Regulation of intracellular pH during anoxia in rice coleoptiles in acidic and near neutral conditions

Rice coleoptiles, renowned for anoxia tolerance, were hypoxically pretreated, excised, 'healed', and then exposed to a combination of anoxia and pH 3.5. The putative acid load was confirmed by net effluxes of K(+) to the medium, with concurrent net decreases of H(+) in the medium, presumably mainly due to H(+) influx. Yet the coleoptiles survived the combination of anoxia and pH 3.5 for at least 90 h, and even for at least 40 h when the energy crisis, inherent to anoxia, had been aggravated by supplying the coleoptiles with 2.5 mM rather than 50 mM glucose. Even in the case of coleoptiles with 2.5 mM glucose, an accumulation ratio of 6 for Cl(-) was attained at 4 h after the start of re-aeration, implying plasma membrane integrity was either maintained during anoxia, or rapidly restored after a return to aerated conditions. Cytoplasmic pH and vacuolar pH were measured using in vivo (31)P nuclear magnetic resonance spectroscopy with 50 mM glucose in the basal perfusion medium. After 60 h in anoxia, external pH was suddenly decreased from 6.5 to 3.5, but cytoplasmic pH only decreased from 7.35 to 7.2 during the first 2 h and then remained steady for the next 16 h. During the first 3 h at pH 3.5, vacuolar pH decreased from 5.7 to 5.25 and then stabilized. After 18 h at pH 3.5, the initial values of cytoplasmic pH and vacuolar pH were rapidly restored, both upon a return to pH 6.5 while maintaining anoxia and after subsequent return to aerated solution. Summing up, rice coleoptiles exposed to a combination of anoxia and pH 3.5 retained pH regulation and cellular compartmentation, demonstrating tolerance to anoxia even during the acid load imposed by exposure to pH 3.5.