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Heat-responsive ONSEN long terminal repeats integrate heat shock factor motifs, DNA methylation and natural sequence variation in Arabidopsis.

ONSEN is a heat-activated Ty1/copia retrotransposon in Arabidopsis thaliana controlled by heat shock factors (HSFs) and epigenetic silencing. Heat shock element (HSE)-like sequences in ONSEN long terminal repeats (LTRs) contribute to heat responsiveness, but relationships among sequence architecture, basal DNA methylation and natural variation remain unclear. We combined transcription-factor motif prediction, transposable-element comparisons, methylome and RNA sequencing (RNA-seq) data, and Arabidopsis genome assemblies. In silico disruption of five HSE cores eliminated HSF-family motif compatibility in the selected design and all 5119 exact-guanine-cytosine (GC) alternatives. Across 16 curated Columbia-0 terminal windows, ONSEN contained 33-49 non-redundant HSF motif-coordinate placements per 800 bp window and was strongly enriched relative to 1930 non-ONSEN transposable elements across score thresholds and continuous metrics. Direct comparison with 779 non-ONSEN LTR retrotransposons showed selectively elevated basal CHH methylation (where H = A, C or T) at ONSEN termini. Genome-wide RNA-seq analysis revealed broad heat-responsive gene and transposable-element changes, including strong ONSEN induction, whereas candidate-window analysis distinguished ONSEN from most HSF-rich non-ONSEN outliers. ONSEN-like variants across eight accessions generally retained HSF-compatible motifs while altering predicted DNA binding with one finger-family motif composition. Together, these findings define ONSEN terminal regions as HSF-rich regulatory sequences that retain heat-responsive potential within a methylated chromatin context and identify candidates for functional analysis.

DNA Methylation

Mining the sHSP20 (small heat-shock protein) gene family in finger millet (Eleusine coracana (L.) Gaertn.): structural, evolutionary and predicted abiotic-stress-responsive insights.

Small heat-shock proteins (sHSPs, the HSP20 family) are ATP-independent molecular chaperones that hold partially unfolded substrates and protect the proteome during heat and other abiotic stresses; every member is defined by a conserved &#x3b1;-crystallin domain (ACD). Finger millet (Eleusine coracana) is a climate-resilient, calcium-rich allotetraploid cereal of the semi-arid tropics whose HSP20 repertoire had not been catalogued. The present study is an entirely computational (in silico) analysis of the chromosome-scale reference genome of finger millet (NCBI GenBank assembly GCA_032690845.1, cultivar KNE 796-S). Mining the predicted proteome with the ACD profile (Pfam PF00011) and confirming every candidate by NCBI CD-search recovered 76 non-redundant ACD-bearing HSP20 genes (EcHSP20-1-EcHSP20-76). Based on phylogeny and TargetP-predicted localization, the members were classified into ten subfamilies: seven cytosolic/nuclear classes (C-I to C-VII, 60 members) together with chloroplastic (11), mitochondrial (3) and endoplasmic-reticulum (2) groups. The proteins ranged from 110 to 355 amino acids (12.1-39.2&#xa0;kDa) with theoretical pI of 4.85-9.69. The 76 loci were distributed over 14 of the 18 chromosomes and were conspicuously absent from chromosomes 8&#xa0;A, 8B, 9&#xa0;A and 9B, with pronounced clustering on chromosomes 1, 2, 3 and 6. Duplication analysis detected 149 paralogous pairs (49 homoeologous, 80 segmental/dispersed and 18 tandem); 147 of 148 pairs for which substitution rates could be calculated returned Ka/Ks&#x2009;<&#x2009;1 (mean 0.20), indicating strong purifying selection consistent with retention after whole-genome/allopolyploid duplication. Promoter analysis (PlantCARE) revealed enrichment of abscisic-acid-responsive (ABRE), MYB/MYC drought-related, STRE, DRE, low-temperature (LTR) and methyl-jasmonate/salicylic-acid elements, whereas canonical heat-shock elements (HSE) were not recovered. Expression profiling against a public drought transcriptome (SRP081350) showed that about half of the genes (39 of 76) are transcribed in leaf tissue, the expressed fraction being dominated by the cytosolic class C-I. This first finger-millet HSP20 catalogue provides a verified, reproducible framework and nominates computationally predicted candidate genes for future functional work on thermotolerance in cereals.

Allotetraploid

Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response.

The heat shock response (HSR) is the major defense mechanism against proteotoxic stress in the cytosol and nucleus of eukaryotic cells. Initiation and attenuation of the response are mediated by stress-dependent regulation of heat shock transcription factors (HSFs). Saccharomyces cerevisiae encodes a single HSF (Hsf1), facilitating the analysis of HSR regulation. Hsf1 is repressed by Hsp70 chaperones under non-stress conditions and becomes activated under proteotoxic stress, directly linking protein damage and its repair to the HSR. J-domain proteins (JDPs) are essential for targeting of Hsp70s to their substrates, yet the specific JDP(s) regulating Hsf1 and connecting protein damage to HSR activation remain unclear. Here, we show that the yeast nuclear JDP Apj1 primarily controls the attenuation phase of the HSR by promoting Hsf1's displacement from heat shock elements in target DNA. In apj1&#x394; cells, HSR attenuation is significantly impaired. Additionally, yeast cells lacking both Apj1 and the major JDP Ydj1 exhibit increased HSR activation even in non-stress conditions, indicating their distinct regulatory roles. Apj1's role in both nuclear protein quality control and Hsf1 regulation underscores its role in directly linking nuclear proteostasis to HSR regulation. Together, these findings establish the nucleus as key stress-sensing signaling hub.

Saccharomyces cerevisiae Proteins

Comparison of heat shocking and acridine orange treatment in phage typing of nontypable strains of Staphylococcus aureus.

Forty-five percent of 108 nontypable strains of Staphylococcus aureus from clinical and environmental sources were phage typable after heat shock and acridine orange treatment. Although phage patterns were produced by different types of treatment, the same phage groups were present, showing specificity. The newly typable strains appear to be similar in phage group distribution to the primary typable strains typed at the Peter Bent Brigham Hospital during the same time period. Ten of the originally nontypable S. aureus strains that showed lytic patterns after heat shock or acridine orange treatment were subcultured four times from the treated culture and then tested again with bacteriophages. Phage patterns were still present in 8 of the 10 strains without any further treatment. These findings are compatible with the findings of other workers, who have shown that the insensitivity of primary nontypable S. aureus strains to phage is linked to phage-resistant genes in extrachromosomal genetic elements that can be "cured" by both heat shock and acridine treatment.

Acridines

The clustered and scrambled arrangement of moderately repetitive elements in Drosophila DNA.

An examination of cloned Drosophila DNA has revealed large clusters of densely spaced, short (less than or equal to 1 kb), moderately repetitive elements. Different clusters have many of the same repetitive elements, but these elements are arranged differently in each cluster. It is improbable that this clustered arrangement can be detected by conventional reassociation kinetic and electron microscopic techniques, but it can be detected and features of its fine structure can be determined by a two-dimensional version of Southern's blotting technique. The genomic organization of these clustered repetitive elements was investigated by hybridizing restriction fragments of cloned DNA to polytene chromosomes, to filter-bound recombinant DNA clones and to Southern blots of total Drosophila DNA. These studies demonstrated that clusters occur in euchromatic regions of the chromosomes and that at least one of the clusters has the same repetitive element organization in cloned and in chromosomal DNA. These studies also demonstrated that copies of the elements from one cluster are scattered in at least 1000 chromosomal regions. These regions appear to have differing concentrations of repetitive DNA, but together they account for a large fraction of Drosophila's moderately repetitive DNA. Aside from indicating the genomic organization of cluster elements, this work has identified cluster elements throughout a 9 kb region neighboring one of the heat shock genes, throughout the intron of the major rDNA repeat and within the apparently transposable element, 412.

Animals

Targeting STK17B kinase activates ferroptosis and suppresses drug resistance in multiple myeloma.

The progression of multiple myeloma (MM), an incurable malignancy of plasma cells, is often associated with the suppression of ferroptosis, a type of cell death driven by iron-dependent lipid peroxidation. The mechanisms underlying this suppression remain largely unknown. Here, we identified serine/threonine kinase 17b (STK17B) kinase as a critical suppressor of ferroptosis in MM. Elevated levels of STK17B are associated with poor overall survival in patients with MM, and STK17B expression is significantly higher in relapsed vs newly diagnosed MM cases. We found that inhibiting STK17B in MM cells increased the labile iron pool, enhanced lipid peroxidation, and sensitized cells to conventional anti-MM therapies. Notably, an orally available, in-house-generated STK17B inhibitor induced ferroptosis and significantly reduced tumor growth in MM xenograft mouse models. Mechanistically, proximity labeling assay combined with the phospho-proteomic analysis identified 2 major regulators of iron uptake and transport as direct targets of STK17B: iron-responsive element binding protein 2 (IREB2), and heat shock protein family B member 1 (HSPB1). We demonstrated that STK17B phosphorylates critical regulatory sites on IREB2 (S157) and HSPB1 (S15), thereby modulating the balance between IREB2 and HSPB1 downstream effectors, proferroptotic transferrin receptor, and antiferroptotic ferritin heavy chain proteins. Furthermore, we demonstrated that STK17B indirectly maintains activating phosphorylation of STAT3, a ferroptosis suppressor and a major driver of MM pathobiology. Our findings uncovered a clinically relevant and targetable STK17B-pIREB2S157/pHSPB1S15 signaling axis that suppresses ferroptosis and contributes to drug resistance in MM.

Ferroptosis

A novel arrangement of tandemly repeated genes at a major heat shock site in D. melanogaster.

Three cloned segments of Drosophila melanogaster DNA have been isolated that derive from the major heat shock site at 87C in chromosome 3. Each of these segments contains sequences homologous to a class of polysomal poly(A)+ RNAs whose synthesis is induced by heat shock of cultured cells. A combination of R loop, heteroduplex and restriction fragment maps of these segments reveals that their RNA-homologous sequences are arranged in tandemly repeated units, each unit consisting of an alpha element (0.49 kb) joined to a beta element (1.10 kb). The polysomal RNAs homologous to these alphabeta units (1.59 kb) are distributed into three size classes exhibiting approximate lengths of 1, 2 and 3 kb. R loop mapping demonstrated that the sequence of the 2 kb RNA is alphabetaalpha, indicating that it, and presumably the 3 kb RNA, derive from transcripts covering more than one repeated unit. One of the cloned segments contains a variant repeat unit, alphagamma, located between two alphabeta units. This unit has the same alpha element, but the beta element has been replaced by a nonhomologous gamma element (0.87 kb). Analysis of the total D. melanogaster DNA indicates that the 87C locus contains at least 21 tandemly repeated units, twelve of which were identified as alphabeta units and six as alphagamma units. The 21 or more units are distributed among at least three different tandem arrays separated by different spacer regions, one of which is within a cloned segment. Sequences in the gamma element, but not those in the alpha or beta elements, are also found at 87A, which contains another heat shock site. The possible roles of the alphabeta-type RNAs and of the gamma sequences are discussed in the light of these results.

Animals

Organization of the multiple genes for the 70,000-dalton heat-shock protein in Drosophila melanogaster.

The organization and number of 70,000-dalton heat-shock protein genes of Drosophila melanogaster has been investigated in a wild-type Oregon R fly stock and in a KC cell line. Six copies were found in the KC cells, and slightly more were found in the Oregon R population examined. In both cases, the basic gene element consisting of the mRNA coding region plus a short 5' "noncoding" sequence element was conserved. Two gene variants distinguished by specific restriction sites were found in both genomic DNAs. Restriction maps of the six genes in KC cells showed that these two gene variants are arranged differently. Restriction analysis of Oregon R embryonic DNA revealed polymorphism in the organization of the genes, which is not observed in KC cells. The data suggest that the arrangement as well as the number of genes for the 70,000-dalton heat-shock protein in D. melanogaster is subject to variations at both the 87A and 87C cytogenetic loci.

Animals

A long noncoding RNA with enhancer-like function in pig zygotic genome activation.

The zygotic genome activation (ZGA) is crucial for the development of pre-implantation embryos. Long noncoding RNAs (lncRNAs) play significant roles in many biological processes, but the study on their role in the early embryonic development of pigs is limited. In this study, we identify lncFKBPL as an enhancer-type lncRNA essential for pig embryo development. lncFKBPL is expressed from the 4-cell stage to the morula stage in pig embryos, and interference with lncFKBPL leads to a developmental arrest at the 8-cell stage. Mechanistic investigations uncover that lncFKBPL is able to bind to MED8, thereby mediating enhancer activity and regulating FKBPL expression. Additionally, FKBPL interacts with the molecular chaperone protein HSP90AA1, stabilizing CDK9 and boosting its protein-level expression. Elevated CDK9 levels enhance Pol II phosphorylation, facilitating ZGA. Our findings illuminate the role of lncFKBPL as an enhancer lncRNA in pig ZGA regulation and early embryo development, providing a foundation for further exploration in this area.

Animals

Conserved HSFA1-dependent chromatin dynamics drive heat stress responses in plants.

Eukaryotic organisms remodel chromatin landscapes to regulate gene expression in response to environmental stress. In plants, heat stress (HS) induces widespread chromatin changes, yet the role of heat shock transcription factors (HSFs) in chromatin remodeling and their evolutionary conservation remains unclear. Using Marchantia polymorpha Mphsf mutants and Arabidopsis thaliana Athsfa1s mutants, we identify HSFA1 as a key regulator of HS-induced cis-regulatory element (CRE) accessibility, a mechanism conserved across land plants, mice, and humans. Gene regulatory network modeling reveals parallel transcription factor subnetworks, with MpWRKY10 and MpABI5B acting as indirect and negative HS regulators. We further showed that ABA modulates gene expression in an HSFA1-dependent manner without inducing chromatin remodeling. Finally, we develop a machine learning framework integrating chromatin accessibility and CRE information to predict gene expression across species, revealing stress-responsive regulatory logic at the transcriptional level. These findings provide insights into how TFs coordinate chromatin architecture to drive stress adaptation.

Heat-Shock Response

Heat shock protein 40 enhances axon regeneration in a mouse model of traumatic optic neuropathy.

Retinal ganglion cell death occurs following injury to the optic nerve either by trauma or in disease such as glaucoma, leading to severe vision loss. Recent innovations have demonstrated that optic nerve regeneration is feasible; however, the regeneration is limited. The aim of the present study is to identify genomic elements enhancing axon regeneration. We have taken a forward genetics approach using the BXD recombinant mouse strains to identify a gene that increases the extent of optic nerve regeneration. Axon regeneration was induced by knocking down Pten in retinal ganglion cells using adeno-associated virus to deliver an shRNA followed by an intravitreal injection of Zymosan with CPT-cAMP that produced a mild inflammatory response. Retinal ganglion cell axons were damaged by optic nerve crush. Following a 12-day survival period, regenerating axons were labeled by intravitreal injection of Cholera Toxin B conjugated with Alexa Fluor 647. Two days later, labeled axons within the optic nerve were examined to determine the number of regenerating axons and the distance they traveled down the optic nerve. The analysis revealed a surprising difference in the amount of axonal regeneration across all 33 BXD strains. There was a 7.5-fold difference in the number of regenerating axons and a 4-fold difference in the distance traveled by regenerating axons. These data were used to generate an interval map defining genomic loci that modulate enhanced axonal regeneration. A quantitative trait locus modulating axon regeneration was identified on Chromosome 14 (115 to 119 Mb). Within this locus were 16 annotated genes. Subsequent testing revealed that one candidate gene, Dnajc3, modulated axonal regeneration. Dnajc3 encodes heat shock protein 40 (HSP40), a molecular chaperone. Knocking down Dnajc3 in the high regenerative strain (BXD90) led to a decreased regeneration response, whereas, overexpression of Dnajc3 in a low regenerative strain (BXD34) resulted in an increased regeneration response. These findings reveal that Dnajc3 not only increases the number of regenerating axons, but also increases the distance that axons travel. The enhanced regeneration will prove to be critical for functional recovery in humans, where the distance axons travel to their targets is considerably longer than that of mice.

axon regeneration

Genes for the 70,000 dalton heat shock protein in two cloned D. melanogaster DNA segments.

Two hybrid plasmids, 56H8 and 132E3, which contain D. melanogaster (Dm) DNA segments coding for the 70,000 dalton heat shock protein, have been isolated (Schedl et al., 1978). The analysis of the sequence arrangement presented in the accompanying paper (Moran et al., 1979) shows that both Dm segments contain homologous regions composed of three distinct sequence elements which together define a common unit. We report here that the sequences complementary to the 70,000 dalton protein mRNA appear to be confined to a major portion of the largest element of the common unit and that the other sequence elements are located at the 5' end of the gene. We have also determined the cytogenetic location of the 70,000 dalton protein genes and have investigated whether these sites are transcriptionally active in salivary gland chromosomes.

Animals

Genome-wide identification of the HSP70 superfamily in tropical sea cucumber Stichopus monotuberculatus and their expression analysis under low-salinity stress.

Heat shock proteins (HSPs) are a group of evolutionarily conserved molecular chaperones that serve as indispensable core regulators in preserving cellular homeostasis and orchestrating organismal stress responses. The tropical sea cucumber Stichopus monotuberculatus, a high-value aquaculture species, is sensitive to fluctuations in environmental salinity-a challenge that has emerged as a critical bottleneck limiting its large-scale commercial cultivation. However, no systematic investigation has been conducted to characterize the HSP70 superfamily in S. monotuberculatus and elucidate its functional roles in salinity adaptation. In the present study, we performed a comprehensive genome-wide scan and identified 19 HSP70 superfamily genes in the S. monotuberculatus genome, with the HSP70IV subfamily showing remarkable gene expansion, containing 8 distinct copies. Phylogenetic analysis, conserved motif identification, and gene structure characterization demonstrated high evolutionary conservation within each HSP subfamily. These genes were unevenly distributed across the chromosomes of S. monotuberculatus, and prediction of cis-acting elements revealed that their upstream regulatory regions were enriched with numerous functional elements associated with stress response and immune regulation. Salinity stress experiments revealed that under severe low-salinity conditions (18&#x2030;), the expression levels of SmHSPA14L and multiple HSP70IV subfamily members were significantly elevated, while SmHYOU1D was significantly downregulated; in contrast, only subtle changes were detected in the expression of most HSP70 genes under moderate low-salinity stress (24&#x2030;). These findings strongly suggest that HSP70 genes, particularly the expanded HSP70IV subfamily, may act as key modulators in the low-salinity stress response. This work provides valuable insight into the molecular mechanisms underlying salinity adaptation in tropical sea cucumbers.

Animals

Transposable Elements Drive Regulatory and Functional Innovation of F-box Genes.

Protein domains of transposable elements (TEs) and viruses increase the protein diversity of host genomes by recombining with other protein domains. By screening 10 million eukaryotic proteins, we identified several domains that define multicopy gene families and frequently co-occur with TE/viral domains. Among these, a Tc1/Mariner transposase helix-turn-helix (HTH) domain was captured by F-box genes in the Caenorhabditis genus, creating a new class of F-box genes. For specific members of this class, like fbxa-215, we found that the HTH domain is required for diverse processes including germ granule localization, fertility, and thermotolerance. Furthermore, we provide evidence that Heat Shock Factor 1 (HSF-1) mediates the transcriptional integration of fbxa-215 into the heat shock response by binding to Helitron TEs directly upstream of the fbxa-215 locus. The interactome of HTH-bearing F-box factors suggests roles in post-translational regulation and proteostasis, consistent with established functions of F-box proteins. Based on AlphaFold2 multimer proteome-wide screens, we propose that the HTH domain may diversify the repertoire of protein substrates that F-box factors regulate post-translationally. We also describe an independent capture of a TE domain by F-box genes in zebrafish. In conclusion, we identify two independent TE domain captures by F-box genes in eukaryotes and provide insights into how these novel proteins are integrated within host gene regulatory networks.

Animals

Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein.

The isolation of the two hybrid plasmids 56H8 and 132E3, which contain D. melanogaster (Dm) DNA sequences complementary to the mRNA coding for the 70,000 dalton heat shock protein, has been reported (Schedl et al., 1978). Here we compare the sequence arrangement in the two cloned Dm DNA segments by restriction, cross-hybridization and heteroduplex analysis. The results show that the two cloned DNA segments derive from nonoverlapping regions of the Dm genome; that they contain homologous regions present once in 56H8 and twice in 132E3; and that each homologous region is composed of three distinct contiguous sequence elements, x, y and z, which together define a 3 kb common unit. While the 2.5 kb z elements show a high degree of sequence homology in all three common units, the three x and y elements display an intriguing relationship. The localization of the mRNA coding sequences within each of these common units is presented in the accompanying paper (Artavanis-Tsakonas et al., 1979).

Animals

An experimental analysis of bivalent interlocking in spermatocytes of the new Triturus vulgaris.

Interlocked bivalents at 1st meiotic metaphase are relatively uncommon in spermatocytes of the newt Triturus vulgaris, but their frequency of occurrence can be significantly increased by subjecting newts to a 24-h heat shock. Newt spermatocytes are sensitive to a heart shock at any stage between the end of premeiotic S and mid to late pachytene. The heat shock does not cause evidence desynapsis, nor does it significantly affect chiasma frequency; therefore the interlocked condition induced in spermatocytes which were subjected to a heat shock when they were in zygotene or pachytene is unlikely to be a consequence of synaptic trapping. By way of explanation it is suggested that a heat shock may cause telomers to detach from the nuclear membrane, or from the synaptonemal complex where the latter is attached to the membrane, thus allowing non-homologous chromonemata to become interwined before chiasmata have formed. If this explanation is valid, it is then further suggested that the recombination process which results in chiasma formation probably takes place in chromosomal regions lying outside the synaptonemal complex, rather than inside, between its 2 lateral elements.

Animals

Comparative Characterization of &#x3c3;32-Dependent Promoters for the Heat-Inducible Expression of FAST-PETase in Escherichia coli.

Efficient regulation of recombinant enzyme expression is an important consideration for the development of microbial biocatalysts. Heat-inducible promoters regulated by the alternative sigma factor &#x3c3;32 provide an inducer-free strategy for controlling gene expression in Escherichia coli. In this study, four &#x3c3;32-dependent promoters (PdnaK, PgrpE, PibpA, and PclpB) were comparatively characterized using the PET-degrading enzyme FAST-PETase fused to superfolder green fluorescent protein as a model recombinant protein. Promoter performance was evaluated based on basal leakage, induction kinetics, and expression strength following heat induction. Among the promoters examined, PdnaK exhibited the strongest heat-inducible expression and was dissected to examine the autonomous and combinatorial behavior of its promoter-derived elements. Molecular docking analysis further supported the experimental observations by showing qualitative agreement between predicted &#x3c3;32-DNA interactions and promoter performance. Together, these findings provide a comparative characterization of &#x3c3;32-dependent promoters and identify promoter architectures that may facilitate the development of heat-inducible recombinant enzyme expression systems in E. coli.

Escherichia coli

Identification of the BrSK gene family in flowering Chinese cabbage and functional characterization of BrSK2 subfamily involvement in heat stress.

Glycogen synthase kinase 3 (GSK3) kinases are evolutionarily conserved regulators of plant development and stress signaling, yet their contributions to thermotolerance in cool-adapted Brassica crops remain poorly understood. Here, we identified 16 BrSK genes in the Caixin (Brassica rapa ssp. chinensis var. parachinensis) genome, all harboring intact catalytic motifs indicative of functional kinase activity. Spatiotemporal expression profiling revealed preferential accumulation of BrSK transcripts in stem apices and floral organs during reproductive transition, while promoter analysis identified abundant heat- and abiotic stress-responsive cis-elements. Under heat stress, BrSK21, BrSK22, and BrSK23 displayed striking genotype-specific expression dynamics. BrSK21/22/23 transcripts were stably suppressed in the heat-tolerant cultivar '49-19' but transiently declined before rapidly rebounding in the heat-sensitive 'Liuye 50', mirroring RNA-seq profiles. Protein-protein interaction assays (Y2H, BiFC, and LCI) demonstrated specific associations between BrSK kinases and BrHSFA1. Functional validation via VIGS revealed that silencing of BrSK21 significantly enhanced thermotolerance, with triple silencing of BrSK21/22/23 conferring additive protection, indicating functional redundancy within the BrSK2 subfamily. Collectively, these findings establish the BrSK2 subfamily as negative regulators of heat tolerance in Caixin, likely via modulation of BrHSFA1 expression. This work identifies high-priority targets for molecular breeding of climate-resilient Brassica vegetables.

Plant Proteins