SlMKK4 is responsible for pollen development in tomato.

The development of viable pollen is a determinant of male fertility and plays an essential role in the reproductive process of angiosperms. Mitogen-activated protein kinase (MAPK) cascades modulate diverse aspects of plant growth, but their involvement in post-meiotic pollen development is unclear. In this study, SlMKK4 was identified as a crucial regulator in overseeing pollen development in tomatoes (Solanum lycopersicum). Utilizing CRISPR-associated protein 9 to disrupt SlMKK4 resulted in an obvious decrease in pollen viability. The results of cell biology and transcriptomic analyses demonstrated that SlMKK4 specifically regulates auxin and sugar metabolism as well as signal transduction during post-meiotic pollen development. This is supported by the finding that protein-protein interaction assays and in vitro phosphorylation assays indicate that SlMKK4 serves as the upstream MAPKK for SlMPK20, which exhibits a distinct function in regulating the uninucleate (UN) to binucleate (BN) transition during microgametogenesis in tomatoes. Moreover, pollen from transgenic plants experienced significant arrest predominantly at the BN stage, accompanied by subcellular abnormalities manifesting during the late UN microspore phase. Furthermore, transcriptomic analyses indicated that SlMKK4 knockout remarkably downregulated the expression of numerous genes regulating auxin and sugar metabolism as well as signal transduction in anthers. Therefore, our findings suggest that SlMKK4 may serve as one of the upstream SlMAPKKs of SlMPK20 and also play a pivotal role in modulating post-meiotic pollen development in tomato plants.