The rice microRNA159–SPOROCYTELESS EAR2 module regulates starch biosynthesis during pollen development and maintains male fertility

Abstract Starch is an indispensable energy reserve for pollen and failure of starch biosynthesis in pollen leads to male sterility in flowering crops. Nonetheless, the regulatory mechanisms underlying starch biosynthesis in rice (Oryza sativa) pollen remain unclear. Here, we identified a target of the microRNA OsmiR159, SPOROCYTELESS ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR-ASSOCIATED AMPHIPHILIC-REPRESSION 2 (OsSPEAR2). OsSPEAR2 is predominantly expressed in mature pollen and OsSPEAR2 possesses transcriptional repressor activity and localizes in the nucleus. Disruption of OsSPEAR2 results in severely shrunken pollen grains and male sterility. OsSPEAR2 interacts with multiple OsTCPs, including OsTCP14. OsTCP14 is a target of OsmiR319 and a knockout mutation in OsTCP14 partially rescues the defective pollen phenotype of Osspear2. In addition, transcriptome analyses revealed significant downregulation of numerous genes associated with carbohydrate metabolism, specifically in Osspear2 anthers, including several genes critical for starch biosynthesis. Moreover, OsTCP14 directly represses the expression of the essential starch biosynthesis gene OsUGP2; however, this repression could be alleviated by OsSPEAR2. Noteworthily, embryophyte-specific SPEAR2 and SPOROCYTELESS were also identified as miR159 targets involved in regulating plant growth and development in Arabidopsis (Arabidopsis thaliana), indicating that the miR159-SPEAR regulatory module may be conserved among embryophytes. Collectively, our findings reveal OsmiR159-OsSPEAR2-OsTCP14-OsUGP2 as a regulatory cascade that modulates starch biosynthesis during pollen development in rice.