Effects of anoxia and flooding on alcohol dehydrogenase in roots of Glyceria maxima and Pisum sativum

Flood tolerant Glyceria maxima and intolerant Pisum sativum were compared in respect of the effects of anoxia and flooding on the maximum catalytic activities of alcohol dehydrogenase in their roots. Small (<73%) increases in enzyme activity occurred when excised roots of both species were incubated in nitrogen for up to 2 days. Further incubation in nitrogen rapidly and permanently damaged the roots of both species. Enzyme activity in flooded roots of Glyceria was about double that in corresponding non-flooded roots. A marginally greater difference was found for roots of Pisum. It was concluded that the two species respond so similarly to the above treatments that variation in the extent of induction of alcohol dehydrogenase is unlikely to be a significant factor in determining their ability to tolerate flooding.     

Alcohol Dehydrogenase Activity in Relation to Flooding Tolerance in Roots

The ability of a number of plants to grow under conditions ofexperimental flooding has been examined. There was an increasein ethanol production under anaerobic conditions in those specieswhose growth was reduced by flooding. The period of floodinginduced a marked increase in alcohol dehydrogenase activityof the roots of these plants. Plants not adversely affectedby flooding showed no increase in ethanol production and noinduction of alcohol dehydrogenase activity. It is suggestedthat species in which such activation occurs are excluded fromwet areas because of the accumulation of toxic quantities ofethanol.     

VARIATION IN ALCOHOL DEHYDROGENASE ACTIVITY AND FLOOD TOLERANCE IN WHITE CLOVER,TRIFOLIUM REPENS

Flooding results in induction of anaerobic metabolism in many higher plants. As an important component of anaerobic energy production, alcohol dehydrogenase (ADH) activity increases markedly in response to flooding in white clover, Trifolium repens. Significant inter-individual variation in flood-induced ADH activity exists in natural populations of T. repens. The genetic basis of this variation was analyzed by offspring-midparent regression of data from 75 greenhouse reared families; the estimated heritability of flood-induced ADH activity was 0.55 (±0.13). Genetic variation in flood-induced ADH activity has pronounced effects on physiological response and flood tolerance in this species. ADH activity is positively correlated with the rate of ethanol production, indicating that observed in vitro activity differences are manifested in in vivo physiological function. T. repens plants with higher ADH activities during flooding have greater flood tolerance (measured as growth rate when flooded/unflooded growth rate). Variation in ADH activity during flooding accounts for more than 79% of the variance in flood tolerance. On the basis of a limited field survey of populations occupying three sites differing in exposure to flooding conditions, individuals from site C, the most frequently flooded site, expressed significantly higher average ADH activity when flooded than individuals from site A, a site with no history of flooding. Since ADH activity levels are not correlated with electrophoretic mobility variation in T. repens, this work supports previous suggestions that regulatory variation in enzyme activity may play a central role in biochemical adaptations to environmental stress.     

Flooding Induced Increase in Alcohol Dehydrogenase Activity in Timothy and Ryegrass Seedlings

Two forage grasses, timothy (Phleum pratense L.) and ryegrass (Lolium multiflorum Lam.) were exposed to flooding, and activities of alcohol dehydrogenase (ADH) and their isozyme profiles were determined. The flooding stress increased ADH activities in both species. This increase was 2-times greater in timothy than in ryegrass. Only one ADH isozyme was found in non-flooded seedlings of both species, whereas two and four bands were identified in ryegrass and timothy seedlings, respectively, under flooding stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Elicitor-Induced Cinnamyl Alcohol Dehydrogenase Activity in Lignifying Wheat (Triticum aestivum L.) Leaves

The substrate-specific induction of wheat (Triticum aestivum L. cv Fenman) leaf cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was examined in relation to its role in regulating the composition of defensive lignin induced at wound margins. Treatment of wounds with a partially acetylated chitosan hydrolysate or spores of the nonpathogen Botrytis cinerea elicited lignification at wound margins and invoked significant increases in phenylalanine ammonia-lyase (EC 4.3.1.5), peroxidase (EC 1.11.1.7), and CAD activities. The substrate-specific induction of CAD with time was determined in elicitor-treated leaves and in excised lignifying wounds. In whole leaf extracts no significant increases in p-cou-maryl and coniferyl alcohol dehydrogenase activities were detectable, but a significant 5-fold increase in sinapyl alcohol dehydrogenase activity was evident 32 h after elicitor treatment. Similarly, fungal challenge resulted in elevated levels of only sinapyl alcohol dehydrogenase in whole-leaf extracts. In excised lignifying tissues p-coumaryl alcohol dehydrogenase levels were similar to those observed in healthy tissue. A small yet significant increase in coniferyl alcohol dehydrogenase was apparent, but the most dramatic increase occurred in sinapyl alcohol dehydrogenase activity, which increased to values approximately 10 times higher than the untreated controls. Our results show for the first time that CAD induction in lignifying tissues of wheat is predominantly attributable to highly localized increases in sinapyl alcohol dehydrogenase activity.     

Aerenchyma development and elevated alcohol dehydrogenase activity as alternative responses to hypoxic soils in the Piriqueta caroliniana complex

The ability of plants to make morphological or physiological adjustments in response to environmental cues allows them to survive and reproduce under a wide range of conditions. One stress that plants are often exposed to is soil oxygen depletion due to flooding. Plants can respond to hypoxic soils by producing oxygen-conducting aerenchymous tissue or through induction of enzymes in the ethanolic fermentation pathway. Here we use greenhouse experiments to examine flood responses in plants of the Piriqueta caroliniana (Turneraceae) complex, which occupy a range of moisture regimes. Morphotypes and hybrids in this complex exhibited contrasting responses to hypoxic conditions. Genotypes from flooded habitats developed aerenchyma and did not substantially elevate levels of alcohol dehydrogenase (ADH) activity, an enzyme associated with anaerobic respiration. Plants from drier sites, on the other hand, did not develop aerenchyma but had much higher levels of ADH activity. Plants with aerenchymous tissue had substantially higher rates of growth under sustained flooding. Results are consistent with the hypothesis that aerenchyma development is an effective strategy in habitats subject to persistent flooding, while elevating activity of enzymes for ethanolic fermentation is effective only under ephemeral flooding. The range of phenotypic responses observed illustrates contrasting adaptive strategies that can lead to habitat isolation and evolutionary divergence.     

涝渍胁迫对不同树种生长和能量代谢酶活性的影响

为了解涝渍条件下不同树种的耐涝性和适应性,通过田间模拟试验,分析了涝渍胁迫对1年生落羽杉、美国山核桃和乌桕实生苗生长及能量代谢酶的影响.试验分为对照、渍水和淹水3个处理,处理时间为60 d.结果表明：在渍水和淹水条件下,3种树种的相对生长率均表现为落羽杉>美国山核桃>乌桕,落羽杉耐涝性最强,乌桕耐涝性最弱.涝渍条件下,3种树种的根冠比显著增加,更多的光合产物被分配到根系.3树种乙醇脱氢酶和乳酸脱氢酶活性显著升高,其中耐涝性强的落羽杉增加幅度不大,但一直维持在较高水平;而乌桕和美国山核桃在处理初期增幅较大,在处理后期呈下降趋势.3种树种的苹果酸脱氢酶、磷酸己糖异构酶和葡萄糖-6-磷酸脱氢酶-6-磷酸葡萄糖酸脱氢酶的活性均低于对照,其中落羽杉降幅最低,淹水条件下分别下降35.6%、21.0%和22.7%.耐涝性强的树种能够通过自身的调节,维持各种能量代谢途径的强度,为树种在低氧条件下的生命活动提供能量,进而维持一定的生长量.     

Distinctive Features of the Functioning of Lactic Acid and the Alcohol Fermentation Enzyme in Sorghum and Pea Leaves under the Conditions of Oxygen Deficiency

Dynamic changes in the enzymatic activity of lactate dehydrogenase, L-lactate-cytochrome-c-coxidoreductase, and alcohol dehydrogenase were analyzed in the seedlings of sorghum, a plant resistant to oxygen deficiency, and peas, a plant vulnerable to oxygen deficiency, cultivated under the conditions of flooding. The subcellular localization and isoform composition of the enzymes were characterized. The mechanism underlying the adaptive reaction of cell metabolism was devised from analysis of the results obtained. The reaction is supposed to involve lactate dehydrogenase and L-lactate-cytochrome-c-oxidoreductase enzymes that suppress cytoplasm acidification in sorghum and pea roots in the case of flooding.     

Parameters affecting the early seedling development of four neotropical trees under oxygen deprivation stress

Some of the parameters that determine flooding resistance-and consequently habitat zonation-were investigated in four neotropical trees (Schizolobium parahyba, Sebastiania commersoniana, Erythrina speciosa and Sesbania virgata). The constitutive parameters of seeds (size, nature and amount of reserves) only partly influenced resistance to flooding, mainly through a high carbohydrate : size ratio. Parameters describing metabolic efficiency under stress conditions were more important. Among them, fermentation capacity and levels of ATP and of total adenylates played a key role. The highest resistance to anoxia was associated with increased availability of free sugars, elevated alcohol dehydrogenase activity and corresponding mRNA levels, more efficient removal of ethanol and lactate, and higher adenylate levels. Finally, as a lethal consequence of energy shortage, free fatty acids were released on a massive scale in the flooding-sensitive species Schizolobium parahyba, whereas lipid hydrolysis did not occur in the most resistant species Sesbania virgata.     

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.     

Inter-specific analysis of Drosophila alcohol dehydrogenase by an immunoenzymatic assay using monoclonal antibodies

Three Drosophila alcohol dehydrogenase monoclonal antibodies have been prepared and characterized. These antibodies cross-react with alcohol dehydrogenase from different species as revealed by immunoblotting assay. An enzyme-linked immunosorbent assay has been devised to quantify alcohol dehydrogenase in several species, different strains and individual larval organs. The assay detects alcohol dehydrogenase via a double-antibody sandwich assay technique giving strictly proportional values for antigen concentration and optical densities in the range of 3-30 ng of antigen per 100 microliters of sample. When alcohol dehydrogenase specific activity is compared in different larval organs a remarkable similarity is observed, whereas protein distribution varies substantially. Larval fat body and larval alimentary canal contribute 63% and 26% respectively to recovered alcohol dehydrogenase.     

Anaerobic metabolism enzymes as markers of flooding stress in maize seeds

Maize (Zea mays L.) seeds differ in their relative tolerance to the anaerobic environment caused by flooding. Seed tolerance to flooding stress depends on cellular and metabolic processes since gross anatomical responses have not developed at the pre-emergence stage. The study reported here characterizes the activities of four anaerobic respiratory enzymes: pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and malic enzyme (ME) in the flood-tolerant A632 and floodsusceptible Mo 17 inbred maize seeds during flooding at 10 and 25°C. Each inbred consisted of two seed lots possessing 95% and 75% germination levels. Flooding increased the activities of all four enzymes. However, no consistent correlation between anaerobic enzyme activity and flood tolerance was observed across genotype, seed quality and flooding temperature. The results indicate that it may not be feasible to use whole-seed anaerobic enzyme activities to predict maize seed performance under flooding stress. Contribution from the Soil Drainage Research Unit, USDA-ARS, Columbus, OH, in cooperation with the Ohio Agricultural Research and Development Center, The Ohio State University. OARDC Journal Article No. 66–86.     

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.     

Activities of enzymes of fermentation pathways in the leaves and roots of contrasting cultivars of sorghum (Sorghum Bicolor L.) during flooding

Flooding evoked a differential response in the activities of enzymes of fermentation pathway in leaves and roots of flood sensitive (S-308) and flood-tolerant (SSG-59-3) cultivars of sorghum. Activities of alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) and alanine aminotransferase (AlaAT) enhanced in roots of SSG-59-3 during 72 h of flooding. In contrast, a transient increase in the activities was discerned in roots of S-308 up to 24 h flooding followed by a decline in activities of these enzymes. In leaves of SSG-59-3, the activities of ADH and LDH increased to about three fold during flooding stress as compared to that in the non-flooded control plants. Though elevation in activities of these enzymes was observed in leaves of S-308 up to 48 h of flooding, the magnitude of enhancement was much lower than that in SSG-59-3. Alanine aminotranferase activity depressed in leaves of both the cultivars but the level of decline was more pronounced in sensitive cultivar S-308 as compare to tolerant SSG-59-3. The amount of alcohol, lactic acid and alanine were higher in both roots and leaves of SSG-59-3 than that in S-308 during flooding stress. It is thus apparent that roots and leaves of flood tolerant variety tends to attain greater capacity to perform reactions of various fermentation pathways to sustain production of ATP under flooded conditions.     

Physiological and morphological responses of red alder and sitka alder to flooding

Red alder (Alnus rubra Bong.) and sitka alder (A. viridis ssp. sinuata [Regel] Löve & Löve) are nitrogen-fixing woody species that grow sympatrically along the Pacific coast of North America. Red alder is found in poorly drained lowlands, as well as in soils of moist upland slopes, whereas sitka alder generally colonizes well-drained soils. To identify factors that contribute to flood tolerance, we conducted greenhouse experiments subjecting both species to a 20-day flood and 10-day recovery and red alder to a 50-day flood and 20-day recovery. We determined the effect of this stress on nitrogenase activity, root and nodule alcohol dehydrogenase (ADH) activity, lenticel and adventitious root development, relative growth rate (RGR), and leaf gas exchange. After 24 h of flooding, nitrogenase activity could not be detected in either species. Limited nitrogenase activity did return in red alder at the end of a 10-day recovery following the 20-day flood, but sitka alder showed no recovery of nitrogenase activity. After 50 days of continuous flooding, red alder nitrogenase activity returned to pretreatment levels. Red alder root and nodule ADH activity was more than twice that of sitka alder under flooded conditions. Sitka alder showed extensive root mortality and leaf abscission over the same 20-day flooding period. Flooded red alder exhibited an initial decline in root RGR, but recovered between days 10 and 20 with the formation of adventitious roots. Furthermore, initiation of adventitious roots in red alder coincided with an increase in stomatal conductance without a similar recovery of carbon dioxide exchange rate. Sitka alder formed few adventitious roots, lost much of its root and leaf biomass, and showed no restoration of growth during flooding or recovery. Different responses of red and sitka alder to flooding serve as a partial explanation for the different patterns of distribution of these species and suggest some adaptations of red alder that permit flood tolerance.     

Pathways of carbohydrate fermentation in the roots of marsh plants

We did this work to discover the pathways of carbohydrate fermentation in unaerated roots of three species of flood-tolerant plants, Ranunculus sceleratus, Glyceria maxima, and Senecio aquaticus. The experiments were done with the apical 1–2 cm of the roots and the results for the three species were similar. The maximum catalytic activities of alcohol dehydrogenase, lactate dehydrogenase, phosphoenolpyruvate carboxylase, NADP-dependent malic enzyme, and phosphofructokinase were appreciable and roughly comparable. Reduced aeration of the roots led to 1.5 to 5-fold increases in the maximum catalytic activities of alcohol dehydrogenase, small increases in those of lactate dehydrogenase in two species, and no increase in those of phosphoenolpyruvate carboxylase and phosphofructokinase. Phosphoenolpyruvate carboxykinase could not be detected. Metabolism of [U-¹⁴C]sucrose under anaerobic conditions by excised roots, grown without aeration, led to appreciable labelling of ethanol and alanine, slight but significant labelling of lactate, and minimal labelling of malate and related organic acids. Incubation of similar excised roots under anaerobic conditions for 4 h caused marked accumulation of ethanol, smaller accumulation of lactate, and no detectable accumulation of malate. We conclude that in all three species fermentation of carbohydrate results in the accumulation of predominant amounts of ethanol, smaller amounts of lactate, no significant quantities of malate, and probably appreciable amounts of alanine. Crawford's metabolic theory of flooding tolerance is held to be incompatible with these results.Abbreviations MES 2-(N-morpholino)ethanesulphonic acid - MOPS 2-(N-morpholino)propanesulphonic acid     

Alcohol dehydrogenase from Drosophila funebris and Drosophila immigrans: Molecular and evolutionary aspects

Alcohol dehydrogenase from Drosophila funebris and D. immigrans is evident at all developmental stages. The highest activity level appears in third-instar larvae and declines to a lower level at all later stages of development. Both species are monomorphic. The enzyme is a dimer consisting of two identical subunits with molecular weight 27,600. The pI values are 8.6 for D. funebris and 9.02 for D. immigrans. The optimum pH is 8.6 and 8.7 for D. funebris and D. immigrans, respectively. The Km values for NAD+, propan-2-ol, and butan-2-ol are 0.15, 2.90, and 2.08 mM, respectively, for D. funebris and 0.16, 1.53, and 1.49 mM, respectively, for D. immigrans. The half-life for the purified enzyme is 45 days for D. funebris and 18 days for D. immigrans at 4 degrees C. Data on the amino acid composition of both enzymes and peptide maps of alcohol dehydrogenase of D. immigrans reveal that they have marked homologies between them and also with alcohol dehydrogenases of other species. D. funebris shows reduced levels of alcohol dehydrogenase synthesis but has the highest specific activity reported to date for a Drosophila species. D. immigrans synthesises six times more enzyme but the specific activity is comparable to that of other species of Drosophila. This evidence could explain their different alcohol tolerance. The molecular properties of these alcohol dehydrogenases together with other species of Drosophila suggest that the alcohol dehydrogenase of Drosophila has arisen by divergent evolution from a common ancestral gene.     

Ethanol synthesis and loss from flooded roots of Medicago sativa L. and Lotus corniculatus L.

Abstract Field flooding of established alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) for up to 12 d resulted in a significant increase in alcohol dehydrogenase activity (ADH) and an increase in the K_m of ADH in both species. Root concentration of ethanol increased throughout the flooding regime in alfalfa roots. No ethanol was detected in any trefoil root samples. Alfalfa plants which had shoots removed 5 d prior to flooding accumulated significantly higher levels of root ethanol and showed flooding injury sooner, indicating a significant effect of shoots on development of flooding injury. Alfalfa and trefoil plants grown in the greenhouse were flooded and ethanol in the transpiration effluent was trapped and measured. Alfalfa transpired measurable quantities of ethanol which peaked just prior to development of shoot injury symptoms. No ethanol was detected in the transpiration effluent from trefoil shoots. Flooded roots of both alfalfa and trefoil excreted ethanol but alfalfa roots synthesized more total ethanol and retained a larger proportion in the roots than did trefoil. While the ethanol accumulation response in alfalfa and trefoil are consistent with the ethanol ‘self-poisoning’ hypothesis of flooding injury, the very small quantities of ethanol found in these roots still raises questions as to its absolute effect in the plant.     

Characterization of alcohol dehydrogenase in young soybean seedlings

Molecular properties of alcohol dehydrogenase (ADH) were examined in young soybean seedlings. Soybean radicle tissue is ADH-rich. Enzyme specific activity decreases slowly with the development of roots and becomes almost undetectable when the first true leaves appear. Soybean ADH was not found to be inducible by flooding. 2,4-Dichlorophenoxyacetic acid (2,4-D) treatment increased ADH specific activity as much as 14-fold. Only one ADH isozyme was detected by isoelectric focusing. By DNA-DNA hydridization, soybean ADH genomic sequences were shown to be partly homologous to maize ADH1 cDNA. The presence of more than one Adh gene in soybean is discussed.