Heat Shock Transcription Factor Research Articles

Transcriptome Differences Suggest Novel Mechanisms for Intrauterine Growth Restriction Mediated Dysfunction in Small Intestine of Neonatal Piglets.

Impaired intestinal function is frequently detected in newborns with intrauterine growth restriction (IUGR), whereas the mechanism between transcriptome profiles and small intestinal dysfunction is still unclear. Therefore, this study was conducted by using IUGR neonatal piglets to uncover the mechanism underlying intestinal dysfunction. Neonatal piglets with IUGR and normal birth weight (NBW) were sacrificed at birth. Transcriptomic sequencing was performed on jejunum samples and generated 18,997 and 17,531 genes in NBW and IUGR groups, respectively. A total of 10 differentially expressed genes (DEGs) were identified; of note, only seven were mapped to the genome reference database, with two up-regulated (HSF4 and NR1H4; heat shock transcription factor 4 and nuclear receptor subfamily 1 group H member 4, respectively) and five down-regulated (SLC35C1, BTNL3, BPI, NLRP6, and SLC5A8; Solute carrier family 35 member C1, butyrophilin like 3, bactericidal permeability increasing protein, NLR family pyrin domain containing 6, and solute carrier family 5 member 8, respectively). Combining an enrichment analysis and reverse transcriptase–quantitative polymerase chain reaction validation of DEGs, our results proved the lipid metabolism disorder, intestinal dysfunction, and inflammatory response in IUGR piglets. Here, IUGR piglets presented lower concentration of glucose and triglyceride and higher concentration of total cholesterol and low-density lipoprotein cholesterol in plasma, compared with NBW piglets. Histological analysis revealed decreased mucins and increased apoptosis in both jejunum and ileum for IUGR piglets. Collectively, we found that IUGR induced intestinal dysfunction by altering lipid metabolism, intestinal barrier, and inflammatory response in neonatal piglets at birth, which provides new insights into the prevention and treatment of IUGR that protects against metabolic disorders and inflammatory-related diseases.

Identification of Heat-Responsive Genes in Guar [Cyamopsis tetragonoloba (L.) Taub

The threat of heat stress on crop production increased dramatically due to global warming leading to the rise on the demand of heat-tolerant crops and understanding their tolerance. The leguminous forage crop Guar [Cyamopsis tetragonoloba (L.) Taub] is a high-temperature tolerant plant with numerous works on its tolerance at morph-physiological levels but lack on molecular thermotolerance level. In the current study, the differential gene expression and the underlying metabolic pathways induced by heat treatment were investigated. An RNA-Seq study on Guar leaves was carried out to estimate gene abundance and identify genes involved in heat tolerance to better understand the response mechanisms to heat stress. The results uncovered 1551 up- and 1466 downregulated genes, from which 200 and 72 genes with unknown function could be considered as new genes specific to guar. The upregulated unigenes were associated with 158 enzymes and 102 KEGG pathways. Blast2GO, InterProScan, and Kyoto Encyclopaedia of Genes and Genomes packages were utilized to search the functional annotation, protein analysis, enzymes, and metabolic pathways and revealed hormone signal transduction were enriched during heat stress tolerance. A total of 301 protein families, 551 domains, 15 repeats, and 3 sites were upregulated and matched to those unigenes. A batch of heat-regulated transcription factor transcripts were identified using the PlantTFDB database, which may play roles in heat response in Guar. Interestingly, several heat shock protein families were expressed in response to exposure to stressful conditions for instance small HSP20, heat shock transcription factor family, heat shock protein Hsp90 family, and heat shock protein 70 family. Our results revealed the expressional changes associated with heat tolerance and identified potential key genes in the regulation of this process. These results will provide a good start to dissect the molecular behaviour of plants induced by heat stress and could identify the key genes in stress response for marker-assisted selection in Guar and reveal their roles in stress adaptation in plants.

Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70‐mediated trimer unzipping and dissociation from DNA

The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of Hsf1 activity regulation remains poorly understood at the molecular level. In metazoa, Hsf1 trimerizes upon heat shock through a leucine‐zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, using purified proteins, we demonstrate that unmodified trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 binds to multiple sites in Hsf1 with different affinities. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucine‐zipper. Starting this unzipping at several protomers of the Hsf1 trimer results in faster monomerization. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation, Hsc70 first binds to a high‐affinity site in the transactivation domain, leading to partial attenuation of the response, and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state.

Heat shock transcription factor 2 predicts mucosal healing and promotes mucosal repair of ulcerative colitis

Background: Mucosal healing(MH) is a treatment goal in ulcerative colitis (UC). Our previous studies showed heat shock transcription factor 2 (HSF2) was positively correlated with the activity of UC and had anti-inflammatory potential in DSS-induced colitis, but the role of HSF2 in MH remains unknown. This study aimed to reveal the predictive value and mechanisms of HSF2 in the MH of UC.Methods: Fecal samples were collected from 51 UC patients and 10 healthy controls. Correlation analyses among HSF2, fecal calprotectin(FC) and Mayo endoscopic subscore(MES) were conducted by Pearson correlation coefficient. Diagnostic accuracy and cutoffs to predict MH were analyzed by ROC curves. 231 UC patients were enrolled to verify the diagnostic validity of the cutoffs. HSF2 siRNA and HSF2-FLAG recombinant plasmids were transfected into HT-29 cells. IL-1β, TNF-α and TGF-β levels in supernatants were determined by ELISA. The expression and phosphorylation levels of MAPKs and Smad2/3 were detected by Western blotting.Results: Positive correlations existed between HSF2 and MES (r = 0.81), FC and MES (r = 0.85), and HSF2 and FC (r = 0.91). Optimal cutoffs of HSF2 was 1.97 ng/ml (AUC 0.919) and that of FC was 678 µg/g (AUC 0.958). HSF2 and FC achieved high sensitivity (73.7% vs 84.2%) and negative predictive value (89.1% vs 93.9%). HSF2 decreased IL-1β and TNF-α secretion via suppression of MAPK signaling pathway activation. HSF2 promoted the expression of TGF-β via increasing phosphorylation of Smad2/3.Conclusions: HSF2 may be a predictor of MH in UC patients. HSF2 inhibited inflammation and promoted mucosal repair.

42-OR: The Impact of GSNOR-Mediated Nitroso-Redox Signaling on Immuno-Metabolic Interaction in the Brown Adipose Tissue

The thermogenic function of brown adipose tissue (BAT) is tightly regulated by cellular redox status, however, the molecular mechanism underlying this regulation is largely unknown. S-nitrosoglutathione reductase (GSNOR, Adh5) is a major denitrosylase that balances intracellular nitroso-redox status. Although GSNOR dysregulation has been implicated in the pathogenesis of many chronic diseases, the pathophysiological role of GSNOR in the BAT is completely unknown. We found cold exposure increased murine BAT GSNOR while diet-induced obesity (DIO) suppressed it, indicating a potential role for GSNOR in metabolic adaptation in the BAT. Mechanistically, we found that the transcription factor heat shock factor 1 (HSF1) occupies the Adh5 promoter activating Adh5 expression, while DIO inhibited the HSF1-GSNOR axis in the BAT. Notably, administration of a HSF1 enhancer to mice with DIO significantly improved glucose intolerance and BAT mitochondrial dysfunciton. Furthermore, GSNOR deletion in the BAT (GSNORBKO) suppressed UCP1-dependent respiration under both thermoneutral and cold acclimation conditions, as well as elevated immune cell infiltration in the BAT. This was in part mediated by increased BAT nitrosative stress and UCP1 S-nitrosylation induced by GSNOR deletion. Moreover, GSNORBKO mice have worsened DIO-induced glucose intolerance and decreased UCP1-mediated oxygen consumption. In contrast, gain-of GSNOR function in the BAT improved DIO-associated glucose intolerance, increased UCP1-dependent oxygen consumption and decreased DIO-induced inflammation. These data provide novel evidence that GSNOR plays a protective role in BAT against metabolic stressors. Ultimately, insights into the mechanisms by which dysregulation of nitroso-redox signaling impairs BAT metabolic homoestasis and drives the pathologies associated with obesity are expected to accelerate the development of novel therapeutic targets for metabolic diseases. Disclosure S. Sebag: None. Q. Qian: None. Z. Zhang: None. M. Li: None. V.A. Lira: None. M.J. Potthoff: None. L. Yang: None. Funding American Diabetes Association (1-18-IBS-149 to L.Y.)

Abstract A19: Modeling intrapatient pharmacotranscriptomic heterogeneity with organoids derived from colorectal cancer liver metastases

Abstract Patients with metastatic colorectal cancer (CRC) have few targeted treatment options compared with other major malignancies, and both over- and undertreatment with cytostatic drugs remain a challenge. Recent studies show that patient-derived organoids (PDOs) can predict clinical responses to systemic therapies in a personalized manner, but tumor heterogeneity may limit the clinical benefit of anticancer therapies, as well as the accuracy of preclinical predictions. PDO lineage establishment from multiple distinct lesions per patient presents a novel opportunity for preclinical investigation of intrapatient pharmacotranscriptomic heterogeneity. From September 2017 until November 2019, we have established a living biobank of 107 PDOs from 53 patients who underwent resection of CRC liver metastases at Oslo University Hospital, Norway, 31 of whom had multiple (2-5) metastases. All PDOs were screened for sensitivity to 40 anticancer agents, including clinically relevant targeted drugs and conventional chemotherapies. Molecular profiling by gene expression and mutation analyses is ongoing and currently completed for 27 PDOs. Recapitulation of known pharmacogenomic/transcriptomic associations was confirmed in PDOs for EGFR inhibition and RAS/BRAFV600E mutation status, as well as TP53-MDM2 inhibition and TP53 mutation status and TP53 transcriptional activity. Antimetabolites such as gemcitabine and methotrexate, or small-molecule inhibitors in late-stage clinical development targeting Aurora A, PLK1, and HSP90, showed strong differential activities, enabling identification of sensitive subgroups. Strong sensitivity to HSP90 inhibition was associated with low protein expression of Heat Shock Transcription Factor 1, which had a homogenous intrapatient intermetastatic expression pattern. Principal component analyses revealed a clear patient-wise separation of PDOs based on both their pharmacologic and transcriptomic profiles separately, indicating only modest intrapatient heterogeneity among distinct metastatic lesions. Accurate prediction of clinical responses to 5-FU and oxaliplatin was shown in PDOs from one patient treated for recurrent liver metastases. Additionally, PDOs from recurrent liver metastases showed an increased sensitivity to off-label drugs compared to PDOs from the first liver resection, supporting therapy repurposing in later lines of treatment. In summary, patient-derived models of CRC liver metastases reveal modest intrapatient pharmacotranscriptomic heterogeneity—an encouraging result for further efforts to develop personalized drug repurposing strategies for this poor-prognosis patient group. Citation Format: Kushtrim Kryeziu, Jarle Bruun, Peter W. Eide, Seyed H. Moosavi, Ina A. Eilertsen, Jonas Langerud, Bård Røsok, Tuva H. Brunsell, Marianne G. Guren, Andreas Abildgaard, Arild Nesbakken, Bjørn Atle Bjørnbeth, Anita Sveen, Ragnhild A. Lothe. Modeling intrapatient pharmacotranscriptomic heterogeneity with organoids derived from colorectal cancer liver metastases [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A19.

S-Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid

Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.

Genome-wide identification, transcriptome analysis and alternative splicing events of Hsf family genes in maize

Heat shock transcription factor (Hsf) plays a transcriptional regulatory role in plants during heat stress and other abiotic stresses. 31 non-redundant ZmHsf genes from maize were identified and clustered in the reference genome sequenced by Single Molecule Real Time (SMRT). The amino acid length, chromosome location, and presence of functional domains and motifs of all ZmHsfs sequences were analyzed and determined. Phylogenetics and collinearity analyses reveal gene duplication events in Hsf family and collinearity blocks shared by maize, rice and sorghum. The results of RNA-Seq analysis of anthesis and post-anthesis periods in maize show different expression patterns of ZmHsf family members. Specially, ZmHsf26 of A2 subclass and ZmHsf23 of A6 subclass were distinctly up-regulated after heat shock (HS) at post-anthesis stage. Nanopore transcriptome sequencing of maize seedlings showed that alternative splicing (AS) events occur in ZmHsf04 and ZmHsf17 which belong to subclass A2 after heat shock. Through sequence alignment, semi-quantitative and quantitative RT-PCR, we found that intron retention events occur in response to heat shock, and newly splice isoforms, ZmHsf04-II and ZmHsf17-II, were transcribed. Both new isoforms contain several premature termination codons in their introns which may lead to early termination of translation. The ZmHsf04 expression was highly increased than that of ZmHsf17, and the up-regulation of ZmHsf04-I transcription level were significantly higher than that of ZmHsf04-II after HS.

Heat shock transcription factor 2 inhibits intestinal epithelial cell apoptosis through the mitochondrial pathway in ulcerative colitis

UC is a chronic inflammatory disease of the colonic mucosa and lacks effective treatments because of unclear pathogenesis. Excessive apoptosis of IECs damages the intestinal epithelial barrier and is involved in the progression of UC, but the mechanism is unknown. HSPs are important in maintaining homeostasis and regulate apoptosis through the mitochondrial pathway. In our previous studies, HSF2, an important regulator of HSPs, was highly expressed in UC patients and negatively correlated with inflammation in mice and IECs. Therefore, we hypothesized that HSF2 may protect against intestinal mucositis by regulating the apoptosis of IECs. In this study, a DSS-induced colitis model of hsf2−/− mice was used to explore the relationship between HSF2 and apoptosis in IECs for the first time. The expression of HSF2 increased in the WT + DSS group compared with that in the WT + H2O group. Moreover, the extent of apoptosis was more severe in the KO + DSS group than in the WT + DSS group. The results showed that HSF2 was negatively correlated with apoptosis in vivo. The expression of HSF2 in Caco-2 cells was changed by lentiviral transfection, and the expression of Bax, cytoplasmic Cyto-C, Cleaved Caspase-9 and Cleaved Caspase-3 were negatively correlated with the different levels of HSF2. These results suggest that HSF2 negatively regulates apoptosis of IECs through the mitochondrial pathway. This may be one of the potential mechanisms to explain the protective role of HSF2 in UC.

AsHSP26.8a, a creeping bentgrass small heat shock protein integrates different signaling pathways to modulate plant abiotic stress response

BackgroundSmall heat shock proteins (sHSPs) are critical for plant response to biotic and abiotic stresses, especially heat stress. They have also been implicated in various aspects of plant development. However, the acting mechanisms of the sHSPs in plants, especially in perennial grass species, remain largely elusive.ResultsIn this study, AsHSP26.8a, a novel chloroplast-localized sHSP gene from creeping bentgrass (Agrostis stolonifera L.) was cloned and its role in plant response to environmental stress was studied. AsHSP26.8a encodes a protein of 26.8 kDa. Its expression was strongly induced in both leaf and root tissues by heat stress. Transgenic Arabidopsis plants overexpressing AsHSP26.8a displayed reduced tolerance to heat stress. Furthermore, overexpression of AsHSP26.8a resulted in hypersensitivity to hormone ABA and salinity stress. Global gene expression analysis revealed AsHSP26.8a-modulated expression of heat-shock transcription factor gene, and the involvement of AsHSP26.8a in ABA-dependent and -independent as well as other stress signaling pathways.ConclusionsOur results suggest that AsHSP26.8a may negatively regulate plant response to various abiotic stresses through modulating ABA and other stress signaling pathways.

Functional characterization of maize heat shock transcription factor gene ZmHsf01 in thermotolerance.

BackgroundHeat waves can critically influence maize crop yields. Plant heat shock transcription factors (HSFs) play a key regulating role in the heat shock (HS) signal transduction pathway.MethodIn this study, a homologous cloning method was used to clone HSF gene ZmHsf01 (accession number: MK888854) from young maize leaves. The transcript levels of ZmHsf01 were detected using qRT-PCR in different tissues and treated by HS, abscisic acid (ABA), hydrogen peroxide (H2O2), respectively, and the functions of gene ZmHsf01 were studied in transgenic yeast and Arabidopsis.ResultZmHsf01 had a coding sequence (CDS) of 1176 bp and encoded a protein consisting of 391 amino acids. The homologous analysis results showed that ZmHsf01 and SbHsfA2d had the highest protein sequence identities. Subcellular localization experiments confirmed that ZmHsf01 was localized in the nucleus. ZmHsf01 was expressed in many maize tissues. It was up-regulated by HS, and up-regulated in roots and down-regulated in leaves under ABA and H2O2treatments. ZmHsf01-overexpressing yeast cells showed increased thermotolerance. In Arabidopsis seedlings, ZmHsf01 compensated for the thermotolerance defects of mutant athsfa2, and ZmHsf01-overexpressing lines showed enhanced basal and acquired thermotolerance. When compared to wild type (WT) seedlings, ZmHsf01-overexpressing lines showed higher chlorophyll content and survival rates after HS. Heat shock protein (HSP) gene expression levels were more up-regulated in ZmHsf01-overexpressing Arabidopsis seedlings than WT seedlings. These results suggest that ZmHsf01 plays a vital role in response to HS in plant.

Pre-harvest climate and post-harvest acclimation to cold prevent from superficial scald development in Granny Smith apples

Superficial scald is one of the most serious postharvest physiological disorders that can affect apples after a prolonged cold storage period. This study investigated the impact of pre- and post-harvest climatic variations on superficial scald in a susceptible apple cultivar. Fruit batches with contrasting phenotypes for superficial scald incidence were identified among several years of “Granny Smith” fruit production. The “low scald” year pre-harvest climate was characterised by a warm period followed by a sudden decrease in temperature, playing the part of an in vivo acclimation to cold storage. This was associated with many abiotic stress responsive genes which were induced in fruit peel. In particular 48 Heat Shock Proteins (HSPs) and 5 Heat Shock transcription Factors (HSFs) were strongly induced at harvest when scald incidence was low. For “high scald” year, a post-harvest acclimation of 1 week was efficient in reducing scald incidence. Expression profiles of stress related genes were affected by the acclimation treatment and indicate fruit physiological adaptations to cold storage. The identified stress-responsive genes, and in particular HSPs, could be useful indicators of the fruit physiological status to predict the risk of scald occurrence as early as harvest.

Protein level variability determines phenotypic heterogeneity in proteotoxic stress response.

Cell-to-cell variability in stress response is a bottleneck for the construction of accurate and predictive models which could guide clinical diagnosis and treatment of certain diseases, for example, cancer. Indeed, such phenotypic heterogeneity can lead to fractional killing and persistence of a subpopulation of cells which are resistant to a given treatment. The heat shock response network plays a major role in protecting the proteome against several types of injuries. Here, we combine high-throughput measurements and mathematical modeling to unveil the molecular origin of the phenotypic variability in the heat shock response network. Although the mean response coincides with known biochemical measurements, we found a surprisingly broad diversity in single-cell dynamics with a continuum of response amplitudes and temporal shapes for several stimulus strengths. We theoretically predict that the broad phenotypic heterogeneity is due to network ultrasensitivity together with variations in the expression level of chaperones controlled by the transcription factor heat shock factor 1. Furthermore, we experimentally confirm this prediction by mapping the response amplitude to chaperone and heat shock factor 1 expression levels.

Endogenous siRNAs promote proteostasis and longevity in germline-less Caenorhabditis elegans.

How lifespan and the rate of aging are set is a key problem in biology. Small RNAs are conserved molecules that impact diverse biological processes through the control of gene expression. However, in contrast to miRNAs, the role of endo-siRNAs in aging remains unexplored. Here, by combining deep sequencing and genomic and genetic approaches in Caenorhabditis elegans, we reveal an unprecedented role for endo-siRNA molecules in the maintenance of proteostasis and lifespan extension in germline-less animals. Furthermore, we identify an endo-siRNA-regulated tyrosine phosphatase, which limits the longevity of germline-less animals by restricting the activity of the heat shock transcription factor HSF-1. Altogether, our findings point to endo-siRNAs as a link between germline removal and the HSF-1 proteostasis and longevity-promoting somatic pathway. This establishes a role for endo siRNAs in the aging process and identifies downstream genes and physiological processes that are regulated by the endo siRNAs to affect longevity.

Transcriptomic profiling of Solanum peruvianum LA3858 revealed a Mi-3-mediated hypersensitive response to Meloidogyne incognita

BackgroundThe Mi-1 gene was the first identified and cloned gene that provides resistance to root-knot nematodes (RKNs) in cultivated tomato. However, owing to its temperature sensitivity, this gene does not meet the need for breeding disease-resistant plants that grow under high temperature. In this study, Mi-3 was isolated from the wild species PI 126443 (LA3858) and was shown to display heat-stable resistance to RKNs. However, the mechanism that regulates this resistance remains unknown.ResultsIn this study, 4760, 1024 and 137 differentially expressed genes (DEGs) were enriched on the basis of pairwise comparisons (34 °C vs. 25 °C) at 0 (before inoculation), 3 and 6 days post-inoculation (dpi), respectively. A total of 7035 DEGs were identified from line LA3858 in the respective groups under the different soil temperature treatments. At 3 dpi, most DEGs were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to plant biotic responses, such as “plant-pathogen interaction” and “plant hormone signal transduction”. Significantly enriched DEGs were found to encode key proteins such as R proteins and heat-shock proteins (HSPs). Moreover, other DEGs were found to participate in Ca2+ signal transduction; the production of ROS; DEGs encoding transcription factors (TFs) from the bHLH, TGA, ERF, heat-shock transcription factor (HSF) and WRKY families were highly expressed, which contribute to be involved into the formation of phytohormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), the expression of most was upregulated at 3 dpi at the 25 °C soil temperature compared with the 34 °C soil temperature.ConclusionTaken together, the results of our study revealed reliable candidate genes from wild materials LA3858, that are related to Mi-3-mediate resistance to Meloidogyne incognita. A large number of vital pathways and DEGs were expressed specifically in accession LA3858 grown at 34 °C and 25 °C soil temperatures at 3 dpi. Upon infection by RKNs, pattern-recognition receptors (PRRs) specifically recognized conserved pathogen-associated molecular patterns (PAMPs) as a result of pathogen-triggered immunity (PTI), and the downstream defensive signal transduction pathway was likely activated through Ca2+ signal channels. The expression of various TFs was induced to synthesize phytohormones and activate R proteins related to resistance, resulting in the development of effector-triggered immunity (ETI). Last, a hypersensitive response in the roots occurred, which was probably induced by the accumulation of ROS.

Effects of thermal manipulation of eggs on the response of jejunal mucosae to posthatch chronic heat stress in broiler chickens

In this study, the aim was to investigate effects of chronic heat stress (CHS) on the mRNA levels of proinflammatory cytokines (interleukin [IL]-6, IL-8, IL-1β, and tumor necrosis factor alpha [TNF-α]), toll-like receptors (TLR2 and TLR4), heat shock proteins (Hsp70, heat shock transcription factor [HSF]-1, and HSF3) and antioxidant enzymes (catalase, glutathione peroxidase, NADPH oxidase, and superoxide-dismutase) in the jejunal mucosae of broiler chickens subjected to thermal manipulation (TM) during embryogenesis. TM was carried out at 39°C and 65% relative humidity (RH) for 18 h daily from embryonic days 10 to 18. Control group was incubated at 37.8°C and 56% RH. CHS was induced by raising the temperature to 35°C for 7 D throughout posthatch days 28 to 35. On post-hatch-day 28 (day zero of CHS) and after 1, 3, 5, and 7 D of CHS, the jejunal mucosae were collected from both groups to evaluate the mRNA levels by real-time reverse transcription-PCR analysis. On day zero of CHS, the mRNA levels of antioxidant enzymes, TLRs, HSF3, IL-1β, and TNF-α were not significantly different between TM and control groups, while the levels of IL-6, IL-8, and HSF1 were lower and the level of Hsp70 was higher in TM. However, during CHS, the mRNA levels of antioxidant enzymes, IL-1β, TNF-α, TLR4, and HSF1 were significantly lower in TM than in controls, while the levels of TLR2 and IL-8 were significantly higher in TM than in controls. In addition, TM led to significant increase of mRNA levels of IL-6 and HSF3 after 1 D and Hsp70 after 3 D of CHS and to significant decrease of mRNA levels of IL-6 after 3 and 5 D, HSF3 after 7 D, and Hsp70 after 5 D of CHS. Results of this study suggest that TM led to altered posthatch antioxidant, immunological, and Hsp response to CHS in the jejunal mucosae of broiler chickens, probably indicating that TM may mitigate the adverse effects of CHS.

New Insights into Evolution of Plant Heat Shock Factors (Hsfs) and Expression Analysis of Tea Genes in Response to Abiotic Stresses.

Heat shock transcription factor (Hsf) is one of key regulators in plant abotic stress response. Although the Hsf gene family has been identified from several plant species, original and evolution relationship have been fragmented. In addition, tea, an important crop, genome sequences have been completed and function of the Hsf family genes in response to abiotic stresses was not illuminated. In this study, a total of 4208 Hsf proteins were identified within 163 plant species from green algae (Gonium pectorale) to angiosperm (monocots and dicots), which were distributed unevenly into each of plant species tested. The result indicated that Hsf originated during the early evolutionary history of chlorophytae algae and genome-wide genetic varies had occurred during the course of evolution in plant species. Phylogenetic classification of Hsf genes from the representative nine plant species into ten subfamilies, each of which contained members from different plant species, imply that gene duplication had occurred during the course of evolution. In addition, based on RNA-seq data, the member of the Hsfs showed different expression levels in the different organs and at the different developmental stages in tea. Expression patterns also showed clear differences among Camellia species, indicating that regulation of Hsf genes expression varied between organs in a species-specific manner. Furthermore, expression of most Hsfs in response to drought, cold and salt stresses, imply a possible positive regulatory role under abiotic stresses. Expression profiles of nineteen Hsf genes in response to heat stress were also analyzed by quantitative real-time RT-PCR. Several stress-responsive Hsf genes were highly regulated by heat stress treatment. In conclusion, these results lay a solid foundation for us to elucidate the evolutionary origin of plant Hsfs and Hsf functions in tea response to abiotic stresses in the future.

SaHsfA4c From Sedum alfredii Hance Enhances Cadmium Tolerance by Regulating ROS-Scavenger Activities and Heat Shock Proteins Expression.

The heat shock transcription factor (Hsf) family, an important member in plant stress response, affects cadmium (Cd) tolerance in plants. In this study, we identified and functionally characterized a transcript of the Hsf A4 subgroup from Sedum alfredii. Designated as SaHsfA4c, the open reading frame was 1,302 bp long and encoded a putative protein of 433 amino acids containing a complete DNA-binding domain (DBD). Heterologous expression of SaHsfA4c in yeast enhanced Cd stress tolerance and accumulation, whereas expression of the alternatively spliced transcript InSaHsfA4c which contained an intron and harbored an incomplete DBD, resulted in relatively poor Cd stress tolerance and low Cd accumulation in transgenic yeast. The function of SaHsfA4c under Cd stress was characterized in transgenic Arabidopsis and non-hyperaccumulation ecotype S. alfredii. SaHsfA4c was able to rescue the Cd sensitivity of the Arabidopsis athsfa4c mutant. SaHsfA4c reduced reactive oxygen species (ROS) accumulation and increased the expression of ROS-scavenging enzyme genes and Hsps in transgenic lines. The present results suggest that SaHsfA4c increases plant resistance to stress by up-regulating the activities of ROS-scavenging enzyme and the expression of Hsps.

Identification of the nucleotide exchange factor BmGrpE and its role under high-temperature stress in silkworm, Bombyx mori.

The high-temperature stress gene GrpE plays an important role in coping with high-temperature stress. The mutation of key sites of this gene can improve the high-temperature resistance of organisms. In the present study, using complementary DNAs from the silkworm fat body as the template, the open reading frame sequence of the GrpE gene (BmGrpE) was amplified and was found to be 644 bp in length and encode a protein with a predicted molecular weight of 24.1 kDa. The presence of a binding site for the heat shock transcription factor (Hsf1) at -1440 bp upstream of its coding region indicates that BmGrpE may respond to high-temperature stress. BmGrpE was constitutively expressed throughout developmental stages, with the highest level observed in the 5th instar larvae stage. Moreover, in 5th instar larvae (the 3th day), BmGrpE was expressed in all tissues examined, with the highest levels in the fat body, silk gland, and midgut. Interestingly, under high-temperature stress, TiO2 nanoparticle treatment increased the messenger RNA levels of BmGrpE in the fat body and silk gland. After treatment with dsRNA of BmGrpE, the cell viability of BmN cells was significantly decreased under 34°C and H2 O2 stress (p < .05). Mutation of BmGrpE (H163L) enhanced the resistance of BmN cells under high-temperature stress. These results provide new clues for the study of molecular mechanisms of insect resistance to high temperatures.

TaHsfA2-1, a new gene for thermotolerance in wheat seedlings: Characterization and functional roles

Heat shock transcription factors (Hsfs) play an important role in regulating heat stress response in plants. Our previous study found that there were 82 non-redundant Hsfs in wheat, 18 of which belonged to subclass A2. In this study, we cloned an A2 member, TaHsfA2-1, which encoded a protein of 346 amino acid residues in wheat. The fusion protein TaHsfA2-1-GFP was localized in the nucleus under normal growth conditions. TaHsfA2-1 was expressed in nearly all the measured tissues, most highly in mature leaves. The expression level of TaHsfA2-1 can be enhanced by heat stress, PEG stress, and signal molecules such as H2O2 and SA. Yeast cells transformed with TaHsfA2-1 improved thermotolerance compared to those with the empty vector. TaHsfA2-1-overexpressing Arabidopsis displayed a better growth state with more green leaves than wild-type seedlings after heat stress. Accordingly, the chlorophyll content and survival rate in the transgenic lines were higher than in the wild type, and relative conductivity in the transgenic lines was lower than in the wild type. Further research found that TaHsfA2-1-overexpressing Arabidopsis up-regulated the expression of some heat shock protein genes (Hsps) compared to wild type after heat stress. These results suggested that TaHsfA2-1 is a new gene that improves thermotolerance in plants by mediating the expression of Hsps. A functional gene was provided for molecular breeding in the subsequent research.