Study Of Hydrogen Peroxide And Calcium Signaling In ABA-induced Stomatal Movement In Vicia Faba

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signaling cascade in guard cells that results in stomatal closure. A network of ABA-induced stomatal closing has been characterized, but the signal transduction events leading to alertion to the stomatal aperture remain incompletely understood. Previous evidence showed that an increase in [Ca2+]cyt has been shown to be an early event in the ABA signal transduction pathway, and Ca2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and ABA. Furthermore, ABA may activate multiple Ca2+ flux in stomatal guard cells, triggering vacuolar K+ (Rb+) release. Besides these known components, hydrogen peroxide is a ubiquitous second messenger which has been researched in plants. This paper showed ABA-induced H2O2 generation in guard cells of vicia faba , and activation of Ca2+ channels by H2O2.Changes in H2O2 generation in guard cells of Vicia faba induced by ABA were measured by using fluorescence probe, 8-hydroxypyrene-l,3,6-trisulfonic acid (HPTS). Examination of epidermis peel was performed using a laser scanning confocal microscope (LSCM) and spectrofluorometer, set to an excitation light of 405 nm and an emission light of 512 ran. The fluorescence image before stimulation showed dye accumulation in guard cells, distributed homogeneously, with the exception of the chloroplast and nucleus where the fluorescence intensity was stronger, and there was almost no fluorescence in the epidermal cells. We also showed that the fluorescence was not significantly fading throughout the experiment. The retention time was long enough for analyses when guard cells were treated with ABA, fluorescence quenching of HPTS occurred. A does-dependent study of the ABA-induced oxidative burst was observed, in which both the magnitude and rate of dye quenching was directly related to the concentration of ABA. And the treatment of the guard cells with catalase of100 units/mL nearly completely obliterated the quenching reaction induced by 10|amol/LABA(final concentration). Furthermore, when 10 \imo\IL H2O2 (final concentration) was added to the suspension after above treatment caused little reduction in HPTS fluorescence. The results obtained by laser scanning confocal microscopy suggested that addition of exogenous ABA resulted in a rapid decrease in fluorescence in most cellular compartments of the guard cells. Above data support the hypothesis that H2O2 is involved in the signal transduction pathway of ABA-induced stomatal closure.The NADPH oxidase is likely involved in ABA-induced stomatal guard cell movement in viciafaba. When guard cells were treated with ABA, fluorescene quenching of HPTS occurred, and the treatment of the guard cells with diphenyleneiodonium (DPI) at lO^mol/mL (final concentration) which is known as an inhibitor of superoxide-generating NADPH oxidases nearly reversed the quenching reaction induced by ABA 0.2mmol/L (final concentration). ABA and H2O2 might induce stomatal closure. Furthermore, when DPI or CAT (103unit/mL) was added to the suspension after above treatment reversed ABA-induced stomatal closure. These results suggest that a novel O2~ generating NADPH oxidase in the plasma membrane of guard cell of vicia faba may be activated by ABA, and subsequently H2O2, which involved in the signal transduction pathway of ABA-induced stomatal closure.Changes in Ca2+ influx across the plasma membrane in guard cells of Vicia faba by ABA-induced hydrogen peroxide generation were performed by patch clamp. We used Ba2+ ions which are permeant to Ca2+ channels with lOmmol/L and 40mmol/L in the pipette and bath solutions (see methods). In whole-cell patch clamp recordings, The inward currents were inhibited by 1 mmol/L La3*, and A23187 which is vector of Ca2T increased inward currents. Soour recordings were inward Ca currents. On adding 1 mmol/L H2O2 and ABA, The inward Ca current was increased, and a does-dependent study of the...