Three PP2C phosphatases modulate FERONIA receptor kinase activity to regulate pollen–stigma interaction in Arabidopsis

Pollen hydration represents the initial and critical step in pollen–stigma interactions and is necessary for successful plant fertilization. The FERONIA (FER) receptor kinase regulates pollen hydration by modulating stigmatic reactive oxygen species (ROS) accumulation through rapid alkalinization factor 23/33 (RALF23/33) and pollen coat protein B-class peptide (PCP-B) signaling. However, the function and regulatory mechanism of FER’s receptor kinase activity in pollen hydration remain poorly understood. In this study, we found that the kinase-dead form of FER^K565R fails to restore stigmatic ROS accumulation and pollen hydration in the fer-4 mutant. By integrating RNA sequencing database analyses with yeast two-hybrid assays, we identified three type 2C phosphatases (PP2Cs)—protein phosphatase 2C clade H 1 (PP2CH1) and clade-E Growth-Regulating 1 and 2 (EGR1 and EGR2)—that interact with FER at the plasma membrane. These PP2Cs dephosphorylate FER at Ser695 and Thr696 within the activation segment, thereby inhibiting its kinase activity. Mutations at these two residues reduced ROS levels in the stigma and increased pollen hydration rates. Altogether, this study reveals a crucial regulatory mechanism of FER signaling, demonstrating that PP2CH1, EGR1, and EGR2 act as negative regulators of FER kinase activity to modulate stigmatic ROS accumulation and promote pollen hydration.