Triphasic regulation of ZmMs13 encoding an ABCG transporter is sequentially required for callose dissolution, pollen exine and anther cuticle formation in maize

INTRODUCTION: ATP Binding Cassette G (ABCG) transporters are associated with plant male reproduction, while their regulatory mechanisms underlying anther and pollen development remain largely unknown. OBJECTIVES: Identify and characterize a male-sterility gene ZmMs13 encoding an ABCG transporter in modulating anther and pollen development in maize. METHODS: Phenotypic, cytological observations, and histochemistry staining were performed to characterize the ms13-6060 mutant. Map-based cloning and CRISPR/Cas9 gene editing were used to identify ZmMs13 gene. RNA-seq data and qPCR analyses, phylogenetic and microsynteny analyses, transient dual-luciferase reporter and EMSA assays, subcellular localization, and ATPase activity and lipidomic analyses were carried out to determine the regulatory mechanisms of ZmMs13 gene. RESULTS: Maize ms13-6060 mutant displays complete male sterility with delayed callose degradation, premature tapetal programmed cell death (PCD), and defective pollen exine and anther cuticle formation. ZmMs13 encodes a plasm membrane (PM)- and endoplasmic reticulum (ER)-localized half-size ABCG transporter (ZmABCG2a). The allele of ZmMs13 in ms13-6060 mutant has one amino acid (I311) deletion due to a 3-bp deletion in its fourth exon. The I311 and other conserved amino acid K99 are essential for the ATPase and lipid binding activities of ZmMS13. ZmMs13 is specifically expressed in anthers with three peaks at stages S5, S8b, and S10, which are successively regulated by transcription factors ZmbHLH122, ZmMYB84, and ZmMYB33-1/-2 at these three stages. The triphasic regulation of ZmMs13 is sequentially required for callose dissolution, tapetal PCD and pollen exine development, and anther cuticle formation, corresponding to transcription alterations of callose-, ROS-, PCD-, sporopollenin-, and anther cuticle-related genes in ms13-6060 anthers. CONCLUSION: ms13-6060 mutation with one key amino acid (I311) deletion greatly reduces ZmMS13 ATPase and lipid binding activities and displays multiple effects during maize male reproduction. Our findings provide new insights into molecular mechanisms of ABCG transporters controlling anther and pollen development and male fertility in plants.