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  1. Abstract

    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 and 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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    The anther‐enriched phased, small interfering RNAs (phasiRNAs) play vital roles in sustaining male fertility in grass species. Their long non‐coding precursors are synthesized by RNA polymerase II and are likely regulated by transcription factors (TFs). A few putative transcriptional regulators of the 21‐ or 24‐nucleotide phasiRNA loci (referred to as21‐or24‐PHASloci) have been identified in maize (Zea mays), but whether any of the individual TFs or TF combinations suffice to activate anyPHASlocus is unclear. Here, we identified the temporal gene coexpression networks (modules) associated with maize anther development, including two modules highly enriched for the21‐or24‐PHASloci. Comparisons of these coexpression modules and gene sets dysregulated in several reported male sterile TF mutants provided insights into TF timing with regard to phasiRNA biogenesis, including antagonistic roles for OUTER CELL LAYER4 and MALE STERILE23.Trans‐activation assays in maize protoplasts of individual TFs using bulk‐protoplast RNA‐sequencing showed that two of the TFs coexpressed with21‐PHASloci could activate several 21‐nucleotide phasiRNA pathway genes but not transcription of21‐PHASloci. Screens for combinatorial activities of these TFs and, separately, the recently reported putative transcriptional regulators of24‐PHASloci using single‐cell (protoplast) RNA‐sequencing, did not detect reproducible activation of either21‐PHASor24‐PHASloci. Collectively, our results suggest that the endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductivePHASloci.

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  3. Summary

    In maize, 24‐nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known.

    Using laser capture microdissection, we analyzed tapetal cells, meiocytes and other somatic cells at several stages of anther development to establish the timing of 24‐PHASprecursor transcripts and the 24‐nt phasiRNA products.

    By integrating RNA and small RNA profiling plus single‐molecule and small RNA FISH (smFISH or sRNA‐FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24‐PHASprecursor andDcl5transcripts and the resulting 24‐nt phasiRNAs. Interestingly, 24‐nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum.

    Our data support the conclusion that 24‐nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24‐nt phasiRNAs in anther cell types.

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  4. Summary

    Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial–temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive.

    Here, through histological and high‐throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis.

    AGO1d preferentially binds to miR2118‐triggered 21‐nucleotide (nt) phasiRNAs with a 5′‐terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes a reduction of 21‐ and 24‐nt phasiRNAs and temperature‐sensitive male sterility. At lower temperatures, anthers of theago1dmutant predominantly show excessive tapetal cells with little starch accumulation during pollen formation, possibly caused by the dysregulation of cell metabolism.

    These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function.

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  5. Abstract

    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.

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  6. Abstract

    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.

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  7. Summary

    In grasses, two types of phased, small interfering RNAs (phasiRNAs) are expressed largely in young, developing anthers. They are 21 or 24 nucleotides (nt) in length and are triggered by miR2118 or miR2275, respectively. However, most of their functions and activities are not fully understood.

    We performed comparative genomic analysis of their source loci (PHAS) in fiveOryzagenomes and combined this with analysis of high‐throughput sRNA and degradome datasets. In total, we identified 8216 21‐PHASand 626 24‐PHASloci. Local tandem and segmental duplications mainly contributed to the expansion and supercluster distribution of the 21‐PHASloci. Despite their relatively conserved genomic positions,PHASsequences diverged rapidly, except for the miR2118/2275 target sites, which were under strong selection for conservation.

    We found that 21‐nt phasiRNAs with a 5′‐terminal uridine (U) demonstratedcis‐cleavage atPHASprecursors, and thesecis‐acting sites were also variable among close species. miR2118 could trigger phasiRNA production from its own antisense transcript and the derived phasiRNAs might reversibly regulate miR2118 precursors.

    We hypothesised that successful initiation of phasiRNA biogenesis is conservatively maintained, while phasiRNA products diverged quickly and are not individually conserved. In particular, phasiRNA production is under the control of multiple reciprocal regulation mechanisms.

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  8. Summary

    Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescentin situhybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescentin situdetection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetricin situhybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.

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  9. 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 for revised estimates. 
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  10. 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. 
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