Overexpression of heat shock protein gene PfHSP21.4 in Arabidopsis thaliana enhances heat tolerance

Nuclear-encoded chloroplast small heat shock proteins (Cp-sHSPs) play important roles in plant stress tolerance due to their abundance and diversity. Their functions in Primula under heat treatment are poorly characterized. Here, expression analysis showed that the Primula Cp-sHSP gene, PfHSP21.4, was highly induced by heat stress in all vegetative and generative tissues in addition to constitutive expression in certain development stages. PfHSP21.4 was introduced into Arabidopsis, and its function was analysed in transgenic plants. Under heat stress, the PfHSP21.4 transgenic plants showed increased heat tolerance as shown by preservation of hypocotyl elongation, membrane integrity, chlorophyll content and photosystem II activity (Fv/Fm), increased seedling survival and increase in proline content. Alleviation of oxidative damage was associated with increased activity of superoxide dismutase and peroxidase. In addition, the induced expression of HSP101, HSP70, ascorbate peroxidase and Δ1-pyrroline-5-carboxylate synthase under heat stress was more pronounced in transgenic plants than in wild-type plants. These results support the positive role of PfHSP21.4 in response to heat stress in plants.     

Contrasting Physiological Responses of Jatropha curcas Plants to Single and Combined Stresses of Salinity and Heat

In this study we evaluated the contrasting major physiological responses of Jatropha curcas L. to salinity alone and in combination with high temperature. The plants were subjected to salinity (100 mM NaCl) before and after exposure to 43 °C (heat stress) for 6 h. The effects of salinity were more harmful than heat stress, and the effects of salt stress were increased when both stress factors were combined. The negative effects of the combined treatments included strong impairment of the CO₂ assimilation rate and stomata conductance and increased Na⁺ and Cl^? accumulation in the leaves associated with increased membrane damage and lipid peroxidation. Heat favorably stimulated the accumulation of glycine betaine and chlorophyll in the salt-stressed leaves. Treatments with salt, heat, and their combination stimulated the antioxidant enzymatic defense system, that is, the expression of ascorbate peroxidase (APX) and superoxide dismutase (SOD), whereas the expression of catalase (CAT) was stimulated through treatments with salt alone and in combination with heat; treatment with heat alone did not affect CAT expression. The ascorbate redox state was decreased under salinity stress alone and in combination with heat but remained unaffected when treated with heat alone. Overall, the leaf H₂O₂ concentration did not change in response to these stresses, but lipid peroxidation and membrane damage was increased. Moreover, high temperature increases the negative effects of salt stress on key physiological processes, but treatment with heat alone is favorable for several metabolic indicators of young J. curcas plants. In contrast with heat, these plants exhibit higher physiological disturbances under isolated salinity stress.     

Physiological response to heat stress of tomato ‘Micro-Tom’ plants expressing high and low levels of mitochondrial sHSP23.6 protein

Tomato ‘Micro-Tom’ plants were transformed for high or low expression of the mitochondrial small “heat shock” protein (HSP) (MT-sHSP23.6) to evaluate their response to high temperature. The plants were raised for 59 days under a controlled temperature, photoperiod and photon flow density and then subjected to heat stress for 24 h at 37 °C, followed by a recovery period under normal conditions (21 ± 2 °C). The cycle was repeated. The chlorophyll a fluorescence intensity was measured, and the parameters of the JIP-test were calculated. The gas exchange was also evaluated. The JIP-test showed significantly different responses of the genotypes to heat stress. The parameters of photosystem I activity and the net assimilation of CO₂ increased during the first stress cycle in genotypes with a high expression of MT-sHSP23.6 and in non-transformed plants; however, the net assimilation of CO₂ decreased in genotypes with a low expression of MT-sHSP23.6. The data suggest that MT-sHSP23.6 participates in the heat tolerance mechanism, considering that the suppression of this protein resulted in greater physiological damage during heat stress.     

Response of Pteris vittata to different cadmium treatments

Pteris vittata is known as an arsenic hyperaccumulator, but there is little information about its tolerance to cadmium and on its ability to accumulate this heavy metal. Our aim was to analyse the accumulation capacity, oxidative stress and antioxidant response of this fern after cadmium treatments. Cadmium content, main markers of oxidative stress and antioxidant response were detected in leaves of plants grown in hydroponics for both short- (5 days) and long- (15 days) term exposure to 0 (control) 60 and 100 μM CdCl₂. In leaves, the concentration of cadmium and oxidative stress were parallel with the increase of cadmium exposure. In the short-term exposure, antioxidant response was sufficient to contrast cadmium phytotoxicity only in 60 μM cadmium-treated plants. In the long-term exposure all treated plants, in spite of the increase in activity of some peroxide-scavenging enzymes, showed a significant increase in oxidative damage. As in the long-term stress markers were comparable in all treated plants, with no clear correlation with hydrogen peroxide content, at least part of cadmium-induced oxidative injury seems not mediated by H₂O₂. Based on our studies, P. vittata, able to uptake relatively high concentrations of cadmium, is only partially tolerant to this heavy metal.     

甜椒细胞质小分子量热激蛋白基因(CaHSP18)的cDNA克隆与表达

用RT-PCR和RACE-PCR技术,从热激处理的甜椒叶片总RNA中扩增出了细胞质小分子量热激蛋白(sHSP)全长779 bp的cDNA基因序列,包含一个480 bp开放阅读框,编码159个氨基酸.Southern杂交结果表明在甜椒基因组中有该基因的小的多基因家族.Northern结果显示该基因在甜椒根、茎、叶中的表达受热激和低温的诱导.原核表达分析表明该基因在高温以及低温条件下可以提高大肠杆菌的生存能力.     

Expression of hsp70, hsp100 and ubiquitin in Aloe barbadensis Miller under direct heat stress and under temperature acclimation conditions

Key message

The study determined the tolerance of Aloe vera to high temperature, focusing on the expression of hsp70 , hsp100 and ubiquitin genes. These were highly expressed in plants acclimated at 35 °C prior to a heat shock of 45 °C.

Abstract

Aloe barbadensis Miller (Aloe vera), a CAM plant, was introduced into Chile in the semiarid IV and III Regions, which has summer diurnal temperature fluctuations of 25 to 40 °C and annual precipitation of 40 mm (dry years) to 170 mm (rainy years). The aim of this study was to investigate how Aloe vera responds to water and heat stress, focusing on the expression of heat shock genes (hsp70, hsp100) and ubiquitin, which not studied before in Aloe vera. The LT₅₀ of Aloe vera was determined as 53.2 °C. To study gene expression by semi-quantitative RT-PCR, primers were designed against conserved regions of these genes. Sequencing the cDNA fragments for hsp70 and ubiquitin showed a high identity, over 95 %, with the genes from cereals. The protein sequence of hsp70 deduced from the sequence of the cDNA encloses partial domains for binding ATP and the substrate. The protein sequence of ubiquitin deduced from the cDNA encloses a domain for interaction with the enzymes E2, UCH and CUE. The expression increased with temperature and water deficit. Hsp70 expression at 40–45 °C increased 50 % over the controls, while the expression increased by 150 % over the controls under a water deficit of 50 % FC. The expression of all three genes was also studied under 2 h of acclimation at 35 or 40 °C prior to a heat shock at 45 °C. Under these conditions, the plants showed greater expression of all genes than when they were subjected to direct heat stress.     

Influence of arbuscular mycorrhiza on the growth and antioxidative activity in cyclamen under heat stress

The influence of the arbuscular mycorrhizal (AM) fungus, Glomus fasciculatum, on the growth, heat stress responses and the antioxidative activity in cyclamen (Cyclamen persicum Mill.) plants was studied. Cyclamen plants (inoculated or not with the AM fungus) were placed in a commercial potting media at 17–20 °C for 12 weeks in a greenhouse and subsequently subjected to two temperature conditions in a growth chamber. Initially, plants were grown at 20 °C for 4 weeks as a no heat stress (HS?) condition, followed by 30 °C for another 4 weeks as a heat stress (HS+) condition. Different morphological and physiological growth parameters were compared between G. fasciculatum-inoculated and noninoculated plants. The mycorrhizal symbiosis markedly enhanced biomass production and HS + responses in plants compared to that in the controls. A severe rate of leaf browning (80–100 %) was observed in control plants, whereas the mycorrhizal plants showed a minimum rate of leaf browning under HS + conditions. The mycorrhizal plants showed an increase activity of antioxidative enzymes such as superoxide dismutase and ascorbate peroxidase, as well as an increase in ascorbic acid and polyphenol contents. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity also showed a greater response in mycorrhizal plants than in the control plants under each temperature condition. The results indicate that in cyclamen plants, AM fungal colonisation alleviated heat stress damage through an increased antioxidative activity and that the mycorrhizal symbiosis strongly enhanced temperature stress tolerance which promoted plant growth and increased the host biomass under heat stress.     

Pepper Heat Shock Protein 70a Interacts with the Type III Effector AvrBsT and Triggers Plant Cell Death and Immunity

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.The heat shock protein HSP70 is a ubiquitous essential protein chaperone and one of the most abundant and diverse heat stress proteins in plants. HSP70s are induced by environmental stresses and are required for plants to cope with heat. HSP70s are involved in protein folding, synthesis, translocation, and macromolecular assemblies such as microtubules (Mayer et al., 2001; Hartl and Hayer-Hartl, 2002). HSP70s protect cells from heat stress by preventing protein aggregation and by facilitating the refolding of denatured proteins. Protein stability can decrease under heat stress conditions and expose hydrophobic patches that cause the aggregation of denatured proteins. HSP70s bind to hydrophobic patches of partially unfolded proteins in an ATP-dependent manner and prevent protein aggregation (Mayer and Bukau, 2005). The modular HSP70 structure consists of a N-terminal ATPase domain and a C-terminal peptide-binding domain that contains a β-sandwich subdomain with a peptide-binding cleft and an α-helical latch-like segment (Zhu et al., 1996; Hartl and Hayer-Hartl, 2002).HSP70s are involved in microbial pathogenesis, cell death responses, and immune responses. Diverse RNA viruses induce HSP70 expression in Arabidopsis (Arabidopsis thaliana; Whitham et al., 2003). Cytoplasmic HSP70s enhance the infection of Nicotiana benthamiana by Tobacco mosaic virus, Potato virus X, Cucumber mosaic virus, and Watermelon mosaic virus (Chen et al., 2008). Recently, the coat protein of Tomato yellow leaf curl virus was suggested to recruit host plant HSP70 during virus infection (Gorovits et al., 2013). HSP70s appear to be involved in regulating viral reproduction, protein folding, and movement, which ultimately promotes viral infection (Boevink and Oparka, 2005; Hafrén et al., 2010). The Pseudomonas syringae effector protein Hopl1 directly binds and manipulates host HSP70, which promotes bacterial virulence (Jelenska et al., 2010). The cytosolic/nuclear heat shock cognate 70 (HSC70) chaperone, which is highly homologous to HSP70 (Tavaria et al., 1996), regulates Arabidopsis immune responses together with SGT1 (for the suppressor of the G2 allele of S-phase kinase-associated protein1 [skp1]; Noël et al., 2007). Cytoplasmic HSP70 is required for the Phytophthora infestans INF1-mediated hypersensitive response (HR) and nonhost resistance to Pseudomonas cichorii in N. benthamiana (Kanzaki et al., 2003). HSP70 is proposed to be involved in both positive and negative regulation of cell death. Selective HSP70 depletion from human cell lines activates a tumor-specific death program that is independent of known caspases and p53 tumor-suppressor protein (Nylandsted et al., 2000), whereas HSP70 promotes tumor necrosis factor-mediated apoptosis by binding IkB kinase γ and impairing nuclear factor-κB signaling in Cos-1 cells (Ran et al., 2004). In N. benthamiana, HSP70 is required for tabtoxinine-β-lactam-induced cell death (Ito et al., 2014). However, HSP70 expression is shown to decrease the cell death triggered by salicylic acid (SA) in Nicotiana tabacum protoplasts (Cronjé et al., 2004). Overexpression of mitochondrial HSP70 suppresses heat- and hydrogen peroxide (H₂O₂)-induced programmed cell death in rice (Oryza sativa; Qi et al., 2011).The genus Xanthomonas YopJ-like AvrBsT protein activates effector-triggered immunity (ETI) in Arabidopsis Pitztal 0 plants (Cunnac et al., 2007). AvrBsT is a member of the YopJ/AvrRxv family identified in Xanthomonas campestris pv vesicatoria (Xcv; Lewis et al., 2011). AvrBsT alters phospholipid signaling and activates defense responses in Arabidopsis (Kirik and Mudgett, 2009). AvrBsT is an acetyltransferase that acetylates Arabidopsis ACETYLATED INTERACTING PROTEIN1 (ACIP1), a microtubule-associated protein required for plant immunity (Cheong et al., 2014). Xcv strain Bv5-4a secretes the AvrBsT type III effector protein that induces hypersensitive cell death and strong defense responses in pepper (Capsicum annuum) and N. benthamiana (Orth et al., 2000; Escolar et al., 2001; Kim et al., 2010). AvrBsT-induced HR-like cell death in pepper is likely part of the typical ETI-mediated defense response cascade (Jones and Dangl, 2006; Eitas et al., 2008; Eitas and Dangl, 2010). AvrBsT overexpression in Arabidopsis triggers plant cell death and defense signaling, leading to both disease and defense responses to diverse microbial pathogens (Hwang et al., 2012). Type III effectors such as Hopl1 and AvrBsT are used to identify unknown components of plant defense cascades (Nomura et al., 2006; Block et al., 2008; Jelenska et al., 2010; Kim et al., 2014) that modulate host innate immunity to achieve disease resistance. The pepper SGT1 was identified recently as a host interactor of AvrBsT (Kim et al., 2014). Pepper SGT1 has features of a cochaperone (Shirasu and Schulze-Lefert, 2003), interacts with AvrBsT, and promotes hypersensitive cell death associated with the pepper receptor-like cytoplasmic protein kinase1 (PIK1) phosphorylation cascade.In this study, we used a yeast (Saccharomyces cerevisiae) two-hybrid screen to identify the pepper HSP70a (CaHSP70a) as an interacting partner of the Xanthomonas spp. type III effector AvrBsT. Coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) analyses verify that CaHSP70a interacts with AvrBsT in planta. Transient CaHSP70a overexpression in pepper leaves enhances heat stress sensitivity and leads to a cell death response. Cytoplasmic localization of the AvrBsT-CaHSP70a complex strongly elevates cell death. CaHSP70a expression is rapidly and strongly induced by avrBsT (for avirulent Xcv Dukso1 [Ds1]) infection in pepper. CaHSP70a silencing enhances susceptibility to Xcv infection, attenuates the reactive oxygen species (ROS) burst and cell death response, reduces SA and jasmonic acid (JA) levels, and disrupts expression of the defense response genes C. annuum pathogenesis-related protein1 (CaPR1; Kim and Hwang, 2000), CaPR10 (Choi et al., 2012), and CaDEF1 (for defensin; Do et al., 2004). Taken together, this study demonstrates that CaHSP70a is a target of the Xanthomonas spp. type III effector AvrBsT and acts as a positive regulator of plant cell death and immunity signaling.