Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between elongation growth and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000g pelletable (2KP) fraction from lettuce (Lactuca sativa L., cv. Arctic) hypocotyl sections has been examined. Sections were loaded with incremental amounts of GA₁ under conditions where growth was arrested (5° C) or permitted (30° C) and, after 16 h, all were transferred to a GA-free medium at 30° C. Growth and 2KP radioactivity were measured at this point and after a further 24 h in the chase medium. Uptake was reduced by 80% at 5° C, as compared to 30° C, but 2KP labelling and protein synthesis were only reduced by half. The growth rate of the 5° C pretreated sections during the chase period was comparable to that observed during the pulse in the 30° C material but the dose/response relationship was flatter. Low temperature sections incorporated a much higher percentage of GA₁ uptake into the 2KP fraction (27% at maximum) but the absolute levels of labelling at this temperature were lower than those measured at 30° C. The data are interpreted as showing that 2KP labelling is not a consequence of growth. It must either precede response or be an unconnected concurrent process.     

Effects of short-term heat stress on oxidative damage and responses of antioxidant system in Lilium longiflorum

This paper aims to determine the changes in reactive oxygen species (ROS) and the responses of the lily (Lilium longiflorum L.) antioxidant system to short-term high temperatures. Plants were exposed to three levels of heat stress (37°C, 42°C, 47°C) for 10 h when hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rate along with membrane injury indexes, and changes in antioxidants were measured. Compared with the control (20°C), electrolyte leakage and MDA concentration varied slightly after 10 h at 37°C and 42°C, while increased significantly at 47°C. During 10 h at 37°C and 42°C, antioxidant enzyme activities, such as SOD, POD, CAT, APX and GR, were stimulated and antioxidants (AsA and GSH concentrations) maintained high levels, which resulted in low levels of O₂⁻ and H₂O₂ concentration. However, after 10 h at 47°C, SOD, APX, GR activities and GSH concentration were similar to the controls, while POD, CAT activities and AsA concentration decreased significantly as compared with the control, concomitant with significant increase in O₂⁻ and H₂O₂ concentrations. In addition, such heat-induced effects on antioxidant enzymes were also confirmed by SOD and POD isoform, as Cu/ZnSOD maintained high stability under heat stress and the intensity of POD isoforms reduced with the duration of heat stress, especially at 47°C. It is concluded that in lily plants, the oxidative damage induced by heat stress was related to the changes in antioxidant enzyme activities and antioxidants.     

Effects of circadian rhythms of fluctuating temperature on growth and biochemical composition of <Emphasis Type="Italic">Ulva pertusa</Emphasis>

The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20 ± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function of the thermal amplitude. Such a growth model can be described by G = β ₀ + β ₁(TA) + β ₂(TA)², where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β ₀ is the intercept on the G axis, and β ₁ and β ₂ are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at 20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga at the diel fluctuating temperatures in the present study. Handling editor: S. M. Thomaz     

Respiration rates of subitaneous eggs from a marine calanoid copepod: monitored by nanorespirometry

The oxygen consumption rate during embryogenesis of Acartia tonsa subitaneous eggs were measured at different temperatures (10, 15, 17, 21, 24 and 28°C) with nanorespirometry. The oxygen consumption was constant during the embryogenesis but increased rapidly at hatching time. The mean ± SD oxygen consumption rate increased exponentially with temperature and ranged from 0.09 ± 0.04 (10°C) to 0.54 ± 0.09 nmol O₂ egg⁻¹ h⁻¹ (28°C). The mean ± SD Q₁₀-value was 2.51 ± 0.15. Calculations of energy consumption during embryogenesis ranged from 1.86 to 18.28 mJ depending on temperature and development time. We conclude that the effect of temperature on oxygen consumption rate was far less important than the prolonged development time when calculating the energy consumed during embryogenesis.     

Encarsia formosa parasitizing the Poinsettia-strain of the cotton whitefly,Bemisia tabaci, on Poinsettia: bionomics in relation to temperature

Adult longevity, developmental time and juvenile mortality ofEncarsia formosa Gahan (Hymenoptera:Aphelinidae) parasitizing the Poinsettia-strain ofBemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) on Poinsettia (Euphorbia pulcherrima Willd.) were investigated in laboratory experiments at three temperatures: 16 °C, 22 °C and 28 °C. Furthermore, the parasitoid's preference for different larval stages of the whitefly was determined at 24.5 °C. The lifespan ofE. formosa decreased with temperature from one month at 16 °C to nine days at 28 °C. A lower temperature threshold of 11 °C for adult development was found. The development of juvenile parasitoids inB. tabaci lasted more than two months at the lowest temperature, but was only 14 days when temperature was 28 °C. The lower temperature threshold for immature development was 13.3 °C, yielding an average of 207 day-degrees for the completion of development into adults. Juvenile mortality was high, varying from about 50% at 16 °C to about 30% at 22 °C and 28 °C.E. formosa preferred to oviposit in the 4th instar and prepupal stages ofB. tabaci followed by the 2nd and 3rd instars. The preference for the pupal stage was low. The parasitoid used all instars of the whitefly for hostfeeding, with no apparent differences between the stages. The average duration of the oviposition posture was four minutes. Demographic parameters were calculated from life tables constructed from the data. The intrinsic rate of increase (r _m) and the net reproductive rate (R ₀) increased with temperature from 0.0279 day⁻¹ at 16 °C to 0.2388 day⁻¹ at 28 °C and from about 12 at 16 °C to about 66 at 28 °C, respectively.     

PHOSPHOLIPASE ACTIVITY IN SQUID AND FROG AXONS

Evidence for the presence of phosphatide acylhydrolase activity (EC 3.1.1) in centrifuged homogenate supernatants and extracts of squid giant axons and centrifuged homogenate supernatants of frog sciatic nerve bundles is reported. The enzyme was assayed by measurement of the rate of deacylation of [U-¹⁴C]phosphatidyl choline. The deacylation activity in the nerve homogenate supernatants exhibits: a pH maximum at 7.2–7.4 (25°C); a calcium ion maximum at 12–13 mM-CaCl₂(aq); a K_m value of 3.4 × 10^?4 M (25°C); and a temperature maximum at 37°C. The activation energy over the range 8–37°C is 5.7 ± 0.2kcal-mol^?1.     

The influence of temperature and nitrogen source on growth and nitrogen uptake of two polar-desert species,Saxifraga caespitosa and Cerastium alpinum

Polar-desert plants experience low average air temperatures during their short growing season (4–8 °C mean July temperature). In addition, low availability of inorganic nitrogen in the soil may also limit plant growth. Our goals were to elucidate which N sources can be acquired by polar-desert plants, and how growth and N-uptake are affected by low growth temperatures. We compared rates of N-uptake and increases in mass and leaf area of two polar-desert species (Cerastium alpinum L. and Saxifraga caespitosa L.) over a period of 3 weeks when grown at two temperatures (6 °C vs. 15 °C) and supplied with either glycine, NH₄ ⁺ or NO₃ ⁻. At 15 °C, plants at least doubled their leaf area, whereas there was no change in leaf area at 6 °C. Measured mean N-uptake rates varied between 0.5 nmol g⁻¹ root DM s⁻¹ on glycine at 15 °C and 7.5 nmol g⁻¹ root DM s⁻¹ on NH₄ ⁺ at 15 °C. Uptake rates based upon increases in mass and tissue N concentrations showed that plants had a lower N-uptake rate at 6 °C, regardless of N source or species. We conclude that these polar-desert plants can use all three N sources to increase their leaf area and support flowering when grown at 15 °C. Based upon short-term (8 h) uptake experiments, we also conclude that the short-term capacity to take up inorganic or organic N is not reduced by low temperature (6 °C). However, net N-uptake integrated over a three-week period is severely reduced at 6 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxygen consumption by ammocoetes of the lampreyGeotria australis in air

 When covered by moistened lint-free gauze, the larvae (ammocoetes) of the lamprey Geotria australis survived, without apparent discomfort, for 4 days in water-saturated air at 10, 15 and 20 °C. In air, the mean standard rates of O₂ consumption of medium to large ammocoetes of G. australis (xˉ=0.52 g) at 10, 15 and 20 °C were 14.5, 35.7 and 52.1 μl⋅g^-1⋅h^-1, respectively. At 15 °C, the slope of the relationship between log O₂ consumption (μl O₂⋅h^-1) and log body weight for ammocoetes over a wide range in body weight was 0.987. The Q ₁₀s for rate of O₂ consumption between 10 and 15 °C, 15 and 20 °C and 10 and 20 °C were 4.9, 2.9 and 3.6, respectively. Our results and observations of the ammocoetes suggest that, when out of water, larval G. australis derives most of its O₂ requirements from cutaneous respiration, particularly at lower temperatures. This would be facilitated by the small size and elongate shape (and thus a relatively high surface-to-volume ratio), low metabolic rate, thin dermis, extensive subdermal capillary network and high haemoglobin concentration of larval G. australis. Accepted: 28 March 1996     

Effects of ambient temperature and of temperature acclimation on nitrogen excretion and differential catabolism of protein and nonprotein resources in the intertidal snails,Littorina saxatilis (Olivi) and L. obtusata (L.)

Nitrogenous excretion in two snails, Littorina saxatilis (high intertidal) and L. obtusata (low intertidal) was studied in relation to temperature acclimation (at 4° and 21°C), including total N excretion rates, the fraction of urea in N excretion, corresponding O:N ratios and the partitioning of deaminated protein between catabolic and anabolic processes at 4°, 11° and 21°C. Aggregate N excretion rates in both species showed no significant compensatory adjustments following acclimation. Total weight specific N excretion rates at 21°C were higher in standard 3 mg L. saxatilis (739 ng N mg⁻¹ h⁻¹) than standard 5 mg L. obtusata (257 ng N mg⁻¹ h⁻¹) for snails acclimated to 21°C. Comparisons of Q₁₀ values of total weight specific N excretion to Q₁₀ values for weight specific oxygen consumption ({xxV}O₂) between 4° to 11 °C and 11° to 21°C indicated that, while total rates of catabolic metabolism ({xxV}O₂) and protein deamination in L. obtusata were essentially parallel, the relationship between N excretion and {xxV}O₂ in L. saxatilis revealed the partitioning of a larger share of deaminated protein carbon into anabolism at 4° and 21°C than at 11°C. Urea N accounted for a larger share of aggregate N excreted in L. saxatilis than in L. obtusata, but in both species urea N is a greater proportion of total N excreted when acclimated at 4°C (urea N: ammonia N ratio range: 1 to 2.15) than in snails acclimated to 21°C (urea N: ammonia N ratio range: 0.46 to 1.39). Molar O:N ratios indicate that the proportion of metabolism supported by protein catabolism is greater in L. saxatilis (O:N range: 2.5–8.4) than in L. obtusata (O:N range: 7.3–13.0). In both species, regardless of acclimation temperature, the O:N ratios are generally lowest (high protein catabolism) at 4°C and highest at 21°C.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

黄河口湿地柽柳灌丛土壤盐渍化特征

为探究黄河三角洲湿地柽柳灌丛下土壤的盐渍化特征,在黄河三角洲国家级自然保护区(37°35''-12''N,118°33''-119°20''E)黄河入海口附近,根据长势基本一致的原则分别在碱蓬群落、柽柳群落和芦苇群落各选3株柽柳,采集柽柳灌丛下土壤样品,分析土壤盐分和盐碱化参数的空间分布以及距基茎不同距离处研究对象(土壤总盐(TS)、电导率(EC)、pH、交换性钠百分率(ESP))和环境因子(Na⁺、K⁺、Ca²⁺、Mg²⁺、Cl^-、HCO₃^-、SO^2-₄)之间的关系。结果表明：(1)研究区土壤为弱碱化盐土,离子含量由高到低依次为Cl^->Na⁺>SO^2-₄ >Ca²⁺>Mg²⁺>HCO^-₃>K⁺。除pH在土壤表层数值最低外,表层土壤TS、EC、ESP和盐分离子大于深层土壤,显示表聚性。(2)土壤盐分和盐碱化参数空间分布总体为：在柽柳基茎周围形成"盐谷"、"碱谷"效应, Na⁺、Mg²⁺、Cl^-表现为"盐谷",K⁺ 、SO^2-₄ 、Ca²⁺ 表现为"盐岛"。(3)在整个土壤剖面中,与TS、EC相关性最强的阴阳离子为Mg²⁺、Cl^-,从灌丛中心到灌丛间裸地Ca²⁺、SO^2-₄与TS、EC的相关性逐渐减弱,Mg²⁺、Cl^-与TS、EC的相关性逐渐增强。Ca²⁺和SO^2-₄与pH表现为较强的负相关性;与ESP相关性最强的阴离子为HCO^-₃,与之相关性最强的阳离子为Na⁺和K⁺,并且Na⁺和K⁺与ESP的相关性表现出从灌丛中心向外逐渐增强。(4)土壤盐渍化主要受控于Na⁺,从灌丛下到灌丛间裸地Cl^-对盐渍化程度的影响逐渐增加,SO^2-₄的影响逐渐降低。     

Effect of temperature on ion content, ion fluxes and energy metabolism in Chara corallina

Abstract Effects of temperature on the ionic relations and energy metabolism of Chara corallina were investigated. Measurements were made of the ionic content, tracer ion fluxes, and photosynthetic and dark CO₂ fixation in isolated cells, and of O₂ exchange in photosynthesis and respiration in isolated shoot apices. The total intracellular concentration of K⁺, Na⁺ and Cl^? was the same in cells held for 5 days in non-growing medium at 15°C (the growth temperature) as in those held at 25°C or 5°C. The tracer influx in the light of all ions tested (Rb⁺, Na⁺, CH₃NH₃⁺, Cl^? and H₂PO₄^?) was lower at 5°C than at 15°C in experiments in which cells were subjected to 5°C for less than 24 h in toto. The influx at 25°C was greater than that at 15°C for H₂PO^?₄, there was no difference between the two temperatures for Na⁺, while the influx at 25°C was less than that at 15°C for Cl^?, Rb⁺ and CH₃NH₃⁺ For Cl^? and H₂PO^?₄ similar results were found in later experiments with cells grown at 20—23°C. Photosynthetic CO₂ fixation and O₂ evolution, and respiratory O₂ uptake, are greater at 25°C, and lower at 5°C, than they are at the growth temperature of 15°C. In longer-term pretreatments at the different temperatures, tracer Cl^? influx at 15°C and particularly at 25°C were lower than in short-term experiments, while the influx at 5°C was higher. It was concluded from these experiments, and from previous data on H⁺ free energy differences across the plasmalemma, that (1) the maintenance of internal ion concentrations involves a close balancing of influx and efflux of K⁺, Na⁺ and Cl^? at all experimental temperatures; (2) the regulation of the tracer fluxes of the ions is kinetic rather than thermodynamic and (3) that the tracer fluxes at low temperatures are not restricted by the rate at which respiration or photosynthesis can supply energy to them.     

Diurnal course of photochemical activity of winter-adapted Scots pine at subzero temperatures

The photochemical activity of native Central Siberian Scots pine trees (Pinus sylvestris L) was estimated from the middle of February to the middle of March 2001. We measured chlorophyll (Chl) fluorescence in attached intact needles from trees located approx. 30 km west of the Yenisey river (60°44′N, 89°09′E) near the village of Zotino. In this period, the air temperature varied between −39 °C and +7 °C. At temperatures below −10 °C, P. sylvestris needles did not exhibit any variable Chl fluorescence during the daylight period. During the night, however, the effective quantum yield of photosystem 2 (PS2) photochemistry, Φ₂ [Φ₂ = (F_m′ − F_t)/F_m′), increased from values near zero to values between 0.05 and 0.20 depending on the needle temperature and sample investigated. The increase started soon after dusk and lasted for 3–6 h depending on the temperature. A faster increase of Φ₂ was found for temperatures around −16 °C, and lower rates occurred at lower temperatures. Irrespective of the temperature, Φ₂ decreased rapidly to near zero values at dawn, when the photosynthetic photon flux density increased to about 1–5 μmol m⁻² s⁻¹, and remained near zero throughout the day. At temperatures higher than −10 °C, the diurnal decrease and the nocturnal increase of Φ₂ were less distinct or disappeared completely. Hence the winter-adapted Scots pine maintains some photochemical activity of PS2 even at extremely cold temperatures. The capacity of photochemical reactions below −10 °C is, however, very limited and PS2 photochemistry is saturated by an extremely low irradiance (less than 5 μmol m⁻² s⁻¹).     

Isolation and characterization of a <Emphasis Type="Italic">Mastigocladus</Emphasis> species capable of growth,N<Subscript>2</Subscript>-fixation and N-assimilation at elevated temperature

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2–3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2–3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO₃ ⁻ and NH₄ ⁺, respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO₃ ⁻-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH₄ ⁺)-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.     

Effects of light,temperature and salinity on the growth rate of harmful marine diatoms,Chaetoceros convolutus and C. concavicornis that kill netpen salmon

Chaetoceros convolutus and C. concavicornis have been implicated in the death of salmon in netpens in the Pacific Northwest by damaging the salmon's gills. To better understand how environmental factors affect the distribution of these two species, the interacting effects of light, temperature and salinity on growth rate were examined by growing these species under a range of temperatures (4–18 °C), light (10–175 μmol photon m⁻² s⁻¹) and salinities (10–30‰). For C. convolutus, the growth rate showed a hyperbolic relationship with irradiance at 8, 14 and 18 °C and light saturation occurred at 9, 14 and 20 μmol photon m^t s⁻¹ respectively. At 4 °C for C. convolutus and 8 °C for C. concavicornis, cells grew at μ_max, even at the lowest irradiances tested (10 μmol photon m⁻² s⁻¹). For C. convolutus, the amount of light required to saturate growth rate increased with temperature in an approximately linear fashion. The Q₁₀ was 1.88, calculated by averaging over both species. C. concavicornis was the more euryhaline species growing at salinities as low as 17.5‰, while C. convolutus grew only at 25‰ and above.     

Components of natural gas resulting from thermal degradation of the blue-green alga (cyanobacterium)Oscillatoria tenuis

Cultures of the blue-green alga (cyanobacterium)Oscillatoria tenuis were used to simulate thermal degradation and gas formation by heating without oxygen at 250° and 350 °C for 100 h. Analysis through gas chromatography showed that the gases were mainly CH₄, C₂H₆, C₃H₈, iC₄ (isobutane), nC₄ (normal butane), iC₅ (isopentane), nC₅ (normal pentane), H₂, C0₂ and N₂. The volume of gases per g dry weight of alga was 44 ml at 250 °C and 100 ml at 350 °C. Alkane gas comprised only 2.04% of the total at 250 °C and rising to 40.0% at 350 °C. The fraction of C0₂ decreased from 83.3% at 250 °C to 40.0% at 350 °C. The quantity of alkane in the soluble organic matter doubled with rising temperature but the H/C atomic ratio in the ‘kerogen’, insoluble organic matter, decreased sharply. Infrared spectra of the ‘kerogen’ showed that the peak of adipose radical at 2900 cm⁻¹ disappeared gradually with rising temperature, which reflects the gradual break of CH₄ or C₂H₆ from ‘kerogen’. This demonstrates that insoluble organic matter rather than soluble organic matter in blue-green algae are the main sources of the gas alkanes in the process of simulated thermal degradation.     

Effect of temperature on the ATP pool and adenylate energy charge in Escherichia coli

The effect of cultivation temperature on the ATP pool and adenylate energy charge (EC) in Escherichia coli has been studied in both batch and continuous cultures. In batch culture, μ_max and the ATP pool increased with increasing growth temperatures between 27–42°C (from 0.26 to 0.62 h⁻¹, and from 5.1 to 8.2 nmol/mg dry wt., respectively). In continuous culture at a constant dilution rate (D = 0.2 h⁻¹), with increasing growth temperatures between 28–43°C, the ATP pool increased about 2-fold (from 4.2 to 8.1 nmol/mg dry wt) and the EC from 0.80 to 0.99.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

Low Root Temperature Retardation of the Mineral Nitrogen Induced Decline in N2Fixation by a Northern Ecotype of White Clover

Nodulated white clover plants (Trifolium repensL.) of a Norwegian ecotype from Pasvik (70°N) were grown in flowing solution culture. Root temperature was 17°C until 51d after sowing, when it was lowered decrementally over 5d to 7°C in four of the eight plant culture units. After a further 24h, mineral N was supplied automatically at 20μMNH₄NO₃in three culture units at each root temperature (7 and 17°C) over 17d. The remaining two units provided control plants solely dependent on N₂fixation at 7 and 17°C.The supply of NH₄NO₃greatly reduced the nodule biomass per plant at 17°C over 17d compared with control plants, but had little effect at 7°C. The nodule decline at 17°C accompanied an acute and progressive decrease in specific rate of N₂fixation, from 9mmolN d^-1g^-1nodule d.wt on day 0 to zero by day 10. Whilst initial rates of N₂fixation were lower at 7°C, the mineral N-induced decrease in fixation rates was also less severe than at 17°C and specific fixation rates recovered after reaching a minimum on day 11. N₂fixation accounted for 36% of the total uptake of N by +min.N plants during the treatment period at 7°C as opposed to only 13% at 17°C. The total N₂fixed at 7°C was 86% of that fixed at 17°C, although the specific growth rate (d.wt) at 7°C was only 55% of that at 17°C. Addition of NH₄NO₃at 7°C had little effect on the gross amount of N₂fixed subsequently. In contrast, total N₂fixation by +min.N plants at 17°C was only 24% of that fixed by the corresponding controls. The possible mechanisms by which mineral N affects N₂fixation are discussed.     

A highly thermostable trehalase from the thermophilic bacterium <Emphasis Type="Italic">Rhodothermus marinus</Emphasis>

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K _m of 0.16 mM and a V _max of 81 μmol of trehalose (min)⁻¹ (mg of protein)⁻¹ at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K _i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.