Effect of dichlorophenolindophenol, dichlorophenolindophenol-sulfonate, and cytochrome c on redox capacity and simultaneous net H+/K+ fluxes in aeroponically grown seedling roots of sunflower (Helianthus annuus L.): new evidence for a plasma membrane CN--resistant redox chain

Excised roots from axenically grown sunflower seedlings reduced or oxidized exogenously added 2,6-dichrorophenolindophenol (DCIP), DCIP-sulfonate (DCI P-S), and cytochrome c, and affected simultaneous H+/K+ net fluxes. Experim ents were performed with nonpretreated "living" and CN--pretreated "poisone d" roots (control and CN--roots). CN--roots showed no H+/K+ net flux activi ty but still affected the redox state of the compounds tested. The hydropho bic electron acceptor DCIP decreased the rate of H+ efflux in control roots with extension of the maximum rate and optimal pH ranges, then the total n et H+ efflux (integralH(+)) equalled that of the roots without DCIP. The si multaneously measured K+ influx rate was first inhibited, then inverted int o efflux, and finally influx recovered to low rates. This effect could not be due to uptake of the negatively charged DCIP, but due to the lower H+ af flux and the transmembrane electron efflux caused by DCIP,which would depol arize the membrane and open outward K+ channels. The different H+ efflux ki netics characteristics, together with the small but significant DCIP reduct ion by CN--roots were taken as evidence that an alternative CN--resistant r edox chain in the plasma membrane was involved in DCIP reduction. The hydro philic electron acceptor DCIP-S enhanced both H+ and K+ flux rates by contr ol roots. DCIP-S was not reduced, but slightly oxidized by control roots, a fter a lag, while CN--roots did not significantly oxidize or reduce DCIP-S. Perhaps the hydrophobic DCIP could have access to and drain electrons from an intermediate carrier deep inside the membrane, to which the hydrophilic DCIP-S could not penetrate. Also cytochrome c enhanced integralH(+) and in tegralK(+), consistent with the involvement of the CN--resistant redox chai n. Control roots did not reduce but oxidize cytochrome c after a 15 min lag , and CN--roots doubled the rate of cytochrome c oxidation without any lag. NADH in the medium spontaneously reduced cytochrome c, but control or CN-- roots oxidized cytochrome c, despite of the presence of NADH. Ln this case CN--roots were less efficient, while control roots doubled the rate of cyto chrome c oxidation by CN--roots, after a 10 min lag in which cytochrome c w as reduced at the same rate as the medium plus NADH did. CN--roots seemed t o have a fully activated CN resistant branch. The described effects on K+ f lux were consistent with the current hypothesis that redox compounds change d the electric membrane potential (de- or hyperpolarization), which induces the opening of voltage-gated in- or outward K+ channels.