Skip to main content
Download PDF

Epitranscriptome profiles reveal participation of the RNA methyltransferase gene OsMTA1 in rice seed germination and salt stress response

BMC Plant Biology volume 25, Article number: 115 (2025) Cite this article

Abstract

Background

RNA m6A methylation installed by RNA methyltransferases plays a crucial role in regulating plant growth and development and environmental stress responses. However, the underlying molecular mechanisms of m6A methylation involved in seed germination and stress responses are largely unknown. In the present study, we surveyed global m6A methylation in rice seed germination under salt stress and the control (no stress) using an osmta1 mutant and its wild type.

Results

The knockout of OsMTA1 resulted in a decreased level of m6A methylation and delayed seed germination, together with increased oxidative damage in the osmta1-1 mutant, especially under salt stress, indicating that OsMTA1 performs a crucial function in rice seed germination and salt stress response. Comparative analysis of m6A profiling using methylated RNA immunoprecipitation sequencing revealed that a unique set of genes that functioned in seed germination, cell growth, and development, including OsbZIP78 and OsA8, were hypomethylated in osmta1-1 embryos and germinating seeds. Numerous genes involved in plant growth and stress response were hypomethylated in the osmta1-1 mutant during seed germination under salt stress. Further combined analysis of the m6A methylome and transcriptome revealed that the loss of function of OsMTA1 had a more complex impact on gene expression in osmta1-1. Several hypomethylated genes with a negative role in growth and development, such as OsHsfA7 and OsHDAC3, were highly up-regulated in the osmta1-1 mutant under the control condition. In contrast, several hypomethylated genes positively associated with stress response were down-regulated, whereas a different set of hypomethylated genes that functioned as negative regulators of growth and stress response were up-regulated in the osmta1-1 mutant under salt stress. These results further demonstrated that OsMTA1-mediated m6A methylation modulated rice seed germination and salt stress response by regulating transcription of a unique set of genes with diverse functions.

Conclusion

Our results reveal a crucial role for the m6A methyltransferase gene OsMTA1 in regulating rice seed germination and salt stress response, and provide candidate genes to assist in breeding new stress-tolerant rice varieties.

Peer Review reports

Introduction

Plant seed germination is a complex biological process regulated by a network of coordinated physiological, metabolic, cellular, and molecular events [1]. Seed germination comprises seed imbibition followed by a plateau phase with water uptake, then a late phase with visible radicle protrusion through the seed coat [2]. With seed imbibition, immediate changes in metabolites associated with energy metabolism are observed, followed by substantial transcriptome rearrangement in germinating seeds [3]. To date, many genes and metabolites have been functionally implicated in seed germination [3,4,5]; in particular, genes involved in abscisic acid and gibberellin biosynthesis and catabolism play crucial roles in the seed germination process [6]. Adverse environmental stresses strongly impact on seed germination, for example, salt stress suppresses seed germination by inhibiting water uptake and ionic toxicity [7, 8]. Several quantitative trait loci and genes associated with salt tolerance during rice seed germination have been identified [9,10,11,12]. However, the genetic and molecular basis of seed germination under salt stress in rice is unclear and requires further elucidation.

N6-methyladenosine (m6A) RNA methylation is the most prevalent and reversible modification in diverse RNAs in eukaryotic organisms. The m6A methylation in mRNA regulates the transcript abundance by affecting its stability [13,14,15], alternative splicing, transport [16], and translation efficiency [17]. The m6A methylation is installed and removed by N6-adenosine methyltransferase complex (writer) and demethylase (eraser), respectively. The writer complex members comprising methyltransferase A (MTA), MTB, FKBP12 INTERACTING PROTEIN 37 (FIP37), VIRILIZER, and HAKAI were identified in Arabidopsis, and down-regulation of these genes causes genome-wide changes in the m6A level and abnormal effects on development [18,19,20,21]. Several writer components in the rice genome have been identified by homologous gene searches. Several writers, including OsMTA, OsMTB, OsFIP, and OsVIR, are differentially regulated by abiotic stress [22]. Further study revealed that the methyltransferases OsEDM2L (ENHANCED DOWNY MILDEW 2-LIKE), OsFIP, and OsMTA2 are involved in microspore development [23,24,25]. Although these results suggest that a methyltransferase complex is conserved in rice, the establishment and function of m6A methylation and their effect on rice growth and development remain largely unknown.

With development of m6A-specific antibodies for immunoprecipitation combined with high-throughput RNA-sequencing (meRIP-Seq), transcriptome-wide m6A profiling has been widely used to examine the molecular roles of m6A methylation in response to abiotic stresses in plants. Increased m6A levels induced by over-expression of PtrMTA enhance drought stress tolerance with improved development of trichomes and roots in transgenic poplar [26]. Knockout of the eraser gene ALKBH10B results in increased m6A levels in the Arabidopsis alkbh10b mutant, and the mutant exhibits delayed seed germination and enhanced seedling growth compared with those of the wild type (WT) under salt stress. Consistent with these results, a salt-sensitive phenotype has been observed in all reported mutants of writer components in Arabidopsis, including MTA, MTB, VIR, and HAKAI, accompanied by genome-wide loss of m6A methylation, revealing a positive relationship between m6A level and salt stress tolerance [27]. The mRNA m6A methylation plays an important role in stress response to low temperature in tomato [28], salt in rice, sweet sorghum, and sugar beet [29,30,31], high temperature in pak choi [32], and drought in cotton and foxtail millet [15, 31]. However, the roles of mRNA m6A modification and associated genes in responses to diverse environmental stresses in rice require further investigation.

In the present study, a CRISPR/Cas9 mutant of the m6A writer OsMTA1 and the WT were used to elucidate the molecular mechanisms of m6A methylation involved in rice seed germination and salt stress. The results revealed the general features of m6A modification and the related transcripts in rice seed germination under control and salt stress conditions. The findings provide a framework for elucidating the impacts of m6A modification on seed germination, and improve our understanding of the post-transcriptional regulatory mechanisms of rice seed germination and salt stress response.

Materials and methods

Rice materials and growth conditions

The rice genotype ‘Zhonghua 11’ (WT, Oryza sativa L. subsp. Geng) was used in this study. The m6A writer mutants (osmta1-1 and osmta1-2) were constructed using CRISPR/Cas9 technology (Figure S1A). Seeds of the WT and mutants were surface-sterilized with 3% sodium hypochlorite solution for 1 h and then rinsed with distilled water three times. Four replicates of 50 seeds each were placed in petri dishes (diameter 9 cm) lined with two sheets of filter paper for germination in distilled water as the control or moistened with 120 mM NaCl, and incubated at 30 °C (12 h light/12 h dark) in a phytotron. The seeds were checked at 24 h intervals and a radicle length of 2 mm was used as the criterion for completion of germination as described in a previous study [33]. The percentage germination was determined at 24 h intervals for 7 consecutive days.

Physiological indicators of rice seed germination under control and salt stress conditions

To investigate physiological differences between the WT and the mutants during seed germination, the malondialdehyde (MDA) content and the activities of peroxidase (POD) and catalase (CAT) in the germinated seeds under the control and salt stress conditions were examined. The MDA content was assayed using the thiobarbituric acid (TBA) method as described by Xie et al. [34]. MDA was extracted using chilled TBA and quantified by determining the absorbance of the supernatant at 532 nm. The POD and CAT activities were measured as described in a previous study [35]. All data were measured with three replicates and the significance of differences between the mean was analyzed using Student’s t-test.

Determination of m6A content

The total RNAs were extracted from germinating seeds of the WT and mutant incubated for 60 h (hereafter ‘60 h germinating seeds’) using the Tiangen RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit (TIANGEN, DP441, China). The m6A/A ratio was determined using the EpiQuik m6A RNA Methylation Quantification Kit in accordance with the manufacturer’s instructions (EpiGentek, P-9005, NY, USA). Approximately 200 ng total RNA was reacted with the specific m6A capture antibody and detection antibody, and the absorbance was measured at 450 nm. The m6A content in the sample is proportional to the optical density and the specific m6A level was calculated according to the standard curve provided with the kit. The experiment was repeated three times.

RNA isolation and high-throughput meRIP-seq

Total RNAs in dry seed embryos and 60 h germinating seeds under the control and salt stress conditions were isolated and purified using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The RNA amount and purity of each sample were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed with a Bioanalyzer 2100 (Agilent, CA, USA) with RIN number > 7.0, and confirmed by denaturing agarose gel electrophoresis. Approximately 25 µg total RNA representing a specific adipose type was used to deplete ribosomal RNA (rRNA) using the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, CA, USA). Following purification, the rRNA-depleted RNA was fragmented into small pieces using the Magnesium RNA Fragmentation Module (NEB, Cat. e6150, USA) at 86 ℃ for 7 min. The cleaved RNA fragments were incubated for 2 h at 4 ℃ with the m6A-specific antibody (no. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl, and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to generate the cDNA with SuperScript™ II Reverse Transcriptase (Invitrogen, Cat. 1896649, USA), which was used to synthesize U-labeled second-stranded DNAs with Escherichia coli DNA polymerase I (NEB, Cat. M0209, USA), RNase H (NEB, Cat. M0297, USA) and dUTP Solution (Thermo Fisher, Cat. R0133, USA. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligation of the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified by PCR under the following conditions: initial denaturation at 95 ℃ for 3 min; eight cycles of denaturation at 98 ℃ for 15 s, annealing at 60 ℃ for 15 s, and extension at 72 ℃ for 30 s; and then final extension at 72 ℃ for 5 min. The average insert size for the final cDNA library was 300 ± 50 bp. Ultimately, we performed 2 × 150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China) following the manufacturer’s recommended protocol.

Data analysis of meRIP-seq and transcriptome sequencing

The fastp software (https://github.com/OpenGene/fastp) was used to remove reads that contained adaptors, low-quality bases, and undetermined bases with the default parameters. The sequence quality of the IP and input samples were verified using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and RseQC (http://rseqc.sourceforge.net/). The HISAT2 software (http://daehwankimlab.github.io/hisat2) was used to map reads to the Oryza sativa reference genome (MSU Release 7.0). Peak calling and differential peak analysis were performed using the R package exomePeak2 (https://bioconductor.org/packages/release/). Peaks were annotated by intersection with the gene architecture using the R package ANNOVAR [36]. The differential m6A peaks (DMPs) between samples were identified using the criterion |log2(fold change) | ≥ 1 and false discovery rate (FDR) < 0.05. MEME (http://meme-suite.org) and HOMER (http://homer.ucsd.edu/homer/motif) were used for detection of de novo and known motifs followed by localization of the motif with respect to the peak summit. StringTie (https://ccb.jhu.edu/software/stringtie) was used to estimate the expression level for all transcripts and genes from the input libraries by calculating the FPKM value (total exon fragments per mapped reads (millions) × exon length (Kb)). The differentially expressed genes (DEGs) were screened according to the criteria |log2(fold change)| ≥ 1 and p-value < 0.05 using the R package edgeR (https://bioconductor.org/packages/edgeR). Gene function enrichment analysis for the DEGs and the genes with DMPs was performed using the agriGO tool kit [37].

Validation of transcriptome data by quantitative real-time PCR

The transcriptome data were validated by qRT-PCR analysis. The analysis was performed on an Applied Biosystems® 7500 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). The OsUBQ5 gene was used as the internal reference gene. Three technical replicates were analyzed to calculate the standard error.

Results

Seed germination phenotype of WT and mutant under control and salt stress conditions

Previous results have shown that OsMTA1 contains a methyltransferase domain and is widely expressed in all rice tissues with higher expression levels in the leaf and seed ([38]; https://ngdc.cncb.ac.cn/red/index). To further examine the function of OsMTA1 in rice, two mutants were generated by using CRISPR/Cas9-mediated target mutagenesis and the homozygous lines (osmta1-1 and osmta1-2) were confirmed by genomic DNA sequencing (Figure S1A). When the WT and mutant lines were grown under control conditions, no significant differences were observed in seedling growth, plant height, and leaf morphology, but the flowering time of the mutant lines was delayed compared with that of the WT (Figure S1B), showing that OsMTA1 plays a role in the reproductive development of rice.

To investigate the role of OsMTA1 in response to salt stress, seeds of the WT and the mutant lines were germinated under control and salt stress conditions. The germination rates of osmta1-1 and osmta1-2 seeds were significantly delayed compared with that of WT seeds under the control condition, e.g., the percentage germination of osmta1-1 and WT seeds was 55% and 70%, respectively, at day 3 (Fig. 1A). The germination rate of mutant seeds was further inhibited and was distinctly slower than that of WT seeds under the salt stress condition. Strikingly, the germination of osmta1-1 seeds was markedly delayed under salt stress and 60% of osmta1-1 seeds germinated, whereas approximately 70% of WT seeds germinated after 7 d under the salt stress treatment (Fig. 1B). These results indicated that OsMTA1 plays a positive role in rice seed germination, especially under salt stress. Both osmta1-1 and osmta1-2 showed a similar defective phenotype of delayed flowering time and seed germination. Therefore, we chose to use osmta1-1 for the following experiments.

Fig. 1
figure 1

Seed germination of the osmta1 mutant and WT under control and salt stress conditions. A. Seedings of osmta1 mutants and WT grown under control and salt stress conditions after germination for 7 d. B. Seed germination rates of osmta1 mutants and WT under control and salt stress conditions. The germination rates were recorded after 1–7 days in the presence of water or 120 mM NaCl. Three independent experiments were conducted, with at least 50 seeds per genotype in each biological replicate. Error bars represent the standard deviation of three replicates. Asterisks indicate a significant difference compared with the WT at each time-point (* p < 0.05; ** p < 0.01; Student’s t-test). WT, wild type

Full size image

Physiological characteristics of seed germination of WT and osmta1-1 mutant under control and salt stress conditions

To test whether OsMTA1 potentially plays a role in the functions of the m6A mRNA methyltransferase complex, we measured the global m6A levels in mRNA extracted from 60 h germinating seeds of WT and the osmta1-1 mutant using a colorimetric method. The total m6A content in osmta1-1 seeds was marginally lower than that of WT seeds under the control condition, whereas the global m6A level of the germinating seeds of osmta1-1 was decreased compared with that of the WT under salt stress (Fig. 2A), indicating that OsMTA1 deficiency resulted in a decrease in m6A methylation.

Fig. 2
figure 2

Physiological indicators during seed germination of osmta1-1 mutant and WT under control and salt stress conditions. The m6A/A ratio (A), activities of catalase (CAT) (B) and peroxidase (POD) (C), and malondialdehyde (MDA) content (D) were determined in 60 h germinating seeds of the osmta1-1 mutant and WT under control (C) and salt stress (S) conditions. Data and error bars are the mean ± SD (n = 3 biological replicates). Different letters indicate a significant difference (p < 0.01). WT, wild type

Full size image

To investigate physiological differences in the osmta1-1 mutant and WT during seed germination under the control and salt stress conditions, the MDA contents and CAT and POD activities were determined. The CAT and POD activities in the osmta1-1 mutant were lower than those of the WT during seed germination under both conditions, especially under salt stress (Fig. 2B, C). The MDA content in the osmta1-1 mutant was significantly higher than that of the WT during seed germination under salt stress (Fig. 2D). These results suggested that the loss of function of OsMTA1 caused increased oxidative damage during germination of osmta1-1 seeds, especially under salt stress.

Profiling of m6A methylation during seed germination of WT and osmta1-1 mutant under control and salt stress conditions

To investigate whether m6A is involved in seed germination, we conducted and sequenced the m6A-IP and corresponding input (non-IP control) libraries of the WT and osmta1-1 mutant seed embryos, and of 60 h germinating seeds under the salt stress and control conditions. A total of 31–49 million reads were generated for each sample and approximately 84–92% clean reads were mapped to the rice reference genome (Table S1). We obtained 41,465 to 47,534 enriched m6A peaks (Fisher’s exact test, p ≤ 0.05) in the six samples, representing 18,781 to 21,465 expressed genes, with an average of 2.2 peaks per transcript (Fig. 3A, Table S2).

Fig. 3
figure 3

Overview of m6A methylation in rice during seed germination under control and salt stress conditions. A, Number of detected m6A peaks and m6A methylated genes in samples using m6A-IP sequencing. B, Distribution of m6A peaks in transcript regions of 5′ untranslated region (UTR), coding sequence (CDS), and 3′ UTR in six samples. C, Sequence logos representing the most common consensus motif in the m6A peaks in rice

Full size image

The m6A peak distribution was investigated according to gene annotations in the MSU rice database [39]. The m6A peaks were highly enriched in the 3′ untranslated region (UTR) regions and a summit of m6A peaks was observed close to the start codon of the coding sequence (CDS) in all samples (Fig. 3B). HOMER software was used to screen the sequence motifs that contained m6A peaks, revealing that two high consensus motifs were significantly enriched in these regions: UDCAG (D = A/G/U) and YUACW (Y = C/U, W = A/U) (Fig. 3C). The UDCAG motif has been identified as a conserved site for RNA binding [40, 41].

Mutation of MTA1 caused genome-wide modification of m6A methylation in rice seeds

To examine the global alteration of m6A methylation caused by knockout of OsMTA1, the difference in m6A methylation in embryos of WT and osmta1-1 seeds was analyzed using m6A-IP sequencing. In total, 1147 hypomethylated and 1735 hypermethylated peaks (fold change ≥ 2 and FDR < 0.05), within 1045 and 1581 genes, respectively, were identified in osmta1-1 embryos compared with WT embryos (Table S3). To investigate the distribution of m6A peaks in genomic regions, all identified differential m6A peaks were mapped to the annotated 5′-UTR, exonic, and 3′-UTR regions (MSU Release 7.0). An evident increase in hypomethylated peaks within exons and a concomitant reduction in the 3′-UTR and 5′-UTR regions were observed in the osmta1-1 mutant (Fig. 4A), indicating that knockout of OsMTA1 caused a distinct alteration in m6A modification in osmta1-1 embryos.

Fig. 4
figure 4

Analysis of the differentially m6A methylated peaks detected in osmta1-1 seed embryos compared with those of the WT. A. Percentage distribution of hypomethylated and hypermethylated m6A peaks within the exons, 5′ untranslated (UTR) and 3′-UTR regions in osmta1-1 seed embryos. B. GO enrichment analysis for the genes with hypomethylated m6A peaks in osmta1-1 seed embryos. WT, wild type

Full size image

We performed GO enrichment analysis of the genes with m6A hypomethylation in the osmta1-1 mutant using agriGO [37]. The genes with hypomethylated m6A peak(s) were functionally enriched in response to stress, protein modification process, cell death, nucleotide binding, and hydrolase activity (Fig. 4B). We further investigated the functions of the genes with m6A hypomethylation in the osmta1-1 embryo. A set of genes, including OsZYGO1, OsMCM2, OsMER3, OsMETS2, OsACO6, OsUAP2, OsACD6, and OsDRW1, were associated with the cell cycle; several of the m6A hypomethylated genes, such as OsIG1, OsESA1, OsDMC1A, OsbZIP78, and OsARF5, were involved in seed development; and a subset of the m6A hypomethylated genes, including OsGGP, OsWAK11, OsARF10, OsNHX2, OsTSD2, and OsIAA6, were functionally associated with stress response (Table 1). These results suggested that OsMTA1 deficiency resulted in m6A hypomethylation of genes that function in growth and development as well as stress response.

Table 1 Top 50 genes with m6A hypomethylation differentially expressed in embryos of osmta1-1 mutant and WT
Full size table

Difference in m6A methylation between WT and osmta1-1 mutant during seed germination under the control condition

To investigate whether m6A modification is involved in seed germination, we compared the m6A methylation profiling data of WT and osmta1-1 seeds after germination for 60 h. In total, 1400 hypomethylated and 1534 hypermethylated m6A peaks (fold change ≥ 2 and FDR < 0.05), within 1282 and 1380 genes, respectively, were detected in the osmta1-1 mutant compared with the WT (Table S4). A GO enrichment analysis revealed that the genes with m6A hypomethylation in the osmta1-1 mutant were functionally prevalent in the protein modification process, response to stress, transmembrane transport, nucleotide binding, kinase activity, protein binding, and hydrolase activity (Fig. 5A). Thus, deficiency of OsMTA1 resulted in extensive loss of m6A methylation in genes with diverse biological functions during rice seed germination.

Fig. 5
figure 5

Comparative analysis of hypomethylated m6A peaks detected in germinating osmta1-1 seeds compared with WT. A. GO enrichment analysis for the genes with hypomethylated m6A peaks in osmta1-1 seeds. B. Venn diagram analysis of the genes with hypomethylated m6A peak(s) in osmta1-1 seed embryos and seeds after 60 h germination. C, D. Genomic visualization of m6A density maps for OsbZIP78 and OsA8 in WT and osmta1-1 seeds under the control condition

Full size image

Further comparison of the m6A hypomethylated genes in dry seed embryos and 60 h germinating seeds of the osmta1-1 mutant revealed that 108 genes were hypomethylated in both samples (Fig. 5B, Table S5). A GO enrichment analysis showed that these 108 genes were functionally enriched in small molecule metabolic process, nucleotide binding, and ATPase activity. Importantly, several of these hypomethylated genes were involved in seed germination, and cell growth and development. For example, OsbZIP78 and OsA8 (Fig. 5C and D) are reported to play crucial roles in rice seed germination [42, 43], and OsMER3, OsSCD1, OsACD6, OsD3, and OsSPL17 are functionally associated with cell growth and development [44,45,46,47,48]. These results suggested that these genes may be associated with delayed seed germination in the osmta1-1 mutant.

Salt-induced m6A methylation changes of WT and osmta1-1 mutant during seed germination

Modification of m6A on mRNA is involved in the plant response to abiotic stresses [49]. To investigate whether OsMTA1 is implicated in the stress response in rice during seed germination, we analyzed the m6A methylation profile in osmta1-1 and WT seeds after 60 h germination under salt stress. A total of 2067 hypomethylated and 1406 hypermethylated m6A peaks (fold change ≥ 2 and FDR < 0.05), within 1881 and 1292 genes, respectively, were identified in osmta1-1 seeds compared with the WT seeds (Table S6). A GO enrichment analysis revealed that the genes with m6A hypomethylation in the osmta1-1 mutant were highly enriched in response to stress, phosphorus metabolic process, protein modification process, nucleotide binding, kinase activity, oxidoreductase activity, and transmembrane transporter activity (Fig. 6A). Thus, many genes with diverse molecular functions were m6A hypomethylated in the osmta1-1 mutant during seed germination under salt stress.

Fig. 6
figure 6

Comparative analysis of hypomethylated m6A peaks in germinating osmta1-1 seeds compared with WT under salt stress. A. GO enrichment analysis for the genes with hypomethylated m6A peaks in osmta1-1 seeds under salt stress. B. Venn diagram analysis of the genes with hypomethylated m6A peak(s) in osmta1-1 seeds under control (C) and salt stress (S) conditions. C, D. Genomic visualization of m6A density maps for OsIAA9, OsAGO7, and OsAUX3 in WT and osmta1-1 seeds under salt stress

Full size image

Further analysis of the hypomethylated genes in germinating seeds of the osmta1-1 mutant under the control and salt stress conditions showed that only a small number of genes were hypomethylated under both conditions (Fig. 6B), indicating that salt stress resulted in specific m6A hypomethylation in osmta1-1 germinating seeds. Functional analysis of these hypomethylated genes revealed that a notable proportion functioned in stress responses, growth, and development, including the genes OsIAA9, OsAGO3 (Fig. 6C), OsRLCK8, OsRLCK46, OsACS5, OsPOD2, OsATL15, OsOFP6, OsLAC8, OsPP2C19, and OsALN, are involved in plant response to stresses [50,51,52,53,54,55,56,57,58]. Genes such as OsAUX3 (Fig. 6D), OsWS1, OsHUS1, OsMRE11, GS5, HDA716, OsSMC4, OsRR20, OsFTL12, and Hd18 are associated with growth and development in plants [59,60,61,62,63,64,65]. These results suggested that loss of function of OsMTA1 causes m6A hypomethylation in genes with diverse functions, especially in stress response and growth and development, in osmta1-1 germinating seeds under salt stress.

Association analysis between m6A modification and gene expression change

To investigate the potential role of m6A modification at the transcriptome level, global gene expression profiling was performed for 60 h germinating seeds of the WT and osmta1-1 mutant under the control and salt stress conditions. We identified 958 (658 up-regulated and 300 down-regulated) and 527 (362 up-regulated and 165 down-regulated) DEGs in the osmta1-1 mutant compared with WT seeds under the salt stress and control conditions, respectively (Fig. 7A, Tables S7 and S8). To evaluate the accuracy of the transcriptome sequencing data, several DEGs in the osmta1-1 mutant and WT were selected for validation by RT-qPCR, which verified the reliability of the transcriptome sequencing results (Figure S2).

Fig. 7
figure 7

Association analysis between m6A modification and gene expression change. A. Differentially expressed genes in osmta1-1 seeds compared with those in the WT under the control and salt stress conditions. B, C. Correlation analysis between changes in m6A methylation and gene expression in the seeds of osmta1-1 vs. WT under the control and salt stress conditions

Full size image

To examine the association between m6A methylation and gene expression, the global transcriptome and m6A-IP sequencing data for the two genotypes under the control and salt stress conditions were analyzed. The expression of more than two-thirds of the genes was reversely affected by m6A modification, e.g., the expression of genes showing evident m6A hypomethylation was significantly up-regulated in the osmta1-1 mutant under both control and salt stress conditions, and vice versa, whereas the expression of one-third of the genes was positively regulated by m6A modification (Fig. 7B, C; Tables S9 and S10). These results indicated that m6A methylation has a complex impact on gene expression in rice plants.

We further examined the DEGs with m6A hypomethylation in the osmta1-1 mutant. In total, 41 hypomethylated m6A peaks were associated with six down-regulated and 35 up-regulated genes in osmta1-1 germinating seeds under the control condition (Table S9). Among these hypomethylated DEGs in the osmta1-1 mutant, several of the up-regulated genes, including OsHsfA7, OsMKKK62, OsHDAC3, CYP714B1, and OsTPP7, have negative functions in growth and development [66,67,68,69,70], suggesting these up-regulated growth negative regulators contributed to delayed seed germination in osmta1. Under the salt stress condition, 93 hypomethylated genes were differentially expressed in osmta1-1 seeds (Table S10). Of these genes, several down-regulated genes, such as OsAUX3, OsProT1, and OsXTH4, are associated with stress response [64, 71, 72]. A unique set of hypomethylated genes were up-regulated in osmta1-1 seeds. Among these genes, OsIAA9, GLO1, OsAGO3, OsRHC, OsPROPEP5, and OsbZIP65 are reported to be negative regulators of growth and stress response [73,74,75,76,77,78]. These results suggested that OsMTA1 might respond to salt stress by affecting the transcription of genes involved in growth and stress response during rice seed germination.

Discussion

RNA m6A modification is broadly involved in plant growth and development and response to environmental stresses [49, 79]. However, the function of m6A in plant seed germination remains largely unknown. In the present study, a CRISPR/Cas9 mutant of the m6A writer OsMTA1 and the corresponding WT were used to systematically analyze the role of m6A-mediated epitranscriptomic regulation in rice seed germination and salt stress response. The results indicated that OsMTA1 plays a positive role in seed germination and salt tolerance by regulating m6A modification of a unique set of genes with diverse biological functions.

Previous reports have shown that m6A methylation plays positive roles in abiotic stress tolerance by enhancing the ability to scavenge reactive oxygen species (ROS) [80, 81]. The present analysis showed that OsMTA1 impacted on rice developmental growth, and that knockout of OsMTA1 resulted in elevated oxidative damage and delayed seed germination under the control and salt stress conditions. The activities of antioxidant enzymes in the osmta1-1 mutant were significantly lower than those of the WT during seed germination under both conditions, indicating that OsMTA-mediated m6A methylation plays an active role in the ROS-scavenging system during seed germination.

The m6A modification is installed by a conserved methyltransferase complex that includes MTA and MTB as core subunits, of which MTA shows major catalytic activity for m6A deposition in Arabidopsis [82]. Loss-of-function of MTA causes a global decrease in m6A methylation and salt sensitivity in Arabidopsis [27]. The present results indicated that knockout of OsMTA1 resulted in a reduced m6A level in osmta1-1 seeds and delayed seed germination, especially under salt stress. Remarkably, the present data showed that seed germination in the osmta1-1 mutant was delayed relative to that of the WT under the control condition. This result differed from a previous finding that the seed germination of MTA mutants in Arabidopsis was not affected under the non-stress condition [27]. These results demonstrate that OsMTA1 has the function of m6A methyltransferase and is essential for rice seed germination.

Further global m6A methylation profiling revealed that the m6A distribution in WT and osmta1-1 seeds was prevalent in 3′-UTR and CDS regions, consistent with previous results in plants [80, 83]. Two sequence motifs that contained m6A peaks have been identified, of which the UDCAG motif is a conserved sites for RNA binding as reported previously [40, 41]. However, YUACW is a new motif identified in the present study, suggesting that this motif may be unique and play an important role in m6A installation during seed germination in rice.

Comparative analysis of m6A methylation between the osmta1-1 mutant and WT revealed that m6A methylation of a unique subset of genes was decreased in osmta11-1 embryos and germinating seeds under the control condition. These genes include those encoding crucial proteins with functions in growth and development, e.g., OsESA is required for embryo development [84], the transcription factor OsbZIP78 plays a positive role in regulating rice seed dormancy by inducing the expression of OsDOG1L-3 [42], and a plasma membrane H+-ATPase OsA8 is involved in seed germination [43]. These results indicate that knockout of OsMTA1 has a negative impact on embryo development through hypomethylation of these unique functional genes.

MTA is the major active subunit of the m6A methyltransferase complex. Impairment of the MTA component results in defective plant growth and development and impaired abiotic stress response [83]. Consistent with the phenotype of delayed seed germination of the osmta1-1 mutant under the non-stress condition, a number of genes with diverse functions were m6A hypomethylated; in particular, a small set of these hypomethylated genes with negative roles in growth and development were up-regulated in osmta1-1 germinating seeds (Table S9). Of these genes, OsHDAC3 is reported to be involved in delayed heading date by repression of Ghd7 expression [68], higher expression of OsHsfA7 inhibits root growth in rice [66], and OsMKKK70 plays a role in impairing seed set and pollen fertility [67]. Loss-of-function of OsMTA1 caused up-regulation of these m6A-hypomethylated genes and delayed osmta1-1 seed germination.

Mutation of m6A writers in plants results in alteration of m6A modification and gene expression in response to abiotic stresses, as revealed in previous studies [27, 85]. We identified many genes that exhibited significant changes in m6A modification and expression in the osmta1-1 mutant compared with the WT during seed germination under salt stress. The genes that function in stress response, growth, and development were distinctly hypomethylated in geminating seeds of the osmta1-1 mutant in response to salt stress. Strikingly, a unique subset of these genes with functions as negative regulators in growth and stress response were up-regulated in the osmta1-1 mutant; e.g., OsIAA9 is reported to play an inhibitory role in auxin-regulated root growth in rice [73], OsAGO3 is involved in modulating rice susceptibility to biotic stress [75], and the transcription factor bZIP65 delays heading date via suppression of Ehd1 expression in rice [78]. These up-regulated hypomethylated genes with negative functions in growth and stress response establish a link between m6A modification and rice seed germination under salt stress.

Conclusion

The present study has demonstrated that OsMTA1 is involved in rice seed germination and response to salt stress. Comparative analysis of m6A profiling between the WT and the osmta1-1 mutant revealed that knockout of OsMTA1 caused hypomethylation of a unique set of genes and delayed seed germination under the control condition. Under salt stress, genes with functions in plant growth and stress response were hypomethylated and differentially expressed in the osmta1-1 mutant, and seed germination was distinctly delayed. The present results not only improve our understanding the molecular regulatory mechanisms of seed germination but also provide valuable information for the improvement of direct seeding and salt tolerance in rice.

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA020555) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

References

  1. Weitbrecht K, Müller K, Leubner-Metzger G. First off the mark: early seed germination. J Exp Bot. 2011;62(10):3289–309.

    Article  PubMed  CAS  Google Scholar 

  2. Bewley JD. Seed germination and dormancy. Plant Cell. 1997;9(7):1055–66.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Howell KA, Narsai R, Carroll A, Ivanova A, Lohse M, Usadel B, Millar AH, Whelan J. Mapping metabolic and transcript temporal switches during germination in rice highlights specific transcription factors and the role of RNA instability in the germination process. Plant Physiol. 2009;149(2):961–80.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Sano N, Rajjou L, North HM. Lost in translation: physiological roles of stored mRNAs in seed germination. Plants (Basel). 2020;9(3):347.

    PubMed  CAS  Google Scholar 

  5. Xu H, Lantzouni O, Bruggink T, Benjamins R, Lanfermeijer F, Denby K, Schwechheimer C, Bassel GW. A Molecular Signal Integration Network Underpinning Arabidopsis seed germination. Curr Biol. 2020;30(19):3703–e37124.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Shu K, Meng YJ, Shuai HW, Liu WG, Du JB, Liu J, Yang WY. Dormancy and germination: how does the crop seed decide? Plant Biol (Stuttg). 2015;17(6):1104–12.

    Article  PubMed  CAS  Google Scholar 

  7. Daszkowska-Golec A. Arabidopsis seed germination under abiotic stress as a concert of action of phytohormones. OMICS. 2011;15(11):763–74.

    Article  PubMed  CAS  Google Scholar 

  8. Ganie SA, Molla KA, Henry RJ, Bhat KV, Mondal TK. Advances in understanding salt tolerance in rice. Theor Appl Genet. 2019;132(4):851–70.

    Article  PubMed  CAS  Google Scholar 

  9. Park GG, Park JJ, Yoon J, Yu SN, An G. A RING finger E3 ligase gene, Oryza sativa delayed seed germination 1 (OsDSG1), controls seed germination and stress responses in rice. Plant Mol Biol. 2010;74(4–5):467–78.

    Article  PubMed  CAS  Google Scholar 

  10. Wang Z, Wang J, Bao Y, Wu Y, Zhang H. Quantitative trait loci controlling rice seed germination under salt stress. Euphytica. 2011;178:297–307.

    Article  Google Scholar 

  11. He Y, Yang B, He Y, Zhan C, Cheng Y, Zhang J, Zhang H, Cheng J, Wang Z. A quantitative trait locus, qSE3, promotes seed germination and seedling establishment under salinity stress in rice. Plant J. 2019;97(6):1089–104.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Zeng P, Zhu P, Qian L, Qian X, Mi Y, Lin Z, Dong S, Aronsson H, Zhang H, Cheng J. Identification and fine mapping of qGR6.2, a novel locus controlling rice seed germination under salt stress. BMC Plant Biol. 2021;21(1):36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Wei LH, Song P, Wang Y, Lu Z, Tang Q, Yu Q, Xiao Y, Zhang X, Duan HC, Jia G. The m6A reader ECT2 controls Trichome morphology by affecting mRNA Stability in Arabidopsis. Plant Cell. 2018;30(5):968–85.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Anderson SJ, Kramer MC, Gosai SJ, Yu X, Vandivier LE, Nelson ADL, Anderson ZD, Beilstein MA, Fray RG, Lyons E, Gregory BD. N6-Methyladenosine inhibits local ribonucleolytic cleavage to stabilize mRNAs in Arabidopsis. Cell Rep. 2018;25(5):1146–57.

    Article  PubMed  CAS  Google Scholar 

  15. Luo W, Tang Y, Li S, Zhang L, Liu Y, Zhang R, Diao X, Yu J. The m6 a reader SiYTH1 enhances drought tolerance by affecting the messenger RNA stability of genes related to stomatal closure and reactive oxygen species scavenging in Setaria italica. J Integr Plant Biol. 2023;65(12):2569–86.

    Article  PubMed  CAS  Google Scholar 

  16. Roundtree IA, Luo GZ, Zhang Z, Wang X, Zhou T, Cui Y, Sha J, Huang X, Guerrero L, Xie P, He E, Shen B, He C. YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs. Elife. 2017;6:e31311.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Kramer MC, Janssen KA, Palos K, Nelson ADL, Vandivier LE, Garcia BA, Lyons E, Beilstein MA, Gregory BD. N6-methyladenosine and RNA secondary structure affect transcript stability and protein abundance during systemic salt stress in Arabidopsis. Plant Direct. 2020;4(7):e00239.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Zhong S, Li H, Bodi Z, Button J, Vespa L, Herzog M, Fray RG. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell. 2008;20(5):1278–88.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Bodi Z, Zhong S, Mehra S, Song J, Graham N, Li H, May S, Fray RG. Adenosine Methylation in Arabidopsis mRNA is Associated with the 3’ end and reduced levels cause developmental defects. Front Plant Sci. 2012;3:48.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Shen L, Liang Z, Gu X, Chen Y, Teo ZW, Hou X, Cai WM, Dedon PC, Liu L, Yu H. N(6)-Methyladenosine RNA modification regulates shoot stem cell fate in Arabidopsis. Dev Cell. 2016;38(2):186–200.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Růžička K, Zhang M, Campilho A, Bodi Z, Kashif M, Saleh M, Eeckhout D, El-Showk S, Li H, Zhong S, De Jaeger G, Mongan NP, Hejátko J, Helariutta Y, Fray RG. Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI. New Phytol. 2017;215(1):157–72.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Hu J, Manduzio S, Kang H. Epitranscriptomic RNA methylation in Plant Development and Abiotic stress responses. Front Plant Sci. 2019;10:500.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Zhang F, Zhang YC, Liao JY, Yu Y, Zhou YF, Feng YZ, Yang YW, Lei MQ, Bai M, Wu H, Chen YQ. The subunit of RNA N6-methyladenosine methyltransferase OsFIP regulates early degeneration of microspores in rice. PLoS Genet. 2019;15(5):e1008120.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Ma K, Han J, Zhang Z, Li H, Zhao Y, Zhu Q, Xie Y, Liu YG, Chen L. OsEDM2L mediates m6A of EAT1 transcript for proper alternative splicing and polyadenylation regulating rice tapetal degradation. J Integr Plant Biol. 2021;63(11):1982–94.

    Article  PubMed  CAS  Google Scholar 

  25. Huang Y, Zheng P, Liu X, Chen H, Tu J. OseIF3h regulates Plant Growth and Pollen Development at Translational Level presumably through Interaction with OsMTA2. Plants (Basel). 2021;10(6):1101.

    PubMed  CAS  Google Scholar 

  26. Lu L, Zhang Y, He Q, Qi Z, Zhang G, Xu W, Yi T, Wu G, Li R. MTA, an RNA m6A methyltransferase, enhances Drought Tolerance by regulating the development of trichomes and roots in Poplar. Int J Mol Sci. 2020;21(7):2462.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Hu J, Cai J, Park SJ, Lee K, Li Y, Chen Y, Yun JY, Xu T, Kang H. N6 -Methyladenosine mRNA methylation is important for salt stress tolerance in Arabidopsis. Plant J. 2021;106(6):1759–75.

    Article  PubMed  CAS  Google Scholar 

  28. Yang D, Xu H, Liu Y, Li M, Ali M, Xu X, Lu G. RNA N6-Methyladenosine responds to low-temperature stress in Tomato anthers. Front Plant Sci. 2021;12:687826.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Wang Y, Du F, Li Y, Wang J, Zhao X, Li Z, Xu J, Wang W, Fu B. Global N6-Methyladenosine profiling revealed the tissue-specific epitranscriptomic regulation of Rice responses to salt stress. Int J Mol Sci. 2022;23(4):2091.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Zheng H, Sun X, Li J, Song Y, Song J, Wang F, Liu L, Zhang X, Sui N. Analysis of N6-methyladenosine reveals a new important mechanism regulating the salt tolerance of sweet sorghum. Plant Sci. 2021;304:110801.

    Article  PubMed  CAS  Google Scholar 

  31. Li B, Zhang M, Sun W, Yue D, Ma Y, Zhang B, Duan L, Wang M, Lindsey K, Nie X, Zhang X, Yang X. N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner. Plant Biotechnol J. 2023;21(6):1270–85.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Liu G, Wang J, Hou X. Transcriptome-wide N6-Methyladenosine (m6A) methylome profiling of heat stress in pak-choi (Brassica rapa ssp. chinensis). Plants (Basel). 2020;9(9):1080.

    PubMed  CAS  Google Scholar 

  33. Liu SJ, Xu HH, Wang WQ, Li N, Wang WP, Møller IM, Song SQ. A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment. Physiol Plant. 2015;154(1):142–61.

    Article  PubMed  CAS  Google Scholar 

  34. Xie Z, Wang J, Wang W, Wang Y, Xu J, Li Z, Zhao X, Fu B. Integrated Analysis of the Transcriptome and Metabolome revealed the Molecular mechanisms underlying the enhanced salt tolerance of Rice due to the application of exogenous melatonin. Front Plant Sci. 2021;11:618680.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Ouyang SQ, Liu YF, Liu P, Lei G, He SJ, Ma B, Zhang WK, Zhang JS, Chen SY. Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants. Plant J. 2010;62(2):316–29.

    Article  PubMed  CAS  Google Scholar 

  36. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38(16):e164.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Tian T, Liu Y, Yan H, You Q, Yi X, Du Z, Xu W, Su Z. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res. 2017;45(W1):W122–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Davidson RM, Gowda M, Moghe G, Lin H, Vaillancourt B, Shiu SH, Jiang N, Robin Buell C. Comparative transcriptomics of three Poaceae species reveals patterns of gene expression evolution. Plant J. 2012;71(3):492–502.

    Article  PubMed  CAS  Google Scholar 

  39. Ouyang S, Zhu W, Hamilton J, Lin H, Campbell M, Childs K, Thibaud-Nissen F, Malek RL, Lee Y, Zheng L, Orvis J, Haas B, Wortman J, Buell CR. The TIGR Rice Genome Annotation Resource: improvements and new features. Nucleic Acids Res. 2007;35(Database issue):D883–7.

    Article  PubMed  CAS  Google Scholar 

  40. Zhang J, Lieu YK, Ali AM, Penson A, Reggio KS, Rabadan R, Raza A, Mukherjee S, Manley JL. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities. Proc Natl Acad Sci U S A. 2015;112(34):E4726–34.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Satterwhite ER, Mansfield KD. RNA methyltransferase METTL16: targets and function. Wiley Interdiscip Rev RNA. 2022;13(2):e1681.

    Article  PubMed  CAS  Google Scholar 

  42. Wang Q, Lin Q, Wu T, Duan E, Huang Y, Yang C, Mou C, Lan J, Zhou C, Xie K, Liu X, Zhang X, Guo X, Wang J, Jiang L, Wan J. OsDOG1L-3 regulates seed dormancy through the abscisic acid pathway in rice. Plant Sci. 2020;298:110570.

    Article  PubMed  CAS  Google Scholar 

  43. Liu G, Zhang MC, Zeng HQ, Zhu YY, Sun SB, Xu GH. Overexpression of a plasma membrane H+ATPase gene promotes rice seed germination. Chin J Rice Sci. 2012;26(6):651–5.

    CAS  Google Scholar 

  44. Wang K, Tang D, Wang M, Lu J, Yu H, Liu J, Qian B, Gong Z, Wang X, Chen J, Gu M, Cheng Z. MER3 is required for normal meiotic crossover formation, but not for presynaptic alignment in rice. J Cell Sci. 2009;122(Pt 12):2055–63.

    Article  PubMed  CAS  Google Scholar 

  45. Wang F, Cheng Z, Wang J, Zhang F, Zhang B, Luo S, Lei C, Pan T, Wang Y, Zhu Y, Wang M, Chen W, Lin Q, Zhu S, Zhou Y, Zhao Z, Wang J, Guo X, Zhang X, Jiang L, Bao Y, Ren Y, Wan J. Rice STOMATAL CYTOKINESIS DEFECTIVE2 regulates cell expansion by affecting vesicular trafficking in rice. Plant Physiol. 2022;189(2):567–84.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Kim JA, Cho K, Singh R, Jung YH, Jeong SH, Kim SH, Lee JE, Cho YS, Agrawal GK, Rakwal R, Tamogami S, Kersten B, Jeon JS, An G, Jwa NS. Rice OsACDR1 (Oryza sativa accelerated cell death and resistance 1) is a potential positive regulator of fungal disease resistance. Mol Cells. 2009;28(5):431–9.

    Article  PubMed  CAS  Google Scholar 

  47. Rong C, Liu Y, Chang Z, Liu Z, Ding Y, Ding C. Cytokinin oxidase/dehydrogenase family genes exhibit functional divergence and overlap in rice growth and development, especially in control of tillering. J Exp Bot. 2022;73(11):3552–68.

    Article  PubMed  CAS  Google Scholar 

  48. Yan Y, Wei M, Li Y, Tao H, Wu H, Chen Z, Li C, Xu JH. MiR529a controls plant height, tiller number, panicle architecture and grain size by regulating SPL target genes in rice (Oryza sativa L). Plant Sci. 2021;302:110728.

    Article  PubMed  CAS  Google Scholar 

  49. Zhou L, Gao G, Tang R, Wang W, Wang Y, Tian S, Qin G. m6A-mediated regulation of crop development and stress responses. Plant Biotechnol J. 2022;20(8):1447–55.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Vij S, Giri J, Dansana PK, Kapoor S, Tyagi AK. The receptor-like cytoplasmic kinase (OsRLCK) gene family in rice: organization, phylogenetic relationship, and expression during development and stress. Mol Plant. 2008;1(5):732–50.

    Article  PubMed  CAS  Google Scholar 

  51. Yu M, Yau CP, Yip WK. Differentially localized rice ethylene receptors OsERS1 and OsETR2 and their potential role during submergence. Plant Signal Behav. 2017;12(8):e1356532.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Xiao Y, Zhang H, Li Z, Huang T, Akihiro T, Xu J, Xu H, Lin F. An amino acid transporter-like protein (OsATL15) facilitates the systematic distribution of thiamethoxam in rice for controlling the brown planthopper. Plant Biotechnol J. 2022;20(10):1888–901.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Ma Y, Yang C, He Y, Tian Z, Li J. Rice OVATE family protein 6 regulates plant development and confers resistance to drought and cold stresses. J Exp Bot. 2017;68(17):4885–98.

    Article  PubMed  CAS  Google Scholar 

  54. Liu Q, Luo L, Wang X, Shen Z, Zheng L. Comprehensive Analysis of Rice Laccase Gene (OsLAC) family and ectopic expression of OsLAC10 enhances tolerance to copper stress in Arabidopsis. Int J Mol Sci. 2017;18(2):209.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Sahoo SA, Raghuvanshi R, Srivastava AK, Suprasanna P. Phosphatases: the critical Regulator of Abiotic stress tolerance in plants. In: Pandey GK, editor. Protein phosphatases and Stress Management in plants: functional genomic perspective. Cham: Springer International Publishing; 2020. pp. 163–201.

    Chapter  Google Scholar 

  56. Luo S, Li Q, Liu S, Pinas NM, Tian H, Wang S. Constitutive expression of OsIAA9 affects Starch Granules Accumulation and Root Gravitropic response in Arabidopsis. Front Plant Sci. 2015;6:1156.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Kapoor M, Arora R, Lama T, Nijhawan A, Khurana JP, Tyagi AK, Kapoor S. Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analysis during reproductive development and stress in rice. BMC Genomics. 2008;9:451.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Lee DK, Redillas MCFR, Jung H, Choi S, Kim YS, Kim JK. A Nitrogen Molecular Sensing System, comprised of the ALLANTOINASE and UREIDE PERMEASE 1 genes, can be used to monitor N status in Rice. Front Plant Sci. 2018;9:444.

    Article  PubMed  PubMed Central  Google Scholar 

  59. Xia K, Ou X, Gao C, Tang H, Jia Y, Deng R, Xu X, Zhang M. OsWS1 involved in cuticular wax biosynthesis is regulated by osa-miR1848. Plant Cell Environ. 2015;38(12):2662–73.

    Article  PubMed  CAS  Google Scholar 

  60. Che L, Wang K, Tang D, Liu Q, Chen X, Li Y, Hu Q, Shen Y, Yu H, Gu M, Cheng Z. OsHUS1 facilitates accurate meiotic recombination in rice. PLoS Genet. 2014;10(6):e1004405.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Shen M, Nie Y, Chen Y, Zhang X, Zhao J. OsMre11 is required for mitosis during Rice Growth and Development. Int J Mol Sci. 2020;22(1):169.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Li Y, Fan C, Xing Y, Jiang Y, Luo L, Sun L, Shao D, Xu C, Li X, Xiao J, He Y, Zhang Q. Natural variation in GS5 plays an important role in regulating grain size and yield in rice. Nat Genet. 2011;43(12):1266–9.

    Article  PubMed  CAS  Google Scholar 

  63. Yoon H, Seong G, Lee SJ, Lim C, Paek NC. Histone deacetylases in rice development and stress responses. J Plant Biology. 2022;65(3):175–85.

    Article  CAS  Google Scholar 

  64. Wang M, Qiao J, Yu C, Chen H, Sun C, Huang L, Li C, Geisler M, Qian Q, Jiang A, Qi Y. The auxin influx carrier, OsAUX3, regulates rice root development and responses to aluminium stress. Plant Cell Environ. 2019;42(4):1125–38.

    Article  PubMed  CAS  Google Scholar 

  65. Shibaya T, Hori K, Ogiso-Tanaka E, Yamanouchi U, Shu K, Kitazawa N, Shomura A, Ando T, Ebana K, Wu J, Yamazaki T, Yano M. Hd18, encoding histone acetylase related to Arabidopsis FLOWERING LOCUS D, is involved in the control of Flowering Time in Rice. Plant Cell Physiol. 2016;57(9):1828–38.

    Article  PubMed  CAS  Google Scholar 

  66. Liu AL, Zou J, Liu CF, Zhou XY, Zhang XW, Luo GY, Chen XB. Over-expression of OsHsfA7 enhanced salt and drought tolerance in transgenic rice. BMB Rep. 2013;46(1):31–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Mei E, Tang J, He M, Liu Z, Tian X, Bu Q. OsMKKK70 negatively regulates Cold Tolerance at Booting Stage in Rice. Int J Mol Sci. 2022;23(22):14472.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  68. Wang H, Jiao X, Kong X, Liu Y, Chen X, Fang R, Yan Y. The histone deacetylase HDA703 interacts with OsBZR1 to regulate rice brassinosteroid signaling, growth and heading date through repression of Ghd7 expression. Plant J. 2020;104(2):447–59.

    Article  PubMed  CAS  Google Scholar 

  69. Magome H, Nomura T, Hanada A, Takeda-Kamiya N, Ohnishi T, Shinma Y, Katsumata T, Kawaide H, Kamiya Y, Yamaguchi S. CYP714B1 and CYP714B2 encode gibberellin 13-oxidases that reduce gibberellin activity in rice. Proc Natl Acad Sci USA. 2013;110(5):1947–52.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Wang K, Li M, Zhang B, Chang Y, An S, Zhao W. Sugar starvation activates the OsSnRK1a-OsbHLH111/OsSGI1-OsTPP7 module to mediate growth inhibition of rice. Plant Biotechnol J. 2023;21(10):2033–46.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  71. Lin JH, Xu ZJ, Peng JS, Zhao J, Zhang GB, Xie J, Yi ZX, Zhang JH, Gong JM, Ye NH, Meng S. OsProT1 and OsProT3 function to Mediate Proline- and γ-aminobutyric acid-specific transport in yeast and are differentially expressed in Rice (Oryza sativa L). Rice (N Y). 2019;12(1):79.

    Article  PubMed  Google Scholar 

  72. He H, Serraj R, Yang Q. Changes in OsXTH gene expression, ABA content, and peduncle elongation in rice subjected to drought at the reproductive stage. Acta Physiol Plant. 2009;31:749–56.

    Article  CAS  Google Scholar 

  73. Song YL. The gene OsIAA9 encoding auxin/indole-3-acetic acid proteins is a negative regulator of auxin-regulated root growth in rice. Biol Plant. 2019;63:210–8.

    Article  CAS  Google Scholar 

  74. Chern M, Bai W, Chen X, Canlas PE, Ronald PC. Reduced expression of glycolate oxidase leads to enhanced disease resistance in rice. PeerJ. 2013;1:e28.

    Article  PubMed  PubMed Central  Google Scholar 

  75. Wang Z, Chen D, Sun F, Guo W, Wang W, Li X, Lan Y, Du L, Li S, Fan Y, Zhou Y, Zhao H, Zhou T. ARGONAUTE 2 increases rice susceptibility to rice black-streaked dwarf virus infection by epigenetically regulating HEXOKINASE 1 expression. Mol Plant Pathol. 2021;22(9):1029–40.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Ding Y, Zhang F, Sun F, Liu J, Zhu Z, He X, Bai G, Ni Z, Sun Q, Su Z. Loss of OsHRC function confers blast resistance without yield penalty in rice. Plant Biotechnol J. 2023;21(8):1516–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  77. Harshith CY, Pal A, Chakraborty M, Nair A, Raju S, Shivaprasad PV. Wound-induced small-peptide-mediated signaling cascade, regulated by OsPSKR, dictates balance between growth and defense in rice. Cell Rep. 2024;43(7):114515.

    Article  PubMed  CAS  Google Scholar 

  78. Pan T, He M, Liu H, Tian X, Wang Z, Yu X, Miao X, Li X. Transcription factor bZIP65 delays flowering via suppressing Ehd1 expression in rice. Mol Breed. 2022;42(10):63.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Wang S, Wang H, Xu Z, Jiang S, Shi Y, Xie H, Wang S, Hua J, Wu Y. m6A mRNA modification promotes chilling tolerance and modulates gene translation efficiency in Arabidopsis. Plant Physiol. 2023;192(2):1466–82.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  80. Hou N, Li C, He J, Liu Y, Yu S, Malnoy M, Mobeen Tahir M, Xu L, Ma F, Guan Q. MdMTA-mediated m6 A modification enhances drought tolerance by promoting mRNA stability and translation efficiency of genes involved in lignin deposition and oxidative stress. New Phytol. 2022;234(4):1294–314.

    Article  PubMed  CAS  Google Scholar 

  81. Guo T, Liu C, Meng F, Hu L, Fu X, Yang Z, Wang N, Jiang Q, Zhang X, Ma F. The m6 a reader MhYTP2 regulates MdMLO19 mRNA stability and antioxidant genes translation efficiency conferring powdery mildew resistance in apple. Plant Biotechnol J. 2022;20(3):511–25.

    Article  PubMed  CAS  Google Scholar 

  82. Tang J, Chen S, Jia G. Detection, regulation, and functions of RNA N6-methyladenosine modification in plants. Plant Commun. 2023;4:100546.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  83. Wong CE, Zhang S, Xu T, Zhang Y, Teo ZWN, Yan A, Shen L, Yu H. Shaping the landscape of N6-methyladenosine RNA methylation in Arabidopsis. Plant Physiol. 2023;191(3):2045–63.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  84. Hou J, Cao C, Ruan Y, Deng Y, Liu Y, Zhang K, Tan L, Zhu Z, Cai H, Liu F, Sun H, Gu P, Sun C, Fu Y. ESA1 is involved in embryo sac abortion in interspecific hybrid progeny of Rice. Plant Physiol. 2019;180(1):356–66.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  85. Dhingra Y, Gupta S, Gupta V, Agarwal M, Katiyar-Agarwal S. The emerging role of epitranscriptome in shaping stress responses in plants. Plant Cell Rep. 2023;42(10):1531–55.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Robert McKenzie, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing a draft of this manuscript.

Funding

This study was supported by the Scientific Innovation 2030 Project (2022ZD0401703); the CAAS Innovation Program; the Hainan Yazhou Bay Seed Lab Project (B23CJ0208); the National High-level Personnel of Special Support Program; Nanfan special project, CAAS (YBXM2322, YYLH2309).

Author information

Author notes

  1. Yingbo Li and Ming Yin contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Crop Gene Resources and Breeding/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, South Zhong-Guan-Cun Street 12#, Beijing, 100081, China

    Yingbo Li, Ming Yin, Juan Wang, Xiuqin Zhao, Jianlong Xu, Wensheng Wang & Binying Fu

  2. National Nanfan Research Institute (Sanya), Chinese Academy of Agricultural Sciences, Sanya, 572024, China

    Wensheng Wang

Authors
  1. Yingbo Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Ming Yin
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Juan Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Xiuqin Zhao
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Jianlong Xu
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Wensheng Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Binying Fu
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Y.L., M.Y. and B.F. conducted the experiments, performed data analysis and wrote the manuscript. W.W., J.W., X.Z. and J.X. participated in material development, sample preparation and data analysis. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Wensheng Wang or Binying Fu.

Ethics declarations

Ethics approval and consent to participate

No applicable.

Consent for publication

No applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

12870_2025_6134_MOESM1_ESM.xlsx

Supplementary Material 1: Table S1. Summary of m6A-seq data in WT and osmta1-1 seed embryos, and 60 h germinating seeds under the control and salt stress conditions.

12870_2025_6134_MOESM2_ESM.xlsx

Supplementary Material 2: Table S2. Summary of detected m6A peaks and expressed genes in WT and osmta1-1 seed embryos and germinating seeds under the control and salt stress conditions.

12870_2025_6134_MOESM3_ESM.xlsx

Supplementary Material 3: Table S3. List of differentially methylated m6A peaks in osmta1-1 seed embryos compared with the WT.

12870_2025_6134_MOESM4_ESM.xlsx

Supplementary Material 4: Table S4. List of differential methylated m6A peaks detected in osmta1-1 seeds compared with the WT after 60 h germination.

12870_2025_6134_MOESM5_ESM.xlsx

Supplementary Material 5: Table S5. List of hypomethylated genes detected both in seed embryos and 60 h germinating seeds of the osmta1-1 mutant.

12870_2025_6134_MOESM6_ESM.xlsx

Supplementary Material 6: Table S6. List of differential methylated m6A peaks detected in seeds of osmta1-1 mutant compared with the WT after 60 h germination under salt stress.

12870_2025_6134_MOESM7_ESM.xlsx

Supplementary Material 7: Table S7. List of differentially expressed genes in seeds of osmta1-1 mutant compared with the WT after 60 h germination under the control condition.

12870_2025_6134_MOESM8_ESM.xlsx

Supplementary Material 8: Table S8. List of differentially expressed genes in seeds of osmta1-1 mutant compared with the WT after 60 h germination under salt stress.

12870_2025_6134_MOESM9_ESM.xlsx

Supplementary Material 9: Table S9. Association analysis between differential m6A peaks and differentially expressed genes in seeds of osmta1-1 mutant compared with the WT after 60 h germination under the control condition.

12870_2025_6134_MOESM10_ESM.xlsx

Supplementary Material 10: Table S10. Association analysis between differential m6A peaks and differentially expressed genes in seeds of osmta1-1 mutant compared with the WT after 60 h germination under salt stress.

12870_2025_6134_MOESM11_ESM.jpg

Supplementary Material 11: Figure S1. CRISPR/Cas9 mutant of OsMTA1 and its phenotype. A, Construction of osmta1 using CRISPR/Cas9 editing; B, Phenotypes of the mutants (osmta1-1 and osmta1-2) and the wild type (WT).

12870_2025_6134_MOESM12_ESM.jpg

Supplementary Material 12: Figure S2. Validation of transcriptome data by quantitative real-time PCR. The left of the y-axis indicates relative expression of the candidate gene detected by quantitative real-time PCR, with Ct values normalized to the OsUBQ5 gene as the internal reference. Statistical significance was assessed using Student’s t-test (*** P < 0.001, ** P < 0.01) for comparisons between the osmta1-1 seeds and wild-type (WT) seeds. Three technical replicates were analyzed, and the standard error of the mean (SEM) was used to represent variability. The right of the y-axis shows the expression change (FC, fold change) in osmta1-1 seeds compared with WT detected by transcriptome sequencing analysis.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, Y., Yin, M., Wang, J. et al. Epitranscriptome profiles reveal participation of the RNA methyltransferase gene OsMTA1 in rice seed germination and salt stress response. BMC Plant Biol 25, 115 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-025-06134-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-025-06134-4

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us