Identification and Functional Exploration of the ALKBH Gene Family in Oriental Melon Fruit Ripening

Abstract

N6-methyladenosine (m⁶A) methylation functions as a vital post-transcriptional and epigenetic modification in higher plants regulated by α-ketoglutarate-dependent dioxygenases (ALKBH). However, the role of ALKBH genes in oriental melon (Cucumis melo L.) fruit ripening has not been explored. Therefore, we treated oriental melon with an exogenous m⁶A demethylase inhibitor (mechlorfenamic acid) then analyzed endogenous ethylene production and ripening-related indicators to explore the effects of m⁶A methylation on ripening. Bioinformatics and real-time quantitative PCR analyses were used to determine the impact of ALKBH genes on key ethylene synthesis gene expression. Treatment effectively inhibited endogenous ethylene production, firmness changes, and soluble solid contents, thereby extending fruit ripening. Eight ALKBH gene family members belonging to five major groups were identified in the melon genome. All members were expressed in ripening fruits, with different expression patterns during ripening. CmALKBH6, CmALKBH7, and CmALKBH8 expression was inhibited by an ethylene inhibitor (1-methylcyclopropene). The transient overexpression (OE) of CmALKBH8 in oriental melon led to the increased expression of the ethylene synthesis genes CmACS1, CmACS2, and CmACO1. In summary, the ethylene-regulated gene CmALKBH8 may participate in oriental melon fruit ripening regulation by modulating the methylation levels of ethylene synthesis-related genes. These findings help us better understand how m⁶A methylation regulates melon ripening.

Keywords: ALKBH; Cucumis melo; RNA methylation; ethylene synthesis; fruit ripening.

Figure 1

Effects of direct injection of meclofenamic acid on oriental melon fruit ripening. (A) Phenotypes of the oriental melon fruits after injection of MA. (B) The firmness, soluble solid content, and ethylene production of the oriental melon fruits were measured after injection. Data are represented as means ± standard deviation of three biological replicates (n = 6), and different lowercase letter indicate significant differences (Student’s test, p < 0.05).

Figure 2

The neighbor-joining (NJ) phylogenetic tree of ALKBH proteins from Arabidopsis thaliana, Solanum lycopersicum, Cucumis melo, Cucumis sativu, and Citrullus lanatus. Black stars represent Arabidopsis thaliana, the red, green, pink, blue circles represent Cucumis melo, Cucumis sativu, and Citrullus lanatus.

Figure 3

Distribution of conserved motifs and exon/intron structures in the predicted CmALKBH protein. Each motif is represented by a colored box. The length of box corresponds to the motif length. Red boxes represent exon, and green/blue lines represent upstream/downstream sequences.

Figure 4

Synteny analysis of ALKBH genes in Cucumis melo (Cme), Arabidopsis thaliana (At), Solanum lycopersicum (Sl), Cucumis sativus (Csa), and Citrullus lanatus (Cla). Red lines display the collinear ALKBH genes among four pairs of plant genomes. The light gray lines denote collinear blocks.

Figure 5

The statistics of cis-regulatory elements of each melon CmALKBH regulatory gene. The number represents the number of responsive elements in the promoter.

Figure 6

Effects of the 1-MCP treatment on the expression of the CmALKBH gene family during melon fruit storage. Values are shown as the means ± SE obtained from three independent experiments, and different lowercase letter above columns indicate significant differences (p ≤ 0.05).

Figure 7

LUC fluorescence signal detection of transient overexpression of CmALKBH8 in oriental fruit and expression analysis. (A) Photos of fruit fluorescence signals in different days after agrobacterium-mediated transient injection. (B) Expression levels of CmALKBH8 in overexpression and the control fruit. (C) Expression levels of CmACS in CmACO family gene expression heatmap in overexpression and the control fruit. From inside to outside, the heatmap represents the control fruit at 0 d after injection, the overexpression fruit at 0 d after injection, the control fruit at 6 d after injection, and the overexpression fruit at 6 d after injection. Values are shown as the means ± SE obtained from three independent experiments, and different lowercase letter above columns indicate significant differences (p ≤ 0.05).