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First published online January 5, 2007; 10.1104/pp.106.090894 Plant Physiology 143:1110-1118 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Phytochelatin Synthases of the Model Legume Lotus japonicus. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants1,[OA]Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, 50080 Zaragoza, Spain (J.R., M.R.C., L.N., J.L., C.P.-R., M.B.); and Kazusa DNA Research Institute, Kisarazu, Chiba 292–0818, Japan (S.S., S.T.)
The biosynthesis of phytochelatins and homophytochelatins has been studied in nodulated plants of the model legume Lotus (Lotus japonicus). In the first 6 to 24 h of treatment with cadmium (Cd), roots started to synthesize elevated amounts of both polypeptides, with a concomitant increase of glutathione and a decrease of homoglutathione, indicating the presence of active phytochelatin synthase (PCS) genes. Screening of transformation-competent artificial chromosome libraries allowed identification of a cluster of three genes, LjPCS1, LjPCS2, and LjPCS3, which were mapped at 69.0 cM on chromosome 1. The genes differ in exon-intron composition and responsiveness to Cd. Gene structures and phylogenetic analysis of the three protein products, LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N, are consistent with two sequential gene duplication events during evolution of vascular plants. Two sites for alternative splicing in the primary transcripts were identified. One of them, involving intron 2 of the LjPCS2 gene, was confirmed by the finding of the two predicted mRNAs, encoding LjPCS2-7R in roots and LjPCS2-7N in nodules. The amino acid sequences of LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share 90% identity, but have only 43% to 59% identity with respect to the typical PCS1 enzymes of Lotus and other plants. The unusual LjPCS2-7N and LjPCS3-7N proteins conferred Cd tolerance when expressed in yeast (Saccharomyces cerevisiae) cells, whereas the alternatively spliced form, LjPCS2-7R, differing only in a five-amino acid motif (GRKWK) did not. These results unveil complex regulatory mechanisms of PCS expression in legume tissues in response to heavy metals and probably to other developmental and environmental factors.
Phytochelatins (PCs) are Cys-rich polypeptides of general structure (  Glu-Cys)2-11-Gly, which play an essential role in the detoxification of some heavy metals (cadmium [Cd], copper [Cu], zinc [Zn], mercury [Hg], and lead [Pb]) and metalloids (arsenic) in fungi, plants, nematodes, and other organisms (Grill et al., 1987 ; Clemens et al., 1999; Cobbett and Goldsbrough, 2002; Vivares et al., 2005). Synthesis of PCs is catalyzed by phytochelatin synthase (PCS) in a stepwise reaction in which a -Glu-Cys unit from glutathione (GSH; Glu-Cys-Gly) is first added to the GSH molecule itself and then to the elongating PC chain (Grill et al., 1989; Rea et al., 2004).
Some legumes produce homoglutathione (hGSH;
Expression of PCS genes has only been examined in Arabidopsis (Arabidopsis thaliana) and Indian mustard (Brassica juncea). However, results are fragmentary and, in most cases, contradictory. The two PCS genes of Arabidopsis, AtPCS1 and AtPCS2, appear to be constitutively expressed and not transcriptionally regulated by Cd (Ha et al., 1999 In this article, three functional LjPCS genes have been identified, mapped, and characterized. We have found that the genes are differentially expressed in the roots of Lotus in response to Cd and that alternative splicing of intron 2 in the primary transcript of the LjPCS2 gene gives rise to two protein products that, when expressed in yeast (Saccharomyces cerevisiae) cells, confer contrasting resistance to Cd in vivo. These findings unravel previously unsuspected complex regulatory mechanisms of PCS expression in plants.
Synthesis of GSH and (h)PCs in Roots Is Activated in Response to Cd
The first necessary step of this work was to determine whether Lotus plants produce PCs and hPCs upon exposure to heavy metals as an indication of the presence of active PCS genes. We selected Cd for comparative purposes because it is the metal activator commonly used for PCS studies in Arabidopsis. The (h)PC precursors, GSH and hGSH, were also quantified. Lotus plants were grown in vermiculite until the late vegetative stage and were treated with 100 or 200 µM Cd for 3 to 96 h to detect rapid responses in PCS mRNA levels as well as slower changes in thiol metabolites. None of the plants exhibited symptoms of heavy metal toxicity, such as reduction of root or shoot growth and leaf chlorosis. Preliminary experiments showed that (h)PC synthesis was undetectable in leaves and low (<12%) in nodules compared to roots, and hence further work was performed only in root tissue. A lack of accumulation of (h)PCs in the leaves was also observed in chickpea (Cicer arietinum) plants treated with Cd (Gupta et al., 2004
Roots of control (untreated) plants contained 9 nmol g–1 of GSH and 178 nmol g–1 of hGSH (Fig. 1
), confirming that hGSH is the major thiol produced by Lotus (Matamoros et al., 2003
However, the most remarkable effect of Cd was on the content of (h)PCs (Fig. 1). Roots of control plants had no detectable (h)PCs, but started to synthesize them within 3 h of Cd treatment, in agreement with the strict metal requirement for PCS activation (Zenk, 1996 ; Clemens et al., 1999; Rea et al., 2004). The total hPC content was 3 nmol g–1 after 3 h with 100 µM Cd and reached 250 nmol g–1 after 96 h with 200 µM Cd. Roots were also able to rapidly synthesize significant amounts of PCs (18%–25% of hPCs). Notably, in roots treated with 200 µM Cd, the content of PC3 and hPC3 polypeptides was 69 and 192 nmol g–1, whereas hPC2 and hPC4 were found at levels in the range of 25 to 35 nmol g–1 (Fig. 1) and their homologs, PC2 and PC4, at levels of <3 nmol g–1. Thus, PC3 and hPC3 accounted for 70% to 80% and 94% to 97% of the total PCs and hPCs formed, respectively, regardless of Cd exposure. This suggests that, in our experimental conditions, LjPCS activity is more efficient at synthesizing hPC3 and/or that these polypeptides are more stable than their structural variants. Unfortunately, we could not detect significant PCS activity in Lotus root extracts by using HPLC with either precolumn or postcolumn derivatization. Parallel control extracts of two other legumes, pea and bean (Phaseolus vulgaris), showed specific activities of 59 and 235 pmol of total PCs (GSH equivalents) min–1 mg–1 of protein, respectively, in Cd-treated roots. Given the strong accumulation of (h)PCs in Lotus roots, PCS activity may be very labile or low in Lotus as compared with the other two legumes.
The structure and function of PCS genes have so far been studied in detail only in Arabidopsis (Ha et al., 1999
Comparison between the LjPCS and AtPCS gene structures revealed interesting features (Fig. 2). Whereas AtPCS1 and AtPCS2 contain nine exons (Ha et al., 1999 ; Cazalé and Clemens, 2001), LjPCS1 consists of eight exons and LjPCS2 and LjPCS3 consist of seven exons. Exons 1 to 6 of all five genes have identical sizes, except for exon 1 of AtPCS2 (3 bp shorter) and exon 2 of LjPCS2 (15 bp shorter). Also, introns of LjPCS2 and LjPCS3 are very close in size and show high sequence identity (77%–94%), except for intron 1 (62%). In contrast, intron sizes and sequences largely differed between LjPCS1 and LjPCS2 or LjPCS3. These findings led us to propose that the three LjPCS genes resulted from two sequential gene duplication events. The first duplication originated LjPCS1 and another gene that, in turn, underwent a second duplication. This gave rise to LjPCS2 and LjPCS3 and probably took place once legumes had diverged from other vascular plants. This hypothesis would explain why three genes are present in Lotus and raises the question as to whether it is also the case for other legumes. A search in the plant databases of The Institute for Genomic Research (http://www.tigr.org/tdb/tgi/plant.shtml) provided three tentative consensus sequences or ESTs encoding PCS in Medicago truncatula (TC105496, TC112156, and BE325953) and soybean (Glycine max; TC208399, TC219312, and TC234831). Because some of these partial sequences do not overlap and legume genomes have not been completely sequenced, we can only conclude at this stage that there are at least two PCS genes in each of those species.
Further support for two duplications of the PCS genes in Lotus was provided by sequence analysis of the derived proteins. The LjPCS1-8R, LjPCS2-7R, LjPCS2-7N, and LjPCS3-7N proteins are predicted to contain 501 (55.5 kD), 477 (53.0 kD), 482 (53.7 kD), and 479 (53.2 kD) amino acids, respectively, which are in the range (421–504) reported for other higher plant PCS enzymes. Comparisons of amino acid sequences of known PCS proteins, along with analysis of mutated and truncated proteins, have led to the hypothesis that the enzymes are functionally organized into an N-terminal (conserved) domain involved in catalysis and a C-terminal (variable) domain conferring metal-sensing ability and enzyme stability (Cobbett and Goldsbrough, 2002
Phylogenetic analysis of PCS proteins faithfully reflects taxonomic groupings, with the exceptions of LjPCS2-7R, LjPCS2-7N, and LjPCS3-7N (Fig. 4 ). The tree shows separate clades for the PCS of nematodes, cyanobacteria, yeasts, ferns, and the higher plant families Typhaceae, Liliaceae, Poaceae, Leguminosae, Brassicaceae, and Solanaceae. The high sequence identity (84%) and similarity (90%) between LjPCS1-8R and the previously reported PCS1 of soybean (GmhPCS1; Oven et al., 2002 ) strongly suggest that they are functional homologs. In sharp contrast, LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share high identity (90%) with each other, but poor identity (approximately 55%) with AtPCS1, AtPCS2, GmhPCS1, and LjPCS1-8R.
An essential prerequisite to study gene expression is the highly sensitive and specific quantification of mRNAs. We used quantitative reverse transcription (qRT)-PCR because the ORFs of the LjPCS genes have sequence identities ranging from 63% (between LjPCS1 and the other two genes) to 90% (between LjPCS2 and LjPCS3), and because the mRNA levels of the three genes were only 0.014% of that of the housekeeping gene. Specific primers were designed based on the genomic sequences described in this article and were used to demonstrate that the three LjPCS genes are transcribed in roots (Fig. 5
), nodules, and leaves (data not shown). Furthermore, following the same criterion for significant gene up-regulation (mRNA level >2-fold of control) in the qRT-PCR data as that considered in cDNA array studies (El Yahyaoui et al., 2004
Further experiments were designed to prove that the mRNAs encode functional proteins. Indeed, the presence of the catalytic triad in the proteins LjPCS1-8R, LjPCS2-7R, LjPCS2-7N, and LjPCS3-7N strongly suggested that this is the case. Previous work from our laboratory showed that LjPCS1 (LjPCS1-8R) has PCS activity when expressed in Escherichia coli (Loscos et al., 2006 ). A similar attempt was made to produce the other three recombinant proteins, but they were invariably found in insoluble form and hence were not amenable to PCS assay. In silico analysis of the amino acid sequences (TMpred; http://www.expasy.org/tools) predicts an additional transmembrane segment in LjPCS2-7R, LjPCS2-7N, and LjPCS3-7N, which might be related to their different solubility properties compared with the typical LjPCS1-8R. To circumvent this problem, ORFs encoding the three anomalous proteins, and also AtPCS1 and LjPCS1-8R as positive controls, were introduced in yeast cells. These were then exposed to toxic Cd concentrations and growth was visualized in solid medium (Fig. 6A
) or quantified in liquid medium (Fig. 6B). Cells transformed to express LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N were tolerant to Cd in both media, whereas those transformed with the empty plasmid were unable to grow, thus demonstrating that the three proteins are active. Specifically, LjPCS1-8R and LjPCS3-7N reached similar levels after 20 h, with LjPCS2-7N significantly lagging behind them after 20 and 40 h (Fig. 6B). Most interestingly, cells transformed with the LjPCS2-7R construct were unable to grow in solid (Fig. 6A) or liquid (Fig. 6B) medium containing Cd, indicating that this protein, unlike the other gene product, LjPCS2-7N, is not functional in yeast.
Alternative Pre-mRNA Splicing Accounts for the Unusual LjPCS2-7R and LjPCS3-7N Protein Sequences
A closer examination of the DNA sequences and proteins encoded by the three LjPCS genes allowed us to identify two sites of alternative splicing for introns 2 and 7, respectively (Fig. 7
). All intron-exon splice junctions conformed to the GT/AG rule described for eukaryotic genes (Shapiro and Senapathy, 1987
Alternative splicing is most often associated with differences in tissue localization or stress responses (Lorkovic et al., 2000 ). Interestingly, the pre-mRNA encoding the GSH S-transferase of maize is alternatively spliced in response to Cd (Marrs and Walbot, 1997). Because the mRNA levels of the LjPCS genes are differentially affected by Cd and small differences in the sequences of the proteins may have, when expressed in a heterologous system, important effects on Cd tolerance in vivo (Fig. 6), it will be most interesting to investigate the expression patterns of the genes in various plant tissues and stress conditions. Also, the observation that LjPCS2-7N and LjPCS3-7N, bearing a C-terminal domain very different from that found in typical higher plant PCS proteins, are fully functional, opens a new prospect for the study of the role of specific Cys residues (Fig. 3) and other amino acid residues or motifs in enzyme catalysis. In conclusion, our results provide an unprecedented view of the multiple regulatory mechanisms of (h)PC biosynthesis in legume tissues and of the large potential for variation, and probably adaptation, of PCS genes and their protein products in response to heavy metals and to other, as yet unidentified, developmental or environmental cues.
Plant Material
Lotus (Lotus japonicus) plants (cv Gifu B-129) were nodulated by Mesorhizobium loti (strain NZP2235) to mimic their natural state and were grown in pots containing vermiculite under controlled environment conditions for 48 to 52 d (Matamoros et al., 2003
Concentrations of GSH and hGSH in root extracts were determined by HPLC after precolumn derivatization with monobromobimane using a Nova-Pak C18 column (3.9 x 150 mm, 4 µm; Waters) and fluorescence detection (model 474; Waters). The protocol of Fahey and Newton (1987)
Total RNA was extracted from roots and nodules with the RNAqueous isolation kit (Ambion). RNA was treated with DNaseI (Roche), and first-strand cDNA was synthesized using oligo(dT)17 primers and Moloney murine leukemia virus reverse transcriptase (Promega). cDNA concentrations for PCR reactions were normalized using Lotus ubiquitin as the reference gene. qRT-PCR analysis was carried out using SYBR Green supermix reagents and the iCycler IQ (Bio-Rad). Oligonucleotide primers (Table I ) were designed using Primer Express, version 2.0 (Applied Biosystems). The PCR program comprised an initial step for polymerase activation (5 min at 95°C), 40 cycles of amplification and quantification (15 s at 95°C, 1 min at 60°C), and a final melting curve (55°C–95°C with a reading every 0.5°C).
Identification, Mapping, and Characterization of LjPCS Genes
A genomic clone (LjT27M11) was isolated by screening transformation-competent artificial chromosome libraries of Lotus with primers based on the GmhPCS1 sequence (AF411075). The clone was entirely sequenced in the context of the Lotus genome-sequencing project and was mapped by using a simple sequence repeat marker (TM0332) found in LjT27M11, as described (Sato et al., 2001
The ORFs of AtPCS1, LjPCS1-8R, LjPCS2-7R, LjPCS2-7N, and LjPCS3-7N were introduced in the Champion pET directional TOPO bacterial expression vector (Invitrogen). The resulting DNA fragments, encoding fusion proteins with an N-terminal poly-His tag, were introduced in pYES2.1 TOPO TA (Invitrogen) to express the recombinant proteins in yeast INVSc1 under the control of the GAL1 promoter (Giniger et al., 1985 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY270030 (LjPCS1), AY928091 (LjPCS2), and DQ013040 (LjPCS3).
We thank J.I. Schroeder, J. Webb, and M. Oven for providing yeast strain INVSc1, Lotus seed, and the E. coli AtPCS1 construct, respectively. We also acknowledge M.H. Zenk for encouragement and advice. Received October 6, 2006; accepted December 26, 2006; published January 5, 2007.
1 This work was supported by Ministerio de Educación y Ciencia (MEC)-Fondos Europeos de Desarrollo Regional (grant no. AGL2005–01404), European Commission (grant no. FP6–2003–INCO–DEV2–517617), and Gobierno de Aragón-Fondo Social Europeo (group E33). J.R., L.N., and J.L. are the recipients of a postdoctoral contract ("Juan de la Cierva" program) from MEC, a predoctoral fellowship from MEC, and a predoctoral fellowship from Gobierno de Aragón, respectively. This work is part of the Ph.D. thesis of L.N. (supervised by J.R. and M.B).  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: Manuel Becana (becana{at}eead.csic.es).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.090894 * Corresponding author; e-mail becana{at}eead.csic.es; fax 34–976–716145.
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ABSTRACT
RESULTS AND DISCUSSION
Ala) instead of or in addition to GSH, and are thus able to synthesize homophytochelatins (hPCs) with general structure (
, Cys residues present in all known higher plant PCS proteins, except in the anomalous LjPCS2-7R/N and LjPCS3-7R; 
, Cys residues present in LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7R, but absent in all other known higher plant PCS proteins.
3') are indicated.
