PhasiRNAs in Plants: Their Biogenesis, Genic Sources, and Roles in Stress Responses, Development, and Reproduction

Abstract

Phased secondary small interfering RNAs (phasiRNAs) constitute a major category of small RNAs in plants, but most of their functions are still poorly defined. Some phasiRNAs, known as trans-acting siRNAs, are known to target complementary mRNAs for degradation and to function in development. However, the targets or biological roles of other phasiRNAs remain speculative. New insights into phasiRNA biogenesis, their conservation, and their variation across the flowering plants continue to emerge due to the increased availability of plant genomic sequences, deeper and more sophisticated sequencing approaches, and improvements in computational biology and biochemical/molecular/genetic analyses. In this review, we survey recent progress in phasiRNA biology, with a particular focus on two classes associated with male reproduction: 21-nucleotide (accumulate early in anther ontogeny) and 24-nucloetide (produced in somatic cells during meiosis) phasiRNAs. We describe phasiRNA biogenesis, function, and evolution and define the unanswered questions that represent topics for future research.

Figure 1.

Mechanisms That Generate the Phased Patterns of PhasiRNAs. (A) Cleavage is responsible for establishing the phasing pattern. During miRNA-mediated secondary siRNA biogenesis, RDR6, recruited by AGO (with the assistance of SGS3), converts the mRNA substrate into dsRNA, followed by processing by DCL4 or DCL5. Cleavage by the miRNA-AGO complex at a consistent nucleotide position marks the defined initiation point of the resulting siRNAs, establishing the phasing pattern. Conversely, the absence of precise cleavage on the mRNA substrate yields dsRNAs with undefined initiation points, producing out-of-phase siRNAs. The dark-blue “Pac-Man” represents the SKI2-3-8 complex that directs 3′→5′ exonucleolysis, while the light-blue Pac-Man represents a 5′→3′-exoribonuclease (e.g., XRN4). The question mark indicates possible degradation. (B) Alternative phasing patterns. At a locus with a primarily 21-nucleotide (nt) phasing pattern (blue phasing signal in the line plot), the resulting phasiRNAs can target and slice the PHAS/TAS precursor in cis, giving rise to a new round of phasiRNA production, with a phasing pattern (red phasing signal in the line plot) shifted 9 nt relative to the original miRNA-mediated phasing pattern (Tamim et al., 2018). Superposition of two phasing patterns creates a new 12-nt/9-nt phasing pattern. Similarly, from a locus generating a primary 24-nt phasing pattern, phasiRNAs targeting a precursor in cis yield a new 12-nt phasing pattern (Xia et al., 2019).

Figure 2.

Conservation of PhasiRNA Pathways in Land Plants. A phylogenetic tree of representative plant species is shown on the left; major groups in the top left are Lw, liverworts; Ms, mosses; Fe, ferns; Gy, gymnosperms; and BA, basal angiosperms. Components of phasiRNA pathways, including trigger miRNAs, PHAS loci, phasiRNAs, and relevant proteins are listed across the top, grouped by pathways. Below these headings, colored circles indicate that the component is present in the species, while empty circles indicate their absence. A circle with a question mark indicates that the triggering miRNA is still unknown.

Figure 3.

Roles of PhasiRNAs in Plant Stress Responses and Development. Regulatory cascades mediated by phasiRNAs, largely described in this review, are represented as three layers for each pathway, in which the yellow, blue, and pink highlighting indicate the trigger miRNA(s), the primary target transcripts that are the precursors generating phasiRNAs, and the secondary target genes that are regulated by phasiRNAs, respectively.

Figure 4.

Schematic Representation of the Accumulation and Roles of Reproductive PhasiRNAs. (A) to (C) Premeiotic 21-nucleotide (nt) phasiRNAs are dependent on a functional epidermis; OCL4, an epidermis-constrained basic leucine zipper–type transcription factor, and the miR2118 triggers are expressed only in epidermal cells (indicated in dark blue); the 21-nt phasiRNAs are enriched in all cell types (indicated in light blue). The lengths of maize (orange) and rice (brown) anthers at the corresponding stages are shown above each panel. In rice and maize, these cells are among those in three-cell-layer (A) and four-cell-layer (B) anthers. The blue circles in the bottom left corner of each panel indicate the abundance of 21-nt phasiRNAs at each stage, with peak levels in all premeiotic stages (see [B] to [D]); data from Zhai et al. (2015). (D) to (F) Meiotic, 24-nucleotide (nt) phasiRNAs are dependent on a functional tapetal layer (indicated in yellow). These phasiRNAs accumulate during these later stages (abundance indicated by the size of the gold circles in the bottom right corner) and peak in (F); data from Zhai et al. (2015). The miR2275 triggers for 24-nt phasiRNA production are most abundant in the tapetum of premeiotic (1.0 mm in maize) anthers (D) and drop slightly in early meiotic (1.5 mm in maize) anthers (E). The bHLH-type TFs, including MS23 in maize and EAT1 in rice, are expressed specifically in the tapetal cells of early meiotic anthers (E). The 24-PHAS precursors and DCL5 transcripts accumulate in tapetal cells at the same stage (E), with DCL5 protein detected slightly later during this stage (2.0 mm in maize), and are likely constrained to the tapetum (F) when the cell wall of the tapetal cells facing the meiocytes degenerates. (G) to (K) Under male-sterile-inducing conditions, rice line Nongken 58S shows altered mitochondria, ER, and premature PCD in the tapetum during premeiotic to early meiotic stages (G). The absence of MEL1/AGO5 in rice stops meiotic progression before pachytene (H). The absence of DCL5 causes defective tapetal development and male sterility (I). The defective tapetum of 58S and dcl5, and the defective meiocytes of mel1 and dcl5, are indicated in dark gray. Bar in each image = 10 μm. (J) and (K) represent environmentally induced male sterility associated with perturbed reproductive phasiRNAs; in each of the four quadrants in (J) and (K), the x axis indicates the stages of anther development and the y axis indicates the transcript abundance of direct or indirect phasiRNA targets; in both cases, longer days or higher temperatures (to the right) yield male sterility in the absence of regulation by phasiRNAs (bottom right). These phenotypes are rescued when phasiRNAs are unperturbed (top quadrants) or in a slower growth environment (i.e., shorter days or lower temperatures, left quadrants), perhaps due to the suppressive activity of phasiRNAs or in their absence, a factor related to growth conditions. (J) In Nongken 58N rice (fertile), optimal (long-day) and permissive (short-day) growth conditions may cause different rates of metabolism/development/growth with no impact on fertility; SNPs in two 21-PHAS loci in 58S cause male sterility under the same conditions. (K) In a maize dcl5 mutant, typical temperatures or photoperiod conditions for maize growth cause male sterility; lower temperatures or shorter days yield male-fertile mutant plants.