To produce mature seed, flowering plants must undergo successful male and female gametogenesis and pollination followed by fruit set, growth, and ripening. This sequential process involves complex genetic programming and less understood epigenetic reprogramming. Here we report a previously unidentified CHROMOMETHYLASE3-directed epi-control in pollen mother cell (PMC)-to-microspore transition that determines male fertility to affect seed formation. We generated and characterized hairpin RNA-mediated RNAi and CRISPR/Cas9 transgenic tomato lines in which CHROMOMETHYLASE3 (CMT3) was either knockdown (KD) or knockout (KO). CHROMOMETHYLASE3 has pleiotropic effects on vegetative and reproductive growth, including leaf, flower, and seed development, besides its influence on tomato ripening and fruit size. However, CMT3 KD plants exhibited stronger effects than KO plants in terms of these vegetative and reproductive processes. Real-time quantitative PCR analysis suggested that genetic compensation might contribute to the less impact of KO plants on pollen and seed development. Integrated RNA-seq and whole-genome bisulfite sequencing reveal that CMT3 functions as an epi-switch via a self-feedback mechanism to modulate gene expression and governs early development of microspores from PMCs prior to the tetrad stage during microsporogenesis to microgametogenesis, possibly through the pectin catabolic process, to establish pollen fertility that affects seed production in tomato.

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