Over-expression of chickpea metallothionein 1 gene confers tolerance against major toxic heavy metal stress in Arabidopsis

Abstract

Heavy metals are ubiquitously present in nature, including soil, water, and thus in plants, thereby causing a potential health risk. This study has investigated the role and efficiency of the chickpea metallothionein 1 (MT1) gene against the major toxic heavy metals, i.e., As [As(III) and As(V)], Cr(VI), and Cd toxicity. MT1 over-expressing transgenic lines had reduced As(V) and Cr(VI) accumulation, whereas Cd accumulation was enhanced in the L3 line. The physiological responses (WUE, A, Gs, E, ETR, and qP) were noted to be enhanced in transgenic plants, whereas qN was decreased. Similarly, the antioxidant molecules and enzymatic activities (GSH/GSSG, Asc/DHA, APX, GPX, and GRX) were higher in the transgenic plants. The activity of antioxidant enzymes, i.e., SOD, APX, GPX, and POD, were highest in the Cd-treated lines, whereas higher CAT activity was observed in As(V)-L1 and GRX in Cr-L3 line. The stress markers TBARS, H₂O_2, and electrolyte leakage were lower in transgenic lines in comparison to WT, while RWC was enhanced in the transgenic lines, and the transcript of MT1 gene was accumulated in the transgenic lines. Similarly, the level of stress-responsive amino acid cysteine was higher in transgenic plants as compared to WT plants. Among all the heavy metals, MT1 over-expressing lines showed a highly increased accumulation of Cd, whereas a non-significant effect was observed with As(III) treatment. Overall, the results demonstrate that Arabidopsis thaliana transformed with the MT1 gene mitigates heavy metal stress by regulating the defense mechanisms in plants.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-021-01103-1.

Keywords: Arabidopsis thaliana; Chickpea; Heavy metals; Metallothionein; Oxidative stress; Transgenics.

Fig. 1

The total transcript level of MT1 gene.Relative expression of MT1 gene in transgenic Arabidopsis under metal stress. The treatment abbreviation is treatment-genotype/line: Arsenite to WT (As(III)-WT); arsenate to WT (As(V)-WT), chromium to WT (Cr-WT); cadmium to WT (Cd-WT); arsenite to L1, L2,L3 (As(III)-L1, As(III)-L2, As(III)-L3); arsenate to L1, L2,L3 (As(V)-L1, As(V)-L2, As(V)-L3);chromium to L1, L2,L3 (Cr-L1, Cr-L2, Cr-L3);cadmium to L1, L2,L3 (Cd-L1, Cd-L2, Cd-L3).Significance of the mean values have been compared for each parameter separately (Duncan’s test, p < 0.05) where bars marked with same letters are not significantly different

Fig. 2

Antioxidant enzyme activities in WT and transgenic Arabidopsis plants under different metal stresses. A SOD (U mg⁻¹ P), B GPX (μM mg⁻¹ P), C APX (μM mg⁻¹ P), D CAT (μM mg⁻¹ P), E POD (μM mg⁻¹ P), F GRX (μM mg⁻¹ P), Significance of the mean values have been compared for each parameter separately (Duncan’s test, p < 0.05) where bars marked with different letters are significantly different

Fig. 3

Physiological performance in transgenic lines and wild-type plants under metal stress. A Photosynthesis rate (A; µmol m⁻² s⁻¹), B Stomatal conductance (G; mol H₂O m⁻² s⁻¹), C Leaf transpiration (E; mmol H₂O m⁻² s⁻¹), D Water use efficiency (WUE; µmol CO₂ mmol H₂O⁻¹), E Photochemical quenching (qP), F Non-photochemical quenching (qN), G Electron transport rate (ETR; µmol e⁻ m⁻² s⁻¹)

Fig. 4

The total accumulation of heavy metals. Total accumulation of As(III), As(V), Cr(VI), and Cd in wild-type and transgenic Arabidopsis thaliana plants. Significance of the mean values have been compared for each parameter separately (Duncan’s test, p < 0.05) where bars marked with different letters are significantly different