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Article

Genome-Wide Methylation Landscape Uncovers the Role of DNA Methylation in Ramie (Boehmeria nivea L.) Bast Fiber Growth

by
Fu Li
1,2,3,†,
Bingbing Luo
1,†,
Yanzhou Wang
3,†,
Jing Rao
3,
Song Gao
2,
Qingzhong Peng
1,
Touming Liu
2,* and
Langbo Yi
1,*
1
College of Biology and Environmental Sciences, Jishou University, Jishou 416000, China
2
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
3
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this article.
Agronomy 2024, 14(7), 1467; https://doi.org/10.3390/agronomy14071467 (registering DOI)
Submission received: 29 May 2024 / Revised: 27 June 2024 / Accepted: 5 July 2024 / Published: 6 July 2024
(This article belongs to the Special Issue Genomics and Genetic Improvement of Bast Fiber Plants)
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Abstract

:
Ramie is one of the most important fiber crops in China, with fibers extracted from stem barks having been used as textile materials for thousands of years. DNA methylation is an important epigenetic modification involved in plant growth and development. However, the role of methylation in ramie fiber growth remains poorly understood. In the present study, we investigated the DNA methylation landscape of the nuclear genome in bark sections taken from the top (TPS) and the middle (MPS) of the stems of ramie plants, which represent different stages of fiber growth, using whole-genome bisulfite sequencing. We detected 7,709,555 and 8,508,326 5-methylcytosines in the TPS and MPS genomes, respectively. The distribution of methylation across three sequence contexts, CG, CHG, and CHH, varied greatly among gene elements, with methylation at CHH being the most prevalent. Comparison of methylation levels between the TPS and MPS genomes revealed 23.162 Mb of differentially methylated genomic regions, encompassing 9485 genes. Among these differentially methylated genes, 841 exhibited altered expression in the MPS genome. Notably, an SND2 ortholog Bni05G006779 showed a negative correlation between its expression and methylation levels. Overexpression of Bni05G006779 in Arabidopsis dramatically increased the number of xylem fibers and the secondary wall thickness of the fibers in the stems of transgenic plants. These findings provide important insights into the involvement of DNA methylation in regulating ramie fiber growth.

1. Introduction

Fibers, which are typically found in various organs of vascular plants, including roots, stems, and leaves, possess specialized secondary cellular walls primarily composed of cellulose, hemicelluloses (xylan and glucomannan), and lignin [1]. In Arabidopsis, hundreds of genes have been identified that regulate fiber growth, influencing cellulose, lignin, and hemicellulose biosynthesis, as well as the patterned deposition of secondary walls [2]. Recent studies suggest that these genes represent the core of a more complex, multilevel regulatory system involving post-transcriptional and post-translational regulation, one which coordinates with pathways controlling primary growth and responses to environmental stimuli [3].
Ramie (Boehmeria nivea L. Gaud), cultivated in China for over 4700 years, is one of the oldest fiber crops in China [4]. It is the second-most-important fiber crop in China, second only to cotton in terms of cultivation acres and fiber production quantity. Ramie fiber is a bast fiber extracted from the stem bark. Recent genetic and molecular studies have markedly improved our understanding of the mechanisms underlying bast fiber growth [5]. Additionally, many studies have indicated that ramie fiber growth is regulated by an epigenetic mechanism involving non-coding small RNAs and circular RNAs [6,7]. Recently, a model for the mechanism of the fiber growth was proposed [8], and studies have revealed the involvement of phosphorylation and ubiquitination modifications in the regulation of fiber growth [9,10]. Collectively, the findings of these studies indicate that fiber growth in ramie is controlled by a complex regulatory system.
DNA methylation is an important epigenetic modification that typically produces 5-methylcytosine (5mC) by adding a methyl group to the fifth carbon of cytosine in eukaryotic genomes [11,12]. In plants, cytosine methylation primarily occurs in three sequence contexts: CG, CHG, and CHH (where H = A, T, or C) [13]. DNA methylation plays key roles in the regulation of various biological processes during plant growth and development [14,15], such as sex determination [16,17], morphogenesis [18], vegetative propagation [19], and stress response [20,21]. Recent studies have also shown that DNA methylation plays a distinct role in fiber growth in cotton [22,23] and poplar [24]. However, its role in the regulation of fiber growth remains unexplored. Fibers in the bast bark from the top stem section (TPS) and the middle stem section (MPS) represent two different stages of growth: the secondary walls of fiber cells in TPS have not initiated growth, while those in MPS are thickening [5]. Numerous genes involved in fiber growth have been identified based on a multi-omics comparison between TPS and MPS [7,8,9]. Therefore, the major objective of this study was to characterize and compare the methylomes of TPS and MPS to gain a primary understanding of the role of methylation in the regulation of fiber growth. This study provides an important basis for understanding how methylation modifications are involved in the regulation of ramie fiber growth.

2. Materials and Methods

2.1. Experimental Material, Tissue Sampling, and DNA Extraction

The elite cultivar Zhongzhu 1 was used for the methylation analysis in the present study. Seedlings were planted on an experimental farm of the Institute of Bast Fiber Crops, Yuanjiang, China, in June 2016. Following the method described by Li et al. [8], TPS and MPS from five 30-day-old plants were separately collected in May 2018. As shown in Figure S1, a 10 cm section of the bark below the midpoint of the stem and a separate 10 cm section from the top 10 to 20 cm of the stem were collected as the MPS and TPS samples, respectively. Three biological replicates were performed for both TPS and MPS tissue collection. TPS and MPS were designated as the control and experimental groups, respectively. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until further use. Genomic DNA was extracted from these samples using a DNA extraction kit (Tiangen, Beijing, China). Total RNA was isolated from each sample using an E.Z.N.A. Plant RNA Kit (OMEGA Bio-Tek, Norcross, GA, USA).

2.2. Whole Genome Bisulfite Sequencing (WGBS)

For WGBS, three biological replicates each of the TPS and MPS samples were collected. Genomic DNA was fragmented to 200–300 bp lengths using sonication (Covaris, Woburn, MA, USA) and purified using a Mini Elute PCR Purification Kit (Qiagen, Hilden, Germany). Following end-repair, a single adenosine nucleotide was added to the 3′ ends of the blunt fragments. The genomic fragments were then ligated to methylated sequencing adapters. Subsequently, purified ligation products underwent bisulfite conversion, followed by treatment with sodium bisulfite using an EZ DNA Methylation Gold Kit (Zymo Research, Tustin, CA, USA). The resulting DNA fragments were subjected to paired-end sequencing on an Illumina HiSeq Xten platform (Illumina Inc., San Diego, CA, USA). Methylome sequencing and analyses were performed by OE Biotech Co., Ltd. (Shanghai, China).

2.3. Methylation Analysis

After filtering the low-quality reads using the FastQC software (v0.11.5) with default parameters, clean reads were used for subsequent methylation analysis. Briefly, in silico conversion using Bismark software (v0.15) [25] (default parameters) converted cytosines in the forward strands of clean reads and the reference genome to thymines, and guanines on the reverse strands were changed to adenines. The converted reads were then aligned to the reference genome of ramie (Zhongsizhu 1 variety; sequence downloaded from https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA663425 (accessed on 4 July 2024)) using the HISAT2 software (v2.2.1.0) [26], with default parameters.
Methylated cytosine (mC) sites were identified using the R package “methykit” 16 (version 1.2). Only cytosine sites covered by at least ten reads were reserved for further analysis. Confirmation of true mC sites employed the binomial distribution of methylated and unmethylated cytosines, with a false discovery rate (FDR) of ≤0.05. Differentially methylated regions (DMRs) were identified using a sliding-window approach. Briefly, methylation levels of different cytosine sites within 1000-bp tiling windows were integrated. The integrated data for each window were then used to detect DMRs using the logistic regression model. Only regions exhibiting a 10% difference in methylation level (FDR ≤ 0.05) were designated as DMRs. Promoter regions were defined as the 2000 bp length upstream of the transcription start site, while downstream regions encompassed 1000 bp following the transcription termination for the corresponding gene.

2.4. Gene Expression Analysis

Our previous study performed RNA sequencing of the TPS and MPS tissues from the Zhongzhu 1 variety, using the wild Boehmeria nivea var. tenacissima genome as a reference for expression analysis [27]. This study employed the cultivar’s genome (Zhongsizhu 1) as the reference for methylation analysis. This difference in reference genomes presents a challenge in directly comparing transcriptome and methylome data. Therefore, to identify genes potentially regulated by methylation, we estimated the transcript abundance of genes using the Zhongsizhu 1nuclear genome as the reference (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA663425 (accessed on 4 July 2024). Briefly, clean reads (obtained from the NCBI GEO database, accession number GSE130587) were aligned to the cultivar reference using HISAT2 software (v2.2.1.0) [26]. Fragments per kilobase per million reads (FPKM) values were calculated to quantify gene expression levels [28]. The DESEQ program (v1.18.0) [29] was used to analyze expression differences between TPS and MPS tissues. Genes with a p value of less than 0.05 and a fold-change greater than two were considered a significantly differential expression.

2.5. Overexpression of Bni05G006779

Polymerase chain reaction amplified the full-length cDNA of Bni05G006779 using a high-fidelity thermostable DNA polymerase, with the following the primer sequences: 5′-ATGACATGGTGCAACGACTCATC-3′ (forward primer) and 5′-CTACATCTTCCTCTCAAGCTTTCCC-3′ (reverse primer). The amplified cDNA was then introduced into the pBI121 vector downstream of the CaMV 35S promoter. The resulting vector (35S: Bnt08G012573) was introduced into Agrobacterium tumefaciens strain GV3101 by heat shock. Subsequently, the resulting Agrobacterium strain was used to infiltrate an Arabidopsis plant using the floral dip method [30]. Both transgenic plants and wild-type control were grown in a greenhouse, under controlled conditions of 22 °C with a 15 h light/9 h dark cycle. After 40 days, the Arabidopsis stems were sectioned, stained with safranin O-fast green, and observed under a transmission-light microscope (CSOIF, Shanghai, China).

3. Results

3.1. Characterization of the DNA Methylation Landscape in the Ramie Nuclear Genome

To investigate the genomic DNA methylation landscape of the ramie, we performed bisulfite sequencing of the bast bark tissues from TPS and MPS using the Illumina HiSeq 2500 platform. Approximately 511 million clean reads, with a total length of 76.7 Gb, were obtained from six libraries (i.e., three biological replicates each for TPS and MPS) (Table 1). These reads provided an average coverage depth of approximately 47.9 folds for each sample across the entire ramie reference genome. Subsequently, the reads were aligned to the reference genome using the Bismark program [25], achieving a mapping rate between 48.7% and 52.5%. During bisulfite sequencing, unmethylated cytosines were changed to thymines, and the conversion rates estimated for the six samples exceeded 99.8%. Based on bisulfite conversion, 7,709,555 (51.1%) and 8,508,326 (50.2%) mCs were identified in the TPS and MPS genomes, respectively, corresponding to average methylation levels of 43.3% and 42.2% (Table 2).
In plants, cytosine methylation occurs primarily in three sequences: CG, CHG, and CHH [13]. Our analysis revealed that CHH was the most abundant methylation sequence context (~70.0%) in the ramie genome, followed by CG (20.6%) and CHG (11.4%) (Figure 1a). CG displayed the highest methylation level (~86.0%), while CHG and CHH exhibited methylation levels of approximately 62.3% and 27.8%, respectively (Figure 1b, Table 2). Notably, the methylation level of CHG sequence in TPS was ~3.1% higher than that in MPS, whereas the other sequence contexts displayed lower methylation levels in TPS than in MPS (Table 2). The density levels of mC in chromosomes 2 and 11 were 57.17 and 56.77 mCs per kb, while chromosome 12 exhibited a density of 92.02 mCs per kb, indicating an uneven distribution of mC among chromosomes (Table 3). Interestingly, all ramie chromosomes were telocentric, and we observed that the mCs clustered dramatically at the ends of the telocentric regions (Figure 1c).
We also investigated the methylation levels in various gene elements and found that all three sequence contexts showed high methylation levels in the gene promoter and intron regions. Specially, CG was highly methylated in the exon region, while CHG and CHH were highly methylated in the downstream regions of the genes (Figure 1d). Comparison of methylation levels between the genomes of TPS and MPS revealed distinct patterns in the genic region: MPS tissue, associated with fiber development, exhibited lower methylation levels for CG and CHH sequences, but higher methylation levels for CHG sequences (Table 4). This suggests that DNA methylation probably plays a role in regulating fiber growth. However, little difference in the methylation of the CG and CHH sequences was observed between the TPS and MPS tissues (Table 5). Notably, the methylation levels in the genic region were far lower than those in repetitive sequences. Interestingly, minimal methylation was identified in the short, interspersed nuclear elements, which are components of repetitive sequences (Table 4).
Taken together, our DNA methylation results revealed distinct features in the ramie genome, and the large-scale identification of methylated sites provided an important basis for the epigenetic study of this fiber crop.

3.2. Identification of Differentially Methylated Genes Associated with Fiber Growth

To identify DMRs in the genomes of TPS and MPS, we employed a sliding-window approach (window size: 1 kb) to scan genomic regions which exhibited significant methylation differences (≥10% in methylated level, p-value < 0.05) between MPS and TPS. This analysis revealed 9.831 Mb, 13.634 Mb, and 2.081 Mb of genomic regions with significant differences in the levels of mCG, mCHG, and mCHH, respectively (Figure 2a, Tables S1–S3). Additionally, 2.244 Mb of the genome displayed significant methylation differences in at least two of the three sequence contexts (Figure 2b). In total, 23.162 Mb of genomic sequence, encompassing 9485 genes, was identified as having significant differences in methylation levels (Table S4).
Expression analysis of TPS and MPS tissues identified 1695 genes with differential expression (Table S5). Among these differentially expressed genes (DEGs), 841 overlapped with differentially methylated genes (Table S6). Furthermore, 443 of these genes displayed a statistically significant correlation (Pearson correlation analysis, p-value < 0.05) between methylation changes and gene expression levels (Table S6). For example, we observed a cellulose synthase (CesA) gene, Bni05G008018, with a significant increase in transcript abundance in MPS tissues, suggesting its role in cellulose biosynthesis during fiber growth (Figure 2c). Notably, a methylated site located downstream of this gene displayed a distinct decrease in methylation level in the MPS tissues, and the methylation and gene expression levels were significantly negatively correlated (correlation coefficient = −0.839, p-value < 0.05). These findings indicate that the expression of Bni05G008018 is potentially regulated by DNA methylation. Beyond Bni05G008018, the expression levels of two other differentially expressed cellulose synthase genes, Bni01G000021 and Bni04G005289, also exhibited significant correlations with changes in the methylation level of the genic regions (p-value < 0.05; Table S6). Additionally, we identified three ramie genes, Bni04G006553, Bni10G014747, and Bni03G005033, encoding homologs of IRX15 (a key enzyme in hemicellulose biosynthesis) [31], HCT, and COMT (essential for lignin biosynthesis) [32], respectively. These genes displayed distinct differences in both gene expression and methylation levels between TPS and MPS tissues. Enrichment analysis of the 841 gene revealed significant enrichment for functional Gene Ontology (GO) terms related to secondary-wall biosynthesis of fibers (Figure 2d), including lignin catabolic process (GO:0046274; p-value = 0.00076), carbohydrate metabolic process (GO:0005975; p-value = 0.00249), cellulose biosynthetic process (GO:0030244; p-value = 0.00604), and cellulose synthase (UDP-forming) activity (GO:0016760; p-value = 0.00395). Collectively, these results strongly support the involvement of DNA methylation in the regulation of fiber growth in ramie.

3.3. Involvement of DNA Methylation in the Regulation of Fiber Growth

Secondary-wall biosynthesis during fiber growth is mediated by a regulatory network involving NAC and MYB transcription factors. In Arabidopsis, at least 16 MYB and 13 NAC genes have been identified as being involved in this network [2]. Notably, among the 841 genes exhibiting differential methylation and expression levels in the MPS tissues, 17 were putative MYB genes. Phylogenetic analysis revealed four MYB transcription factors (Bni06G008563, Bni11G015705, Bni10G014673, and Bni03G004672) as homologs of Arabidopsis MYB proteins involved in secondary-wall biosynthesis. One of these MYB genes, Bni06G008563, is the ortholog of Arabidopsis AtMYB4, which is known to be involved in secondary-wall biosynthesis [2] (Figure S2). Interestingly, the methylation level of the mCG sequence in the promoter region of Bni06G008563 displayed a significant negative correlation with its expression level during fiber development (correlation coefficient = −0.842, p-value < 0.05). In addition, three of the 841 identified genes encoded NAC proteins, including Bni05G006779, which encodes an ortholog of SND2 (Figure 3a), a key regulator of Arabidopsis secondary-wall biosynthesis [33]. Two CHG sites flanking Bni05G006779, one upstream and one downstream, displayed a distinct decrease in methylation levels in the MPS tissues (Figure 3b). However, the expression level of Bni05G006779 was significantly enhanced in the MPS tissues (Figure 3c), displaying a significant negative correlation with the methylation level of the upstream methylated site (correlation coefficient = −0.985, p-value < 0.05). These findings suggest that the methylation of the upstream CHG site might play a role in regulating the expression of Bni05G006779. Overexpression of Bni05G006779 in Arabidopsis dramatically increased the stem xylem fiber number and secondary wall thickness of fibers (Figure 3d,e), suggesting that Bni05G006779 regulates fiber growth. Based on these results, we propose the following model for the function of Bni05G006779 (Figure 3f): (1) DNA methylation represses the expression of the NAC gene Bni05G006779, and (2) during fiber development, demethylation of the upstream region activates the expression of Bni05G006779, thereby triggering fiber growth.

4. Discussion

In plants, DNA methylation plays an essential role in maintaining genomic stability and regulating gene expression during developmental and stress-response processes [13]. WGBS is a powerful tool, allowing researchers to investigate genome-wide DNA methylation patterns at single-nucleotide resolution. WGBS has been applied in a growing number of species, including model plants like Arabidopsis [15] and poplar [17,24], and agriculturally important crops like rice [34,35], maize [36], soybean [37], cotton [22,23,38], tomato [39], spruce [40], and oil palm [41]. In the present study, we used WGBS to decipher the single-base methylation pattern in the stem bark of ramie. This study is the first overview of the overall cytosine methylation landscape in this fiber crop. Of the reads from WGBS, approximately 50% can be mapped within the ramie genome, and this mapping rate is accordance with previous studies using WGBS [42,43]. It is probable that the conversion of unmethylated cytosine caused an alteration in the sequence from WGBS, which is likely a reason for the relatively low mapping rate found in aligning these WGBS reads onto the genome. We found that ramie has higher overall methylation levels compared to cotton, another fiber crop, for all three sequence contexts [38]. Additionally, we identified 841 genes with differential methylation patterns that are potentially involved in fiber growth. These findings indicate the potential involvement of DNA methylation in controlling ramie fiber growth.
NAC and MYB proteins constitute two major classes of transcription factors that modulate fiber growth by regulating the expression of numerous genes involved in secondary-wall biosynthesis [2]. Recent studies have shown that several NAC and MYB genes in ramie are involved in fiber growth. For example, the MYB protein whole_GLEAN_10015497 significantly promotes fiber growth in transgenic Arabidopsis, and its ubiquitination level is significantly reduced in the MPS tissue [10]. In Arabidopsis, NST1, NST2, VND4, and VND5 are master-switch proteins that trigger secondary-wall biosynthesis [2]. In ramie, Bnt08G012573 encodes the ortholog of NST1/NST2, and Bnt03G004997 encodes the ortholog of VND4/VND5; overexpression of these genes remarkably increases the number of xylem fibers in Arabidopsis [27,44]. These studies indicate that NAC and MYB proteins play similar roles in the regulation of fiber growth in ramie. In the present study, we identified 20 MYB and three NAC genes related to fiber growth. Notably, one NAC gene, Bni05G006779, was found to significantly promote the fiber formation in transgenic Arabidopsis. Additionally, these genes exhibited differential methylation in the bark tissue at different stages of fiber development, suggesting the potential involvement of methylation in regulating ramie fiber growth. The identification of these fiber growth-related genes provides a valuable genetic resource for improving fiber traits in ramie and holds great potential for application in its molecular breeding.
Previous studies have demonstrated that DNA methylation regulates numerous MYB genes and plays critical roles in regulating plant growth and development. These include the regulation of fruit and seed coat color [45,46], anthocyanin biosynthesis [47,48], and stress responses [49]. Additionally, several NAC genes can also be regulated by methylation. For instance, the insertion of a miniature inverted-repeat transposable element into the promoter of maize ZmNAC111, and its subsequent methylation, represses ZmNAC111 expression, ultimately affecting drought tolerance [50]. A recent study revealed that N6-methyladenosine mRNA modification is crucial for the regulation of fiber elongation in cotton by controlling the stability of transcripts of GhMYB44, a negative regulatory gene [51]. In the present study, the identification of methylation within the fiber growth-related NAC and MYB genes provides novel insights into the regulatory role of DNA methylation in fiber development. These findings establish an important foundation for further exploration of the mechanisms underlying fiber formation in plants.

5. Conclusions

This study revealed the methylation landscape of ramie bark fibers under different stages of growth, resulting in the identification of 23.162 Mb of differentially methylated genomic regions, encompassing 841 genes potentially related to fiber growth. The NAC transcription factor encoded by Bni05G006779 was shown to facilitate the formation of xylem fibers in Arabidopsis. Methylation of two CHG sites, in the upstream and downstream regions of Bni05G006779, resulted in a negative correlation with its expression. These findings provide important insights into the involvement of methylation in the control of ramie fiber growth.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14071467/s1. Figure S1: Microscopic observation of fiber cells from the stem barks of ramie [5]. The barks from the top part of stem (TPS) and from the middle part of stem (MPS), places where the secondary cellular walls have not initiated their growth, and where the walls are thickening, respectively, were used for methylome analysis in this study. Figure S2: Phylogenetic relation of 17 ramie MYB members and Arabidopsis secondary wall-biosynthetic MYB proteins. Bni06G008563 is the ortholog of AtMYB4. Table S1: Genomic regions with significant differences in the methylation levels of CG. Table S2: Genomic regions with significant differences in the methylation levels of CHG. Table S3: Genomic regions with significant differences in the methylation levels of CHH. Table S4: Genes containing differentially methylated sites. Table S5: Genes differentially expressed between the TPS and MPS. Table S6: Genes with differences in both methylation level and transcript abundance.

Author Contributions

F.L., B.L. and J.R. performed the experiment designed to analyze the function of Bni05G006779. Q.P. performed the sample and extracted genomic DNA. Y.W. carried out WGBS analysis and performed the bioinformatic analysis. S.G. managed the project and contributed novel reagents. L.Y. and T.L. designed this study and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Natural Science Foundation of Hunan Province (2022JJ30649), and the China Agriculture Research System of MOF and MARA (CARS-16).

Data Availability Statement

The raw sequence reads of whole genome bisulfite sequencing have been deposited in the National Genomics Data Center (NGDC), under the project number of PRJCA027196, and the other data are contained within the article or Supplementary Materials.

Conflicts of Interest

The authors declare no competing interests.

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Figure 1. Characteristics of the methylome of ramie bast bark. (a) Percentages of methylation according to three sequence contexts, CG, CHG, and CHH, in the bast bark. Among these, methylation is most prevalent at mCHH. (b) Methylation levels of CG, CHG, and CHH. The y-axis indicates the percentage of mCs compared to the total number of cytosines for each sequence context. (c) Distribution of mCs compared to the corresponding total number of cytosines (both methylated and unmethylated) across the 14 chromosomes of ramie. (d) Methylation levels of the three sequence contexts within different gene elements.
Figure 1. Characteristics of the methylome of ramie bast bark. (a) Percentages of methylation according to three sequence contexts, CG, CHG, and CHH, in the bast bark. Among these, methylation is most prevalent at mCHH. (b) Methylation levels of CG, CHG, and CHH. The y-axis indicates the percentage of mCs compared to the total number of cytosines for each sequence context. (c) Distribution of mCs compared to the corresponding total number of cytosines (both methylated and unmethylated) across the 14 chromosomes of ramie. (d) Methylation levels of the three sequence contexts within different gene elements.
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Figure 2. Comparison of the methylation levels between the top (TPS) and middle (MPS) stem sections of ramie. (a) Identification of differentially methylated context sequences, mCG, mCHG, and mCHH, in the ramie genome. A red dashed line indicates the threshold for differences. (b) The Venn diagram identifies genomic regions that contain two or three differentially methylated context sequences. (c) The expression of the cellulose synthase gene Bni05G008018 shows a significant increase, while its methylation level displays distinct decrease in the MPS tissues. The y-axis in the left panel indicates the methylation level (%) and the y-axis on the right panel represents the fragments per kilobase per million reads value. (d) Gene Ontology terms enriched among the 841 genes that exhibit differential methylation and expression. The red arrow points to functional terms associated with fiber growth.
Figure 2. Comparison of the methylation levels between the top (TPS) and middle (MPS) stem sections of ramie. (a) Identification of differentially methylated context sequences, mCG, mCHG, and mCHH, in the ramie genome. A red dashed line indicates the threshold for differences. (b) The Venn diagram identifies genomic regions that contain two or three differentially methylated context sequences. (c) The expression of the cellulose synthase gene Bni05G008018 shows a significant increase, while its methylation level displays distinct decrease in the MPS tissues. The y-axis in the left panel indicates the methylation level (%) and the y-axis on the right panel represents the fragments per kilobase per million reads value. (d) Gene Ontology terms enriched among the 841 genes that exhibit differential methylation and expression. The red arrow points to functional terms associated with fiber growth.
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Figure 3. Functional characterization of ramie NAC gene Bni05G006779. (a) Phylogenetic tree showing the relationships among three ramie NAC proteins (Bni10G014175, Bni10G014904, and Bni05G006779) and Arabidopsis NAC proteins involved in secondary-wall biosynthesis. Bni05G006779 is an ortholog of SND2. (b) Two mCs located upstream (3407 bp) and downstream (406 bp) of Bni05G006779 exhibit significantly reduced methylation levels in the MPS tissues. (c) The expression of Bni05G006779 is up-regulated in the MPS compared to in the TPS. The y-axis represents the fragments per kilobase per million reads value. (d,e) Light microscopy findings of transected stems of wild-type and Bni05G006779-overexpressing Arabidopsis. Secondary walls are stained red, and most of these stained cells represent xylem fiber cells. Arrows in (d,e) indicate fiber cells and their cell wall, respectively. The scale bar represents 200 μm (d) and 20 μm (e). (f) A proposed model illustrating the role of Bni05G006779 in promoting fiber growth. This model suggests that changes in methylation influence the expression of Bni05G006779, thereby regulating fiber growth.
Figure 3. Functional characterization of ramie NAC gene Bni05G006779. (a) Phylogenetic tree showing the relationships among three ramie NAC proteins (Bni10G014175, Bni10G014904, and Bni05G006779) and Arabidopsis NAC proteins involved in secondary-wall biosynthesis. Bni05G006779 is an ortholog of SND2. (b) Two mCs located upstream (3407 bp) and downstream (406 bp) of Bni05G006779 exhibit significantly reduced methylation levels in the MPS tissues. (c) The expression of Bni05G006779 is up-regulated in the MPS compared to in the TPS. The y-axis represents the fragments per kilobase per million reads value. (d,e) Light microscopy findings of transected stems of wild-type and Bni05G006779-overexpressing Arabidopsis. Secondary walls are stained red, and most of these stained cells represent xylem fiber cells. Arrows in (d,e) indicate fiber cells and their cell wall, respectively. The scale bar represents 200 μm (d) and 20 μm (e). (f) A proposed model illustrating the role of Bni05G006779 in promoting fiber growth. This model suggests that changes in methylation influence the expression of Bni05G006779, thereby regulating fiber growth.
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Table 1. Statistical data associated with the whole-genome bisulfite sequencing of ramie.
Table 1. Statistical data associated with the whole-genome bisulfite sequencing of ramie.
Clean ReadsTotal Length (Gb)Mapped ReadsMapping Rate (%)
TPS 187,732,990 13.244,221,36850.4
TPS 281,849,044 12.339,854,31448.7
TPS 383,089,394 12.541,034,46849.4
MPS 179,946,258 12.041,992,67052.5
MPS 285,472,562 12.843,255,79650.6
MPS 392,933,364 13.947,537,84851.2
Average85,170,602 12.842,982,74450.5
Total511,023,612 76.7--
Table 2. Summary of data for the methylated cytosine identified from the genomes of stem barks.
Table 2. Summary of data for the methylated cytosine identified from the genomes of stem barks.
SampleCovered CmCmC Percent
(%)		Methylation Level (%)
mCmCGmCHGmCHH
TPS 116,170,7138,209,01150.844.085.964.427.0
TPS 215,021,4047,663,63251.043.185.663.927.2
TPS 314,112,9217,256,02351.442.884.863.227.5
TPS (average) 15,101,6797,709,55551.143.385.463.827.2
MPS 116,500,3458,323,91250.445.087.061.328.9
MPS 216,575,9538,264,59949.943.686.260.828.2
MPS 317,800,2848,936,46750.243.986.260.028.3
MPS (average)16,958,8618,508,32650.244.286.560.728.4
Table 3. Distribution of methylated cytosines in the chromosomes of ramie.
Table 3. Distribution of methylated cytosines in the chromosomes of ramie.
ChromosomemCGmCHGmCHHTotalDensity (per kb)
Chr1229,140211,9241,226,8911,667,95575.33
Chr2157,216149,944854,3911,161,55157.17
Chr3233,205210,1341,219,9971,663,33682.76
Chr4199,460186,9811,063,6111,450,05275.80
Chr5209,529197,6501,154,0451,561,22474.67
Chr6199,774195,8161,159,8791,555,46987.53
Chr7152,426140,522852,6651,145,61364.22
Chr8178,186159,877955,6421,293,70576.68
Chr9210,234186,0181,094,0001,490,25281.82
Chr10146,555132,524748,4701,027,54967.64
Chr11134,106118,233685,194937,53356.77
Chr12217,319194,8641,110,1471,522,33092.02
Chr13131,877119,331696,751947,95962.24
Chr1496,74286,758521,924705,42463.76
Scaffold123,23892,430593,679809,34743.09
Total2,619,0072,383,00613,937,28618,939,29971.04
Table 4. Statistics of methylation levels in gene regions.
Table 4. Statistics of methylation levels in gene regions.
mCG mCHG mCHH
TPSMPSTPSMPSTPSMPS
Promoter0.6960.6800.5120.5300.3180.296
Exon0.6830.6540.3460.3520.1590.147
Intron0.7820.7640.5340.5530.2890.272
Downstream0.6600.6440.4830.5020.2920.270
Intergenic0.8280.8160.5660.6030.2310.219
Table 5. Statistics of methylation levels in repetitive sequences.
Table 5. Statistics of methylation levels in repetitive sequences.
mCG mCHG mCHH
MPSTPSMPSTPSMPSTPS
DNA0.881 0.882 0.618 0.663 0.321 0.303
LINE0.911 0.914 0.693 0.732 0.218 0.214
SINE0.000 0.028 0.024 0.000 0.038 0.078
LTR0.914 0.912 0.651 0.696 0.227 0.220
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Li, F.; Luo, B.; Wang, Y.; Rao, J.; Gao, S.; Peng, Q.; Liu, T.; Yi, L. Genome-Wide Methylation Landscape Uncovers the Role of DNA Methylation in Ramie (Boehmeria nivea L.) Bast Fiber Growth. Agronomy 2024, 14, 1467. https://doi.org/10.3390/agronomy14071467

AMA Style

Li F, Luo B, Wang Y, Rao J, Gao S, Peng Q, Liu T, Yi L. Genome-Wide Methylation Landscape Uncovers the Role of DNA Methylation in Ramie (Boehmeria nivea L.) Bast Fiber Growth. Agronomy. 2024; 14(7):1467. https://doi.org/10.3390/agronomy14071467

Chicago/Turabian Style

Li, Fu, Bingbing Luo, Yanzhou Wang, Jing Rao, Song Gao, Qingzhong Peng, Touming Liu, and Langbo Yi. 2024. "Genome-Wide Methylation Landscape Uncovers the Role of DNA Methylation in Ramie (Boehmeria nivea L.) Bast Fiber Growth" Agronomy 14, no. 7: 1467. https://doi.org/10.3390/agronomy14071467

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