Pollen-expressed methionine synthases in Nicotiana tabacum regulate pollen development and germination

Methionine synthases (MetEs) are essential for plant metabolism and are highly expressed during reproductive processes. Despite their importance, the subcellular localization of MetEs and their specific roles and regulatory mechanisms in plant reproduction remain unclear. To address these knowledge gaps, we conducted subcellular localization and phylogenetic analyses of MetEs in the model species tobacco (Nicotiana tabacum). Additionally, we explored their functions through morphological, metabolomic, and transcriptomic analyses. The results showed that six NtMetEs belonging to dicot subclade 2 (DS2) localized in the endoplasmic reticulum, nucleus and cytosol, while NsylMetE4 of DS1 with a transit peptide targeted to the cytosol and nucleus. Knockout of four NtMetEs abundantly expressed in anthers reduced the pollen germination ratio by an average of 45.15% and methionine (Met) and S-adenosylmethionine (Sam) levels by 32.55% and 47.58%, respectively. Exogenous Met and Sam partially restored pollen germination in mete1234, and Met mainly rescued germination of pollen with normal size both in vivo and in vitro. In mete1234 pollen, sucrose, starch, and energy metabolism were impaired, accompanied by reduced amyloplast accumulation and increased intine deposition. These results indicated that the localization of MetEs was diverse, and the transit peptide did not necessarily determine their plastid localization. NtMetE1-4 synergistically regulated pollen development and germination through modulating carbohydrate metabolism and amyloplast deposition. These findings expand our understanding of MetEs localization, Met synthesis, and pollen fertility regulation, offering implications for plant reproduction.