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Competing Interests: Conflict of interest statement. None declared.

Reproductive phasiRNAs (phased, small interfering RNAs), produced from numerous PHAS loci, are essential for plant anther development. PHAS transcripts are enriched on endoplasmic reticulum-bound ribosomes in maize (Zea mays), but the impact of ribosome binding on phasiRNA biogenesis remains elusive. Through ribosome profiling of maize anthers at 10 developmental stages, we demonstrated that 24-PHAS transcripts are bound by ribosomes, with patterns corresponding to the timing and abundance of 24-PHAS transcripts. Ribosome binding to 24-PHAS transcripts is conserved among different maize inbred lines, with ribosomes enriched upstream of miR2275 target sites. We detected short open reading frames (sORFs) in the ribosome-binding regions of some 24-PHAS transcripts and observed a 3-nt periodicity in most sORFs, but mass spectrometry failed to detect peptides corresponding to the sORFs. Deletion of the entire ribosome-binding region of 24PHAS_NO296 locus eliminated ribosome binding and decreased 24-nt phasiRNA production, without affecting 24PHAS_NO296 transcript levels. In contrast, disrupting only the sORFs in 24PHAS_NO296 did not substantially affect the generation of 24-nt phasiRNAs. A newly formed sORF in these mutants may have re-directed ribosome binding to its transcripts. Overall, these findings demonstrate that sORFs facilitate ribosome binding to 24-PHAS transcripts, thereby promoting phasiRNA biogenesis in meiotic anthers., (© 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.)

P/TGMS (Photo/thermo-sensitive genic male sterile) lines are crucial resources for two-line hybrid rice breeding. Previous studies revealed that slow development is a general mechanism for sterility-fertility conversion of P/TGMS in Arabidopsis. However, the difference in P/TGMS genes between rice and Arabidopsis suggests the presence of a distinct P/TGMS mechanism in rice. In this study, we isolated a novel P/TGMS line, ostms19, which shows sterility under high-temperature conditions and fertility under low-temperature conditions. OsTMS19 encodes a novel pentatricopeptide repeat (PPR) protein essential for pollen formation, in which a point mutation GTA(Val) to GCA(Ala) leads to ostms19 P/TGMS phenotype. It is highly expressed in the tapetum and localized to mitochondria. Under high temperature or long-day photoperiod conditions, excessive ROS accumulation in ostms19 anthers during pollen mitosis disrupts gene expression and intine formation, causing male sterility. Conversely, under low temperature or short-day photoperiod conditions, ROS can be effectively scavenged in anthers, resulting in fertility restoration. This indicates that ROS homeostasis is critical for fertility conversion. This relationship between ROS homeostasis and fertility conversion has also been observed in other tested rice P/TGMS lines. Therefore, we propose that ROS homeostasis is a general mechanism for the sterility-fertility conversion of rice P/TGMS lines., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co-expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage-specific manner. Members of the miR482/miR2118 superfamily and their 21-nucleotide phasiRNAs originating from resistance genes show a robust co-expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage-specific manner, related to modulated hormonal responses and transcription factor expression., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

The origin and functionality of long noncoding RNA (lncRNA) remain poorly understood. Here, we show that multiple quantitative trait loci modulating distinct domestication traits in soybeans are pleiotropic effects of a locus composed of two tandem lncRNA genes. These lncRNA genes, each containing two inverted repeats, originating from coding sequences of the MYB genes, function in wild soybeans by generating clusters of small RNA (sRNA) species that inhibit the expression of their MYB gene relatives through post-transcriptional regulation. By contrast, the expression of lncRNA genes in cultivated soybeans is severely repressed, and, consequently, the corresponding MYB genes are highly expressed, shaping multiple distinct domestication traits as well as leafhopper resistance. The inverted repeats were formed before the divergence of the Glycine genus from the Phaseolus-Vigna lineage and exhibit strong structure-function constraints. This study exemplifies a type of target for selection during plant domestication and identifies mechanisms of lncRNA formation and action., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., Competing Interests: Competing interests statement:Reviewer X. Chen and corresponding author B.C.M. have co-authored editorial and commentary papers in 2022 and 2023, but have not had research collaborations in more than 48 mo.

Duckweeds are among the fastest reproducing plants, able to clonally divide at exponential rates. However, the genetic and epigenetic impact of clonality on plant genomes is poorly understood. 5-methylcytosine (5mC) is a modified base often described as necessary for the proper regulation of certain genes and transposons and for the maintenance of genome integrity in plants. However, the extent of this dogma is limited by the current phylogenetic sampling of land plant species diversity. Here we analyzed DNA methylomes, small RNAs, mRNA-seq, and H3K9me2 histone modification for Spirodela polyrhiza. S. polyrhiza has lost highly conserved genes involved in de novo methylation of DNA at sites often associated with repetitive DNA, and within genes, however, symmetrical DNA methylation and heterochromatin are maintained during cell division at certain transposons and repeats. Consequently, small RNAs that normally guide methylation to silence repetitive DNA like retrotransposons are diminished. Despite the loss of a highly conserved methylation pathway, and the reduction of small RNAs that normally target repetitive DNA, transposons have not proliferated in the genome, perhaps due in part to the rapid, clonal growth lifestyle of duckweeds., Competing Interests: Conflicts of interest. The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2023 Science China Press. Published by Elsevier B.V. All rights reserved.)

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In Arabidopsis thaliana , ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. AGO1 associates to the rough endoplasmic reticulum to conduct miRNA-mediated translational repression, mRNA cleavage, and biogenesis of phased siRNAs. Here, we show that a 37°C heat stress (HS) promotes AGO1 protein accumulation in cytosolic condensates where it colocalizes with components of siRNA bodies and of stress granules. AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which is sufficient to undergo phase separation independently of the presence of SGS3. HS only moderately affects the small RNA repertoire, the loading of AGO1 by miRNAs, and the signatures of target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting or impairing its activity in reprogramming gene expression during stress. Collectively, our work sheds new light on the impact of high temperature on a main effector of RNA silencing in plants., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Plant small RNAs are 21-24 nucleotide, noncoding RNAs that function as regulators in plant growth and development. Colorimetric detection of plant small RNAs was made possible with the introduction of locked nucleic acid probes. However, fluorescent detection of plant small RNAs has been challenging due to the high autofluorescence from plant tissue. Here we report a fluorescent in situ detection method for plant small RNAs. This method can be applied to most plant samples and tissue types and also can be adapted for single-molecule detection of small RNAs with super-resolution microscopy., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Hybrid breeding for increased vigour has been used for over a century to boost agricultural outputs without requiring higher inputs. While this approach has led to some of the most substantial gains in crop productivity, breeding barriers have fundamentally limited soybean (Glycine max) from reaping the benefits of hybrid vigour. Soybean flowers self-pollinate prior to opening and thus are not readily amenable to outcrossing. In this study, we demonstrate that the barnase/barstar male sterility/rescue system can be used in soybean to produce hybrid seeds. By expressing the cytotoxic ribonuclease, barnase, under a tapetum-specific promoter in soybean anthers, we are able to completely block pollen maturation, creating male sterile plants. We show that fertility can be rescued in the F1 generation of these barnase-expressing lines when they are crossed with pollen from plants that express the barnase inhibitor, barstar. Importantly, we found that the successful rescue of male fertility is dependent on the relative dosage of barnase and barstar. When barnase and barstar were expressed under the same tapetum-specific promoter, the F1 offspring remained male sterile. When we expressed barstar under a relatively stronger promoter than barnase, we were able to achieve a successful rescue of male fertility in the F1 generation. This work demonstrates the successful implementation of a biotechnology approach to produce fertile hybrid offspring in soybean., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis., Competing Interests: Conflict of interest statement: J.M.S. is Chief Scientific Officer of Plastomics Inc. J.M.S. and B.C.M. have filed a patent application based on results reported in this paper. The remaining authors declare no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Key Message: We analyzed the evolutionary pattern of cysteine-rich peptides (CRPs) to infer the relationship between CRP copy number and plant ecotype, and the origin of bi-domains CRPs. Plants produce cysteine-rich peptides (CRPs) that have long-lasting broad-spectrum antimicrobial activity to protect themselves from various groups of pathogens. We analyzed 240 plant genomes, ranging from algae to eudicots, and discovered that CRPs are widely distributed in plants. Our comparative genomics results revealed that CRP genes have been amplified through both whole genome and local tandem duplication. The copy number of these genes varied significantly across lineages and was associated with the plant ecotype. This may be due to their resistance to changing pathogenic environments. The conserved and lineage-specific CRP families contribute to diverse antimicrobial activities. Furthermore, we investigated the unique bi-domain CRPs that result from unequal crossover events. Our findings provide a unique evolutionary perspective on CRPs and insights into their antimicrobial and symbiosis characteristics., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Key Message: A QTL associated with BPH resistance at the early seedling stage was identified on chromosome 3. Functional Bph14 in Rathu Heenati was associated with BPH resistance at the early seedling stage. Brown planthopper (BPH; Nilaparvata lugens Stål) is considered the most important rice pest in many Asian countries. Several BPH resistance genes have previously been identified. However, there are few reports of genes specific for BPH resistance at the early seedling stage, a crucial stage for direct-seeding cultivation. In this study, we performed a QTL-seq analysis using two bulks (20 F 2 lines in each bulk) of the F 2 population (n = 300) derived from a cross of Rathu Heenati (RH) × HCS-1 to identify QTL/genes associated with BPH resistance at the early seedling stage. An important QTL was identified on chromosome 3 and Bph14 was identified as a potential candidate gene based on the differences in gene expression and sequence variation when compared with the two parents. All plants in the resistant bulks possessed the functional Bph14 from RH and all plants in the susceptible bulk and HCS-1 contained a large deletion (2703 bp) in Bph14. The functional Bph14 gene of RH appears to be important for BPH resistance at the early seedling stage of rice and could be used in conjunction with other BPH resistance genes in rice breeding programs that confer resistance to BPH at the early and later growth stages., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 as 21- or 24-PHAS loci) have been identified in maize (Zea mays), but whether any of the individual TFs or TF combinations suffice to activate any PHAS locus is unclear. Here, we identified the temporal gene coexpression networks (modules) associated with maize anther development, including two modules highly enriched for the 21- or 24-PHAS loci. 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 with 21-PHAS loci could activate several 21-nucleotide phasiRNA pathway genes but not transcription of 21-PHAS loci. Screens for combinatorial activities of these TFs and, separately, the recently reported putative transcriptional regulators of 24-PHAS loci using single-cell (protoplast) RNA-sequencing, did not detect reproducible activation of either 21-PHAS or 24-PHAS loci. Collectively, our results suggest that the endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductive PHAS loci., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Small RNAs, including microRNAs (miRNAs), phased secondary small interfering RNAs (phasiRNA), and heterochromatic small interfering RNAs (hc-siRNA) are an essential component of gene regulation. To establish a broad potato small RNA atlas, we constructed an expression atlas of leaves, flowers, roots, and tubers of Desiree and Eva, which are commercially important potato ( Solanum tuberosum ) cultivars. All small RNAs identified were observed to be conserved between both cultivars, supporting the hypothesis that small RNAs have a low evolutionary rate and are mostly conserved between lineages. However, we also found that a few miRNAs showed differential accumulation between the two potato cultivars, and that hc-siRNAs have a tissue specific expression. We further identified dozens of reproductive and non-reproductive phasiRNAs originating from coding and noncoding regions that appeared to exhibit tissue-specific expression. Together, this study provides an extensive small RNA profiling of different potato tissues that might be used as a resource for future investigations., Competing Interests: The authors declare no conflicts of interest associated with the work described in this manuscript., (© 2022 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Previous research has identified E. coli populations that persist in freshwater beach sand distinct from fecal pollution events. This work identifies factors that influence the survival of E. coli in sand using laboratory microcosms to replicate beach conditions. Microcosms were deployed to examine the effect of genetic background, competition with native microbial community, and increased nutrient concentrations on E. coli survival. Survival was comparable between the phylotypes B1 and B2, however, deficiency of stress response greatly reduced survival. In the absence of the native community under nutrient conditions comparable to those observed in sand, E. coli cell densities remained within an order of magnitude of initial concentrations after 5 weeks of incubation. Increased nitrogen was associated with decreased decay rates in the first 2 weeks, and increased carbon appeared to provide an advantage at later time points. However, the highest survival was found with the addition of both carbon and nitrogen. Native sand seeded with fresh Cladophora maintained higher concentrations of E. coli, compared to sand containing decayed Cladophora or no Cladophora . Our findings demonstrate persistent E. coli populations in sand can be affected by the availability of carbon and nitrogen, the ability to regulate stress, and the presence of algal mats (i.e., Cladophora ). Further, this work suggests that the native microbial communities may modulate survival by outcompeting E. coli for nutrients. IMPORTANCE Current monitoring for fecal pollution does not account for persistent E. coli populations in freshwater sand, which can result in higher concentrations in water when no threat to human health is present. This work examined the drivers for persistent E. coli populations in sand to aid beach management techniques. We examined the influence of nutrients, including localized sources such as stranded Cladophora , on E. coli populations. We found the major determinant of E. coli survival in freshwater beach sand was the addition of nutrients, specifically carbon and nitrogen concentrations 10-fold higher than baseline concentrations on beaches. This work provides the framework for identifying pollution sources that can promote E. coli survival in sand through the characterization of carbon and nitrogen content, which can be incorporated into beach management techniques. Through this improved knowledge, we can begin to understand E. coli fluctuations in water due to resuspension from sand into water.

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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Duckweeds are the smallest angiosperms, possessing a simple body architecture and highest rates of biomass accumulation. They can grow near-exponentially via clonal propagation. Understanding their reproductive biology, growth, and development is essential to unlock their potential for phytoremediation, carbon capture, and nutrition. However, there is a lack of non-laborious and convenient methods for spatially and temporally imaging an array of duckweed plants and growth conditions in the same experiment. We developed an automated microscopy approach to record time-lapse images of duckweed plants growing in 12-well cell culture plates. As a proof-of-concept experiment, we grew duckweed on semi-solid media with and without sucrose and monitored its effect on their growth over 3 days. Using the PlantCV toolkit, we quantified the thallus area of individual plantlets over time, and showed that L. minor grown on sucrose had an average growth rate four times higher than without sucrose. This method will serve as a blueprint to perform automated high-throughput growth assays for studying the development patterns of duckweeds from different species, genotypes, and conditions., Competing Interests: The authors do not have any conflict of interests to declare., (© 2022 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues ( Hordeum vulgare ), nematode ( Caenorhabditis elegans ) and yeast ( Saccharomyces cerevisiae ). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms., 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 © 2022 Bélanger, Berensmann, Baena, Duncan, Meyers, Narayan and Czymmek.)

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-PHAS precursor 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-PHAS precursor and Dcl5 transcripts 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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

RNA silencing is a conserved mechanism in eukaryotes involved in development and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA- and small interfering RNA-directed silencing, and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 assembles an SCF complex that selectively targets for proteolysis AGO1 when it is unloaded and mutated. Although FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA-silencing activity. Interestingly, under conditions in which small-RNA accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Accordingly, the non-degradable AGO1 protein assembles high-molecular-weight complexes and binds illegitimate small RNA, leading to off-target cleavage. Therefore, control of AGO1 homeostasis by FBW2 plays an important role in quality control of RNA silencing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The spatiotemporal development of somatic tissues of the anther lobe is necessary for successful fertile pollen production. This process is mediated by many transcription factors acting through complex, multi-layered networks. Here, our analysis of functional knockout mutants of interacting basic helix-loop-helix genes Ms23, Ms32, basic helix-loop-helix 122 (bHLH122), and bHLH51 in maize (Zea mays) established that male fertility requires all four genes, expressed sequentially in the tapetum (TP). Not only do they regulate each other, but also they encode proteins that form heterodimers that act collaboratively to guide many cellular processes at specific developmental stages. MS23 is confirmed to be the master factor, as the ms23 mutant showed the earliest developmental defect, cytologically visible in the TP, with the most drastic alterations in premeiotic gene expression observed in ms23 anthers. Notably, the male-sterile ms23, ms32, and bhlh122-1 mutants lack 24-nt phased secondary small interfering RNAs (phasiRNAs) and the precursor transcripts from the corresponding 24-PHAS loci, while the bhlh51-1 mutant has wild-type levels of both precursors and small RNA products. Multiple lines of evidence suggest that 24-nt phasiRNA biogenesis primarily occurs downstream of MS23 and MS32, both of which directly activate Dcl5 and are required for most 24-PHAS transcription, with bHLH122 playing a distinct role in 24-PHAS transcription., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: The authors declare no conflict of interest associated with the work described in this manuscript., (© 2022 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant cells communicate information for the regulation of development and responses to external stresses. A key form of this communication is transcriptional regulation, accomplished via complex gene networks operating both locally and systemically. To fully understand how genes are regulated across plant tissues and organs, high resolution, multi-dimensional spatial transcriptional data must be acquired and placed within a cellular and organismal context. Spatial transcriptomics (ST) typically provides a two-dimensional spatial analysis of gene expression of tissue sections that can be stacked to render three-dimensional data. For example, X-ray and light-sheet microscopy provide sub-micron scale volumetric imaging of cellular morphology of tissues, organs, or potentially entire organisms. Linking these technologies could substantially advance transcriptomics in plant biology and other fields. Here, we review advances in ST and 3D microscopy approaches and describe how these technologies could be combined to provide high resolution, spatially organized plant tissue transcript mapping., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

In this work, we sequenced and annotated the genome of Streptochaeta angustifolia , one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size. We find good collinearity with the rice genome and have captured most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions., 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 © 2021 Seetharam, Yu, Bélanger, Clark, Meyers, Kellogg and Hufford.)

Colletotrichum species are globally distributed and well known as members of a destructive phytopathogenic genus, causing the anthracnose disease in a wide variety of crops and fruits. Colletotrichum sublineola is the causal agent of the anthracnose disease in sorghum, causing losses of up to 50% in yield. Here, we used PacBio sequencing combined with RNA-seq to generate a chromosome-level assembly and annotation of the Colletotrichum sublineola strain CsGL1.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Small RNAs play important roles in plant growth and development by modulating expression of genes and transposons. In many flowering plant species, male reproductive organs, the anthers, produce abundant phased small interfering RNAs (phasiRNAs). Two classes of reproductive phasiRNAs are generally known, mostly from monocots: (i) pre-meiotic 21-nucleotide (nt) phasiRNAs triggered by miR2118 and (ii) meiotic 24-nt phasiRNAs triggered by miR2275. Here, we describe conserved and non-conserved triggers of 24-nt phasiRNAs in several eudicots. We found that the abundant 24-nt phasiRNAs in the basal eudicot columbine (Aquilegia coerulea) are produced by the canonical trigger miR2275, as well as by other non-canonical triggers, miR482/2118 and miR14051. These triggering microRNAs (miRNAs) are localized in microspore mother cells and tapetal cells of meiotic and post-meiotic stage anthers. Furthermore, we identified a lineage-specific trigger (miR11308) of 24-nt phasiRNAs and an expanded number of 24-PHAS loci in wild strawberry (Fragaria vesca). We validated the presence of the miR2275-derived 24-nt phasiRNA pathway in rose (Rosa chinensis). Finally, we evaluated all eudicots that have been validated for the presence of 24-nt phasiRNAs as possible model systems in which to study the biogenesis and function of 24-nt phasiRNAs. We conclude that columbine (Aquilegia coerulea) would be a strong model because of its extensive number of 24-PHAS loci and its diversity of trigger miRNAs., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ . However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers. Graphic abstract: Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands ( i.e. , a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing., Competing Interests: Competing interestsNo competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

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., (© 2021. The Author(s).)

Maize (Zea mays L.) Ufo1-1 is a spontaneous dominant mutation of the unstable factor for orange1 (ufo1). We recently cloned ufo1, which is a Poaceae-specific gene highly expressed during seed development in maize. Here, we have characterized Ufo1-1 and a loss-of-function Ds insertion allele (ufo1-Dsg) to decipher the role of ufo1 in maize. We found that both ufo1 mutant alleles impact sugars and hormones, and have defects in the basal endosperm transfer layer (BETL) and adjacent cell types. The Ufo1-1 BETL had reduced cell elongation and cell wall ingrowth, resulting in cuboidal shaped transfer cells. In contrast, the ufo1-Dsg BETL cells showed a reduced overall size with abnormal wall ingrowth. Expression analysis identified the impact of ufo1 on several genes essential for BETL development. The overexpression of Ufo1-1 in various tissues leads to ectopic phenotypes, including abnormal cell organization and stomata subsidiary cell defects. Interestingly, pericarp and leaf transcriptomes also showed that as compared with wild type, Ufo1-1 had ectopic expression of endosperm development-specific genes. This study shows that Ufo1-1 impacts the expression patterns of a wide range of genes involved in various developmental processes., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)