Effect of dichlorophenolindophenol, dichlorophenolindophenol-sulfonate, and cytochromec 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

Abstract

Excised roots from axenically grown sunflower seedlings reduced or oxidized exogenously added 2,6-dichlorophenolindophenol (DCIP), DCIP-sulfonate (DCIP-S), and cytochrome c, and affected simultaneous H+/K+ net fluxes. Experiments were performed with nonpretreated "living" and CN(-)-pretreated "poisoned" roots (control and CN(-)-roots). CN(-)-roots showed no H+/K+ net flux activity but still affected the redox state of the compounds tested. The hydrophobic 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 net H+ efflux ([symbol: see text]H+) equalled that of the roots without DCIP. The simultaneously measured K+ influx rate was first inhibited, then inverted into 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+ efflux and the transmembrane electron efflux caused by DCIP, which would depolarize the membrane and open outward K+ channels. The different H+ efflux kinetics characteristics, together with the small but significant DCIP reduction by CN(-)-roots were taken as evidence that an alternative CN(-)-resistant redox chain in the plasma membrane was involved in DCIP reduction. The hydrophilic electron acceptor DCIP-S enhanced both H+ and K+ flux rates by control roots. DCIP-S was not reduced, but slightly oxidized by control roots, after 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 [symbol: see text]H+ and [symbol: see text]K+, consistent with the involvement of the CN(-)-resistant redox chain. 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. In this case CN(-)-roots were less efficient, while control roots doubled the rate of cytochrome c oxidation by CN(-)-roots, after a 10 min lag in which cytochrome c was reduced at the same rate as the medium plus NADH did. CN(-)-roots seemed to have a fully activated CN(-)-resistant branch. The described effects on K+ flux were consistent with the current hypothesis that redox compounds changed the electric membrane potential (de- or hyperpolarization), which induces the opening of voltage-gated in- or outward K+ channels.

Parameters for Cellular Viability and Membrane Function in Chenopodium Cells Show a Specific Response of Extracellular pH to Heat Shock with Extreme Q10

“…DCPIP is a lipophilic compound and has access to electron carriers inside the membrane. Reduced DCPIP remains inside or attached to the membrane or may accumulate in the cell (Garrido et al, 2001). The increase in the reduction of DCPIP at 47 -50 8C may be explained by facilitated access to reducing compounds.…”

“…The biochemical responses of cells from Chenopodium rubrum to heat shock were investigated by measuring extracellular pH, osmolality, alkaline phosphatase, protein, betalain, and the reduction of MTT and DCPIP. The latter two are used as vitality markers: MTT is reduced in living cells by mitochondrial reductases (Bernas and Dobrucki, 2000) and DCPIP is reduced by plasmalemma electron transport chains (Garrido et al, 2001). The reduction of MTT and DCPIP was only inhibited by heat shock over 45 8C (Fig.…”

“…Plasma membrane reductase activity (Askerlund and Larsson, 1991;Garrido et al, 2001) was estimated photometrically at 595 nm in an assay containing 10 mM MES/NaOH pH 5.5, 70 μM DCPIP, and Chenopodium cells (20 mg FW) in 1 ml with an incubation time of 15 min at RT.…”

“…In addition to MTT, we tested the redox indicator DCPIP. DCPIP has been reported to be reduced by plasmalemma electron transport chains (Garrido et al, 2001). Thus, assays for the reduction of DCPIP can detect perturbations or damage to the cell membrane.…”

“…MTT is a membrane-permeable tetrazolium salt and can be reduced in living cells by mitochondrial reductases (Bernas and Dobrucki, 2000). DCPIP has been reported to be a substrate for plasmalemma electron transport chains (Garrido et al, 2001). Given these earlier findings, the aim of the present investigation was to study heat shock effects on plant cellular parameters, including pH and vitality.…”