Hypoxic Induction of Anoxia Tolerance in Root Tips of Zea mays

When root tips of fully aerobic, intact maize (Zea mays L.) seedlings are made anaerobic, viability normally is only 24 hours or less at 25°C. We find that viability can be extended to at least 96 hours if seedlings are given a hypoxic pretreatment for 18 hours by sparging the solution with 4% O₂ in nitrogen (v/v) before anoxia. Fully aerobic root tips (sparged with 40% O₂) had very low alcohol dehydrogenase (ADH) activity (per gram root fresh weight), and the level remained low under anoxia. In hypoxically pretreated roots, however, high levels of ADH activity were induced, and activity rose further during the initial 24 hours of anoxia, and then remained high at about 20 times that of controls in 40% O₂. ADH activity in roots in solution sparged with air (21% O₂) was about three times that in 40% O₂. Improved viability of hypoxically pretreated root tips was associated with maintenance of a high energy metabolism (ATP concentration, total adenylates, and adenylate energy charge). Roots that were not pretreated lost 94% of the total adenylates and ATP at 24 hours of anoxia. The relation between induced ADH activity, energy metabolism, and improved anoxia-tolerance in acclimated maize root tips is discussed.     

Hypoxic induction of alcohol and lactate dehydrogenases in lupine seedlings

Seedlings of lupine (Lupinus luteus L. cv. Juno) were exposed for up to 96 hours to 1 to 2 kPa partial pressure oxygen (hypoxic treatment) and activities of alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) and their isoform profiles were determined. Roots of lupine seedlings were grown in a nitrogen flushed nutrient solution while their shoots were in air. Prolonged hypoxia led to a reduction of root elongation. This was accompanied by reduced increase in dry weight suggesting that insufficient carbohydrate supply was the cause of retarded growth of lupine roots. Hypoxically treated roots showed induction of ADH and LDH acivities. The maximum increase in LDH activity was low (2-fold) in contrast to ADH activity, which increased up to 7-fold. Hypoxic treatment of roots did not affect the activities of ADH and LDH in hypocotyls and cotyledons. Analysis of ADH and LDH activity gels indicated in roots 1 and 2 isoforms, respectively. The level of isozymes of both enzymes increased in roots upon exposure to hypoxic stress. Differences in isoenzymatic spectrum of ADH and LDH between roots, hypocotyls and cotyledons indicate organ specificity of isozymes of both enzymes. The importance of alcohol and lactate fermentation in roots to cope with hypoxic stress is discussed.     

Effects of Anoxia on Wheat Seedlings: II. INFLUENCE OF O2 SUPPLY PRIOR TO ANOXIA ON TOLERANCE TO ANOXIA, ALCOHOLIC FERMENTATION, AND SUGAR LEVELS

Prior exposure of roots of intact wheat seedlings for 15–30h to hypoxia (0016-006 mol m 02) greatly increased their toleranceto subsequent anoxia, as assessed by the ability of the rootsto elongate upon return to air. Such hypoxically pretreatedroots had 2–4-fold higher activities of pyruvate decarboxylase(PDC) and 35–l7-fold higher activities of alcohol dehydrogenase(ADH) in their 0–1 mm apices and 0–5 mm root tipsthan in apices and tips of roots pretreated in air (026–031mol m3 02). The ADH/ PDC ratio increased I 3–5-fold duringhypoxic pretreatment. Furthermore, the rate of alcoholic fermentationby 0–5 mm tips of the hypoxically pretreated roots was14-4-fold faster than in tips from aerobically pretreated roots.No consistent difference between 02 pretreatment was found foralcoholic fermentation by tissues taken between 10 and 20 mmfrom the root tip. The observed activities of PDC and rates of alcoholic fermentationindicate that alcoholic fermentation is usually rate-limitedby PDC in 0–1 mm apices and 0–5 mm tips of wheatroots. Comparisons with data in the literature indicate thatwheat has at most a small Pasteur effect, which may explainwhy wheat is more intolerant to anoxia than rice. Exogenous glucose delayed the loss of elongation potential inboth aerobically and hypoxically pretreated roots. In the absenceof glucose, more than 85% of aerobically pretreated roots hadlost their elongation potential after 9 h anoxia, compared with30% in the presence of glucose. After 21 h anoxia nearly allaerobically pretreated roots had lost their elongation potential,compared with 10% and 0% of hypoxically pretreated roots inthe absence and presence, of glucose, respectively. The protective effect of glucose was presumably not due to anendogenous sugar deficiency; at the start of anoxia, 0–1mm apices of aerobically pretreated roots contained sufficientsugar for 23 h of their measured rate of ethanol synthesis yet,85% of these apices had lost their elongation potential afteronly 9 h of anoxia. It is suggested that in wheat roots, lowrates of synthesis of ethanol and hence of ATP, lead to injuryof cells, in turn generating a requirement for exogenous glucose,despite high endogenous sugar concentrations. Key words: Wheat seedlings, anoxia, glucose, O₂ pretreatment, alcoholic fermentation     

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 NO₃⁻. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO₃⁻ to NO₂⁻. 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.     

Flooding induction of alcohol dehydrogenase in shoots and roots of barley seedlings

Barley (Hordeum vulgare) seedlings were exposed to flooding and activities of alcohol dehydrogenase (ADH) and their isoform profiles were determined. The flooding increased ADH activities in shoots and roots of the seedlings. By day 3, the activity increased to 4 and 3 times that of the initial level for the shoots and the roots, respectively. Only two bands of ADH isoform were found in the shoots and the roots of non-induced seedling, whereas five bands were identified in those of induced seedlings.     

Effect of anaerobiosis on alcohol dehydrogenase in oat seedlings

In order to clarify the induction of alcohol dehydrogenase (ADH) by anaerobiosis in oat (Avena sativa L.), the seedlings were exposed to anaerobiosis and activity of ADH and ADH isozyme profiles were determined. The anaerobiosis increased ADH activities in shoots and roots of the seedlings. By day 2, the activity increased 5 and 4 times in the roots and the shoots, respectively, compared with those under aerobic condition. Based on nondenaturing electrophoresis, ADH isozyme composition analysis revealed six bands consisting of a dimmer enzyme with submits encoded by three different Adh genes. Changes in staining intensity of the isozymes indicated that the increase in ADH activity in oat under anaerobiosis resulted from increased enzyme synthesis.     

Osmotic Stress Increases Alcohol Dehydrogenase Activity in Maize Seedlings

Maize (Zea mays L.) seedlings were exposed to osmotic stress, and alcohol dehydrogenase (ADH) activity and abscisic acid (ABA) concentration were determined. The osmotic stress increased ADH activities in both roots and shoots, whereas the increase was 2-fold greater in roots than the shoots. The stress also increased ABA concentration in both roots and shoots and the increase was greater in the roots than in the shoots.     

Metabolic adaptation to flooding stress in upland and lowland rice seedlings

Ability of metabolic adaptation in upland and lowland rice (Oryza sativa L.) seedlings to flooding stress was compared. Flooding stress increased alcohol dehydrogenase (ADH) activity and ethanol concentration in shoots and roots of the upland and lowland rice seedlings. The difference in ADH activity and ethanol concentration in shoots between the upland and lowland rice was not apparent. However, both ADH activity and ethanol concentration in roots of the lowland rice were 2-fold greater than those in roots of the upland rice, suggesting that flooding-induction of ethanolic fermentation in lowland rice roots may be significantly greater than that in the upland rice roots. Since flooding often causes the anaerobic conditions in rooting zone than aerial part of plants and ethanolic fermentation is essential to survive in the anaerobic conditions, the ability of metabolic adaptation in lowland rice seedlings to flooding stress may be greater than that in upland rice seedlings.     

Low-temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings

Low-temperature stress was shown to cause a rapid increase in steady-state levels of alcohol dehydrogenase-1 message (Adh1) and protein activity (ADH1) in maize (Zea mays) (B37N, A188) and rice (Oryza sativa) (Taipei 309, Calmochi 101) seedlings. Maize roots and rice shoots and roots from 7-day seedlings shifted to low temperature (10°C) contained as much as 15-fold more Adh1 mRNA and 8-fold more ADH1 protein activity than the corresponding tissues from untreated seedlings. Time-course studies showed that these tissues accumulated Adh1 mRNA and ADH1 activity severalfold within 4- to 8-hour, levels plateaued within 20 to 24 hours, and remained elevated at 4 days of cold treatment. Within 24 hours of returning cold-stressed seedlings to ambient temperature, Adh1 mRNA and ADH1 activity decreased to pretreatment levels. Histochemical staining of maize and rice tissue imprints showed that ADH activity was enhanced along the lengths of cold-stressed maize primary roots and rice roots, and along the stems and leaves of rice shoots. Our observations suggest that short-term cold stress induces Adh1 gene expression in certain plant tissues, which, reminiscient of the anaerobic response, may reflect a fundamental shift in energy metabolism to ensure tissue survival during the stress period.     

The expression and anaerobic induction of alcohol dehydrogenase in cotton

The alcohol dehydrogenase (ADH) system in cotton is characterized, with an emphasis on the cultivated allotetraploid speciesGossypium hirsutum cv. Siokra. A high level of ADH activity is present in seed of Siokra but quickly declines during germination. When exposed to anaerobic stress the level of ADH activity can be induced several fold in both roots and shoots of seedlings. Unlike maize andArabidopsis, ADH activity can be anaerobically induced in mature green leaves. Three major ADH isozymes were resolved in Siokra, and it is proposed that two genes,Adh1 andAdh2, are coding for these three isozymes. The genes are differentially expressed. ADH1 is predominant in seed and aerobically grown roots, while ADH2 is prominent in roots only after anaerobic stress. Biochemical analysis demonstrated that the ADH enzyme has a native molecular weight of approximately 81 kD and a subunit molecular weight of approximately 42 kD, thus establishing that ADH in cotton is able to form and is active as dimers. Comparisons of ADH activity levels and isozyme patterns between Siokra and other allotetraploid cottons showed that the ADH system is highly conserved among these varieties. In contrast, the diploid species of cotton all had unique isozyme patterns.This work was generously supported by an Australian Cotton Research Council Postgraduate Studentship.     

Hypoxic Induction of Anoxia Tolerance in Roots of Adh1 Null Zea mays L

Seedlings of alcohol dehydrogenase 1 null mutants (Adh1-) of Zea mays L., which fail to synthesize alcohol dehydrogenase 1 (ADH1) isozymes, were hypoxically acclimated by 18 h of exposure to an atmosphere of 4% (v/v) O2 in N2 at 25[deg]C. Their ability to tolerate subsequent anoxia by exposure to anaerobic (O2-free) conditions was compared with that of unacclimated seedlings that were transferred immediately from an atmosphere of 40% (v/v) O2 to anaerobic conditions. Only 10% of the root tips of unacclimated seminal roots survived 6 h of anoxia, whereas 70% of the hypoxically acclimated root tips were viable at 24 h. During anoxia, acclimated root tips had enhanced ADH activity compared with unacclimated root tips, through induction of Adh2. Despite this, enzyme activity was still only about 5% that of acclimated, wild-type root tips and about half that of unacclimated, wild-type root tips. During anoxia, acclimated Adh1- root tips showed a higher rate of anaerobic respiration and ethanol production, greater concentrations of ATP and total adenylates, and a greater adenylate energy charge compared with unacclimated root tips. These results suggest that although enhanced ADH activity may have raised fermentation rates in acclimated Adh1- tissues and thereby contributed to energy metabolism and viability, the high levels of ADH activity inducible in acclimated, wild-type maize root tips appear to be in excess of that required to increase rates of fermentation.     

Expression of alcohol dehydrogenase in rice embryos under anoxia

Summary Alcohol dehydrogenase (ADH) activity was present in roots and shoots of 48-h rice embryos and rose in response to anoxia. The increase was accompanied by changes in the ADH isozyme pattern. Translatable levels of mRNA for two ADH peptides increases as early as 1 h after the beginning of anoxic treatment. Adh mRNA was detected in aerobically grown rice embryos by hybridization to maize Adh1 cDNA: its level increased significantly after 3 h of anoxia.     

Evaluation of the importance of lactate for the activation of ethanolic fermentation in lettuce roots in anoxia

According to the Davies–Roberts hypothesis, plants primarily respond to oxygen limitation by a burst of lactate production and the resulting pH drop in the cytoplasm activates ethanolic fermentation. To evaluate this system in lettuce ( Lactuca sativa L.), seedlings were subjected to anoxia and in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) and concentrations of ethanol, acetaldehyde and lactate were determined in roots of the seedlings. The in vitro activities of ADH and PDC in the roots increase in anoxia, whereas no significant increase was measured in LDH activity. At 6 h, the ADH and PDC activities in the roots kept in anoxia were 2.8- and 2.9-fold greater than those in air, respectively. Ethanol and acetaldehyde in the roots accumulated rapidly in anoxia and increased 8- and 4-fold compared with those in air by 6 h, respectively. However, lactate concentration did not increase and an initial burst of lactate production was not found. Thus, ethanol and acetaldehyde production occurred without an increase in lactate synthesis. Treatments with antimycin A and salicylhydroxamic acid, which are respiratory inhibitors, to the lettuce seedlings in the presence of oxygen increased the concentrations of ethanol and acetaldehyde but not of lactate. These results suggest that ethanolic fermentation may be activated without preceding activation of lactate fermentation and may be not regulated by oxygen concentration directly.     

Regulation of root anaerobiosis and carbon translocation by light and root aeration in Isoetes alpinus

The activity of alcohol dehydrogenase (ADH) was measured in corms and roots of the submerged freshwater macrophyte Isoetes alpinus Kirk. growing in situ, and related to its capacity for internal oxygen transport and to carbohydrate translocation. ADH activity was present in roots but not corms at uniform activity (0.15–0.35 × 10^?6 mol g^?1 fresh weight s^?1) over the entire plant depth range (3–7 m depth), and was intermediate to that developed in excised roots after 1‐week exposure to either dissolved oxygen at air‐saturation or to anoxia. Responses of photosynthesis and root oxygen release to light intensity confirmed that shoot‐to‐root oxygen transport saturated at similar light intensities to photosynthetic oxygen evolution, but was positive in the dark and at irradiances below the compensation point for photosynthesis, due to contributions to transport by oxygen diffusion from the external medium. Transport of ¹⁴C‐labelled photo‐assimilates to roots nevertheless ceased when intact plants were exposed to a combination of leaf darkness and root external anoxia, even when high ¹⁴C concentrations were present in shoots, but remained high when the roots were provided with external oxygen. The lack of any control over permeability of the root surface to gases in this species suggested that ADH activity and reduced translocation is most likely caused by development of hypoxic tissues in the apical tissue. These results suggest that reductions in ambient light intensity may have indirect effects on I. alpinus viability by increasing the degree of root hypoxia and impairing carbon partitioning.     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

Distribution of alcohol dehydrogenase in roots and shoots of rice (Oryza sativa) and Echinochloa seedlings

Abstract Aerobically germinated seedlings of rice and Echinochloa were found to survive when placed in an anaerobic environment for 4 d, whereas pea and maize seedlings did not. Although root and shoot growth were inhibited in rice and Echinochloa under anaerobiosis, growth resumed when the seedlings were returned to aerobic conditions. Alcohol dehydrogenase (ADH) activity increased more, and protein synthesis was greater, in the shoots than in the roots under anaerobic conditions. These results suggest that, in anaerobiosis-tolerant species, ADH activity and protein synthesis in the shoots represents or results from metabolic adaptations to low oxygen. These results are discussed in terms of plant establishment and growth in a low-oxygen environment.     

Alcohol dehydrogenase activity in the roots of marsh plants in naturally waterlogged soils

The aim of this work was to discover whether oxygen tensions in the roots of marsh plants in flooded soils are high enough to allow fully acrobic metabolism. Activity of alcohol dehydrogenase (ADH), a protein synthesised in anoxic plants, was measured in roots of marsh plants growing in habitats where the availability of oxygen to the roots would be expected to differ. Roots of Carex riparia in standing water had ADH activities about 2.5 times higher than those of phosphofructokinase, and comparable to ADH activities of Poa trivialis, Urtica dioica and Ranunculus repens roots in dry soil. Removal of the oxygen supply via aerenchyma to Carex roots caused a 30-fold increase in ADH activity relative to that of phosphofructokinase. There was no change in ADH activity with depth in Carex roots in waterlogged soil, but in Filipendula ulmaria roots activity was 14 times higher below 10 cm depth than near the surface. Urtica roots in waterlogged soil had alcohol dehydrogenase activities 26 times higher than roots in dry soil, but for Poa and Ranunculus roots this figure was only 1.7 and 4.2, respectively. These results indicate that the oxygen tensions in the roots of marsh plants in waterlogged soil differ considerably among species. Ethanol was the major product of fermentation in roots of all species studied. There was no correlation between ADH activity and the rate of ethanol production under anoxia of Urtica roots. The physiological significance of high ADH activities in roots is thus unclear.Abbreviations ADH alcohol dehydrogenase - PFK phosphofructokinase - PFP pyrophosphate:fructose 6-phosphate phosphotransferase     

Der Einfluß der Wurzelanaerobiose auf Äthanol-, Laktat- und Glukosegehalte sowie auf die Aktivitäten von ADH und LDH in Weizenkeimpflanzen

The effect of anaerobiosis of wheat seedling roots during 6 consecutive days on contents of ethanol, lactate and glucose in roots and shoots and on the exudation of ethanol from roots to the medium was examined. Activities of alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) were determined. After 36 h of anaerobiosis the concentration of ethanol in roots increased temporarily about 6 times and after 6 days it decreased to the level of control plants. The exudation of ethanol from roots to the medium showed similar pattern. The content of lactate was unaffected by anaerobiosis. In contrast, the content of glucose in roots of seedlings increased already after 1 day of anaerobiosis about 2 times and this higher level of glucose was noticed during consecutive 5 days. Anaerobiosis of roots caused an increase in the activity of ADH in both roots and shoots but the increase was not related to the content of ethanol in tissues, or exudated to the medium. The activity of LDH was unaffected by this factor. The results are discussed in relation to the limitation of energy supply of plants grown under root anaerobiosis.