The Effect of Growth and Measurement Temperature on the Activity of the Alternative Respiratory Pathway

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19°C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28°C but was unchanged in soybean cotyledons grown at 14°C compared with plants grown at 28°C. When the short-term response of tissue respiration was measured over the temperature range of 35°C to 9°C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19°C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.     

TEMPERATURE CHARACTERISTICS FOR THE OXYGEN CONSUMPTION OF GERMINATING SEEDS OF LUPINUS ALBUS AND ZEA MAYS

The rate of oxygen consumption by germinating seeds of Lupinus albus and of Zea mays was studied as a function of temperature (7–26°C.). The Warburg manometer technique was used, with slight modifications. Above and below a critical temperature at 19.5°C. the temperature characteristic for oxygen consumption by Lupinus albus was found to be µ = 11,700± and 16,600 respectively. The same critical temperature was encountered in the case of Zea mays, with temperature characteristics µ = 13,100± above and µ = 21,050 below that temperature.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen.     

TEMPERATURE CHARACTERISTICS FOR THE PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS AND ZEA MAYS

The rates of production of CO₂ by germinating seeds of Lupinus albus and Zea mays were studied between temperatures 12.5° and 25°C. with the HCl-Ba(OH)₂ titration method. The temperature characteristics found are different from those previously obtained for the oxygen consumption of the same seeds germinated in the same manner. For Lupinus, the temperature characteristics above and below the critical temperature of 20° are 16,100 ± and 24,000 ± calories respectively. For Zea, no evidence of a critical temperature was found in this region, and the temperature characteristic is 20,750 ± calories throughout the range of temperature tested. The possible interpretations of the difference in the values of temperature characteristics for oxygen consumption and for production of CO₂ are noted.     

THE TEMPERATURE CHARACTERISTIC OF RESPIRATION OF AZOTOBACTER

The temperature characteristic of respiration of Azotobacter vinelandii possesses a constant value of 19,330 ± 165 over the temperature range 20–30°C. This value is independent of pH, oxygen tension, age of culture, and other factors within the limits studied. The optimum temperature of respiration is 34–35°C., with limits at about 10° and 50°C.     

Catabolite Inactivation in the Methylotrophic Yeast Pichia pastoris

Inactivation of the alcohol oxidase enzyme system of Pichia pastoris, during the whole-cell bioconversion of ethanol to acetaldehyde, was due to catabolite inactivation. Electron microscopy showed that methanol-grown cells contained peroxisomes but were devoid of these microbodies after the bioconversion. Acetaldehyde in the presence of O₂ was the effector of catabolite inactivation. The process was initiated by the appearance of free acetaldehyde, and was characterized by an increase in the level of cyclic AMP, that coincided with a rapid 55% drop in alcohol oxidase activity. Further enzyme inactivation, believed to be due to proteolytic degradation, then proceeded at a constant but slower rate and was complete 21 h after acetaldehyde appearance. The rate of catabolite inactivation was dependent on acetaldehyde concentration up to 0.14 mM. It was temperature dependent and occurred within 24 h at 37°C and by 6 days at 15°C but not at 3°C. Alcohol oxidase activity was psychrotolerant, with only a 17% decrease in initial specific activity over a temperature drop from 37 to 3°C. In contrast, protease activity was inhibited at temperatures below 15°C. When the bioconversion was run at 3°C, catabolite inactivation was prevented. In the presence of 3 M Tris hydrochloride buffer, 123 g of acetaldehyde per liter was produced at 3°C, compared with 58 g/liter at 30°C. By using 0.5 M Tris in a cyclic-batch procedure, 140.6 g of acetaldehyde was produced.     

Acclimation to low temperature by microsomal membranes from tomato cell cultures

Sealed vesicles were prepared from microsomal membranes from cell suspension cultures of tomato (Lycopersicon esculentum Mill cv VF36). ATP-dependent proton transport activity by the vesicles was measured as quenching of fluorescence of acridine orange. Measurements of proton transport were correlated with the activity of a nitrate-inhibitable ATPase. The initial rate of proton influx into the vesicles was strongly temperature dependent with a Q₁₀ of 2 and a maximum rate near 35°C. The data suggest that passive permeability did not increase at chilling temperatures but did increase rapidly with temperatures above 30°C. A comparison was made between membranes from cell cultures grown at 28°C and 9°C. The temperature optimum for proton transport broadened and shifted to a lower temperature range in membranes from cells maintained at 9°C.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.     

Nitrogen Fixation by Thermophilic Blue-Green Algae (Cyanobacteria): Temperature Characteristics and Potential Use in Biophotolysis

Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biophotolysis. Three strains of Mastigocladus laminosus were tested and were found to be equally effective in biophotolysis as judged by nitrogenase activity. The alga, M. laminosus NZ-86-m, which was chosen for further study, grew well in the temperature range from 35 to 50°C, with optimum growth at 45°C, at which temperature acetylene reduction activity was also greatest. The maximum tolerable temperature was 55°C. Acetylene reduction activity was saturated at a light intensity of 1 × 10⁴ ergs cm⁻² s⁻¹. Atmospheric oxygen tension was found to be slightly inhibitory to acetylene reduction of both slowly growing and exponentially growing cultures. Nonsterile continuous cultures, which were conducted to test problems of culture maintenance, could be operated for 2 months without any significant decrease in nitrogenase activity or contamination by other algae. Nitrogen-starved cultures of M. laminosus NZ-86-m produced hydrogen at comparable rates to Anabaena cylindrica. The conversion efficiency of light to hydrogen energy at maximum rates of hydrogen production was 2.7%.     

Seedling growth, mitochondrial characteristics, and alternative respiratory capacity of corn genotypes differing in cold tolerance

Four maize (Zea mays L.) inbreds representing genetic differences in seedling cold tolerance were used to determine the effect of growth temperatures on dry weight accumulation and mitochondrial properties, especially the alternative oxidase capacity. Seedlings were grown in darkness at 30°C (constant), 14°C (constant), and 15°C for 16 hours and 8°C for 8 hours. Inbreds B73 and B49 were characterized as cold tolerant while G50 and G84 were cold sensitive. Shoot growth rate of cold-sensitive inbreds in the lower temperatures was slower relative to the tolerant inbreds. Mesocotyl tissue was particularly sensitive to low temperatures during growth after germination. There were no significant differences in relative rates of mitochondrial respiration in the cold-tolerant compared to cold-sensitive inbreds measured at 25°C. Mitochondria from all seedlings grown at all temperatures had the ability to phosphorylate as indicated by the observation of respiratory control. This result indicated that differences in low temperature growth were probably not related to mitochondrial function at low temperatures. Alternative oxidase capacity was higher in mitochondria from seedlings of all inbreds grown at 14°C compared to 30°C. Capacities in seedlings of 14°C-grown B73 and G50 were higher than in B49 and G84. Capacities in seedlings grown for 16 hours at 15°C and 8 hours at 8°C were similar to those from 14°C-grown except in G50 which was lower and similar to those grown at 30°C. Mesocotyl tissue was the most responsive tissue to low growth temperature. Coleoptile plus leaf tissue responded similarly but contained lower capacities. Antibody probing of western blots of mitochondrial proteins confirmed that differences in alternative oxidase capacities were due to differences in levels of the alternative oxidase protein. Male sterile lines of B73 were also grown under the three different temperature regimes. These lines grew equally as well as the normal B73 at all temperatures and the response of alternative oxidase capacity and protein to low growth temperature was similar to normal B73.     

Effects of Temperature on Electron Transport in Arum maculatum Mitochondria

The effects of temperature upon the respiratory pathways of Arum maculatum mitochondria have been studied. The alternate oxidase sustained a greater proportion of the total respiration at low temperatures than at higher temperatures. Arrhenius plots of respiratory activities show two discontinuities, one at 14°C and one at 21°C. The lower temperature discontinuity was associated with electron transport from succinate dehydrogenase to the alternative oxidase, enzymes that face the inner side of the membrane while the higher temperature discontinuity was associated with electron transport from the external NADH dehydrogenase to cytochrome c oxidase, which face the outer side of the membrane. Both discontinuities resulted in a decrease in the activation energy for electron transport on one side of the membrane. Arrhenius plots of transmembrane electron transport showed discontinuities at both 14° and 21°C but the upper discontinuity resulted in an increase in the activation energy. Activation energies determined for the respiratory activities show that above 21°C the exogenous NADH-cytochrome pathway and the succinate-alternative oxidase pathway were lower than those for the NADH-alternative pathway or the succinate cytochrome pathway.     

Effect of temperature on electrolyte metabolism of isolated frog skin

A study is presented on the effect of temperature on unidirectional active ion transport, resting electrolyte equilibrium (electrolyte composition), and oxygen consumption in isolated frog skin. The aims were twofold: first, to find out whether the rate of active transport can be changed without affecting the Na⁺ and K⁺ balance of skin itself; second, to arrive at minimal ΔNa/ΔO₂ values by correlating quantitatively inhibition of active ion transport with inhibition of O₂ consumption. NaCl transport was maximal at 20°C. At 28° and at temperatures below 20°, rate of NaCl transport was diminished. In many instances NaCl transport was diminished in skins which maintained their normal Na⁺ and K⁺ content. In several cases, however, neither rate of transport nor resting electrolyte equilibrium was affected; in other cases, both were. O₂ consumption decreased when lowering the temperature over the range from 28 to 10°C. From a plot of log Q_OO2 against 1/T an activation energy of µ 13,700 cal. was calculated, valid for the range from 10 to 20°C. It appeared that µ was smaller for temperatures above 20°C. Working between 10 and 20°, it was found that, on the average, 4 to 5 equivalents of Na⁺ were transported for one mole of O₂ consumed in skins with undisturbed resting electrolyte equilibrium.     

Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.     

The Effect of Diel Temperature and Light Cycles on the Growth of Nannochloropsis oculata in a Photobioreactor Matrix

A matrix of photobioreactors integrated with metabolic sensors was used to examine the combined impact of light and temperature variations on the growth and physiology of the biofuel candidate microalgal species Nannochloropsis oculata. The experiments were performed with algal cultures maintained at a constant 20°C versus a 15°C to 25°C diel temperature cycle, where light intensity also followed a diel cycle with a maximum irradiance of 1920 µmol photons m⁻² s⁻¹. No differences in algal growth (Chlorophyll a) were found between the two environmental regimes; however, the metabolic processes responded differently throughout the day to the change in environmental conditions. The variable temperature treatment resulted in greater damage to photosystem II due to the combined effect of strong light and high temperature. Cellular functions responded differently to conditions before midday as opposed to the afternoon, leading to strong hysteresis in dissolved oxygen concentration, quantum yield of photosystem II and net photosynthesis. Overnight metabolism performed differently, probably as a result of the temperature impact on respiration. Our photobioreactor matrix has produced novel insights into the physiological response of Nannochloropsis oculata to simulated environmental conditions. This information can be used to predict the effectiveness of deploying Nannochloropsis oculata in similar field conditions for commercial biofuel production.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Role of Glucose in Enhancing the Temperature-Dependent Growth Inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp.

Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25°C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp. found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition. The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested. The pathogen grew better as the temperature increased from 10 to 15 and 25°C and grew better in pure culture than in mixed cultures. Pseudomonas sp. inhibited E. coli O157:H7 in cocultures with glucose at 10°C, while at 15°C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period. Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15°C and, irrespective of glucose, at 25°C. These results indicate that glucose enhances the growth inhibition of E. coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (≤15°C) temperatures.