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, SlYTHDF2, 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. SlYTHDF2 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 SlYTHDF2 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 CR-slythdf2 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 SlYTHDF2 in regulating tomato leaf senescence.