ABSTRACT31P nuclear magnetic resonance spectroscopy was used to measure intracellular pH in living tissues. Oxygen deprivation caused fast cytoplasmic acidification from pH 7.4 to 7.0 in shoots of rice, Oryza sativa L. var arborio, a species highly resistant to anoxia. Acidification was complete after 10 minutes of anoxia. Alkalinization of both cytosplasm and vacuole followed thereafter. In the anoxia intolerant wheat shoots, Triticum aestivum L. var MEK, the same treatment caused a sharper cytoplasmic acidification, from pH 7.4 to 6.6, which occurred during a period of 2 hours. Cytoplasmic acidification continued with progress of anoxia and there was no vacuolar alkalinization comparable to the one observed in rice. In wheat oxygen, withdrawal also caused the reduction of both glucose-6-phosphate level and of metabolic rate. It also induced heavy losses of inorganic phosphate from tissues. Conversely, in rice, glucose-6-phosphate level and metabolic rate were increased and inorganic phosphate leakage from tissues was completely absent. These results are discussed in relation to the mechanisms of plant resistance to anoxia.According to studies on maize root tips, resistance to anoxia resides in the ability of some plants in exploiting the initial, lactate-based cytoplasmic acidification, to stop further lactate production and to activate at the same time alcoholic fermentation (22). The the synthesis of new isozymes and lasted for days, suggesting a long-term function for LDH in plants subjected to ROA. In the highly anoxia-tolerant rice shoots, alcoholic fermentation started in the absence of (21) or with limited lactate production (14). Instead, a prolonged though low succinate production was found (16).The response of plants to ROA has been shown to be accompanied by a revolution in the expression of genetic information. In particular, it results in the increase in several glycolytic and fermentative enzyme activities (24). Apparently, the success of resisting to anoxia is mediated by an increase in carbon flux through glycolytic and fermentative chains. Such an increase may be enhanced by acclimatization of plants through pretreatment in hypoxia (10).Anoxia caused acidification of cell sap in shoot tissues of sensitive plants like wheat and barley. In contrast, cell sap alkalinization has been observed in resistant species like rice and Echinochloa crus-galli (14). Metabolic proton consumption has been indicated as an important device used by plants to counteract or prevent cell sap acidosis (14). In the present study we evaluated whether and to what extent cytoplasmic acidification was still induced by ROA in a resistant plant like rice in spite of the previously reported alkalinization of cell sap in this species (14,15 RESPONSE TO ANOXIA IN RICE AND WHEAT SEEDLINGSand circulation of incubation media (6). The central glass tube of the airlift system for the upward flow of gas and medium had a diameter of 5 mm. Gases were delivered inside this tube, just above the signal detecting zone at the rate of 5 ...

In Arabidopsis thaliana cells, hypoosmotic treatment initially stimulates Ca2+ influx and inhibits its efflux and, concurrently, promotes a large H2O2 accumulation in the external medium, representative of reactive oxygen species (ROS) production. After the first 10-15 min, Ca2+ influx rate is, however, lowered, and a large rise in Ca2+ efflux, concomitant with a rapid decline in H2O2 level, takes place. The drop of the H2O2 peak, as well as the efflux of Ca2+, are prevented by treatment with submicromolar concentrations of eosin yellow (EY), selectively inhibiting the Ca2+-ATPase of the plasma membrane (PM). Comparable changes of Ca2+ fluxes are also induced by hyperosmotic treatment. However, in this case, the H2O2 level does not rise, but declines below control levels when Ca2+ efflux is activated. Also K+ and H+ net fluxes across the PM and cytoplasmic pH (pH(cyt)) are very differently influenced by the two opposite stresses: strongly decreased by hypoosmotic stress and increased under hyperosmotic treatment. The H2O2 accumulation kinetics, followed as a function of the pH(cyt) changes imposed by modulation of the PM H+-ATPase activity or weak acid treatment, show a close correlation between pH(cyt) and H2O2 formed, a larger amount being produced for changes towards acidic pH values. Overall, these results confirm a relevant role for the PM Ca2+-ATPase in switching off the signal triggering ROS production, and propose a role for the PM H+-ATPase in modulating the development of the oxidative wave through the pH(cyt) changes following the changes of its activity induced by stress conditions.

The effects of extracellular K+ concentration ([K+]o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H+ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K+ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K+ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K+ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H+ extrusion and of K+ uptake. The correlation between these effects of [K+]o on intracellular pH and those on H+ extrusion indicates that changes in extracellular K+ concentration, and thus in K+ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H+ extrusion by the plasmalemma H+ pump.