Coordination of m6A mRNA methylation and gene transcriptome in rice response to cadmium stress

Abstract

N⁶-methyladenosine (m⁶A) is the most prevalent internal modification present in the mRNAs of all higher eukaryotes. However, the role of the m⁶A methylomes in rice is still poorly understood. With the development of the MeRIP-seq technique, the in-depth identification of mRNAs with m⁶A modification has become feasible. A study suggested that m⁶A modification is crucial for posttranscriptional regulation related to Cd²⁺-induced malignant transformation, but the association between m⁶A modification in plants and Cd tolerance has not been reported. We investigated the m⁶A methylomes in the roots of a cadmium (Cd)-treated group and compared them with the roots in the control (CK) group by m⁶A sequencing of cv. 9311 and cv. Nipponbare (NIP) plants. The results indicated that Cd leads to an altered modification profile in 3,406 differential m⁶A peaks in cv. 9311 and 2,065 differential m⁶A peaks in cv. NIP. KEGG pathway analysis of the genes with differentially modified m⁶A peaks indicated that the "phenylalanine", "tyrosine and tryptophan biosynthesis", "glycine", "adherens junctions", "glycerophospholipid metabolism" and "threonine metabolism" signalling pathways may be associated with the abnormal root development of cv. 9311 rice due to exposure to Cd. The "arginine", "proline metabolism", "glycerolipid", and "protein processing in endoplasmic reticulum" metabolism pathways were significantly enriched in genes with differentially modified m⁶A peaks in cv. NIP. Unlike that in Arabidopsis, the m⁶A-modified nucleotide position on mRNAs (m⁶A peak) distribution in rice exhibited a preference towards both the stop codon and 3' untranslated regions (3' UTRs). These findings provide a resource for plant RNA epitranscriptomic studies and further increase our knowledge on the function of m⁶A modification in RNA in plants.

Keywords: Cadmium stress; N6-methyladenosine; Posttranscriptional regulation; Rice; Seedling.

Fig. 1

Phenotypes of cv. 9311 and cv. NIP under CK (control) and Cd (cadmium)groups. (A) The 3-day-old seedlings of cv. 9311 and cv. NIP under control condition and Cd stress conditions. (B) and (C) Comparison of root length, in 3-day-old seedlings of cv. 9311 and cv. NIP under under control condition and Cd stress conditions, respectively. Data are presented as means ± SE. n = 15. Statistical analysis was conducted using the Student’s t-test. *, P-value < 0.05; **, P-value < 0.01; ***, P value < 0.001. scale bar = 1 cm

Fig. 2

Overview of m⁶A methylome in rice. (A) Annotation of identified m⁶A high confident peaks (B)-(E) canonical RRACH and motif URUAY motif in four samples

Fig. 3

The m⁶A methylome changes under Cd stress in rice. (A) Genomic distribution of high confidience peaks (hcpeaks) in four groups. The reference genome is split into 100 kb bins and the frequencies of genes or peaks located in each 100 kb bin was counted and plot as line. The tracks are “gene (heatmap)”, “peaks in cv. 9311_cd”, “peaks in cv. 9311_ck”, “peaks in cv. NIP_cd” and “peaks in cv. NIP_ck” from inside outwards. (B) Comparison of high confidence peaks (hcpeaks) in 4 groups. (C) Cumulative distribution function of log2 peak intensity of m⁶A-modified sites under CK and Cd groups in cv. 9311 and cv. NIP

Fig. 4

Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of coding genes containing differentially enriched m⁶A hcpeaks in cv. 9311_CK vs. cv. 9311_Cd. (A) Volcano plot showing logFC against statistical significance. Hcpeaks with FDR < 0.05 are coloured red with select points at the extremities of the plot labelled for easier visualisation (top 10 DE peaks or logFC < -4 or logFC > 4) (B) Major gene ontology terms were significantly enriched for these genes. (C) Major Kyoto Encyclopedia terms were significantly enriched for these genes

Fig. 5

Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of coding genes containing differentially enriched m⁶A hcpeaks in cv. NIP_CK vs. cv. NIP_Cd. (A) Volcano plot showing logFC against statistical significance. Hcpeaks with FDR < 0.05 are coloured red with select points at the extremities of the plot labelled for easier visualisation (top 10 DE peaks or logFC < -4 or logFC > 4) (B) Major gene ontology terms were significantly enriched for these genes. (C) Major Kyoto Encyclopedia terms were significantly enriched for these genes

Fig. 6

Functional characteristics of differentially methylated peaks (DMPs) in the context of genic location in cv. 9311 and cv. NIP. (A) top 10 over-represented GO term in each category at their 5’-UTR in cv. 9311 (B) top 10 over-represented GO term in each category at their 3’-UTR in cv. 9311(C) top 10 over-represented GO term in each category at their 5’-UTR in cv. NIP(D) top 10 over-represented GO term in each category at their 3’-UTR in cv. NIP

Fig. 7

Compare the differential hcpeaks and DE genes. (A) comparison of genes overlapping with differential hcpeaks and DE genes in cv. 9311_cd vs. cv. 9311_ck (B) comparison of genes overlapping with differential hcpeaks and DE genes in cv. NIP _ cd vs. cv. NIP _ck

Fig. 8

The KEGG terms of differential m⁶A methylation genes in cv. NIP and cv. 9311. (A) the KEGG terms of common genes with differential m⁶A methylation in indica and japonica under cadmium stress (B) the KEGG terms of unique genes with differential m⁶A peaks in indica (C) the KEGG terms of unique genes with differential m⁶A peaks in japonica