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Article|03 Aug 2022|OPEN
The bHLH1-DTX35/DFR module regulates pollen fertility by promoting flavonoid biosynthesis in Capsicum annuum L.
Zhishuo Zhang1,2 ,† , Yi Liu1,3 ,† , Qiaoling Yuan1 , Cheng Xiong1 , Hao Xu1 , Bowen Hu1 , Huan Suo1 , Sha Yang1 , Xilin Hou2 , Fang Yuan1 , Zhenming Pei1 and Xiongze Dai1 , Xuexiao Zou1 , Feng Liu,1 ,
1Engineering Research Center of Education Ministry for Germplasm Innovation and Breeding New Varieties of Horticultural Crops, Key Laboratory for Vegetable Biology of Hunan Province, College of Horticulture, Hunan Agricultural University, Changsha 410125, China
2College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
3Longping Branch, Graduate School of Hunan University, Changsha 410125, China
*Corresponding author. E-mail:
Both authors contributed equally to the study.

Horticulture Research 9,
Article number: uhac172 (2022)
Views: 82

Received: 18 Mar 2022
Accepted: 25 Jul 2022
Published online: 03 Aug 2022


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 (rpf1) mutant of Capsicum annuum with reduced pollen fertility and yellow anthers from an EMS (ethyl methanesulfonate)-mutagenized pepper population. Through construction of an F2 population followed by BSA (bulked segregant analysis) mapping and KASP genotyping, we identified CabHLH1 as a candidate gene for control of this trait. A G → A mutation at a splice acceptor site in CabHLH1 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 CabHLH1 also has an important effect on pollen fertility. Pollen vigor, anther flavonoid content, and seed number were lower in CabHLH1-silenced pepper plants, whereas anther H2O2 and MDA (malondialdehyde) contents were higher. RNA-seq analyses showed that expression of the flavonoid synthesis genes DFRANS, and RT was significantly reduced in anthers of CabHLH1-silenced plants and rpf1 plants, as was the expression of DTX35, 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 DTX35 and DFR 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.