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Several protein families participate in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to the American sweet flag (Acorus americanus), the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained, or further duplicated in subgroups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17, and AGO18. Analyses of nuclear localization signal sequences andmore »catalytic triads of AGO proteins shed light on the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways.

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Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. 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 been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. 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 an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.

Small RNAs play important roles during plant development by regulating transcript levels of target mRNAs, maintaining genome integrity, and reinforcing DNA methylation.Dicer-like 5(Dcl5) is proposed to be responsible for precise slicing in many monocots to generate diverse 24-nt phased, secondary small interfering RNAs (phasiRNAs), which are exceptionally abundant in meiotic anthers of diverse flowering plants. The importance and functions of these phasiRNAs remain unclear. Here, we characterized several mutants ofdcl5, including alleles generated by the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9system and a transposon-disrupted allele. We report thatdcl5mutants have few or no 24-nt phasiRNAs, develop short anthers with defective tapetal cells, and exhibit temperature-sensitive male fertility. We propose that DCL5 and 24-nt phasiRNAs are critical for fertility under growth regimes for optimal yield.

Plant cells accumulate small RNA molecules that regulate plant development, genome stability, and environmental responses. These small RNAs fall into three major classes based on their function and mechanisms of biogenesis—microRNAs, heterochromatic small interfering RNAs, and secondary small interfering RNAs—plus several other less well-characterized categories. Biogenesis of each small RNA class requires a pathway of factors, some specific to each pathway and others involved in multiple pathways. Diverse sequenced plant genomes, along with rapid developments in sequencing, imaging, and genetic transformation techniques, have enabled significant progress in understanding the biogenesis, functions, and evolution of plant small RNAs, including those that had been poorly characterized because they were absent or had low representation in Arabidopsis ( Arabidopsis thaliana). Here, we review recent findings about plant small RNAs and discuss our current understanding of their biogenesis mechanisms, targets, modes of action, mobility, and functions in Arabidopsis and other plant species, including economically important crops. Expected final online publication date for the Annual Review of Plant Biology, Volume 74 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Abstract Plant microRNAs (miRNAs) are short, noncoding RNA molecules that restrict gene expression via posttranscriptional regulation and function in several essential pathways, including development, growth, and stress responses. Accurately identifying miRNAs in populations of small RNA sequencing libraries is a computationally intensive process that has resulted in the misidentification of inaccurately annotated miRNA sequences. In recent years, criteria for miRNA annotation have been refined with the aim to reduce these misannotations. Here, we describe miRador, a miRNA identification tool that utilizes the most up-to-date, community-established criteria for accurate identification of miRNAs in plants. We combined target prediction and Parallel Analysis of RNA Ends (PARE) data to assess the precision of the miRNAs identified by miRador. We compared miRador to other commonly used miRNA prediction tools and found that miRador is at least as precise as other prediction tools while being substantially faster than other tools. miRador should be broadly useful for the plant community to identify and annotate miRNAs in plant genomes.

Abstract Anthers express the most genes of any plant organ, and their development involves sequential redifferentiation of many cell types to perform distinctive roles from inception through pollen dispersal. Agricultural yield and plant breeding depend on understanding and consequently manipulating anthers, a compelling motivation for basic plant biology research to contribute. After stamen initiation, two theca form at the tip, and each forms an adaxial and abaxial lobe composed of pluripotent Layer 1-derived and Layer 2-derived cells. After signal perception or self-organization, germinal cells are specified from Layer 2-derived cells, and these secrete a protein ligand that triggers somatic differentiation of their neighbors. Historically, recovery of male-sterile mutants has been the starting point for studying anther biology. Many genes and some genetic pathways have well-defined functions in orchestrating subsequent cell fate and differentiation events. Today, new tools are providing more detailed information; for example, the developmental trajectory of germinal cells illustrates the power of single cell RNA-seq to dissect the complex journey of one cell type. We highlight ambiguities and gaps in available data to encourage attention on important unresolved issues.

Abstract The Solanaceae or “nightshade” family is an economically important group with remarkable diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the family’s small RNA (sRNA) genomic landscape, we downloaded over 255 publicly available sRNA data sets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major sRNA classes: (1) microRNAs (miRNAs), typically 20- to 22-nucleotide (nt) RNAs generated from a hairpin precursor and functioning in gene silencing and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs and trans-acting siRNAs generated via miRNA-directed cleavage of a polymerase II-derived RNA precursor. Our analyses described thousands of sRNA loci, including poorly understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these sRNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar sRNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities.