The Studies On The Functional Mechanism Of HY5in Response To Methanol And Ethanol Stimulation In The Arabidopsis

Our laboratory’studies have demonstrated that the application of methanol and ethanol can promote the growth of model plants, crops and ornamental plants, and improve chlorophyll content, photosynthetic rate, intercellular CO2concentration, stomatal conductance and transpiration rate. In Xuan Xiuxia’s thesis, the Arabidopsis were treated with methanol or ethanol for24hours, and then the cDNA microarray analysis showed the largest proportion of up-regulated genes were signal transduction genes and the genes related to transcriptional regulation. Pathway analysis showed that methanol and ethanol both raised the expression of genes which involved in chlorophyll biosynthesis and chlorophyll a/b binding protein, photoreaction of photosynthesis and CO2assimilation pathway, photorespiration pathway and so on, such illustrated that methanol and ethanol may be used as signal molecules to promote the expression of photosynthesis-related genes in some unknown way, and thus can contribute to the biomass accumulation of plants. Yu Xuesong was a member of our labotatory and his study showed that methanol and ethanol can induce the exoprssion of HY5(LONG HYPOCOTYL5) which is a central transcription factor in photomorphogenesis. In this study hy5mutant and wild-type(WT) Arabidopsis were used as expetiment materisls. The two type arabidopsis growing in agar MS medium were treated with low concentration methanol and ethanol, and then the effects of methanol and ethanol on physiological characteristics and the expression of photosynthesis-related genes were compared, and the effects of methanol and ethanol on the binding between HY5and the promoter regions of its downstream genes were analyzed. The functional mechanism of HY5in response to methanol and ethanol stimulation in the Arabidopsis was investigated. The main results were as follows:Wide-type and hy5mutant Arabidopsis grew in the MS agar medium adding2mM or6mM methanol and ethanol for three weeks, and we observed that the stimulation of ethanol was better than methanol, and the effect of high concentration (6mM) was worse than that of low concentration (2mM), and the relative growth rate of hy5mutant was slower than that of wild-type. The results showed the mutation of HY5weakened the stimulation of methanol and ethanol. Methanol and ethanol both can increase the chlotophyll content, and the stimulation effect of low concentration (2 mM) was better than that of high concentration (6mM), but the chlotophyll content in hy5mutant was lower than that in wild-type. The results showed the mutation of HY5reduced the simulation of methanol and ethanol to chlotophyll. The two Arabidopsis were transferred onto the MS agar medium containing5mM NaH13CO3and2mM methanol or ethanol,24hours later, the cell sap of plants was extracted and used to perform13C-NMR analysis. The results showed that methanol and ethanol both can promote CO2assimilation in hy5mutant and WT, but the sugars and amino acids which were by CO2assimilation in hy5mutant were lower than that in WT, such illustrated the HY5redudced the effects that methanol and ethanol promoted CO2assimilation. These results verified that HY5involved in the process of methanol and ethanol stimulating plants growth.WT and hy5mutant Arabidopsis were treated with2mM and6mM methanol or ethanol for12hours or72hours, and then photosynthetic genes expression profiles were analyzed using RT-PCR. The results showed the treatment can dramaticlly induced the expression of genes related to photosynthesis----RBCS, chloroplast structural protein genes----Lhcb3, Lhca4, Lhca2, Cab3, Cab2and Cab I and another two genes which were regulted by HY5, but the induction in hy5mutant Arabidopsis was lower than in wide-type, such showed HY5mutation reduced the response effects that methanol and ethanol induced the expression of RBCS and chloroplast structural protein genes. And then the vector connected with Lhca4promoter that can expression Luc was conctrcted. Later, the transgenic Arabidopsis was successfully screened. The two Arabidopsis were transferred onto the MS agar medium containing2mM or6mM methanol and ethanol, and treated for12hours and72hours, respectively. Afterwards, we used RT-PCR to analyze the transcript of Luc and the luciferase concentrations were measured by ELIASA. The results showed that methanol and ethanol caused transcript of Luc and the luciferase activity rising markedly, which stated there was response element to methanol and ethanol in Lhca4promoter regions. However, the patterns that the transcript of Luc and the luciferase activity responsed to methanol and ethanol were different. Using CHIP, the effects of methanol and ethanol on the binding between HY5and the promoter sequences of Lhca4, LZF1and RBCS were analyzed, and the results manifested different concentrations of methanol and ethanol enhanced their binding, but stimulation modes of different promoter under different treamteat were different. The above indicated methanol and ethanol can improved the expression of chlorophll synthesis gene and photosynthesis-related gene by enhancing the binding between HY5and its regulation genes, such resulted in enhancing photosynthesis of the plants.