Plants are frequently subjected to ROA,' yet the mechanisms that permit some species to tolerate oxygen deprivation are poorly understood (7,8). Most studies have focused on plant roots (7,12), since these organs experience oxygen deprivation more frequently, as a result of waterlogged soil, for example. Seed germination and early growth of seed plants inhabiting particular ecological niches, such as rice fields and natural wetlands, are also affected by ROA. The same may be true of seedlings of crop plants, due to temporary soil waterlogging. It has been reported (15) that shoots resist ROA better than roots. In the case of rice and Echinochloa (3), shoot but not root growth occurred in the absence of oxygen. Therefore, biochemical mechanisms relevant to ROA resistance are likely to be better expressed in shoot rather than in root tissues (3,8,9).In the absence of oxygen the main pathway for energy production in plants is alcoholic fermentation. Lactic fermentation, however, was maintained in plants for purposes not completely understood. In maize root tips, transient lactate production was the induction signal for alcoholic fermentation (12). This model, however, seems inadequate to explain the prolonged lactate production in barley roots (7) and the limited cytoplasmic acidosis in maize root tips (1 1). Lactate, as opposed to ethanol, was a negligible source of ATP in maize and pea root tips, but appreciable in the roots of 'Abbreviations: ROA, restricted oxygen availability; GABA, yaminobutyric acid.Ranunculus sceleratus and Senecius aquaticus (13 and references therein).In animals with marked resistance to anoxia, lactic fermentation is frequently replaced by pathways leading to succinate (6). Advantages of this substitution are an increased amount of ATP generated per mole of substrate consumed and a reduced proton production per mole of ATP cycled (6).Succinate accumulation in higher plants under oxygen deficiency has been reported in only three cases: Fagopyrum aesculentum seedlings (4), Rhododendron ponticum leaves (2), and rice seedlings (9). In the last work, succinate accumulated in shoots, but not in roots. The succinate-lactate ratio was 5 to 1, suggesting that, in plants with high resistance to ROA, as in animals, succinate production may replace that of lactate. Here we report marked differences of anaerobic succinate-lactate production and of concomitant changes in cell sap pH between seedling shoots ofROA resistant and sensitive plants. MATERIALS AND METHODS

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 ...

Anaerobic production of succinate, a common feature in animals able to sustain anoxia, has seldom been reported in plants. By the use of 1H‐nuclear magnetic resonance spectroscopy we show here that succinate is produced by rice seedlings (Oryza sativa L. cv. Arborio) subjected to anoxic conditions. Starting from levels below I μmol (g fresh weight)−1 in air, after 48 h of anoxia the levels of alanine, succinate and lactate had increased to 23.8, 5.2 and 1.0 μmol (g fresh weight) −1, respectively, in shoot tissues. Succinate was accumulated in shoots, notably in the coleoptiles, but not in roots of the rice seedlings, suggesting its involvement in rice coleoptile elongation under anoxia. Other possible functions of succinate production in rice seedling, an organism highly tolerant to anoxia, are discussed.

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Seeds of the water plant Trapa natans L. (water chestnut) can germinate in strict anoxia. The seedlings show seminal roots growing upwards while shoot buds remain quiescent until O2 becomes available. Trapa seedlings are highly tolerant to anoxia. The rate of ethanol fermentation was 21.2 μmol (g FW)−1 h−1, while production of lactate was negligible and lower than that of succinate. The seminal root of Trapa compares better to the rice coleoptile rather than to the rice root, both functionally and as to the metabolic response to anoxia. The anaerobic germination of Nuphar luteum L. and Scirpus mucronatus L. was also characterized by a limited developmental program.