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Fig. 10 | Genome Biology

Fig. 10

From: RNA methylomes reveal the m6A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

Fig. 10

SlALKBH2 is necessary for normal tomato fruit ripening. a Genotyping of mutations mediated by CRISPR/Cas9 gene-editing system in slalkbh2-23, slalkbh2-25, and slalkbh2-28 mutants. Diagram showing the single guide RNAs (sgRNAs) containing different target sequences (T1, T2, and T3), which were designed to specifically target the exons of SlALKBH2. The red letters indicate the protospacer adjacent motif (PAM). The transgenic plants in the second generation were genotyped by sequencing genomic regions flanking the target sites. Red arrows indicate the editing sites. Two mutants (slalkbh2-23 and slalkbh2-28) have a homozygous 1-bp insertion, and one (slalkbh2-25) has a homozygous 5-bp deletion caused by target T2 in the fourth exon of SlALKBH2. b Ripening phenotype of slalkbh2 mutants. Fruit from wild-type (WT) and slalkbh2 mutants (slalkbh2-23, slalkbh2-25, and slalkbh2-28) at 39, 42, 47, and 52 days post-anthesis (DPA) are shown. c LC-MS/MS assay showing the amount of mRNA m6A in WT and slalkbh2 mutant fruit at 39 DPA. Data are presented as mean ± standard deviation (n = 3). d m6A-IP-PCR assay showing the relative m6A enrichment in SlDML2 mRNA in WT and slalkbh2 mutant fruit at 39 DPA. e SlDML2 gene expression in WT and slalkbh2 mutant fruit at 39 and 42 DPA. The ACTIN gene was used as an internal control. d, e Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (*P < 0.05, **P < 0.01; Student’s t test). f Model for the relationship between DNA methylation and m6A mRNA methylation in fruit ripening. DNA methylation negatively regulates SlALKBH2 to mediate overall m6A mRNA methylation. The m6A modification promotes SlDML2 mRNA decay, thereby affecting DNA methylation and fruit ripening

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