Molecular Insights into the Role of PcHSP20s in Mediating Thermotolerance in Polygonatum cyrtonema

Abstract

Small heat shock proteins (HSP20s) are known to function as molecular chaperones that bind to denatured proteins under high-temperature stress and assist in their conformational recovery, thereby contributing to plant thermotolerance. In the present study, three HSP20 genes—PcHSP12.8, PcHSP12.9, and PcHSP13.4—were identified in the transcriptome of Polygonatum cyrtonema Hua. Bioinformatics analysis indicated their phylogenetic relationships, conserved domains, and potential tertiary structures. RT-qPCR analysis revealed up-regulation of all three genes in response to heat stress. Subcellular localization studies further suggested that PcHSP12.8, PcHSP12.9, and PcHSP13.4 are predominantly localized in the nucleus. Heterologous expression of these genes in a heat-sensitive yeast mutant appeared to improve cell survival under heat stress relative to the control strain. In Arabidopsis thaliana overexpressing these genes, moderate improvements in germination rate, root elongation, and stress survival were observed compared to wild-type plants under heat stress. Transgenic lines also showed a tendency toward reduced reactive oxygen species accumulation, as reflected by decreased 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, together with increased activities of catalase (CAT) and peroxidase (POD), as well as higher chlorophyll retention under thermal stress. Taken together, these findings imply that the three PcHSP20 genes could be involved in thermotolerance in P. cyrtonema.

1. Introduction

Heat stress poses a serious threat to medicinal plants, significantly inhibiting their growth and reducing medicinal yield. During seed germination, high temperatures decrease germination rates and suppress radicle and plumule development. At the vegetative stage, prolonged heat stress restrains shoot growth, induces leaf scorching, browning, and wilting, and in severe cases, leads to plant dehydration and death [1]. In root-derived medicinal crops such as Polygonatum sibiricum and Polygonatum odoratum, high temperatures disrupt photosynthesis and limit biomass accumulation in medicinal organs, ultimately resulting in yield loss or even crop failure. Nevertheless, they possess defense mechanisms to counteract heat stress. Medicinal plants recognize high-temperature signals through a sophisticated perception system that does not rely on the classical ligand–receptor interaction model. Instead, thermal signals are primarily perceived via mechanisms such as phase transitions in plasma membrane lipid–protein complexes, conformational changes in photoreceptors (e.g., phytochrome B, phyB), and alterations in RNA secondary structure [2]. Once perceived, complex signal transduction networks are activated. Among these, the calcium ion (Ca²⁺) signaling pathway plays a central role: heat stimulates Ca²⁺ influx through plasma membrane-localized cyclic nucleotide-gated channels (CNGCs). The increased cytosolic Ca²⁺ concentration subsequently activates downstream components such as calmodulin 3 (CaM3) and calmodulin-binding kinase 3 (CBK3), which further phosphorylate and activate heat shock factors (HSFs) [3,4,5]. The activated HSFs bind to heat shock elements (HSEs) in the promoters of heat shock protein (HSP) genes, inducing HSP expression to maintain proteostasis.

Heat shock proteins (HSPs) function as central regulators of plant thermotolerance. Under high-temperature stress, heat-induced genes are rapidly transcribed and translated into HSPs, which act as molecular chaperones to prevent protein denaturation, facilitate proper folding, and maintain structural and functional integrity of cellular proteins, thereby assisting plants in restoring physiological homeostasis [6]. Based on molecular weight, plant HSPs are classified into five major families: HSP100, HSP90, HSP70, HSP60, and HSP20/sHSPs (small heat shock proteins) [7]. These chaperones play essential roles under stress conditions by correcting misfolded proteins, stabilizing nascent polypeptides, and maintaining proteostasis. Among these families, HSP20/sHSPs exhibit the most pronounced heat inducibility and high sensitivity to temperature fluctuations—their expression is often suppressed under low-temperature conditions [8]. HSP20 subunits, ranging from 12–43 kDa, contain an α-crystallin domain that enables ATP-independent binding to denatured proteins, serving as a temporary reservoir during stress to prevent protein aggregation [9,10]. The Arabidopsis genome harbors 19 HSP20 genes, and under heat stress, these proteins bind to denatured substrates. During recovery, they release these substrates for refolding assisted by ATP-dependent chaperones such as HSP70 and HSP60 [11]. The accumulation of HSP20 is critical for both basal and acquired thermotolerance in plants. For instance, Arabidopsis AtHSP17.6II mutants fail to develop acquired thermotolerance, while overexpression of the lily LimHSP16.45 gene rescues this heat-sensitive phenotype [12]. Similarly, RNAi lines targeting six cytosolic HSP20 genes exhibit enhanced thermosensitivity, a defect that can be reversed by HSP20 overexpression [13]. In wheat, chloroplast-localized HSP26 is essential for thermotolerance during seed maturation and germination [14], and analogous functions have been reported in tobacco and rice [15,16]. Beyond heat stress, HSP20 also enhances plant tolerance to salt, drought, and cold stresses [14].

In summary, Polygonatum cyrtonema has high medicinal value and market demand, but high temperatures severely impair its cultivation. Research on its heat tolerance mechanisms remains scarce—particularly, the functions of sHSPs genes are unclear, leading to a lack of scientific support for heat-tolerant breeding. Thus, targeted studies in this area are urgently needed. This study, through transcriptomic analysis and functional validation, investigated the roles of HSP20 genes in P. cyrtonema under high-temperature stress. We identified three small heat shock protein genes—PcHSP12.8, PcHSP12.9, and PcHSP13.4—and demonstrated their potential contribution to thermotolerance via heterologous expression in yeast and transgenic Arabidopsis. These findings not only provide novel insights into the molecular basis underlying heat tolerance in Curculigo orchioides but also lay a solid foundation for further dissection of the HSP20-mediated regulatory network in medicinal plants, while additionally holding substantial implications for medicinal plant biology and crop improvement applications: they elucidate the critical role of HSP20 in mediating heat tolerance in medicinal plants, thereby establishing a reference paradigm for heat tolerance research in other medicinal plant species (e.g., Dendrobium officinale, Panax ginseng); furthermore, the cloned heat-tolerant PcHSP20 genes can serve as excellent candidate genes for the transgenic breeding of both food crops and medicinal crops (e.g., Lycium barbarum, Astragalus membranaceus), facilitating the development of heat-resistant cultivars to safeguard yield and quality under extreme climatic conditions.

2. Results

2.1. Transcriptome Analysis of Polygonatum cyrtonema Hua Under Short-Term Heat Stress

To investigate the transcriptional response of P. cyrtonema to short-term heat stress, uniformly grown three-year-old cultivated plants were subjected to a 3 h heat treatment at 42 °C, with control plants maintained at 23 °C. Transcriptome sequencing and library construction were performed by LC-Bio using the Illumina platform. Raw sequencing data were processed with fqtrim to remove low-quality reads and adapter sequences. After filtering, the heat-treated (0 h and 3 h) samples yielded 35,024,104; 39,356,246; 38,209,406; and 38,535,140 clean reads, respectively. All samples exhibited high data quality, with Q20 ≥ 98.11% and Q30 ≥ 94.35%. Due to the lack of a reference genome for P. cyrtonema, de novo transcriptome assembly was conducted with Trinity v2.15.1 to generate transcripts, which were then clustered and filtered into unigenes; expression was subsequently quantified by Salmon v1.10.0 and normalized to TPM for cross-gene and cross-sample comparisons.

Differential gene expression analysis between the 0 h and 3 h heat stress samples was performed using the edgeR package. Genes exhibiting a |log₂(fold change)| > 1 with a p-value < 0.05 were defined as differentially expressed genes (DEGs). A total of 2316 DEGs were identified (Table S1), comprising 1203 up-regulated and 1113 down-regulated genes. The overall distribution of these DEGs was visualized using a volcano plot (Figure 1A). Further stratification based on the magnitude of fold change (Figure 1B) revealed 83 genes with extreme expression differences (Log₂(Fold Change) > 8), among which 76 were up-regulated and 7 were down-regulated. The expression patterns of (DEGs) between the 0 h and 3 h heat stress groups are displayed in a clustered heatmap (Figure 1C). To validate the transcriptome data, we selected ten DEGs (including PcHSP70, PcNSUN5, PcMBF1C, PcDREB2B, PcBI-1, PcCYP76B10, PcCht1, PcSALR, PcPGIP2, PcCHS and PcHSP20) for qRT-PCR analysis using 18S rRNA (Table S2) as the reference. The expression trends were consistent with the RNA-seq results (Figure S1), confirming the reliability of our sequencing data.

2.2. Functional Enrichment Analysis of Heat-Responsive DEGs in P. cyrtonema

For the functional annotation of the screened DEGs, the protein sequences of the DEGs were aligned against the GO and KEGG databases using DIAMOND v2.1.8 software. Enrichment analysis of the DEGs was subsequently conducted via the LC-Bio Cloud Platform. KEGG pathway analysis highlighted 20 significantly enriched pathways among the heat-responsive DEGs (Figure 1D). Key pathways included plant–pathogen interaction (map04626), protein processing in endoplasmic reticulum (map04141), flavonoid biosynthesis (map00941), spliceosome (map03040), plant hormone signal transduction (map04075), and MAPK signaling pathway–plant (map04016). The most significantly enriched pathway was protein processing in the endoplasmic reticulum, involving 81 DEGs. These results suggest that P. cyrtonema may transduce heat signals through plant hormone and MAPK signaling pathways, modulate protein folding and functional maturation via endoplasmic reticulum processing, and regulate flavonoid biosynthesis to cope with high-temperature stress.

To systematically annotate the functional roles of DEGs in Polygonatum cyrtonema under heat stress, gene ontology (GO) enrichment analysis was conducted with the threshold set at p-value < 0.05 (Figure 1E, Table S3). In the BP category, The top enriched terms included “response to heat “ (GO:0009408, ~90 DEGs, p < 0.01), “response to high light intensity” (GO:0009644), “protein folding” (GO:0006457), indicates that P. cyrtonema prioritizes activating heat-specific stress response pathways to counteract high-temperature damage, and the enrichment of protein folding-related terms suggests maintaining protein structural stability is a core heat stress process, consistent with plants’ well-documented thermal stress transcriptional response. CC was enriched in subcellular compartments including “chloroplast stroma” (GO:0009570, ~50 DEGs, p < 0.01), “cytoplasm ribosome” (GO:0022626), “plastid stroma” (GO:0009532). MF was primarily enriched in binding, catalytic, and chaperone activities such as “DNA binding transcription factor activity” (GO:0003700), “monooxygenase activity” (GO:0004497). Notably, “heat shock protein binding” (GO:0031072) and “unfolded protein binding” (GO:0051082, p < 0.01) were strongly enriched, directly correlating with HSP family proteins in recognizing and refolding misfolded proteins under heat stress.

2.3. Expression Pattern of PcHSP20 Genes

KEGG enrichment analysis demonstrated significant enrichment of heat shock proteins (HSPs) in the “protein processing in endoplasmic reticulum” pathway (map04141). Notably, the prominent induction of HSP20 family genes under heat stress prompted our attention. To systematically explore the role of this gene family in the heat stress response of Polygonatum cyrtonema, we further identified the complete set of PcHSP20s family genes, encompassing 24 members in total. Expression profiling showed that 16 of these genes were significantly upregulated under heat stress, while the remaining 8 showed no significant expression changes—indicating that the majority of PcHSP20s members are involved in mediating the heat stress response (Table S4). We therefore selected three representative genes from these upregulated PcHSP20s for further in-depth analysis. These three genes exhibited remarkably high differential expression, with fold changes of 982.29, 760.08, and 508.46, respectively (Figure 2A). The coding sequences (CDSs) of these genes were predicted via TransDecoder and subsequently translated into amino acid sequences (Table S5). Structural alignment confirmed that all three proteins share the same conserved domain, namely the characteristic alpha-crystallin domain (ACD). Accordingly, these three genes were designated as PcHSP12.8, PcHSP12.9, and PcHSP13.4 based on their predicted molecular weights. Subsequently, to verify whether the expression patterns of these three genes were consistent with the transcriptome sequencing data, quantitative real-time PCR (qRT-PCR) was performed. The results showed that three PcHSP20 genes were significantly up-regulated (p < 0.05) under high-temperature stress (Figure 2A), which aligns consistently with the transcriptomic data.

To investigate the subcellular localization of PcHSP20 proteins, Three full-length coding regions of PcHSP20 lacking the termination codon were cloned and individually fused in-frame with the green fluorescent protein (GFP) gene to construct the fusion expression vectors 35S::PcHSP20-GFP, where the cauliflower mosaic virus (CaMV) 35S promoter drives the constitutive expression of the corresponding chimeric genes. Subsequently, in accordance with the established protocol described by Sparkes et al. [17]. The 35S::PcHSP20-GFP vectors and the nuclear-specific marker NLS-mCherry (red fluorescence; the nuclear localization signal [NLS] ensures its exclusive nuclear targeting) were co-transformed into the epidermal cells of Nicotiana benthamiana via Agrobacterium tumefaciens strain GV3101-mediated transient transformation. Fluorescent signals were visualized using an Olympus FV3000 confocal laser scanning microscope with the following optimized parameters: GFP was excited at 488 nm, and its emission was collected within the range of 498–548 nm; mCherry was excited at 543 nm, and its emission was detected within the range of 560–620 nm. Image overlay and colocalization analysis revealed that the green fluorescent signals of PcHSP20-GFP completely overlapped with the red fluorescent signals of NLS-mCherry, producing orange-yellow merged signals (Figure 2B). Collectively, these results demonstrate that all three PcHSP20 proteins are specifically localized to the nucleus. Notably, PcHSP13.4 exhibited distinct punctate green fluorescent signals, whereas such punctate green fluorescent structures were absent in the GFP signals of the other two PcHSP20 proteins. Their green fluorescent signals exclusively overlapped with that of NLS-mCherry in the nucleus, generating orange-yellow merged signals, with no additional cytoplasmic fluorescent features observed.

2.4. Phylogenetic and Structural Analysis of PcHSP20 Genes

To characterize the structural features of the three selected genes, their tertiary protein structures were predicted using AlphaFold 3 based on the corresponding amino acid sequences (Figure 3A). Pairwise structural comparisons revealed a high degree of conservation among the three proteins. The greatest structural similarity was observed between PcHSP12.8 and PcHSP12.9 (RMSD 0.645 Å over 78 atom pairs). Both proteins exhibited moderate divergence from PcHSP13.4, with RMSD values of 0.855 Å and 0.882 Å (over 52 atom pairs), respectively. All three of these PcHSP20 models exhibit the presence of a conserved alpha-crystallin domain (ACD)—a hallmark of small heat shock proteins (sHSPs)—which folds into an antiparallel β-sandwich conformation critical for chaperone function. For comparative analysis, we also included representative HSP20 proteins from Arabidopsis thaliana (AtHSP17.6B, light brown) and Oryza sativa (OsHSP17.9, light purple), both of which similarly harbor the ACD; notably, PcHSP13.4 displays a β-strand count most structurally analogous to that of these two orthologs. While the core β-strand region is structurally conserved and facilitates oligomerization, the intrinsically disordered regions (IDRs) exhibit conformational flexibility that is essential for substrate recognition. Variations in β-strand topology and IDR properties across HSP20 proteins from distinct species indicate that such underlying structural adaptations may enable these sHSPs to perform their functions across a broad spectrum of physiological and stress conditions.

Furthermore, conserved domain analysis confirmed that all three PcHSP20s’ motifs belong to the sHSP family (Figure 3B)—defined by the ACD, which is linked to chaperone activity. This aligns with our prior Gene Ontology (GO) enrichment results, where “heat shock protein binding” (GO:0031072) and “response to heat” (GO:0009408) were significantly enriched in the gene set. We also incorporated the conserved domains of Arabidopsis thaliana AtHSP17.6B and Oryza sativa OsHSP17.9 for comparative analysis: the identified domains include several variants and superfamilies, such as ACD_sHsps_p23_like (unique to AtHSP17.6B) and ACD_sHspB9_like (unique to OsHSP17.9)—both absent in PcHSP20 proteins. This structural divergence plus the shared core ACD reflects cross-species evolutionary conservation (core ACD) and functional diversification (lineage-specific variants). Finally, to elucidate the evolutionary relationships of these genes, we constructed a phylogenetic tree using HSP20 protein sequences from Arabidopsis thaliana, Oryza sativa, and the three P. cyrtonema HSP20s (Figure 3C). The analysis showed that PcHSP12.8 clusters closely with OsHSP20-33, while PcHSP12.9 exhibits the highest sequence identity (91%) with AtHSP20-16, and PcHSP20-13.4 (PcHSP13.4) shares 73% identity with OsHSP20-12. Notably, PcHSP20-12.8 (PcHSP12.8) and PcHSP20-12.9 (PcHSP12.9) group within the same clade, indicating a close evolutionary relationship between them.

2.5. Heterologous Expression of PcHSP20 in Yeast Confers Thermotolerance

To investigate whether these three PcHSP20 genes function as key determinants of thermotolerance, we utilized a Saccharomyces cerevisiae strain with a knockout of the SKN7 gene (SKN7Δ). Previous studies have confirmed that compared to the wild type, SKN7Δ renders yeast significantly hypersensitive to both heat stress and reactive oxygen species (ROS). Thus, SKN7Δ is commonly used as an ideal stress-sensitive model to verify the stress-resistant functions of target genes, by assessing whether these genes can rescue the mutant’s stress-sensitive phenotype (e.g., improved survival under heat stress) [18]. Subsequently, heterologous expression vectors for the three genes were constructed and successfully transformed into the SKN7Δ yeast strain. Yeast cells were subjected to heat stress at 52 °C for 15, 20, and 25 min. Spot assay results revealed that the heat-sensitive SKN7Δ mutant carrying the empty vector pYES2 exhibited significantly reduced cell viability under heat stress (Figure 4), while wild-type yeast grew normally, consistent with established findings. In contrast, the three SKN7Δ yeast strains heterologously expressing PcHSP12.8, PcHSP12.9, or PcHSP13.4 consistently displayed better survival than the pYES2-transformed SKN7Δ control strain. This indicates that the PcHSP20 family proteins, acting as molecular chaperones, can confer a degree of thermotolerance to cells, However, compared with the pYES2 empty vector control, yeast expressing PcHSP12.8 exhibited a significantly increased survival rate, showing a clear heat protection effect. In contrast, PcHSP13.4 and PcHSP12.9 did not significantly improve yeast survival, indicating that their heat protection effects are mild and limited under the experimental conditions.

2.6. PcHSP20 Overexpression on Arabidopsis thaliana Grow Better Under Heat Stress

To functionally validate the thermotolerance conferred by the three target genes, transgenic Arabidopsis lines overexpressing 35S::PcHSP12.8, 35S::PcHSP12.9, and 35S::PcHSP13.4 were generated (Figure S2). Studies have confirmed that 50 °C is effective in differentiating the heat tolerance phenotypes between transgenic and wild-type Arabidopsis seeds [19,20]. Thus, wild-type (WT) and transgenic Arabidopsis seeds were exposed to 50 °C for 2 h immediately after sowing, with untreated seeds serving as controls. Germination rates were scored on day 3, and root lengths were measured on day 10. For survival assays, 10-day-old seedlings were subjected to 50 °C for 30 min, then allowed to recover for 72 h at 22 °C before survival rates were evaluated. Heat treatment (HT) suppressed germination in both WT and transgenic plants compared to the control group. However, the germination rates of transgenic Arabidopsis seeds were significantly higher than those of WT seeds (Figure S3A,B). Root lengths showed no significant difference between transgenic and WT plants under control conditions. In contrast, under HT conditions, root growth was inhibited in both WT and transgenic plants compared to controls, but transgenic Arabidopsis exhibited significantly longer roots than WT plants (Figure 5A,B). These results suggest that PcHSP20 likely confers enhanced tolerance to the inhibitory effects of heat stress on root growth, enabling plants to maintain relatively normal growth and development under thermal stress. Regarding the statistical results of survival rates, all plants grew normally under control conditions. Following heat treatment, transgenic plants displayed significantly higher survival rates compared to WT plants (Figure S3C,D). This indicates that PcHSP20 overexpression enhances thermotolerance in plants, leading to improved survival under high-temperature stress.

2.7. Physiological and Biochemical Changes Following Heat Stress

Heat stress typically induces changes in reactive oxygen species (ROS) levels within plants. The capacity to scavenge ROS is a key indicator of plant stress tolerance [21]. Therefore, we performed DAB and NBT staining on leaves from transgenic plants before and after stress treatment to detect H₂O₂ and O₂⁻ accumulation, respectively. Seedlings in the treatment group were exposed to 42 °C for 3 h, while the control group remained at 22 °C. Leaves from 35S::PcHSP12.8, 35S::PcHSP12.9, and 35S::PcHSP13.4 transgenic lines consistently exhibited less intense DAB and NBT staining compared to WT leaves following heat stress (Figure 6A–D). This result indicates that the accumulation of ROS (H₂O₂ and O₂⁻) was lower in the leaves of all three types of transgenic plants under heat stress conditions.

The accumulation of reactive oxygen species (ROS) that can damage cellular components. To mitigate this damage, plants activate antioxidant defense systems, including enzymes like catalase (CAT) and peroxidase (POD), which scavenge harmful ROS such as H₂O₂ [22]. Concurrently, heat stress frequently disrupts photosynthetic machinery, leading to chlorophyll degradation and reduced photosynthetic efficiency [23]. To assess the impact of PcHSP20 overexpression on these key physiological responses under heat stress, we conducted biochemical analyses. Three-week-old hydroponically grown wild-type (WT) and transgenic Arabidopsis plants were used. Plants in the treatment group were exposed to heat stress at 42 °C for 3 h, while the control group remained at 22 °C. Following treatment, CAT and POD activities were quantified using specific enzyme activity assay kits according to the manufacturer’s protocols. Chlorophyll content was determined spectrophotometrically using the acetone extraction method [24]. Under normal growth conditions (22 °C), CAT and POD activities remained relatively stable and showed no significant differences between WT and the various transgenic lines. However, after heat stress, both CAT and POD activities were significantly higher in all transgenic lines compared to WT plants (Figure 6E,F). This elevated antioxidant enzyme activity suggests that PcHSP20 overexpression enhances the plant’s capacity to rapidly detoxify excess ROS generated during high-temperature stress. Consistent with the antioxidant enzyme results, chlorophyll content measurements also revealed a significant difference under heat stress. Transgenic plants overexpressing PcHSP20 genes maintained markedly higher levels of chlorophyll compared to WT plants following the heat treatment (Figure 6G). Taken together, these physiological and biochemical data—increased antioxidant enzyme activity and preserved chlorophyll content—demonstrate that PcHSP20 overexpression confers enhanced thermotolerance to transgenic Arabidopsis plants relative to the wild type under heat stress conditions.

2.8. PcHSP20 Enhances Arabidopsis Thermotolerance via HSF-HSP Pathway

The heat shock factor-heat shock protein (HSF-HSP) pathway is a conserved core regulatory mechanism in the heat stress response (HSR) of various plant species. It not only mediates chaperone synthesis but also balances thermotolerance and plant growth. To investigate whether PcHSP20 acts through the HSF-HSP pathway and additional heat-responsive mechanisms, we employed RT-qPCR to examine the expression profiles of heat stress-related genes in wild-type Arabidopsis thaliana and three PcHSP20 overexpression lines (PcHSP12.8-OE, PcHSP12.9-OE, and PcHSP13.4-OE) under normal conditions (25 °C) and heat stress (42 °C for 3 h). These analyses systematically elucidated the regulatory role of PcHSP20 genes in enhancing plant thermotolerance.

This study systematically elucidated the regulatory role of PcHSP20 genes in enhancing plant thermotolerance by comparing the expression profiles of heat stress-related genes between wild-type Arabidopsis and three PcHSP20-overexpressing lines under normal and heat-shock conditions (Figure 7). The results showed that several overexpression lines already exhibited basal upregulation of protective genes under non-stress conditions, including the heat shock protein AtHSP90 and the heat-related transcription factor AtWRKY1, suggesting that PcHSP20 may pre-activate the cellular protective network through a “pre-adaptation” mechanism [25,26]. Following heat-shock treatment, the expression of these genes was further significantly enhanced. In particular, the expression levels of key HSPs such as AtHSP17.6, AtHSP18.2, and AtHSP101 [27] were markedly higher than those in the wild type (Figure 7A). Concurrently, the core heat-signal transcription factor AtHSFA2 maintained sustained high expression, collectively driving a more efficient heat stress response. Moreover, expression changes of transcription factors such as WRKY1, WRKY33, and DREB2A [28,29], as well as stress-responsive functional proteins AtGolS1 and AtRD29A [30,31], implied that PcHSP20 may orchestrate synergistic regulation through multiple pathways, not only enhancing heat tolerance but also improving antioxidant capacity and cross-stress adaptability (Figure 7B). In summary, overexpression of PcHSP20 genes can systematically reprogram the heat stress response network in Arabidopsis, spanning from pre-activation of gene expression and signal amplification to multi-level physiological protection, thereby providing important insights into the molecular functions of plant small heat shock proteins and the mechanisms underlying plant thermotolerance.

To further dissect the heat stress signaling pathway mediated by PcHSP12.9, and due to the lack of published whole-genome data for Polygonatum cyrtonema. we investigated whether its Arabidopsis homolog AtHSP22.0 (91% amino acid identity) is directly transcriptionally regulated by key heat shock factors (HSFs). Given that the HsfA1 subfamily and HsfA2 are key regulators of the heat stress response and are closely associated with AtHSP22.0 expression, coupled with STRING database predictions indicating high-confidence interactions between AtHSP22.0 and six Hsfs (AtHsfA1a/1b/1d/1e, AtHsfA2, and AtHsfA3) (Figure S4), we therefore performed a yeast one-hybrid assay to validate these interactions. In this assay, the AtHSP22.0 promoter cloned into the pAbAi (A specialized vector for yeast one-hybrid assays, used for genomic integration and positive clone selection) (Figure S5) vector was used as bait, co-transformed with individual Hsfs constructed in the pGADT7 prey vector for analysis. All controls performed as expected, confirming system validity. However, none of the candidate Hsfs showed detectable binding to the AtHSP22.0 promoter (Figure S6), as evidenced by the absence of yeast growth on selective medium containing Aureobasidin A in three independent experiments. These results suggest that the regulation of AtHSP22.0 by these Hsfs may not be direct under the tested conditions.

3. Discussion

Heat stress is a key adverse environmental factor limiting the growth, development, and accumulation of secondary metabolites in medicinal plants. This study, through transcriptome analysis, molecular cloning, heterologous expression, and transgenic functional validation, revealed the critical roles of three small heat shock protein genes, PcHSP12.8, PcHSP12.9, and PcHSP13.4, from Polygonatum cyrtonemain the response to heat stress.

3.1. High-Throughput Screening of Heat-Tolerance Related Genes in Polygonatum cyrtonema on Transcriptome Analysis

In this study, transcriptome sequencing of P. cyrtonema under heat stress identified a total of 2316 differentially expressed genes (Figure 1A, Table S1). Among these, heat shock protein-related genes were significantly enriched in the “protein processing in endoplasmic reticulum” pathway (Figure 1D). This result suggests that under short-term heat stress, P. cyrtonema rapidly activate the protein quality control system to maintain cellular homeostasis. The three significantly upregulated HSP20 genes selected for further study showed expression levels increased several hundred-fold after 3 h of heat stress, indicating they are likely core components of the early heat response in this species. Notably, Gene Ontology (GO) terms including “response to heat,” “heat shock protein binding,” and “DNA binding transcription factor activity” were significantly enriched, suggesting that the heat stress signal may coordinate the heat stress response by mediating the refolding of misfolded proteins and regulating transcriptional remodeling (Figure 1E). This systematic gene expression profile not only provides a candidate gene pool for heat tolerance research in the Polygonatumgenus but also offers insights into the transcriptional-level adaptation strategies of non-model medicinal plants to high temperatures.

3.2. PcHSP20 Genes Exhibit Conserved Thermotolerant Functions in Yeast and Arabidopsis

To further validate the functions of the candidate genes, we conducted heterologous expression experiments in the heat-sensitive yeast mutant SKN7Δ and generated transgenic Arabidopsis lines. In yeast, all three PcHSP20 genes significantly enhanced cell survival rates at 52 °C, indicating their possession of cross-species conserved chaperone functions, likely by preventing protein aggregation and maintaining proteostasis to enhance cellular thermotolerance. The mild protective effects of PcHSP13.4 and PcHSP12.9 may stem from limitations of the heterologous expression system. Plant sHSPs typically exert functions by forming complexes with other HSPs (e.g., HSP70, HSP90) [32], but evolutionary differences in sequence and function between yeast and plant HSP family members may hinder effective interaction with PcHSP13.4/PcHSP12.9. Notably, similar phenomena have been reported: Oryza sativa OsHSP17.0 and Arabidopsis thaliana AtHSP17.6B showed significantly weaker protective effects in yeast than in their native systems [33,34], supporting the impact of heterologous systems on plant sHSP function and the tissue specificity of sHSPs.

In Arabidopsis, overexpression lines exhibited improved germination rates, enhanced root elongation, and increased survival rates under heat stress (Figure 5A,B and Figure S3A–D), accompanied by reduced reactive oxygen species accumulation, elevated antioxidant enzyme activities, and improved chlorophyll retention (Figure 6A–G). These results collectively demonstrate that PcHSP20 genes not only directly participate in protein protection but may also enhance comprehensive plant thermotolerance at multiple levels by bolstering the antioxidant defense system and maintaining the stability of photosynthetic apparatus.

Particularly noteworthy are the subcellular localization results: PcHSP12.8 and PcHSP12.9 were primarily localized to the nucleus, whereas PcHSP13.4, in addition to nuclear localization (Figure 2B), displayed distinct punctate signals in the cytoplasm, suggesting potential localization to organelles such as peroxisomes. This compartmentalization difference may reflect functional specialization within the cell: nuclear-localized HSP20s might be associated with protecting the transcriptional machinery and chromatin structure, while the cytoplasm/peroxisome-localized PcHSP13.4 could be involved in regulating redox homeostasis. This provides new structural evidence for explaining the functional diversity of the HSP20 family in plants.

3.3. Insights into the Regulatory Mechanism of PcHSP20 Genes

To investigate the upstream regulatory mechanism of the PcHSP20 genes, we used the yeast one-hybrid assay to test the binding capacity of six heat shock transcription factors to the promoter of their Arabidopsis homolog, AtHSP22.0 (Figure S4). However, no significant direct interaction signals were observed. This negative result might stem from the following reasons: the transcriptional regulation of HSP genes in plants often relies on the formation of multi-protein complexes and modulation by post-translational modifications (e.g., phosphorylation), conditions which might be difficult to fully recapitulate in the yeast one-hybrid system [35,36]. Furthermore, beyond the canonical heat shock elements, the HSP20 promoters might contain other cis-acting elements requiring synergistic activation by multiple factors. Future studies could utilize chromatin immunoprecipitation (ChIP), dual-luciferase reporter assays, or CRISPR/Cas9-mediated promoter editing to further dissect their transcriptional regulatory network. Simultaneously, exploring their expression regulation from epigenetic and non-coding RNA perspectives will also contribute to a deeper understanding of their thermotolerance mechanisms.

4. Materials and Methods

4.1. Plant Materials

Uniform three-year-old cultivated plants of Polygonatum cyrtonema were collected from a cultivation base in Jiujiang, China. Plants were acclimatized in a growth chamber at 23 °C under an 8/16 h (light/dark) photoperiod, under a photosynthetic photon flux density (PPFD) of 60 μmol m⁻² s⁻¹ and 65% relative humidity for 14 days prior to experimentation. Wild-type Arabidopsis thaliana (ecotype Columbia-0, Col-0) used in this study was provided by Nanjing Agricultural University.

4.2. Strains and Plasmids

Escherichia coli DH5α: TaKaRa (Otsu, Shiga, Japan); Agrobacterium tumefaciens GV3101: Weidi Biotechnology (Shanghai, China); Saccharomyces cerevisiae BY4741 and SKN7Δ mutant: South China Botanical Garden, CAS (Guangzhou, Guangdong, China); pCAMBIA1302: Nanjing Agricultural University (Nanjing, Jiangsu, China); pGreenII-35S-mGFP6: Wuhan Miaoling Biotechnology (Wuhan, Hubei, China); pYES2: Qiyunbio (Nanchang, Jiangxi, China); Y1HGold strains [p53-AbAi+pGADT7-p53] (positive control) and [p53-AbAi+pGADT7-Rec2] (negative control): Coolaber (Beijing, China); Plasmids pGADT7 and pAbAi: Qiyunbio (Nanchang, Jiangxi, China).

4.3. Heat Stress Treatment of Polygonatum cyrtonema

For heat stress treatment, seedlings were transferred to a growth chamber set at 42 °C for 3 h, while control plants were maintained at 23 °C. The experimental design included two independent treatment groups and two control groups, with each group comprising three biological replicates (n = 3). Following treatment, all aerial tissues located at and above the position ~1 cm distal to the root crown were immediately collected, flash-frozen in liquid nitrogen, and stored at -80 °C for subsequent analysis.

4.4. RNA Extraction and Quality Assessment

Total RNA was isolated from frozen tissue samples using a polysaccharide-polyphenol plant total RNA extraction kit (Biosharp, Hefei, Anhui, China). RNA integrity was verified by 1.5% agarose gel electrophoresis, showing clear 28S and 18S rRNA bands with a ratio ≥ 1.5. RNA purity and concentration were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with all samples exhibiting A260/A280 ratios between 1.8 and 2.0.

4.5. Transcriptome Sequencing and Data Processing

High-throughput transcriptome sequencing was performed by LC-Bio (Hangzhou, China). mRNA was enriched, fragmented, and used to construct cDNA libraries. Paired-end (PE150) sequencing was conducted on the Illumina platform. Raw sequencing data were processed to remove low-quality reads and adapter sequences using fqtrim, and subsequent transcriptome assembly and expression quantification were performed using Trinity and Salmon, respectively.

4.6. Quantitative RT-PCR Analysis

Uniform three-year-old cultivated plants of Polygonatum cyrtonema were acclimatized in a growth chamber for 14 days under the same conditions as previously described. Plants were then exposed to 42 °C for 3 h for heat treatment, while control plants were maintained at the optimal temperature of 23 °C. After treatment, All aerial tissues located above the ~1 cm mark from the root crown were harvested for total RNA extraction and cDNA synthesis. Amplifications were performed on a LightCycler^® 480 System (Roche) under: 95 °C for 5 min; 40 cycles of 95 °C/10 s, 60 °C/30 s, 72 °C/15 s; melting curve analysis (65–95 °C). Using 18S rRNA as the internal reference gene (Table S2), gene expression was quantified on a LightCycler 480 system, and relative expression levels were calculated using the 2^−ΔΔCt method.

4.7. Vector Information and Screening Method

The vectors used for gene expression were 35S::PcHSP12.8, 35S::PcHSP12.9, and 35S::PcHSP13.4, constructed based on the pCAMBIA1302 vector. Vectors for subcellular localization of the three genes were pGreenII-35S-mGFP6, which contains the reporter gene GFP. Both pCAMBIA1302 and pGreenII-35S-mGFP6 harbor the CaMV 35S promoter, with kanamycin resistance (Kan_r) for bacterial selection and hygromycin resistance (Hyg_r) for plant selection. The vector used for yeast heterologous expression was pYES2, carrying the GAL1 promoter and the URA3 gene (for screening ura3 genotype yeast transformants by complementing uracil auxotrophy). Positive screening was performed using the uracil-deficient SG-Ura medium.

4.8. Cloning Procedure

Primer design: Upstream primers contained 15–20 bp homologous arms at the 5′ end, complementary to the upstream region of the linearized vector; downstream primers had 15–20 bp homologous arms at the 5′ end, complementary to the downstream region of the linearized vector.

PCR amplification: Target fragments were amplified from Polygonatum sibiricum cDNA using Qizyme Multi One Step high-fidelity cloning enzyme, with an annealing temperature of 58 °C for 35 cycles. Amplicons were verified by agarose gel electrophoresis, then recovered and purified.

Vector linearization: pGreenII-35S-mGFP6 was linearized by double digestion with EcoRI and SacI (37 °C, 30 min); pYES2 by EcoRI and BamHI; pCAMBIA1302 by BglII and SpeI. All digested products were verified by agarose gel electrophoresis, followed by recovery and purification.

Homologous recombination ligation: Purified target fragments and linearized vectors were mixed at the optimal ratio, supplemented with homologous recombination enzyme (4× MultiS Enzyme Premix), and incubated at 37 °C for 30 min.

Transformation: Recombinant products were transformed into competent Escherichia coli/SKN7∆ cells by heat shock (42 °C, 90 s). Positive clones were obtained via resistance screening.

4.9. Seed Germination Assay

Approximately 40 surface-sterilized seeds per genotype (two independent transgenic lines with three biological replicates) were sown on tripartite MS plates. Plates were: Treated at 50 °C for 2 h (heat stress) or 22 °C (control). Stratified at 4 °C for 48 h. Cultivated at 22 °C under 16 h light/8 h dark cycle. Germination rates were scored after 72 h.

4.10. Root Length Measurement

Seeds were sown vertically on square MS plates (n = 9 seedlings/line, three replicates). After identical heat/stress treatment and stratification as above, roots were measured after 10 days using ImageJ v1.53.

4.11. Plant Survival Assay

10-day-old seedlings (two true leaves expanded) were exposed to 50 °C for 0.5 h. Following 72 h recovery at 22 °C, survival rates were quantified based on turgor maintenance and chlorophyll retention.

4.12. Histochemical Staining (DAB/NBT)

Hydroponically grown 3-week-old plants were heat-stressed (42 °C/3 h) or maintained at 22 °C. Fully expanded leaves were vacuum-infiltrated with: DAB (1 mg/mL, pH 3.8) for H₂O₂ detection (4 h, 28 °C dark); NBT (0.5 mg/mL) for O₂⁻ detection (2 h, 28 °C dark). Destained in 75% ethanol until chlorophyll cleared. Stained areas were quantified using ImageJ.

4.13. Chlorophyll Quantification

Fresh tissue (100 mg) from identically treated plants was extracted in acetone:ethanol (4:1, v/v) for 24 h at 22 °C in darkness. Absorbance of supernatants (12,000× g, 10 min) was measured at 663 nm and 646 nm. Chlorophyll content was calculated using Lichtenthaler’s formulae.

4.14. RNA Extraction and qRT-PCR Analysis for Heat-Stressed Arabidopsis

Two-week-old wild-type and genotype-specific Arabidopsis seedlings grown in Hoagland’s nutrient solution were heat-treated at 42 °C for 3 h in an illuminated incubator, with 25 °C grown seedlings as controls. Total RNA was extracted post-treatment using the Universal Total RNA Extraction Kit (BG0050-L, BIOGROUND). Each biological sample (treatment/control) consisted of three uniformly grown seedlings to reduce experimental error.

First-strand cDNA was synthesized from 1 μg total RNA with a Reverse Transcription Kit (Biosharp). The cDNA was fivefold diluted and used as the template for qRT-PCR. The reaction system was prepared following the manufacturer’s protocol for SYBR Green qPCR Mix (Biosharp). Three technical replicates were set per sample, and qRT-PCR was performed as described above, with Actin2 (AT3G18780) used as the reference.

5. Conclusions

In summary, transcriptomic analysis of Polygonatum cyrtonema under short-term heat stress identified PcHSP12.8, PcHSP12.9, and PcHSP13.4 as candidate genes potentially associated with the heat shock response. Functional validation through heterologous expression in yeast and genetic transformation in Arabidopsis thaliana demonstrated that these three PcHSP20 genes contribute to thermotolerance under certain conditions, as evidenced by relative improvements in yeast survival under heat stress, along with enhanced germination rate, root elongation, and stress survival in transgenic Arabidopsis lines.