MicroRNAs (miRNAs), short non-coding RNAs, intricately regulate gene expression during plant reproductive development, influencing inflorescence architecture, flower organ identity, flowering time, and yield components in crops. Despite extensive studies in model plants like Arabidopsis thaliana and rice, comprehensive genome-wide information on miRNA functions in barley, a major crop, is elusive. This study characterizes potential miRNAs involved in barley reproduction across key inflorescence developmental stages.
Samples from three critical stages of barley inflorescence development—spikelet initiation, floral organ differentiation, and floral organ growth—were collected, along with a seedling stage. Small RNA sequencing generated 91,926,979 clean reads (18-44 nt), identifying 38 known miRNA families. GO term analysis indicated known miRNA target genes are involved in DNA-templated transcription, RNA biosynthetic processes, and nucleobase-containing compound metabolic processes, emphasizing transcriptional regulation's importance in subsequent stages.
Focusing on miR397, which showed reduced expression in the reproductive phase, we explored the role of hvu-miR397a in barley inflorescence development. Overexpressing hvu-pre-miR397a via the 35S promoter resulted in OXmiR397 lines with pleiotropic phenotypes, including multiple spikes per tiller, male sterility, and an extended pre-senescence period. Notably, OXmiR397 lines displayed sterility in anthers, contrasting with increased seed number and size observed in rice and Arabidopsis.
In conclusion, our study contributes to understanding of miRNA-mediated regulation in barley, emphasizing miR397's crucial role in male reproductive development. It also highlights some conservation of hvu-miR397a's role in both barley and Arabidopsis, with additional implications in barley's male fertility, providing valuable insights into complex regulatory networks governing barley reproductive processes.