The Role Of Different Subunits Of Heterotrimeric G Protein In The Gene Expression Of Arabidopsis Leaves Induced By Different Irradiance UV-B Radiation

Previous studies show that UV-B radiation,functioning as an important environmental signal,plays important roles in plant growth and development,including morphogenesis.There are at least two UV-B signaling transduction pathways.One isa UV-B-specific signaling pathway which is UVR8-dependent,and the other one is a UV-B-nonspecific signaling pathway which is UVR8-independent.Heterotrimeric G protein,which is composed of alpha(Ga),beta(G?)and gamma(G?)subunits,is a kind of important and conserved signal molecules in eukaryotic cells.Previous studies in animals show that Ga and G?? subnunits are involved in a variety of physiological responseinduced by UV-B radiation,but whether the G protein is involved in the physiological response induced by UV-B radiation in plant is not clear.Previous results show that HY5,HYH,CHS,CRYD and ELIP1 are UVR8-dependent genes,and FAD,UDP and WRKY are UVR8-independent genes,and thus we choose the two group genes as molecular markers of UV-B-specific signaling pathway and UV-B-nonspecific signaling pathway,respectively.Then we detect the expression levels of these marker genes in the leaves of Arabidopsis thaliana wild-type and heterotrimeric G protein loss-of-function mutants,using RT-PCR(Reverse Transcription-Polymerase Chain Reaction)and qRT-PCR(quantitative Real-Time PCR)techniques.In this way,we mainly discuss the role of different heterotrimeric G protein subunits in the gene expression regulation of UV-B signaling transduction pathways,and the relationship betweenheterotrimeric G protein and UVR8-dependent/independent signaling pathways.The following are our main results and conclusions:1.We first found thatthe suitable treatment time for UV-B radiationto induce the expression of all the marker genes is 3 hours.Then we detected the expression levels of these marker genesinduced by different UV-B irradiance(0.02,0.04,0.1,0.2 Wm-2).Our results show that 0.02 Wm-2 dose of UV-B radiation is enough toinitiate the expression of UV-B-specific marker genes(HY5,HYH,CHS,CRYD and ELIP1),while at least 0.1 Wm-2 dose of UV-B radiation is needed toinitiate the expression UV-B-non-specific marker genes(WRKY,UDP and FAD).Therefore,we choose 0.02 Wm-2 and 0.2 Wm-2 as the low dose and high dose of UV-B radiation treatment,respectively.2.We found that in uvr8,cop]and hy5/hyh loss-of-function mutants,UV-B radiation could induce the expression UV-B-non-specific marker genes(WRKY,UDP and FAD),but not the expression of UV-B-specific marker genes(HY5,HYH,CHS,CRYD and ELIP1).Previous results show that UVR8,COP1 and HY5/HYH are key components of UVR8 signaling pathways,so our results confirm that HY5,HYH,CHS,CRYD and ELIP1 are UVR8-dependent genes,and FAD,UDP and WRKY are UVR8-independent genes,and the two group genes are suitable to be chosen as marker genes.3.Compared with wild-type,the heterotrimeric G protein alpha subunit GPA1 loss-of-function mutant gpa1-1 and gpa1-2 do not affect the expression of UV-B-specific marker genes(HY5,HYH,CHS,CRYD and ELIP1)induced by low doses of UV-B radiation(0.02 Wm-2),but they inhibitboth the expression levels of UV-B-specific marker genes(HY5,HYH,CHS,CRYD and ELIP1)and UV-B-non-specific marker genes(WRKY,UDP and FAD)induced by high UV-B irradiance(0.2 Wm-2).The results suggested that the G protein alpha subunit GPA1 is a positive regulatory factor in UV-B-non-specific signaling pathways.GPA1 not only mediated UV-B-non-specific signaling pathways by regulating gene expression,but also interacted with UV-B-specific signaling pathways to promote the UV-B-specific genes expression under high doses of UV-B.4.Compared with wild-type,the heterotrimeric G protein beta subunit AGB1 loss-of-function mutant agb1-1 and agb1-2 do not affect the expression of UV-B-specific marker genes(HY5,HYH,CHS,CRYD and ELIP1)induced by low dose of UV-B radiation(0.02 Wm-2),but they inhibitboth the expression levels of UV-B-specific marker genes(HY5,HYH,CHS,CRYD and ELIP1)and UV-B-non-specific marker genes(WRKY,UDP and FAD)induced by high UV-B radiation dose(0.2 Wm-2).The results suggested AGB1 is also a positive regulatory factor in UV-B-nonspecific signaling pathways.AGB1 not only mediated UV-B-non-specific signaling pathways by regulating gene expression,but also interacted with UV-B-specific signaling pathwaysto promote the UV-B-specific genes expression under high doses of UV-B.5.Compared with wild-type,the G protein gamma subunit AGG1,AGG2 and AGG3 loss-of-function mutants do not affect the expression of UV-B-specific marker genes(HY5,HYH,CHS,CRYD and ELIP1)induced neither by low dose(0.02 Wm-2)nor high dose(0.2 Wm-2)of UV-B radiation,but promote the expression of the UV-B-non-specific marker genes(WRKY,UDP and FAD)induced by high dose(0.2 Wm-2)of UV-B radiation.The results suggested that G protein gamma subunit AGG1,AGG2 and AGG3 are all negative regulatory factors in UV-B-nonspecific signaling transduction pathways,and may play no or little roles in the UV-B-specific signaling transduction pathways.