Geographic and temporal genetic structure in <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis> from four salmon farms along the northwest and west coasts of Ireland: results from a microsatellite analysis

The marine ectoparasitic copepod of salmonids, Lepeophtheirus salmonis (Krøyer), is a major pest of farmed Atlantic salmon (Salmo salar L.) causing great economic impact. The spatial scales over which L. salmonis populations in different salmon farms are typically connected, and the temporal scales over which L. salmonis from the same farm typically undergo genetic change are largely unknown. These questions were posed in a small-scale geographic study of population structure in L. salmonis from four salmon farms, along the northwest and west coasts of Ireland, using two outgroups from Norway and Canada. The temporal stability of genetic composition was also studied in samples collected quarterly during one year from one salmon farm in Ireland. Genetic composition in L. salmonis was characterised using four nuclear microsatellites. Significant but low genetic differentiation was observed between all sites (F _ST = 0.08), with no evidence that differentiation was correlated with geographic distance. Temporal genetic differentiation was also evident (F _ST = 0.07). An analysis of all L. salmonis samples except the ones from Norway detected two separate clusters. Each cluster contained both geographical and temporal samples. These results are consistent with a population model in which L. salmonis in salmon farms along the northwest and west coasts of Ireland are not isolated, but are potentially subject to (i) localised ecological factors at the particular farm sites or (ii) selection post-settlement or a combination thereof.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

Molecular cloning and characterization of the <Emphasis Type="Italic">MsHSP17.7</Emphasis> gene from <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial roles in plant development and adaptation to stress, and the aim of this study is to characterize the HSP gene in alfalfa. Here we isolated a small heat shock protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7 contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and drought stress. Prokaryotic expression indicated that the salt and peroxide tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis plants overexpressing MsHSP17.7 exhibited increased root length of transgenic Arabidopsis lines under salt stress compared to the wild-type line. The malondialdehyde (MDA) levels in the transgenic lines were significantly lower than in wild-type, although proline levels were similar between transgenic and wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might play a key role in response to high salinity stress.     

Adaptation of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45°C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. A 60-min preincubation of cells at 37°C or pretreatment with low concentrations of H₂O₂ (0.5 mM) or menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

ERK1/2 regulates heat stress-induced lactate production via enhancing the expression of HSP70 in immature boar Sertoli cells

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1?×?10^?6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.     

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.     

Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from <Emphasis Type="Italic">Chilo suppressalis</Emphasis>

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from ?11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to ?3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.     

Effect of salicylic acid on the development of induced Thermotolerance and induction of heat shock protein synthesis in the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> cell culture

Salicylic acid (SA) could be involved in the development of tolerance to abiotic stresses, to heat shock in particular. Under normal conditions (26°C), treatment with SA improved the tolerance of heterotrophic Arabidopsis thaliana (L.) Heynh culture to severe heat shock (50°C). Under mild heat shock (37°C) inducing the development of thermotolerance, the presence of SA, in contrast, reduced the capability of arabidopsis cells to tolerate high temperature (50°C) and simultaneously suppressed induction of HSP synthesis (Hsp101 and Hsp17.6) important for the development of induced thermotolerance. Since SA suppressed cell respiration and activated the alternative pathway of electron transport, SA is supposed, by modulating mitochondria functions, to be an endogenous regulator of plant stress gene expression.     

Heat susceptibility of grain filling in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) linked with rapid chlorophyll loss during a 3-day heat treatment

Brief heat events (1–3 days, >30 °C) commonly reduce wheat (Triticum aestivum L.) grain size and consequently yield. To identify mechanisms of tolerance to such short heat events, 36 wheat genotypes were treated under day/night temperatures of 37 °C/27 °C for 3-days in a growth chamber, at 10 days after anthesis, and a range of developmental, chlorophyll and yield-related traits monitored. The degree of flag leaf chlorophyll loss during the treatment was the variable that showed the highest correlation to grain weight loss (r = 0.63; p < 0.001), identifying chlorophyll stability during this brief period as a potential determinant or indicator of grain weight stability under heat. Variables summarizing the combined during- and post-heat chlorophyll losses showed similar or lower correlations with heat tolerance of grain filling, despite the fact that genotypes varied in their ability to resume normal chlorophyll loss rates after the heat treatment. Additionally, heat tolerance of grain size showed no correlation with grain filling duration or traits relating to utilization of stem carbon reserves under heat stress. Measurement of chlorophyll loss over a forecasted heat wave was thereby identified as a potential basis for developing tools to help breeders select heat tolerant genotypes.     

Identification of HSP70 gene in <Emphasis Type="Italic">Corythucha ciliata</Emphasis> and its expression profiles under laboratory and field thermal conditions

Previous laboratory studies have demonstrated that insects can tolerate high temperatures by expressing inducible heat shock proteins (HSPs). This HSP-based tolerance, however, has seldom been studied under field conditions. Here, we cloned the HSP70 gene of Corythucha ciliata (Cchsp70), an invasive insect species with substantial thermal tolerance in subtropical China. We also compared the relative mRNA expression levels of Cchsp70 in response to controlled temperature treatments (2 h at 33–43 °C at 2 °C intervals in the laboratory) and to natural increases in temperature (08:00–14:00 at 2-h intervals, 29.7–37.2 °C) on a hot summer day in the field. The complete cDNA of Cchsp70 is 2256 bp long and has a 1917 bp open reading frame that encodes a protein (CcHSP70) with 639 amino acids. The expression levels of Cchsp70 significantly increased in response to high temperatures in both laboratory and field. At similar temperatures, however, the expression levels were much higher in the field than in the laboratory. These results suggest that CcHSP70 contributes to the thermal tolerance of C. ciliata and that factors in addition to thermal stress may induce Cchsp70 expression in the field.     

Application of phytohormones during seed hydropriming and heat shock treatment on sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) chilling resistance and changes in soluble carbohydrates

The objective of this study was to analyze the mechanism of some physiological processes accompanying acquisition of sunflower (Helianthus annuus L.) chilling resistance due to seeds hydropriming in the presence of salicylic acid, jasmonic acid, 24-epibrassinolide followed exposition of seeds to short-term heat shock treatment. The seeds were hydroprimed at 25 °C in limited amounts of water or solution of salicylic or jasmonic acid at 10^?2, 10^?3 and 10^?4 M concentration, 24-epibrassinolide at 10^?6, 10^?8 and 10^?10 M concentration. The seeds were incubated for 2 days, subjected to short-term heat shock (45 °C, 2 h) and chilled for 21 days at 0 °C. Sunflower chilling susceptibility and physiological responses were evaluated according to the inhibition of radicle growth, the inhibition of the number of lateral roots formation, the activity of catalase and changes in soluble carbohydrates in seedlings developing for 72 h at 25 °C. Hydropriming and short-term heat shock application explicitly reduced inhibition of roots as well as lateral roots development by allowing the germinating seeds to recover from the growth-inhibiting effects of chilling. Seeds hydropriming in solutions containing salicylic acid, jasmonic acid and 24-epibrassinolide followed heat shock treatment additionally promoted the activity of catalase and sugars metabolism, which stimulated seedlings development and alleviated the decrease of F _v/F _m caused by chilling conditions. These beneficial effects contributed to increased resistance of sunflower seedlings to chilling stress. The present study demonstrated that the most profitable effect on reducing negative effect of chilling may be achieved by short-term heat shock applied during hydropriming in water supplemented with 24-epiBL (10^?8 and 10^?10 M) or salicylic acid (10^?3 and 10^?4 M).     

A purine-type heat shock protein 90 inhibitor promotes the heat shock response in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Geldanamycin is a macrocyclic heat shock protein 90 (HSP90) inhibitor that suppresses cancer cell proliferation. Since geldanamycin also promotes the heat shock response (HSR) in cells, this compound is used as a chemical inducer of the HSR in Arabidopsis. Although many types of HSP90 inhibitors that are different from the macrocyclic types have been developed in pharmaceutical research, non-macrocyclic HSP90 inhibitors have not been investigated in terms of whether they can induce the HSR in plants. Here, we determined the HSR-inducing activities in Arabidopsis of 10 non-macrocyclic HSP90 inhibitors including 2 benzamide derivatives, 3 purine derivatives, and 5 resorcinol derivatives. Among the tested inhibitors, PU-H71, which is a purine derivative, showed the highest HSR-inducing activity. The activity of PU-H71 was significantly higher than that of geldanamycin. The application of PU-H71 induced the HSR in all Arabidopsis seedlings. The HSP17.6C-CI and HSP70 proteins accumulated after the treatment with PU-H71. The seedlings treated with PU-H71 maintained more chlorophyll than the control seedlings after the heat stress. These results suggest that the purine-derivative HSP90 inhibitor PU-H71 enhanced the heat tolerance of Arabidopsis by promoting the HSR in the plant.     

The synthesis of nucleic acids in cultivated <Emphasis Type="Italic">Polyscias filicifolia</Emphasis> cells exposed to oxidative stress

The content of nucleic acids in the cell culture of fern-leaf aralia Polyscias filicifolia (Moore ex Fournter) Bailey (Araliaceae) exposed to heat shock (3 h at 45°C) decreased significantly (by 20–30%). The decrease in DNA and RNA contents was even larger (30–40%) after longer heat shock (24 h). Cold (24 h at 7°C) caused an even more dramatic decrease in DNA (by 34.2%) and total RNA (by 48%) contents. To judge from the DNA production rate, the presence of hydrogen peroxide and phenazine methosulfate in the culture exerted a dose-dependent and differently directed action on cell proliferation.