Some effects of oxygen concentration on levels of respiratory intermediates in buckwheat seedlings

Changes in levels of glycolytic and some related respiratory intermediates in young seedlings of buckwheat (Fagopyrum esculentum) following transfer A) from air to anoxia, B) from air to 1.5% O₂, C) from 1.5% O₂ to anoxia and D) from anoxia to air, are recorded and discussed in relation to other measurements made with these seedlings.

On transfer from air to anoxia the changes are similar to those recorded for pea seeds in which it is inferred that glycolysis is faster in anoxia than in air. The results with buckwheat however can be explained in terms of a decreased rate of glycolysis in anoxia. An alternative hypothesis is developed which states that glycolysis is faster in anoxia than in air and that it is the contribution of the pentose phosphate cycle to the total respiratory catabolism which decreases as the oxygen concentration is reduced towards zero.

     

Effect of Water Stress on the Carbohydrate Metabolism of Citrullus lanatus Seeds during Germination

Gluconeogenesis in Citrullus lanatus seeds is a post germinative event. Increases in isocitrate lyase activity and incorporation of radioactivity from [2-¹⁴C]acetate into sugars occur only after radicle emergence. During germination, the seeds appear to rely on carbohydrate as the respiratory substrate. At this time, glycolysis, the pentose phosphate pathway, and the tricarbocyclic acid cycle seem to be functional. Utilization of raffinose during germination appears to be important.

Water stress, which completely inhibits germination, has a marked effect on carbohydrate metabolism. The rate of ¹⁴CO₂ release from [2-¹⁴C]acetate, [1-¹⁴C]glucose, and [6-¹⁴C]glucose is lower in the stressed seeds than the control seeds during the respiratory lag phase. However, in the stressed seeds neither glycolysis, the pentose phosphate pathway, nor the tricarboxylic acid cycle is completely inhibited. In contrast to the control seeds in which raffinose content sharply declines after 12 h of incubation, raffinose content in the stressed seeds remains fairly constant.

The respiratory lag phase of the control seeds coincides with a lower reducing substance content, glucose content, and fructose content than in the stressed seeds during the corresponding incubation period.

     

Effects of Oxygen Concentration on the Respiration of Excised Root-Tip Segments of Maize and Rice, and of Germinating Grains of Rice and Buckwheat

In view of the known tolerance of rice (Oryza sativa) to wet soils, in contrast to maize (Zea mays), a comparison of the respiratory responses of the two plants to different O₂ concen trations was made. It was found that if the O₂ concentration was raised to 100%, an increase was observed of the O₂ input and the CO₂ output in maize root-tip segments and in germinating grains of rice and also in buckwheat (Fagopyrum sp.). In marked contrast rice root-tip segments, when treated with 100% O₂, exhibited no such increase over the air controls. Increased O₂ concentrations decreased RQ values ranging from 2.0 (with 5% O₂) to 0.86 (with 100% O₂). Air-control RQ values were lowest with maize roots (0.9) and highest with germinating rice grains (1.0). Rice grains also exhibited the highest F/R ratio (0.85), and buckwheat grains the lowest (0.45). The addition of sucrose to maize root tips tended to promote fermentation (CO₂ output) rather than aerobic respiration (O₂ input).     

A new mathematical approach to the metabolism of [14C]glucose, with applications to sensory ganglia of chicken embryos

Abstract— The available models of carbohydrate metabolism are not suitable for analysis of experiments on dorsal root ganglia of chicken embryos because they assume that certain products of the pentose cycle mix freely with those of glycolysis, which appears not to be true in this tissue, and because full isotopic equilibration, needed before the start of measurements, is not achieved while the excised ganglia are reasonably fresh. Therefore, new equations were developed which assume only a steady state of relevant metabolic intermediates and make use of the process of isotopic equilibration as a source of information. It is also assumed that an initially unknown but calculable fraction of the products of each pentose cycle re-enters the next cycle, the remainder leaking either to glycolysis or to the incubation medium. From measurements of the time course of output of labelled CO₂ in the presence of [1-¹⁴C]- and [6-¹⁴C]glucose and the incorporation and release in lactate of labelled C from [l-¹⁴C]glucose, the equations permit the estimation of many features of carbohydrate metabolism, such as the partitioning of material between the pentose cycle and glycolysis, the partitioning of CO₂ output between the pentose and citric acid cycles, the partitioning of the products of glycolysis between CO₂ and other destinations, such as lactate, and the degree of recycling from one pentose cycle into the next. In addition, the time course of labelled CO₂ output from [2-¹⁴C]glucose can be predicted; this, by comparison with the observed output, serves to support some variants of the basic model, while invalidating others. In dorsal root ganglia from 15-day chicken embryos, the assumption of a metabolic steady state was supported by a constant output of labelled CO² from [l-¹⁴C]glucose for 15 or more hours, except for the initial period of isotopic equilibration. By use of the new equations, it is concluded that in these ganglia (a) recycling in the pentose cycle can be 100% efficient in some incubation conditions, but not in others, (b) more CO₂ is released from the pentose cycle than from the citric acid cycle, (c) large, quantifiable differences exist between the utilization of the various carbon atoms of glucose, and (d) a pool of intermediates within the pentose cycle, with a time constant of about 1 h, explains a large delay observed in the output of C-6 of glucose into CO², which occurs with a time constant as long as 5 h under some conditions. Under conditions where recycling is complete in the pentose cycle, this cycle must operate in isolation from glycolysis, which would otherwise convert much of the output of the pentose cycle to lactate. This may explain the role of fructose-1,6-diphospha-tase in the tissue, without recourse to the oft-proposed, puzzling, and ATP-degrading‘futile cycle’between fructose-6-P and fructose-1,6-diP. It is proposed that the new equations may be suitable for similar analyses on some, but not all, other tissues.     

Photosynthesis in Rhodospirillum rubrum. I. Autotrophic Carbon Dioxide Fixation

The incorporation and distribution of activity from ¹⁴CO₂ was investigated under autotrophic conditions in the facultative photoautotroph, Rhodospirillum rubrum, with cells cultured on hydrogen, carbon dioxide, and ammonium sulfate. In 1 second ¹⁴CO₂ fixation experiments essentially all of the activity was found in 3-phosphoglyceric acid: plotted against time percent incorporation into phosphate esters has a strikingly negative slope. These results suggest that under autotrophic conditions the reductive pentose phosphate cycle or the key reactions of the cycle play a major role in carbon metabolism in this photosynthetic bacterium. Incorporation into amino acids and into intermediates of the tricarboxylic acid cycle was quite low.     

Variations in short term products of inorganic carbon fixation in exponential and stationary phase cultures of Aphanocapsa 6308

Aphanocapsa 6308 metabolizes both NaHCO₃ and Na₂CO₃. The short term incorporation (5-s) metabolic pattern and the patterns of incorporation of bicarbonate for exponential versus stationary phase cultures differ, however. Cells were equilibrated for 10 min in air and distilled water prior to injection of either NaH¹⁴CO₃ at pH 8.0, or Na₂ ¹⁴CO₃ at pH 11.0. Hot ethanol extracts were analyzed via paper chromatography and autoradiography for products of CO₂ fixation. At 5 s, malate (51.5%) predominates slightly as a primary bicarbonate fixation product over 3-phosphoglycerate (40.3%); 3-phosphoglycerate is the primary product of carbonate fixation. At 60 s, the carbonate and bicarbonate labelling patterns are similar. Cells in stationary phase fix in 5 s a greater proportion of bicarbonate into malate (36% vs. 14% for 3-phosphoglycerate) than do cells in exponential growth. Likewise, 60 s incorporations show a large amount of bicarbonate fixed into aspartate (30.9%) in stationary phase cells over that of exponential phase (11.6%). These data suggest an operative C₄ pathway for purposes not related to carbohydrate synthesis but rather as compensation for the incomplete tricarboxylic acid cycle in cyanobacteria. The enhancement of both aspartate fixation and CO₂ fixation into citrulline in stationary phase correlates with an increase in cyanophycin granule production which requires both aspartate and arginine.Nonstandard Abbreviations 3-PGA 3-phosphoglyceric acid - TCA tricarboxylic acid     

Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) Seedlings : Intermediary Metabolism and Ethanol Tolerance

Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O₂.

Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O₂. Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.

Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [¹⁴C]sucrose primarily to CO₂ and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [¹⁴C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O₂. Lipids were also labeled when seeds were supplied with [¹⁴C]ethanol or [¹⁴C]acetate. Labeling experiments using the above compounds plus [¹⁴C]NaHCO₃, showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.

The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O₂. In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O₂.

     

Role of carbon dioxide in germination of spores of Streptomyces viridochromogenes

CO₂ in required continuously during germination of Streptomyces viridochromogenes spores. Spores incubated in a defined germination medium in the absence of CO₂ remain phase bright and do not release spore carbon. In the presence of CO₂, the spores initiate germination accompanied by loss of refractility and spore carbon. The CO₂ requirement is replaced by oxaloacetate or a mixture of tricarboxylic acid cycle (TCA) intermediates. Labeled CO₂ is taken up by germinating spores, and is incorporated into protein and RNA. TCA cycle intermediates and related amino acids contain most of the acid-soluble label following short term exposures of germinating spores to ¹⁴CO₂. TCA cycle inhibitors repress germination and ¹⁴CO₂ uptake whereas folic acid antagonists do not. The results indicate that CO₂ is incorporated into oxaloacetate which is converted to biosynthetic intermediates required for germination. Operation of the TCA cycle appears to be essential for spore germination. The conclusion is reached that CO₂ is required during germination in order to maintain the cycle by an anaplerotic reaction.Abbreviations SN sucrose-nitrate medium - TX buffer Trisbuffer pH 7.3 containing-Triton X-100 - DGM defined germination medium - TX salts TX buffer plus Mg and Ca ions - TA trichloroacctic acid - TCA tricarboxylic acid     

Effects of anoxia and post-anoxia recovery on carbohydrate metabolism in the jaw muscle of the crab Chasmagnathus granulatus maintained on carbohydrate-rich or high-protein diets

The present study assesses the effects of 1-h anoxia and 3-h post-anoxia recovery period on the activity of pyruvate kinase (PK), ¹⁴CO₂ produced from ¹⁴C-glucose and ¹⁴C-lactate, ATP, and glycogen levels in the jaw muscle of Chasmagnathus granulatus fed either a carbohydrate-rich (HC) or high-protein (HP) diet.In the HC control group the jaw muscle PK activity was higher than in HP-fed crabs. In jaw muscle from control HP-fed crabs the lactate oxidation was higher than in HC-fed animals. We observed increased PK activity and ATP concentration and a reduction in the glycogen concentration, ¹⁴CO₂ production from ¹⁴C-lactate in HP-fed crab jaw muscle during anoxia. In crabs fed an HC diet the PK activity decreased in anoxia, the ¹⁴CO₂ production from ¹⁴C-glucose increased, and the ¹⁴CO₂ production from ¹⁴C-lactate did not change.During recovery, a low oxidation capacity for lactate was found in jaw muscle of both dietary groups. PK activity and ¹⁴CO₂ production from ¹⁴C-glucose were high during the recovery period only in the jaw muscle from HP-fed crabs.Recovery decreased the ATP concentration of both dietary groups as compared to anoxia and normoxia, and did not restore the glycogen concentration in the jaw muscle.     

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 CO2 absorption on the measurement of CO2 evolution rate in aerobic and anaerobic continuous cultures

A general and simple equation is presented to account for the effect of CO₂ absorption and the dissociation of carbonic acid in liquid on the measurement of the CO₂ evolution rate (QCO₂) of both anaerobic and aerobic continuous cultures. For aerobic cultures the same equation applies to the measurement of the respiratory quotient (RQ). The deviation of QCO₂ and RQ, calculated from gas-phase measurements, from their true values can be assessed with two parameters: one accounts for the influence of pH, resulting from dissociation of carbonic acid, the other for the influence of operating conditions. Plots are given to show the influences of culture and operating conditions; they may be used as a guideline for choosing proper operating conditions for a reliable measurement of CO₂ evolution rate and RQ value.Dedicated to Prof. Dr. Fritz Wagner on the occasion of his 65th birthday     

Germination of Echinochloa crus-galli (Barnyard Grass) Seeds under Anaerobic Conditions : Respiration and Response to Metabolic Inhibitors

Echinochloa crus-galli L. Beauv., a rice-field weed, can germinate and grow for extended periods of time in an anaerobic environment. Compared to pea, which does not germinate under anaerobiosis, the evolution of CO₂ in Echinochloa and rice is lower and the peak rate of CO₂ evolution is delayed when germinated without oxygen. The plants studied also differ with respect to their respiration ratio ([CO₂] N₂/[CO₂] air) and metabolism used during the early stages of germination. Echinochloa does not increase its glycolytic rate under anaerobiosis, whereas pentose phosphate pathway activity appears to increase during the first 40 to 50 hours of germination.

Based on its response to metabolic inhibitors (NaF, dinitrophenol, and malonate), anaerobic metabolism in Echinochloa proceeds primarily through glycolysis, with partial operation of the tricarboxylic acid cycle and little or no oxidative phosphorylation. Also, Echinochloa is sensitive to CN during aerobic germination, whereas rice appears to be able to shift to CN-insensitive electron transport. Finally, the effectiveness of cyanide and azide on inhibiting germination of Echinochloa in N₂, but not CO, suggests that cytochrome oxidase is not used to reoxidize pyridine nucleotides in the absence of oxygen. The possible existence of an alternate electron acceptor is discussed.

     

Carbon Flux Analysis by 13C Nuclear Magnetic Resonance To Determine the Effect of CO2 on Anaerobic Succinate Production by Corynebacterium glutamicum

Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO₂ on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO₂ caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO₂ was estimated by using ¹³C-labeled glucose and ¹³C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO₂. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H⁺:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.     

Alternative pathways of glucose utilization in brain. Changes in the pattern of glucose utilization in brain during development and the effect of phenazine methosulfate on the integration of metabolic routes.

The activities of alternative pathways of glucose metabolism in developing rat brain were evaluated by measurement of the yields of ¹⁴CO₂ from glucose labeled with ¹⁴C on carbons 1, 2, 3 + 4, 6 and uniformly labeled glucose, from the detritiation of [2-³H]glucose and from the incorporation of ¹⁴C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. The glycolytic route and tricarboxylic acid cycle (¹⁴CO₂ yield from carbons 3, 4, and 6 of glucose) increased during development. The flux through the glutamate-γ-aminobutyric route (¹⁴CO₂ yield from carbon 2-carbon 6 of glucose) also showed an increase with development. In contrast, the proportion of glucose metabolized via the pentose phosphate pathway was markedly decreased as development progressed. The artificial electron acceptor, phenazine methosulfate, was used as a probe to investigate the effect of alterations in the redox state of $\text{NADP}{}^{\mathrm{+}}\text{NADPH}$ couple on a number of NADP-linked systems in developing brain. Phenazine methosulfate produced a massive (20- to 50-fold) stimulation of the pentose phosphate pathway, in contrast, the incorporation of glucose carbon into fatty acids and flux through the glutamate-γ-aminobutyrate shunt were sharply decreased. The effects of phenazine methosulfate on the incorporation of glucose into glyceride glycerol, on the flux of glucose through the pyruvate dehydrogenase reaction and tricarboxylic acid cycle, all processes linked to the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ couple, appeared to be minimal in the brain at the stages of development studied, i.e., 1, 5, 10, 20 days, and in the adult rat. The significance of the massive reserve potential of the pentose phosphate pathway in the developing brain is discussed.     

Compartmentation of organic acids in corn roots I. Differential labeling of 2 malate pools

Bicarbonate-¹⁴C and acetate-³H were simultaneously provided to corn roots to give 2 isotopic forms of malate in the tissue, malate-¹⁴C produced by dark fixation reactions and malate-³H produced by reactions of the tricarboxylic acid cycle. Following a short pulse of exposure to the isotopes, the dissimilation of both isotopic forms of malic acid was followed. The rate of utilization of malate-³H was much faster than that of malate-¹⁴C.

These results are interpreted as showing that the malate produced from ¹⁴CO₂ is in a pool physically separated from that in the tricarboxylic acid cycle. The introduction of the 2 isotopes through distinct metabolic pathways produced the differential labeling of 2 distinct pools of malate.

     

Twenty-four-hour variations in activity,core temperature,metabolic rate,and respiratory quotient in captive chinese pangolins

The circadian rhythms in activity, core temperature (T_c), O₂ consumption, CO₂ production, and respiratory quotient (RQ) were monitored in four captive Chinese pangolins (Manis pentadactyla). The pangolins were strictly nocturnal, never emerging from their nest before 1600 h, and their intermittent activity continued no later than 0230. As is usual in nocturnal mammals, the highest values observed in T_c, O₂ consumption, and CO₂ production occurred during the night; the lowest values occurred during the day. The magnitude of the variation in T_c, O₂ consumption, CO₂ production, and RQ averaged 1.2°C, 1.3 ml O₂ kg^?1 min^?1, 1.2 ml CO₂ kg^?1 min^?1, and 0.24, respectively. The circadian pattern in RQ was independent of activity, T_c, and the metabolic parameters and was of a different character than the patterns exhibited in the other variables. RQ remained constant at either a high or low level for long periods (8–10 h) and then increased or decreased relatively rapidly (1–2h) to the other level as in a square wave, whereas the rhythms in the other variables are similar to sine waves. The sharp increase in RQ was followed by a slow decline in T_c, and the sharp decline in RQ was followed by a slow increase in T_c.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate

Growth on glycerol has already been a topic of research for several yeast species, and recent publications deal with the regulatory mechanisms of glycerol assimilation by the fission yeast Schizosaccharomyces pombe. We investigated glycerol metabolism of S. pombe from a physiological point of view, characterizing growth and metabolism on a mixture of glycerol and acetate and comparing it to growth on glucose under respirative growth conditions in chemostat experiments. On glycerol/acetate mixtures, the cells grew with a maximum specific growth rate of 0.11 h^?1 where 46 % of the carbon was channeled into biomass and the key fermentation product ethanol was not detectable. ¹³C-assisted metabolic flux analysis resolved substrate distributions through central carbon metabolism, proving that glycerol is used as a precursor for glycolysis, gluconeogenesis, and the pentose phosphate pathway, while acetate enters the tricarboxylic acid cycle via acetyl-CoA. Considering compartmentalization between cytosol and mitochondria in the metabolic model, we found compartmentalization of biosynthesis for the amino acids aspartate and leucine. Balancing of redox cofactors revealed an abundant production of cytosolic NADPH that must be finally regenerated via the respiratory chain shown by the simulated and measured CO₂ production and oxygen consumption rates which were in good agreement.     

Dark CO(2) Fixation and its Role in the Growth of Plant Tissue

Experiments were designed to determine the significance of dark CO₂ fixation in excised maize roots, carrot slices and excised tomato roots grown in tissue culture. Bicarbonate-¹⁴C was used to determine the pathway and amounts of CO₂ fixation, while leucine-¹⁴C was used to estimate protein synthesis in tissues aerated with various levels of CO₂.

Organic acids were labeled from bicarbonate-¹⁴C, with malate being the major labeled acid. Only glutamate and aspartate were labeled in the amino acid fraction and these 2 amino acids comprised over 90% of the ¹⁴C label in the ethanol-water insoluble residue.

Studies with leucine-¹⁴C as an indicator of protein synthesis in carrot slices and tomato roots showed that those tissues aerated with air incorporated 33% more leucine-¹⁴C into protein than those aerated with CO₂-free air. Growth of excised tomato roots aerated with air was 50% more than growth of tissue aerated with CO₂-free air. These studies are consistent with the suggestion that dark fixation of CO₂ is involved in the growth of plant tissues.

     

Characterization of Ethanol Production from Xylose and Xylitol by a Cell-Free Pachysolen tannophilus System

Whole cells and a cell extract of Pachysolen tannophilus converted xylose to xylitol, ethanol, and CO₂. The whole-cell system converted xylitol slowly to CO₂ and little ethanol was produced, whereas the cell-free system converted xylitol quantitatively to ethanol (1.64 mol of ethanol per mol of xylitol) and CO₂. The supernatant solution from high-speed centrifugation (100,000 × g) of the extract converted xylose to ethanol, but did not metabolize xylitol unless a membrane fraction and oxygen were also present. Fractionation of the crude cell extract by gel filtration resulted in an inactive fraction in which ethanol production from xylitol was fully restored by the addition of NAD⁺ and ADP. The continued conversion of xylose to xylitol in the presence of fluorocitrate, which inhibited aconitase, demonstrated that the tricarboxylic acid cycle was not the source of the electrons for the production of xylitol from xylose. Therefore, the source of the electrons is indirectly identified as an oxidative pentose-hexose cycle.