RNA sequencing in parthenocarpic chili pepper (Capsicum annuum) fruit reveals genes involved in transcriptional regulation of capsaicinoid biosynthesis genes responsible for the fluctuation of pungency

Abstract

• RNA-sequencing was used to assess transcriptome changes associated with fluctuation of the pungency in chili pepper fruits. • Pungent fluctuation was associated with changes in gene expressions responsible for metabolic pathways around the biosynthesis of capsaicinoids. • Clustering analysis in genes related to transcriptional regulation was performed using disclosed RNA sequencing data. • The nine genes were noted to exhibit expression patterns similar to those of capsaicinoid biosynthesis genes. • Transcript levels in most of these nine genes were proportional to the pepper pungency levels and are inferred to contribute to the fluctuation of pungency. Chili peppers ( Capsicum spp.) are a popular horticultural crop; the characteristic hot taste of chili peppers is the result of a class of compounds called capsaicinoids. In the horticultural production of such peppers, fluctuations in pungency (changes in capsaicinoid content) are an important issue, given that this fluctuation can cause confusion in the distribution or deterioration of pepper quality. Here, we sought to clarify the mechanism of this variation in pungency, specifically by investigating this variation at the transcriptome level. In the present study, we used the pungency-variable sweet chili pepper ‘Shishito’ ( C. annuum ) and created high-pungency seedless fruits by parthenocarpic treatment. RNA-sequencing analysis then was used to comprehensively compare gene expression in these fruits with that in less-pungent seeded fruits. We firstly profiled differentially expressed genes (DEGs) contributing to metabolic pathways involved with capsaicinoid biosynthesis. Subsequently, we employed clustering analysis (using previously published RNA-sequencing data) to explore changes in the expression of genes encoding putative transcription factors that might serve as regulators of the capsaicinoid biosynthesis genes (CABGs). Specifically, we screened nine DEGs ( CaMYB31, WRKY9, TF62308, TF60920, TF62062, TF75838, ACOT9, TF62804 , and TF64623 ) that have gene ontology related to the regulation of transcription, and found that the expression patterns of these DEGs were similar to those of several CABGs. We also employed quantitative reverse transcriptional polymerase chain reaction to investigate the expression (in the pungency-varied fruits) of these DEGs with putative roles in transcription. Accordingly, we observed that the transcript levels in most of these DEGs correlated positively with the amount of capsaicinoid in the placental septum. Therefore, we postulate that these DEGs with putative transcriptional regulatory roles are involved in capsaicinoid biosynthesis, such that the transcript levels of these DEGs may contribute to the fluctuation of pepper pungency.