Mechanisms of cytoplasmic pH regulation in hypoxic maize root tips ...

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Feb 22, 1984 - JUSTIN K. M. ROBERTS*, JUDY CALLISt, DAVID WEMMER*t, VIRGINIA ..... sion, Dr. P. M. Ray for access to laboratory equipment and advice,.
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.

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.

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 (,0o

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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 0.8

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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). mm

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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

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

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

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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