The bHLH1-DTX35/DFR module regulates pollen fertility by promoting flavonoid biosynthesis in Capsicum annuum L.

High pollen fertility can ensure the yield and efficiency of breeding work, but factors that affect the fertility of pepper pollen have not been studied extensively. In this work, we screened the reduced pollen fertility 1 () mutant of with reduced pollen fertility and yellow anthers from an EMS (ethyl methanesulfonate)-mutagenized pepper population. Through construction of an population followed by BSA (bulked segregant analysis) mapping and KASP genotyping, we identified as a candidate gene for control of this trait. A G → A mutation at a splice acceptor site in causes a frameshift mutation in the mutant, and the translated protein is terminated prematurely. Previous studies on CabHLH1 have focused on the regulation of flavonoid synthesis. Here, we found that also has an important effect on pollen fertility. Pollen vigor, anther flavonoid content, and seed number were lower in -silenced pepper plants, whereas anther HO and MDA (malondialdehyde) contents were higher. RNA-seq analyses showed that expression of the flavonoid synthesis genes , , and was significantly reduced in anthers of -silenced plants and plants, as was the expression of , a gene related to pollen fertility and flavonoid transport. Yeast one-hybrid and dual-luciferase reporter assays showed that CabHLH1 can directly bind to the promoters of and and activate their expression. These results indicate that CabHLH1 regulates reactive oxygen species homeostasis by promoting the synthesis of anther flavonoids and acts as a positive regulator of pepper pollen fertility.