The Molecular Mechanism Of Methanol And Ethanol Enhanced-growth And-photosynthesis In Brassica Napus L.

It has been repeatedly confirmed that exogenous methanol/ethanol can stimulate plant growth. A lot of work has been developed in our lab focusing on the mechanisms of methanol/ethanol stimulating plants growth. Basing on the previous studies, signals-mediated mechanism was proposed responses to methanol/ethanol-stimulated, the methanol/ethanol might be used as a signal molecule to stimulate the expression of photosynthesis-related genes, thus enhance photosynthesis and promote growth of plants. Rape (Brassica napus L.) is an important edible oil crop next to soybean, which is used as experimental materials in this study. Firstly, the effect of different concentrations of methanol/ethanol on growth and photosynthetic characteristics of rape was analyzed to determine the optimal spraying concentration. Using cDNA microarray technology identified responses to methanol/ethanol-stimulated differentially expressed genes of rape leaves and to verify the reliability of microarray data by RT-PCR, further analyzed methanol/ethanol-affected photosynthetic characteristics of rape potential signal pathways. According to analyze the effects of single use of exogenous hydrogen peroxide (H2O2) and abscisic acid (ABA) or co-processing of methanol/ethanol on leaf photosynthetic rate, stomatal physiological-biochemical characteristics of rape, it clarified whether methanol/ethanol regulated H2O2signal pathway and influenced the stomatal physiological characteristics and further increased the photosynthetic rate. The effect of single use of PM H+-ATPase inhibitor vanadate (VA) and adenosine5’-monophosphate (AMP) or co-processing of methanol/ethanol on photosynthetic rate and stomatal physiological and biochemical characteristics of rape, which revealed the role of PM H+-ATPase responses to methanol/ethanol-stimulated in rape. Using overexpression and suppression expression PM H+-ATPase or14-3-3protein of transgenic tobacco verified the responded role of PM H+-ATPase. Finally according to analyze the effects of single use of Ca2+/CaM inhibitor/antagonist or co-processing of methanol/ethanol on stomatal physiological and biochemical characteristics of rape and expression level of CaM, which clarified the role of responses to methanol/ethanol-stimulated in rape. The results showed that the molecular mechanism of methanol/ethanol stimulating and promoting the growth and photosynthesis of rape, further exploited that potential application of methanol/ethanol as a plant growth regulator in agricultural production as well as provided a theoretical basis for their application. The following main research results were obtained:1. Analyzing the effect of the different concentration methanol/ethanol on rape, the results showed that the methanol/ethanol stimulated growth in rapes, foliar spray of2%and5%methanol caused a53%and145%increase in the fresh weight; foliar spray of2%and5%ethanol caused a107%and223%increase in the fresh weight; The results showed that foliar spray with5%methanol/ethanol had a significant stimulatory on the growth and photosynthesis, and foliar spray of ethanol had better effects than methanol. The methanol/ethanol also affected the stomatal physiological characteristics, foliar spray with5%methanol led to an increase in stomatal conductance by25%,20%,23%and25%in four rape varieties, respectively; Foliar spray with5%ethanol led to an increase in stomatal conductance by32%,27%,31%and32%in four rape varieties, respectively; Foliar spray5%methanol/ethanol led to an increase in total chlorophyll content by27.57%and32.06%respectively. There were differences in different rape varieties responses to methanol/ethanol-stimulated. Comprehensive growth and photosynthesis indicators, in this study Qinyou2variety was selected as material for subsequent experiments. In addition, foliar spray of5%methanol/ethanol reduced the leave H2O2content and significantly increased three antioxidant enzyme (SOD, CAT and POD) activity in rape leaves. From the above results it might be speculated that an increase in antioxidant enzyme activity might be a direct factor of H2O2reduction in leaves, and the increase of stomatal conductance might have a relationship with the H2O2content decreased.2. To understand the mechanism of methanol/ethanol to stimulate and promote the growth and photosynthesis of rapes at the transcription level, Using cDNA microarray analyzed the differentially expression genes with5%methanol/ethanol-sprayed in rape leaves. The results showed that the number of the methanol/ethanol-responsive up-regulated genes were much greater than the down-regulated genes. These up-regulated and down-regulated genes were294and 199by foliar spraying of methanol, and up-regulated and down-regulated genes were358and147by foliar spraying of ethanol, respectively. RT-PCR analysis verified that three genes related to the calmodulin were induced to express at12h after methanol/ethanol spray, and33genes related to photosynthesis, two14-3-3proteins encoding genes and one plasma membrane (PM) H+-ATPase encoding gene were strongly induced to express from24to48h. The data indicated that methanol/ethanol stimulation might regulate the expression of photosynthesis-related genes by affecting calmodulin signaling pathways. Western blot and immune co-precipitation (CO-P) analysis showed that the expression and interaction of PM H+-ATPase and14-3-3protein in rape leaves were enhanced with the increase of methanol/ethanol processing time. These results showed that the expression and interaction of PM H+-ATPase and14-3-3protein were also involved in response to methanol/ethanol-stimulation in rape.3. Stomatal is an important place of plant gas exchange with the external environment, the physiological and biochemical characteristics of stomatal is closely related to photosynthetic efficiency. H2O2is one of the most important signal molecules in regulating plant stomatal movement, the effects on leaf photosynthetic rate, stomatal physiological and biochemical characteristics of rape were analyzed by single use of exogenous H2O2or co-treated with methanol/ethanol. The results showed that the photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2concentration gradually decreased with increasing of exogenous H2O2concentration (10-10000μM), and H2O2content increased in leaves and the guard cells. Using1mM H2O2smearing leaves for lh and smearing with2mM methanol/ethanol for11h, the H2O2content has significantly decreased in guard cells, simultaneously alleviated the inhibition of H2O2on stomatal aperture, stomatal conductance and photosynthetic rate. The single use of2mM methanol/ethanol treatment increased the activity of PM H+-ATPase and H+-pump, however the results of single use of H2O2were contrary to single use of methanol/ethanol. Combined use of methanol/ethanol with H2O2could alleviate the inhibition of PM H+-ATPase and H+-pump activity by using H2O2only, but it could not completely reverse the effects by exogenous H2O2. CO-IP analysis showed that methanol/ethanol also could alleviate the inhibition of interaction between PM H+-ATPase and14-3-3protein by exogenous H2O2. These results indicated that methanol/ethanol might antagonize the inhibition of photosynthetic rate and stomatal physiological and biochemical characteristics in rape by exogenous H2O2treatment. Thus it can be suspected that methanol/ethanol might increase the photosynthetic rate through regulating H2O2signaling pathways to influence the porosity of rape physiological and biochemical characteristics.4. H2O2is a downstream member in ABA regulation of stomatal movement signaling pathways. To understand whether ABA participated in methanol/ethanol regulation function of H2O2signaling pathways, the effects on leaf photosynthetic rate, stomatal physiological and biochemical characteristics were analyzed in rape leaves by single use of exogenous ABA or co-treated with methanol/ethanol. The results showed that the photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2concentration were a significantly downward trend with the increasing of ABA concentration (10-1000μM), H2O2content increased simultaneously in leaves and guard cells and PM H+-ATPase and H+-pump activity decreased. Using10μM ABA smearing leaves for1h and smearing with2mM methanol/ethanol for11h alleviated the inhibition of ABA on photosynthetic rate, and led to a decrease in leaves H2O2content by51%and58%. The H2O2content has significantly decreased in guard cells, but led to an increase in PM H+-ATPase and H+-pump activity by9%and33%than single use of ABA. However it can’t make them recover the level of non-treated controls. CO-IP analysis showed that the methanol/ethanol could significantly decrease the inhibition of PM H+-ATPase and14-3-3proteins interaction by ABA treatment. These results indicated that methanol/ethanol might antagonize the inhibition of photosynthetic rate and stomatal physiological and biochemical characteristics in rape by exogenous ABA treatment. Thus it can be suspected that methanol/ethanol might also reduce the accumulation of H2O2though affecting ABA signal transduction and increase the PM H+-ATPase and H+-pump activity, and promote stomatal opening, thus increase stomatal conductance and leaves photosynthesis.5. PM H+-ATPase is a key member and effecter in regulation signaling pathways of stomatal movement. To understand the roles of stomata physiological characteristics response to methanol/ethanol-stimulation, the effect on photosynthetic rate and stomatal physiological and biochemical characteristics were analyzed by single use of PM H+-ATPase inhibitor VA and AMP or co-treated with methanol/ethanol. The results showed that the photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2concentration gradually decreased and H2O2content gradually increased in leaves and guard cells with the increasing of VA (30-120μM) and AMP (50-200μM) concentration. Using90μM VA or150μM AMP smearing leaves for1h and smearing with2mM methanol/ethanol for llh significantly alleviated the inhibition of PM H+-ATPase and H+-pump activity and H2O2content by VA or AMP treatment, and alleviated the decreasing degree of stomatal aperture and stomatal conductivity by VA or AMP treatment. But the improvement effects of AMP treatment groups were inferior to VA treatment groups. According to the Western blot and CO-IP analysis results, it can be suspected that methanol/ethanol might increase PM H+-ATPase and14-3-3protein interaction to antagonize the effect of VA or AMP.6. Four transgenic tobaccos were used to verify the above speculation, the overexpression PM H+-ATPase (GHA5) and14-3-3proteins (S23) and suppression expression PM H+-ATPase (RP1) and14-3-3proteins (RE10) of transgenic tobaccos have been prepared by M.S. Guo Chuan-long in our lab. The results showed that2mM methanol/ethanol smearing leaves caused an increase in the photosynthetic efficiency, transpiration rate, stomatal conductance and intercellular CO2concentration in four transgenic tobaccos, but there were differences in growth stimulatory effect between different types of transgenic tobaccos. The effect on GHA5and S23were better than RE10and RP1. The ethanol had better effects than methanol. The stimulatory effect on the PM H+-ATPase and H+-pump activity of four transgenic tobaccos was consistent with the effect on photosynthesis. Western blot and CO-IP results confirmed that enhancing the expression and interaction of PM H+-ATPase and14-3-3protein in GHA5and S23was greater than RE10and RP1by methanol/ethanol treatment.7. To understand the role of calmodulin in regulation stomatal physiological characteristics by methanol/ethanol, single using Ca2+/CaM inhibitors trifluoroacetic piperazine (TFP), calcium channel inhibitor nifedipine (NIF), non-specific protease inhibitor staurosporine (ST) and calmodulin signal antagonists (W7/KN62) or co-treated with methanol/ethanol in rape leaves, the changes were analyzed in stomatal physiological-biochemical characteristics and CaM expression levels. The results showed that all five kinds of different antagonists inhibited the photosynthesis and reduced stomatal aperture and stomatal conductance in rape, and caused a decrease in three antioxidant enzyme activities and H2O2accumulation in leaves, thus reduced the CaM expression level. Using five inhibitor/antagonists smearing leaves for1h and smearing with2mM methanol/ethanol for11h, the results showed that they improved the inhibition effects of photosynthetic rate, stomatal aperture and stomatal conductance. The biggest relieved degree was the inhibition caused by KN62, next came of the inhibition caused by W7and ST, but the improving effect was the smallest caused by NIF and TFP. At the same time, it showed that the CaM expression levels were improved, three antioxidant enzyme activities were increased and H2O2content was reduced in leaves. Thus basing on these results, it could be suspected that methanol/ethanol might influence the antioxidant enzyme activities though improving the CaM expression level and enhancing the role of Ca+/CaM signaling pathways, further antagonized the influencing effect of Ca2+/CaM inhibitors or antagonists on stomatal physiological characteristics in rape.