Anaerobic stimulation of root exudates and disease of peas

Summary The relationships between root exudation, root disease and anaerobic root stresses were investigated. Sand culture and mist chamber studies demonstrated that low O₂ and high CO₂ reduced plant growth and increased the exudation of ethanol, amino acids, and sugars by pea roots. The relative loss of ethanol by roots was much greater in treatments with atmospheres of N₂ containing 30% CO₂ than in treatments of air containing 30% CO₂ or N₂. Ethanol was not detected in the nutrient solution of aerated plant roots. Atmospheres of N₂ plus 30% CO₂ caused 500% greater mycelial growth ofFusarium solani f. sp.pisi and 400% more disease of inoculated pea roots. Relative losses of four amino acids and four sugars were much greater in atmospheres of N₂ plus 30% CO₂ than in N₂ or air.     

CO2- and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different Listeria monocytogenes Strains

Although carbon dioxide (CO₂) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO₂ concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N₂, and 100% CO₂. The CO₂-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO₂ as to other anaerobic, slightly acidic environments (100% N₂, pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO₂ and N₂ at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO₂-containing atmospheres. Together, our findings demonstrate that the CO₂-response is a partly strain-dependent complex mechanism. The possible links between the CO₂-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.     

Glucose metabolism in anaerobic rice seedlings

More than 80% of the radioactivity from [U-¹⁴C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO₂; less than 5% was recovered as water-soluble acidic components. Rates of ¹⁴CO₂ formation from [U-¹⁴C]glucose were similar in roots and coleoptiles in both N₂ and air atmospheres. More ¹⁴CO₂ was formed from [U-¹⁴C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ¹⁴CO₂ from [6-¹⁴C]glucose and [1-¹⁴C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Metabolic evidence for stelar anoxia in maize roots exposed to low o(2) concentrations

This investigation presents metabolic evidence to show that in 4- to 5-day-old roots of maize (Zea mays hybrid GH 5010) exposed to low external O₂ concentrations, the stele receives inadequate O₂ for oxidative phosphorylation, while the cortex continues to respire even when the external solution is at zero O₂ and the roots rely solely on aerenchyma for O₂ transport. Oxygen uptake rates (micromoles per cubic centimeter per hour) declined at higher external O₂ concentrations in excised segments from whole roots than from the isolated cortex; critical O₂ pressures for respiration were greater than 0.26 moles per cubic meter O₂ (aerated solution) for the whole root and only 0.075 moles per cubic meter O₂ for the cortex. For plants with their shoots excised and the cut stem in air, ethanol concentrations (moles per cubic meter) in roots exposed to 0.06 moles per cubic meter O₂ were 3.3 times higher in the stele than in the cortex, whereas this ethanol gradient across the root was not evident in roots exposed to 0 moles per cubic meter O₂. Alanine concentrations (moles per cubic meter) in the stele of roots exposed to 0.13 and 0.09 moles per cubic meter O₂ increased by 26 and 44%, respectively, above the levels found for aerated roots, whereas alanine in the cortex was unchanged; the increase in stelar alanine concentration was not accompanied by changes in the concentration of free amino acids other than alanine. For plants with their shoots intact, alcohol dehydrogenase and pyruvate decarboxylase activities (micromoles per gram protein per minute) in roots exposed to 0.13 moles per cubic meter O₂ increased in the stele by 40 to 50% over the activity in aerated roots, whereas there was no appreciable increase in alcohol dehydrogenase and pyruvate decarboxylase activity in the cortex of these roots. More convincingly, for roots receiving O₂ solely from the shoots via the aerenchyma, pyruvate decarboxylase in the cortex was in an “inactive” state, whereas pyruvate decarboxylase in the stele was in an “active” state. These results suggest that for roots in O₂-free solutions, the aerenchyma provides adequate O₂ for respiration in the cortex but not in the stele, and this was supported by a change in pyruvate decarboxylase in the cortex to an active state when the O₂ supply to the roots via the aerenchyma was blocked.     

Expression of Different Levels of Ethanologenic Enzymes from Zymomonas mobilis in Recombinant Strains of Escherichia coli

The expression of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II in Escherichia coli converted this organism from the production of organic acids to the production of ethanol. Ethanol was produced during both anaerobic and aerobic growth. The extent to which these ethanologenic enzymes were expressed correlated with the extent of ethanol production. The replacement of organic acids with ethanol as a metabolic product during aerobic and anaerobic growth resulted in dramatic increases in final cell density, indicating that these acids (and the associated decline in pH) are more damaging than the production of ethanol. Of the plasmids examined, the best plasmid for growth and ethanol production expressed pyruvate decarboxylase and alcohol dehydrogenase II at levels of 6.5 and 2.5 IU/mg of total cell protein, respectively.     

Effect of oxygen on photosynthesis by spinach leaf protoplasts

The photosynthetic CO₂ fixation by spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts was inhibited by substituting an atmosphere of N₂ with one of either air (21% O₂) or 100% O₂. The inhibitory effect of 100% O₂ was greater than that of air. The mode of inhibition by 100% O₂ and air was competitive with respect to CO₂; Ki(O₂) value was 0.32 mM at pH 7 and 0.28 mM at pH 8.5 The labeling patterns of compounds in protoplasts exposed to ¹⁴CO₂ in light after transferring them from N₂ to O₂ atmospheres were examined. There was no detectable ¹⁴CO₂ incorporation into glycolate under anaerobic and O₂ atmospheres; a more marked labeling of glycine occurred under an oxidative environment compared to that under the anaerobic condition, presumably because of a rapid transformation of glycolate to glycine in the protoplasts.     

淹水对两种甜樱桃砧木根系无氧呼吸酶及发酵产物的影响

以美早/东北山樱桃、美早/马哈利为试材,研究了淹水过程中两种甜樱桃砧木生长根、褐色木质根中无氧呼吸酶——丙酮酸脱羧酶(PDC)、乙醇脱氢酶(ADH)和乳酸脱氢酶(LDH)活性及褐色木质根的发酵产物——乙醛、乙醇和乳酸含量变化,结果表明:两类根系PDC、LDH活性均呈先升后降趋势,ADH活性变化在生长根中亦先升后降,而在褐色木质根中为上升趋势,三种酶活性变化幅度表现为生长根大于褐色木质根;美早/东北山樱桃两类根系中ADH和LDH活性增加幅度大于美早/马哈利,PDC则相反;两种砧木褐色木质根乙醛、乙醇含量呈升高趋势,乳酸含量先升后降;最终美早/东北山樱桃褐色木质根中乙醛含量低于美早/马哈利,乙醇含量则相反,而乳酸含量前者较早达峰值且高于后者峰值。     

Effects of anaerobiosis on carbohydrate oxidation by roots of Pisum sativum

The aim of this work was to discover the effects of anaerobiosis on the breakdown of sugars by the apical 6 mm of the roots of 5-day-old seedlings of Pisum sativum. Estimates of the maximum catalytic activities of alcohol dehydrogenase, lactate dehydrogenase, phosphoenolpyruvate carboxylase and NADP-specific malic enzyme showed them to be comparable to that of phosphofructokinase. Metabolism of sucrose-[U-¹⁴C] by excised apices was restricted by anoxia mainly to conversion to ethanol, CO₂ alanine and glycolytic intermediates. Measurements of metabolites over a period of 240 min after transfer of excised apices to nitrogen showed a marked and continual accumulation of ethanol, a smaller continual accumulation of alanine, a small initial rise in lactate and no detectable accumulation of malate or pyruvate. The rates of CO₂ production, of accumulation of ethanol and alanine, and of the labelling of these compounds by sucrose-[¹⁴C] declined markedly during the first 240 min of anaerobiosis. The conclusion is that under anaerobic conditions carbohydrate metabolism in the pea root apex is largely restricted to alcoholic fermentation, and, to a lesser degree, to alanine production.     

Effect of Anaplerotic Pathways Activation on CO<Subscript>2</Subscript>-dependent Anaerobic Glucose Utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains Deficient in the Main Pathways of Mixed Acid Fermentation

The effect of anaplerotic pathways activation on CO₂-dependent anaerobic glucose utilization by Escherichia coli strains deficient in the main fermentation pathways and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO₂ generation in the strains was ensured resulting from oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Sodium bicarbonate dissolved in the medium was used as an external source of CO₂. The genes of heterologous pyruvate carboxylase and native NADH-dependent malic enzyme were overexpressed in the strains to allow anaplerotic carboxylation of pyruvic acid to oxaloacetic or malic acid. The ability of the strains to reoxidize NADH utilizing carboxylation products was additionally increased due to enhanced expression of malate dehydrogenase gene. In the case of endogenous CO₂ formation, the activation of anaplerotic pathways did not cause a notable increase in the anaerobic glucose consumption by the constructed strains. At the same time, the expression of pyruvate carboxylase led to a pronounced decrease in the secretion of pyruvic acid with the concomitant increase in the yield of four-carbon metabolites. Further enhancement of NADH-dependent malic enzyme expression provoked activation of a pyruvate–oxaloacetate–malate–pyruvate futile cycle in the strains. The availability in the medium of the external CO₂ source sharply increased the anaerobic utilization of glucose by strains expressing pyruvate carboxylase. The activity of the futile cycle has raised with the increased malic enzyme expression and dropped upon enhancement of malate dehydrogenase expression. As a result, the efficiency of CO₂-dependent anaerobic glucose utilization coupled to the formation of four-carbon carboxylation products increased in the studied strains resulting from the primary anaplerotic conversion of pyruvic acid into oxaloacetic acid followed by the involvement of the precursor formed in NADH-consuming biosynthetic reactions dominating over the reactions of the revealed futile cycle.     

Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions

In Euglena gracilis, pyruvate:NADP⁺ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP⁺ oxidoreductase. In contrast, pyruvate:NADP⁺ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.     

Roles of alcohol dehydrogenase, lactate dehydrogenase and pyruvate decarboxylase in low-temperature sweetening in tolerant and susceptible varieties of potato (Solanum tuberosum)

Low-temperature sweetening (LTS) results when tubers of potato ( Solanum tuberosum ) are stored at temperatures below 9–10°C with the accumulation of sucrose and reducing sugars glucose and fructose. Our earlier study showed that the LTS-tolerant varieties have higher ethanol and lactate tissue levels compared with the LTS-susceptible variety Monona ( Blenkinsop et al. 2003 ), which led us to investigate the role of the anaerobic respiratory pathway in LTS tolerance. The anaerobic respiratory enzymes alcohol dehydrogenase (ADH), l -lactate dehydrogenase (LDH) and pyruvate decarboxylase (PDC) were, therefore, investigated in LTS-tolerant and -susceptible potato varieties. A positive correlation ( P ≤ 0.05) was observed between reducing sugar concentration and the K _M of PDC, with the LTS-tolerant ND 860-2 possessing a lower K _M and reducing sugar content than the LTS-susceptible Monona variety. The moderately LTS-tolerant variety, Snowden, exhibited intermediate behavior between the two aforementioned cultivars at 4°C. The isozyme profile of the tolerant varieties differed from the susceptible variety. Two groups of LDH isozyme families were observed in all varieties with the exception of ND 860-2, where the second group appeared only during low-temperature exposure. Moreover, the tolerant variety possessed one additional ADH isozyme. Gene expression levels of these enzymes were higher in ND 860-2 as compared with Monona at 4°C. The above results suggest that the anaerobic respiratory enzymes contribute to LTS-tolerance.     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

Effect of extra- and intracellular sources of CO<Subscript>2</Subscript> on anaerobic utilization of glucose by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains deficient in carboxylation-independent fermentation pathways

The effect of extra- and intracellular CO₂ sources on anaerobic glucose utilization by Escherichia coli strains deficient in the main pathways of mixed acid fermentation and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO₂ generation in the strains was ensured resulting from the oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Endogenous CO₂ formation by pyruvate dehydrogenase stimulated anaerobic glucose consumption by the strains due to the involvement in the fermentation process of condensation reactions between oxaloacetic acid and acetyl-CoA. The availability of an external CO₂ source (dissolved in medium sodium bicarbonate) promoted utilization of carbohydrate substrate by favoring the predominant participation in the fermentation of reactions directly dependent on phosphoenolpyruvate carboxylation. The positive effect of the availability of exogenous СО₂ was sharply decreased in recombinant strains with the impaired functionality of the reductive branch of the tricarboxylic acid cycle. As a result, intracellular СО₂ generation coupled to acetyl-CoA formation promoted anaerobic glucose utilization by cells of the corresponding mutants more markedly than the presence in the medium of dissolved sodium bicarbonate.     

Alcohol production from glucose and xylose usingEscherichia coli containingZymomonas mobilis genes

Summary AnEscherichia coli strain containing a recombinant plasmid encoding the pyruvate decarboxylase and alcohol dehydrogenase genes fromZymomonas mobilis metabolized glucose and xylose to near theoretical yields of ethanol. Enzyme activity measurements indicate high expression levels of both plasmid-encodedZymomonas proteins in the recombinantE. coli. The expression inE. coli is under the control of a promoter in theZymomonas sequence upstream of the pyruvate decarboxylase gene. The maximum ethanol level, using 4% glucose as substrate, was 1.8% (w/v) in anaerobic conditions. In aerobic conditions the natural repression ofE. coli alcohol dehydrogenase results in less ethanol production from clones expressing onlyZymomonas pyruvate decarboxylase.     

Transient releases of acetaldehyde from tree leaves − products of a pyruvate overflow mechanism?

Emissions of acetaldehyde from tree leaves were investigated by proton‐transfer‐reaction mass spectrometry (PTR‐MS), a technique that allows simultaneous monitoring of different leaf volatiles, and confirmed by derivatization and high‐performance liquid chromatography analysis. Bursts of acetaldehyde were released by sycamore, aspen, cottonwood and maple leaves following light–dark transitions; isoprene emission served as a measure of chloroplastic processes. Acetaldehyde bursts were not accompanied by ethanol, but exposure of leaves to inhibitors of pyruvate transport or respiration, or anoxia, led to much larger releases of acetaldehyde, accompanied by ethanol under anoxic conditions. These same leaves have an oxidative pathway for ethanol present in the transpiration stream, resulting in acetaldehyde emissions that are inhibited in vivo by 4‐methylpyrazole, an alcohol dehydrogenase (Adh) inhibitor. Labelling of leaf volatiles with ¹³CO₂ suggested that the pools of cytosolic pyruvate, the proposed precursor of acetaldehyde bursts, were derived from both recent photosynthesis and cytosolic carbon sources. We hypothesize that releases of acetaldehyde during light–dark transitions result from a pyruvate overflow mechanism controlled by cytosolic pyruvate levels and pyruvate decarboxylase activity. These results suggest that leaves of woody plants contribute reactive acetaldehyde to the atmosphere under different conditions: (1) metabolic states that promote the accumulation of cytosolic pyruvate, triggering the pyruvate decarboxylase reaction; and (2) leaf ethanol oxidation resulting from ethanol transported from anoxic tissues.     

Purification and characterization of pyruvate decarboxylase from sweet potato roots.

Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting.     

Effects of abscisic acid on rooting stem cuttings of sweet potato in open top chambers under enriched CO2 environment

Ten centimeter long stem cuttings of sweet potato (Ipomoea batatas L. cv. Georgia Jet) with intact apex and leaves were cultured in distilled water as well as in varying concentrations of abscisic acid (ABA) in open top chambers at 364, 438 and 666 cm³ m{-3}CO₂. Low concentration of ABA promoted rooting and elongation of roots at 364 cm₃ m{-3} CO₂ while rooting was suppressed at enriched levels of CO₂. However, biomass production in shoots and roots was higher in 666 than in 364 cm³ m^-3 CO₂.     

Performance and allocation patterns of the perennial herb,Plantago lanceolata,in response to simulated herbivory and elevated CO2 environments

Summary We tested the prediction that plants grown in elevated CO₂ environments are better able to compensate for biomass lost to herbivory than plants grown in ambient CO₂ environments. The herbaceous perennial Plantago lanceolata (Plantaginaceae) was grown in either near ambient (380 ppm) or enriched (700 ppm) CO₂ atmospheres, and then after 4 weeks, plants experienced either 1) no defoliation; 2) every fourth leaf removed by cutting; or 3) every other leaf removed by cutting. Plants were harvested at week 13 (9 weeks after simulated herbivory treatments). Vegetative and reproductive weights were compared, and seeds were counted, weighed, and germinated to assess viability.Plants grown in enriched CO₂ environments had significantly greater shoot weights, leaf areas, and root weights, yet had significantly lower reproductive weights (i.e. stalks + spikes + seeds) and produced fewer seeds, than plants grown in ambient CO₂ environments. Relative biomass allocation patterns further illustrated differences in plants grown in ambient CO₂ environments. Relative biomass allocation patterns further illustrated differences in plant responses to enriched CO₂ atmospheres: enriched CO₂-grown plants only allocated 10% of their carbon resources to reproduction whereas ambient CO₂-grown plants allocated over 20%. Effects of simulated herbivory on plant performance were much less dramatic than those induced by enriched CO₂ atmospheres. Leaf area removal did not reduce shoot weights or reproductive weights of plants in either CO₂ treatment relative to control plants. However, plants from both CO₂ treatments experienced reductions in root weights with leaf area removal, indicating that plants compensated for lost above-ground tissues, and maintained comparable levels of reproductive output and seed viability, at the expense of root growth.