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 O₂ 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 O₂ 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 O₂. It is concluded that the basal H₂O₂ production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O₂^?-producing oxidase.