Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-Pconfers multiple abiotic stress tolerance in tobacco via ROS scavenging
- Gautam Kumar†1,
- Hemant Ritturaj Kushwaha1,
- Vaishali Panjabi-Sabharwal1,
- Sumita Kumari1,
- Rohit Joshi1,
- Ratna Karan1,
- Shweta Mittal1,
- Sneh L Singla Pareek2 and
- Ashwani Pareek1Email author
© Kumar et al.; licensee BioMed Central Ltd. 2012
Received: 8 May 2012
Accepted: 25 June 2012
Published: 10 July 2012
Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented.
We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P.
We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species.
Metallothioneins (MTs) are low molecular weight, cysteine-rich metal chelators with an ability to bind heavy metal ions. MTs are able to bind a variety of metal ions by the formation of mercaptide bonds between numerous Cys residues (present in the proteins) and the metal, and thus contribute to metal detoxification by buffering cytosolic metal concentration . MTs typically contain two metal-binding, cysteine-rich domains that give these metalloproteins a dumbbell conformation. They are widely distributed in animals, plants, fungi as well as cyanobacteria. Based on sequence similarities and their phylogenetic relationships, MTs have been broadly classified into three types [2–4]. Class I MTs are widespread in vertebrates and have 20 conserved cysteine residues giving them the dumbbell conformation. Class II MTs do not have this strict arrangement of cysteine residues and are widespread in plants, fungi and invertebrates. On the other hand, phytochelatins, which are enzymatically synthesized metal binding peptides, are described as Class III MTs.
Plant MTs identified so far contain two cysteine-rich domains and a large spacer region (30–50 a.a. residues, devoid of cysteine) [5, 6]. These MTs have only a few histidines, while their Cys content varies between 10 and 17 residues. On the other hand, the number of aromatic amino acids in plant MTs varies from none to several. Based on the distribution of cysteine residues, number of aromatic amino acids as well as length of the spacer region, plant MTs are further classified into four types, type 1 through 4 [1, 6, 7]. Analysis of various EST database shows that MTs are amongst the highly abundant transcripts in plants . Recent studies have established important roles for plant MTs in fruit development, root development and suberization besides heavy metal tolerance [9–12]. Furthermore, the role of plant MTs in abiotic stresses such as oxidative, dehydration, senescence as well as hormonal alterations have also been shown [10, 13, 14]. The antioxidant function of MTs is attributed to the presence of a large number of cysteine residues, which besides metal binding are also capable of ROS scavenging . Recently, a type 1 MT from mustard i.e. LSC54 has been reported to be induced by ROS production . Further, LSC54 has also been documented to be related to ROS imbalance during leaf senescence. Similarly, in rice, several type 1 and type 2 MTs have been found to play a direct role in antioxidation .
Abiotic stresses, a major factor in reducing plant productivity, are proving to be an increasing threat to agriculture. Development of genetically modified abiotic stress tolerant varieties may provide a solution to this problem . Recently, we have characterized the molecular response of rice seedlings towards salinity stress based on their subtractive transcriptome profiling . One of the key members of this response, OsMT1e-P – a type 1 MT isolated from a salinity tolerant genotype i.e. O. sativa cv. Pokkali has been reported to be induced strongly in response to salinity stress. In the present communication, we provide detailed investigations on OsMT1e-P. In essence, we have found that OsMT1e-P is transcriptionally induced in response to salinity stress. Ectopic expression of OsMT1e-P in transgenic tobacco (under the control of 35 S constitutive promoter) provides stress tolerance against salinity, drought, cold, heat and heavy metals (Cu2+ and Zn2+). OsMT1e-P over expressing plants accumulate lower amount of ROS (H2O2) under salinity stress. Further, we also show that at least five members of type 1 MT are tightly clustered on the distal arm of chromosome XII which show co-regulation in response to various abiotic stress conditions. Based on these results, we propose that OsMT1e-P may serve as an important “candidate gene” for raising ‘multi-stress tolerant’ crops.
Results and discussion
MT gene family in rice: highly conserved gene family with 5 members clustered on chromosome XII
Members of metallothionein family in rice
Earlier proposed name
23467876 – 23468536 (+)
23467876 – 23468352 (+)
9936480 – 9937064 (+)
23350565 – 23349930 (-)
23481607 – 23482236 (+)
28269831 - 28270196 (+)
23319560 – 23318879 (-)
23479046 – 23479680 (+)
2691774 – 2690331 (-)
584010 – 584488 (+)
584010 – 584488 (+)
43374103 – 43374519 (+)
5475423 – 5476251 (+)
20838860 – 20839209 (+)
The multiple sequence alignment of the various rice MT protein sequences (based on in silico predictions) with the protein sequence of MT gene cloned in our laboratory from Indica rice genotype Pokkali, OsMT1e-P [Genbank: EU684548] has been shown in Additional file 1: Figure S1A. Based on this analysis, it could be concluded that rice MTs share a very high degree of conservation (as high as 98% within their respective types). Two of them i.e. OsMT1a1 and OsMT1a2 have an additional stretch of 17 amino acids which is not present in any other type 1 members. The rooted phylogenetic tree of MTs reported from Arabidopsis and rice (based on the amino acid sequence) showed a very close relationship between them (Additional file 1: Figure S1B). OsMT1e-P of Pokkali was observed to have 100% identity with OsMT1e member of O. sativa sp. Japonica and both of them clustered with other type I members.
Quantitative real-time PCR analysis indicated co-regulation of clustered metallothionein genes
Since OsMT1a, OsMT1c, OsMT1d, OsMT1f and OsMT1g genes were found tightly clustered on chromosome XII, we wanted to check if these genes are ‘transcriptionally co-regulated’ as well. For this purpose, qRT-PCR was carried out with cDNA prepared from shoots of rice seedlings subjected to salinity, drought and ABA stress. Figure 1B shows transcript abundance for three of these genes namely OsMT1a, OsMT1c and OsMT1d. This analysis indicated that these three “tightly placed” genes are down regulated under salinity stress as well as exogenous ABA application while up regulated after 24 h of drought stress (barring OsMT1a and OsMT1c where down regulation in transcript was seen just after 8 h of stress). However, we could not quantify the transcripts for other two genes namely OsMT1g and OsMT1f which may be due to the extremely low abundance of transcripts of these genes under the tested conditions (the low abundance of these transcripts was also confirmed through the analysis of rice MPSS database). This data clearly indicates that these clustered metallothionein genes are co-regulated under various stresses in the shoots of rice seedlings. Similar observation has been made previously where co-expression of nuclear genes, coding for different subunits of photosystem or proteins involved in the transcription/translation of plastome having overlapping functions, has been documented as a novel mechanism for co-ordinated regulation of gene expression .
Salinity and development regulated expression of OsMT1e-P
It has been established in literature that both ‘very early’ and ‘late’ responses associated with salinity stress have their own significance. The very early response is generally the ‘shock response’ while the late responses may often have genetic basis [18, 24]. Since the OsMT1e-P gene was obtained in a salinity stress specific subtractive library , RNA gel blot analysis was carried out to study the effect of salt stress on transcript abundance in rice seedlings. We have carried out this transcript abundance analysis twice but data from only one such representative set is presented in Figure 2B. Transcripts for OsMT1e P could be detected in rice seedlings even under non-stress conditions which were further induced during salinity stress (10’, 20’ or 30’). The transcript levels further increased in response to 24 h of stress treatment and continued to increase up to 48 h of stress (as high as 6 folds - as compared to non stress conditions, Figure 2B). Even after 72 h of stress, induced levels of OsMT1e-P could be detected, though lesser as compared to that in response to 48 h of stress.
The salinity stress inducibility of OsMT1e-P was also observed at the tillering stage as its transcript increases within 30’ and 24 h of salinity stress (Figure 2C). In this case, we could again see a clear time-dependent transcript accumulation pattern in response to salinity stress. We have further extended this analysis to various tissues of mature rice plants grown under standard agronomic practices. In Figure 2D, it could be seen that the various tissues of the mature rice plants maintain differential levels for OsMT1e-P transcripts which in turn are differentially regulated by salinity stress. All the tissues analyzed in this study i.e. inflorescence, upper stem, lower stem, upper leaf, lower leaf and roots showed a constitutive level of expression for OsMT1e-P, with roots and inflorescence showing the highest levels. Moreover, the constitutive levels of transcripts were higher at maturity stage than seedling and tillering stage. OsMT1e-P was further found to be induced by salinity in all the tissues except roots and inflorescence, where down regulation of the transcripts could be seen in response to salinity stress. It is interesting to note that both the apical tissue of the mature plant behave differently from all other tissue.
The expression patterns obtained in mature plant tissues are in agreement with the previous studies where type 1 MT-like transcripts have been detected primarily in roots, senescing leaves, stems, leaves and flowers [25–27]. Several rice MT1 and MT2 genes show high transcript accumulation in roots, seedlings as well as stem [26–28]. Probably, the down-regulation of OsMT1e-P expression in the early phase of stress results in an oxidative burst phase, which potentiates ROS to function as a signal in salinity stress tolerance. Roots are the most appropriate site for initiating stresses response as they are the primary site of perception and injury for several types of water-limiting stresses, including salinity and drought. Under many circumstances, it is the stress-sensitivity of the root that limits the productivity of the entire plant. A high level of OsMT1e-P induction was detected in the leaves, while relatively low induction was detected in stem (Figure 2D). Leaves are more sensitive than stem due to the presence of highly developed photosynthetic apparatus which is an active site for ROS production  and hence metallothionein induction in leaves is important for protection under abiotic stress.
OsMT1e-P transgenic plants show enhanced tolerance to multiple abiotic stresses including salinity
The response of plants to abiotic stress consists of a coordinated function of several biochemical pathways to regulate cellular ion homeostasis, enabling the plant to show tolerance and yield stability under saline environments. Reports have suggested that although abiotic stress is a multigenic trait, stress tolerant plants could be produced by transgenic approaches by the transfer of a single or multiple genes. Several genes such as barley HVA1, rice CDPK, alfalfa Alfin1, tobacco NPK1, and Brassica GlyI and rice GlyII[34–37] have been expressed in transgenic plants to enhance their stress tolerance .
Since, OsMT1e-P is also a putative metal binding protein, multiple stress tolerance ability of OsMT1e-P ectopic expressing lines (T1) were assayed at the seedling stage by transferring them to various concentrations of NaCl (salinity), PEG (dehydration), ZnSO4 and CuSO4 (heavy metal stress) and monitoring their growth for 15 days. All OsMT1e-P transgenic seedlings showed comparable growth in the absence of NaCl (Figure 3H). However, in the presence of mild salinity (100 mM NaCl), a strong difference in seedling growth was observed between WT and transgenic lines. When exposed to 200 mM NaCl, strong contrast was observed in growth pattern where the transgenic seedlings grew much better as compared to WT seedlings. WT seedlings just could not grow in the presence of higher levels of salinity (300 mM NaCl), but most of the seedlings of transgenic lines were able to grow well under similar conditions. Similarly, transgenic seedlings outperformed WT plants under 5% PEG or ZnSO4. However, in the presence of 5 mM CuSO4, the WT and transgenic lines behaved almost the same way where growth of the seedlings was almost arrested. Histograms depicting the differences in the fresh weight of transgenic vis-a-vis wild type tobacco lines show a clear advantage of OsMT1e-P ectopic expression, particularly under salinity, PEG and zinc stress (Figure 3I). Stress mitigating capacity of several plant metallothionein genes is mediated through their metal binding capacity [11, 13, 14]. When released from metallothionein proteins, Zn2+/Cu2+ ions become available for antioxidant metal binding enzymes e.g. Cu, Zn-superoxide dismutase and may also help in spatial regulation of oxidoreductive environment in the cell . MTs have been reported to initiate Zn2+ mediated antioxidant response in mammals and fungi . Improved growth and survival of OsMT1e-P transgenic plants under multiple stresses is proposed to be mediated through maintenance of redox balance and thereby reducing ROS induced injury.
Leaf disc assay has been shown to be one of the quick assays for assessing the tolerance of plants towards salinity stress . We also performed this assay with T1 plants in the presence of high salinity (200 mM and 300 mM NaCl). All the transgenic lines (L1, L2 and L7) showed considerably better chlorophyll retention under high salinity (after five days of stress), while WT lines showed excessive bleaching under similar conditions (Additional file 3: Figure S3A). To explore if the ectopic expression of OsMT1e P is contributing towards better ion homeostasis, ionic measurements were carried out in leaves of the salt-stressed WT and transgenic plants. This study revealed that there was relatively less disturbance in the ion balance in transgenic plants as compared to the WT plants under salinity stress. Under control conditions, a K+/Na+ ratio in the range of 11.5 to 12 was observed for both WT and transgenic plants while a drastic decline was observed in WT when subjected to 200 mM NaCl (4.36) and 300 mM NaCl (0.74) for 5 days (Additional file 3: Figure S3B, statistically significant differences have been marked as different alphabets on the top of the bars). In contrast, the transgenic lines maintained this ratio between 7.6-9.9 and 3.3-4.5 under 200 mM and 300 mM NaCl, respectively. Maintenance of K+/Na+ homeostasis is considered as an important aspect of salinity tolerance in plants and the higher K+/Na+ levels are very well correlated with enhanced salinity tolerance [35, 41, 42].
Reduced accumulation of H2O2in the transgenic lines shows their enhanced antioxidant activity
Ectopic expression of OsMT1e-P in tobacco provides multiple stress tolerance in subsequent generations
OsMT1e-P transgenic plants flower normally and set viable seeds under high salinity
Expression of OsMT1e-P gene is regulated by multiple abiotic stresses in a development and organ specific manner and is therefore an important candidate gene for abiotic stress tolerance. It is proposed that OsMT1e-P helps in detoxification and cellular repair while maintaining the cellular homeostasis via ROS scavenging directly or indirectly via other antioxidants. However, further studies are warranted to work out the precise mechanism to link their metal binding affinity with abiotic stress tolerance. OsMT1e-P probably interacts with other physiological processes in the cell for selective uptake and sequestration of ions during salinity stress.
Isolation of cDNA clone and sequencing
The OsMT1e-P clone (acc. no. EU684548) was isolated during subtractive hybridization of differentially expressed cDNAs from 4-day old seedlings of O. sativa L. cv Pokkali treated with 200 mM NaCl for 30 min .
Sequence analysis of rice MT genes
The MT sequence protein profile was built using MT sequences obtained from NCBI database using psi-BLAST . The sequences thus obtained were used to make profile using hmmbuild program of HMMER2 package (version 2.3.2; http://hmmer.janelia.org). This profile was further used for searching the presence of MT proteins in rice [TIGR version 6.1]. The sequences were aligned using MUSCLE  and the parsimonious tree was plotted using the alignment with Phylip package (version 3.6) with default parameters . Pairwise alignment of the OsMT genomic sequences were carried out using Needle program of EMBOSS package (5.0.0) . Mummer software package (v 3.20)  was used for analyzing the duplication of genes present on chromosome XII. The final figures for alignment were prepared using Jalview .
Expression analysis based on microarray data
Genevestigator 4 (http://www.genevestigator.com) was used to fetch the expression data for OsMT1e P using default parameters . The gene query was the LOC (Os.3445.1.S1_at). The output of the analysis was exported in the pdf format for presentation.
Plant material and stress treatments for transcript analysis of OsMT1e-P under various conditions
For the analysis of transcript abundance under various stress conditions at the seedling stage, rice seeds were rinsed with distilled water and germinated in a hydroponic system for 7-days in half strength Yoshida medium. Salinity stress treatment was given by transferring the seedlings to 200 mM NaCl solution for 10 min, 20 min, 30 min, 8 h, 24 h, 48 h, and 72 h. Further, seedlings were exposed to drought/dehydration (air drying), and ABA (100 μM) for 8 h and 24 h. The untreated samples were taken as control.
Leaves of field grown plants at the tillering stage, while in the mature plant, tissues from various plant parts viz. stem and leaves (upper and lower), inflorescence and roots subjected to 200 mM salinity stress for 30 min or 24 h in ½ Yoshida medium and untreated samples were taken as control .
Total RNA isolation, mRNA purification and cDNA synthesis
Total RNA was isolated using Raflex solution (Bangalore Genei) according to the manufacturer’s instructions. The concentration of RNA was determined using spectrophotometer and its integrity was checked on agarose gel. Enrichment of polyA+ RNA and cDNA synthesis was carried out as described earlier .
Primers for real time PCR analysis of the OsMT gene members were designed using Primer Express 3.0 software (PE Applied Biosystems, USA). The sequences for these primers are given in additional file 4: Table S2 and real time PCR was performed as described earlier . The specificity of amplification was tested by dissociation curve analysis and agarose gel electrophoresis. The expression of each gene in different RNA samples was normalized with the expression of internal control gene, actin. The mRNA levels for each candidate gene in different tissue samples were calculated relative to its expression in control seedlings using ΔΔCT method of SDS 1.4 software (Applied Biosystems). Three technical as well as biological replicates were analyzed for each sample (n = 3).
Northern blot was prepared using 20 μg total RNA and probes were prepared by labeling the PCR amplicons with α32P-dATP using HexaLabel DNA labeling kit (Fermentas Life Sciences, USA). Hybridization, washing and scanning of RNA blots were carried out as described .
Construction of plant transformation vector and generation of transgenic plants
For ectopic expression of OsMT1e-P, the complete ORF (513 bp) was PCR amplified by using primers OsMT1e P-F1 (5′-GAAGATCTTCATGTCTTGCAGCTGTGGATC-3′) and OsMT1e P-R1 (5′-GACTAGTCTTAACAGTTGCAAGGGTTGC-3′), and cloned at the BglII and SpeI sites of plant expression vector pCAMBIA1304. For tobacco transformation, the pCAMBIAOsMT1e-P construct was mobilized into Agrobacterium tumefaciens (GV3101) by liquid nitrogen freeze-thaw method. Tobacco leaf discs (Nicotiana tabaccum L. cv Xanthi) were transformed using the standard protocol  and the transformants were selected on hygromycin (25 mg/L).
Genomic DNA PCR of transgenic plants
Putative transformants were screened by PCR analysis using tobacco genomic DNA (from WT and various transgenic lines) as template and vector specific primers (forward primer 5'-CAAGACCCTTCCTCTATATAAG-3' and reverse primer 5'-CAAGAATTGGGACAACTCCAG-3'). The pCAMBIAOsMT1e-P vector was used as template for positive control.
Testing transgenic tobacco seedlings for their stress tolerance
To assess the relative stress tolerance of various plants, WT and transgenic seeds ectopically expressing OsMT1e-P were germinated on MS medium supplemented with NaCl (100, 200 or 300 mM) or 5 mM CuSO4 or ZnSO4 (5 or 10 mM) or 5% PEG for imposing different abiotic stresses or onto plain MS medium that served as the experimental control. The seedlings were maintained under culture room conditions (28 ± 1°C, 16 h light/8 h dark) and their growth was monitored for 15 days under different stresses and fresh weight of surviving seedlings was measured.
Leaf disc assay and measurement of chlorophyll contents
Leaf discs of 1 cm diameter were excised from healthy and fully expanded tobacco leaves of similar age from transgenic and WT plants were kept in half strength Hoagland media containing 200 mM NaCl or 5 mM CuSO4 or 10 mM ZnSO4 or 5% PEG. For cold stress, leaf discs were exposed to 4°C for 5 days while heat stress was given for 8 h at 42°C. Leaf discs kept in half strength Hoagland were taken as control. The chlorophyll content was measured spectrophotometrically after extraction in 80% acetone . The experiment was repeated thrice with three different transgenic lines (n = 3).
In planta histochemical estimation of H2O2
Accumulation of H2O2 was examined based on histochemical staining by 3, 3'-diaminobenzidine (DAB) as described earlier . WT and transgenic leaves of 15 days old seedlings were kept in 200 mM NaCl stress for different time intervals (30 min, 1 h, 2 h, 4 h, 6 h and 21 h). These leaves were vacuum infiltrated into 1 mg/ml fresh DAB solution (pH 3.8) prepared in 10 mM phosphate buffer (pH 7.8) and placed in a plastic box under high humidity and light until brown spots were observed (5 to 6 h). The stained leaves were then fixed with a solution of 3:1:1 ethanol: lactic acid: glycerol and photographed. The experiment was repeated thrice with three different transgenic lines (n = 3).
Testing transgenic tobacco plants for their tolerance towards salinity stress
In addition to the experiments at seedling stage, we carried out the assessment for the tolerance of transgenic plants when they are grown in the presence of salinity throughout their life cycle. For this purpose, the seedlings were transferred to earthern pots and grown in a greenhouse until maturity (16 h light/8 h dark and 25°C ± 2°C) either in absence or presence of 200 mM NaCl. Various parameters such as plant height, total fresh weight of plant, number of days to flower and pod weight were taken into consideration for assessment of salinity stress tolerance. Three plants from each transgenic line were randomly picked up for this analysis.
Three plants each of transgenic lines and WT grown under control or salinity stress conditions (as described above) were harvested and washed with distilled water three times. Plants were separated into root, lower leaf, upper leaf and pod. Equal amount of these plant tissue samples were dried at 105 °C in an oven, crushed to fine powder using a mortar-pestle grinder. Dry plant powder thus obtained was pressed by using 15-ton pressure and tablets of 100.0 mg were made. EDXRF measurements were performed on the EDXRF spectrometer (PANalytical, Netherland) with a Ge solid state detector. The source of X-ray was 100 keV Gadolinium tube which allows fluorescence efficiency for K-lines higher than for L-lines of elements. All samples were measured for a period of 2000 seconds on the sample holders made of Titanium rings. For relative quantitative analysis of element, Epsilon software was used. The experiment was repeated thrice with three different transgenic lines (n = 3).
Data analysis was performed in Microsoft excel and analysis of variance (ANOVA) and presented as mean ± standard error (SE) of three biological replicates. Statistical analysis was performed using One-way-Analysis-of Variance (ANOVA). This was followed by Tukey’s post-hoc multiple comparison test using SPSS (version 19.0) for data statistics at different time points. Different letters were used (p < 0.05) to present statistically significant differences and similar letters were considered as statistically non significant.
Reactive oxygen species
Polymerase chain reaction
Work carried out in this paper has been supported by funds available from UGC Resource Networking, Capacity building funds from JNU, Department of Science and Technology, Department of Biotechnology, Ministry of Science and Technology, Government of India. Authors acknowledge AIRF, JNU for EDXRF measurements. Award of research fellowship from UGC (GK and RK) and CSIR (HRK and SK) is also gratefully acknowledged.
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