Functional Verification Of Tomato Steroidal Glycoalkaloids Synthetase SlERT1b And Its Transcriptional Regulation

Steroidal glycoalkaloids are the unique alkaloids in Solanaceae,including tomatoes,potatoes and eggplants.In tomatoes,steroidal glycoalkaloids mainly exist in the form ofα-tomatine and dehydrotomatine.They often have a bitter taste and are found in non-edible tissues.Fruit ripening process is accompanied by changes in metabolites.Steroidal glycoalkaloids also change during the ripening process.Steroidal glycoalkaloids in green tomatoes mainly exist in the form of α-tomatine,which gives tomatoes bitter taste and is not suitable for consumption.During the fruit ripening process,α-tomatine will generate Esculeoside A through a series of enzymatic reactions,which make fruits eatable.Whereas,this metabolic pathway has not been completely elucidated.In the previous research of metabolite Genome Wide Association Study(m GWAS),we identified a glycosyltransferase Sl ERT1 b that was involved in this metabolic pathway.In current study,we conducted a tomato genetic transformation experiment about Sl ERT1 b including overexpression and RNAi to study its function.We used High Performance Liquid Chromatograph-Mass Spectrometer(HPLC-MS)to detect the contents of steroidal glycoalkaloids in leaves,mature green fruits,breaker fruits,fruits 5 days after breaker stage and fruits 10 days after breaker stage.These experiments verified that Sl ERT1 b can catalyze the conversion of Acetoxy-hydroxytomatine to Esculeoside A,which can act as a glycosyltransferase.Furthermore,in order to study whether hormones can regulate Sl ERT1 b and affect the contents of steroidal glycoalkaloids,we applyed exogenous ethylene and ethylene inhibitor1-methylcyclopropene(1-MCP),and then found that ethylene can significantly change the expression of Sl ERT1 b and the synthesis of steroidal glycoalkaloids.We applyed exogenous abscisic acid(ABA)and the abscisic acid inhibitor nordihydroguaiaretic acid(NDGA),and then found that ABA can changed the expression of Sl ERT1 b but did not significantly affect the content of steroidal glycoalkaloids.Through co-expression analysis,yeast one-hybrid and tobacco dual-luciferase interaction experiments,we identified the transcription factors Sl JERF1 and Sl AREB2 that can regulate the expression of Sl ERT1 b and the synthesis of steroidal glycoalkaloids by responding to hormones.The analysis of the content of steroidal glycoalkaloids in different tissues found that during the entire fruits development process,steroidal glycoalkaloids were orderly synthesized from α-tomatine to esculeoside A at different times.In addition,it is possible that there is a new metabolic pathway from α-tomatine,Hydroxytomatine to Di-hydroxytomatine.This study resolved the biological functions of Sl ERT1 b involved in the metabolic pathway of steroidal glycoalkaloids,and clarified the complete biological process from hormone signals such as ethylene and abscisic acid,to hormone-responsive transcription factors,and then to the metabolic pathway of steroidal glycoalkaloids.It will provide theoretical basis for future research of steroidal glycoalkaloids and the tomato genetic breeding.