Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori

Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90‐, 84‐, 62‐, 60‐, 52‐ and 33‐kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2‐DE with MALDI‐TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1‐DE gel was identified as HSP83 by MALDI‐TOF‐MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock‐induced protein spots were excised from 2‐DE gels for MALDI‐TOF‐MS analysis. The Mascot search results are for HSP68, HSC70‐1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC‐3 and HSP83 in NB₄D₂. Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB₄D₂. Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.     

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.     

Partial sequence and characterization of nitric oxide synthase gene from Hyphantria cunea

Nitric oxide synthase (NOS) gene has been partially sequenced from Hyphantria cunea and compared with those already determined from insects. Hyphantria cunea NOS possesses putative recognition sites for co‐factors heme, BH₄, CaM, FMN, FAD, and NADPH common to NOS. The deduced amino acid sequence of H. cunea NOS cDNA showed 70.3% identity to Manduca sexta NOS and 57.6–69.5% identity to NOS sequences from other insects. Nitric oxide synthase is expressed in all tissues of H. cunea, except in hemocytes. The NOS expression in midgut, fat body, epidermis, and Malpighian tubule strongly increased against Gram‐positive and Gram‐negative bacterial infection. These results suggest that NOS may play an important role in insect defense system against bacterial infection.     

Expression of selected heat shock proteins after individually applied and combined drought and heat stress

Drought and heat stress are among the abiotic factors causing the most severe damage on plant crops. Their combination is quite common in dry and semi-dry regions worldwide and little is known about its effect on heat shock protein (HSP) profile in wheat plants. The expression of four HSP genes (Hsp 17.8, Hsp 26.3, Hsp 70 and Hsp 101b) in Triticum aestivum L. plants subjected to individually applied water deprivation or high temperature and their combination was monitored via one-step RT-PCR analysis. Changes in the expression levels of small HSPs (smHSPs), HSP70 and HSP100 were established also by SDS-PAGE. The combination of drought and heat induced HSP expression more effectively than the individually applied stresses. The induction of HSPs displayed greater rate in the drought-tolerant wheat variety Katya than in the drought-sensitive cv. Sadovo. The results obtained in wheat plants suggested that the effect of separately applied drought and heat shock cannot be extrapolated to their combination.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Genome‐wide identification and characterization of HSP gene superfamily in whitefly (Bemisia tabaci) and expression profiling analysis under temperature stress

Heat shock proteins (HSP)are essential molecular chaperones that play important roles in the stress stimulation of insects.Bemisia tabaci,a phloem feeder and invasive species,can cause extensive crop damage through direct feeding and transmission of plant viruses.Here we employed comprehensive genomics approaches to identity HSP superfamily members in the Middle East Asia Minor 1 whitefly genome.In total,we identified 26 Hsp genes,including three Hsp90,17 Hsp70,one Hsp60 and five sHSP (small heat shock protein)genes.The HSP gene superfamily of whitefly is expanded compared with the other five insects surveyed here.The gene structures among the same families are relatively conserved.Meanwhile,the motif compositions and secondary structures of BtHsp proteins were predicted.In addition,quantitative polymerase chain reaction analysis showed that the expression patterns of BtHsp gene superfamily were diverse across different tissues of whiteflies.Most Hsp genes were induced or repressed by thermal stress (40℃)and cold treatment (4℃)in whitefly.Silencing the expression of BtHsp70-6 significantly decreased the survival rate of whitefly under 45℃.All the results showed the Hsps conferred thermo-tolerance or cold-tolerance to whiteflies that protect them from being affected by detrimental temperature conditions.Our observations highlighted the molecular evolutionary properties and the response mechanism to temperature assaults of Hsp genes in whitefly.     

Changes in the chlorophyll content of grape leaves could provide a physiological index for responses and adaptation to UV‐C radiation

Stilbenes are a group of phytoalexins that play an important role in grapevine (Vitis) basal immunity and can be induced by biotic and abiotic stresses. The levels of chlorophylls, the main pigments in plant cells, can also indicate the tolerance of plants to various stresses. Here, the response of different grapevine genotypes to UV‐C radiation treatment was tested and the abundance of chlorophyll in the Hoe29 and Ke53 genotypes was observed to increase significantly within 6 h of UV‐C treatment. Conversely, chlorophyll levels decreased markedly in the Augster Weiss and Müller–Thurgau genotypes. Furthermore, stilbene abundance increased substantially in the Hoe29, Ke53, Ke83 and Pinot Blanc genotypes, but increased only slightly in Augster Weiss and Müller–Thurgau. The expression of resveratrol synthase, which encodes a key enzyme in the stilbene synthesis pathway, increased in Hoe29, Ke53, Ke83 and Pinot Blanc following UV‐C treatment, in a manner consistent with stilbene accumulation. In addition, we observed that reactive oxygen species (ROS) provide a key trigger in physiological responses and changes in secondary metabolite contents. In summary, the results from this study support a link between ROS, chlorophyll levels and genetic diversity for stilbene abundance in different grape genotypes, providing insights into mechanisms for plant physiological and biochemical responses and adaptations to stress.     

Effects of systemic potato response to wounding and jasmonate on the aphid Macrosiphum euphorbiae (Sternorryncha: Aphididae)

Plant induced responses are activated by multiple biotic and abiotic stresses, and may affect the interactions between a plant and phytophagous insects. The objective of this work was to evaluate the effects of different stresses inflicted to potato plants (Solanum tuberosum) on the potato aphid (Macrosiphum euphorbiae). Abiotic wounding, biotic wounding by Leptinotarsa decemlineata and treatment with volatile methyl jasmonate (MeJA) were evaluated with regard to the orientation behaviour, the feeding behaviour and the development of the potato aphids. Dual‐choice olfactometry showed that plants treated with MeJA lost their attractiveness for the potato aphids, while both abiotic and biotic wounding did not alter the orientation of aphids. Electropenetrography revealed that the feeding behaviour of aphids was only slightly disturbed by a previous L. decemlineata wounding, while it was highly disturbed by mechanical wounding and MeJA treatment. Aphid nymph survival was reduced on mechanically wounded plants, the pre‐reproductive period was lengthened and the fecundity reduced on plants treated with MeJA. Our results bring new information about the effects of various stresses inflicted to S. tuberosum on M. euphorbiae. We showed that wounding and MeJA treatment induced an antixenosis resistance in potato plants against M. euphorbiae, which may influence aphid colonization processes.     

Cardiac and hepatic role of r‐AtHSP70: basal effects and protection against ischemic and sepsis conditions

Heat shock proteins (HSPs), highly conserved in all organisms, act as molecular chaperones activated by several stresses. The HSP70 class of stress‐induced proteins is the most studied subtype in cardiovascular and inflammatory disease. Because of the high similarity between plant and mammalian HSP70, the aim of this work was to evaluate whether recombinant HSP70 of plant origin (r‐AtHSP70) was able to protect rat cardiac and hepatic function under ischemic and sepsis conditions. We demonstrated for the first time that, in ex vivo isolated and perfused rat heart, exogenous r‐AtHSP70 exerted direct negative inotropic and lusitropic effects via Akt/endothelial nitric oxide synthase pathway, induced post‐conditioning cardioprotection via Reperfusion Injury Salvage Kinase and Survivor Activating Factor Enhancement pathways, and did not cause hepatic damage. In vivo administration of r‐AtHSP70 protected both heart and liver against lipopolysaccharide‐dependent sepsis, as revealed by the reduced plasma levels of interleukin‐1β, tumour necrosis factor alpha, aspartate aminotransferase and alanine aminotransferase. These results suggest exogenous r‐AtHSP70 as a molecular modulator able to protect myocardial function and to prevent cardiac and liver dysfunctions during inflammatory conditions.     

Cross‐Species Alleviation of Biotic and Abiotic Stresses by the Endophyte Pseudomonas aeruginosa PW09

A wheat endophytic bacterium (Pseudomonas aeruginosa PW09) was evaluated for its ability to trigger an induced systemic resistance response in cucumber against biotic and abiotic stresses. PW09 was applied to cucumber seeds, and the seedlings were subjected to Sclerotium rolfsii infection and NaCl (150 mm ). The role of PW09 was evaluated in alleviating the stresses by assessing plant mortality due to S. rolfsii infection and biomass accumulation under NaCl stress as well as at the physiological level through phenylpropanoid metabolism, antioxidant activities and proline accumulation. The endophyte reduced seedling mortality by 60% and increased biomass accumulation significantly under S. rolfsii (7%) and NaCl (18%) stresses, respectively, compared with endophyte‐untreated seedlings. Application of PW09 also induced higher accumulation of proline (1.3‐ and 1.4‐fold) and total phenolics (1.2‐ and 1.1‐fold) and activities of polyphenol oxidase (4.3‐ and 1.5‐fold), phenylalanine ammonia lyase (1.29‐ and 1.27‐fold) and superoxide dismutase (2.5‐ and 1.39‐fold) under S. rolfsii and NaCl stresses, indicating the ability of the wheat endophyte PW09 in alleviating both biotic and abiotic stresses in cucumber.