SO₂-Induced Stomatal Movement And Guard Cell Death In Vicia Faba Leaves

Sulfur dioxide (SO2) is a common air pollutant. When entered into cells, SO2 became SO32- and HSO3-, which had harmful effects to tissue, cells and biomacromolecules. SO2 exposure influences stomatal movement, blocks the normal stomatal open, and affects plant physiological activity. SO2 exposure could cause visible foliar damage such as chlorosis and necrosis, inhibit cell division, induce micronucleus, and lead to cell death. But how SO2 induces stomatal movement and guard cell death, and which kinds of signals involved in the procedure have not been reported.Stomatal guard cells, with sensitive and accurate responses to environmental changes, are model experimental system to study signal transduction. In this study, the effect of SO2 derivatives (a mixture of sodium sulfite and sodium bisulfite,3:1 mmol·L-1) on stomatal movement and guard cell death was investigated in Vicia faba leaves. The expermential results indicated that SO2 induced stomatal closure at low concentrations, in which reactive oxygen species (ROS) and Ca2+ played important roles. Moreover, SO2 induced guard cell death at high concentrations, in which ROS generation, Ca2+ level increase and caspase-like proteases taken part in the death procedure and played important roles.Stomatal aperture decreased after epidermal strips exposed to 7.5 to 150μmol·L-1 SO2 derivatives for 3 h. Treated with 0.1,1.0 and 10μmol·L-1 H2O2 or 10,100 and 200μmol·L-1 CaCl2 respectively, stomatal aperture also decreased. The stomatal aperture increased after epidermal strips exposed to SO2 derivatives in the presence of 1 mmol·L-1 AsA or LaCl3 (a Ca2+ channel inhibitor). LaCl3 can relieve stomatal aperture decrease caused by H2O2.SO2 exposure caused guard cell viability decrease and induced significant cell death in Vicia faba guard cells exposed to 1 to 4 mmol·L-1 SO2 derivatives for 3 h. Detected with ROS fluorescence indicator DCFH-DA and Ca2+ fluorescence indicator Fluo-3AM, the intracellular ROS and Ca2+ levels in guard cells showed significant increase in SO2 groups. However, ROS fluorescence intensity is not obvious difference when the strips exposed to 3 mmol·L-1 SO2 derivatives combined with 0.1 mmol·L-1 LaCl3·Ca2+ fluorescence intensity is obviously weaker when the strips esposed to the mixture of 3 mmol·L-1 SO2 derivatives and 0.1 mmol·L-1 AsA. The intracellular ROS level and cell death rate decreased after epidermal strips exposed to SO2 derivatives combined with 0.1 and 1 mmol·L-1 AsA or with 200U·mL-1 CAT. EGTA, a Ca2+ chelator, decreased SO2-induced cell death at concentrations of 0.1 and 1 mmol·L-1. LaCl3 (a Ca2+ channel inhibitor) decreased SO2-induced cell death and intracellular Ca2+ level, as well as H2O2 caused cell death at 0.1 mmol·L-1.Stained with PI and Schiff's reagent, the dead cells showed typical features of apoptosis such as nuclear condensation, fragmentation and apoptotic bodies. Caspase inhibitors Z-Asp-CH2-DCB (0.1 and 0.5μmol·L-1) and TLCK (0.1μmol·L-1) also effectively inhibited SO2-induced cell death.The results of the present study indicated that SO2 induced stomatal closure in Vicia faba leaves at low concentrations, and induced guard cell death at high concentrations. SO2 exposure induced intracellular ROS generation. Increased ROS level in guard cells activated Ca2+ channels in plasma membrane, lead to Ca2+ influx and intracellular Ca2+ level increase, and mediated a series of signal responses to regulate stomatal aperture and cell death. Our results also suggest that SO2 exposure can induce guard cell apoptosis, and caspase-like proteases in leaf guard cells may involve in the procedure.