Critical oxygen pressure for growth and respiration of excised and intact roots

A method based on the measurement of ATP/ADP ratios is described. It permits the determination of the critical respiratory oxygen pressure of any organ, or part of any organ, of an intact plant. The data obtained by this method with intact maize (Zea mays L. INRA 508) root tips are compared with polarographic determinations on similar excised tissues.

When internal O₂ transport from the aerial part was prevented, the critical oxygen pressure found for the respiration of intact tips was similar to that found with excised tips. It was close to 10 kilopascals in a humid atmosphere and about 30 kilopascals in a liquid medium. Flooding of the gas spaces by vacuum infiltration did not modify these results. When internal O₂ transport from the aerial parts of the plant occurred, significantly lower values were obtained in liquid medium for the critical oxygen pressure, which shifted from more than 21 to 6 kilopascals. The higher values observed with excised root tips, compared to those obtained with intact tissues, can be explained by the lack of internal O₂ transport, rather than by the flooding of gas spaces.

Data are presented which show that root growth started to be limited at a significantly higher pressure than the respiration. These results are attributed to nonrespiratory oxidative processes with a low affinity for O₂ involved in root elongation.

     

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).     

Larger adenylate energy charge and ATP/ADP ratios in aerenchymatous roots of Zea mays in anaerobic media as a consequence of improved internal oxygen transport

Internal transport of O₂ from the aerial tissues along the adventitious roots of intact maize plants was estimated by measuring the concentrations of adenine nucleotides in various zones along the root under an oxygen-free atmosphere. Young maize plants were grown in nutrient solution under conditions that either stimulated or prevented the formation of a lysigenous aerenchyma, and the roots (up to 210 mm long) were then exposed to an anaerobic (oxygen-free) nutrient solution. Aerenchymatous roots showed higher values than non-aerenchymatous ones for ATP content, adenylate energy charge and ATP/ADP ratios. We conclude that the lysigenous cortical gas spaces help maintain a high respiration rate in the tissues along the root, and in the apical zone, by improving internal transport of oxygen over distances of at least 210 mm. This contrasted sharply with the low energy status (poor O₂ transport) in non-aerenchymatous roots.Abbreviation AEC adenylate energy charge     

Oxygen Dependence of Glucose and Acetylcholine Metabolism in Slices and Synaptosomes from Rat Brain

Abstract: Previous studies have shown that a reduction in the O₂ tension of the blood from 120 torr to 57 torr (hypoxic hypoxia) decreases brain acetylcholine (ACh) synthesis. To determine if this decrease is due to a direct impairment of ACh metabolism or to an indirect effect mediated by other neurotransmitter systems, we studied ACh formation in rat brain slices and synaptosomes. At O₂ tensions ranging from 760 to less than 1 torr, ¹⁴CO₂ production and [¹⁴C]ACh synthesis from [U-¹⁴C]glucose, the levels of lactate and ATP, and the ATP/ADP ratio were determined. In slices, the first decreases were observed in the rate of ¹⁴CO₂ production and [¹⁴C]ACh synthesis at an O₂ tension of 152 torr. The ATP level started to decline at 53–38 torr, and a reduction in the ATP/ADP ratio was first found at and below 19 torr. Lactate formation was maximally stimulated at 38–19 torr. Synaptosomes responded differently than brain slices to reduced O₂ tensions. In synaptosomes, ¹⁴CO₂ production and [¹⁴C]ACh synthesis from [U-¹⁴C]glucose, the levels of lactate and ATP, and the ATP/ADP ratio were unaltered if a minimum O₂ tension of 19 torr was maintained. Despite the difference in sensitivities to decreases in O₂ levels, there is a curvilinear relationship between [U-¹⁴C]glucose decarboxylation and [¹⁴C]ACh synthesis at various O₂ tensions for both tissue preparations with a high coefficient of determination (R²= 0.970). The difference in the metabolic sensitivity of slices and synaptosomes to a reduced O₂ level may be explained by the greater distance O₂ must diffuse in slices. The results are discussed in comparison with hypoxia in vivo.     

Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays

Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1⁻ mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO₃⁻. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO₃⁻ to NO₂⁻. These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl⁻ mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.     

Enhancement of Anaerobic Respiration in Root Tips of Zea mays following Low-Oxygen (Hypoxic) Acclimation

Root tips (10-millimeter length) were excised from hypoxically pretreated (HPT, 4% [v/v] oxygen at 25°C for 16 hours) or nonhypoxically pretreated (NHPT, 40% [v/v] oxygen) maize (Zea mays) plants, and their rates of respiration were compared by respirometry under aerobic and anaerobic conditions with exogenous glucose. The respiratory quotient under aerobic conditions with 50 millimolar glucose was approximately 1.0, which is consistent with glucose or other hexose sugars being utilized as the predominant carbon source in glycolysis. Under strictly anaerobic conditions (anoxia), glycolysis was accelerated appreciably in both HPT and NHPT root tips, but the rate of anaerobic respiration quickly declined in NHPT roots. [U-¹⁴C]Glucose supplied under anaerobic conditions was taken up and respired by HPT root tips up to five times more rapidly than by NHPT roots. When anaerobic ethanol production was measured with excised root tips in 50 millimolar glucose, HPT tissues consistently produced ethanol more rapidly than NHPT tissues. These data suggest that a period of low oxygen partial pressure is necessary to permit adequate acclimation of the root tip of maize to subsequent anoxia, resulting in more rapid rates of fermentation and generation of ATP.     

Respiration in Adipocytes is Inhibited by Reactive Oxygen Species

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat‐fed mice. Maximum O₂ consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Δψ_M) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N‐acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Δψ_M and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O₂ consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O₂ consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O₂ consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O₂ consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.     

Nitrogen Fixation and Hydrogen Metabolism in Relation to the Dissolved Oxygen Tension in Chemostat Cultures of the Wild Type and a Hydrogenase-Negative Mutant of Azorhizobium caulinodans

Both the wild type and an isogenic hydrogenase-negative mutant of Azorhizobium caulinodans growing ex planta on N₂ as the N source were studied in succinate-limited steady-state chemostat cultures under 0.2 to 3.0% dissolved O₂ tension. Production or consumption of O₂, H₂, and CO₂ was measured with an on-line-connected mass spectrometer. In the range of 0.2 to 3.0%, growth of both the wild type and the mutant was equally dependent on the dissolved O₂ tension: the growth yield decreased, and the specific O₂ consumption and CO₂ production increased. A similar dependency on the dissolved O₂ tension was found for the mutant with 2.5% H₂ in the influent gas. The H₂/N₂ ratio (moles of H₂ evolved per mole of N₂ consumed via nitrogenase) of the mutant, growing with or without 2.5% H₂, increased with increasing dissolved O₂ tensions. This increase in the H₂/N₂ ratio was small but significant. The dependencies of the ATP/N₂ ratio (moles of ATP consumed per mole of N₂ fixed) and the ATP/2e^- ratio [moles of ATP consumed per mole of electron pairs transferred from NAD(P)H to nitrogenase] on the dissolved O₂ tension were estimated. These dependencies were interpreted in terms of the physiological concepts of respiratory protection and autoprotection.     

Hypoxic Induction of Anoxia Tolerance in Root Tips of Zea mays

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

Correlation between Oxygen Availability, Energy Charge, and Protein Synthesis in Squash Cotyledons Isolated from Germinating Seeds

The influence of O₂ availability on the rate of protein synthesis, the levels of RNA and of adenylates, and the value of the energy charge in squash (Cucurbita maxima) cotyledons isolated from seeds germinated for 15 or 28 hours at different O₂ concentration (3% or 20% O₂) has been investigated.

The rate of protein synthesis is five times lower in cotyledons maintained in 3% O₂ than in those maintained in 20% O₂. Also net RNA synthesis is almost blocked in 3% O₂, while in 20% O₂ it proceeds almost linearly for 48 hours.

The different RNA contents cannot explain the different rates of protein synthesis.

The results of shift experiments (cotyledons shifted from 20% to 3% O₂ or vice versa) show that the rate of protein synthesis is strictly correlated with actual O₂ availability and is largely independent of the one in the previous period. O₂ controls the development of the adenylate pool and particularly the increase of ATP level. Thus, both the adenylate pool and the values of the energy charge ratio are lower in cotyledons grown in 3% than in 20% O₂.

The shifts of O₂ availability induce rapid changes of ATP, ADP, and AMP levels and thus of the values of the energy charge, which are about 0.7 at 3% O₂ and higher than 0.8 at 20% O₂, independent of previous O₂ availability.

The rate of protein synthesis appears to be largely independent of the levels of the single nucleotides and better correlated to the energy charge values.

     

Respiration of barley protoplasts before and after illumination

Respiratory O₂ consumption was investigated in dark-adapted barley (Hordeum vulgare L. cv. Gunilla) protoplasts and after illumination for 10 min at high and very low CO₂ in the presence of respiratory and photorespiratory inhibitors. In dark-adapted protoplasts no difference was observed between inhibitor treatments in high and very low CO₂. The respiratory rate increased somewhat after illumination and a difference in responce to inhibitors was in some cases observed between high and very low CO₂. Thus, the operation of the mitochondrial electron transport chain is affected following a period of active photosynthesis. In all situations tested, oligomycin inhibited respiratiory O₂ uptake indicating that respiration of mitochondria in protoplasts is not strictly ADP limited. Antimycin A inhibited respiration more in dark-adapted protoplasts than after illumination whereas SHAM gave the opposite response. Rotenone inhibited respiration both in dark-adapted protoplasts (about 30%) and after illumination where the inhibition was much greater in very low CO₂ (50%) than in high CO₂ (10%). After illumination in very low CO₂. SHAM + rotenone inhibited respiration almost completely (70%). Photorespiratory inhibitors had very small effect on O₂ consumption in darkness. After illumination the effect of aminoacetonitrile (AAN) was also very low whereas α-hydroxypyridine-2-methane sulphonate (HPMS) in photorespiratory conditions inhibited O₂ uptake much stronger (35%). The addition of glyoxylate enhanced respiration in the presence of HPMS up to the control level suggesting that alternative pathways of glyoxylate conversion might be operating. The differences in inhibitor responses may reflect fine mechanisms for the regulation of energetic balance in the plant cell which consists of switching from electron transport coupled to ATP production to non-coupled transport. Photorespiratory flux is also very flexible, and the suppression of glycine decarboxylation can induce bypass reactions of glyoxylate metabolism.     

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5–25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H₂O₂, did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H₂O₂ signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H₂O₂ in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.     

Oxygen distribution and movement,respiration and nutrient loading in banana roots (Musa spp. L.) subjected to aerated and oxygen-depleted environments

Excessive soil wetness is a common feature where bananas (Musa spp.) evolved. Under O₂ deficiency, a property of wet soils, root growth and functions will be influenced by the respiratory demand for O₂ in root tissues, the transport of O₂ from the shoot to root and the supply of O₂ from the medium. In laboratory experiments with nodal roots of banana, we examined how these features influenced the longitudinal and radial distributions of O₂ within roots, radial O₂ loss, solute accumulation in the xylem, root hydraulic conductivity, root elongation and root tip survival. In aerated roots, the stele respired about 6 times faster than the cortex on a volume basis. Respiratory O₂ consumption decreased substantially with distance from the root apex and at 300–500 mm it was 80% lower than at the apex. Respiration of lateral roots constituted a sink for O₂ supplied via aerenchyma, and reduced O₂ flow towards the tip of the supporting root. Stelar anoxia could be induced either by lowering the O₂ partial pressure in the bathing medium from 21 to 4 kPa (excised roots) or, in the case of intact roots, by reducing the O₂ concentration around the shoot. The root hair zone sometimes extended to 1.0 mm from the root surface and contributed up to a 60% drop in O₂ concentration from a free-flowing aerated solution to the root surface. There was a steep decline in O₂ concentration across the epidermal-hypodermal cylinder and some evidence of a decline in the O₂ permeability of the epidermal-hypodermal cylinder with increasing distance from the root apex. The differences in O₂ concentration between cortex and stele were smaller than reported for maize and possibly indicated a substantial transfer rate of dissolved O₂ from cortex to stele in banana, mediated by a convective water flow component. An O₂ partial pressure of 4 kPa in the medium reduced net nutrient transfer into the vascular tissue in the stele within 1 or 2 h. Hypoxia also caused a temporary decrease in radial root hydraulic conductivity by an order of magnitude. In O₂ deficient environments, the stele would be among the first tissues to suffer anoxia and O₂ consumption within the root hair zone might be a major contributor to root anoxia/hypoxia in banana growing in temporarily flooded soils.     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

H Efflux and Hexose Transport under Imposed Energy Status in Maize Root Tips

The relationship between changes in H⁺ flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H⁺ efflux was abolished in anoxia but not by the 2-deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H⁺-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H⁺ extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H⁺ secretion under the conditions of this investigation.     

The Site of Oxygen Limitation in Soybean Nodules

In legume nodules the [O₂] in the infected cells limits respiration and nitrogenase activity, becoming more severe if nodules are exposed to subambient O₂ levels. To identify the site of O₂ limitation, adenylate pools were measured in soybean (Glycine max) nodules that were frozen in liquid N₂ before being ground, lyophilized, sonicated, and separated on density gradients of nonaqueous solvents (heptane/tetrachloroethylene) to yield fractions enriched in bacteroid or plant components. In nodules maintained in air, the adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) was lower in the plant compartment (0.65 ± 0.04) than in the bacteroids (0.76 ± 0.095), but did not change when the nodulated root system was exposed to 10% O₂. In contrast, 10% O₂ decreased the bacteroid AEC to 0.56 ± 0.06, leading to the conclusion that they are the primary site of O₂ limitation in nodules. To account for the low but unchanged AEC in the plant compartment and for the evidence that mitochondria are localized in O₂-enriched microenvironments adjacent to intercellular spaces, we propose that steep adenylate gradients may exist between the site of ATP synthesis (and ADP use) in the mitochondria and the extra-mitochondrial sites of ATP use (and ADP production) throughout the large, infected cells.     

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

Nodulated soybeans (Glycine max L. Merr, cv. Maple Arrow) were exposed to various physiological and environmental treatments to determine the relationship between nodule adenylate pools and the degree of O₂ limitation of nitrogenase. Adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) and ATP/ADP ratios declined under conditions of decreased (10%) external pO₂ but increased in nodules exposed to elevated (30%) external pO₂. Nitrogenase activity was inhibited by both pO₂ treatments, but recovered towards initial levels within 45 min. AEC also returned to initial levels during this period. To account for these and related data in the literature, it was hypothesized that 1) legume nodules regulate infected cell O₂ concentration (Oi) to maintain adenylate pools at levels which limit respiratory metabolism: 2) treatments which decrease Oi alter the adenylate pools and further limit nodule metabolism; 3) treatments which increase Oi to levels in excess of a narrow range alter the adenylate pools and activate biochemical pathways which are not conducive to nitrogenase activity. In a preliminary test of these hypotheses, changes in AEC and ATP/ADP ratio were studied in nodules in which nitrogenase activity was inhibited by stem girdling, nitrate fertilization and exposure to an Ar:O₂ atmosphere. All three treatments caused an increased O₂ limitation of nodule respiration and nitrogenase activity. However, decreases in AEC were observed only in the stem girdling and nitrate fertilization treatment: Ar:O₂ exposure had no effect on whole nodule AEC. While this result challenged the hypotheses suggesting a central role for adenylates in the regulation of O₂-limited metabolism, it was noted that the Ar:O₂ treatment would differ from the other treatments in that it would have a specific effect on the ATP demands for NH₃ assimilation in the plant fraction. Since AEC and ATP/ADP ratio would be affected by both the rate of ATP synthesis (potentially an O₂-limited process) and the demand for ATP, changes in these parameters in the whole nodule may not be a reliable indicator of adenylate-mediated O₂ limitation. Futher studies are needed to examine in vivo changes in adenylate pools in the plant and bacteroid fractions in nodules which vary in their degree of O₂-limited metabolism.     

Rapid Respiratory Changes Due to Red Light or Acetylcholine during the Early Events of Phytochrome-mediated Photomorphogenesis

Two millimeter long secondary root tips of etiolated mung bean (Phaseolus aureus) plants were given 4 minute consecutive treatments of darkness, red light, far red light, and acetylcholine during darkness. We studied the effects of these treatments on exogenous (H⁺) changes, ATP utilization, O₂ uptake, P₁ levels, and ATPase activity. Red light and acetylcholine increased the level of P₁, O₂ uptake, and exogenous H⁺, but decreased ATP concentrations. Darkness and far red light caused the amount of ATP to increase and decreased the O₂ uptake and P₁ level. O₂ uptake of both excised root tips and isolated mitochondria was promoted by acetylcholine levels of the same order of magnitude that promoted the other photomimetic phenomena. ADP-O ratios indicated that acetylcholine did not cause an appreciable decrease in ATP synthesis. The total ATPase activity remained constant throughout all treatments. Ouabain caused no adhesion to negatively charged glass in the dark, while the inhibitors valinomycin, atractyloside, digitoxin, gramicidin, and oligomycin caused immediate adhesion. All of the inhibitors prevented release from the glass. In red light ouabain increased adhesion, whereas the other inhibitors caused caused immediate and complete adhesion.     

Effect of Ethylene and Oxygen on the Development of Cyanide-resistant Respiration in Whole Plant Mitochondria

Mitochondria from whole potatoes (Solanum tuberosum) ordinarily fail to oxidize respiratory substrates and to consume molecular O₂ in the presence of cyanide. Mitochondrial preparations obtained from tubers previously held for 24 hours in ethylene (10 microliters per liter) in air are only partially inhibited by cyanide. Application of ethylene in 100% O₂ led to an additional increase in the resistance of the mitochondrial respiration to cyanide. The resistance to cyanide was accompanied by a decrease in the respiratory control but no change in oxidative phosphorylation as shown by the measurement of ATP synthesis.     

The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts : I. INTERACTION OF ADENINE NUCLEOTIDES AND ENERGY TRANSFER INHIBITORS WITH THE COUPLING FACTOR COMPLEX

The rate of nonphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in well-coupled (ATP/2e⁻ = 0.9-1.1) maize mesophyll chloroplasts is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin and Hg²⁺ ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations in medium pH, and to the presence of adenine nucleotides and energy transfer inhibitors in the assay medium. Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in C₃ and C₄ mesophyll chloroplasts.