Evolution and post-transcriptional regulation insights of m6A writers, erasers, and readers in plant epitranscriptome.

As a dynamic and reversible post-transcriptional marker, N6-methyladenosine (m6A) plays an important role in the regulation of biological functions, which are mediated by m6A pathway components including writers (MT-A70, FIP37, VIR and HAKAI family), erasers (ALKBH family) and readers (YTH family). There is an urgent need for a comprehensive analysis of m6A pathway components across species at evolutionary levels. In this study, we identified 4062 m6A pathway components from 154 plant species including green algae, utilizing large-scale phylogenetic to explore their origin and evolution. We discovered that the copy number of writers was conserved among different plant lineages, with notable expansions in the ALKBH and YTH families. Synteny network analysis revealed conserved genomic contexts and lineage-specific transpositions. Furthermore, we used Direct RNA Sequencing (DRS) to reveal the Poly(A) length (PAL) and m6A ratio profiles in six angiosperms species, with a particular focus on the m6A pathway components. The ECT1/2-Poeaece4 sub-branches (YTH family) with unique genomic contexts exhibited significantly higher expression level than genes of other ECT1/2 poeaece sub-branches (ECT1/2-Poeaece1-3), accompanied by lower m6A modification and PAL. Besides, conserved m6A sites distributed in CDS and 3'UTR were detected in the ECT1/2-Poaceae4, and the dual-luciferase assay further demonstrated that these conserved m6A sites in the 3'UTR negatively regulated the expression of Firefly luciferase (LUC) gene. Finally, we developed transcription factor regulatory networks for m6A pathway components, using yeast one-hybrid assay demonstrated that PheBPC1 could interact with the PheECT1/2-5 promoter. Overall, this study presents a comprehensive evolutionary and functional analysis of m6A pathway components and their modifications in plants, providing a valuable resource for future functional analysis in this field.

Integrating Physiological Features and Proteomic Analyses Provides New Insights in Blue/Red Light-Treated Moso Bamboo (Phyllostachys edulis).

Bamboo is one of the most important nontimber forestry products in the world. Light is not only the most critical source of energy for plant photosynthesis but also involved in regulating the biological processes of plants. However, there are few reports on how blue/red light affects Moso bamboo. This study investigated the growth status and physiological responses of Moso bamboo (Phyllostachys edulis) to blue/red light treatments. The growth status of the bamboo plants was evaluated, revealing that both blue- and red-light treatments promoted plant height and overall growth. Gas exchange parameters, chlorophyll fluorescence, and enzyme activity were measured to assess the photosystem response of Moso bamboo to light treatments. Additionally, the blue light treatment led to a higher chlorophyll content and enzyme activities compared to the red light treatment. A tandem mass tag quantitative proteomics approach identified significant changes in protein abundance under different light conditions with specific response proteins associated with distinct pathways, such as photosynthesis and starch metabolism. Overall, this study provides valuable insights into the physiological and proteomic responses of Moso bamboo to blue/red light treatments, highlighting their potential impact on growth and development.

Insight into gene expression associated with DNA methylation and small RNA in the rhizome-root system of Moso bamboo.

Moso bamboo (Phyllostachys edulis), typically a monopodial scattering bamboo, is famous for its rapid growth. The rhizome-root system of Moso bamboo plays a crucial role in its clonal growth and spatial distribution. However, few studies have focused on rhizome-root systems. Here we collected LBs, RTs, and RGFNSs, the most important parts of the rhizome-root system, to study the molecular basis of the rapid growth of Moso bamboo due to epigenetic changes, such as DNA modifications and small RNAs. The angle of the shoot apical meristem of LB gradually decreased with increasing distance from the mother plant, and the methylation levels of LB were much higher than those of RT and RGFNS. 24 nt small RNAs and mCHH exhibited similar distribution patterns in transposable elements, suggesting a potential association between these components. The miRNA abundance of LB gradually increased with increasing distance from the mother plant, and a negative correlation was observed between gene expression levels and mCG and mCHG levels in the gene body. This study paves the way for further exploring the effects of epigenetic factors on the physiology of Moso bamboo.

Inhibition of DNA and RNA methylation disturbs root development of moso bamboo.

DNA methylation (5mC) and N6-methyladenosine (m6A) are two important epigenetics regulators, which have a profound impact on plant growth development. Phyllostachys edulis (P. edulis) is one of the fastest spreading plants due to its well-developed root system. However, the association between 5mC and m6A has seldom been reported in P. edulis. In particular, the connection between m6A and several post-transcriptional regulators remains uncharacterized in P. edulis. Here, our morphological and electron microscope observations showed the phenotype of increased lateral root under RNA methylation inhibitor (DZnepA) and DNA methylation inhibitor (5-azaC) treatment. RNA epitranscriptome based on Nanopore direct RNA sequencing revealed that DZnepA treatment exhibits significantly decreased m6A level in the 3'-untranslated region (3'-UTR), which was accompanied by increased gene expression, full-length ratio, higher proximal poly(A) site usage and shorter poly(A) tail length. DNA methylation levels of CG and CHG were reduced in both coding sequencing and transposable element upon 5-azaC treatment. Cell wall synthesis was impaired under methylation inhibition. In particular, differentially expressed genes showed a high percentage of overlap between DZnepA and 5-azaC treatment, which suggested a potential correlation between two methylations. This study provides preliminary information for a better understanding of the link between m6A and 5mC in root development of moso bamboo.

Development of an efficient expression system with large cargo capacity for interrogation of gene function in bamboo based on bamboo mosaic virus.

Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions. Although bamboo has high economic value and produces much biomass quickly, gene functional research is hindered by the low efficiency of genetic transformation in this species. We therefore explored the potential of a bamboo mosaic virus (BaMV)-mediated expression system to investigate genotype-phenotype associations. We determined that the sites between the triple gene block proteins (TGBps) and the coat protein (CP) of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species. Moreover, we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1, which resulted in the promotion and suppression of internode elongation, respectively. In particular, this system was able to drive the expression of three 2A-linked betalain biosynthesis genes (more than 4 kb in length) to produce betalain, indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future. Since BaMV can infect multiple bamboo species, we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.

Transcriptome Profiling of Stem-Differentiating Xylem in Response to Abiotic Stresses Based on Hybrid Sequencing in Cunninghamia lanceolata.

Cunninghamia lanceolata (C. lanceolata) belongs to Gymnospermae, which are fast-growing and have desirable wood properties. However, C. lanceolata's stress resistance is little understood. To unravel the physiological and molecular regulation mechanisms under environmental stresses in the typical gymnosperm species of C. lanceolata, three-year-old plants were exposed to simulated drought stress (polyethylene glycol 8000), salicylic acid, and cold treatment at 4 °C for 8 h, 32 h, and 56 h, respectively. Regarding the physiological traits, we observed a decreased protein content and increased peroxidase upon salicylic acid and polyethylene glycol treatment. Superoxide dismutase activity either decreased or increased at first and then returned to normal under the stresses. Regarding the molecular regulation, we used both nanopore direct RNA sequencing and short-read sequencing to reveal a total of 5646 differentially expressed genes in response to different stresses, of which most had functions in lignin catabolism, pectin catabolism, and xylan metabolism, indicating that the development of stem-differentiating xylem was affected upon stress treatment. Finally, we identified a total of 51 AP2/ERF, 29 NAC, and 37 WRKY transcript factors in C. lanceolata. The expression of most of the NAC TFs increased under cold stress, and the expression of most of the WRKY TFs increased under cold and SA stress. These results revealed the transcriptomics responses in C. lanceolata to short-term stresses under this study's experimental conditions and provide preliminary clues about stem-differentiating xylem changes associated with different stresses.

The pattern of DNA methylation alteration, and its association with the expression changes of non-coding RNAs and mRNAs in Moso bamboo under abiotic stress.

Epigenetic changes play an important role in plant growth and development and in stress response. However, DNA methylation pattern and its relationship with the expression changes of non-coding RNAs and mRNAs of Moso bamboo in response to abiotic stress is still largely unknown. In this work, we used whole-genome bisulfite sequencing in combination with whole-transcriptome sequencing to analyze the DNA methylation and transcription patterns of mRNAs and non-coding RNAs in Moso bamboo under abiotic stresses such as cold, heat, ultraviolet (UV) and salinity. We found that CHH methylation in the promoter region was positively correlated with gene expression, while CHG and CHH methylations in the gene body regions were negatively associated with gene expression. Moreover, CG and CHG methylations in the promoter regions were negatively correlated with the transcript abundance of long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs). Similarly, the methylation levels of three contexts in the genic regions were negatively correlated with the transcript abundance of lncRNAs and miRNAs but positively correlated with that of circRNAs. In addition, we suggested that the reduction of 21-nt and 24-nt small interfering RNA (siRNA) expression tended to increase methylation levels in the genic regions. We found that stress-responsive genes such as CRPK1, HSFB2A and CIPK were differentially methylated and expressed. Our results also proposed that DNA methylation may regulate the expression of the transcription factors (TFs) and plant hormone signalling genes such as IAA9, MYC2 and ERF110 in response to abiotic stress. This study firstly reports the abiotic stress-responsive DNA methylation pattern and its involvement of expression of coding RNAs and non-coding RNAs in Moso bamboo. The results expand the knowledge of epigenetic mechanisms in Moso bamboo under abiotic stress and support in-depth deciphering of the function of specific non-coding RNAs in future studies.

Analysis of Transcriptome and Epitranscriptome in Plants Using PacBio Iso-Seq and Nanopore-Based Direct RNA Sequencing.

Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses.

Genome-Wide Profiling of Circular RNAs in the Rapidly Growing Shoots of Moso Bamboo (Phyllostachys edulis).

Circular RNAs, including circular exonic RNAs (circRNA), circular intronic RNAs (ciRNA) and exon-intron circRNAs (EIciRNAs), are a new type of noncoding RNAs. Growing shoots of moso bamboo (Phyllostachys edulis) represent an excellent model of fast growth and their circular RNAs have not been studied yet. To understand the potential regulation of circular RNAs, we systematically characterized circular RNAs from eight different developmental stages of rapidly growing shoots. Here, we identified 895 circular RNAs including a subset of mutually inclusive circRNA. These circular RNAs were generated from 759 corresponding parental coding genes involved in cellulose, hemicellulose and lignin biosynthetic process. Gene co-expression analysis revealed that hub genes, such as DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), MAINTENANCE OF METHYLATION (MOM), dicer-like 3 (DCL3) and ARGONAUTE 1 (AGO1), were significantly enriched giving rise to circular RNAs. The expression level of these circular RNAs presented correlation with its linear counterpart according to transcriptome sequencing. Further protoplast transformation experiments indicated that overexpressing circ-bHLH93 generating from transcription factor decreased its linear transcript. Finally, the expression profiles suggested that circular RNAs may have interplay with miRNAs to regulate their cognate linear mRNAs, which was further supported by overexpressing miRNA156 decreasing the transcript of circ-TRF-1 and linear transcripts of TRF-1. Taken together, the overall profile of circular RNAs provided new insight into an unexplored category of long noncoding RNA regulation in moso bamboo.

The interplay between microRNA and alternative splicing of linear and circular RNAs in eleven plant species.

MOTIVATION: MicroRNA (miRNA) and alternative splicing (AS)-mediated post-transcriptional regulation has been extensively studied in most eukaryotes. However, the interplay between AS and miRNAs has not been explored in plants. To our knowledge, the overall profile of miRNA target sites in circular RNAs (circRNA) generated by alternative back splicing has never been reported previously. To address the challenge, we identified miRNA target sites located in alternatively spliced regions of the linear and circular splice isoforms using the up-to-date single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) and Illumina sequencing data in eleven plant species. RESULTS: In total, we identified 399 401 and 114 574 AS events from linear and circular RNAs, respectively. Among them, there were 64 781 and 41 146 miRNA target sites located in linear and circular AS region, respectively. In addition, we found 38 913 circRNAs to be overlapping with 45 648 AS events of its own parent isoforms, suggesting circRNA regulation of AS of linear RNAs by forming R-loop with the genomic locus. Here, we present a comprehensive database of miRNA targets in alternatively spliced linear and circRNAs (ASmiR) and a web server for deposition and identification of miRNA target sites located in the alternatively spliced region of linear and circular RNAs. This database is accompanied by an easy-to-use web query interface for meaningful downstream analysis. Plant research community can submit user-defined datasets to the web service to search AS regions harboring small RNA target sites. In conclusion, this study provides an unprecedented resource to understand regulatory relationships between miRNAs and AS in both gymnosperms and angiosperms. AVAILABILITY AND IMPLEMENTATION: The readily accessible database and web-based tools are available at http://forestry.fafu.edu.cn/bioinfor/db/ASmiR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications.

BACKGROUND: Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date. RESULTS: Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA3, suggesting that post-transcriptional regulation might be also involved in the GA3 response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA3-induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition. CONCLUSIONS: This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.

A novel double-antigen sandwich enzyme-linked immunosorbent assay for Measurement of Antibodies against rabies virus.

A novel double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) was developed to measure rabies antibodies in dogs. In contrast to the 4 days required for detecting rabies antibody with conventional rabies antibody virus neutralization assays, this ELISA can be completed in hours, without using live virus, in routine laboratories.