CRISPR/Cas9-mediated knockout of GhAMS11 and GhMS188 reveals key roles in tapetal development and pollen exine formation in upland cotton

Abstract

The ABORTED MICROSPORES (AMS) gene is crucial for tapetal cell development and pollen formation, but its role in Upland cotton (Gossypium hirsutum) has not been previously documented. This study identified GhAMS11 as a key transcription factor, with its high expression specifically observed during the S4-S6 stages of anther development, a critical period for tapetal activity and pollen formation. Subcellular localization confirmed that GhAMS11 was located in the nucleus. CRISPR/Cas9 knockout of GhAMS11 resulted in pollen inviability, with mutants displaying abnormal tapetal development and defective pollen exine formation. TUNEL assays highlighted GhAMS11's involvement in proper tapetal programmed cell death (PCD). Additionally, GhAMS11 was found to activate GhMS188 expression, as demonstrated by dual-luciferase assays and EMSA assays, with their interaction confirmed through LCI assays, yeast two-hybrid assays and GST pull down assays. Deletion of GhMS188 led to pollen sterility, grain collapse, and impaired pollen exine formation. Thus, this research identified the bHLH transcription factor GhAMS11, addressing a gap in AMS gene research in Upland cotton, and elucidated its key regulatory role in pollen development in cooperation with GhMS188.

Keywords: Cotton male sterile; Sexine; Tapetum.