Respiration of barley roots: Assessment of activity of the alternative path using SHAM

The activity of the alternative path of O₂ consumption in detached and intact roots of barley Hordeum distichum (L.) Lam. cv. Maris Mink] was determined by titration with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. In the absence of cyanide, only high concentrations were inhibititory (> 5 m M ). whilst in its presence low SHAM concentrations (2.5–5.0 m M ) gave maximum inhibition: the resulting ϱ V_alt plots were non-linear. A SHAM-stimulated peroxidase could readily be washed from these roots, but non-linearity cannot be explained in terms of SHAM-stimulation of this peroxidase as it is not active in the absence of an exogenous supply of NADH. In detached roots the degree of inhibition of respiration with 25 m M SHAM was nearly double the capacity of the alternative path (measured as the degree of inhibition by SHAM in the presence of cyanide), suggesting non-specific inhibition. Effects of SHAM on cytochrome path activity in intact roots were examined by reverse titration with cyanide in the presence and absence of SHAM. At 5 m M SHAM had no effect on the cytochrome path, but at 25 m M it inhibited. We conclude that the only factor causing non-linearity of ϱV_alt plots in barley roots is non-specific inhibition of the cytochrome path by high concentrations of SHAM; consequently only low concentrations of SHAM (2.5–5.0 m M ) are suitable for estimating alternative path activity in barley roots.