The Role Of MAPK, H₂O₂ And NO In The Regulation Of Gene Expression In Arabidopsis Leaves By UV-B Radiation

Ultraviolet B(UV-B),as a component of sunlight,is essential for plant growth and development.Previous study showed that UV-B can induce two distinct categories of responses in plants:UVR8(specific UV-B receptor)-dependent and UVR8-independent signaling pathways.As a UV-B-specific signaling pathway,UVR8-dependent signaling pathway mainly includes UVR8,COP1,HY5 and HYH,and initiates at low doses of UV-B radiation.And UVR8-dependent signaling pathway mediates UV-B regulation of plant morphogenesis and UV light defense gene expression.Meanwhile,UVR8-independent signaling pathway,which initiates at high doses of UV-B,mainly includes DNA damage,reactive oxygen species(ROS),nitric oxide(NO)and signal molecules in defense reactions.UVR8-independent signaling pathway does not rely on UV-B,and can mediate gene expression related to other stress.Both the UVR8-dependent signaling pathway and UVR8-independent signaling pathway are activated by high doses of UV-B,so we wonder whether the UVR8-dependent and UVR8-independent signaling pathway interact with each other in the gene expression regulation induced by UV-B.Thus we choose two groups of genes as markers in the UVR8-dependent and UVR8-independent signaling pathway,respectively.The first group of genes,which are dependent on UVR8,includes HY5,HYH,CHS,CRYD and ELIP1.And the second group of genes,which are independent on UVR8,includes FAD and WRKY.In order to unravel the roles of MEK1,MEK4,MAPK6,MAPK3,MKP1,H2O2 from AtrbohD/F and NO from Nial/2 in UV-B-specific signaling pathway and UVR8-independent signaling pathway,we detect the expression levels of the marker genes induced by UV-B in the Arabidopsis loss-of-function mutants by semi-quantitative PCR and realtime quantitative PCR.The main results and conclusions are as follows:1.By detecting the genes expression of Arabidopsis wild-type,cop]-4,uvr8-xl,hy5-x and hy5/hyh mutants,we found that the UV-B induced-expression levels of HY5,HYH,CHS,CRYD and ELIP1 are suppressed obviously in these four mutants,while the UV-B induced-expression levels of FAD and WRKY are not effected by the mutation of COP1,VVR8 HY5 and HYH.These results show that genes expression of HY5,HYH.CHS,CRYD and ELIP1 induced by UV-B depend on UVR8,COP1,HY5 and HYH.But genes expression of FAD and WRKY induced by UV-B are independent of UVR8,COP1,HY5 and HYH.Also these results confirm that the genes we had chosen as marker genes of UV-B signaling pathways are suitable.2.By detecting the genes expression levels of Arabidopsis mapk3-1,mapk6-4 and mek4-1 mutants,we found that the marker genes in both UVR8-dependent signaling pathway and UVR8-independent are not affected by the mutation of MAPK3,MAPK6 and MEK4 under low dose of the UV-B(0.02 Wm-2).But when the UV-B dose increased to 0.2 Wm-2,the marker genes in both UVR8-dependent signaling pathway and UVR8-independent were significantly upregulated by the mutation of MAPK3,MAPK6 and MEK4.The above results show that MAPK3 and MAPK6 only function at high dose of UV-B radiation,suggesting that MEK4,MAPK3,MAPK6 may be involved in the same signal cascade pathway,and they are negative control components of UVR8-independent signaling pathway.Meanwhile they interact with UVR8-dependent signaling pathway and inhibit genes expression regulated by UVR8 signaling pathway.3.By detecting the gene expression levels of Arabidopsis mekl-1 and mek1-3 mutants,we found that the marker genes in both UVR8-dependent signaling pathway and UVR8-independent signaling pathway are not affected by the mutation of MEK1 under low dose of the UV-B(0.02 Wm-2).But at high dose of UV-B(0.2 Wm-2),the marker genes in both UVR8-dependent signaling pathway and UVR8-independent are downregulated by the mutation of MEK1.The above results show that MEK1 only participate in gene expression in high dose of UV-B radiation.It suggests that MEK1 is a positive regulator of UVR8-independent signaling pathway.Meanwhile MEK1 interacts with UVR8-dependent signaling pathway and promotes genes expression regulated by UVR8 signaling pathway.4.We also examine the changes of gene expression levels of Arabidopsis mkp1-1 and wild-type.Interestingly,the mutation of MPK1 inhibit the expression of the marker genes in both UVR8-dependent signaling pathway and UVR8-independent signaling pathway,either under low dose(0.02 Wm-2)or under high dose(0.2 Wm-2)of UV-B radiation treatment.We conclude that MKP1 may be a positive regulator of UV-B-specific signaling pathway.MKP1 also participate in gene expression regulated by UVR8-independent signaling pathway by inactivating its target protein include MAPK3 and MAPK6.5.We also compare the changes of gene expression of Arabidopsis rbohD/F mutant and wild-type.We found that the marker genes in both UVR8-dependent signaling pathway and UVR8-independent signaling pathway are not affected by the mutation of AtrbohD/F under low dose of the UV-B(0.02 Wm-2).But the mutation of rbohD/F obviously upregulated the marker genes in both UVR8-dependent signaling pathway andUVR8-independent signaling pathway under high dose of the UV-B(0.2 Wm-2).These results indicate that H2O2 from NADPH oxidase only participates in gene expression in high dose of UV-B radiation and function as negative factor,and it is a component of UVR8-independent signaling pathway.Meanwhile H2O2 from NADPH oxidase interacts with UVR8-dependent signaling pathway and inhibits genes expression regulated by UVR8 signaling pathway.6.We also studied the gene expression of Arabidopsis nial-2/2-5 mutant and wild-type,and found that the marker genes in both UVR8-dependent signaling pathway and UVR8-independent signaling pathway are not affected by the mutation of NIA1/2 under low dose of the UV-B(0.02 Wm-2).But the mutation of NIA1/2 obviously upregulated the marker genes in both UVR8-dependent signaling pathway and UVR8-independent signaling pathway under high dose of the UV-B(0.2 Wm-2).These results suggest that NO from nitrate reductase only participates in gene expression in high dose of UV-B radiation and function as negative factor,and it is a component of UVR8-independent signaling pathway.Meanwhile NO from nitrate reductase interacts with UVR8-dependent signaling pathway and inhibits genes expression regulated by UVR8 signaling pathway.In conclusion,the results suggest that H2O2 from NADPH oxidase,NO from nitrate reductase,MEK1,MEK4,MAPK3 and MAPK6 are involved in UV-B non-specific signaling pathway,and they are negative regulators except MEK1,And they may function in the same signal cascade.These signal componets not only participate in genes expression regulation in UV-B non-specific signaling pathway,but also interact with UVB specific signaling pathway at high dose of UV-B radiation.Meanwhile MKP1 may be a positive regulatory factor in UV-B-specific signaling pathway.MKP1 may also participate in gene expression regulation in UVR8-independent signaling pathway by inactivating its target protein,including MAPK3 and MAPK6.