Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H2O2 production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H2O2 production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H2O2 production. In the soybean root H2O2 production can be stimulated 10-fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase-catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O-2 to H2O2. Comparative measurements showed that the NADH-induced H2O2 production of the roots resembles the H2O2-forming activity of horseradish peroxidase with respect to NADH and O-2 concentration requirements and sensitivity to inhibition by KCN, NaN3, superoxide dismutase and catalase. NADH-induced H2O2 production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H2O2 production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH-induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O-2. It is concluded that the basal H2O2 production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O-2(-)-producing oxidase.