Reversible phosphorylation of NPH3/RPT2-like proteins regulates phototropin receptor signaling

Phototropin receptor kinases (phot1 and phot2) enhance photosynthesis by coordinating light responses such as phototropism and chloroplast repositioning under low blue light conditions. These processes rely on NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)/ROOT PHOTOTROPISM 2-like (NRL) proteins. The Arabidopsis (Arabidopsis thaliana) NRL protein NPH3 is phosphorylated by phot1 at S744, a modification that alters its localization and promotes hypocotyl phototropism. Although reversible phosphorylation of S744 is necessary for reassembling an active phot1-NPH3 complex at the plasma membrane, the mechanisms driving NPH3 dephosphorylation remain unclear. Here, we show that clade L TYPE 2C PROTEIN PHOSPHATASES (PP2C19 and PP2C35) regulate S744 phosphorylation and NPH3 function. Mutants lacking PP2C19 exhibit sustained S744 phosphorylation and reduced phototropism, with greater defects observed in pp2c19 pp2c35 double mutants, indicating functional redundancy between these phosphatases. Moreover, clade L pp2c mutants display altered RPT2 phosphorylation and abundance and modulate the phosphorylation status of NRL PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1), suggesting broader regulatory control over phot1-NRL targets. Consistent with this, phototropin-dependent chloroplast accumulation is impaired in both Arabidopsis and Marchantia pp2c19 mutants. These findings identify clade L PP2Cs as key regulators of auxin-dependent (phototropism) and auxin-independent (chloroplast accumulation movement) light responses, which, together with their additional role in regulating hypocotyl gravitropism, are particularly important for seedling emergence and establishment from beneath the soil surface.