Effect of acetaldehyde, arsenite, ethanol, and heat shock on protein synthesis and chilling sensitivity of cucumber radicles

Exposure to a chilling temperature of 2.5°C for 96 h inhibited the subsequent growth of cucumber seedling radicles at 25°C by 92%. Exposing seedling with 5 ± 1 mm long radicles to acetaldehyde vapour (275 µl l⁻¹) or to an aqueous ethanol solution (0.6  M ) for 2 h, or to 45°C for 10 min before chilling, increased chilling tolerance so that the chilling treatment reduced growth by only 47, 39 or 36%, respectively. All of these effective treatments induced the synthesis of a number of proteins, and suppressed de novo protein synthesis (i.e. the incorporation of [³⁵S]-methionine) by about 70%. In contrast, treatment for 2 h with an aqueous arsenite solution (100 µ M ) had no effect on chilling sensitivity or the incorporation of [³⁵S]-methionine, yet it induced the synthesis of a complement of proteins that were similar to that induced by the effective heat-shock treatment. A unique protein or set of proteins may be responsible for heat-shock-induced chilling tolerance, but none was detected. The ability of various abiotic stresses to suppress protein synthesis may be more important in increasing tolerance to chilling injury than their ability to induce the synthesis of specific proteins.     

The heat shock response in meso- and thermoacidophilic chemolithotrophic bacteria

Abstract The heat shock response was studied in a chemolithotrophic thermoacidophilic archaebacterium Sulfolobus acidocaldarius (shifted from 70° to 85°C) and a mesoacidop0ilic microorganism Thiobacillus ferrooxidans (from 30° to 41°C). When transferred from their normal growth temperature to the stress temperature, cells showed a decrease in the incorporation of Na₂¹⁴CO₃ into proteins, and at the same time, the synthesis of a specific subset of heat shock proteins was observed. Ethanol (4%) at 30°C, also caused a response similar to the heat shock upon T. ferrooxidans cells, whereas Sulfolobus cells at 70°C did not incorporate radioactive CO₂ in the presence of ethanol, apparently being damaged by the organic solvent.     

Wound-induced PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45°C reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R² > 0.90) in tissue developing the normal wound response and in tissue subjected to 0–180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45°C for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.     

Cloning and characterization of a relA/spoT homologue from Bacillus subtilis

A PCR-amplified DNA fragment of the relA gene from genomic Bacillus subtilis DNA was used to isolate the entire relA / spoT homologue and two adjacent open reading frames (ORFs) from a λ ZAP Express library. The relA gene, which encodes a protein of 734 amino acid residues (aa), is flanked by an ORF (170 aa) that shares high similarity to adenine phosphoribosyltransferase genes ( apt ), and downstream by an ORF (131 aa) of unknown function. This genetic organization is similar to that in Streptomyces coelicolor A3(2) and Streptococcus equismilis H46A. relA shows significant similarity to the Escherichia coli relA and spoT genes, which are responsible for the synthesis and degradation of the highly phosphorylated guanosine nucleotides (p)ppGpp, triggering the stringent response. Deletion of the relA gene generated a (p)ppGpp⁰ phenotype that demonstrated its essential role in the response to amino acid deprivation and resulted in impaired/lowered induction of proteins involved in stress response as well as amino acid biosynthesis, as judged by two-dimensional gel electrophoresis. The same effects of impaired induction of some σ^B-independent proteins could also be shown in a sigB/relA double mutant, supporting the role of relA in derepression/induction of catabolic and anabolic genes during stringent response.     

Temperature-dependent photoinhibition and recovery of photosynthesis in the green alga Chlamydomonas reinhardtii acclimated to 12 and 27°C

Photoinhibition of photosynthesis and subsequent recovery were studied in cultures of the unicellular green alga Chlamydomonas reinhardtii L. (wt strain 137 c mating type +) acclimated at high (27°C) and low (12°C) temperature, Photoinhibition was assayed by fluorescence kinetics (77K) and oxygen evolution measurements under growth temperature conditions Inhibition of 50% was obtained by exposing cultures acclimated at high temperature to a photosynthetic photon flux density (PPFD) of 1 600 μmol m⁻² S⁻¹ at. 27°C. and cultures acclimated at low temperature to a PPFD of 900 μmol m⁻² s⁻¹ at 12°C When the photoinhibitory conditions were shifted it was revealed that algae acclimated at low temperature had acquired an increased resistance to photoinhibition at both 12 and 27°C. Furthermore, acclimation at low temperature increased the capacity to recover from 50% photoinhibition at both 12 and 27°C Studies of photoinhibition in the presence of the protein synthesis inhibitor, chloramphenicol, revealed that in response to acclimation at low temperature during growth the algae became more dependent on protein synthesis to avoid photoinhibition. It is suggested that acclimation at low temperature rendered C. reinhardtii an increased resistance to photoinhibition by. increasing the rate of turnover of photodamaged proteins in photosystem II (PS II). However, we cannot exclude the possibility that the increased resistance to photoinhibition of C. reinhardtii acclimated at low temperature also involves modifications of the mechanism of photoinhibition.     

Influence of nutrient-limited growth on pathogenesis-associated outer membrane proteins of Yersinia enterocolitica

From studies based on batch culture, it has been postulated that the expression of the virulence-associated proteins of Yersinia spp. is controlled by temperature and Ca²⁺, such that these proteins are synthesized only at the higher temperature (37°C) and calcium-scarce conditions of the intracellular environment. It was found, however, that in Yersinia enterocolitica one of these proteins (140 kDa) is not synthesized at submaximal growth rates under any of the relevant conditions, and that another of the implicated proteins (34 kDa), is synthesized even at 28°C during nutrient-limited growth. Thus, temperature and Ca²⁺ influence the synthesis of these proteins differently under growth conditions that better approximate the natural environments than do batch cultures.     

Regulation of the heat-shock response depends on divalent metal ions in an hfIB mutant of Escherichia coli

HfIB, also called FtsH, is an essential Escherichia coli protein involved in the proteolysis of the heat-shock regulator σ³² and of the phage regulator λcll. The hfIB1 (Ts) allele (formerly called ftsH1 ) conferring temperature-sensitive growth at 42°C is suppressed by loss of the ferric-uptake repressor Fur and by anaerobic growth. We show here that suppression requires TonB-dependent Fe(III) transport in the hfIB1 (Ts) fur mutant during aerobic growth at 42°C and Feo-dependent Fe(II) transport during anaerobic growth at 42°C. Temperature-resistant growth of hfIB1 (Ts) strains is also observed at 42°C in the presence of a high concentration of Fe(II), Ni(II), Mn(II) or Co(II) salts, but not in the presence of Zn(II), Cd(II), Cu(II), Mg(II), Ca(II) or Cr(III) salts. However, neither Ni(II) nor a fur mutation permits growth in the complete absence of HfIB. The heat-shock response, evaluated by an htpG :: lacZ fusion, is overinduced in hfIB1 (Ts) strains at 42°C because of stabilization of σ³². Growth in the presence of Ni(II) or in the absence of the Fur repressor abolishes this overinduction in the hfIB1 (Ts) strain, and, in the hfIB1 (Ts) fur mutant, σ³² is no longer stabilized at 42°C. These results reinforce the recent observation that HfIB is a metalloprotease active against σ³² in vitro and suggest that it can associate functionally in vivo with Fe(II), Ni(II), Mn(II) and Co(II) ions.     

Cold-induced changes in the polysome pattern and protein synthesis in winter rye (Secale cereale) leaves

Cold-induced changes in the polysome pattern and protein synthesis were analyzed in winter rye, Secale cereale L. cv. Voima, during one week's cold stress treatment, which was performed by transferring the 7-day-old plants from the greenhouse (25°C, long-day conditions) to 3°C and a photoperiod of 10. 5 h. Freezing resistance determined by electrolyte leakage increased significantly upon cold stress starting from LT₅₀ value –5°C. and reaching –9°C on the day 7 of cold exposure. After 4 weeks at low temperature, plants reached an LT₅₀ of –12°C. The polysome content increased markedly during cold stress compared to the control plants. After 2 weeks of cold treatment the polysome content decreased to the same level as that in control plants. The size-class distribution of polysomes showed a high proportion of large protein synthesizing polysomes in cold-stressed plants. After 2 weeks the values were comparable to those in control plants. Cold-induced proteins were detected using ³⁵S-labelled methionine for in vitro translations. At least 2 new polypeptides, M_r 30000 and 18000, were induced on the first day of cold stress and continued to be expressed at low temperatures 4 weeks later.     

Thermal tolerance and heat shock protein synthesis during development in the anuran Lepidobatrachus laevis

Development of the Paraguayan anuran Lepidobatrachus laevis is unusual in that the larvae are obligate carnivores, facultative cannibals and apparently exist at high environmental temperatures in their natural habitat. In the present study, the effect of environmental temperature on the rate of anuran development was investigated. The larvae have a thermotolerance range of 18°C for normal development between 19 and 37°C. The effect of temperature on the rate of development was dramatic; larvae that were incubated at 36.8°C develop to stage 24 (Gosner) in approximately 9 h compared with 24 h for larvae incubated at 19°C. The ability of larvae to survive heat shock was also examined; larvae did not survive a shock of 45°C for 15 min when it was administered at stages 3, 5, 9, 10 or 20. However, using the same heat shock conditions, 50% survival was observed when larvae were shocked at stage 16. To study protein synthesis during heat shock, larvae were pulsed with [³⁵S]-methionine during heat shock and labeled proteins were analyzed by electrophoresis under reducing and denaturing conditions. Larvae synthesized two sets of heat-shock proteins at doublet molecular weights of 83/78 and 62/59 kDa. These proteins were synthesized independently of the stage of development at which the shock was administered or the magnitude of the heat shock.     

CO2, light and temperature influence senescence and protein degradation in wheat leaf segments

Effects of environmental conditions influencing photosynthesis and photorespiration on senescence and net protein degradation were investigated in segments from the first leaf of young wheat ( Triticum aestivum L. cv. Arina) plants. The segments were floated on H₂O at 25, 30 or 35°C in continuous light (PAR: 50 or 150 µmol m⁻² s⁻¹) in ambient air and in CO₂‐depleted air. Stromal enzymes, including phosphoglycolate phosphatase, glutamine synthetase, ferredoxin‐dependent glutamate synthase, phosphoribulokinase, and the peroxisomal enzyme, glycolate oxidase, were detected by SDS‐PAGE followed by immunoblotting with specific antibodies. In general, the net degradation of proteins and chlorophylls was delayed in CO₂‐depleted air. However, little effect of CO₂ on protein degradation was observed at 25°C under the lower level of irradiance. The senescence retardation by the removal of CO₂ was most pronounced at 30°C and at the higher irradiance. The stromal enzymes declined in a coordinated manner. Immunoreactive fragments from the degraded polypeptides were in most cases not detectable. However, an insolubilized fragment of glycolate oxidase accumulated in vivo, especially at 25°C in the presence of CO₂. Detection of this fragment was minimal after incubation at 30°C and completely absent on blots from segments kept at 35°C. In CO₂‐depleted air, the fragment was only weakly detectable after incubation at 25°C. The results from these investigations indicate that environmental conditions that influence photosynthesis may interfere with senescence and protein catabolism in wheat leaves.     

Changes in protein synthesis during stratification and dormancy release in embryos of sugar maple (Acer saccharum)

Protein synthesis in dormant embryos of sugar maple ( Acer saccharum ) was investigated in seeds stratified at 4°C or incubated at 15°C. Seeds stratified at 4°C germinated after 27 days; seeds incubated at 15°C failed to germinate. Stratification increased the embryo's capacity for protein synthesis by day 11 as measured by in vivo incorporation of [³⁵S]-methionine into purified protein. At 4°C protein synthesis in the embryonic axis rose in a linear fashion prior to germination, whereas in cotyledons it increased until day 20 and then declined. Analysis of radiolabelled proteins by two-dimensional gel electrophoresis revealed that the levels of specific proteins were altered by temperature, primarily in the cotyledons. Several proteins were expressed in the cotyledons at 15°C but were absent in unstratified embryos and in embryos stratified at 4°C. That is, the expression of these proteins was repressed during stratification and release from dormancy. Levels of other proteins in the cotyledons declined at 4°C during stratification. We suggest that one or more of these proteins may be associated with the inhibition of growth of the embryonic axis imposed by the cotyledons.     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.     

Thermoregulation and metabolism in the Cape golden mole (Insectivora: Chrysochloris asiatica)

The Cape golden mole, Chrysochloris asiatica is an insectivore which excavates superficial foraging burrows as it searches for its food. It has a mean (±S.D.) resting metabolic rate (RMR) when newly captured of 1–17±0.17 cm³ O₂g^-1 h^-1 ( n = 14), within the thermoneutral zone (TNZ) of 30–32°C.
The body temperature (Tb) of the mole in the TNZ is low 32.9 ± 0.36 ( n = 14) and remains stable at ambient temperatures (Tas) from 28–32°C. Above 32°C (range 34–37°C), Tb increases albeit slightly to 36 ± 1.75°C ( n = 14). The conductance is high 0.27 ± 006cm³ O₂g^-1 h^-l°C^-1 ( n = 46) at the lower limit of thermoneutrality. The mean RMR at 9°C (the lowest Ta tested) was 4.82±11 cm³ O₂g^-1h^-1, which is 4.1 times that of the RMR in the TNZ.
At an ambient temperature of 9°C, three of the golden moles entered a state of torpor where the RMR was reduced from 5.9±0.56 to 10 1.0 ± 0.69cm³O₂g^-1h^-1.     

Effect of temperature and dissolved oxygen concentration on the metabolic rate of the turbot and the relationship between metabolic scope and feeding demand

Turbot Scophthalmus maximus maximum oxygen uptake following feeding and exhaustive exercise increased from 107 mg O2 kg⁻¹ h⁻¹ at 6° C to c . 218 mg O2 kg⁻¹ h⁻¹ at 18° C, then increased slightly from 18 to 22° C to 224 mg O2 kg⁻¹ h⁻¹. Standard oxygen uptake increased exponentially as a function of temperature from 11 mg O2 kg ⁻¹ h⁻¹ at 6° C to 66 mg O2 kg⁻¹ h⁻¹ at 22° C. Gradual reduction in oxygen concentration to 87–90% air saturation at 6, 10. 18° C and <80% at 14 and 22° C limited the maximum metabolic rate but, supersaturation (>100% saturation) had little effect. Metabolic scope attained a maximum of 176 mg O₂ kg⁻¹ h⁻¹ at 18° C. Interpolation of the results showed that this value changed little between 16 and 20° C. It is suggested that this temperature range is optimal for turbot of c . 500 g. A comparison with a previous study on feeding demand in intensive farming conditions showed a linear relationship between appetite and metabolic scope. It is concluded that the ability of a fish to supply energy (including the energy requirement of digestive metabolism) above a standard level is a limiting factor in the manifestation of its feeding demand.     

Photosynthetic response of Eragrostis tef to temperature

Photosynthetic characteristics of leaves of tef, Eragrostis tef (Zucc.) Trotter, plants, grown at 25/15°C (day/night), were measured at temperatures from 18 to 48°C. The highest carbon exchange rates (CER) occurred between 36 and 42°C. and averaged 27 μmol m⁻² s⁻¹. At lower or higher temperatures, CER was reduced, but the availability of CO₂ to the mesophyll, measured as internal CO₂ concentration, was highest when temperatures were above or below the optimum for CER. In addition, CER and stomatal conductance were not correlated, but residual conductance was highly correlated with CER (r = 0.98). In additional experiments, relative ¹³C composition for leaf tissue grown at 25, 35 and 45°C averaged -14.4 per mille, confirming that tef is a C₄ grass species. Dry matter accumulation was higher at 35 than at 25, and lowest at 45°C. Leaf CER rates increased hyperbolically with increased light when measured from 0 to 2000 μmol m⁻² s⁻¹ PPFD. The highest CER, 31.8 μ-mol m_-2 s⁻¹, occurred at 35°C and 2000 μmol m⁻² s⁻¹ PPFR. At high light, CER at 25 and 35°C were nearly equal because of higher stomatal conductance at 25°C. Residual conductance was, however, clearly highest at 35°C compared to 25 and 45°C treatments. Stomatal conductance and residual conductance were not correlated in either set of experiments, yet residual conductance was always highest when temperatures were between 35 and 42°C across experiments, suggesting that internal leaf photosynthetic potential was highest across that temperature range.     

High temperature causes arrest of cell cycle in G2 phase in BmN cells derived from the silkworm, Bombyx mori

Abstract.  The influence of temperature on the insect cell line, BmN, derived from the silkworm, Bombyx mori is investigated. These cells proliferate at an accelerated pace as the temperature increases from 22 to 30 °C, but the growth rate slows at 34 °C, and proliferation stops at 38 °C. At high temperatures, abnormal cellular morphology is observed. Cells treated at 38 °C have cytoplasmic bilateral protrusions and they gradually aggregate and float in the medium. BmN cells without proliferation at 38 °C are viable but have reduced DNA synthesis. At high temperatures, the cell cycle of BmN cells halts at the G₂ phase. After heat treatment of the larvae, an accumulation of larval haemocytes with high DNA content is found, which suggests that the cell cycle arrest at G₂ also occurs in the silkworm at high temperatures.