Identification and characterization of LjGPX genes
The first aim of this study was to isolate and characterize the GPX genes of L. japonicus. A search in the gene databases of this model legume allowed us to identify three TC sequences that contained the complete open reading frames (ORFs), as well as some 5′- and 3′-untranslated region (UTR) sequences, of LjGPX1, LjGPX3 and LjGPX6 (Table 1). Database mining also resulted in the identification of two singletons that were assigned to LjGPX4 and LjGPX5. An additional gene, LjGPX2, was identified by screening the TAC libraries using the TC sequence of LjGPX1. The LjGPX2 gene was found to be transcribed by determination of mRNA levels, as there were no ESTs available for this gene. In fact, the number of ESTs for each gene (Table 1), which were obtained by reverse transcription of total RNA from young plants, immature flowers, pods, roots and nodules of L. japonicus, strongly suggests that the expression of LjGPX2, LjGPX4 and LjGPX5 is much lower than that of LjGPX1 and LjGPX3. The EST sequence information was also used to isolate TAC clones and to map five of the genes on chromosomes 4 and 5 (Table 1). Interestingly, LjGPX1 and LjGPX2 are tandemly arranged with the same orientation on the chromosome, and their ORFs are separated by only 1477 bp. However, these two genes show only 71% identity in their ORFs, indicating that they did not originate from a recent duplication event.
To establish the exon–intron organization of the less expressed LjGPX genes, namely, LjGPX2, LjGPX4 and LjGPX5, their ORFs were completely sequenced using information based on the L. japonicus genome project (Sato et al., 2001). All the genes, except LjGPX4, contain six exons interrupted by five introns (Fig. 1). The same exon composition was observed for the eight GPX genes of A. thaliana (Rodríguez Milla et al., 2003), the six GPX genes of poplar (Navrot et al., 2006) and the single GPX gene of citrus (CsGPX1) so far examined (Avsian-Kretchmer et al., 2004). Thus, all the AtGPX and PtGPX genes have six exons, and a previous annotation (Rodríguez Milla et al., 2003) that AtGPX3 and AtGPX7 have seven and five exons, respectively, was found to be incorrect. There is also a high degree of conservation among plant species in the exon lengths. Exons 2 to 5 have identical size for all the LjGPX and AtGPX genes, with the exception of exon 5 in LjGPX4. The length of exon 1 in the LjGPX, AtGPX, OsGPX and PtGPX genes is variable, as expected for genes encoding GPX isoforms lacking or bearing N-terminal signal peptides. Exon 6 is absent in LjGPX4 and has a similar size in the other LjGPX genes (30–45 bp) and in the AtGPX genes (30–39 bp).
Figure 1. Exon–intron organization of Lotus japonicus glutathione peroxidase (LjGPX) genes. The untranslated regions (UTRs) are depicted in hatched boxes, open reading frames (ORFs) in black boxes and introns in white boxes. Disrupted lines denote intron lengths not drawn to scale. All other lengths are drawn to scale.
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The promoter regions of the six LjGPX genes were searched for the presence of putative cis-acting regulatory elements as an indication of the responsiveness of the genes to hormones or environmental cues. Most or all the LjGPX promoters contain elements that are responsive to light, biotic stress and abiotic stress, whereas only some of them contain elements that are involved in the response to anaerobiosis and hormones (Table S2). A similar conclusion was drawn from the in silico analysis of the AtGPX promoters (Rodríguez Milla et al., 2003). The LjGPX1 and LjGPX6 promoters also contain cis-sequences, with one or two mismatches, that may be responsive to oxidative stress. These sequences include the antioxidant responsive element (ARE), which also has been found in the promoters of the three catalase genes of maize (Zea mays; Scandalios et al., 1997).
Predicted properties of LjGPX proteins
The derived amino acid sequences of the six LjGPX proteins contain the three motifs present in most plant and mammalian GPXs (Criqui et al., 1992; Depège et al., 1998; Churin et al., 1999). These motifs contain residues that are proposed to be part of the catalytic site (LjGPX1 numbering): Cys-111, Cys-140, Gln-142 and Trp-200. The presence of Cys-111 indicates that none of the six LjGPX proteins is a selenium-dependent enzyme, as this amino acid residue is replaced by selenocysteine in the homologue proteins of mammals (Ursini et al., 1995) and C. reinhardtii (Fu et al., 2002). From the alignment of the LjGPX sequences shown in Fig. 2 and the data presented in Table 2, three types of protein have been differentiated based on the amino acid sequence length: LjGPX1 and LjGPX6 (c. 235 amino acids; 26 kDa); LjGPX3 (211 amino acids; 24 kDa); and LjGPX2, LjGPX4 and LjGPX5 (c. 170 amino acids; 19 kDa). These differences, and the poor homology among the first 40 or 70 amino acid residues of LjGPX1, LjGPX3 and LjGPX6 (Fig. 2), clearly suggest that these three proteins bear signal peptides for organelle targeting. Prediction programs for subcellular localization suggest that LjGPX6 has a chloroplastic N-terminal transit peptide and that LjGPX1 has an ambiguous N-terminal peptide for targeting to mitochondria and/or plastids. The same programs predicted that LjGPX2, LjGPX4 and LjGPX5 are localized in the cytosol, and LjGPX3 in the cytosol and secretory pathway (Table 2).
Figure 2. Amino acid sequences of Lotus japonicus glutathione peroxidase (LjGPX) proteins. The three conserved domains found in most animal and plant glutathione peroxidases (GPXs) are marked in green, red and blue, respectively. The amino acid residues that form part of the proposed catalytic site of the GPXs are marked with asterisks. The cysteine residue marked with a green asterisk is replaced by selenocysteine in mammalian phospholipid hydroperoxide GPXs. Amino acid residues that are identical in at least five of the sequences are marked in white lettering on a black or coloured background.
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Table 2. Predicted properties of glutathione peroxidases (GPXs) of Lotus japonicus
|Protein||Length (aa)*||Molecular mass (kDa)*||pI*||TP (aa)†||Subcellular localization†||Putative homologues‡|
|LjGPX1||236 (156)||26.2 (17.3)||8.78 (5.58)||80||Mito/chloro||At6, Os1, Os3, Pt3.2|
|LjGPX3||211 (175)||24.2 (19.6)||7.61 (7.84)||36||Sec (cyt)||At2, At3, Os5, Pt2|
|LjGPX4||170||19.0||9.58||–||Cyt||At4, At5, Os4, Pt4|
|LjGPX5||171||19.1||9.37||–||Cyt||At4, At5, Os4, Pt4|
|LjGPX6||235 (164)||26.3 (18.6)||9.01 (7.85)||71||Chloro||At1, At7, Os2, Pt1|
The full-length amino acid sequences of plant GPXs available in the databases were used to build an unrooted phylogenetic tree (Fig. 3). Inclusion of the complete sequences rather than mature sequences was preferred for constructing the tree because there was uncertainty about the cleavage sites for many of the proteins. Alignment of the whole sequences by ClustalW was consistent with the homologies among the predicted mature proteins. The putative subcellular localizations are marked with different colours in the same figure. The tree is composed of five clades and a separated branch for barley GPX3. Clades I and II are hypothesized to contain, respectively, chloroplastic and cytosolic isoforms; clades III and IV, both cytosolic and secreted proteins; and clade V, cytosolic proteins and proteins with N-terminal transit peptides for targeting either to the mitochondria or to both the mitochondria and chloroplasts. This phylogenetic analysis updates a previous version and is fully consistent with the assignments made for clades I and II (Navrot et al., 2006). However, careful inspection of amino acid sequences by us and others (Rodríguez Milla et al., 2003) strongly suggests that at least some proteins of clades III and V may be targeted to multiple subcellular compartments. This hypothesis is based on the presence of ambiguous N-terminal signal peptides in LjGPX1 and of putative alternative translation sites in the mRNAs of LjGPX1, LjGPX3 and AtGPX6 (Table 2; Rodríguez Milla et al., 2003).
Figure 3. Phylogenetic analysis of glutathione peroxidase (GPX) proteins of vascular plants. Only complete sequences were considered. The tree was constructed using the neighbour-joining method of ClustalW, with 1000 bootstraps, and the bar indicates 0.1 substitution per site. Predicted localizations of proteins are denoted by different colours: green, chloroplasts; red, cytosol; blue, mitochondria; brown, secretory pathway. Abbreviations of plant species: At, Arabidopsis thaliana; Ca, Cicer arietinum; Cs, Citrus sinensis; Gm, Glycine max; Ha, Helianthus annuus; Hv, Hordeum vulgare; Le, Lycopersicon esculentum; Lj, Lotus japonicus; Mc, Mesembryantemum crystallinum; Md, Malus ×domestica; Mt, Medicago truncatula; Ns, Nicotiana sylvestris; Os, Oryza sativa; Pt, Populus trichocarpa; Ps, Pisum sativum; So, Spinacia oleracea; Tm, Triticum monococcum; Vv, Vitis vinifera; Zm, Zea mays. Accession numbers for L. japonicus genes are indicated in Table 1 and those of other plant species are as follows (in brackets): At1 (At2g25080); At2 (At2g31570); At3 (At2g43350); At4 (At2g48150); At5 (At3g63080); At6 (At4g11600); At7 (A4g31870); At8 (At1g63460); Ca (CAD31839); Cs (CAA47018); Gm1 (TC203397); Gm2 (TC203326); Gm3 (TC203271); Ha1 (CAA74775); Ha2 (CAA75009); Hv1 (CAB59895); Hv2 (CAB59893); Hv3 (CAB59894); Hv4 (BAC55016); Le1 (CAA75054); Le2 (AAP59427); Mc (CAB96145); Md (AAQ03092); Ns (CAA42780); Mt1 (TC94412); Mt2 (BM814275); Mt3 (TC106328); Mt4 (TC94581); Os1 (Os04g0556300); Os2 (Os06g0185900); Os3 (Os02g0664000); Os4 (Os03g0358100); Os5 (Os11g0284900); Ps (CAA04142); Pt1 (ABK96776); Pt2 (DT518382); Pt3.1 (ABK96047); Pt3.2 (ABK94488); Pt4 (ABK95195); Pt5 (2P5Q_A); So (BAA22194); Tm (AAQ64633); Vv (CB978870); Zm (AAM88847).
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Expression analyses of LjGPX genes in plant organs
The steady-state mRNA levels of the LjGPX genes were determined by qRT-PCR in leaves, roots and nodules of L. japonicus. This study allowed us to compare the abundance of the six LjGPX mRNAs within each plant organ (Fig. 4a) as well as the abundance of a specific LjGPX mRNA among the different plant organs (Fig. 4b). The first comparison was made by normalizing all mRNA levels with respect to those of one gene with significant expression, such as LjGPX6. It can be clearly observed that LjGPX1 and LjGPX3 are the most abundantly expressed genes in all three organs. Relative to LjGPX6, the mRNA level of LjGPX1 was 6-fold greater in the leaves and nodules and 25-fold greater in the roots; also, the mRNA levels of LjGPX3 were 6-, 30- and 43-fold greater, respectively, in the leaves, roots and nodules. In contrast, the less abundant transcripts (< 0.5-fold relative to LjGPX6) were those of LjGPX2 and LjGPX5 in the leaves and those of LjGPX4 in all three plant organs (Fig. 4a). The second comparison was made by normalizing the mRNA levels with respect to those found in the roots (Fig. 4b). It can be seen that there was high expression of LjGPX6 (4-fold) in leaves and of LjGPX3 (2.5-fold) and LjGPX6 (3-fold) in nodules. In contrast, LjGPX4 expression was negligible (< 0.01-fold) in both leaves and nodules (Fig. 4b). Interestingly, a consistent up-regulation (6.8-fold) of the LjGPX3 gene in nodules with respect to uninfected roots was also detected in a transcriptomic study using cDNA arrays (Colebatch et al., 2002).
Figure 4. Expression of glutathione peroxidase (LjGPX) genes in leaves, roots and nodules of Lotus japonicus. (a) Relative expression of each gene in the three plant organs. Steady-state mRNA levels were normalized with respect to ubiquitin and are expressed relative to the values of the LjGPX6 gene, which were given an arbitrary value of 1. (b) Relative expression of each gene in leaves and nodules with respect to roots. Steady-state mRNA levels were normalized with respect to ubiquitin and are expressed relative to the values found in the roots, which were given an arbitrary value of 1. All data are means ± SE of six replicates.
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Expression analyses of GPX genes in plants exposed to stressful conditions
The expression at the transcriptional level of the GPX genes was studied in L. japonicus and L. corniculatus plants exposed to several stress treatments involving production of ROS and/or RNS. We included L. corniculatus in this expression analysis to extend the molecular information gained with L. japonicus to other legume species, as we confirmed by melting curve analysis of the PCR products that the gene-specific primers were also valid for L. corniculatus.
Short-term exposure of L. japonicus plants to salt stress (150 mM NaCl) did not affect the expression of LjGPX genes, except for LjGPX4, which was down-regulated between 1 and 24 h (Fig. 5). This result is at variance with the increase of mRNA levels observed for some AtGPX genes between 3 and 12 h of treatment with 500 mM NaCl (Sugimoto & Sakamoto, 1997; Rodríguez Milla et al., 2003). This discrepancy can be attributed to the different plant species used or, more probably, to variations in the growth and stress conditions used in the experiments. A transient increase of the CsGPX1 mRNA level after 4–7 h of treatment with 200 mM NaCl was also observed in a salt-sensitive line, but not in a salt-tolerant line, of citrus cells (Avsian-Kretchmer et al., 1999). Assuming a similar link between salt sensitivity and up-regulation of GPX genes, the failure of salt stress to induce expression of LjGPX genes suggests that L. japonicus plants are relatively salt tolerant and do not experience oxidative stress. This suggestion is supported by the finding that the same salt stress treatment neither affects expression of many other antioxidant genes nor causes accumulation of lipid peroxides (data not shown).
Figure 5. Time-course expression analysis of Lotus japonicus glutathione peroxidase (LjGPX) genes in roots of L. japonicus exposed to NaCl (150 mM), sodium nitroprusside (SNP, 100 µM) or potassium ferricyanide (100 µM). Steady-state mRNA levels were normalized with respect to ubiquitin and are expressed relative to the values at time 0 (control), which were given an arbitrary value of 1. Data are means ± SE of three to six replicates. Asterisks denote significant up-regulation (>2.0–fold) or down-regulation (<0.5–fold).
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In sharp contrast with salinity, the exposure of plants to SNP, a compound capable of releasing NO for at least 40 h (Bethke et al., 2006), caused significant up-regulation of LjGPX2 after 24 h, LjGPX4 after 1, 3 and 24 h, and LjGPX5 after 3, 6 and 24 h (Fig. 5). However, the most striking effect of SNP was, by far, on the expression of the LjGPX6 gene. After only 1 h of SNP treatment, the steady-state mRNA level of LjGPX6 increased by 30-fold and by 14-, 3- and 8-fold after 3, 6 and 24 h, respectively (Fig. 5). Because decomposition of SNP yields ferricyanide in addition to NO (Bethke et al., 2006), a control treatment with potassium ferricyanide, at the same concentration as SNP, was included in the study. The only changes observed with ferricyanide were a decrease in the LjGPX4 mRNA level after 24 h (Fig. 5), confirming that the effects of SNP were indeed caused by NO. Previously, the effect of NO on GPX expression in plants had not been examined, but NO was found to up-regulate two other enzymes related to thiol metabolism, γ-glutamylcysteine synthetase and glutathione synthetase (Innocenti et al., 2007). Our finding that NO triggers, within 1 h, the expression of specific LjGPX genes, in particular LjGPX6, strongly suggests an important role of NO in the modulation of GPX function and also that at least some GPXs, in turn, may be involved in signalling pathways downstream of NO.
The expression of GPX genes was examined in L. corniculatus plants treated with Cd or Al (Fig. 6), two metals with potent phytotoxic effects. A recent report has shown that, in addition to its general inhibitory effects on plant growth and photosynthesis, Cd induces oxidative stress, as evidenced by an increase in ROS production and in the amounts of oxidized lipids and proteins (Romero-Puertas et al., 2004). In addition, two other pieces of evidence argue in favour of a role of GPX in the response to Cd. First, the protein level of an A. thaliana GPX is increased in response to Cd, and secondly, the resolution of the structure of poplar GPX5, in which 32 Cd atoms are bound to a dimer, suggests that at least some plant GPXs could act as a sink for Cd (Navrot et al., 2006; Koh et al., 2007). We found that Cd caused down-regulation of GPX1, GPX2, GPX4 and GPX6 between 3 and 24 h of treatment, and of GPX5 only after 3 h (Fig. 6). In contrast, there was significant up-regulation (2.7-fold) of GPX3, GPX4 and GPX5 after 1 h of treatment and of GPX3 after 24 h. Thus, Cd triggers a very rapid and transient activation (1 h) of specific GPX genes and a subsequent decrease (within 24 h) of the mRNA levels for all the genes (except GPX3) to control (or below control) values.
Figure 6. Time-course expression analysis of glutathione peroxidase (GPX) genes in roots of Lotus corniculatus exposed to 20 µM cadmium (Cd) or 20 µM aluminium (Al). Steady-state mRNA levels were normalized with respect to ubiquitin and are expressed relative to the values at time 0 (control), which were given an arbitrary value of 1. Data are means ± SE of three to six replicates. Asterisks denote significant up-regulation (>2.0–fold) or down-regulation (<0.5–fold).
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The effect of Al was also studied, for comparison, at the same concentration as Cd. The first symptom of Al toxicity, which develops at acid pH, is the inhibition of root growth (Barceló & Poschenrieder, 2002). Moreover, Al causes oxidative stress, an increase in antioxidant enzyme activities and lipid peroxidation (Sharma & Dubey, 2007; and references therein). We found that Al caused a general decrease of GPX transcripts after 6 and 24 h of treatment (Fig. 6). An earlier report showed that the treatment of A. thaliana plants with a combination of 100 µM Al and 100 µM Fe (pH 4.0) increased the AtGPX6 mRNA level (Sugimoto & Sakamoto, 1997). However, this experiment is not comparable to ours because of major differences in the growth and treatment conditions of plants.
Immunoblot analyses and immunolocalization of GPX proteins
To gain further information on LjGPXs and also as a prerequisite for immunolocalization studies, we performed immunoblot analyses in leaves, roots and nodules of L. japonicus using an antibody raised against poplar GPX3.2 (Navrot et al., 2006). This antibody was not isoform specific and recognized at least two LjGPX isoforms in the three plant organs (Fig. S1). The apparent molecular masses of the immunoreactive proteins were 20.5 and 24 kDa, in the range expected for GPX proteins (Table 2). The antibody also recognized two immunoreactive bands in extracts from L. corniculatus, A. thaliana, and poplar (data not shown) and a single immunoreactive band of c. 20.5 kDa when the antibody was challenged with poplar GPX3.2 (Fig. S1). An antiserum raised against synthetic peptides, based on the sequence of tomato GPXLe-1 (Herbette et al., 2004), recognized the same two immunoreactive bands in leaf, root and nodule extracts of L. japonicus, further confirming that the two immunoreactive bands are genuine GPXs. No major changes were observed in the LjGPX protein levels of L. japonicus or L. corniculatus exposed to the stresses described above for up to 24 h (data not shown), although they were increased in plants treated with 150 mM NaCl for 7 d (Fig. S1).
To our knowledge, there are only two reports on the subcellular localization of GPXs in plants. First, the antiserum against GPXLe-1 was used to detect the protein in the cytoplasm and cell wall of tomato internodes (Herbette et al., 2004). Secondly, a fusion of the N-terminal extension of GPX3.2 with the green fluorescent protein was used to show that GPX3.2 is targeted to the mitochondria and chloroplasts of tobacco (Nicotiana tabacum) cells (Navrot et al., 2006). We have used the GPX3.2 antibody, which was affinity-purified and hence more suitable for EM studies than the GPXLe-1 antiserum, to immunolocalize GPX proteins in L. japonicus. Plant material was high-pressure frozen for optimal preservation of antigenicity (Moran et al., 2003). Most surprisingly, GPX was found exclusively in the chloroplasts or amyloplasts of leaves, roots and nodules (Fig. 7). Furthermore, the labelling was associated with the starch grains in the uninfected cells of the cortex (Fig. 7a) and in the interstitial cells of the infected region (data not shown). The same localization of GPX was found in the proplastids of root tips (Fig. 7c) and in the chloroplasts of leaves (Fig. 7d). For comparison purposes, the localization of GPX was also examined in alfalfa, a crop legume with indeterminate nodules, and in S. rostrata, a tropical legume with determinate nodules on the roots and stems. Thus, abundant labelling was found in the starch grains in the infected cells of alfalfa nodules (Fig. 7e), as well as in the cortical chloroplasts of stem nodules (Fig. 7f) and in the leaf chloroplasts (Fig. 7g) of S. rostrata. Plastids in the nonphotosynthetic root nodules on S. rostrata were also labelled (not shown). Negligible labelling was observed on sections incubated in nonimmune serum, such as those of L. japonicus nodules (Fig. 7b) and S. rostrata leaves (Fig. 7h).
Figure 7. Immunogold localization of glutathione peroxidase (GPX) proteins within plastids of leaves, roots and nodules of legumes. (a) Plastid (p) within a cortical cell in a nodule of Lotus japonicus. The immunogold labelling (arrow) is specifically localized on the starch grain (s) in the plastid. (b) Serial section to (a) but treated with nonimmune serum substituted for the GPX antibody. There is no significant labelling on the starch grain (s) or elsewhere in the cortical cell. (c) Immunogold-labelled (arrow) starch grain (s) in a plastid (p) within an L. japonicus root tip cell. Note that the adjacent mitochondria (m) are not labelled. (d) Immunogold labelling (arrow) of starch grains (s) in a leaf chloroplast of L. japonicus. (e) Immunogold labelling (arrows) of starch (s) in a plastid within an infected cell of an alfalfa nodule. (f) Chloroplast (c) in a Sesbania rostrata stem nodule with immunogold labelling (arrows) of the starch grains (s) within it. (g) Immunogold labelling of starch grains (s) within chloroplasts (c) in a leaf of S. rostrata. (h) Serial section to (g) but treated with nonimmune serum substituted for the GPX antibody. There is no significant labelling on the starch grain (s) or elsewhere in the leaf cell. b, bacteroid; c, chloroplast; is, intercellular space; p, plastid; v, vacuole. Bars, 1 µm.
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The presence of GPX in association with starch grains had not been reported earlier but is consistent with the detection of thioredoxins f and m and of peroxiredoxin BAS1 in the amyloplasts (Balmer et al., 2006; Barajas-López et al., 2007). Thus, thioredoxins are substrates for both GPXs and peroxiredoxins, which are closely related enzymes (Navrot et al., 2006). Our finding of GPX in the amyloplasts suggests that peroxides (their other substrates) are also formed in these organelles and points to a regulatory role of this enzyme, in connection with thioredoxins, in heterotrophic tissues. In this context, at least two questions are worthy of further research. First, are some GPX and peroxiredoxin isoforms involved in the regulation of starch biosynthesis and/or mobilization, perhaps through changes in the thiol redox state mediated by ROS and/or RNS? Secondly, why was the antibody unable to detect any GPX in the cytosol or other subcellular compartments, despite the finding that the LjGPX3 gene, encoding a putative cytosolic or secretory protein (Table 2), is highly expressed at the mRNA level (Fig. 4)? It is possible that LjGPX3 is subjected to post-transcriptional regulation or that the localization of the corresponding protein is confined to certain plant tissues or cells. In any case, the results of the immunolocalization studies along with the surprisingly rapid and strong induction of specific GPX genes by NO underscore the complex regulation of the GPX genes in plants and strongly support the hypothesis that the GPX proteins play an important role not only as antioxidants but also in stress or metabolic signalling.