Integration of C3H15-mediated transcriptional and post-transcriptional regulation confers plant thermotolerance in Arabidopsis.

Heat stress is an environmental factor that significantly threatens crop production worldwide. Nevertheless, the molecular mechanisms governing plant responses to heat stress are not fully understood. Plant zinc finger CCCH proteins have roles in stress responses as well as growth and development through protein-RNA, protein-DNA, and protein-protein interactions. Here, we reveal an integrated multi-level regulation of plant thermotolerance that is mediated by the CCCH protein C3H15 in Arabidopsis. Heat stress rapidly suppressed C3H15 transcription, which attenuated C3H15-inhibited expression of its target gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), a central regulator of heat stress response (HSR), thereby activating HEAT SHOCK COGNATE 70 (HSC70.3) expression. The RING-type E3 ligase MED25-BINDING RING-H2 PROTEIN 2 (MBR2) was identified as an interacting partner of C3H15. The mbr2 mutant was susceptible to heat stress compared to wild-type plants, whereas plants overexpressing MBR2 showed increased heat tolerance. MBR2-dependent ubiquitination mediated the degradation of phosphorylated C3H15 protein in the cytoplasm, which was enhanced by heat stress. Consistently, heat sensitivities of C3H15 overexpression lines increased in MBR2 loss-of-function and decreased in MBR2 overexpression backgrounds. Heat stress-induced accumulation of HSC70.3 promoted MBR2-mediated degradation of C3H15 protein, implying that an auto-regulatory loop involving C3H15, HSFA2, and HSC70.3 regulates HSR. Heat stress also led to the accumulation of C3H15 in stress granules (SGs), a kind of cytoplasmic RNA granule. This study advances our understanding of the mechanisms plants use to respond to heat stress, which will facilitate technologies to improve thermotolerance in crops.

Arabidopsis cell suspension culture and RNA sequencing reveal regulatory networks underlying plant-programmed cell death.

Programmed cell death (PCD) facilitates selective, genetically controlled elimination of redundant, damaged, or infected cells. In plants, PCD is often an essential component of normal development and can mediate responses to abiotic and biotic stress stimuli. However, studying the transcriptional regulation of PCD is hindered by difficulties in sampling small groups of dying cells that are often buried within the bulk of living plant tissue. We addressed this challenge by using RNA sequencing and Arabidopsis thaliana suspension cells, a model system that allows precise monitoring of PCD rates. The use of three PCD-inducing treatments (salicylic acid, heat, and critical dilution), in combination with three cell death modulators (3-methyladenine, lanthanum chloride, and conditioned medium), enabled isolation of candidate core- and stimuli-specific PCD genes, inference of underlying regulatory networks and identification of putative transcriptional regulators of PCD in plants. This analysis underscored a disturbance of the cell cycle and mitochondrial retrograde signaling, and repression of pro-survival stress responses, as key elements of the PCD-associated transcriptional signature. Further, phenotyping of Arabidopsis T-DNA insertion mutants in selected candidate genes validated the potential of generated resources to identify novel genes involved in plant PCD pathways and/or stress tolerance.

Genome-Wide Identification, Characterization, and Expression Analysis of NF-Y Gene Family in Ginkgo biloba Seedlings and GbNF-YA6 Involved in Heat-Stress Response and Tolerance.

Nuclear factor Y (NF-Y) transcription factors play an essential role in regulating plant growth, development, and stress responses. Despite extensive research on the NF-Y gene family across various species, the knowledge regarding the NF-Y family in Ginkgo biloba remains unknown. In this study, we identified a total of 25 NF-Y genes (seven GbNF-YAs, 12 GbNF-YBs, and six GbNF-YCs) in the G. biloba genome. We characterized the gene structure, conserved motifs, multiple sequence alignments, and phylogenetic relationships with other species (Populus and Arabidopsis). Additionally, we conducted a synteny analysis, which revealed the occurrence of segment duplicated NF-YAs and NF-YBs. The promoters of GbNF-Y genes contained cis-acting elements related to stress response, and miRNA-mRNA analysis showed that some GbNF-YAs with stress-related cis-elements could be targeted by the conserved miRNA169. The expression of GbNF-YA genes responded to drought, salt, and heat treatments, with GbNF-YA6 showing significant upregulation under heat and drought stress. Subcellular localization indicated that GbNF-YA6 was located in both the nucleus and the membrane. Overexpressing GbNF-YA6 in ginkgo callus significantly induced the expression of heat-shock factors (GbHSFs), and overexpressing GbNF-YA6 in transgenic Arabidopsis enhanced its heat tolerance. Additionally, Y2H assays demonstrated that GbNF-YA6 could interact with GbHSP at the protein level. Overall, our findings offer novel insights into the role of GbNF-YA in enhancing abiotic stress tolerance and warrant further functional research of GbNF-Y genes.

AtPUB48 E3 ligase plays a crucial role in the thermotolerance of Arabidopsis.

As the global temperature gradually increases, thermotolerance is vital to the growth and survival for plants. Ubiquitin-mediated protein degradation is a central regulator of many key cellular and physiological processes, including responses to biotic and abiotic stresses. E3 Ubiquitin-ligases, as the major components in the ubiquitination pathway, confer specificity of substrate recognition. Herein, we report that AtPUB48 expression was induced by heat stress, including basal and acquired thermotolerance. AtPUB48-overexpressing lines (OEs) of plants were generated to detect the functions of AtPUB48 in the heat response signaling pathway in Arabidopsis. Seeds of Atpub48-2 mutant had a lower germination rate than those of wild-type (WT) and OE plants when suffered from high temperatures. On the contrary, overexpression of AtPUB48 in Arabidopsis enhanced basal and acquired thermotolerance in seed germination and seedling growth. Moreover, the transcript expression levels of several heat-related downstream genes were highly improved in the OE lines under heat stress, although there were lower levels in the Atpub48-2 mutant compared with that of WT. An in vitro ubiquitination assay confirmed that AtPUB48 with U-box and ARM-repeats functioned as an E3 ubiquitin ligase. The subcellular localization showed that AtPUB48 localized to the nucleus. Collectively, these data imply that AtPUB48 acts as a novel regulator in the heat response signaling pathway. AtPUB48 may target the unknown substrate receptor to 26S proteasome proteolysis.

Heat-Responsive Proteomics of a Heat-Sensitive Spinach Variety.

High temperatures seriously limit plant growth and productivity. Investigating heat-responsive molecular mechanisms is important for breeding heat-tolerant crops. In this study, heat-responsive mechanisms in leaves from a heat-sensitive spinach (Spinacia oleracea L.) variety Sp73 were investigated using two-dimensional gel electrophoresis (2DE)-based and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics approaches. In total, 257 heat-responsive proteins were identified in the spinach leaves. The abundance patterns of these proteins indicated that the photosynthesis process was inhibited, reactive oxygen species (ROS) scavenging pathways were initiated, and protein synthesis and turnover, carbohydrate and amino acid metabolism were promoted in the spinach Sp73 in response to high temperature. By comparing this with our previous results in the heat-tolerant spinach variety Sp75, we found that heat inhibited photosynthesis, as well as heat-enhanced ROS scavenging, stress defense pathways, carbohydrate and energy metabolism, and protein folding and turnover constituting a conservative strategy for spinach in response to heat stress. However, the heat-decreased biosynthesis of chlorophyll and carotenoid as well as soluble sugar content in the variety Sp73 was quite different from that in the variety Sp75, leading to a lower capability for photosynthetic adaptation and osmotic homeostasis in Sp73 under heat stress. Moreover, the heat-reduced activities of SOD and other heat-activated antioxidant enzymes in the heat-sensitive variety Sp73 were also different from the heat-tolerant variety Sp75, implying that the ROS scavenging strategy is critical for heat tolerance.

Deciphering source and sink responses of potato plants (Solanum tuberosum L.) to elevated temperatures.

Potato is an important staple food with increasing popularity worldwide. Elevated temperatures significantly impair tuber yield and quality. Breeding heat-tolerant cultivars is therefore an urgent need to ensure sustainable potato production in the future. An integrated approach combining physiology, biochemistry, and molecular biology was undertaken to contribute to a better understanding of heat effects on source- (leaves) and sink-organs (tubers) in a heat-susceptible cultivar. An experimental set-up was designed allowing tissue-specific heat application. Elevated day and night (29°C/27°C) temperatures impaired photosynthesis and assimilate production. Biomass allocation shifted away from tubers towards leaves indicating reduced sink strength of developing tubers. Reduced sink strength of tubers was paralleled by decreased sucrose synthase activity and expression under elevated temperatures. Heat-mediated inhibition of tuber growth coincided with a decreased expression of the phloem-mobile tuberization signal SP6A in leaves. SP6A expression and photosynthesis were also affected, when only the belowground space was heated, and leaves were kept under control conditions. By contrast, the negative effects on tuber metabolism were attenuated, when only the shoot was subjected to elevated temperatures. This, together with transcriptional changes discussed, indicated a bidirectional communication between leaves and tubers to adjust the source capacity and/or sink strength to environmental conditions.

Thermally Reversible and Irreversible Phase Transition Behaviors in Poly(ethylene oxide)/Ionic Liquid Mixtures.

The irreversible and reversible phase transition behaviors during phase separation-recovery (heating-cooling) cycles for poly(ethylene oxide)/1-ethyl-3-methylimidazolium tetrafluoroborate ionic liquid (PEO/[EMIM][BF4 ]) mixtures with a lower critical solution temperature phase diagram are reported for the first time. The evident differential scanning calorimetry endothermic and exothermic peaks are observed during the heating-cooling scan cycles near the phase boundary, in which the large heat loss for samples below the critical composition (60 wt% PEO) and obvious downward shift of phase transition temperature for all the compositions between the first and second cycles are particularly attractive. After the first recovery process, a reversible behavior during the next cycles is expected. These interesting phenomena are further confirmed by optical microscopy and Fourier-transform infrared measurements. It is demonstrated that the disruption and partial recovery of the hydrogen bonds, combined with the conformational change of PEO chains, can contribute to this irreversible behavior as well as a conversion to reversible phase transition behavior.

Heat-Responsive Photosynthetic and Signaling Pathways in Plants: Insight from Proteomics.

Heat stress is a major abiotic stress posing a serious threat to plants. Heat-responsive mechanisms in plants are complicated and fine-tuned. Heat signaling transduction and photosynthesis are highly sensitive. Therefore, a thorough understanding of the molecular mechanism in heat stressed-signaling transduction and photosynthesis is necessary to protect crop yield. Current high-throughput proteomics investigations provide more useful information for underlying heat-responsive signaling pathways and photosynthesis modulation in plants. Several signaling components, such as guanosine triphosphate (GTP)-binding protein, nucleoside diphosphate kinase, annexin, and brassinosteroid-insensitive I-kinase domain interacting protein 114, were proposed to be important in heat signaling transduction. Moreover, diverse protein patterns of photosynthetic proteins imply that the modulations of stomatal CO2 exchange, photosystem II, Calvin cycle, ATP synthesis, and chlorophyll biosynthesis are crucial for plant heat tolerance.

Time course of natural heat acclimatization in well-trained cyclists during a 2-week training camp in the heat.

The aim of this study was to determine the time course of physiological adaptations and their relationship with performance improvements during 2 weeks of heat acclimatization. Nine trained cyclists completed 2 weeks of training in naturally hot environment (34 ± 3 °C; 18 ± 5% relative humidity). On days 1, 6, and 13, they performed standardized heat response tests (HRT-1, 2, 3), and 43.4-km time trials in the heat (TTH-1, 2, 3) were completed on days 2, 7, and 14. Within the first 5-6 days, sweat sodium concentration decreased from 75 ± 22 mmol/L to 52 ± 24 mmol/L, sweat rate increased (+20 ± 15%), and resting hematocrit decreased (-5.6 ± 5.4%), with no further changes during the remaining period. In contrast, power output during TTHs gradually improved from TTH-1 to TTH-2 (+11 ± 8%), and from TTH-2 to TTH-3 (+5 ± 4%). Individual improvements in performance from TTH-1 to TTH-2 correlated with individual changes in hematocrit (assessed after the corresponding HRT; r = -0.79, P < 0.05), however, were not related to changes in performance from TTH-2 to TTH-3. In trained athletes, sudomotor and hematological adaptations occurred within 5-6 days of training, whereas the additional improvement in performance after the entire acclimatization period did not relate to changes in these parameters.

Heat-Resistant Inbred Lines Coordinate the Heat Response Gene Expression Remarkably in Maize (Zea mays L.).

High temperatures are increasingly becoming a prominent environmental factor accelerating the adverse influence on the growth and development of maize (Zea mays L.). Therefore, it is critical to identify the key genes and pathways related to heat stress (HS) tolerance in maize. Great challenges have been faced in dissecting genetic mechanisms and uncovering master genes for HS tolerance. Here, Z58D showed more thermotolerance than AF171 at the seedling stage with a lower wilted leaf rate and H2O2 accumulation under HS conditions. Transcriptomic analysis identified 3006 differentially expressed genes (DEGs) in AF171 and 4273 DEGs in Z58D under HS treatments, respectively. Subsequently, GO enrichment analysis showed that commonly upregulated genes in AF171 and Z58D were significantly enriched in the following biological processes, including protein folding, response to heat, response to temperature stimulus and response to hydrogen peroxide. Moreover, the comparison between the two inbred lines under HS showed that response to heat and response to temperature stimulus were significantly over-represented for the 1234 upregulated genes in Z58D. Furthermore, more commonly upregulated genes exhibited higher expression levels in Z58D than AF171. In addition, maize inbred CIMBL55 was verified to be more tolerant than B73, and more commonly upregulated genes also showed higher expression levels in CIMBL55 than B73 under HS. These consistent results indicate that heat-resistant inbred lines may coordinate the remarkable expression of genes in order to recover from HS. Additionally, 35 DEGs were conserved among five inbred lines via comparative transcriptomic analysis. Most of them were more pronounced in Z58D than AF171 at the expression levels. These candidate genes may confer thermotolerance in maize.

Genes and pathways correlated with heat stress responses and heat tolerance in maize kernels.

Global warming leads to frequent extreme weather, especially the extreme heat events, which threating the safety of maize production. Here we selected a pair of maize inbred lines, PF5411-1 and LH150, with significant differences in heat tolerance at kernel development stage. The two maize inbred lines were treated with heat stress at kernel development stage. Compared with the control groups, transcriptomic analysis identified 770 common up- and down-regulated genes between PF5411-1 and LH150 under heat stress conditions, and 41 putative TFs were predicted. Based on the interaction term of the two-factorial design, we also identified 6,744 differentially regulated genes between LH150 and PF5411-1, 111 common up-regulated and 141 common down-regulated genes were overlapped with the differentially regulated genes, respectively. Combined with proteins and metabolites data, several key pathways including seven differentially regulated genes were highly correlated with the heat tolerance of maize kernels. The first is the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ko04141: protein processing in endoplasmic reticulum, four small heat shock protein (sHSP) genes were enriched in this pathway, participating with the process of ER-associated degradation (ERAD). The second one is the myricetin biosynthesis pathway, a differentially regulated protein, flavonoid 3',5'-hydroxylase [EC:1.14.14.81], catalyzed the synthesis of myricetin. The third one is the raffinose metabolic pathway, one differentially regulated gene encoded the raffinose synthase controlled the synthesis of raffinose, high level of raffinose enhances the heat tolerance of maize kernels. And the last one is the ethylene signaling pathway. Taken together, our work identifies many genes responded to heat stress in maize kernels, and finds out seven genes and four pathways highly correlated with heat tolerance of maize kernels.

Too hot to defend: a tale of salicylic acid.

Extreme temperatures threaten plant immunity by suppressing the salicylic acid (SA) biosynthesis via unknown mechanisms. Kim et al. demonstrated that suppression of the SA pathway and plant immunity can be rescued by optimised expression of two master immune regulator(s), advancing our prospects for better protecting plants in a warming climate.

cAMP Is a Promising Regulatory Molecule for Plant Adaptation to Heat Stress.

With gradual warming or increased frequency and magnitude of high temperature, heat stress adversely affects plant growth and eventually reduces plant productivity and quality. Plants have evolved complex mechanisms to sense and respond to heat stress which are crucial to avoiding cell damage and maintaining cellular homeostasis. Recently, 33″,55″-cyclic adenosine monophosphate (cAMP) has been proved to be an important signaling molecule participating in plant adaptation to heat stress by affecting multi-level regulatory networks. Significant progress has been made on many fronts of cAMP research, particularly in understanding the downstream signaling events that culminate in the activation of stress-responsive genes, mRNA translation initiation, vesicle trafficking, the ubiquitin-proteasome system, autophagy, HSPs-assisted protein processing, and cellular ion homeostasis to prevent heat-related damage and to preserve cellular and metabolic functions. In this present review, we summarize recent works on the genetic and molecular mechanisms of cAMP in plant response to heat stress which could be useful in finding thermotolerant key genes to develop heat stress-resistant varieties and that have the potential for utilizing cAMP as a chemical regulator to improve plant thermotolerance. New directions for future studies on cAMP are discussed.

Genome-Wide Identification of Long Non-coding RNAs in Crassostrea gigas and Their Association with Heat Stress.

Oysters face a complex and changeable environment in the intertidal zone. Heat stress is the main cause of their mass summer deaths. Several important genes are identified to be associated with heat response for oysters. However, regulation of these heat response genes in oysters remains largely unknown. In this study, 27 RNA-seq datasets are used to give a relatively comprehensive of long noncoding RNA (lncRNA) sets for C. gigas. Then, the differential expressed genes and lncRNAs are identified under heat stress. Among all the heat shock proteins (HSPs) and inhibitor of apoptosis proteins (IAPs) in the C. gigas genome, 25 heat shock proteins and 14 IAPs are differential expressed. The Gene Ontology analysis reveals that differential expressed genes (DEGs) are enriched in 6, 7, and 7 GO terms in cellular components, molecular function, and biological process, respectively. Within these terms, cellular response to stimulus is the most abundant term. Furthermore, the potential cis target of the differential expressed lncRNAs (DELs) are predicted to investigate their functions. Of the 394 DELs, there are 80 DELs being found to be corresponded to 113 protein-coding genes. Of them, eight HSPs are found to be regulated by their lncRNA regulators under heat stress. This work provides valuable resource of lncRNA and their regulatory roles under heat stress in C. gigas and gives new insights into adaptive evolution in marine mollusks.

Extraspecific Manifestation of Nanoheater's Position Effect on Distinctive Cellular Photothermal Responses.

Subcellular localization of nanoparticles plays critical roles in precision medicine that can facilitate an in-depth understanding of disease etiology and achieve accurate theranostic regulation via responding to the aiding stimuli. The photothermal effect is an extensively employed strategy that converts light into heat stimulation to induce localized disease ablation. Despite diverse manipulations that have been investigated in photothermal nanotheranostics, influences of nanoheaters' subcellular distribution and their molecular mechanism on cellular heat response remain elusive. Herein, we disclose the biological basis of distinguishable thermal effects at subcellular resolution by localizing photothermal upconversion nanoparticles into specific locations of cell compartments. Upon 808 nm light excitation, the lysosomal cellular uptake initialized by poly(ethylenimine)-modified nanoheaters promoted mitochondria apoptosis through the activation of Bid protein, whereas the cell surface nanoheaters anchored via metabolic glycol biosynthesis triggered necrosis by direct perturbation of the membrane structure. Intriguingly, these two different thermolyses revealed similar levels of heat shock protein expression in live cells. This study stipulates insights underlying the different subcellular positions of nanoparticles for the selective thermal response, which provides valuable perspectives on optimal precision nanomedicine.

Plant Hormone-Mediated Regulation of Heat Tolerance in Response to Global Climate Change.

Agriculture is largely dependent on climate and is highly vulnerable to climate change. The global mean surface temperatures are increasing due to global climate change. Temperature beyond the physiological optimum for growth induces heat stress in plants causing detrimental and irreversible damage to plant development, growth, as well as productivity. Plants have evolved adaptive mechanisms in response to heat stress. The classical plant hormones, such as auxin, abscisic acid (ABA), brassinosteroids (BRs), cytokinin (CK), salicylic acid (SA), jasmonate (JA), and ethylene (ET), integrate environmental stimuli and endogenous signals to regulate plant defensive response to various abiotic stresses, including heat. Exogenous applications of those hormones prior or parallel to heat stress render plants more thermotolerant. In this review, we summarized the recent progress and current understanding of the roles of those phytohormones in defending plants against heat stress and the underlying signal transduction pathways. We also discussed the implication of the basic knowledge of hormone-regulated plant heat responsive mechanism to develop heat-resilient plants as an effective and efficient way to cope with global warming.

Comparative Analysis of miRNA Expression Profiles Between Heat-Tolerant and Heat-Sensitive Genotypes of Flowering Chinese Cabbage Under Heat Stress Using High-Throughput Sequencing.

Heat stress disturbs cellular homeostasis, thus usually impairs yield of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). MicroRNAs (miRNAs) play a significant role in plant responses to different stresses by modulating gene expression at the post-transcriptional level. However, the roles that miRNAs and their target genes may play in heat tolerance of flowering Chinese cabbage remain poorly characterized. The current study sequenced six small RNA libraries generated from leaf tissues of flowering Chinese cabbage collected at 0, 6, and 12 h after 38 °C heat treatment, and identified 49 putative novel miRNAs and 43 known miRNAs that differentially expressed between heat-tolerant and heat-sensitive flowering Chinese cabbage. Among them, 14 novel and nine known miRNAs differentially expressed only in the heat-tolerant genotype under heat-stress, therefore, their target genes including disease resistance protein TAO1-like, RPS6, reticuline oxidase-like protein, etc. might play important roles in enhancing heat-tolerance. Gene Ontology (GO) analysis revealed that targets of these differentially expressed miRNAs may play key roles in responses to temperature stimulus, cell part, cellular process, cell, membrane, biological regulation, binding, and catalytic activities. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified their important functions in signal transduction, environmental adaptation, global and overview maps, as well as in stress adaptation and in MAPK signaling pathways such as cell death. These findings provide insight into the functions of the miRNAs in heat stress tolerance of flowering Chinese cabbage.

Analyses of functional conservation and divergence reveal requirement of bHLH010/089/091 for pollen development at elevated temperature in Arabidopsis.

The Arabidopsis bHLH010/089/091 (basic helix-loop-helix) genes are functionally redundant and are required for both anther development and normal expression of DYT1-activated anther-related genes. These three genes are conserved in Brassicaceae, suggesting that each of them is under selection pressure; however, little is known about the possible functional differences among these bHLH genes and between the bHLH and DYT1 genes. Here, we compared novel anther transcriptomic data sets from bHLH010/089/091 single and double mutants, with an anther transcriptomic data set from the wild type (WT) and a previously obtained anther transcriptomic data set from the bhlh010 bhlh089 bhlh091 triple mutant. The results revealed molecular phenotypes that support the functional redundancy and divergence of bHLH010, bHLH089, and bHLH091, as well as the functional overlap and difference between them and DYT1. DNA-binding analyses revealed that DYT1 and bHLH089 specifically recognize the TCATGTGC box to activate the expression of target genes, including ATA20, EXL4, and MEE48. In addition, among genes whose expression was affected in the bhlh010 bhlh089 double and bhlh010 bhlh089 bhlh091 triple mutants, genes that are involved in the stress response and cell signaling were enriched, which included 256 genes whose expression was preferentially induced by heat during early flower development. Moreover, the bhlh double mutants exhibited defective pollen development when the plants were grown under elevated temperature, suggesting that bHLH genes are important for anther gene expression under such conditions. These results are consistent with the observation that the heat-induced expression of several genes is less in the bhlh mutants than that in the WT. Therefore, our results provide important insights into the molecular mechanism underlying the activation of direct targets by DYT1-bHLH089 heterodimers and demonstrate the protective roles of bHLH010/089/091 in maintaining fertility upon heat stress.

Heat induces myogenic transcription factors of myoblast cells via transient receptor potential vanilloid 1 (Trpv1).

Exercise generates heat, blood flow, and metabolic changes, thereby inducing hypertrophy of skeletal muscle cells. However, the mechanism by which heat incudes hypertrophy in response to heat is not well known. Here, we hypothesized that heat would induce differentiation of myoblast cells. We investigated the underlying mechanism by which myoblast cells respond to heat. When mouse myoblast cells were exposed to 42 °C for over 30 min, the phosphorylation level of protein kinase C (PKC) and heat shock factor 1 (Hsf1) increased, and the mRNA and protein expression level of heat shock protein 70 (Hsp70) increased. Inhibitors of transient receptor potential vanilloid 1 (Trpv1), calmodulin, PKC, and Hsf1, and the small interfering RNA-mediated knockdown of Trpv1 diminished those heat responses. Heat exposure increased the phosphorylation levels of thymoma viral proto-oncogene 1 (Akt), mammalian target of rapamycin (mTOR), eukaryotic translation initiation factor 4E binding protein 1 (Eif4ebp1), and ribosomal protein S6 kinase, polypeptide 1 (S6K1). The knockdown of Trpv1 decreased these heat-induced responses. Antagonists of Hsp70 inhibited the phosphorylation level of Akt. Finally, heat increased the protein expression level of skeletal muscle markers such as myocyte enhancer factor 2D, myogenic factor 5, myogenic factor 6, and myogenic differentiation 1. Heat also increased myotube formation. Knockdown of Trpv1 diminished heat-induced increases of those proteins and myotube formation. These results indicate that heat induces myogenic transcription factors of myoblast cells through the Trpv1, calmodulin, PKC, Hsf1, Hsp70, Akt, mTOR, Eif4ebp1, and S6K1 pathway. Moreover, heat increases myotube formation through Trpv1.

Characterizing the heat response of Arabidopsis thaliana from the perspective of codon usage bias and translational regulation.

mRNA translation is carefully regulated at both the initiation and the elongation step. Under heat stress, it is known that particular genes change their expression and translation levels to respond to the environment. Attention has been paid to the detailed mechanisms of how a few proteins work, and little is done to analyze whether the global evolutionary patterns affect the translational changes. Determinants like codon usage bias and its related evolutionary features are less studied in heat stress experiments, especially for plants. Utilizing the RNA-seq and Ribo-seq data of normal and heat-stressed Arabidopsis thaliana generated from a previous study, we conducted gene-level (global) and codon-resolution (local) translation analyses. We studied how codon usage bias and other evolutionary features could impact the translation patterns in the heat response of the plant. We found that the evolutionary features including codon usage bias, tAI, nitrogen cost, and conservation (identity) could affect the global and local translation efficiency. Under heat stress, the optimal and conserved codons are more likely to alter their local translation elongation speed to modulate the global translation of host genes. Meanwhile, we also verified the widely accepted notions that the secondary structures and proline codons could largely slow down the translation rate. Our results revealed the effect of codon usage bias and other evolutionary patterns on the translation regulation under heat stress. Unveiling the effect of these features on translational regulation of plants might be helpful in understanding the relationship and interaction between plants and the environment.