Abstract

Capsaicinoids are alkaloids, mainly synthesized in the placental tissues of pepper fruit, which render a pungent flavor to it. Fruit development is one of the key endogenous processes influencing the biosynthesis and accumulation of capsaicinoids. Nevertheless, a systematic understanding of the molecular mechanism governing the production of capsaicinoids during fruit development has not yet been established. Homeodomain-leucine zipper (HD-ZIP) transcription factors (TFs) are key regulators of plant growth, development, and secondary metabolism. In the present study, we employed transcriptomic data and capsaicinoid accumulation profiles of placentae from pepper fruits collected across developmental stages to analyze the expression patterns of HD-ZIP TFs. We found that CaHAT5 , an HD-ZIP transcription factor gene, correlated most significantly with capsaicinoid levels and used it as a key candidate for further functional research. Transient overexpression of CaHAT5 in pepper fruits significantly upregulated the capsaicinoid synthase gene, CaAT3 , and increased the capsaicinoid content. In contrast, virus-induced silencing of CaHAT5 resulted in significant downregulation of CaAT3 and decrease in the capsaicinoid content. Yeast one-hybrid, dual-luciferase reporter, and electrophoretic mobility shift assays revealed that CaHAT5 could directly bind to the CaAT3 promoter region and enhanced its transcription. Overall, this study showed that CaHAT5 positively regulates CaAT3 expression during fruit development, thereby promoting capsaicinoid biosynthesis. These findings provide theoretical support for a CaHAT5-targeted molecular breeding approach aimed at improving the pepper quality.