTRA and NTRB, the two cytosolic TRs. As expected, the genes that encode plastidial FTR, NTRC and GR2 display a preferential expression in green tissues, whereas GR1 and NTRA/ NTRB, encoding cytosolic proteins, are strongly expressed in roots. The reductase genes are remarkably not affected by abiotic stresses and hormones, except for GR1 that appears slightly up-regulated in response to cold, osmotic, salt and ultraviolet (UV) stresses in shoots, as well as 3 h of ABA treatment. Some pathogens induce the expression of the cytosolic-encoding NTRA/NTRB and GR1, while slightly inhibiting the plastidial-encoding genes (FTR genes, NTRC, GR2), which is in agreement with the common response of plastidial TRX and GRX-encoding genes (see above). GSH1 and GSH2, encoding the two enzymes responsible for glutathione biosynthesis, are ubiquitously expressed, except in rare tissues such as germinating pollen and procambium (Supporting Information Fig. S5). Their expression is also remarkably stable, except in response to biotic stresses and MeJa which induce both genes.

DISCUSSION This work is the first attempt to present a clear, exhaustive but concise map of TRX and GRX gene expression in A. thaliana. We were first interested in the subcellular localization of TRX and GRX proteins. Although we could confirm or update some predictions by a careful analysis of proteomic data and other experimental arguments, it is obvious that some work is still required in order to identify the exact subcellular localization of many of these proteins. Among the ROXY family, for example, we only have vague predictions for 16 or 17 members out of 21. Moreover, we should be careful when analysing subcellular localization. Indeed, more and more proteins previously predicted or published as cytosolic proteins are in fact associated to the PMs or endomembranes, or take part of the secretory pathway (at least a fraction of these proteins). Techniques of transient overexpression in heterologous systems (e.g. onion epidermal cells) or in Arabidopsis protoplasts are often not sufficient to detect such subtle localizations. The generation of stable Arabidopsis transgenic lines expressing the protein (fused to any fluorescent protein) under its endogenous promoter should be favoured, together with immunolocalization experiments. Biochemical approaches, such as subcellular © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

Arabidopsis redoxins gene expression fractionation or in vitro import assays, have also proven to be helpful (Laloi et al. 2001; Chew et al. 2003; Pfalz et al. 2006; Cain et al. 2009; Lorang et al. 2012). Efforts to get better information on GRX and TRX subcellular localization will undoubtedly provide significant clues in order to understand their functions.We can observe that many GRX and TRX are finally predicted (or shown) to be targeted to membranes or to the secretory pathway. This has particularly been shown for the myristoylated/palmitoylated TRXhII and TRXhIII subfamilies (Meng et al. 2010; Traverso et al. 2013). This is consistent with a recent report showing that several Arabidopsis membrane proteins could be regulated by redox mechanisms, including important Ca2+-sensing proteins (Ueoka-Nakanishi et al. 2013). Correlations between subcellular localization and gene expression pattern could help us in better predicting both of these parameters. Indeed, we can observe that core chloroplastic redoxins, essential for the regulation of the photosynthesis machinery, display a unique pattern of expression, with high expression in green tissues, a weak decrease in response to abiotic and biotic stresses, and almost no response to phytohormones in seedlings. In contrast, we can observe that plastidial redoxins whose expression does not display this specific pattern have been shown to be localized to specific compartments and to ensure functions not directly related to photosynthesis. For example, TRXm3 is localized in non-green plastids of meristems and primordia, and seems essential for the regulation of cell-to-cell communication in these tissues (Benitez-alfonso & Jackson 2009; Benitez-Alfonso et al. 2009). Some plastidial redoxins, such as TRXy1 (found only in one chloroplast proteome, even though it has been shown to be localized into plastids during in vitro import assay) or ACHT3, which do not display the unique profile of chloroplastic gene expression, might also be localized to non-green or other specific plastids (Marchand et al. 2010; Bohrer et al. 2012). They might not be involved in the general control of the photosynthetic machinery, but most probably ensure other specific functions, such as the control of other chloroplastic metabolic pathways (Marchand et al. 2010). Conversely, some genes display the specific expression pattern of core chloroplastic genes, although they are not predicted to encode plastidial proteins. The TRXh-encoding CXXS1 or the GRX-encoding ROXY10, for example, is expressed according to this unique pattern. We have little indication of their subcellular localization, with only one experiment of 35S:CXXS1-GFP in onion epidermal cells (Serrato et al. 2008) suggesting a general cytosolic localization. However, we can postulate from their expression pattern either a chloroplastic localization or a function tightly related with photosynthesis (e.g. the coordination of photosynthesis with some other metabolic process in the cell). The validity of our high-throughput analysis is confirmed by the retrieval of many results previously reported. Indeed, we clearly detect, for example, the induction of TRXh5 by wounding, pathogens and ABA, as previously shown (Reichheld et al. 2002; Laloi et al. 2004). Another example is GRXS17, with an expression stronger in roots, stems and © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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flowers than in leaves, which corresponds to the reported expression levels determined by qRT-PCR (Cheng et al. 2011). The last example is ROXY18/GRXS13, which we find induced by pathogens, including Botrytis cinerea, as already reported (La Camera et al. 2011). Finally, our report allows the identification of new putative research perspectives. For example, the GRX-encoding 4CXXC4 is responsive to auxin treatment. Interestingly, it is predominantly expressed in the columella, the pericycle and the lateral root primordia in roots, three tissues where auxin transport is finely tuned, via the complex coordination of several membrane transporters. Moreover, the 4CXXC4 protein has been identified in many PM proteomes (Table S2). Altogether, these data suggest an interaction between 4CXXC4 and auxin function in roots. Because this gene is also induced specifically by salt stress and pathogens, 4CXXC4 might therefore play a critical role in root developmental adaptation to these environmental constraints. Another example is CPFA3, a gene specifically expressed in the lateral root cap, root procambium, senescent leaves, and mature and germinating pollen. Such a very specific expression pattern provides a powerful way to identify putative functions of the protein by using the co-expression tools available online. The use of Genevestigator Co-expression Tool (https://www.genevestigator.com/gv/plant.jsp; Hruz et al. 2008) allows to identify several leucine-rich repeat (LRR) kinase protein-encoding genes as anatomically tightly co-expressed with CPFA3. Interestingly, LRR kinases have been implicated in pollen development and pollen–pistil interactions, organ abscission, and vasculature differentiation (Torii 2004). Furthermore, LRR-type kinases are also important for response to pathogens and CPFA3 is highly induced in response to biotic stresses. Finally, a lot of LRR-related proteins display an LRR domain flanked by two wellconserved motifs composed of paired cysteines spaced by six amino acids (CX6C; Torii 2004). Although the enzymatic activity of CPFA proteins will have to be further investigated, all these arguments sustain the hypothesis that CPFA3 might directly regulate LRR kinases. Of course, such co-expression analyses are useful mainly for genes displaying a very specific pattern of expression. However, this technique applied to our data can provide very interesting new perspectives in identifying the functions of TRX and GRX genes. This work tries to give a clear picture of all TRX and GRX gene expression and subcellular localization in Arabidopsis. Clearly, we cannot refer to every data in the literature, and we provide only a general view of gene expression, probably missing some specificity for certain genes (like responses to missing treatments, such as high light, salicylic acid or strigolactones, or cell-specific expression such as guard cells). Moreover, our current knowledge is only partial and some information has not been investigated yet (such as fine subcellular localization of many of these genes, or transcriptomic data in specific cells or conditions, e.g. in female gametophyte). Finally, the expression redundancy between some TRX or GRX from the same subfamily (such as the cluster of genes ROXY11 to ROXY15) or between members of TRX and GRX families may prevent the identification of

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their specific functions. Interestingly, recent genetic data have emphasized functional redundancies between TRX and GRX in several developmental functions (Reichheld et al. 2007; Xing & Zachgo 2008;Li et al. 2009;Marty et al. 2009;Bashandy et al. 2010). However, for some of these functions, the respective TRX and GRX involved have not yet been identified. For example, pollen fertility is dramatically perturbed in a ntr gr mutant, suggesting that TRX and GRX are involved in pollen fertility (Marty et al. 2009). A careful analysis of transcriptional profiles of TRX and GRX can identify a limited number of TRX and GRX potentially involved in pollen (i.e. TRXh9, GRXC2).Therefore, we are quite confident that this report of all redoxin members in Arabidopsis will allow the identification of new research perspectives, as illustrated above.

ACKNOWLEDGMENTS This work was supported by Agropolis Fondation through the ‘Investissements d’Avenir’ programme (ANR-10-LABX0001-01) under reference ID 1300-006 (contract funding of J.C.), by the Centre National de la Recherche Scientifique and the Agence Nationale de la Recherche (ANR-Blanc Cynthiol 12-BSV6–0011). We have no conflict of interest to declare.

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Received 27 September 2013; received in revised form 3 January 2014; accepted for publication 6 January 2014

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. TRXh3 subcellular localization. 35S:TRXh3GUS-GFP stable tobacco transgenic line. (a) fluorescence picture; (b) superimposition of the fluorescence and the transmission pictures. Figure S2. TRXh5 response to flagellin. Transcriptional fusions of TRX promoters with GUS reporter gene. Leaves have been infiltrated with mock solution (left) or 50 nm Flg22 (right) 8 h prior to GUS revelation. Figure S3. GRXC1 and GRXC2 expression in the root tip. Transcriptional fusions of GRX promoters with GUS reporter gene. Figure S4. GRXC2, GRXC3 and GRXC4 expression in seeds and siliques. Transcriptional fusions of GRX promoters with GUS reporter gene. (a) Expression in embryo from dry seeds.

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Dry seeds have been shortly imbibed (1 h) prior to dissection and GUS revelation. (b) Expression in developing siliques. Figure S5. Analysis of reductases and glutathione biosynthesis gene expression. (a) tissue-specific expression; (b,c) details of gene expression in roots; (d) stress responses; (e) hormone responses.

Table S1. Experimental arguments for TRX subcellular localization. Question mark indicates a predictive localization, not validated by experimental data. Table S2. Experimental arguments for GRX subcellular localization. Question mark indicates a predictive localization, not validated by experimental data.

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment