Roles And Relationships With H₂O₂/NO Of Cytokinin/auxin, MEK/CDPK And CO In The Stomatal Movement Of Vicia Faba

Guard cells control transpiration in plants and regulate gas exchange in leaves by opening and closing stomatal pores.Stomatal movements are regulated by both internal and external factors. Previous studies have shown that cytokinins and auxins can affect stomatal behaviour,and MAPKs/CDPKs are involved in the stomatal movement.However,little was still known about these exact mechanisms.From being molecules of somewhat novelty interest,in the last few years,H₂O₂ and NO have emerged to be central players in the world of plant guard cells signalling,articularly under various stressful situations.Our previous studies provided evidence that light/dark regulate stomatal movement via influencing hydrogen peroxide(H₂O₂) and nitric oxide(NO) production in guard cells of Vicia faba,and H₂O₂ and NO cross talk in the process.Up to now,there is little report to insight into the relation between mitogen-activated protein kinase kinase(MEK)/ calcium-dependent protein kinase(CDPK) and H₂O₂/NO,no report between cytokinins,auxins, carbon monoxide(CO) and H₂O₂/NO during light/darkness-regulated stomatal movement.Here,we select Vicia faba as plant material,through pharmacological and surgical approaches combined with laser scanning confocal microscope(LSCM) method to seek for these above problems.The main results are as follows.1.Cytokinins at concentrations of≥0.05μM significantly induced stomatal opening in darkness, as did auxins at concentrations of≥0.1μM.The optimum concentrations of cytokinins and auxins were 0.2 and 10μM,respectively.Both cytokinins and auxins reduced the levels of H₂O₂ in guard cells and induced stomatal opening in darkness.Additionally,cytokinins not only reduced exogenous H₂O₂ levels in guard cells caused by exposure to light,but also abolished H₂O₂ that had been generated during a dark period,and promoted stomatal opening,as did ascorbic acid(ASA,an important reducing substrate for H₂O₂ removal).However,unlike cytokinins,auxins did not reduce exogenous H₂O₂,did not abolish H₂O₂ that had been generated in the dark,and therefore did not promote reopening of stoma induced to close in the dark.The above-mentioned effects of auxins were similar to that of diphenylene iodonium(DPI,an inhibitor of the H₂O₂-generating enzyme NADPH oxidase).Taken together our results indicate that cytokinins probably reduce the levels of H₂O₂ in guard cells by scavenging,whereas auxins limit H₂O₂ levels through restraining H₂O₂ generation,inducing stomatal opening in darkness.2.Both cytokinins and auxins reduced the levels of NO in guard cells and induced stomatal opening in darkness.Additionally,cytokinins not only reduced NO levels in guard cells caused by sodium nitroprusside(SNP) in light but also abolished NO that had been generated by dark,and then promoted the closed stomata reopening,as did NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO).However,unlike cytokinins,auxins not only had incapability to reduce NO levels by SNP but also could not abolish NO having been generated by dark,so auxins could not promote the closed stomata to reopen.The above-mentioned effects of auxins were similar to that of nitric oxide synthase(NOS,enzyme commission 1.14.13.39) inhibitor N^G-nitro-L-Arg-methyl ester(L-NAME).Hence,it is concluded that cytokinins reduced probably the levels of NO in guard cells via scavenging,and auxins reduced NO levels through restraining NO generation in all probability,and then induced stomatal opening in darkness.3.Both 2`-amino-3`-methoxyflavone(PD98059)(an inhibitor of mitogen-activated protein kinase kinase,MEK) and Trifluoperazine(TFP)(a specific inhibitor of calcium-dependent protein kinase,CDPK) reduced the levels of H₂O₂ in guard cells and promoted stomatal opening significantly in dark,implying that MEK/CDPK mediate dark-induced stomatal closure by influencing H₂O₂ levels of guard cells.In addition,like ASA,but unlike DPI,PD98059 and TFP not only reduced exogenous H₂O₂ levels in guard cells in light,but also abolished H₂O₂ that had been generated during a dark period,and promoted stomatal opening,the results suggest MEK and CDPK are probably relational to restraining the H₂O₂ scavenging enzyme to elevate H₂O₂ levels in guard cells during dark-induced stomatal closure,of course,the probability of MEK and CDPK acting as the target downstream of H₂O₂ in the signaling transduction chain is not excluded.4.Both PD98059 and TFP reduced the levels of NO in guard cells and promoted stomatal opening significantly in dark,implying that MEK/CDPK mediate dark-induced stomatal closure by affecting NO levels in guard cells.In addition,like NO scavenger cPTIO,but unlikeL-NAME, PD98059 and TFP not only reduced 4,5-diaminofluorescein diacetate(DAF-2 DA) fluorescence in guard cells by sodium nitroprusside(SNP) in light,but also abolished NO that had been generated during a dark period,and promoted stomatal opening,suggesting MEK and CDPK are probably relational to restraining the NO scavenging to elevate NO levels in guard cells during dark-induced stomatal closure.5.Recently,carbon monoxide(CO) was implicated as another important physiological messenger or bioactive molecule in animals.Previous researches indicate that heine oxygenase-1 (HO-1,EC 1.14.99.3) catalyzes the oxidative conversion of heine to CO and biliverdinâ…¨a with the concomitant release of iron.However,little is known about the physiological roles of CO in plant.In the portion,the regulatory role of CO during stomatal movement in Vicia faba was surveyed.Results indicated that,like hydrogen peroxide(H₂O₂),CO donor Hematin induced stomatal closure in dose-and time-dependent manners.These responses were also proven by the addition of gaseous CO aqueous solution with different concentrations,showing the first times that CO and H₂O₂ exhibit the similar regulation role in the stomatal movement.Moreover,our data showed that ascorbic acid (ASA,an important reducing substrate for H₂O₂ removal) and diphenylene iodonium(DPI,an inhibitor of the H₂O₂-generating enzyme NADPH oxidase) not only reversed stomatal closure by CO, but also suppressed the H₂O₂ fluorescence induced by CO,implying that CO induced-stomatal closure probably involves H₂O₂ signal.Additionally,the CO/NO scavenger hemoglobin(Hb) and CO specific synthetic inhibitor ZnPPIX,ASA and DPI reversed the darkness-induced stomatal closure and H₂O₂ fluorescence.These results show that,maybe like H₂O₂,the levels of CO in guard cells of Vicia faba is higher in dark than that in light,HO-1 and NADPH oxidase are the enzyme systems responsible for generating endogenous CO and H₂O₂ in darkness respectively,and that CO being from HO-1 mediated darkness-induced H₂O₂ synthesis in guard cells stomatal closure of Vicia faba.6.Like SNP,CO donor Hematin induced stomatal closure in dose- and time-dependent manners.These responses were also proven by the addition of gaseous CO aqueous solution with different concentrations,showing the first times that CO and NO exhibite the same regulation role in the stomatal movement.Moreover,our data showed that cPTIO/L-NAME not only reversed stomatal closure by CO,but also suppressed the NO fluorescence induced by CO,implying that CO induced-stomatal closure probably involves NO/NOS signal system.Additionally,the CO/NO scavenger hemoglobin(Hb) and CO specific synthetic inhibitor ZnPPIX,NO scavenger cPTIO and nitric oxide synthase(NOS) inhibitor L-NAME reversed the darkness-induced stomatal closure and NO fluorescence.These results show that,maybe like NO,the levels of CO in guard cells of Vicia faba is higher in dark than that in light,HO-1 and NOS are the enzyme systems responsible for generating endogenous CO and NO in darkness respectively,and that CO being from HO-1 mediated darkness-induced NO synthesis in guard cells stomatal closure of Vicia faba.All together,the rough outlines of light/darkness,cytokinins/auxins,MAPKs/CDPKs,CO and H₂O₂/NO signalling of guard cell were drawn in the conclusions and future perspectives section (P.152-Figureâ…§-1).