RabA4b is a RabGTPase which localizes to the tips of growing root hairs, where it may function in the membrane trafficking steps required for polarized secretion.
While EYFP-RabA4b is localized consistently to the tips of growing root hairs over time (A.), this consistent localization is lost in the root hair development mutant rhd4-1, a mutant which forms short, bulged root hairs. In rhd4-1, EYFP-RabA4b tip localization is correlated with tip growth, whereas a loss of EYFP-RabA4b is observed during bulge formation (B.). The gene responsible for the rhd4-1 phenotype was identified using a map based cloning approach, and was found to encode a Sac1p-like phosphoinositide phosphatase.
In order to determine whether the rhd4-1 mutation resulted in abnormal phosphoinositide levels, we used 3H-inositol labeling of wild type and rhd4-1 plants to quantify phosphoinositides in root tissue, and we found that rhd4-1 roots accumulates PI(4)P (C.). Additionally, using an antibody specific for PI(4)P, we see that PI(4)P is localized to the tips of wild type root hairs, but that PI(4)P is greatly accumulated in rhd4-1 root hairs (D.). We then created transgenic plants expressing a native promoter-YFP-RHD4 construct, and we see that RHD4 is localized to the tips of root hairs (E.). These results help us to construct a model (F.), suggesting that PI(4)P is important for maintaining proper polarized secretion; in wild type RHD4 balances levels of PI4K-?1 generated PI(4)P at the root hair tip whereas in rhd4-1, a loss of RHD4 phosphatase activity results in increased PI(4)P and therefore increased vesicle fusion events, ultimately causing wider, bulging root hairs.
