See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/232229883 Genome-wide identification of rice class i metallothionein gene: Tissue expression patterns and induction in respons.... Article in Functional & Integrative Genomics · October 2012 DOI: 10.1007/s10142-012-0297-9 · Source: PubMed CITATIONS READS 27 149 7 authors, including: Neelam Gautam Pankaj Verma National Botanical Research Institute - India Council of Scientific and Industrial Research … 5 PUBLICATIONS 47 CITATIONS 16 PUBLICATIONS 79 CITATIONS SEE PROFILE SEE PROFILE Shikha Verma Prabodh Trivedi National Botanical Research Institute - India Council of Scientific and Industrial Research … 12 PUBLICATIONS 60 CITATIONS 223 PUBLICATIONS 3,867 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: arsenic detoxification via biovolatilization View project Molecular characterization of genes involved in biosynthesis of specific withanolides from Withania somnifera View project All content following this page was uploaded by Debasis Chakrabarty on 22 July 2015. The user has requested enhancement of the downloaded file. Funct Integr Genomics (2012) 12:635–647 DOI 10.1007/s10142-012-0297-9 ORIGINAL PAPER Genome-wide identification of rice class I metallothionein gene: tissue expression patterns and induction in response to heavy metal stress Neelam Gautam & Pankaj Kumar Verma & Shikha Verma & Rudra Deo Tripathi & Prabodh Kumar Trivedi & Bijan Adhikari & Debasis Chakrabarty Received: 23 April 2012 / Revised: 3 September 2012 / Accepted: 24 September 2012 / Published online: 10 October 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract 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. Electronic supplementary material The online version of this article (doi:10.1007/s10142-012-0297-9) contains supplementary material, which is available to authorized users. N. Gautam : P. K. Verma : S. Verma : R. D. Tripathi : P. K. Trivedi : D. Chakrabarty (*) CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001 UP, India e-mail: debasis1972@rediffmail.com B. Adhikari Rice Research Station, Department of Agriculture, Chinsurah, Hooghly, Government of West Bengal 712 102, India Keywords Alternative splicing . Arsenic . Heavy metal . Rice . Stress . Metallothionein Introduction 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 metalloenzymes 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; 636 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(γGlu-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 Funct Integr Genomics (2012) 12:635–647 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 proteinencoding 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 μmolm−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 Funct Integr Genomics (2012) 12:635–647 637 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). 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. qRT-PCR analysis In silico identification and analysis of rice metallothioneins 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. Database search and sequence analysis resulted in the identification of 11 class I MTs in rice. The multiple sequence alignments of full-length MT protein sequences showed that N-terminal and C-terminal of these MTs are highly conserved having two CRD connected by a spacer characteristic to all plant MTs (Fig. 1a). Out of all the MTs, one OsMT-IIIa (Os01g05585) is a duplicated form of OsMT-I-IIb (Os01g05650), which does not contain one N-terminal CRD. Rice genome may have lost this domain during evolution. Another MT located on chromosome 12, OsMT-I-If (Os12g38300) also lacks C-terminal CRD. The localization of MTs on the chromosomes depicted the clustering of five genes on the 12 chromosome (Fig. 1b). It has been reported that 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) which resulted in identifying two genes that are tandem duplicates, OsMT-I-Ic (Os12g38010) and OsMT0I-Id (Os12g38051) (Supplementary Fig. S1). Different MTs identified with their gene name, gene length, open reading frame length, protein length, chromosomal location and other related information are provided in Table 1. 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). Statistical analysis 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 Results Expression of MTs in various tissues and developmental stages We studied the expression of different rice MTs using microarray data available at the GEO database under accession numbers GSE6893 and GSE7951, respectively (Jain et al. 2007; Li et al. 2007). All the tissues/organs and developmental stages for which microarray data were analysed in this study are shown in Fig. 2a. It is clear from our analysis that MTs play differential role during different developmental stages. Out of 11 MTs, four are root specific (OsMT-I-If: Os12g38300, OsMT-I-Ie:Os12g38290, OsMT-I-Id: Os12g38051 and OsMT-I-Ic:Os12g38010). The validation of differential gene expression of these selected MT genes in root/shoot tissues by real-time PCR analysis showed very good agreement with the microarray results (Fig. 2b). OsMT-I-Ic gene showed highest expression in root tissue followed by OsMT-I-If and OsMT-I-If. OsMT-I-Ic gene is also root specific but its expression level is lower than the other root-specific MTs (Fig. 3b). Seven MTs expressed 638 Funct Integr Genomics (2012) 12:635–647 a CRD CRD b Fig. 1 a, b Alignment of protein sequences of OsMT class I gene family members by ClustalX software. Conserved cysteine-rich domains (CRD) are shown in black line (a). Graphical representation of the location of different MT class 1 genes (TIGR locus IDs of rice MTs) on rice chromosomes (b). The chromosome number is indicated at the bottom of each chromosome during seed development (OsMT-1-IIa, OsMT-1-IIb, OsMT1-IIc, OsMT-1-Ia, OsMT-1-IIIa, OsMT-1-Ib and OsMT-1-If), among them four MTs also expressed in leaves (OsMT-1IIa, OsMT-1-IIb, OsMT-1-IIIa and OsMT-1-Ib). 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-IIIa (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 To study responsiveness of MT gene family to different heavy metal treatments in rice, real-time PCR was performed with total RNA isolated from the roots and shoots of IR-64 rice treated with Pb, As(V), Cr and Cd (Figs. 3 and Funct Integr Genomics (2012) 12:635–647 639 Table 1 Class I metallothionine genes (OsMTs) in rice Sl no. Locus ID MT type cDNA No. of Protein Mol wt pIf Genea length (bp) lengthb (bp) intronsc lengthd (kDa)e Chromosome NCBI Acch. no.g 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. OsMT-I-IIa OsMT-IIb OsMT-I-IIc OsMT-I-Ia OsMT-I-IIIa OsMT-I-Ib OsMT-I-Ic OsMT-I-Id OsMT-I-Ie OsMT-I-IId OsMT-I-If 1,047 1,952 1,007 585 2,217 838 999 8,444 981 1,026 652 1 1 1 3 5 11 12 12 12 12 12 195 249 243 219 198 225 237 240 231 237 240 2 2 3 2 3 2 3 3 3 2 3 64 82 81 73 66 75 79 80 77 79 80 6.307 7.85 7.6 7.13 6.85 7.48 7.59 7.71 7.54 7.64 7.72 4.79 4.5 4.29 4.85 4.68 6.88 5.68 6.42 4.53 5.62 8.61 NP_001042028 NP_001042028/D15602 NP_001045552/AB002820 NP_001049782/AK059587 A3B0Y1/AF001396 NP_001068544/U43529 NP_001067063 Q2QNE8/BE039194 ABA99660 ABA99658/AK103445 ABG22056/BE039221 Length of genomic fragment TIGR release 7 d Length (no. of amino acids) of the deduced polypeptide e Molecular weight of the deduced polypeptide in kilodalton f Isoelectric point of the deduced polypeptide g Chromosome number on which the gene is present h Locus ID from Release 7 of the MSU Rice Genome Annotation Project was BLASTN in NCBI S5 P6 P3 P4 P5 P1 P2 Ovary Stigma R Fig. 2 a, b Expression profiles of OsMT class I gene family in different tissues and developmental stages using Affymetrix Rice Genome Array. The profiles in germinating seedlings, seedling roots (R), mature leaves (ML), young leaves (YL; leaf subtending the shoot apical meristem), shoot apical meristem (SAM), stigma, ovary and various stages of panicle (P1–P6) and seed (S1–S5) development are presented by cluster display. The colour scale (representing log signal values) is shown at the bottom (a). Quantitative real-time PCR analysis to study the rootspecific expression pattern of MT gene family members (b) significantly increased expression in roots (OsMT-I-IIa after 24 h and OsMT-I-IIIa after 7 days) but others showed YL ML SAM 4). In Pb treatment, only two genes (OsMT-I-IIa: Os01g05585 and OsMT-I-IIIa:Os05g11320) showed S4 No. of introns within cDNA S3 Length of cDNA c S2 b S1 a Os01g05585 Os01g05650 Os01g74300 Os03g17870 Os05g11320 Os11g47809 Os12g38010 Os12g38051 Os12g38290 Os12g38270 Os12g38300 a Os.35005.1.S1_x_at Os.35005.1.S1_at Os.46398.1.S1_s_at OsMT-1-IIa Os.46398.1.S1_s_at Os.5523.1.S1_x_at Os.35005.1.S1_at OsMT-1-IIb Os.12410.1.S1_x_at Os.12410.2.S1_at Os.12410.3.S1_x_at OsMT-1-IIc OsAffx.7826.2.S1 at OsAffx.7826.1.S1_at Os.54914.1.S1_at Os.37783.2.S1_x_at Os.37783.1.S1 a at OsAffx.32080.1.S1_at Os.17004.1.S1_at OsMT-1-Ia OsMT-1-IIIa OsMT-1-Ib OsMT-1-IId OsMT-1-If b 900 Relative transcript level (Genes/Actin) 800 SHOOT ROOT 700 600 500 400 300 200 100 0 OsMT-I-Ie OsMT-I-Id OsMT-I-If OsMT-I-Ic OsMT-1-Ic OsMT-1-Id OsMT-1-Ie Root 15 10 5 Bc 0 300 250 200 150 100 50 0 140 120 100 80 60 40 20 0 c B A B B a a b c A a b b B B b 2.5 2 1.5 a 300 200 b c 100 c 0 Control Pb As Cr Cd B 1 b b 0.5 B b B Cc 0 30 15 b A A b 10 0 c Cd B C A 12 10 B 8 6 4 2 A 6 5 B 4 B 3 2 C 1 c 0 C C a a a a a 0 A A a 8 b 6 b 4 2 B Cc 0 D d 25 A 20 a 15 b 10 5 B c C 0 Control a b b Cc 10 20 5 7 12 a 25 1000 400 A Relative transcript level (OsMT-I-IIIa/Actin) b a Relative transcript level (OsMT-I-IIb/Actin) Shoot 20 3 Relative transcript level (OsMT-I-IId/Actin) a a Relative transcript level (OsMT-I-Ib/Actin) 25 Relative transcript level Relative transcript level (OsMT-I-IIa/Actin) (OsMT-I-Ia/Actin) Funct Integr Genomics (2012) 12:635–647 Relative transcript level (OsMT-I-Ic/Actin) Relative transcript level (OsMT-I-If/Actin) Relative transcript level (OsMT-I-Ie/Actin) Relative transcript level (OsMT-I-Id/Actin) Relative transcript level (OsMT-I-IIc/Actin) 640 Pb B d d As Cr Cd 800 600 400 200 b 0 control Pb As Cr Cd Fig. 3 Quantitative real-time PCR analysis to study the expression pattern of MT gene family members exposed to various heavy metal stresses after 24 h. 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 μmolm−2 s−1) and 8-h dark photoperiod at 25±2 °C for 24 h and 7 days. The grey bars represent root and black bars represent shoot. Y-axis represents relative mRNA level in stressed or treated samples as compared to control samples and various treatments are given on X-axis, namely lead (Pb), arsenate (AsV), chromium (Cr) and cadmium (Cd). Actin expression was used as internal control each time. The error bars indicate standard error of mean values for three biological replicates. Three technical replicates have been employed for each biological replicate decreased expression revealing that Pb responsive MT genes have different induction kinetics. The transcript levels of 11 genes continuously increased throughout the time course for other heavy metal [As(V), Cr and Cd] treatments. Two MTs, OsMT-I-Ie and OsMT-I-IIa (Os12g38290 and Os01g05585, respectively) were highly upregulated in roots a f t e r 24 h A s ( V ) t r e a t m e n t w h e r e a s O s M T-I- Ib (Os11g47809), OsMT-I-IId (Os12g38270), OsMT-I-IIb (Os01g05650) and OsMT-I-IIIa (Os05g11320) showed higher accumulation in shoots. Interestingly OsMT-I-Ia (Os12g38051) showed no change in expression after 24 h treatment, however was significantly upregulated after 7 day of As(V) treatment. After 7 days, ten out of 11 MTs were upregulated especially in roots except OsMT-I-Ib (Os11g47809) which was upregulated both in shoots and roots after As(V)-treatment. In case of Cr, OsMT-I-Id, OsMT-I-If and OsMT-I-Ic (Os12g38051, Os12g38300 and Os12g38010, respectively) were downregulated after 24 h whereas all the MTs were upregulated after 7 days of treatment. However, after 7 days of Cr-treatment OsMT-I-Ic (Os12g38010) showed sharp increase in their transcript level indicating that this MT is highly responsive to Cr and thus is Cr specific. Interestingly, OsMT-I-Ib, OsMT-IIIb, OsMT-I-IIIa and OsMT-I-IId (Os11g47809, Os01g5650, Os05g11320 and Os12g38270, respectively) showed higher level of expression in shoots whereas OsMT-I-Ia and OsMTI-IIa (Os03g17870 and Os01g05585, respectively) showed higher expression level both in shoots and roots of Crtreated rice seedlings. This suggests that Cr strongly regulates the expression pattern of MTs as compared to other heavy metals. In the Cd treatment, out of 11 MTs, seven MTs, OsMT-I-IIc, OsMT-I-Ia, OsMT-I-IIIa, OsMT-I-IIa, OsMT-I-IIb, OsMT-I-If and OsMT-I-IId (Os01g74300, Os03g17870, Os05g11320, Os01g05585, Os01g05650, 40 20 0 b A Cc B C a 800 a 600 400 b 200 c A B 0 1400 1200 1000 800 600 400 200 0 a b c A A d 2500 a 2000 1500 b 1000 500 c 0 Control d Pb A B As Cr c Cd Relative transcript level (OsMT-I-IIIa/Actin) b a 40 30 20 10 0 b A B Cc Bc 250 Relative transcript level (OsMT-I-IIb/Actin) 60 50 a 200 150 100 b b 50 0 c c A A A 20 A 15 B a 10 a C 5 b D b D c 0 Relative transcript level (OsMT-I-IId/Actin) Root Relative transcript level Relative transcript level (OsMT-I-IIa/Actin) (OsMT-I-Ia/Actin) 80 a Shoot Relative transcript level (OsMT-I-Ib/Actin) 120 100 641 Relative transcript level (OsMT-I-Ic/Actin) Relative transcript level (OsMT-I-If/Actin) Relative transcript level (OsMT-I-Ie/Actin) Relative transcript level (OsMT-I-Id/Actin) Relative transcript level (OsMT-I-IIc/Actin) Funct Integr Genomics (2012) 12:635–647 50 a 40 30 b b 20 A 10 0 Bd B A A c 250 a 200 150 100 50 0 400 350 300 250 200 150 100 50 0 b c A Bc d a b b B c Control Pb A C As Cr Cd 500 a 400 300 200 100 b 0 control Pb As Cr Cd Fig. 4 Quantitative real-time PCR analysis to study the expression pattern of MT gene family members exposed to various heavy metal stresses after 7 days. 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 μmolm−2 s−1) and 8-h dark photoperiod at 25±2 °C for 24 h and 7 days. The grey bars represent root and black bars represent shoot. Y-axis represents relative mRNA level in stressed or treated samples as compared to control samples and various treatments are given on X-axis, namely lead (Pb), arsenate (AsV), chromium (Cr) and cadmium (Cd). Actin expression was used as internal control each time. The error bars indicate standard error of mean values for three biological replicates. Three technical replicates have been employed for each biological replicate Os12g38300 and Os12g38270, respectively) were highly upregulated in roots. Only OsMT-I-Ie (Os12g38290) showed shoot-specific expression pattern and OsMT-I-IIIa (Os05g11320) was highly expressed both in shoot and root after 24 h Cd treatment. After 7 days of Cd treatment, similar expression pattern was noticed except for OsMT-IIIb (Os01g05650) and OsMT-Id (Os12g38051). cultivar as compared to Azucena indicating their role in AsV tolerance (Fig. 5a). In all the cases studied above, it is clear that alternative splicing plays an important role in regulating some of the MTs expression during different stresses. 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 Expression of MTs in contrasting lines of As-tolerant rice cultivars Expression analysis from rice varieties Azucena (arsenate sensitive) and Bala (arsenate tolerant) grown in the presence or absence of 13.3 μm sodium arsenate for 7 days was studied by Norton et al. (2008). We also analyzed the expression pattern of the 11 MTs in the same database and observed that OsMT-I-IIc, OsMT-I-Ic, OsMT-I-Id, OsMT-I-Ie and OsMT-I-IId genes are highly upregulated in tolerant 642 Funct Integr Genomics (2012) 12:635–647 Relative transcript level (OsMT-I-IIa/Actin) Bala (AsV) Os.46398.1.S1_s_at Os.5523.1.S1_x_at Os.35005.1.S1 at OsMT-1-IIb Os.12410.1.S1_x_at Os.12410.2.S1_at Os.12410.3.S1 x at OsMT-1-IIc OsAffx.7826.2.S1 at OsMT-1-Ic OsAffx.7826.1.S1_at Os.54914.1.S1_at Os.37783.2.S1_x_at Os.37783.1.S1_a_at OsAffx.32080.1.S1_at Os.17004.1.S1 at 2.5 * 2 1.5 * 2 1.5 1 1 0.5 0.5 0 2.5 0 OsMT-1-Id ** 3 2.5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 ** ** Relative transcript level (OsMT-I-Id/Actin) OsMT-1-IIa ** 3 * * 2 1.5 1 0.5 IET4786 C IET4786 AS(V) 0 TRIGUNA C TRIGUNA AS(V) Relative transcript level (OsMT-I-IId/Actin) Os.35005.1.S1_x_at Os.35005.1.S1_at Os.46398.1.S1 s at Relative transcript level (OsMT-I-IIc/Actin) 3.5 3.5 Relative transcript level (OsMT-I-Ie/Actin) Azucena (AsV) b Bala (C) Azucena (C) a OsMT-1-Ie OsMT-1-Ia OsMT-1-IIIa OsMT-1-Ib OsMT-1-IId OsMT-1-If 9 7 * 6 5 4 3 2 1 0 IET4786 C Fig. 5 a, b Expression profile of OsMT class I gene family in rice varieties Azucena (arsenate-sensitive) and Bala (arsenate-tolerant) grown in the presence or absence of 13.3 μm sodium arsenate for 7 days. The colour scale (representing log signal values) is shown at cultivar Triguna as compare to sensitive cultivar IET 4786 (Fig. 5b). Expression of MTs during cold, drought and salinity The role of MTs is well documented in the protection of plants from abiotic stresses (Yang et al. 2009). Considering the role of MTs in abiotic stresses, microarray data available under series accession no. GSE6901 (Jain et al. 2007) for abiotic stresses (desiccation, salt and cold) were analysed. This analysis showed that most of the MTs are differentially expressed in different abiotic stresses. OsMT-I-IIa and OsMT-I-IIb are highly upregulated in both desiccation and salt stress. However, except these two MTs others are highly downregulated in desiccation stress. Similarly, OsMT-I-IId is highly upregulated in salt stress. It is also interesting to observe that alternative splicing plays an important role for regulating the expression pattern of OsMT-I-Id as processed transcript is not accumulating in drought stress (higher accumulation of pre-mRNA in drought stress) whereas the final processed transcript is accumulating in higher amount in cold stress (Fig. 6). ** 8 IET4786 AS(V) TRIUNA C TRIGUNA AS(V) the bottom (a). Quantitative real-time PCR analysis to study the rootspecific expression pattern of selected MT gene family members in Triguna and IET 4786 rice varieties treated with 50 μM of arsenate (AsV) for 7 days (b) C CS DS SS Os.35005.1.S1_x_at Os.35005.1.S1_at Os.46398.1.S1_s_at OsMT-1-IIa Os.46398.1.S1_s_at Os.5523.1.S1_x_at Os.35005.1.S1_at OsMT-1-IIb Os.12410.1.S1_x_at Os.12410.2.S1_at Os.12410.3.S1_x_at OsMT-1-IIc OsAffx.7826.2.S1_at OsMT-1-Ic OsAffx.7826.1.S1_at Os.54914.1.S1_at OsMT-1-Id Os.37783.2.S1_x_at Os.37783.1.S1_a_at OsAffx.32080.1.S1_at OsMT-1-Ie Os.17004.1.S1_at OsMT-1-Ia OsMT-1-IIIa OsMT-1-Ib OsMT-1-IId OsMT-1-If Expression of MTs during biotic stresses Ascomycete fungus M. grisea causes serious and widespread diseases of rice blast. Recently, a transcriptome Fig. 6 Expression profile analysis of OsMT class I gene family during cold (CS), drought (DS), and salinity (SS) stresses. The colour scale (representing log signal values) is shown at the bottom Funct Integr Genomics (2012) 12:635–647 643 analysis of a fully susceptible infection of rice (cultivar Nipponbare) by a compatible M. grisea isolate (FR13) was performed to understand the molecular mechanism involved in their interaction (Ribot et al. 2008). We analysed these data to establish role of the rice MT gene family in this host– pathogen interaction (Fig. 7). The data included microarray analysis of 2-week-old rice seedlings (cultivar Nipponbare) treated with M. grisea (virulent isolate FR13) spore suspension on gelatine or gelatine alone after 3 days [3 days postinoculation (dpi), without disease symptoms) and 4 days (4 dpi, with disease symptoms] post inoculation. Our analysis suggests that OsMT-I-IId, OsMT-I-IIa and OsMT-I-IIb genes are significantly upregulated after 4 dpi indicating MTs role in biotic stress tolerance (Fig. 7). 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, OsMTI-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 We also used the data from rice MPSS database (http:// mpss.udel.edu/in9311/) to quantify the expression of individual members of the MT gene family. MPSS technology provides a quantitative measure of transcript accumulation of virtually all the genes in a tissue sample in terms of the Discussion 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 Nipponbare IAC165 Mg-Mock-3dpi Mg-3dpi Mg-Mock-4dpi Mg-4dpi Sh-Mock-2dpi Sh-2dpi Sh-Mock-4dpi Sh-4dpi Sh-Mock-11dpi Sh-11dpi Sh-Mock-2dpi Sh-2dpi Sh-Mock-4dpi Sh-4dpi Sh-Mock-11dpi Sh-11dpi Fig. 7 Differential expression of OsMT class I gene family in response to various biotic stress conditions. The colour scale for fold change values is shown at the bottom. Numbers on each panel represent days postinoculation (dpi) number of small signature sequences corresponding to each gene (Brenner et al. 2000). A survey of 46 rice MPSS libraries representing different tissue samples as well as biotic and abiotic stress conditions showed that all the MT genes have corresponding 17-base signatures in these, suggesting that expression of different members of MTs vary in different tissues and stresses (Fig. 8). OsMT-I-Ic, OsMT-I-Id, OsMT-I-Ie and OsMT-I-If are root specific and they expressed during different stressed conditions. This result supports our microarray and q-RT-PCR data. Our MPSS analysis also suggests OsMT-I-IIc is specifically expressed in panicle and seed development which is well correlated with the microarray analysis. However, OsMT-I-IIc is also highly expressed in meristematic tissue. Otherwise, our analysis of the transcript abundance of the MT gene family from rice in different tissues and stress-specific libraries revealed that they are differentially expressed which are well correlated with our microarray and qRT-PCR data. Os.35005.1.S1_x_at Os.35005.1.S1_at Os.46398.1.S1 s at OsMT-1-IIa Os.46398.1.S1_s_at Os.5523.1.S1_x_at Os.35005.1.S1 at OsMT-1-IIb Os.12410.1.S1_x_at Os.12410.2.S1_at Os.12410.3.S1 x at OsMT-1-IIc OsAffx.7826.2.S1_at OsMT-1-Ic OsAffx.7826.1.S1_at Os.54914.1.S1_at Os.37783.2.S1_x_at Os.37783.1.S1_a_at OsMT-1-Id OsAffx.32080.1.S1_at Os.17004.1.S1 at OsMT-1-Ie OsMT-1-Ia OsMT-1-IIIa OsMT-1-Ib OsMT-1-IId OsMT-1-If 644 Funct Integr Genomics (2012) 12:635–647 14000 90000 NYR NPO NSL XC06 XR48 MR03 MS06 MC24 12000 12000 NRA NOS NDR XC24 XS03 MR06 MS12 PLA NST NIP NDL XR03 XS06 MR12 MS24 PLW NYL NGS NCR XR06 XS12 MR24 MS48 PLC NLA NCA NCL XR12 XS24 MR48 MS96 NME NSR XC00 XR24 XS48 MS03 MC00 10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 00 OsMT-I-IIb OsMT-I-IIc OsMT-I-Ia OsMT-I-IIIa OsMT-I-Ib OsMT-I-Ic OsMT-I-Id OsMT-I-Ie OsMT-I-IId OsMT-I-If Fig. 8 Transcript abundance of OsMT gene family members in plant growth and development and salinity, drought and cold libraries from the MPSS database. Different libraries in the MPSS database were analysed for the expression level of OsMT genes. NYR 14 days— young roots, NRA 60 days—mature roots A, NST 60 days—stem, NYL 14 days—young leaves, NYL 60 days—mature leaves A, NLA 60 days—mature leaves A, NME 60 days—crown vegetative meristemetic tissue, NPO mature pollen, NOS ovary and mature stigma, NIP 90 days—immature panicle, NGS 3 days—germinating seed, NCA 35 days—callus, NSR 14 days—young leaves stressed in 250 mM NaCl for 24 h, NSL 14 days—young roots stressed in 250 mM NaCl for 24 h, NDR 14 days—young roots stressed in drought for 5 days, NDL 14 days—young leaves stressed in drought for 5 days, NCR 14 days—young roots stressed in 4C cold for 24 h, NCL 14 days— young leaves stressed in 4C cold for 24 h, XC00 unwounded control— Nipponbare Xa21-0h, XC06 mock treatment—6 h, XC24 mock treatment—24 h, XR03 X. oryzae-R—3 h, XR06 X. oryzae-R—6 h, XR12 X. oryzae-R—12 h, XR24 X. oryzae-R—24 h, XR48 X. oryzae-R—48 h, XS03 X. oryzae-S—3 h, XS06 X. oryzae-S—6 h, XS12 X. oryzae-S— 12 h, XS24 X. oryzae-S—24 h, XS48 X. oryzae-S—48 h, MR03 M.grisea-R—3 h, MR06 M. grisea-R—6 h, MR12 M. grisea-R— 12 h, MR24 M. grisea-R—24 h, MR48 M. grisea-R—48 h, MS03 M. grisea-S—3 h, MS06 M. grisea-S—6 h, MS12 M. grisea-S—12 h, MS24 M. grisea-S—24 h, MS48 M. grisea-S—48 h, MS96 M. grisea-S —96 h, MC00 mock treatment—0 h, MC24 mock treatment—24 h, PLA rice leaf, beet armyworm damaged, 24 h, PLW rice leaf, water weevil damaged, 24 h, PLC rice leaf, mechanical damaged, 24 h 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 Funct Integr Genomics (2012) 12:635–647 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 645 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. Acknowledgements 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. 646 References Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59:95–104 Bratić AM, Majić DB, Samardžć JT, Maksimović VR (2009) Functional analysis of the buckwheat metallothionein promoter: tissue specificity pattern and up-regulation under complex stress stimuli. J Plant Physiol 166:996–1000 Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. 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