Increased de novo shoot regeneration efficiency in the met1-3 mutant. (A) Shoot regeneration phenotypes. (B) Number of regenerated shoots. In (A) and (B), calli preincubated for 7 days on CIM were transferred to SIM. The number of regenerated shoots from calli was measured (n > 30) at 3 weeks after incubation on SIM. Error bars indicate the SEM. Statistically significant differences between wild-type and mutant calli are indicated by asterisks (Student’s t-test, **P < 0.01). Scale bars = 10 mm.|@|~(^,^)~|@|Marginal changes in CG methylation during leaf-to-callus transition. (A) Fractions of methylated and unmethylated cytosines in the CG context in wild-type and met1-3. (B) Average CG methylation levels over genic regions in wild-type and met1-3. TSS, transcription start site; TTS, transcription termination site. (C) Hierarchical clustering of genome-wide CG methylation patterns. The color bar indicates the methylation level. (D) Venn diagram of regions hypomethylated in each tissue of met1-3 relative to that of the wild-type.|@|~(^,^)~|@|MET1-dependent CG methylation at the CRY loci. (A) CG methylation patterns at the chromatin of CRY1 and CRY2 genes. DNA methylation states and mRNA levels are depicted. (B) Transcript level of CRY1 in met1-3 determined by RT-qPCR. Three independent biological replicates were averaged. Asterisks indicate significant differences (Student’s t-test; *P < 0.05). DAC, days after incubation on CIM. (C and D) Shoot regeneration capacity of the cry1-1 mutant. Calli preincubated for 7 days on CIM were transferred to SIM. The number of regenerated shoots from calli was measured (n > 30) at 3 weeks after incubation on SIM. Statistically significant differences between wild-type and mutant calli are indicated by asterisks (Student’s t-test, *P < 0.05). Scale bars = 10 mm.|@|~(^,^)~|@|Potential connection of CRY1 and cytokinin signaling components. (A) Coexpression network of genes hypomethylated and up-regulated in met1-3. Cytokinin signaling genes are indicated by purple nodes. (B) Close neighbor genes of CRY1 in the coexpression subnetwork. Cytokinin signaling genes are indicated by purple nodes. (C and D) Transcript levels of type-B ARR genes in met1-3 (C) and cry1-1 (D) calli determined by RT-qPCR. Three independent biological replicates were averaged. Asterisks indicate significant differences (Student’s t-test; *P < 0.05).|@|~(^,^)~|@|The role of MET1 in de novo shoot regeneration. The CRY1 gene is silenced by MET1-dependent DNA methylation to ensure homeostasis of light signaling in wild-type leaves. In met1-3, the CG methylation-free CRY1 locus is transcriptionally activated, which stimulates the expression of cytokinin signaling genes, such as type-B ARRs. Increased cytokinin signaling promotes de novo shoot regeneration.

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