Fig. 6

m⁶A methyltransferases positively regulate the ripening of strawberry fruit. a Ripening phenotypes of MTA/MTB RNA interference (RNAi-MTA/RNAi-MTB) and overexpression (OE-MTA/OE-MTB) fruits. Strawberry fruit agroinfiltrated with empty plasmid were used as controls. The experiments were performed with more than three highly reproducible biological replicates and the representative results are presented. Scale bar = 1 cm. b Transcript levels of MTA and MTB, as well as the two important ripening genes CHS and PG1, in the RNAi and overexpression fruits determined by quantitative RT-PCR. The ACTIN gene served as an internal control. c LC-MS/MS assay revealing the changes in global m⁶A methylation levels in the MTA-silenced (RNAi-MTA) or MTA-overexpressed (OE-MTA) fruits. d RNA immunoprecipitation (RIP) assay revealing the binding of MTA protein to the transcripts of NCED5, ABAR, and AREB1. The protein-RNA complexes were extracted from strawberry fruit expressing the MTA-eGFP fusion protein and subjected to immunoprecipitation with anti-GFP monoclonal antibody or mouse IgG (negative control). e–h The changes in relative m⁶A enrichment (e), gene expression (f), mRNA stability (g), and translation efficiency (h) of NCED5, ABAR, and AREB1 in the MTA-silenced (RNAi-MTA) or MTA-overexpressed (OE-MTA) fruits. The relative m⁶A enrichment and gene expression were determined by m⁶A-IP-qPCR and quantitative RT-PCR, respectively. For mRNA stability assay, the total RNAs were extracted after actinomycin D treatment at an indicated time point and submitted to quantitative RT-PCR assay. Translation efficiency was expressed as the abundance ratio of mRNA in the polysomal RNA versus the total RNA. Data are presented as mean ± standard deviation (n = 3). Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test). NS, no significance