Thermal stress evaluation of suspension cell cultures in winter wheat

Summary The objectives of this study were to compare thermotolerance in whole plants vs. suspension cell cultures of winter wheat, and to evaluate the synthesis of heat shock proteins in relation to genotypic differences in thermotolerance in suspension cells. Whole plant genetic differences in the development of heat tolerance were identified for three wheat genotypes (ND 7532, KS 75210 and TAM 101). Suspension cell cultures of these genotypes were used to evaluatein vitro response to heat stress. Viability tests by triphenyl tetrazolium chloride (TTC) and by fluorescein diacetate (FD) were utilized to determine the relationship of cellular response to heat stress (37°C/24 h, 50°C/1h). KS 75210 and ND 7532 are relatively heat susceptible. TAM 101 is heat tolerant. Both tests at the cellular level were similar to the whole plant response. Thus, cellular selection for enhancing heat tolerance seems feasible. Heat shock protein (HSP) synthesis of two genotypes, ND 7532 and TAM 101 were determined for suspension cultured cells. In suspension cultures, HSPs of molecular weight 16 and 17 kD were found to be synthesized at higher levels in the heat tolerant genotype (TAM 101) than the susceptible genotype (ND 7532), both at 34° and 37°C treatments for 2 hours and 5 hours. HSP 22 kD was synthesized more at 34°C for TAM 101 than ND 7532, but not at 37°C; whereas, HSP 33 kD was synthesized at 37°C at similar abundance for both genotypes, but not at 34°C.These results indicated that there is a differential expression of HSP genes in wheat suspension cells at different temperature stress durations and between heat tolerant and heat susceptible genotypes. It appears that the levels of synthesis of HSPs 16 and 17 kD are correlated with genotypic differences in thermal tolerance at the cellular level in two genotypes of wheat.     

Synthesis of heat shock proteins inThermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes EM1)

The response to heat stress was examined inThermoanaerobacterium thermosulfurigenes EM1. Upon a temperature shift-up from 50° to 62°C, four heat shock proteins (hsps) were synthesized at an elevated level. Two proteins were found to be immunologically related to theEscherichia coli GroEL protein and theMycobacterium tuberculosis hsp71 (DnaK similar protein), and the correspondinggroE anddnaK homologous sequences were detected in the chromosome ofT. thermosulfurigenes EM1. The heat shock response in this thermophile was transient, with a maximum synthesis of hsps between 10 and 15 min after the shock. The enhanced synthesis of DnaK and GroEL was consistent with increased mRNA levels of the genes, which reached a maximum 15 min after heat treatment.     

Localization of the 70000 Dalton Heat-induced protein in the nuclear matrix of BHK cells

The exposure of exponentially growing BHK cells to supranormal temperatures (41–44 °C, for 15 min to 1 h) induces the synthesis of a new set of proteins, the heat shock proteins, while the synthesis of proteins made before heat shock is repressed at 43 °C. Among the two major heat shock proteins induced, of molecular weight 70 K and 68 K, only the 70 kD protein is found bound to the nuclear matrix. This protein is resolved differently from the normal matrix proteins by isoelectric focusing and, when blotted, does not react with antibodies directed against nuclear matrices. These results show that the 70 kD heat shock protein is a new protein transferred from the cytoplasm to the nucleus, where it binds to the nuclear matrix, suggesting a structural role for this protein.     

Effect of temperature on translation of mRNA coding for an extracellular proteinase and cell proteins inBacillus megaterium

The content and the half-life of mRNA coding for the Ca²⁺-dependent metalloproteinase were measured by determining the enzyme activity excreted into the medium by cells pregrown in the absence of Ca²⁺ after addition of Ca²⁺ and actinomycin D. The content of the functional proteinase mRNA was highest at 31°C, which is the optimal temperature for the synthesis of this enzyme. Its half-life was 15 min, 7 min, and less than 2 min at 24°, 35°, and 42°C, respectively. Only the third of mRNA molecules synthesized at 31°C was translated in vivo into an active enzyme at 42°C, when compared with the translation proceeding at 24°C. Two-thirds of mRNA molecules synthesized at 31°C were translated into stable cell proteins at 42°C when compared with translation at 24°C. The mean half-life of mRNAs coding for cell proteins was 6–7 min at 24°C, 3 min at 35°C and 2 min at 42°C.     

TMV protein synthesis is not translationally regulated by heat shock

Summary Tobacco mosaic virus (TMV) protein synthesis in tobacco leaf tissue was not translationally regulated under conditions of heat shock as were most of the other proteins that were produced at 25°C. Upon shift from 25°C to 37–40°C, most host protein synthesis was inhibited followed by initiation of synthesis of heat shock proteins. In contrast, TMV protein synthesis continued after the temperature shift. This phenomenon allowed the enhancement of detection of TMV protein synthesis in tobacco leaves. The most prominent proteins labeled were viral when tissue was labeled during the first hr following the shift to 40°C, a period after heat shock repression of host protein synthesis, but before the onset of most heat shock protein synthesis. Another method to predominately label viral proteins was to incubate infected leaves for periods at 35°C which induced repression of preexisting host protein synthesis without inducing synthesis of heat shock proteins.     

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.     

Induction of cold shock proteins in Bacillus subtilis

The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described. Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37°C. The B. subtilis cells were cold shocked at 25°C, 20°C, 15°C, and 10°C. A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis. General stress proteins were identified by a comparative analysis with the heat shock response of B. subtilis. Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced. Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.     

Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.     

Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27°C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32°C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20°C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34°C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20°C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.     

Effect of Different Temperature Downshifts on Protein Synthesis by <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The psychrotrophic bacterium Aeromonas hydrophila 7966 was subjected to cold shocks from 30°C to 20°C, 15°C, 10°C, or 5°C, or were incubated at low temperature to determine its adaptative response. The cell protein patterns analyzed by two-dimensional electrophoresis revealed that only a few proteins were underexpressed, whereas numerous new proteins appeared with the decrease of temperature, and some others were overexpressed. Among them, a few constituted cold shock proteins because they were transiently induced, whereas others belong to the acclimatation family proteins. Two cold shock proteins of 11 kDa were synthesized at low level because they were visualized only after radiolabeling or silver staining. Moreover, under our experimental conditions, no major cold shock protein of a molecular mass similar to that of E. coli (7.4 kDa) could be identified.     

Heat shock protein induction and the acquisition of thermotolerance in the psychrotrophic yeastTrichosporon pullulans

Two-dimensional gel electrophoretic analysis of the heat shock response in the psychrotrophic yeastTrichosporon pullulans revealed the induction of 11 heat shock proteins (hsps) after a 5° to 21°C heat shock, 12 hsps after a 5° to 26°C heat shock, and 12 hsps after a 5° to 29°C heat shock. Heat shock from 5° to 26° or 29°C resulted in a statistically significant increase in thermotolerance to a lethal heat challenge at 45°C for 5 min. When the protein synthesis inhibitor, cycloheximide, was added prior to the heat shock, no statistically significant thermotolerance was acquired. To confirm the correlation between the synthesis of hsps and the acquisition of thermotolerance, protein extracts of cells that had been heat shocked in the presence or absence of cycloheximide were electrophoretically analyzed. Addition of the same concentration of cycloheximide that prevented the acquisition of thermotolerance also inhibited the synthesis of any hsps.     

Physiological adaptations in the survival of rotifers (Brachionus plicatilis,O. F. Müller) at low temperatures

The biology of the rotifer Brachionus plicatilis has been studied extensively in recent years, due to its importance in aquaculture. Today, the culture of several marine fish species relies completely on the daily production of live rotifers.In the present paper we explore the conditions that facilitate maintaining live rotifers for extensive periods at low temperatures. In addition to its possible contribution in providing reserve stocks for commercial application, these studies may be of ecological importance. They could explain some of the physiological adaptive mechanisms that are involved in the survival of rotifers under adverse environmental conditions.Experimental results showed relatively high survival rates (82–85%) in rotifers that were cultured at 25 °C and exposed later to -1 °C for 12–14 days. During this period, rotifers were kept without food and their media were not changed. The survival was found to depend on the rotifer culture conditions, prior to exposure to -1 °C. These included the type of food fed to rotifers (yeast or algae), the salinity in which they were cultured, and an essential acclimation period of 2–6 day at 4 °C or 10 °C. The acclimation period was associated with the synthesis of at least one specific protein and accumulation of lipids. Profiles of protein synthesis in rotifers incubated at 10 °C revealed a 94 kD protein, which did not appear in rotifers cultured at 25 or 37 °C. Immunoisolation, using a polyclonal antibody that was prepared against HSP60, revealed that this protein was synthesized in rotifers kept at 10, 25 or 37 °C. However, this antibody did not react with the 94 kD peptide.In addition, rotifers kept at 10 °C accumulated substantial amounts of lipids, including eicosapentaenoic acid (EPA), which is found in the algae fed to them. These results support the hypothesis of specific adaptations to survival at low temperatures during an acclimation period.     

Basic features of the Streptococcus thermophilus heat shock response

The heat shock response was investigated in the thermophilic acid bacterium Streptococcus thermophilus. The heat resistance (58°C, 30 min) of log-phase cells grown at 42°C was enhanced by pretreatment at 52°C for 15 or 30 min. Concurrently to this acquired thermotolerance, two-dimensional gel electrophoresis indicates that the cells induced the synthesis of at least 22 heat shock proteins after temperature upshift. Furthermore, following SDS-PAGE, Western blotting, and immunological analysis, six proteins were found to be antigenically related to the Escherichia coli heat shock proteins DnaK, DnaJ, GroEL, GrpE, and La and to the Bacillus subtilis ⁴³ factor Among these six proteins, two related to DnaK and GroEL, are clearly overexpressed during this stress. It is concluded that S. thermophilus possesses a heat shock response similar to that known to occur in mesophilic microorganisms.     

The proteomic response of sea squirts (genus Ciona) to acute heat stress: a global perspective on the thermal stability of proteins

Congeners belonging to the genus Ciona have disparate distributions limited by temperature. Ciona intestinalis is more widespread with a cosmopolitan distribution ranging from tropical to sub-arctic zones, while Ciona savignyi is limited to temperate-latitudes of the northern Pacific Ocean. To compare the heat stress response between congeners, we quantified changes in protein expression using proteomics. Animals were exposed to 22 °C, 25 °C, and 28 °C for 6 h, then recovered at a control temperature (13 °C) for 16 h (high heat stress experiment). In a second experiment we exposed animals to lower levels of heat stress at 18 °C, 20 °C, and 23 °C, with a 16 °C control. A quantitative analysis, using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry (with a 69% and 93% identification rate for Ciona intestinalis and Ciona savignyi, respectively), showed changes in a number of protein functional groups, including molecular chaperones, extracellular matrix proteins, calcium-binding proteins, cytoskeletal proteins and proteins involved in energy metabolism. Our results indicate that C. intestinalis maintains higher constitutive levels of molecular chaperones than C. savignyi, suggesting that it is prepared to respond faster to thermal stress. Systematic discrepancies between estimated versus predicted molecular masses of identified proteins differed between protein families and were more pronounced under high heat conditions, suggesting that thermal sensitivities are lower for cytoskeletal proteins and ATP-synthase than for any other protein group represented on 2D gels.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

Physiological compensation in unilateral eyestalk ablated crayfish, Cherax quadricarinatus

The eyestalks of crustaceans contain neurosecretory cells involved in the regulation of molting. In crayfish, bilateral ablation results in increased molting frequency and weight gain whereas unilateral ablation typically has no effect on molting frequency and weight gain. The effects of unilateral ablation were examined in juvenile Australian freshwater crayfish, Cherax quadricarinatus. As observed for other crayfish species, molting frequency and weight gain of unilateral ablated crayfish were not significantly different from control (intact) crayfish. Survival of unilateral ablated crayfish, however, was reduced compared to controls and was likely due to stress associated with the surgical procedure itself. Using radiolabeling techniques, protein synthesis was determined for neural tissues from the remaining eyestalk of ablated crayfish and compared to protein synthesis of neural tissues from eyestalks of control, non-ablated crayfish. Protein synthesis of ablated crayfish neural tissues was significantly higher (ca. 45%) than protein synthesis of control neural tissues. Electrophoretic analysis (SDS-PAGE and autoradiography) further demonstrated that protein synthesis increased linearly for all proteins in the remaining eyestalk of ablated crayfish. Together, these results suggest that a compensatory response occurred in unilateral ablated crayfish allowing normal physiological functions, particularly those involved in regulating growth cycles, to be maintained. J. Exp. Zool. 289:184-189, 2001.     

Proteomic analysis of response to long-term continuous stress in roots of germinating soybean seeds

Germination is a complex process, highly dependent on various environmental factors, including temperature and water availability. Germinating soybean seeds are especially vulnerable to unfavorable environmental conditions and exposure to long-term abiotic stresses may result in diminishing much of the yield and most importantly – restrained germination. In the present study, a proteomic approach was employed to analyze influence of cold and osmotic stress on roots of germinated soybean (Glycine max, L.) seeds. Seeds were germinating under continuous conditions of cold stress (+10 °C/H₂O), osmotic stress (+25 °C/−0.2 MPa) as well as cold and osmotic stress combined (+10 °C/−0.2 MPa). Proteome maps established for control samples and stress-treated samples displayed 1272 CBB-stained spots. A total of 59 proteins, present in both control and stress-treated samples and showing significant differences in volume, were identified with LC/nanoESI-MS. Identified proteins divided into functional categories, revealed 9 proteins involved in plant defense, 8 proteins responsible for plant destination and storage and 10 proteins involved in various tracks of carbohydrate metabolism. Furthermore, a number of proteins were assigned to electron transport, range of metabolic pathways, secondary metabolism, protein synthesis, embryogenesis and development, signal transduction, cellular transport, translocation and storage. By analyzing differences in expression patterns, it was possible to trace the soybean response to long-term abiotic stress as well as to distinguish similarities and differences between response to cold and osmotic stress.