Heat-induced Expression Research Articles

Combination of magnetic hyperthermia and gene therapy for breast cancer.

This study presented a novel breast cancer therapy model that uses magnetic field-controlled heating to trigger gene expression in cancer cells. We created silica- and amine-modified superparamagnetic nanoparticles (MSNP-NH2) to carry genes and release heat under an alternating current (AC) magnetic field. The heat-inducible expression plasmid(pHSP-Azu) was designed to encode anti-cancer azurin and was delivered by magnetofection. MCF-7 cells demonstrated over 93% cell viability and 12% transfection efficiency when exposed to 75µg/ml of MSNP-NH2, 3µg of DNA, and PEI at a 0.75 PEI/DNA ratio (w: w), unlike non-tumorigenic cells (MCF-10A). Magnetic hyperthermia (MHT) increased azurin expression by heat induction, leading to cell death in dual ways. The combination of MHT and heat-regulated azurin expression induced cell death, specifically in cancer cells, while having negligible effects on MCF-10A cells. The proposed strategy clearly shows that simultaneous use of MHT and MHT-induced azurin gene expression may selectively target and kill cancer cells, offering a promising direction for cancer therapy.

Distinct induction pathways of heat shock protein 27 in human keratinocytes: Heat stimulation or capsaicin through phosphorylation of heat shock factor 1 at serine 326 and/or suppression of ΔNp63

Epidermal keratinocytes, forming the outermost layer of the human body, serve as a crucial barrier against diverse external stressors such as ultraviolet radiation. Proper keratinocyte differentiation and effective responses to external stimuli are pivotal for maintaining barrier integrity. Heat is one such stimulus that triggers the synthesis of heat shock proteins (HSPs) when cells are exposed to temperatures above 42 °C. Additionally, activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) occurs at 42 °C. Here, we explore the interplay between TRPV1 signaling and HSP induction in human keratinocytes. Both heat and capsaicin, a TRPV1 agonist, induce expression of HSP27, HSP70, and HSP90 in keratinocytes. Interestingly, pharmacological inhibition of TRPV1 attenuates heat-induced HSP27 expression, but not that of HSP70 or HSP90. Furthermore, both heat and capsaicin stimulation result in distinct phosphorylation patterns of heat shock factor 1 (HSF1), with phosphorylation at serine 326 being a common feature. Notably, genetic manipulation to mimic dephosphorylation of HSF1 at serine 326 reduces HSP27 levels. Additionally, ΔNp63, a key regulator of epidermal differentiation, negatively modulates HSP27 expression independently of HSF1 phosphorylation status. While heat stimulation has no effect on ΔNp63 expression, capsaicin reduces its levels. The precise role of TRPV1 signaling in keratinocytes warrants further investigation for a comprehensive understanding of its impact on barrier function.

NEAR INFRARED-RESPONSIVE HYDROGELS CONTAINING ADENOVIRAL VECTORS: APPLICATION IN BONE REGENERATION

We have developed plasmonic fibrin-based hydrogels that incorporate gold nanoparticles which transduce incident near-infrared (NIR) light into heat. Human adenovirus serotype type-5 vectors encoding a firefly luciferase (fLuc) coding sequence driven by a heat-inducible promoter were incorporated into the hydrogels. Transmission electronic microscopic analysis revealed that the adenoviral vectors were associated to the fibrin fibers. In vitro experiments in which human cells were cultured with plasmonic hydrogels showed that the adenoviral vectors can diffuse from the hydrogels, transduce the cells, and stimulate heat-induced transgene expression upon NIR irradiation. The hydrogels were implanted in 4.2 mm drill hole defects generated in the humerus of male rabbits. Three days after implantation, the defects were NIR-irradiated. Six h later, the animals were euthanized and samples from the bone defect zone were processed for immunohistochemical analyses using a specific fLuc antibody. The results showed strong expression of fLuc in tissues surrounding the implants of NIR-irradiated rabbits, while non- irradiated animals exhibited negligible expression. We next aimed to use the temperature increase to induce the production of transgenic bone morphogenetic protein 6 (BMP-6), using safe gene switches that can provide tighter control of in vivo transgene expression than heat-inducible promoters. These switches are only activated by heat in the presence of rapamycin and maintain a high level of targeted transgene expression for several days after heat activation. Adenoviral vectors encoding the safe switches that control the expression of BMP-6 were incorporated to the composites. The resulting NIR-responsive hydrogels were implanted in the bone defects generated in rabbits and used as a platform to transduce host cells, generate local hyperthermia and stimulate BMP-6 production.Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033).

A heat-inducible expression system for external control of gene expression in plastids.

Inducible expression systems can overcome the trade-off between high-level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature-inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature-sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light-dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature-dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pRpromoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat-inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost-effective and does not depend on chemical inducers.

Genome-wide identification and expression analysis reveal the role of histone methyltransferase and demethylase genes in heat stress response in potato (Solanum tuberosum L.)

BackgroundPotato (Solanum tuberosum L.), the third most important non-cereal crop, is sensitive to high temperature. Histone modifications have been known to regulate various abiotic stress responses. However, the role of histone methyltransferases and demethylases remain unexplored in potato under heat stress. MethodsPotato genome database was used for genome-wide analysis of StPRMT and StHDMA gene families, which were further characterized by analyzing gene structure, conserved motif, domain organization, sub-cellular localization, promoter region and phylogenetic relationships. Additionally, expression profiling under high-temperature stress in leaf and stolon tissue of heat contrasting potato genotypes was done to study their role in response to high temperature stress. ResultsThe genome-wide analysis led to identification of nine StPRMT and eleven StHDMA genes. Structural analysis, including conserved motifs, exon/intron structure and phylogenetic relationships classified StPRMT and StHDMA gene families into two classes viz. Class I and Class II. A variety of cis-regulatory elements were explored in the promoter region associated with light, developmental, hormonal and stress responses. Prediction of sub-cellular localization of StPRMT proteins revealed their occurrence in nucleus and cytoplasm, whereas StHDMA proteins were observed in different sub-cellular compartments. Furthermore, expression profiling of StPRMT and StHDMA gene family members revealed genes responding to heat stress. Heat-inducible expression of StPRMT1, StPRMT3, StPRMT4 and StPRMT5 in leaf and stolon tissues of HS and HT cultivar indicated them as probable candidates for enhancing thermotolerance in potato. However, StHDMAs responded dynamically in leaf and stolon tissue of heat contrasting genotypes under high temperature. ConclusionThe current study presents a detailed analysis of histone modifiers in potato and indicates their role as an important epigenetic regulators modulating heat tolerance. General significanceUnderstanding epigenetic mechanisms underlying heat tolerance in potato will contribute towards breeding of thermotolerant potato varieties.

GSDME has prognostic and immunotherapeutic significance in residual hepatocellular carcinoma after insufficient radiofrequency ablation

BackgroundHeat stress can induce programmed cell death (PCD). Pyroptosis is a gasdermin-mediated PCD. This study hypothesized that insufficient radiofrequency ablation (IRFA) induced pyroptosis in hepatocellular carcinoma (HCC) and investigated its underlying mechanism and clinical significance. MethodsThermostatic water bath was used to stimulate IRFA in vitro. Cell viability was assessed by MTT assay. IL-1β and HMGB1 were measured by ELISA assay. LDH level was measured by LDH cytotoxicity detection kit. Permeability of cell membrane was assessed by Hoechst33342/PI fluorescence staining. RNA expression was evaluated by qRT-PCR, and protein was assessed by Western Blotting or immunofluorescence or immunohistochemistry. Gene expression with clinicopathological characteristics from HCC patients treated by RFA were analyzed for associations between GSDME expression and prognosis. ResultsOur study revealed that IRFA induced pyroptosis in HCCLM3 and HepG2 cells. GSDME, rather than GSDMD, was cleaved in heat stress-induced pyroptosis in HCCLM3 and HepG2 cells due to caspase-3 activation. However, GSDME overexpression promoted HCC growth in vivo and predicted poor PFS and OS in HCC patients treated by RFA. Heat stress modulated gene expression related to PD-L1 signaling and caspase inhibitors inhibited heat-induced PD-L1 expression in residual HCC after IRFA. Gsdme overexpression caused resistance to PD-L1 inhibitor in residual HCC after IRFA by increasing infiltrating of CD3+PD-1+ or CD3+CTLA-4+ exhausted T cells. ConclusionsThis study indicated that GSDME could serve as a potential prognostic biomarker and help to prescribe personalized sequential immunotherapy for HCC patients receiving RFA.

The ability to induce heat shock transcription factor-regulated genes in response to lethal heat stress is associated with thermotolerance in tomato cultivars.

Heat stress is a severe challenge for plant production, and the use of thermotolerant cultivars is critical to ensure stable production in high-temperature-prone environments. However, the selection of thermotolerant cultivars is difficult due to the complex nature of heat stress and the time and space needed for evaluation. In this study, we characterized genome-wide differences in gene expression between thermotolerant and thermosensitive tomato cultivars and examined the possibility of selecting gene expression markers to estimate thermotolerance among different tomato cultivars. We selected one thermotolerant and one thermosensitive cultivar based on physiological evaluations and compared heat-responsive gene expression in these cultivars under stepwise heat stress and acute heat shock conditions. Transcriptomic analyses reveled that two heat-inducible gene expression pathways, controlled by the heat shock element (HSE) and the evening element (EE), respectively, presented different responses depending on heat stress conditions. HSE-regulated gene expression was induced under both conditions, while EE-regulated gene expression was only induced under gradual heat stress conditions in both cultivars. Furthermore, HSE-regulated genes showed higher expression in the thermotolerant cultivar than the sensitive cultivar under acute heat shock conditions. Then, candidate expression biomarker genes were selected based on the transcriptome data, and the usefulness of these candidate genes was validated in five cultivars. This study shows that the thermotolerance of tomato is correlated with its ability to maintain the heat shock response (HSR) under acute severe heat shock conditions. Furthermore, it raises the possibility that the robustness of the HSR under severe heat stress can be used as an indicator to evaluate the thermotolerance of crop cultivars.

Membrane Fluidization Governs the Coordinated Heat-Inducible Expression of Nucleus- and Plastid Genome-Encoded Heat Shock Protein 70 Genes in the Marine Red Alga Neopyropia yezoensis.

Heat shock protein 70 (HSP70) is an evolutionarily conserved protein chaperone in prokaryotic and eukaryotic organisms. This family is involved in the maintenance of physiological homeostasis by ensuring the proper folding and refolding of proteins. The HSP70 family in terrestrial plants can be divided into cytoplasm, endoplasmic reticulum (ER)-, mitochondrion (MT)-, and chloroplast (CP)-localized HSP70 subfamilies. In the marine red alga Neopyropia yezoensis, the heat-inducible expression of two cytoplasmic HSP70 genes has been characterized; however, little is known about the presence of other HSP70 subfamilies and their expression profiles under heat stress conditions. Here, we identified genes encoding one MT and two ER HSP70 proteins and confirmed their heat-inducible expression at 25 °C. In addition, we determined that membrane fluidization directs gene expression for the ER-, MT-, and CP-localized HSP70 proteins as with cytoplasmic HSP70s. The gene for the CP-localized HSP70 is carried by the chloroplast genome; thus, our results indicate that membrane fluidization is a trigger for the coordinated heat-driven induction of HSP70 genes harbored by the nuclear and plastid genomes in N. yezoensis. We propose this mechanism as a unique regulatory system common in the Bangiales, in which the CP-localized HSP70 is usually encoded in the chloroplast genome.

Population divergence in heat and drought responses of a coastal plant: from metabolic phenotypes to plant morphology and growth.

Studying intraspecific variation in multistress responses is central for predicting and managing the population dynamics of wild plant species under rapid global change. Yet, it remains a challenging goal in this field to integrate knowledge on the complex biochemical underpinnings for the targeted 'non-model' species. Here, we studied divergence in combined drought and heat responses among Northern and Southern European populations of the dune plant Cakile maritima, by combining comprehensive plant phenotyping with metabolic profiling via FT-ICR-MS and UPLC-TQ-MS/MS. We observed pronounced constitutive divergence in growth phenology, leaf functional traits, and defence chemistry (glucosinolates and alkaloids) among population origins. Most importantly, the magnitude of growth reduction under drought was partly weaker in southern plants and associated with divergence in plastic growth responses (leaf abscission) and the modulation of primary and specialized metabolites with known central functions not only in plant abiotic but also in biotic stress responses. Our study indicates that divergent selection has shaped the constitutive and drought-/heat-induced expression of numerous morphological and biochemical functional traits to mediate higher abiotic stress resistance in southern Cakile populations, and highlights that metabolomics can be a powerful tool to explore the underlying mechanisms of local adaptation in 'non-model' species.

Remote control of transgene expression using noninvasive near-infrared irradiation.

This study investigated whether noninvasive near-infrared (NIR) energy could be transduced into heat in deep-seated organs in which adenovirus type-5 vectors tend to accumulate, thereby activating heat shock protein (HSP) promoter-mediated transgene expression, without local administration of photothermal agents. NIR irradiation of the subdiaphragmatic and left dorsocranial part of the abdominal cavity of adult immunocompetent C3H/HeNRj mice with an 808-nm laser effectively increased the temperature of the irradiated regions of the liver and spleen, respectively, resulting in the accumulation of the heat-inducible HSP70 protein. Spatial control of transgene expression was achieved in the NIR-irradiated regions of the mice administered an adenoviral vector carrying a firefly luciferase (fLuc) coding sequence controlled by a human HSP70B promoter, as assessed by bioluminescence and immunohistochemistry analyses. Levels of reporter gene expression were modulated by controlling NIR power density. Spatial control of transgene expression through NIR-focused activation of the HSP70B promoter, as well as temporal regulation by administering rapamycin was achieved in the spleens of mice inoculated with an adenoviral vector encoding a rapamycin-dependent transactivator driven by the HSP70B promoter and an adenoviral vector carrying a fLuc coding sequence controlled by the rapamycin-activated transactivator. Mice that were administered rapamycin and exposed to NIR light expressed fLuc activity in the splenic region, whereas no activity was detected in mice that were only administered rapamycin or vehicle or only NIR-irradiated. Thus, in the absence of any exogenously supplied photothermal material, remote control of heat-induced transgene expression can be achieved in the liver and spleen by means of noninvasive NIR irradiation.

Expression of Heat Shock Genes in Hepatocytes is Affected by Age and Food Restriction in Rats

The objective of this study was to determine how food restriction altered the age-related changes in the basal and heat-induced expression of heat shock genes (hsc70, grp78, and ubiquitin) by hepatocytes isolated from young (4 to 6 mo old) and old (26 to 28 mo old) male Fischer 344 rats. The basal levels of the mRNA transcripts for hsc70 and ubiquitin were similar for hepatocytes isolated from young and old rats fed with free access to the diet (control) or from rats fed a restricted diet (40% restriction of food intake). However, the induction of the mRNA transcripts for hsc70 and ubiquitin by a heat shock (42.5°C for 30 min) was significantly higher for hepatocytes isolated from old rats fed the restricted diet compared with old rats fed with free access to the diet. The changes in hsc70 mRNA levels were paralleled by similar changes in hsc70 transcription by isolated nuclei; therefore, the greater induction of hsc70 expression by food restriction results from changes in the transcription of hsc70 gene. In contrast with hsc70 and ubiquitin, the basal levels of grp78 were reduced with age and by food restriction. Therefore, the effect of aging and food restriction on the basal and heat-induced expression of heat shock genes varies considerably from gene to gene.

Lipid extract derived from newly isolated Rhodotorula toruloides LAB-07 for cosmetic applications

Rhodotorula toruloides is a non-conventional yeast with a natural carotenoid pathway. In particular, R. toruloides is an oleaginous yeast that can accumulate lipids in high content, thereby gaining interest as a promising industrial host. In this study, we isolated and taxonomically identified a new R. toruloides LAB-07 strain. De novo genome assembly using PacBio and Illumina hybrid platforms yielded 27 contigs with a 20.78 Mb genome size. Subsequent genome annotation analysis based on RNA-seq predicted 5296 protein-coding genes, including the fatty acid production pathway. We compared lipid production under different media; it was highest in the yeast extract salt medium with glycerol as a carbon source. Polyunsaturated α-linolenic acid was detected among the fatty acids, and docking phosphatidylcholine as a substrate to modeled Fad2, which annotated as Δ12-fatty acid desaturase showed bifunctional Δ12, 15-desaturation is structurally possible in that the distances between the diiron center and the carbon-carbon bond in which desaturation occurs were similar to those of structurally identified mouse stearoyl-CoA desaturase. Finally, the applicability of the extracted total lipid fraction of R. toruloides was investigated, demonstrating an increase in filaggrin expression and suppression of heat-induced MMP-1 expression when applied to keratinocytes, along with the additional antioxidant activity. This work presents a new R. toruloides LAB-07 strain with genomic and lipidomic data, which would help understand the physiology of R. toruloides. Also, the various skin-related effect of R. toruloides lipid extract indicates its potential usage as a promising cosmetic ingredient.

Positive selection and heat-response transcriptomes reveal adaptive features of the Brassicaceae desert model, Anastatica hierochuntica.

Plant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat-tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts. We generated an A. hierochuntica reference transcriptome and identified extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat, and detecting positively selected genes in A. hierochuntica. The two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat 'stress-ready' state. Furthermore, the A. hierochuntica global transcriptome as well as heat-responsive orthologs, display a lower basal and higher heat-induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV-B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod-insensitive, early-flowering phenotype. We suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.

Genome-wide identification of PEBP gene family members in potato, their phylogenetic relationships, and expression patterns under heat stress

The phosphatidylethanolamine-binding protein (PEBP) gene family is involved in regulating many plant traits. Genome-wide identification of PEPB members and knowledge of their responses to heat stress may assist genetic improvement of potato (Solanum tuberosum). We identified PEBP gene familymembers from both the recently-updated, long-reads-based reference genome (DM v6.1) and the previous short-reads-based annotation (PGSC DM v3.4) of the potato reference genome and characterized their heat-induced gene expression using RT-PCR and RNA-Seq. Fifteen PEBP familygenes were identified from DM v6.1 and named as StPEBP1 to StPEBP15 based on their locations on 6 chromosomes and were classified into FT, TFL, MFT, and PEBP-like subfamilies. Most of the StPEBP genes were found to have conserved motifs 1 to 5. Tandem or segmental duplications were found between StPEBP genes in seven pairs. Heat stress induced opposite expression patterns of certain FT and TFL members but involving different members in leaves, roots and tubers. The long-reads-based genome assembly and annotation provides a better genomic resource for identification of PEBP familygenes. Heat stress tends to decrease FT gene activities but increases TFL gene activities, but this opposite expression involves different FT/TFL pairs in leaves, roots, and tubers. This tissue-specific expression pattern of PEBP members may partly explain why different potato organs differ in their sensitivities to heat stress. Our study provides candidate PEBP familygenes and relevant information for genetic improvement of heat tolerance in potato and may help understand heat-induced responses in other plants.

A MITE variation-associated heat-inducible isoform of a heat-shock factor confers heat tolerance through regulation of JASMONATE ZIM-DOMAIN genes in rice.

High temperatures cause huge yield losses in rice. Heat-shock factors (Hsfs) are key transcription factors which regulate the expression of heat stress-responsive genes, but natural variation in and functional characterization of Hsfs have seldom been reported. A significant heat response locus was detected via a genome-wide association study (GWAS) using green leaf area as an indicative trait. A miniature inverted-repeat transposable element (MITE) in the promoter of a candidate gene, HTG3 (heat-tolerance gene on chromosome 3), was found to be significantly associated with heat-induced expression of HTG3 and heat tolerance (HT). The MITE-absent variant has been selected in heat-prone rice-growing regions. HTG3a is an alternatively spliced isoform encoding a functional Hsf, and experiments using overexpression and knockout rice lines showed that HTG3a positively regulates HT at both vegetative and reproductive stages. The HTG3-regulated genes were enriched for heat shock proteins and jasmonic acid signaling. Two heat-responsive JASMONATE ZIM-DOMAIN (JAZ) genes were confirmed to be directly upregulated by HTG3a, and one of them, OsJAZ9, positively regulates HT. We conclude that HTG3 plays an important role in HT through the regulation of JAZs and other heat-responsive genes. The MITE-absent allele may be valuable for HT breeding in rice.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Evaluation of the effects of temperature, light, and UV-C radiation on HSP70A expression in Chlamydomonas reinhardtii

In this study, various physical parameters (temperature, light intensity and UV-C radiation) which could be effective in heat shock response on C.reinhardtii by using molecular tools were investigated. In total, 256 transformants were obtained, among them, 160 transformants had continuous expression while 96 of them had heat-inducible expression. In these transformants, arylsulfatase activities were detected qualitatively and quantitatively. The best two transformants were selected and used in studies. To determine the effect of temperature, the cells were shifted from 23 °C to 35 °C, 37 °C, 40 °C and 42 °C. The heat shock response was induced at all temperatures. In investigating the effect of light intensity, 0, 14, 28, 70, 140 µmol E.m-2s-1 were used. It was found that the light intensity of 28 µmol E.m-2s-1 and above increased ARS activity. On the other hand, ultraviolet C radiation application was carried out for periods of 2, 6 and 12 h, and no significant change in ARS activity was observed. In order to compare the selected arylsulfatase activity results in the study, real-time polymerase chain reaction trials were conducted at the transcript level, and parallel results were obtained. As a result of the study, it was determined that the heat shock response was triggered by temperature and light intensity. These might be also important for plant stress and ecological studies.

Analysis of proline accumulation, antioxidant capacity and HSP expression in mutant rice lines with different heat tolerance

Three mutant rice (Oryza sativa L.) lines (AG1, AG2 and AG3) were selected as heat tolerant mutants from a gamma-ray-irradiated population of a heat-susceptible line (AG), based on their floret fertility grown under high temperatures. They were subjected to heat stress treatment (45°C, 22 hrs) at the 5-leaf stage, together with a heat-tolerant cultivar N22 and AG. Analysis of seedling root growth by WinRHIZO scanning revealed that N22 and AG3 were more heat-tolerant than the other lines (AG being the most heat susceptible). Following heat stress, a significantly higher level of oxidative damage, as indicated by TBARS, was observed in AG than in N22 and AG3. The proline accumulation was significantly higher in N22 and AG3（12- to 13.5- fold）than AG (2.5- fold). Similarly, significantly greater increases of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity were observed in N22 and AG3 than in AG. The expression of four heat shock proteins was also investigated using qPCR: OsHSP16.9, OsHSP80.3 and OsHSP100.9were induced by heat stress to various levels while OsHSP72.6was down-regulated in all tested lines. The heat-induced expression of OsHSP16.9 and OsHSP100.9 in N22 and AG3 was about twice that of AG. Higher proline accumulation and expression of the three HSP genes, as well as elevation ofT-AOC and SOD activity were observed in the heat-tolerant N22 and the mutant line AG3 under heat stress

Genome-wide identification, evolutionary estimation and functional characterization of two cotton CKI gene types

BackgroundCasein kinase I (CKI) is a kind of serine/threonine protein kinase highly conserved in plants and animals. Although molecular function of individual member of CKI family has been investigated in Arabidopsis, little is known about their evolution and functions in Gossypium.ResultsIn this study, five cotton species were applied to study CKI gene family in cotton, twenty-two species were applied to trace the origin and divergence of CKI genes. Four important insights were gained: (i) the cotton CKI genes were classified into two types based on their structural characteristics; (ii) two types of CKI genes expanded with tetraploid event in cotton; (iii) two types of CKI genes likely diverged about 1.5 billion years ago when red and green algae diverged; (iv) two types of cotton CKI genes which highly expressed in leaves showed stronger response to photoperiod (circadian clock) and light signal, and most two types of CKI genes highly expressed in anther showed identical heat inducible expression during anther development in tetraploid cotton (Gossypium hirsutum).ConclusionThis study provides genome-wide insights into the evolutionary history of cotton CKI genes and lays a foundation for further investigation of the functional differentiation of two types of CKI genes in specific developmental processes and environmental stress conditions.

Transcriptome analysis of the role of autophagy in plant response to heat stress.

Autophagy plays a critical role in plant heat tolerance in part by targeting heat-induced nonnative proteins for degradation. Autophagy also regulates metabolism, signaling and other processes and it is less understood how the broad function of autophagy affects plant heat stress responses. To address this issue, we performed transcriptome profiling of Arabidopsis wild-type and autophagy-deficient atg5 mutant in response to heat stress. A large number of differentially expressed genes (DEGs) were identified between wild-type and atg5 mutant even under normal conditions. These DEGs are involved not only in metabolism, hormone signaling, stress responses but also in regulation of nucleotide processing and DNA repair. Intriguingly, we found that heat treatment resulted in more robust changes in gene expression in wild-type than in the atg5 mutant plants. The dampening effect of autophagy deficiency on heat-regulated gene expression was associated with already altered expression of many heat-regulated DEGs prior to heat stress in the atg5 mutant. Altered expression of a large number of genes involved in metabolism and signaling in the autophagy mutant prior to heat stress may affect plant response to heat stress. Furthermore, autophagy played a positive role in the expression of defense- and stress-related genes during the early stage of heat stress responses but had little effect on heat-induced expression of heat shock genes. Taken together, these results indicate that the broad role of autophagy in metabolism, cellular homeostasis and other processes can also potentially affect plant heat stress responses and heat tolerance.