Brassinosteroid And Hydrogen Peroxide Interdependently Promote Guard Cell Starch Degradation And Stomatal Opening

Stomata are microscopic pores in the plant epidermis bounded by a pair of guard cells.The stomatal movement,which is essential for plants to exchange gas and water during photosynthesis and transpiration,is attributed to the swell and shrink of guard cells due to an increase or decrease in their osmotic pressure.In classical plant physiology,there are two hypotheses about the mechanism of stomatal movement:the ion pump hypothesis and the starch-sugar hypothesis.With the in-depth study of molecular biology,more and more experimental data support the ion pump hypothesis,while the research of starch-sugar is shortageStarch is the main carbohydrate storage in plants,which can be synthesized and degraded correspondingly in plant fitness under challenging environmental conditions The starch in guard cells plays an essential role in regulating the stomatal movement The abnormal or failed degradation of guard cell starch can prevent the stomatal opening.The time and extent of starch degradation are precisely regulated by environmental factors,different hormones and developmental signals.However,the mechanism remains unclear.Brassinosteroids(BRs)are a group of plant steroid hormones that play fundamental roles in a series of plant growth and development processes.In this study,we investigated the molelular mechanism that BR induces stomatal opening by promoting guard cell starch degradation using the methods of molecular biology,biochemistry,cell biology,and genetics.The results and conclusions are as follows:BR and BZR1 involve in guard cell starch degradation and stomatal openingTo investigate the molecular mechanism of BR regulating stomatal movement,we systematically analyzed the phenotypes in BR-deficient mutants,BR-insensitive mutants and BR signal enhanced mutants.The results showed that BR deficient mutants det2,rot3 and BR insensitive mutants bril-116,bzrl and besl led to a decrease in stomatal opening,while BR BR signal enhanced materials D WF4-Ox,BRI1-Ox and the dominant BR gain-of-function mutant bzrl-1D showed a larger stomatal opening phenotype.These results suggested that BR promotes stomatal opening.The starch degradation in guard cells plays an essential role in regulating the stomatal movement.To explore whether BR-mediated stomatal opening is assiocated with the starch degradation in guard cells.The experiments of guard cells starch mentabolism and stomatal movement were carried out.The results displayed that in wild-type plants,starch started to be synthesized one hour after light exposure,and its synthesis continued until midnight,when the content of starch peaked.In the BR-insensitive mutant bril-116 and the BR-deficient mutant det2,the starch content of guard cells was significantly higher than that in wild-type plants,and the degradation rate was slower after light exposure.The high levels of starch in guard cells and the starch degradation rate of bril-116 and det2 were partially suppressed by the dominant gain-of-function mutant bzrl-1D.The starch granules in the whole leaves of bril-116 and det2 at the end of day(EoD)were less accumulated than that in wild-type plants and this decreased-starch phenotypes were not rescued by bzrl-1D.In addition,the bril-116 mutant opened stomata slowly with the extension of white light exposure time,but the stomatal aperture was significantly smaller than that of the wild type.The small stomata phenotypes of bril-116 and det2 were partially restored by bzrl-1D.These results suggested that BR involved in guard cells starch degradation and stomatal opening.Furthermore,we showed that two independent sets of BZR1 and BES1 loss-of-function mutants displayed decreased starch degradation ratios in guard cells,reduced stomatal opening phenotypes and slightly reduced BR sensitivity.These results indicated that BR promotes starch breakdown in guard cells through a BZR1-dependent pathway,while BR induces starch metabolism in other cells through a BZR1-independent pathway.H2O2 plays an important role in guard cell starch degradation and stomatal openingNow,some studies thought that H2O2,as a major signaling molecule,is required for the action of BR in the regulation of cell elongation,root stem cell maintenance,as well as plant responses to biotic and abiotic stresses.To examine whether H2O2 is involved in BR-mediated guard cell starch degradation and stomatal opening.The experiment was performed that BR treated the wild-type rosette leaves,co-treatment with diphenyleneiodonium(DPI)and potassium iodide(KI),which reduced the accumulation of H2O2 in plants,strongly counteracted BR effects to promote stomatal opening.Consistent with these results,the H2O2-deficient plant materials including rbohdrbohf double mutants,and catalase gene overexpression plants(CAT2-Ox)failed to effectively open their stomata in response to BR,and the guard cell starch degradation had no response to BR.The results suggested that H2O2 is required for BR-promoted guard cell starch degradation.In addition,BR was also required for H2O2-mediated guard cell starch degradation.H2O2 was initially considered to be a toxic byproduct of oxidative plant aerobic metabolism,which could regulate stomatal closure.In this study,H2O2 has a double effect on stomatal movement,promoting stomatal opening at low concentrations,but inhibiting stomatal opening at high concentrations.The stomatal aperture of bril-116,bril-301 and det2 were insensitive to exogenous H2O2.H2O2 had weak effects on bzrl-crl and besl-crl mutants.Exogenous application of BR and H2O2 both promoted starch degradation in guard cells of wild type.Therefore,BR and H2O2 depended on each other to induce stomatal opening by promoting guard cell starch degradation.BZR1 and GBF2 synergistically promote guard cell starch degradation and stomatal openingBased on the fact that BZR1 can integrate BR and H2O2 to promote cell elongation,we speculated that these two signals may control starch degradation and promote stomatal opening by regulating the interaction between BZRI and other transcription factors.To investigate this possibility,we performed a yeast two-hybrid(Y2H)screen to identify the transcription factors that interact with BZR1,and then analyzed the functions of these putative BZRI-interacting transcription factors.In this study,we selected a bZIP transcription factors for further in-depth study.The phenotype of guard cell starch degradation and stomatal aperture were tested The stomatal aperture in gbf2-1 exhibited a dramatic reduction.By contrast,GBF2-Ox displayed significantly larger stomatal apertures than wild-type plants.The BR and H2O2 treatments significantly induced stomatal opening in wild type Col-0 plants,but had weak effects on gbf2-1.Besides,gbf2-1 showed a slow guard cell starch degradation rate,and GBF2-Ox displayed normal guard cell starch degradation under light exposure.However,the mutation of GBF2 in both wild-type plants and in the bzr1-ID background impaired the rapid degradation of guard cell starch in response to light.Therefore,GBF2 plays an important role in BZR1-promoted starch degradation and stomata movement.BZR1 and GBF2 synergistically promote guard cell starch degradation and stomata opening.To decipher the molecular mechanism through which BR and H2O2 regulate starch levels in guard cells,the transcriptional level was analyzed and the result showed BZR1 and GBF2 up-regulated BAM1 mRNA expression.We then performed chromatin immunoprecipitation-quantitative PCR(ChIP-qPCR)experiments and showed BAM1 was the direct target of BZR1 and GBF2.Together,these results suggested that BZR1 and GBF2 directly induce BAM1 expression to promote starch breakdown in guard cells and stomatal opening.BR and H2O2 promote stomatal opening through a BAM1-dependent pathway?-amylasel(BAM1)and a-amylase3(AMY3)are preferentially and highly expressed in guard cells and are responsible for the majority of starch degradation.After BAM1 was knocked out,BR and H2O2 had no effect on the stomatal aperture of bam1-1 and baml-1 amy3 mutants.A mutation in BAM1 also prevented bzr1-1D and GBF2-Ox from inducing stomatal opening.Furthermore,exogenous application of BL and H2O2 at a certain concentration up-regulated the expression of BAM1 mRNA,and promoted the accumulation of ProBAM1:GFP fluorescent signals.In summary,BR and H2O2 have a dual role in stomatal movement.Low concentrations can promote stomatal opening,and high concentrations can induce stomatal closure.In wild type plants,binding of BR to the receptor kinase BRI1 triggers the accumulation of H2O2 that enhances the interaction between BZR1 and GBF2,which directly promotes the expression of BAM1 at the transcription level,increasing content or activity P-amylase,which causes guard cell starch degradation and stomatal opening.When the mutants with no or weak BR signals,the reduced H2O2 content and decreased expression level of BAM1 lead to the accumulation of starch in guard cells and impairing the starch degradation rate,subsequently reducing stomatal opening.In this study,we revealed a new mechanism of BR and H2O2 regulated stomatal movement,which provides a scientific basis for BR and H2O2 to balance plant growth and drought resistance,and supplies crop scientists with a theoretical basis for improving crop yields.