Overexpression of CmMYBS3 decreases the cold tolerance in the ground-cover chrysanthemum

Overexpression of CmMYBS3 decreases the cold tolerance in the ground-cover chrysanthemum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Overexpression of CmMYBS3 decreases the cold tolerance in the ground-cover chrysanthemum Jiayi Zhou, Li Zhao, Yingjie Quan, Peng Liu, Xintong Li, Yansong Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7107028/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Low temperature constitutes a critical abiotic stress that significantly impairs plant growth and development, particularly for species in cold regions. In Northeast China, the persistently low winter temperatures over an extended period pose significant challenges to the survival of chrysanthemums. Results This study employed the ground cover plant "Yingjie" as the experimental material and cloned the CmMYBS3 . The CmMYBS3 protein lacks transcriptional activity and is localized exclusively in the nucleus. Under low-temperature treatment, the activities of SOD, CAT, and POD were significantly lower in chrysanthemums overexpressing CmMYBS3 than in the wild-type line. Additionally, the MDA content in the CmMYBS3 overexpression lines was higher than in the wild-type lines. To elucidate the mechanism by which CmMYBS3 regulates the response to low temperature, we conducted transcriptome sequencing analysis and identified a total of 5425 differentially expressed genes, comprising 2646 upregulated genes and 2779 downregulated genes. The GO analysis reveals that the primary enrichment occurs in the "biological process", "cellular component", and "molecular function". The KEGG enrichment analysis identified significant alterations in several pathways associated with plant growth and development, as well as stress responses. Through yeast single-hybrid analysis, it was demonstrated that CmMYBS3 specifically binds to the promoter region of CmDREB1 and inhibiting the expression of the CmDREB1 . Conclusion This study demonstrates that CmMYBS3 reduces the cold tolerance of ground cover chrysanthemums by suppressing the expression of the CmDREB1 gene, providing an important theoretical basis for the breeding of cold-tolerant ground cover chrysanthemum varieties. Ground cover chrysanthemum CmMYBS3 RNA-Seq Cold stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Abiotic stresses, such as temperature stress, salinity stress, drought, and waterlogging stress, significantly affect plant growth and development. Among these, low-temperature stress plays a crucial role in the geographical distribution of plants and influencing their yield and quality, especially in cold regions such as high latitudes or high altitudes. Low temperature significantly impacts plant compromises the stability of the cell membrane system, and inhibits photosynthesis. Under low temperature conditions, the plants exhibit a reduced growth rate, leaf waterlogging, wilting and abscission, even the death of the plant [ 1 ]. In response to low-temperature stress, plants enhance the activity of antioxidant enzymes and promote the accumulation of osmoregulatory sbustances, effectively mitigating stress-induced damage [ 2 ]. Many studies have reported that superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) play crucial roles in scavenging Reactive Oxygen Species (ROS), and their enzyme activities are often used as important indicators of plant antioxidant capacity [ 3 , 4 ] In addition, plants mitigate the damage caused by low-temperature stress to organelles and cell membranes through enhanced accumulation of osmoregulatory substances. Collectively, these mechanisms contribute to maintaining intracellular ROS homeostasis and enable plants to adapt to low-temperature environments [ 5 , 6 ]. In recent years, research into the mechanisms of plant responses to low-temperature stress has progressively deepened from the physiological level to the molecular level, leading to a series of significant breakthroughs. Notably, the ICE-CBF/DREB-COR signaling pathway has garnered extensive attention as a pivotal mechanism enabling plants to withstand low-temperature stress. When plants perceive cold signals, Inducer of CBF Expression (ICE) functions as an upstream transcription factor by binding to the C-repeat binding factor/dehydration responsive element binding (CBF/DREB) response element to promote the expression of CBF/DREB genes. Subsequently, CBF/DREB binds to the dehydration response element (DRE) to activate cold-responsive ( COR ) gene expression and induce the synthesis of a series of antifreeze proteins and metabolites, thereby enhancing plant cold tolerance [ 7 ]. In addition, several transcription factor families have been identified to be involved in the regulation of plant cold stress. These families of transcription factors include, MYB [ 8 ], AP2/ERF [ 9 ], WRKY [ 10 ], NAC [ 11 ], and bHLH [ 12 ]. Notably, MYB transcription factor family is not only involved in the regulation of plant growth and development, but also plays an important role in the response to low temperature stress. Based on the different number of repetitions (R) in a sequence, the MYB family can be divided into four subfamilies: 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB, with each R repetition consisting of approximately 50–52 amino acid residues [ 13 , 14 ]. These R domains are composed of tandem repeats, forming a distinctive helix-loop-helix (HLH) topological conformation that facilitates their involvement in the DNA binding process [ 15 ]. AtMYB15 suppresses CBF gene expression in Arabidopsis , leading to reduced antioxidant enzyme activity and increased cold sensitivity in plants [ 16 ]. The expression of OsMYB3R-2 and OsMYBS3 in rice, as well as MpMYBS3 in banana, was up-regulated under low-temperature stress. Furthermore, their heterologous overexpression in transgenic plants conferred robust cold tolerance through modulating stress-responsive gene network [ 17 ]. Our previous transcriptome analysis revealed that the expression of CmMYBS3 in ground cover chrysanthemum 'Yingjie' was significantly downregulated under low-temperature treatment compared to normal-temperature treatment [ 18 ]. This suggests that CmMYBS3 may play a role in the plant's cold resistance mechanism. In Chrysanthemum , CmMYBS3 inhibits the expression of CmMYB121 by forming a complex with CmHSFA4 and CmTPL , thereby enhancing the salinity tolerance [ 19 ]. Nevertheless, whether CmMYBS3 also functions in regulating cold tolerance remains to be elucidated. Ground cover chrysanthemum ( Chrysanthemum × morifolium ) is a herbaceous perennial species in the genus Chrysanthemum (family Asteraceae), which has excellent characteristics such as rich flower colors, dense flower clusters, rounded and compact crowns, as well as a high tolerance to cold and drought. In northern China, the low temperature and prolonged winter duration pose significant challenges to the overwintering survival of ground cover chrysanthemums, resulting in a relatively low survival rate [ 20 ]. This environmental conditions severely limited the large-scale deployment of ground cover chrysanthemums in the region. Therefore, in this study, the CmMYBS3 gene was cloned to investigate its spatial expression pattern, subcellular localization, self-activation activity, and biological functions in cold stress responses. Additionally, transcriptomic data were employed to analyze the downstream regulatory network of CmMYBS3 . These results clarify the function of the CmMYBS3 gene and the molecular mechanism underlying cold tolerance regulation, and providing a solid theoretical foundation for the molecular genetic breeding of ground cover chrysanthemums. Materials and methods Plant materials and growth conditions Cuttings of the ground-cover chrysanthemum variety ‘Yingjie’ was obtained from the greenhouse at Yanbian University Teaching Base, located in Yanji City, Jilin Province, China. Aseptically excised shoot tips of ground cover chrysanthemum 'Yingjie' (2.0 cm in length) were cultured on Murashige and Skoog (MS) medium supplemented with 30 g/L sucrose and 0.8% agar, maintained under 16-h photoperiod (30 µmol·m⁻²·s⁻¹) at 25 ± 2°C for 4-week subculture cycles. The obtained tissue culture seedlings were used as materials for gene cloning and genetic transformation. The transgenic lines were transplanted to the greenhouse at Yanbian University's teaching base. The environmental conditions were stringently controlled, with a temperature range of 25°C–27°C, a photoperiod of 14 hours light/10 hours dark, and relative humidity maintained between 60% – 70%. Plants that developed approximately 7–8 leaves and exhibited robust growth were selected for subsequent experiments. Cloning and sequence analyses of CmMYBS3 Total RNA was extracted from the leaves of the ground cover plant 'Yingjie' using the Trans-Zol Plant Reagent Kit (TIANGEN, Beijing, China). The extracted RNA was then reverse-transcribed into cDNA using the FastKing gDNA Eliminating RT SuperMix Reagent Kit (TIANGEN, Beijing, China). Based on the CDS sequence of the CmMYBS3 gene previously screened and annotated by our research group from the transcriptome database of the 'Yingjie', specific primers MYBS3-F/R were designed using Primer 5.0 software (Table S1 ). The PCR reaction system consisted of 2.5 µL of 10 × PCR Buffer (containing Mg 2+ ), 1.6 µL dNTP (2.5 mmol/L), 1.0 µL cDNA, 1.0 µL each of the upstream and downstream primers (10 µmol/L), 0.2 µL rTaq, and 17.3 µL ddH2O, in a total volume of 20 µL. The PCR reaction was programmed as follows: 94°C for 30 s, 57°C for 30 s, 72°C for 90 s, with 35 cycles. Finally, the reaction was extended at 72°C for 10 min and stored at 4°C. Subsequently, the PCR products were recovered and ligated with the vector PMD-T19, and then sent to Nanjing Sipujin Biotechnology Co., Ltd.. for sequencing. Download the MYBS3 protein sequences of various plants from NCBI, perform multiple sequence alignment using DNAMAN software, analyze the conserved domains of CmMYBS3 protein via NCBI CD-Search. The phylogenetic tree was constructed by using MEGA7.0 software. Gene expression analysis The 'Yingjie' plants were subjected to cultivation temperatures of 25°C, 10°C, 5°C, and − 5°C for 2 hours each. Following the treatment, 3–4 leaves from the upper portion of the plant were harvested and immediately immersed in liquid nitrogen for long-term preservation at -80°C, for subsequent evaluation of cold-responsive biomarkers (such as SOD, POD and CAT activities). Collect the roots, stems, leaves and flower organs of 'Yingjie', and analyze the spatio-temporal expression pattern of CmMYBS3 . The quantitative Real-Time PCR (qRT-PCR) reaction system consisted of 5 µL of cDNA, 5 µL of 2 × SuperReal PreMix Plus, 0.4 µL of 50 × ROX Reference DyeΔ, and 1 µL of each of the forward and reverse primers (Table S1 ), and the addition of 2.6 µL of deionized water. The cycling program was set as follows: pre-denaturation at 95 ℃ for 2 min; 35 cycles were performed, including denaturation at 95 ℃ for 15 s, annealing at 58 ℃ for 15 s, and extension at 72 ℃ for 30 s. The melting curves were prepared in the range of 55 to 95 ℃. The CmEF1α gene was used as an internal reference (Table S1 ). Each treatment was performed with three biological replicates, and the relative expression levels of the target genes were calculated using the 2 −∆∆Ct method. Subcellular localization To investigate the subcellular localization of the CmMYBS3 protein, the constructed pORE-R4-35S- CmMYBS3 vector was introduced into Agrobacterium EHA105 via the heat shock method. The Agrobacterium tumefaciens suspension containing the target construct was subsequently infiltrated into tobacco leaves. The infiltrated plants were maintained under controlled environmental conditions (25 ± 2°C, 16 h photoperiod) for 72–96 hours. Finally, the subcellular localization of CmMYBS3 was determined by observing the fluorescence signals using confocal microscopy. Transcriptional activation assay The pGBKT7- CmMYBS3 bait construct was generated through Gateway cloning technology (Invitrogen) and validated by restriction enzyme analysis ( Nde I / BamH I). Recombinant plasmids including the experimental construct (pGBKT7- CmMYBS3 ), negative control (pGBKT7-Lam, Clontech), and positive control (pGBKT7-53, Clontech) were introduced into Y2HGold yeast cells via lithium acetate-mediated transformation. Subsequently, culture the transformed yeast strains in synthetic dextrose dropout medium without tryptophan (SD/-Trp) medium at 30°C for 3–4 days. Select individual colonies from the growing yeast strains and inoculate them onto SD/-His-Ade medium supplemented with 5-bromo-4-chloro-3-indolyl α-D-galactopyranoside (X-α-gal) and the growth of the colonies was observed. Expression vector construction and identification of transgenic chrysanthemums The coding sequence of CmMYBS3 was PCR-amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs) with gene-specific primers containing BamH I and Sal I restriction sites. The amplicon was purified via AxyPrepTM Biospin Gel Extraction Kit (Axygen, Hangzhou, China) and ligated into pORE-R4-35S vector (Clontech) digested with corresponding restriction enzymes. Recombinant clones were verified by colony PCR followed by sequencing. The recombinant plasmid was introduced into Agrobacterium EHA105 to genetically transform chrysanthemums with reference to the method of Gao et al [ 21 ]. The recombinant pORE-R4-35S- CmMYBS3 plasmid was introduced into Agrobacterium tumefaciens EHA105 by freeze-thaw transformation. Bacterial cultures were incubated in YEP liquid medium supplemented with 50 mg/L kanamycin for 16 hours at 28°C with shaking (180 rpm). Plant transformation was performed using the leaves dip method [ 21 ] with minor modifications. To identify the positive strain, Genomic DNA was isolated from putative transgenic chrysanthemum leaves, and specific primers (Table S1 ) were designed for PCR detection. Total RNA was isolated from the transgenic lines, and the expression level of CmMYBS3 was detected through reverse transcription into cDNA followed by qRT-PCR analysis. Relative expression levels were calculated using the 2 −ΔΔCt method with three biological replicates. Cold treatment of CmMYBS3 overexpression plants and their oxidative responses Three CmMYBS3 overexpression lines (OE3, OE4, and OE5) and wild-type (WT) 'Yingjie' at the five-leaf stage were selected for differential temperature stress treatments in growth chamber (MGZ-200L-2). The temperatures were set at 25 ℃, 10 ℃, 5 ℃, and − 5 ℃, respectively. After each treatment lasting for 2 hours, the morphological of the CmMYBS3 overexpression chrysanthemum were observed. At the same time, the topmost 3–4 leaves were harvested for the measurement of SOD, POD, and CAT enzyme activities, as well as Pro and MDA contents. Specific assays were conducted following the method described by Guo et al [ 22 ]. To evaluate the accumulation of H 2 O 2 and O 2 − under cold treatment, histochemical staining was conducted using 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), following the method described by Wei et al [ 23 ]. Transcriptome analysis Four-week-old CmMYBS3 overexpressing lines (OE3) and WT at the four-leaf stage were exposed to cold stress in a climate chamber (MGZ-200L-2) at 4°C for 2 hours, with 65% relative humidity and 16 h photoperiod. Then, the third leaf was selected for RNA-seq. Total RNA was extracted from frozen samples using the Trans-Zol Plant Kit (TIANGEN, Beijing, China) following the manufacturer's protocol. The integrity and quality of the RNA were assessed by agarose gel electrophoresis. The library was sequenced on the Illumina HiSeq2000 platform by Biomarker Technologies Co., Ltd. (Beijing, China). The raw sequencing data was processed through Cutadapt to remove low-quality sequences and adapter contaminants, yielding high-quality clean reads. Subsequently, the HISAT2 aligner was employed for efficient and precise alignment of the processed reads against the reference genome ( Chrysanthemum lavandulifolium Genome, https://cgdnjau.edu.cn/asteraceae/browse/genomepage/cl ). Gene expression levels were quantified using fragments per kilobase of transcript per million mapped reads (FPKM) values. During the detection of differentially expressed genes (DEGs), DESeq2 software was utilized for differential analysis, and FC ≥ 1.5 and FDR < 0.05 would be used as the screening criteria. Differentially expressed genes (DEGs) were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. The results were visualized using TBtools. Data analysis All experiments were independently replicated three times. The data were statistically analyzed using SPSS statistical software (version 21.0) and visualized using GraphPad Prism (version 8.3.0). Significant differences were analyzed using Duncan's new multiple range test, and the level of significance was set at p < 0.05. Results Cloning and bioinformatics analysis of CmMYBS3 CmMYBS3 is a gene with a length of 1151 bp, containing an open reading frame (ORF) that encodes a protein consisting of 358 amino acids. The relative molecular weight of CmMYBS3 protein was predicted to be 38992.64 kDa, isoelectric point was 7.72, instability index was 52.27, grand average hydrophilicity was − 0.660, and aliphatic index was 65.39 by ExPASy website (Table S2). The functional domain of CmMYBS3 protein was predicted using the NCBI CD-search tool, and it was identified as containing the SANT-MYB-SHAQKYE functional domain (Fig. 1 a). Phylogenetic analysis of MYB proteins revealed that CmMYBS3 exhibits the highest degree of homology with TcMYBS3 and is classified as a member of the MYB CCA1-like family (Fig. 1 b). Analysis of the expression level of CmMYBS3 The CmMYBS3 gene is expressed in various organ of the 'Yingjie', including roots, stems, flowers, and leaves. Notably, the expression level CmMYBS3 is highest in the roots (2.15), followed by flowers and stems (Fig. 2 a). To investigate the expression characteristics of the CmMYBS3 gene under low temperature stress, the 'Yingjie' plants were subjected to low-temperature treatment. The results indicated that the expression level of CmMYBS3 initially decreased and subsequently increased. At 5°C, the expression level of CmMYBS3 was at its lowest. At -5°C, the expression level of CmMYBS3 increased slightly (Fig. 2 b). These results suggest that low temperature significantly affects the expression of CmMYBS3 gene and plays an important role in the cold stress response of 'Yingjie'. Analysis of subcellular localization and transcriptional activation activity of CmMYBS3 To analyze the subcellular localization of CmMYBS3 protein, the Agrobacterium strain harboring the CmMYBS3 -GFP fusion gene was infiltrated into tobacco leaves. The results demonstrated that CmMYBS3 -GFP emitted a strong green fluorescence signal exclusively in the cell nucleus, whereas the positive control GFP exhibited uniformly distributed fluorescence throughout the entire cell (Fig. 3 a). These findings confirm that the CmMYBS3 protein is localized specifically in the cell nucleus. The transcriptional activation activity of CmMYBS3 was analyzed using yeast single-hybrid experiments. As shown in Fig. 3 b, all yeast cells grew robustly on the SD/-Trp medium, suggesting that the pGBKT7- CmMYBS3 plasmid had been successfully transformed into the Y2H yeast strain. The transformed bacterial solution was plated onto the SD/-His-Ade + X-α-gal medium. It was observed that the pGBKT7- CmMYBS3 colonies failed to grow and did not exhibit a blue coloration. Conversely, the positive control pGBKT7-53 formed colonies that grew successfully and displayed a blue color, suggesting that the transcription factor CmMYBS3 lacks transcriptional activation activity. Screening and identification of CmMYBS3 overexpression lines An overexpression vector was constructed and Agrobacterium -mediated transformation was performed. Consequently, five independent transgenic seedlings (designated as OE1, OE2, OE3, OE4, and OE5) were successfully obtained. DNA was extracted independently from each seedlings. PCR analysis revealed the presence of specific bands in the OE1, OE3, OE4, and OE5 lines, whereas no bands were detected in OE2 and the WT lines (Fig. 4 a). At the same time, we analyzed the expression levels of CmMYBS3 in each seedling. Our results showed that the expression levels of CmMYBS3 in the OE3, OE4, and OE5 lines were significantly higher than those in the WT. Notably, the relative expression level of CmMYBS3 in the OE3 line was 4.20 times higher than that in the WT (Fig. 4 b). These findings further confirm that the OE3, OE4, and OE5 lines are overexpression lines. Changes in ROS accumulation and antioxidant enzyme activities in the CmMYBS3 overexpressing line under low-temperature stress As the temperature decreased, the degree of injury in both the overexpression lines and the WT plants was significantly increased. At 5°C, the leaves of the overexpression lines became markedly softer and drooped, with an increased angle at the leaf axil compared to the WT plants. When the temperature dropped to -5°C, the CmMYBS3 overexpression lines displayed severe wilting (Fig. 5 a). Following low-temperature stress treatment and histochemical staining with NBT and DAB, it was observed that the CmMYBS3 overexpression lines exhibited a more intense blue color after NBT staining, indicating a higher accumulation of O₂⁻ compared to the WT plants. DAB staining revealed that the leaves of the CmMYBS3 overexpression lines exhibited a dark brown coloration, indicating a significantly higher H 2 O 2 content compared to the WT (Fig. 5 b). To further investigate the cold resistance of the CmMYBS3 overexpression lines, we analyzed the activities of antioxidant enzymes. At low temperatures (10 ℃ ~ -5 ℃), the activities of SOD, POD, and CAT in the CmMYBS3 overexpression lines were significantly lower than those in the WT (Fig. 5 c-e). At the same time, we also measured and analyzed the contents of MDA and Pro in the CmMYBS3 overexpression lines. The MDA content exhibited a gradual increase as the temperature decreased. At -5 ℃, the MDA content in the CmMYBS3 overexpression lines was significantly higher than that in the WT. Specifically, the MDA content of OE3 was 57 µg.g − 1 , which was 1.16 times higher than that of the WT. The proline content first decreased and then increased. Under the same low-temperature conditions, the proline content in the CmMYBS3 overexpression lines were lower than that in the WT. Specifically, at 5°C, the proline content in the WT was 1.25 times higher than that in the OE3 (Fig. 5 g). These results suggest that the CmMYBS3 overexpression lines exhibits lower cold tolerance compared to the WT. Transcriptome analysis of CmMYBS3 overexpressing lines under cold stress The molecular mechanism of CmMYBS3-mediated cold tolerance regulation was investigated via comprehensive transcriptome profiling of CmMYBS3 overexpression lines and wild-type plants following cold stress treatment. A total of 36.29 Gb of clean data was obtained, with an average of 5.79 Gb per sample and a Q30 value of at least 97.38%. The alignment rate of reads to the reference genome varied between 60.17% and 66.12% across samples (Table S3). Principal Component Analysis (PCA) was conducted to evaluate the similarity among the six samples. The first principal component (PC1) explained 35.61% of the total variance, whereas the second principal component (PC2) accounted for 18.41% of the variance (Fig. 6 a). The Pearson correlation coefficient was employed to perform the correlation analysis on the samples. The correlations between the three replicates were all higher than 0.810, which demonstrates the high reliability of the data (Fig. 6 b). A total of 5,425 differentially expressed genes (DEGs) were identified between CmMYBS3 overexpression lines and WT plants. Specifically, 2,646 genes were up-regulated and 2,779 genes were down-regulated (Fig. 6 d). GO enrichment analysis revealed that the DEGs were significantly enriched and annotated into three main categories: "biological process", "cellular component", and "molecular function". Specifically, 1920 DEGs were enriched in " biological process ", 2776 DEGs were enriched in " cellular component ", and 3468 DEGs were enriched in " molecular function " (Table S4). Notably, the DEGs were predominantly enriched in the cellular anatomical entitiy within the cellular component category, cellular processes within the biological process category, and binding functions within the molecular function category (Fig. 6 c). KEGG enrichment analysis revealed significant alterations in pathways associated with plant development and stress response, including "plant hormone signal transduetion", "circadian rhythm -plant", "beta-Alanine metabolism", "alpha-Linolenic acid mefabolism", "biosynthesis of amino acids", "carbon metabolism", "arginine and proline metabolism", as well as "monoterpenoid biosynthesis" (Fig. 6 e). These findings suggest that under low-temperature stress, CmMYBS3 is involved in multiple metabolic pathway reactions. Validation of transcriptome data Twelve genes including members of the CmbHLH , CmMYB , and CmWRKY transcription factor families, were randomly selected for qRT-PCR validation (Table S1 ). At 4°C, the qRT-PCR results demonstrated that the expression levels of the EVM0068325 and EVM0066486 genes in the CmMYBS3 overexpression lines were reduced compared to those in the WT. In contrast, the expression levels of the EVM0018803, EVM0023763, EVM0006868, EVM0056947, EVM0021029, EVM0059112, EVM0060714, EVM0022717, EVM0078075, and EVM0020482 genes were increased (Fig. 7 ). These findings are consistent with the trends observed in the FPKM data from the transcriptome analysis, thereby reinforcing the reliability of the results. Discussion Low-temperature stress is a critical abiotic stress factor that significantly affects plant growth and development[ 24 , 25 ]. Under low-temperature conditions, the stability of the plant cell membrane system is initially impaired. The phase transition of membrane lipids induces structural damage to the membranes, which in turn results in cellular substance leakage. Meanwhile, low temperatures disrupt the normal metabolic processes in plants, leading to an imbalance in the electron transport chain and a substantial accumulation of reactive oxygen species (ROS). The excessive production of ROS intensifies oxidative stress, causing damage to biological macromolecules such as proteins, nucleic acids, and lipids. This subsequently impairs cellular structures and functions, and if prolonged or severe, may ultimately result in plant mortality[ 26 ]. However, over the course of prolonged evolution, plants have developed a series of complex and sophisticated adaptive survival mechanisms to withstand harsh environmental conditions. For instance, they alleviate the toxic effects of reactive oxygen species (ROS), facilitate the accumulation of osmoregulatory substances, and synthesize antifreeze proteins[ 27 – 29 ]. At the molecular regulatory level, transcription factors (TFs) serve as pivotal hubs in modulating plant cold tolerance. The TFs specifically recognize and bind to the cis-acting elements of downstream target genes, thereby activating or repressing the expression of associated genes and collectively regulating the aforementioned physiological and biochemical processes[ 30 , 31 ]. The transcription factors involved in plant cold stress responses include MYB , bHLH , ICE , and others. Among these, the MYB family has garnered significant attention owing to its frequent appearance in studies exploring cold resistance mechanisms[ 32 ]. The study by Liao et al[ 33 ]. revealed that soybean plants overexpressing the GmMYB76 and GmMYB177 genes exhibited significantly elevated proline contents following exposure to low temperature, accompanied by a marked improvement in survival rates. These findings suggest that the GmMYB76 and GmMYB177 genes are critical regulators in the soybean's response to low temperature stress. The study by Lee and Seo[ 34 ]demonstrated that in the myb96 mutant, the expression levels of CBF and its downstream COR genes were markedly decreased. Further investigation revealed that the interaction between MYB96 and the HHP protein suppresses the expression of downstream CBF genes[ 34 ]. The DgMYB1 and DgMYB2 genes were successfully cloned from chrysanthemum. Compared with the wild type, the DgMYB1/2 overexpression lines exhibited significantly reduced REL and MDA content, along with markedly enhanced activities of antioxidant enzymes, including SOD, POD, and CAT. Additionally, the accumulation contents of osmotic adjustment substances, such as soluble sugar, soluble protein, and Pro, were also significantly increased. These physiological alterations collectively contributed to the enhanced cold tolerance of chrysanthemum[ 35 , 36 ]. In this study, the Agrobacterium -mediated transformation method was utilized to successfully generate the CmMYBS3 overexpression 'Yingjie'. Following exposure to low temperature stress, phenotypic analysis demonstrated that, compared with the WT, the angle between the leaf axils of the CmMYBS3 overexpression significantly increased, resulting in a drooping leaf posture (Fig. 5 a), suggesting its heightened sensitivity to low-temperature conditions. The physiological index tests demonstrated that the activities of SOD, POD, and CAT in the CmMYBS3 overexpression lines were significantly lower compared to those in the WT. Furthermore, the relative expression analysis of CmSOD , CmPOD , and CmCAT revealed that their transcriptional levels in the CmMYBS3 overexpression lines were also significantly reduced relative to those in the WT (Fig. S1 ). Based on these findings, it can be inferred that the CmMYBS3 gene suppresses the antioxidant capacity of the ground cover chrsanthemum, thereby exacerbating membrane lipid peroxidation damage and reducing the plant's tolerance to low-temperature stress. The ICE-CBF/DREB-COR signaling pathway is widely recognized as one of the most extensively studied and well-documented regulatory pathways in the field of cold tolerance. This pathway utilizes the inducible transcription factor ICE (Inducer of CBF Expression) as an upstream activation element. Upon perception of low-temperature signals, ICE activates the expression of CBF (C-repeat Binding Factor) or DREB (Dehydration Responsive Element Binding) transcription factors. The activated CBF/DREB then bind to the CRT/DRE cis-acting element located in the promoter region of downstream COR (Cold-Responsive) genes, thereby promoting the expression of COR genes and enhancing plant cold tolerance[ 37 ]. In Arabidopsis thaliana , DREB/CBF proteins are classified into six subgroups according to their binding domains. Among these, the three genes DREB1B/CBF1 , DREB1A/CBF3 , and DREB1C/CBF2 are capable of responding to low temperature signals. Studies have demonstrated that under cold stress conditions, transgenic Arabidopsis plants overexpressing the DREB1B/CBF1 or DREB1C/CBF2 genes exhibit significantly higher survival rates compared to wild-type plants[ 38 ]. Under the induction of exogenous methy jasmonic acid, the expression level of the COR gene in wheat was significantly upregulated. Further investigation demonstrated that under low-temperature stress conditions, the antioxidant enzyme including CAT, SOD, and POD in COR overexpressing wheat plants exhibited markedly enhanced activity compared to those in WT plants [ 39 ]. In the low-temperature regulation network, jasmonic acid (JA) has a significant impact on the ICE-CBF/DREB-COR pathway. When plants do not perceive the JA signal, the (JASMONATE ZIM-DOMAIN 1) JAZ1 protein is highly expressed. It can interact with the ICE1 of the cold signal pathway, thereby inhibiting the transcriptional activation activity of ICE1 on downstream CBF genes [ 40 , 41 ]. Under low-temperature stress conditions, the biosynthesis pathway of JA in plants is rapidly activated, catalyzing the synthesis of the key signaling molecule jasmonoyl-isoleucine (JA-Ile). JA-Ile functions as a specific ligand that efficiently binds to the F-box protein CORONATINE INSENSITIVE 1 (COI1), inducing a conformational change in COI1[ 42 ]. The altered COI1 subsequently forms a stable co-receptor complex with JAZ1 proteins. This complex triggers the degradation of JAZ1 proteins via the 26S proteasome-mediated ubiquitination process [ 43 ]. The degradation of JAZ1 proteins effectively alleviates their inhibitory effect on ICE transcription factors, thereby initiating the expression of downstream cold-responsive genes and enabling plants to acquire cold tolerance (Fig. 8 ). Hu et al. [ 44 ]demonstrated that the overexpression of JAZ1 and JAZ4 significantly downregulated the expression levels of CBF genes by inhibiting ICE expression, thereby reducing the cold stress tolerance of Arabidopsis thaliana . This study revealed that the expression levels of CmDREB1 and CmCOR were reduced in the CmMYBS3 overexpression lines. It is possible that the expression of CmMYBS3 suppresses the expression of CmDREB1 , consequently inhibiting the expression of downstream CmCOR genes. We performed a yeast single-hybrid experiment with CmMYBS3 as the prey and the CmDREB1 promoter as the bait. All yeast cells exhibited robust growth on SD/-Trp/-His/-Leu medium. However, upon the addition of 3-AT to the medium, only the yeast cells harboring both the prey and the bait demonstrated normal growth (Fig. 8 b), indicating that CmMYBS3 may directly interact with the CmDREB1 promoter. These preliminary findings suggest that CmMYBS3 may decrease the cold tolerance of ground cover chrysanthemum by repressing the expression of CmDREB1 . At the same time, it was observed that the expression level of CmJZA1 was upregulated, suggesting that CmMYBS3 may positively regulate the expression of CmJAZ1 . However, no significant changes were detected in the expression level of CmICE1 between the CmMYBS3 overexpression lines and the wild type. These results indicate that CmMYBS3 does not suppress the expression of CmCOR via the CmJZA1 pathway to reduce cold tolerance (Fig. 8 a). This finding offers critical insights into the regulatory mechanism of CmMYBS3 within the plant cold tolerance network. Conclusion In summary, the cold-related gene CmMYBS3 was isolated from the ground cover chrysanthemum 'Yingjie', and its overexpression resulted in increased sensitivity to cold stress in the ground cover chrysanthemum. Under low-temperature stress, the induction of antioxidant enzyme activity was reduced in the overexpressed lines, leading to increased levels of reactive oxygen species (ROS) in the transgenic lines. Additionally, the transgenic lines exhibited lower Pro content and higher MDA content compared to the wild-type. After low-temperature treatment, the transgenic lines showed wilting and drooping leaves compared to the wild-type, indicating that the overexpression of CmMYBS3 reduces the cold tolerance of the groundcover chrysanthemum. Transcriptome and yeast one-hybrid data indicate that CmMYBS3 binds to the CmDREB1 promoter and inhibits transcription, thereby increasing sensitivity to low temperatures. We will further validate CmMYBS3 and continue to explore its relationship with the JA pathway. This will enhance our understanding of the role of the CmMYBS3 protein in cold stress responses and lay the foundation for further exploration of the molecular mechanisms underlying its negative regulation of cold tolerance. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The raw transcriptome data used during this study has been deposited in NCBI SRA with the accession number PRJNA1293879. GenBank accession number for the nucleotide sequence of the CmMYBS3 gene: BankIt2984616 Seq1 PV982153. Declaration of competing interest The authors declare that they have no conflicts of interest. Funding This work is supported by National Natural Science Foundation of China (NSFC) (32060696), Project funded by the Science and Technology Department of Jilin Province (YDZJ202501ZYTS556), Science and Technology Project of Education Department of Jilin Province (JJKH20220541KJ). 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Effect of long-chain saturated and unsaturated fatty acids on hypothalamic fatty acid sensing in Chinese perch ( Siniperca chuatsi ). Comp Biochem Phys B. 2020;241:110395. Kyu SY, Naing AH, Pe PPW, Park KI, Kim CK. Tomato seeds pretreated with Antifreeze protein type I (AFP I) promotes the germination under cold stress by regulating the genes involved in germination process. Plant Signal Behav. 2019;14:1682796. Liu X-Y, Bulley SM, Varkonyi-Gasic E, Zhong C-H, Li D-W. Kiwifruit bZIP transcription factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress. Plant Physioy. 2023;192:982–99. Han D-G, Du M, Zhou Z-Y, Wang S, Li T-M, Han J-X et al. An NAC transcription factor gene from Malus baccata , MbNAC29 , increases cold and high salinity tolerance in Arabidopsis . In Vitro Cell Dev-Pl. 2020;56:588–599. Wang Z-Z, Peng Z, Khan S, Qayyum A, Rehman A, Du X-M. Unveiling the power of MYB transcription factors: Master regulators of multi-stress responses and development in cotton. Int J Biol Macromol. 2024;276:133885. Liao Y, Zou H-F, Wang H-W, Zhang W-K, Zhang MB. Soybean GmMYB76 , GmMYB92 , and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants. Cell Res. 2008;18:1047–60. Lee H-G, Seo PJ. The MYB96–HHP module integrates cold and abscisic acid signaling to activate the CBF–COR pathway in Arabidopsis . Plant J. 2015;82:962–77. Chen Q-B, Gao K, Xu Y-R, Sun Y-H, Pan B, Chen D-L, et al. Research advance on cold tolerance in chrysanthemum. Front Plant Sci. 2023;14:1259229. Yang X-H, Luo Y-C, Bai H-R, Li X, Tang S, Liao X-Q, et al. DgMYB2 improves cold resistance in chrysanthemum by directly targeting DgGPX1 . Hortic Res-England. 2022;9:uhab028. Akhtar M, Jaiswal A, Taj G, Jaiswal JP, Qureshi MI, Sing NK. DREB1/CBF transcription factors: their structure, function and role in abiotic stress tolerance in plants. J Genet. 2012;91:385–95. Medina J, Catalá R, Salinas J. The CBFs: Three arabidopsis transcription factors to cold acclimate. Plant Sci. 2011;180:3–11. Repkina N, Ignatenko A, Holoptseva E, MiszalskI Z, Kaszycki P, Talanova V. Exogenous Methyl Jasmonate Improves Cold Tolerance with Parallel Induction of Two Cold-Regulated ( COR ) Genes Expression in Triticum aestivum L. Plants-Basel. 2021;10:1421. An J-P, Wang X-F, Zhang X-W, You C-X, Hao Y-J. Apple B-box protein BBX37 regulates jasmonic acid mediated cold tolerance through the JAZ-BBX37-ICE1-CBF pathway and undergoes MIEL1-mediated ubiquitination and degradation. New Phytol. 2021;229:2707–29. Hu Y-R, Jiang L-Q, Wang F, Yu D-Q. Jasmonate Regulates the INDUCER OF CBF EXPRESSION–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 Cascade and Freezing Tolerance in Arabidopsis . Plant Cell. 2013;25:2907–24. Fonseca S, Chini A, Hamberg M, Adie B, Porzel A, Kramell R, et al. (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nat Chem Biol. 2009;5:344–50. Yan J-B, Zhang C, Gu M, Bai Z-Y, Zhang W-G, Qi T-C, et al. The Arabidopsis CORONATINE INSENSITIVE1 Protein Is a Jasmonate Receptor. Plant Cell. 2009;21:2220–36. Hu Y-R, Jiang Y-J, Han X, Wang H-P, Pan J-J, Yu D-Q. Jasmonate regulates leaf senescence and tolerance to cold stress: crosstalk with other phytohormones. J Exp Bot. 2017;68:1361–9. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7107028","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494526191,"identity":"d58119aa-69b3-43ff-abab-5df7a7871b1b","order_by":0,"name":"Jiayi Zhou","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Zhou","suffix":""},{"id":494526192,"identity":"27ffc616-2825-45e6-be18-a255bca643a4","order_by":1,"name":"Li Zhao","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhao","suffix":""},{"id":494526194,"identity":"1a424f3c-8397-42e6-9665-82a34155e145","order_by":2,"name":"Yingjie Quan","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Yingjie","middleName":"","lastName":"Quan","suffix":""},{"id":494526196,"identity":"c4d007f4-8fa8-4efd-8e4b-a44ead8ca331","order_by":3,"name":"Peng Liu","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Liu","suffix":""},{"id":494526198,"identity":"e6a2ac82-6a4e-423e-b7a6-e8badaa9beb1","order_by":4,"name":"Xintong Li","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Xintong","middleName":"","lastName":"Li","suffix":""},{"id":494526201,"identity":"84042bcc-be92-4a68-9663-6630a2789006","order_by":5,"name":"Yansong Wang","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Yansong","middleName":"","lastName":"Wang","suffix":""},{"id":494526202,"identity":"b4406e87-9735-4219-8886-5ce3fe62ddde","order_by":6,"name":"Zimeng Li","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Zimeng","middleName":"","lastName":"Li","suffix":""},{"id":494526204,"identity":"c60804ff-0d83-4847-8405-ee361e5683f8","order_by":7,"name":"Chunxin Dong","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Chunxin","middleName":"","lastName":"Dong","suffix":""},{"id":494526206,"identity":"5ffd9181-97a1-4b26-a1ac-dd030fb37568","order_by":8,"name":"Hongbo Liu","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Liu","suffix":""},{"id":494526207,"identity":"2bfb9b35-36cb-4a1e-b637-5d8ef08bec1c","order_by":9,"name":"Ri Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYLCChAqGBBAtQYKWMyRrYWwjRYu8++FjHx7OO5xncID54G0eBrs8gloMz6Qlz0jcdrjY4ABbsjUPQ3IxYS0NOcYMQC2JGw7wmEnzMBxIbCCopf8NUMsckBb+b8RpkZcA2dIAtoWNOC0GEs+SGRKOpRdLHmYztpxjkEyELf3Jhxl/1Fjn8R1vfnjjTYUdEbYcAFPNDAzMYC4h9SBbIIbWEaF0FIyCUTAKRiwAAPMzPIrefOUSAAAAAElFTkSuQmCC","orcid":"","institution":"Yanbian University","correspondingAuthor":true,"prefix":"","firstName":"Ri","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2025-07-12 09:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7107028/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7107028/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88427174,"identity":"adda522d-4f58-429d-a17f-586eb5f6c729","added_by":"auto","created_at":"2025-08-06 10:07:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32515139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmMYBS3\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Sequence comparison of CmMYBS3 and MYB homologous proteins from other plant species. b: Phylogenetic tree analysis of CmMYBS3. (TcMYBS3ID:GEZ75247.1; CcMYBS3ID:XP_024970566.1; EcMYBS3 ID:XP_043615384.1; HaMYBS3 ID:\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/XP_021984471.1?report=genbank\u0026amp;log$=prottop\u0026amp;blast_rank=6\u0026amp;RID=2DSFXZZE013\" title=\"Show report for XP_021984471.1\"\u003eXP_021984471.1\u003c/a\u003e; LaMYBS3 ID:\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/XP_023729377.1?report=genbank\u0026amp;log$=prottop\u0026amp;blast_rank=8\u0026amp;RID=2DSFXZZE013\" title=\"Show report for XP_023729377.1\"\u003eXP_023729377.1\u003c/a\u003e; ItMYBS3 ID:\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/XP_031104989.1?report=genbank\u0026amp;log$=prottop\u0026amp;blast_rank=13\u0026amp;RID=2DSFXZZE013\" title=\"Show report for XP_031104989.1\"\u003eXP_031104989.1\u003c/a\u003e; VvMYB1R1 ID:NC_012022.3; HaMYB1R1 ID:NC_035449.2; ItMYB1R1 ID:NC_044923.1;SsREV8 ID:TKY45616.1; GhREV8 ID:XP_016671119.1; GhMYB133ID:NC_053437.1;ItMYB6 ID:NC_044925.1; EcMYB6 ID:NC_057767.1; HaMYB6 ID:NC_035433.2; HaMYB14 ID:NC_035435.2; CcvMYB14 ID:NC_037529.1)\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/117d8024fb1a7ed37bfb9433.png"},{"id":88427155,"identity":"e5782cee-8f99-4573-85c3-d880ab36cf98","added_by":"auto","created_at":"2025-08-06 10:07:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1115705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression level of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmMYBS3\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Expression patterns of CmMYBS3 in different organs. b: Expression patterns of \u003cem\u003eCmMYBS3\u003c/em\u003e in different temperature treated\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/ffdd01842a4a0c82963e7f41.png"},{"id":88427143,"identity":"980cb666-ac5c-4573-bc68-836015542118","added_by":"auto","created_at":"2025-08-06 10:07:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11318126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization and transcriptional activation activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmMYBS3\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Subcellular localization of CmMYBS3 in tobacco leaves. (35S::GFP: negative control; DAPI: excitation light at 480 nm; GFP: excitation light at 465 nm (DAPI); Bright: bright field; Merged: GFP with Bright superimposed on the field; Scale bar = 20 μm. b: Transcriptional activation assay of CmMYBS3. pGBKT7-53 is a positive control and pGBKT7-Lam is a negative control.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/a13b761ca73ad939647edc35.png"},{"id":88427163,"identity":"692b985a-fe68-4aa4-a9eb-62fea4311f81","added_by":"auto","created_at":"2025-08-06 10:07:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3902009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening and identification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmMYBS3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpression lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: Detection of DNA levels. OE1, OE2, OE3, OE4, OE5 represents the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpressed lines. WT represents the wild type. b: Relative expression levels of \u003cem\u003eCmMYBS3\u003c/em\u003e. Different lowercase letters in the graphs indicate significant differences (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/272960057b7771b0e8c912ac.png"},{"id":88427177,"identity":"17801829-36bd-403a-a942-699818a74c8d","added_by":"auto","created_at":"2025-08-06 10:07:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14264241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and physiological changes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmMYBS3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpressing under low-temperature stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: Morphological of \u003cem\u003eCmMYBS3 \u003c/em\u003eoverexpressing following exposure to low temperature. b: NBT and DAB staining of \u003cem\u003eCmMYBS3 \u003c/em\u003eoverexpressing after cold stress. c-g: Activity of SOD, POD, and CAT, as well as the content of MDA and Pro in \u003cem\u003eCmMYBS3\u003c/em\u003e overexpressing plants after 2 h of treatment at various temperatures. OE3, OE4, OE5 represents the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpressed lines. Different lowercase letters in the graphs indicate significant differences (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/121829bbff04101b1dbf8b80.png"},{"id":88428448,"identity":"d87b666b-7616-4cff-b882-3d49d35f5629","added_by":"auto","created_at":"2025-08-06 10:15:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":33090355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of DEGs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCmMYBS3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e overexpressed plants under cold stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: PCA plots of WT and \u003cem\u003eCmMYBS3\u003c/em\u003eoverexpressed lines. b: Correlation analysis of sample gene expression levels. c: GO enrichment analysis of DEGs between wild-type and \u003cem\u003eCmMYBS3 \u003c/em\u003eoverexpressed plants. The Y and X axes correspond to GO terms and the number of DEGs. d: Volcano plot of the DEGs between wild-type and \u003cem\u003eCmMYBS3 \u003c/em\u003eoverexpressed plants. Green and red colors represent down-regulated and up-regulated genes, respectively. e: KEGG enrichment of DEGs between wild-type and \u003cem\u003eCmMYBS3 \u003c/em\u003eoverexpressed plants. The Y axis corresponds to the KEGG pathway, and the X axis shows the enrichment ratio between the number of DEGs enriched in a particular pathway. The colour of the dot represents the p value, and the size of the dot represents the number of DEGs mapped to the reference pathway.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/e38f5a6a6acde29534a9d13b.png"},{"id":88427122,"identity":"94f48a9a-aea8-460d-a948-a79e0d7e401c","added_by":"auto","created_at":"2025-08-06 10:07:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10957521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of transcriptome data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative expression level of OE3. MYB74 (EVM0018803), MYB111 (EVM0023763), MYB306 (EVM006832), MYB15 (EVM0006868, EVM0056947), bHLH148 (EVM0020129), bHLH157 (EVM0066486), WRKY6 (EVM0059112), WRKY24 (EVM0060714), WRKY2 (EVM0020482), ZAT10 (EVM0022717).\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/13f359f8e1e7c925d1fc6435.png"},{"id":88428454,"identity":"060ca094-fe04-4954-96f8-9d8fb3df8b42","added_by":"auto","created_at":"2025-08-06 10:15:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3592680,"visible":true,"origin":"","legend":"\u003cp\u003ea: Model of the regulatory network of \u003cem\u003eCmMYBS3\u003c/em\u003e in response to cold stress. b: Yeast one-hybrid assay. \u003cem\u003eCmMYBS3\u003c/em\u003e represses \u003cem\u003eCmDREB1\u003c/em\u003e by directly binding to its promoter.\u003c/p\u003e\n\u003cp\u003eYeast cell solutions containing different plasmids were diluted at different ratios.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/ceefc5ab6e382312bdea9c5f.png"},{"id":103507676,"identity":"ec3fbe9b-42fe-4e1f-81f7-56d7c6942364","added_by":"auto","created_at":"2026-02-26 13:43:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":97165835,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/4e7c9055-9af9-49fe-a451-15e7d8a24331.pdf"},{"id":88427131,"identity":"40bfe12e-9b66-43a3-8875-0e6be3b87fba","added_by":"auto","created_at":"2025-08-06 10:07:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":281968,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7107028/v1/6a61acd0d0991781959ac888.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Overexpression of CmMYBS3 decreases the cold tolerance in the ground-cover chrysanthemum","fulltext":[{"header":"Background","content":"\u003cp\u003eAbiotic stresses, such as temperature stress, salinity stress, drought, and waterlogging stress, significantly affect plant growth and development. Among these, low-temperature stress plays a crucial role in the geographical distribution of plants and influencing their yield and quality, especially in cold regions such as high latitudes or high altitudes. Low temperature significantly impacts plant compromises the stability of the cell membrane system, and inhibits photosynthesis. Under low temperature conditions, the plants exhibit a reduced growth rate, leaf waterlogging, wilting and abscission, even the death of the plant [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In response to low-temperature stress, plants enhance the activity of antioxidant enzymes and promote the accumulation of osmoregulatory sbustances, effectively mitigating stress-induced damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Many studies have reported that superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) play crucial roles in scavenging Reactive Oxygen Species (ROS), and their enzyme activities are often used as important indicators of plant antioxidant capacity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] In addition, plants mitigate the damage caused by low-temperature stress to organelles and cell membranes through enhanced accumulation of osmoregulatory substances. Collectively, these mechanisms contribute to maintaining intracellular ROS homeostasis and enable plants to adapt to low-temperature environments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent years, research into the mechanisms of plant responses to low-temperature stress has progressively deepened from the physiological level to the molecular level, leading to a series of significant breakthroughs. Notably, the ICE-CBF/DREB-COR signaling pathway has garnered extensive attention as a pivotal mechanism enabling plants to withstand low-temperature stress. When plants perceive cold signals, Inducer of CBF Expression (ICE) functions as an upstream transcription factor by binding to the C-repeat binding factor/dehydration responsive element binding (CBF/DREB) response element to promote the expression of \u003cem\u003eCBF/DREB\u003c/em\u003e genes. Subsequently, \u003cem\u003eCBF/DREB\u003c/em\u003e binds to the dehydration response element (DRE) to activate cold-responsive (\u003cem\u003eCOR\u003c/em\u003e) gene expression and induce the synthesis of a series of antifreeze proteins and metabolites, thereby enhancing plant cold tolerance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition, several transcription factor families have been identified to be involved in the regulation of plant cold stress. These families of transcription factors include, MYB [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], AP2/ERF [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], WRKY [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], NAC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and bHLH [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, MYB transcription factor family is not only involved in the regulation of plant growth and development, but also plays an important role in the response to low temperature stress. Based on the different number of repetitions (R) in a sequence, the MYB family can be divided into four subfamilies: 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB, with each R repetition consisting of approximately 50\u0026ndash;52 amino acid residues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These R domains are composed of tandem repeats, forming a distinctive helix-loop-helix (HLH) topological conformation that facilitates their involvement in the DNA binding process [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eAtMYB15\u003c/em\u003e suppresses CBF gene expression in \u003cem\u003eArabidopsis\u003c/em\u003e, leading to reduced antioxidant enzyme activity and increased cold sensitivity in plants [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The expression of \u003cem\u003eOsMYB3R-2\u003c/em\u003e and \u003cem\u003eOsMYBS3\u003c/em\u003e in rice, as well as \u003cem\u003eMpMYBS3\u003c/em\u003e in banana, was up-regulated under low-temperature stress. Furthermore, their heterologous overexpression in transgenic plants conferred robust cold tolerance through modulating stress-responsive gene network [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our previous transcriptome analysis revealed that the expression of \u003cem\u003eCmMYBS3\u003c/em\u003e in ground cover chrysanthemum 'Yingjie' was significantly downregulated under low-temperature treatment compared to normal-temperature treatment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This suggests that \u003cem\u003eCmMYBS3\u003c/em\u003e may play a role in the plant's cold resistance mechanism. In \u003cem\u003eChrysanthemum\u003c/em\u003e, \u003cem\u003eCmMYBS3\u003c/em\u003e inhibits the expression of \u003cem\u003eCmMYB121\u003c/em\u003e by forming a complex with \u003cem\u003eCmHSFA4\u003c/em\u003e and \u003cem\u003eCmTPL\u003c/em\u003e, thereby enhancing the salinity tolerance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nevertheless, whether \u003cem\u003eCmMYBS3\u003c/em\u003e also functions in regulating cold tolerance remains to be elucidated.\u003c/p\u003e\u003cp\u003eGround cover chrysanthemum (\u003cem\u003eChrysanthemum\u003c/em\u003e \u0026times; \u003cem\u003emorifolium\u003c/em\u003e) is a herbaceous perennial species in the genus \u003cem\u003eChrysanthemum\u003c/em\u003e (family Asteraceae), which has excellent characteristics such as rich flower colors, dense flower clusters, rounded and compact crowns, as well as a high tolerance to cold and drought. In northern China, the low temperature and prolonged winter duration pose significant challenges to the overwintering survival of ground cover chrysanthemums, resulting in a relatively low survival rate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This environmental conditions severely limited the large-scale deployment of ground cover chrysanthemums in the region. Therefore, in this study, the \u003cem\u003eCmMYBS3\u003c/em\u003e gene was cloned to investigate its spatial expression pattern, subcellular localization, self-activation activity, and biological functions in cold stress responses. Additionally, transcriptomic data were employed to analyze the downstream regulatory network of \u003cem\u003eCmMYBS3\u003c/em\u003e. These results clarify the function of the \u003cem\u003eCmMYBS3\u003c/em\u003e gene and the molecular mechanism underlying cold tolerance regulation, and providing a solid theoretical foundation for the molecular genetic breeding of ground cover chrysanthemums.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003ePlant materials and growth conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCuttings of the ground-cover chrysanthemum variety \u0026lsquo;Yingjie\u0026rsquo; was obtained from the greenhouse at Yanbian University Teaching Base, located in Yanji City, Jilin Province,\u003c/p\u003e\u003cp\u003eChina. Aseptically excised shoot tips of ground cover chrysanthemum 'Yingjie' (2.0 cm in length) were cultured on Murashige and Skoog (MS) medium supplemented with 30 g/L sucrose and 0.8% agar, maintained under 16-h photoperiod (30 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1;) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 4-week subculture cycles. The obtained tissue culture seedlings were used as materials for gene cloning and genetic transformation. The transgenic lines were transplanted to the greenhouse at Yanbian University's teaching base. The environmental conditions were stringently controlled, with a temperature range of 25\u0026deg;C\u0026ndash;27\u0026deg;C, a photoperiod of 14 hours light/10 hours dark, and relative humidity maintained between 60% \u0026ndash; 70%. Plants that developed approximately 7\u0026ndash;8 leaves and exhibited robust growth were selected for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCloning and sequence analyses of\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from the leaves of the ground cover plant 'Yingjie' using the Trans-Zol Plant Reagent Kit (TIANGEN, Beijing, China). The extracted RNA was then reverse-transcribed into cDNA using the FastKing gDNA Eliminating RT SuperMix Reagent Kit (TIANGEN, Beijing, China). Based on the CDS sequence of the \u003cem\u003eCmMYBS3\u003c/em\u003e gene previously screened and annotated by our research group from the transcriptome database of the 'Yingjie', specific primers MYBS3-F/R were designed using Primer 5.0 software (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The PCR reaction system consisted of 2.5 \u0026micro;L of 10 \u0026times; PCR Buffer (containing Mg\u003csup\u003e2+\u003c/sup\u003e), 1.6 \u0026micro;L dNTP (2.5 mmol/L), 1.0 \u0026micro;L cDNA, 1.0 \u0026micro;L each of the upstream and downstream primers (10 \u0026micro;mol/L), 0.2 \u0026micro;L rTaq, and 17.3 \u0026micro;L ddH2O, in a total volume of 20 \u0026micro;L. The PCR reaction was programmed as follows: 94\u0026deg;C for 30 s, 57\u0026deg;C for 30 s, 72\u0026deg;C for 90 s, with 35 cycles. Finally, the reaction was extended at 72\u0026deg;C for 10 min and stored at 4\u0026deg;C. Subsequently, the PCR products were recovered and ligated with the vector PMD-T19, and then sent to Nanjing Sipujin Biotechnology Co., Ltd.. for sequencing. Download the MYBS3 protein sequences of various plants from NCBI, perform multiple sequence alignment using DNAMAN software, analyze the conserved domains of CmMYBS3 protein via NCBI CD-Search. The phylogenetic tree was constructed by using MEGA7.0 software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene expression analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 'Yingjie' plants were subjected to cultivation temperatures of 25\u0026deg;C, 10\u0026deg;C, 5\u0026deg;C, and \u0026minus;\u0026thinsp;5\u0026deg;C for 2 hours each. Following the treatment, 3\u0026ndash;4 leaves from the upper portion of the plant were harvested and immediately immersed in liquid nitrogen for long-term preservation at -80\u0026deg;C, for subsequent evaluation of cold-responsive biomarkers (such as SOD, POD and CAT activities). Collect the roots, stems, leaves and flower organs of 'Yingjie', and analyze the spatio-temporal expression pattern of \u003cem\u003eCmMYBS3\u003c/em\u003e. The quantitative Real-Time PCR (qRT-PCR) reaction system consisted of 5 \u0026micro;L of cDNA, 5 \u0026micro;L of 2 \u0026times; SuperReal PreMix Plus, 0.4 \u0026micro;L of 50 \u0026times; ROX Reference DyeΔ, and 1 \u0026micro;L of each of the forward and reverse primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and the addition of 2.6 \u0026micro;L of deionized water. The cycling program was set as follows: pre-denaturation at 95 ℃ for 2 min; 35 cycles were performed, including denaturation at 95 ℃ for 15 s, annealing at 58 ℃ for 15 s, and extension at 72 ℃ for 30 s. The melting curves were prepared in the range of 55 to 95 ℃. The \u003cem\u003eCmEF1α\u003c/em\u003e gene was used as an internal reference (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each treatment was performed with three biological replicates, and the relative expression levels of the target genes were calculated using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubcellular localization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the subcellular localization of the CmMYBS3 protein, the constructed pORE-R4-35S-\u003cem\u003eCmMYBS3\u003c/em\u003e vector was introduced into \u003cem\u003eAgrobacterium EHA105\u003c/em\u003e via the heat shock method. The \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e suspension containing the target construct was subsequently infiltrated into tobacco leaves. The infiltrated plants were maintained under controlled environmental conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 16 h photoperiod) for 72\u0026ndash;96 hours. Finally, the subcellular localization of CmMYBS3 was determined by observing the fluorescence signals using confocal microscopy.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptional activation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe pGBKT7-\u003cem\u003eCmMYBS3\u003c/em\u003e bait construct was generated through Gateway cloning technology (Invitrogen) and validated by restriction enzyme analysis (\u003cem\u003eNde\u003c/em\u003e I / \u003cem\u003eBamH\u003c/em\u003e I). Recombinant plasmids including the experimental construct (pGBKT7-\u003cem\u003eCmMYBS3\u003c/em\u003e), negative control (pGBKT7-Lam, Clontech), and positive control (pGBKT7-53, Clontech) were introduced into Y2HGold yeast cells via lithium acetate-mediated transformation. Subsequently, culture the transformed yeast strains in synthetic dextrose dropout medium without tryptophan (SD/-Trp) medium at 30\u0026deg;C for 3\u0026ndash;4 days. Select individual colonies from the growing yeast strains and inoculate them onto SD/-His-Ade medium supplemented with 5-bromo-4-chloro-3-indolyl α-D-galactopyranoside (X-α-gal) and the growth of the colonies was observed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression vector construction and identification of transgenic chrysanthemums\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe coding sequence of \u003cem\u003eCmMYBS3\u003c/em\u003e was PCR-amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs) with gene-specific primers containing \u003cem\u003eBamH\u003c/em\u003e I and \u003cem\u003eSal\u003c/em\u003e I restriction sites. The amplicon was purified via AxyPrepTM Biospin Gel Extraction Kit (Axygen, Hangzhou, China) and ligated into pORE-R4-35S vector (Clontech) digested with corresponding restriction enzymes. Recombinant clones were verified by colony PCR followed by sequencing. The recombinant plasmid was introduced into \u003cem\u003eAgrobacterium EHA105\u003c/em\u003e to genetically transform chrysanthemums with reference to the method of Gao et al [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The recombinant pORE-R4-35S-\u003cem\u003eCmMYBS3\u003c/em\u003e plasmid was introduced into \u003cem\u003eAgrobacterium tumefaciens EHA105\u003c/em\u003e by freeze-thaw transformation. Bacterial cultures were incubated in YEP liquid medium supplemented with 50 mg/L kanamycin for 16 hours at 28\u0026deg;C with shaking (180 rpm). Plant transformation was performed using the leaves dip method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] with minor modifications. To identify the positive strain, Genomic DNA was isolated from putative transgenic chrysanthemum leaves, and specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed for PCR detection. Total RNA was isolated from the transgenic lines, and the expression level of \u003cem\u003eCmMYBS3\u003c/em\u003e was detected through reverse transcription into cDNA followed by qRT-PCR analysis. Relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method with three biological replicates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCold treatment of\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e \u003cb\u003eoverexpression plants and their oxidative responses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThree \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines (OE3, OE4, and OE5) and wild-type (WT) 'Yingjie' at the five-leaf stage were selected for differential temperature stress treatments in growth chamber (MGZ-200L-2). The temperatures were set at 25 ℃, 10 ℃, 5 ℃, and \u0026minus;\u0026thinsp;5 ℃, respectively. After each treatment lasting for 2 hours, the morphological of the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression chrysanthemum were observed. At the same time, the topmost 3\u0026ndash;4 leaves were harvested for the measurement of SOD, POD, and CAT enzyme activities, as well as Pro and MDA contents. Specific assays were conducted following the method described by Guo et al [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To evaluate the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e under cold treatment, histochemical staining was conducted using 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), following the method described by Wei et al [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFour-week-old \u003cem\u003eCmMYBS3\u003c/em\u003e overexpressing lines (OE3) and WT at the four-leaf stage were exposed to cold stress in a climate chamber (MGZ-200L-2) at 4\u0026deg;C for 2 hours, with 65% relative humidity and 16 h photoperiod. Then, the third leaf was selected for RNA-seq.\u0026nbsp;Total RNA was extracted from frozen samples using the Trans-Zol Plant Kit (TIANGEN, Beijing, China) following the manufacturer's protocol. The integrity and quality of the RNA were assessed by agarose gel electrophoresis. The library was sequenced on the Illumina HiSeq2000 platform by Biomarker Technologies Co., Ltd. (Beijing, China). The raw sequencing data was processed through Cutadapt to remove low-quality sequences and adapter contaminants, yielding high-quality clean reads. Subsequently, the HISAT2 aligner was employed for efficient and precise alignment of the processed reads against the reference genome (\u003cem\u003eChrysanthemum lavandulifolium\u003c/em\u003e Genome, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cgdnjau.edu.cn/asteraceae/browse/genomepage/cl\u003c/span\u003e\u003cspan address=\"https://cgdnjau.edu.cn/asteraceae/browse/genomepage/cl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene expression levels were quantified using fragments per kilobase of transcript per million mapped reads (FPKM) values. During the detection of differentially expressed genes (DEGs), DESeq2 software was utilized for differential analysis, and FC\u0026thinsp;\u0026ge;\u0026thinsp;1.5 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 would be used as the screening criteria. Differentially expressed genes (DEGs) were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. The results were visualized using TBtools.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eAll experiments were independently replicated three times. The data were statistically analyzed using SPSS statistical software (version 21.0) and visualized using GraphPad Prism (version 8.3.0). Significant differences were analyzed using Duncan's new multiple range test, and the level of significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCloning and bioinformatics analysis of\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCmMYBS3\u003c/em\u003e is a gene with a length of 1151 bp, containing an open reading frame (ORF) that encodes a protein consisting of 358 amino acids. The relative molecular weight of CmMYBS3 protein was predicted to be 38992.64 kDa, isoelectric point was 7.72, instability index was 52.27, grand average hydrophilicity was \u0026minus;\u0026thinsp;0.660, and aliphatic index was 65.39 by ExPASy website (Table S2). The functional domain of CmMYBS3 protein was predicted using the NCBI CD-search tool, and it was identified as containing the SANT-MYB-SHAQKYE functional domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Phylogenetic analysis of MYB proteins revealed that \u003cem\u003eCmMYBS3\u003c/em\u003e exhibits the highest degree of homology with \u003cem\u003eTcMYBS3\u003c/em\u003e and is classified as a member of the MYB CCA1-like family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of the expression level of\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eCmMYBS3\u003c/em\u003e gene is expressed in various organ of the 'Yingjie', including roots, stems, flowers, and leaves. Notably, the expression level \u003cem\u003eCmMYBS3\u003c/em\u003e is highest in the roots (2.15), followed by flowers and stems (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To investigate the expression characteristics of the \u003cem\u003eCmMYBS3\u003c/em\u003e gene under low temperature stress, the 'Yingjie' plants were subjected to low-temperature treatment. The results indicated that the expression level of \u003cem\u003eCmMYBS3\u003c/em\u003e initially decreased and subsequently increased. At 5\u0026deg;C, the expression level of \u003cem\u003eCmMYBS3\u003c/em\u003e was at its lowest. At -5\u0026deg;C, the expression level of CmMYBS3 increased slightly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These results suggest that low temperature significantly affects the expression of \u003cem\u003eCmMYBS3\u003c/em\u003e gene and plays an important role in the cold stress response of 'Yingjie'.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of subcellular localization and transcriptional activation activity of\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo analyze the subcellular localization of CmMYBS3 protein, the Agrobacterium strain harboring the \u003cem\u003eCmMYBS3\u003c/em\u003e-GFP fusion gene was infiltrated into tobacco leaves. The results demonstrated that \u003cem\u003eCmMYBS3\u003c/em\u003e-GFP emitted a strong green fluorescence signal exclusively in the cell nucleus, whereas the positive control GFP exhibited uniformly distributed fluorescence throughout the entire cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). These findings confirm that the CmMYBS3 protein is localized specifically in the cell nucleus. The transcriptional activation activity of \u003cem\u003eCmMYBS3\u003c/em\u003e was analyzed using yeast single-hybrid experiments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, all yeast cells grew robustly on the SD/-Trp medium, suggesting that the pGBKT7-\u003cem\u003eCmMYBS3\u003c/em\u003e plasmid had been successfully transformed into the Y2H yeast strain. The transformed bacterial solution was plated onto the SD/-His-Ade\u0026thinsp;+\u0026thinsp;X-α-gal medium. It was observed that the pGBKT7-\u003cem\u003eCmMYBS3\u003c/em\u003e colonies failed to grow and did not exhibit a blue coloration. Conversely, the positive control pGBKT7-53 formed colonies that grew successfully and displayed a blue color, suggesting that the transcription factor \u003cem\u003eCmMYBS3\u003c/em\u003e lacks transcriptional activation activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eScreening and identification of\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e \u003cb\u003eoverexpression lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAn overexpression vector was constructed and \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation was performed. Consequently, five independent transgenic seedlings (designated as OE1, OE2, OE3, OE4, and OE5) were successfully obtained. DNA was extracted independently from each seedlings. PCR analysis revealed the presence of specific bands in the OE1, OE3, OE4, and OE5 lines, whereas no bands were detected in OE2 and the WT lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). At the same time, we analyzed the expression levels of \u003cem\u003eCmMYBS3\u003c/em\u003e in each seedling. Our results showed that the expression levels of \u003cem\u003eCmMYBS3\u003c/em\u003e in the OE3, OE4, and OE5 lines were significantly higher than those in the WT. Notably, the relative expression level of \u003cem\u003eCmMYBS3\u003c/em\u003e in the OE3 line was 4.20 times higher than that in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These findings further confirm that the OE3, OE4, and OE5 lines are overexpression lines.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges in ROS accumulation and antioxidant enzyme activities in the\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e \u003cb\u003eoverexpressing line under low-temperature stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs the temperature decreased, the degree of injury in both the overexpression lines and the WT plants was significantly increased. At 5\u0026deg;C, the leaves of the overexpression lines became markedly softer and drooped, with an increased angle at the leaf axil compared to the WT plants. When the temperature dropped to -5\u0026deg;C, the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines displayed severe wilting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Following low-temperature stress treatment and histochemical staining with NBT and DAB, it was observed that the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines exhibited a more intense blue color after NBT staining, indicating a higher accumulation of O₂⁻ compared to the WT plants. DAB staining revealed that the leaves of the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines exhibited a dark brown coloration, indicating a significantly higher H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). To further investigate the cold resistance of the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines, we analyzed the activities of antioxidant enzymes. At low temperatures (10 ℃ ~ -5 ℃), the activities of SOD, POD, and CAT in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines were significantly lower than those in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). At the same time, we also measured and analyzed the contents of MDA and Pro in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines. The MDA content exhibited a gradual increase as the temperature decreased. At -5 ℃, the MDA content in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines was significantly higher than that in the WT. Specifically, the MDA content of OE3 was 57 \u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was 1.16 times higher than that of the WT. The proline content first decreased and then increased. Under the same low-temperature conditions, the proline content in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines were lower than that in the WT. Specifically, at 5\u0026deg;C, the proline content in the WT was 1.25 times higher than that in the OE3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). These results suggest that the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines exhibits lower cold tolerance compared to the WT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome analysis of\u003c/b\u003e \u003cb\u003eCmMYBS3\u003c/b\u003e \u003cb\u003eoverexpressing lines under cold stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe molecular mechanism of CmMYBS3-mediated cold tolerance regulation was investigated via comprehensive transcriptome profiling of \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines and wild-type plants following cold stress treatment. A total of 36.29 Gb of clean data was obtained, with an average of 5.79 Gb per sample and a Q30 value of at least 97.38%. The alignment rate of reads to the reference genome varied between 60.17% and 66.12% across samples (Table S3). Principal Component Analysis (PCA) was conducted to evaluate the similarity among the six samples. The first principal component (PC1) explained 35.61% of the total variance, whereas the second principal component (PC2) accounted for 18.41% of the variance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The Pearson correlation coefficient was employed to perform the correlation analysis on the samples. The correlations between the three replicates were all higher than 0.810, which demonstrates the high reliability of the data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). A total of 5,425 differentially expressed genes (DEGs) were identified between \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines and WT plants. Specifically, 2,646 genes were up-regulated and 2,779 genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). GO enrichment analysis revealed that the DEGs were significantly enriched and annotated into three main categories: \"biological process\", \"cellular component\", and \"molecular function\". Specifically, 1920 DEGs were enriched in \" biological process \", 2776 DEGs were enriched in \" cellular component \", and 3468 DEGs were enriched in \" molecular function \" (Table S4). Notably, the DEGs were predominantly enriched in the cellular anatomical entitiy within the cellular component category, cellular processes within the biological process category, and binding functions within the molecular function category (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). KEGG enrichment analysis revealed significant alterations in pathways associated with plant development and stress response, including \"plant hormone signal transduetion\", \"circadian rhythm -plant\", \"beta-Alanine metabolism\", \"alpha-Linolenic acid mefabolism\", \"biosynthesis of amino acids\", \"carbon metabolism\", \"arginine and proline metabolism\", as well as \"monoterpenoid biosynthesis\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). These findings suggest that under low-temperature stress, \u003cem\u003eCmMYBS3\u003c/em\u003e is involved in multiple metabolic pathway reactions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eValidation of transcriptome data\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwelve genes including members of the \u003cem\u003eCmbHLH\u003c/em\u003e, \u003cem\u003eCmMYB\u003c/em\u003e, and \u003cem\u003eCmWRKY\u003c/em\u003e transcription factor families, were randomly selected for qRT-PCR validation (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). At 4\u0026deg;C, the qRT-PCR results demonstrated that the expression levels of the EVM0068325 and EVM0066486 genes in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines were reduced compared to those in the WT. In contrast, the expression levels of the EVM0018803, EVM0023763, EVM0006868, EVM0056947, EVM0021029, EVM0059112, EVM0060714, EVM0022717, EVM0078075, and EVM0020482 genes were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings are consistent with the trends observed in the FPKM data from the transcriptome analysis, thereby reinforcing the reliability of the results.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLow-temperature stress is a critical abiotic stress factor that significantly affects plant growth and development[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Under low-temperature conditions, the stability of the plant cell membrane system is initially impaired. The phase transition of membrane lipids induces structural damage to the membranes, which in turn results in cellular substance leakage. Meanwhile, low temperatures disrupt the normal metabolic processes in plants, leading to an imbalance in the electron transport chain and a substantial accumulation of reactive oxygen species (ROS). The excessive production of ROS intensifies oxidative stress, causing damage to biological macromolecules such as proteins, nucleic acids, and lipids. This subsequently impairs cellular structures and functions, and if prolonged or severe, may ultimately result in plant mortality[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, over the course of prolonged evolution, plants have developed a series of complex and sophisticated adaptive survival mechanisms to withstand harsh environmental conditions. For instance, they alleviate the toxic effects of reactive oxygen species (ROS), facilitate the accumulation of osmoregulatory substances, and synthesize antifreeze proteins[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. At the molecular regulatory level, transcription factors (TFs) serve as pivotal hubs in modulating plant cold tolerance. The TFs specifically recognize and bind to the cis-acting elements of downstream target genes, thereby activating or repressing the expression of associated genes and collectively regulating the aforementioned physiological and biochemical processes[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The transcription factors involved in plant cold stress responses include \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eICE\u003c/em\u003e, and others. Among these, the \u003cem\u003eMYB\u003c/em\u003e family has garnered significant attention owing to its frequent appearance in studies exploring cold resistance mechanisms[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The study by Liao et al[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. revealed that soybean plants overexpressing the \u003cem\u003eGmMYB76\u003c/em\u003e and \u003cem\u003eGmMYB177\u003c/em\u003e genes exhibited significantly elevated proline contents following exposure to low temperature, accompanied by a marked improvement in survival rates. These findings suggest that the \u003cem\u003eGmMYB76\u003c/em\u003e and \u003cem\u003eGmMYB177\u003c/em\u003e genes are critical regulators in the soybean's response to low temperature stress. The study by Lee and Seo[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]demonstrated that in the \u003cem\u003emyb96\u003c/em\u003e mutant, the expression levels of \u003cem\u003eCBF\u003c/em\u003e and its downstream \u003cem\u003eCOR\u003c/em\u003e genes were markedly decreased. Further investigation revealed that the interaction between MYB96 and the HHP protein suppresses the expression of downstream CBF genes[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The \u003cem\u003eDgMYB1\u003c/em\u003e and \u003cem\u003eDgMYB2\u003c/em\u003e genes were successfully cloned from chrysanthemum. Compared with the wild type, the \u003cem\u003eDgMYB1/2\u003c/em\u003e overexpression lines exhibited significantly reduced REL and MDA content, along with markedly enhanced activities of antioxidant enzymes, including SOD, POD, and CAT. Additionally, the accumulation contents of osmotic adjustment substances, such as soluble sugar, soluble protein, and Pro, were also significantly increased. These physiological alterations collectively contributed to the enhanced cold tolerance of chrysanthemum[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, the \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation method was utilized to successfully generate the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression 'Yingjie'. Following exposure to low temperature stress, phenotypic analysis demonstrated that, compared with the WT, the angle between the leaf axils of the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression significantly increased, resulting in a drooping leaf posture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), suggesting its heightened sensitivity to low-temperature conditions. The physiological index tests demonstrated that the activities of SOD, POD, and CAT in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines were significantly lower compared to those in the WT. Furthermore, the relative expression analysis of \u003cem\u003eCmSOD\u003c/em\u003e, \u003cem\u003eCmPOD\u003c/em\u003e, and \u003cem\u003eCmCAT\u003c/em\u003e revealed that their transcriptional levels in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines were also significantly reduced relative to those in the WT (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Based on these findings, it can be inferred that the \u003cem\u003eCmMYBS3\u003c/em\u003e gene suppresses the antioxidant capacity of the ground cover chrsanthemum, thereby exacerbating membrane lipid peroxidation damage and reducing the plant's tolerance to low-temperature stress. The ICE-CBF/DREB-COR signaling pathway is widely recognized as one of the most extensively studied and well-documented regulatory pathways in the field of cold tolerance. This pathway utilizes the inducible transcription factor \u003cem\u003eICE\u003c/em\u003e (Inducer of CBF Expression) as an upstream activation element. Upon perception of low-temperature signals, ICE activates the expression of \u003cem\u003eCBF\u003c/em\u003e (C-repeat Binding Factor) or \u003cem\u003eDREB\u003c/em\u003e (Dehydration Responsive Element Binding) transcription factors. The activated \u003cem\u003eCBF/DREB\u003c/em\u003e then bind to the \u003cem\u003eCRT/DRE\u003c/em\u003e cis-acting element located in the promoter region of downstream \u003cem\u003eCOR\u003c/em\u003e (Cold-Responsive) genes, thereby promoting the expression of \u003cem\u003eCOR\u003c/em\u003e genes and enhancing plant cold tolerance[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, DREB/CBF proteins are classified into six subgroups according to their binding domains. Among these, the three genes \u003cem\u003eDREB1B/CBF1\u003c/em\u003e, \u003cem\u003eDREB1A/CBF3\u003c/em\u003e, and \u003cem\u003eDREB1C/CBF2\u003c/em\u003e are capable of responding to low temperature signals. Studies have demonstrated that under cold stress conditions, transgenic Arabidopsis plants overexpressing the \u003cem\u003eDREB1B/CBF1\u003c/em\u003e or \u003cem\u003eDREB1C/CBF2\u003c/em\u003e genes exhibit significantly higher survival rates compared to wild-type plants[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Under the induction of exogenous methy jasmonic acid, the expression level of the \u003cem\u003eCOR\u003c/em\u003e gene in wheat was significantly upregulated. Further investigation demonstrated that under low-temperature stress conditions, the antioxidant enzyme including CAT, SOD, and POD in COR overexpressing wheat plants exhibited markedly enhanced activity compared to those in WT plants [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the low-temperature regulation network, jasmonic acid (JA) has a significant impact on the ICE-CBF/DREB-COR pathway. When plants do not perceive the JA signal, the (JASMONATE ZIM-DOMAIN 1) JAZ1 protein is highly expressed. It can interact with the \u003cem\u003eICE1\u003c/em\u003e of the cold signal pathway, thereby inhibiting the transcriptional activation activity of \u003cem\u003eICE1\u003c/em\u003e on downstream \u003cem\u003eCBF\u003c/em\u003e genes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Under low-temperature stress conditions, the biosynthesis pathway of JA in plants is rapidly activated, catalyzing the synthesis of the key signaling molecule jasmonoyl-isoleucine (JA-Ile). JA-Ile functions as a specific ligand that efficiently binds to the F-box protein CORONATINE INSENSITIVE 1 (COI1), inducing a conformational change in COI1[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The altered COI1 subsequently forms a stable co-receptor complex with JAZ1 proteins. This complex triggers the degradation of JAZ1 proteins via the 26S proteasome-mediated ubiquitination process [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The degradation of JAZ1 proteins effectively alleviates their inhibitory effect on \u003cem\u003eICE\u003c/em\u003e transcription factors, thereby initiating the expression of downstream cold-responsive genes and enabling plants to acquire cold tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Hu et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]demonstrated that the overexpression of \u003cem\u003eJAZ1\u003c/em\u003e and \u003cem\u003eJAZ4\u003c/em\u003e significantly downregulated the expression levels of \u003cem\u003eCBF\u003c/em\u003e genes by inhibiting \u003cem\u003eICE\u003c/em\u003e expression, thereby reducing the cold stress tolerance of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. This study revealed that the expression levels of \u003cem\u003eCmDREB1\u003c/em\u003e and \u003cem\u003eCmCOR\u003c/em\u003e were reduced in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines. It is possible that the expression of \u003cem\u003eCmMYBS3\u003c/em\u003e suppresses the expression of \u003cem\u003eCmDREB1\u003c/em\u003e, consequently inhibiting the expression of downstream \u003cem\u003eCmCOR\u003c/em\u003e genes. We performed a yeast single-hybrid experiment with \u003cem\u003eCmMYBS3\u003c/em\u003e as the prey and the \u003cem\u003eCmDREB1\u003c/em\u003e promoter as the bait. All yeast cells exhibited robust growth on SD/-Trp/-His/-Leu medium. However, upon the addition of 3-AT to the medium, only the yeast cells harboring both the prey and the bait demonstrated normal growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb), indicating that \u003cem\u003eCmMYBS3\u003c/em\u003e may directly interact with the \u003cem\u003eCmDREB1\u003c/em\u003e promoter. These preliminary findings suggest that \u003cem\u003eCmMYBS3\u003c/em\u003e may decrease the cold tolerance of ground cover chrysanthemum by repressing the expression of \u003cem\u003eCmDREB1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the same time, it was observed that the expression level of \u003cem\u003eCmJZA1\u003c/em\u003e was upregulated, suggesting that \u003cem\u003eCmMYBS3\u003c/em\u003e may positively regulate the expression of \u003cem\u003eCmJAZ1\u003c/em\u003e. However, no significant changes were detected in the expression level of \u003cem\u003eCmICE1\u003c/em\u003e between the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines and the wild type. These results indicate that \u003cem\u003eCmMYBS3\u003c/em\u003e does not suppress the expression of \u003cem\u003eCmCOR\u003c/em\u003e via the \u003cem\u003eCmJZA1\u003c/em\u003e pathway to reduce cold tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). This finding offers critical insights into the regulatory mechanism of \u003cem\u003eCmMYBS3\u003c/em\u003e within the plant cold tolerance network.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the cold-related gene \u003cem\u003eCmMYBS3\u003c/em\u003e was isolated from the ground cover chrysanthemum 'Yingjie', and its overexpression resulted in increased sensitivity to cold stress in the ground cover chrysanthemum. Under low-temperature stress, the induction of antioxidant enzyme activity was reduced in the overexpressed lines, leading to increased levels of reactive oxygen species (ROS) in the transgenic lines. Additionally, the transgenic lines exhibited lower Pro content and higher MDA content compared to the wild-type. After low-temperature treatment, the transgenic lines showed wilting and drooping leaves compared to the wild-type, indicating that the overexpression of \u003cem\u003eCmMYBS3\u003c/em\u003e reduces the cold tolerance of the groundcover chrysanthemum. Transcriptome and yeast one-hybrid data indicate that \u003cem\u003eCmMYBS3\u003c/em\u003e binds to the \u003cem\u003eCmDREB1\u003c/em\u003e promoter and inhibits transcription, thereby increasing sensitivity to low temperatures. We will further validate \u003cem\u003eCmMYBS3\u003c/em\u003e and continue to explore its relationship with the JA pathway. This will enhance our understanding of the role of the CmMYBS3 protein in cold stress responses and lay the foundation for further exploration of the molecular mechanisms underlying its negative regulation of cold tolerance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw transcriptome data used during this study has been deposited in NCBI SRA with the accession number PRJNA1293879. GenBank accession number for the nucleotide sequence of the \u003cem\u003eCmMYBS3\u003c/em\u003e gene: BankIt2984616 Seq1 PV982153.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by National Natural Science Foundation of China (NSFC) (32060696), Project funded by the Science and Technology Department of Jilin Province (YDZJ202501ZYTS556), Science and Technology Project of Education Department of Jilin Province (JJKH20220541KJ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJZ: Conceptualization, Investigation, Writing – original draft,Writing – review \u0026amp; editing, Methodology, Validation. LZ: Resources, Validation, Writing – review \u0026amp; editing.YQ: Investigation,Writing – review \u0026amp; editing. PL: Methodology, Writing – original draft,Writing – review \u0026amp; editing. XL: Investigation, Software, Writing –original draft. YW: Software, Writing – original draft. ZL: Investigation, Methodology, Software, Writing – original draft. CD and HL: Investigation, Methodology, Software, Writing – original draft. RG: Conceptualization, Funding acquisition, Investigation, Resources,Writing – original draft, Writing – review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYadav SK. Cold stress tolerance mechanisms in plants. A review. Agron Sustain Dev. 2010;30:515\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDoğru A, \u0026Ccedil;akirlar H. 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(+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nat Chem Biol. 2009;5:344\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYan J-B, Zhang C, Gu M, Bai Z-Y, Zhang W-G, Qi T-C, et al. The \u003cem\u003eArabidopsis\u003c/em\u003e CORONATINE INSENSITIVE1 Protein Is a Jasmonate Receptor. Plant Cell. 2009;21:2220\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu Y-R, Jiang Y-J, Han X, Wang H-P, Pan J-J, Yu D-Q. Jasmonate regulates leaf senescence and tolerance to cold stress: crosstalk with other phytohormones. J Exp Bot. 2017;68:1361\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ground cover chrysanthemum, CmMYBS3, RNA-Seq, Cold stress","lastPublishedDoi":"10.21203/rs.3.rs-7107028/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7107028/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eLow temperature constitutes a critical abiotic stress that significantly impairs plant growth and development, particularly for species in cold regions. In Northeast China, the persistently low winter temperatures over an extended period pose significant challenges to the survival of chrysanthemums.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThis study employed the ground cover plant \"Yingjie\" as the experimental material and cloned the \u003cem\u003eCmMYBS3\u003c/em\u003e. The CmMYBS3 protein lacks transcriptional activity and is localized exclusively in the nucleus. Under low-temperature treatment, the activities of SOD, CAT, and POD were significantly lower in chrysanthemums overexpressing \u003cem\u003eCmMYBS3\u003c/em\u003e than in the wild-type line. Additionally, the MDA content in the \u003cem\u003eCmMYBS3\u003c/em\u003e overexpression lines was higher than in the wild-type lines. To elucidate the mechanism by which \u003cem\u003eCmMYBS3\u003c/em\u003e regulates the response to low temperature, we conducted transcriptome sequencing analysis and identified a total of 5425 differentially expressed genes, comprising 2646 upregulated genes and 2779 downregulated genes. The GO analysis reveals that the primary enrichment occurs in the \"biological process\", \"cellular component\", and \"molecular function\". The KEGG enrichment analysis identified significant alterations in several pathways associated with plant growth and development, as well as stress responses. Through yeast single-hybrid analysis, it was demonstrated that \u003cem\u003eCmMYBS3\u003c/em\u003e specifically binds to the promoter region of \u003cem\u003eCmDREB1\u003c/em\u003e and inhibiting the expression of the \u003cem\u003eCmDREB1\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study demonstrates that \u003cem\u003eCmMYBS3\u003c/em\u003e reduces the cold tolerance of ground cover chrysanthemums by suppressing the expression of the \u003cem\u003eCmDREB1\u003c/em\u003e gene, providing an important theoretical basis for the breeding of cold-tolerant ground cover chrysanthemum varieties.\u003c/p\u003e","manuscriptTitle":"Overexpression of CmMYBS3 decreases the cold tolerance in the ground-cover chrysanthemum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 10:07:33","doi":"10.21203/rs.3.rs-7107028/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3855ba5-da34-47f2-b073-0098e189aa86","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-25T08:11:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 10:07:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7107028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7107028","identity":"rs-7107028","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}