The relationship between the differences in frost resistance of Arabidopsis and Thellungiella and heat shock proteins and dehydrins

Plants of extremophile Thellungiella (Thellungiella salsuginea (Pall.) OE Schulz) withstood freezing at ?15°C for 2 h without hardening, whereas plants of Arabidopsis (Arabidopsis thaliana (L.) Heinh.) were damaged at ?10°C and died at ?15°C under these conditions. The content of heat shock proteins (HSPs) HSP101, HSP60 and constitutive HSC70 was significantly higher in unhardened Thellungiella plants than in unhardened Arabidopsis plants. The spectrum of dehydrins (DHNs) in unhardened Thellungiella plants was more diverse and their total content was higher than in unhardened Arabidopsis plants. Frost resistance of Arabidopsis increased after hardening (4°C, 7 days), and there was an increase in the content of HSP101 and HSP60, as well as in the content of the DHN with a mol wt of 70 kD. Thellungiella plants survived after hardening at ?18°C, and the increase in the content of HSP101, HSP70, and HSP60 was significantly less pronounced than in Arabidopsis. At the same time, the content of DHNs in Thellungiella increased significantly during the hardening primarily because of the appearance of two DHNs (mol wts of 42 and 45 kD). It is assumed that an increased content of HSPs and DHNs and their greater diversity can be one of the factors of Thellungiella resistance to low temperatures as compared to Arabidopsis.     

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.     

The natural alkaloid sanguinarine promotes the expression of heat shock protein genes in Arabidopsis

Small-molecule heat shock response inducers are known to enhance heat tolerance in plants. In this paper, we report that a plant alkaloid enhances the heat tolerance of Arabidopsis. We investigated 12 commercially available alkaloids to determine whether they enhance the heat tolerance of Arabidopsis seedlings using an in vitro assay system with geldanamycin, which is a known heat shock response inducer, as a positive control. Accordingly we found that the isoquinoline alkaloid sanguinarine can enhance heat tolerance in Arabidopsis. No such effect was shown for the other 11 alkaloids. The sanguinarine treatment increased the expression of heat shock protein genes such as HSP17.6C-CI, HSP70, and HSP90.1, which were up-regulated by geldanamycin. Treatments with other isoquinoline alkaloids (berberine and papaverine), which showed few heat tolerance-enhancing effects, did not promote the expression of the heat shock protein genes. These results suggest that sanguinarine influenced the heat tolerance of Arabidopsis by enhancing the expression of heat shock protein genes.     

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.     

Systematic identification and characterization of stress-inducible heat shock proteins (HSPs) in the salmon louse (<Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>)

Salmon lice (Lepeophtheirus salmonis) are parasitic copepods, living mainly on Atlantic salmon and leading to large economical losses in aquaculture every year. Due to the emergence of resistances to several drugs, alternative treatments are developed, including treatment with hydrogen peroxide, freshwater or thermal treatment. The present study gives a first overview of the thermotolerance and stress response of salmon lice. Sea lice nauplii acclimated to 10 °C can survive heat shocks up to 30 °C and are capable of hardening by a sublethal heat shock. We searched in the genome for heat shock protein (HSP) encoding genes and tested their inducibility after heat shock, changes in salinity and treatment with hydrogen peroxide, employing microfluidic qPCRs. We assessed 38 candidate genes, belonging to the small HSP, HSP40, HSP70 and HSP90 families. Nine of these genes showed strong induction after a non-lethal heat shock. In contrast, only three and two of these genes were induced after changes in salinity and incubation in hydrogen peroxide, respectively. This work provides the basis for further work on the stress response on the economically important parasite L. salmonis.     

Involvement of Arabidopsis CPR5 in thermotolerance

The constitutive expressor of pathogenesis-related genes 5 (CPR5) plays a role in pathogen defence responses, programmed cell death, cell wall biogenesis, seed generation and senescence regulation in plants. Here, we investigated the functional characteristics of CPR5 to long-term heat stress in Arabidopsis with different genotypes: wild type (WT), cpr5 mutant and cpr5/CPR5 complementary transgenic plant. The cpr5 mutant showed increased susceptibility to long-term heat stress, displaying significant decreases in hypocotyl elongation, seedling and inflorescence survival, membrane integrity and photosystem II activity (Fv/Fm) during heat stress. However, the thermotolerance was recovered when cpr5 mutant was transformed with a CPR5 gene. H₂O₂ accumulation and lipid peroxidation were lower in cpr5/CPR5 plants and WT than in cpr5 mutants after exposure to 36?°C for 5?days. The alleviated oxidative damage was associated with increased activities of superoxide dismutase, catalase, and ascorbate peroxidase. Furthermore, the induced expression of HSP17.6A-CI, HSP101 and HSP70B under long-term heat stress was more substantial in cpr5/CPR5 plants and WT than in cpr5 mutants. These findings suggest that CPR5 plays an important role in thermotolerance of Arabidopsis by regulating the activities of antioxidant enzymes and the expressions of heat shock protein genes.     

Identification of an Arabidopsis thaliana cDNA encoding a HSP70-related protein belonging to the HSP110/SSE1 subfamily

Heat-shock protein 70 (HSP70)-related proteins are classified in two main subfamilies: the DnaK subfamily and the HSP110/SSE1 subfamily. We have characterized the first plant member of the HSP110/SSE1 subfamily, HSP91. At least two, tightly linked genes encoding HSP91 are present per haploid Arabidopsis genome. HSP91 is constitutively expressed in non-stressed Arabidopsis plants and is transiently induced by heat shock.     

Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants

Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6–9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16–17 and 14–15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.     

The influence of exogenous carbohydrate provision and pre-exercise alkalosis on the heat shock protein response to prolonged interval cycling

The aim of this study was to observe the intracellular heat shock protein 72 (HSP72) and heme oxygenase-1 (HSP32) response to prolonged interval cycling following the ingestion of carbohydrates (CHO) and sodium bicarbonate (NaHCO₃). Six recreationally active males (mean ± SD; age 23.2 ± 2.9 years, height 179.5 ± 5.5 cm, body mass 76.5 ± 6.8 kg, and peak power output 315 ± 36 W) volunteered to complete a 90 min interval cycling exercise on four occasions. The trials were completed in a random and blinded manner following ingestion of either: placebo and an artificial sweetener (P–P), NaHCO₃ and sweetener (B–P), placebo and CHO (P–CHO), and NaHCO₃ and CHO (B–CHO). Both HSP72 and HSP32 were significantly increased in monocytes and lymphocytes from 45 min post-exercise (p ≤ 0.039), with strong relationships between both cell types (HSP72, r = 0.83; HSP32, r = 0.89). Exogenous CHO had no influence on either HSP72 or HSP32, but the ingestion of NaHCO₃ significantly attenuated HSP32 in monocytes and lymphocytes (p ≤ 0.042). In conclusion, the intracellular stress protein response to 90 min interval exercise is closely related in monocytes and lymphocytes, and HSP32 appears to be attenuated with a pre-exercise alkalosis.     

The Arabidopsis HSP18.2 promoter/GUS gene fusion in transgenic Arabidopsis plants: a powerful tool for the isolation of regulatory mutants of the heat-shock response

A detailed study of the expression of the promoter of the HSP18.2 gene from Arabidopsis fused to the bacterial gene for β-glucuronidase (GUS) in transgenic Arabidopsis plants is described. High levels of GUS activity were induced in all organs of transformants except for seeds during heat shock. The optimum temperature for expression of GUS in Arabidopsis was 35°C regardless of the plant growth temperature. Heat shock of 40°C did not induce any detectable levels of GUS activity. Pre-incubation at 35°C was found to have a protective effect on the induction of GUS activity at 40°C. GUS activity was also increased in response to a gradual increase in temperature. Histochemical analysis revealed that basal levels of GUS activity were induced in the vascular tissue of leaves and sepals, as well as at the tips of carpels, at the normal growth temperature. Heat treatment of a limited part of the plant tissue did not appear to cause systemic induction of GUS activity. To extend the analysis of the plant heat-shock response, we attempted to screen mutations in genes involved in the regulation of the induction of heat-shock protein (HSP) genes, using the GUS gene as a selection marker in transgenic Arabidopsis plants, and the results of this analysis are described.     

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.     

Winter wheat cells subjected to freezing temperature undergo death process with features of programmed cell death

Programmed cell death is a process defined as genetically regulated self-destruction or cell suicide. It can be activated by different internal and external factors, but few studies have investigated whether this process occurs under cold and freezing temperatures. In this study, a freezing treatment (?8 °C for 6 h) induced cell death with features of programmed cell death in suspension cultures of winter wheat (Triticum aestivum L.). This process occurred for 10 days after cold exposure. The death of cells in culture was slow and prolonged, and was accompanied by protoplast shrinkage, DNA fragmentation, and an increase in the level of reactive oxygen species. Other changes observed after the freezing treatment included an increase in the respiration rate, changes in mitochondrial transmembrane potential (?Ψ _m ), and the release of cytochrome c from mitochondria into the cytosol. These findings indicated that mitochondria are involved in the cell death process that occurs after a freezing treatment in cells of winter wheat.     

The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells

The objective of this study is to investigate the signal transduction pathways that regulate heat shock protein 27 (HSP27) phosphorylation and migration of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) induced by angiotensin II (AngII) and platelet derived growth factor-BB (PDGF-BB). The activity of HSP27 was evaluated by Western blot with specific phospho-HSP27 antibody. F-actin polymerization was detected by FITC-Phalloidine staining using confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration assessment. Within a given concentration, the phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the specific P38MAPK inhibitor SB202190, the specific PI3K inhibitor LY294002 and the specific ERK1/2 inhibitor U0126 in a concentration-dependent manner, with a peak inhibition rate at 87.2%, 78.4% and 37.3%, respectively, induced by AngII (P < 0.01), with a peak inhibition rate at 85.0%, 55.3% and 41.0%, respectively, induced by PDGF-BB (P < 0.01).The migration of VSMCs induced by AngII and PDGF-BB was inhibited by 100 μmol/l SB202190, 30 μmol/l LY294002, and 30 μmol/l U0126, with a inhibition rate at 60.1%, 71.7% and 47.3%, respectively, provoked by AngII (P < 0.01), with a inhibition rate at 55.3%, 55.6% and 38.1%, respectively, provoked by PDGF-BB (P < 0.01). P38MAPK and PI3 K/Akt are important pathways that contribute to the phosphorylation of HSP27 and migration of VSMCs in response to AngII and PDGF-BB. ERK1/2 might be involved in HSP27 phosphorylation and migration of VSMCs provoked by AngII and PDGF-BB.     

Gene Silencing of Selenoprotein K Induces Inflammatory Response and Activates Heat Shock Proteins Expression in Chicken Myoblasts

In the present study, specific small interfering RNA (siRNA) for selenoprotein K (Selk) gene was designed and transfected into chicken myoblasts. Then, the expressions of inflammatory factors (including induced nitric oxide synthase [iNOS], nuclear factor-kappa B [NF-κB], heme-oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and prostaglandin E synthase [PTGEs]), inflammation-related cytokines (including interleukin [IL]-1β, IL-6, IL-7, IL-8, IL-17, and interferon [IFN]-γ), and heat shock proteins (HSPs) (including HSP27, HSP40, HSP60, HSP70, and HSP90) were examined at 24 and 72 h after transfection. The results showed that messenger RNA (mRNA) expressions of iNOS, NF-κB, HO-1, COX-2, IL-6, IL-7, IL-8, HSP 27, HSP 40, HSP 60, HSP 70, and HSP 90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection, and the mRNA expressions of PTGEs, IL-1β, IL-17, and IFN-γ were significantly increased and decreased (p < 0.05) at 24 and 72 h after siRNA transfection. The results also showed that the protein expressions of iNOS, NF-κB, HO-1, COX-2, HSP60, HSP70, and HSP90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection. The correlation analysis and principal component analysis (PCA) showed that PTGEs, IL-1β, IL-17, IFN-γ, HSP40, and HSP90 might play special roles in response to Selk silencing in chicken myoblasts. These results indicated that Selk silencing induced inflammation response by affecting the expression levels of inflammatory factors and inflammation-related cytokines, and the heat shock proteins might play protective roles in this response in chicken myoblasts.