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Plant Physiol, February 2001, Vol. 125, pp. 912-925
Origin of the Cytoplasmic pH Changes during Anaerobic Stress in
Higher Plant Cells. Carbon-13 and Phosphorous-31 Nuclear Magnetic
Resonance Studies
Elisabeth
Gout,
Anne-Marie
Boisson,
Serge
Aubert,
Roland
Douce, and
Richard
Bligny*
Laboratoire de Physiologie Cellulaire Végétale,
Unité Mixte de Recherche 5019 (Commissariat à l'Energie
Atomique, Centre National de la Recherche Scientifique,
Université Joseph Fourier), Département de Biologie
Moléculaire et Structurale, CEA-Grenoble, 17 rue des
Martyrs, 38054 Grenoble cedex 9, France
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 31P- and 13C-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 1 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.
*
Corresponding author; e-mail rbligny{at}cea.fr.; fax
33-4-76- 88-50-91.
© 2001 American Society of Plant Physiologists
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