Identification of HSF gene family and functional analysis of two HSFA1 genes with tandem repeat features in Fritillaria cirrhosa D.Don

Abstract

Background

Heat stress (HS) is a growing environmental factor impacting the growth and medicinal value of plateau medicinal plants due to global climate change. Plant heat shock factors (HSFs) are key transcriptional regulators in HS responses, yet the mechanisms of HSFs in plateau medicinal plants remain largely unexplored.

Results

In this study, we identified 17 HSF genes from the plateau medicinal plant Fritillaria cirrhosa D.Don. All FcHSF members were divided into two different phylogenetic groups. Moreover, the distribution of conserved motifs among these genes reveals subfamily-specific divergence. PCR-based cloning was further used to amplify two transcript variants of FcHSFA1, designated as FcHSFA1a and FcHSFA1b, which display distinct tandem repeat configurations at their C-termini regions. Both variants were upregulated under HS, with FcHSFA1b showing higher expression. Subcellular localization showed both variants in the nucleus and cytoplasm of tobacco epidermal cells. FcHSFA1b exhibited stronger transcriptional activation activity than FcHSFA1a in yeast cells. Overexpression of both variants in tobacco enhanced HS-related gene expression, increased peroxidase activity and chlorophyll content, and thereby improved thermotolerance.

Conclusions

These findings suggest that FcHSFA1 variants contribute to heat tolerance, with distinct transcriptional responses, offering strategies to enhance basal thermotolerance in F. cirrhosa.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12621-1.

Background

Climate change increasingly threatens plant survival on earth. Global warming has led to extreme climatic conditions, such as high temperatures, which severely reduce the yield and quality of economic plants, including highland medicinal plants [1, 2]. Exploring the molecular mechanism of plant responses to heat stress (HS) is important for improving the productivity of agricultural plants under changing climate conditions [3]. HS activates complex transcriptional regulatory networks in plants to reduce damage and improve survival [4]. Among these, heat shock factors (HSFs) play a crucial role in regulating plant responses to HS and have been extensively studied for their critical role in transcriptional control [4, 5].

HSFs have a modular structure with multiple conserved domains, such as N-terminal DNA binding domain (DBD), polymerization domain (OD or HR-A/B motif), C-terminal nuclear localization signal (NLS), and nuclear export signal (NES) [5]. Among these, DBD and OD are responsible for recognizing and binding to the promoter regions of target genes, respectively, while the NLS and NES domains regulate the shuttling of HSF between the cytoplasm and the nucleus, thereby maintaining its homeostatic distribution in both compartments [5, 6]. HSFs are classified into A, B, and C classes based on differences in the sequences of conserved structural domain proteins [6]. HSFAs contain activating groups or aromatic and hydrophobic amino acid residues (AHAs) at the C-terminus near the NES, which are essential for transcriptional activation [5]. Acting as central regulators, HSFs modulate downstream genes in response to HS, including heat shock proteins (HSPs) and other chaperones, reactive oxygen species (ROS) scavengers, enzymes involved in protective metabolic responses and osmotic stress biosynthesis, apoptosis regulators, or other transcription factors [5]. Given the wide impact of HSFs on cell survival and function under HS, their expression and activation are tightly regulated at multiple levels, including genomic, transcriptional, and post-transcriptional mechanisms [3, 4]. Prior research has indicated that the plant HSFs are highly susceptible to splicing regulation under HS, demonstrating the potential of heat-induced HSFs selective splicing regulatory mechanisms in promoting plant heat tolerance [7].

As the canonical master regulators of the heat shock response, HsfA1s are essential for the coordination of the downstream transcriptional network [3, 4]. In Arabidopsis thaliana, HSFA1s acts as a pivotal regulator of high-temperature signaling, coordinating thermo-stress responses with heat shock responses (HSRs). Specifically, HSFA1d facilitates thermomorphogenesis by stabilizing the photosensitive pigment-interacting factor 4 (PIF4). Under HS, HSFA1s are activated and directly upregulate HSFA2, a principal regulator of HSR, thereby conferring thermotolerance. This creates a cross-regulatory network that links thermomorphogenesis with heat tolerance [8–10]. Additionally, HSFA1b acts as a thermosensor by regulating OST1-mediated stomatal movement through the suppression of adenylate cyclase activity, while concurrently activating heat shock gene expression through changes in nuclear localization [11]. In rice, the miR444b.2-HsfA1-AOC1 module couples thermosensing with rice blast resistance. HS downregulates miR444b.2, alleviating repression of HsfA1 and enabling AOC1 activation, which enhances jasmonic acid (JA) biosynthesis and bolsters disease resistance. This work provides genetic tools—such as the native HsfA1 alleles and CRISPR-uORF knockouts—for developing heat-tolerant and blast-resistant rice, safeguarding yield stability under climate change [12]. These insights underscore the potential of targeting HsfA1-related pathways in molecular breeding programs to develop crops with enhanced resilience to combined heat and biotic stresses.

Tandem repeats (TRs) are abundant in the proteomes of various species, including plants. Based on the size of repeat units, TRs are classified into three categories: shorter repeats (2–20 amino acids), longer repeats (20–40 amino acids), and lengthier repeats (> 100 amino acids) [13]. Most TRs are associated with protein-protein interactions, with longer repeats and lengthier repeats mediating these interactions through peptide folding [13, 14]. In plants, proteins containing TRs play important roles in cell physiology, growth, development, and stress response. Examples include Ammadilo (ARM), Ankyrin (ANK), HEAT, Kelch-like repeats, Tetratricopeptide (TPR), Leucine rich repeats (LRR), WD40, and Pentatricopeptide repeats (PPR), which are categorized as longer repeats [15]. Meanwhile, many lower frequency TRs are present in the plant proteome and contribute to diverse biological functions [14].

Proteins within the same gene family may exhibit variations in activity due to the distinct structural properties and functions conferred by TRs. Evolutionary processes such as gene duplications, genetic recombination, segmental duplications, and retrotransposition have increased the abundance of TR domain gene family members in plants, resulting in variations in repeat length and number that confer distinct functions [14, 15]. For example, a pan-genomic analysis of 17 high-quality Arabidopsis genomes revealed that the evolution of NLR genes is maintained at the population level through a variety of genetic variation mechanisms, sustaining a large and highly variable immune gene pool. This can be seen as a population-level ‘adaptive immune system’ to cope with constantly changing pathogen threats. It also provides important theoretical guidance for the future design and utilization of durable disease resistance in crops [16]. Deletions in repeat sequences can also generate TR proteins with different transcripts and potentially different functions. The Arabidopsis R gene RPS4 provides specific resistance to Pseudomonas syringae DC3000 expressing AvrRps4. Removal of introns 2 and 3 from the functional RPS4-Ler gene eliminated avrRps4-specific resistance, as the truncated transcripts encoded fewer or no LRR repeats [17]. The Arabidopsis ALFIN-LIKE 6 (AL6) encodes a regulatory protein with a poly-E short TR region located upstream of a plant homeodomain-type zinc finger domain [18]. In vivo studies demonstrate that changes in the length of coding region repeats directly influence the regulatory activity of AL6 on promoters and its function in protein interactions [18]. However, the effect of rare repeat sequences present in plant proteins remains poorly understood and requires further elucidation.

Fritillaria cirrhosa D.Don, commonly known as ‘Chuan-Bei-Mu’, is a valuable medicinal plant from China, classified under the genus Fritillaria in the Liliaceae family. It is predominantly found in areas around the Tibetan Plateau [19]. Renowned for its ability to moisturize the lungs and alleviate coughs, F. cirrhosa faces increasing threats to its wild populations due to specific habitat requirements. F. cirrhosa prefers humid conditions and avoids both drought and waterlogged high temperatures [20]. As a cold-season medicinal plant, it is highly sensitive to elevated temperatures, particularly during flowering and bulb filling. Environmental stresses such as salinity, high temperatures, and drought lead to growth retardation and mortality. HS is primary factor limiting the growth and yield of F. cirrhosa. Therefore, enhancing the heat tolerance of F. cirrhosa is beneficial for its successful domestication at low altitudes and for promoting artificial cultivation practices. In the related Liliaceae species Lilium longiflorum, LlHSFA1 interacts with LlHSFA2 to co-activate multiple downstream genes associated with HS response, thereby enhancing the plant’s thermotolerance [21]. LlHSFC2 also cooperates with HSFAs to modulate the heat stress response, orchestrating a transcriptional program crucial for thermotolerance [22]. These findings suggest that members of the HSFA subfamily likely play a crucial role in regulating the heat stress response in F. cirrhosa, although studies directly addressing this function remain limited at present.

In this study, a total of 17 genes were identified, followed by phylogenetic profiling, conserved motif analysis, and characterization of conserved domains. Notably, two FcHSFA1 transcripts with different TR events were cloned, designated as FcHSFA1a (OR345533.1) and FcHSFA1b (OR345532.1). The proteins encoded by these two transcripts were localized to both the nucleus and cytoplasm in Nicotiana benthamiana epidermal cells. Yeast transcriptional activation assays showed that FcHSFA1b exhibited stronger transcriptional activation activity than FcHSFA1a. Furthermore, heterologous expression of LlHSFA1a and FcHSFA1b in Nicotiana tabacum upregulated downstream HS response genes and improved basal thermotolerance.

Materials and methods

Identification, sequence analyses, and phylogenetics of FcHSFs

The full-length transcriptome data of F. cirrhosa were obtained from Figshare (10.6084/m9.figshare.21900003.v1.) [23]. The cDNA sequence of FcHSF17 (HSFA1b) was obtained by PCR amplification from F. cirrhosa plant tissue. Gene screening was performed using a Hidden Markov Model (HMM). The HSF domain (PF00447) was downloaded from the Pfam database (http://pfam.xfam.org/). HMMER 3.0 was used to identify predicted HSFs in F. cirrhosa, with an E-value < 1 × 10^{− 5} [24, 25]. The candidate HSF protein sequences were further confirmed using the National Center for Biotechnology Information (NCBI) Batch CD-Search tools (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). Protein physicochemical properties were calculated using TBtools [26]. Subcellular localization was predicted by WOLF PSORT (https://www.genscript.com/wolf-psort.html). The conserved motifs were predicted using the MEME suite 5.5.8 software (https://meme-suite.org/meme/) [27]. The full-length amino acid sequences of HSF members from Arabidopsis thaliana, Oryza sativa, and Salvia miltiorrhiza a were searched using the same method for phylogenetic analysis and subfamily classification. Phylogenetic trees were constructed using MEGA 11 with the neighbor-joining method and 1000 boot-strap replicates [28].

Growth conditions and heat treatment of F. cirrhosa

For tissue culture, explants of Fritillaria. cirrhosa D. Don were collected from Kangding City, Sichuan Province (30°03′44.90″ N, 101°58′03.81″ E) and identified by Prof. Qi Zhao from Chengdu University. The voucher specimen (No. F-200725-1) was deposited at the Engineering Research Center of the Sichuan-Tibet Traditional Medicinal Plant, Chengdu, China. We certify that that the research program adheres to all relevant institutional, national, and international guidelines and regulations, and that we have received the necessary authorization to collect F. cirrhosa. Tissue-cultured F. cirrhosa plantlets were used for gene cloning and heat treatment experiments. Sterile F. cirrhosa plantlets were prepared according to Zhao et al. [29] and maintained in a standard greenhouse at 18 °C with a 16 h/8 h light/dark photoperiod. The reproductive stage of F. cirrhosa plantlets with uniform size (bulb perimeter: 2.0 cm; plant height: 6.0 cm) was used for FcHSFA1s expression analysis experiments. To evaluate FcHSFA1s expression across organs, roots, bulbs, and leaves were sampled from three individual F. cirrhosa plantlets at 18 ℃ or 37 °C for 3 h. For heat treatment, F. cirrhosa plantlets were cultured on MS medium in a temperature controlled incubator (RuihuaHP400G, Wuhan, China) at 37 °C without light for different durations (0 h, 1 h, 3 h, 6 h, 12 h, and 24 h). Leaves were collected immediately after heat treatment from three biological replicates.

Isolation and identification of FcHSFA1 TR variants

Total RNA was extracted from leaves of tissue-cultured F. cirrhosa D.Don plantlets incubated at 37 °C for 3 h using Trizol reagent (Invitrogen, USA). cDNA synthesis was performed with the HiScript III Q RT SuperMix Kit (Vazyme, Nanjing, China). Transcriptome data indicated that FcHSFA1s may have TRs, and the ORFs of FcHSFA1s were amplified using special primer. Quantitative real-time PCR (qRT-PCR) was used to analyze FcHSFA1s expression levels, using the Cwbio SYBR Master Mix kit (Cwbio, Jiangsu, China) and a Bio-Rad CFX96 Real-time PCR system. For TR variant identification, semiquantitative RT-PCR was performed with 2×Rapid Taq Master Mix (Vazyme, Nanjing, China), using F. cirrhosa 18 S rRNA as the internal control. Relative quantification of the gel bands was performed using ImageJ software. All primer sequences are listed in Supplementary Table 1. Each RT-PCR analysis was performed with three biological replicates and three technical replicates. Protein structure prediction of FcHSFA1a/FcHSFA1b was used the RoseTTAFold program (https://robetta.bakerlab.org/). Sequence alignment of FcHSFA1s proteins with the reference protein (Lilium longiflorum LlHSFA1) from NCBI database was aligned by DNAMAN v.7.0.2.

Subcellular localization of FcHSFA1a/FcHSFA1b protein

The ORFs of FcHSFA1a and FcHSFA1b without termination codon were amplified by primers containing BamH I and Kpn I sites (Supplementary Table 1) and subcloned into the pCAMBIA1300-35 S-yellow fluorescent protein (YFP) vector. RFP-NLS_SV40 was used as a nuclear marker protein, and the empty vector was used as a control [30]. Transient transformation of N. benthamiana leaves was performed by infiltrating leaves with Agrobacterium solution, followed by fluorescence signals in transiently transformed leaves, as previously described [23].

Transcriptional activity analysis of FcHSFA1a/FcHSFA1b in yeast

The ORFs of FcHSFA1a and FcHSFA1b were amplified by PCR and cloned into the pGBKT7 vector between the Nde I and EcoR I sites, generating pGBKT7-FcHSFA1a and pGBKT7-FcHSFA1b. The pGBKT7 and GAL4 were used as negative and positive controls, respectively. All plasmids were then transformed into the yeast strain AH109 and cultured on yeast synthetic drop-out media (SD/-Trp, SD/-Trp/-His, and SD/-Trp/-His + X-α-gal) for 3 days at 30 °C before photographing. β-Galactosidase activity was measured by β-GAL assay kits (Coolaber Technology Co. Ltd, Beijing, China). Primer sequences for vector construction are provided in Supplementary Table 1.

Stable transformation of tobacco

The full-length cDNAs of FcHSFA1a and FcHSFA1b were amplified by PCR and cloned into pKSE401 vector, which was transformed into Agrobacterium rhizogenes LBA4404. The recombinant vector was transformed into tobacco plants (N. tabacum K326, purchased from Wuhan Biorun Biotechnology Co., Wuhan, China) using the leaf disc transformation method [31]. Transgene-positive plants were screened for resistance, and the expression of FcHSFA1a and FcHSFA1b was verified by qRT-PCR. Two T1 generation homozygous lines were selected for gene function analysis. Primer sequences are provided in Supplementary Table 1.

Transgenic tobacco HS test

Transgenic and wild-type tobacco plants were placed in a light incubator at 42 °C for 48 h, followed by a recovery period of 72 h at 25 °C. Three biological replicates were conducted for each group to observe the stress phenotypes of both transgenic and wild-type tobacco. Leaf samples were collected before and after the stress treatment to measure chlorophyll content, peroxidase (POD), ascorbic acid (APX), and other physiological parameters. The expression of downstream heat-responsive target genes was analyzed using qRT-PCR. Chlorophyll content was determined via the ethanol acetone extraction method [32]. Antioxidant enzyme activities of leaf samples were measured using POD and APX activity assay kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China) according to the protocols of manufacturer.

Results

Identification and physical parameters of HSF gene family members in F. cirrhosa

A total of 16 putative HSF genes were identified through systematic analysis of the full-length transcriptome of F. cirrhosa and were designated as FcHSF1 to FcHSF16. Furthermore, a novel transcript (FcHSF17) was isolated via cDNA amplification from F. cirrhosa tissues. The predicted open reading frames (ORFs) for the 17 FcHSF genes ranged from 756 bp (FcHSF16) to 1755 bp (FcHSF17), encoding deduced proteins consisting of 251 to 584 amino acids (Table 1). The relative molecular weights of the FcHSF proteins varied between 28.26 and 65.03 KDa, with predicted theoretical isoelectric points ranging from 4.8 to 7.68. The instability index values ranged from 48.44 to 68.86, while the aliphatic index values of FcHSF proteins ranged from 65.78 to 82.16. The grand average of hydropathicity (GRAVY) value was used to assess the overall hydrophobicity or hydrophilicity of a protein. Negative GRAVY values for all 17 FcHSF proteins indicated that they were predominantly hydrophilic (Table 1). Subcellular localization predictions revealed that all FcHSF proteins were localized in the nucleus (Table 1).

Table 1.

Physicochemical properties of proteins encoded by the 17 FcHSF genes in F. cirrhosa

Sequence ID	Protein name	Length of CDS (bp)	Size (aa)	MW (kDa)	pI	I.I.	A.I.	GRAVY	Localization Predicted
transcript/16,587	FcHSF1	1668	555	61.91	4.83	65.9	70.43	-0.636	Nucleus
transcript/16,743 (HSFA1a)	FcHSF2	1692	563	62.61	4.83	63.56	72.38	-0.606	Nucleus
transcript/22,921	FcHSF3	1515	504	55.29	5.76	61.1	74.56	-0.445	Nucleus
transcript/27,903	FcHSF4	954	317	36.78	5.31	53.42	66.12	-0.762	Nucleus
transcript/28,101	FcHSF5	1050	349	40.44	5.83	54.13	68.71	-0.734	Nucleus
transcript/28,229	FcHSF6	891	296	32.94	5.1	66.53	77.2	-0.491	Nucleus
transcript/28,642	FcHSF7	1305	434	48.98	5.29	60.23	66.47	-0.735	Nucleus
transcript/28,690	FcHSF8	927	308	34.52	6.79	57.78	75.78	-0.51	Nucleus
transcript/35,025	FcHSF9	1119	372	41.72	5.73	58.07	67.63	-0.641	Nucleus
transcript/35,896	FcHSF10	1104	367	41.16	5.72	58.82	68.04	-0.62	Nucleus
transcript/36,342	FcHSF11	813	270	30.29	7.68	48.44	65.78	-0.741	Nucleus
transcript/38,325	FcHSF12	960	319	34.73	6.53	61.23	75.96	-0.356	Nucleus
transcript/38,524	FcHSF13	891	296	33.00	5.03	64.85	76.22	-0.463	Nucleus
transcript/39,003	FcHSF14	900	299	32.52	4.97	60.36	72.31	-0.442	Nucleus
transcript/39,920	FcHSF15	1047	348	38.84	5.49	68.86	82.16	-0.43	Nucleus
transcript/45,852	FcHSF16	756	251	28.26	6.49	48.56	68.41	-0.747	Nucleus
HSFA1b	FcHSF17	1755	584	65.03	4.8	68.48	72.45	-0.64	Nucleus

MW Molecular weight, pI Isoelectric point, I.I. Instability index, A.I. Aliphatic index, GRAVY Grand average of hydropathicity

Phylogenetic analysis, conserved motifs, and domains of FcHSFs

To investigate the evolutionary relationships of FcHSF proteins, a phylogenetic tree was constructed by based on sequence alignment of 21 AtHSFs, 25 OsHSFs, 30 SmHSFs, and 17 FcHSFs (Fig. 1). Following established HSF protein classifications from A. thaliana, O. sativa, and S. miltiorrhiza, HSF proteins are typically grouped into three major subfamilies: HSFA, HSFB, and HSFC [33, 34]. In F. cirrhosa, ten FcHSF proteins were classified into group A, representing the largest subfamily, while seven FcHSFs were categorized into group B (Figs. 1 and 2A). No FcHSF members belonging to the group C were identified here, possibly due to the limitations of transcriptome sequencing.

Fig. 1.

Phylogenetic analysis of HSF gene family members in F. cirrhosa (Fc), A.thaliana (At), O. sativa (Os), and S. miltiorrhiza (Sm). Different colors represent different HSF classes

Fig. 2.

Phylogenetic relationship, motif distribution, and conserved domain of FcHSF proteins. A Phylogenetic relationships of FcHSF proteins with different colors indicating different subgroups. B Protein motifs in FcHSF members with each colorful box representing a unique motif. C Conserved domain analysis of FcHSF proteins. The conserved HSF domains are shown in the indicated colors

The conserved motif map of the FcHSF proteins was generated through MEME analysis and integrated with the phylogenetic tree (Figs. 2A, B). Seven conserved motifs were identified, with motifs 1 and 2 being the most conserved in F. cirrhosa. These motifs are present in all FcHSFs except FcHsf16, which contains only motif 1 (Fig. 2B). In group A, FcHSF1, FcHSF2, FcHSF3, and FcHSF17 shared the same motif structure (motifs 1–7), while other members retained at least the motifs 1–3 structure. The HSF proteins in group B all contained motifs 1 and 4, with FcHSF11, FcHSF12, FcHSF14, and FcHSF15 have the same motif structure (motifs 1, 2, 4, 6) (Fig. 2B). Conserved motif patterns were characteristic of HSF family members, with similar structural compositions observed within each phylogenetic group. Conserved domain analysis further demonstrated that all 17 FcHSFs harbored domains from either the HSF1 or HSF_DNA-binding superfamily. Notably, the four FcHSFs in group B exclusively contained additional domains from the PRK13992 or MreC superfamily, implying potential functional differentiation between groups A and B (Fig. 2C).

Characterization of two FcHSFA1 TR variants

In Arabidopsis, HSF family member HsfA1s serve as master regulators, modulating the expression of downstream genes under HS [34, 35]. To investigate the role of HSFA1 in F. cirrhosa thermotolerance, we obtained the cDNA sequences of FcHSF2 and FcHSF17 through PCR. Sequence analysis revealed that both proteins belong to the HSFA1 family and exhibit high sequence similarity, differing only in the C-terminal region due to distinct TR events. Therefore, we designated these two genes as FcHSFA1a and FcHSFA1b (Figs. 3A and 4). RoseTTAFold protein structure analysis revealed that FcHSFA1a lacked a 21-amino acid peptide TR enriched in glutamate (E) amino acid residues at its C-terminus compared with FcHSFA1b. This glutamate-rich peptide is predicted to form an amphipathic helix (Fig. 3B). Multiple sequence alignments of FcHSFA1a, FcHSFA1b, and LlHSFA1 indicated that both FcHSFA1 variants possess all functional domains of HSF [34], including DBD, HR-A/B, NLS, AHA, and NES regions (Fig. 4A). Phylogenetic analysis of HSF members from Arabidopsis and lily categorized the FcHSFA1 proteins within the HSF class A1 family, closely related to LlHSFA1 (Fig. 4B). These findings suggest that FcHSFA1a and FcHSFA1b are members of the HSFA1 subfamily.

Fig. 3.

Isolation and identification of FcHSFA1s from F. cirrhosa. A The PCR amplification and schematic diagram of protein structures of FcHSFA1a and FcHSFA1b. Unprocessed data corresponding to the gel image are displayed in Source data. B Protein structure prediction of FcHSFA1a and FcHSFA1b using the RoseTTAFold program. The unique regions of the two proteins are highlighted in yellow and red, representing the first two TRs and the third TR, respectively, with differing lengths and conformations

Fig. 4.

Amino acid sequence alignment and phylogenetic analysis of FcHSFA1s and other plant HSF proteins. A Alignment of the deduced amino acid sequences of FcHSFA1s and LlHSFA1 proteins using DNAMAN software. Completely and partly conserved amino acids in proteins are shaded in black and gray, respectively. Protein sequences used for this alignment are LlHSFA1 acquired from GenBank. The letters are as follows: DBD, DNA-binding domain; HR-A and HR-B hydrophobic repeat regions A/B; NLS, nuclear localization signal; AHA, activator peptide motifs; NES, nuclear export signal. B Phylogenetic tree of FcHSFA1s and HSFs from Arabidopsis and lily, constructed using neighbor-joining method in MEGA11.0 software. The red triangle indicates FcHSFA1 proteins. Bootstrap values indicate the degree of differentiation of each branch, and the scale indicates branch length

Expression of FcHSFA1a and FcHSFA1b in response to HS

To investigate the roles of FcHSFA1 variants in HS response in F. cirrhosa tissue culture seedlings, their organ-specific and heat-induced expression patterns of FcHSFA1s were analyzed. qRT-PCR results revealed that FcHSFA1s were expressed in all examined organs. Under normal growth conditions at 18 °C, the highest expression level was observed in the roots. In contrast, upon exposure to 37 °C, the relative expression of FcHSFA1s was significantly upregulated in both bulbs and leaves, with the most pronounced induction occurring in leaves. These results suggest that FcHSFA1s may play an important role in mediating heat stress responses specifically in the bulbs and leaves of F. cirrhosa. (Fig. 5A, B). HSFA1 is induced by heat and acts as a master regulator in the heat response signaling pathway [36]. Furthermore, we analyzed the temporal expression patterns of FcHSFA1s in leaves of F. cirrhosa following HS treatment at multiple time points. Under 37 °C, the expression of FcHSFA1s rapidly increased, peaking at 3 h, then gradually decreased, with another significant increase observed 24 h of treatment (Fig. 5C). Given that proteins from the same gene family may exhibit activity variations due to distinct structural properties conferred by TRs [37, 38], semiquantitative RT-PCR was used to quantify the expression levels of each FcHSFA1 variant at 37 ℃ and 18 ℃. The results showed that both FcHSFA1a and FcHSFA1b were upregulated under heat treatment, but the relative expression of FcHSFA1a was lower than that of FcHSFA1b (Fig. 5D).

Fig. 5.

Expression analysis of FcHSFA1s under HS. A F. cirrhosa plantlets in tissue culture. B Transcriptional expression of FcHSFA1s in roots, bulbs, and leaves under normal and HS conditions, analyzed using qRT-PCR at 37 °C for 3 h. Samples from roots under normal conditions (control, Con) were used for normalization. C Expression levels of FcHSFA1s in leaves at different time points at 37 °C. The 18 S rRNA gene of F. cirrhosa was used as an internal control for normalization of PCR efficiency. Values are mean ± SD of biological triplicates. Significant differences compared with the control are determined using Student’s t-test (ns: p value > 0.05, ∗∗p value < 0.01, ∗∗∗∗p value < 0.0001). D Detection of FcHSFA1a and FcHSFA1b transcripts in F. cirrhosa under normal and HS conditions using semiquantitative RT-PCR (30 cycles). These bands were confirmed by sequencing. Unprocessed data corresponding to the gel image are displayed in Source data

Subcellular localization and transcriptional activity of FcHSFA1a and FcHSFA1b

Subcellular localization of FcHSFA1a or FcHSFA1b was analyzed by transiently expressing YFP-fused constructs in N. benthamiana leaves, along with the nuclear localization marker RFP-NLS_SV40 (Fig. 6A). YFP observations showed that the fluorescence of FcHSFA1a-YFP and FcHSFA1b-YFP partially overlapped with that of RFP-NLS_SV40, and both proteins were localized in the nucleus and cytoplasm of N. benthamiana leaves. This nucleocytoplasmic shuttling was similar to that observed in L. longiflorum HsfA1 [21]. For transactivation analysis, yeast cells transformed with FcHSFA1a or FcHSFA1b grew on the SD/-Trp and SD/-Trp/-His media and catalyzed β-galactosidase degradation, demonstrating transactivation activity (Fig. 6B). When compared with the activity of β-galactosidase of FcHSFA1a, FcHSFA1b exhibited a stronger β-galactosidase activity in the yeast cells (Fig. 6C), suggesting that TRs may influence the activation potential of FcHSFA1a and FcHSFA1b.

Fig. 6.

Subcellular localization and transactivation assays of FcHSFA1a and FcHSFA1b proteins. A Transient expression profiles of FcHSFA1a and FcHSFA1b in N. benthamiana leaves. The transient expression of 35 S::FcHSFA1a-YFP and 35 S::FcHSFA1b-YFP was monitored in N. benthamiana leaves, with the YFP empty vector serving as the control. RFP-NLS_SV40 fusion protein is a nuclear marker. Bars = 50 μm. B Transactivation activity of FcHSFA1a and FcHSFA1b in yeast cells. The transformation of yeast cells was detected in SD/-Trp medium; the growth of transformants was detected in SD/-Trp-His medium; and the β-galactosidase activity of transformants was detected by X-α-gal staining. GAL4 and pGBKT7 were used as positive and negative controls, respectively. C The β-galactosidase activity of the transformants was measured by enzyme assay. Data are mean ± SD from three independent experiments

Heterologous expression of FcHSFA1a and FcHSFA1b in N. tabacum enhanced basal thermotolerance

FcHSFA1a and FcHSFA1b transgenic N. tabacum lines were identified by qRT-PCR, and positive lines OE-a-4, OE-a-8, OE-b-4, and OE-b-9 were selected for further study (Supplementary Fig. 1). To assess basal thermotolerance, wild-type (WT) and transgenic seedlings were exposed to 42 °C for 48 h, followed by a 24 h recovery culture period at 25 °C. It was observed at 72 h after treatment that the OE-b lines showed better basal thermotolerance than OE-a lines, as indicated by higher chlorophyll content (Fig. 7A, B). POD and APX are important antioxidant enzymes in plants, which mitigate oxidative damage by scavenging reactive oxygen species during HS [39]. The activities of POD and APX in OE-a and OE-b transgenic lines under HS were significantly higher than those in the WT control, with OE-b lines exhibiting greater POD activity than OE-a lines (Fig. 7C, D). These findings therefore implied that both FcHSFA1a and FcHSFA1b could improve the basal thermotolerance in transgenic tobacco, with FcHSFA1b exhibiting superior performance.

Fig. 7.

Transgenic N. tabacum plants of FcHSFA1a and FcHSFA1b enhanced basal thermotolerance. A Phenotypic comparison of two transgenic tobacco lines and the wild type (K326) under normal conditions, after HS treatment (42℃ for 48 h) and following recovery (25℃ for 72 h). B Chlorophyll content analysis of wild type and transgenic plants. C POD activity in wild type and transgenic plants. D APX activity in wild type and transgenic plants. E-H Relative expression levels of genes downstream of NtHSFA1s under normal and HS conditions. Values are mean ± SD of biological triplicates. Significant differences compared with the control are determined using Student’s t-test (∗p value < 0.05, ∗∗p value < 0.01, ∗∗∗p value < 0.001, ∗∗∗∗p value < 0.0001)

To investigate the molecular mechanism by which FcHSFA1s enhance basal thermotolerance in transgenic tobacco, we examined the expression of downstream HS responsive genes in transgenic lines using qRT-PCR [40]. Under HS, transcript levels of the NtHSFA1 target gene, NtDREB2A, and three HSP genes (NtHSP18, NtHSP201, and Ntsmall HSP) were significantly upregulated in the transgenic lines compared with the WT control (Fig. 7E-H). This enhanced expression of heat-responsive genes may contribute to the improved heat tolerance observed in transgenic tobacco plants.

Discussion

Although the mechanism of HSF function has been extensively studied in other model plants, research on high-altitude plants with sensitivity to high temperatures remains limited [3, 39]. In this study, we identified 17 HSF genes through transcriptome database mining and PCR cloning from the F. cirrhosa. Comprehensive analyses including phylogenetic tree analysis, conserved motifs identification, and domains architecture characterization were performed. Two tandemly duplicated FcHSFA1 variants were further functionally characterized. These variants displayed differential transcriptional responses to HS. Furthermore, it was shown that FcHSFA1s play a crucial role in HS response by upregulating the expression of DREB2A and HSPs, as well as modulating the chlorophyll content and antioxidant activity (Fig. 8). These results suggest that the response of FcHSFA1 to HS in plants from specialized habitats may involve a more complex regulatory mechanism.

Fig. 8.

A simple working model of the FcHSFA1s-mediated regulatory mechanism in F. cirrhosa under HS. HS induces elevated expression of two FcHSFA1 variants, FcHSFA1a and FcHSFA1b, which differ in their tandem repeats. FcHSFA1b transcripts accumulate to higher levels than FcHSFA1a to mediate the response mechanism to HS. Specifically, FcHSFA1s regulate the upregulation of downstream genes such as DREB2 and HSPs, which in turn increase chlorophyll content and the production of peroxidase. These changes collectively contribute to the enhancement of thermotolerance in F. cirrhosa plantlets

Plant HSFs are encoded by large gene families exhibiting diverse structures, expression patterns, and functions [5]. The plant HSF family is typically classified into three categories: A, B, and C, with their size and composition shaped by evolutionary processes [6]. A notable evolutionary divergence is observed in the HSFC subfamily, which shows substantially greater complexity in monocots compared to dicots [6]. In this study, 17 HSF genes were identified from the transcript data of F. cirrhosa based on their evolutionary relationships and characteristics. These genes were classified into two subfamilies, A and B, containing 10 and 7 genes, respectively (Fig. 1). Notably, unlike garlic (Allium sativum) [41], also a Liliaceae species, we found no HSFC subfamily members detected in the F. cirrhosa HSF gene family. This apparent absence may be related to insufficient transcriptome sequencing coverage. Future studies should combine genomic data to systematically analyze the Fritillaria HSF gene family. Conserved motif and domain analysis revealed that FcHSF proteins from the same subfamily exhibit similar characteristics (Fig. 2), suggesting that these conserved motifs and domains are closely associated with the regulatory functions of different subfamilies.

Gene duplication and whole genome duplication (WGD) at different stages of evolution have likely contributed to the diversity of HSF genes in angiosperms [6]. For example, Arabidopsis, soybean, and tomato contain four to five HSFA1 members, whereas rice contains only one [6]. In Arabidopsis, HsfA1a, HsfA1b, and HsfA1d are major positive regulators of HS-responsive gene expression and play important roles in normal plant growth [42]. Similarly, among the four HsfA1 factors in tomato, HsfA1a is a master regulator of heat tolerance, maintaining pollen viability and cellular homeostasis [43, 44]. Here, we identified two FcHSFA1 transcript variants from F. cirrhosa, both of which responded to HS (Figs. 3 and 5). Multiple sequence alignment between FcHSFA1s and LlHSFA1 indicated that FcHSFA1s have five key structural domains and motifs typical of HSFA1 proteins (Fig. 4A). Phylogenetic analysis also showed that FcHSFA1s were most closely related to LlHSFA1 and clustered together with AtHSFA1 branches, indicating that FcHASFA1 was in the HSFA1 group (Fig. 4B). Given the complexity of the two transcript variants of FcHSFA1, we hypothesize that the F. cirrhosa HSFA1 group consists of at least two distinct members.

Protein sequence TRs are frequently found in the plant proteome, with their length or number often closely linked to protein function [14]. The occurrence of TRs in plant proteins is a complex process driven by mechanisms including replication slippage, recombination, transposition, and selective pressures that favor the maintenance or expansion of these repeats for functional purposes [15]. Evolutionarily, TR emergence appears to be a continuous process across the plant kingdom. Interestingly, highly variable TRs may serve as a source of variation for rapid adaptation, such as the LRR repeats in plant disease resistance R genes, where TR unit gain or loss may respond to pathogen resistance [6, 45]. Here, we identified two variants of FcHSFA1s containing different numbers of TRs at the C-terminus, where FcHSFA1a lacks a 21-amino acid TR compared with FcHSFA1b, potentially resulting in the absence of an amphipathic helix in FcHSFA1a (Fig. 3). Subcellular localization analysis revealed that both FcHSFA1a-YFP and FcHSFA1b-YFP localized to the nucleus and cytoplasm in tobacco epidermal cells, but the transcriptional activation activity of FcHSFA1b was shown to be stronger than that of FcHSFA1a in yeast cells (Fig. 6). These results imply that TR differences may not affect subcellular localization of FcHSFA1s but are directly linked to transcriptional activation. Similar correlations between gene expression and short TR length variation have been reported in the model plant A. thaliana [18]. Moreover, heterologous overexpression of FcHSFA1b in transgenic tobacco also appeared to exhibit better basal heat tolerance than FcHSFA1a (Fig. 7A-D), indicating that FcHSFA1b with longer TRs may play a predominant role in the heat tolerance response of F. cirrhosa.

Upon exposure to high-temperature stress, plants activate a suite of coordinated molecular and physiological mechanisms to mitigate damage, including heat shock response, reactive oxygen species (ROS) scavenging, photosynthesis regulation, and maintenance of cell membrane stability and so on [39, 46]. Overexpression of NtHSP70-8b conferred enhanced thermotolerance in tobacco by modulating stomatal conductance and augmenting antioxidant capacity [47]. In Brassica campestris, BcHsfA1 overexpression in transgenic tobacco increased chlorophyll content and enhanced the activities of SOD and POD under HS, compared to wild-type plants. Additionally, 11 candidate heat response genes were significantly up-regulated in the transgenic lines [40]. Herein, under HS, transgenic tobacco lines overexpressing FcHSFA1a or FcHSFA1b exhibited greater chlorophyll accumulation, enhanced activities of POD and APX, and up-regulated expression of the heat-responsive genes NtDREB2A and three HSPs compared to the wild-type. These coordinated responses collectively conferred enhanced thermotolerance (Fig. 7B-H), consistent with findings in tobacco lines overexpressing BcHsfA1 [40]. Beyond the observed increases in chlorophyll content and peroxidase activity, it is likely that additional physiological responses contribute to FcHSFA1s-mediated thermotolerance. Moreover, functional divergence between FcHSFA1a and FcHSFA1b is conceivable, which warrants further investigation.

Due to its well-characterized genetic toolkit and physiological sensitivity, the annual plant N. tabacum is widely employed as a model system to elucidate the molecular mechanisms underlying abiotic stress tolerance [46–48]. The absence of mature genetic transformation systems in many plant species makes tobacco the preferred heterologous host for expressing key genes, which are essential for investigating their role in the regulation of heat tolerance [46]. For instance, heterologous expression of the grass PpEXP1 gene or the barley HvSHN1 transcription factor in transgenic N. tabacum conferred enhanced heat tolerance or multi-stress (heat, drought, and salt) tolerance, respectively [49, 50]. Moreover, for N. tabacum, the optimal growth temperature is around 23.5 °C, while heat stress is typically induced by exposure to 42–45 °C [47, 49, 51]. This approach allows for a more comprehensive validation of the biological functions of key genes in response to HS. In this study, heterologous overexpression transgenic lines of FcHSFA1s were generated in N. tabacum. A heat stress treatment of 42 °C was then applied to identify and characterize the phenotypic responses of these transgenic plants to thermal stress (Fig. 7). However, as the tissue-cultured plantlets of F. cirrhosa originate from alpine environments and have an optimal growth temperature of 18 °C, a temperature of 37 °C was selected in this study to impose HS (Fig. 5). Should a robust and efficient genetic transformation system for F. cirrhosa be established in the future, it would enable the functional validation of FcHSA1s through knockdown or overexpression studies in its native host. This approach is essential to elucidate precisely the regulatory role of FcHSA1s in the HS response mechanism of F. cirrhosa.

HSFA1 has been shown to have biological functions in controlling the response to abiotic and biotic stresses, particularly under HS [5]. Overexpression of HSFA1 has been shown to enhance basal thermotolerance in several plant species, including A. thaliana [52], Solanum lycopersicum [43], L. longiflorum [21], and B. campestris [40]. Under HS, activation of HSFA1 can rapidly induce the upregulation of downstream target genes, such as DREB2A, HSFA2, and HSRs, promoting cellular antioxidant capacity, protein repair, degradation, and secondary metabolite synthesis [3, 53]. Similarly, overexpression of FcHSFA1a and FcHSFA1b in transgenic tobacco upregulation of HS-responsive target genes, increased chlorophyll content and antioxidant enzyme activities, thereby enhancing basal thermotolerance (Fig. 7). This observation further supports the functional conservation of A1-type HSF members. In addition, different members of the plant HSF family may exhibit distinct functional roles in the response to stress, which are shaped by their unique expression patterns, regulatory mechanisms, and target gene specificity. In Arabidopsis, HsfA1a, HsfA1b, and HsfA1d primarily act as positive regulators in HS responses, while HsfA1d and HsfA1e also play crucial roles in response to high light and salt stress [42, 54]. Similarly, in L. longiflorum, overexpression of LlHsfA3A enhances both basal and acquired thermotolerance in transgenic Arabidopsis, whereas overexpression of LlHsfA3B improves only acquired thermotolerance [55]. Following HS, the expression levels of FcHSFA1 genes were significantly upregulated in both bulbs and leaves of F. cirrhosa, with higher transcript levels in leaves (Fig. 5B). These results indicate that FcHSFA1s are predominantly involved in the HS response of above-ground tissues, particularly the leaves. This pattern aligns with observations in lily, where LlHSFA1 expression was most strongly induced in the leaves upon HS exposure [21]. Futhermore, we observed significantly higher transcript levels of FcHSFA1b than those of FcHSFA1a under HS (Fig. 5D), indicating potential functional differentiation between the two variants in regulating heat tolerance. This suggests that the heat-responsive regulatory mechanisms of FcHSFA1 may differ from those of the model plant, warranting further detailed studies to better understand its thermotolerance mechanisms.

Conclusions

In this study, 17 members of the HSF gene family were identified from both transcriptome data and PCR cloning of F. cirrhosa. Analysis of phylogenetic relationships, conserved motifs, and structural domains indicated that FcHSF proteins maintain a highly conserved structural architecture, while displaying characteristic sequence distribution patterns among subfamilies. Notably, HSFA1 members FcHSFA1a and FcHSFA1b, which exhibit different TR events, showed distinct transcriptional responses to HS. Nonetheless, both variants enhanced basal heat tolerance in heterologous-overexpressing plants, suggesting potential functional partitioning under HS.

However, the findings of this study are accompanied by significant limitations. The identification of HSF members was based on transcriptome data, potentially omitting low-expression or condition-specific genes from the genomic profile. Furthermore, functional validation was performed in a heterologous tobacco system. Therefore, the roles of FcHSFA1s in F. cirrhosa must ultimately be confirmed through genetic transformation in the native host.

Abbreviations

HMM

Hidden Markov Model

MW

Molecular weight

pI

Isoelectric point

I.I.

Instability index

A.I.

Aliphatic index

GRAVY

Grand average of hydropathicity

Authors’ contributions

R.L., M.X. and Z.Z. devised the experiment, analyzed statistical, and wrote the paper; X.Q., Y.Y. and Q.Z. collected data; R.L., M.X., Z.Z. and D.C. analyzed data and performed the experiments. All authors reviewed and edited the final manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (Grant No. 32470397), Natural Science Foundation of Sichuan (Grant No. 2025ZNSFSC1125), and the Sichuan Province “three districts” talent support project.

Data availability

The full-length transcriptome datasets generated and/or analysed during the current study are available in the Figshare repository, [10.6084/m9.figshare.21900003.v1]. The cDNA and protein sequences of FcHSFA1a and FcHSFA1b have been deposited in the NCBI GenBank database under accession numbers OR345533.1 and OR345532.1. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable. The sampling of plant material was performed in compliance with institutional guidelines. The research conducted in this study required neither approval from an ethics committee, nor did it involve any human or animal subjects.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.