Origin of the Cytoplasmic pH Changes during Anaerobic Stress in Higher Plant Cells. Carbon-13 and Phosphorous-31 Nuclear Magnetic Resonance Studies
Abstract
We tested the contribution of nucleoside triphosphate (NTP) hydrolysis, ethanol, and organic acid syntheses, and H-pump ATPases activity in the acidosis of anoxic sycamore (Acer pseudoplatanus) plant cells. Culture cells were chosen to alter NTP pools and fermentation with specific nutrient media (phosphate [Pi]-deprived and adenine- or glycerol-supplied). In vivo P- and C-nuclear magnetic resonance (NMR) spectroscopy was utilized to noninvasively measure intracellular pHs, Pi, phosphomonoesters, nucleotides, lactate, and ethanol. Following the onset of anoxia, cytoplasmic (cyt) pH (7.5) decreased to 6.8 within 4 to 5 min, whereas vacuolar pH (5.7) and external pH (6.5) remained stable. The NTP pool simultaneously decreased from 210 to <20 nmol g cell wet weight, whereas nuceloside diphosphate, nucleoside monophosphate, and cyt pH increased correspondingly. The initial cytoplasmic acidification was at a minimum in Pi-deprived cells containing little NTP, and at a maximum in adenine-incubated cells showing the highest NTP concentration. Our data show that the release of H ions accompanying the Pi-liberating hydrolysis of NTP was the principal cause of the initial cyt pH drop and that this cytoplasmic acidosis was not overcome by H extrusion. After 15 min of anoxia, a partial cyt-pH recovery observed in cells supplied with Glc, but not with glycerol, was attributed to the H-consuming ATP synthesis accompanying ethanolic fermentation. Following re-oxygenation, the cyt pH recovered its initial value (7.5) within 2 to 3 min, whereas external pH decreased abruptly. We suggest that the H-pumping ATPase located in the plasma membrane was blocked in anoxia and quickly reactivated after re-oxygenation.
The fall in cytoplasmic (cyt) pH following the onset of anoxia is a common phenomenon observed in most organisms, including plants (Roberts, 1984; Raven, 1986; Kennedy et al., 1992; Ratcliffe, 1997). However, the origin of the pH changes occurring in plant cells during anoxia is not yet clearly established as outlined by Ratcliffe (1995). Several parameters are involved in the regulation of intracellular pHs in anoxic plant cells, as recently reviewed by Saglio et al. (1999). Several parameters may contribute to increase the cyt H concentration: the passive influx of H ions from the external (ext) medium or from the vacuole (vac), the hydrolysis of the pools of Mg nucleoside triphosphate (NTP) and sugar phosphates (Gevers, 1977; Busa and Nuccitelli, 1984), the accumulation of non-processed acidic intermediates like glycolytic compounds (Felle, 1996), the synthesis of lactate (Davies et al., 1974), and a poor CO2 removal. Other parameters have the opposite effect: the synthesis of Ala and γ-aminobutyrate (Menegus et al., 1989), the decarboxylation of organic acids (Roberts et al., 1992), the activation of H extrusion at acidic cyt pH (Guern et al., 1991; Xia and Roberts, 1996), and the functioning of the H-pumping ATPases located at the plasma membrane and tonoplast (Gout et al., 1992). The apparent coincidence between the fall in pH and the synthesis of lactic acid in anoxic tissues suggested that the accumulation of lactate participates in the acidification of the cytoplasm (Roberts et al, 1984; Rivoal and Hanson, 1993). However, anoxia also triggers a cyt acidosis in rice shoots, although the lactate synthesis is very low in this material (Menegus et al., 1991). Furthermore, the fall of cyt pH following the onset of anoxia in maize root tips precedes the accumulation of lactate (Saint-Gès et al., 1991). It was also reported that there is a correlation between the cyt pH and the size of the NTP pool during anoxia. The hydrolysis of NTP liberates H ions, contributing to the decrease of the cyt pH, and conversely, the synthesis of NTP should alkalize the cyt pH. On the other hand, the decrease of NTP can also limit the activity of the ATP-dependent H pumps of the plasma membrane and tonoplast that have apparent Km for ATP of 0.1 to 0.2 and 0.3 to 0.5 mm, respectively (Sze, 1984), thereby preventing the recovery of the normoxic cyt pH after the initial acidification. Until now the evaluation of the contribution of these different parameters to the initial cyt acidosis was impeded by the absence of data obtained with a unique material in which NTP level could be easily varied and fermentation could be prevented.
In this work experiments were set up to address the following questions: To what extent does the H-liberating hydrolysis of NTP participate in the initial cyt pH fall? Do the H pump ATPases extrude H from the cytoplasm in anoxia? What is the contribution of the synthesis of ethanol and organic acids to the regulation of intracellular pHs during anoxia?
To answer these questions heterotrophic cell suspensions from higher plants were utilized in preference to dense tissues. This facilitated the diffusion of gas and substrates during anoxia and improved the homogeneity of the incubation conditions and allowed specific modifications of the physiological state of plant material (Bligny and Leguay, 1987). For example, the importance of NTP hydrolysis in cyt acidosis during anoxia was assessed by diminishing or increasing the cell NTP pool by incubating cells either in a phosphate- (Pi) free (Rébeillé et al., 1982) or in an adenine-supplied nutrient medium (Dorée et al., 1970). The contribution of the synthesis of lactate to the cyt acidosis was assessed by incubating cells on glycerol as a unique carbon source (Aubert et al., 1994), thereby inhibiting fermentation processes.