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Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Paclitaxel (trade name Taxol) is a rare diterpenoid with anticancer activity isolated from Taxus. At present, paclitaxel is mainly produced by the semi-synthetic method using extract of Taxus tissues as raw materials. The studies of regulatory mechanisms in paclitaxel biosynthesis would promote the production of paclitaxel through tissue/cell culture approaches. Here, we systematically identified 990 transcription factors (TFs), 460 microRNAs (miRNAs), and 160 phased small interfering RNAs (phasiRNAs) in Taxus chinensis to explore their interactions and potential roles in regulation of paclitaxel synthesis. The expression levels of enzyme genes in cone and root were higher than those in leaf and bark. Nearly all enzyme genes in the paclitaxel synthesis pathway were significantly up-regulated after jasmonate treatment, except for GGPPS and CoA Ligase. The expression level of enzyme genes located in the latter steps of the synthesis pathway was significantly higher in female barks than in male. Regulatory TFs were inferred through co-expression network analysis, resulting in the identification of TFs from diverse families including MYB and AP2. Genes with ADP binding and copper ion binding functions were overrepresented in targets of miRNA genes. The miRNA targets were mainly enriched with genes in plant hormone signal transduction, mRNA surveillance pathway, cell cycle and DNA replication. Genes in oxidoreductase activity, protein-disulfide reductase activity were enriched in targets of phasiRNAs. Regulatory networks were further constructed including components of enzyme genes, TFs, miRNAs, and phasiRNAs. The hierarchical regulation of paclitaxel production by miRNAs and phasiRNAs indicates a robust regulation at post-transcriptional level. Our study on transcriptional and posttranscriptional regulation of paclitaxel synthesis provides clues for enhancing paclitaxel production using synthetic biology technology. [ABSTRACT FROM AUTHOR]

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known. [ABSTRACT FROM AUTHOR]

Summary: MicroRNA482/2118 (miR482/2118) is a 22‐nt miRNA superfamily, with conserved functions in disease resistance and plant development. It usually instigates the production of phased small interfering RNAs (phasiRNAs) from its targets to expand or reinforce its silencing effect. Using a new high‐quality reference genome sequence and comprehensive small RNA profiling, we characterized a newly evolved regulatory pathway of miR482/2118 in litchi. In this pathway, miR482/2118 cleaved a novel noncoding trans‐acting gene (LcTASL1) and triggered phasiRNAs to regulate the expression of gibberellin (GA) receptor gene GIBBERELLIN INSENSITIVE DWARF1 (GID1) in trans; another trans‐acting gene LcTASL2, targeted by LcTASL1‐derived phasiRNAs, produced phasiRNAs as well to target LcGID1 to reinforce the silencing effect of LcTASL1. We found this miR482/2118‐TASL‐GID1 pathway was likely involved in fruit development, especially the seed development in litchi. In vivo construction of the miR482a‐TASL‐GID1 pathway in Arabidopsis could lead to defects in flower and silique development, analogous to the phenotype of gid1 mutants. Finally, we found that a GA‐responsive transcription factor, LcGAMYB33, could regulate LcMIR482/2118 as a feedback mechanism of the sRNA‐silencing pathway. Our results deciphered a lineage‐specifically evolved regulatory module of miR482/2118, demonstrating the high dynamics of miR482/2118 function in plants. [ABSTRACT FROM AUTHOR]

[Display omitted] Small RNAs (sRNAs), found extensively in plants, play an essential role in plant growth and development. Although various sRNA analysis tools have been developed for plants, the use of most of them depends on programming and command-line environments, which is a challenge for many wet-lab biologists. Furthermore, current sRNA analysis tools mostly focus on the analysis of certain type of sRNAs and are resource-intensive, normally demanding an immense amount of time and effort to learn the use of numerous tools or scripts and assemble them into a workable pipeline to get the final results. Here, we present sRNAminer, a powerful stand-alone toolkit with a user-friendly interface that integrates all common functions for the analysis of three major types of plant sRNAs: microRNAs (miRNAs), phased small interfering RNAs (phasiRNAs), and heterochromatic siRNAs (hc-siRNAs). We constructed a curated or "golden" set of MIRNA and PHAS loci, which was used to assess the performance of sRNAminer in comparison to other existing tools. The results showed that sRNAminer outperformed these tools in multiple aspects, highlighting its functionality. In addition, to enable an efficient evaluation of sRNA annotation results, we developed Integrative Genomics Viewer (IGV)-sRNA, a modified genome browser optimized from IGV and we incorporated it as a functional module in sRNAminer. IGV-sRNA can display a wealth of sRNA-specific features, enabling a more comprehensive understanding of sRNA data. sRNAminer and IGV-sRNA are both platform-independent software that can be run under all operating systems. They are now freely available at https://github.com/kli28/sRNAminer and https://gitee.com/CJchen/IGV-sRNA. [ABSTRACT FROM AUTHOR]

Abstract Background Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.

Plants will face increased heat stress due to rising global temperatures. Heat stress affects plant reproductive development and decreases productivity; however, the underlying molecular mechanisms of these processes are poorly characterized. Plant small RNAs (sRNAs) have important regulatory roles in plant reproductive development following abiotic stress responses. We generated sRNA transcriptomes of reproductive bud stages at three different time points to identify sRNA-mediated pathways responsive to heat stress in flax (Linum usitatissimum). With added sRNA transcriptomes of vegetative tissues, we comprehensively annotated miRNA and phasiRNA-encoding genes (PHAS) in flax. We identified 173 miRNA genes, of which 42 are newly annotated. Our analysis revealed that 141 miRNA genes were differentially accumulated between tissue types, while 18 miRNA genes were differentially accumulated in reproductive tissues following heat stress, including members of miR482/2118 and miR2275 families, known triggers of reproductive phasiRNAs. Furthermore, we identified 68 21-PHAS flax loci from protein-coding and noncoding regions, four 24-PHAS loci triggered by miR2275, and 658 24-PHAS-like loci with unknown triggers, derived mostly from noncoding regions. The reproductive phasiRNAs are mostly downregulated in response to heat stress. Overall, we found that several previously unreported miRNAs and phasiRNAs are responsive to heat stress in flax reproductive tissues.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

BackgroundSmall RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action.ResultsWe investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line "B73") and rice (Oryza sativa, cv. "Nipponbare") by high-throughput sequencing of polysome-associated sRNAs and 3' cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy.ConclusionsMBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

Background: Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results: We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion: We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato. [ABSTRACT FROM AUTHOR]

Microspore embryogenesis is a model for developmental plasticity and cell fate decisions. To investigate the role of miRNAs in this development, we sequenced sRNAs and the degradome of barley microspores collected prior to (day 0) and after (days 2 and 5) the application of a stress treatment known to induce embryogenesis. Microspores isolated at these timepoints were uniform in both appearance and in their complements of sRNAs. We detected 68 miRNAs in microspores. The abundance of 51 of these miRNAs differed significantly during microspore development. One group of miRNAs was induced when the stress treatment was applied, prior to being repressed when microspores transitioned to embryogenesis. Another group of miRNAs were up-regulated in day-2 microspores and their abundance remained stable or increased in day-5 microspores, a timepoint at which the first clear indications of the transition toward embryogenesis were visible. Collectively, these miRNAs might play a role in the modulation of the stress response, the repression of gametic development, and/or the gain of embryogenic potential. A degradome analysis allowed us to validate the role of miRNAs in regulating 41 specific transcripts. We showed that the transition of microspores toward the embryogenesis pathway involves miRNA-directed regulation of members of the ARF, SPL, GRF, and HD-ZIPIII transcription factor families. We noted that 41.5% of these targets were shared between day-2 and day-5 microspores while 26.8% were unique to day-5 microspores. The former set may act to disrupt transcripts involved in pollen development while the latter set may drive the commitment to embryogenesis.

Phasic small interfering RNAs are plant secondary small interference RNAs that typically generated by the convergence of miRNAs and polyadenylated mRNAs. A growing number of studies have shown that miRNA-initiated phasiRNA plays crucial roles in regulating plant growth and stress responses. Experimental verification of miRNA-initiated phasiRNA loci may take considerable time, energy and labor. Therefore, computational methods capable of processing high throughput data have been proposed one by one. In this work, we proposed a predictor (DIGITAL) for identifying miRNA-initiated phasiRNAs in plant, which combined a multi-scale residual network with a bi-directional long-short term memory network. The negative dataset was constructed based on positive data, through replacing 60% of nucleotides randomly in each positive sample. Our predictor achieved the accuracy of 98.48% and 94.02% respectively on two independent test datasets with different sequence length. These independent testing results indicate the effectiveness of our model. Furthermore, DIGITAL is of robustness and generalization ability, and thus can be easily extended and applied for miRNA target recognition of other species. We provide the source code of DIGITAL, which is freely available at https://github.com/yuanyuanbu/DIGITAL.

Summary: Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER‐LIKE 4 (DCL4), or DCL5 to produce 21‐ or 24 nucleotide (nt) double‐strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21‐ and 24‐nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5‐A/B becoming non‐functioning and TaRDR6‐A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub‐genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding. [ABSTRACT FROM AUTHOR]

Lignan is an important functional health care substance. At present, research on the molecular mechanism of flax lignan biosynthesis is primarily focused at the transcriptional level, with no research on the regulatory mechanism of small RNAs, particularly phased small interfering RNAs (phasiRNAs) related to lignan content. In this study, we used flax seeds at different developmental stages as materials to construct small RNA libraries and degradation libraries. Eighteen known and 4 new miRNA families containing 40 and 4 miRNA members, respectively, were identified. A total of 104 target genes were identified. Using small RNA data for a genome-wide search, a total of twenty-two 21-nt PHAS loci and twenty-eight 24-nt PHAS loci were obtained. Five miRNAs capable of triggering target genes to produce phasiRNAs were identified. These 5 miRNAs triggered their target genes to produce 39 phasiRNAs. Three complete miRNA-PHAS loci-phasiRNA-target gene functional regulatory pathways were identified. Furthermore, qRT‒PCR confirmed an obvious regulatory relationship among the regulatory pathways. In this study, the molecular regulatory network among flax miRNAs, phasiRNAs and target genes was described from the perspective of lignan synthesis, providing an important scientific basis for the study of flax molecular breeding for high lignan content.

Phased small interfering RNAs (phasiRNAs) are abundantly expressed in anthers and linked to environment-related male fertility in grasses, yet how they function under different environmental conditions remains unclear. Here, we identified a rice (Oryza sativa) low temperature-induced Argonaute (AGO) protein, OsAGO1d, that is responsible for generating phasiRNAs and preserving male fertility at low temperature. Loss of OsAGO1d function causes low-temperature male sterility associated with delayed programmed cell death of tapetal cells during anther development. OsAGO1d binds miR2118 and miR2275 family members and triggers phasiRNA biogenesis; it also binds 21-nt phasiRNAs with a 5′ terminal U. In total, phasiRNAs from 972 loci are OsAGO1d-dependent. OsAGO1d protein moves from anther wall cells into meiocytes, where it loads miR2275 to produce 24-nt phasiRNAs. Together, our results show that OsAGO1d acts as a mobile signal to fine-tune phasiRNA production and this function is important for male fertility at low temperature. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Small RNAs are short non-coding nucleotides and regulate various biological processes in eukaryotes. During past decades, numerous studies have demonstrated the biogenesis of small RNAs and their modes of action in regulating gene expression at the transcriptional and post-transcriptional level. Based on the involvement of genes in biogenesis and function, small RNAs can be classified into three categories, miRNA, hc-siRNA, and phasiRNA. With the continuous advances in cell sorting/isolation technology and next-generation sequencing, recent discoveries provided evidence that all three categories play important roles in reproductive development in plants including model plants and crops. This review summarizes the recent findings on small RNAs, their potential targets, and genes involved in small RNA biogenesis in plant reproductive development and mainly focuses on gametogenesis.

Plants produce small RNAs that accomplish a surprisingly versatile number of functions. The heterogeneity of functions of plant small RNAs is evident at the tissue-specific level. In particular, in the last years, the study of their activity in reproductive tissues has unmasked an unexpected diversity in their biogenesis and roles. Here, we review recent findings about the biogenesis pathways and roles of small RNAs during plant sexual reproduction. [ABSTRACT FROM AUTHOR]

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 the ago1d mutant 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. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs), a group of small noncoding RNAs (approximately 20-24 nucleotides), act as essential regulators affecting endogenous gene expression in plants. MiR482/2118 is a unique miRNA superfamily in plants and represses NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT (NBS-LRR)genesto function in plant resistance to pathogens. In addition, over the past several years, it has been found that miR482/2118 not only targets NBS-LRRs but also acts on other molecular mechanisms to affect plant resistance. miR482/2118- 5ps, phased small interfering RNAs (phasiRNAs) and long noncoding RNAs (lncRNAs) play important roles in plant disease resistance. This review summarizes the current knowledge of the interactions and links between miR482/2118 and its new interacting molecules, miR482/2118-5p, phasiRNAs and lncRNAs, in plant disease resistance. Here, we aim to provide a comprehensive view describing the new molecular mechanism associated with miR482/2118 in the plant immune system. [ABSTRACT FROM AUTHOR]

PhasiRNAs (phased, small interfering RNAs) play a crucial role in supporting male fertility in grasses. Earlier work in maize (Zea mays) and rice (Oryza sativa) - and subsequently many other plant species - identified premeiotic 21-nt and meiotic 24-nt phasiRNAs. More recently, a group of premeiotic 24-nt phasiRNAs was discovered in the anthers of two Pooideae species, barley (Hordeum vulgare) and bread wheat (Triticum aestivum). Whether premeiotic 24-nt phasiRNAs and other classes of reproductive phasiRNAs are conserved across Pooideae species remains unclear. We conducted comparative RNA profiling of three anther stages in six Pooideae species and one Bambusoideae species. We observed complex temporal accumulation patterns of 21-nt and 24-nt phasiRNAs in Pooideae and Bambusoideae grasses. In Bambusoideae, 21-nt phasiRNAs accumulated during meiosis, whereas 24-nt phasiRNAs were present in both premeiotic and postmeiotic stages. We identified premeiotic 24-nt phasiRNAs in all seven species examined. These phasiRNAs exhibit distinct biogenesis mechanisms and potential Argonaute effectors compared to meiotic 24-nt phasiRNAs. We show that specific Argonaute genes co-expressed with stage-specific phasiRNAs are conserved across Bambusoideae and Pooideae species. Our degradome analysis identified a set of conserved miRNA target genes across species, while 21-nt phasiRNA targets were species-specific. Cleavage of few targets was observed for 24-nt phasiRNAs. In summary, this study demonstrates that premeiotic 24-nt phasiRNAs are present across Bambusoideae and Pooideae families, and the temporal accumulation of other classes of 21-nt and 24-nt phasiRNA differs between bamboo and Pooideae species. Furthermore, targets of the three classes of phasiRNAs may be rapidly evolving or undetectable., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Key message: miR6024 acts as a negative regulator of R genes, hence of Tomato plant immunity, and facilitates disease by the necrotrophic pathogen A. solani. Plant resistance genes or Nucleotide-binding leucine-rich repeat (NLR) genes, integral components of plant disease stress-signaling are targeted by variable groups of miRNAs. However, the significance of miRNA-mediated regulation of NLRs during a pathogen stress response, specifically for necrotrophic fungus, is poorly understood. A thorough examination of Tomato NLRs and miRNAs could map substantial interactions of which half the annotated NLRs were targets of Solanaceae-specific and conserved miRNAs, at the NB subdomain. The Solanaceae-specific miR6024 and its NLR targets analysed in different phytopathogenic stresses revealed differential and mutually antagonistic regulation. Interestingly, miR6024-targeted cleavage of a target NLR also triggered the generation of secondary phased siRNAs which could potentially amplify the defense signal. RNA-seq analysis of leaf tissues from miR6024 overexpressing Tomato plants evidenced a perturbation in the defense transcriptome with the transgenics showing unwarranted immune response-related genes' expression with or without infection with necrotrophic Alternaria solani, though no adverse effect could be observed in the growth and development of the transgenic plants. Transgenic plants exhibited constitutive downregulation of the target NLRs, aggravated disease phenotype with an enhanced lesion, greater ROS generation and hypersusceptibility to A. solani infection, thus establishing that miR6024 negatively impacts plant immune response during necrotrophic pathogenesis. Limited knowledge about the outcome of NLR-miRNA interaction during necrotrophic pathogenesis is a hindrance to the deployment of miRNAs in crop improvement programs. With the elucidation of the necrotrophic disease-synergistic role played by miR6024, it becomes a potent candidate for biotechnological manipulation for the rapid development of pathogen-tolerant solanaceous plants. [ABSTRACT FROM AUTHOR]

Summary: Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood.Here, transcriptome and small RNA high‐throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit.Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C‐terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4‐D4(−) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F‐box1, all of which are repressors of MYB10, was observed.We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species. [ABSTRACT FROM AUTHOR]

Abstract Background In plants, microRNAs (miRNAs) are pivotal regulators of plant development and stress responses. Different computational tools and web servers have been developed for plant miRNA target prediction; however, in silico prediction normally contains false positive results. In addition, many plant miRNA target prediction servers lack information for miRNA-triggered phased small interfering RNAs (phasiRNAs). Creating a comprehensive and relatively high-confidence plant miRNA target database is much needed. Results Here, we report TarDB, an online database that collects three categories of relatively high-confidence plant miRNA targets: (i) cross-species conserved miRNA targets; (ii) degradome/PARE (Parallel Analysis of RNA Ends) sequencing supported miRNA targets; (iii) miRNA-triggered phasiRNA loci. TarDB provides a user-friendly interface that enables users to easily search, browse and retrieve miRNA targets and miRNA initiated phasiRNAs in a broad variety of plants. TarDB has a comprehensive collection of reliable plant miRNA targets containing previously unreported miRNA targets and miRNA-triggered phasiRNAs even in the well-studied model species. Most of these novel miRNA targets are relevant to lineage-specific or species-specific miRNAs. TarDB data is freely available at http://www.biosequencing.cn/TarDB . Conclusions In summary, TarDB serves as a useful web resource for exploring relatively high-confidence miRNA targets and miRNA-triggered phasiRNAs in plants.

SUMMARY: Tomato has undergone extensive selections during domestication. Recent progress has shown that genomic structural variants (SVs) have contributed to gene expression dynamics during tomato domestication, resulting in changes of important traits. Here, we performed comprehensive analyses of small RNAs (sRNAs) from nine representative tomato accessions. We demonstrate that SVs substantially contribute to the dynamic expression of the three major classes of plant sRNAs: microRNAs (miRNAs), phased secondary short interfering RNAs (phasiRNAs), and 24‐nucleotide heterochromatic siRNAs (hc‐siRNAs). Changes in the abundance of phasiRNAs and 24‐nucleotide hc‐siRNAs likely contribute to the alteration of mRNA gene expression in cis during tomato domestication, particularly for genes associated with biotic and abiotic stress tolerance. We also observe that miRNA expression dynamics are associated with imprecise processing, alternative miRNA‐miRNA* selections, and SVs. SVs mainly affect the expression of less‐conserved miRNAs that do not have established regulatory functions or low abundant members in highly expressed miRNA families. Our data highlight different selection pressures on miRNAs compared to phasiRNAs and 24‐nucleotide hc‐siRNAs. Our findings provide insights into plant sRNA evolution as well as SV‐based gene regulation during crop domestication. Furthermore, our dataset provides a rich resource for mining the sRNA regulatory network in tomato. Significance Statement: The change of epigenetic regulation during crop domestication, particularly with regard to the dynamic expression and function of small RNAs (sRNAs), has rarely been characterized in a systematic manner. Here, we report the comprehensive comparative investigation of sRNA expression dynamics during tomato domestication, which reveals differential selection pressures over distinct classes of sRNAs and structural variants as a driving force for the dynamic expression of sRNAs in the history of tomato breeding. [ABSTRACT FROM AUTHOR]

Abstract Background Small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action. Results We investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy. Conclusions MBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

Phased small-interfering RNAs (phasiRNAs) are a special class of small RNAs, which are generated in 21- or 24-nt intervals from transcripts of precursor RNAs. Although phasiRNAs have been found in a range of organisms, their biological functions in plants have yet to be uncovered. Here we show that phasiRNAs generated by the photopheriod-sensetive genic male sterility 1 (Pms1) locus were associated with photoperiod-sensitive male sterility (PSMS) in rice, a germplasm that started the two-line hybrid rice breeding. The Pms1 locus encodes a long-noncoding RNA PMS1T that was preferentially expressed in young panicles. PMS1T was targeted by miR2118 to produce 21-nt phasiRNAs that preferentially accumulated in the PSMS line under long-day conditions. A single nucleotide polymorphism in PMS1T nearby the miR2118 recognition site was critical for fertility change, likely leading to differential accumulation of the phasiRNAs. This result suggested possible roles of phasiRNAs in reproductive development of rice, demonstrating the potential importance of this RNA class as regulators in biological processes.

Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway. In this study, we analyzed small RNA and mRNA changes in different stages of spikelets from wild-type rice, and from msp1 and ostdl1a mutants. Analysis of the small RNA data identified miRNAs demonstrating differential abundances. miR2275 was depleted in the two rice mutants; this miRNA is specifically enriched in anthers and functions to trigger the production of 24-nt phased secondary siRNAs (phasiRNAs) from PHAS loci. We observed that the 24-nt phasiRNAs as well as their precursor PHAS mRNAs were also depleted in the two mutants. An analysis of co-expression identified three Argonaute-encoding genes (OsAGO1d, OsAGO2b, and OsAGO18) that accumulate transcripts coordinately with phasiRNAs, suggesting a functional relationship. By mRNA in situ analysis, we demonstrated a strong correlation between the spatiotemporal pattern of these OsAGO transcripts and phasiRNA accumulations.

Abstract Background The non-coding small RNA tRFs (tRNA-derived fragments) and phasiRNAs (plant-specific) exert important roles in plant growth, development and stress resistances. However, whether the tRFs and phasiRNAs respond to the plant important stress hormone abscisic acid (ABA) remain enigma. Results Here, the RNA-sequencing was implemented to decipher the landscape of tRFs and phasiRNAs in tomato (Solanum lycopersicum) leaves and their responses when foliar spraying exogenous ABA after 24 h. In total, 733 tRFs and 137 phasiRNAs were detected. The tRFs were mainly derived from the tRNAAla transporting alanine, which tended to be cleaved at the 5 ’ terminal guanine site and D loop uracil site to produce tRFAla with length of 20 nt. Most of phasiRNAs originated from NBS-LRR resistance genes. Expression analysis revealed that 156 tRFs and 68 phasiRNAs expressed differentially, respectively. Generally, exogenous ABA mainly inhibited the expression of tRFs and phasiRNAs. Furthermore, integrating analysis of target gene prediction and transcriptome data presented that ABA significantly downregulated the abundance of phsaiRNAs associated with biological and abiotic resistances. Correspondingly, their target genes such as AP2/ERF, WRKY and NBS-LRR, STK and RLK, were mainly up-regulated. Conclusions Combined with the previous analysis of ABA-response miRNAs, it was speculated that ABA can improve the plant resistances to various stresses by regulating the expression and interaction of small RNAs (such as miRNAs, tRFs, phasiRNAs) and their target genes. This study enriches the plant tRFs and phasiRNAs, providing a vital basis for further investigating ABA response-tRFs and phasiRNAs and their functions in biotic and abiotic stresses.

Abstract Plants will face increased heat stress due to rising global temperatures. Heat stress affects plant reproductive development and decreases productivity; however, the underlying molecular mechanisms of these processes are poorly characterized. Plant small RNAs (sRNAs) have important regulatory roles in plant reproductive development following abiotic stress responses. We generated sRNA transcriptomes of reproductive bud stages at three different time points to identify sRNA‐mediated pathways responsive to heat stress in flax (Linum usitatissimum). With added sRNA transcriptomes of vegetative tissues, we comprehensively annotated miRNA and phasiRNA‐encoding genes (PHAS) in flax. We identified 173 miRNA genes, of which 42 are newly annotated. Our analysis revealed that 141 miRNA genes were differentially accumulated between tissue types, while 18 miRNA genes were differentially accumulated in reproductive tissues following heat stress, including members of miR482/2118 and miR2275 families, known triggers of reproductive phasiRNAs. Furthermore, we identified 68 21‐PHAS flax loci from protein‐coding and noncoding regions, four 24‐PHAS loci triggered by miR2275, and 658 24‐PHAS‐like loci with unknown triggers, derived mostly from noncoding regions. The reproductive phasiRNAs are mostly downregulated in response to heat stress. Overall, we found that several previously unreported miRNAs and phasiRNAs are responsive to heat stress in flax reproductive tissues.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding. [ABSTRACT FROM AUTHOR]

Since co-suppression was discovered as a pioneer silencing phenomenon of RNA interference (RNAi) in petunia in 1990, many types of small RNAs have been identified in the RNAi pathway among various eukaryotes. In plants, a large number of 21- or 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs) are produced via processing of long RNA precursors by Dicer-like proteins. However, the roles of phasiRNAs remain largely unknown. The development of imaging technology and RNA profiling has clarified the spatiotemporal regulation of phasiRNAs, and subsequently the different functions of 21-nt trans-acting phasiRNAs and 24-nt cis-regulatory phasiRNAs during male organ development. This review focuses on the biogenesis, diversification, spatiotemporal expression pattern and function of phasiRNAs in plants. [ABSTRACT FROM AUTHOR]

In eudicot plants, the miR482/miR2118 superfamily regulates and instigates the production of phased secondary small interfering RNAs (siRNAs) from NB-LRR (nucleotide binding leucine-rich repeat) genes that encode disease resistance proteins. In grasses, this miRNA family triggers siRNA production specifically in reproductive tissues from long noncoding RNAs. To understand this functional divergence, we examined the small RNA population in the ancient gymnosperm Norway spruce (Picea abies). As many as 41 miRNA families in spruce were found to trigger phasiRNA (phased, secondary siRNAs) production from diverse PHAS loci, with a remarkable 19 miRNA families capable of targeting over 750 NB-LRR genes to generate phasiRNAs. miR482/miR2118, encoded in spruce by at least 24 precursor loci, targets not only NB-LRR genes to trigger phasiRNA production (as in eudicots) but also noncoding PHAS loci, generating phasiRNAs preferentially in male or female cones, reminiscent of its role in the grasses. These data suggest a dual function of miR482/miR2118 present in gymnosperms that was selectively yet divergently retained in flowering plants. A few MIR482/MIR2118 precursors possess an extremely long stem-loop structure, one arm of which shows significant sequence similarity to spruce NB-LRR genes, suggestive of an evolutionary origin from NB-LRR genes through gene duplication. We also characterized an expanded miR390-TAS3 (TRANS-ACTING SIRNA GENE 3)-ARF (AUXIN RESPONSIVE FACTOR) pathway, comprising 18 TAS3 genes of diverse features. Finally, we annotated spruce miRNAs and their targets. Taken together, these data expand our understanding of phasiRNA network in plants and the evolution of plant miRNAs, particularly miR482/miR2118 and its functional diversification.

Plants are frequently attacked by a variety of pathogens and thus have evolved a series of defense mechanisms, one important mechanism is resistance gene (R gene)-mediated disease resistance, but its expression is tightly regulated. NBS-LRR genes are the largest gene family of R genes. microRNAs (miRNAs) target to a number of NBS-LRR genes and trigger the production of phased small interfering RNAs (phasiRNAs) from these transcripts. phasiRNAs cis or trans regulate NBS-LRR genes, which can result in the repression of R gene expression. In this study, we screened for upregulated miR482 in the susceptible apple cultivar 'Golden Delicious' (GD) after inoculation with the fungal pathogen Alternaria alternata f. sp. mali (ALT1). Additionally, through combined degradome sequencing, we identified a gene targeted by miR482, named MdTNL1 , a gene encoding a TIR-NBS-LRR (Toll/interleukin1 receptor-nucleotide binding site-leucine-rich repeat) protein. This gene exhibited a significant down-regulation post ALT1 inoculation, suggesting an impact on gene expression mediated by miRNA regulation. miR482 could cleave MdTNL1 and generate phasiRNAs at the cleavage site. We found that overexpression of miR482 inhibited the expression of MdTNL1 and thus reduced the disease resistance of GD, while silencing of miR482 increased the expression of MdTNL1 and thus improved the disease resistance of GD. This work elucidates key mechanisms underlying the immune response to Alternaria infection in apple. Identification of the resistance genes involved will enable molecular breeding for prevention and control of Alternaria leaf spot disease in this important fruit crop. • MdTNL1 belonging to the TIR type significantly down-regulated after infection with ALT1. • miR482 generates phasi RNAs after cleavage of MdTNL1. • miR482- MdTNL1 pathway involved in apple resistance to apple Alternaria leaf spot disease. • Overexpression of MdTNL1 or silencing of miR482 improves apple resistance to Alternaria leaf spot disease. [ABSTRACT FROM AUTHOR]

Abstract Background Argonaute proteins (AGOs) are important players in the regulation of plant development by directing sRNAs to target mRNAs. In maize (Zea mays), AGO18b is a tassel-enriched and grass-specific AGO. Previous studies have shown that AGO18b is highly expressed in tassels during meiosis and negatively regulates determinacy of spikelet meristems. However, binding profile on RNAs and acting mechanisms of AGO18b remain unknown. Results In this study, we explored the binding profile of AGO18b in maize tassel by UV cross-linking RNA immunoprecipitation, followed by deep sequencing of these cDNA libraries (cRIP-seq), and systematically studied AGO18b-associated small RNAs and mRNAs by bioinformatics analysis. By globally analyzing the phased small-interfering RNA (phasiRNA) and miRNA abundance bound by AGO18b, we found AGO18b primarily binds to 21-nt phasiRNAs/miRNAs with a 5′-uridine and binds less strongly to 24-nt phasiRNAs with a 5′-adenosine in the premeiotic tassels. The abundance profile of AGO18b-associated miRNAs was different from their expression profile. Moreover, AGO18b strongly binds to miR166a-3p. We then obtained the AGO18b-bound mRNA targets of miR166a-3p by cRIP-seq, and confirmed the molecular function of AGO18b in regulating spikelet meristems. Conclusions Our results indicated that AGO18b binds to phasiRNAs with obvious 5 prime end bias under different sRNA length. MiRNAs and their target mRNAs associated with AGO18b indicated the molecular mechanisms of AGO18b as a negative regulator of inflorescence meristem and tassel development through integrating both phasiRNAs and miRNA pathways, which extended our view of sRNA regulation in flower development and provided potential methods to control pollination in the future.

Summary: The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, but the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates production of secondary small interfering RNAs (siRNAs) in plants. We show that the double-stranded RNA-binding protein SGS3 interacts directly with the 3′ end of the microRNA in an Argonaute protein, resulting in ribosome stalling. Importantly, microRNA-mediated ribosome stalling correlates positively with efficient production of secondary siRNAs from target mRNAs. Our results illustrate a role of paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.

Key message: Maize Outer cell layer 4 (ocl4) encodes an HD-ZIP IV transcription factor required for robust male fertility and 21-nt phasiRNA biogenesis. ocl4 fertility is favored in warm conditions, and phasiRNAs are partially restored. Environment-sensitive male-sterile plants have been described before and can result from different molecular mechanisms and biological processes, but putative environment-conditioned, transgenerational rescue of their male fertility is a rather new mystery. Here, we report a derivative line of the male-sterile outer cell layer 4 (ocl4) mutant of maize, in which fertility was restored and perpetuated over several generations. Conditioned fertile ocl4 anthers exhibit the anatomical abnormality of a partially duplicated endothecial layer, just like their sterile counterparts. We profiled the dynamics of phased, small interfering RNAs (phasiRNAs) during pre-meiotic development in fully sterile and various grades of semi-fertile ocl4 anthers. The conditioned fertile anthers accumulated significantly higher 21-nt phasiRNAs compared to ocl4 sterile samples, suggesting a partial restoration of phasiRNAs in conditioned fertility. We found that the biogenesis of 21-nt phasiRNAs is largely dependent on Ocl4 at three key steps: (1) production of PHAS precursor transcripts, (2) expression of miR2118 that modulates precursor processing, and (3) accumulation of 21-nt phasiRNAs. [ABSTRACT FROM AUTHOR]

Background: In plants, microRNAs (miRNAs) are pivotal regulators of plant development and stress responses. Different computational tools and web servers have been developed for plant miRNA target prediction; however, in silico prediction normally contains false positive results. In addition, many plant miRNA target prediction servers lack information for miRNA-triggered phased small interfering RNAs (phasiRNAs). Creating a comprehensive and relatively high-confidence plant miRNA target database is much needed. Results: Here, we report TarDB, an online database that collects three categories of relatively high-confidence plant miRNA targets: (i) cross-species conserved miRNA targets; (ii) degradome/PARE (Parallel Analysis of RNA Ends) sequencing supported miRNA targets; (iii) miRNA-triggered phasiRNA loci. TarDB provides a user-friendly interface that enables users to easily search, browse and retrieve miRNA targets and miRNA initiated phasiRNAs in a broad variety of plants. TarDB has a comprehensive collection of reliable plant miRNA targets containing previously unreported miRNA targets and miRNA-triggered phasiRNAs even in the well-studied model species. Most of these novel miRNA targets are relevant to lineage-specific or species-specific miRNAs. TarDB data is freely available at http://www.biosequencing.cn/TarDB. Conclusions: In summary, TarDB serves as a useful web resource for exploring relatively high-confidence miRNA targets and miRNA-triggered phasiRNAs in plants. [ABSTRACT FROM AUTHOR]

Summary: Plant phased small interfering RNAs (phasiRNAs) contribute to robust male fertility; however, specific functions remain undefined. In maize (Zea mays), male sterile23 (ms23), necessary for both 24‐nt phasiRNA precursor (24‐PHAS) loci and Dicer‐like5 (Dcl5) expression, and dcl5‐1 mutants unable to slice PHAS transcripts lack nearly all 24‐nt phasiRNAs.Based on sequence capture bisulfite‐sequencing, we find that CHH DNA methylation of most 24‐PHAS loci is increased in meiotic anthers of control plants but not in the ms23 and dcl5 mutants.Because dcl5‐1 anthers express PHAS precursors, we conclude that the 24‐nt phasiRNAs, rather than just activation of PHAS transcription, are required for targeting increased CHH methylation at these loci.Although PHAS precursors are processed into multiple 24‐nt phasiRNA products, there is substantial differential product accumulation. Abundant 24‐nt phasiRNA positions corresponded to high CHH methylation within individual loci, reinforcing the conclusion that 24‐nt phasiRNAs contribute to increased CHH methylation in cis. [ABSTRACT FROM AUTHOR]

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 five Oryza genomes and combined this with analysis of high‐throughput sRNA and degradome datasets. In total, we identified 8216 21‐PHAS and 626 24‐PHAS loci. Local tandem and segmental duplications mainly contributed to the expansion and supercluster distribution of the 21‐PHAS loci. Despite their relatively conserved genomic positions, PHAS sequences 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) demonstrated cis‐cleavage at PHAS precursors, and these cis‐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. [ABSTRACT FROM AUTHOR]

Correct anther development is essential for male fertility and subsequently agricultural yield. Defects in anther development range from the early stage of stamen formation until the late stage of tapetum degeneration. In particular, the specification of the four distinct somatic layers and the inner sporogenous cells need perfect orchestration relying on precise cell–cell communication. Up to now, several signals, which coordinate the anther´s developmental program, have been identified. Among the known signals are phytohormones, environmental conditions sensed via glutaredoxins, several receptor-like kinases triggered by ligands like MAC1, and small RNAs such as miRNAs and the monocot-prevalent reproductive phasiRNAs. Rather than giving a full review on anther development, here we discuss anther development with an emphasis on mobile elements like ROS/oxygen, secreted proteins and small RNAs (only briefly touching on phytohormones), how they might act and interact, and what the future of this research area might reveal. [ABSTRACT FROM AUTHOR]

Small noncoding RNAs (sRNA/sncRNAs) are generated from different genomic loci and play important roles in biological processes, such as cell proliferation and the regulation of gene expression. Next-generation sequencing (NGS) has provided an unprecedented opportunity to discover and quantify diverse kinds of sncRNA, such as tRFs (tRNA-derived small RNA fragments), phasiRNAs (phased, secondary, small-interfering RNAs), Piwi-interacting RNA (piRNAs) and plant-specific 24-nt short interfering RNAs (siRNAs). However, currently available web-based tools do not provide approaches to comprehensively analyze all of these diverse sncRNAs. This study presents a novel integrated platform, sRNAtools (https://bioinformatics.caf.ac.cn/sRNAtools), that can be used in conjunction with high-throughput sequencing to identify and functionally annotate sncRNAs, including profiling microRNAss, piRNAs, tRNAs, small nuclear RNAs, small nucleolar RNAs and rRNAs and discovering isomiRs, tRFs, phasiRNAs and plant-specific 24-nt siRNAs for up to 21 model organisms. Different modules, including single case, batch case, group case and target case, are developed to provide users with flexible ways of studying sncRNA. In addition, sRNAtools supports different ways of uploading small RNA sequencing data in a very interactive queue system, while local versions based on the program package/Docker/virtureBox are also available. We believe that sRNAtools will greatly benefit the scientific community as an integrated tool for studying sncRNAs. [ABSTRACT FROM AUTHOR]

Paclitaxel (trade name Taxol) is a rare diterpenoid with anticancer activity isolated from Taxus . At present, paclitaxel is mainly produced by the semi-synthetic method using extract of Taxus tissues as raw materials. The studies of regulatory mechanisms in paclitaxel biosynthesis would promote the production of paclitaxel through tissue/cell culture approaches. Here, we systematically identified 990 transcription factors (TFs), 460 microRNAs (miRNAs), and 160 phased small interfering RNAs (phasiRNAs) in Taxus chinensis to explore their interactions and potential roles in regulation of paclitaxel synthesis. The expression levels of enzyme genes in cone and root were higher than those in leaf and bark. Nearly all enzyme genes in the paclitaxel synthesis pathway were significantly up-regulated after jasmonate treatment, except for GGPPS and CoA Ligase . The expression level of enzyme genes located in the latter steps of the synthesis pathway was significantly higher in female barks than in male. Regulatory TFs were inferred through co-expression network analysis, resulting in the identification of TFs from diverse families including MYB and AP2. Genes with ADP binding and copper ion binding functions were overrepresented in targets of miRNA genes. The miRNA targets were mainly enriched with genes in plant hormone signal transduction, mRNA surveillance pathway, cell cycle and DNA replication. Genes in oxidoreductase activity, protein-disulfide reductase activity were enriched in targets of phasiRNAs. Regulatory networks were further constructed including components of enzyme genes, TFs, miRNAs, and phasiRNAs. The hierarchical regulation of paclitaxel production by miRNAs and phasiRNAs indicates a robust regulation at post-transcriptional level. Our study on transcriptional and posttranscriptional regulation of paclitaxel synthesis provides clues for enhancing paclitaxel production using synthetic biology technology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Jia, Chen, Chen, Guo, Fu and Xue.)

Gene regulation involves the orchestrated action of multiple regulators to fine-tune the expression of genes. Hierarchical interactions and co-regulation among regulators are commonly observed in biological systems, leading to complex regulatory networks. Small RNA (sRNAs) have been shown to be important regulators of gene expression due to their involvement in multiple cellular processes. In plants, microRNA (miRNAs) and phased small interfering RNAs (phasiRNAs) correspond to two well-characterized types of sRNAs involved in the regulation of posttranscriptional gene expression, although information about their targets and interactions with other gene expression regulators is limited. We describe an extended sRNA-mediated regulatory network in Arabidopsis thaliana that provides a reference frame to understand sRNA biogenesis and activity at the genome-wide level. This regulatory network combines a comprehensive evaluation of phasiRNA production and sRNA targets supported by degradome data. The network includes ~17% of genes in the A. thaliana genome, representing ~50% annotated gene ontology (GO) functional categories. Approximately 14% of genes with GO annotations corresponding to regulation of gene expression were found to be under sRNA control. The unbiased bioinformatic approach used to produce the network was able to detect 107 PHAS loci (regions of phasiRNA production), 5,047 active phasiRNAs (~70% of which were non-canonical), and reconstruct 17 regulatory modules resulting from complex regulatory interactions between different sRNA-regulatory pathways. Known regulatory modules like miR173-TAS-PPR/TPR and miR390-TAS3-ARF/F-box were faithfully reconstructed and expanded, illustrating the accuracy and sensitivity of the methods and providing confidence for the validity of findings of previously unrecognized modules. The network presented here includes a 2X increase in the number of identified PHAS loci, a large complement (~70%) of non-canonical phasiRNAs, and the most comprehensive evaluation of sRNA cleavage activity in A. thaliana to date. Structural analysis showed similarities to networks of other biological systems and demonstrated connectivity between phasiRNA regulatory modules with extensive co-regulation of transcripts by miRNAs and phasiRNAs. The described regulatory network provides a reference that will facilitate global analyses of individual plant regulatory programs such as those that control homeostasis, development, and responses to biotic and abiotic environmental changes.

Background: The non-coding small RNA tRFs (tRNA-derived fragments) and phasiRNAs (plant-specific) exert important roles in plant growth, development and stress resistances. However, whether the tRFs and phasiRNAs respond to the plant important stress hormone abscisic acid (ABA) remain enigma. Results: Here, the RNA-sequencing was implemented to decipher the landscape of tRFs and phasiRNAs in tomato (Solanum lycopersicum) leaves and their responses when foliar spraying exogenous ABA after 24 h. In total, 733 tRFs and 137 phasiRNAs were detected. The tRFs were mainly derived from the tRNAAla transporting alanine, which tended to be cleaved at the 5'terminal guanine site and D loop uracil site to produce tRFAla with length of 20 nt. Most of phasiRNAs originated from NBS-LRR resistance genes. Expression analysis revealed that 156 tRFs and 68 phasiRNAs expressed differentially, respectively. Generally, exogenous ABA mainly inhibited the expression of tRFs and phasiRNAs. Furthermore, integrating analysis of target gene prediction and transcriptome data presented that ABA significantly downregulated the abundance of phsaiRNAs associated with biological and abiotic resistances. Correspondingly, their target genes such as AP2/ERF, WRKY and NBS-LRR, STK and RLK, were mainly up-regulated. Conclusions: Combined with the previous analysis of ABA-response miRNAs, it was speculated that ABA can improve the plant resistances to various stresses by regulating the expression and interaction of small RNAs (such as miRNAs, tRFs, phasiRNAs) and their target genes. This study enriches the plant tRFs and phasiRNAs, providing a vital basis for further investigating ABA response-tRFs and phasiRNAs and their functions in biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]