Redox activity and peroxidase activity associated with the plasma membrane of guard-cell protoplasts

Redox systems have been reported in the plasma membrane of numerous cell types and in cells from various species of higher plant. A search for a redox system in the plasma membrane of guard cells was therefore made in efforts to explain how blue light stimulates stomatal opening, a process which is coupled to guard cell H⁺ efflux and K⁺ uptake. The rates of O₂ uptake by intact guard-cell protoplasts (GCP) of Commelina communis L., in the dark, were monitored in the presence of NAD(P)H since the stimulation of O₂ consumption by reduced pyridine nucleotides is used as an indicator of the presence of a redox system in the plasma membrane. Oxygen consumption by intact GCP increased two- to threefold in the presence of NAD(P)H. The NAD(P)H-stimulation of O₂ uptake was dependent on Mn²⁺ and was stimulated 10- to 15-fold by salicylhydroxamic acid (SHAM). Catalase, cyanide and ascorbate, a superoxide scavenger, all individually inhibited the SHAM-stimulated O₂ uptake. These are all characteristics of peroxidase activity although some of these features have been used to imply the presence of a redox system located in the plasma membrane. High levels of peroxidase activity (using guaiacol as a substrate) were also detected in the GCP and in the supernatant. The activity in the supernatant increased with time indicating that peroxidase was being excreted by the protoplasts. The properties of O₂ uptake by the incubation medium after separation from the protoplasts were similar to those of the protoplast suspension. It is concluded that our observations can be more readily explained by peroxidase activity associated with the plasma membrane and secreted by the GCP than by the presence of a redox system in the plasma membrane of the protoplasts.Abbreviations EDTA ethylenediaminetetraacetic acid - GCP guard cell protoplast - Mes 2-(N-morpholino)ethanesulphonic acid - SHAM salicylhydroxamic acid