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Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (41) and K+ (MRb) influx of corn root tissue over a wide range of K+ activity. N,N'Dicyclohexylcarbodimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K+ influx and depolarized 4 at low, but not at high, K+ activity (K°). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K+ influx over a wide range of K0, with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (4p) In ?revious papers we have examined the K+ dependence of the 'p in 4-h washed corn roots (11) and the relationship of potential to K4 influx (12). The membrane potential had an electrogenic component which was strongly dependent in magnitude on K°and which had a minimum in the range where 4 = EK (0.5-2 mM K°) (11). This was the same range in which the passive contribution to K+ influx became noticeable (12).In these experiments ip and K+ influx were inhibited by cutting off the energy supply through uncoupling or anoxia. If a membrane ATPase is involved as we (12, 29, 30) and others (19, 23, 31, 33, 35, 36) (31), to reduce the pH sensitivity of K+ influx in rat liver mitochondria (15), and to inhibit the H+ translocation associated with Cyt oxidase (9). Some caution is needed in interpreting DCCD responses as due to ATPase inhibition unless confirmed by other ATPase inhibitors.Oligomycin, on the other hand, is highly specific for the ATPase proton channel of mitochondrial, bacterial, and chloroplast membranes (25). It is also effective in blocking energy-linked ion uptake by higher plant cells (2, 21). Although oligomycin does not inhibit the K+ stimulated, Mg2+-requiring ATPase of isolated plasmalemma fractions (27, 28) histochemical staining indicates that it blocks both plasmalemma and mitochondrial ATPases in vivo (32).DES has been reported to be a much more effective inhibitor of the plasmalemma ATPase than of the mitochondrial ATPase (2). However, DES also inhibits oxidative phosphorylation (2). Severe membrane damage has been reported for both plant cells, in which DES causes rapid loss of previously acquired 'Rb label (3), and mitochondria, in which DES caused solubilization of 25-30%o of the mitochondrial protein and nearly complete release of bound Ca2`and Mg24 (8).In the work reported here, we find that all three inhibitors effectively reduce K+ influx and electrical potential at low K°(in the mechanism I range), but have smaller effects at higher K°(in the mechanism II range). A detailed examination of the action of DCCD shows that while mechanism I is nearly completely inhibited, as evidenced by the reduction of net H+ efflux, active K+ influx, and 4p, the electrogenic system of mechanism II is DCCD insensitive. Possibilities as to the nature of mechanism II are discussed briefly.Some of these data have been given in a preliminary report (10). MATERIALS AND ...

A comparison has been made of the effects of fusicoccin with those of washing on segments of corn (Zea mays L.) root tissue. Both fusicoccin and washing caused increases in K+(86Rb) influx, net H+ efflux, and electrogenic cell membrane potential, but with no effect on respiration rate. The similarity was most evident with fresh tissue during the initial phases of washing, prior to the developmental changes which augment the anion and general solute transport rates of the tissue. After the development of enhanced transport capacity the proportional response to fusicoccin was much diminished. It is suggested that the fusicoccin-like response to washing may be a manifestation of recovery from injury.There are three major, interrelated responses of plant tissue to the fungal phytotoxin FC2: enhancement of energy-linked net H+ efflux, K+ influx, and cell membrane potential (18). Fusicoccin also causes minor increases in anion, glucose, and amino acid uptake, but these are not generally considered to be direct responses. Marre (18) Root tissue of corn (4, 12, 18), barley (23, 24), and oat (3) manifest the major responses to FC. We have been intrigued by the observation that washing corn root segments produces responses which resemble those produced by FC: rapid increases in K+(m'Rb) influx (6, 22) net H+ efflux (this paper) and electrogenic cell membrane potential (15). In the study reported here we have compared the responses of corn root tissue to washing and FC. We have also examined whether the effectiveness of FC in stimulating H+ efflux, K+ influx, and electrogenic membrane potential is modified by washing.A preliminary report of this work has been given (10). MATERIALS AND METHODSMethods for raising corn seedlings (Zea mays L.), washing 0.5 to 2.5-cm root segments, and following ion influx were in general those described by Leonard and Hanson (14 (Fig. 1) or 50 ml where FC was used (Table I).FC was dissolved in absolute ethanol and added to the standard solution to give a final concentration of 10 ,UM. This concentration has been reported to elicit maximal stimulation of H+ efflux and hyperpolarization of cell membrane potential in corn (4, 12) and barley roots (24). Controls (no FC) contained equivalent ethanol (0.5%) which did not appreciably affect any of the metabolic parameters investigated in this study.Net H+ efflux (Fig. 2) was determined with 0.8 g of tissue in 200 ml of an aerated medium of 0.2 mm K-phosphate plus 0.2 mm CaSO4 at 30 C (pH 6.0). Root segments were washed in the same medium for the prescribed time and then transferred to fresh medium to determine H+ efflux. A Beckman Century SS pH meter was used to record solution pH. After transferring the tissue, 5 min were allowed for equilibration, after which the pH was adjusted to pH 6.0. After 30 min, the pH of the solution was backtitrated to pH 6.0 with 20 mM NaOH to determine net H+ efflux. The maximum pH change before back-titration was 0.15 pH unit.Simultaneous measurements of net H+ efflux and K+ ("6Rb) influx were made with 0...

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The adenine nucleotide content of the 2-centimeter segments excised from tray-grown corn (Zea mays L., WF9 x MoI7) roots declines for the first hour after excision. Concomitant with the loss of adenine nucleotides is a decline in respiration and a leakage of K+. With continued washing, these parameters partially or completely recover and increased phosphate influx develops. Increasing the wound effect by cutting 0.5-centimeter segments gives a more rapid and pronounced degradation of adenine nucleotides and slower recovery. Conversely, the mild injury caused by submerging intact roots induces less degradation and produces greater net adenine nucleotide synthesis during recovery, adding auxin to the washing medium produces a similar result. With all treatments, there is stabilization of energy charge at about 0.85.Brief submersion or rubbing of intact roots, as well as recutting washed and recovered root segments, will initiate the transient loss of adenine nucleotides but will not induce increased phosphate influx.It is suggested that the loss in adenine nucleotides may reflect homeostasis in energy charge via catabolism arising from membrane permeablity changes.Washing (or 'aging') of excised root tissue leads to increased ion influx (9, 10, 13, 16, 24), H+ efflux (13, 14), and electrogenic cell potential (14,20). To a large extent these changes reflect recovery from injury inflicted by cutting, cold shock, or handling the tissue (10, 13, 14). There are two phases to the washing response: an initial phase lasting about I h during which there is rapid (no lag) reinitiation of the H+ and K+ fluxes and cell potential, much as with fusicoccin but slower, followed by a general augmentation of solute transport, typified by increase in phosphate influx and microsomal K+-ATPase activity (14,16,17). These phases have been termed 'inductive ' and 'developmental' (14, 16).During the inductive phase, there is a decline in the level of ATP which recovers if washing is continued for 3 to 4 h (19). Similar changes in adenine nucleotides (and other metabolites) during washing or aging of excised plant tissue have been reported by other investigators (7,21,23,25). The phenomenon must reflect some important metabolic change in the cells which is linked to wounding or other injury, and in corn roots may have a role in inducing the developmental phase.In the study reported here, we have examined the changes in A preliminary report on part of this work has been given (12). MATERIALS AND METHODSPlant Material and Treatments. Corn seeds (Zea mays L., WF9 x Mol7) were germinated for 3 d in the dark at 28°C on paper towels saturated with 0.1 mm CaCl2, and 2-cm segments were excised 0.5 to 2.5 cm from the tip of the primary root as described (16). Segments were assayed for AdN or phosphate influx immediately after cutting, and after various periods of washing in aerated 0.2 mm CaC12 + 0.2 mm KH2PO4 (adjusted to pH 6.0) at 30°C (16). In some experiments, the 2-cm segment was additionally wounded by cutting into four 0.5-cm segme...