Knockout of SlYTH2, encoding a YTH domain-containing protein, caused plant dwarfing, delayed fruit internal ripening, and increased seed abortion rate in tomato.

The m6A is one of the most abundant and widespread modifications in eukaryotic mRNAs, which regulates gene expression at the post-transcriptional level and plays key roles in many physiological processes. The YT521-B homologous (YTH) domain-containing proteins act as m6A readers to regulate m6A-RNA metabolism processes and mediate the various functions of m6A modification. Previously, we reported tomato contains 9 YTH genes, among which SlYTH2 is relatively highly expressed. This study reports the physiological functions of SlYTH2 in tomato. SlYTH2 protein is distributed in the nucleus and cytoplasm, and its three-dimensional structure is highly similar to human HsYTHDF1. SlYTH2 knockout through Crispr/Cas9 gene editing technology leaded to pleiotropic phenotypes, including dwarfing plant, delayed fruit internal ripening process, and increased seed abortion rate. The deletion of SlYTH2 gene increased the accumulation of endogenous ABA, decreased the content of endogenous GA3, and enhanced the sensitivity of seed germination to exogenous ABA and seedling growth to exogenous GA3. RNA-Seq data showed that the expression levels of multiple hormone-related genes were altered in SlYTH2 knockout line. These facts suggested SlYTH2 plays its physiological roles related to ABA, gibberellin and other hormones in tomato.

Tomato SlYTH1 encoding a putative RNA m6A reader affects plant growth and fruit shape.

N6-methyladenosine (m6A), the most abundant and common modification on eukaryotic mRNA, plays crucial roles in multiple biological processes through controlling endogenous gene activity in organisms. The m6A reader specifically recognizes the m6A mark to mediate the regulation of m6A on mRNA, and determines the fate of its target mRNA. In plants, the currently confirmed m6A readers are YTH (YT521B homology) domain-containing proteins. We previously reported that tomato contains 9 YTH genes, of which SlYTH1 has the strongest expression. The present study reports the functional characterization of SlYTH1 in tomato. SlYTH1 mutants generated via CRISPR/Cas9 technology exhibited pleiotropic phenotypes, including low seed germination rate, shortened seedling root, retarded plant growth and development during vegetative development, and elongated and longitudinally flattened fruit with reduced the locule number. SlYTH1 knockout reduced GA3 content and downregulated the expression of related genes in gibberellin biosynthesis pathway. Moreover, exogenous GA3 application could partially restore the phenotypic defects caused by SlYTH1 mutations. SlYTH1 knockout could alleviate the inhibition of seedling root elongation by exogenous GA3 application at relatively low concentration. These facts indicated SlYTH1 is involved in regulating gibberellin biosynthesis and plays important roles in multiple physiological processes in tomato.

Genome-wide identification and characterization of YTH domain-containing genes, encoding the m6A readers, and their expression in tomato.

KEY MESSAGE: 9 YTH genes in tomato were identified and cloned, and their expression patterns were comprehensively analyzed, which reveal potential multiple roles in development and fruit ripening. N6-methyladenosine (m6A) is an abundant and pervasive post-transcriptional modification in eukaryotic mRNAs. The YTH domain-containing proteins act as m6A readers to read m6A marks and transduce their downstream regulatory effects by altering m6A-mRNA metabolism processes. Identification of YTH proteins is essential for understanding the regulatory mechanisms of m6A in physiological processes, but little is known about YTH proteins in tomato, a model system for fruit development. Here, we report that tomato genomes contain a total of 9 SlYTH genes. While YTH proteins of both tomato and Arabidopsis can be classified into two subfamilies, the member distributions in subfamilies are very different between the two species. Homology modeling exhibited the similar three-dimensional structures of SlYTH proteins to human YTHDF1 or YTHDC1. Multiple hormone-response elements locating on the promoters of SlYTH genes indicate that they are involved in the physiological processes related to phytohormone. SlYTH genes are ubiquitous and spatiotemporal dynamic expression in tomato. Eight SlTYH genes have the strongest expression in stamens among the parts of flowers. Throughout fruit ontogeny, most of the SlYTH genes display obvious high mRNA levels during the developmental phases (4 dpa to mature green); moreover, SlYTH1 and SlYTH2 have absolute predominant expressions demonstrated by RNA-seq. The results lay a foundation for future characterizations on the functions of YTH proteins and m6A regulatory mechanism in tomato.