Functional loss of pAMT results in biosynthesis of capsinoids, capsaicinoid analogs, in Capsicum annuum cv. CH-19 Sweet.

Capsaicinoids are responsible for the spicy flavor of pungent peppers (Capsicum). The cultivar CH-19 Sweet is a non-pungent pepper mutant derived from a pungent pepper strain, Capsicum annuum CH-19. CH-19 Sweet biosynthesizes capsaicinoid analogs, capsinoids. We determined the genetic and metabolic mechanisms of capsinoid biosynthesis in this cultivar. We analyzed the putative aminotransferase (pAMT) that is thought to catalyze the formation of vanillylamine from vanillin in the capsaicinoid biosynthetic pathway. Enzyme assays revealed that pAMT activity catalyzing vanillylamine formation was completely lost in CH-19 Sweet placenta tissue. RT-PCR analysis showed normal mRNA transcription of the pAMT gene; however, SNP analysis of the cDNA sequence showed a T nucleotide insertion at 1291 bp in the pAMT gene of CH-19 Sweet. This insertion formed a new stop codon, TGA, that prevented normal translation of the gene, and the pAMT protein did not accumulate in CH-19 Sweet as determined using Western blot analysis. We developed a dCAPS marker based on the T insertion in the pAMT gene of CH-19 Sweet, and showed that the pAMT genotype co-segregated with the capsinoid or capsaicinoid fruit phenotype in the F(2) population. The T insertion was not found in other pungent and non-pungent Capsicum lines, suggesting that it is specific to CH-19 Sweet. CH-19 Sweet's pAMT gene mutation is an example of a nonsense mutation in a single gene that alters a secondary metabolite biosynthetic pathway, resulting in the biosynthesis of analogs. The dCAPS marker will be useful in selecting lines with capsinoid-containing fruits in pepper-breeding programs.