Characterisation of the Arabidopsis thaliana telomerase TERT-TR complex.

Most eukaryotic organisms employ a telomerase complex for the maintenance of chromosome ends. The core of this complex is composed of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) subunits. The TERT reverse transcriptase (RT) domain synthesises telomeric DNA using the TR template sequence. The other TERT domains contribute to this process in different ways. In particular, the TERT RNA-binding domain (TRBD) interacts with specific TR motif(s). Using a yeast 3-hybrid system, we show the critical role of Arabidopsis thaliana (At) TRBD and embryophyta-conserved KRxR motif in the unstructured linker preceding the TRBD domain for binding to the recently identified AtTR subunit. We also show the essential role of the predicted P4 stem and pseudoknot AtTR structures and provide evidence for the binding of AtTRBD to pseudoknot and KRxR motif stabilising interaction with the P4 stem structure. Our results thus provide the first insight into the core part of the plant telomerase complex.

Epitranscriptome insights into Riccia fluitans L. (Marchantiophyta) aquatic transition using nanopore direct RNA sequencing.

BACKGROUND: Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition. RESULTS: A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant's resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions. CONCLUSIONS: These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant's dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

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.

Transcriptome dynamics of Gossypium purpurascens in response to abiotic stresses by Iso-seq and RNA-seq data.

Gossypium purpurascens is a member of the Malvaceae family, holds immense economic significance as a fiber crop worldwide. Abiotic stresses harm cotton crops, reduce yields, and cause economic losses. Generating high-quality reference genomes and large-scale transcriptomic datasets across diverse conditions can offer valuable insights into identifying preferred agronomic traits for crop breeding. The present research used leaf tissues to conduct PacBio Iso-seq and RNA-seq analysis. We carried out an in-depth analysis of DEGs using both correlations with cluster analysis and principal component analysis. Additionally, the study also involved the identification of both lncRNAs and CDS. We have prepared RNA-seq libraries from 75 RNA samples to study the effects of drought, salinity, alkali, and saline-alkali stress, as well as control conditions. A total of 454.06 Gigabytes of transcriptome data were effectively validated through the identification of differentially expressed genes and KEGG and GO analysis. Overwhelmingly, gene expression profiles and full-length transcripts from cotton tissues will aid in understanding the genetic mechanism of abiotic stress tolerance in G. purpurascens.

RNA Isolation Method in Marginal Crops with High Agronomic Potential.

Ribonucleic Acid (RNA) isolation is a basic technique in the field of molecular biology. The purpose of RNA isolation is to acquire pure and complete RNA that can be used to evaluate gene expression. Many methods can be used to perform RNA isolation, all of them based on the chemical properties of nucleic acids. However, some of them do not achieve high RNA yields and purity levels when used in a number of marginally studied crops of agronomic importance, such as grain and vegetable amaranth plants. In the method described here, the use of guanidinium thiocyanate and two additional precipitation steps with different reagents designed to obtain high yields and RNA purity levels from diverse plant species employed for plant functional genomics studies is described.

Systematic identification and characterization of microRNAs with target genes involved in high night temperature stress at the filling stage of rice.

High night temperature stress is one of the main environmental factors affecting rice yield and quality. More and more evidence shows that microRNA (miRNA) plays an important role in various abiotic stresses. However, the molecular network of miRNA regulation on rice tolerance to high night temperatures remains unclear. Here, small RNA, transcriptome and degradome sequencing were integrated to identify differentially expressed miRNAs, genes, and key miRNA-target gene pairs in rice heat-sensitive and heat-tolerant lines at the filling stage suffering from high night temperature stress. It was discovered that there were notable differences in the relative expression of 102 miRNAs between the two rice lines under stress. Meanwhile, 5263 and 5405 mRNAs were differentially expressed in the heat-sensitive line and heat-tolerant line, and functional enrichment analysis revealed that these genes were involved in heat-related processes and pathways. The miRNAs-mRNAs target relationship was further verified by degradome sequencing. Eventually, 49 miRNAs-222 mRNAs target pairs with reverse expression patterns showed significant relative expression changes between the heat-tolerant and the heat-sensitive line, being suggested to be responsible for the heat tolerance difference of these two rice lines. Functional analysis of these 222 mRNA transcripts showed that high night temperature-responsive miRNAs targeted these mRNAs involved in many heat-related biological processes, such as transcription regulation, chloroplast regulation, mitochondrion regulation, protein folding, hormone regulation and redox process. This study identified possible miRNA-mRNA regulation relationships in response to high night temperature stress in rice and potentially contributed to heat resistance breeding of rice in the future.

Predictive Role of Cluster Bean (Cyamopsis tetragonoloba) Derived miRNAs in Human and Cattle Health.

MicroRNAs (miRNAs) are small non-coding conserved molecules with lengths varying between 18-25nt. Plants miRNAs are very stable, and probably they might have been transferred across kingdoms via food intake. Such miRNAs are also called exogenous miRNAs, which regulate the gene expression in host organisms. The miRNAs present in the cluster bean, a drought tolerant legume crop having high commercial value, might have also played a regulatory role for the genes involved in nutrients synthesis or disease pathways in animals including humans due to dietary intake of plant parts of cluster beans. However, the predictive role of miRNAs of cluster beans for gene-disease association across kingdoms such as cattle and humans are not yet fully explored. Thus, the aim of the present study is to (i) find out the cluster bean miRNAs (cb-miRs) functionally similar to miRNAs of cattle and humans and predict their target genes' involvement in the occurrence of complex diseases, and (ii) identify the role of cb-miRs that are functionally non-similar to the miRNAs of cattle and humans and predict their targeted genes' association with complex diseases in host systems. Here, we predicted a total of 33 and 15 functionally similar cb-miRs (fs-cb-miRs) to human and cattle miRNAs, respectively. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the participation of targeted genes of fs-cb-miRs in 24 and 12 different pathways in humans and cattle, respectively. Few targeted genes in humans like LCP2, GABRA6, and MYH14 were predicted to be associated with disease pathways of Yesinia infection (hsa05135), neuroactive ligand-receptor interaction (hsa04080), and pathogenic Escherichia coli infection (hsa05130), respectively. However, targeted genes of fs-cb-miRs in humans like KLHL20, TNS1, and PAPD4 are associated with Alzheimer's, malignant tumor of the breast, and hepatitis C virus infection disease, respectively. Similarly, in cattle, targeted genes like ATG2B and DHRS11 of fs-cb-miRs participate in the pathways of Huntington disease and steroid biosynthesis, respectively. Additionally, the targeted genes like SURF4 and EDME2 of fs-cb-miRs are associated with mastitis and bovine osteoporosis, respectively. We also found a few cb-miRs that do not have functional similarity with human and cattle miRNAs but are found to target the genes in the host organisms and as well being associated with human and cattle diseases. Interestingly, a few genes such as NRM, PTPRE and SUZ12 were observed to be associated with Rheumatoid Arthritis, Asthma and Endometrial Stromal Sarcoma diseases, respectively, in humans and genes like SCNN1B associated with renal disease in cattle.

Frontiers in plant RNA research in ICAR2023: from lab to innovative agriculture.

The recent growth in global warming, soil contamination, and climate instability have widely disturbed ecosystems, and will have a significant negative impact on the growth of plants that produce grains, fruits and woody biomass. To conquer this difficult situation, we need to understand the molecular bias of plant environmental responses and promote development of new technologies for sustainable maintenance of crop production. Accumulated molecular biological data have highlighted the importance of RNA-based mechanisms for plant stress responses. Here, we report the most advanced plant RNA research presented in the 33rd International Conference on Arabidopsis Research (ICAR2023), held as a hybrid event on June 5-9, 2023 in Chiba, Japan, and focused on "Arabidopsis for Sustainable Development Goals". Six workshops/concurrent sessions in ICAR2023 targeted plant RNA biology, and many RNA-related topics could be found in other sessions. In this meeting report, we focus on the workshops/concurrent sessions targeting RNA biology, to share what is happening now at the forefront of plant RNA research.

Fluorescent In Situ Detection of Small RNAs in Plants Using sRNA-FISH.

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.

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation.

KEY MESSAGE: Barley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5' terminal nucleotide of small RNAs, demonstrating functional conservation and divergence. The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5' adenine residue, while also accepting 5' guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5' adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

Plant non-coding RNAs: The new frontier for the regulation of plant development and adaptation to stress.

Most plant transcriptomes constitute functional non-coding RNAs (ncRNAs) that lack the ability to encode proteins. In recent years, more research has demonstrated that ncRNAs play important regulatory roles in almost all plant biological processes by modulating gene expression. Thus, it is important to study the biogenesis and function of ncRNAs, particularly in plant growth and development and stress tolerance. In this review, we systematically explore the process of formation and regulatory mechanisms of ncRNAs, particularly those of microRNAs (miRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Additionally, we provide a comprehensive overview of the recent advancements in ncRNAs research, including their regulation of plant growth and development (seed germination, root growth, leaf morphogenesis, floral development, and fruit and seed development) and responses to abiotic and biotic stress (drought, heat, cold, salinity, pathogens and insects). We also discuss research challenges and provide recommendations to advance the understanding of the roles of ncRNAs in agronomic applications.

Harnessing the potential of non-coding RNA: An insight into its mechanism and interaction in plant biotic stress.

Plants have developed diverse physical and chemical defence mechanisms to ensure their continued growth and well-being in challenging environments. Plants also have evolved intricate molecular mechanisms to regulate their responses to biotic stress. Non-coding RNA (ncRNA) plays a crucial role in this process that affects the expression or suppression of target transcripts. While there have been numerous reviews on the role of molecules in plant biotic stress, few of them specifically focus on how plant ncRNAs enhance resistance through various mechanisms against different pathogens. In this context, we explored the role of ncRNA in exhibiting responses to biotic stress endogenously as well as cross-kingdom regulation of transcript expression. Furthermore, we address the interplay between ncRNAs, which can act as suppressors, precursors, or regulators of other ncRNAs. We also delve into the regulation of ncRNAs in response to attacks from different organisms, such as bacteria, viruses, fungi, nematodes, oomycetes, and insects. Interestingly, we observed that diverse microorganisms interact with distinct ncRNAs. This intricacy leads us to conclude that each ncRNA serves a specific function in response to individual biotic stimuli. This deeper understanding of the molecular mechanisms involving ncRNAs in response to biotic stresses enhances our knowledge and provides valuable insights for future research in the field of ncRNA, ultimately leading to improvements in plant traits.

PlantC2U: deep learning of cross-species sequence landscapes predicts plastid C-to-U RNA editing in plants.

In plants, C-to-U RNA editing mainly occurs in plastid and mitochondrial transcripts, which contributes to a complex transcriptional regulatory network. More evidence reveals that RNA editing plays critical roles in plant growth and development. However, accurate detection of RNA editing sites using transcriptome sequencing data alone is still challenging. In the present study, we develop PlantC2U, which is a convolutional neural network, to predict plastid C-to-U RNA editing based on the genomic sequence. PlantC2U achieves >95% sensitivity and 99% specificity, which outperforms the PREPACT tool, random forests, and support vector machines. PlantC2U not only further checks RNA editing sites from transcriptome data to reduce possible false positives, but also assesses the effect of different mutations on C-to-U RNA editing based on the flanking sequences. Moreover, we found the patterns of tissue-specific RNA editing in the mangrove plant Kandelia obovata, and observed reduced C-to-U RNA editing rates in the cold stress response of K. obovata, suggesting their potential regulatory roles in plant stress adaptation. In addition, we present RNAeditDB, available online at https://jasonxu.shinyapps.io/RNAeditDB/. Together, PlantC2U and RNAeditDB will help researchers explore the RNA editing events in plants and thus will be of broad utility for the plant research community.

From environmental responses to adaptation: the roles of plant lncRNAs.

As sessile organisms, plants are continuously exposed to heterogeneous and changing environments and constantly need to adapt their growth strategies. They have evolved complex mechanisms to recognize various stress factors, activate appropriate signaling pathways, and respond accordingly by reprogramming the expression of multiple genes at the transcriptional, post-transcriptional, and even epigenome levels to tolerate stressful conditions such as drought, high temperature, nutrient deficiency, and pathogenic interactions. Apart from protein-coding genes, long non-coding RNAs (lncRNAs) have emerged as key players in plant adaptation to environmental stresses. They are transcripts larger than 200 nucleotides without protein-coding potential. Still, they appear to regulate a wide range of processes, including epigenetic modifications and chromatin reorganization, as well as transcriptional and post-transcriptional modulation of gene expression, allowing plant adaptation to various environmental stresses. LncRNAs can positively or negatively modulate stress responses, affecting processes such as hormone signaling, temperature tolerance, and nutrient deficiency adaptation. Moreover, they also seem to play a role in stress memory, wherein prior exposure to mild stress enhances plant ability to adapt to subsequent stressful conditions. In this review, we summarize the contribution of lncRNAs in plant adaptation to biotic and abiotic stresses, as well as stress memory. The complex evolutionary conservation of lncRNAs is also discussed and provides insights into future research directions in this field.

The miR159a-PeMYB33 module regulates poplar adventitious rooting through the abscisic acid signal pathway.

As sessile organisms, plants experience variable environments and encounter diverse stresses during their growth and development. Adventitious rooting, orchestrated by multiple coordinated signaling pathways, represents an adaptive strategy evolved by plants to adapt to cope with changing environmental conditions. This study uncovered the role of the miR159a-PeMYB33 module in the formation of adventitious roots (ARs) synergistically with abscisic acid (ABA) signaling in poplar. Overexpression of miR159a increased the number of ARs and plant height while reducing sensitivity to ABA in transgenic plants. In contrast, inhibition of miR159a (using Short Tandem Target Mimic) or overexpression of PeMYB33 decreased the number of ARs in transgenic plants. Additionally, miR159a targets and cleaves transcripts of PeMYB33 using degradome analysis, which was further confirmed by a transient expression experiment of poplar protoplast. We show the miR159a-PeMYB33 module controls ARs development in poplar through ABA signaling. In particular, we demonstrated that miR159a promotes the expression of genes in the ABA signaling pathway. The findings from this study shed light on the intricate regulatory mechanisms governing the development of ARs in poplar plants. The miR159a-PeMYB33 module, in conjunction with ABA signaling, plays a crucial role in modulating AR formation and subsequent plant growth.

Nonsense-mediated mRNA decay pathway in plants under stress: general gene regulatory mechanism and advances.

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.

Review: Plant microRNAs in pathogen defense: A panacea or a piece of the puzzle?

Plant microRNAs (miRNAs) control key agronomic traits that are associated with their conserved role(s) in development. However, despite a multitude of studies, the utility of miRNAs in plant-pathogen resistance remains less certain. Reviewing the literature identifies three general classes of miRNAs regarding plant pathogen defense. Firstly, a number of evolutionary dynamic 22 nucleotide miRNA families that repress large numbers of plant immunity genes, either directly, or through triggering the biogenesis of secondary siRNAs. However, understanding of their role in defense and of their manipulation to enhance pathogen resistance are still lacking. Secondly, highly conserved miRNAs that indirectly impact disease resistance through their targets that are primarily regulating development or hormone signaling. Any alteration of these miRNAs usually results in pleiotropic impacts, which may alter disease resistance in some plant species, and against some pathogens. Thirdly, are the comparatively diverse and evolutionary dynamic set of non-conserved miRNAs, some of which contribute to pathogen resistance, but whose narrow evolutionary presence will likely restrict their utility. Therefore, reflecting the diverse and evolving nature of plant-pathogen interactions, a complex interplay of plant miRNAs with pathogen responses exists. Any miRNA-based solution for pathogen resistance will likely be highly specific, rather than a general panacea.

RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.