NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP ((R) under bar HO (o) under barf (p) under bar lants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.