Evidence for a specific and critical role of mitogen‐activated protein kinase 20 in uni‐to‐binucleate transition of microgametogenesis in tomato

Summary Mitogen‐activated protein kinases ( MAPK s) regulate diverse aspects of plant growth. However, their potential role in reproductive development remains elusive. Here, we discovered an unique role of Sl MPK 20, a plant‐specific group D MAPK , in pollen development in tomato. RNA i‐mediated suppression of Sl MPK 20 or its knockout using CRISPR /Cas9 significantly reduced or completely abolished pollen viability, respectively, with no effects on maternal fertility. Cell biology and gene expression analyses established that Sl MPK 20 exerts its role specifically at the uni‐to‐binucleate transition during microgametogenesis. This assertion is based on the findings that the transgenic pollen was largely arrested at the binucleate stage with the appearance of subcellular abnormality at the middle uninucleate microspore stage; and Sl MPK 20 mRNA and Sl MPK 20‐ GUS signals were localized in the tetrads, uninuclear microspores and binuclear pollen grains but not in microspore mother cells or mature pollen grains. Transcriptomic and proteomic analyses revealed that knockout of Sl MPK 20 significantly reduced the expression of a large number of genes controlling sugar and auxin metabolism and signaling in anthers. Finally, protein–protein interaction assays identified Sl MYB 32 as a putative target protein of Sl MPK 20. We conclude that Sl MPK 20 specifically regulates post‐meiotic pollen development through modulating sugar and auxin metabolism and signaling.