Authors: Jiao Yu, Yihang Zhou, Chaotian Xu, Fang Wang, Yanling Zheng
Categories: Research, Cycas panzhihuaensis, Freezing stress, Plant hormone signal transduction, Transcriptome
Source: BMC Plant Biology
Authors: Jiao Yu, Yihang Zhou, Chaotian Xu, Fang Wang, Yanling Zheng
Global warming intensifies climate extremes, with rising temperatures and more frequent heatwaves. This situation may make it more difficult for species in high-temperature regions, such as the Cycas panzhihuaensis. Introduction and domestication are vital for conservation, but low temperatures limit the spread of tropical and subtropical plants to higher latitudes and altitudes. Phytohormones mediate cold adaptation through complex signaling networks that regulate physiological and molecular responses. However, the hormonal regulation and molecular mechanisms underlying freezing tolerance in C. panzhihuaensis remain poorly understood.
This study used C. panzhihuaensis as the research subject. We exposed the plant to freezing stress and measures leaf hormone levels under CK (control group), F1(-5 °C for 1.5 h), and F2(-5 °C for 6 h). Additionally, we conducted transcriptomic analysis to explore gene expression differences in plant hormone signal transduction pathways. The findings indicated that with prolonged freezing treatment, the contents of cis-12-oxophytodienoic acid (cis-OPDA), gibberellin A4 (GA4), and salicylic acid (SA) initially increased significantly but subsequently decreased markedly. After F2 treatment, abscisic acid (ABA) levels significantly decreased, whereas castasterone (CS), 1-aminocyclopropanecarboxylic acid (ACC), and cytokinin (CTK) exhibited notable increases. Through transcriptomic analysis, a total of 4,036 differentially expressed genes (DEGs) associated with freezing stress were identified. In the plant hormone signal transduction pathway, 69 DEGs were enriched as determined by KEGG enrichment analysis. Further analysis indicated that the altered gene expression in this pathway was closely associated with hormonal level variations and the freezing resistance of C. panzhihuaensis. This research offers essential understanding of the molecular processes behind the freezing resistance of C. panzhihuaensis and provides theoretical advice for its introduction and cultivation.
This study revealed that C. panzhihuaensis adapted to freezing stress through dynamic hormonal regulation and transcriptional reprogramming. Key phytohormones (cis-OPDA, GA4, SA, ABA, ACC, CS, and CTK) exhibited stage-specific accumulation patterns, and the plant hormone signal transduction pathway may be involved in regulating the cold resistance mechanism in C. panzhihuaensis. These findings provide crucial insights into cold resistance mechanisms of C. panzhihuaensis and establish a preliminary theoretical foundation for introducing this species to higher latitudes.
The online version contains supplementary material available at 10.1186/s12870-025-07907-7.
Cycads are among the oldest extant seed plants, having persisted and flourished to this day. They have undergone significant environmental changes and contain rich genetic data, which makes them extremely important for the research on the origin and early evolution of seed plants [1]. Despite their ancient lineage, cycads have become one of the most endangered species due to the destruction of plant resources by human activities (habitat loss, overexploitation, grazing, invasive alien plants, etc.) and the intensification of global climate change [2]. Conserving cycads is not only about protecting biodiversity, but also about safeguarding a living record of Earth’s evolutionary history. This endeavor holds profound social significance for maintaining ecological balance and advancing our scientific understanding of life’s evolution. C. panzhihuaensis is a plant of the Cycadaceae family. As an ancient relic species endemic to China, it holds great importance for research in plant geography, phytogeography, paleoclimatology, and paleogeography. C. panzhihuaensis is primarily distributed in the arid-hot valley along the middle reaches of the Jinsha River, representing the northernmost natural distribution of extant Cycas species worldwide [3]. Yet it is precisely this unique distribution pattern that has placed its survival under severe threat. The natural habitat of this species occurs in semi-arid valley climate zones within the northern subtropical region, where extreme temperatures frequently exceed 40 °C. The combination of highly variable spatiotemporal precipitation patterns, nutrient-poor soil conditions, and low water retention capacity creates exceptionally challenging growth conditions, rendering these arid-hot valley ecosystems particularly vulnerable [4]. Although the prolonged stability of tropical climates, the rapid formation of species in tropical regions results in numerous species with limited range and population sizes, making them vulnerable to habitat disturbances and seemingly more prone to natural extinction [5]. With the intensification of global warming, climate fluctuations are becoming more severe. The average temperature is rising, leading to more frequent instances of hot weather. Extreme high-temperature events may make species living in hot areas, such as C. panzhihuaensis, even more difficult to adapt, thereby accelerating the process of extinction. This highlights the urgent need for effective conservation interventions. Conservation strategies such as assisted introduction and domesticated cultivation hold promise for mitigating these risks. However, low temperature poses a significant barrier to the expansion of tropical and subtropical plants into higher latitude and altitude regions. Our previous physiological studies have shown that C. panzhihuaensis exhibits relatively strong cold resistance among Cycas species, indicating its potential for introduction to higher latitudes and altitudes [6]. Therefore, an in-depth investigation and elucidation of its inherent cold-tolerance mechanisms are essential for expanding its suitable range and mitigating its endangered status through scientifically guided introduction. Nevertheless, the molecular mechanisms governing this cold tolerance remain entirely unknown, severely limiting our capacity to develop science-based conservation or breeding programs.
Among the main abiotic stresses, low temperature is particularly detrimental to plant growth and development, which in turn hampers the sustainable advancement of agriculture and forestry. To counteract this adverse condition, plants possess a comprehensive regulatory system, including photosynthetic adjustments, antioxidant defenses, cell membrane modifications, and alterations in metabolic and protein production pathways [7]. Transcriptional regulation is an important biological process during plant growth, and these physiological responses are often precisely regulated at the level of transcription [8]. In recent years, RNA sequencing (RNA-seq) technology has dramatically accelerated research into plant cold response mechanisms. This technology enables not only rapid identification of key transcription factors, but also reveals metabolic pathways involving differentially expressed genes and facilitates reconstruction of gene regulatory networks [9]. Research demonstrates that plants employ transcriptional cascade reactions to convert external low-temperature stimuli into internal signals, thereby activating defense mechanisms to deal with harsh environmental circumstances [10]. To cope with cold stress, plants have established several signaling pathways that activate particular transcription factors and functional proteins, resulting in physiological and biochemical modifications that boost cold stress resistance [11, 12]. Among the many signaling pathways, the regulatory role of plant hormone signaling pathways is of central importance. Temperature-induced molecular and physiological reactions in plants are modulated by hormone regulation, a process that critically governs immune responses, stress adaptation, and developmental processes [13].
A series of plant hormones regulate stress responses in plants, including auxin (IAA), ABA, CTK, SA, gibberellins (GAs), jasmonic acid (JA), ethylene (ETH), and brassinosteroids (BR) [14]. The content and activity of these hormones are altered under low-temperature stress and hormonal homeostasis is regulated through their biosynthesis, degradation, and transport [15]. As an essential plant hormone, auxin mediates plant morphogenesis through regulating embryogenesis, elongation of stems and roots, meristem and vascular differentiation, and also participates in plant responses to various environmental signals [16]. Under various stress conditions, plants regulate the content and distribution of auxin, auxin receptor proteins, and auxin transporter proteins, which in turn modulate downstream auxin-responsive genes to confer stress adaptation [17]. Abscisic acid is critical for enhancing plant stress tolerance by modulating ABA-responsive gene expression through signal transduction pathways during various ecological challenges, including freezing, drought, high-salinity/osmotic stress and other abiotic threats [18]. It has been demonstrated that cold stress inhibits the regulation of MdGH3-2/12 and activates the regulation of MdNCED2 by MdHY5, thereby suppressing IAA accumulation, promoting ABA synthesis and anthocyanin accumulation, and enhancing the capacity for reactive oxygen species (ROS) scavenging, which is involved in the cold acclimation process of Malus domestica [19]. Cytokinins are essential at every stage of the plant growth cycle. They promote cell division and differentiation while suppressing chlorophyll and macromolecular degradation to delay senescence, and function as pivotal signaling regulators in plant stress adaptation [20]. During the response to imbibitional chilling stress in Arachis hypogaea, the abundance of cytokinin oxidase decreases, leading to an increase in cytokinin content under cold stress, and exogenous cytokinin application may alleviate chilling injury during water absorption [21]. Phytohormones including JA, SA, and ETH mediate endogenous signaling in plants, coordinately regulating metabolic processes and activating both defense responses and growth regulation to enhance abiotic stress tolerance [22]. Jasmonic acid, a hormone derived from lipids, has been evidenced to aid in the reduction of cold stress in numerous plants [23]. For example, in Arabidopsis thaliana, JA signaling is positively regulated in response to freezing stress through the interaction between jasmonate ZIM-domain (JAZ) proteins and ICE1/2, and blocking endogenous jasmonic acid synthesis and signaling increases plants sensitivity to freezing stress [24]. In banana, the stress-activated transcription factor MusaDREB1G boosts ABA and JA levels by inducing their biosynthetic genes, while enhancing ROS scavenging, thereby synergistically improving drought and cold tolerance [25]. Furthermore, in Citrullus lanatus, the synergistic interaction between SA signaling and C-repeat binding factor (CBF) pathways confers enhanced cold defense responses [26]. In Arabidopsis, the NAC member ATAF2 enhances stress adaptation by activating genes that promote both ethylene biosynthesis and response [27]. In summary, low temperature significantly modulates phytohormone metabolic pathways, while these hormones reciprocally regulate plant cold responses through intricate signaling networks. Therefore, systematic investigation of cold-induced hormonal biosynthesis and associated gene expression changes in signaling transduction pathways is crucial for deciphering plant cold resistance mechanisms.
Due to the overexploitation of ornamental plants in nature, C. panzhihuaensis has also been facing potential endangered challenges. Therefore, it is crucial for the conservation of C. panzhihuaensis to study its potential mechanisms of environmental adaptability. Despite these well-documented mechanisms in model plants, the molecular basis of cold adaptation in ancient gymnosperms like cycads remains a scientific blind spot. Current research on abiotic stress tolerance in C. panzhihuaensis has primarily focused on physiological biochemistry, lipid metabolism, and energy metabolism [6, 28, 29], while molecular mechanisms underlying its freezing stress response remain largely unexplored. This gap not only hinders the development of effective conservation strategies for this endangered species but also limits our understanding of the evolution of cold adaptation mechanisms in seed plants. Based on the above, we propose that the freezing stress response in C. panzhihuaensis is mediated by phytohormone signaling pathways. To test this hypothesis, we subjected C. panzhihuaensis to freezing stress and investigated the differences in plant hormone concentrations and the transcription levels of key genes to uncover the molecular regulatory mechanisms of freezing stress response. Our findings are expected to provide a molecular-level foundation for the conservation and climate-resilient cultivation of this endangered cycad, while also offering evolutionary insights into how ancient seed plants respond to low-temperature stress.
The seeds of the C. panzhihuaensis were collected in Panzhihua with the approval of the Administration Bureau of Panzhihua Cycas National Nature Reserve, Sichuan province. The seedlings of this species were identified by Shuangzhi Li, a taxonomist expert at Southwest Forestry University. Voucher specimens of C. panzhihuaensis have been deposited in the herbarium of Southwest Forestry University with an accession number No. ZYL-001, respectively. The C. panzhihuaensis used in this study were all cultivated in the greenhouse of Southwest Forestry University. The potted soil was composed of sandy loam, humus, and red soil (mixed in a ratio of 1:1). A total of three treatment groups were established for this experiment. During the experimental period, the temperature was approximately 15–25 ℃. The specific temperature parameters and duration for the freezing stress treatment were determined based on preliminary studies. We placed healthy and uniformly growing 6-year-old plants of C. panzhihuaensis into a controlled-environment chamber. The temperature was set to decrease from 4 ℃ to − 1 ℃ at a rate of 2 ℃/h. After maintaining at − 1 ℃ for 1 h, frost was sprinkled on the surface of the potted soil to induce freezing. The temperature was subsequently reduced at the same cooling rate to − 5 °C and held constant for 1.5 h (F1). Another treatment was kept at − 5 ℃ for 6 h (F2). The light intensity during the entire treatment was 300 µmol m^− 2^ s^− 1^. Plants without freezing treatment served as the control group (CK). For each treatment, five independent biological replicates (n = 5) were established. Each biological replicate sample was created by pooling leaf tissues from three uniformly grown seedlings. Samples were immediately collected after the cold stress treatment, frozen in liquid nitrogen, and stored at − 80 ℃.
The types and contents of plant hormones were determined according to the method of Liu et al. [30] with modifications. The hormones analyzed in this study included ABA, ACC, CS, cis-OPDA, N6-isopentenyladenine (iP), isopentenyladenine riboside (iPR), trans-zeatin (tZ), trans-zeatin-riboside (tzR), cis-zeatin (cZ), cis-zeatin-riboside (czR), GA4, IAA and SA. The total content for each major hormone category was subsequently calculated. The detailed procedures are described below.
The homogenized sample (100 mg) was weighed after liquid nitrogen grinding and sequentially mixed with 1170 µL of acetonitrile-water-formic acid solution (80:19:1, v/v), 10 µL of ISMix-A (internal standard), and 20 µL of ISMix-B. The mixture was vortexed (60 s), sonicated under dark conditions at low temperature (25 min), and incubated at − 20 °C overnight. Following centrifugation (14,000 rcf, 20 min, 4 °C), 900 µL of supernatant was processed through an Ostro 25 mg 96-well plate using positive pressure filtration. The filtrate was washed with 200 µL of fresh extraction solvent (same composition) before nitrogen evaporation. The dried extract was stored at − 80 °C pending LC-MS/MS analysis.
The chromatographic separation was performed on an Agilent 1290 Infinity LC UHPLC system with the autosampler maintained at 4 °C and the column temperature set at 45 °C. The mobile phase consisted of 0.05% formic acid in water (A) and 0.05% formic acid in acetonitrile (B), delivered at a flow rate of 400 µL min^− 1^ with an injection volume of 4 µL. The gradient elution program was optimized as 0–1 min, 2–10% B; 1–10 min, 10–70% B; 10–11 min, 70–95% B; 11–11.1 min, 95 − 2% B; and 11.1–13 min, 2% B for column re-equilibration. Quality control (QC) samples were interspersed at regular intervals throughout the sample sequence to monitor system stability and reproducibility, while standard mixtures of target analytes were analyzed for retention time alignment and method validation.
Mass spectrometric analysis was performed using a 5500 QTRAP mass spectrometer (SCIEX) operating in both positive and negative ion modes. For the positive ion mode electrospray ionization (ESI) source, the parameters were set as source 550 ℃;ion Source Gas1(Gas1)༚55༛Ion Source Gas2 (Gas2)༚50༛Curtain gas (CUR)༚30༛ionSapary Voltage Floating(ISVF)༚4500 V. The negative ion mode ESI source employed identical gas and temperature conditions (GS1: 55 psi; GS2: 50 psi; CUR: 30 psi; source 550 °C) with an ISVF of − 4500 V. All analytes were detected using multiple reaction monitoring (MRM) mode with optimized transitions for target ion pairs.
Total RNA was isolated from fifteen samples using the RNAprep Pure Plant Kit (Tiangen, China). RNA integrity and quantity were assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) to ensure suitability for cDNA library construction. Poly(A) + mRNA was enriched from total RNA using Oligo(dT) magnetic beads and subsequently fragmented to approximately 300 bp. First-strand cDNA was synthesized using random hexamer primers and reverse transcriptase, followed by second-strand synthesis. The resulting cDNA libraries were amplified by PCR, and size selection was performed to obtain fragments with an average size of 450 bp. Final library quality and concentration were confirmed using the Agilent 2100 Bioanalyzer. The qualified libraries were then subjected to paired-end (PE) sequencing on an Illumina NovaSeq 6000 platform (Illumina, USA).After sequencing, the raw transcriptome sequencing data were obtained and compared against the C. panzhihuaensis genome database (https://db.cngb.org/codeplot/datasets/public_dataset?id = PwRftGHfPs5qG3gE). The raw sequencing data have been deposited in the NCBI database under the BioProject accession number PRJNA1279757.
We performed GO and KEGG enrichment analyses to better understand the functions and pathway enrichment of DEGs in C. panzhihuaensis following freezing stress. The categories of GO annotation include biological process (BP), molecular function (MF), and cellular component (CC). In this study, DESeq2 was used for differential gene expression analysis, and genes with |log2FoldChange| >1 and P-value < 0.05 were defined as significantly differentially expressed genes.
To confirm the RNA-Seq findings, 11 DEGs associated with plant hormone signal transduction were selected for qRT-PCR validation. Total RNA was reverse-transcribed into cDNA using the FastKing RT Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. Gene-specific primers were designed using Primer Premier v6.0 (Premier Biosoft Inc., CA, USA) and are listed in Table S1. Primer specificity was confirmed by melting curve analysis and agarose gel electrophoresis, showing a single amplification product. The amplification efficiency of the primers was assessed using a ten-fold serial dilution of the standard template, yielding efficiencies between 95% and 100% with R² >0.99. qRT-PCR was performed using the BIO-RAD CFX384 real-time PCR detection system (Bio-Rad Laboratories, Shanghai, China) with Power qPCR PreMix (Genecopoeia, USA). Each reaction was performed in triplicate, and ACTB was used as the internal reference gene. According to the method by Livak and Schmittgen [31], the relative expression levels of these genes were quantified using the 2^−ΔΔCT^ approach.
Phytohormones serve as indispensable signaling molecules that enable plants to perceive environmental changes, modulate growth and development, and enhance stress resilience to ensure survival. We quantitatively measured the plant hormone content in the leaves of C. panzhihuaensis under different cold stress durations and found that the content of different endogenous hormones showed distinct patterns of change with the extended treatment time (Fig. 1a). As the treatment time increased, ABA content decreased, with CK and F1 groups showing significantly higher levels than the F2 group. In contrast, the contents of ACC and CTK showed the opposite trend to that of ABA. Extended treatment time led to an increase in ACC and CTK levels, with the F2 group having significantly higher levels than the CK and F1 groups. No significant differences were observed in the levels of these three hormones between the CK and F1 treatments. The concentrations of cis-OPDA, GA4, and SA increased significantly by 197.3%, 32.9%, and 110.4% respectively following F1 treatment, but decreased markedly by 59.2%, 64.2%, and 95.1% after F2 treatment. With the extended cold stress duration, the level of CS first decreased and then increased. After the F2 treatment, its level was significantly higher than that in the CK and F1 treatments, although there was no significant difference between the CK and F1 treatments. Additionally, the content of auxin showed no significant difference after F1 and F2 treatments. Correlation analysis (Fig. 1b) revealed a significant correlation network among the tested phytohormones. ABA exhibited negative correlations with ACC, CS, and CTK, and positive correlations with cis-OPDA, GA4, and SA. ACC correlated positively with CS and CTK but negatively with GA4 and SA. Conversely, cis-OPDA correlated positively with GA4 and SA but negatively with CS. CS demonstrated negative associations with GA4, SA, and IAA, alongside a positive correlation with CTK. Notably, GA4 and SA showed a positive correlation with each other and a concurrent negative correlation with CTK. IAA and CTK were also significantly negatively correlated.Fig. 1Plant hormone contents (a) and correlation heatmap of hormone levels (b) in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). Different letters indicate significant difference between treatments based on Fisher’s least significant difference at P ≤ 0.05. Values shown are the mean ± SD, n = 5. Red indicates positive correlations, while blue represents negative correlations. ABA: abscisic acid; ACC: 1-aminocyclopropanecarboxylic acid; cis-12-oxophytodienoic acid; CS: castasterone; GA4: gibberellin A4; SA: salicylic acid; IAA: auxin; CTK: cytokinin
To identify DEGs significantly enriched in C. panzhihuaensis leaves under varying freezing durations, we applied selection criteria of |log2FoldChange| >1 with adjusted P-value < 0.05 when comparing freezing-stressed samples to controls. The results showed that in the CK vs. F1 comparison, the number of up-regulated DEGs reached 911, while the down-regulated DEGs numbered 1,387. In contrast, the CK vs. F2 comparison exhibited fewer up-regulated DEGs (646) but a higher number of down-regulated DEGs (1,509). Furthermore, between F1 and F2 treatments, there were 785 up-regulated DEGs and 1,093 down-regulated DEGs (Fig. 2a). To characterize global expression changes, we performed hierarchical clustering analysis of all DEGs and visualized the results as a heatmap (Fig. 2b). The Figure shows that the expression levels of DEGs in C. panzhihuaensis samples under CK, F1, and F2 treatments are highly consistent. An upset plot was generated to visualize the distribution of shared and unique DEGs among the three experimental groups (Fig. 2c). The results showed that a total of 2298 DEGs were identified in the CK vs. F1 treatment, 2155 in the CK vs. F2 treatment, and 1878 in the F1 vs. F2 treatment. In addition, 820 common genes were identified in both the CK vs. F1 and CK vs. F2 groups, 572 common genes in both the CK vs. F1 and F1 vs. F2 groups, and 591 common genes in both the CK vs. F2 and F1 vs. F2 groups. A total of 156 shared genes were identified across all three comparison groups (CK vs. F1, CK vs. F2, and F1 vs. F2). The expression of these differentially expressed genes reveals the unique response mechanisms of C. panzhihuaensis to freezing stress. For the detailed expression profiles of the most significant top 50 up and down-regulated genes across comparisons, please refer to supplementary Fig. S1. The complete list of all up and down-regulated DEGs from all comparison groups, with detailed annotations, is provided in supplementary Table S2.Fig. 2Analysis of DEGs in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Up-regulated, and down-regulated DEGs for each group. b: Clustering heatmap of DEGs for each treatment. The red color represents high expression, and the green color represents low expression. c: Upset plot of DEGs for each group. The connecting line of all points on the x-axis represents the number of common genes identified across all samples, while individual points or connected points represent the number of unique genes identified in the corresponding sample
To determine the functions of DEGs, we conducted GO enrichment analysis. In this study, a significance level of P-value < 0.05 was used as the criterion for comparing the sample data between low-temperature stress and control treatments. The lists of significantly enriched GO terms for each comparison group are provided in Table S3. The top 10 results were selected to create the GO enrichment analysis chart (Fig. 3). Compared to CK, the 17,234 DEGs in F1 are enriched in 502 GO terms, of which 18 are related to the CC category, with the most significantly enriched term being nuclear nucleosome (GO: 0000788); 96 are related to the MF category, with the most significantly enriched term being transcription regulator activity (GO: 0140110); and 388 are related to the BP category, with the most significantly enriched term being regulation of nucleic acid-templated transcription (GO: 1903506). In the CK vs. F2 comparison, 20,001 DEGs were enriched in 602 GO terms. These included 22 CC terms, 134 MF terms, and 446 BP terms. The most significantly enriched terms were plasma membrane (GO: 0005886) in CC, DNA-binding transcription factor activity (GO: 0003700) in MF, and response to oxygen-containing compound (GO: 1901700) in BP. In the F1 versus F2 comparison, 12,730 DEGs were significantly enriched in 506 GO terms, including 20 CC terms (most significantly enriched plasma membrane, GO: 0005886), 106 MF terms (most significant molecular transducer activity, GO: 0060089), and 380 BP terms (most significantly enriched defense response, GO: 0006952).Fig. 3GO enrichment analysis of DEGs in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: CK vs. F1; b: CK vs. F2; c: F1 vs.F2. CC: cellular component; MF: molecular function; BP: biological process
The lists of significantly enriched KEGG pathways for each comparison group are provided in Table S4. The top 10 most significantly enriched pathways were selected to create the KEGG enrichment analysis plot (Fig. 4). The KEGG enrichment analysis results indicated that all DEGs involved in plant hormone signal transduction (ko04075), plant-pathogen interaction (ko04626), MAPK signaling pathway - plant (ko04016), phenylpropanoid biosynthesis (ko00940), flavonoid biosynthesis (ko00941), glutathione metabolism (ko00480), alpha-Linolenic acid metabolism (ko00592), and linoleic acid metabolism (ko00591) were significantly enriched under different durations of freezing stress treatment. Moreover, compared to the CK group, we found that these DEGs were also commonly enriched in the F1 and F2 groups in the following cyanoamino acid metabolism (ko00460), starch and sucrose metabolism (ko00500), stilbenoid, diarylheptanoid and gingerol biosynthesis (ko00945), and ubiquinone and other terpenoid-quinone biosynthesis (ko00130). Furthermore, in both the F1 and F2 groups, DEGs involved in diterpenoid biosynthesis (ko00904) also exhibited significant enrichment. These results suggest that the freeze resistance of C. panzhihuaensis may be associated with the DEGs participating in these pathways.Fig. 4KEGG enrichment analysis of DEGs in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: CK vs. F1; b: CK vs. F2; c: F1 vs. F2. The size of the dots indicates the quantity of DEGs, and the color of the dots reflects the false discovery rate (FDR), with deeper red indicating higher enrichment significance
KEGG enrichment analysis revealed that plant hormone signaling pathways were significantly enriched in all freezing stress treatment groups compared to the CK group (Fig. 4), demonstrating the complexity of phytohormone regulatory networks in cold stress responses. We analyzed the expression patterns of DEGs in plant hormone signaling pathways and generated a clustering heatmap to visualize their transcriptional profiles (Fig. 5). It can be seen that these DEGs exhibit distinct expression patterns, with the expression patterns under F1 and F2 treatments being the most similar.Fig. 5Clustering heatmap of DEGs in plant hormone signal transduction pathway in Cycas panzhihuaensis under -5°C freezing stress for 1.5 h (F1) and 6 h (F2). The red color represents high expression, and the blue color represents low expression
Twenty-three DEGs were identified to be associated with auxin signal transduction (Fig. 6a), including CYCAS_000373 encoding an auxin influx carrier (AUX1/LAX) that was upregulated during both early and late stages of freezing stress, while CYCAS_024887 was specifically upregulated only in the early stage. The genes CYCAS_005761 and CYCAS_005762, which encode transport inhibitor response 1 (TIR1), were significantly upregulated in F2 compared with CK and F1. In addition, CYCAS_004428 and CYCAS_025840 both encode auxin-responsive protein IAA. The former was upregulated only in F2 compared with F1, while the latter was significantly upregulated in both F1 and F2 compared with CK. Meanwhile, among the genes encoding the auxin-responsive GH3 gene family (GH3), compared with CK, CYCAS_005084 was significantly downregulated in both F1 and F2, CYCAS_006649 was significantly downregulated in F1, and CYCAS_021100 was significantly upregulated in both F1 and F2. Among the eight identified SAUR family proteins (SAUR) genes, the majority exhibited downregulation under freezing stress. A key finding was the consistent downregulation of CYCAS_011297 throughout the stress period. The expression of another key gene, CYCAS_018325, was only transiently induced during the early stage. We also found that among the DEGs related to auxin signal transduction, six genes encoding protein phosphatase 2 C (PP2C) underwent significant changes, which are involved in the response to abscisic acid. We performed a correlation analysis to examine the relationship between the expression levels of these DEGs and the IAA content. The results showed that CYCAS_004428, CYCAS_004050, and CYCAS_012147 had the strongest correlation with IAA, all showing significant negative correlation (Fig. 6b).Fig. 6DEGs involved in auxin signal transduction in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Auxin signal transduction pathway. b: Correlation heatmap between IAA content and expression of DEGs in auxin signal transduction pathway (Red: positive correlation; Blue: negative correlation). AUX1,LAX: auxin influx carrier; TIR1: transport inhibitor response 1; IAA: auxin-responsive protein; GH3: auxin responsive GH3 gene; SAUR: SAUR family proteins; PP2C: protein phosphatase 2C
The DEGs involved in cytokinin signal transduction are shown in Fig. 7a. As can be seen from the figure, a total of 11 DEGs are involved in the encoding. Among the genes encoding arabidopsis histidine kinase 2/3/4 (AHK2/3/4), CYCAS_007097 showed significant downregulation in F2 compared to F1, while CYCAS_013006 exhibited significant downregulation in F1 versus CK but significant upregulation in F2 relative to F1. Additionally, CYCAS_014789 demonstrated significant upregulation in F2 compared to both CK and F1 groups. The two genes encoding histidine-containing phosphotransfer protein (AHP) showed different trends. Compared with F1, CYCAS_020776 was upregulated in F2, and compared with CK, CYCAS_030665 was downregulated in F1, with no significant differences in the other groups. In addition, three genes encoding GARP family transcription factor, ARR-B subfamily (ARR-B) were all upregulated in F1 vs. F2. Notably, CYCAS_025340 was downregulated in F1 (compared to CK) and then upregulated in F2. The two-component response regulator ARR-A family (ARR-A) is encoded by three genes, two of which (CYCAS_020397 and CYCAS_02250) showed significant downregulation in F2 compared to both CK and F1, while the third gene (CYCAS_022323) exhibited significant upregulation in F2 relative to CK. As shown in Fig. 7b, the expression levels of CYCAS_013006, CYCAS_014789, CYCAS_020776, CYCAS_013420, CYCAS_021486, CYCAS_025340, and CYCAS_022323 exhibited a significant positive correlation with CTK content.Fig. 7DEGs involved in cytokinin signal transduction in *Cycas panzhihuaensis *under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Cytokinin signal transduction pathway. b: Correlation heatmap between CTK content and expression of DEGs in cytokinin signal transduction pathway (Red: positive correlation; Blue: negative correlation). AHK2/3/ arabidopsis histidine kinase 2/3/4; AHP: histidine-containing phosphotransfer protein; ARR-B: GARP family transcription factor, ARR-B subfamily; ARR-A: two-component response regulator ARR-A family; CTK: cytokinin
In the gibberellin signal transduction pathway, the two genes encoding gibberellin receptor GID1 were both upregulated. Compared with CK and F1, CYCAS_022440 was significantly upregulated in F2, and compared with F1, CYCAS_030993 was significantly upregulated in F2 (Fig. 8a). Furthermore, both CYCAS_029265 (encoding DELLA protein) and CYCAS_001732 (encoding F-box protein GID2/SLY1) were significantly upregulated in F1 compared to CK, but showed significant downregulation in F2 relative to F1. The other gene encoding GID2/SLY1 (CYCAS_019902), displayed a distinct expression pattern, showing significant downregulation in F2 compared to CK. The correlation results between GA4 and DEGs indicate that the two genes encoding GID1 (CYCAS_022440 and CYCAS_030993) are significantly correlated with GA4, showing a negative correlation (Fig. 8b).Fig. 8DEGs involved in gibberellin signal transduction in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Gibberellin signal transduction pathway. b: Correlation heatmap between GA4 content and expression of DEGs in gibberellin signal transduction pathway (Red: positive correlation; Blue: negative correlation). GID1: gibberellin receptor; DELLA: DELLA protein; GID2/SLY1: F-box protein; GA4: Gibberellin A4
As illustrated in Fig. 9a, all genes encoding abscisic acid receptor PYR/PYL family (PYL) proteins exhibited significant upregulation in F1 compared to CK. Additionally, both CYCAS_001054 and CYCAS_028458 showed significant downregulation in F2 relative to F1, while CYCAS_027455 was significantly downregulated in F2 compared to both CK and F1. All PP2C-encoding genes exhibited significant upregulation in F2 compared to F1, except for CYCAS_014625. Notably, CYCAS_004050 and CYCAS_012147 were significantly downregulated in F1 relative to CK, while CYCAS_014625 and CYCAS_028494 demonstrated significant downregulation in both F1 and F2 when compared to CK. CYCAS_018925 encodes serine/threonine-protein kinase SRK2 (SNRK2), which is significantly downregulated in F2 compared with CK and F1. ABA responsive element binding factor (ABF) is encoded only by CYCAS_021444, which is downregulated in F1 compared with CK, and upregulated in F2 compared with F1. According to the correlation analysis results (Fig. 9b), it can be seen that CYCAS_004050, CYCAS_004909, and CYCAS_013366 are significantly negatively correlated with ABA.Fig. 9DEGs involved in abscisic acid signal transduction in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Abscisic acid signal transduction pathway. b: Correlation heatmap between ABA content and expression of DEGs in abscisic acid signal transduction pathway (Red: positive correlation; Blue: negative correlation). PYL: abscisic acid receptor PYR/PYL family; PP2C: protein phosphatase 2C; SNRK2: serine/threonine-protein kinase SRK2; ABF: ABA responsive element binding factor; ABA: abscisic acid
The DEGs related to ethylene signal transduction are shown in Fig. 10a. The Figure indicates that CYCAS_008768, which encodes an ethylene receptor (ETR, ERS), was significantly downregulated in F1 compared to CK but significantly upregulated in F2 compared to F1. In contrast, CYCAS_024227 was significantly upregulated in both F1 and F2 compared to CK. Ethylene-insensitive protein 3 (EIN3) is encoded by CYCAS_010234. Compared with F1, this gene is downregulated in F2. The expression of genes encoding ethylene-responsive transcription factor 1 (ERF1) was broadly suppressed by freezing stress, exhibiting a predominant downregulation trend. Furthermore, correlation analysis revealed that CYCAS_024227 exhibited a significant positive correlation with ACC, whereas CYCAS_010234 showed a significant negative correlation with ACC (Fig. 10b).Fig. 10DEGs involved in ethylene signal transduction in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Ethylene signal transduction pathway. b: Correlation heatmap between ACC content and expression of DEGs in ethylene signal transduction pathway (Red: positive correlation; Blue: negative correlation). ETR, ERS: ethylene receptor; EIN3: ethylene-insensitive protein 3; ERF1: ethylene-responsive transcription factor 1; ACC: 1-aminocyclopropanecarboxylic acid
In the plant hormone signal transduction pathways, xyloglucosyl transferase TCH4 is the only enzyme related to BR, which is encoded by three genes (Fig. 11a). Among them, compared with CK, CYCAS_003468 was significantly upregulated in F1, while CYCAS_003470 was significantly downregulated in F1. Compared with F1, CYCAS_003468 was downregulated in F2, whereas CYCAS_003471 was upregulated in F2. The three genes annotated to this pathway showed no significant correlation with CS (Fig. 11b).Fig. 11DEGs involved in brassinosteroid signal transduction in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Brassinosteroid signal transduction pathway. b: Correlation heatmap between CS content and expression of DEGs in brassinosteroid signal transduction pathway (Red: positive correlation; Blue: negative correlation). TCH4: xyloglucosyl transferase TCH4; CS: castasterone
As shown in Fig. 12a, most genes associated with the jasmonic acid signal transduction pathway were downregulated, with no genes showing significant upregulation. Among the 10 genes encoding JAZ, half of the genes were significantly downregulated in F1 and F2 compared to CK, while two genes were downregulated only in F1. The remaining three genes were significantly downregulated in F2 (compared to CK and F1). CYCAS_007122 and CYCAS_021982 both encode the transcription factor myelocytomatosis oncogene homolog 2 (MYC2). Compared with CK, the former was downregulated in both F1 and F2, while the latter was downregulated only in F2. At the same time, we found that CYCAS_019000 was significantly negatively correlated with cis-OPDA (Fig. 12b).Fig. 12DEGs involved in jasmonic acid signal transduction in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Jasmonic acid signal transduction pathway. b: Correlation heatmap between cis-OPDA content and expression of DEGs in jasmonic acid signal transduction pathway (Red: positive correlation; Blue: negative correlation). JAZ: jasmonate ZIM domain-containing protein; MYC2: myelocytomatosis oncogene homolog 2; cis-OPDA, cis-12-oxophytodienoic acid
In the salicylic acid signaling pathway, a total of 2 DEGs were involved in encoding (Fig. 13a). In this signaling pathway, only downregulated genes were observed, with no upregulated genes detected. Compared to CK, the gene CYCAS_021866 encoding the transcription factor TGA was significantly downregulated in F2, while the gene CYCAS_026526 encoding pathogenesis-related protein 1 (PR1) was significantly downregulated in both F1 and F2. SA showed no significant correlation with CYCAS_021866 and CYCAS_026526 (Fig. 13b).Fig. 13DEGs involved in salicylic acid signal transduction in Cycas panzhihuaensis under freezing stress at -5°C for 1.5 h (F1) and 6 h (F2). a: Salicylic acid signal transduction pathway. b: Correlation heatmap between SA content and expression of DEGs in salicylic acid signal transduction pathway (Red: positive correlation; Blue: negative correlation). TGA: transcription factor; PR1: pathogenesis-related protein 1; SA: salicylic acid
To validate the transcriptome data, we selected 11 genes from the plant hormone signal transduction pathway for qRT-PCR analysis. The results showed that the expression patterns of these target genes were largely consistent with the transcriptome sequencing data (Fig. S2), indicating the reliability of the transcriptome data and the credibility of the derived analytical results. Among the 11 selected genes, the majority exhibited a consistent trend of initial decrease followed by an increase in both RNA-seq expression levels and qRT-PCR relative expression levels over the treatment duration. A minority of genes displayed distinct both expression levels for CYCAS_002672 consistently decreased, those for CYCAS_024227 consistently increased, and those for CYCAS_001054 showed an initial increase followed by a decrease.
The survival and adaptability of plants largely depend on their sophisticated mechanisms that integrate external stimuli with endogenous regulatory networks. However, as climate change intensifies and extreme weather events become more frequent, these abiotic stresses are posing increasingly severe challenges to plant survival. To thoroughly elucidate the plant molecular regulatory network under stress, transcriptomics serves as a pivotal technique for identifying key transcription factors and functional genes, thereby elucidating the mechanistic basis of abiotic stress adaptation. Earlier studies demonstrated that C. panzhihuaensis exhibited fully reversible physiological impairments after F2 treatment, confirming that the exposure intensity had not yet reached its lethal threshold [6]. In this study, KEGG enrichment analysis of DEGs in C. panzhihuaensis under freezing stress revealed significant enrichment in the plant hormone signal transduction pathway. This result indicates that plant hormone signaling emerges as a critical regulator of the response to freezing stress in C. panzhihuaensis. We measured the hormone levels of C. panzhihuaensis under different treatments (Fig. 1a). During the later phase of freezing stress, we observed a significant accumulation of CTK, ACC, and CS, whereas ABA accumulation decreased. Initially, freezing stress caused a significant increase in GA4, SA, and cis-OPDA levels, but these levels declined markedly in the later phase. The changes in the contents of CS and SA are consistent with the changes observed in the cold-tolerant Gossypium hirsutum variety XLZ16 during low-temperature stress at the seedling phase [32]. The variations in the contents of ABA and CTK are in line with the changes observed in Triticum aestivum when it resists low-temperature stress [33]. Since CTK has a positive effect on plant photosynthesis, such as in detached tomato leaves, where cytokinin can promote photosynthesis by alleviating photoinhibition and increasing CO₂ assimilation [34], its increase after F2 treatment may help in energy formation and enhance cold tolerance [35]. Under normal circumstances, abiotic stress triggers ABA accumulation in plants, thereby activating a series of response mechanisms [36]. However, during the initial phase of freezing stress, ABA content in C. panzhihuaensis remained stable, but it decreased significantly in the late phase. Another study of ours also found that the content of ABA in C. panzhihuaensis decreased after high-temperature stress treatment (unpublished data). We hypothesize that C. panzhihuaensis may employ an atypical response mechanism to adapt to low-temperature environments. The observed decrease in ABA levels suggests that this species potentially enhances cold tolerance through alternative pathways rather than relying on ABA-mediated signaling cascades. However, this proposed mechanism requires further experimental validation. Variations in the concentrations of these essential hormones are initial reactions to abiotic stress and affect the metabolic activities that adjust to environmental stress [37]. To elucidate hormonal regulation in C. panzhihuaensis freezing resistance, we investigated plant hormone signal transduction pathways and analyzed DEGs associated with IAA, CTK, GAs, ABA, ETH, BR, JA and SA signal transduction.
Under multiple environmental stresses, plants regulate resource allocation through hormonal signaling to prioritize responses to the most severe stress, while simultaneously activating distinct signaling pathways to maintain growth-defense homeostasis [38, 39]. Plants are capable of quickly detecting and reacting to fluctuating levels of auxin, which requires the control of AUX/IAA, ARF, SAUR, GH3, and other factors [40, 41]. The perception of auxin is mediated by a transient co-receptor complex, which is composed of TIR1/AFB proteins and Aux/IAA proteins. Following co-receptor binding, auxin triggers ubiquitin-mediated proteolysis of Aux/IAA proteins via the 26 S proteasome, which releases ARF transcription factors from repression and modulates downstream transcriptional networks [42]. The AUX1/LAX family is a class of auxin influx carriers that are responsible for transporting auxin into cells. They are essential for maintaining auxin distribution patterns, dynamically adjusting them according to environmental or developmental signals, promoting cellular auxin uptake, and modulating endogenous free auxin distribution [43, 44]. TIR1/AFB proteins serve as the primary auxin receptors that initiate downstream signaling cascades upon auxin binding [45]. After suffering from freezing stress, the expression levels of AUX1 and TIR1/AFB genes in C. panzhihuaensis were significantly upregulated (Fig. 6a), indicating that the cells were actively taking up auxin at this time, the ability to perceive auxin was enhanced, and the auxin signal transduction pathway was activated. Most Aux/IAA proteins act as transcriptional repressors within the auxin signaling pathway, suppressing auxin-responsive gene expression in the nucleus [46]. Furthermore, auxin may also affect the transcription of early-response genes by modifying the interactions between ARFs and Aux/IAAs [40]. The F1 and F2 treatments significantly upregulated AUX/IAA gene expression, which may influence downstream gene expression by modulating the stability of AUX/IAA proteins. Additionally, we found that the genes encoding TIR1/AFB were significantly positively correlated with the genes encoding AUX/IAA (Fig. 6b). Thus, we speculate that the upregulation of AUX/IAA expression may act in concert with the upregulation of TIR1/AFB receptors to maintain a balance with the activity of TIR1/AFB, ensuring that the auxin signaling pathway can respond to stress without being overactivated. The GH3 gene family represents classical early auxin-responsive genes that regulate both plant development and stress adaptation through biosynthesis of hydrolyzable auxin conjugates and inactivated auxin [47–49]. F1 treatment significantly downregulated the expression of most GH3-encoding genes, indicating a reduction in auxin metabolism and decreased auxin deactivation, which is consistent with the observed increase in IAA content under this treatment. When the duration of freezing stress was extended to 6 h, the IAA content decreased significantly, but the expression levels of GH3 genes showed no clear pattern, which may be related to crosstalk (synergistic or antagonistic effects) with other hormones. SAUR represents the most extensive group of early auxin-responsive genes and is vital in numerous functions such as cell enlargement, leaf morphogenesis and senescence, auxin movement, tropic growth, and stress responses [50, 51]. The downregulation of SAUR genes in F1 and F2 treatments likely reflects attenuated auxin-mediated growth promotion, leading to alterations in the rate or direction of cell growth. This phenomenon could be associated with the upregulation of AUX/IAA gene expression, which may suppress the expression of downstream auxin-responsive genes.
Cytokinin has been closely linked to cold stress responses in numerous studies [52–54]. The cytokinin signal is perceived by AHK located on the plasma membrane or endoplasmic reticulum membrane [55]. Upon cytokinin binding to its receptor, the receptor undergoes autophosphorylation, with the phosphate group being transferred from a histidine residue in the kinase domain to an aspartate residue in the receiver domain [56]. The phosphorylated receptor subsequently transfers the phosphate group to AHP in the cytoplasm, which in turn pass the phosphate group to the response regulators ARRs [57]. ARRs are categorized into type-A and type-B. Type-B ARRs are transcription factors that activate the transcription of downstream target genes to regulate the physiological effects of cytokinin, while type-A ARRs function as negative regulators by inhibiting type-B ARRs through feedback mechanisms to modulate cytokinin signaling intensity [58, 59]. The F1 treatment significantly downregulated the gene expression of CRE1, AHP, and B-ARR (Fig. 7a), indicating that the cytokinin signal transduction pathway was suppressed during the initial phase of freezing stress. Under F2 treatment, the expression levels of CRE1 and B-ARR genes were significantly upregulated, while most A-ARR genes exhibited low expression levels. This expression pattern indicates activation of the cytokinin signal transduction pathway during the later phase of freezing stress. In A. thaliana, the regulation of freezing stress by EIN3 is negative, achieved by directly modifying the expression of CBF and type-A ARR genes, while the crosstalk between ethylene and cytokinin signaling pathways modulates their responses to cold stress [60]. Therefore, the significant upregulation of another A-ARR-encoding gene in this study may indicate its involvement in other regulatory processes. The variation in gene expression levels may be an adaptive response of C. panzhihuaensis to freezing stress in its later stages. By enhancing cytokinin signaling, these changes could regulate intracellular physiological processes and promote cell recovery.
The gibberellin signaling pathway is a key regulatory mechanism in plant growth and development, with its core components including the GA receptor GID1, DELLA proteins, and F-box proteins (such as SLY1 or GID2) [61]. The initial step of gibberellin signaling transduction involves the binding of bioactive GA to its receptor GID1, a soluble protein capable of specifically recognizing and binding active GA molecules [62]. When GA binds to GID1, it causes a structural change that reveals the site for DELLA protein binding [63]. In the gibberellin signaling pathway, DELLA proteins are essential negative regulators that inhibit downstream gene expression to restrict plant growth, thereby assisting plants in adapting to cold environments [64, 65]. After GA binds to GID1, the GID1-DELLA complex is formed, allowing the DELLA protein to be identified by an F-box protein (such as SLY1 or GID2) and subsequently degraded through the ubiquitin-proteasome pathway [61, 66]. DELLA degradation relieves their repressive effect on downstream transcription, triggering GA signaling activation and transcriptional reprogramming of GA-responsive genes. This cascade modulates plant growth, development, and environmental adaptation. In this study, during the initial period of freezing stress, the expression levels of genes encoding DELLA and GID2 were both upregulated (Fig. 8a). We speculate that C. panzhihuaensis may accumulate DELLA proteins to inhibit growth, thereby reducing energy consumption and enhancing cold stress responsiveness. The accumulation of DELLA proteins under low-temperature conditions indicates their role in the initial phases of the cold stress response pathway [64]. Studies have also shown that growth inhibition is a vital part of the initial stress response in plants, likely due to the need to redirect energy towards producing various protective compounds under stress conditions [35]. GID2 serves as a positive regulator in gibberellin signaling and participates in the degradation of DELLA proteins. However, the upregulation of GID2 gene expression in the beginning of freezing stress suggests that C. panzhihuaensis might attempt to maintain gibberellin signaling through enhanced GID2 expression. But at this time, the accumulation of DELLA proteins may have exceeded the degradation capacity of GID2, resulting in the continued accumulation of DELLA proteins. In the later phase of freezing stress, the high expression of the gene encoding GID1 indicates that C. panzhihuaensis may have enhanced its ability to perceive gibberellin under cold stress, attempting to regulate growth and development by activating the gibberellin signaling pathway. The low expression of the gene encoding DELLA suggests a reduction in DELLA protein accumulation, which may relieve the inhibition of growth through the degradation of DELLA proteins [62]. The downregulation of the gene encoding GID2 indicates a weakened gibberellin signaling capacity, which may be a self-regulation mechanism of C. panzhihuaensis to avoid overactivation of the gibberellin signaling pathway, thereby preventing excessive growth under low temperatures and balancing growth with stress response. In summary, the changes in gene expression indicate that C. panzhihuaensis balances growth and stress response under freezing stress by regulating the gibberellin signaling pathway, thereby enhancing its adaptability to low temperature.
Abscisic acid regulates key developmental processes from seed dormancy to plant senescence, and is essential for plant resistance to cold, drought, and salt stresses, making it a vital stress-response hormone [67]. Under stress conditions, ABA is activated and regulates the expression of stress-responsive genes through its signaling pathway, thereby enhancing plant stress tolerance [68]. The core components of abscisic acid signaling include the ABA receptor PYR/PYL, PP2C, SnRK2, and the ABA responsive element binding factor ABF [69]. After perceiving ABA, the ABA receptors PYR/PYLs/RCARs interact with the negative regulators PP2C, inhibiting the activity of PP2C and thereby releasing SnRK2 [70]. The activated SnRK2 undergoes autophosphorylation and subsequently phosphorylates downstream targets, including transcription factors (e.g., ABF family members) and ion channels, thereby regulating ABA-responsive gene expression and initiating plant stress responses [67]. Under ABA-deficient conditions, PP2C directly interacts with SnRK2 and suppresses its kinase activity through dephosphorylation, thereby inhibiting ABA signal transduction [71]. Early freezing stress triggers PYR/PYL gene upregulation in C. panzhihuaensis, which subsequently inhibits PP2C expression while decreasing ABF gene expression (Fig. 9a). During the late stage of freezing stress in C. panzhihuaensis, the expression levels of PYR/PYL genes were downregulated, leading to the release of PP2C inhibition and subsequent upregulation of PP2C gene expression. The increased PP2C activity then inhibited SnRK2 kinase activity through dephosphorylation, resulting in suppression of ABA signal transduction. Interestingly, although ABA signaling pathway activity was suppressed during this phase, ABF gene expression exhibited significant upregulation. Despite the downregulation of SnRK2 gene expression, the ABA signaling pathway may be activated through other mechanisms. Freezing stress may indirectly activate the expression of ABF through other signaling pathways (such as the MAPK signaling pathway) [70], rather than entirely relying on the ABA-mediated SnRK2 signaling pathway. However, this hypothesis requires further validation. Research on Ammopiptanthus mongolicus revealed that low-temperature stress significantly upregulates PP2C gene expression, while the expression of SNRK2 generally shows a downward trend and the transcription levels of most ABF transcripts are elevated [72]. This pattern of gene expression for PP2C, SNRK2, and ABF is similar to that observed in the present study. Additionally, similar to the findings in banana under biotic stress, ABA signal suppression under stress conditions is often associated with the elevated expression of ABA-negative regulatory genes (e.g., PP2C) and crosstalk with other hormone signaling pathways [73]. This suggests that the abnormal activation of ABF in C. panzhihuaensis may also be involved in the coordination with other hormone signals, which together constitute the adaptive regulatory network under freezing stress. This phenomenon reflects the complex signaling regulatory networks in plants under low-temperature stress, as well as their multi-mechanistic adaptive responses to environmental challenges.
Ethylene is produced under various environmental stresses and acts as a bridge between constantly changing environments and developmental adaptations [74]. Plants rely on ethylene signaling to modulate their responses to low-temperature stress. For example, cold treatment in Tetrastigma hemsleyanum increases ethylene levels, with multiple ethylene biosynthesis and signaling genes participating in the cold stress response, indicating that ethylene positively regulates cold tolerance in this species [75]. In addition, research on low-temperature stress in grapevine leaves has further revealed the diverse regulatory mechanisms of ethylene. It regulates cellular metabolic processes while also promoting ABA and IAA accumulation and downstream signaling by modulating their biosynthesis and signaling-related gene expression, thereby enhancing low-temperature stress response [76]. In C. panzhihuaensis under freezing stress, the expression levels of genes encoding ETR, EIN3, and ERF1/2 undergo significant changes (Fig. 10a), all of which participate in the perception and transduction of ethylene signaling. ETR functions as an ethylene receptor and negatively regulates ethylene signal transduction [77]. Under ethylene-deficient conditions, ethylene receptors activate constitutive triple response 1 (CTR1) to phosphorylate and suppress downstream signaling, whereas ethylene binding inhibits receptor activity to relieve this suppression and trigger ethylene responses [78]. In F1, the two genes encoding ETR are differentially regulated, with one being upregulated and the other downregulated. This suggests that ethylene receptor family members (such as ETR1 and ETR2) may balance signal sensitivity through differential regulation. Furthermore, expression of ETR-encoding genes was significantly upregulated in the F2 group compared to both CK and F1, accompanied by increased ACC content. This may be because low temperature stimulates ethylene synthesis, and C. panzhihuaensis needs to increase the number of receptors to better perceive ethylene signals, thereby initiating a cold-resistant response. In addition to the changes in the gene encoding ETR, the gene encoding EIN3 is also significantly downregulated in F2 (compared to F1), and the genes encoding ERF1/2 are significantly downregulated in all treatments. The altered expression patterns of these genes after freezing stress suggest that C. panzhihuaensis may regulate its response to cold through the modulation of the ethylene signaling transduction pathway following freezing stress.
In plants, the jasmonic acid signaling pathway is critical for responding to both biotic and abiotic stresses, serving as a primary mechanism for environmental adaptation. The biologically active jasmonoyl-isoleucine conjugate (JA-Ile) is specifically recognized by the receptor protein coronatine insensitive 1 (COI1) [79]. Under normal conditions, JAZ proteins bind to the transcription factor MYC2 and inhibit its activity [80]. Upon JA-Ile accumulation, the COI1-JA-Ile complex mediates JAZ protein ubiquitination and degradation, releasing MYC2 and other transcription factors to activate jasmonate-responsive genes (including defense-related and secondary metabolism genes), thereby coordinating plant defense responses and adaptive growth [81, 82]. Compared to CK, the expression levels of most genes encoding JAZ and MYC2 are significantly downregulated in both F1 and F2 treatments (Fig. 12a). However, compared to F1, only a small portion of the genes encoding JAZ show significant downregulation in F2. The downregulation of these genes indicates that low-temperature stress inhibits the jasmonic acid signaling pathway. This may be a regulatory mechanism employed by C. panzhihuaensis to adapt to low-temperature environments by modulating its own physiological metabolism and defense responses. Research has indicated that jasmonic acid interacts with multiple hormone signaling pathways to modulate plant growth and abiotic stress tolerance, with JAZ and MYC2 serving as critical components in this crosstalk [83]. Plant hormones interact in complex ways to coordinate responses to environmental stresses. In banana, the NAC transcription factor MusaNAC29-like specifically elevates JA and SA content, and these two hormones synergistically activate downstream stress-responsive genes (e.g., antioxidant genes、WRKY) to enhance drought and salt tolerance [84]. Therefore, the observed alterations in the jasmonic acid signaling pathway under freezing stress may involve crosstalk with other hormonal regulatory networks.
Salicylic acid regulates metabolic, osmotic, and secondary metabolic responses to abiotic stress, its signaling being species-specific and dependent on application mode, SA levels (exogenous/endogenous), and stress characteristics [85, 86]. Under normal physiological conditions, SA accumulation promotes the formation of NPR1-TGA complexes through monomeric NPR1 binding, thereby activating the expression of pathogenesis-related PR-1 genes [87]. However, after freezing stress, the expression levels of TGA and PR-1 genes in C. panzhihuaensis are significantly downregulated (Fig. 13a). This suggests that low temperature may inhibit the salicylic acid signaling pathway, thus hindering the plant’s defense against pathogens. In addition, in the brassinosteroid signaling pathway, only the expression level of the gene encoding TCH4 has changed significantly (Fig. 11a), but there is no obvious pattern. In many cases, these plant hormone signaling pathways do not act alone but interact with other hormone signaling pathways and metabolic pathways in complex ways. For example, the interaction between sucrose biosynthesis and jasmonate signaling is related to the cold adaptation of A. thaliana [88], reflecting the complex signaling regulatory networks in plants.
This study has preliminarily established a theoretical framework for the response of C. panzhihuaensis to freezing stress and identified several key candidate genes, laying a foundation for elucidating the molecular mechanisms of its freeze tolerance. The results revealed a close correlation between hormone levels and gene expression, suggesting that these genes may be involved in potential physiological regulation pathways. It should be noted that transcriptomic evidence is inherently correlative; therefore, follow-up studies should employ genetic and functional experiments (such as gene overexpression or knockout) to clarify the regulatory roles of these candidate genes. In addition, the potential of exogenous hormones in enhancing freeze tolerance warrants experimental verification. Although this study uncovers important mechanisms under controlled conditions, successful field introduction requires addressing complex ecological interactions. To bridge this gap, future research should conduct field adaptability trials across latitudinal gradients to comprehensively evaluate its adaptation potential. These steps are crucial for translating our molecular understanding into strategies for promoting the broader cultivation and conservation of C. panzhihuaensis.
This study subjected C. panzhihuaensis to freezing stress treatment, quantitatively measured the content of plant hormones, and analyzed differentially expressed genes through transcriptomics, aiming to elucidate the molecular mechanisms that govern the response of C. panzhihuaensis to freezing stress. Our research found that with the extension of freezing treatment time, the contents of cis-OPDA, GA4, and SA first significantly increased and then significantly decreased. After F2 treatment, the content of ABA significantly decreased, while the contents of ACC, CS, and CTK significantly increased. At the transcriptional level, 2,298 DEGs were identified in the CK vs. F1 comparison, 2,155 DEGs in the CK vs. F2 comparison, and 1,878 DEGs in the F1 vs. F2 comparison. These changes in hormone levels and differential gene expression indicated that C. panzhihuaensis adapted to low temperatures by reprogramming its metabolic activity under stress conditions. Our KEGG enrichment analysis of DEGs revealed that the plant hormone signal transduction pathway is a significant differentially grouped pathway. We speculate that this signaling pathway critically regulates downstream genes and associated physiological processes. Despite the complexity of natural environments, plant adaptation arises from the coordinated interplay among intrinsic developmental programs, endogenous hormonal regulation, and external environmental responses. In summary, based on transcriptomic analysis, this study identified differences between the control and treated groups of C. panzhihuaensis. We proposed that the plant hormone signal transduction pathway may mediate essential functions in the cold resistance of this species. However, the key regulatory genes and the effects of exogenous phytohormones on its freezing tolerance require further investigation and experimental validation. These findings significantly advance our understanding of the molecular mechanisms underlying freeze tolerance in C. panzhihuaensis.
Supplementary Material 1.