ALKBH10B, an mRNA m6A Demethylase, Modulates ABA Response During Seed Germination in Arabidopsis

Abstract

As the most abundant and reversible chemical modification in eukaryotic mRNA, the epitranscriptomic mark N ⁶-methyladenine (m⁶A) regulates plant development and stress response. We have previously characterized that ALKBH10B is an Arabidopsis mRNA m⁶A demethylase and regulates floral transition. However, it is unclear whether ALKBH10B plays a role in abiotic stress response. Here, we found that the expression of ALKBH10B is increased in response to abscisic acid (ABA), osmotic, and salt stress. The alkbh10b mutants showed hypersensitive to ABA, osmotic, and salt stress during seed germination. Transcriptome analysis revealed that the expression of several ABA response genes is upregulated in alkbh10b-1 than that of wild type, indicating ALKBH10B negatively affects the ABA signaling. Furthermore, m⁶A sequencing showed that ABA signaling genes, including PYR1, PYL7, PYL9, ABI1, and SnRK2.2 are m⁶A hypermethylated in alkbh10b-1 after ABA treatment. Taken together, our work demonstrated that ALKBH10B negatively modulates ABA response during seed germination in Arabidopsis.

Keywords: ABA; ALKBH10B; Arabidopsis; N6-methyladenine; osmotic stress; seed germination.

FIGURE 1

The expression of ALKBH10B is induced by virous external stimuli. (A,B) ALKBH10B expression in Col-0 plants in response to osmotic stress, salt stress, ABA, and MeJA. Seven-day-old seedlings were transferred to solid 1/2 MS medium, medium with 300 mM mannitol or 150 mM NaCl, or liquid 1/2 MS medium, medium with 50 μM ABA or 50 μM MeJA, respectively. After treated with the indicated time, seedlings were harvested followed by RNA extraction and cDNA synthesis for qRT-PCR. Asterisks indicate statistically significant differences as compared to the mock of every time-point (*P < 0.05; **P < 0.01; Student’s t-test). (C) ALKBH10B expression in abi1-1 mutant in response to ABA and mannitol. Seven-day-old seedlings were transferred to solid 1/2 MS medium or medium containing 50 μM ABA or 300 mM mannitol for 24 h, respectively. Then seedlings were harvested followed by qRT-PCR. The transcript level of ALKBH10B were normalized to the At2G28390 expression. Error bars represent the standard deviation of three replicates. Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01; Student’s t-test).

FIGURE 2

ALKBH10B modulates ABA sensitivity during seed germination. The germination rates of indicated genotypes were recorded from 1–6 days after stratification in the presence of various ABA contents (0, 0.5, or 1.0 μM). Cotyledon-greening percentages of the 10th day were recorded. Three independent experiments were conducted, with at least 100 seeds per genotype in each replicate. Error bars represent the standard deviation of three replicates. Asterisks indicate statistically significant differences as compared to the mock of every time-point (*P < 0.05; **P < 0.01; Student’s t-test). (A) Photographs of seedings grown on different media at day 6 after stratification. (B) Seed germination rates of indicated genotypes grown on different medium. (C) Green cotyledon ratio at day 10 after stratification.

FIGURE 3

ALKBH10B modulates osmotic and salt stress response during seed germination. The germination rates of indicated genotypes were recorded from 1–6 days after stratification in the presence of 300 mM mannitol or 150 mM NaCl. Cotyledon-greening percentages of the 8th day were recorded. Three independent experiments were conducted, with at least 100 seeds per genotype in each replicate. Error bars represent the standard deviation of three replicates. Asterisks indicate statistically significant differences as compared to the mock of every time-point (*P < 0.05; **P < 0.01; Student’s t-test). (A) Photographs of seedings grown on different media at day 6 after stratification. (B) Seed germination rates of indicated genotypes grown on different medium. (C) Green cotyledon ratio at day 8 after stratification.

FIGURE 4

Differential expression genes identified in alkbh10b-1 compared with Col-0. (A) Volcano plots showing the differentially expressed genes identified in alkbh10b-1. (B) Heatmap plots showing the normalized RPKM of differentially expressed genes in alkbh10b-1. (C) Biological process enrichment analysis of the upregulated and downregulated genes in alkbh10b-1.

FIGURE 5

ALKBH10B regulates the expression of ABA response genes. The expression patterns of ABA signaling-related ABI3, ABI5, and RD29B, and embryogenesis-related EM1 and EM6 were determined in seven-day-old seedlings treated with 50 μM ABA for 3 h. The transcript level of each gene was normalized to that of At2G28390, and the relative level of each gene in mock-treated Col-0 was set to 1. Values are mean ± SD of three replications. Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01; Student’s t-test).

FIGURE 6

Characterization of m⁶A modification in Col-0 and alkbh10b-1 under ABA treatment. (A) Overlap of two biological replicates of m⁶A enriched peaks in alkbh10b-1. (B) Overlap of two biological replicates of m⁶A enriched peaks in Col-0. (C) Volcano plot showing the hypermethylated m⁶A peaks identified in alkbh10b-1 compared with Col-0. (D) Pie chart depicting RNA types of hypermethylated m⁶A peaks in alkbh10b-1. (E) Metagene profile showing the density of hypermethylated m⁶A peaks across the transcript. (F) Pie chart depicting the fraction of the hypermethylated m⁶A peaks in the five non-overlapping transcript segments [5′UTR, start codon, coding sequence (CDS), stop codon and 3′UTR]. (G) Biological process enrichment analysis of genes with hypermethylated m⁶A peaks. (H) Pathway analysis of genes with hypermethylated m⁶A peaks.

FIGURE 7

Integrative genomics viewer (IGV) tracks showing the hypermethylated m⁶A peaks of several ABA signaling genes identified in alkbh10b-1.