Genome-wide identification of rice class I metallothionein gene: tissue expression patterns and induction in response to heavy metal stress
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- Gautam, N., Verma, P.K., Verma, S. et al. Funct Integr Genomics (2012) 12: 635. doi:10.1007/s10142-012-0297-9
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Metallothioneins (MTs) are members of a family of cysteine-rich low molecular weight polypeptides which play an important role in heavy metal detoxification and homeostasis of intracellular metal ions in plant. Though MT genes from some selected plants have been characterized with respect to their protein sequences, kinetic properties and tissue-specific localization, no detailed study has been carried out in rice. Here, we present genome-wide identification, structural and expression analyses of rice MT gene family. Our analysis suggests presence of 11 class I MT genes in rice genome (Release 7 of the MSU Rice Genome Annotation Project) which are differentially expressed during growth and development, in various tissues and during biotic and abiotic stresses. Our analyses suggest that class I MT proteins in rice differ in tissue localization as well as in heavy metal coordination chemistry. We also suggest that some MTs have a predominant role in detoxification of As (V) in arsenic-tolerant rice cultivars. Our analysis suggests that apart from transcriptional regulation, post-transcriptional alternative splicing in some members of this family takes place during growth and development, in various tissues and during biotic and abiotic stresses.
KeywordsAlternative splicingArsenicHeavy metalRiceStressMetallothionein
Heavy metal ions, such as cadmium (Cd), arsenic (As), chromium (Cr), lead (Pb), zinc (Zn) and mercury (Hg), are highly reactive and toxic to living cells (Hall 2002). Plants have evolved mechanisms such as chelation and sequestration of heavy metals by particular ligands to counter this problem (Hall 2002). The two best-characterized heavy metal-binding ligands in plant cells are the phytochelatins (PCs), a family of enzymatically synthesized cysteine-rich peptides and metallothioneins (MTs). Metallothioneins (MTs) are low-molecular-weight (7–8 kDa), cysteine-rich (20–30 %), metal-binding proteins (Margoshes and Vallee 1957). These proteins are involved in essential-metal homeostasis and impart protection against heavy metal toxicity by sequestration (Mir et al. 2004; Hassinen et al. 2009a; Huang and Wang 2010), scavenging of reactive oxygen species (Hussain et al. 1996), regulation of metallo-enzymes and transcription factors (Andrews 2000; Bratić et al. 2009), metabolism of metallo-drugs and response to stress conditions. There are more than 50 plant MT-like proteins included in various databases, about one third of all known metallothioneins (Liu et al. 2000).
In plants, the first evidence for the role of MTs in Cu and Cd tolerance was provided through functional complementation of MT-deficient yeast by two Arabidopsis MT genes (Zhou and Goldsbrough 1994). Further evidence for the function of various MTs in plants was elucidated using knock-down and over-expression lines, tissue-specific and metal-regulated expression, as well as from the characterisation of MT–metal complexes (Guo et al. 2003, 2008; Domènech et al. 2007a, b; Freisinger 2007; Yuan et al. 2008; Bratić et al. 2009; Hassinen et al. 2009a, b). Studies on the localisation of MTs in specific plant parts, tissues and subcellular organelles have also provided the role of these proteins in metal detoxification and plant development (Nakajima et al. 1991).
Plant MTs have been classified into classes I, II and III based on the arrangement of Cys residues (Cobbett and Goldsbrough 2002). Most of the plant MTs are class I proteins containing two smaller Cys-rich domains (CRD) and a large spacer region devoid of this amino acid. In class II MTs cysteine residues are grouped into three cysteine-rich domains separated by 10 to 15 amino acid residues (Lane et al. 1987; Zhou et al. 2005). Class III consists of phytochelatins, enzymatically synthesized peptides with a poly(γ-Glu-Cys)-glycine structure (Cobbett and Goldsbrough 2002).
Class I MTs can be further divided into different types (type I–IV) and differ in the arrangement and distribution of cysteine residues. Class I MTs have the capacity to bind both physiological (such as zinc, copper and selenium) and xenobiotic heavy metals (such as cadmium, mercury, silver and arsenic) through the thiol group of its cysteine residues. The organization/distribution of cysteine residues confers on different MT isoforms the ability to bind and sequester different metal ions for detoxification and homeostasis. Further, the four types of MTs are known to express in different plant organs (Cobbett and Goldsbrough 2002). Expression of metallothionein genes is regulated by abiotic stress including metals and plays an important role in metal detoxification and homeostasis (Usha et al. 2007, 2009; Huang and Wang 2009, 2010; Singh et al. 2011). In addition, some plant MT-2 genes have been expressed in E. coli to determine its metal-binding properties (Tommey et al. 1991; Jin et al. 2006; Huang and Wang 2010). These functional capabilities of MTs allow them to play a role in mobilization of metal ions from senescing leaves and the sequestration of excess metal ions in trichomes.
In rice, class I MTs, especially OsMT1a (Os11g47809) have been characterized and it has been suggested that it plays the pivotal role in zinc homeostasis and drought tolerance (Yang et al. 2009). Yuan et al. (2008) characterized another MT from Oryza sativa (Indica variety), OsMT2b (Os01g74300), and reported that its expression was downregulated by cytokinins and it was expressed in rice immature panicles, scutellum of germinating embryos and primordium of lateral roots. Dong et al. (2010) characterized the OsMT-I-4b (Os12g38051) gene promoter which expresses in the roots and the buds with its activity highly upregulated by abscisic acid (ABA), drought, dark and heavy metals including Cu, Zn, Pb, Al, Co and Cd. In this study, 11 class I MT genes identified in Release 7 of the MSU Rice Genome Annotation Project were shown to be differentially expressed during plant growth, development and during various stresses in order to establish a model for the detoxification of heavy metals such as As (V).
Materials and methods
Database search and sequence analysis
Genes encoding MT proteins were identified by keyword, domain name and BLASTP searches available at Rice Genome Annotation Project (Release 7 of the MSU Rice Genome Annotation Project). The model Metallothio_2 (accession PF01439.11) of the Pfam database was used as a query to search the protein and nucleotide databases of NCBI (The National Center for Biotechnology Information) and the matching genes were confirmed by previous searches in Release 7 of the MSU Rice Genome Annotation Project. A self BLAST of these sequences followed by manual editing to remove redundancy finally resulted in the identification of 11 class I MTs in rice (Cobbett and Goldsbrough 2002).
Multiple sequence alignment analyses were performed using ClustalX (version 1.83) programme (Larkin et al. 2007). The intron–exon structure was identified by aligning cDNA sequences of rice MTs with their corresponding genome sequences. All the sequenced contigs of japonica cv Nipponbare have been physically constructed as pseudomolecules at TIGR (http://rice.plantbiology.msu.edu/), representing the 12 rice chromosomes. Each of the MT protein-encoding genes was positioned on these rice chromosome pseudomolecules by the BLASTN search. Genes separated by ≤5 genes were considered to be putative tandem duplicates (Jain et al. 2010; Kumar et al. 2011). Such putative duplicates were analysed using online Vista Tools for Comparative Genomics (Frazer et al. 2004) for further analysis.
Plant Material and RNA isolation
The rice variety IR-64 was germinated and allowed to grow for 5 day at 37 °C and then transferred to Hewitt solution for growth. After 10 days of growth, seedlings of uniform size and growth were treated with different concentrations of Cr (K2Cr2O7), Cd (CdCl2), AsV (Na2HAsO4) and Pb [Pb(NO3)2] (100 μM) under standard physiological conditions of 16-h light (115 μmol m−2 s−1) and 8-h dark photoperiod at 25 ± 2 °C for 24 h and 7 days. In another experiment, one tolerant cultivar Triguna and one sensitive cultivar IET 4786 for As(V) were grown similarly as discussed above and treated with 50 μM As (V). All the samples were ground in liquid N2 and stored at −80 °C till further use. Total RNA was extracted from the treated rice roots using the QIAGEN RNeasy Plant Maxi Kit (QIAGEN, MD). The yield and RNA purity were determined spectrophotometrically (NanoDrop, Wilmington, DE) and by formaldehyde–agarose gel electrophoresis. First strand cDNA was synthesised using 5 μg purified total RNA and RevertAid First Strand cDNA synthesis Kit (Fermantas, Life Sciences, USA).
Total RNA and the first cDNA strand were prepared as described above. qRT-PCR was carried out using the primer pairs listed in Supplementary Table S1 which were designed by using Primique online software http://cgi-www.daimi.au.dk/cgi-chili/primique/front.py. Specificity of each primer to its corresponding gene was checked using the BLASTN program of the NCBI. One microgram aliquots of cDNA were subjected to each qRT-PCR reaction in a final volume of 20 μl containing 12.5 μl SYBR Green Master Mix Reagent (Takara, Japan) and specific primers (Supplementary Table S1). qRT-PCR reactions were carried out in a StepOne realtime PCR machine (Applied Biosystems, USA) as described by Wu et al. (2011). Three biological replicas were performed for each sample. To normalize the total amount of cDNA present in each reaction, the actin was co-amplified as an endogenous control for calibration of relative expression. The comparative Ct method (ΔΔCT method) of relative gene quantification recommended by Applied Biosystems (CA, USA) was used to calculate the expression levels of different treatments.
Microarray data analysis
The microarray data was collected for different rice tissues/organs and developmental stages, including germinating seedling (GS), seedling root (R), mature leaf (ML), leaf (YL; leaf subtending the shoot apical meristem), shoot apical meristem (SAM) and various stages of panicle (P1–P6) and seed (S1–S5) development (Jain et al. 2007; GSE6893). GSE7951 (expression profiling of stigma), GSE6901 (expression data for stress treatment), GSE7256 (expression data for virulent infection by Magnaporthe grisea) and GSE10373 (expression data for interaction with the parasitic plant Striga hermonthica) were selected for the analysis of probe sets corresponding to rice MT gene families. The CEL files were downloaded from the Gene Expression Omnibus database at the National Center for Biotechnology Information and analyzed using dChip software (Li and Wang 2001).
All experiments were repeated three times with three replicates per treatment. Bars represent SD of means. *, ** and *** indicate values that differ significantly from the control at P < 0.05, P < 0.01 and P < 0.001, respectively, according to Duncan’s multiple range test to determine the significant difference between treatments.
In silico identification and analysis of rice metallothioneins
Class I metallothionine genes (OsMTs) in rice
Genea length (bp)
cDNA lengthb (bp)
No. of intronsc
Mol wt (kDa)e
Expression of MTs in various tissues and developmental stages
We also searched the probe IDs assigned for different MT gene families. Interestingly, we observed that out of 11 MTs, five MTs have multiple probe IDs for its single gene. There are three different probe ID present in OsMT-I-IIb (Os01g05650), OsMT-I-IIa (Os01g05585) and OsMT-I-IIc (Os01g74300). In OsMT-I-IIb and OsMT-I-IIc, two probe IDs represent CDS whereas the third one is present in the intron region (Os.35005.1.S1_at and Os.12410.2.S1_at, respectively). Heat map of these genes under various developmental stages and tissues clearly suggests that transcript containing intron is present in large amount in tissues during panicle and seed development whereas the functional transcript (without intron) content decreases (Fig. 2a). Similarly, OsMT-I-Id (Os12g38051) gene also has four different probe set IDs and one of the probe set ID is from intronic region (Os.54914.1.S1_at) showing differential expression pattern. This indicates that promoter during S5 stage is very active and transcribes pre-mRNA (with intron). Surprisingly, processing of intron in OsMT-I-IIb (Os01g05650) and OsMT-I-IIa (Os01g05585) is completely reverted in OsMT-I-IIc (Os01g74300) suggesting that mRNA processing machinery is tightly regulated suggesting functional redundancy for these MTs (Fig. 2a).
Expression of MTs during different heavy metal stress
Expression of MTs in contrasting lines of As-tolerant rice cultivars
Our previous study on large-scale screening of rice germplasm for grain arsenic level and sensitivity to arsenic (Tuli et al. 2010; Tripathi et al. 2012) suggests that two cultivars (Triguna and IET 4786) are contrasting in their arsenic sensitivity (Tripathi et al. 2012; Rai et al. 2011). To study the expression pattern of rice MT genes that are highly expressed during As (V) stress (OsMT-I-IIc, OsMT-I-IIa, OsMT-I-Ic, OsMT-I-Ie and OsMT-I-IId), these two cultivars, Triguna (tolerant) and IET 4786 (sensitive), were grown in 50 μM As (V) for 7 days and studied expression through qRT-PCR. Results of expression in roots showed the similar results as of Bala and Azucena. OsMT-I-IIa, OsMT-I-IIc and OsMT-I-IId showed higher expression pattern in tolerant cultivar Triguna as compare to sensitive cultivar IET 4786 (Fig. 5b).
Expression of MTs during cold, drought and salinity
Expression of MTs during biotic stresses
Similarly, we also studied the expression profiles of rice MT genes in roots of susceptible (IAC165) and highly resistant (Nipponbare) cultivars in response to infection with S. hermonthica after 2, 4 and 11 dpi (Swarbrick et al. 2008). Expression analysis of 11 MTs shows that OsMT-I-IIa, OsMT-I-IIb, OsMT-I-Ia and OsMT-I-Ib genes are highly upregulated in susceptible IAC165 in response to infection with rice roots and hemiparasite (Fig. 7).
MPSS-based expression analysis of MT gene family
Plants have evolved multiple strategies to tolerate and response various stresses. Some of these strategies include immobilization, exclusion, chelation and compartmentalization especially for heavy metal stress (Gasic and Korban 2007). It has been demonstrated that MTs and PCs are important molecules for metal detoxification in plants (Huang and Wang 2010; Shukla et al. 2012). Though information about these small proteins and their involvement in stress response is known in some plants, no detailed study has been carried out in rice. The objectives of this study were to determine the expression patterns of members of class I OsMT gene family during plant growth and development and under different stresses especially during heavy metal stresses with aim to select the most appropriate candidate genes which can be exploited for improving stress tolerance in future.
In this study, 11 class I OsMT genes were identified in Release 7 of the MSU Rice Genome Annotation Project which is in agreement with earlier reports for presence of multiple copies of rice MTs (Zhou et al. 2006). From our study, it is clear that the number of class I OsMT genes increased rapidly during the course of evolution and whole genome duplication and/or tandem/segmental duplication played an important role in the expansion of MT genes in rice (Kumar et al. 2011).
Differential expression of class I OsMT genes in various organs/developmental stages
The gene expression patterns can provide important clues for the gene function. We studied expression of all the class I OsMT genes in MPSS databases as well as microarray data from 51 arrays representing 17 stages of development throughout the life cycle of rice. The hierarchical cluster analysis based on average log signal values as well as MPSS signatures for 11 class I OsMT indicated that this gene family display diverse expression patterns. In our study, the preferential expression of class I OsMT was found especially in roots, leaves and developing seeds indicating its specific role in developmental stages which was not reported earlier (Zhou et al. 2006).
Role of class OsMT under different stresses
In the present study, we also established that class I MT genes were differentially regulated by different heavy metal stresses and might play a crucial role in different heavy metal metabolism. Our result revealed that some of rice MTs was regulated by a specific heavy metal only; however, most of the class I MTs were regulated by more than one heavy metal stress condition. Our study also indicates that MTs (OsMT-I-IIa, OsMT-I-IIc and OsMT-I-IId) might play very crucial role for detoxification of As (V) in tolerant rice cultivars. There were plenty of evidence that MT plays a role in heavy metal tolerance in fungi and animals (Hamer 1986). Although, it has been reported that expression of some MT is strongly induced by Cu, Zn, Pb, Al, Co and Cd in Arabidopsis and rice, the role of MTs in heavy metal detoxification in plants remains to be established. Grispen et al. (2009) overexpressed AtMT2b in Nicotiana tabacum and found that the highest AtMT2b expressing line exhibited a significantly decreased arsenic accumulation in roots with increased accumulation in shoots, while the total amount of arsenic taken up remained unchanged indicating that AtMT2b expression enhanced the arsenic root to shoot transport. In our previous study, we also reported that three (Os12g38300; Os12g38290 and Os12g38051) and two class I (Os12g38290 and Os12g38051) MTs were upregulated in As (V) and As (III) stresses, respectively (Chakrabarty et al. 2009).
To understand the role of class I OsMT genes during various abiotic stress conditions, their expression patterns were investigated in rice seedlings subjected to desiccation, salt and cold. Our results revealed that OsMT-I-IId gene showed response to salt stress only, while others were responsive to two or more stress treatments. The role of MTs is not so well documented in the protection of plants from biotic (Shim et al. 2004; Choi et al. 1996) and abiotic stresses (Yang et al. 2009). Xue et al. (2009) reported a cotton MT (GhMT3a) was upregulated not only by high salinity, drought and low temperature stresses, but also by heavy metal ions, ABA, ethylene and reactive oxygen species (ROS) in cotton seedlings. They concluded that GhMT3a could function as an effective ROS scavenger and its expression could be regulated by abiotic stresses through ROS signalling. Similarly, Yang et al. (2009) also reported that a rice class I MT, OsMT1a (Os11g47809) plays an important role in drought tolerance in rice. Microarray data from two earlier studies on transcriptome analysis of rice (Nipponbare) after infection with M. grisea and gene expression profiling in roots of susceptible (IAC165) and highly resistant (Nipponbare) cultivars after infection with S. hermonthica revealed that OsMT-I-IIa, OsMT-I-IIb, OsMT-I-IId, OsMT-I-Ia and OsMT-I-Ib genes showed response to biotic stress conditions. It has already been reported that metallothionein was induced during M. grisea infection in wild rice (Oryza minuta) (Shim et al. 2004). Choi et al. (1996) reported a wound and pathogen inducible metallothionein-like cDNA from Nicotiana glutinosa while cloning plant disease resistance–response genes by subtractive hybridization. Induced expression MTs with other stress-related genes which evoke stress responses have also been observed during banana fruit ripening (Kesari et al. 2007) suggesting importance of MTs in defence response. Our findings may lead in a way to develop counter defences against biotic and abiotic stress factors.
Regulation of expression of class I OsMT genes: post-transcriptional alternative splicing
Apart from transcriptional regulation, post-transcriptional alternative splicing in some members of this family takes place during growth and development, in various tissues and various abiotic and biotic stresses. Similar observations were also made by Kumar et al. (2011) with alternative splicing of members of sulphate transporter gene family in different tissues indicating that alternative splicing plays an important role to modulate the gene expression during different developmental stages in regulating the level of functional transcripts via a mechanism termed regulated unproductive splicing and translation (Lewis et al. 2003; Lareau et al. 2007). It has already been reported that intron retention is involved in important plant processes, such as floral development, where alternative splicing of Arabidopsis FCA pre-mRNA regulates the switch from the vegetative to the reproductive phase (Quesada et al. 2003; Razem et al. 2006; Reddy 2007). Although an earlier report indicated tissue-specific expression of rice MTs (EST database of all MTs from NCBI including japonica and indica subspecies, Zhou et al. 2006) no information was given about alternative splicing which we argue is an important step in gene regulation. Several reports demonstrate that alternative splicing can be influenced by abiotic and biotic stresses (Kumar et al. 2011). It was suggested that genes required to ameliorate various stress responses respond to changing environmental conditions by evolving rapidly, and the acquisition of alternative splice isoforms (in our case accumulation of pre-mRNA) may provide an additional mechanism to facilitate such behaviour.
The authors are thankful to Director, National Botanical Research Institute, Lucknow for the facilities and for the financial support from CSIR Network Project, New Delhi, India. NG acknowledges the financial support from ICMR. PKV acknowledge the financial support from CSIR. The authors declare that they have no conflict of interest associated with this work.