Pre-meiotic, 21-nucleotide Reproductive PhasiRNAs Emerged in Seed Plants and Diversified in Flowering Plants

Abstract

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. Here, we describe additional variants of 21-nt reproductive phasiRNAs, including those triggered by miR11308 in wild strawberry, a eudicot, and we validate the presence of this pathway in rose. We 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, our 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.

Conserved and non-conserved triggers of 24-nt reproductive phasiRNAs in eudicots

“…Out of the 11 clusters, six are polycistronic precursors; Figure 1B shows an example of a polycistronic cluster. To study 24-nt phasiRNA expression in columbine, we analyzed sRNAs data derived from vegetative tissues (leaf and root) and reproductive tissues (four unopened bud stages; Pokhrel et al, 2020). We found that mature miR2275 sequences highly accumulate in bud stages two and three (Figure 1C) which represent meiotic/post-meiotic stages of anther development.…”

“…In the genome browser, it is apparent that the first 24-nt phasiRNAs are generated from the cleavage site of aco_can81 and miR2118 (Figure 2E and 2F), respectively. Interestingly, members of both of these miRNA families also trigger production of 21-nt reproductive phasiRNAs in columbine (Pokhrel et al, 2020).…”

“…To investigate the conservation of this pathway, we analyzed sRNA data of vegetative tissues, and anthers from four unopened bud stages of rose (Guo et al, 2019; Pokhrel et al, 2020). We found 112 24- PHAS loci triggered by miR2275 variants, accumulating to the greatest abundance at bud stage one (Figure 5D, Supplemental Table 4).…”

“…miR2275 is deeply conserved in angiosperms, including the characteristic of its polycistronic precursors, and the only known role of miR2275 is the generation of reproductive 24-nt phasiRNAs (Xia et al, 2019). Interestingly, a well-studied miRNA, miR2118/482, known for triggering 21-nt phasiRNAs from both protein-coding and non-coding loci in eudicots and monocots (Zhai et al, 2015b; Bélanger et al, 2020; Pokhrel et al, 2020), has a function in generating 24-nt phasiRNAs in columbine and wild strawberry, adding to the data from Solanaceous species (Xia et al, 2019), barley and wheat (Bélanger et al, 2020) that there are multiple mechanisms for activation of the 24- PHAS pathway independent of miR2275. However, there were few 24- PHAS loci triggered by miR2118/482 compared to the total number 24- PHAS loci in each species.…”

“…However, there were few 24- PHAS loci triggered by miR2118/482 compared to the total number 24- PHAS loci in each species. miRNAs that may be lineage-specific, aco_cand81 in columbine and miR11308 in wild strawberry, trigger both 24- and 21-nt reproductive phasiRNAs (Pokhrel et al, 2020). The 21-nt phasiRNAs are produced by the action of DCL4 while DCL3 and DCL5 are responsible for 24-nt phasiRNA production in eudicots and monocots, respectively.…”