The Function Study Of Transcription Factor SlERF.D6 In Regulating SGAs Metabolism And Fruit Ripening In Tomato

Steroidal alkaloids(SAs)and their glycosylated forms(steroidal glycoalkaloids,SGAs)are a group of nitrogenous organic molecules derived from cholesterol.They are present in Solanaceae plants and beneficial to the plant against pathogens,but are toxic to humans so they are considered to be antinutritional compounds.In green tissues,it exists mainly in the form of the toxic tomatine,which is converted to the non-toxic esculeoside A during fruit maturation.Several structural genes have been reported to be involved in the SGAs synthesis pathway,while little is known about the influence of transcription factors on SGAs.In this study,349 tomato accessions were analyzed for metabolic profiling,and metabolite-based genome-wide association analysis(m GWAS)was used to identify an AP2/ERF transcription factor,Sl ERF.D6,which is closely associated with esculeoside A.Through transient overexpression,virus-induced gene silencing(VIGS)and interaction verification,we speculated that Sl ERF.D6 is involved in tomato SGAs metabolism and fruit ripening.To investigate the function of Sl ERF.D6,the expression profile was determined by quantitative Real-Time PCR(q RT-PCR).It was found that the expression level of Sl ERF.D6 was highest in breaker stage,suggesting that Sl ERF.D6 might act a pivotal part in fruit ripening.In this study,transient overexpression and virus-induced gene silencing(VIGS)in tomato fruits were carried out,and the results showed that the increase of Sl ERF.D6 expression level could promote fruit ripening.We measured the expression levels of genes related to SGAs synthesis and discovered that GAME12 expression decreased while GAME2,GAME5 and GAME18 increased.The content of SGAs associated with these four genes were detected by liquid chromatography-mass spectrometry(LC-MS),showing that the content of tomatidine was reduced,while the levels of furostanol-26-aldehyde,β1-tomatine,α-tomatine,and esculeoside A were raised.When Sl ERF.D6 was silenced,fruit ripening was delayed and the change trend of SGAs pathway gene expression and related SGAs content were opposite to that of Sl ERF.D6 overexpression.To understand the transcriptional regulation of Sl ERF.D6,yeast one hybrid(Y1H)assay and dual-luciferase transient expression assay(LUC)were performed.Sl ERF.D6 was identified to bind to the promoter region of GAME12 and negatively regulate the expression of GAME12.While for GAME2,GAME5,and GAME18,Sl ERF.D6 did not directly bind to their promoter regions and had no transcriptional activation activity to them.This study resolved the biological functions of a new transcription factor,Sl ERF.D6,involved in the metabolism of SGAs during fruit development and its effects on fruit ripening.And it provides new insights into the study of SGAs,fruit development-related transcription factors and new resources for tomato breeding and improvement.