Ozone-induced Vicia Faba Stomatal Movement And Signal Regulation Mechanisms

There had been a lot of researches both on the signal regulation and ozone induced movement in stomata, while the mechanisms of signal regulation and ozone-induced movement are still remains unclear. The regularity of stomatal movement response to different concentration of ozone (O3) was examined using isolated epidermal strip bioassay and 03-fumigation in open-top chamber (OTC) with model plant- broad bean (Vicia faba) in this research.Based on the principles bout reactive oxygen species (ROS), and signal molecule of stomatal movement-regulation underlying mechanisms and combined with the prevailing methodologies currently applied in the research fields of Plant Physiology, Biochemistry and Molecular Biology, at the same time the laser-scanning confocal microscopy was used. The origin of ROS signal molecule, which mediated 03-induced stomatal movement and underlying signal conduction mechanism were examined. The results varied as following:1. The manner of stomatal movement altered by ozoneIn 03-inhibited stomatal opening experiments, abaxial epidermal strips were immediately peeled from stomata-closed V.faba (5-week-old) which were kept in the dark overnight (12 h) and then incubated in different concentrations of O3 with or without catalase (CAT), diphenylene iodonium (DPI) or ascorbic acid (Vc) under opening conditions, and stomatal apertures were determined in different time after the incubation. For promotion of stomatal closing experiments, freshly prepared abaxial epidermal strips were first incubated in Mes-KCL for 3 h under conditions promoting stomatal opening, and then the open stomata induced by Mes-KCL were transferred to Mes-KCL containing different concentrations of O3 with or without CAT, DPI or ascorbic acid for another 3 h. Stomatal apertures were determined as above.As epidermal strips were treated as above description, the manner of stomatal movement which treated only with O3 was significantly different from that of control. O3 treatment inhibited stomatal opening and promoted stomatal closure significantly. Little effect was observed at concentrations of O3< 150 nL L-1, but at concentrations = 150 nL L-1, O3 significantly (P< 0.05) inhibited stomatal opening. Interestingly, the concentrations of O3 that significantly (P< 0.05) promoted stomatal closure were higher than that of O3 significantly inhibited stomatal opening, and should to be= 450 nL L-1. We also found that O3 promoted stomatal closure in a dose-dependent manner. The maximum promotion of stomatal closure was observed at 2.5 h after treatment with 850 nL L-1 O3. The stomatal apertures were 7.7 [(width/length)x100%], only 13% of the control value. Nevertheless, under these conditions, the effects of O3 on stomatal aperture were completely reversible. On the contrary, as we elevated the concentration of O3= 1500 nL L-1, the reversibility of stomatal aperture was abolished and most of guard cells lost viability after 3 h O3 treatment.In addition to epidermal strip bioassay, the whole intact plants were exposed to different concentrations of gaseous O3 epidermal strips for 1 h in OTC, from which epidermal strips were immediately peeled. For these isolated epidermal strips treated with CAT, ascorbic acid or DPI as above, we obtained the same results.When epidermal strips were treated with different concentrations of O3 and with CAT, Vc or DPI, CAT and Vc inhibited 03-promoted stomatal close effectively. DPI reduced effect of 03-promoted stomatal close somewhat. At the same time, CAT, Vc and DPI reduced 03-inhibited stomatal opening significantly.The results from above show that:O3 was able to not only promote stomatal close, but also inhibit stomatal opening, i.e. O3 had significant effect on stomatal movement. The effect of O3 on stomatal movement was reduced significantly during the O3 treatment with ROS scavenger or ROS-producing inhibitor demonstrated that ROS (mainly existing in the form of long-life span H2O2 in living cell) was involved in O3-induced stomatal movement as a signal molecule.2. ROS burst could be induced by O3 in guard cellsNonfluorescent compound H2DCF oxidated by H2O2 and catalyzed by peroxidases can yield the highly fluorescent DCF. The quantity of ROS in cells is well represented by DCF fluorescence intensity. Epidermal strips loaded with H2DCF were transferred to different concentrations of O3 for treatment for 2.5 minutes, and then observed with laser-scanning confocal microscopy. For epidermal strips loaded with H2DCF, the O3 treatment enhanced relative fluorescence intensity of DCF only in guard cells. The DCF fluorescence intensity in O3-treated guard cells showed a dose-dependent increase, i.e. DCF fluorescence intensity enhanced as O3 concentration increased. But DCF fluorescence in the area of epidermal pavement cells and intercellular space was as low as control. Nevertheless, when the epidermal tissues were treated with O3 at the concentration=1500 nL L-1, the DCF fluorescence intensity in guard cells decreased sharply. But the DCF fluorescence intensity in epidermal pavement cells and intercellular space was enhanced intensively. This phenomenon showed that high concentration of O3 exposure resulting in the death of guard cells so as to cell membrane collapse, hence the DCF fluorescence spread all over the epidermal strips.The results from above indicated that the accumulation of ROS (H2O2) in guard cells resulted from O3 elicitation rather than from O3 breakdown, otherwise the DCF fluorescence spread all over the epidermal strip. Furthermore, ROS appeared only in guard cells when challenged with moderate concentration of O3. This result showed that the components producing ROS in guard cells was absent in epidermal pavement cells.3. Chloroplasts were key players in producing ROS in guard cells when epidermal strips challenged with O3 When the epidermal tissues were treated with O3+CAT and O3+Vc respectively, the fluorescence intensity of guard cells strongly decreased in contrast to control. As the epidermal tissues were treated with O3+DPI, the fluorescence intensity of guard cells was decreased a little. It showed that NADP-oxidase was a component but not the only one involved in ROS producing. Enhanced ROS concentrated at guard cells not at epidermal pavement cells when challenged with O3. At the same time, there is a lack of chloroplasts in epidermal pavement cells in contrast to guard cells. We speculated that chloroplast was the key player in ROS producing. So diuron (DCMU), an inhibitor which can block the electron transporting from Q_A to Q_B in the thylakoid membrane was used to examine if the chloroplast was involved in ROS producing. DCMU pretreatment almost completely inhibited 03-induced ROS burst. There was no significant difference in fluorescence intensity between control and DCMU+ O3 treated guard cells.All of these indicated that chloroplasts play a pivotal role in 03-induced ROS production. Our data also showed that ROS-producing in other components was obviously affected by chloroplast. Once the ROS-producing in chloroplast was inhibited, little ROS produced from other sources.To sum up, the stomatal movement behavior altered greatly under the stress of O3. O3 could not only promote stomatal close but also inhibit stomatal opening. Moreover, the threshold concentration that O3 significantly inhibits stomatal opening is lower than that of that O3 significantly promotes stomatal closure; under the stress of O3, the accumulation of H2O2 in guard cells resulted from O3 elicitation rather than from O3 breakdown; ROS was an important signal molecule in mediating O3-induced stomatal movement behavior changes; chloroplast was the key player in O3-elicited ROS burst and ROS-producing in other components was obviously affected by chloroplast..