The Mechanism Of Arabidopsis Rubisco Activase Response On Methanol And Ethanol Stimulation

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key emzyme of the first step reaction in Catalytic photosynthetic carbon assimilation(CO2fixation), which plays an important role in regulating the photosynthesis efficiency. Rubisco activate enzymes (RCA) is a kind of chloroplast positioning enzyme, which catalyze activation of Rubisco that need to carbamoylase with divalent magnesiumion binding in plant. Many studies have shown that application of methanol and ethanol promote the growth of plants,especially methanol exogenous administration promoting effect in the model plant tobacco and Arabidopsis has been repeatedly confirmed, while the effect of exogenous administration of ethanol to stimulate plant growth has been also confirmed by some researchers. We had shown that the application of methanol in plants significantly induced RCA expression, so this study combines different components of DNA fragments in RCA promoter with transcription factor, and analyzes response under the application of methanol and ethanol, moreover,we compare several physiological biochemical indexes and photosynthesis related transcription gene level in the RCA mutant Arabidopsis (rca mutant) and the wild type Arabidopsis (WT Arabidopsis), in order to study the mechanism of RCA response under the application of methanol and ethanol, the experimental results are as follows:This study constructed plant expression vector pKm-Prca330-luc and pKm-Prca983-luc to control report gene luc expression level using330bp and983bp DNA fragments in rca gene promoter, then transformed them into tobacco to obtain transgenic plants, and analyzed the response situation of luc gene transcription and LUC protein expression in transgenic tobacco under the stimulation of methanol and ethanol. The results shown that330bp and983bp DAN fragments in rca gene promote controled luc transcription and translation, which could reply to methanol and ethanol’s stimulate, but their response patterns were different. The response of luc expression by330fragments happened in72h of methanol and ethanol treatment, but in12h by983fragments, it illustrated that the response of983fragments containing promoter element were faster than that of330fragments. The different transcription factors (ARF1/ARF7:Auxin response factor, ASF-1/ASF-2:Auxin or salicylic acid response factor, LCR1:Low-molecular-weight cysteine-rich1, GAmyb:Gibberellin myb, WRKY71:WRKY DNA-binding protein71) of rca promoter were analyzed expression of spectrum after methanol and ethanol treatment with RT-PCR in Arabidopsis, this result shown that ASF-1and ARF7transcription could reply to methanol and ethanol’s stimulate, and their response patterns was similar to RCA, these indicated these two transcription factors might be the same regulate transcription factor in RCA transcription responsed with methanol and ethanol. LCR1and WRKY71transcription only maked response to methanol, while Gamyb transcription only maked response to ethanol, these indicated those transcription factors might be regulate transcription factor in RCA transcription responsed with methanol and ethanol.Compared the growth response of stimulation effect in rca Arabidopsis and WT Arabidopsis in MS culture medium containing sucrose with methanol and ethanol, we found the growth effect of rca Arabidopsis under methanol was similar to WT Arabidopsis, this shown the growth response ability of rca Arabidopsis was same with WT Arabidopsis with methanol, however, worse than WT Arabidopsis with ethanol, it indicated the growth response ability of rca Arabidopsis was worse than WT Arabidopsis with ethanol.Meantime, we analyze chlorophyll and anthocyanin content to respond methanol and ethanol stimulation in MS culture medium containing sucrose and3%glucose, found that response patterns of chlorophyll and anthocyanin content in rca Arabidopsis was the same to those of WT Arabidopsis with methanol in MS culture medium containing sucrose:the anthocyanin content of these two kinds plant descended in containing methanol, and ascended in containing ethanol. But the response patterns of chlorophyll a and b content were different from rca Arabidopsis and WT Arabidopsis with methanol and ethanol:chlorophyll a and b content were reduced in containing methanol and ethanol of WT Arabidopsis, but were increased of rca Arabidopsis. Anthocyanin content was both decreased of WT Arabidopsis in glucose culture medium containing methanol and ethanol, while increased of rca Arabidopsis. Chlorophyll a content significantly was decreased in medium containing methanol and ethanol, and chlorophyll b was not changed, however, chlorophyll a content and chlorophyll b content both was decreased in rca Arabidopsis.We used RT-PCR to analyze the expression spectrum of rca Arabidopsis and WT Arabidopsis photosynthetic related gene responsing methanol and ethanol stimuliation, the result shown the response patterns of the Calvin cycle related gene such as FBP/SBP、rbcS transcriptional is the same in rca and WT Arabidopsis under the stimulation of methanol and ethanol:the transcription of genes could be upregulated by methanol and ethanol, but chlorophyll a/b binding protein (CAB) transcription response patterns were different. Used13C-NMR analysis NaH13CO3metabolic spectrum under the stmulation methanol and ethanol of rca and WT Arabidopsis, the result shown the effect of NaH13CO3assimilate to [U-13C]Gluc and [U-13C]Fruc in rca Arabidopsis was smaller than WT Arabidopsis, but the [3-13C]Ser synthesis made the same response pattern in them both under the stimulation of methanol and ethanol:the[3-13C]Ser synthesis were reduced by methanol, but were increased by ethanol in rca and WT Arabidopsis. The response pattern of [U-13C]Glu synthesis were the same both by methanol and ethanol:[U-13C]Glu synthesis were increased by methanol and ethanol. But The response pattern of [U-13C]Gln synthesis were different:[U-13C]Gln synthesis were unchanged in WT Arabidopsis, but increased in rca Arabidopsis.