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.     

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : II. CONTRIBUTION FROM ELECTRON TRANSPORT AND PHOTOPHOSPHORYLATION

As part of an analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C₃ grass, the response of electron transport and photophosphorylation to temperature in isolated chloroplast thylakoids has been examined. The response of the light reactions to temperature was found to depend strongly on the preincubation time especially at temperatures above 35°C. Using methyl viologen as a noncyclic electron acceptor, coupled electron transport was found to be stable to 38°C; however, uncoupled electron transport was inhibited above 38°C. Photophosphorylation became unstable at lower temperatures, becoming progressively inhibited from 35 to 42°C. The coupling ratio, ATP/2e⁻, decreased continuously with temperature above 35°C. Likewise, photosystem I electron transport was stable up to 48°C, while cyclic photophosphorylation became inhibited above 35°C. Net proton uptake was found to decrease with temperatures above 35°C supporting the hypothesis that high temperature produces thermal uncoupling in these chloroplast thylakoids. Previously determined limitations of net photosynthesis in whole leaves in the temperature region from 35 to 40°C may be due to thermal uncoupling that limits ATP and/or changes the stromal environment required for photosynthetic carbon reduction. Previously determined limitations to photosynthesis in whole leaves above 40°C correlate with inhibition of photosynthetic electron transport at photosystem II along with the cessation of photophosphorylation.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

Factors associated with the instability of nitrate-insensitive proton transport by maize root microsomes

Proton transport catalyzed by the nitrate-insensitive, vanadate-sensitive H⁺-ATPase in microsomes from maize (Zea mays L.) roots washed with 0.25 molar KI decreased as a function of time at 0 to 4°C. The rate of proton transport was approximately one-half of that by freshly isolated microsomes after 6 to 18 hours of cold storage. The decrease in proton transport coincided with losses in membrane phosphatidylcholine and was not associated with a change in vanadate-sensitive ATP hydrolysis. A technique based on a protocol developed for the reconstitution of Neurospora crassa plasma membrane H⁺-ATPase (DS Perlin, K Kasamo, RJ Brooker, CW Slayman 1984 J Biol Chem 259: 7884-7892) was employed to restore proton transport activity to maize microsomes. These results indicated that the decline in proton transport by maize root membranes during cold storage was not due to degradation of the protein moiety of the H⁺-ATPase, but was due to the loss of phospholipids.     

Effects of dicyclohexylcarbodiimide (DCCD) treatment on coupled activities of vanadate-sensitive ATPase from plasma membrane of maize roots

The presence of dicyclohexylcarbodiimide (DCCD) inhibited the activities of vanadate-sensitive H⁺ -ATPase in both native and reconstituted plasma membrane of maize (Zea mays L. cv. WF9 × Mo 17) roots. Concentration dependence of DCCD inhibition on adenosine triphosphate (ATP) hydrolysis of native plasma membrane vesicles suggested that the molar ratio of effective DCCD binding to ATPase was close to 1. The DCCD inhibition of ATP hydrolysis could be slightly reduced by the addition of ATP, Mg:ATP, adenosine monophosphate (AMP), Mg:AMP and adenosine diphosphate (ADP). More hydrophilic derivatives of DCCD such as l-ethyl-N^?-3-trimethyl ammonium carbodiimide (EDAC) or 1-ethyl-3-3-dimethyl-aminopropyl carbodiimide (EDC) gave no inhibition, indicating that the effective DCCD binding site was located in a hydrophobic region of the protein. The proton transport activity of reconstituted plasma membrane at a temperature below 20°C or above 25°C was much sensitive to DCCD treatment. Build-up of the proton gradient was analyzed according to a kinetic model, which showed that proton leakage across de-energized reconstituted plasma membranes was not affected by DCCD, but was sensitive to the method employed to quench ATP hydrolysis. Reconstituted plasma membrane vesicles treated with DCCD exhibited a differential inhibition of the coupled H⁺-transport and ATP hydrolysis. The presence of 50 μM DCCD nearly abolished transport but inhibited less than 50% of ATP hydrolysis. The above results suggest that the link between proton transport and vanadate-sensitive ATP hydrolysis is indirect in nature.     

Temperature dependence of energy-transducing functions and inhibitor sensitivity in chloroplasts

A comparative analysis of the temperature dependence of energy-transducing reactions in spinach (Spinacia oleracea) chloroplasts and their sensitivity for uncouplers and energy-transfer inhibitors at different temperatures is presented. Arrhenius plots reveal two groups of transitions, around 19°C and around 12°C. Activities that show transitions around 19°C include linear electron flow from water to ferricyanide, its coupled photophosphorylation, the dark-release of the fluorescent probe atebrin, and the slow component of the 515 nm (carotenoid) absorbance decay after a flash. The transitions around 12°C are observed with pyocyanine-mediated cyclic photophosphorylation, light- and dithioerythritol-activated ATP hydrolysis, the dark-release of protons, and the fast 515 nm decay component. It is suggested that both groups of temperature transitions are determined by proton displacements in different domains of the exposed thylakoid membranes. The effects of various uncouplers and an energy-transfer inhibitor are temperature dependent. Some uncouplers also show a different relative inhibition of proton uptake and ATP synthesis at lower temperatures. The efficiency of energy transduction (ATP/e₂) varied with temperature and was optimal around 10°C.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

Temperature Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of BASF 13.338 (4-Chloro-5-Dimethylamino-2-Phenyl-3(2H)Pyridazinone)

When Triticum vulgare cv HD 2189 seedlings were grown in the presence of 125 micromolar BASF 13.338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone), the rate of electron transport (H₂O → methyl viologen) in chloroplast thylakoids isolated from the treated seedlings was higher (by 50%) as compared to the control at assay temperatures above 30°C. Below 30°C, however, the rate with the treated seedlings was lower than the control rate. The temperature dependence of the rate of photosystem I electron transport (2-6-dichlorophenol indophenol-reduced → methyl viologen) in the treated system was similar to that in the control. At high temperatures (>30°C), with diphenyl carabazide as electron donor, the rates of electron transfer (diphenyl carbazide → methyl viologen) were similar in the treated and in the control thylakoids. Direct addition of BASF 13.338 to the assay mixture for the measurement of rate of electron transport (H₂O → methyl viologen) in the thylakoids isolated from the control plants did not cause any change in the temperature dependence of photosynthetic electron transport. These results suggested that the donor side of photosystem II became tolerant to heat in the treated plants. Chlorophyll a fluorescence emission was monitored continuously in the leaves of control and BASF 13.338 treated wheat seedlings during continuous increase in temperature (1°C per minute). The fluorescence-temperature profile showed a decrease in the fluorescence yield above 55°C; this decrease was biphasic in the control and monophasic in the treated plants.     

Effect of temperature on the plasma membrane and tonoplast ATPases of barley roots : comparison of results obtained with acridine orange and quinacrine

The effect of temperature on the rate of proton transport and ATP hydrolysis by plasma membrane (PM) and tonoplast (TN) ATPases from barley (Hordeum vulgare L. cv CM 72) roots were compared. Rates of proton transport were estimated using the fluorescent amine dyes quinacrine and acridine orange. The ratio between rate of transport and ATP hydrolysis was found to depend on the dye, the temperature, and the type of membrane. The PM ATPase had an estimated Arrhenius energy of activation (Ea) of approximately 18 kilocalories per mole for ATP hydrolysis, and the Ea for proton transport was best estimated with acridine orange, which gave an Ea of 19 kilocalories per mole. The TN ATPase had an Ea for ATP hydrolysis of approximately 10 kilocalories per mole and the Ea for proton transport was best estimated with quinacrine, which gave an Ea of 10 kilocalories per mole. Acridine orange did not give an accurate estimate of Ea for the TN ATPase, nor did quinacrine for the PM ATPase. Reasons for the differences are discussed. Because it was suggested (AJ Pope, RA Leigh [1988] Plant Physiol 86: 1315-1322) that acridine orange interacts with anions to dissipate the pH gradient in TN vesicles, the complex effects of NO₃⁻ on the TN ATPase were also examined using acridine orange and quinacrine and membranes from oats and barley. Fluorescent amine dyes can be used to evaluate the effects of ions, substrates, inhibitors, and temperature on transport but caution is required in using rates of quench to make quantitative estimates of proton fluxes.     

When an ATPase Is Not an ATPase: at Low Temperatures the C-Terminal Domain of the ABC Transporter CvaB Is a GTPase

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from Escherichia coli is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg²⁺ and Mn²⁺ dependent. However, BCTD possesses similar GTPase and ATPase activities at 37°C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10°C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.     

Phospholipid requirement of the vanadate-sensitive ATPase from maize roots evaluated by two methods

The activation of the vanadate-sensitive ATPase from maize (Zea mays L.) root microsomes by phospholipids was assessed by two different methods. First, the vanadate-sensitive ATPase was partially purified and substantially delipidated by treating microsomes with 0.6% deoxycholate (DOC) at a protein concentration of 1 milligram per milliliter. Vanadate-sensitive ATP hydrolysis by the DOC-extracted microsomes was stimulated up to 100% by the addition of asolectin. Of the individual phospholipids tested, phosphatidylserine and phosphatidylglycerol stimulated activity as much as asolectin, whereas phosphatidylcholine did not. Second, phospholipid dependence of the ATPase was also assessed by reconstituting the enzyme into proteoliposomes of differing phospholipid composition. In these experiments, the rate of proton transport and ATP hydrolysis was only slightly affected by phospholipid composition. DOC-extracted microsomes reconstituted with dioleoylphosphatidylcholine had rates of proton transport similar to those found with microsomes reconstituted with asolectin. The difference between the two types of assays is discussed in terms of factors contributing to the interaction between proteins and lipids.     

Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence

The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32°C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30°C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32°C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14°C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32°C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.     

Studies on Freezing Injury in Plant Cells : II. Protein and Lipid Changes in the Plasma Membranes of Jerusalem Artichoke Tubers during a Lethal Freezing in Vivo

Plasma membranes were isolated from both unfrozen and frozen tissues of Jerusalem artichoke tubers (Helianthus tuberosus L.) in high purity utilizing an aqueous two-polymer phase partition system. Although the recovery of the plasma membranes was decreased significantly by freezing of tissues even at the nonlethal temperature (−5°C), the isolated plasma membrane samples were considered to be representative of the plasma membranes in situ. Freezing of the tissues at sublethal temperatures resulted in marked changes in the chemical composition of the plasma membrane. Those are losses of sterols and phosphatidylethanolamine from the plasma membranes, and a change of specific proteins with relatively high molecular weights into low molecular weight peptides. These specific proteins were designated as frost susceptible proteins. The properties of the plasma membrane ATPase seem to be not affected so much by the in vivo freezing of cells. However, inhibition of the plasma membrane ATPase by N,N′-dicyclohexylcarbodiimide (DCCD) was relatively low before and after freezing in vivo at the nonlethal temperature at −5°C, but was markedly enhanced by freezing in vivo at sublethal temperatures below −10°C. From the results, it is assumed either that the enzyme molecule was partially modified, especially at the presumed DCCD binding sites or that the DCCD had become more accessible to the enzyme as a result of increased permeability of the plasma membranes. These observed changes are discussed in connection with the mechanism of cell injury.     

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

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.     

THE RELATION BETWEEN TEMPERATURE AND THE PEDAL RHYTHM OF BALANUS

1. The relation of temperature to the pedal rhythm of Balanus balanoides L. has been studied under otherwise constant conditions. 2. The frequency of movement increases with temperature, showing three groups of thermal increments and three critical temperatures. Five animals yielded µ = 5,700 above 14.5° C. and 12,100 below; 3 gave µ = 7,800 above 9.3° and 22,500 below; while 9 showed µ = 9,500 above 8.1° and 22,100 below. 3. The upper critical temperatures, above which different effects appeared in different animals were 23.4°, 26.0°, and 27.0°. Above 27.0° none of the valves remained open. 4. Excepting the values 5,700 and 9,500, the increments are similar to those previously found to be associated with respiratory and with neuromuscular activities. 5. Dilution of the sea water with from 3 to 4 per cent fresh water decreases the rate without altering the increments. More than 4 per cent dilution causes irregularity.     

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : III. Responses of Protoplasts and Intact Chloroplasts

Protoplasts and intact chloroplasts isolated from Agropyron smithii Rybd. were utilized in an effort to determine the limiting factor(s) for photosynthesis at supraoptimal temperatures. Saturated CO₂-dependent O₂ evolution had a temperature optimum of 35°C for both protoplasts and intact chloroplasts. A sharp decline in activity was observed as assay temperature was increased above 35°C, and at 45°C only 20% of the maximal rate remained. The temperature optimum for 3-phosphoglycerate reduction by intact chloroplasts was 35°C. Above this temperature, 3-phosphoglycerate reduction was more stable than CO₂-dependent O₂ evolution. Reduction of nitrite in coupled intact chloroplasts had a temperature optimum of 40°C with only slight variation in activity between 35°C and 45°C. Reduction of nitrite in uncoupled chloroplasts had a temperature optimum of 40°C, but increasing the assay temperature to 45°C resulted in a complete loss of activity. Reduction of p-benzoquinone by protoplasts and intact chloroplasts had a temperature optimum of 32°C when measured in the presence of dibromothymoquinone. This photosystem II activity exhibited a strong inhibition of O₂ evolution as assay temperature increased above the optimum. It is concluded that, below the temperature optimum, ATP and reductant were not limiting photosynthesis in these systems or intact leaves. Above the temperature optimum, photosynthesis in these systems is limited in part by the phosphorylation potential of the stromal compartment and not by the available reductant.     

Effect of Temperature and Retention Time on Methane Production from Beef Cattle Waste

The effect of temperature and retention time on the rate of methane production from waste of beef cattle fed a finishing diet was investigated by using continuously mixed 3-liter working volume anaerobic fermentors. The temperatures ranged from 30 to 65°C with 5°C increments between fermentors. The fermentors were fed once per day with 6% volatile solids (organic matter). Retention time for each temperature was varied from 18 to 2.5 days. After 3-volume turnovers, samples were obtained on 4 consecutive days. The highest methane production rate (liters/liter of fermentor per day) and methane yield at that rate (liters/gram of volatile solids) were 1.27 and 0.19 at 9 days and 30°C, 1.60 and 0.16 at 6 days and 35°C, 2.28 and 0.23 at 6 days and 40°C, 2.42 and 0.24 at 6 days and 45°C, 2.83 and 0.14 at 3 days and 50°C, 2.75 and 0.14 at 3 days and 55°C, 3.18 and 0.14 at 2.5 days and 60°C, and 1.69 and 0.17 at 6 days and 65°C. Volatile solids degradation at these retention times and temperatures was between 46 and 54%. The concentrations of volatile acids in the 30 to 55°C fermentors were generally below 2,000 mg/liter, with the exception of the 3-day retention time. The 60 and 65°C fermentors were usually above this level for all retention times. These studies indicate potential rates of methane production from the fermentation of untreated waste of beef cattle fed high-grain finishing diets. This information should serve as preliminary guidelines for various kinetic analyses and aid in economic evaluations of the potential feasibility of fermenting beef cattle waste to methane.     

Carbohydrate Level and Growth of Tomato Plants: II. The Effect of Irradiance and Temperature

The growth response of tomato (Lycopersicon esculentum L.) to temperature and irradiance may be related to carbohydrate concentration. Plants in the exponential phase of vegetative growth were grown under temperatures ranging from 9 to 36°C and under low or high irradiances of approximately 110 or 370 microeinsteins per square meter per second photosynthetically active radiation for a 12 hour photoperiod. The relative growth rate, leaf area ratio, net assimilation rate and whole plant carbohydrate levels were measured. At high irradiance, relative growth rate was 43% faster and total nonstructural carbohydrate concentration was 41% greater than at low irradiance. The change in carbohydrate with irradiance could explain the growth response. Plant growth was fastest at 25°C and decreased parabolically at lower and higher temperatures with a half-maximal rate at 13 and 36°C. Total nonstructural carbohydrate decreased between 13 and 23°C and remained constant at higher temperatures. Soluble sugar concentrations varied little with temperature above 13°C except for sucrose, whose level rose above 30°C. The change in carbohydrate with temperature could not explain the growth response. Above 23°C tomato plants appeared to regulate growth rate to maintain a relatively constant nonstructural carbohydrate concentration.     

Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNO₃-insensitive ATPase. The ATPase has high specificities both for substrate and Mg²⁺ ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl⁻. Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9°C) and lower (6.7°C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5°C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.