Knockdown of SlYTHDF2 Accelerates Dark–Induced Tomato Leaf Senescence by Affecting the ABA Pathway

N6-methyladenosine (m⁶A) is a widespread post-transcriptional modification in eukaryotic mRNAs. Proteins with the YTH structural domain act as m⁶A-binding proteins by recognizing the m⁶A modification and regulating mRNA through this recognition. In this study, <i>SlYTHDF2</i>, a prototypical m⁶A -binding protein gene in the YTH family was expressed in various tissues, and subcellular localization analyses indicated that the SlYTHDF2 protein was localized in the nucleus and cytoplasm. <i>SlYTHDF2</i> knockout lines were obtained using CRISPR/Cas9 technology and showed the senesced leaves prematurely increased endogenous ABA accumulation compared with the wild type. Moreover, we found that dark promoted leaf senescence in <i>SlYTHDF2</i> knockout lines and exogenous ABA further accelerated leaf senescence under dark conditions. The qRT-PCR analysis revealed significant alterations in the expression of genes associated with the ABA pathway. Relative to the wild type, the <i>CR-slythdf2</i> plants exhibited reduced levels of photosynthetic pigments, higher accumulation of reactive oxygen species, and increased damage to cell membranes. Additionally, we discovered that SlYTHDF2 interacts with the chloroplast-binding protein SlRBCS3 through yeast two-hybrid and BiFC experiments. Overall, our data suggest the important role of <i>SlYTHDF2</i> in regulating tomato leaf senescence.