Differential induction of pyruvate decarboxylase subunits and transcripts in anoxic rice seedlings.

In 2-d-old rice (Oryza sativa L.) seedlings subjected to anoxic stress, pyruvate decarboxylase (PDC) activity increased 9-fold during a 168-h period. A polyclonal PDC antiserum that recognized alpha- and beta-subunits was used to quantify PDC protein by an enzyme-linked immunosorbant assay and showed a 5.6-fold increase, suggesting that the anoxically induced enzyme has a higher specific activity than the PDC isoform present under normoxia. Immunoblot analysis showed that levels of both PDC subunits were induced by anoxia. Immunoprecipitation of proteins labeled in vivo during anoxic treatment demonstrated that the alpha-subunit was preferentially synthesized at the onset of anoxia. Two partial cDNAs, including a novel sequence, were cloned from a cDNA library made from seedlings subjected to anoxia for 6 h. Gene-specific probes used to quantify northern blots showed that two or three PDC mRNAs are differentially induced by anoxia in rice seedlings. Immunoprecipitation of in vitro translation products of mRNAs isolated a different times of anoxic treatment confirmed this findings Our results suggest that anoxic induction of rice PDC involves transcriptional and posttranscriptional regulation of gene expression as well as differences in enzyme characteristics.

Purification and biochemical characterization of a vacuolar serine endopeptidase induced by glucose starvation in maize roots.

An endopeptidase (designated RSIP, for root-starvation-induced protease) was purified to homogeneity from glucose-starved maize roots. The molecular mass of the enzyme was 59 kDa by SDS/PAGE under reducing conditions and 62 kDa by gel filtration on a Sephacryl S-200 column. The isoelectric point of RSIP was 4.55. The purified enzyme was stable, with no auto-proteolytic activity. The enzyme activity was strongly inhibited by proteinaceous trypsin inhibitors, di-isopropylfluorophosphate, 3,4-dichloroisocoumarin and PMSF, suggesting that the enzyme is a serine protease. The maximum proteolytic activity against different protein substrates occurred at pH 6.5. With the exception of succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin, no hydrolysis was detected with synthetic tryptic, chymotryptic or peptidylglutamate substrates. The determination of the cleavage sites in the oxidized B-Chain of insulin showed specificity for hydrophobic residues at the P2 and P3 positions, indicating that RSIP is distinct from other previously characterized maize endopeptidases. Both subcellular fractionation and immuno-detection in situ indicated that RSIP is localized in the vacuole of the root cells. RSIP is the first vacuolar serine endopeptidase to be identified. Glucose starvation induced RSIP: after 4 days of starvation, RSIP was estimated to constitute 80% of total endopeptidase activity in the root tip. These results suggest that RSIP is implicated in vacuolar autophagic processes triggered by carbon limitation.

Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose.

Metabolic pathways of the intermediate metabolism of maize root tips were identified and quantified after labeling to isotopic and metabolic steady state using glucose labeled on carbon-1, -2, or -6 with 14C or 13C. The specific radioactivity of amino acids and the 13C-specific enrichment of specific carbons of free glucose, sucrose, alanine and glutamate were measured and used to calculate metabolic fluxes. The non-triose pathways, including synthesis of polysaccharides, accumulation of free hexoses, and to a lesser extent starch synthesis, were found to consume 75% of the glucose entering the root tips. The cycle of synthesis and hydrolysis of sucrose was found to consume about 70% of the ATP produced by respiration. The comparison of the specific radioactivities of amino acids and phospholipid glycerol phosphate after labeling with [1-(14)C] or [6-(14)C]glucose revealed the operation of the pentose phosphate pathway. The transfer of label from [2-(14)C]glucose to carbon-1 of starch glucosyl units confirmed the operation of this pathway and indicated that it is located in plastids. It was found to consume 32% of the hexose phosphates entering the triose pathways. The remaining 68% were consumed by glycolysis. The determination of the specific enrichment of carbohydrate carbons -1 and -6 after labeling with [1-(13)C]glucose indicated that both the conversion of triose phosphates back to hexose phosphates and the transaldolase exchange contributed to this randomization. Of the triose phosphates produced by glycolysis and the pentose phosphate pathway, about 60% were found to be recycled to hexose phosphates, and 28% were directed to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to be directed through the pyruvate kinase branch and one-third through the phosphoenolpyruvate branch. The latter essentially has an anaplerotic function since little malate was found to be converted to pyruvate (malic enzyme reaction).

Molecular cloning and characterization of six cDNAs expressed during glucose starvation in excised maize (Zea mays L.) root tips.

In order to isolate glucose-starvation-related cDNAs in maize (Zea mays L.) root tips, a cDNA library was constructed with poly(A)+ mRNA from 24 h starved root tips. After differential screening of the library, we isolated six different cDNAs (named pZSS2 and pZSS7) which were expressed during glucose starvation. Time course analysis revealed that maximum expression of five of these genes occurs 30 h after the onset of the starvation treatment. On the contrary, the expression of mRNAs corresponding to pZSS4 was maximal at an early stage of starvation and then dramatically decreased. The expression of this gene did not seem to be specific for glucose starvation. The pattern of induction of the genes corresponding to pZSS2, pZSS3, pZSS5, pZSS6 and pZSS7 revealed that non-metabolizable sugars such as L-glucose and mannitol induce mRNA transcription similarly to glucose starvation. When D-glucose or any other metabolizable sugar was supplied, the level of transcripts was reduced. Nucleotide sequence analyses of the six cDNAs allowed identification of five of them by comparison with sequence data bases. The protein encoded by clone pZSS2 is analogous to a wound-induced protein from barley. Clones pZSS4 to pZSS7 encode, respectively, a transmembrane protein, a cysteine protease, a metallothionein-like protein and a chymotrypsin/subtilisin-like protease inhibitor. Clone pZSS3 shares no significant homology with any known sequence.

Effects of glucose starvation on the oxidation of fatty acids by maize root tip mitochondria and peroxisomes: evidence for mitochondrial fatty acid beta-oxidation and acyl-CoA dehydrogenase activity in a higher plant.

Fatty acid beta-oxidation was studied in organellar fractions from maize root tips by h.p.l.c. and radiometric analysis of the products of incubations with [1-14C]octanoate and [1-14C]palmitate. In crude organellar fractions containing both mitochondria and peroxisomes, octanoate and palmitate beta-oxidation, as determined by the production of acetyl-CoA, was functional and, for palmitate, was activated 4-12-fold after subjecting the root tips to 48 h of glucose starvation. The sensitivity to a 'cocktail' of respiratory-chain inhibitors containing cyanide, azide and salicylhydroxamate depended on the conditions of incubation, with no inhibition in a medium facilitating peroxisomal beta-oxidation and a significant inhibition in a medium potentially facilitating mitochondrial beta-oxidation. Indeed, preparations of highly purified mitochondria from glucose-starved root tips were able to oxidize octanoate and palmitate to give organic acids of the tricarboxylic acid cycle. This activity was inhibited 5-10-fold by the above cocktail of respiratory-chain inhibitors, with no parallel accumulation of acetyl-CoA, thus showing that the inhibition affected beta-oxidation rather than the pathway from acetyl-CoA to the organic acids. This provides the first evidence that the complete beta-oxidation pathway from fatty acids to citrate was functional in mitochondria from a higher plant. Moreover, an acyl-CoA dehydrogenase activity was shown to be present in the purified mitochondria. In contrast with the peroxisomal activity, mitochondrial beta-oxidation showed the same efficiency with octanoate and palmitate and was strictly dependent on glucose starvation.

Effects of glucose starvation on mitochondrial subpopulations in the meristematic and submeristematic regions of maize root.

Mitochondria isolated from 3-mm long maize (Zea mays L. var Dea) root tips were found to be heterogeneous on Percoll density gradients. The ultrastructure of these isolated mitochondria correlated well with that of mitochondria observed in situ and was consistent with the existence of mitochondria at different stages of maturation during cell development. The mitochondria of higher density presented an ultrastructure with many cristae and a dense matrix. These mitochondria showed classic respiratory properties, although with low ADP/O ratios. In contrast, the mitochondria of lower density showed few cristae and a clear matrix and did not seem to be fully functional because their rate of respiration was low and showed weak respiratory control. Lower- and higher- density mitochondria were shown to be differentially affected during the first stages of glucose starvation. The higher-density mitochondria from glucose-starved maize root tips retained the ultrastructure and most of the respiratory properties of nonstarved mitochondria, whereas lower- and intermediate-density mitochondria were absent in the mitochondrial preparations from glucose-starved maize root tips and were not observed in situ. Quantitatively, there was a decrease of the total mitochondrial pool when expressed as the amount of mitochondrial protein per root tip. However, this decrease affected low- and intermediate-density mitochondria, but not higher-density mitochondria. Thus, it was shown that a significant pool of functional mitochondria is maintained in maize root tips during the first stages of glucose starvation. The reasons for these apparently selective effects of glucose starvation on mitochondria are discussed in relation to effects on mitotic and differentiation processes.

Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips.

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO(2) by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the beta-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall beta-oxidation activity was assayed as the formation of [(14)C]acetyl-CoA from [(14)C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the beta-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.

Effects of anoxia on mitochondrial biogenesis in rice shoots: modification of in organello translation characteristics.

Shoots of germinating rice (Oryza sativa L.) seedlings are able to grow under anoxia and to withstand long periods of anoxic treatment. Mitochondria were purified from aerobically germinated and anaerobically treated rice shoots by differential and isopycnic centrifugation and were found to consist of two subpopulations. The mitochondrial subpopulation of higher density was used for further characterization. Ultrastructural studies showed anaerobic mitochondria to be significantly different from aerobic mitochondria, with a matrix of lower density and more developed cristae. Aerobic and anaerobic mitochondria also differed in their specific activities for fumarase and succinate dehydrogenase, which were significantly lower after the anoxic treatment. In vivo labeling of seedlings with l-[(35)S]methionine and subsequent isolation of the mitochondria indicated that anoxia induced a drastic decrease, but not a total inactivation, of the synthesis of mitochondrial proteins. In organello protein synthesis showed that anaerobic mitochondria were able to synthesize most of the polypeptides synthesized by aerobic mitochondria, although only in the presence of exogenous ATP, as would occur under anoxia. Anaerobic mitochondria, but not aerobic mitochondria, could carry out protein synthesis without a functional respiratory chain. Thus, mitochondrial protein synthesis was found to be potentially functional in the rice shoot under anoxia.

Asparagine metabolism and nitrogen distribution during protein degradation in sugar-starved maize root tips.

Excised maize (Zea mays L.) root tips were used to monitor the effects of prolonged glucose starvation on nitrogen metabolism. Following root-tip excision, sugar content was rapidly exhausted, and protein content declined to 40 and 8% of its initial value after 96 and 192 h, respectively. During starvation the contents of free amino acids changed. Amino acids that belonged to the same "synthetic family" showed a similar pattern of changes, indicating that their content, during starvation, is controlled mainly at the level of their common biosynthetic steps. Asparagine, which is a good marker of protein and amino-acid degradation under stress conditions, accumulated considerably until 45 h of starvation and accounted for 50% of the nitrogen released by protein degradation at that time. After 45 h of starvation, nitrogen ceased to be stored in asparagine and was excreted from the cell, first as ammonia until 90-100 h and then, when starvation had become irreversible, as amino acids and aminated compounds. The study of asparagine metabolism and nitrogen-assimilation pathways throughout starvation showed that: (i) asparagine synthesis occurred via asparagine synthetase (EC 6.3.1.1) rather than asparagine aminotransferase (EC 2.6.1.14) or the ß-cyanoalanine pathway, and asparagine degradation occurred via asparaginase (EC 3.5.1.1); and (ii) the enzymic activities related to nitrogen reduction and assimilation and amino-acid synthesis decreased continuously, whereas glutamate dehydrogenase (EC 1.4.1.2-4) activities increased during the reversible period of starvation. Considered together, metabolite analysis and enzymic-activity measurements showed that starvation may be divided into three phases: (i) the acclimation phase (0 to 30-35 h) in which the root tips adapt to transient sugar deprivation and partly store the nitrogen released by protein degradation, (ii) the survival phase (30-35 to 90-100 h) in which the root tips expel the nitrogen released by protein degradation and starvation may be reversed by sugar addition and (iii) the cell-disorganization phase (beyond 100 h) in which all metabolites and enzymic activities decrease and the root tips die.

Kinetic studies of the variations of cytoplasmic pH, nucleotide triphosphates (31P-NMR) and lactate during normoxic and anoxic transitions in maize root tips.

We have followed the dynamic evolution of intracellular pH and of the intracellular concentration of nucleotides (NDP, NTP), Pi and lactate in maize root tips during the course of normoxia and anoxia transition. The intracellular pH, determined from the 31P-NMR chemical shift of the cytoplasmic P1 peak, dropped from 7.5 to 6.9 during the first few minutes after anaerobiosis. It increased again, then settled to a steady-state value of 7.1-7.2, 25 min after the beginning of the anoxic treatment. Following oxygenation, the chemical shift of the cytoplasmic Pi peak drifted gradually to its initial value. The cytoplasmic pH followed an oscillatory time course which was almost identical to the time course of NTP. Intracellular lactate accumulated steadily during the first 30 min after anaerobiosis, then its intracellular concentration remained almost constant. Following oxygenation, the intracellular concentration of lactate decreased slowly. The cytoplasmic pH followed a time course which was not identical to the time course of lactate. Following hypoxia, the pH dropped to low values long before the intracellular lactate concentration reached a steady-state equilibrium. Conversely, subsequent to oxygenation, the pH returned to normal values long before lactate. These results do not agree with the statement that cytoplasmic acidification in hypoxic maize root tips is necessarily associated with lactic acid synthesis.

Study of glucose starvation in excised maize root tips.

Excised maize (Zea mays) root tips were used to follow the effects of a prolonged glucose starvation. Respiration rate began to decrease immediately after excision, reaching 30 to 40% of its initial value after 20 hours, and then declined more slowly until death of the tissues, which occurred after 200 hours of starvation. During the whole process, respiration could be uncoupled by 2,4-dinitrophenol and the energy charge remained high. These results suggest that in excised maize root tips, respiration rate is essentially limited by the rate of biosyntheses (ATP-utilizing processes) rather than mitochondrial number. During starvation the sugar content sharply decreased for the first 20 hours and reached zero at 120 hours. Following root excision, proteins and lipids were continuously degraded and were virtually the only substrates for respiration and biosyntheses after 20 hours of starvation. Over the first 90 hours of starvation, enzymic activities related to sugar metabolic pathways and the Krebs cycle decreased to 20% or less of their initial activity. Starvation was reversible only for the first 80 to 90 hours. Between 80 and 100 hours, there was a sharp fall in intracellular osmolarity and a 25% loss in the dry weight. The irreversibility may be due, as in senescence, to a change in membrane selective permeability.

Anaerobic stress induces the transcription and translation of sucrose synthase in rice.

Sucrose synthase activity increased in 2-day-old rice (Oryza sativa) seedlings submitted to anaerobic stress. Likewise, both denaturing and native Western blot analysis detected a rise in the cellular concentration of sucrose synthase protein. Significantly higher steady-state levels of sucrose synthase mRNA, as determined by Northern blots and by the ability of total RNA to direct in vitro synthesis of sucrose synthase, were also induced by anaerobic treatment. Analysis of run-on transcripts showed increased transcription of sucrose synthase genes as early as 60 minutes after initiation of anaerobic stress. Together, these results indicate that sucrose synthase is a typical anaerobic protein in rice.

Lactate Dehydrogenase in Oryza sativa L. Seedlings and Roots: Identification and Partial Characterization.

A lactate dehydrogenase activity is present in rice (Oryza sativa L.) seedlings and roots. Under aerobic conditions, lactate dehydrogenase activity is barely detectable in rice seedlings and is very low in rice roots. In 30 day old roots, the activity is increased two to three times by an anoxic or hypoxic treatment and can be detected on immunoblots by an antiserum raised against barley lactate dehydrogenase. The activity present in aerobic seedlings was partially purified. The native enzyme has a molecular mass of 160 kilodaltons, and is a tetramer of 2 subunit (38 and 39 kilodaltons) randomly associated. Studies of substrate specificity, native gel electrophoresis, and immunoblot analysis indicate that the partially purified enzyme is a typical lactate dehydrogenase. However, no increase of lactate dehydrogenase activity or protein was observed in seedlings transferred to anoxia.

Oxidative phosphorylation by mitochondria extracted from dry sunflower seeds.

The role of mitochondria in the phosphorylation of ADP to ATP in the early steps of seed germination has been studied. Mitochondria were extracted from dry sunflower (Helianthus annuus) seeds. Adenylate kinase-dependent ATP synthesis was inhibited by p(1),p(5)-di(adenosine-5')pentaphosphate. Synthesis of ATP was observed with the different substrates: citrate, alpha-ketoglutarate, succinate, malate, pyruvate or NADH. This synthesis was activated by cytochrome c, and inhibited by cyanide, oligomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and carboxyatractyloside. The ATP/O values with succinate were 0.85 and 1.2 in the absence or presence, respectively, of cytochrome c. Electron micrographs showed that mitochondria of dry tissues have different structures when observed in situ or in vitro after aqueous extraction, suggesting that profound changes occurred after the contact with the aqueous medium. These results confirm previous data obtained in vivo showing that mitochondria present in dry seeds are able to synthesize ATP as soon as the seeds are rehydrated.

Purification and partial characterization of pyruvate decarboxylase from Oryza sativa L.

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 mu kat/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC, the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxia and would be activated by small changes in pyruvate concentration.

Effects of metabolic inhibitors and hypoxia on the ATP, ADP and AMP content of the rabbit carotid body in vitro: the metabolic hypothesis in question.

The effects of metabolic inhibitors (cyanide, antimycin) and hypoxia on the nucleotide content of the carotid body were investigated in vitro. The mean ATP content of carotid bodies superfused for 1 h in normoxic conditions was around 200 pmol/organ. Whereas metabolic inhibitors induced a decrease in ATP and an increase in AMP, hypoxia (10% O2 in N2, either 4 or 30 min) did not induce any significant change in nucleotide content. The significance of these results is discussed with regard to the metabolic hypothesis.

Artefactual Origins of Cyclic AMP in Higher Plant Tissues.

A highly sensitive radioimmunoassay has been used to determine the levels of adenosine 3',5'-cyclic monophosphate (cAMP) in five higher plants (Lactuca sativa, Helianthus annuus, Oryza sativa, Pinus pinaster, Nicotiana tabacum). Particular attention was paid to the three main sources of errors in the characterization of cAMP in plants: presence of interfering substances in plant tissues; possible artefactual formation of cAMP from endogenous ATP during extraction, purification, and assay; and microbial origin of cAMP. In all the tested tissues, the cAMP level was below the detection limit of 0.5 picomole per gram fresh weight, a value much lower than those reported for similar materials of the same species in many previous studies. This result is not in favor of cAMP-dependent regulations in higher plants.

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase contains two subunits differentially regulated by anaerobiosis.

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is composed of two subunits of different molecular weights. Cytosolic GAPDH activity and protein both decreased immediately after transfer of 48-h rice seedlings to anaerobic conditions. Subsequent increase in activity and protein was accompanied by a change in isoenzyme profile and was preceded by an increase in steady-state messenger levels. One and two-dimensional electrophoretic analyses of in vivo and in vitro labeled GAPDH suggested that the change in isoenzyme profile under anaerobic conditions is due to preferential synthesis of one of the two GAPDH subunits caused by a specific increase in its mRNA.

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos.

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.

Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays.

Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger 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 transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1(-) 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 NO(3) (-). Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO(3) (-) to NO(2) (-). These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl(-) mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.