Heat Shock Proteins of Sorghum [Sorghum bicolor (L.) Moench] and Pearl Millet [Pennisetum glaucum (L.) R.Br.] Cultivars with Differing Heat Tolerance at Seedling Establishment Stage

The production of heat shock proteins was compared in sorghumand pearl millet genotypes differing in seedling establishmentcharacteristics under heat stress. Two major heat shock proteins(hsps) of apparent mol. wt. 65 kD and 62 kD were seen in allthe genotypes of sorghum tested when the incubation temperatureof the 40 h seedlings was altered from 35 ?C to 45 ?C for 2h. Under identical conditions, pearl millet genotypes showedmore hsps and the apparent mol. wt. of these ranged from 30–70kD. The hsp bands were more prominent in whole seedlings androots as compared to plumules. Differences in the productionof hsps were seen in sorghum and pearl millet genotypes withcontrasting heat tolerance at seedling establishment stage butthe significance of these needs to be studied further. Key words: Heat shock proteins, sorghum, genotypic differences     

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.     

Heat shock induces changes in the expression and binding of ubiquitin in senescent Drosophila melanogaster

We examined the effect of aging on the expression of ubiquitin RNA and the binding of the ubiquitin polypeptide to proteins following heat shock in Drosophila melanogaster. Heat-shocked adult flies transcribe two major RNA species-one of 4.4 kb and one of about 6 kb that hybridize to the polyubiquitin-encoding probe. Several less abundant RNAs were also observed but the 4.4-kb band was present as the major RNA species in both stressed and nonstressed flies of both ages. The 6-kb fragment was more abundant in heat shocked aged flies than in younger flies. The quantitative expression of the polyubiquitin gene increased in proportion to the duration of the heat stress. Moreover, the induction of the polyubiquitin RNA was markedly elevated during aging following heat shock. Hybridization of Northern blots with the monoubiquitin gene probe revealed a band of 0.9 kb that was not significantly affected by heat stress. We also investigated the relationship between the changes in polyubiquitin gene expression and the formation of ubiquitin-protein complexes in aging heat-shocked flies. Heat shock to old flies results in a significant increase in the level of proteins immunoprecipitated by anti-ubiquitin antibodies. In the case of proteins synthesized 2 h before heat shock, most of the ubiquitinated proteins were of high molecular weight. For those proteins synthesized during a 30-min heat shock and the 2 h following heat shock, two major immunoprecipitated bands were observed: an 80-kD and a 70-kD polypeptide. The ubiquitination of a 60 kD protein was also observed in nonstressed flies, but its for mation was drastically reduced following heat shock. For proteins synthesized during and after heat shock from both age groups, the major ubiquitinated polypeptide is 70 kD. In all age groups, more ubiquitin complexes were formed with proteins synthesized before heat shock, than with proteins synthesized either during or after heat shock. This suggests that cellular proteins synthesized at physiological temperatures are more sensitive to heat induced damage than those synthesized during stress. These data support the hypothesis that in aging flies, heat shock induces an unusually high concentration of abnormal proteins which are targeted for degradation by the ubiquitin-dependent proteolytic system. © 1993Wiley-Liss, Inc.     

Induction of stress proteins by sodium chloride treatment in Bacillus subtilis

In Bacillus subtilis, heat shock proteins can be classified into two main groups: specific heat shock proteins (about 5) and general stress proteins (at least 14). Salt stress was very effective in the induction of general stress proteins (5 to 50-fold stimulation), but the synthesis of heat-specific stress proteins was not stimulated. Furthermore there were some proteins whose synthesis was accelerated only by salt stress.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

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     

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.     

Arabidopsis: Sensitivity of growth to high temperature

Arabidopsis thaliana seedlings as measured by an electrolyte leakage assay, have been found to be extremely sensitive to high temperature stress as compared to a high temperature tolerant variety (Tracy) of soybean. Over 50% ion leakage occurred in Arabidopsis leaves during a 15-minute exposure to 50°C, indicating a heat killing time of less than 15 minutes. In contrast, the heat killing time for soybean at 50°C was over five times longer. When soybean or Arabidopsis seedlings in culture plates were exposed to 37°C for 2 hours and then returned to 23°C, they suffered no apparent short-term or long-term damage. Soybean seedlings given a 42°C, treatment for 2 hours also showed no damage. Arabidopsis seedlings after a 42°C treatment for 2 hours showed no apparent immediate damage, but 48 hours after return to 23°C severe damage symptoms were visible and after 96 hours all the seedlings were dead. Both soybean and Arabidopsis seedlings synthesize heat shock proteins (hsps) when exposed to 42°C for 2 hours. The hsps synthesized are of similar molecular weights, although the relative abundances of the different size classes are very different in the two plants. Even though hsps are produced in Arabidopsis seedlings after a 2 hour exposure to 42°C their presence is not sufficient for the seedlings to recover from the effects of rhe heat shock when returned to 23°C. Our results show that Arabidopsis has a heat sensitive genotype. This along with its other characteristics should make it a good model system in which to assay in transgenic plants, the functions of homologous and heterologous genes that might be candidates for determining heat tolerance in plants.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.     

Heat shock proteins in willow (Salix viminalis)

The heat shock response in three vegetatively propagated clones of Salix viminalis L. was studied. In the clone 78198, synthesis of a total of 58 proteins was induced or increased by heat shock. Of these proteins, 39 were found in both leaves and callus, 8 only in leaves, and 11 only in callus. The number of heat shock proteins differed between the three clones studied. The molecular weights of the heat shock proteins ranged from 18000 to over 94000. The optimal synthesis of heat shock proteins took place at 37–40°C, but several proteins could be induced at 25–30°C. The synthesis of the majority of the proteins present at a normal growth temperature (20°C) was not completely blocked by the heat shock. More than 12 h was needed for the reappearance of the normal protein synthesis pattern after heat shock.     

菜豆下胚轴质膜微囊和液泡膜微囊体外热稳定性的研究

从抗热潜势较高的“８５ＣＴ－４９７６２”菜豆（Ｐｈａｓｅｏｌｕｓ ｖｕｌｇａｒｉｓ Ｌ．）的下胚轴分离出微粒体、质膜微囊和液泡膜微囊。通过比较正常细胞的膜微囊、热锻炼细胞的膜微囊以及热锻炼过程中放线菌酮抑制了蛋白质合成的细胞膜微囊的质子泵在体外的热稳定性,发现热锻炼可使细胞膜微囊质子泵体外热稳定性提高,热锻炼过程中合成的蛋白质与此效应有关。进一步分析膜外周蛋白对质子泵体外热稳定性的影响,证明热激蛋白ＨＳＰ７０ 和小分子量热激蛋白（ＬＭＷ ＨＳＰ）对质子泵有热保护功能     

In Vitro Heat Stability of Plasma Membrane Vesicles and Tonoplast Membrane Vesicles Isolated From the Hypocotyle of Phaseolus vulgaris

Microsome, plasma membrane vesicles and tonoplast membrane vesicles were isolated from the hypocotyles of Phaseolus vulgaris L. 85CT-49762, with very high heat tolerance potential. Comparing the H+-pump heat stability in vitro of the vesicles from the heat acclimated cells and the cells in which protein synthesis was inhibited by actidion during heat acclimation with that of normal cells, the authors found that heat acclimation could increase the heat stability of membrane vesicles, and that the heat shock proteins synthesized during heat acclimation were related to the effect. The authors further analysed the role of membrane peripheral proteins on H+-pump thermotolerance of membrane vesicles, and proved that heat shock protein HSP 70 and low molecular weight heat shock protein (LMW HSP) were able to protect H+-pump from heat destruction.     

Heat-shock-induced changes in the phosphorylation of ribosomal and ribosome-associated proteins in the filamentous fungus Achlya ambisexualis

In the filamentous fungus Achlya ambisexualis, heat shock resulted in a rapid reduction in the rate of protein synthesis. This was accompanied by dephosphorylation of a prominent basic 30 kD protein associated with the small subunit of Achlya ribosomes and which may be analogous to ribosomal protein S6 of vertebrates. A large ribosomal subunit protein with a relative molecular weight (MW) of 24,500 exhibited increased phosphorylation during heat shock, while a second large subunit protein having a relative MW of 22,000 was dephosphorylated. Several proteins which could be dissociated from Achlya ribosomes by 0.5 M KCl also exhibited altered patterns of phosphorylation during heat shock. These KCl-soluble proteins included proteins at 50, 21, 20 and 19 kD, which exhibited decreased phosphorylation with heat shock and proteins at 32 and 23.5 kD, which exhibited increased phosphorylation with heat shock. Such alterations in the phosphorylation of components of the Achlya translational apparatus may be involved in the qualitative and quantitative changes in protein synthesis which are observed with heat shock in Achlya.     

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.     

HeLa细胞热休克蛋白的代谢动力学

 HeLa细胞经45℃,15min应激后,可诱导一组分子量为100,85,73,70,54kD的热休克蛋白,其中分子量为73/70kD的HSP产量最高。在产生HSP的同时,正常蛋白质合成受到抑制,并在随后数小时内恢复。HSP73/70在应激后能迅速诱导产生,应激后4—6小时为其合成高峰,10小时后明显减少,24小时恢复正常。其分解遵循指数规律,半衰期为49.9小时。     

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.Abbreviations IP isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.