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31, 1992
Effect
of D20 on maize
plasmalemma coupled proton
Qlaf Dijring
Received
Universitiit
Hamburg,
November
11,
ATPase and electron pumping
and Michael
870-876
transport
Biittger
Institut fiir Allgemeine Botanik, D-2000 Hamburg 52, FRG
Ohnhorststrz&e
18,
1991
Heavy water (DzO] has been used as a utative inhibitor of the plasma membrane H+-ATPase and the plasma mem E rane redox system. Concentrations above 50% D,O inhibited H+ secretion and the lasma membrane redox system of Zea rnays L. roots. Inhibition of H+ secretion f.!)y vanadate was reduced in resence of D,O. The plasma membrane of roots was transiently depolarized af! er the addition of heavy water in concentrations above 5%. The repolarization of the plasma membrane that takes place while the H+ secretion is still reduced by heavy water indicates that, despite the overall inhibiting effect of D,O, the plant 0 1992 Academic P;ess, Inc. is still able to regulate the membrane potential.
The H+-secretion of roots is due to a plasma membrane (PM) bound ATPase and, as recently discussed, somehow linked to the activity of a PM e--transport system (redox system]] l-31. H+ extrusion produces an electrochemical gradient across the PM that is used for ion uptake via different carrier systems [4,5]. An increased H+-pumping activity caused by the activity of a PM redox system is only detectable after treatment with external e- acceptors such as hexacyanoferrate III (HCF III] [ 1,2,6-81. The reduction of the acceptor together with PM depolarization (AE,,Jis most probably due to an e--transport across the plasmalemma [9]. The protons observed concomitant with the reduction may be transported by the PM ATPase [lo-121 or by the redox system itself [2,3]. Recently, effects of deuterium (D] on H+ (D+] pumping of yeast cells have been shown by Kotyk et al. [131 using D+ as competitive inhibitor of the PM ATPase. The experiments described in this article were carried out in order to investigate a possible difference in the action of D20 on H+ (D+) pumping of the PM ATPase and the redox induced H+ (D+) secretion, and the consequences of such an inhibition on PM energy state. Such a difference in reaction of the ATPase and the redox system with regard to H+ secretion may give evidence for or against a redox coupled H+ pump distinct from the PM ATPase. Abbreviations: AE, = Membrane HCF III = Potassiumhexacyanoferrate 0006-291x/92 Copyright All rights
Depolarization: (III) : PM = Plasma Membrane.
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Chemicals: All chemicals were obtained from Merck (Darmstadt, FRG). D,O was used NMR grade. Plant Material: Seeds of Zea n-rays L. cv. Gold rinz (C. Sperling. Liineburg. FRG) were soaked in tap water for 2 hours an B germinated for 48 hours on wet filter pa er at 26°C in the dark. Prior to measurement, the seedlings were preincu %ated at 22°C for 12 hours in an aerated medium containing 3 mM KCl, 0.5 mM CaCl, and 0.125 mM MgSO.+ in distilled H,O or D,O (NMR grade) respectively. Membrane potential measurements: Measurement of membrane potential (Awl was erformed as described elsewhere [ 141. Usually the plants were preincubated Por 30 minutes. Incubation medium for measurement of Av was adjusted to pH 5.5 (H,O) or 5.9 (D,O). Medium flow was maintained by a peristaltic pump, flow rate was adjusted to 5 ml/mm. Measurement of reduction rate and I-I+ secretion rate of corn roots: Redox activi was measured simultaneously with H+-extrusion in a pH-oxidostat as descri t ed elsewhere (151. In this system the acidification of the medium caused by the roots was compensated by titration with KOH. In D20 experiments solid KOH dissolved in D,O was used for titration. The error resulting from the H in KOH is negligible compared with the error resulting from the water content of the plant tissue investigated. Experiments were carried out in a growth chamber at 22°C at pH 5.5 (in H,O) or pH 5.9 (in D,O). The concentration of oxidized HCF III in the pH-oxidostat was held constant throughout the e eriment. Because in the pH-oxidostat pH was measured with a glass pH-e3) e&rode the pH had to be adjusted up to 0.4 units higher when measured in D,O [16].
Results H+ secretion: Net H+ secretion was inhibited by D20 concentrations above 5%. This inhibition was dependent on concentration and transient below 50% D20 (data not shown). Fig. 1 illustrates this inhibition for 95% D20. The inhibition was not strictly reversible, a change from 95% D20 to H20 caused net H+ extrusion to drop to zero for about two hours rather than restoring the original pumping rate (not shown). Redox activity and redox induced I-I+ secretion: As recently shown, addition of HCF III (1mM) in H20 caused H+ secretion to increase [ 151. Such an increase in H+ secretion can be measured during incubation with D20 (Fig. 2). Replacing H20 with D20 during incubation with HCF III (ImM), while holding HCF III constant, inhibited both HCF III reduction and H+ secretion (Fig. 3). Electron transport is inhibited partially at D20 concentrations of 50% while inhibition of H+-extrusion is more pronounced at 75% D20 (Fig. 3). Effect of vanadate: Inhibition of net H+ secretion by vanadate was partially reverted after replacing H20 with D20 (Fig. 4). Addition of vanadate (0. 1mM) in the presence of D20 altered net H+ secretion to a lesser extend than in H20 (Fig. 5). Further addition of vanadate to a final concentration of (0.2mM) caused a decrease in H+ secretion. Membrane Potential: The plasma membrane depolarized after replacing H20 in the incubation medium with D20. This AE, was transient, concentration de871
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Finure 1. The net H+ secretion rate of maize roots measured in H,O-medium at a constant pH of 5.5 is inhibited in medium containing 95% D,O (n=4). Figure 2. Adding HCF III (final concentration 1 mM i.A.) in the presence of D,O caused an increase of H+ secretion. The left art of the curve shows the H+-pumping rate of maize roots already inhi #. ited by 95% DzO (n=4).
pendent and detectable at D20 10 min. in the presence of D20 value. Replacing D20 with H20 depolarization of the membrane in the presence of 95% D20 by
concentrations as low as 5%. (Fig. 6). After the membrane had repolarized to the original after that time caused a second transient (Fig. 7). HCF III (1mM) still depolarized the PM 5+ 3mV.
Discussion
Physico-chemical properties of water are of major importance for all biological processes. Although isotopes are known to have similar physical and chemical properties, this is not true for the isotopes of hydrogen. Hydrogen, deuterium
1.5
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Electrons
Figure 3. Substitution of H 0 with D,O (50% and 75% respectively) in the resence of 1 mM HCF III inhibits e--flux and net H+ secretion. The HCF III in a)uced H+ secretion is less sensitive to D,O than the HCF III reduction rate (n=4). 872
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Figure 4. Inhibition of net H+ secretion of maize roots by vanadate (0.1 mM) was partially reverted 30 minutes after replacing Hz0 with D,O (90%) (n=3). Without D,O H+ secretion remained inhibited (data not shown). Figure 5. Addition of vanadate (0.1 mM) in the presence of D,O (90%) did not alter net H+ secretion as vanadate concentrations sufficient to inhibit H+ secretion in H,O-medium do (Fig. 4, n=3).
and tritium are isotopes physico-chemically very different from each other, which leads to strong isotope effects. Lower D20 concentrations generally inhibit or slow down the processes under investigation. Uphaus et al. [ 171 therefore called D20 a “chaotropic reagent”, which paraphrases the fact that a certain single mode of action of D20 does not exist. Beside the various effects of heavy water on morphology and histology [ 17,181 D20 is known to affect a multitude of physiological processes [ 19-301. Recently, effects of heavy water on the activity of the PM ATPase have been shown by Kotyk et al. [ 131 using D as competitive inhibitor. Such an inhibition of the primary H+ pump explains well the loss of H+-pumping activity and the
Addition
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20 Il-lV
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Figure 6. Depolarization of the plasma membrane by different concentrations of D,O. Track (a) = 5%. (b) = 15%. (c) = 50%. and (d) = 90%. Tracks a-d all have a starting otential between - 100 and - 105 mV. Rising curves indicate depolarization Peach track n=5).
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Figure 7. After 10 minutes in the presence of D,O the membrane repolarized to the original value. Replacing D,O with H 0 after that time caused a second transient AE, of the membrane. Rising o Pthe curve indicates depolarization (n=4).
initial AE, of the PM. Inhibition of ATP hydrolysis of yeast ATPase, however, is minute [ 131. As described above the action of D20 on enzyme activity is generally inhibitory. We therefore cannot be sure that the D20 action is confined to competitive inhibition of the ATPase. Direct inhibition of enzyme activity by D20 has been documented for different enzymes after D20 pretreatment for various times and after addition of D20 to the assay of non pretreated enzymes [251. These authors, nevertheless, found that this inhibition alone is not sufficient to explain the effects observed in living plants. According to Cooke and coworkers [251 a slowdown of enzyme biosynthesis has also to be taken into account to explain the effects observed over some hours. Such inhibition of protein synthesis, which may account to the effects observed in long time incubation experiments, is most likely not the reason for the initial inhibition of net H+ extrusion and redox activity, measurable after some minutes (the time resolution of the oxidostat), and should not account for the AE,, which is detectable within seconds. A slow exchange of H with D in proteins 13 11. resulting in loss of enzyme activity, may contribute to effects observed over a longer time scale. Beside isotope effects, which in this case is a relatively vague explanation, we lack an idea of what the reason for the observed transient inhibition of H+-pumping activity after replacement of D20- with H20-medium might be. Since the AE, caused by HCF III in H20 does not exceed 1OmV (usually between 4 and 8mV,[ 141) under the conditions given, and varies greatly for individual roots, the AE, found in D20 cannot be considered as significantly different from the depolarization in H20. The consequences of H substitution with D and effects of D20 on the vicinity of enzymes by altering their hydration, the latter acting faster than the former, must, in view of the immediate depolarization, not be the only influences of D20 on the membrane. Andjus and coworkers [30] proposed solvent isotope effects to be responsible for alteration of some membrane characteristics in Chara and NiteUa cells, the most important of which for interpreting our obser-
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vations appears to be altered channel gating. Increased K+ efilux as observed for C. gymnophylla, would be an explanation for the repolarization of the membrane after D20 addition. However, since all membrane channels investigated so far change their behaviour after D20 addition [30], it is difficult to speculate on the reasons for the observed de- and repolarization. Further evidence for altered activity of membrane transport systems can be found in the loss of influence of vanadate on H+-pumping after D20 treatment. According to Ulhich-Eberius et al. 1321 vanadate is taken up via the phosphate/H+ symport carrier. Cytoplasmic reductive detoxification might to some extent prevent the inhibition of the PM ATPase by vanadate, found in in vitro experiments 1331. After D20 treatment, inhibition of H+ pumping caused by vanadate in H20 diminished within about 30 minutes (Fig. 4). In the presence of D20, decreased inhibition of net H+-secretion by vanadate was found (Fig. 5). Both results may be due to an inhibition of vanadate uptake. An inhibition of H+ requiring uptake processes by D20 has already been reported by Kotyk et al. [ 131. Cytoplasmic detoxification could be the reason for the slow recovering of H+ pumping activity in vanadate-treated roots some time after switching to D20-medium without vanadate. Another possible way to explain the recovery of H+-pumping activity in the presence of vanadate may be an altered phosphate/vanadate binding site of the ATPase after D20 treatment. In this case higher concentrations of vanadate would be needed to achieve the same inhibition of the ATPase in the presence of D20. The ability of HCF III to depolarize the plasma membrane and to stimulate net H+ secretion in the presence of high concentrations of D20 indicates that the H+ extrusion process responsible for redox induced H+-secretion as well as e--transport is not completely inhibited. However, e--transport appears to be more sensitive to D20 than total net H+ secretion (Fig. 3). Acknowledgments, in the Faculty of Biology,
This article is based on a doctoral University of Hamburg.
study by 0. Doring
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