Positional differences of intronic transposons in pAMT affect the pungency level in chili pepper through altered splicing efficiency

Abstract

Capsaicinoids are unique compounds that give chili pepper fruits their pungent taste. Capsaicinoid levels vary widely among pungent cultivars, ranging from low‐pungency to extremely pungent. However, the molecular mechanisms underlying its quantitative variation have not been elucidated. Our previous study identified various loss‐of‐function alleles of the pAMT gene, which led to low‐pungency. The mutations in these alleles are commonly defined by Tcc transposon insertion and its footprint. In this study, we identified two leaky pamt alleles (pamtL1 and pamtL2) with different levels of pAMT activity. Notably, both alleles had a Tcc transposon insertion in intron 3, but the locations of the insertions within the intron were different. Genetic analysis revealed that pamtL1, pamtL2 and a loss‐of‐function pamt allele reduced capsaicinoid levels to about 50%, 10%, and less than 1%, respectively. pamtL1 and pamtL2 encoded functional pAMT proteins, but they exhibited lower transcript levels compared with the functional‐type. RNA‐seq analysis showed that intronic transposons disrupted splicing in intron 3, which resulted in simultaneous expression of functional pAMT mRNA and non‐functional splice variants containing partial sequences of Tcc. The non‐functional splice variants were more dominant in pamtL2 than that in pamtL1. This suggested that the difference in position of the intronic transposons could alter splicing efficiency, which led to different pAMT activities and reduced capsaicinoid content to different levels. Our results provide a striking example where intronic transposons caused allelic variations, which contributed to quantitative differences in secondary metabolite contents.