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.     

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.     

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.     

Introduction of Alcohol Dehydrogenase in Lettuce Seedlings by Flooding Stress

Alcohol dehydrogenase (ADH), its isozyme profiles and ethanol concentration in lettuce (Lactuca sativa L.) seedlings subjected to flooding stress were determined. Flooding stress caused increases in ADH activity and ethanol concentration. By 48 h, ADH activity and ethanol concentration in the flooded seedlings increased 3.2- and 7.0-fold, respectively, in comparison with those in non-stressed seedlings. Five electrophoretically separable ADH bands were found in extract of the flooded seedlings, whereas only two or three ADH bands were found in extract of non-stressed seedlings. These results indicate that lettuce ADH may have a system of three-gene and six-isozyme, and the increase in ADH activity in the flooded seedlings may be due to increased synthesis of the enzyme.     

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.     

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.     

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.     

Hypoxia induces anoxia tolerance in roots and shoots of lupine seedlings

Lupine seedlings were exposed to 4 kPa partial pressure oxygen (hypoxically pretreated) for 18 hours before treatment with strictly anaerobic conditions (anoxia). Seedlings previously exposed to hypoxia were more tolerant than the controls (not hypoxically pretreated) to anoxic stress in both roots and shoots. Hypoxic pretreatment induced roots and shoots survival in anoxia. Improved viability of roots, following hypoxic pretreatment, was associated with increased activity of ADH. In nonacclimated roots and shots significant increase in LDH activity occurd during the first hours under anoxia but the in vitro activity of LDH was two orders of magnitude lower than that of ADH. The results are discussed in relation to the ability of lupine seedlings to survive anoxia.     

Organ-specific analysis of the anaerobic primary metabolism in rice and wheat seedlings. I: Dark ethanol production is dominated by the shoots

During anaerobiosis in darkness the main route for ATP production in plants is through glycolysis in combination with fermentation. We compared the organ-specific anaerobic fermentation of flooding-tolerant rice (Oryza sativa) and sensitive wheat (Triticum aestivum) seedlings. A sensitive laser-based photoacoustic trace gas detection system was used to monitor emission of ethanol and acetaldehyde by roots and shoots of intact seedlings. Dark-incubated rice seedlings released 3 times more acetaldehyde and 14 times more ethanol than wheat seedlings during anaerobiosis. Ninety percent of acetaldehyde originated from shoots of both species. In comparison to wheat shoots, the high ethanol production of rice shoots correlated with larger amounts of soluble carbohydrates, and higher activities of fermentative enzymes. After 24 h of anaerobiosis in darkness rice shoots still contained 30% of aerated ATP level, which enabled seedlings to survive this period. In contrast, ATP content declined almost to zero in wheat shoots and roots, which were irreversibly damaged after a 24-h anaerobic period. When plants were anaerobically and dark incubated for 4 h and subsequently transferred back to aeration, shoots showed a transient peak of acetaldehyde release indicating prompt re-oxidation of ethanol. Post-anoxic acetaldehyde production was lower in rice seedlings than in wheat. This observation accounts for a more effective acetaldehyde detoxification system in rice. Compared to wheat the greater tolerance of rice seedlings to transient anaerobic periods is explained by a faster fermentation rate of their shoots allowing a sufficient ATP production and an efficient suppression of toxic acetaldehyde formation in the early re-aeration period.Angelika Mustroph and Elena I. Boamfa contributed equally to the paper.     

Alcohol dehydrogenase and an inactivator from rice seedlings

Alcohol dehydrogenase (ADH) was measured in the various organs of rice seedlings (Oryza sativa) growing in air. In extracts from ungerminated seeds, the ADH is stable, but in extracts from seedlings more than 2 days old the enzyme initially present loses activity in a time- and temperature-dependent fashion, due to the presence of an inactivating component which increases with age in roots and shoots. The inactivation can be prevented completely by dithiothreitol, and when this is included in the extraction medium the apparent loss of total ADH in roots and shoots with age is not observed. In seedlings grown in N₂, ADH levels in coleoptile extracts are higher than those in air, the enzyme is stable, and no inactivator can be detected. When seedlings grown for 5 days in air were transferred to N₂ for 3 days, ADH levels increased and there was a decline in inactivator activity. Transfer back to air after 1 day in N₂ led to loss of the accumulated ADH and increase in inactivator. These reciprocal changes and the fact that the inactivator is absent from coleoptiles of seedlings grown in N₂ appear to suggest a regulatory role for the inactivator in vivo. However, it is clear that high levels of inactivator and ADH can exist in cells of seedlings grown in air for long periods without loss of enzyme activity, and it is argued that they must normally be separately compartmented.     

Germination of Wild Rice, Zizania aquatica, Seeds and the Activity of Alcohol Dehydrogenase in Young Seedlings

Seeds of wild rice (Zizania aquatka L.) which were stored at 3°C for c. 3 months after harvest did not germinate until portions of seed coat above the embryo region were removed. During the iirst few days following germination there was no apparent difference between seedlings germinated under normal, aerated and anaerobic conditions. After 14 days, plants maintained under well aerated conditions were the most fully developed. Unlike shoot growth, roots did not develop under reduced aeration. The activity of alcohol dehydrogenase (ADH) in extracts obtained from young Zizania seedlings was highly specific for ethanol as a substrate, for NAD as the coenzyme, and the optimum pH was c. 9.1. Enzyme activity changed with stage of growth and development, reaching maximum activity 8 days after germination. Aeration strongly inhibited ADH activity. Under anaerobic conditions the activity of ADH increased several fold over the activity of ADH extracted from shoots of seedlings maintained under normal conditions.     

Differential biochemical responses of wheat shoots and roots to nickel stress: antioxidative reactions and proline accumulation

Wheat (Triticum aestivum L. cv. ‘Zyta’) seedlings were treated with 10, 100 and 200 μM Ni. Tissue Ni accumulation, length, relative water content (RWC), proline and H₂O₂ concentrations as well as the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD) and glutathione S-transferase (GST) were studied in the shoots and roots after 6 days of Ni exposure. Treatment with Ni, except for its lowest concentration, resulted in a significant reduction in wheat growth. In comparison to the shoots, the roots showed greater inhibition of elongation, which corresponded with higher accumulation of Ni in these organs. Both shoots and roots responded to Ni application with a decrease in RWC and enhancement in proline concentration. Greater dehydration of the shoot tissue was accompanied by more intense accumulation of proline. Treatment of the wheat seedlings with the highest concentration of Ni led to about 60% increase in H₂O₂ concentration in both studied organs. Apart from CAT, constitutive activities of antioxidative enzymes were much higher in the roots than in the shoots. Exposure of the seedlings to Ni resulted in SOD activity decline, which was more marked in the roots. While the shoots showed a substantial decrease (up to 30%) in CAT activity, in the roots the activity of this enzyme remained unchanged. After Ni application APX, POD and GST activities increased several-fold in the shoots, whereas in the roots they were not significantly altered. The results suggest that differential antioxidative responses of the shoots and roots of wheat seedlings to Ni stress might be related to diverse constitutive levels of antioxidant enzyme activities in both organs.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

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.     

Rapid elicitation of second messengers by nanomolar doses of triacontanol and octacosanol

Triacontanol (TRIA) increases the dry weight and alters the metabolism of rice (Oryza sativa L.) seedlings within 10 min of application to either the shoots or roots. This activity is prevented if octacosanol (OCTA, C₂₈ primary alcohol) is applied with the TRIA on the roots or shoots. Triacontanol activity is also stopped if the OCTA is applied at least 1 min before the TRIA on the opposite part of the seedling.Triacontanol rapidly elicits a second messenger that moves rapidly throughout the plant resulting in stimulation of growth (dry-weight increase) and water uptake. Octacosanol also produces a second messenger that inhibits TRIA activity. We have named the putative secondary messengers elicited by TRIA and OCTA, TRIM and OCTAM, respectively. The water-soluble TRIM extracted from plants treated with TRIA increases the growth of rice seedlings about 50% more than extracts from untreated plants, within 24 h of application. Both OCTAM and OCTA inhibit the activity of TRIA but not of TRIM.The TRIA messenger was isolated from rice roots within 1 min of a foliar application of TRIA. The TRIM elicited by TRIA will pass through a 4-mm column of water connecting cut rice shoots with their roots and can also be recovered from water in which cut stems of TRIA-treated plants have been immersed. Triacontanol applied to oat (Avena sativa L.) or tomato (Lycopersicon esculentum Mill.) shoots connected to rice roots by a 4-mm water column also results in the appearance of TRIM in rice roots.Abbreviations OCTA octacosanol - OCTAM second messenger elicited by OCTA - TAS tallow alkyl sulfate - TRIA triacontanol - TRIM second messenger elicited by TRIA Michigan Agricultural Experiment Station Journal Article No. 12001     

In vitro translation of maize ADH: Evidence for the anaerobic induction of mRNA

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amounts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.Research was supported by NSF Grant PCM 76-11009. M.D.B. is supported by National Institute of Health Grant PHS 5 T32 GM07227-04. R.J.F. is a Predoctoral Trainee in Genetics supported by National Institute of Health Training Grants 82 and 7757 from the National Institute of General Medical Sciences.     

Induction of Nitrate Reductase and Peroxidase Activity in the Seedlings of Normal and High Lysine Maize

Steady state levels of in vivo nitrate reductase activity in the endosperm, scutella, roots and shoots of maize seedlings were higher in normal maize than those in high lysine maize. Activity of peroxidase in the roots, however, was higher in the high lysine cultivar. The nitrate reductase activity increased with the supply of nitrate in all parts of the seedlings of both cultivars, although the rates of increment in the endosperm were lower than those in scutella, roots and shoots. In relation to substrate concentration, a saturation was achieved at 5 to 10 mM of nitrate except in the endosperm, where activity increased slowly up to 100 mM at least. Final levels of enzyme activity were significantly higher in the scutella of normal than in that of high lysine seedlings. In vitro enzyme activity in the roots also increased with the supply of nitrate in both cultivars, reaching maximum at 5 to 10 mM nitrate.     

Structure,expression, chromosomal location and product of the gene encoding ADH1 inPetunia

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.     

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.