The Effect Of Different Doses Of UV-B Radiation On The Expression Of ACC Synthase Gene In Arabidopsis Leaves And Its Relationship With UVR8 Signaling Pathway

As an important environmental signal,UV-B radiation significantly effects plant growth and development.Previous studies have shown that UV-B radiation could induce the expression of ethylene biosynthesis keying enzyme(ACC synthase,ACS)gene,increase the enzyme activity of ACS,and thus increase ethylene production.In Arabidopsis thaliana ACS was encoded by a multi-gene family,which contains twelve homologous ACS genes(ACS1-12).Among these ACS genes,ACSl,ACS3,ACS10 and ACS 12 do not encode proteins with ACC synthase activities,but the rest were likely to encode functional ACC synthases.Meanwhile,considerable evidence showed that there were at least two UV-B signal transduction pathways in plants,including UVR8(UV-B receptor)-dependent UV-B specific signaling pathway and UVR8-independent UV-B non-specific signaling pathway.Although both UVR8-dependent and UVR8-independent pathways could mediate the UV-B signaling,they are initiated by different dose of UV-B.The UVR8-dependent pathway was stimulated at the low dose of UV-B radition,and the main signal transduction molecules include positive factor UVR8,COP1 and HY5/HYH and negative factor RUP1/RUP2.But UVR8-independent signaling pathway was only initiated by a much higher dose of UV-B radiation,and it included a number of signal components of oxidative stress and defense responses.In addition,previous studies also showed that the ethylene signal transduction components and photomorphogenesis signaling molecules COP1 and HY5 have many crosstalks.Based on these backgrounds,we raised the following questions.(1)Which of ACS gene family members are involved in ethylene biosynthesis induced by UV-B radiation?(2)Whether the same kinds of ACS are involved in ethylene biosynthesis induced by different doses of UV-B radiation?(3)Which UV-B signaling pathway induces the expression of ACS genes?(4)Whether the UV-B signal transduction pathways for different doses of UV-B radiation induce the same ACS gene expression?(5)Whether there exists a crosstalk between the ethylene signal pathway and UVR8 signaling pathway under the UV-B radiation?To answer these questions,we examine the gene expression levels in the loss-of-function mutants of UVR8 signaling pathway members and ethylene signal transduction components,by RT-PCR(Reverse Transcription PCR)and qRT-PCR(quantitative Real-Time PCR)techniques.We studied the effects of different doses of UV-B radiation on the ACS gene expression levels,and tried to find their relationship with UVR8 signaling pathway.Also we want to know the effects of ethylene signaling pathway on the expression levels of HY5 and HYH which were key members of UVR8 signaling pathway under UV-B radiation.The following main results and conclusions were obtained:1.Based on previous studies,we chose HYH,CHS genes and FAD,WRKY genes as molecular markers of UVR8-dependent UV-B signaling pathway and UVR8-independent UV-B signaling pathway,respectively.Then we studied the effects of different doses of UV-B radiation on the expression of these genes.We found that low dose of UV-B radiation(0.02 W·m-2)can only stimulate UVR8-dependent signaling pathway,and high dose of UV-B radiation(0.5 W·m-2)can both stimulate UVR8-dependent signaling pathway and UVR8-independent signaling pathway.So we chose 0.02 and 0.5 W·m-2 as two different doses of UV-B radiation.2.1n wild-type Arabidopsis leaves,the gene expression levels of ACS2,ACS6,ACS7,ACS8 and ACSll were higher at 0.02 and 0.5 W·m-2 dose of UV-B radiation than at visible light,while ACS4 gene expression decreased compared with its expression level at visible light,indicating that in varying levels,all the above six ACS were affected by different doses of UV-B radiation.However,under the low dose of UV-B radiation,the peak of gene expression of ACS2,ACS4,ACS6 and ACS7 reached at 3h,while ACS8 and ACS11 genes showed the highest expression at 1h.Moreover,under the high dose of UV-B radiation,only ACS8 gene showed the highest expression at 1h,while other ACS gene expression levels peaked at 3h.In addition,under different doses of UV-B radiation,the increasing extent and duration of ASC2 and ACS6 expression were significan,tly-higher and longer than the other ACS genes,suggesting that different ACS in UV-B radiation-induced ethylene biosynthesis may played a minor role,but ACS2 and ACS6 may.play the major role in different stages.3.Under the low dose of UV-B radiation(0.02 W·m-2),the mutation of UVR8,COP1 and HY5/HYH,which 'are UVR8 signaling pathway positive regulators,inhibited ACS2,ACS6,ACS7,ACS8 and ACS11 gene expression,but promoted ACS4 gene expression.On the contrast,the mutation of RUP1/2,which are UVR8 signaling pathway negative regulator factors,promoted ACS2,ACS6,ACS7,ACS8 and ACS11 gene expression,but inhibited ACS4 gene expression.So UVR8 signaling pathways was involved in the regulation of ACS gene expression by low dose of UV-B radiation,and UVR8 signaling pathway played a positive role in ACS2,ACS6,ACS7,ACS8 and ACS11 gene expression,whereas played a negative regulatory role in ACS4 gene expression induced by low dose of UV-B radiation.4.Under the high dose of UV-B radiation(0.5 W·m-2),the mutation of UVR8,COP1 and HY5/HYH inhibited ACS4,ACS8 and ACSll gene expression,but promoted ACS2,ACS6 and ACS7 gene expression levels.On the contrast,the mutation of RUP1/2,promoted ACS4,ACS8 and ACS11 gene expression,but inhibited ACS2,ACS6 and ACS7 gene expression under the high dose of UV-B radiation(0.5 W·m-2).So,UVR8 signaling pathway played a positive role in ACS4,ACS8 and ACS 11 gene expression,whereas plays a negative role in ACS4,ACS6 and ACS7 gene expression induced by high dose of UV-B radiation.Compared with the effects of UVR8 signaling pathway on each ACS gene expression in low dose of UV-B radiation,the effects of UVR8 signaling pathway in ACS2,ACS4,ACS6 and ACS7 gene expression induced by high dose of UV-B radiation was completely different from its effects in low dose of UV-B radiation,suggesting that,to mediate the ACS gene expression,UVR8-independent UV-B signaling pathway in high dose of UV-B radiation may interact with UVR8-dependent signaling pathway,but it requires further study to clarify the mechanism.5.Different doses of UV-B radiation could significantly induce gene expression of HY5 and HYH,which are key components of UVR8 signaling pathway,in wild-type Arabidopsis leaves,but the effects were significantly suppressed in etrl-9 and ersl-3(ethylene receptor mutants),as well as ein2-5 and ein3-1(ethylene signal transduction element mutants),indicating that ethylene signal transduction pathway was involved in UV-B radiation-induced gene expression of HY5 and HYH of UVR8 signaling pathways members.All in all,we concluded that Arabidopsis,after feeling UV-B signalling,initiates ethylene production by UVR8 signal pathway,and during the UVR8 signal pathway,the ethylene production has positive feedback regulation in HY5,element of UVR8 signal pathway,and promotes expression of its homology gene HYH,thus further promotes transferring UV-B signal.