Jul 2, 1986 - ELEANOR NEUFELD2 AND ALAN W. BowN*. Department ..... LEONG T, WR BRIGGS 1981 Partial purification and characterization ofa blue.
Plant Physiol. (1987) 83, 895-899 0032-0889/87/83/0895/05/$0 1.00/0
A Plasmamembrane Redox System and Proton Transport in Isolated Mesophyll Cells' Received for publication July 2, 1986 and in revised form November 11, 1986
ELEANOR NEUFELD2 AND ALAN W. BowN*
Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3AI ABSTRACT Potassium ferricyanide (K3FeICNIe) was added to aerated and stirred nonbuffered suspensions of mechanically isolated photosynthetically competentAsparagussprenger Regel mesophyll cells. Rates of Fe(CN)63 reduction and H' efflux were measured with or without illumination. On the addition of I millimolar Fe(CN)*3 to nonilluminated cell suspensions acidification of the medium indicated an H' efflux of 1.54 nanomoles H'/10' cells per minute. Simultaneous Fe(CN)e- reduction occurred at a rate of 1.55 nanomoles Fe(CN),'/10' cells per minute. Illumination stimulated these rates 14 to 17 times and corresponding values were 26.1 nanomoles H'/10' cells per minute and 22.9 nanomoles Fe(CN)*3/10' cells per minute. These two processes appeared to be tightly coupled and were rapidly inhibited when illuminated suspensions were transferred to darkness or treated with 1 micromolar 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Addition of 0.1 millimolar diethylstilbestrol eliminated ATP dependent H' efflux in illuminated or nonilluminated cells but had no influence on Fe(CN),> dependent H' efflux. Recent reports indicate that a transmembrane redox system spans the plasma membrane of root cells and is coupled to the efflux of H'. The present report extends these observations to photosynthetically competent mesophyUl cells. The results indicate a transport process independent of ATP driven H' efflux which operates with a H+/e- stoichiometry of one.
Substantial evidence indicates that a plasma membrane located, ATP requiring outwardly directed proton pump establishes a proton electrochemical gradient that is used to drive the transport of solutes into and out of plant cells ( 17, 23, 24). However, a proton electrochemical gradient across biological membranes may also be established by redox reactions involving the sequential operation of hydrogen and electron carriers (7, 14). Electrogenic redox driven proton movements across chloroplast and mitochondrial membranes have been well characterized and do not require ATP. Recent work with root tissue suggests that plant cells may have a plasma membrane located redox chain that establishes proton gradients. Reduction of exogenous Fe(CN)63- and an associated apparent net proton efflux have been demonstrated with corn roots (9, 12, 18), bean roots (21), and oat roots (19). The decline in endogenous NADPH levels when bean roots or corn roots are exposed to Fe(CN)63- suggests that NADPH is the physiological reductant (16, 22). The physiological oxidant may be ferric salt (21, 22) or 02 (7, 14). Oxidoreductase activity has been detected 'Supported by a grant from the Natural Sciences and Engineering Research Council of Canada. 2Prmnt address: Health Sciences Centre, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4NI 895
in plasma membrane enriched fractions from root cells (9, 19) and hypocotyl segments (8), and evidence for a flavoprotein Cyt complex in the plasma membrane has been obtained (13, 25). Thus H4 efflux may, to some extent, be driven by a plasma membrane located redox system which utilizes reducing equivalents from NADPH and is independent of ATP energy. The temporal relationships and stoichiometry between Fe(CN)63- reduction and Fe(CN)63- dependent H4 efflux are not clear. Work with Elodea leaves and carrot and yeast cells indicates a H+/e- ratio very close to one suggesting an electroneutral redox driven transport process (7, 1 1). Work with iron-deficient bean roots indicates a H+/e- ratio of 0.5 indicating a redox process which involves the net export of negative charge (21). This model would explain the rapid depolarization observed when bean roots and Elodea leaf cells are exposed to Fe(CN)62 (1 1, 21) and also the Fe(CN)63- stimulation of K4 efflux (12, 21). A third model has recently been proposed in which plasma membrane redox activity transports only electrons to an exogenous acceptor. The resulting depolarization then activates the proton ATPase and accounts for the increase in H4 efflux observed on addition of Fe(CN)631 (18). Very little is known concerning the existence of plasma membrane redox activities in photosynthetic cells. The reduction of exogenous Fe(CN)63- by Elodea leaf cells and an associated decline in membrane potentials has been reported (1 1). Active ATP driven proton efflux and related transport processes are currently being investigated using isolated Asparagus mesophyll cells (3, 5, 20). It is not clear whether redox contributes to H+ efflux from these cells. In the present study, illuminated or nonilluminated suspensions of photosynthetically competent Asparagus mesophyll cells (6) were exposed to Fe(CN)63'. The objectives were to determine (a) the influence of ferricyanide, if any, on net H+ efflux; (b) the rate, if any, of ferricyanide reduction; (c) the stoichiometry and temporal relationships between these two processes; and (d) the relationship, if any, between redox dependent H+ efflux and ATP dependent H4 efflux.
MATERIALS AND METHODS Asparagus sprengeri Regel was grown under greenhouse conditions. Mesophyll cells were isolated from cladophylls using a technique of gentle mechanical disruptions (6). Cell numbers were measured using a light microscope and hemocytometer. From 50 to 100 x 106 cells were prepared daily. Net H4 efflux was normally measured using a recording pH meter as described previously (5, 20). Rates were calculated as nmol H4/106 cells-min after determination of the rate of pH decline and the buffering capacity of the cell suspension medium. Cells were incubated at 30°C in 10 or 20 ml of 1 mm CaSO4. When required the rate of pH change due to Fe(CN)63--dependent acidification was slowed by suspending the cells in 1 mm CaSO4 and 3 mM Mes (Ca[OH]2) buffer (pH 6.2). In addition,
NEUFELD AND BOWN
when Fe(CN)63--independent H+efflux was studied, this rate was stimulated by suspending the cells in 5 mM KCl, 5 mM NaCl, and 0.2 mm CaC12. These concentrations of Na+and K+stimulate Fe(CN)63 independent H+ efflux. Net H+ efflux was also measured using a recording pH stat apparatus as described previously (3). The pH-stat technique was used to determine the effect of Fe(CN)63- concentration on the rate of Fe(CN)63--dependent acidification in illuminated and nonilluminated cell suspensions. Rates of Fe(CN)63- reduction were determined spectrophotometrically. Stock solutions of 100 mm K3Fe(CN)6 were prepared daily and Fe(CN)63- was added to the cell suspension medium to give a final concentration of 1.0 mM. At zero time and subsequent 5 min intervals 2.5 ml aliquots of the cell suspension were removed and centrifuged in a clinical centrifuge to sediment the cells. The absorbance of the supernatant fluid was measured at 420 nm. Using an extinction coefficient of 1.0 mm-' cm-', the rate of reduction was calculated as nmol Fe(CN)63- cells. min. Alternatively, the rate of Fe(CN)63- reduction was determined by measuring Fe(CN)64- spectrophotometrically at 535 nm (1). Fe(CN)64- production was calculated as nmol Fe(CN)64 produced/106 cell min using an extinction coefficient of 10.8 mm-1 cm-'. The rate of Fe(CN)64- production was found to be equivalent to the rate of Fe(CN)63- loss from the medium. Cell suspensions were illuminated with a 300 W reflector lamp (Sylvania) which gave an irradiance at the surface of the vessel of 4.6 x 10-4 mol* m2 * s'. Stock solutions of DCMU and DES3 were made up in 80% (v/v) ethanol. All concentrations quoted are final values after dilution with the cell suspension.
RESULTS When cells were suspended in 1 mM CaSO4 in the absence of Na+ or K+ ions, H+ efflux was not observed. On addition of 1 mM K3Fe(CN)6 to illuminated cells, acidification of the medium rapidly ensued at a rate which was too fast to measure accurately (Fig. 1). After approximately 5 min during which the pH dropped from about 6.1 to 4.7 acidification ceased. Inhibition of acidification was due to the decline in the pH since weak buffers allowed the acidification to continue for 30 min or more and back titration with alkali to the original pH resulted in the resumption of acidification. Fe(CN)63- addition to nonilluminated cells resulted in a much lower measurable rate of acidification, which increased rapidly with the onset of illumination (Fig. 1). An objective measure of the delay before acidification was obtained by measuring the time between addition of Fe(CN)63- and the point at which an extrapolation forward of the initial rate intersected with the extrapolation backwards of the stimulated rate. In the light, Fe(CN)63--dependent acidification commenced within 4 (±3) s; in the dark, the lag period was 1 1 (±4) s. This longer lag period may be only apparent and result from lower rates of acidification in the dark (Fig. 1). The rapid response to Fe(CN)62- indicates that acidification does not result from a process in which it is taken up into the cell prior to the release of protons to the external medium. With a pH-stat apparatus and illuminated or nonilluminated cells, initial acidification rates were characterized as a function of Fe(CN)63 concentration at a constant pH of 6.2. In both cases, the acidification response appeared to saturate at a concentration of approximately 1 mm. However, rates were approximately 15 times greater in illuminated cells. In nonilluminated cells 1 mm Fe(CN)631 resulted in mean rates of 1.2 nmol H+/ 106 cells min, whereas in illuminated cells the corresponding value was 18 nmol H+/106 cells.min. (Fig. 2). In subsequent experiments, 1 mM Fe(CN)63- was routinely used in conjunction with pH versus time measurements. Unlike the pH stat apparatus, this technique allowed measurement of both acidification and alkalinization 'Abbreviation: DES, diethylstilbestrol.
Plant Physiol. Vol. 83, 1987
responses within one experiment. The relationship between Fe(CN)63 stimulated acidification and Fe(CN)63- reduction was investigated. Consecutive measurements of reduction and acidification were made over 30 min periods using identical conditions and the same cell preparation. Mes buffer pH 6.2 was used to reduce the rate of pH decline. With illuminated cells, Fe(CN)63- concentrations were measured at 420 nm; without illumination, reduction rates were considerably slower and were obtained using the more sensitive Fe(CN)64determination method. Both the decline in Fe(CN)63- and the increase in Fe(CN)64- appeared approximately linear over 30 min (data not shown). From the absorbance changes, rates of reduction in nmol/106 cells min were calculated and compared to the comparable acidification rates (Table I). The data demonstrate that the rates of acidification and reduction were 14 to 17 times greater in the presence of illumination. However, the H+/e- ratio with illuminated cells was found to be 1.21 whereas the corresponding ratio in the dark was 0.99 suggesting a 1:1 stoichiometry between acidification and reduction in both cases. To determine the influence of photosynthesis on this apparent redox process cell suspensions were illuminated for 15 min in 4 mM Mes buffer (pH 6.2) before being transferred into darkness or exposed to 1 ,uM DCMU, an inhibitor of noncyclic photophosphorylation. This concentration of DCMU had no influence on the buffering capacity of the medium. Darkness eliminated measurable Fe(CN)63- reduction and DCMU resulted in a decline in reduction rate from 22.9 (±8.9) to 6.06 (±3.2) nmol
Fe(CN)37/106 cells.min (Fig. 3). Similarly acidification was reduced 94% by DCMU and darkness resulted in inhibition of acidification and a significant rate of alkalinization of 19 (± 11) nmol OH-/106 cells. min (Fig. 4). The change in reduction rates occurred within 5 min, and acidification rates appeared to change within a few seconds (Fig. 1). The data suggest that acidification and reduction are tightly coupled processes. To investigate the relationship between Fe(CN)63-dependent and ATP-dependent (Fe[CN]63--independent) acidification cells were suspended in a 5 mM KCI, 5 mM NaCl, 0.2 mM CaCl2 medium. The presence of univalent cations stimulates the rate of Fe(CN)63--independent acidification (Figs. 1 and 4; cf Fig. 5). The plasma membrane proton ATPase inhibitor DES inhibited acidification and resulted in alkalinization of the medium indicating a passive influx of protons. Once the pH had stabilized, Fe(CN)63- was added and normal rates of Fe(CN)63--stimulated acidification were obtained. With illuminated cells the initial Fe(CN)63--independent rate was 0.44 (±0.20 SD) nmol H+/106 cells- min. This rate was completely inhibited by 100 gM DES and subsequent ferricyanide addition resulted in H+ efflux of 25.4 (±2.2) nmol H/lO6 cells- min. With nonilluminated cells the Fe(CN)63-independent DES inhibited process gave a rate of 0.73 (±0.09 SD) nmol H+/ 106 cells min. Subsequent addition of Fe(CN)63- resulted in resumption of acidification at a rate of 2.16 (±0.78 SD) nmol H+/106 cells.min (Fig. 5). These results indicate the presence of Fe(CN)63- dependent and independent mechanisms of acidification in the presence or absence of illumination. Although DES addition by itself does not contribute to the buffering capacity of the medium, an increase in buffering results from an apparent stimulated efflux of metabolites from the cell (Fig. 5). Various control experiments were performed to eliminate facile explanations of the results described. In the absence of cells, neither acidification nor Fe(CN)63- reduction was observed. HI efflux from cells exposed to 3 mM KCI was 0.6 and 11%, respectively, of those obtained after addition of 1 mM K3 Fe(CN)6 to illuminated and nonilluminated cells. The decline in Fe(CN)63 concentration in the medium of illuminated cells was stoichiometrically equivalent to the accumulation of Fe(CN)64' demonstrating that the decline was not due to Fe(CN)631 break-
H+ EFFLUX AND REDOX ACTIVITY IN MESOPHYLL CELLS
.50 nmoles H +
C Fe(CN)pH60 pH 6.09 -
* pH 6.17 A
FIG. 1. Ferricyanide-dependent acidification in illuminated and nonilluminated cells. The stirred and aerated cell suspensions containing 7 x 106 cells in a 10 mL volume of 1 mm calcium sulfate were titrated to determine buffering capacity before the addition of 1 mm Fe(CN)63-. Suspensions were illuminated throughout (A) or illumination began 1 min after the addition of the Fe(CN)63- to a nonilluminated suspension (B). Lag periods were determined by drawing two tangent lines to rates before and after treatment and determining the time between the treatment change and the intersect of the two lines. The acidification rate is expressed in nmol H+/ 106 cells -min.
L :-"& gnt
down or its accumulation in the cells. When cells were removed from the medium after 60 min with or without illumination, subsequent addition of Fe(CN)63- did not result in acidification. This result indicates that acidification does not arise from the release of a reductant to the medium. Similarly, the process does not involve the release of a reductant and a redox catalyst functioning exterior to the plasma membrane, since incubation of illuminated or nonilluminated cells for extended periods prior to the addition of Fe(CN)63- did not result in a stimulation of acidification. Fe(CN)64 was not oxidized when added to illuminated or nonilluminated cell suspensions. Since Fe(CN)64- accumulates in the medium and no evidence for the release of a reductant can be obtained, the data indicate the presence of a trans-plasma membrane redox system in Asparagus mesophyll cells.
DISCUSSION The present report indicates that the reduction of exogenous Fe(CN)63 is coupled to H+ efflux with a H+/e- ratio of one (Table I; Figs. 3 and 4), and extends the evidence for a redox system spanning the plasma membrane to Asparagus mesophyll cells. Extensive data on this proposition has been obtained in root tissue and various authors have demonstrated that Fe(CN)63- reduction is associated with H+ efflux (9, 12, 18, 19, 21). In contrast, only one group using the aquatic plant Elodea canadensis has investigated similar phenomena in a photosynthetic tissue (1 1). A recent proposal suggests that the plasma membrane redox system of root tissues transfers only electrons to an external acceptor (18). The consequent depolarization of the plasma membrane was suggested to result in the stimulation of the ATPdependent proton pump and H+ efflux. This proposal is sup-
ported by observations indicating that Fe(CN)63- is reduced immediately on addition whereas Fe(CN)63--dependent H+ efflux begins after a 5 min lag period, and the observed inhibition of Fe(CN)63--dependent H+ efflux by inhibitors ofthe plasma membrane ATP-dependent proton pump (18). The results described here do not support this model. The lag period before detection of Fe(CN)63--dependent acidification in this study was 4 s in the light and 11 s in the dark (Fig. 1). The H+/e- stoichiometry of one (7, 1 1; Table I) is consistent with tight coupling between H+ efflux and reduction and the following reaction: AH2 + 2Fe(CN)63- - A + 2H+ + 2Fe(CN)64An H+/e- ratio of one is not predicted by a model in which stimulation of a redox system results in an indirect stimulation of ATP driven H+ efflux. Data demonstrating that the proton ATPase inhibitors DES, dicyclohexyl-carbodiimide, and vanadate inhibit the rate of Fe(CN)63--dependent acidification (18) does not by itself indicate that acidification is mediated by the proton ATPase. Inhibition of net redox driven acidification would also result if inhibitors increased passive leakage of protons back into the cell as indicated by the alkalinization response to DES in this study (Fig. 5). In addition, a decline in acidification rate on application of a potential inhibitor cannot be equated with a decline in the rate of H+ efflux until it has been demonstrated that the inhibitor does not increase the buffering capacity of the medium. This necessitates the determination of buffering capacity prior to and after the addition of the inhibitor (Fig. 5). Vanadate which is analogous to phosphate with three reversibly protonated groups will increase the buffering capacity of the medium particularly if its pH is adjusted with Mes which has a pKa of 6.1 (15, 18). Similarly, DES addition increased the buffering capacity of the 10 ml suspension from 680 nmol/pH
Plant Physiol. Vol. 83, 1987
NEUFELD AND BOWN 1.05.
10 20 Time (min.)
0.4) 0.0 Fe(CN)36
FIG. 3. Effect of darkness on ferricyanide reduction in illuminated cell suspensions. Fe(CN)63- (1 mM) was added to stirred, aerated, and illuminated cell suspensions containing 10 to 12 x 106 cells suspended in a 20 mL volume of 1 mM calcium sulfate and 3 mm MES-Ca(OH)2 buffer (pH 6.2). Aliquots were removed at 5 min intervals over a 30 min period. The aliquots were centrifuged and the absorbance of the supernatant was determined spectrophotometrically at 420 nm. The cells were continuously illuminated over a 30 min period (0) or the cells were placed in the dark after 15 min (A). The results shown were obtained from representative experiments.
FIG. 2. Effect of ferricyanide concentration on the rate of ferricyanide-dependent acidification. Various concentrations of Fe(CN)63- were added to stirred and aerated cells suspended in 10 mL of 1 mm calcium sulfate. The pH stat technique was used to determine the H+ efflux over the initial 5 min at a preset pH of 6.2. Acidification rates are expressed in nmol H+/I06 cells- min. Each point is the mean obtained from 4 to 6 trials. The error bars indicate standard deviation. Illuminated cell suspensions contained 5 x 106 cells (A); nonilluminated cell suspensions contained 15 x 106 cells (B). Rates ofFerricyanide Reduction and Ferricyanide-Dependent Acidification in the Light and in the Dark In the light, 10 to 12 x 106 cells were suspended in pH 6.2 3 mM Mes buffer containing 1 mm CaSO4, and Fe(CN63- reduction was measured at 420 nm. In the dark, 22 to 24 x 106 cells were suspended in pH 6.2 1 mM Mes buffer containing 1 mm CaSO4, and Fe(CN)63- reduction was measured using the determination of Fe(CN)64-. In both cases, 1 mM Fe(CN)63- was added and rates of acidification were calculated using the rate of pH change with time. The H+/e- ratio is the ratio of acidification to reduction. Values in the light are the mean of 10 measurements, in the dark they are the mean of 3 measurements. Conditions Acidification Reduction H+/eratio nmol/106 cells -min ± SD 22.9 (8.9) 1.21 26.1 (8.0) Light Dark 1.54 (0.38) 1.55 (0.28) 0.99
unit to approximately 1300 nmol/pH unit (Fig. 5). DES itself does not have a reversibly protonated group in the physiological range and increased buffering presumably results from the release of cellular metabolites having acid base properties. Thus, data demonstrating inhibition of Fe(CN)63 stimulated acidification by plasma membrane proton ATPase inhibitors may result from increased buffering, not inhibition of the ATPase. The present
FeN L ight
p9;H 6.3 23.2 _
L ight ,
1 nmoles OH-
FIG. 4. Effect of darkness and DCMU on the rate of acidification. Fe(CN)63- (1 mM) was added to stirred, aerated, and illuminated cell suspensions containing 10 to 12 x 106 cells suspended in a 10 mL volume of I mm calcium sulfate and 3 mm MES-Ca(OH)2 buffer (pH 6.2). The cells were illuminated for 15 min and then the cells were either placed in the dark or 1 ;&M DCMU was added. The acidification rates are expressed in nmol H+/I06 cells-min.
data show that Fe(CN)63- stimulated normal acidification rates after the usual ATP-dependent rate of H+ efflux was eliminated with DES (Fig. 5). These results indicate the presence of two independent mechanisms of H+ efflux. Photosynthesis stimulates both Fe(CN)63- reduction and Fe(CN)63--dependent H+ efflux 14 to 17 times (Table I; Figs. 3 and 4). These data are consistent with a stimulation by light of Fe(CN)63- reduction and associated H+ effiux in leaves ofElodea ( 11). The export of photosynthetically generated reducing equiv-
H+ EFFLUX AND REDOX ACTIVITY IN MESOPHYLL CELLS 1 mM
< 100 nmoles 014
FIG. 5. Separation of ATP-dependent and redox driven acidification in nonilluminated cells. The stirred, aerated, and nonilluminated cell suspension containing 10 x IO6 cells in a 10 mL volume of 5 mm KCI, 5 mM NaCl, and 0.2 mm CaCl2 was titrated to determine buffering capacity before the addition of 0.1 mM DES and also after the Fe(CN)63 addition. The addition of ethanol rather than DES in a control run did not result in inhibition of the Fe(CN)W--independent acidification rate. The acidification rates are expressed in nmol H+/ 106 cells min.
alents from the chloroplasts to the cytosol may involve the phosphoglyceraldehyde/dihydroxyacetone phosphate shuttle or a malate/oxaloacetate shuttle (10). The presence of redox activity in the absence of illumination demonstrates the process is light stimulated but not light dependent. The alternative oxidase pathway has been detected in Asparagus mesophyll cells (4) and has been implicated in redox driven H+ efflux in nonphotosynthetic tissue (2). LITERATURE CITED 1.
AVRON H, N SHAVIT 1969 A sensitive and simple method for the determination of ferrocyanide. Anal Biochem 6: 549-554
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