AGD1, a class 1 ARF-GAP, acts in common signaling pathways with phosphoinositide metabolism and the actin cytoskeleton in controlling Arabidopsis root hair polarity.

The Arabidopsis thaliana AGD1 gene encodes a class 1 adenosine diphosphate ribosylation factor-gtpase-activating protein (ARF-GAP). Previously, we found that agd1 mutants have root hairs that exhibit wavy growth and have two tips that originate from a single initiation point. To gain new insights into how AGD1 modulates root hair polarity we analyzed double mutants of agd1 and other loci involved in root hair development, and evaluated dynamics of various components of root hair tip growth in agd1 by live cell microscopy. Because AGD1 contains a phosphoinositide (PI) binding pleckstrin homology (PH) domain, we focused on genetic interactions between agd1 and root hair mutants altered in PI metabolism. Rhd4, which is knocked-out in a gene encoding a phosphatidylinositol-4-phosphate (PI-4P) phosphatase, was epistatic to agd1. In contrast, mutations to PIP5K3 and COW1, which encode a type B phosphatidylinositol-4-phosphate 5-kinase 3 and a phosphatidylinositol transfer protein, respectively, enhanced the root hair defects of agd1. Enhanced root hair defects were also observed in double mutants to AGD1 and ACT2, a root hair-expressed vegetative actin isoform. Consistent with our double-mutant studies, targeting of tip growth components involved in PI signaling (PI-4P), secretion (RABA4b) and actin regulation (ROP2), were altered in agd1 root hairs. Furthermore, tip cytosolic calcium ([Ca²⁺](cyt) ) oscillations were disrupted in root hairs of agd1. Taken together, our results indicate that AGD1 links PI signaling to cytoskeletal-, [Ca²⁺](cyt-) , ROP2-, and RABA4b-mediated root hair development.

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