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Article

Coordination of m6A mRNA Methylation and Gene Transcriptome in Sugarcane Response to Drought Stress

by
Jinju Wei
1,2,
Haibi Li
3,
Yiyun Gui
1,2,
Hui Zhou
1,2,
Ronghua Zhang
1,2,
Kai Zhu
1,2,* and
Xihui Liu
1,2,*
1
Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
2
Guangxi Key Laboratory of Sugarcane Genetic Improvement, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
3
Guangxi South Subtropical Agricultural Science Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 532415, China
*
Authors to whom correspondence should be addressed.
Plants 2023, 12(21), 3668; https://doi.org/10.3390/plants12213668
Submission received: 7 October 2023 / Revised: 20 October 2023 / Accepted: 23 October 2023 / Published: 24 October 2023
(This article belongs to the Special Issue Abiotic Stress Responses in Plants)
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Abstract

:
The N6-methyladenosine (m6A) methylation of mRNA is involved in biological processes essential for plant growth. To explore the m6A modification of sugarcane and reveal its regulatory function, methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to construct the m6A map of sugarcane. In this study, m6A sites of sugarcane transcriptome were significantly enriched around the stop codon and within 3′-untranslated regions (3′UTR). Gene ontology (GO) analysis showed that the m6A modification genes are associated with metabolic biosynthesis. In addition, the m6A modification of drought-resistant transcript mRNA increased significantly under drought (DR) treatment, resulting in enhanced mRNA stability, which is involved in regulating sugarcane drought resistance. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results showed that differentially methylated peak (DMP) modification of differentially expressed genes (DEGs) in DR were particularly associated with abscisic acid (ABA) biosynthesis. The upregulated genes were significantly enriched in the ABA metabolism, ethylene response, fatty acid metabolism, and negative regulation of the abscisic acid activation signaling pathway. These findings provide a basis and resource for sugarcane RNA epigenetic studies and further increase our knowledge of the functions of m6A modifications in RNA under abiotic stress.

1. Introduction

N6-methyl-adenosine (m6A) is a prevalent RNA epigenetic modification that affects mRNA in various ways and is critical in regulating plant development and evolution [1]. The m6A methyltransferase complex regulates early rice spikelet degeneration, thereby modulating sporogenesis [2]. Adenosine methylase deposits m6A on pri-miRNAs to regulate miRNA biosynthesis in Arabidopsis. The m6A modification in Arabidopsis is closely related to photosynthesis [3,4], indicating that m6A participates in regulating photosynthesis, affecting the plant circadian rhythm, flowering time, and polyadenylation site selection [5]. m6A demethylation regulates fruit ripening [6] and promotes the proliferation of root meristem tissue cells in rice and the formation of tiller buds, thereby enhancing photosynthetic efficiency and drought tolerance and increasing the yield of rice and potatoes [7]. The level of m6A modification in plants is dynamically regulated by methyltransferases and demethylases and participates in important biological processes.
Growing evidence indicates that m6A plays a role in regulating plant responses to various biotic and abiotic stressors [7]. The overall level of m6A in plants increases under stress from pathogenic microorganisms and other factors [3]. m6A also mediates mRNA decay, affecting the function of signaling and regulatory factors in biochemical reactions within plants [8]. In addition, m6A participates in regulating salt [1,9,10], cadmium [11], and cold stress [12] responses. Drought is the most significant abiotic stress factor affecting plant growth, development, and yield. Many studies have been conducted on m6A modifications induced by drought stress. In sea buckthorn, m6A modification can regulate transcription levels under drought stress [13]. Gene expression and protein relatedness decrease under drought stress and m6A modifications may negatively regulate the expression of genes related to wood development (cellulose, hemicellulose, and lignin) [14]. In summary, these studies demonstrated that m6A modifications play an important role in plant responses to stress; however, their regulatory mechanisms are complex and require further clarification.
The effect of drought stress on m6A methylation in sugarcane has not yet been reported. In this study, we used the methylated RNA immunoprecipitation (MeRIP) method to study the effect of drought stress on m6A methylation in sugarcane leaves of the variety ROC22. In the present study, we found that the overall m6A levels in sugarcane leaves significantly increased after drought stress. Therefore, we analyzed the m6A methylation profile of sugarcane leaves in response to drought stress using whole-genome m6A-seq and bioinformatic methods. In addition, we used RNA sequencing to analyze the response pattern and speculated on the functions of differentially expressed genes (DEGs) modified by m6A in the transcriptional profile of drought-stressed sugarcane leaves. Additionally, we validated the effectiveness of some target genes using qPCR (qPCR). This study represents the first comprehensive characterization of m6A modifications in the sugarcane transcriptome. Furthermore, we elucidated the potential roles and regulatory mechanisms of m6A-mediated sugarcane production in response to drought stress.

2. Results

2.1. m6A Methylation Is Abundant in Sugarcane Leaf mRNA

An m6A-targeted antibody was used to obtain the transcriptome-wide m6A map of sugarcane leaves, methylated RNA immunoprecipitation sequencing (MeRIP-seq) was performed, and a series of m6A-immunoprecipitation (IP) and matched input (non-IP control) libraries were constructed. We obtained almost 44 million reads per library (input and IP libraries) and detected 2826 m6A-enriched peaks in sugarcane leaf samples, which have an average length of 405 bp. The minimum peak length was 192 bp. Based on these results, we found that the sugarcane transcriptome contained 0.1–2.0 m6A peaks per 1000 nucleotides, which are similar to those obtained in mammals. Most of the genes contained one or two m6A peaks (Figure 1a).
m6A peak distribution was investigated based on gene annotation. Most reads of m6A peaks were located around the 3′UTRs (Figure 1b). Similar distribution patterns of m6A peaks have also been observed in Arabidopsis [15], sweet sorghum [9] and sea buckthorn [13]. The results of the locations of m6A on transcripts showed that m6A peaks were abundant around the stop codon (60.30%), followed by the 3′UTRs (21.76%), coding regions (14.93%), and the start codon (2.76%) (Figure 1c). The distribution pattern of m6A peaks in sugarcane was similar to that observed in maize [16] and Arabidopsis [17].
We also observed that m6A peaks were significantly enriched around the 3′UTRs (enrichment score > 90) using segment normalization of each gene portion (Figure 1d). The genome-wide density of m6A modification peaks showed that the m6A peaks had different distribution patterns across chromosomes (Figure 1e). The analysis revealed that the average density of chromosome 4C was the highest, and that of chromosome 6A was the lowest. The distribution patterns among these chromosomes suggested that m6A modifications may be related to chromatin status.
According to the analysis of the m6A consensus sequence, 15,387 (94.39%) m6A peaks had at least one RRACH motif in the CK group, and 10,873 (94.15%) m6A peaks in the DR group had at least one RRACH motif (Figure 1f). The same sequence motif observed in eukaryotic mRNA may be essential for m6A modifications.
Through analysis of m6A consensus sequences, we also observed that 2367 (83.76%) m6A peaks included at least one typical conserved sequence, RRACH, and 2564 (90.73%) m6A peaks included one typical conserved sequence, DRACH, in the CK group. The same sequence motifs have been observed in eukaryotic mRNA and may be crucial for m6A modification.

2.2. Characterization of m6A mRNA Methylation in Sugarcane

Previous studies demonstrated that m6A is crucial for plant development. To further analyze the function of m6A methylation in sugarcane, GO and KEGG enrichment analyses of genes modified by m6A under drought stress were performed. The result showed that most of these genes were involved in metabolic biosynthesis (Figure 2a,b). The genes were divided into three subgroups (Peak I: m6A peaks around the start codon; Peak II: m6A peaks around the stop codon; Peak III: m6A peaks around both the start and stop codons) based on the distribution of m6A peak values in different gene functional elements to determine the m6A distribution patterns of the genes related to their GO categories. The GO enrichment analysis of each subgroup showed that m6A-containing genes in Peak I were mainly enriched in photosynthesis-related processes (Figure 2c), those in Peak II were mainly enriched in protein synthesis and translation processes (Figure 2d), and those in Peak III exhibit high enrichment of organic compound biosynthetic and metabolic processes (Figure 2e). We identified a series of metabolism-related genes containing m6A peaks; for example, acetaldehyde dehydrogenase (ScALDH7B4) and CLYI-1, which contained m6A peaks near the stop codon and 3′UTR, playing important roles in the oxidation and detoxification of aldehydes and the carbohydrate metabolism, respectively. Glycerol-3-phosphate dehydrogenase A (GAPA) and citrate synthase (CSY3) play important roles in carbohydrate biosynthesis and also contain m6A peaks in the stop codon and 3′UTR (Figure 2f,g). These results indicated that m6A mRNA methylation may regulate the biosynthesis of plant metabolites.

2.3. The m6A Methylation Responses to Drought Stress in Sugarcane

The m6A methylomes in sugarcane leaves under CK and DR treatments were profiled to investigate changes in m6A methylation in sugarcane under drought stress. After comparing the distribution of m6A methylation in CK and DR, we identified 2826 and 6620 m6A methylation peaks in CK and DR, respectively. Venn diagram analysis revealed 1945 (25.90%) peaks in both CK and DR, with 881 peaks unique to CK and 4675 peaks unique to DR (Figure 3a). This indicates that m6A methylation increases under drought stress. Differential analysis showed that 2138 m6A peaks were differentially methylated peaks (DMPs) (p < 0.05, |log2FC| > 1), including 1373 upregulated and 765 downregulated DMPs (Figure 3b). Then, we detected 2208 transcripts that have DMPs in DR compared to CK. Among the 1373 upregulated DMPs related genes, including 150 up-regulated genes, 94 down-regulated genes, and 1168 unaltered genes; and among the 765 downregulated DMPs related genes, including 126 up-regulated genes, 32 down-regulated genes, and 637 unaltered genes, more than 40% of DMPs were located in the stop codon region (Figure 3c). Further analysis of the whole transcriptomes of CK and DR groups revealed that m6A modification affected gene expression levels (Figure 3d). GO enrichment analysis of genes containing DMPs was performed to explore the functional characteristics of DMPs in the context of their genetic location. GO enrichment analysis of the upregulated DMPs-containing genes showed that the biological processes of cell metabolism (GO:0044237), gene expression (GO:0010467), nitrogen compound metabolic processes (GO:0006807), and photosynthesis and light reaction (GO:0019684) were significantly enriched (Figure 3e). GO enrichment analysis of the downregulated DMPs-containing genes showed that the biological processes of protein ubiquitination (GO:0016567), nuclear-transcribed mRNA catabolic process (GO:0000956), and cell wall modification (GO:0042545) were significantly enriched. In addition, the KEGG analysis showed that the upregulated and downregulated DMPs contained genes that were mainly enriched in biological metabolic pathways (Figure 3g,h).

2.4. m6A Modification Regulates mRNA Abundance by Regulating the Stability of Drought-Tolerance Transcripts

A transcriptomic analysis of the sugarcane response to drought was performed to explore the effect of m6A methylation on the drought tolerance of sugarcane. Subsequently, the correlations between DMPs and differentially expressed genes (DEGs) was analyzed. A total of 10,994 DEGs, including 8436 upregulated and 2558 downregulated genes, were identified by comparing CK and DR treatments. There were 2138 DMPs in total (FDR < 0.05); 1373 upregulated DMPs were associated with 1326 upregulated DEGs, and 765 downregulated DMPs were associated with 769 downregulated DEGs (Figure 4a).
mRNA expression is affected by m6A modifications; therefore, there is a potential trend for co-regulation between gene expression in the transcriptome and the abundance of m6A peaks. We analyzed the DEGs identified in the transcriptome using the differentially identified peak-related genes in m6A. In total, 12,675 genes, including 364 differentially expressed genes, were identified (Figure 4b). Interestingly, when we analyzed transcripts with significant changes in m6A modification in sugarcane leaves, the mRNA abundance of some drought-resistant genes with significantly increased m6A modifications also increased significantly. For example, there were significant changes in m6A methylation around the stop codons of OAT (Sspon.01G0026110-2B), NAC (Sspon.01G0042440-1P), and ALDH (Sspon.02G0013900-3D), accompanied by a significant increase in mRNA abundance (Figure 4c).
Research has shown that increasing the proline content in plant cells can effectively enhance plant drought resistance; therefore, proline accumulation is closely related to plant drought stress. Proline biosynthesis in higher plants can be divided into two pathways, glutamate, and ornithine. ScOAT is a key enzyme in the ornithine pathway of proline synthesis and plays an important role in the response of sugarcane plants to drought stress [18]. NAC transcription factors not only play important roles in plant embryogenesis, flower patterning, secondary wall formation, leaf senescence, and lateral root development; they also participate in responses to biotic and abiotic stressors, such as pathogens, drought, salt, and cold stress [19,20]. With the assistance of NAD(P)+, ALDH converts a series of endogenous and exogenous aromatic and aliphatic aldehydes into their corresponding carboxylic acids, which are important enzymes for clearing reactive oxygen species in organisms and play an important role in maintaining material balance [21]. m6A modification of these key drought-resistant transcriptional transcripts increased mRNA stability and thus increased mRNA abundance (Figure 4d).

2.5. Changes in m6A Methylation-Related Genes in Response to Drought Stress in Sugarcane

To further explore the effect of drought stress on m6A methylation in sugarcane leaves, 12 drought-resistant genes in the CK and DR groups that were previously reported [18,21,22,23,24,25,26,27,28,29,30,31,32,33] were analyzed. Six genes were downregulated in the DMGs and upregulated in the DEGs (Table 1). Changes in methylation modification and up/downregulated gene expression of these drought-resistant genes may explain the phenotypic differences in sugarcane under drought stress.
We analyzed the role of the aldehyde dehydrogenase gene (ALDH7) with differential methylation and expression in the two sets of association analyses based on sup8 m6A+ transcriptome correlation analysis to further explore the relationship between m6A methylation and the transcriptome. Under drought and other stress conditions, plants may increase their abscisic acid (ABA) content and accumulate large amounts of free proline and arginine. Arginine and proline can be converted to putrescine (Spd) by PAQ1 and PAQ5, and putrescine can be converted to 4-aminobutanal by PAQ1. 4-aminobutanal can be converted into 4-aminobutyric acid by the action of the ALDH7 enzyme, which is finally converted into succinate succinic acid. Arginine and proline can be converted into beta-amino propanal by the action of PAQ1 and AOC3, and beta-amino propanal can be converted into beta-alanine by the action of ALDH7. Beta-alanine is further converted into pantothenic acid and pantothenic acid salts by PANC. In addition, the acetic acid salt in the cell can be converted into acetaldehyde by ALDH7, and acetaldehyde can be deaminated and converted into ethanol by ALDH (Figure 5). Previous studies have shown that butyric acid, pantothenic acid, and ethanol can improve drought resistance [18,25,34,35,36,37,38].
Aldehyde dehydrogenase plays a significant role as the core component of these three metabolic processes. It is noteworthy that the m6A level of the aldehyde dehydrogenase gene (ALDH7) is downregulated (down arrow), while the mRNA level is upregulated (up arrow). This indicates that the downregulation of m6A level may lead to an increase in mRNA expression level, thus resulting in the accumulation of a large amount of aldehyde dehydrogenase protein (ALDHs), which oxidizes aldehyde substances into the corresponding pantothenic acid, butyric acid, and ethanol, reduces lipid peroxidation, and ultimately participates in plant abiotic stress and developmental regulation.

2.6. Real-Time Quantitative PCR

In this study, nine DGEs were randomly selected to verify the expression of genes in the CK and DR sugarcane plants. Six upregulated (up arrow) and three downregulated (down arrow) genes involved in plant biotic metabolism, photosynthesis, and defense responses were selected. The upregulated and downregulated genes encoded aldehyde dehydrogenase, heat shock protein, ornithine aminotransferase, dehydration-responsive element-binding protein, fructose-bisphosphate aldolase cytoplasmic isozyme, pyrophosphate-energized vacuolar membrane proton pump, peptidylprolyl isomerase, and lactoylglutathione lyase. The results of the RT-PCR analysis are shown in Figure 6. Quantitative real-time analysis of the nine differential genes was compared to transcriptome data, and the results supported the m6A-seq quantification results.

3. Discussion

In the present study, we used m6A-seq technology to generate a transcriptome-wide RNA m6A modification profile in sugarcane leaves. We found that the distribution pattern of m6A in sugarcane is similar to that of other plants, such as Arabidopsis, sorghum, and rice [3,16,39], which are mainly enriched in the 3′UTR region and also have a similar RRm6ACH methylation motif in each transcript [3,40,41]. These results indicate that the distribution pattern of m6A methylation in plants is conserved. However, some differences were found between sugarcane, Arabidopsis, and sorghum, which may represent differences in m6A methylation levels and unique patterns between species or may be the result of different depths of m6A-seq in the studies. Previous studies with lower m6A resolutions may not be sufficient to identify weak m6A signals in most mRNA coding sequences, and earlier reports have suggested that their estimates of m6A sites may not be sufficient [40,42].
The deposition of m6A is positively correlated with mRNA levels, plays a regulatory role in plant gene expression, and regulates plant development and evolution [1]. Studies have shown that m6A deposition is negatively correlated with transcript abundance and interacts with DNA methylation; m6A demethylation regulates fruit ripening [6]. In the present study, drought stress increased the degree of m6A modification in crops and upregulated the expression of drought stress response genes, confirming that m6A modification of drought-stress-related transcripts is closely related to regulating plant responses to drought stress [9].
In the present study, we found that m6A modifications in sugarcane leaves changed significantly under drought stress. Some genes, such as NAC002, HSP70, ALDH7B4, GLYI-11, and DREB1C, showed decreased methylation levels but increased expression of drought-responsive genes, indicating that m6A may play an important role in the drought response. m6A modification can enhance the mRNA stability of some key drought-resistant genes, thereby increasing mRNA abundance, which is consistent with the conclusion of methylation changes in the salt tolerance of sorghum [9]. This study also suggests that m6A modification significantly changes plant development and abiotic stress. In addition, GO and KEGG analyses showed that the upregulated genes were significantly enriched in regulating abscisic acid metabolism and signaling pathways, such as glycine metabolism and activation of the abscisic acid signaling pathway (Figure 4d). For example, one of the members of the Arabidopsis ALDH family, ALDH7B4, and the key enzyme gene OAT in rice proline are important transcription factors in the ABA signaling pathway and play an important regulatory role in response to stress. In this study, compared to the CK samples, the mRNA m6A methylation levels of OAT and ALDH7B4 in the DR samples increased four-fold, whereas the transcription levels of OAT and ALDH7B4 in the DR samples were higher than those in the CK samples, responding to water shortage and enhancing the tolerance of sugarcane plants to drought stress. PP2C50 is a serine/threonine residue protein phosphatase in the protein phosphatase family, which is involved in regulating ABA permeation and stress signaling pathways. It has hyper- and hypo-m6A peaks in the DR, which affect ABA expression levels. SODA is a superoxide dismutase family member involved in arginine and proline metabolism, whereas cFBP is involved in carbon metabolism. SODA and cFBP participate in the synthesis of abscisic acid metabolism. These results suggested that m6A methylation regulates the expression of ABA-related genes in sugarcane in response to drought stress.
These key drought resistance genes play a positive role in regulating the response of sugarcane plants to drought stress. The expression of drought-responsive genes is the basis for plant drought resistance [43]. Most genes induced via drought stress are closely related to cellular regulation, protein metabolism, signal transduction, and transcription [44]. Although only a few key drought-resistant transcripts can be accurately detected to directly regulate mRNA abundance through m6A modification, the regulatory effects of m6A modification on mRNA are complex, and most m6A modifications may regulate mRNA by affecting RNA metabolism. It can be speculated that more m6A-modified critical drought-resistant transcripts will provide more complex water stress regulatory pathways, such as RNA splicing, RNA export, 3′UTR processing, and translation, thus making plants more drought-resistant.
Under water stress, sugarcane can adjust the expression of downstream related genes and directly act as functional proteins to protect plant cells from the impact of water stress, ultimately enabling plants to exhibit adaptability or resistance to water stress [45,46,47,48,49]. Studies have shown that ABA not only regulates the entire development process of plants but also plays a bridging role in different environmental signals. ABA is a key factor for plant cells in adapting to drought and plays an important role in plant stress responses by initiating drought response mechanisms [50,51]. At the same time, it has been found that in the drought response process, ABA can not only change the transcription levels of plant cells but also affect other important basic metabolic processes of plants, including plant lipid and carbon metabolism, which also implies the central role of ABA in plant responses to drought [17]. In this study, ALDH7B4, a gene related to ABA metabolism and synthesis, showed a decrease in m6A methylation levels, indicating that m6A modification of the ABA metabolic synthesis pathway is a stress response of plant drought resistance caused by environmental drought stress. Upregulation of ALDH7B4 transcription promotes the ability of the cell to regulate permeability under adverse conditions. For plant drought resistance, regulating drought-resistant genes is a quantitative trait, an alternative to relying on a single gene for a decisive role. Therefore, the m6A modification of drought-resistant transcripts may provide more signaling pathways for regulating plants under water stress.

4. Materials and Methods

4.1. Plant Materials and RNA Isolation

The sugarcane variety ROC22 (Saccharum spp. hybrid) was provided by the Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China (108°19′ E, 22°49′ N). Sugarcane seed pieces with single buds were cultivated using a substrate, and healthy cane seedlings were selected and planted in 10 L pots 35 cm × 30 cm (height × diameter) in size. Drought stress was applied to sugarcane after 3 months of growth. Sugarcane was naturally drought-stressed in a greenhouse with controlled light, temperature, and humidity conditions (16 h dark, 8 h light; 29–31 °C under day and dark, 60–70% relative humidity). Soil water content in the drought resistance (DR) group was 7–9% after 10 days of drought treatment, whereas it was maintained at 25–28.5% in the control check (CK) group. The +1 leaves of CK and DR plants were randomly sampled, rapidly frozen in liquid nitrogen, and stored at −80 °C for RNA extraction and sequencing.

4.2. RNA Isolation and Library Construction

Total RNA was extracted from leaves using the TRIzol (Qiagen, Beijing, China) method, and the quality of RNA samples was determined using a Nanodrop microspectrophotometer and gel electrophoresis. Polyadenylated RNA was extracted and fragmented to 100 nt using a fragmentation reaction system. The fragment RNA and m6A-specific antibody were incubated in IP buffer at 4 °C for 2 h for immunoprecipitation. The mixture was eluted with IP buffer, the precipitated RNA was recovered, and immunoprecipitation mRNA (IP) and pre-immunoprecipitation mRNA (Input) libraries were constructed for each sample using an Illumina Ribo-Zero Gold kit (MRZG12324, Illumina, San Diego, CA, USA). We performed m6A-seq and input RNA-seq with NovaSeq 6000 using a paired-end strategy.

4.3. Processing of Raw Data

The reads obtained from the sequencing machines included raw reads containing adapters or low-quality bases which would affect the following assembly and analysis. Raw reads would be processed to get high quality clean reads according to four stringent filtering standards use fastp software (version: 0.20.0) [40]. Clean reads were mapped to the sugarcane (Saccharum spontaneum L.) reference genome (https://www.life.illinois.edu/ming/downloads/Spontaneum_genome/ accessed on 9 December 2021) using HISAT2 software (version:2.1.0). We used the R package (exomePeak2) (version:1.0.0) to identify m6A peaks (p-value < 0.05); then, the distribution of m6A in the different parts of the gene and transcripts was estimated.
Motif analysis was performed using MEME Suite (http://meme-suite.org/ accessed on 11 December 2021) to obtain the precise location and motif regions of the m6A peaks. The Integrated Genome Browser was used for visual analysis of m6A peaks [52]. All m6A modification sites were assigned to different transcriptional regions, including transcription start sites (TSS), 5′UTR, coding sequences (CDS), 3′UTR, and introns. Gene expression levels were calculated using the reads per kilobase per million mapped reads (RPKM) method. The m6A differential site algorithm was used to determine the differential m6A peaks between CK and DR sugarcane leaves [53], with a p-value of <0.05. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify the biological processes of differentially modified genes.

4.4. Quantitative Real-Time PCR (qRT-PCR) Validation

Previous studies have shown that quantitative real-time PCR (qRT-PCR) can be used to validate RNA-seq results. We analyzed the expression patterns of the selected genes. RNA extraction, cDNA synthesis, and qRT-PCR were performed as previously described. Transcription levels of the selected genes were quantified using the cycle threshold 2−ΔΔCt method. The sugarcane GAPDH gene (GenBank accession number: EF189713) was used to normalize the expression values. The primers used for qRT-PCR are listed in Supplementary Table S1. Experiments were conducted using three biological replicates.

5. Conclusions

This study reports the effects of drought stress on m6A mRNA methylation and the expression of related genes in sugarcane leaves. Using MERIP-seq, we constructed a transcriptome-wide m6A modification map of sugarcane and demonstrated the role of m6A modification in the response to drought stress. Our results suggest that the number and extent of m6A modifications in drought-resistance-related mRNA may be important parameters in assessing the drought tolerance of crops.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12213668/s1.

Author Contributions

Conceptualization, J.W.; methodology and formal analysis, H.L. and Y.G.; validation, H.Z.; writing-original draft preparation, J.W.; writing—review and editing, K.Z. and X.L.; supervision, X.L. and K.Z.; funding acquisition, R.Z. and X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32101696, 32160486 and 32201766), Guangxi Natural Science Foundation (2023GXNSFBA026194), Foundation for Guangxi Academy of Agricultural Sciences (GNK2021YT006), and the earmarked fund for CARS (170105).

Data Availability Statement

The data presented in this study are available within the article and its Supplementary Materials.

Acknowledgments

We are grateful to/thank Guangzhou Genedenovo Biotechnology Co., Ltd. (Guangzhou, China) for assisting in sequencing and/or bioinformatics analysis.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Overview of m6A methylome in sugarcane. (a) Number of genes with different m6A peak numbers. (b) Accumulation of m6A-IP and input reads of transcripts. Each transcript contained three parts: 5ʹUTRs, CDS, and 3′UTRs. (c) The m6A peak distribution in different genic regions. (d) The m6A peak distribution along a metagene. Enrichment scores are calculated as (n/N)/p; n, number of peaks belonging to each category; N, number of total peaks; p, proportion of each category within the genome by length. (e) Distribution of m6A peaks across sugarcane chromosomes. (f) The canonical RRACH and DRACH conserved sequence motifs of m6A peak regions in the CK sample.
Figure 1. Overview of m6A methylome in sugarcane. (a) Number of genes with different m6A peak numbers. (b) Accumulation of m6A-IP and input reads of transcripts. Each transcript contained three parts: 5ʹUTRs, CDS, and 3′UTRs. (c) The m6A peak distribution in different genic regions. (d) The m6A peak distribution along a metagene. Enrichment scores are calculated as (n/N)/p; n, number of peaks belonging to each category; N, number of total peaks; p, proportion of each category within the genome by length. (e) Distribution of m6A peaks across sugarcane chromosomes. (f) The canonical RRACH and DRACH conserved sequence motifs of m6A peak regions in the CK sample.
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Figure 2. m6A methylation enrichment analysis. (a) GO enrichment of genes with m6A peaks. (b) KEGG enrichment of genes with m6A peaks. (c) GO enrichment of genes with m6A peaks in Peak I. (d) GO enrichment of genes with m6A peaks in Peak II. (e) GO enrichment of genes with m6A peaks in Peak III. (f) Examples of genes with m6A peaks at the stop codon. (g) Examples of genes with m6A peaks at the stop codon.
Figure 2. m6A methylation enrichment analysis. (a) GO enrichment of genes with m6A peaks. (b) KEGG enrichment of genes with m6A peaks. (c) GO enrichment of genes with m6A peaks in Peak I. (d) GO enrichment of genes with m6A peaks in Peak II. (e) GO enrichment of genes with m6A peaks in Peak III. (f) Examples of genes with m6A peaks at the stop codon. (g) Examples of genes with m6A peaks at the stop codon.
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Figure 3. Overview of m6A methylation of sugarcane leaf under CK and DR treatment. (a) Venn diagram of m6A peaks between CK and DR. (b) Volcano analysis of differentially m6A methylation peaks. “nosig” is no significant changes. (c) Distribution of differentially m6A-methylated peaks across the mRNAs. (d) Cumulative distribution of mRNA expression changes between CK and DR for m6A-modified genes and non-target genes. (e) GO enrichment terms for the upregulated genes with DMPs between CK and DR. (f) GO enrichment terms for the downregulated genes with DMPs between CK and DR. (g) KEGG enrichment pathways of the upregulated genes with DMPs between CK and DR. (h) KEGG enrichment pathways of the downregulated genes with DMPs between CK and DR.
Figure 3. Overview of m6A methylation of sugarcane leaf under CK and DR treatment. (a) Venn diagram of m6A peaks between CK and DR. (b) Volcano analysis of differentially m6A methylation peaks. “nosig” is no significant changes. (c) Distribution of differentially m6A-methylated peaks across the mRNAs. (d) Cumulative distribution of mRNA expression changes between CK and DR for m6A-modified genes and non-target genes. (e) GO enrichment terms for the upregulated genes with DMPs between CK and DR. (f) GO enrichment terms for the downregulated genes with DMPs between CK and DR. (g) KEGG enrichment pathways of the upregulated genes with DMPs between CK and DR. (h) KEGG enrichment pathways of the downregulated genes with DMPs between CK and DR.
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Figure 4. Analysis of DMP-related gene and DEGs between sugarcane leaf under CK and DRtreatment. (a) Venn map of DMP-related genes and DEGs. (b) Volcano map of DMP-related genes and DEGs. (c) Examples of DEGs with DMPs at the stop codon. (d) Expression level of DEGs with DMPs at the entire gene functional element.
Figure 4. Analysis of DMP-related gene and DEGs between sugarcane leaf under CK and DRtreatment. (a) Venn map of DMP-related genes and DEGs. (b) Volcano map of DMP-related genes and DEGs. (c) Examples of DEGs with DMPs at the stop codon. (d) Expression level of DEGs with DMPs at the entire gene functional element.
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Figure 5. The action pattern of aldehyde dehydrogenase-related gene ALDH7B4 under drought stress.
Figure 5. The action pattern of aldehyde dehydrogenase-related gene ALDH7B4 under drought stress.
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Figure 6. Quantitative real-time PCR analysis of selected differentially expressed genes.
Figure 6. Quantitative real-time PCR analysis of selected differentially expressed genes.
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Table 1. The m6A level and methylation-related gene expression of 12 drought-resistant genes under drought stress.
Table 1. The m6A level and methylation-related gene expression of 12 drought-resistant genes under drought stress.
IDGene_IDGene
Name		DMGDEGReference
Sspon.01G0036970-2CAGT17406.1bZIPUPUP[22]
Sspon.01G0042440-1PAGU13503.1NACDOWNUP[23]
Sspon.01G0052010-1PXP_002465468.1HSP70DOWNUP[24]
Sspon.02G0013900-1AAMS36872.1ALDH7DOWNUP[21,25]
Sspon.04G0016680-1TXP_002453419.1AVPDOWNDOWN[26]
Sspon.06G0009730-1AXP_021321469.1GLYI-11DOWNUP[27]
Sspon.07G0010240-3COEL18927.1WRKYUPUP[28]
Sspon.07G0024880-3DAFO59568.1SODADOWNUP[29]
Sspon.08G0024020-1BXP_002436386.1DREBDOWNUP[30,31]
Sspon.01G0026110-2BXP_002464174.1OATUPUP[18]
Sspon.07G0003250-3DXP_002441446.2PP2CDOWNUP[32]
Sspon.07G0007070-2BXP_021304140.1cFBPDOWNUP[33]
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Wei, J.; Li, H.; Gui, Y.; Zhou, H.; Zhang, R.; Zhu, K.; Liu, X. Coordination of m6A mRNA Methylation and Gene Transcriptome in Sugarcane Response to Drought Stress. Plants 2023, 12, 3668. https://doi.org/10.3390/plants12213668

AMA Style

Wei J, Li H, Gui Y, Zhou H, Zhang R, Zhu K, Liu X. Coordination of m6A mRNA Methylation and Gene Transcriptome in Sugarcane Response to Drought Stress. Plants. 2023; 12(21):3668. https://doi.org/10.3390/plants12213668

Chicago/Turabian Style

Wei, Jinju, Haibi Li, Yiyun Gui, Hui Zhou, Ronghua Zhang, Kai Zhu, and Xihui Liu. 2023. "Coordination of m6A mRNA Methylation and Gene Transcriptome in Sugarcane Response to Drought Stress" Plants 12, no. 21: 3668. https://doi.org/10.3390/plants12213668

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