Distinct classes of 21 and 24-nt phasiRNAs suggest diverse mechanisms of biogenesis and function in rice anther development

PhasiRNAs (phased small interfering RNAs) are a major class of plant small RNAs known to be key regulators in male reproductive development of maize (Zea mays) and rice (Oryza sativa), among other plants. Earlier research focused primarily on premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNAs, while new studies uncovered a premeiotic class of 24-nt phasiRNAs. The biogenesis and function of these phasiRNAs remain unclear. We conducted an integrative analysis combining small RNA (sRNA) sequencing and transcriptomic profiling of sRNA-associated genes across ten developmental stages of anther in Kitaake rice to map associations between sRNA-related genes and phasiRNA classes. We identified previously undescribed classes of postmeiotic 21-nucleotide (nt) and 24-nt phasiRNA-producing loci and characterized their unique accumulation patterns. Additionally, our findings reveal distinct nucleotide composition and register accumulation among the phasiRNA classes, suggesting the presence of diverse mechanisms of biogenesis and function. Our results provide new insights into the regulatory complexity of phasiRNAs, establishing a foundation for further functional studies and advancing our understanding of their roles in anther development and their underlying mechanisms. Plain Language SummarySmall RNAs play important roles in controlling how plants develop, but their function in plant reproduction is still not well understood. In rice, small RNAs called phasiRNAs help regulate how pollen develops, but past research has mostly focused on just two types: one found before meiosis and one found during meiosis. In this study, we analyzed small RNA activity across ten stages of rice anther development and found new phasiRNA-producing genes that are active after meiosis. We also discovered that different types of phasiRNAs have distinct patterns of accumulation and composition, suggesting that they have specialized roles in reproductive development. These findings reveal new layers of complexity in how small RNAs function and set the stage for future research to understand their exact roles in pollen formation. This work may help improve our understanding of plant fertility and contribute to advancements in crop breeding. Core ideasO_LIStage-specific accumulation patterns suggest more specialized functions of phasiRNAs. C_LIO_LIMeiotic 24-nt phasiRNAs are compositionally distinct from other 24-nt phasiRNAs. C_LIO_LINewly described phasiRNA clusters may differ in mechanism of biogenesis and function. C_LI