Correction: Genome-wide identification of the HSF gene family in Chinese chestnut and functional characterization of CmHSF4 under temperature stress

Chinese chestnut (Castanea mollissima Blume) is an important woody tree species in China. The fruit of the tree are rich in nutrients and have significant economic and ecological value. However, Chinese chestnut is often threatened by various abiotic stresses during its growth and development, such as high temperature, drought, and salt. Among these stresses, extreme high-temperature caused by global warming is becoming increasingly frequent and severe, and it is one of the key environmental factors restricting the improvement of yield and quality. To breed superior stressresistant varieties of Chinese chestnut through the use of molecular breeding methods and ensuring the sustainable development of the industry, it is necessary to determine stress-resistant genetic resources and identify the key stress-resistance genes. Adverse environmental stresses such as cold, heat, drought, and salt can severely affect seed germination, growth, development, yield, and plant quality (Kerchev et al. 2020;Kowalczewski et al. , 2020;de Medeiros et al. , 2023). Heat-shock transcription factors (HSFs) play a core regulatory role in the complex molecular networks of plant responses to high-temperature stress. When plants are exposed to heat stress, denatured proteins accumulate in the cytoplasm, activating HSFs, and causing them to form trimers and translocate to the nucleus. HSFs contain five special structures: the N-terminal DNA-binding domain, oligomerization domain, nuclear localization signal, C-terminal transcriptional activation domain, AHA, and nuclear export signal (Scharf et al. , 2012), which are also known as HSF domains. The first HSF gene was cloned from tomato (Lycopersicon esculentum) (Scharf et al. , 1990) in plants. In the nucleus, HSF specifically recognizes and binds to heat shock elements (HSE, with the conserved sequence 5'-AGAAnnTTCT-3') in the promoters of downstream target genes through its highly conserved N-terminal DNA-binding domain (DBD) (JJiang, 2021). This initiates the transcription of numerous stress response genes, including heat shock proteins, which jointly assist in the repair of damaged proteins and maintaining intracellular homeostasis, thus establishing heat tolerance (Baniwal, 2004). Typical plant HSF proteins have a modular structure, including a DBD, an HR-A/B region responsible for protein oligomerization (Oligomerization Domain, OD), a nuclear localization signal, a nuclear export signal, and a C-terminal transcriptional activation domain, where the short peptide motif specific to class A HSF is called the AHA motif (Guo et al. , 2008). Based on the structural characteristics of the HR-A/B region, plant HSFs are classified into three major classes: A, B, and C. Of these, class A is generally regarded as the main positive regulator of the high-temperature response. The AHA motif is a specific transcriptional activation motif in HSFA, whereas HSFB and HSFC lack transcriptional activation activity because of the absence of the AHA motif (Scharf et al. , 1990). The heat stress elements (HSEs) of HSFs are formed by the repetitive pattern of the palindromic binding motif upstream of the HS-induced gene TATA box, and they usually require two or more HSE motifs (Nover et al. , 2001). Many reports of functional studies on members of the HSF gene family have been published. For example, the HSF family in Camellia sinensis comprises 22 members. Of these, both CsHsf15 andCsHsf16 have high induction rates under light and low-temperature stress, and contain cis-acting elements related to light and low-temperature responses, which indicates that they may play a role in light resistance and low-temperature stress (Li et al. , 2025). Seventeen members have been identifiedin the carnation (Dianthus caryophyllus), and each member is upregulated by high temperatures and drought. Among them, four members, DCAhSCF-A2A, DCAhSCF-A5, DCAhSCF-B2B, and DCAhSCF-C1 are slightly upregulated at low temperatures, which indicates that DcaHsf plays a key role in different stress response pathways (Li et al. , 2019). Twenty-five HSF members have been identified in Malus domestica. At high temperatures, 12 were significantly higher than the reference sample, whereas only MdHsfA9b and Mdhsfb4A-b were strongly downregulated in response to an increase in temperature (Giorno et al. , 2012). Therefore, members of the HSF gene family respond to various stressors through the activation or inhibition of plant growth. Many studies have confirmed that HSF proteins play important roles in plant responses to high-temperature stress. For example, the silencing of HSFA1a in tomatoes reduces the synthesis of chaperone proteins and HsfA1a proteins induced by heat stress, thereby increasing the sensitivity of HSFA1A-silenced tomato plants to heat stress. Furthermore, compared to wild-type plants, HSFA2 mutant plants of Arabidopsis thaliana are more sensitive to heat stress at 37 ℃. In addition to heat stress, HSFs are involved in plant growth and other abiotic stresses (heat, cold, salt, and drought). For example, HSFA9 is involved in the embryonic development and seed maturation of Arabidopsis thaliana and sunflower (Almoguera et al. , 2002), and the four HSF genes (HSFA1e, HSFA3, HSFA4a, HSFB2a) of Arabidopsis thaliana are strongly induced by salt, low temperature and osmotic stress (Kilian et al. , 2007) With the extensive development of plant genome sequencing, the HSF gene family has been systematically identified at the whole-genome level in various plants, such as rice (Oryza sativa L. ) (Baniwal et al. , 2004), Arabidopsis (Guo et al. , 2008), maize (Zea mays) (Lin et al. , 2011), apple (Malus domestica) (Giorno et al. , 2012), soybean (Glycine max), and cucumber (Cucumis sativus L. ) (Chung et al. , 2013). These studies have shown that the number of HSF family members varies significantly among different species and that they play diverse roles in response to high temperature and other abiotic stresses (such as cold, drought, and salt), presenting the coexistence of subfunctionalization and functional redundancy in the HSF family. These studies have provided an important foundation for understanding the roles and biological functions of the HSF gene family in the molecular mechanisms of high-temperature stress responses. Based on this, this study employed bioinformatics methods to conduct the first genome-wide (Wang et al. , 2021). Subsequently, the HSF domain and all AtHSF sequences were compared using the Simple HMM Search and Blast Several Sequences in the large database program of the TBtools software (Su et al. , 2023), and the intersection of the comparison results of the two programs was uploaded to the InterProScan database (https: //www. ebi. ac. uk/interpro/result/interprosca/). Finally, all members of the CmHSF gene family were identified (Chen et al. , 2023). After identifying all members, chromosome mapping and drawing of chromosome mapping diagrams for each CmHSF gene family member were conducted using the Gene Location Visualization (Advanced) program in TBtools software. They were named according to their chromosomal distributions. To better understand the physicochemical properties of these CmHSFs, the amino acid quantity, molecular weight, isoelectric point, instability coefficient, and other indicators of CmHSF gene family member proteins were analyzed using the ExPASy ProtParam online tool (https: //web. expasy. org/protparam/). The specific location of each family member protein in the cell, where it plays a specific role, was predicted using the BUSCA online tool (https: //busca. biocomp. unibo. it/). To better understand the grouping of the family members, we constructed phylogenetic trees for AtHSF and CmHSF. We first aligned all protein sequences using the MAFFT v7. 471 program and employed MEGA7 software to construct a phylogenetic tree, which is displayed on the Chiplot website (https: //www. chiplot. online/) (Xie et al. , 2023). CmHSF was grouped based on the grouping of 21 AtHSF protein sequences in the TAIR database (https: //www. arabidopsis. org/) and the phylogenetic tree. The Simple MEME Wrapper program of TBtools software was used to analyze the motifs in the CmHSF protein sequences. Ten conserved motif sequences were obtained and uploaded to the InterProScan database (https: //www. ebi. ac. uk/interpro/result/interprosca/) for functional annotation. Finally, the Gene Structure View of the TBtools software was used to visualize the motifs and gene structures (Chen et al. , 2023). Using the Fasta Extract program of the TBtools software, the 2000 bp promoter sequences preceding the ATG start codon of all CmHSF genes were extracted and uploaded to the PlantCARE database (http: //bioinformatics. psb. ugent. be/webtools/plantcare/) for cis-regulatory element prediction. The number of such elements was counted, and the final results were presented as a heatmap using the HeatMap program in TBtools (Chen et al. , 2023;Wang et al. , 2012;Qiao et al. , 2019). The collinearity of CmHSF gene family members within the Chinese chestnut genome was analyzed using MCScanX software, and a collinearity map was created using the Advanced Circos program in TBtools. The non-synonymous substitution rate/synonymous substitution rate (Ka/Ks) of the collinear gene pairs was calculated using the Simple Ka/Ks Calculator (NG) in TBtools. The replication types of the CmHSF gene family were analyzed using the DupGenfinder program and classified into four categories: whole-genome duplication (WGD), transposed duplication (TRD), dispersed duplication (DSD), and tandem duplication (TD) (Chen et al. , 2023). The proximal duplication (PD) pattern was not observed in the CmHSF gene family members. The genome and gff annotation files of Chinese chestnut, grape, and corn were downloaded from the National Genome Database of China (https: //ngdc. cncb. ac. cn/gwh), and the genome and gff annotation files of Arabidopsis thaliana and rice were downloaded from the Ensembl Plants database (https: //plants. ensembl. org/). The genome and annotation files of Castanopsis tibetana were downloaded from the CNGB database (https: //db. cngb. org/) ; the genome and gff annotation files of Japanese chestnut were downloaded from the PlantGenomePortal database (https: //plantgarden. jp/en/index) ; and the genome and gff annotation files of American chestnut were downloaded from the Phytozome13 database (https: //phytozome-next. jgi. doe. gov/info/cdentataᵥ1₁. ). MCScanX software was used to conduct a collinearity analysis between the Chinese chestnut genome and those of these species, and to determine the number of collinearity gene pairs. The formation time of each species was analyzed using the Timetree database (https: //timetree. org/) to infer the time point at which the members of the CmHSF gene family were generated. The transcriptome FASTQ file with the accession number GSA: CRA022911 was obtained from the Chinese National Genome Database (https: //ngdc. cncb. ac. cn/gsa), and the transcriptome was analyzed to explore transcriptome data of Yanbao under low light stress (0%, 50%, 75%, and 95%, which represents the gene expression status of two-year-old Yanbao chestnut seedlings after 10 d under shading intensities of 0%, 50%, 75%, and 95%, respectively). The transcriptome FASTQ file with the accession number PRJNA1166987 was obtained from the NCBI database (https: //www. ncbi. nlm. nih. gov/). The transcriptome was analyzed to explore the gene expression status of Yanshan Zao Feng chestnuts under low-temperature (D0h, D5h, D10h, and D15h) and high-temperature (G0h, G4h, G8h, and G12h) stress. D0h, D5h, D10h, and D15h represent the transcriptome data of Chinese chestnut seedlings at -15℃ after 0, 5, 10, and 15 h, and G0h, G4h, G8h, and G12h represent the transcriptome data of Chinese chestnut seedlings at 45℃ after 0, 4, 8, and 12 h. The transcriptome FASTQ file (accession number: PRJNA731244) was obtained from the NCBI for Biotechnology Information database (https: //www. ncbi. nlm. nih. gov/). The transcriptome was analyzed to explore the gene expression status of Yanshan Zao Feng chestnuts under drought (0d, 10d, 20d, 30d, 40d) stress, where 0d, 10d, 20d, 30d, and 40d represent the transcriptome data of Chinese chestnut seedlings under continuous water-free conditions for 0, 10, 20, 30, and 40 days. All transcriptome data were sequenced using the Illumina platform in the paired-end mode. After obtaining the reads, they were aligned to the reference genome of Chinese chestnut N11-1, and the fragments per kilobase of transcript per million fragments mapped (FPKM) was used as the gene expression level. Leaves of one-year-old Chinese chestnut seedlings grown in a 45 °C high-temperature stress environment for 0 h (G0h), 4 h (G4h), 8 h (G8h), and 12 h (G12h) were rapidly collected, frozen with liquid nitrogen, and stored at -80 °C for subsequent gene cloning and qRT-PCR experiments. RNA was extracted from the tender leaves using the Plant RNA Extraction Kit (Takara, Beijing, China), and RNA purity was determined using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA was reverse-transcribed into cDNA using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara, Beijing, China). RT-qPCR was performed on 12 samples (G0h, G4h, G8h, and G12h, four periods, with three replicates for each treatment, totaling 12 samples) using a SYBR PrimeScript RT-PCR Kit (Takara, Beijing, China). An ABI 7500 Real-Time PCR system (ABI 7500; Thermo Fisher Scientific, Singapore) was employed. The relative expression levels of the genes were calculated using the 2 -∆∆CT method. Fluorescent quantitative PCR primers were designed using the Batch qPCR Primer Design of the TBtools software. 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