Heat stress responses in cultured plant cells : development and comparison of viability tests

The response of suspension-cultured pear (Pyrus communis cv Bartlett) cells to heat stress was studied using three viability tests: regrowth (culture growth during 10 days after stress); triphenyltetrazolium chloride reduction; and electrolyte leakage. Critical (50% injury) temperatures for a 20-minute exposure were 42°, 52°, and 56°C, respectively, for these viability tests. Electrolyte leakage had the lowest temperature coefficient. Heat stress inhibition of triphenyltetrazolium chloride reducing capacity was much greater if the viability test was conducted 3 days, rather than immediately, after the stress treatment. Consistent with a major role for indirect metabolic strain in heat injury, treatment with 3.6 micromolar cycloheximide and heat stress (20 minutes at 43°C) affected culture regrowth similarly. We conclude that the measurements of direct response are not adequate substitutes for regrowth tests in assessing heat injury to cultured plant cells.     

Roles of the Escherichia coli Small Heat Shock Proteins IbpA and IbpB in Thermal Stress Management: Comparison with ClpA, ClpB, and HtpG In Vivo

We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene. All mutants exhibited growth defects at 46°C, but not at lower temperatures. However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50°C. When cultures were allowed to recover from transient exposure to 50°C, all mutations except Δibp led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42°C when combined with the dnaK756 or groES30 alleles, while the Δibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46°C compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.     

Interaction of heat and salt shock in cultured tobacco cells

Cultured tobacco cells (Nicotiana tabacum L. var Wisconsin-38) developed tolerance to otherwise nonpermissive 54°C treatment when heat-shocked at 38°C (2 h) but not at 42°C. Heat-shocked cells (38°C) exhibited little normal growth when the 54°C stress came immediately after heat shock and normal growth when 54°C stress was administered 8 hours after heat shock. Heat shock extended the length of time that the cells tolerated 54°C. Tobacco cells developed tolerance to otherwise lethal 2% NaCl treatment when salt-shocked (1.2% NaCl for 3 hours). The time course for salt tolerance development was similar to that of thermotolerance. Heat-shocked cells (38°C) developed tolerance of nonpermissive salt stress 8 hours after heat shock. Alternatively, cells heat-shocked at 42°C exhibited immediate tolerance to lethal salt stress followed by a decline over 8 hours. Radioactive methionine incorporation studies demonstrated synthesis of heat shock proteins at 38°C. The apparent molecular weights range from 15 to 115 kilodaltons with a protein complex in the 15 to 20 kilodalton range. Synthesis of heat shock proteins appeared to persist at 42°C but with large decreases in incorporation into selected heat shock protein. During salt shock, the synthesis of normal control proteins was reduced and a group of salt shock proteins appeared 3 to 6 h after shock. Similarities between the physiology and salt shock proteins/heat shock proteins suggest that both forms of stress may share common elements.     

Starvation-Induced Thermal Tolerance as a Survival Mechanism in a Psychrophilic Marine Bacterium

Carbon-starved cultures of strain Ant-300, a psychrophilic marine vibrio isolated from the Antarctic Convergence, were compared with their nonstarved counterparts for resistance to heat. Specifically, starved and unstarved cells were exposed to 17°C, which is 4°C above the maximum growth temperature, and compared with cells maintained at the optimum temperature (5 to 7°C). Total cell counts, direct viable-cell counts, and plate counts were monitored. At a temperature of 17°C, viability (as indicated by plate counts) was lost within 40 h, with direct viable-cell counts indicating less than 5% viability at this time. However, when cells were carbon starved for 1 week prior to heat challenge, significant plateability was maintained for more than 6 days; direct viable-cell counts of starved cells maintained at 17°C indicated the presence of viable cells for at least 12 days. Because starvation is the normal physiological state of copiotrophic, heterotrophic bacteria in oligotrophic marine waters, these data suggest that starvation conditions may be a significant factor in providing heat tolerance to psychrophiles.     

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.     

Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle

Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26°C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42°C. At 34 and 38°C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42°C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38°C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38°C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau.     

Thermal Tolerance of Zymomonas mobilis: Temperature-Induced Changes in Membrane Composition

The membrane composition of Zymomonas mobilis changed dramatically in response to growth temperature. With increasing temperature, the proportion of vaccenic acid declined with an increase in myristic acid, the proportion of phosphatidylcholine and cardiolipin increased with decreases in phosphatidylethanolamine and phosphatidylglycerol, and the phospholipid/protein ratio of the membrane declined. These changes in membrane composition were correlated with changes in thermal tolerance and with changes in membrane fluidity. Cells grown at 20°C were more sensitive to inactivation at 45°C than were cells grown at 30°C, as expected. However, cells grown at 41°C (near the maximal growth temperature for Z. mobilis) were hypersensitive to thermal inactivation, suggesting that cells may be damaged during growth at this temperature. When cells were held at 45°C, soluble proteins from cells grown at 41°C were rapidly lost into the surrounding buffer in contrast to cells grown at lower temperatures. The synthesis of phospholipid-deficient membranes during growth at 41°C was proposed as being responsible for this increased thermal sensitivity.     

Effect of temperature on freezing and heat stress tolerance of cultured plant cells

The relationship between freezing and heat tolerance was investigated with suspension-cultured pear (Pear cammunis cv. Bartlett) cells. This culture showed considerable capacity for both cold and heat acclimation. Growth at 2 °C (Cold acclimation) and at 30 °C (heat acclimation) both increased the freezing tolerance [measured via triphenyltetrazolium chloride (TTC) reduction]of pear cells. However, heat acclimation induced by heat shock treatment did not significantly effect freezing tolerance. Although growth at 30 °C increased freezing tolerance (relative to 22 °C-grown controls), growth at 2 °C (cold acclimation) decreased heat tolerance substantially. Thus, the only similarity detected between cold and heat acclimation was that both processes conferred freezing resistance to TTC-reducing system(s) in pear cells. The pear suspension culture will be a useful tool to further investigate cold acclimation via comparisons between heat and freezing acclimation and injury.     

Heat Shock Response in the Thermophilic Enteric Yeast Arxiozyma telluris

Heat stress tolerance was examined in the thermophilic enteric yeast Arxiozyma telluris. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.     

Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins

Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [³⁵S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M_r of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M_r 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M_r 79,000, pI 4.8) similar to that of a HSP (M_r 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.     

Thermotolerance is developmentally dependent in germinating wheat seed

During the initial 9 to 12 hours of imbibition, the imbibing wheat (Triticum aestivum L.) seed was found to exhibit substantial tolerance to high temperature relative to later times of imbibition. Tolerance was assessed by seed viability and seedling growth. This initial high temperature tolerance gradually declines with increasing time of seed imbibition. A range of 2 hour heat pretreatments (38-42°C) prior to imposition of a 2 hour heat shock (51-53°C) during this same 9 to 12 hour interval was unable to increase survival or seedling growth over that of seed that did not receive a pretreatment. However, after 9 to 12 hours of imbibition the pretreatment provided both increased survival and increased seedling growth, measured 120 hours later, i.e., classical thermotolerance could be acquired. This response is called a `thermotolerance transition.' Isolated embryos responded in a similar manner using a 2,3,5-triphenyltetrazolium chloride assay for viability determination following heat treatments. The high temperature tolerance during early imbibition indicates that the thermotolerance transition involves the loss of an existing thermotolerance coincident with acquiring the ability to become thermotolerant following heat pretreatment. Despite the inability to acquire thermotolerance, heat shock protein synthesis was induced by heat shock immediately upon imbibition of wheat seed or isolated embryos. Developmentally regulated heat shock proteins of 58 to 60, 46, 40, and 14 kilodaltons were detected at 1.5 hours of imbibition following heat shock, but were absent or greatly reduced by 12 hours. Constitutive synthesis of 70 and 90 kilodalton hsp groups appeared to be greater at 1.5 hours of imbibition than at 12 hours of imbibition.     

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37°C for 6 hours followed by 4 days at 2.5 or 12.5°C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5°C, but not to 12.5°C, a nonchilling temperature. Exposure to 37°C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5°C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37°C and held at the nonchilling temperature of 12.5°C. There was no relationship between abscisic acid content following exposure to 12.5 or 37°C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42°C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42°C.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.     

Cold Shock Induction of Thermal Sensitivity in Listeria monocytogenes

Cold shock at 0 to 15°C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60°C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8°C for controls and 7.7°C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28°C followed by heating at 60°C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D₆₀ values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Heat stress enhances phytohemagglutinin synthesis but inhibits its transport out of the endoplasmic reticulum

In this study we examined the effect of heat stress (up to 6 hours at 43°C) on the biosynthesis and transport of phytohemagglutinin (PHA) in cotyledons of developing seeds of the common bean, Phaseolus vulgaris. Heat stress resulted in a decrease of total protein synthesis and an enhancement of the synthesis of heat shock proteins and PHA. Pulse chase experiments showed that a considerable proportion of the newly synthesized PHA was present in the endoplasmic reticulum (ER)/Golgi fraction and did not readily chase-out. Analysis with endoglycosidase H showed that the oligosaccharide sidechains of PHA were almost entirely in the high mannose configuration, indicating that most of the newly synthesized PHA was in the ER. However, some of the PHA became fucosylated at 43°C, indicating fucosyltransferase activity. That the biosynthesis and secretion of fucosyl-containing cell wall polymers proceeded normally at 43°C provided evidence that certain Golgi functions (i.e. transport to the cell wall) remained unaffected by heat stress. The ER obtained from these heat stress cotyledons had a greater density (1.16 g· cm⁻³ at 43°C instead of 1.14 g·cm⁻³ at 22°C) in sucrose gradients. Ultrastructural observations showed that the width of the lumen of the ER cisternae had increased from 20 nanometers at 22°C to 60 to 80 nanometers at 43°C; the lumen was filled with electrondense material presumed to be protein. The experiments are interpreted as evidence that heat stress imposes a block in the transport of PHA out of the ER. Whether heat stress affects the ER itself or alters the conformation of PHA, thereby preventing its transport, is not clear.     

Prototheca zopfii Krüger Strain UMK-13 Growth on Acetate or n-Alkanes

A new strain of Prototheca zopfii Krüger was grown on acetate or on pure n-alkanes. A maximum acetate-supported exponential growth of 12 divisions day⁻¹ occurred at pH 5 and 30°C. At 25°C, growth on n-alkanes was almost as fast, but no growth occurred at 30°C. After 4 days at 25°C, 34 to 45% of the n-alkanes had been removed, whereas at 21°C and slower growth, utilization was twofold greater after 15 days. Rates of growth and utilization increased markedly after a point of sudden emulsification.