Ferricyanide Reduction by Guard Cell Protoplasts

Guard cell protoplasts of Commelina communis L. reduced exogenousferricyanide at pH values lower than 5?0; upon addition of NADH,reduction of ferricyanide by guard cell protoplasts was stimulatedover the pH range 4?0 to 9?0 with two peaks of activity at pH5?0 and between pH 8?0 and pH 9?0. Calcium chloride (1?0 molm^–3) and MgCl2 (1?0 mol m^–3) increased the NADH-stimulatedreduction of ferricyanide. Superoxide dismutase and cyanidehad little effect on the NADH-stimulated reduction of ferricyanideby guard cell protoplasts, but, salicylhydroxamic acid completelyinhibited this activity. The NADH-stimulated reduction of ferricyanidealso occurred in the cell-free supernatant. Horseradish peroxidasedid not reduce ferricyanide in the absence of NADH over a broadrange of pH (4?0 to 9?0). However, in the presence of NADH,horseradish peroxidase reduced ferricyanide over the pH range5?0 to 9?0 with maximal activity at pH 8?0. The NADH-stimulatedreduction of ferricyanide by horseradish peroxidase showed similarproperties to those observed with guard cell protoplasts. Mannitol,superoxide dismutase, and cyanide did not inhibit the NADH-stimulatedreduction of ferricyanide by horseradish peroxidase; SHAM, however,completely inhibited the reduction of ferricyanide by horseradishperoxidase. Catalase inhibited the NADH-stimulated reductionof ferricyanide by horseradish peroxidase by 20%, while absenceof oxygen in the assay medium stimulated this activity over60%. We propose that the reduction of ferricyanide in the presenceof NADH by guard cell protoplasts, can be explained in termsof peroxidase activity associated with the plasma membrane andsecreted to the extracellular medium. However, the capacityof guard cell protoplasts to reduce ferricyanide at acid pHvalues where little peroxidase activity occurs may indicatethe presence of a plasma membrane redox system in guard cellsof C. communis. Key words: Commelina, guard cell protoplasts, ferricyanide reduction, peroxidase, redox system