Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Graphical (scaled) representation of location of putative genes for OsMTs on O. sativa chromosomes and qRT PCR analysis of clustered MT genes localized on chromosome XII. (A) The centromeres on chromosomes have been marked in circles. The position of first exon of genes (in Mb) has been marked in the parentheses along with their names at same location on chromosomes. Arrow marks the direction of the ORF specific to the OsMT genes. Enlarged view of a portion of chromosome XII (inset) shows a cluster of MT genes and their distribution revealing the striking similarity in the gene structure of the members. Intron/exon(s) are shown as thin and thick line, respectively. (B) Real-time PCR analysis of OsMT1a, OsMT1c, and OsMT1d expression in shoots of 4-day old rice seedlings under various abiotic stresses such as salinity (200 mM NaCl), drought (air drying) and ABA (100µM) for various durations i.e. 8 h or 24 h. Expression of various MTs have been shown relative to that of the MT1d which is the lowest expressed gene under control conditions. The controls were grown in half strength Yoshida medium. Data are means ± SE. Each data set represents an average of minimum three separate experiments.

Figure 2

Salinity and development regulated expression of OsMT1e-P in rice (Pokkali) grown in green house under standard agronomic practices. (A) Expression pattern of OsMT1e-P at various developmental stages of Oryza sativa based on microarray data (www.genevestigator.com). The expression data shown here is normalized, high quality and manually curated data obtained from several hundreds of independent experiments. The stage of development of plant is also shown diagrammatically in the figure. (B) Time course RNA gel blot analysis of OsMT1e-P transcripts in shoots of Pokkali rice seedlings stressed with 200 mM NaCl for 10′, 20′, 30′, 24 h, 48 h or 72 h. (C) Time course RNA gel blot analysis of OsMT1e-P transcripts in shoots of Pokkali rice tillers stressed with 200 mM NaCl for 30′ or 24 h. (D) RNA gel blot analysis of the OsMT1e-P expression profile in various tissues of mature rice plants (stressed with 200 mM NaCl for 24 h), grown in green house. The 0 h time point served as the control in all the analyses. The RNA gel blot analysis was performed twice with similar results. The EtBr stained RNA gel is shown below each RNA blot as loading control. The scanned intensity of each band is shown above the corresponding blot in the form of a histogram.

Figure 3

Confirmation of OsMT1e-P transgenic tobacco plants and assessment of their tolerance towards salinity stress. (A) Schematic representation of construct (pCAMBIA OsMT1e-P ) used to ectopically express OsMT1e-P in tobacco plants. (B) PCR analysis of the wild type (WT) and T₀ transgenic lines (L1, L2 and L7) to confirm the integration of the OsMT1e-P gene. (C) Northern blot analysis of OsMT1e-P transcript in shoots of seven days old WT and T₀ transgenic lines (L1, L2 and L7) under unstressed condition. (D) Ethidium bromide stained RNA gel used as loading control for northern blot analysis of samples used in C. (E) Northern blot analysis of OsMT1e-P transcript in shoots of seven days old WT and T₀ transgenic lines (L1, L2 and L7) under salinity (200 mM NaCl, 48 h) stress condition. (F) Ethidium bromide stained RNA gel used as loading control for northern blot analysis of samples used in E. (G) Histogram depicting signal intensity of OsMT1e-P transcript in WT and T₀ transgenic lines (L1, L2 and L7) under unstressed and salinity (200 mM NaCl) stress conditions as obtained from C and E above. (H) Germination assay of OsMT1e-P transgenic seeds (T₁) on MS media supplemented with various stressors. For this purpose, medium was supplemented with either NaCl (100 or 200 or 300 mM) or CuSO₄ (5 mM) or PEG (5%) or ZnSO₄ (5 mM or 10 mM). Seeds sown on plain MS medium served as control. (I) Histograms depicting fresh weight of WT and OsMT1e-P transgenic seedlings grown under various stress conditions. The seedlings were maintained under culture room conditions (28 ± 1⁰ 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. The data represent means ± SE of three biological replicates (n = 3). Bars with different letters are statistically significant and those with the same letters are not significantly different (p < 0.05).

Figure 4

Assessment of relative H₂O₂ levels in tobacco plants under salinity stress. (A) Constitutive and salinity stress (200 mM NaCl) induced levels of H₂O₂ in leaves of WT as well as OsMT1e-P ectopically expressing lines (T₁ - 15-d-old plants) were visualized by DAB staining. (B) Histograms depicting relative quantity of H₂O₂ in these leaves in terms of their colouration intensity. The data represents the means ± SE of three biological replicates (n = 3). Bars with different letters are statistically significant and those with same letter are not significantly different (p < 0.05).

Figure 5

Assessment of OsMT1e-P transgenic tobacco plants for their tolerance towards various abiotic stresses and heavy metal toxicity. (A) Floating leaf disc senescence assay for abiotic stress tolerance in transgenic tobacco plants (T₂). Experiments were performed on three transgenic lines (L1, L2 and L7) subjected to either 42⁰ C for heat stress, 10 mM CuSO_4, 10 mM ZnSO_4, 4⁰ C for cold stress, 5% PEG or 200 mM NaCl stress in Hoagland medium. Heat stress was given for 8 h only while other stresses were given for 5 days. Leaf discs floated in Hoagland medium served as the experimental controls. (B) Histogram depicting chlorophyll content retained in corresponding leaf discs shown in A. The data represent mean ± SE of three biological replicates (n = 3). Bars with different letters are statistically significant and those with same letter are not significantly different (p < 0.05).

Figure 6

Comparison of various yield parameters of WT and OsMT1e-P ectopically expressing tobacco plants, continuously grown under saline conditions. (A) Growth of WT and OsMT1e-P tobacco plants grown in the presence 200 mM NaCl as assessed visually (B) Various growth and yield parameters of the WT and OsMT1e-P tobacco plants grown in the presence of water or 200 mM NaCl, respectively. (C) Na⁺, K⁺ and Ca²⁺ (D) Cu²⁺ and Zn²⁺ content [conc. as ppm], in various tissues of the OsMT1e-P ectopically expressing transgenic plants grown under continued presence of 200 mM NaCl, measured using EDXRF analysis. For each analysis, roots, old leaf, young leaf, or pods were collected from three different plants of each line. Similar data for WT plants could not be obtained as these plants did not grow further in the presence of 200 mM NaCl. The data represents mean ± SE of three biological replicates (n = 3). Bars with different letters are statistically significant and those with same letter are not significantly different (p < 0.05).