Search

ABSTRACT Modifications to RNA have recently been recognized as a pivotal regulator of gene expression in living organisms. More than 170 chemical modifications have been identified in RNAs, with N6‐methyladenosine (m6A) being the most abundant modification in eukaryotic mRNAs. The addition and removal of m6A marks are catalyzed by methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively. In addition, the m6A marks in mRNAs are recognized and interpreted by m6A‐binding proteins (referred to as “readers”), which regulate the fate of mRNAs, including stability, splicing, transport, and translation. Therefore, exploring the mechanism underlying the m6A reader‐mediated modulation of RNA metabolism is essential for a much deeper understanding of the epigenetic role of RNA modification in plants. Recent discoveries have improved our understanding of the functions of m6A readers in plant growth and development, stress response, and disease resistance. This review highlights the latest developments in m6A reader research, emphasizing the diverse RNA‐binding domains crucial for m6A reader function and the biological and cellular roles of m6A readers in the plant response to developmental and environmental signals. Moreover, we propose and discuss the potential future research directions and challenges in identifying novel m6A readers and elucidating the cellular and mechanistic role of m6A readers in plants. [ABSTRACT FROM AUTHOR]

Numerous invasive viral infections pose a growing threat to rice yield. Rice viruses encode multifunctional effector proteins to evade or counter plant immunity and eventually establish a successful infection. Phytohormones play an important role in the innate immune system, restricting broad-spectrum viral infections. Recent advances have revealed similar pathogenic strategies that have evolved convergently from several distinct plant RNA viruses by dampening host phytohormone signaling networks. Plants have developed very diverse strategies to defend themselves against viral pathogens, among which plant hormones play pivotal roles. In response, some viruses have also deployed multifunctional viral effectors that effectively hijack key component hubs to counter or evade plant immune surveillance. Although significant progress has been made toward understanding counter-defense strategies that manipulate plant hormone regulatory molecules, these efforts have often been limited to an individual virus or specific host target/pathway. This review provides new insights into broad-spectrum antiviral responses in rice triggered by key components of phytohormone signaling, and highlights the common features of counter-defense strategies employed by distinct rice-infecting RNA viruses. These strategies involve the secretion of multifunctional virulence effectors that target the sophisticated phytohormone system, dampening immune responses by engaging with the same host targets. Additionally, the review provides an in-depth exploration of various viral effectors, emphasizing tertiary structure-based research and shared host targets. Understanding these conserved characteristics in detail may pave the way for molecular drug design, opening new opportunities to enhance broad-spectrum antiviral trials through precise engineering. [ABSTRACT FROM AUTHOR]

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) is a high-tech method for characterizing the expression patterns of heterogeneous cells in the same tissue and has changed our evaluation of biological systems by increasing the number of individual cells analyzed. However, the full potential of scRNA-seq, particularly in plant science, has not yet been elucidated. To explore the utilization of scRNA-seq technology in plants, we firstly conducted a comprehensive review of significant scRNA-seq findings in the past few years. Secondly, we introduced the research and applications of scRNA-seq technology to plant tissues in recent years, primarily focusing on model plants, crops, and wood. We then offered five databases that could facilitate the identification of distinct expression marker genes for various cell types. Finally, we analyzed the potential problems, challenges, and directions for applying scRNA-seq in plants, with the aim of providing a theoretical foundation for the better use of this technique in future plant research. [ABSTRACT FROM AUTHOR]

Virus-induced drought tolerance presents a fascinating facet of biotic–abiotic interaction in plants, yet its molecular intricacies remain unclear. Our study shows that cowpea mild mottle virus (CPMMV) infection enhances drought tolerance in common bean (Phaseolus vulgaris) plants through a virus-derived small interfering RNA (vsiRNA)-activated autophagy pathway. Specifically, a 21 nt vsiRNA originating from the CPMMV Triple Gene Block1 (TGB1) gene targeted the 5′ untranslated region (UTR) of the host Teosinte branched 1, Cycloidea, Proliferating Cell Factor (TCP) transcription factor gene PvTCP2, independent of the known role of TGB1 as an RNA silencing suppressor. This targeting attenuated the expression of PvTCP2, which encodes a transcriptional repressor, and in turn upregulated the core autophagy-related gene (ATG) PvATG8c, leading to activated autophagy activity surpassing the level induced by drought or CPMMV infection alone. The downstream EARLY RESPONSIVE TO DEHYDRATION (ERD) effector PvERD15 is a homologue of Arabidopsis thaliana AtERD15, which positively regulates stomatal aperture. PvERD15 was degraded in PvATG8c-mediated autophagy. Therefore, we establish a TGB1-PvTCP2-PvATG8c-PvERD15 module as a trans-kingdom fine-tuning mechanism that contributes to virus-induced drought tolerance in plant–drought–virus interactions. A virus-derived small interfering RNA targets a plant transcriptional repressor, which results in the activation of autophagy, ultimately enhancing drought tolerance in common bean. [ABSTRACT FROM AUTHOR]

CANTATAdb 3.0 is an updated database of plant long non-coding RNAs (lncRNAs), containing 571,688 lncRNAs identified across 108 species, including 100 Magnoliopsida (flowering plants), a significant expansion from the previous version. A notable feature is the inclusion of 112,980 lncRNAs that are expressed specifically in certain plant organs or embryos, indicating their potential role in development and organ-specific processes. In addition, CANTATAdb 3.0 includes 74,886 pairs of evolutionarily conserved lncRNAs found across 47 species and inferred from genome–genome alignments as well as conserved lncRNAs obtained using a similarity search approach in 5,479 species pairs, which would further aid in the selection of lncRNAs for functional studies. Interestingly, we find that conserved lncRNAs with tissue-specific expression patterns tend to occupy the same plant organ across different species, pointing toward conserved biological roles. The database now offers extended search capabilities and downloadable data in popular formats, further facilitating research on plant lncRNAs. [ABSTRACT FROM AUTHOR]

Gene-editing technology, specifically the CRISPR-Cas13a system, has shown promise in breeding plants resistant to RNA viruses. This system targets RNA and, theoretically, can also combat RNA-based viroids. To test this, the CRISPR-Cas13a system was introduced into tomato plants via transient expression and into Nicotiana benthamiana through transgenic methods, using CRISPR RNAs (crRNAs) targeting the conserved regions of both sense and antisense genomes of potato spindle tuber viroid (PSTVd). In tomato plants, the expression of CRISPR-Cas13a and crRNAs substantially reduced PSTVd accumulation and alleviated disease symptoms. In transgenic N. benthamiana plants, the PSTVd levels were lower as compared to wild-type plants. Several effective crRNAs targeting the PSTVd genomic RNA were also identified. These results demonstrate that the CRISPR-Cas13a system can effectively target and combat viroid RNAs, despite their compact structures. [ABSTRACT FROM AUTHOR]

Summary: Positive‐strand RNA viruses co‐opt organellar membranes for biogenesis of viral replication organelles (VROs). Tombusviruses also co‐opt pro‐viral cytosolic proteins to VROs. It is currently not known what type of molecular organization keeps co‐opted proteins sequestered within membranous VROs.In this study, we employed tomato bushy stunt virus (TBSV) and carnation Italian ringspot virus (CIRV) – Nicotiana benthamiana pathosystems to identify biomolecular condensate formation in VROs.We show that TBSV p33 and the CIRV p36 replication proteins sequester glycolytic and fermentation enzymes in unique condensate substructures associated with membranous VROs. We find that p33 and p36 form droplets in vitro driven by intrinsically disordered region. The replication protein organizes partitioning of co‐opted host proteins into droplets. VRO‐associated condensates are critical for local adenosine triphosphate production to support energy for virus replication. We find that co‐opted endoplasmic reticulum membranes and actin filaments form meshworks within and around VRO condensates, contributing to unique composition and structure. We propose that p33/p36 organize liquid–liquid phase separation of co‐opted concentrated host proteins in condensate substructures within membranous VROs.Overall, we demonstrate that subverted membranes and condensate substructures co‐exist and are critical for VRO functions. The replication proteins induce and connect the two substructures within VROs. See also the Commentary on this article by May, 243: 1636–1638. [ABSTRACT FROM AUTHOR]

Summary RNA‐dependent RNA Polymerases (RdRPs) synthesize double‐stranded RNA (dsRNA) from a single‐stranded RNA (ssRNA) template. In plants, dsRNAs produced by RdRPs can be further processed into small interfering RNA (siRNAs) with different lengths, ranging from 21 to 24 nucleotides (nt). These siRNAs play a pivotal role in various biological processes, including antiviral responses, transposable elements silencing, DNA methylation, and the regulation of plant reproduction and development. Recent research has reported significant progress in uncovering the molecular mechanisms of plant RNA‐DEPENDENT RNA POLYMERASE 2 (RDR2), a representative RdRP involved in the RNA‐directed DNA methylation (RdDM) pathway. These discoveries provide a molecular basis underlying the principles of RdRP function and offer insights into potential advancements in crop breeding and antiviral defense strategies. [ABSTRACT FROM AUTHOR]

The epitranscriptomic mark N6-methyladenosine (m6A) is the most common type of messenger RNA (mRNA) post-transcriptional modification in eukaryotes. With the discovery of the demethylase FTO (FAT MASS AND OBESITYASSOCIATED PROTEIN) in Homo Sapiens, this modification has been proven to be dynamically reversible. With technological advances, research on m6A modification in plants also rapidly developed. m6A modification is widely distributed in plants, which is usually enriched near the stop codons and 3'-UTRs, and has conserved modification sequences. The related proteins of m6A modification mainly consist of three components: methyltransferases (writers), demethylases (erasers), and reading proteins (readers). m6A modification mainly regulates the growth and development of plants by modulating the RNA metabolic processes and playing an important role in their responses to environmental signals. In this review, we briefly outline the development of m6A modification detection techniques; comparatively analyze the distribution characteristics of m6A in plants; summarize the methyltransferases, demethylases, and binding proteins related to m6A; elaborate on how m6A modification functions in plant growth, development, and response to environmental signals; and provide a summary and outlook on the research of m6A in plants. [ABSTRACT FROM AUTHOR]

Plant organelle transcription has been studied for decades. As techniques advanced, so did the fields of mitochondrial and plastid transcriptomics. The current view is that organelle genomes are pervasively transcribed, irrespective of their size, content, structure, and taxonomic origin. However, little is known about the nature of organelle noncoding transcriptomes, including pervasively transcribed noncoding RNAs (ncRNAs). Next‐generation sequencing data have uncovered small ncRNAs in the organelles of plants and other organisms, but long ncRNAs remain poorly understood. Here, we argue that publicly available third‐generation long‐read RNA sequencing data from plants can provide a fine‐tuned picture of long ncRNAs within organelles. Indeed, given their bloated architectures, plant mitochondrial genomes are well suited for studying pervasive transcription of ncRNAs. Ultimately, we hope to showcase this new avenue of plant research while also underlining the limitations of the proposed approach. [ABSTRACT FROM AUTHOR]

Summary: RNA interference (RNAi) has emerged as an efficient technology for pest control by silencing the essential genes of targeted insects. Owing to its nucleotide sequence‐guided working mechanism, RNAi has a high degree of species‐specificity without impacts on non‐target organisms. However, as plants are inevitably under threat by two or more insect pests in nature, the species‐specific mode of RNAi‐based technology restricts its wide application for pest control. In this study, we artificially designed an intermediate dsRNA (iACT) targeting two β‐Actin (ACT) genes of sap‐sucking pests Bemisia tabaci and Myzus persicae by mutual correction of their mismatches. When expressing hairpin iACT (hpiACT) from tobacco nuclear genome, transgenic plants are well protected from both B. tabaci and M. persicae, either individually or simultaneously, as evidenced by reduced fecundity and suppressed ACT gene expression, whereas expression of hpRNA targeting BtACT or MpACT in transgenic tobacco plants could only confer specific resistance to either B. tabaci or M. persicae, respectively. In sum, our data provide a novel proof‐of‐concept that two different insect species could be simultaneously controlled by artificial synthesis of dsRNA with sequence optimization, which expands the range of transgenic RNAi methods for crop protection. [ABSTRACT FROM AUTHOR]

Plant viruses may behave differently in glasshouse than in nature, especially those that are naturally transmitted by vector. Biological assays, as the oldest and still used virus detection methods, involve inoculation of host plant and monitoring symptoms in glass- or green-house. While maintaining Peanut stunt virus (PSV) in glasshouse we came across with symptom attenuation of PSV strain B (PSV-B) in Nicotiana benthamiana after eight successive passages. To determine if any changes have occurred in the PSV-B genome, the virus particles were purified from the plant and subjected to RNA extraction with phenol/ chloroform to remove the virus coat protein (CP), and followed by precipitation with ethanol to purify the virus RNAs. Then, electrophoresis of the virus RNAs on agarose gel showed that the RNA3 component of the PSV-B isolate was longer than that in the original reference PSV-B, suggesting occurrence of insertion(s) in the RNA3. To confirm this observation, the full length cDNA corresponding to the RNA3 was synthesized by RT-PCR with the use of the 5' and 3' terminal primers which were designed according to sequences of the PSV RNA3 terminal ends (accession PSU31366). The RT-PCR product was subjected to dideoxy cycle sequencing and the sequence data was compared with the corresponding PSV RNA3 sequences. This revealed occurrence of a 183- nucleotide repeat at the 3' untranslated region of the RNA3 and suggested an association between this mutation (repeat) and the symptom attenuation. This emphasizes that in glasshouse studies, sequential passages of a virus can lead to emergence of mutants. This observation also has implication as to plant protection because the mutated variant may be used in cross protection. [ABSTRACT FROM AUTHOR]

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis. Author summary: The symmetrical structures of plant RNA viruses are usually composed of numerous identical coat protein (CP) subunits encapsidated one molecule of viral genomic RNA. Interestingly, a large number of rod-shaped and filamentous plant viruses encode a cysteine-rich protein (CRP), which usually function as viral suppressors of RNA silencing and pathogenicity determinants during viral infection. However, their roles in viral life cycle remain poorly understood. Here, our study reveals the essential role of CRPs in virus morphogenesis. The cysteine-rich γb protein binds to the surface of rod-shaped barley stripe mosaic virus (BSMV) virion by interacting with CP in a Zn2+-dependent manner, thereby facilitating virion assembly and stability. Both His-85 site in γb predicted to generate a potential CCCH motif and His-13 site in CP exposed to the surface of the virions are key amino acids in γb-CP interaction and the zinc-binding activity. Attractively, the phenomenon of CRPs being tightly bound to their rod-shaped virions occurs in a broad range of distantly related viruses, implying a conserved role for the CRPs in orchestrating virus morphogenesis. [ABSTRACT FROM AUTHOR]

Plant viruses cause substantial losses in crop yield and quality; therefore, devising new, robust strategies to counter viral infections has important implications for agriculture. Virus inhibitory protein endoplasmic reticulum-associated interferoninducible (Viperin) proteins are conserved antiviral proteins. Here, we identified a set of Viperin and Viperin-like proteins from multiple species and tested whether they could interfere with RNA viruses in planta. Our data from transient and stable overexpression of these proteins in Nicotiana benthamiana reveal varying levels of interference against the RNA viruses tobacco mosaic virus (TMV), turnip mosaic virus (TuMV), and potato virus x (PVX). Harnessing the potential of these proteins represents a novel avenue in plant antiviral approaches, offering a broader and more effective spectrum for application in plant biotechnology and agriculture. Identifying these proteins opens new avenues for engineering a broad range of resistance to protect crop plants against viral pathogens. [ABSTRACT FROM AUTHOR]

Plants are among the many creatures that have benefited from the widespread application of the CRISPR-associated Cas system as a genome-editing tool for investigating gene function, identifying disease, and enhancing agricultural yields. Although the CRISPR/Cas systems for DNA editing are widely employed, post-transcriptional manipulation of RNA remains difficult despite the prevalence of Cas9. Type VI CRISPR/Cas systems, which were recently found, allow for precise RNA editing without permanently affecting the genome. Cas13d has been put to good use in RNA-related studies across a wide range of RNA knock-down, and RNA detection without affecting DNA. Regulation of cas13d specificity and activity helps to avoid the off-target effects and immune responses in plants. Cas13d as highly efficient RNA-targeting tools for the virus resistance, gene function studies, disease diagnostics, and crop improvement in plants. However, CRISPR/Cas13d applications in plant RNA biology are just getting started. This article discusses how RNA editing tools derived from the CRISPR/Cas13d system are currently being used and where they may be used in the future for plant research. [ABSTRACT FROM AUTHOR]

Ilarviruses are a relatively understudied but important group of plant RNA viruses that includes a number of crop pathogens. Their genomes comprise three RNA segments encoding two replicase subunits, movement protein, coat protein (CP), and (in some ilarvirus subgroups) a protein that suppresses RNA silencing. Here we report that, in many ilarviruses, RNA3 encodes an additional protein (termed CP-RT) as a result of ribosomal readthrough of the CP stop codon into a short downstream readthrough (RT) ORF. Using asparagus virus 2 as a model, we find that CP-RT is expressed in planta where it functions as a weak suppressor of RNA silencing. CP-RT expression is essential for persistent systemic infection in leaves and shoot apical meristem. CP-RT function is dependent on a putative zinc-finger motif within RT. Replacing the asparagus virus 2 RT with the RT of an ilarvirus from a different subgroup restored the ability to establish persistent infection. These findings open up a new avenue for research on ilarvirus silencing suppression, persistent meristem invasion and vertical transmission. Author summary: Ilarviruses comprise a group of plant-infecting RNA viruses. Until now, ilarvirus genomes were thought to only encode two components of the viral replicase, a movement protein, the capsid protein, and the 2b suppressor of RNA silencing. Interestingly, whereas most plant viruses encode one or more proteins that suppress the host antiviral RNA silencing pathway, the gene encoding 2b is only present in some ilarviruses. Here, we identify an additional ilarvirus protein that is expressed via low efficiency ribosomal readthrough of the capsid protein (CP) stop codon. Readthrough ribosomes translate a short downstream readthrough (RT) domain to produce the capsid-readthrough fusion protein, CP-RT. We show that CP-RT is a second ilarvirus suppressor of silencing protein and is required for the model ilarvirus asparagus virus 2 to maintain persistent infection in the model plant Nicotiana benthamiana. These findings advance our understanding of ilarvirus molecular virology and virus-host interaction. Interestingly, there are substantial differences in the RT peptide between divergent groups of ilarviruses and, in at least two cases, a separate RT peptide appears to have evolved independently. Thus it will be of future interest to investigate the function of CP-RT in different ilarvirus lineages. [ABSTRACT FROM AUTHOR]

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells. [ABSTRACT FROM AUTHOR]

Mitochondrial genomes of land plants are large and exhibit a complex mode of gene organization and expression, particularly at the post-transcriptional level. The primary organellar transcripts in plants undergo extensive maturation steps, including endo- and/or exo-nucleolytic cleavage, RNA-base modifications (mostly C-to-U deaminations) and both 'cis'- and 'trans'-splicing events. These essential processing steps rely on the activities of a large set of nuclear-encoded factors. RNA helicases serve as key players in RNA metabolism, participating in the regulation of transcription, mRNA processing and translation. They unwind RNA secondary structures and facilitate the formation of ribonucleoprotein complexes crucial for various stages of gene expression. Furthermore, RNA helicases are involved in RNA metabolism by modulating pre-mRNA maturation, transport and degradation processes. These enzymes are, therefore, pivotal in RNA quality-control mechanisms, ensuring the fidelity and efficiency of RNA processing and turnover in plant mitochondria. This review summarizes the significant roles played by helicases in regulating the highly dynamic processes of mitochondrial transcription, RNA processing and translation in plants. We further discuss recent advancements in understanding how dysregulation of mitochondrial RNA helicases affects the splicing of organellar genes, leading to respiratory dysfunctions, and consequently, altered growth, development and physiology of land plants. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs in plants. They play critical functions in various biological processes during plant growth and development. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant with significant medicinal, economic, and academic values. In order to elucidate the role of miRNAs in S. miltiorrhiza, six small RNA libraries from mature roots, young roots, stems, mature leaves, young leaves and flowers of S. miltiorrhiza and one degradome library from mixed tissues were constructed. A total of 184 miRNA precursors, generating 137 known and 49 novel miRNAs, were genome-widely identified. The identified miRNAs were predicted to play diversified regulatory roles in plants through regulating 891 genes. qRT-PCR and 5′ RLM-RACE assays validated the negative regulatory role of smi-miR159a in SmMYB62, SmMYB78, and SmMYB80. To elucidate the function of smi-miR159a in bioactive compound biosynthesis, smi-miR159a transgenic hairy roots were generated and analyzed. The results showed that overexpression of smi-miR159a caused a significant decrease in rosmarinic acid and salvianolic acid B contents. qRT-PCR analysis showed that the targets of smi-miR159a, including SmMYB62, SmMYB78, and SmMYB80, were significantly down-regulated, accompanied by the down-regulation of SmPAL1, SmC4H1, Sm4CL1, SmTAT1, SmTAT3, SmHPPR1, SmRAS, and SmCYP98A14 genes involved in phenolic acid biosynthesis. It suggests that smi-miR159a is a significant negative regulator of phenolic acid biosynthesis in S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

Background: Nitrogen (N) is essential for plant growth and development. In Lithocarpus polystachyus Rehd., a species known for its medicinal and food value, phlorizin is the major bioactive compound with pharmacological activity. Research has revealed a positive correlation between plant nitrogen (N) content and phlorizin synthesis in this species. However, no study has analyzed the effect of N fertilization on phlorizin content and elucidated the molecular mechanisms underlying phlorizin synthesis in L. polystachyus. Results: A comparison of the L. polystachyus plants grown without (0 mg/plant) and with N fertilization (25, 75, 125, 175, 225, and 275 mg/plant) revealed that 75 mg N/plant fertilization resulted in the greatest seedling height, ground diameter, crown width, and total phlorizin content. Subsequent analysis of the leaves using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detected 150 metabolites, including 42 flavonoids, that were differentially accumulated between the plants grown without and with 75 mg/plant N fertilization. Transcriptomic analysis of the L. polystachyus plants via RNA sequencing revealed 162 genes involved in flavonoid biosynthesis, among which 53 significantly differed between the N-treated and untreated plants. Fertilization (75 mg N/plant) specifically upregulated the expression of the genes phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), and phlorizin synthase (PGT1) but downregulated the expression of trans-cinnamate 4-monooxygenase (C4H), shikimate O-hydroxycinnamoyltransferase (HCT), and chalcone isomerase (CHI), which are related to phlorizin synthesis. Finally, an integrated analysis of the transcriptome and metabolome revealed that the increase in phlorizin after N fertilization was consistent with the upregulation of phlorizin biosynthetic genes. Quantitative real-time PCR (qRT‒PCR) was used to validate the RNA sequencing data. Thus, our results indicated that N fertilization increased phlorizin metabolism in L. polystachyus by regulating the expression levels of the PAL, PGT1, 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase (C3'H), C4H, and HCT genes. Conclusions: Our results demonstrated that the addition of 75 mg/plant N to L. polystachyus significantly promoted the accumulation of flavonoids, including phlorizin, and the expression of flavonoid synthesis-related genes. Under these conditions, the genes PAL, 4CL, and PGT1 were positively correlated with phlorizin accumulation, while C4H, CHI, and HCT were negatively correlated with phlorizin accumulation. Therefore, we speculate that PAL, 4CL, and PGT1 participate in the phlorizin pathway under an optimal N environment, regulating phlorizin biosynthesis. These findings provide a basis for improving plant bioactive constituents and serve as a reference for further pharmacological studies. [ABSTRACT FROM AUTHOR]

Background: Cassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce. Results: The effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.–vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods. Conclusion: Our method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions. [ABSTRACT FROM AUTHOR]

N6‐methyladenosine (m6A), as the most abundant and well‐studied RNA modification, can be reversibly added or removed by m6A methyltransferase and demethylase. The further molecular and biological function of m6A is achieved by the recognition of its binding protein. m6A functions in the diverse progress of RNA processing, including transcription regulation, splicing, nuclear export, stability, and translation, to regulate the fate of cells. Although been extensively studied in various animal cell systems, research on m6A's regulatory functions in plant cells lags. In recent years, with a deepening understanding of the functions of m6A and the development of various sequencing technologies, researches on m6A in plant cells have gradually increased. In this review, we focused on discussing the molecular functions of m6A in the nucleus and cytoplasm, aiming to elucidate the specific molecular mechanisms by which m6A regulates the fate of RNAs in plants. Finally, we provide some perspectives on future investigations of the detailed molecular mechanism of m6A‐mediated regulation in plants, which might provide insights into future strategies for achieving multiple growth regulatory processes in crops. [ABSTRACT FROM AUTHOR]

SUMMARY: RNA editing is a crucial post‐transcriptional modification process in plant organellar RNA metabolism. rRNA removal‐based total RNA‐seq is one of the most common methods to study this event. However, the lack of commercial kits to remove rRNAs limits the usage of this method, especially for non‐model plant species. DSN‐seq is a transcriptome sequencing method utilizing duplex‐specific nuclease (DSN) to degrade highly abundant cDNA species especially those from rRNAs while keeping the robustness of transcript levels of the majority of other mRNAs, and has not been applied to study RNA editing in plants before. In this study, we evaluated the capability of DSN‐seq to reduce rRNA content and profile organellar RNA editing events in plants, as well we used commercial Ribo‐off‐seq and standard mRNA‐seq as comparisons. Our results demonstrated that DSN‐seq efficiently reduced rRNA content and enriched organellar transcriptomes in rice. With high sensitivity to RNA editing events, DSN‐seq and Ribo‐off‐seq provided a more complete and accurate RNA editing profile of rice, which was further validated by Sanger sequencing. Furthermore, DSN‐seq also demonstrated efficient organellar transcriptome enrichment and high sensitivity for profiling RNA editing events in Arabidopsis thaliana. Our study highlights the capability of rRNA removal‐based total RNA‐seq for profiling RNA editing events in plant organellar transcriptomes and also suggests DSN‐seq as a widely accessible RNA editing profiling method for various plant species. Significance Statement: Nowadays, the assembly of plant organellar genomes has seen significant progress, yet the lack of suitable methods hampers RNA editing profiling in these genomes. Our study presents an effective solution by exploring rRNA removal‐based NGS methods, which will facilitate accurate and high‐throughput RNA editing profiling in various plant species. [ABSTRACT FROM AUTHOR]

RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation pathway that relies on the atypical RNA polymerases Pol IV-RDR2 complex and Pol V to produce the siRNA precursor and lncRNA, respectively. Both Pol IV and Pol V have evolved from Pol II, but adopt unique biochemical features relevant for their functions in siRNA precursor (Pol IV) and scaffold lncRNA (Pol V) production. Recent structural studies suggest that the Pol IV and Pol V NRPD(E)2 subunit-induced enhanced backtracking plays an important role in their ncRNA production function. A dual measurement mechanism was proposed for the siRNA precursor production by Pol IV-RDR2 holoenzyme in RdDM. In plants, two atypical DNA-dependent RNA polymerases, RNA polymerase IV (Pol IV) and Pol V, and an RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) together produce noncoding RNAs (ncRNAs) to guide the plant-specific RNA-directed DNA methylation (RdDM). Although both Pol IV and Pol V have evolved from the canonical Pol II, they have adapted to different roles in RdDM. The mechanisms of their adaptation are key to understanding plant DNA methylation and the divergent evolution of polymerases. In this review, we summarize insights that have emerged from recent structural studies of Pol IV, Pol V, and RDR2 and discuss their structural features critical for efficient ncRNA production in RdDM. [ABSTRACT FROM AUTHOR]

RNAs play important roles in regulating biological growth and development. Advancements in RNA-imaging techniques are expanding our understanding of their function. Several common RNA-labeling methods in plants have pros and cons. Simultaneously, plants' spontaneously fluorescent substances interfere with the effectiveness of RNA bioimaging. New technologies need to be introduced into plant RNA luminescence. Aggregation-induced emission luminogens (AIEgens), due to their luminescent properties, tunable molecular size, high fluorescence intensity, good photostability, and low cell toxicity, have been widely applied in the animal and medical fields. The application of this technology in plants is still at an early stage. The development of AIEgens provides more options for RNA labeling. Click chemistry provides ideas for modifying AIEgens into RNA molecules. The CRISPR/Cas13a-mediated targeting system provides a guarantee of precise RNA modification. The liquid–liquid phase separation in plant cells creates conditions for the enrichment and luminescence of AIEgens. The only thing that needs to be looked for is a specific enzyme that uses AIEgens as a substrate and modifies AIEgens onto target RNA via a click chemical reaction. With the development and progress of artificial intelligence and synthetic biology, it may soon be possible to artificially synthesize or discover such an enzyme. [ABSTRACT FROM AUTHOR]

Main conclusion: Nonsense-mediated mRNA decay in eukaryotes is vital to cellular homeostasis. Further knowledge of its putative role in plant RNA metabolism under stress is pivotal to developing fitness-optimizing strategies. Nonsense-mediated mRNA decay (NMD), part of the mRNA surveillance pathway, is an evolutionarily conserved form of gene regulation in all living organisms. Degradation of mRNA-bearing premature termination codons and regulation of physiological RNA levels highlight NMD's role in shaping the cellular transcriptome. Initially regarded as purely a tool for cellular RNA quality control, NMD is now considered to mediate various aspects of plant developmental processes and responses to environmental changes. Here we offer a basic understanding of NMD in eukaryotes by explaining the concept of premature termination codon recognition and NMD complex formation. We also provide a detailed overview of the NMD mechanism and its role in gene regulation. The potential role of effectors, including ABCE1, in ribosome recycling during the translation process is also explained. Recent reports of alternatively spliced variants of corresponding genes targeted by NMD in Arabidopsis thaliana are provided in tabular format. Detailed figures are also provided to clarify the NMD concept in plants. In particular, accumulating evidence shows that NMD can serve as a novel alternative strategy for genetic manipulation and can help design RNA-based therapies to combat stress in plants. A key point of emphasis is its function as a gene regulatory mechanism as well as its dynamic regulation by environmental and developmental factors. Overall, a detailed molecular understanding of the NMD mechanism can lead to further diverse applications, such as improving cellular homeostasis in living organisms. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are dynamic structures formed through diverse mechanisms, including liquid-liquid phase separation. These condensates have emerged as crucial regulators of cellular processes in eukaryotic cells, enabling the compartmentalization of specific biological reactions while allowing for dynamic exchange of molecules with the surrounding environment. RNA silencing, a conserved gene regulatory mechanism mediated by small RNAs (sRNAs), plays pivotal roles in various biological processes. Multiple types of biomolecular condensate, including dicing bodies, processing bodies, small interfering RNA bodies, and Cajal bodies, have been identified as key players in RNA silencing pathways. These biomolecular condensates provide spatial compartmentation for the biogenesis, loading, action, and turnover of small RNAs. Moreover, they actively respond to stresses, such as viral infections, and modulate RNA silencing activities during stress responses. This review summarizes recent advances in understanding of dicing bodies and other biomolecular condensates involved in RNA silencing. We explore their formation, roles in RNA silencing, and contributions to antiviral resistance responses. This comprehensive overview provides insights into the functional significance of biomolecular condensates in RNA silencing and expands our understanding of their roles in gene expression and stress responses in plants. [ABSTRACT FROM AUTHOR]

Telosma mosaic virus (TeMV) is a highly infectious and rapidly spreading virus that infects a variety of natural hosts, with passion fruit (Passiflora edulis) being its primary host across parts of Asia. Symptoms of TeMV, which seriously threatens passion fruit production worldwide, include mosaic, deformities and lignification of fruit. We present here a review of the current state of knowledge of TeMV, including its structural characteristics, natural and experimental host range, symptoms and detection, and provide insights into control strategies. The mode of transmission of TeMV remains unclear, but it can be mechanically transmitted through grafting, cuttings and other artificial asexual propagation techniques, which unfortunately constitute the primary means of propagation in the passion fruit industry. Moreover, increasing detection rates of TeMV indicate that this virus probably causes more serious losses to the passion fruit industry than previously reported. No systematic control strategy for TeMV infection currently exists, and no TeMV‐resistant passion fruit varieties are currently available. As a speedy, short‐term solution for dealing with TeMV infection in the absence of a long‐term solution, we propose rapid plant propagation through virus‐free plant tissue culture techniques that can generate a substantial number of virus‐free passion fruit seedlings. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM)-like proteins (CMLs) are the largest family of calcium-binding proteins in plants, yet the functions of most CMLs are unknown. Arabidopsis CML13 and CML14 are closely related paralogs that interact with the isoleucine–glutamine (IQ) domains of myosins, IQ-domain proteins and CaM-binding transcription activators (CAMTAs). Here, we explored the physiological roles of CML13 and CML14 during development by using dexamethasone (Dex)-inducible RNA silencing to suppress either CML13 or CML14 transcript levels. In the absence of inducible suppression, CML13 - and CML14 -RNA-interference lines were indistinguishable from wild-type (WT) plants throughout development. In contrast, induction of silencing treatment led to rapid increases in RNA-hairpin production that correlated with a targeted reduction in CML13 or CML14 transcript levels and a range of developmental and morphological effects. RNA-suppression treatment did not impair the germination of CML13- or 14 -RNA-interference lines, but these seedlings were chlorotic, displayed high mortality and failed to achieve seedling establishment. Under Dex treatment, seeds of CML13- and CML14 -RNA-interference lines exhibited differential sensitivity to exogenous ABA compared to WT seeds. Induced RNA suppression of mature plants led to reduced silique length, shorter roots and rapid leaf senescence in CML13- and 14 -RNA-interference plants, which correlated with increased gene expression of the senescence marker Senescence-Associated Gene13 (SAG13). Plants induced for RNA suppression at 2 weeks post-germination exhibited a much stronger phenotype than treatment of 3-, 4- or 5-week-old plants. Collectively, our data indicate that both CML13 and CML14 are essential for normal development and function across a broad range of tissues and developmental stages. [ABSTRACT FROM AUTHOR]

Introduction Understanding how plants adapt to the space environment is essential, as plants will be a valuable component of long duration space missions. Several spaceflight experiments have focused on transcriptional profiling as a means of understanding plant adaptation to microgravity. However, there is limited overlap between results from different experiments. Differences in experimental conditions and hardware make it difficult to find a consistent response across experiments and to distinguish the primary effects of microgravity from other spaceflight effects. Methods Plant Signaling (PS) and Plant RNA Regulation (PRR) were two separate spaceflight experiments conducted on the International Space Station utilizing the European Modular Cultivation System (EMCS). The EMCS provided a lighted environment for plant growth with centrifugal capabilities providing an onboard 1 g control. Results and discussion An RNA-Seq analysis of shoot samples from PS and PRR revealed a significant overlap of genes differentially expressed in microgravity between the two experiments. Relative to onboard 1 g controls, genes involved in transcriptional regulation, shoot development, and response to auxin and light were upregulated in microgravity in both experiments. Conversely, genes involved in defense response, abiotic stress, Ca++ signaling, and cell wall modification were commonly downregulated in both datasets. The downregulation of stress responses in microgravity in these two experiments is interesting as these pathways have been previously observed as upregulated in spaceflight compared to ground controls. Similarly, we have observed many stress response genes to be upregulated in the 1 g onboard control compared to ground reference controls; however these genes were specifically downregulated in microgravity. In addition, we analyzed the sRNA landscape of the 1 g and microgravity (μ g) shoot samples from PRR. We identified three miRNAs (miR319c, miR398b, and miR8683) which were upregulated in microgravity, while several of their corresponding target genes were found to be downregulated in microgravity. Interestingly, the downregulated target genes are enriched in those encoding chloroplast-localized enzymes and proteins. These results uncover microgravity unique transcriptional changes and highlight the validity and importance of an onboard 1 g control. [ABSTRACT FROM AUTHOR]

Summary: In plants, exoribonuclease‐resistant RNAs (xrRNAs) are produced by many viruses. Whereas xrRNAs contribute to the pathogenicity of these viruses, the role of xrRNAs in the virus infectious cycle remains elusive.Here, we show that xrRNAs produced by a benyvirus (a multipartite RNA virus with four genomic segments) in plants are involved in the formation of monocistronic coat protein (CP)‐encoding chimeric RNAs. Naturally occurring chimeric RNAs, we discovered, are composed of 5′‐end of RNA 2 and 3′‐end of either RNA 3 or RNA 4 bearing conservative exoribonuclease‐resistant 'coremin' region.Using computational tools and site‐directed mutagenesis, we show that de novo formation of chimeric RNAs requires intermolecular base‐pairing interaction between 'coremin' and 3′‐proximal part of the CP gene of RNA 2 as well as a stem‐loop structure immediately adjacent to the CP gene. Moreover, knockdown of the expression of the XRN4 gene, encoding 5′→3′ exoribonuclease, inhibits biogenesis of both xrRNAs and chimeric RNAs.Our findings suggest a novel mechanism involving a unique tropology of the intermolecular base‐pairing complex between xrRNAs and RNA2 to promote formation of chimeric RNAs in plants. XrRNAs, essential for chimeric RNA biogenesis, are generated through the action of cytoplasmic Xrn 4 5′→3′ exoribonuclease conserved in all plant species. [ABSTRACT FROM AUTHOR]

Background: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. Method: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. Results: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. Conclusion: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts. [ABSTRACT FROM AUTHOR]

Coconut cadang-cadang viroid (CCCVd) is an infectious single-stranded RNA (ssRNA) pathogen, which leads directly to the death of a large number of coconut palm trees and heavy economic loss to coconut farmers. Herein, a novel electrochemical impedance RNA genosensor is presented based on highly stable gold nanoparticles (AuNPs) decorated phosphorene (BP) nanohybrid with graphene (Gr) for highly sensitive, low-cost, and label-free detection of CCCVd. BP-AuNPs are environmentally friendly prepared by ultrasonic-assisted liquid-phase exfoliation of black phosphorus, accompanying direct reduction of chloroauric acid. Gr/BP-AuNPs are facilely prepared by the in situ growth of AuNPs onto the BP surface and its nanohybrid with Gr to improve environmental stability of BP. Gr/BP-AuNP-based RNA genosensor is fabricated by immobilizing the thiol-functionalized single-stranded DNA (ssDNA) oligonucleotide probe onto the surface of Gr/BP-AuNP-modified glassy carbon electrode via gold-thiol interactions, which served as an electrochemical genosensing platform for the label-free impedance detection of CCCVd by hybridization between the functionalized ssDNA probe and the complementary CCCVd ssRNA sequence in a wide linear range from 1.0 × 10−11 to 1.0 × 10−7 M with a low limit of detection of 2.8 × 10−12 M. This work supplies an experimental support and theoretical direction for the fabrication of RNA biosensors based on graphene-like materials and potential application for a specific diagnosis of plant RNA viral disease in Arecaceae planting industry. [ABSTRACT FROM AUTHOR]

Plants use RNA interference for basal antiviral immunity, but emerging evidence suggests that additional RNA-targeting defense mechanisms also defend against invading viruses. Recent advancements in the understanding of RNA decay, RNA quality control, and N6-methyladenosine (m6A) RNA modifications have unveiled new insights into the molecular arms race between plants and viruses. [ABSTRACT FROM AUTHOR]

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms. [ABSTRACT FROM AUTHOR]

Increasing plant resistance to biotic and abiotic stresses may help to address global food insecurity. We review small RNA (sRNA) research and consider the potential of sRNA-based technologies as strategies to enhance plant resistance to environmental stresses. sRNAs are essential non-coding signalling molecules 21–24 nucleotides in length that are involved in various reproduction, defence and plant development processes. sRNAs guide regulatory processes during development and environmental adaptation at the DNA or RNA level in various eukaryotic organisms. They control gene expression in eukaryotes via a process commonly termed RNA silencing. sRNAs are responsible for suppressing some pathogenic genes in eukaryotes and pests. This suppression offers the potential to protect plant growth and development through a new generation of eco-friendly RNA-based fungicides or insecticides that are specific in their target and can easily control multiple diseases simultaneously. This review focuses on sRNA production in crop species, the role of sRNAs in plant responses to a range of stresses, and their prospective applications, highlighting sRNA-based technology and applications in crops under stress. This review could serve as a reference for future researchers working on small RNAs and the roles they play in plant response to environmental stresses. This review suggests that small RNA (sRNA) based technologies are promising strategies for increasing plant resistance to biotic and abiotic stresses, thereby helping to address global food insecurity. The technology has the potential to improve plant resistance to pests, diseases and other environmental stresses, resulting in increased crop yield and reliability. A focus on sRNA-based technology and its potential applications for plant improvement suggests a brighter future for agriculture. [ABSTRACT FROM AUTHOR]

Noncoding RNAs constitute a substantial portion of the transcriptome and play pivotal roles in plant growth and development. Among these processes, flowering stands out as a crucial trait, ensuring reproductive success and seed set, and is meticulously controlled by genetic and environmental factors. With remarkable advancements in the identification and characterization of noncoding RNAs in plants, it has become evident that noncoding RNAs are intricately linked to the regulation of flowering time. In this article, we present an overview of the classification of plant noncoding RNAs and delve into their functions in the regulation of flowering time. Furthermore, we review their molecular mechanisms and their involvement in flowering pathways. Our comprehensive review enhances the understanding of how noncoding RNAs contribute to the regulation of flowering time and sheds light on their potential implications in crop breeding. [ABSTRACT FROM AUTHOR]

Plant-infecting RNA viruses from 30 families and floating genera, as well as a great number of uncultured as yet-unclassified plant-associated viruses have been described. Even so, the plant RNA virosphere is still underexplored. RNA extracted from enriched virus particles of 50 L water samples from the Teltow Canal and the Havel River in Berlin, Germany, was sequenced using Illumina next-generation sequencing. Sequences were searched for plant viruses with BLAST and DIAMOND. Phylogenetic analyses were conducted with IQ-TREE 2. Altogether, 647 virus sequences greater than 1 kb were detected and further analyzed. These data revealed the presence of accepted and novel viruses related to Albetovirus, Alphaflexiviridae, Aspiviridae, Bromoviridae, Endornaviridae, Partitiviridae, Potyviridae, Solemoviridae, Tombusviridae and Virgaviridae. The vast majority of the sequences were novel and could not be taxonomically assigned. Several tombus- and endorna-like viruses make use of alternative translation tables that suggest unicellular green algae, ciliates, or diplomonades as their hosts. The identification of 27 albeto-like satellite viruses increases available sequence data five-fold. Sixteen new poty-like viruses align with other poty-like viruses in a link that combines the Astroviridae and Potyviridae families. Further, the identification of viruses with peptidase A6-like and peptidase A21-like capsid proteins suggests horizontal gene transfer in the evolution of these viruses. [ABSTRACT FROM AUTHOR]

Pseudouridine is one of the well-known chemical modifications in various RNA species. Current advances to detect pseudouridine show that the pseudouridine landscape is dynamic and affects multiple cellular processes. Although our understanding of this post-transcriptional modification mainly depends on yeast and human models, the recent findings provide strong evidence for the critical role of pseudouridine in plants. Here, we review the current knowledge of pseudouridine in plant RNAs, including its synthesis, degradation, regulatory mechanisms, and functions. Moreover, we propose future areas of research on pseudouridine modification in plants. [ABSTRACT FROM AUTHOR]

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses. [ABSTRACT FROM AUTHOR]

SUMMARY: The protein factors for the specific C‐to‐U RNA editing events in plant mitochondria and chloroplasts possess unique arrays of RNA‐binding pentatricopeptide repeats (PPRs) linked to carboxy‐terminal cytidine deaminase DYW domains via the extension motifs E1 and E2. The E1 and E2 motifs have distant similarities to tetratricopeptide repeats known to mediate protein–protein interactions but their precise function is unclear. Here, we investigate the tolerance of PPR56 and PPR65, two functionally characterized RNA editing factors of the moss Physcomitrium patens, for the creation of chimeras by variably replacing their C‐terminal protein regions. Making use of a heterologous RNA editing assay system in Escherichia coli we find that heterologous DYW domains can strongly restrict or widen the spectrum of off‐targets in the bacterial transcriptome for PPR56. Surprisingly, our data suggest that these changes are not only caused by the preference of a given heterologous DYW domain for the immediate sequence environment of the cytidine to be edited but also by a long‐range impact on the nucleotide selectivity of the upstream PPRs. Significance Statement: DYW domains affect upstream PPR arrays for RNA recognition. [ABSTRACT FROM AUTHOR]

Plant virology has been at the forefront of major discoveries in RNA biology for over a century. Recent discoveries in gene expression strategies to bypass the monocistronic mRNA dogma have expanded our insights and led to biotechnological applications of viral elements. Work with plant viruses has spearheaded the discoveries of RNA silencing and its suppression. Plant viruses are versatile gene vector tools for various application, including gene editing. Key principles pertaining to RNA biology not infrequently have their origins in plant virology. Examples have arisen from studies on viral RNA-intrinsic properties and the infection process from gene expression, replication, movement, and defense evasion to biotechnological applications. Since RNA is at the core of the central dogma in molecular biology, how plant virology assisted in the reinforcement or adaptations of this concept, while at other instances shook up elements of the doctrine, is discussed. Moreover, despite the negative effects of viral diseases in agriculture worldwide, plant viruses can be considered a scientific treasure trove. Today they remain tools of discovery for biotechnology, studying evolution, cell biology, and host–microbe interactions. [ABSTRACT FROM AUTHOR]

This article discusses the development and optimization of a solid phase-based plant RNA extraction method called Solid Phase Gene Extraction (SPGE). The SPGE method allows for rapid and in situ extraction of RNA from plant cells and tissues, making it suitable for gene expression profiling and screening of genetically modified plants. The article focuses on the application of SPGE in the extraction of mRNA from Iris sanguinea Donn. ex Horn., a cold-tolerant plant native to northeastern China. The authors systematically optimized key parameters of the SPGE method, such as probe size, incubation time, embedding time, oligo dt25 concentration, and probe storage conditions. The study demonstrates the successful extraction of mRNA using the SPGE method and highlights its potential for gene expression studies and transgenic plant detection. [Extracted from the article]

Plant noncoding RNA transcripts have gained increasing attention in recent years due to growing evidence that they can regulate developmental plasticity. In this review article, we comprehensively analyze the relationship between noncoding RNA transcripts in plants and their response to environmental cues. We first provide an overview of the various noncoding transcript types, including long and small RNAs, and how the environment modulates their performance. We then highlight the importance of noncoding RNA secondary structure for their molecular and biological functions. Finally, we discuss recent studies that have unveiled the functional significance of specific long noncoding transcripts and their molecular partners within ribonucleoprotein complexes during development and in response to biotic and abiotic stress. Overall, this review sheds light on the fascinating and complex relationship between dynamic noncoding transcription and plant environmental responses, and highlights the need for further research to uncover the underlying molecular mechanisms and exploit the potential of noncoding transcripts for crop resilience in the context of global warming. [ABSTRACT FROM AUTHOR]

Summary: Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses.Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss‐of‐function mutants in Arabidopsis thaliana.pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous‐like lineage.Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants. [ABSTRACT FROM AUTHOR]

The biological function of RNA can be modulated by base modifications. Here, we unveiled the occurrence of N4-acetylation of cytidine in plant RNA, including mRNA, by employing LC-MS/MS and acRIP-seq. We identified 325 acetylated transcripts from the leaves of 4-week-old Arabidopsis (Arabidopsis thaliana) plants and determined that 2 partially redundant N-ACETYLTRANSFERASEs FOR CYTIDINE IN RNA (ACYR1 and ACYR2), which are homologous to mammalian NAT10, are required for acetylating RNA in vivo. A double-null mutant was embryo lethal, while eliminating 3 of the 4 ACYR alleles led to defects in leaf development. These phenotypes could be traced back to the reduced acetylation and concomitant destabilization of the transcript of TOUGH, which is required for miRNA processing. These findings indicate that N4-acetylation of cytidine is a modulator of RNA function with a critical role in plant development and likely many other processes. The base modification N4-acetylation of cytidine in RNA is essential for microRNA biogenesis as well as leaf and embryo development in plants. [ABSTRACT FROM AUTHOR]

DNA-dependent RNA polymerases (Pols) transfer the genetic information stored in genomic DNA to RNA in all organisms. In eukaryotes, the typical products of nuclear Pol I, Pol II, and Pol III are ribosomal RNAs, mRNAs, and transfer RNAs, respectively. Intriguingly, plants possess two additional Pols, Pol IV and Pol V, which produce small RNAs and long noncoding RNAs, respectively, mainly for silencing transposable elements. The five plant Pols share some subunits, but their distinct functions stem from unique subunits that interact with specific regulatory factors in their transcription cycles. Here, we summarize recent advances in our understanding of plant nucleus-localized Pols, including their evolution, function, structures, and transcription cycles. This review explores the evolution, structure, and functions in transcription of nucleus-localized DNA-dependent RNA polymerases in plants. [ABSTRACT FROM AUTHOR]

Extracellular RNAs are an emerging research topic in fungal-plant interactions. Fungal plant pathogens and symbionts release small RNAs that enter host cells to manipulate plant physiology and immunity. This communication via extracellular RNAs between fungi and plants is bidirectional. On the one hand, plants release RNAs encapsulated inside extracellular vesicles as a defense response as well as for intercellular and inter-organismal communication. On the other hand, recent reports suggest that also full-length mRNAs are transported within fungal EVs into plants, and these fungal mRNAs might get translated inside host cells. In this review article, we summarize the current views and fundamental concepts of extracellular RNAs released by plant-associated fungi, and we discuss new strategies to apply extracellular RNAs in crop protection against fungal pathogens. Key points: • Extracellular RNAs are an emerging topic in plant-fungal communication. • Fungi utilize RNAs to manipulate host plants for colonization. • Extracellular RNAs can be engineered to protect plants against fungal pathogens. [ABSTRACT FROM AUTHOR]

Procuring high-grade RNA from mature coconut tissues is a tricky and labor-intensive process due to the intricate scaffold of polysaccharides, polyphenols, lipids, and proteins that form firm complexes with nucleic acids. However, we have effectively developed a novel method for the first time, letting the retrieval of high-grade RNA from the roots, endosperm, and mesocarp of mature coconut trees take place. In this method, we exploited dichloromethane as a replacement to phenol/chloroform for RNA recovery from mature coconut tissues. The amount of high-grade RNA acquired from the roots of mature coconut trees was 120.7 µg/g, with an A260/280 ratio of 1.95. Similarly, the mature coconut mesocarp yielded 134.6 µg/g FW of quality RNA with A260/280 ratio of 1.98, whereas the mature coconut endosperm produced 120.4 µg/g FW of quality RNA with A260/280 ratio of 2.01. Furthermore, the RNA isolation using the dichloromethane method exhibited excellent performance in downstream experiments, particularly in RT-PCR for cDNA production and amplification. On the contrary, the RNA plant kit, TRIZOL, and Cetyl Trimethyl Ammonium Bromide (CTAB) methods were unsuccessful in isolating substantial quantities of RNA with exceptional purities from the mentioned coconut tissues. In view of these findings, we conclude that the newly developed method will be pivotal in effectively extracting RNA with high purity from mature coconut tissues. [ABSTRACT FROM AUTHOR]