Measurement of the activity and capacity of the alternative pathway in intact plant tissues: Identification of problems and possible solutions

Møller, I. M., Bérczi, A., van der Plas, L. H. W. and Lambers, H. 1988. Measurement of the activity and capacity of the alternative pathway in intact plant tissues: Identification of problems and possible solutions. ‐ Physiol. Plant. 72: 642–649. The cyanide‐insensitive, benzhydroxamic acid‐sensitive (e.g. salicylhydroxamic acid, SHAM) alternative pathway is located in the inner membrane of plant mitochondria and electron flow through it is not coupled to H⁺ pumping and ATP synthesis. When estimating the activity and capacity of the alternative pathway in intact plant tissues three main problems arise: 1) There is almost always a substantial (10–50%) KCN‐insensitive, SHAM‐insensitive residual respiration, which may be due to peroxisomal a‐oxidation of fatty acids, and which must be subtracted from all data in the further analyses. 2) There is a (KCN‐sensitive) peroxidase in many tissues that is stimulated by low SHAM concentrations (1–10 mAf), but inhibited at higher concentrations (15–50 mM). 3) High concentrations of SHAM may inhibit the cytochrome pathway. Means of identifying and alleviating these problems are presented. Provided experimental conditions are chosen such as to minimize the three problems for each new plant organ or species or each new growth condition, SHAM can be used to estimate the size of the alternatively pathway in vivo.