COP1 And HY5 Mediate UV-B-induced Stomatal Closure By Promoting Ethylene Synthesis And Transmitting Ethylene Signaling

UVR8(UV Resistance Locus 8)-dependent UV-B-specific signaling pathway including UVR8,COP1(Constitutively Photomorphogenic 1)and HY5(Elongated Hypocotyl 5)components can be activated at low doses of UV-B radiation in plants.The initial perception of UV-B by receptor UVR8 is followed by interaction with COP1 and HY5,which regulate the expression of many defense genes and enhance the UV-B tolerance for plants.UVR8 signaling pathway also mediates many physiological processes regulated by UV-B radiation.For example,UVR8 mediates UV-B-repressed hypocotyl elongation,UV-B-improved plant disease resistance and UV-B-regulated circadian clock.At present,many studies well documented the mechanism of UVR8 signaling pathway and its regulated-physiological process,but it is less known about the role and interrelationship of UVR8 signaling pathway and other signaling pathways.The phytohormone ethylene plays pivotal roles in plant response to UV-B radiation.UV-B can enhance the expression of ethylene synthase genes and induce ethylene production in tomato leaves;Ethylene as a signaling molecule mediates UV-B-induced the expression of defense genes in Arabidopsis leaves.However,in ethylene-mediated UV-B responses,whether UVR8 signaling pathway is involved and whether or not the UVR8 pathway participates in the UV-B-regulated ethylene biosynthesis are still unclear.Additionally,previous studies have shown that visible light-regulated some photomorphogenesis responses are dependent on the interaction between ethylene signaling elements and the light signal transduction elements COP1 and HY5,but it is still unclear whether UV-B-regulated some responses also depend on the interaction between the ethylene signaling elements and the UVR8 signaling components.In order to explore the above issues,we used Arabidopsis thaliana as the plant material in this paper and studied the role and relationship of UVR8 signaling pathway and ethylene signal pathway in UV-B-induced stomatal closure.By using the methods and techniques of genetics,molecular biology,cell biology and biochemistry,the main results and conclusions are as follows:1.0.5 W·m-2 UV-B induced stomatal closure and H2O2,NO generation in guard cells of wild type Arabidopsis,but didn't do that in uvr8,cop1,hy5 mutants respectively.Exogenous H2O2 could induce stomatal closure in the wild type and uvr8 mutant,but could not induce closure in cop1,hy5 mutants in either the absence or presence UV-B treatment.While,exogenous NO donor SNP could rescue the defect of UV-B-induced stomatal closure in the mutants tested.Taken together,we conclude that the UVR8-specific pathway UVR8-COP1-HY5 mediates UV-B-induced stomatal closure by controlling H2O2 and NO generation,and COP1 and HY5 also function without UVR8 to transduce H2O2 signal and work upstream of NO in guard cells.2.When leaves were exposed to 0.5 W m-2 UV-B radiation for 0-3 h,the expression of ACS2,ACS6 and ACS11 genes are significantly induced in the wild leaves and guard cells,but the expression of ACS4,ACS5,ACS7 and ACS8 were not significantly changed.The maximal effect of UV-B-enhanced expression of ACS2,ACS6 and ACS11 were achieved at 2 h.However,in either leaves or guard cells,these effects of UV-B were highly repressed in uvr8,copl and hy5 mutants,indicating that the UVR8-COP1-HY5 signaling module positively mediates UV-B-induced the expression of ACS2,ACS6 and ACS11 in Arabidopsis leaves as well as in guard cells.3.0.5 W·m-2 UV-B radiation induced a rapid and striking increase in ethylene production in wild-type leaves.However,the UV-B-induced ethylene production was significantly decreased in the uvr8,cop1 and hy5 mutants,the acs2 acs6 double mutant(N16581)and the acs2 acs6 acs1 acs4 acs5 acs7 acs9 septuple mutant(N16650),and completely blocked in the acs2 acs6 acs11 acs1 acs4 acs5 acs7 acs9 octuple mutant(N16651)and wild-type plants treated with aminooxyethyl glycine(AVG,a specific inhibitor of ACS activity).These results indicate that UV-B induces ethylene production by enhancing the expression of ethylene synthase genes ACS2,ACS6 and ACS11,and this process is dependent on the UVR8-COP1-HY5 signaling pathway.4.0.5 W·m-2 UV-B-induced stomatal closure and production of H2O2 and NO were inhibited mainly in N16581 and N16650 mutants,and completely blocked in N16651 mutant and wild-type plants treated with AVG.Furthermore,exogenous H2O2 or NO donor SNP closed stomata of high-order acs mutants and wild-type plants treated with AVG in either the absence or presence of UV-B radiation.These pharmacological and genetic pieces of evidence convincingly indicate that ethylene synthesis is required for UV-B-triggered H2O2 and NO production in guard cells and stomatal closure.5.0.5 W·m-2 UV-B and exogenous ACC could induce stomatal closure in ethylene receptors mutants etr2 and ers2,but didn't in copper transporter mutant ran1 and ethylene receptors mutants etr1,ein4 and ersl as well as ethylene signal components mutants ein2,ein3 and arr2;Both UV-B and exogenous ACC could induce H2O2 generation in ein2,ein3,arr2,etr2 and ers2 mutants,but could not in ran1,etr1,ein4 and ers1 mutants;Exogenous H2O2 rescued the defect UV-B-or exogenous ACC-induced stomatal closure in mutants ran1 and ein4,but could not rescue this defect in mutants etr1,ers1,ein2,ein3 and arr2;UV-B or exogenous ACC didn't induce NO generation in guard cells in all tested mutants except etr2 and ers2;Exogenous NO donor SNP rescued the defects of all mutants in the UV-B-or ACC-induced stomatal closure.Stomata of ethylene negative regulator CTR1 null mutants showed constitutive H2O2 and NO production and closure.These data indicate that copper transporter RAN1,ethylene receptors ETR1,ERS1 and EIN4,ethylene negative regulator CTR1 and ethylene signal components ARR2,EIN2 and EIN3 are all involved in UV-B-triggered guard cell signaling pathways,but ethylene receptors ETR2,ERS2 do not function in this process;Furthermore,in the signal transduction pathway of UV-B-induced stomatal closure,RAN1,EIN4,ETR1,ERS1 and CTR1 all work upstream of H2O2,while ETR1 and ERS1 also function downstream of H2O2 to mediate UV-B-induced NO production in guard cells through an EIN2,EIN3 and ARR2-dependent manner.6.Application of ACC induced the production of H2O2andNO in guard cells and closed stomata in the uvr8 mutants in either the absence or presence of UV-B,as it did that in wild type.Whereas,ACC failed to close stomata and induce NO production but could induce H2O2 generation in the copl and hy5 mutants in either the absence or presence of UV-B.These results suggest that COP1 and HY5 are involved in ethylene-induced NO generation in guard cells and stomatal closure,but not in ethylene-induced H2O2 generation,and UV-B receptor UVR8 didn't participate in ethylene signaling in guard cells;Additionally,the data further indicating that COP1 and HY5 not only act with UVR8 in the UV-B signaling pathway responsible for ethylene production but also act without UVR8 downstream of ethylene to regulate NO generation in UV-B-induced stomatal closure.7.The transgenic plants 35S:COP1/Col-0,35S:HY5/Col-0,35S:CEND/Col-0,35S:EIN3/Col-0,35S:COP1/ein2-1,35S:COP1/ein3-1,35S:HY5/ein2-1 and 35S:HY5/ein3-1 showed higher NO levels in guard cells and smaller stomatal apertures but same levels of H2O2 compared to those in the wild type and ein2 and ein3 mutants under light,while the transgenic plants 35S:CEND/cop1-4,35S:CEND/hy5-ks50,35S:EIN3/cop1-4 and 35S:EIN3/hy5-ks50 displayed the same levels of H2O2 and NO in guard cells and the same stomatal apertures as those in the wild type and mutants copl and hy5-ks50 under light;UV-B induced H2O2 generation in the transgenic plants 35S:COP1/ein2-1,35S:COP1/ein3-1,35S:HY5/ein2-1 and 35S:HY5/ein3-1 but didn't induce H2O2 and NO generation and stomatal closure in the transgenic plants 35S:CEND/cop1-4,35S:CEND/hy5-ks50,35S:EIN3/cop1-4 and 35S:EIN3/hy5-ks50;Exogenous ACC treatment induced H2O2 generation not only in the transgenic plants 35S:COP1/ein2-1,35S:COP1/ein3-1 35S:HY5/ein2-1 and 35S:HY5/ein3-1 but also in the transgenic plants 35S:CEND/cop1-4,35S:CEND/hy5-ks50,35S:EIN3/cop1-4 and 35S:EIN3/hy5-ks50 in the guard cells.However,application of ACC failed to induce NO generation in guard cells and stomatal closure in the transgenic plants 35S:CEND/cop1-4,35S:CEND/hy5-ks50,35S:EIN3/copl-4 and 35S:EIN3/hy5-ks50.These results suggest that COP1,HY5,EIN2 and EIN3 are all located in the downstream of H2O2 and the upstream of NO in the ethylene-mediated UV-B-induced stomatal closure as well as exogenous ethylene-induced stomatal closure in Arabidopsis.Moreover,COP1 and HY5 act on the downstream of EIN2 and EIN3 to regulate the production of endogenous NO in guard cells,and then induce stomatal closure.In conclusion,the signal transduction pathway of UV-B-induced stomatal closure in Arabidopsis is as follows:UV-B is perceived by the UV-B photoreceptor UVR8 dimer and results in the interaction of UVR8 monomer with COP1 in the nucleus,which induces the expression and protein stability of transcription factor HY5.HY5 activates the expression of ACS genes ACS2,ACS6,and ACS11,which induce ethylene production.Ethylene is perceived by its receptor ETR1,ERS1 and EIN4 with the help of cooper transporter RAN 1 to inactivate the CTR1 protein kinase,which then results in H2O2 production.H2O2 signaling in guard cells is initiated by ETR1 and ERS1 and then induces NO production in guard cells through an EIN2-EIN3-COP1 and HY5-dependent pathway(s).Finally,NO activates ion channels in guard cells and subsequently induces stomatal closure.