Arsenic is a highly toxic metalloid for all forms of life including plants. Arsenic enters in plants through phosphate transporters as a phosphate analogue or through aquaglycoporins. Uptake of arsenic in plant tissues can affect plant metabolism, causing various physiological disorders, structural abnormalities and even plant death. Oxidative stress is considered to be a key mechanism of arsenic toxicity.
In this study, the effect of sodium arsenite (NaAsO₂) on guard cell viability was investigated in detached epidermis of V. faba leaves. Epidermal strips were obtained from 4-week-old plants by peeling off the lower epidermis of V. faba leaves and incubated in 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing some chemicals (NaAsO₂ with or without some antagonists) for 3 h in white light at 23 ℃ as the treatments. After treatment, the epidermal strips were stained with fluorescein diacetate (FDA) to show cell viability, or with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluo-3 acetomethoxyester (Fluo-3AM) respectively to indicate intracellular reactive oxygen species (ROS) and calcium ion (Ca²⁺) levels.
The results of our experiments showed that NaAsO₂ treatment significantly decreased cell viability and induced cell death in the concentration range of 0.3 to 10 mg/L. Arsenic provokes synchronous increases in cell death rate and intracellular levels of ROS and Ca²⁺ in V. faba guard cells. The typical nuclear morphological changes including nuclear fragmentation and nuclear condensation were observed in As-treated guard cells, while Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH₂-DCB), a specific inhibitor of mammalian caspases, significantly blocked As-induced cell death. The occurrence of characteristic features of programmed cell death (PCD) and the inhibitory effect of caspase inhibitor Z-Asp-CH₂-DCB on As-induced cell death suggest the activation of a PCD pathway evoked by arsenic exposure. Application of antioxidant catalase significantly inhibited As-induced cell death, PCD-to-total-cells ratio and intracellular Ca²⁺ increase provoked by arsenic. A specific Ca²⁺ chelator ethylene glycol tetraacetic acid also significantly decreased the cell death caused by arsenic. These results clearly demonstrated that arsenic induced cell death associated with obvious increases in intracellular ROS and Ca²⁺ levels, in which ROS positively regulated intracellular Ca²⁺ level.ROS generation and intracellular Ca²⁺ level increase in As-treated stomatal guard cells were involved in the process of As-induced cell death. The results of the present study indicate that arsenite induced guard cell death via a PCD pathway through ROS mediating Ca²⁺ elevation.