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Oxygen uptake and energy charge were monitored during aging of excised maize root tips and related to the soluble sugar content and exogenous sugar supply.Oxygen uptake declined imsediately after excision to 50 to 30% of its initial value after 8 and 24 hours of aging at 25 C. There was also a sharp decline of the total sugar content (glucose, fructose, and sucrose). Starch content was very low at the time of excision and aimost neglgible 5 hours later. During the same period, the respiratory quotient declined from I to 0.75 and then remained stable.The addition of exogenous sugars induced a rapid rise of the respiratory rate which stabilized at a level correlated to the external sugar concentration. Addition of 0.2 molar glucose was necessary to restore the respiratory rate to the initial, also the maximum, level. These results indicate that metabolic activity of root tips is hily reliant on sugar import and carbohydrate reserves at the time of excision cannot compensate for the cessation of import. The control of respiration by substrate supply is in good agreement with the failure for dinitrophenol to stimulate oxygen uptake in aged sugar-depleted root tips.The energy charge remained constant at about 0.9, irrespective of the presence or absence of glucose and in spite of a large decline of respiratory activity in aged, sugar-depleted tissues.In normoxia as defined by Pradet and Bomsel (16), the respiratory rate of tissues is not limited by the 02 partial pressure.Under such conditions, most of the biological energy provided to the cells comes from sugar oxidation through respiratory pathways. The sugar supply of non-chlorophyllous tissues varies depending upon factors which affect the efficiency ofcarbon fixation by leaves and transport of the recently synthesized carbohydrates (5, 7, 13, 21).The question arises as to how metabolism adjusts to such fluctuation. Stimulation of root respiratory rate by light after darkness has been reported previously (6, 9). Hatrick and Bowling (10), using sunflower and barley, reported evidence for a complete dependence of root respiration on the rate of assimilate translocation from the shoot, suggesting that roots of young herbaceous plants have no reserves which might reduce the effect of fluctuations in the rate of translocation of photosynthetic sugars.The present study is part of an effort to understand the factors which limit and control the metabolic activity of roots. Our approach was to relate 02 uptake, taken as a measure of the metabolic activity, to soluble sugar and adenine nucleotide content of the tissues at various times after excision. The data reported demonstrate the influence of exogenous sugars on the metabolic activity of root tips. The significance ofenergy charge as parameter of normoxic cellular metabolism is discussed. MATERIALS AND METHODSPrimary root tips, 0.5 cm, were cut from maize seedlings (Zea mays L. INRA 402) germinated for 3 days at 25 C between sheets of filter paper soaked with 2 mm CaCl2.Determination of Gas Exchange. Measureme...

Severa1 enzyme activities were measured in extracts from acclimated and nonacclimated maize (Zea mays) root tips at pH 6.5 and 7.5, corresponding to cytoplasmic pH in anaerobiosis or aerobiosis, respectively, to determine what causes the decline of the glycolytic flux observed in anoxia in nonacclimated tips. We found that phosphorylation of hexoses by kinases was a major limiting step of glycolysis in anoxia. When fructose was substituted for glucose, glycolysis was slightly enhanced and survival improved, but neither matched that of acclimated tips. Decrease of kinase activities was not the result of proteolytic degradation but was more likely the result of inhibition by interna1 factors (low pH and low ATP). There was no evidence of induction during the hypoxic pretreatment of isoenzymes better adapted to the anoxic cellular environment. A preacclimation in hypoxia allows excised maize (Zea nzays) root tips supplemented with externa1 Glc to survive severa1 days in strict anoxia instead of less than 10 h in the absence of pretreatment Xia and Saglio, 1992). However, in the absence of exogenous Glc, they do not survive significantly longer than nonacclimated tips. We have shown that, below a critica1 threshold of glycolytic flux, survival was compromised (Xia et al., 1995). An hypoxic pretreatment induces an increase of maximal in vitro catalytic activities of many enzymes involved in sugar metabolism, a higher ATP level correlated with a higher energy charge (reviewed by Ricard et al., 1994), and a better regulation of cytoplasmic pH linked to an efficient efflux of lactic acid (Xia and Roberts, 1994;Xia et al., 1995;Xia and Saglio, 1992). However, the role of these modifications in the subsequent survival of tissues in anoxia is not obvious. For example, we have shown that in HPT root tips the level of ATP is not critica1 for survival or for cytosolic pH regulation in anoxia (Xia et al., 1995), and the increase in glycolytic enzyme activities is not correlated with a higher glycolytic rate during the 90 min after the transfer of tissues from air to strictly anoxic conditions. During this period, the glycolytic rate (measured as the sum of ethanol plus lactate) remains strictly identical in HPT and NHPT * Corresponding author; e-mail saglio@bordeaux.inra.fr; fax 56-84-32-45. root tips. It is only after 60 to 90 min that the glycolytic flux declines and then almost stops in NHPT root tips even in the presence of added Glc, whereas it remains sustained in HPT tips (Xia and Saglio, 1992). Such a decline of ethanol production during the hours or days after the transfer to anoxia appears to be common to many plants (Smith and ap Rees, 1979;Raymond et al., 1985; Kimmerer and Mac Donald, 1987). Exceptions are rice seeds and seedlings, which are resistant to anoxia and in which the rate of ethanol production increases during at least the first 2 d in the absence of O, (Raymond et al., 1985).How glycolytic flux is modified in HPT and NHPT root tips and what is responsible for its decline in NHPT tips early in anoxia (...

ABSTRACFYoung intact plants of maize (Zea mays L cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretratment) for 18 hours before excision of the 5 millimeter root apex and treatment with sictly anaerobic conditions (anoxia). Hypoxic accUmation pve rise to lrer amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips trferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was assoted with incsed activity of alcohol dehydrogenase (ADH), and indction of ADH-2 isozymes. Roots of Adbl-mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of N03-. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NOi-to NO2-. These tips (9-11, 25) is strongly suggestive of such acclimation, but the 02 concentrations to which cells were exposed during published experiments often were not controlled or defined closely, and were rarely, if ever, strictly anaerobic. Anaerobic polypeptides were induced by hypoxia in roots of rice (PO2 < 5 kPa) and wheat (PO2 < 2 kPa) continuously maintained at different constant concentrations of 02 (3, 4). However, the significance of these proteins, which include ADH2 and some of the glycolytic enzymes was not examined and remains unclear.Nitrate ion may play a special role during anoxia, by acting possibly as a terminal electron acceptor in respiration in the absence of molecular 02, with NADH-dependent reduction of nitrate to nitrite via NR in 'nitrate respiration' (7,12,18). It's significance lies in the regeneration of NAD+, essential for the continuation of glycolysis. Additionally, it has been suggested that competition for cytoplasmic pools of NADH between NR and ADH can occur, thereby lowering the rate of ethanol production (7,12 Anaerobic Treatment. Root apices, 5 mm long, were excised and placed in groups of 10 in 10 ml-volume glass vials with rubber puncture caps in 2.0 ml of the solution in which the roots had been pretreated, but supplemented with 100 mm glucose.Hypodermic needles through the rubber caps were used to gas the solution and headspace, one needle bubbling gas into the solution, the other to give gas exit from the vial. Equilibrium between these small volumes and a new gaseous atmosphere was attained in a few minutes. Roots from the fully aerobic and the hypoxic pretreatments were gassed either with 40 to 50% (v/v) 02 in N2, or with O2-free N2 (the anaerobic treatment, N2 = 99.99%). There was 3-fold replication.Extraction and Estimation of Adenine Nucleotides. Solution was forced out from each vial by the gas mixture with...

Hypoxic pretreatment (3 kPa oxygen) of maize (Zea mays L.) root tips improved their survival time in a subsequent anoxic incubation from 10 h to more than 3 d, provided that glucose was added to the medium to sustain metabolism. The glycolytic flux (lactate + ethanol) was the same in both pretreated and untreated root tips during the 1st h after transfer to anoxia. It was only after 2 h that it declined sharply in untreated tips, but was sustained in pretreated ones. Right after the transition from normoxia to anoxia of untreated root tips, the only fermentative product detected was lactic acid, which accumulated in a 7:1 proportion after 30 min in tissue and medium, respectively. It took 10 min before ethanol could be detected and 20 min for it to be produced at its maximum rate at the expense of lactate production, which slowed down. In contrast, in hypoxically pretreated root tips, ethanol was produced at a maximum rate right after the transfer to anoxia. Concurrently, low amounts of lactic acid were produced that accumulated in a 1:1 proportion after 30 min in tissue and medium, respectively. This large efflux of lactic acid could account for the higher cytoplasmic pH values always found in pretreated tissues. The presence of cycloheximide during pretreatment abolished this difference, suggesting that the greater efficiency of lactate efflux was linked to protein synthesis. The role of lactate in cytosolic pH regulation and in sensitivity to anoxia is discussed.Most mesophytes, including maize, are intolerant to anoxic conditions. However, pretreatment at low oxygen pressure induces mechanisms that improve their survival capacity under subsequent anoxic conditions. These mechanisms lead to two kinds of survival strategies. One consists of the formation of lysigenous cortical aerenchymas, helping to maintain a high respiration rate in the tissues by improving internal oxygen transport (5). This is a slow (days) and nonreversible mechanism induced by ethylene (4). The second strategy consists of the metabolic acclimation that apparently improves the energy relationships of anoxic tissues and contributes to a more prolonged survival in the absence of oxygen (9,25). This response is fast (hours) and involves the induction of a limited number of 'anaerobic polypeptides' (23). These polypeptides are different from the 'heat shock proteins ' (14), and those that have been identified are enzymes involved in the glycolytic and fermentative pathways (18,19,24). However, the significance of these proteins in tolerance to anoxia has rarely been examined and remains unclear. The results obtained by Hanson and colleagues (6, 7) on hypoxically inducible LDH' in barley roots and aleurone tissues imply some unrecognized function for this enzyme. Furthermore, there is evidence that ADH levels, which are known to increase dramatically during hypoxic acclimation, are not correlated with the better survival capacity of acclimated maize root tips in anoxia (22).Metabolic studies of survival of plant tissues in the absence...

The induction of the sucrose synthase (SuSy) gene (SuSy) by low O 2 , low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in doublemutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O 2 . In this situation, SuSy plays a critical role in anoxic tolerance.

Hypoxic pretreatment of tomato (Lycopersiron esculentum M.)roots induced an acclimation to anoxia. Survival in the absence of oxygen was improved from 10 h to more than 36 h if externa1 sucrose was present. The energy charge value of anoxic tissues increased during the course of hypoxic acclimation, indicating an improvement of energy metabolism. In acclimated roots ethanol was produced immediately after transfer to anoxia and little lactic acid accumulated in the tissues. In nonacclimated roots significant ethanol synthesis occurred after a 1-h lag period, during which time large amounts of lactic acid accumulated in the tissues. Severa1 enzyme activities, including that of alcohol dehydrogenase, lactate dehydrogenase, pyruvate decarboxylase, and sucrose synthase, increased during the hypoxic pretreatment. In contrast to maize, hexokinase activities did not increase and phosphorylation of hexoses was strongly inhibited during anoxia in both kinds of tomato roots. Sucrose, but not glucose or fructose, was able to sustain glycolytic flux via the sucrose synthase pathway and allowed anoxic tolerance of acclimated roots. These results are discussed in relation to cytosolic acidosis and the ability of tomato roots to survive anoxia.

Energy charge and fermentative metabolism under anoxia were monitored in excised maize root tips after various times of aging in air and were related to their soluble sugar content. The energy charge value, which was 0.9 in air irrespective of the time of aging, dropped to a lower value within minutes of transfer to a nitrogen atmosphere. This value was dependent upon sugar content of the tissues which was itself a function of aging. The energy charge value after transfer to nitrogen was 0.6 in freshly excised tissue but only 0.2 in tissue aged for 4 hours. When aged tissues supplied with 0.2 molar glucose were transferred to nitrogen, the energy charge was 0.6, irrespective of the time of aging. When 0.2 molar glucose was added under nitrogen, energy charge rose to 0.6. This rise was faster in root tips aged for 8 hours than those aged for 24 hours.The rate of ethanol plus lactate production (representing 60 and 10%, respectively, of the total sugar consumption in anoxia) was closely correlated to the level of energy charge. It is concluded that, in anoxia, there is a quantitative relationship between the energy charge value and the level of metabolic activity via fermentative pathways.