Genetic And Molecular Mechanisms Of Transcription Factors Regulation Of Capsaicinoids Biosynthesis In Capsicum

Capsaicin(Cap),dihydrocapsaicin(Dh Cap)and other analogs collectively called capsaicinoids(CAPDs),is a group of alkaloids which biosynthesis strictly by the genus of Capsicum.CAPDs confer pepper fruits a pungent flavor,but also assist plants resistance to pathogens and herbivores to attack its fruits.Besides,CAPD also widely used in many other fields.The Capsicum genome sequencing has completed,comparative genomics analysis indicate that capsaicinoid biosynthetic genes(CBGs)also presented in Solanaceae plant species such as the tomato and potato.Comparative transcriptomic revealed that CBGs are highly expressed in the pepper placenta from 16 days post anthesis(DPA)to the mature green stage.Whereas these orthologous genes were weakly expressed in the tomato and potato during these stages.The results indicate that the changes in the expression of key CBGs likely enable CAPD biosynthesis synthesis in hot pepper fruits.Moreover,the coefficient of the key CBG transcriptional level is positive correlation with CAPD contents.However,the genetic and molecular mechanisms of CBGs specific expressed in the placenta of Capsicum,and high levels of CBGs in high CAPD contents genotypes Capsicum plants are not well understood.In this paper,we applied physiology,genetics,bioinformatics,and molecular biology aimed to uncover the underlying mechanisms of genetic variations associated with CAPD content variations,and the results are as follows.(1)The contents of Cap and Dh Cap at fruit different development stages of two pepper genotypes(i.e.,C.annuum 59 inbred line and C.chinense‘CPI'inbred line)were quantified by the high-performance liquid chromatography(HPLC).Results indicate that content of Cap and Dh Cap in placenta and pericarp of two genotypes were development stage regulated.Between two investigated accessions,the highest content of Cap and Dh Cap was detected at45 DPA.In addition,the content of Cap and Dh Cap in placenta,pericarp and whole fruit of thirty-one genotypes from five Capsicum species i.e.,C.annuum,C.baccatum,C.chinense,C.frutescens,C.pubescens were determined.We found that the CAPD content of placenta,pericarp and whole fruit was varied greatly among interspecies and intraspecies of Capsicum.We found that the content of CAPD in the C.chinense species was significantly higher than that of the other four species,this mainly arose from the pericarp accumulation of large amount of CAPD.The ratio of Cap and Dh Cap was range from 1.7 to 4.8 among the investigated accessions.(2)The extremely pungent female parent 740 inbred line(C.chinense)hybridization with slightly pungent male parent CA1 inbred line(C.annuum)to generate intra-specific populations.We use the high throughput based simplified genome sequencing strategy analyzed the SNPs among the 150 F₂ progenies and constructed a high-density genetic map containing 9038 markers.We phenotyping analysis of CAPD content among 150 progenies,and found the content in our populations showed normal distribution,indicate that CAPD content is a quantitative trait under genetic control.Combined with the high-density genetic map with CAPD content phenotypic data we identified a major QTL on chromosome 7 which refers to as Cap1 could explain 37%?42%of phenotypic variations in populations.We pooled the sequencing data of 20 individuals with extreme opposite trait values for CAPD content in our segregating progeny to perform the bulked segregant analysis(BSA).Results also found the highest amount of SNPs also colocalization with Cap1 loci,indicates it is the main region for control of CAPD content.(3)To further identify the candidate gene within Cap1 region and elucidate its function exert to regulate CAPD contents.We fine mapping delimited Cap1 to 510 kb and identified a 6 genes cluster that encodes MYB family transcription factors(TF).Among these 4 of which sequence identity was higher than 84%.Colinearity analysis indicates that 2 to 4 MYB also presented in the corresponding region of solanaceous plants.Among these six TFs only one MYB,termed as Cap1 was specifically expressed in placenta,and expression pattern highly consistent with CBGs.However,the orthologous of Cap1 from were not or rarely expressed other solanaceous plants.We employed the virus-induced gene silencing(VIGS),yeast one-hybrid(Y1H),dual-luciferase reporter system(DLR),and chromatin immunoprecipitation assay(Ch IP)verified its function in the regulation of CAPD biosynthesis.(4)To uncover the CAPD content variations among different Capsicum species,we determined the correlation of Cap1 transcription level and CAPD contents.The results revealed that Cap1 and key CBGs(i.e.,AMT and AT3)transcription level significant positive correlation with CAPD contents.Analyzing the coding sequence of Cap1 found that SNPs rarely presented in the region encodes the protein DNA binding and transactivation domain.However,we found numerous SNP and In Dels presented in the Cap1 promoter of five domesticated Capsicum species.Promoter activity analysis indicates 1102 to 1292 upstream of Cap1 start codon was a major region for its actives higher than the other four species.Notably,we found normal W-box present in C.chiense species,while this deletion or SNP occurred within this region lead formatted a mutated W-box or W-box deleted from this region directly.Further study indicates this W-box was binding and regulated by placenta-specific WRKY TF WRKY9 lead to the transcription level of Cap1 in C.chinense genotype significantly higher than other species.