The Relationship Between UV-B Induced Guard Cell ACC Synthase Gene Expression And UVR8 Signaling Pathway And MPK6

UV-B radiation,as an environmental factor,affects the morphogenesis and physiological reactions of plants.The response of plants to UV-B radiation can be divided into UVR8-dependent UV-B specific signaling pathway and UVR8-independent UV-B non-specific signaling pathway.UVR8 signaling pathway can be activated at very low dose of UV-B,which mainly regulates the plant's morphogenesis,and pathway members mainly compose of positive regulators,such as UVR8,COP1,HY5/HYH,and negative regulators RUP1/RUP2 likewise.UV-B non-specific signaling pathway starts at high dose of UV-B,and mainly regulates stress-related gene expression.The signaling molecules include reactive oxygen species,jasmonic acid,ethylene and MAPK.Previous studies have shown that UV-B radiation can induce gene expression of ACS,which is ethylene biosynthesis rate limiting enzyme,and enhance enzyme activity and facilitate ethylene production.At the same time,our research had revealed that ethylene mediated UV-B-induced stomatal closure.However,it is not clear which ACS genes are regulated by UV-B and the relationship between the expression of ACS gene and the UVR8 signaling pathway in guard cells.Besides,as a signal molecule of UV-B non-specific signaling pathway,MPK6 is involved in UV-B-induced stomatal closure,nevertheless,it still need more study on whether it participates in the regulation of ACS genes in guard cells.In addition,HY5 is a transcription factor of the bZIP family,and it is more unclear whether it can directly bind to the promoter of the ACS gene to regulate its expression.In order to clarify these issues,we test the effects of 0.5 W·m-2 UV-B radiation on the ACS gene expression in abaxial epidermis of leaves and guard cells,and tried to find their relationship with UVR8 signaling pathway and MKP1/MPK6,by using gene expression analysis technique of RT-PCR and promoter fusion GUS reporter gene.We also studied the interaction between HY5 and ACS gene promoters at DNA level via yeast one-hybrid technology.All the above is based on the material of Arabidopsis wild type,loss-of-function mutants of UVR8 signaling pathway members and deficient mutants of MPK6 and MKP1.The main experimental results and conclusions obtained in this paper are as follows:1.After UV-B radiation treatment,the expression of ACS2,ACS4,ACS5,ACS6,ACS7,ACS8 and ACS11 genes in abaxial epidermis and guard cells of Arabidopsis wild-type leaves significantly increased compared with the visible light,indicating that UV-B radiation can induce the expression of these 7 ACS genes in guard cells of Arabidopsis wild-type leaves,suggesting that they may be involved in the process of ethylene production induced by UV-B radiation in guard cells of leaves.2.Compared with the wild type,the uvr8,cop1 and hy5hyh mutants significantly promoted the induction on the expression of ACS2,ACS4,ACS5 and ACS6 genes under UV-B radiation in abaxial epidermis of leaves and guard cells,but inhibited on the expression of ACS7,ACS8 and ACS11 genes.On the contrary,the rupl/rup2 mutant significantly inhibited the induction on the expression of ACS2,ACS4,ACS5 and ACS6 genes under UV-B radiation and promoted on the expression of ACS7,ACS8 and ACS11 genes.It is suggested that UVR8 signaling pathway is involved in the regulation of UV-B on ACS gene expression in guard cells of Arabidopsis thaliana,and the UVR8,COP1 and HY5/HYH as positive regulators,play a negative regulatory role in UV-B induced ACS2,ACS4,ACS5 and ACS6 expression,and are positively regulated in UV-B induced ACS7,ACS8 and ACS11 expression.And RUP1/RUP2 as a negetive regulator of UVR8 signal pathway,positively regulate in the expression of UV-B induced ACS2,ACS4,ACS5,ACS6 expression,negatively regulate in UV-B induced ACS7,ACS8 and ACS11 expressions.3.Yeast one-hybrid test showed that HY5 could directly bind to G-box on ACS2 gene promoter,but it could not directly interact with other ACS gene promoters.It is suggested that the transcriptional factor HY5 of the UVR8 signaling pathway mediates the expression of the ACS2 gene regulated by UV-B radiation in the guard cells via direct interaction,and the expression of other ACS genes mediated by UV-B radiation may through indirect ways.4.Compared with the wild type,the mpk6 mutant significantly inhibited the induction of UV-B radiation on the expression of ACS2,ACS4,ACS5,ACS6,ACS7,ACS8 and ACS11 in abaxial epidermis of leaves and guard cells of Arabidopsis thaliana,and the mkpl mutant significantly promoted the induction of UV-B radiation to the expression of these ACS genes,indicating MKP1 and its target protein MPK6 are involved in the regulation of UV-B radiation on ACS gene expression in guard cells of Arabidopsis thaliana.Moreover,MPK6 plays a positive role in UV-B induced ACS gene expression in guard cells,while MKP1 plays a negative role in UV-B induced ACS gene expression in guard cells.