Effects Of High Temperature On The Metabolism And Gene Methylation Of Megastigmatrienone Precursors In Tobacco

As one of the most important aroma substances in tobacco,megastigmatrienone has a low content in Chinese flue-cured tobacco as a whole,which is an important bottleneck to improve the aroma quality of flue-cured tobacco in China.Carotenoids mainly synthesized and accumulated during the mature stage,the important precursors of megastigmatrienone,determines the types of degradation products through their own kinds,and temperature is one of main ecological factors affecting different kinds of carotenoids.Therefore,it is of great significance to study synthesis and regulation mechanism of carotenoid in response to high temperature stress during mature stage of flue-cured tobacco.Here we analyzed the relationship between meteorological factors and the contents of megastigmatrienone in tobacco,and identified the key genes and methylation sites in response to high temperature by comparative transcriptome(RNA-Seq),target metabolism and whole genome bisulfite sequencing(WGBS)analysis based on the controlled experiments at different temperatures.The main conclusions are as follows:(1)To clarify the role of temperature in the accumulation of megastigmatrienone,the climate characteristics and the content difference of megastigmatrienone in five typical tobacco growing areas were compared and analyzed.Taking Jiangxi flue-cured tobacco as an example,it was found that the higher temperature in the mature period was the key factor affecting the accumulation of megastigmatrienone in tobacco.(2)To avoid the interference of complex field environment,a pot experiment of different temperature stress levels(26°C,29°C,32°C,and 35°C)was established.It was found that the expressions of phytoene synthase gene(PSY)in carotenoid pathway and?-carotene hydroxylase gene(BCH)in lutein pathway were up-regulated under high temperature stresses,resulting in the increasing of lutein,3-oxo-?-ionol and megastigmatrienone contents.Concomitantly,the differentially expressed genes of 11 DNA methyltransferase gene(MET)and 12 domain rearranged methyltransferase gene(DRM)were identified.(3)To further analyze the DNA methylation regulation in different downstream metabolic branches of carotenoids,the integrated analysis of WGBS and RNA-Seq displayed that methylation modification is closely related to photosynthesis and terpenoid synthesis at 26°C and 35°C,of which zeaxanthin epoxidase gene(ZEP)and carotenoid cleavage dioxygenase 4 gene(CCD4)expressions in ?-carotenoid branch were directly and indirectly down regulated by methylation,respectively.As a result,?-carotenoid branch metabolism was weakened.To summary,the temperature was the key meteorological factor affecting the accumulation of megastigmatrienone during maturity stage of flue-cured tobacco.High temperature stress induced the expressed up-regulation of PSY in carotenoid pathway and BCH in lutein pathway,and inhibited the expression of ZEP and CCD4 expressions in ?-carotenoid branch through methylation modification,which led to the accumulation of megastigmatrienone content in tobacco.The results revealed the unique metabolism pathway of high temperature stress responsive megastigmatrienone precursor,along with the identifications of the key response genes and methylation regulation sites.These findings provided theoretical basis and technical support for the formation mechanism of megastigmatrienone in flue-cured tobacco and agricultural production and utilization mode of high aroma flue-cured tobacco in China.