Auxin-stimulated NADH oxidase of the plasma membrane of soybean

The focus of this research was to describe and characterize a hormone-responsive NADH oxidase activity of the plasma membrane of plants. This was the first demonstration of a hormone-sensitive redox activity of the plasma membrane for plant cells. Plasma membrane vesicles were highly purified by aqueous polymer two-phase partition from the elongating region of soybean hypocotyls. The specific activity of NADH oxidase was 1-10 nmoles/min/mg protein and as NADH was oxidized the electrons were transferred to molecular oxygen with a ratio of NADH to oxygen of 2.5. The activity was stimulated 2-3 fold by auxins and only the growth-active auxins were effective. The stimulation was concentration dependent, with maxima at 10 $\mu$M for indole-3-acetic acid, 1 $\mu$M for 2,4-dichlorophenoxyacetic acid and 0.1 $\mu$M for $\alpha$-napthaleneacetic acid. The stimulation of NADH oxidase by auxins correlated strongly with stimulation of cell elongation. Actinomycin D and osmotic stress (sucrose-induced), both of which inhibited auxin-stimulated cell elongation, severely inhibited NADH oxidase activity in parallel. Plasma membrane vesicles isolated by two-phase partition were oriented predominantly with the cytoplasmic-side in. Mg$\sp{2+}$ATPase and NADH-ferricyanide oxido-reductase, two enzymes with active sites on the cytoplasmic side, were 75 and 90% latent respectively based on stimulation of enzyme activity by membrane permeabilizing concentrations of detergent. NADH oxidase was 53% latent in these vesicles indicating that the enzyme could accept electrons from NADH on either surface of the plasma membrane. Partial inactivation (75%) of NADH oxidase in sealed vesicles by trypsin revealed that the enzyme had an externally exposed region sensitive to proteolytic digestion. The proximity of NADH oxidase activity to the primary response site of auxin action was determined by sequentially purifying the NADH oxidase activity away from other plasma membrane components and monitoring the responsiveness of the activity to auxin. NADH oxidase retained the ability to be stimulated (3-5 fold) by auxin (1 $\mu$M, 2,4-D) through solubilization, and purification by both ion exchange and gel filtration chromatography. NADH oxidase activity was purified approximately 2000 fold over the starting homogenate and consistently revealed three protein bands (36, 52, 72 kD) when analyzed by SDS-PAGE. A model supported by these new findings involves NADH oxidase as a rate-limiting component of electron transport across the plasma membrane. Auxin might regulate elongation growth of plant cells by activating NADH oxidase to stimulate transmembrane electron flow and growth response.