Conservation and divergence of reproductive phasiRNAs in flowering plants
Authors:   , Pokhrel Suresh 
Published: May 2022

Plant small RNAs (sRNAs) are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. They have critical roles in most pathways involved in plant growth and reproductive development. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have not been described in eudicots. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. In my dissertation project, I am investigating the origins and diversity in eudicots of these reproductive phasiRNAs and their role in heat stress. Here, I describe unexpected variants of 21-nt reproductive phasiRNAs, including those triggered by miR11308 in wild strawberry, a eudicot, and validate the presence of this pathway in rose. I report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots, with novel biogenesis triggers in the basal eudicot columbine and the rosid flax. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, my work in eudicots supports a singular finding in spruce, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages. Furthermore, I describe conserved and non-conserved triggers of 24-nt phasiRNAs in several eudicots. I find that the abundant 24-nt phasiRNAs in the basal eudicot columbine are produced by the canonical trigger, miR2275, as well as by other non-conserved triggers, miR482/2118 and aco_cand81. These triggering miRNAs are localized in microspore mother cells (MMC) and tapetal cells of meiotic and post-meiotic stage anthers. In addition, I identify a new trigger (miR11308) of 24-nt phasiRNAs and expand the number of 24-PHAS loci in wild strawberry. I also validate the presence of miR2275-derived 24-nt phasiRNAs pathway in rose. Finally, I evaluate all the eudicots that have been validated for the presence of 24-nt phasiRNAs as models to study biogenesis and function of 24-nt phasiRNAs and conclude that columbine would be an excellent model because of its extensive number of 24-PHAS loci and its diversity of trigger miRNAs. Lastly, I identify sRNA-mediated pathways responsive to heat stress in flax. Heat stress affects reproductive development of plants and decreases their productivity. However, underlying molecular mechanisms of heat stress in reproductive development of plants are poorly characterized. Plant sRNAs are implicated to have important regulatory roles in reproductive development of plants under the abiotic stress responses. I aim to comprehensively annotate miRNAs and phasiRNAs spawning genes (PHAS) in flax and characterize their response to heat stress. I identify 173 miRNA genes, of which 42 loci are novel. Differential analysis revealed that 141 miRNA genes differentially expressed between tissue types while 18 miRNA genes were differentially expressed in reproductive tissues due to heat stress including members of miR2118/482 and miR2275, known triggers of reproductive phasiRNAs. Furthermore, I identified 68 21-PHAS loci in flax both from protein coding and non-coding regions. I also identified 4 24-PHAS loci triggered by miR2275 and 658 24-PHAS-like loci with unknown triggers mostly from non-coding regions. The reproductive phasiRNAs are mostly down-regulated due to heat stress. Overall, I found that several previously not reported miRNAs and phasiRNAs are responsive to heat stress in reproductive tissues of flax.