Arabidopsis heat shock factor: isolation and characterization of the gene and the recombinant protein

We have isolated the Arabidopsis heat shock factor gene Athsf1 as genomic and corresponding cDNA sequences via cross-hybridization with tomato clones. Sequence analysis indicates only a partial homology with the HSFs from tomato and other organisms which is confined to the DNA-binding and the oligomerization domains. The gene is constitutively expressed but the level of mRNA for Athsf1 increases two-fold upon heat shock. However, the putative promoter region lacks the canonical heat shock elements. After expression in Escherichia coli the recombinant Athsf1 protein binds specifically to a synthetic oligonucleotide containing five heat shock elements. The native size of recombinant ATHSF1 in vitro is consistent with a trimer as demonstrated by chemical cross-linking and pore exclusion limit analysis.The publication is dedicated to Professor Dr. Wolfram Heumann, Universität Erlangen, on occassion of his 80th birthday.     

Effect of continuous ohmic heating to inactivate Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice

Aims: The purpose of this study was to investigate the efficacy of continuous ohmic heating for reducing Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice. Methods and Results: Orange juice and tomato juice were treated with electric field strengths in the range of 25–40 V cm^?1 for different treatment times. The temperature of the samples increased with increasing treatment time and electric field strength. The rate of temperature change for tomato juice was higher than for orange juice at all voltage gradients applied. Higher electric field strength or longer treatment time resulted in a greater reduction of pathogens. Escherichia coli O157:H7 was reduced by more than 5 log after 60‐, 90‐ and 180‐s treatments in orange juice with 40, 35 and 30 V cm^?1 electric field strength, respectively. In tomato juice, treatment with 25 V cm^?1 for 30 s was sufficient to achieve a 5‐log reduction in E. coli O157:H7. Similar results were observed in Salm. Typhimurium and L. monocytogenes. The concentration of vitamin C in continuous ohmic heated juice was significantly higher than in conventionally heated juice (P < 0·05). Conclusions: Continuous ohmic heating can be effective in killing foodborne pathogens on orange juice and tomato juice with lower degradation of quality than conventional heating. Significance and Impact of the Study: These results suggest that continuous ohmic heating might be effectively used to pasteurize fruit and vegetable juices in a short operating time and that the effect of inactivation depends on applied electric field strengths, treatment time and electric conductivity.     

Heat shock stress in Bacteroides fragilis

The response to heat shock was investigated in the obligate anaerobe Bacteroides fragilis. The cells responded quickly to stress and synthesised seven heat shock proteins immediately upon exposure to heat. The apparent molecular weights of the seven proteins differed from the apparent molecular weights of the proteins induced by UV irradiation, O₂ and H₂O₂. Heat shock did not induce phage reactivation whereas UV irradiation, O₂ and H₂O₂ did induce phage reactivation systems. Ethanol did not elicit the heat shock response. Two heat resistant B. fragilis mutants were isolated. Both mutants lost the ability to synthesise the same two heat shock proteins. It is concluded that the heat shock response and the responses to UV irradiation, O₂ and H₂O₂ represent two independent groups of stress responses in B. fragilis.     

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Changes in gene expression, by application of H₂O₂, O₂°^–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂°^– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.     

Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori

We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB₄D₂, KSO₁, NP₂, CSR₂ and CSR₄and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35°C, 40°C and 45°C for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS‐PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP₂ exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar > second instar > third instar > fourth instar > fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB₄D₂, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.     

Molecular chaperone activity of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) endoplasmic reticulum-located small heat shock protein

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His₆-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

One- and two-dimensional polyacrylamide gel analysis of the heat shock proteins of the virilis group of Drosophila

The heat shock proteins of the virilis group of Drosophila are analyzed by one- and two-dimensional polyacrylamide gel analysis. This group consists of the two closely related but distinct virilis and montana phylads. The analysis reveals that some of the heat shock proteins are highly conserved among the two phylads while others are not. The 83-, 72-, and 69-kdalton proteins comigrate in all species examined. There is, however, a noticable trend toward greater molecular weight variability in the smaller heat shock proteins. In general, the heat shock protein patterns within each phylad follow the proposed phylogenetic relationships with some exceptions. D. ezoana and D. littoralis, both members of the montana phylad, exhibit heat shock protein patterns more similar to those of the virilis phylad. The data also demonstrate that the montana phylad has almost two times the heat shock allele members that the virilis phylad has. It is also shown that F₁ and F₂ hybrid flies of crosses between Drosophila species having different patterns of heat shock proteins show Mendelian segregation of alleles. After several generations of inbred growth, however, the pattern of heat shock protein synthesis in reciprocal hybrids each resembles that of the paternal parent. The implications of these findings are discussed.This research was supported in part by Damon Runyon-Walter Winchell Grant DRG-233F to R.M.S. and NIH Grant GM 27611 to R.V.S. R.V.S. is the recipient of an NIH Research Career Development Award.     

Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

Analysis of the role of mitochondrial and endoplasmic reticulum localized small heat shock proteins in tomato

This communication examines the role of small heat shock proteins (sHsps) targeted to mitochondria (Mt) and endoplasmic reticulum (ER) in tomato plants (Lycopersicon esculentum Mill.) under heat stress. Genetic response of transgenic and wild type plants varied under optimum, moderately elevated and elevated temperature. In optimum temperature higher biomass was recorded in wild type than the transgenic lines, whereas in moderately elevated temperature biomass increased in Mt-sHsp line. Also, net photosynthetic rate (P_N) increased in Mt-sHsp line in both the elevated temperatures, though higher in moderately elevated. Cell membrane stability (CMS) improved in all the lines after exposure to elevated temperatures, but always remained higher in transgenic lines. Transgenic lines expressed sHsps in different temperature regimes in both vegetative and reproductive parts, while wild type expressed such proteins only after 1 h of heat shock.     

Identificazione di Alcuni Fattori di Crescita Nel Succo di Pomodoro

Abstract

Growth-factors in tomato juice. Raw juice of green and red tomato fruits contains 7 amines which can be separated by paper electrophoresis. These were identified as basic amino acids (arginine, lysine and ornithine) and aliphatic diamine and polyamines (putrescine, spermine and spermidine). These amines were found in tomato juice for the first time. One compound (band 6, table 1) was not identified. Putrescine, spermidine and the unidentified band, after elution, were assayed « in vitro » for the possible cellular proliferation of Jerusalem artichoke (« Helianthus tuberosus ») dormant tubers. The results show that the last three coumpounds must be considered natural growth-factors present in the raw juice of tomato fruits.     

Lycopene-rich treatments modify noneosinophilic airway inflammation in asthma: Proof of concept

Antioxidant-rich diets are associated with reduced asthma prevalence. However, direct evidence that altering intake of antioxidant-rich foods affects asthma is lacking. The objective was to investigate changes in asthma and airway inflammation resulting from a low antioxidant diet and subsequent use of lycopene-rich treatments. Asthmatic adults (n=32) consumed a low antioxidant diet for 10 days, then commenced a randomized, cross-over trial involving 3×7 day treatment arms (placebo, tomato extract (45 mg lycopene/day) and tomato juice (45 mg lycopene/day)). With consumption of a low antioxidant diet, plasma carotenoid concentrations decreased, Asthma Control Score worsened, %FEV₁ and %FVC decreased and %sputum neutrophils increased. Treatment with both tomato juice and extract reduced airway neutrophil influx. Treatment with tomato extract also reduced sputum neutrophil elastase activity. In conclusion, dietary antioxidant consumption modifies clinical asthma outcomes. Changing dietary antioxidant intake may be contributing to rising asthma prevalence. Lycopene-rich supplements should be further investigated as a therapeutic intervention.     

番茄SlGRAS4基因特征分析和耐热功能鉴定

GRAS转录因子是调控植物生长发育和非生物胁迫响应的重要转录因子之一,而目前还没有GRAS调控高温胁迫的研究。为了深入研究番茄SlGRAS4生物功能,以耐热番茄LA2093为试验材料,分析番茄SlGRAS4基因结构、启动子序列及进化关系,利用qRT-PCR检测SlGRAS4在不同胁迫和不同激素处理下的表达水平,利用VIGS验证SlGRAS4基因耐热功能。结果表明:(1)生物信息学分析显示,SlGRAS4蛋白长度为666 aa,分子量为75 737.72 Da,理论等电点为6.31,含有GRAS转录因子家族典型的结构域,主要集中在C末端的277～657 aa之间;在SlGRAS4启动子区域发现脱落酸(ABA)和水杨酸(SA)响应元件;SlGRAS4与烟草NtGRAS1蛋白亲缘关系最近,推测SlGRAS4可能与其同源基因具有相似的生物功能。(2)在高温、低温、盐和干旱胁迫处理12 h时番茄SlGRAS4基因表达量升至最高,分别增加到对照的8.86、4.86、55.38和7.63倍;在ABA和SA激素处理8 h时SlGRAS4基因的表达量达到峰值,分别达到对照的120.72和3.55倍,说明SlGRAS4可能参与了多种非生物胁迫响应和激素信号传导。(3)沉默SlGRAS4基因番茄植株(VSlGRAS4)在高温胁迫下较对照植株(Ve)更容易萎蔫,且F_v/F_m与SOD、POD活性显著降低,REL和H_2O_2含量显著升高,说明在高温胁迫下沉默SlGRAS4使番茄植株细胞膜氧化损伤加重,光合能力降低,活性氧(ROS)清除酶活性减弱。(4)qRT-PCR分析显示,VSlGRAS4植株中高温信号应答关键基因HsfA1b、ROS信号应答基因ZAT10和ZAT12以及ROS清除酶编码基因CuZnSOD、FeSOD、APX1、APX2、CAT的表达水平均显著低于Ve植株,表明SlGRAS4转录因子可以通过调控高温和ROS信号转导来影响番茄的耐热性。研究认为,高温、低温、干旱、盐、ABA和SA均可显著诱导番茄SlGRAS4基因的表达,沉默SlGRAS4基因番茄植株的耐热性显著降低,证明番茄SlGRAS4基因具有耐热功能,为进一步解析SlGRAS4参与番茄耐热调控的分子机制奠定基础。     

Atmospheric CO2, plant nitrogen status and the susceptibility of plants to an acute increase in temperature

Abstract. Elevated levels of CO₂ in the atmosphere are expected to affect plant performance and may alter global temperature patterns. Changes in mean air temperatures that might be induced by rising levels of CO₂ and other greenhouse gases could also be accompanied by increased variability in daily temperatures such that acute increases in air temperature may be more likely than at present. Consequently, we investigated whether plants grown in a CO₂ enriched atmosphere would be differently affected by a heat shock than plants grown at ambient CO₂ levels. Plants of a C₃ annual (Abutilon theophrasti), a C₃ annual crop (Sinapis alba) and a C₄ annual (Amaranthus retroflexus) were grown from seed in growth chambers under either 400 or 700cm³ m^?3 CO₂, and were fertilized with either a high or low nutrient regime. Young seedlings of S. alba, as well as plants of all species in either the vegetative or reproductive phase of growth were exposed to a 4-h heat shock in which the temperature was raised an additional 14–23°C (depending on plant age). Total biomass and reproductive biomass were examined to determine the effect of CO₂, nutrient and heat shock treatments on plant performance. Heat shock, CO₂, and nutrient treatments, all had some significant effects on plant performance, but plants from both CO₂ treatments responded similarly to heat shocks. We also found, as expected, that plants grown under high CO₂ had dramatically decreased tissue N concentrations relative to plants grown under ambient conditions. We predicted that high-CO₂-grown plants would be more susceptible to a heat shock than ambient-CO₂-grown plants, because the reduced N concentrations of high-CO₂ grown plants could result in the reduced synthesis of heat shock proteins and reduced thermotolerance. Although we did not examine heat shock proteins, our results showed little relationship between plant nitrogen status and the ability of a plant to tolerate an acute increase in temperature.     

Carbon dioxide enrichment alleviates heat stress by improving cellular redox homeostasis through an ABA‐independent process in tomato plants

Plant responses to elevated CO₂ and high temperature are critically regulated through a complex network of phytohormones and redox homeostasis. However, the involvement of abscisic acid (ABA) in plant adaptation to heat stress under elevated CO₂ conditions has not been thoroughly studied. This study investigated the interactive effects of elevated CO₂ (800 μmol·mol^?1) and heat stress (42 °C for 24 h) on the endogenous level of ABA and the cellular redox state of two genotypes of tomato with different ABA biosynthesis capacities. Heat stress significantly decreased maximum photochemical efficiency of PSII (Fv/Fm) and leaf water potential, but also increased levels of malondialdehyde (MDA) and electrolyte leakage (EL) in both genotypes. Heat‐induced damage was more severe in the ABA‐deficient mutant notabilis (not) than in its parental cultivar Ailsa Craig (Ailsa), suggesting that a certain level of endogenous ABA is required to minimise the heat‐induced oxidative damage to the photosynthetic apparatus. Irrespective of genotype, the enrichment of CO₂ remarkably stimulated Fv/Fm, MDA and EL in heat‐stressed plants towards enhanced tolerance. In addition, elevated CO₂ significantly strengthened the antioxidant capacity of heat‐stressed tomato seedlings towards a reduced cellular redox state for a prolonged period, thereby mitigating oxidative stress. However, elevated CO₂ and heat stress did not alter the endogenous level of ABA or the expression of its biosynthetic gene NCED2 in either genotype, indicating that ABA is not involved in elevated CO₂‐induced heat stress alleviation. The results of this study suggest that elevated CO₂ alleviated heat stress through efficient regulation of the cellular redox poise in an ABA‐independent manner in tomato plants.     

Phytochelatin accumulation and stress tolerance in tomato cells exposed to cadmium

Cell suspension cultures of tomato (Lycopersicon esculentum) adapted to growing continuously in the presence of 0.1 mM CdCl₂ and accumulated phytochelatins (PCs, poly(-Glu-Cys)n-Gly). The highest level of PCs was measured 4 days after inoculation and coincided with the peak of cellular cadmium concentration. At this time there was an 8-fold molar excess of PC (-Glu-Cys) over Cd. PCs could not be detected after 12 days when the cellular concentration of cadmium was 0.2 mM. These results indicate that PCs are produced in excess of that required to bind the cellular cadmium in the early stage of the culture period followed by degradation of PCs during the stationary phase. Adaptation to 0.1 mM CdCl₂ did not increase tolerance to higher concentrations of cadmium when compared with control cells, but did significantly enhance tolerance to both anaerobiosis and heat shock. Exposure of tomato cells to 0.1 mM CdCl₂ resulted in several changes in proteins synthesized.     

Heat Shock Inhibits the Induced Expression of the SOS and SoxRS Regulons in Escherichia coli

Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression ofsfiA and soxS genes induced by oxidative agents H₂O₂, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined inE. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ.The expression of these genes induced by cell treatment with H₂O₂, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion ,O^¯ ₂ it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H₂O₂ is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O^¯ ₂.