In barley, embryogenesis can be induced in the microspore (male gametophyte) to produce a diploid plant that is perfectly homozygous. To change developmental fates in this fashion, microspores need to engage in cellular de-differentiation, interrupting activities leading to pollen formation, and restore totipotency prior to engaging in embryogenesis. In this work, our objective was to characterize the sRNA transcriptome and the degradome of immature barley microspores prior to (day 0) and immediately after (days 2 and 5) the application of a stress pretreatment that induces embryogenesis. A deep sRNA-seq analysis revealed that most of these sRNAs were 24-nt long (19,707;93.8%), the remainder measuring 20, 21 and 22 nt (755; 5.9%). These two classes are typically known as siRNA and miRNA, respectively. As miRNAs are known to play important roles in development, we focused on these and identified a total of 60 miRNA loci (48 known and 12 novel). Among the set of known miRNAs, we observed a total of 20 miRNA families. Among novel miRNAs, 11 unique sequence tags were observed providing candidates for 11 novel miRNA families. An expression analysis identified a total of 12 differentially expressed miRNAs throughout microspore development. A PARE-seq analysis revealed that the transcripts of 148 genes were cleaved by miRNAs over all three time points, of which only 10.8% were shared, suggesting a highly specific miRNA regulation in this system. In response to the pretreatment, we observed the cleavage of transcripts for numerous ARF and PPR genes triggered by miR167 and a novel miRNA, respectively. The transition from the microspore to developing embryo was marked by the cleavage of transcripts for many SPL and GRF genes, respectively triggered by miR156 and miR396. This work sheds light on the miRNA transcriptional changes and their post-transcriptional regulation activities that occur during barley microspore embryogenesis.
The asset you are trying to access is locked. Please enter your access key to unlock.