Brape1 regulates male fertility by influencing the tapetum and pollen wall in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Abstract Key Message：The mutation of Brape1causes male sterility in Chinese cabbage and has been validated by RNAi. The male-sterile line of Chinese cabbage represents an ideal resource to resolve challenges in hybrid seed production by exploiting heterosis. However, the molecular basis for the relationship between the tapetum, pollen wall and male sterility in Chinese cabbage has not been fully elucidated. Here, a male-sterile mutant, M5007, was obtained from the Chinese cabbage ‘FT’ via ethyl methanesulfonate mutagenesis. The tapetum in M5007degraded prematurely, with pollens exhibiting an abnormal concave morphology and a defective exine structure. MutMap sequencing and Kompetitive Allele-Specific PCR sequencing identified BraA09g013200.3.5C as the candidate gene, encoding an acyl-CoA synthetase, involved in pollen exine formation. According to the abnormal pollen exine in M5007, the gene was named Brape1. BrAPE1 and Brape1 were subcellularly localized to the endoplasmic reticulum. qRT-PCR revealed that Brape1 was specifically expressed in buds, but not in flowers, leaves, roots, or stems. The activity of acyl-CoA synthetase was significantly attenuated in M5007 relative to ‘FT’. RNA interference for Brape1 resulted male sterility, with a significant reduction inBrape1 expression levels. Yeast two-hybrid screening of BrAPE1 identified two interacting proteins, BrRGP1 and BrRGP2. The interaction between BrAPE1 and BrRGP1/BrRGP2 was validated through yeast two hybrid screening, luciferase complementation imaging, and pull-down experiments. Transcriptome analysis revealed that the Brape1 mutation affected genes associated with tapetum development and meiosis, while the absence of pollen likely influenced pollen tube gene expression. This study provided new insights into the regulatory mechanisms of male sterility in Chinese cabbage and offered valuable genetic resources for hybrid breeding in Chinese cabbage.