ABA-Responsive Peach PpMYB6 Positively Regulates Freezing Tolerance but Retards Plant Growth Through Direct Transactivation of PpCBF2

1. Introduction

Peach (Prunus persica (L.) Batsch) has a long cultivation history spanning over 4,000 years, making it one of the oldest cultivated fruit trees in China [1]. Due to its strong adaptability to diverse soil and climatic conditions, peach is widely grown across a wide latitudinal range from northern to southern China. Furthermore, peach cultivation holds substantial economic value, characterized by early bearing, rapid economic returns, and manageable orchard maintenance. However, cold stress—comprising chilling stress (<20 °C) and freezing stress (< 0 °C)—represents a major abiotic constraint that hinders the further expansion of the peach industry and limits its northern cultivation boundaries [2].

Low-temperature stress severely impairs various physiological processes in plants, leading to reduced vigor, growth retardation, leaf curling and browning, branch desiccation, pericarp discoloration, and eventual wilting, with extreme freezing ultimately causing plant mortality. Furthermore, cold stress induces cellular membrane damage, alters osmoprotectant levels, and triggers antioxidant defense mechanisms [3,4,5].

Numerous studies have demonstrated that endogenous phytohormones play crucial roles in mediating abiotic stress responses [6,7,8,9]. As a classic stress hormone, abscisic acid (ABA) plays a pivotal role in mediating plant tolerance to cold, drought, and saline-alkali stress [9,10,11,12,13]. Similarly, substantial physiological evidence indicates that exogenous ABA application significantly alleviates stress-induced peroxidative damage and maintains photosynthetic system stability [14,15]. In our previous study, we confirmed that exogenous ABA application enhanced cold tolerance in peach trees, identifying 50 mg·L⁻¹¹ as the optimal concentration [16].

Existing findings reveal that ABA modulates cold stress responses through multi-layered mechanisms, including phytohormone crosstalk, hierarchical transcription factor cascades, and protein phosphorylation [17,18]. However, despite the well-documented protective role of exogenous ABA, how cold and ABA signaling pathways interactively regulate low-temperature tolerance in peach remains poorly understood. Specifically, the transcriptomic mechanisms underlying cold stress responses in peach remain elusive. Unraveling the transcriptomic network mediated by cold and ABA signal crosstalk is therefore of paramount importance.

In recent years, RNA sequencing (RNA-Seq) has emerged as a powerful tool for dissecting transcriptomic regulation under abiotic stress and has been widely applied in crops such as apple, wheat, rice, and citrus [19,20,21,22]. RNA-Seq has also been utilized to explore cold stress regulatory networks in peach. Previous studies revealed that cold-responsive differentially expressed genes (DEGs) in peach are significantly enriched in pathways such as biosynthesis of secondary metabolites, galactose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis [23,24].

Among the established molecular mechanisms governing cold acclimation, the ICE-CBF-COR module forms the core cold-signaling cascade [25,26,27]. Furthermore, multiple transcription factor families, including AP2/ERF, WRKY, NAC, and MYB, participate in the co-regulation of ABA signaling and cold responses [28,29,30,31,32]. Notably, MYB transcription factors serve not only as master switches regulating anthocyanin and flavonoid biosynthesis, but also as key players in phytohormone-mediated stress adaptation [33,34].

Nevertheless, research on ABA-mediated responses to short-term cold stress in peach remains limited. To address this scientific question, we conducted time-course RNA-seq analysis on peach seedlings to identify core DEGs co-responsive to cold stress and ABA treatment.

2. Materials and Methods

2.1. Plant Materials and Treatments

Seedlings of the cold-tolerant peach cultivar ‘Shiji Zhixing’ grown under field conditions were obtained from the Horticulture and Landscape Experimental Station of Hebei Normal University of Science and Technology (39.708° N, 119.176° E). For cold stress treatment, seedlings were placed in a growth chamber at 0 °C, and leaf samples were collected at 0, 1, 2, and 4 h. Concurrently, seedlings exhibiting uniform growth were sprayed with an exogenous ABA solution (50 mg·L⁻¹¹), and leaf samples were harvested at the corresponding time points. All collected leaves were immediately frozen in liquid nitrogen and stored at -80 °C until use. Three biological replicates were performed for each treatment.

2.2. RNA Extraction, Library Construction, and RNA-Seq

Total RNA was extracted using the Polyphenol/Polysaccharide Plant RNA Extraction Kit (Coolaber, Beijing, China) according to the manufacturer’s instructions. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Sequencing libraries were constructed following standard protocols with the NEBNext Ultra RNA Library Prep Kit (NEB, Ipswich, MA, USA). Preliminary quantification and insert size detection were conducted using a Qubit 2.0 Fluorometer (Life Technologies, CA, USA). Effective library concentrations (> 2 nM) were precisely quantified via qRT-PCR prior to high-throughput sequencing on the Illumina platform at Novogene Co., Ltd. (Beijing, China) [35,36].

2.3. Transcriptome Alignment, DEG Identification, and Functional Enrichment

Raw sequencing reads were processed for quality control by filtering out adaptor sequences and low-quality reads to obtain clean reads. Clean reads were aligned to the peach reference genome using HISAT2 software. Gene expression levels were normalized using the fragments per kilobase of transcript per million mapped reads (FPKM) method. Differentially expressed genes (DEGs) responsive to cold stress and ABA treatment were identified using the DESeq2 package, with P-values adjusted using the Benjamini-Hochberg method to control the false discovery rate [37,38].

Hierarchical clustering (H-cluster) was performed on log2(FPKM+1)-transformed and mean-centered expression values, partitioning DEGs into four distinct clusters with similar expression trends across treatments [39]. Functional annotations of DEGs were performed by integrating multiple databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the clusterProfiler package. A threshold of P < 0.05 was defined as statistically significant for GO terms and KEGG pathways, and directed acyclic graphs (DAGs) were constructed to visualize the GO enrichment results [40].

2.4. Quantitative Real-Time PCR (qRT-PCR) Verification

Six DEGs co-responsive to cold stress and ABA treatment were selected for qRT-PCR validation, with the PpActin gene used as the internal reference [41]. Specific forward and reverse primers were designed using Primer3 software and synthesized by Sangon Biotech (Shanghai, China). Quantitative real-time PCR was performed using the Hieff^® SYBR Green Master Mix (Yisheng Biotech, Shanghai, China) on a Bio-Rad CFX Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative expression levels of target genes were calculated using the 2−ΔΔCt method, with the control samples (0 °C treatment) serving as the baseline reference. Three biological and technical replicates were analyzed for each gene [42].

2.5. Bioinformatic and Promoter Analysis

In this study, the physicochemical properties of the PpMYB6 protein sequence were analyzed using the online ExPASy ProtParam tool (https://web.expasy.org/protparam/). The tertiary (3D) structure of the protein was predicted using the SWISS-MODEL online server (https://swissmodel.expasy.org/). Multiple sequence alignment was conducted using DNAMAN software, and phylogenetic tree analysis was performed using MEGA 12 software. For promoter sequence analysis, cis-acting regulatory elements within the promoter region were identified using the PlantPAN 4.0 database (https://plantpan.itps.ncku.edu) and the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

2.6. Generation of PpMYB6-Overexpressing Peach Callus

To construct the PpMYB6 overexpression vector, the full-length coding sequence of PpMYB6 was cloned into the pCAMBIA-2300 vector using the Biomed 2×Seamless Mix (Biomed, Beijing, China). The recombinant construct was transformed into Agrobacterium tumefaciens strain GV3101 competent cells. A 50 μL aliquot of the transformed A. tumefaciens culture was inoculated into 50 mL of liquid YEP medium containing 50 mg·L⁻¹¹ kanamycin and 50 mg⁻¹L⁻¹ rifampicin, and incubated at 220 rpm for 16 h. The bacterial cells were harvested by centrifugation at 5,000 rpm for 10 min and resuspended in infection medium (4.43⁻¹g·L⁻¹ MS salts and 35⁻¹0 g·L⁻¹ sucrose).

Dark-adapted peach calli were immersed in the bacterial suspension and shaken at 220 rpm at 37 °C for 1 h. Following infection, the calli were filtered and blotted dry on sterile filter paper. The calli were then co-cultivated on co-cultivation medium (4.43 g·L⁻¹¹ MS, 35.0 g⁻¹L⁻¹ sucrose, 6.0⁻¹g·L⁻¹ agar, 1.⁻¹ mg·L⁻¹ 6-BA, ⁻¹.2 mg·L⁻¹ IBA, an⁻¹ 0₃1 mg·L⁻¹ GA₃) in the dark for 2–3 days. Subsequently, the calli were transferred to selection medium (co-cultivation medium supplemented⁻¹with 80 mg·L⁻¹ kanamyc⁻¹n and 300 mg·L⁻¹ cefotaxime) under light conditions. Proliferating calli were subcultured onto fresh selection medium, and stable transgenic lines were verified via genomic PCR and qRT-PCR [43,44].

2.7. Generation and Selection of Transgenic Arabidopsis thaliana Lines

Arabidopsis thaliana seeds were sown in pots containing soil substrate and stratified in the dark at 4 °C for 3 days to synchronize germination. The pots were then transferred to an environmental growth chamber and cultivated until plants reached the bolting stage for floral dip transformation.

For primary bacterial culture preparation, a 50 µL aliquot of Agrobacterium tumefaciens culture harboring the PpMYB6 overexpression construct was inoculated into 500 µL of YEP liquid medium (supplemented with 50 mg·L⁻¹¹ rifampicin and 50 mg⁻¹L⁻¹ kanamycin) in a sterile 2 mL microcentrifuge tube and cultured overnight at 28 °C on a shaker. Subsequently, a 100 µL volume of the primary culture was transferred into a sterile 50 mL Erlenmeyer flask containing 25 mL of fresh YEP medium (+ Rif + Kana) and cultivated at 28 °C with shaking for 14 h. Bacterial cells were harvested by centrifugation at 5,000 rpm for 10 min. After discarding the supernatant, the pellet was washed with 10 mL of sterile distilled water and centrifuged at 5,000 rpm for 5 min to recollect the bacterial cells. The resulting pellet was resuspended in floral dip infiltration medium (2.2⁻¹g·L⁻¹ MS salts, ⁻¹0 g·L⁻¹ sucrose, ⁻¹00 µL·L⁻¹ Silwet L-77, and 100µM acetosyringone).

The inflorescences of bolting Arabidopsis plants were submerged in the bacterial suspension for 45 s. The dipped plants were placed horizontally under dark conditions for 24 h and then returned to normal growth conditions in the growth chamber. The floral dip transformation was repeated weekly for a total of three treatments.

Harvested T₀ seeds were collected into 2 mL microcentrifuge tubes and surface-sterilized by vortexing in 75% ethanol for 1 min, followed by 6% sodium hypochlorite solution for 10 min, and rinsed six times with sterile distilled water (2 min per wash). The sterilized seeds were sown onto 1/2 MS solid medium containing 50 mg·⁻¹⁻¹ kanamycin. The plates were stratified at 4 °C in the dark for 3 days and transferred to a growth chamber. Kanamycin-resistant green seedlings were selected to obtain positiv₁ T₁ transgenic lines. Homozygo₃s T₃ transgenic lines were established through three consecutive generations of antibiotic screening and subsequently verified via genomic PCR and qRT-PCR.

2.8. Cold Stress Tolerance Assays of Transgenic Callus and Arabidopsis thaliana

Successfully transformed PpMYB6-overexpressing (OE) peach calli were subcultured on selection medium every 20 days for propagation. Wild-type (WT) calli and three independent OE lines were inoculated onto the same MS medium plate, with each line occupying one quadrant. Plates were incubated at 28 °C under light for 7 days. For cold stress treatment, the plates were exposed to -5 °C for 6 h and subsequently transferred back to 28 °C for a 7-day recovery period. Morphological changes and growth phenotypes were observed to evaluate the differences between WT and transgenic calli under cold stress.

Seeds of selected T₃ generation transgenic Arabidopsis thaliana lines and wild-type (WT) controls were sown. After completing cold stratification (vernalization), the seeds were transferred to an environmental growth chamber for germination and cultivated for 7 days. The 7-day-old seedlings were photographed to document the baseline phenotype of the non-stressed control group. Subsequently, the seedlings were subjected to freezing cold stress at -4℃ for 3.5h in a temperature-controlled growth chamber, after which morphological changes and cold-stress phenotypes were observed and evaluated.

2.9. Yeast One-Hybrid (Y1H) Assay

Carrier DNA was denatured in a boiling water bath for 5 min and immediately chilled on ice for 5 min.

The yeast transformation reaction mixture was assembled as follows: Carrier DNA 10 µL, Recombinant pHIS2 plasmid 500 ng, Y187 yeast competent cells 100.0 µL, PEG/LiAc solution 500 µL.

The mixture was mixed extremely gently and incubated in a 30 °C water bath for 10 min. The tube was removed and mixed gently again before continuing incubation; this step was repeated 3 times for a total incubation time of 30 min.

A volume of 70 µL of DMSO was added to the mixture.

The solution was again mixed extremely gently, incubated in a 42 °C water bath for 5 min, and repeated 3 times for a total of 15 min.

Following incubation, the mixture was briefly centrifuged at 12,000 rpm, and the supernatant was discarded to collect the yeast cell pellet.

The cell pellet was resuspended in 1 mL of liquid YPDA medium and incubated at 30 °C with shaking at 220 rpm for 1 h.

After cultivation, the mixture was centrifuged at 12,000 rpm, the supernatant was discarded, and the yeast pellet was resuspended in 200 µL of 0.9% NaCl solution.

Aliquots of 100 µL suspension were spread onto synthetic dropout medium lacking tryptophan (SD/−Trp) and medium lacking tryptophan and histidine (SD/−Trp/−His) agar plates, followed by incubation at 30 °C for 3–5 days.

Colony growth on SD/−Trp/−His plates was evaluated to test for reporter gene auto-activation; colony formation indicated auto-activation of the promoter construct.

If auto-activation was observed, SD/−Trp/−His agar media supplemented with 3-amino-1,2,4-triazole (3-AT) at concentrations of 20, 30, 40, 50, 60, and 70 mM were prepared.Yeast colonies harboring the recombinant plasmid were resuspended in liquid medium and spot-plated onto SD/−Trp/−His media containing varying 3-AT concentrations. Plates were incubated at 30 °C for 3–5 days to determine the optimal 3-AT concentration required to completely inhibit auto-activation.

Recombinant pHIS2-PpCBF2 reporter vector and pGADT7-PpMYB6 effector vector were co-transformed into Y187 yeast competent cells. Following transformation, cell suspensions were spread onto double dropout media (SD/−Trp/−Leu) and incubated at 30 °C for 3–5 days. Single colonies were picked from SD/−Trp/−Leu plates and spot-plated onto triple selective media (SD/−Trp/−His/−Leu) supplemented with the pre-determined optimal concentration of 3-AT. Plates were incubated at 30 °C for 3–5 days to evaluate yeast growth and confirm promoter-binding activity.

2.10. Dual-Luciferase Reporter Assay

Sequencing-verified recombinant reporter plasmid pGreenII 0800-PpCBF2 was transformed into Agrobacterium tumefaciens strain GV3101 competent cells. A 100 µL aliquot of the transformed cell culture was inoculated into 2 mL of YEP liquid medium supplemented with kanamycin (Kana) and rifampicin (Rif) and cultured overnight at 28 °C with shaking at 220 rpm for 14–16 h. A portion of the culture was transferred into 20 mL of fresh YEP (+ Kana + Rif) medium and grown until reaching an OD600 of 0.8. Cells were harvested by centrifugation at 5,000 rpm for 10 min, and the supernatant was discarded.

MES buffer was prepared (2.033 g·L⁻¹¹ Mg₂l₂₂6H₂O, 2.132⁻¹g·L⁻¹ MES and 100 μM acetosyringone, and adjust the pH to 5.5). The bacterial pellet obtained in the previous step was resuspended in the buffer to a fi₆₀₀al OD₆₀₀ of 0.8–1.0, and incubated at 28 °C in the dark for 1 h for recovery.

Agrobacterium culture harboring the reporter plasmid (pGreenII 0800-PpCBF2) was mixed with Agrobacterium culture harboring the PpMYB6-overexpression effector construct. The mixed suspension was infiltrated into the abaxial surface of Nicotiana benthamiana leaves using a needleless syringe. Infiltrated plants were kept in the dark for 48 h prior to bioluminescence detection using a plant live imaging system.

Leaf discs were sampled from the infiltrated regions using a leaf puncher and homogenized in a mortar. Protein extraction was performed by adding 200 µL of cell lysis buffer (Firefly Luciferase Reporter Gene Assay Kit, Beyotime). The homogenate was centrifuged at 12,000 rpm for 2 min, and the supernatant was collected for analysis.

A 20 µL aliquot of the supernatant was loaded into a microplate well and mixed thoroughly with 50 µL of Firefly luciferase detection reagent by pipetting. Firefly luciferase luminescence (LUC) was measured using a multifunctional microplate reader in chemiluminescence mode.

Subsequently, 100 µL of Renilla luciferase detection reagent was added to the same well and mixed thoroughly by pipetting, after which Renilla luciferase luminescence (REN) was recorded. Luminescence measurements were completed within 5 min.

Relative promoter activity was calculated as the ratio of Firefly luciferase activity to Renilla luciferase activity (LUC/REN).

2.11. Yeast Two-Hybrid (Y2H) Library Screening for PpMYB6-Interacting Proteins

To investigate the molecular mechanisms underlying PpMYB6 function, the full-length coding sequence of PpMYB6 was cloned into the pGBKT7 vector to generate the bait construct, and its autoactivation was evaluated. The recombinant bait plasmid was then co-transformed with a peach cDNA library into yeast competent cells.

Yeast cells harboring the pGBKT7-PpMYB6 construct were cultured in 100 mL of medium until reaching an OD₆₀₀₀₀ of 0.8. The culture was centrifuged at 4,000 rpm for 5 min, and the cell pellet was resuspended in 1 mL of LiAc/TE buffer, transferred to a 1.5 mL microcentrifuge tube, and briefly spun at 12,000 rpm. The resulting pellet was resuspended in 600 μL of LiAc/TE buffer and incubated on ice for 30 min to prepare competent yeast cells, which were used within 4 h.

The peach cDNA library plasmids were transformed into the prepared competent cells and subsequently plated on double-dropout (SD/-Trp/-Leu, DDO) and quadruple-dropout (SD/-Trp/-Leu/-His/-Ade, QDO) synthetic media. After incubation at 30 °C for 5 days, single colonies surviving on QDO plates were picked and resuspended in 0.9% NaCl solution. Aliquots (5 μL) of the cell suspensions were spotted onto fresh QDO plates and cultured at 30 °C for an additional 3–5 days to confirm potential positive interactions. Plasmids from robustly growing colonies were isolated using a Yeast Plasmid Extraction Kit (Coolaber, Beijing, China). Colony PCR was performed using pGADT7-specific primers, and positive inserts were sequenced to identify candidate PpMYB6-interacting proteins [45].

3. Results

3.1. Transcriptome Sequencing and Quality Control

To investigate gene expression dynamics in peach branches under low-temperature stress, time-series transcriptome sequencing was performed. As shown in Table 1, following stringent quality control, a total of 157.01 Gb of clean data were obtained, with each sample yielding over 6.01 Gb. The GC content across all samples exceeded 45%, and the Q30 percentage consistently surpassed 93.12%. These metrics demonstrate the high quality and accuracy of the sequencing data, providing a robust dataset for subsequent analysis of ABA- and cold-responsive transcriptomic changes.

3.2. Sample Correlation and Principal Component Analysis

A sample correlation heatmap was generated by calculating Pearson correlation coefficients of gene expression levels among all samples. As shown in Figure 1, biological replicates within each treatment group exhibited exceptionally high reproducibility, with coefficient of determination (R²) values exceeding 0.95. These results confirmed the reliability and experimental reproducibility of the sampling process. Furthermore, distinct gene expression profiles were observed between different treatment groups, laying a solid foundation for identifying differentially expressed genes (DEGs).

To further evaluate the sample spatial distribution and treatment-specific effects, principal component analysis (PCA) was conducted. The first two principal components accounted for 32.12% (PC1) and 19.24% (PC2) of the total variance, respectively. In the 2D PCA score plot, biological replicates within the same treatment group clustered tightly, further validating high intra-group consistency. Spatially, ABA-treated samples were predominantly localized along the negative PC1 axis. In contrast, cold-treated samples displayed a distinct shift along the positive PC1 axis over time. These findings indicate that both ABA application and low-temperature stress exert profound and distinct impacts on the global transcriptomic landscape of peach.

3.3. Identification and Expression Dynamics of Differentially Expressed Genes

To systematically analyze transcriptomic alterations under cold stress and ABA treatment, differentially expressed genes (DEGs) across comparisons were identified, and their numerical distribution and overlaps were evaluated. Overall, the total number of DEGs displayed a time-dependent increasing trend under both ABA application and low-temperature stress.

As shown in Figure 2, in ABA-treated samples, the number of DEGs peaked at 4 h (ABA4 vs. ABA0), with a total of 2,962 DEGs (1,548 upregulated and 1,414 downregulated), which was significantly higher than those observed at 1 h and 2 h. Similarly, DEG abundance under cold stress reached its maximum at 4 h (Cold4 vs. Cold0), yielding 1,898 DEGs (1,515 upregulated and 383 downregulated). Notably, upregulated DEGs markedly outnumbered downregulated DEGs under cold stress.

Venn diagram analysis revealed that 419 DEGs were consistently differentially expressed across all cold stress time points (1, 2, and 4 h), whereas 641 DEGs responded to ABA across all treatment durations. For both treatments, the overall transcriptomic response culminated at 4 h.

To elucidate the potential synergistic crosstalk between ABA signaling and cold stress responses in peach, DEGs from both treatments were integrated. A total of 1,105 DEGs were co-responsive to both cold stress and ABA treatment. These co-regulated DEGs represented candidate genes mediated by the ABA pathway in response to low temperature, likely serving as pivotal hubs within the ABA-dependent cold signaling network.

As shown in Figure 3, hierarchical clustering of DEG expression profiles classified these genes into four distinct expression patterns (Clusters a–d):

Cluster a: Genes in this cluster exhibited continuous downregulation over time under cold stress, accompanied by minimal responsiveness to ABA treatment.

Cluster b: Genes responded simultaneously to both ABA and cold treatments, displaying progressive upregulation over time. These genes were candidate participants in the ABA-mediated cold tolerance pathway, likely functioning in cross-signal integration.

Cluster c: Genes showed transient upregulation followed by a decline under cold stress, suggesting their involvement in the early perception of cold signals. Conversely, their expression levels steadily decreased following ABA application.

Cluster d: Genes displayed opposite expression trends between the two treatments, characterized by continuous downregulation under cold stress but steady upregulation under ABA treatment.

3.4. Gene Ontology (GO) Functional Enrichment Analysis

To elucidate the functional classifications of DEGs, Gene Ontology (GO) enrichment analysis was conducted across different time points for both ABA and cold treatments.

As shown in Figure 4, in ABA-treated samples, the overall enrichment intensity and the number of significantly enriched GO terms progressively decreased over time.

At 1 h, DEGs were significantly enriched in Molecular Function (MF) terms including transferase activity, transcription factor binding, oxidoreductase activity, and sulfate transmembrane transporter activity. Biological Process (BP) terms primarily encompassed defense response, response to stress, response to biotic stimulus, carbohydrate metabolic process, and protein modification process. Cellular Component (CC) terms were significantly enriched in the ribosome, cell wall, and extracellular matrix.

At 2 h, the number of enriched terms declined, though defense response and response to biotic stimulus maintained high significance.

By 4 h, enrichment intensity diminished further, with transmembrane transport, ion binding, and maintenance of cellular homeostasis becoming the predominant enriched functions.

In contrast, cold-stressed samples exhibited a temporal expansion in both the quantity and functional spectrum of enriched GO terms.

At 1 h, biological processes were dominated by response to endogenous stimulus, response to hormone, and response to chemical, while molecular functions were mainly enriched in glycosyltransferase activity, polysaccharide binding, and DNA-binding transcription factor activity.

At 2 h, iron ion binding and oxidoreductase activity became significantly enriched, with an increasing proportion of DEGs involved in polysaccharide metabolism and signal transduction pathways.

By 4 h, the functional coverage broadened further, highlighting significant enrichment in regulation of enzyme activity, ion binding, transcriptional regulation, polysaccharide metabolism, and extracellular matrix components. This transition indicates a shift in cold response from early stress perception and hormone signaling toward structural reorganization and metabolic remodeling.

Comparative synthesis of GO enrichment profiles between both treatments demonstrated shared enrichment in defense response, redox homeostasis regulation, ion transmembrane transport, and transcription factor-mediated transcriptional reprogramming. Both ABA application and cold stress exhibited a highly conserved response logic, initiated by early defense activation and signal transduction cascades, followed by a transition toward cellular homeostasis maintenance and metabolic acclimation in the later stages.

3.5. KEGG Pathway Enrichment Analysis

To further elucidate the metabolic networks and signal transduction pathways involved, KEGG pathway enrichment analysis was conducted for DEGs under ABA and cold treatments across different time points.

As shown in Figure 5, under ABA application, the enriched pathways exhibited a distinct temporal transition from basal metabolic activation toward hormone signal transduction cascades over time.

At 1 h, DEGs were predominantly enriched in metabolic biosynthesis pathways, including phenylpropanoid biosynthesis and sulfur metabolism.

At 2 h, significantly enriched pathways included flavonoid biosynthesis, ribosome biogenesis, and terpenoid and polyketide biosynthesis.

By 4 h, the most enriched pathways shifted from metabolic biosynthesis to signal transduction and stress response pathways, specifically plant-pathogen interaction, protein processing in endoplasmic reticulum, plant hormone signal transduction, and the MAPK signaling cascade.

Conversely, under cold stress, enriched pathways demonstrated an inverse temporal shift from initial signal transduction toward downstream metabolic biosynthesis.

At 1 h, early-responsive pathways were concentrated on signal perception and relay modules, including plant hormone signal transduction, the MAPK signaling cascade, protein processing in endoplasmic reticulum, and plant-pathogen interaction.

At 2 h and 4 h, enriched pathways progressively transitioned toward metabolic biosynthesis, such as biosynthesis of amino acids, cysteine and methionine metabolism, and flavonoid biosynthesis.

Cross-comparison revealed that both ABA application and cold stress significantly co-regulated secondary metabolism (e.g., phenylpropanoid biosynthesis and flavonoid biosynthesis) and shared core signaling pathways, including plant-pathogen interaction, protein processing in endoplasmic reticulum, plant hormone signal transduction, and the MAPK signaling cascade.

Notably, a temporal divergence was observed between the two stimuli: ABA application initially activated biosynthetic pathways before engaging signal transduction cascades, whereas cold stress immediately triggered signal transduction networks followed by secondary metabolic biosynthesis. These findings highlighted both functional crosstalk and distinct temporal division of labor between ABA- and cold-mediated stress regulatory networks in peach.

3.6. qRT-PCR Validation of Co-Responsive DEGs

Through cluster and enrichment analyses, six genes co-responsive to both cold stress and ABA signals—ERF48, ERF017, MYB6, WRKY46, WRKY40, and BHLH35—were selected for qRT-PCR validation. As shown in Figure 6, the qRT-PCR results indicated that under cold stress, the expression of PpMYB6 in ‘Shiji Zhixing’ peach exhibited an initial increase followed by a decrease over the treatment time series. Its expression peaked at 2 h of cold exposure, reaching a level 4-fold higher than that of the untreated control. Similarly, PpERF017 expression peaked at 2 h before declining slightly, showing a maximum expression level 40-fold higher than the untreated control. In contrast, the transcript levels of PpERF48, PpWRKY40, PpWRKY46, and PpBHLH35 continued to be upregulated throughout the treatment, reaching their maximum levels at 4 h. The qRT-PCR expression patterns of all six genes were highly consistent with the RNA-Seq dataset.

3.7. Bioinformatic Analysis of PpMYB6

As shown in Figure 7 and Figure 8, physicochemical property analysis using ExPASy ProtParam indicated that the PpMYB6 protein comprises 368 amino acids, with a predicted molecular weight of 42.22 kDa and a theoretical isoelectric point (pI) of 5.90. The PpMYB6 gene is located on chromosome 4 of the peach genome (genomic coordinates: 1,629,509–1,678,488 bp), with a total genomic length of 1,577 bp and a coding sequence (CDS) of 1,107 bp. Conserved domain analysis via NCBI CD-Search revealed that residues 15–118 aa harbor typical conserved R2R3-MYB and SANT domains. Secondary structure prediction using SOPMA showed that random coils constituted the largest fraction (56.30%), followed by extended strands (22.36%) and α-helices (21.34%). Tertiary structure modeling via Swiss-Model generated a 3D structure with 70.94% sequence coverage relative to the template model. Multiple sequence alignment and phylogenetic tree analysis demonstrated that PpMYB6 (Accession No. XM_007211402.2) shares high sequence similarity and conserved MYB-binding domains with MYB orthologs from almond (Prunus dulcis MYB102), plum (Prunus domestica MYB6), and pear (Pyrus pyrifolia MYB102).

To further explore the upstream regulatory mechanisms of PpMYB6 in response to cold and ABA signaling, cis-acting elements within the upstream promoter sequence of PpMYB6 were predicted. As shown in Figure 9, the promoter sequence of PpMYB6 contained multiple core cis-acting elements directly responsive to ABA and low temperature. Several DRE/CRT elements harboring the conserved CCGAC core motif were densely localized in the 600–900 bp upstream region of the promoter, suggesting that PpMYB6 can be directly activated by cold stress signaling pathways. Typical ABRE elements containing the ACGT core motif were also densely distributed in the 1100–1300 bp region, which can be directly bound and activated by bZIP transcription factors—the core regulators of ABA signaling—indicating that PpMYB6 directly participates in ABA responsiveness. Additionally, W-box elements recognized by WRKY transcription factors were widely present, which are extensively involved in the crosstalk between ABA signaling and abiotic stress responses. Cis-acting element analysis demonstrated that PpMYB6 is dual-regulated by both ABA and cold signals at the transcriptional promoter level, which is consistent with its functional role in mediating ABA-induced cold tolerance in peach described above.

3.8. Generation and Phenotypic Analysis of PpMYB6-Overexpressing Calli and Arabidopsis thaliana

As shown in Figure 10, following confirmation by genomic PCR and qRT-PCR, three independent positive PpMYB6-overexpressing (OE) callus lines were successfully established. To evaluate cold stress tolerance, wild-type (WT) and transgenic calli were subjected to low-temperature treatment, and their fresh weight (FW) changes were measured. Following cold treatment, the average FW of WT calli decreased by 51.2%, whereas the average FW of PpMYB6-OE calli decreased by only 5.8%. Phenotypic evaluation revealed that under normal growth conditions (28 °C), PpMYB6-OE calli exhibited a slightly slower growth rate compared with WT calli. However, under cold stress, while WT callus growth was severely restricted, the growth of PpMYB6-OE calli remained largely unaffected. These results demonstrated that overexpression of PpMYB6 significantly enhanced cold tolerance in peach callus while repressing callus growth under non-stress conditions.

As shown in Figure 11, following confirmation by genomic PCR and qRT-PCR, three independent positive PpMYB6-overexpression (OE) lines were successfully established. Wild-type (WT) and PpMYB6-OE Arabidopsis thaliana plants were subjected to freezing stress at -4 ℃, and their survival rates were evaluated. Following freezing treatment, the survival rate of WT plants dropped to 56.1%, whereas the survival rates of the three OE lines remained at 81.9%, 77.6%, and 79.3%, respectively, which were significantly higher than that of WT.

Under normal growth conditions (23 °C), WT plants exhibited a significantly greater plant height of 23.5 cm compared to the three OE lines (16.4 cm, 15.7 cm, and 15.1 cm, respectively). These results demonstrate that overexpression of PpMYB6 enhances freezing tolerance in Arabidopsis thaliana under cold stress, while repressing vegetative growth under non-stress conditions, which is highly consistent with the phenotypes observed in peach callus.

3.9. PpMYB6 Directly Binds to and Transcriptionally Activates the PpCBF2 Promoter

As shown in Figure 12, the PpCBF2 promoter region was significantly enriched with numerous cis-acting elements harboring MYB and MYB/SANT binding motifs. These findings suggest that the promoter of PpCBF2 may directly bind to MYB transcription factors. To verify whether PpMYB6 directly interacts w (PpERF48, PpERF017, PpMYB6, PpWRKY46, PpWRKY40, and PpbHLH3 ith the promoter of PpCBF2, a yeast one-hybrid (Y1H) assay was conducted. Yeast Y187 cells co-transformed with the recombinant reporter construct pHIS2-PpCBF2pro and the effector construct pGADT7-PpMYB6 grew normally on triple selective medium (SD/−Trp/−Leu/−His) supplemented with 55mM 3-AT. In contrast, negative control yeast co-transformed with empty pGADT7 vector and pHIS2-PpCBF2pro failed to grow under the same selective conditions.

Live bioluminescence imaging showed that leaf areas co-infiltrated with both 35Spro::PpMYB6 and PpCBF2pro::LUC exhibited significantly higher fluorescence intensity than control combinations (35Spro + LUC, 35Spro + PpCBF2pro::LUC, and 35Spro::PpMYB6 + LUC). Quantitative measurements of relative Firefly to Renilla luciferase activity (LUC/REN ratio) further confirmed that co-expression of 35Spro::PpMYB6 and PpCBF2pro::LUC yielded the highest relative LUC/REN value among all treatments. Altogether, These findings suggest that MYB transcription factors may directly bind to the PpCBF2 promoter and acts as a positive transcriptional activator to upregulate PpCBF2 expression.

3.10. Yeast Two-Hybrid (Y2H) Screening for PpMYB6-Interacting Proteins

To identify protein partners interacting with PpMYB6, a peach cDNA library was screened using a yeast two-hybrid (Y2H) system. Putative positive yeast single colonies were isolated and spot-plated onto quadruple dropout medium (SD/−Ade/−His/−Leu/−Trp, QDO) to validate potential interactions. As shown in Figure 13(a,b), following colony PCR confirmation, 58 positive yeast plasmids were recovered and sequenced.

As shown in Table 1, BLAST alignment and functional annotation against the NCBI database identified 24 non-redundant candidate interacting proteins involved in stress responses and developmental regulation. Notably, the identified candidate interactors included DWARF8: A DELLA protein serving as a master transcriptional repressor in gibberellin (GA) signaling and a negative regulator of plant growth.

The identification of these candidate partners laid a solid foundation for further elucidating the molecular mechanisms by which PpMYB6 coordinates cold stress tolerance and growth balance in peach.

4. Discussion

Cold stress represents one of the major environmental factors restricting peach yield and northern cultivation boundaries, while also exerting profound effects on fruit quality. During plant adaptation to low temperatures, phytohormone-mediated transcriptional regulation constitutes a highly complex molecular mechanism of cold resistance. Among plant hormones, abscisic acid (ABA) serves as a core stress hormone whose pivotal role in regulating abiotic stress responses in peach has been widely established. Furthermore, the protective effect of exogenous ABA application against cold stress in peach was confirmed in our previous study. However, the early molecular mechanisms governing ABA-mediated cold responses in peach remain to be fully elucidated.

Our transcriptomic analysis revealed that a substantial number of genes co-responded to both cold stress and ABA treatments, identifying a total of 1,105 overlapping DEGs. This overlap reinforces the functional role of ABA as a key hormonal regulator mediating cold stress tolerance in peach. These findings corroborate our group’s previous physiological research demonstrating that exogenous ABA enhances cold resistance in peach, while remaining consistent with established literature defining ABA as a primary phytohormone in plant cold responses [14,15,16].

GO and KEGG enrichment analyses demonstrated that the biological functions and responsive pathways under cold stress and ABA treatment exhibited both functional crosstalk and temporal divergence. DEGs from both treatments were significantly enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, oxidoreductase activity, MAPK signaling cascades, and plant hormone signal transduction pathways. These enrichment patterns closely mirror transcriptome characteristics reported in cold-stressed apple and citrus [19,20,21,22]. In particular, the enrichment of phenylpropanoid and flavonoid biosynthetic pathways aligns with earlier findings on cold resistance in peach, indicating that these pathways represent conserved defense mechanisms against cold stress across fruit tree species.

Furthermore, this study uncovered distinct temporal patterns during early responses to cold and ABA signals in peach. At early stress stages, cold treatment and ABA application displayed contrasting response kinetics. Cold stress first activated signal transduction pathways, followed by the regulation of cellular metabolism and biological functions. Conversely, ABA-responsive DEGs exhibited an inverse trend, with enrichment first observed in metabolic pathways before subsequently activating signal transduction cascades [23,24].

From the DEG clusters that co-responded to ABA and cold stress in an upregulated manner, six transcription factors—ERF48, ERF017, PpMYB6, WRKY46, WRKY40, and BHLH35—were selected for further investigation. qRT-PCR validation yielded expression profiles consistent with the RNA-Seq data, confirming that all six genes positively respond to cold stress, likely through an ABA-dependent regulatory pathway. Members of the MYB family have been extensively documented as master regulators mediating hormone signaling and cold stress adaptation [33,34], while numerous MYB transcription factors also act as key switches in flavonoid biosynthesis.

Through bioinformatic analysis and transgenic overexpression in peach calli, we demonstrated that PpMYB6 significantly enhanced cold tolerance in peach calli while suppressing callus growth under room temperature. The overexpressing calli exhibited a phenotype characterized by enhanced stress resistance accompanied by growth inhibition. To rigorously validate its biological function at the whole-plant level, heterologous overexpression lines were established in Arabidopsis thaliana. Under freezing stress, PpMYB6-overexpressing Arabidopsis plants displayed strikingly lower mortality rates and reduced freezing injury compared to wild-type controls. Intriguingly, under non-stress growth conditions, these transgenic lines displayed a distinct dwarfism/growth retardation phenotype. These whole-plant phenotypic observations directly mirror the responses observed in peach calli, providing definitive in vivo genetic evidence that PpMYB6 enhances cold resilience at the expense of vegetative growth. This trade-off aligns with previous findings on MYB-mediated growth-defense balance in wheat, as well as established evidence that abscisic acid (ABA) orchestrates the trade-off between stress tolerance and plant growth [46], suggesting that PpMYB6 serves as a key potential regulator coordinating stress tolerance and growth balance in peach.

To elucidate the downstream molecular mechanisms by which PpMYB6 regulates cold stress tolerance, we analyzed the cis-acting regulatory elements within the promoter of PpCBF2, a candidate downstream cold-responsive gene, and identified multiple MYB transcription factor binding motifs. Based on this finding, we confirmed the direct binding of PpMYB6 to the PpCBF2 promoter using yeast one-hybrid (Y1H) and dual-luciferase reporter assays. As PpCBF2 is a core regulatory component of the canonical ICE-CBF-COR signaling cascade [25,26,27], its direct upregulation by PpMYB6 substantially enhances cold tolerance in peach. Altogether, these results demonstrate that the ABA-responsive transcription factor PpMYB6 acts as a key upstream transcriptional activator governing cold stress tolerance in peach (Prunus persica).

Through Y2H library screening, we identified multiple potential interacting proteins for PpMYB6, offering critical clues into its dual functions in conferring cold tolerance and suppressing growth. Among these candidates, NCED1 acts as a key rate-limiting enzyme in ABA biosynthesis, a role validated in various crops including tomato, wheat, and maize [47,48]. The interaction between PpMYB6 and NCED1 may promote endogenous ABA accumulation, thereby exerting positive feedback control that amplifies ABA signaling under cold stress.

Another candidate, DWARF8, belongs to the DELLA protein family and serves as a major transcriptional repressor in the gibberellin (GA) signaling pathway. Extensive research indicates that DELLA proteins regulate plant growth and development through binding to GID1[49,50]. The interaction between DWARF8 and PpMYB6 may represent the molecular basis underlying the negative growth regulation exerted by PpMYB6. By integrating ABA biosynthesis and GA signaling, PpMYB6 concurrently coordinates stress tolerance and plant growth during cold exposure.Additionally, the identified transcriptional coactivator SRC2 has been reported to regulate abiotic stress resistance in maize and interact with AtRbohF in Arabidopsis to facilitate downstream stress gene expression [51]. Its interaction with PpMYB6 may enhance the transcriptional activation efficiency of downstream target genes.

Altogether, the identification of these interacting proteins outlines a preliminary molecular framework through which PpMYB6 balances cold tolerance and growth in peach, laying a solid foundation for future pathway dissection.

5. Conclusions

Integrative transcriptomic analysis under abscisic acid (ABA) and low-temperature treatments, combined with quantitative real-time PCR (qRT-PCR) validation, identified PpMYB6 as a core transcription factor gene co-responsive to ABA and cold signaling. Bioinformatic characterization and phenotypic assays of PpMYB6-overexpressing (OE) transgenic peach callus and Arabidopsis thaliana revealed the vital function of PpMYB6 in coordinating the balance between plant growth and cold stress tolerance. Furthermore, yeast one-hybrid (Y1H) and dual-luciferase reporter assays confirmed that PpMYB6 directly binds to the promoter of the key downstream cold-tolerance gene PpCBF2 to activate its transcription. Yeast two-hybrid (Y2H) library screening identified candidate protein partners, providing valuable resources for mapping the PpMYB6 regulatory network. Among these candidates, PpDWARF8 represses plant growth by modulating the gibberellin (GA) signaling pathway, serving as a key interactor mediating the negative growth regulation by PpMYB6.