Advanced knowledge of messenger RNA (mRNA) N⁶-methyladenosine (m⁶A) and DNA N⁶-methyldeoxyadenosine (6 mA) redefine our understanding of these epigenetic modifications. Both m⁶A and 6mA carry important information for gene regulation, and the corresponding catalytic enzymes sometimes belong to the same gene family and need to be distinguished. However, a comprehensive analysis of the m⁶A gene family in tomato remains obscure. Here, 24 putative m⁶A genes and their family genes in tomato were identified and renamed according to BLASTP and phylogenetic analysis. Chromosomal location, synteny, phylogenetic, and structural analyses were performed, unravelling distinct evolutionary relationships between the MT-A70, ALKBH, and YTH protein families, respectively. Most of the 24 genes had extensive tissue expression, and 9 genes could be clustered in a similar expression trend. Besides, SlYTH1 and SlYTH3A showed a different expression pattern in leaf and fruit development. Additionally, qPCR data revealed the expression variation under multiple abiotic stresses, and LC-MS/MS determination exhibited that the cold stress decreased the level of N⁶ 2′-O dimethyladenosine (m⁶Am). Notably, the orthologs of newly identified single-strand DNA (ssDNA) 6mA writer–eraser–reader also existed in the tomato genome. Our study provides comprehensive information on m⁶A components and their family proteins in tomato and will facilitate further functional analysis of the tomato N⁶-methyladenosine modification genes.

ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N⁶-methyladenosine (m⁶A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B.

The tomato fruit is a complex organ and is composed of various structures from the inside out, such as columella, septum, and placenta. However, our understanding of the development and function of these internal structures remains limited. In this study, we identified a plant-specific YABBY protein, SlYABBY2a, in the tomato (Solanum lycopersicum). SlYABBY2a exhibits relatively high expression levels among the nine YABBY genes in tomatoes and shows specific expression in the septum of the fruit. Through the use of a gene-editing technique performed by CRISPR/Cas9, we noticed defects in septum development in the Slyabby2a mutant fruits, leading to the inward concavity of the fruit pericarp and delayed septum ripening. Notably, the expression levels of key genes involved in auxin (SlFZY4, SlFZY5, and SlFZY6) and ethylene (SlACS2) biosynthesis were significantly downregulated in the septum of the Slalkbh10b mutants. Furthermore, the promoter activity of SlYABBY2a was regulated by the ripening regulator, SlTAGL1, in vivo. In summary, these discoveries provide insights into the positive regulation of SlYABBY2a on septum development and ripening and furnish evidence of the coordinated regulation of the auxin and ethylene signaling pathways in the ripening process, which expands our comprehension of septum development in the internal structure of the fruit.