Protein Phosphatase TOPP4 Regulates Pavement Cell Morphogenesis In Arabidopsis

In plants, cell morphogenesis is dependent on intercellular auxin accumulation. The polar subcellular localization of PIN-FORMED (PIN) is crucial for the fomation of auxin accumulation. Previous studies have shown that protein kinase PINOID (PID)and protein phosphatase 2A (PP2A) modulates polar localization of PIN proteins and auxin flux via regulating PIN phosphorylation status in an antagonistic manner. The polar localization of PIN 1 modulates leaf PC interdigitation, and PID- and FyPP1-(phytochrome-associated serine/threonine protein phosphatase) dependent PIN 1 phosphorylation affects PIN 1 polarity in this process. However, the underlying mechanisms of PIN 1 dephosphorylation in this process still remain unclear.In this study, we show a new mutant with pavement cell (PC) interdigitation defect by screening ethyl methane sulfonate (EMS)-mutagenized population.Map-based cloning revealed that a G to A single-nucleotide substitution in At2g39840(type one protein phosphatase) resulted in the conversion of threonine (Thr) to methionine (Met) in amino acid 246 near the C terminus of the protein. Therefore, the mutant was designated as topp4-1. Outlines of PCs from topp4-1 were smoother and much less wavy. The lobe number and length as well as the neck width of PCs in topp4-1 were dramatically reduced. Overexpression of TOPP4 in the topp4-1 mutant rescued the PC defects, and its overexpression in wild-type plants promoted PC interdigitated growth. Expression of topp4-1 in wild-type plants could recapitulate the PC defects of the topp4-1 mutant, suggesting that the mutant form of TOPP4 most likely affects PC development in a dominant-negative fashion.The topp4-1 mutant displays other auxin-related phenotypes (i.e., reduced apical dominance, increased number of branches and reduced root length), and exogenously applied auxin dose not rescue PC interdigitation defects. Genetic analyses suggested that TOPP4 and PIN1 likely function in the same pathway to regulate PC morphogenesis, and TOPP4 may act antagonistically with PID in this process.Furthermore, the subcellular co-localization of proteins, in vitro and in vivo protein interaction studies, and dephosphorylation assays revealed that TOPP4 can interact with PIN 1 at plasma membrane,and could dephosphorylate PIN 1. Moreover,analyses of the subcellular localization of PIN 1 in different tissues showed that TOPP4 mutation causes leads to changes in PIN1 polarity. Using live fluorescence microscopy, we observed that TOPP4 mutation decreased BFA-induced PIN internalization in PCs, while TOPP4 overexpression enhanced BFA sensitivity of PIN1 localization. At same time, the localization of Ara7-GFP (the endocytic marker Ara7) was affected in the topp4-1 mutant. Above results suggested that TOPP4 regulates PIN1 polar localization in PCs via affecting PIN1 endocytic trafficking. In addition, in the topp4-1 mutant, auxin-stimulated ROP2 (Rho of plant 2) activity was abolished and the localization of RIC1/4 (ROP-interactive C.RIB rmotif-containing protein 1/4) was compromised. This result suggests that TOPP4 affects cytoskeleton pattern through the ROP GTPase signaling pathway. Taken together, our study elucidated that TOPP4 regulats the mechanism of PC morphogenesis via PIN dephosphorylation in Arabidopsis.