SO₂-Induced Stomatal Movement And Guard Cell Death

Sulfur dioxide (SO2) is a common air pollutant. SO2exposure influences stomatal movement, blocks the normal stomatal open, and affects plant physiological activity. SO2exposure could cause visible foliar damage such as chlorosis and necrosis, lead to cell death. But how SO2induces 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 SO2derivatives (a mixture of sodium sulfite and sodium bisulfite,3:1mmol·L-1) on stomatal movement and guard cell death was investigated. The expermential results indicated that SO2induced stomatal closure at low concentrations, in which NO, reactive oxygen species (ROS), Ca2+, cGMP and MAPK played important roles. Moreover, SO2induced guard cell death at high concentrations, in which NO, ROS, Ca2+, cGMP, MAPK and caspase-like proteases taken part in the death procedure.Stomatal aperture decreased after epidermal strips exposed to7.5to150μmol·L-1SO2derivatives.200U·mL-1of catalase (CAT),0.2mmol·L-1of the NO scavenger (c-PTIO),0.1mmol·L-1of nitrate reductase (NR) inhibitor NaN3,25μmol·L-1nitric oxide synthase (NOS) inhibitor L-NAME,1mmol·L-1of the Ca2+chelator EGTA,0.1mmol·L-1of the MAPK kinase inhibitor PD98059and2μmol·L-1synthesis inhibition of cGMP agent ODQ can relieve stomatal aperture decrease caused by7.5μmol·L-1SO2. nial and nia.2co-regulation synthesis NR in Arabidopsis, test with a single mutation (nia2deletion) and double mutant(nial and nia2deletion both) in the study,25umol·L-1of L-NAME,2μmol·L-1ODQ and0.1mmol·L-1of Ca2+channel inhibitor LaCl3, can relieve stomatal aperture decrease caused by7.5μmol·L-1SO2. Of which,0.1mmol·L-1of NaN3can relieve stomatal aperture decrease caused by7.5umol·L-1SO2, but the double mutant guard cells cann’t; Using the fluorescent indicator DCFH-DA and NO fluorescent indicator DAF-FM DA detect intracellular ROS and NO levels With7.5μmol·L-1SO2in treatment time, found that guard cells SO2can cause elevated levels of ROS and NO, and peaked at15min, that is similar with Time course of SO2-induced stomatal movement.SO2caused cell viability loss associated with typical programmed cell death (PCD)-related changes of cell morphology in guard cells in detached epidermis of V. faba leaves at concentrations of2to10mmol·L-1(3hours exposure). The inhibitory effects of caspase inhibitor Z-Asp-CH2-DCB (0.5μmol·L-1) and cysteine proteinase inhibitor TLCK (0.1μmol·L-1) suggest the activation of a PCD pathway in SO2-stimulated guard cells. NO antagonists (0.2mmol·L-1NO scavenger c-PTIO,25μmol·L-1NO synthase inhibitor L-NAME and0.1mmol·L-1nitrate reductase inhibitor NaN3), antioxidant (ascorbic acid, AsA,1mmol·L-1) and Ca2+channel inhibitor LaCl3(0.1mmol·L-1) all blocked SO2-induced cell death. NaN3also prevented SO2-induced PCD features and elevations of intracellular ROS and Ca2+levels. However, LaCl3and AsA can not suppress SO2-induced intracellular NO elevation. All results suggest that NO production in plant response to SO2stress triggers a PCD process through [Ca2+]cyt elevation. A specific inhibitor of guanylate cyclase, ODQ, blocked SO2-induced cell death, while a novel inhibitor of mitogen-activated protein kinase, PD98059, cannot inhibit SO2-induced cell death, suggesting that NO generation mediated SO2-induced cell death via a cGMP-dependent and MAPK-independent pathway.