Proc. Natl. Acad. Sci. USA Vol. 81, pp.

3379-3383, June 1984 Botany

Mechanism of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia
(in vivo NMR/Zea mays L./flooding tolerance/pH-stat/ethanol fermentation)

JUSTIN K. M. ROBERTS*, JUDY CALLISt, DAVID WEMMER*t, VIRGINIA WALBOTt, AND OLEG JARDETZKY* *Stanford Magnetic Resonance Laboratory and tDepartment of Biological Sciences, Stanford University, Stanford, CA 94305
Communicated by Winslow R. Briggs, February 22, 1984

ABSTRACT We show that a transient lactic fermentation provides the signal triggering ethanol production in hypoxic maize root tips. The signal is cytoplasmic pH. This interaction between lactic and ethanolic fermentation permits tight cytoplasmic pH regulation during hypoxia-cytoplasmic pH remaining near neutrality for several hours. Mutant roots unable to synthesize ethanol can neither regulate cytoplasmic pH nor maintain ATP levels during extended periods of hypoxia and, like vertebrate tissues, are less tolerant of hypoxia than normal maize. This indicates that cytoplasmic pH regulation is an important factor in survival under hypoxia.

Certain higher plant tissues, such as maize roots, although requiring oxygen for normal functioning, can survive long periods (>18 hr) of anaerobiosis (1), with glycolysis continuing during most of this period (2, 3). Most vertebrate tissues, on the other hand, can survive only short periods of hypoxia (<2 hr), after which glycolysis is greatly inhibited, and in most cases irreversible cell damage occurs (4, 5). This difference between plants and animals cannot be due simply to the lower metabolic rates often seen in plants: the maize root tips (at 250C) used in this study respire five times the rate of a resting man (at 370C) and about half the rate of a resting mouse (at 370C) (6). It is possible, however, that the ability of higher plants to undergo a mainly ethanolic fermentation (2, 3, 7-9), rather than the exclusively lactic fermentation seen in higher animals, is to some degree responsible for their ability to withstand long periods of hypoxia. We considered this view after showing that the cytoplasmic pH of maize root tips falls to a stable value -0.5 pH unit below aerobic values within ""20 min after transfer to an anaerobic environment (10). This behavior is in complete contrast to active, hypoxic vertebrate tissues, where cytoplasmic pH falls throughout hypoxia because of continuous lactic acid accumulation (4, 11, 12) until glycolysis ceases. Thus, in hypoxic vertebrate tissues, energy production (glycolysis) leads to cytoplasmic acidosis, which eventually inhibits continued energy production; in plants, continued energy production does not involve generation of acid (other than carbon dioxide), and so no inhibition of glycolysis due to acidosis is observed. We describe here the mechanism by which cytoplasmic pH is regulated, and ethanol production induced, in hypoxic maize root tips. The mechanism is consistent with in vitro data (13, 14). We also show that root tips of mutants that lack the ability to make ethanol during hypoxia-and have decreased viability-cannot regulate cytoplasmic pH but instead, like vertebrate tissues, undergo cytoplasmic acidification throughout hypoxia.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.
3379

MATERIALS AND METHODS Experiments were performed with -=1.5-g samples of 2-mm hybrid maize (WW x Br38) (obtained from Customaize Research, Decateur, IL) root tips excised from 2-day-old seedlings, perfused as described in the figure legends (10). NADH fluorescence (15) was measured in a Perkin-Elmer fluorospectrophotometer; light emitted at 90 to the excitation beam was collected; excitation 366 2 nm, emission 470 5 nm; the excitation and emission spectra of this fluorescence peak are very similar to those of pure NADH in solution. The rate of ethanol production was determined by enzymatic analysis (16) of the effluent, collected by fraction collector; virtually identical results were obtained if flow rates were kept constant throughout the experiment, only oxygen tension being changed (data not shown). Cytoplasmic pH was estimated by 31P NMR (17-19), from the chemical shift (8) of the cytoplasmic inorganic phosphate resonance (20), using titration curves of phosphate solutions approximating to expected intracellular composition (10, 18). 31p chemical shifts are referenced to 0.5 M methylene diphosphonate with 5 mM ethylenediaminetetraacetic acid in H20, brought to pH 8.9 with Tris base. Spectra were obtained on a modified Bruker HXS-360 spectrometer operating at 145.7 MHz. 13C NMR spectra of methyl resonances in 2-mm maize root tips perfused with 50 mM [1-_3C]glucose (90% enrichment) were obtained with the same spectrometer. Spectra were obtained while irradiating the methyl protons. 3C chemical shifts are given relative to tetramethylsilane at 0 ppm. 13C methyl assignments were made on the basis of chemical shifts; the intensities of peaks were copsistent with concentrations of lactate and alanine determined enzymatically (16) in root tip extracts (data not shown). Root tips were perfused with 0.1 mM CaSO4 to deplete endogenous sugar (21) prior to perfusion with [1-13C]glucose, and this perfusion continued for 3 hr before the experiments were initiated. See figure legends for perfusion details. The seed lines described in Figs. 5 and 6 were a gift from M. Freeling (Department of Genetics, University of California, Berkeley); they were propagated at Stanford, in the same field, in the summer of 1982.

RESULTS AND DISCUSSION

Time Course of Fermentation End Products During Hypox-

ia. Although metabolic changes in plants are largely complete after 30 min of hypoxia (8, 22-24), there are no published data on the accumulation of fermentation end products with more than three time points in this period. Here we describe the appearance of these end products in detail. Fig. 1 shows the kinetics of NADH fluorescence, ethanol
Abbreviation: ADH, alcohol dehydrogenase. tPresent address: Department of Chemistry, University of Washington, Seattle, WA 98195.

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j2min
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Proc. NatL. Acad. Sci. USA 81 (1984)

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FIG. 1. Time course of NADH fluorescence (a), rate of ethanol production (b), and cytoplasmic pH (c) in perfused maize root tips during hypoxia, determined in vivo. Root tips were initially perfused at 50 ml-min-' with 02-saturated 50 mM glucose/0.1 mM CaSO4. After 20 min, they were perfused at 4 ml-min-' with the same solution now saturated with N2-

production, and cytoplasmic pH in perfused maize root tips during an aerobic/anaerobic transition. The rapid increase (=2 min) in NADH levels in hypoxia indicates a rapid inhibition of oxidative phosphorylation. Thus, reductant for lactate or alcohol dehydrogenase is available early in hypoxia.

Within the first 2 min of hypoxia, cytoplasmic acidification begins and, as we reported previously (10), within 20 min of hypoxia, cytoplasmic pH falls by -0.5 pH unit to a stable pH value; this new cytoplasmic pH can be maintained (0.1 pH unit) for at least 10 hr (data not shown). After a 10-min lag, ethanol appears in the perfusion effluent; ethanol production increases to a maximum by -30 min, remaining constant thereafter. No lactate is observed in the effluent (data not shown); this contrasts with many animal tissues (e.g., muscle), from which lactate leaks (25) and is metabolized in other aerobic tissues (e.g., liver). The 13C NMR partial spectra in Fig. 2 show that the cytoplasmic acidification described by Fig. 1 is due to a transient lactic fermentation. Thus, the methyl signal due to lactate, arising via glycolysis from [1-13C]glucose, reaches a constant intensity within 20 min of hypoxia. Methyl signals from alanine and ethanol also rapidly reach a constant intensity; this result indicates that intracellular ethanol rapidly equilibrates with the perfusion medium. Alanine most probably arises from transamination of pyruvate via glutamic-pyruvic transaminase; decreases in glutamate and increases in a-ketoglutarate have been observed in anoxic buckwheat seedlings (7). It is clear from Fig. 1 that the rate of ethanol production continues to increase considerably (>2-fold) long after cytoplasmic pH has stabilized-i.e., after lactic acid production has stopped. This indicates that the Pasteur effect in this tissue (2) occurs several minutes after fermentation reactions begin, not at the same time. This phenomenon will be discussed elsewhere. Control of Lactate and Ethanol Production. In vitro studies (13, 14) have shown lactate dehydrogenase to have an alkaline pH optimum, whereas pyruvate decarboxylase (which
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FIG. 2. 13C NMR spectra (90.5 MHz) of methyl resonances in 2maize root tips perfused with 50 mM [1-'3C]glucose (90%o enrichment) at 40 ml-min-'. Hypoxia was induced by perfusing with N2-saturated 50 mM glucose at 4 ml-min-'. Resonances are assigned to lactate (peak 1), ethanol (peak 2), and alanine (peak 3).
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FIG. 3. Time course for rate of ethanol production (a) and cytoplasmic pH (b) in maize root tips perfused initially as in Fig. 1. After 20 min, root tips were perfused with 02-saturated 50 mM glucose/ 0.1 mM CaSO4 acidified to pH 3 with H2SO4 (a) (as a control for low external pH) or with 02-saturated 50 mM glucose/0.1 mM CaSO4 with 2.5 mM (o) or 5 mM (A) acetic acid, all at 40 ml-min-'. After a further 25 min, all three samples were perfused with N2-saturated 50 mM glucose/0.1 mM CaSO4 at 4 ml-min-'.

Botany: Roberts et aL
catalyzes the first reaction leading to ethanol from pyruvate) has an acid pH optimum. This result, and work with pea seed extracts, led Davies et al. (14) to suggest that in hypoxic plants it is cytoplasmic pH that controls which fermentation end product is formed. They postulated that initially lactic acid is formed in the alkaline cytoplasm, the resultant lowered cytoplasmic pH then inhibits further lactic acid production while activating pyruvic decarboxylase, leading to ethanol production. The results presented above are entirely consistent with this view and led us to devise a critical test. If a low cytoplasmic pH is required for ethanol formation, one would predict that acidification of the cytoplasm of aerobic root tips will lead to a shorter lag in ethanol production once NADH becomes available in hypoxia. Fig. 3 shows that the prediction is fulfilled: a 2.5 or 5 mM acetic acid pretreatment reduces or eliminates, respectively, the lag in ethanol production in hypoxia. Normally, ethanol production is not stimulated before cytoplasmic pH falls to pH -6.9 (Figs. 1 and 3). The 2.5 mM acetic acid treatment results in a cytoplasmic pH, prior to hypoxia, that is slightly higher than pH 6.9 and does not completely eliminate the lag in ethanol production (Fig. 3). The 5 mM acetic acid treatment results in a cytoplasmic pH lower than pH 6.9 (Fig. 3) and completely eliminates the lag. This result indicates that there is a sharply defined threshold of cytoplasmic pH above which ethanolic fermentation does not occur. The abrupt acceleration in ethanol production that follows the lag (Figs. 1 and 3) also suggests a sharp threshold. Note that the kinetics of ethanol production after 20 min of hypoxia (when lactic acid production normally ceases) are not affected significantly by the acetic acid pretreatment (Fig. 3). This result, together with the observation that ATP levels decrease by no more than 25% during the acetic acid treatments (data not shown), suggests that the acetic acid treatment is not toxic. Not only does an acetic acid pretreatment result in earlier ethanol production in hypoxia but it also suppresses lactate formation (Fig. 4). Thus, 13C NMR partial spectra of root tips fed [1-13C]glucose and pretreated with 5 mM acetic acid

Proc. NatL. Acad. Sci. USA 81 (1984)

3381

show little or no lactate-methyl signal when made hypoxic, whereas the ethanol-methyl signal is strong. We attribute the further cytoplasmic acidification seen when acetic acidtreated tissue becomes hypoxic (Fig. 3) to the acetic acid; hypoxia will inhibit completely the principal means for consuming protons released when acetic acid enters the cellnamely, its conversion to Krebs cycle intermediates and their oxidation to CO2 (and water), this acid escaping from the cell. We conclude that once NADH becomes available following inhibition of oxidative phosphorylation, the formation of lactate and ethanol is regulated by cytoplasmic pH: low cytoplasmic pH favors ethanol and high pH favors lactate. Thus, these two pathways constitute a "pH-stat" (26, 27) that automatically controls cytoplasmic pH in hypoxia. Only if the CO2 produced during ethanolic fermentation is prevented from escaping from the tissue, by sealing the root tips off completely in a small volume, will this pH-stat fail and cytoplasmic pH fall (10). Adh-1 Is Required for Cytoplasmic pH Regulation During Hypoxia. Although ethanol production is primarily regulated at the level of pyruvate decarboxylase, regeneration ofNAD+ is required for continued ethanolic fermentation. This regeneration is catalyzed by alcohol dehydrogenase (ADH). There are two loci that encode ADH activity in maize. In aerobic roots, ADH-1 is the predominant isozyme present (28). After 3-4 hr of hypoxia, roots begin to synthesize both ADH-1 and ADH-2 (1). Aerobic maize root tips, homozygous for a mutation at the Adh-J locus [denoted S5657 (28)] resulting in no ADH-1 enzyme, contain <1% of normal non0.6-

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20

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Time, min

FIG. 5. Time course of rate of ethanol production (a) and cytoplasmic pH (b) in perfused 4-mm maize root tips from an Adh-J-line [S5657 (28)] (A, A) and one of the parent lines [1s2p (28)] from which the mutant was derived (o, C). The other parent line, Funk G4343 (28), gave identical results to those obtained with 1s2p (data not shown).

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Botany: Roberts et aL

Proc. Natl. Acad. Sci. USA 81 (1984)

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over FIG. 6. 31P NMR spectra (145.7 MHz) of 4-mm maize root tips from Adh-1 - [S5657 (28)] (a) and Adh-J + [1s2p (28)] (b) lines, obtained phosphate vacuolar inorganic 2), (peak phosphate inorganic cytoplasmic 1), (peak 6-phosphate glucose to assigned are 30 min. Resonances (peak 3), rP of ATP (peak 4), a-P of ATP, UDP-Glc, and nicotinamide-adenine nucleotides (peaks 5), and 3-P of ATP (peak 6) (20).

induced ADH activity (data not shown) and make barely detectable amounts of ethanol in hypoxia (Fig. 5). Moreover, when such root tips become hypoxic, the stabilization of cytoplasmic pH at about pH 6.8 seen in normal root tips (Figs. 1 and 5) does not occur. Instead, cytoplasmic pH continues to fall throughout the hypoxic treatment (Fig. 5), presumably due to a continued, though lower, rate of lactate production. This lowered rate of lactate production explains why ATP virtually disappears in the hypoxic mutant (Fig. 6a), instead of the -60% reduction seen in normal hypoxic root tips (Fig. 6b); note that the mutant and normal maize lines have very similar ATP levels in the presence of oxygen (Fig. 6). This result indicates that lactic fermentation can fulfill the energetic needs of hypoxic root tips only for short periods of time (-10 min). The similarity of the sequence of events described for the maize ADH-1 null to that seen in ischemic and/or hypoxic vertebrate tissues (4, 29-31) is striking. In vertebrates, lactic fermentation, likewise the only metabolic option for regeneration of NAD+ necessary for continued glycolysis, results in a continual cytoplasmic acidification to pH values close to 6, until glycolysis ceases and irreversible cell injury results (4, 11, 12). It is interesting to note that some protection of rat hearts from ischemic damage was afforded by preperfusing them with a strong buffer (4). In such hearts, the rate of cytoplasmic acidification was significantly decreased during ischemia, high glycolytic rates were maintained for longer, and ATP and phosphocreatine levels fell more slowly; on reperfusion, complete recovery of cytoplasmic pH, ATP, and phosphocreatine was seen (4). It appears, then, that the greatly diminished resistance of ADH-1 nulls to hypoxia (32) is due primarily to an inability to regulate cytoplasmic pH. The explanation for why roots of plant species differ in their ability to withstand hypoxia-although apparently all undergo a mainly ethanolic fermentation (9, 33, 34)-remains to be determined.

We thank Dr. M. Freeling for seed samples and useful discussion, Dr. P. M. Ray for access to laboratory equipment and advice, and the Carnegie Institution of Washington, Department of Plant Biology, for use of the fluorospectrophotometer. This work was supported by National Science Foundation Grants PCM82-04877 and GP23633 and National Institutes of Health Grant RROO711. J.C. is supported by National Institutes of Health Training Grant GM07276-08. 1. Sachs, M. M., Freeling, M. & Okimoto, R. (1980) Cell 20, 761768. 2. Neal, M. J. & Girton, R. E. (1955) Am. J. Bot. 42, 733-737. 3. Leblova, S., Zima, J. & Perglerovd, E. (1976) Aust. J. Plant Physiol. 3, 755-761. 4. Garlick, P. B., Radda, G. K. & Seeley, P. J. (1979) Biochem. J. 184, 547-554. 5. Hochachka, P. W. (1980) Life Without Oxygen: Closed and Open Systems in Hypoxia Tolerance (Harvard Univ. Press, Cambridge, MA). 6. Goddard, D. R. & Bonner, W. D. (1960) in Plant Physiology: A Treatise, ed. Steward, F. C. (Academic, New York), Vol. 1A, pp. 209-312. 7. Effer, W. R. & Ranson, S. L. (1967) Plant Physiol. 42, 10421052. 8. Kobr, M. J. & Beevers, H. (1971) Plant Physiol. 47, 48-52. 9. Smith, A. M. & ap Rees, T. (1979) Planta 146, 327-334. 10. Roberts, J. K. M., Wemmer, D., Ray, P. M. & Jardetzky, 0. (1982) Plant Physiol. 69, 1344-1347. 11. Dawson, M. J., Gadian, D. G. & Wilkie, D. R. (1978) Nature (London) 274, 861-866. 12. Hochachka, P. W. & Mommsen, T. P. (1983) Science 219, 1391-1397. 13. Davies, D. D. & Davies, S. (1972) Biochem. J. 129, 831-839. 14. Davies, D. D., Grego, S. & Kenworthy, P. (1974) Planta 118, 297-310. 15. Chance, B., Cohen, P., Jobsis, F. & Schoener, B. (1962) Science 137, 499-508. 16. Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis (Academic, New York), 2nd Ed.

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17. Moon, R. B. & Richards, J. H. (1973) J. Biol. Chem. 248, 7276-7278. 18. Roberts, J. K. M., Wade-Jardetzky, N. & Jardetzky, 0. (1981) Biochemistry 20, 5389-5394. 19. Roberts, J. K. M. & Jardetzky, 0. (1981) Biochim. Biophys. Acta 639, 53-76. 20. Roberts, J. K. M., Ray, P. M., Wade-Jardetzky, N. & Jardetzky, 0. (1980) Nature (London) 283, 870-872. 21. Saglio, P. H. & Pradet, A. (1980) Plant Physiol. 66, 516-519. 22. Barker, J., Khan, M. A. A. & Solomos, T. (1967) New Phytol. 66, 577-596. 23. Faiz-ur-Rahman, A. T. M., Trewavas, A. J. & Davies, D. D. (1974) Planta 118, 195-210. 24. Saglio, P. H., Raymond, P. & Pradet, A. (1980) Plant Physiol. 66, 1053-1057. 25. Mainwood, G. W. & Worsley-Brown, P. (1975) J. Physiol. (London) 250, 1-22.

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26. Davies, D. D. (1973) Symp. Soc. Exp. Biol. 27, 513-529. 27. Smith, F. A. & Raven, J. A. (1979) Annu. Rev. Plant Physiol. 30, 289-311. 28. Freeling, M. & Buchler, J. A. (1981) in Genetic Engineering: Principles and Methods, eds. Setlow, J. K. & Hollaender, A. (Plenum, New York), Vol. 3, pp. 223-264. 29. Gadian, D. G., Hoult, 0. I., Radda, G. K., Seeley, P. J., Chance, B. & Barlow, C. (1976) Proc. Natl. Acad. Sci. USA 73, 4446 4448. 30. Hollis, D. P., Nunnally, R. L., Taylor, G. J., Weisfeldt, M. L. & Jacobus, W. E. (1978) J. Magn. Reson. 29, 319-330. 31. Wemmer, D., Wade-Jardetzky, N., Robin, E. & Jardetzky, 0. (1982) Biochim. Biophys. Acta 720, 281-287. 32. Schwartz, D. (1969) Am. Nat. 103, 479-481. 33. Smith, A. M. & ap Rees, T. (1979) Phytochemistry 18, 14531458. 34. Alpi, A. & Beevers, H. (1983) Plant Physiol. 71, 30-34.