Fig. 4

m⁶A modification facilitates mRNA stability or translation of genes in the ABA pathway. a A brief model of the two core ABA signaling pathways in plants. b Integrated Genome Browser (IGB) tracks showing the distribution of m⁶A reads in transcripts of the 9-cis-epoxycarotenoid dioxygenase 5 (NCED5), putative ABA receptor (ABAR), and ABA-responsive element-binding protein 1 (AREB1). The hypermethylated m⁶A peaks (fold change ≥ 1.5; P value < 0.05) in fruit at the RS1 stage compared to those at the S6 stage are indicated by shadow boxes. S6, the growth stage 6; RS1, the ripening stage 1. Rep, replicate. c m⁶A enrichment for NCED5, ABAR, and AREB1 from m⁶A-seq data. d Validations of the m⁶A enrichment by m⁶A-immunoprecipitation (IP)-qPCR. e, f Transcript levels of NCED5, ABAR, and AREB1 determined by RNA-seq (e) and quantitative RT-PCR (f). FPKM, fragments per kilobase of exon per million mapped fragments. The ACTIN gene served as an internal control in f. g Schematic diagram of the expression cassettes used for mRNA stability assay. The intact (WT) or mutated (MU) CDS of NCED5, ABAR, and AREB1 were separately cloned into the pCambia2300 vector driven by the CaMV 35S promoter. The specific m⁶A sites (highlighted in red) identified in m⁶A-seq and validated by SELECT were mutated from adenosine (A) to guanine (G) using site-directed mutagenesis kit. h Determination of mRNA stability for NCED5, ABAR, and AREB1. The intact (WT) or mutated (MU) CDS were expressed in Nicotiana benthamiana leaves. After actinomycin D treatment at an indicated time point, the total RNAs were extracted and submitted to quantitative RT-PCR assay with the N. benthamiana ACTIN gene serving as an internal control. i m⁶A-IP-qPCR assay showing the relative m⁶A enrichment in the intact or mutated transcripts. j Brief workflow for the analysis of translation efficiency. k Translation efficiency of NCED5, ABAR, and AREB1. 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)