Recovery from Heat Shock in Heat-Tolerant and Nontolerant Variants of Creeping Bentgrass

Recovery from the heat-shock response was tested in heat-tolerant (selected bentgrass [SB]) and nontolerant (nonselected bentgrass [NSB]) variants of creeping bentgrass (Agrostis palustris Huds.) SB increased incorporation of radioactive amino acids into protein 2 h earlier than NSB when leaf blades were incubated at the recovery temperature following heat shock. Electrophoresis indicated that heat-shock protein (HSP) synthesis decreased and normal protein synthesis increased at 4 h in SB and at 6 to 8 h in NSB. Increased synthesis of normal proteins was not due to increased abundance of normal mRNAs, which were equivalent in SB and NSB at 4 h. But at 4 h, more of the normal mRNA population was associated with polysomes in SB than in NSB. Synthesis of HSP70 and HSP18 decreased earlier in SB than in NSB. The decreased synthesis of these HSPs appeared to be correlated with decreased mRNA abundance. But at 4 h, some of the HSP18 mRNA may have been associated with heat-shock granules in SB. Synthesis of HSP25 continued through the 8-h recovery in both variants. Although the abundance of HSP25 was equivalent in SB and NSB during heat shock and recovery, more HSP25 mRNA was associated with polysomes in SB than in NSB.     

Heat-shock proteins during pollen development in maize

In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat-shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat-shock responses. One-dimensional Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis technique (SDS-PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat-shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat-stress conditions; however, the consti-tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.     

Characterization and Physiological Function of Class I Low-Molecular-Mass,Heat-Shock Protein Complex in Soybean

Examination of an ammonium sulfate-enriched fraction (70-100% saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular mass complex (280 kD) in soybean (Glycine max) seedlings. This complex cross-reacted with antibodies raised against soybean class I low-molecular-mass (LMW) HSPs. Dissociation of the complex by denaturing polyacrylamide gel electrophoresis showed the complex to contain at least 15 polypeptides of the 15-to 18-kD class I LMW HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiata L.; 295 kD), in pea (Pisum sativum L.; 270 kD), and in rice (Oryza sativa L.; 310 kD). The complex was stable under high salt conditions (250 mM KCI), and the integrity was not affected by 1% Nonidet P-40 and 3 [mu]g/ML RNase treatment. The size of the isolated HSP complex in vitro was conserved to 55[deg]C; however, starting at 37.5[deg]C, it changed to higher molecular forms in the presence of soluble proteins. The isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.     

玉米胚发育过程中热激蛋白的合成

35S-Met标记玉米胚蛋白合成结果表明,热激处理(42℃)与对照(25℃)的蛋白合成趋势相近,热激抑制16 DAP的蛋白合成,增加22和34 DAP蛋白合成.SDS-PAGE自显影图谱表明,热激诱导16DAP的胚合成86.4、80.0、73.2 kD等3种分子量较高的热激蛋白,22DAP后热激诱导合成86.4、80.0、73.2、24.4、18.2、16.8和13.6 kD等7种分子量的热激蛋白.2D-PAGE自显影图谱进一步显示,热激诱导22和28 DAP的胚合成近20种热激蛋白,其中超过10种为小分子热激蛋白.特异热激蛋白BiP(HsP70)、PDI(HsP60)Western blot表明,这2种热激蛋白在玉米胚发育过程均有高水平的表达,热激对其合成影响不明显.     

温度,热激蛋白与高粱育性的变化

高粱不育系3A在热激(43～45℃)诱导下结出了种子,由不育系转变为可育系。比较3A和3B线粒体在热激条件的热激蛋白得知,它们的热激蛋白是由核编码的,在细胞质中合成后才运到线粒体中,热激2h,3A出现70、31、24、18和16kDa 5条蛋白带,3B除出现上述5条蛋白带外还多出现96、94kDa 2条,而且70kDa含量比3A大。热激4h,3B的96、94kDa消失,两系趋于一致。此时,3A和3B线粒体总蛋白比热激前大量增加。此后HSPs急剧降低。热激8h,3B线粒体仅有70、31、24和16kDa 4条蛋白带,70kDa特别明显,而3A则全部消失。从而表明,HSPs在3B中是稳定的,在3A中是缺乏或不稳定的,这些差异可能与3B育性稳定性及3A不育性有关。     

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.     

Heat shock induced changes in the gene expression of terminally differentiating avian red blood cells

Reticulocytes, purified from the blood of quail and chickens recovering from anaemia, respond to heat shock by the new and (or) enhanced synthesis of heat-shock protein (HSPs) with relative molecular masses of greater than 400,000, 90,000, 70,000, and 26,000 (quail) or 24,000 (chicken) and the depressed synthesis of many proteins normally produced at a control temperature. The synthesis of these HSPs is noncoordinate since the expression of each protein depends upon the particular temperature and duration of the time at that temperature. Separation of proteins from quail reticulocytes into Triton X-100 soluble and insoluble fractions demonstrates that the 70,000 and 26,000 Da HSPs are found in both fractions, whereas the greater than 400,000 and 90,000 Da HSPs are located only in the detergent-soluble fraction. Triton X-100 fractionation also reveals that there are three isoelectric variants of the 70,000 Da HSP and that they are constitutively synthesized and selectively partitioned between cellular compartments. Heat shock induced synthesis of the 90,000, 70,000, and 26,000 Da quail HSPs is prevented by actinomycin D, while enhanced synthesis of the greater than 400,000 Da HSP is unaffected by this inhibitor. These results demonstrate that nucleated, terminally differentiating avian red blood cells are capable of responding to heat stress by rapid changes in their highly restricted "program" of gene expression.     

Tissue specificity of the heat-shock response in maize

The tissue specificity of the heat-shock response in maize was investigated. The ability to synthesize heat shock proteins (hsp) at 40°C, as well as the intensity and duration of that synthesis, was analyzed in coleoptiles, scutella, green and etiolated leaves, suspension-cultured cells, germinating pollen grains, and primary root sections at different stages of development. One-dimensional sodium dodecyl sulfate gel electrophoresis of extracted proteins revealed that most of the tissues synthesized the typical set of 10 hsp, but that the exact characteristics of the response depended upon the tissue type. While elongating portions of the primary root exhibited a strong heat shock response, the more mature portions showed a reduced ability to synthesize hsp. Leaves, whether green or etiolated, excised or intact, constitutively synthesized a low level of hsp at 25°C, and high levels could be induced at 40°C. Suspension-cultures of Black Mexican sweet corn synthesized, besides the typical set of hsp, two additional polypeptides. In contrast to all the other tissues, germinating pollen grains could not be induced to synthesize the typical set of hsp but did synthesize two new polypeptides of 92 and 56 kD molecular weight.

The heat shock response was transient for most of the tissues which synthesized the standard set of hsp. Hsp synthesis was detected up to 2 to 3 hours, but not at 10 hours of continuous 40°C treatment. The exception was suspension cultured cells, in which hsp synthesis showed only a slight reduction after 10 hours at 40°C. Tissue-specific differences in the heat-shock response suggest that there are differences in the way a given tissue is able to adapt to high temperature.

We have confirmed the previous suggestion that maize hsp do not accumulate in substantial quantities. Using two-dimensional gel analysis, hsp could be detected by autoradiography but not by sensitive silver staining techniques.

     

The role of oxidative stress in the induction of Drosophila heat-shock proteins

The role of oxidative stress in the induction of heat-shock proteins (HSPs) was studied in Drosophila Kc cells by comparing the effects of two different inducers, temperature stress and reoxygenation following a period of anoxia, on cellular respiration, thiol status, and the accumulation of HSPs. A heat shock from 25 to 37 degrees C caused a 60% increase in the rate of O2 uptake but caused little oxidative stress as indicated by a constant level of reduced glutathione, a slight increase in oxidized glutathione, and no change in protein sulfhydryls. Heat shock resulted in a pronounced accumulation of HSPs which was not inhibited by anoxic conditions. A different HSP inducer, reoxygenation following anoxia, resulted in an overall inhibition of respiration, the appearance of CN -insensitive O2 uptake, a 50% decrease in the level of reduced glutathione and a fourfold increase in the ratio of oxidized to reduced glutathione. Despite these indicators of oxidative stress, HSP synthesis was less pronounced than observed during heat shock and was not affected by antioxidants. Oxidative stress may induce HSP synthesis in some cases but is not responsible for HSP synthesis during a heat shock.     

Heat-shock proteins 70 kDa and 19 kDa are not required for induction of embryogenesis of Brassica napus L. cv. topas microspores

It is currently accepted that 'stress' triggers induction of microspore embryogenesis, and for Brassica napus L. cv. Topas it is heat-shock. It has been postulated that the heat-shock proteins (HSPs) generated during heat stress have a central role in the induction mechanism. To test this hypothesis we developed a microspore induction procedure, using colchicine instead of heat treatment. The level of HSP70 increased significantly during and following the microspore heat treatment while sHSP19 expression was induced at the onset of heat-shock and declined after 8 h. In contrast, induction of embryogenesis with colchicine was not accompanied by elevation of HSP70 nor by induction of sHSP19, indicating that these HSPs are not required for induction of microspore embryogensis in this model system. These data refute the current hypothesis that HSPs have an essential role in triggering microspore embryogenesis.     

HSP_(70)基因表达对高粱育性的影响(英文)

高粱细胞质雄性不育系３１９７Ａ（３Ａ）在常温条件下是不育的（Ｆｉｇｓ．１１＆２）,经热激（４５℃）诱导不同程度地恢复了育性（Ｆｉｇｓ．１３＆４）,为研究其不育机理提供了线索。热激２ｈ后,３Ａ中即可产生一类线粒体热激蛋白（ＨＳＰｓ）。其中,分子量为７０ｋＤ的ＨＳＰ７０含量最高,也最为稳定。不过,３Ａ中ＨＳＰｓ的稳定性弱于保持系３１９７Ｂ（３Ｂ）（Ｆｉｇ．２,Ｐａｎｅｌｓ１～４）。放线菌素Ｄ抑制ＨＳＰｓ的合成,而氯霉素无此作用（Ｆｉｇ．２,Ｐａｎｅｌｓ５＆６）,表明：ＨＳＰｓ是由核基因编码、在细胞质中合成、再跨膜转运到线粒体中的。３Ａ幼穗经热激后,线粒体的总蛋白量猛增了２．７倍（Ｆｉｇ．３）,达到３Ｂ的水平,育性亦变为可育的。Ｆｉｇ．４表明：ＨＳＰ７０反义链ｃＤＮＡ（Ｒ１）能进入到３Ｂ花药细胞中,并与靶ＲＮＡ（ＨＳＣ７０ｍＲＮＡ）结合,而对照、正义链ｃＤＮＡ（Ｄ）链无此反应。由此、再增加一个通用保守序列的反义链ｃＤＮＡ（Ｒ２）、共两个探针（Ｒ１、Ｒ２）,可以检测到：３Ａ在常温下没有能力合成ＨＳＣ７０ｍＲＮＡ（Ｆｉｇ．５）,而在热激条件下,转变为有能力（Ｆｉｇ．６）。启示：３Ａ在热激条件下由不育转变为可育     

Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: localization and possible functional implications

We isolated and sequenced Ha hsp 17.9, a DNA complementary (cDNA) of dry-seed stored mRNA that encodes a low-molecular-weight heat-shock protein (LMW HSP). Sequence analysis identified Ha hsp17.9, and the previously reported Ha hsp17.6, as cDNAs encoding proteins (HSP17.6 and HSP17.9) which belong to different families of cytoplasmic LMW HSPs. Using specific antibodies we observed differential expression of both proteins during zygotic embryogenesis under controlled environment, and a remarkable persistence of these LMW HSPs during germination. Immuno-blot analysis of HSP17.9 proteins in two-dimensional gels revealed that the polypeptides expressed in embryos were indistinguishable from LMW HSPs expressed in vegetative tissues in response to water deficit; but they appeared different from homologeous proteins expressed in response to thermal-stress. Tissue-print immunolocalization experiments showed that HSP17.9 and HSP17.6 were homogeneously distributed in every tissue of desiccation-tolerant dry seeds and young seedlings under non-stress conditions. These results demonstrate developmental regulation of specific, cytoplasmic, plant LMW HSPs, suggesting also their involvement in water-stress tolerance.     

Analytical assays of human HSP27 and thermal-stress survival of Escherichia coli cells that overexpress it

HSP27 is a small heat-shock protein (sHSP). Such proteins are produced in all organisms. These small HSPs exhibit chaperone-like activity that can bind to unfolded polypeptides and prevent uncontrolled protein aggregation in vitro. Cellular anti-apoptosis function and enhanced cell survival are correlated with increased expression of HSPs. This study presents a thermal-stress survival model for cells using the Escherichia coli expression system for which human HSP27, a recombinant protein, is inducible. Results show that E. coli cells overexpressing human HSP27 have enhanced tolerance to 50 degrees C thermal stress.     

Tissue-Type-Specific Heat-Shock Response and Immunolocalization of Class I Low-Molecular-Weight Heat-Shock Proteins in Soybean

A monospecific polyclonal antibody was used to study the tissue-type specificity and intracellular localization of class I low-molecular-weight (LMW) heat-shock proteins (HSPs) in soybean (Glycine max) under different heat-shock regimes. In etiolated soybean seedlings, the root meristematic regions contained the highest levels of LMW HSP. No tissue-type-specific expression of class I LMW HSP was detected using the tissue-printing method. In immunolocalization studies of seedlings treated with HS (40[deg]C for 2 h) the class I LMW HSPs were found in the aggregated granular structures, which were distributed randomly in the cytoplasm and in the nucleus. When the heat shock was released, the granular structures disappeared and the class I LMW HSPs became distributed homogeneously in the cytoplasm. When the seedlings were then given a more severe heat shock following the initial 40[deg]C -> 28[deg]C treatment, a large proportion of the class I LMW HSPs that originally localized in the cytoplasm were translocated into the nucleus and nucleolus. Class I LMW HSPs may assist in the resolubilization of proteins denatured or aggregated by heat and may also participate in the restoration of organellar function after heat shock.     

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).     

QUANTITATIVE AND QUALITATIVE ANALYSES OF PROTEIN SYNTHESIS DURING HEAT SHOCK IN THE MARINE DIATOM NITZSCHIA ALBA (BACILLARIOPHYCEAE)1

Protein synthesis in the diatom Nitzschia alba Lewin and Lewin was drastically altered when the cells were incubated at a supraoptimal temperaeture. Quantitatively, the overall protein synthesis was greatly suppressed as indicated by teh rate of [³⁵S] methionine incorporation. The extent of suppression of protein synthesis was proportional to the severity of the heat-shock treatment which was a combination of elevated temperature and treatment duration. The in viro synthesized proteins were also qualitativelty anlayzed by two-dimensional gel electrophoresis. Dependeing on the treatment condition, a set of heat-shock proteins (HSPs) were induced. They were best detected when the cells were subjected to shocks of 35°C for 60 min or 40°C for 10 min followed by a 60 min labelling at 30°C. The results revealed 16 HSps which had moluecular weights ranging from 24–94 kD and isoelectric points ranging from 5.50–7.10. Some of the HSps were identical in molelcular weights but with differeentr isoelectric points. The induction and accumulation of HSPs in Nitzschia alba were transitory. Prologned heat-shock treatments resulted in a complete cessation of protein syntehsis and no further induction of HSPs. In all aspects, the heat shock response of diatoms was similar to that in higher plants such as soybean, maize and tobacco but differenet from most animal systems.     

Mitochondrial Low-Molecular-Weight Heat-Shock Proteins and the Tolerance of Cereal Mitochondria to Hyperthermia

Relationships between the appearance of low-molecular-weight heat-shock proteins (LMW HSPs) in maize, winter wheat, and winter rye mitochondria and the tolerance of the mitochondria to hyperthermia (42°C, 3 h) were studied using one-dimensional SDS-PAGE, immunochemical methods, and polarography. Heat shock inhibited respiration to a greater extent in the wheat and rye than in the maize mitochondria. A single 20-kD LMW HSP was found both inside and on the surface of mitochondria isolated from heat-treated wheat and rye seedlings. After heating maize seedlings, two LMW HSPs (28 and 23 kD) appeared inside the mitochondria, and three proteins (22, 20, and 19 kD) appeared on their surface. We suppose that the latter three proteins play an essential role in the protection of mitochondria from hyperthermic damage. It seems likely that the diversity of the hyperthermia-induced LMW HSPs in plant mitochondria affects their thermal stability.     

The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast

In S. cerevisiae the induction of heat-shock protein (HSP) synthesis is accompanied by a decrease in the cytoplasmic and vacuolar pH as determined by means of [31P]NMR spectroscopy. The relationship of HSP synthesis and acidification of the cytoplasmic pH is dose-dependent under a variety of treatments (temperature increases (23-32 degrees C), addition of 2,4-dinitrophenol (greater than 1 mM), sodium arsenite (greater than 3.75 X 10(-5) M) or sodium cyanide (greater than 10 mM]. Changes in the intracellular pH occur within 5 min after treatment, attain a maximum within 30 min and are subsequently stable. HSPs 98, 85 and 70 show maximum synthesis rates 1-2 h after a 40 degrees C heat shock. The synthesis rates then decline. HSPs 56, 44 and 33 reveal a smaller and slower increase and almost no decrease in the synthesis rate within 4 h at 40 degrees C. The similar dose dependencies of HSP synthesis and cytoplasmic pH. as well as the immediate response of the pH, can also be demonstrated in the mitochondrial mutant of S. cerevisiae (Q0). This result indicates that the heat-shock response is mainly independent of intact oxidative phosphorylation. No correlation was observed between HSP synthesis rate and total intracellular ATP content.     

Heat shock induced proteins in plant cells

Tobacco (Nicotiana tabacum) and soybean (Glycine max) tissue culture cells were exposed to a heat shock and protein synthesis studied by SDS-polyacrylamide gel electrophoresis after labeling with radioactive amino acids. A new pattern of protein synthesis is observed in heat-shocked cells compared to that in control cells. About 12 protein bands, some newly appearing, others synthesized in greatly increased quantities in heat-shock cells, are seen. Several of the heat-shock proteins (HSPs) in both tobacco and soybean are similar in size. One of the HSPs in soybean (76K) shares peptide homology with its presumptive 25°C counterpart, indicating that the synthesis of at least some HSPs may not be due to activation of new genes. The optimum temperature for maximal induction of most HSPs is 39–40°C. Total protein synthesis decreases as heat-shock temperature is increased and is barely detectable at 45°C. The heat-shock response is maintained for a relatively short time in tobacco cells. After 3 hr at 39°C, a decrease is seen in the synthesis of the HSPs, and after 4 hr practically no HSPs are synthesized. After exposure to 39°C for 1 hr, followed by a return of tobacco cells to 26°C, recovery to the control pattern of synthesis requires greater than 6 hours. These results indicate that cells of flowering plants exhibit a heat-shock response similar to that observed in animal cells.