Temperature-sensitivity of D1 protein metabolism in isolated Zea mays chloroplasts

The effects of low temperature on the synthesis and stability of the 32 kDa D1 protein of photosystem II were investigated in chloroplasts isolated from maize (Zea mays cv. LG11) leaves. The synthesis of D1 by intact chloroplasts in vitro was strongly dependent on temperature; the Q₁₀ for the initial rate of incorporation of [³⁵S]-methionine into D1 was ca. 2.6 over the range 13–25°C. The synthesis of other thylakoid polypeptides exhibited a similar temperature dependence, whilst synthesis of stromal proteins was considerably less temperature-dependent, with the exception of two polypeptides of ca. 56 and 59.5 kDa. The stability of newly-synthesized D1 in the thylakoid membranes was dependent both on the temperature at which the plants were grown and on the temperature during the pulse-labelling period when the protein was synthesized. In chloroplasts isolated from maize leaves grown at 25°C, D1 that was synthesized and assembled at 25 °C in vitro was rapidly degraded during the chase period. At lower chase temperatures the protein was more stable. When chloroplasts from 25°C-grown leaves were pulse-labelled at 13°C, the stability of D1 was markedly enhanced at all temperatures during the chase period. This effect was even more pronounced in chloroplasts isolated from plants grown at 14°C. The implications of these results are discussed with regard to the ability of maize to recover from photoinhibitory damage at low temperatures.     

Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos

The effect of temperature of imbibition on the synthesis and turnover of membrane phosphatidyl choline was studied. Pea seeds (Pisum sativum cv. Alaska) were imbibed in [U-¹⁴C]glycerol and then germinated. Seeds were kept constantly either at 5° or 25°, or were imbibed at one temperature and then germinated at the other one. Glycerol incorporation into phosphatidyl choline in the ER and the plasma membrane, obtained from the embryonic axes after germination, and the glycerol pool were measured. Embryos from seeds kept constantly at 25° showed a rapid incorporation of glycerol into membranes followed by a loss of label; in embryos from seeds kept at 5° incorporation was much lower. Embryos from seeds transferred from 25° to 5° behaved as if continuously kept at 25°, while the behaviour of the embryos from seeds transferred from 5° to 25° resembled embryos from seeds maintained at 5°. The glycerol content of the axes rose during imbibition and fell thereafter. The activities of phospholipases C and D also responded to the initial temperature of imbibition, but the two activities changed differently. The results are discussed in relation to the effect of transient exposure to temperature changes in the seed membranes and the possible way in which such changes are sensed.     

Rapid Decrease in the Glutamine Synthetase Activity during Imbibition of Thermodormant New York Lettuce Seeds

A high glutamine synthetase (GS) activity was detected in dryseeds of New York lettuce but it decreased rapidly during imbibitionat 35°C. The decrease in GS activity was remarkable at 35°C,but not so at 45°C or at 25°C. The activity of extractedGS was relatively constant even at 35°C. The decrease inGS activity stopped immediately after the addition of cycloheximide(CH). This suggests the presence of a GSinactivating systemin the seeds. The amount of ammonia increased abruptly duringthe early stage of imbibition at 35°C, suggesting the blockageof ammonia-assimilation at high temperature. The GS activitythat decreased to a low level during imbibition at 35°Cfor 12 h increased again during the subsequent imbibition atlow temperature (15CC) before the break of thermodormancy. Ahigher GS activity was found in the embryonic axes than in thecotyledons. Partial purification of GS showed that lettuce seedGS was eluted as a single peak on Biogel A1.5m or DEAE-Sephacel(mol wt: 4.4 x 10⁵). Thus the thermodormancy of New York lettuce seeds may be relatedto inactivation of GS during imbibition at high temperatures,and the activity of GS in the embryonic axes may control thegermination of New York lettuce seeds through the regulationof glutamine formation. (Received May 11, 1983; Accepted September 13, 1983)     

Protein Synthesis in Euglena gracilis is Light-and Temperature-Dependent,Oscillating in a Circadian,Temperature-Compensated Manner

Abstract: Incorporation of radiolabelled amino acids into proteins of Euglena gracilis revealed that the amount of labelled protein depends on the conditions of illumination and temperature of cultivation. Protein synthesis was generally lower under dark conditions except at 37 °C. The largest amounts of labelled protein were measured at 21 °C and decreased at higher and lower temperatures. By separating the labelled proteins of the membraneous cell fraction from subcultures under a range of culture conditions, the synthesis of some specific proteins was found to be light- and/or temperature-dependent. On incubating cells taken at different times during a light/dark cycle and under constant conditions, a circadian rhythm of ³⁵S-methionine- as well as ³⁵S-cysteine-incorporation was detected. Thereby the cells incorporated ten-times less cysteine than methionine. Protein synthesis always peaked during the last quarter of the daily light phase, confirming the rhythmic rise in total protein. The length of the rhythm period, approximately 24 h, was nearly independent of the applied temperature in the range of 16 to 27 °C.     

CYANOBACTERIAL DOMINANCE OF POLAR FRESHWATER ECOSYSTEMS: ARE HIGH-LATITUDE MAT-FORMERS ADAPTED TO LOW TEMPERATURE?1

Although it is generally believed that cyanobacteria have high temperature optima for growth (> 20° C), mat-foming cyanobacteria are dominant in many types of lakes, streams, and ponds in the Arctic and Antarctic. We studied the effect of temperature on growth (μ) and relative pigment composition of 27 isolates of cyanobacteria (mat-forming Oscillatoriaceae) from the Arctic, subarctic, and Antarctic to investigate whether they are a) adapted to the low temperature (i.e. psychrophilic) or b) tolerant of the low temperature of the polar regions (i.e. psychrotrophic). We also derived a parabolic function that describes both the rise and the decline of cyanobacterial growth rates with increasing temperature. The cyanobacteria were cultured at seven different temperatures (5°-35° C at 5° C intervals), with continuous illumination of 225 μmol photons.m⁻².s⁻¹. The parabolic function fits the μ-temperature data with 90% confidence for 75% of the isolates. Among the 27 isolates of cyanobacteria studied, the temperature optima (T_opt) for growth ranged from 15° to 35° C, with an average of 19.9° C. These results imply that most polar cyanobacteria are psychrotrophs, not psychrophiles. The cyanobacteria grew over a wide temperature range (typically 20° C) but growth rates were low men at T_opt (average μ_max of 0.23 ± 0.069 d⁻¹). Extremely slow growth rates at low temperature and the high temperature for optimal growth imply that the cyanobacteria are not adapted genetically to cold temperatures, which characterize their ambient environment. Other competitive advantages such as tolerance to desiccation, freeze—thaw cycles, and bright, continuous solar radiation may contribute to their dominance in polar aquatic ecosystems.     

Suppression of amber mutants in vitro induced by low temperature.

Amber mutations are efficiently and specifically suppressed during protein synthesis in vitro in an Su^? S-30 extract at 25 °C, but not at 37 °C. Eight different amber mutations in three different genes have been tested, and all are suppressed. The efficiencies of suppression range from 20 to 35%, when protein synthesis is at the Mg²⁺ concentration optimal for β-galactosidase synthesis at 25 °C. The suppression efficiency increases to approximately 60% at higher Mg²⁺ concentrations, and is reduced to less than 5% at very low concentrations. Ochre and UGA mutations are not suppressed at all under these conditions. The amber suppression is inhibited by addition of a purified protein synthesis release factor to the reaction, or when the protein synthesis reaction takes place in extracts derived from bacteria which are streptomycin-resistant.     

Effect of temperature on yield and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors

Outdoor experiments carried out in Florence, Italy (latitude 43.8° N, longitude 11.3° E), using tubular photobioreactors have shown that in summer the average net productivity of a Spirulina platensis culture grown at the optimal temperature of 35 °C was superior by 23% to that observed in a culture grown at 25 °C. The rates of night biomass loss were higher in the culture grown at 25 °C (average 7.6% of total dry weight) than in the one grown at 35 °C (average 5%). Night biomass loss depended on the temperature and light irradiance at which the cultures were grown, since these factors influenced the biomass composition. A net increase in carbohydrate synthesis occurred when the culture was grown at a low biomass concentration under high light irradiance or at the suboptimal temperature of 25 °C. Excess carbohydrate synthesized during the day was only partially utilized for night protein synthesis.     

INORGANIC CARBON REPLETION DISRUPTS PHOTOSYNTHETIC ACCLIMATION TO LOW TEMPERATURE IN THE CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS1

Acclimation of cyanobacteria to ambient fluctuations in inorganic carbon (Ci) and temperature requires reorganization of the major protein complexes involved in photosynthesis. We grew cultures of the picoplanktonic cyanobacterium Synechococcus elongatus Naegeli across most of its range of tolerable temperatures from 23 to 35°C at both low (<0.1 mM) and high Ci (approximately 4 mM). Over that range of temperatures, the chl‐based doubling time did not differ between low and high Ci grown cells but did increase with decreasing temperature. Cells grown at 23°C high Ci showed an elongated morphology, which was not present in 23°C low Ci cells nor at 35°C high and low Ci. Furthermore, 23°C high Ci cells showed premature senescence and death compared with all other treatments. Phycocyanin per cell was greater in high Ci grown cells at all temperatures but showed a characteristic decrease with decreasing temperature. Functional PSII determination showed that 23°C high Ci cells had 1.5 × 10⁵ PSII·cell^–1 compared with only 6.9 × 10⁴ PSII·cell^–1 for 23°C low Ci. The 35°C high and low Ci cells had 7.7 × 10⁴ and 6.4 × 10⁴ PSII·cell^–1, respectively. These data were supported by immunoblot determinations of PsbA content·cell^–1. As a result of their high PSII·cell^–1, 23°C high Ci cells generated more reductant from PSII than could be accommodated by downstream assimilative metabolism, resulting in early senescence and death of 23°C high Ci cells, probably as a result of the generation of reactive byproducts of electron transport.     

Effect of Combined Salt and Heat Treatments on Germination and Heat-Shock Protein Synthesis in Lentil Seeds

The germination of lentil seeds was gradually reduced when seeds were exposed to temperature of 30 or 40 °C, either alone or combined with 0.1, 0.2 or 0.3 M NaCl or 34.1 % (m/v) PEG 8000, during 6 –12 h imbibition. [³⁵S]-methionine incorporation in 12 h imbibed lentil axes also decreased with increasing NaCl concentration at 20 and 40 °C, whereas at 30 °C only 0.3 M NaCl treatment partially inhibited protein synthesis. An analysis of newly synthesized proteins by 1-D SDS PAGE, showed that the expression of most polypeptides decreased following increasing stress. Among these, low molecular mass heat-shock proteins declining, higher in 40 °C treated axes than those treated at 30 °C, supports the hypothesis that at this temperature maximal level of expression of these proteins was achieved.     

The effect of temperature on feeding,growth, and metabolism during the last larval stadium of the female house cricket, Acheta domesticus

Eighth instar female house crickets at 35°C developed faster, gained slightly more wet weight, and consumed less food, water, and oxygen than at 25°C. The duration of the 8th stadium at 25°C was 13 days (undisturbed), but was 14 days when disturbed by daily weighing. The duration of the 8th stadium at 30°C was 8 days and at 35°C was 6 days. During the first half of the 8th stadium at 25, 30, and 35°C, there was a high rate of food and water consumption resulting in statistically equal maximum dry weight achievement (124 mg). Respiratory quotients greater than one during this time indicated the conversion of ingested carbohydrate to fat. During the latter half of the 8th stadium, food and water consumption declined and the crickets lost weight. The period of weight loss was proportionally much longer at 25°C than at 30 or 35°C. Respiratory quotients lower than 1.0 during the latter half of the 8th stadium at 30 and 35°C indicated the metabolism of stored lipids. The respiratory quotient at 25°C never fell below 1.0, possibly because some food remained in the gut. The absorption efficiency was not influenced by temperature (25–35°C). Though the caloric content of the faeces was lower at 25°C than at 30 or 35°C, which correlated to the much longer time for food passage at 25°C than at 35°C, the difference in total calories egested was insufficient to alter the absorption efficiency. A longer period of reduced feeding and greater dry weight loss during the latter half of the 8th stadium at 25°C resulted in a lower metabolic efficiency at 25°C than at 30 or 35°C. Eighth instar crickets in response to a step-function transfer from 30°C–25 or 35°C showed an immediate (<1 hr) and complete metabolic adjustment which was not affected by the temperature history during the 7th stadium. House crickets did not exhibit temperature acclimation in the range 20–40°C, the metabolic rate being determined by ambient temperature. The Q₁₀ for oxygen consumption in the range 20–40°C was about 2.     

Decrease in sunflower (Helianthus annuus) seed viability caused by high temperature as related to energy metabolism, membrane damage and lipid composition

The aim of the present work was to investigate whether loss of germination ability and viability of sunflower (Helianthus annuus L.) seeds during incubation at a high temperature (45°C) was related to changes in energy metabolism, loss of membrane integrity, and/or changes in lipid composition. Pre‐treatment of seeds at 45°C progressively reduced subsequent germination at the optimal temperature (25°C). Seeds did not germinate at 45°C and almost all of them were dead after 72 h of soaking at this high temperature. This loss of seed viability was associated with a large increase in leakage of K⁺ and total electrolytes into the incubation medium, and with production of malondialdehyde in the embryonic axis and cotyledons, suggesting a loss of membrane integrity probably due to lipid peroxidation. ATP and ADP levels increased sharply during the first hours of imbibition at 45°C, remained high for about 24 h and then decreased. As a consequence, the energy charge followed a similar pattern. If the treatment at 45°C did not exceed 48 h, seeds recovered an apparently normal energy metabolism after transfer to 25°C, even though they lost their ability to germinate at this temperature. Therefore, energy metabolism at the whole embryo level cannot be considered as an indicator of germination ability. Incubation of seeds at 45°C resulted in an increase in triacylglycerols and diacylglycerols without a significant change in their fatty acid composition. It also induced a slight increase in phospholipid content with an increase in C_16:0, C_18:0 and C_18:1, but with no change in C_18:2. In phospholipids, the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios thus declined during treatment at 45°C. The results obtained suggest that deterioration of sunflower seeds during incubation at a high temperature is mainly related to membrane damage and alteration of energy metabolism, and that accumulation of malondialdehyde, which is an index of lipid peroxidation, does not correspond to a decrease in total lipids and phospholipids nor to a significant change in fatty acid composition, except in PL in which the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios slightly declined.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

Temperature sensitive phases during the germination of lettuce (Lactuca sativa) seeds

Lettuce seeds cvs Hilde, Feltham King and Avoncrisp were subjected, at different phases during imbibition at 22°C, to a high temperature (33°C) inhibitory for germination, for periods ranging from 4 to 144 h, before returning them to 22°C. The results showed, that the first 4h of imbibition and also the phase between the commencement of mitosis and the onset of radicle emergence were more sensitive to the effects of high temperature than other phases in the germination process. Short exposures (8–24 h) to 33°C commencing at the latter phase delayed germination by up to 4 days, and at the earlier by up to 8 days. Percentage germination was unaffectd except after prolonged exposures (> 48 h) from the beginning of imbibition, which reduced it. Seedling emergence from moist sieved soil was both delayed and reduced when imbibing seeds were exposed for a short period from the beginning of imbibition to 33°C compared with seeds imbibing continuously at 19°C. Germination was delayed and not reduced when seed was exposed to 33°C at the phase between commencement of mitosis and the onset of radicle emergence.     

Accumulation and turnover of abscisic acid in osmotically stressed cotton leaf tissue in relation to temperature

Leaf discs of cotton (Gossypium hirsutum L. cv. Deltapine 70) were osmotically stressed by floating them on solutions of polyethylene glycol 8000. The tissue produced copious amounts of abscisic acid (ABA) when stressed. Accumulation of ABA depended strongly upon temperature during the incubation, displaying a maximum at 20°C. At 35°C, the amount of ABA accumulated after 24 h was 45–80% less than at 20°C. Temperature did not affect leakage of ABA into the medium. Turnover rate of [¹⁴C]ABA was more than 3 times greater at 35°C than at 20°C. This rapd turnover at 35°C could account for the decreased ABA accumulation. Three ¹⁴C-containing metabolites of ABA were extracted from the tissue. At 20°C, two of these accumulated and retained substantial ¹⁴C over 16 h. At 35°C, though, the ¹⁴C in one of these compounds was almost completely lost during the last 8 h of the incubation. Although the metabolites are not identified, the results show some specific effects of temperature on ABA metabolism. The strong effect of temperature on ABA accumulation may contribute to patterns of ABA-dependent processes (such as stomatal closure) during water stress.     

Ethanolamine incorporation into membranes of pea embryonic axes during germination

Changes in the ethanolamine pool of the embryonic axes of pea seeds exposed to different temperatures during imbibition and germination were followed. The ethanolamine pool decreased except during imbibition at 25°. Label from ethanolamine was incorporated almost entirely into phosphatidylethanolamine with incorporation into phosphatidylcholine being observed only after imbibition and germination at 25°. The incorporation of ethanolamine was apparently less sensitive to temperature than that of choline and glycerol, previously reported. Preliminary results also show an effect of the imbibition temperature on some of the membrane proteins, but most did not seem to be affected.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

Effects of assay and acclimation temperatures on incorporation of amino acids into protein of isolated hepatocytes from the european eel, Anguilla anguilla L.

Hepatocytes of adult eels acclimated to 5° C, 10° C and 20° C, respectively were isolated by perfusion of the liver with collagenase. The liver-somatic index and the protein content of liver cells showed significantly higher values in fish kept at the lower temperatures. However, in the adenine nucleotide content and energy charge no significant differences were observed between the 5° C and the 20° C acclimation groups. The incorporation of radioactivity from a ¹⁴C-labelled amino acid mixture into perchloric acid precipitates was used as an estimate of over-all protein synthesis. When eel hepatocytes were incubated in Hanks' solution containing tracer amounts of amino acids, labelling of perchloric acid precipitates showed linear time courses over at least 60 min at 10° C and 20° C assay temperatures. The total cellular radioactivity, however, exhibited non-linear time courses. In the measurement range from 5° C to 25° C Arrhenius plots of protein labelling exhibited a discontinuity in both groups of fish. Hepatocytes from 10° C-acclimated eel showed almost twice the incorporation rates of amino acids as those from the 20° C-acclimated fish. It is concluded that high temperature dependencies in the low temperature range require an increase in the capacity of the apparatus for protein synthesis during cold acclimation.