The Function And Mechanism Of M~6A Methyltransferase In Arabidopsis Thaliana Under Heat Stress

High temperature,as one of the important abiotic stress factors,seriously affects plant growth,development,photosynthesis,respiration,cellar membrane permeability and nutrient transport,etc.It seriously threatens globe food production and safety.As the abundant internal modification in m RNA,m~6A RNA methylation has been found in yeast,mammals and plants.The reversible m~6A modification affects almost all aspects of plant growth and development,including embryonic/flower development,microspores generation and fruit-ripening.Although m~6A RNA methylation has been studied in animal,human diseases and viruses,however,its function in plant stress response is still largely unknown.In this study,the function and molecular mechanism of m~6A RNA methylation involved in regulating the heat stress process was investigated in Col-0 wild-type and m~6A RNA methyltransferase knockdown mutant Arabidopsis(hakai and vir).The main results are as follows:(1)The m~6A RNA methylation modification was closely related to the heat stress response in Arabidopsis.We found that heat stress induced the expression of m~6A methyltransferase gene,which led to an increase in m~6A levels.In addition,heat stress also increased the expression of m~6A methyltransferase gene in knockdown mutants(mta,mtb,vir,and hakai)in Arabidopsis,indicating that m~6A RNA methylation modification was closely related to the heat stress response in Arabidopsis plants.(2)The m~6A methyltransferase mutants were very sensitive to heat stress.We found that although there was no obvious difference between the mutants and wild type plants under the heat stress treatment at 42℃and heat adaptation at 37℃→42℃,however,under the heat stress treatment at 37℃,hakai and vir mutants displayed significant heat-sensitive phenotype compared with the wild type,such as low survival rate,fresh weight,chlorophyll and high MDA content,during the heat stress recovery period.These results indicated that the defect of m~6A RNA methyltransferase function may lead to a significant increase of heat sensitivity in Arabidopsis.(3)m~6A methylation may affect the heat tolerance of Arabidopsis through the indirect regulation of the expression of heat stress related genes.The q PCR verification showed that among the 36 positive regulated genes in heat response pathway,the expression levels of 29 genes in the hakai and 30 genes in the vir mutants were significantly lower than those in the wild type.By contrast,among 11 negatively regulated genes in heat response pathway,the expression levels of 10 genes in hakai and5 genes in vir mutants were significantly higher than those in the wild type plants.In addition,the results of m~6A-IP-q PCR and m RNA stability experiments further indicated that the gene expression and RNA stability of selected genes(CSD1,CSD2,HSP101,CBK3 and Dj C53)have no significant difference between mutant and wild type plants,implying that m~6A regulated the heat stress responsive gene indirectly.Therefore,the target genes of m~6A should be further surveyed through m~6A Me RIP-seq and RNA-seq experiment in the future.In summary,the defect of the m~6A RNA methyltransferase genes HAKAI and VIRIZER led to the decrease of m~6A level in Arabidopsis,and resulted in the heat-sensitive phenotype.Also,in the m~6A RNA methyltransferase gene function-loss-mutants,the gene expressions of most heat stress related genes were significantly altered during the stress and/or recovery period.However,m~6A may be regulated the heat stress response in Arabidopsis by affecting the heat stress related genes indirectly.Therefor,the target genes that m~6A affects directly under heat stress need to be further explored.This study laid a foundation for further study on the regulation of m~6A RNA methylation under heat stress in plant,and will further promotes our knowledge and understanding on the regulation mechanism of heat stress in plant.