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

PhasiRNAs (phased small interfering RNAs) are a major class of plant small RNAs (sRNA) 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-nucleotide (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 sRNA sequencing and transcriptomic profiling of sRNA-associated genes across 10 developmental stages of anther in Kitaake rice to map associations between expression of the transcripts encoding sRNA-related proteins and accumulation of phasiRNA classes. We identified previously undescribed classes of postmeiotic 21- 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.