Expression of a Conserved Family of Cytoplasmic Low Molecular Weight Heat Shock Proteins during Heat Stress and Recovery

Plants synthesize several families of low molecular weight (LMW) heat shock proteins (HSPs) in response to elevated temperatures. We have characterized two cDNAs, HSP18.1 and HSP17.9, that encode members of the class I family of LMW HSPs from pea (Pisum sativum). In addition, we investigated the expression of these HSPs at the mRNA and protein levels during heat stress and recovery. HSP18.1 and HSP17.9 are 82.1% identical at the amino acid level and are 80.8 to 92.9% identical to class I LMW HSPs of other angiosperms. Heat stress experiments were performed using intact seedlings subjected to a gradual temperature increase and held at a maximum temperature of 30 to 42 degrees Celsius for 4 hours. HSP18.1 and HSP17.9 mRNA levels peaked at the beginning of the maximum temperature period and declined rapidly after the stress period. Antiserum against a HSP18.1 fusion protein recognized both HSP18.1 and HSP17.9 but not members of other families of LMW HSPs. The accumulation of HSP18.1-immunodetected protein was proportional to the severity of the heat stress, and the protein had a half-life of 37.7 ± 8 hours. The long half-life of these proteins supports the hypothesis that they are involved in establishing thermotolerance.     

HSP70-3 Interacts with Phospholipase Dδ and Participates in Heat Stress Defense

Heat shock proteins (HSPs) function as molecular chaperones and are key components responsible for protein folding, assembly, translocation, and degradation under stress conditions. However, little is known about how HSPs stabilize proteins and membranes in response to different hormonal or environmental cues in plants. Here, we combined molecular, biochemical, and genetic approaches to elucidate the involvement of cytosolic HSP70-3 in plant stress responses and the interplay between HSP70-3 and plasma membrane (PM)-localized phospholipase Dδ (PLDδ) in Arabidopsis (Arabidopsis thaliana). Analysis using pull-down, coimmunoprecipitation, and bimolecular fluorescence complementation revealed that HSP70-3 specifically interacted with PLDδ. HSP70-3 bound to microtubules, such that it stabilized cortical microtubules upon heat stress. We also showed that heat shock induced recruitment of HSP70-3 to the PM, where HSP70-3 inhibited PLDδ activity to mediate microtubule reorganization, phospholipid metabolism, and plant thermotolerance, and this process depended on the HSP70-3–PLDδ interaction. Our results suggest a model whereby the interplay between HSP70-3 and PLDδ facilitates the re-establishment of cellular homeostasis during plant responses to external stresses and reveal a regulatory mechanism in regulating membrane lipid metabolism.

The heat shock protein 70-3 interacts with phospholipase Dδ to regulate microtubule organization, lipid metabolism, and plant thermotolerance in Arabidopsis.     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

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 resistance conferred by expression of the human HSP27 gene in rodent cells

Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.     

AsHSP17, a creeping bentgrass small heat shock protein modulates plant photosynthesis and ABA‐dependent and independent signalling to attenuate plant response to abiotic stress

Heat shock proteins (HSPs) are molecular chaperones that accumulate in response to heat and other abiotic stressors. Small HSPs (sHSPs) belong to the most ubiquitous HSP subgroup with molecular weights ranging from 12 to 42 kDa. We have cloned a new sHSP gene, AsHSP17 from creeping bentgrass (Agrostis stolonifera) and studied its role in plant response to environmental stress. AsHSP17 encodes a protein of 17 kDa. Its expression was strongly induced by heat in both leaf and root tissues, and by salt and abscisic acid (ABA) in roots. Transgenic Arabidopsis plants constitutively expressing AsHSP17 exhibited enhanced sensitivity to heat and salt stress accompanied by reduced leaf chlorophyll content and decreased photosynthesis under both normal and stressed conditions compared to wild type. Overexpression of AsHSP17 also led to hypersensitivity to exogenous ABA and salinity during germination and post‐germinative growth. Gene expression analysis indicated that AsHSP17 modulates expression of photosynthesis‐related genes and regulates ABA biosynthesis, metabolism and ABA signalling as well as ABA‐independent stress signalling. Our results suggest that AsHSP17 may function as a protein chaperone to negatively regulate plant responses to adverse environmental stresses through modulating photosynthesis and ABA‐dependent and independent signalling pathways.     

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.     

Nitrogen availability alters patterns of accumulation of heat stress-induced proteins in plants

Mounting evidence suggests that heat-shock proteins (HSPs) play a vital role in enhancing survival at high temperature. There is, however, considerable variation in patterns of HSP production among species, and even among and within individuals of a species. It is not known why this variation exists and to what extent variation in HSPs among organisms might be related to differences in thermotolerance. One possibility is that production of HSPs confers costs and natural selection has worked towards optimizing the cost-to-benefits of HSP synthesis and accumulation. However, the costs of this production have not been determined. If HSP production confers significant nitrogen (N) costs, then we reasoned that plants grown under low-N conditions might accumulate less HSP than high-N plants. Furthermore, if HSPs are related to thermotolerance, then variation in HSPs induced by N (or other factors) might correlate with variation in thermotolerance, here measured as short-term effects of heat stress on net CO₂ assimilation and photosystem II (PSII) function. To test these predictions, we grew individuals of a single variety of corn (Zea mays L.) under different N levels and then exposed the plants to acute heat stress. We found that: (1) high-N plants produced greater amounts of mitochondrial Hsp60 and chloroplastic Hsp24 per unit protein than their low-N counterparts; and (2) patterns of HSP production were related to PSII efficiency, as measured by F _v/F _m. Thus, our results indicate that N availability influences HSP production in higher plants suggesting that HSP production might be resource-limited, and that among other benefits, chloroplast HSPs (e.g., Hsp24) may in some way limit damage to PSII function during heat stress.     

Detailed characterization of heat shock protein synthesis and induced thermotolerance in seedlings of Sorghum bicolor L.

Tolerance of both protein synthesis and seedling growth to apreviously lethal high temperature can be induced by prior exposureto a sub-lethal temperature during which the synthesis of heatshock proteins (HSPs) occurs. In this study, a thermal gradientbar was used to measure the physiological effects of temperatureon seedlings of sorghum (Sorghum bicolor L.) in conjunctionwith studies of gene expression. The duration of HSP synthesis,both during continued high temperature treatment or on returnto normal temperatures, was found to be very finely modulatedand was dependent on the severity of the initial heat shock.The synthesis of heat shock proteins and the induction of thermotolerancewere rapid, reversible and reinducible phenomena. Maximal thermotolerancewas obtained after treatments that induced the full complementof HSPs. Subsequent treatments that repressed HSP synthesis,also abolished thermotolerance. The presence of HSPs, however,was not sufficient for the tissue to be in a thermotolerantstate and the results suggest that either their de novo synthesis,or some other factor, is required for the induction of thermotolerance.Pre-existing HSPs did not inhibit the synthesis of new HSPs.Although the kinetics of the synthesis of HSPs and the developmentof thermotolerance show a tight correlation, the kinetics ofthe decay of thermotolerance and the degradation of HSPs werenot linked. The functional state or distribution of HSPs maywell change during the recovery process. Key words: Heat shock, thermotolerance, Sorghum bicolor, growth, protein synthesis     

Acquisition of thermotolerance in bay scallops, Argopecten irradians irradians, via differential induction of heat shock proteins

Many cells and organisms are rendered transiently resistant to lethal heat shock by short exposure to sublethal temperatures. This induced thermotolerance is thought to be related to increased amounts of heat shock proteins (HSPs) which, as molecular chaperones, protect cells from stress-induced damage. As part of a study on bivalve stress and thermotolerance, work was undertaken to examine the effects of sublethal heat shock on stress tolerance of juveniles of the northern bay scallop, Argopecten irradians irradians, in association with changes in the levels of cytoplasmic HSP70 and 40. Juvenile bay scallops heat-shocked at a sublethal temperature of 32 °C survived an otherwise lethal heat treatment at 35 °C for at least 7 days. As determined by ELISA, acquisition of induced thermotolerance closely paralleled HSP70 accumulation, whereas HSP40 accrual appeared less closely associated with thermotolerance. Quantification of scallop HSPs following lethal heat treatment, with or without conditioning, suggested a causal role for HSP70 in stress tolerance, with HSP40 contributing to a lesser, but significant extent. Overall, this study demonstrated that sublethal heat shock promotes survival of A. irradians irradians juveniles upon thermal stress and the results support the hypothesis that HSPs have a role in this induced thermotolerance. Exploitation of the induced thermotolerance response shows promise as a means to improve survival of bay scallops in commercial culture.     

The Stress of Suicide: Temporal and Spatial Expression of Putative Heat Shock Protein 70 Protect the Cells from Heat Injury in Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

Heat stress adversely affects growth, development, and yield of winter wheat (Triticum aestivum). Plants have, however, evolved mechanisms to adapt to such conditions mainly by the expression of stress-associated chaperones, the heat shock proteins (HSPs), for modulating the tolerance level. Here, we report cloning of cytosolic putative HSP70 of 1678 bp from a thermotolerant cultivar (C306) of wheat (T. aestivum). A BLASTn search showed maximum homology with the predicted HSP70 protein reported from Hordeum vulgare (accession no AK354795.1). In silico characterization showed the presence of a nucleotide-binding domain of the sugar kinase/HSP70/actin superfamily in the sequence. Putative HSP70 showed temporal and spatial variations in the expression under heat stress (HS). We observed the abundance of HSP70 protein, H₂O₂, proline, and guaiacol peroxidase activity during the seed-hardening stage under HS; accumulation was, however, higher in the thermotolerant C306 than in thermosusceptible HD2329 cultivar. A gradual decrease in cell membrane stability (CMS) and an increase in total antioxidant capacity (TAC) were observed in both the cultivars at the different stages of growth. The expression of HSP70 showed a negative correlation with CMS and a positive correlation with TAC under HS; changes were less pronounced in C306 than in HD2329 at all the stages of growth studied. HSP70 seems to play diverse roles associated with thermotolerance, and partially protect wheat from terminal HS. Being the important member of family of the HSPs, HSP70 needs to be studied in detail, to be used for developing climate-smart wheat crops, through genetic engineering/breeding approaches.     

NPR1-dependent salicylic acid signaling is not involved in elevated CO2-induced heat stress tolerance in Arabidopsis thaliana

Elevated CO₂ can protect plants from heat stress (HS); however, the underlying mechanisms are largely unknown. Here, we used a set of Arabidopsis mutants such as salicylic acid (SA) signaling mutants nonexpressor of pathogenesis-related gene 1 (npr1-1 and npr1-5) and heat-shock proteins (HSPs) mutants (hsp21 and hsp70-1) to understand the requirement of SA signaling and HSPs in elevated CO₂-induced HS tolerance. Under ambient CO₂ (380 µmol mol⁻¹) conditions, HS (42°C, 24 h) drastically decreased maximum photochemical efficiency of PSII (Fv/Fm) in all studied plant groups. Enrichment of CO₂ (800 µmol mol⁻¹) with HS remarkably increased the Fv/Fm value in all plant groups except hsp70-1, indicating that NPR1-dependent SA signaling is not involved in the elevated CO₂-induced HS tolerance. These results also suggest an essentiality of HSP70-1, but not HSP21 in elevated CO₂-induced HS mitigation.     

植物热激蛋白的功能及其基因表达的调控

本文介绍了植物热激蛋白的产：生、分布和分类。着重论述了热激反应的特点、植物热激蛋白的功能、热激基因表达与调控的研究进展。     

Effect of Heat Stress on Photosynthetic Characteristics of Different Green Organs of Winter Wheat During Grain-filling Stage

Four winter wheat ( Triticum aestivum L.) varieties (“JD 8”, “Jing 411”,“Centurk” and “Tam 202”) were used to study the effect of heat stress on photosynthetic characteristics of flag leaf blade, flag leaf sheath, peduncle, glume, lemma and awn during grain-filling stage. The results showed that heat acclimation during grain-filling stage increased thermotolerance of wheat with significant differences among different green organs. During heat stress, the decreases of the efficiency of primary light energy conversion ( Fv/Fm ) of PS and pigment (chlorophyll and carotenoid) content were much slower in peduncle, flag leaf sheath and glume than in flag leaf blade, lemma and awn; and the percentage of decrease in net photosynthetic rate ( Pn ) of ear was lower than that of the flag leaf blade. The measured photosynthetic parameters ( Fv/Fm , Pn and pigment content) of “JD 8”, a relatively heat tolerant variety, declined more slowly than those of the other three varieties during the whole heat stress period.      

有棱丝瓜苗期耐热性及其对热胁迫的生理响应

为鉴定有棱丝瓜(Luffa acutangula)的耐热性,对32个品种进行了耐热性筛选,并对不同耐热性品种在热胁迫下的生理响应进行了研究。结果表明,这些品种中有耐热品种3个,热敏品种4个,居中的25个。对耐热品种WB121,较耐热品种YL8和热敏品种WB105进行高温处理,结果表明,随着高温胁迫时间的延长,3个品种叶片的丙二醛含量、相对电导率、可溶性糖含量均逐渐增加。3种保护酶(SOD、CAT和POD)活性未表现出与耐热性一致的变化。3个品种的叶绿素荧光参数Fv/Fm、Yeild和ETR随着高温胁迫时间的延长均下降,以热敏品种下降幅度最大。Pearson相关分析表明,热害指数与相对电导率呈显著正相关(r=0.693*),与丙二醛和可溶性糖含量呈极显著正相关(r=0.814,0.899**),与叶绿素荧光参数(Fv/Fm,Yield,ETR)呈极显著负相关(r=-0.892,-0.896,-0.897**),与3种保护酶活性的相关性不显著。由此可见,叶绿素荧光参数(Fv/Fm,Yield,ETR)可以快速、准确地反映植物热害,建议将其作为有棱丝瓜耐热性鉴定的重要生理指标。     

植物热激蛋白的功能及其基因表达的调控

本文介绍了植物热激蛋白的产生、分布和分类。着重论述了热激反应的特点、植物热激蛋白的功能、热激基因表达与调控的研究进展     

Molecular genetics of heat tolerance and heat shock proteins in cereals

Heat stress is common in most cereal-growing areas of the world. In this paper, we summarize the current knowledge on the molecular and genetic basis of thermotolerance in vegetative and reproductive tissues of cereals. Significance of heat stress response and expression of heat shock proteins (HSPs) in thermotolerance of cereal yield and quality is discussed. Major avenues for increasing thermotolerance in cereals via conventional breeding or genetic modification are outlined.