CpMYB14, a novel R2R3-MYB transcription factor, interacts with Cp1-SST to negatively regulate inulin-type fructan accumulation and modulates plant growth in Codonopsis pilosula

CpMYB14, a novel R2R3-MYB transcription factor, interacts with Cp1-SST to negatively regulate inulin-type fructan accumulation and modulates plant growth in Codonopsis pilosula | 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 CpMYB14, a novel R2R3-MYB transcription factor, interacts with Cp1-SST to negatively regulate inulin-type fructan accumulation and modulates plant growth in Codonopsis pilosula Lifang Zhang, Xiao Guo, Tingting Zhang, Hao Niu, Zheyu Liu, Yarong Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9035409/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Codonopsis polysaccharides (CPPs), the primary bioactive constituents of the edible Chinese medicinal plant Codonopsis pilosula (Franch.) Nannf., are mainly composed of inulin-type fructans. The biosynthesis of these fructans is catalyzed by Cp1-SST (sucrose:sucrose 1-fructosyltransferase), a key enzyme whose gene expression can be regulated by MYB transcription factors. However, the regulatory mechanism of Cp1-SST remained unclear. Here, two MYB cis-elements were found in Cp1-SST promoter which exhibited transcriptional activation activity and responded to cold stress. subsequently a R2R3-MYB transcription factor, CpMYB14, was isolated and functionally characterized. CpMYB14 was most responsive to cold, temperature difference, iron overload stress and was localized in the nucleus. Silencing of CpMYB14 resulted in a significant upregulation of Cp1-SST expression and inulin-type fructan content but a decreased expression of the genes associated with CPPs hydrolysis metabolism, including Cp1-FEH , Cp6-FEH , CpSuSy and CpNI . Concurrently, the plant stem height and root length were markedly increased in CpMYB14 gene-silenced lines. Oppositely, overexpression of CpMYB14 significantly decreased the Cp1-SST expression in C. pilosula but with significantly increased Cp1-FEH , Cp6-FEH and CpSuSy expression. Yeast one-hybrid and chromatin immunoprecipitation assays confirmed that CpMYB14 directly binds to the promoter of Cp1-SST . Therefore, CpMYB14 is a negative regulator of Cp1-SST and inulin-type fructan biosynthesis. These findings provide a theoretical foundation for the molecular breeding aimed at developing high-quality varieties of C. pilosula . Codonopsis pilosula CpMYB14 Cp1-SST inulin-type fructan biosynthesis transcription regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Codonopsis pilosula (Franch.) Nannf. is a perennial herb whose root is utilized both as the traditional Chinese medicinal material known as Codonopsis Radix (CR) and as a functional food ingredient. The main bioactive constituents of CR are Codonopsis polysaccharides (CPPs), which exhibit a range of notable biological activities including immunomodulation, anti-tumor, prebiotic, antifatigue, antiviral, and antioxidative effects[ 1 , 2 ]. Recent studies further shows that CPPs can alleviate diet-induced metabolic disorders; for instance, they help reduce hepatic lipid accumulation by modulating related metabolic pathways [ 3 ]. Interestingly, most CPPs belong to the inulin-type fructans (ITFs) [ 4 ]. ITFs have long been used as sweeteners in diabetic foods and as dietary fiber sources [ 5 , 6 ]. Specifically, ITFs derived from CR have been shown to promote gastrointestinal functions, such as anti-gastric, immunoregulation, and intestinal microbial regulation activities [ 1 , 2 ]. Within the plant itself, fructans play a dual role: they act not only as reserve carbohydrates but also as important regulatory molecules that aid in adapting to environmental stress [ 7 ]. ITFs metabolism involves several key enzymes. In plants, sucrose 1-fructosyltransferase (1-SST) acts as the gateway enzyme for ITFs biosynthesis, catalyzing the transfer of a fructosyl unit between sucrose molecules to yield 1-kestose, the foundational building block of ITFs [ 8 ]. Plant fructan exohydrolases (FEHs) are evolutionarily derived from ancestral cell wall invertases (CW-INVs) [ 9 ]. Various types of plant FEHs have been cloned and identified from species such as chicory, wheat, and perennial ryegrass. Among them, fructan 1-exohydrolase (1-FEH) specifically hydrolyzes the β-(2→1)-linked fructosyl group at the terminal of ITFs or mixed-type fructans [ 10 ], while fructan 6-exohydrolase (6-FEH) cleaves the β-(2→6) fructosyl group at the terminal of levan or mixed-type fructans[ 11 ]. Previous study has demonstrated that Cp1-SST which synthesizes fructooligosaccharides with different degrees of polymerization is the key enzyme for ITFs biosynthesis in C. pilosula . Furthermore, its overexpression enhances cold tolerance by modulating fructan accumulation [ 12 ]. Transcription factors (TFs), which play an important role in plant stress response and metabolic engineering, can coordinately regulate the expression of genes across multiple biosynthetic pathways. In the Asteraceae and Poaceae families, several TFs, primarily from the MYB and DOF families, have been identified as regulators of 1-SST expression [ 13 – 15 ]. Notably, CiMYB17, a stress-induced R2R3-MYB transcription factor in Cichorium intybus , directly activates 1-SST transcription by binding to the conserved DTT HGGt motif in the 1-SST promoter [ 13 ]. Beyond 1-SST, CiMYB17 also coregulates the downstream genes involved in fructan synthesis ( 1-FFT ) and hydrolysis ( 1-FEH ), establishing a bidirectional "synthesis-degradation" network. Genomic analysis further reveals that fructan-active enzymes (FAZYs) such as 1-SST and 1-FFT are spatially clustered with transcription factor genes like MYB17 , MYB3 , and MYB5 in chicory, providing a structural basis for their coordinated regulation [ 13 ]. In barley, Overexpression of a sucrose-induced wheat MYB transcription factor, TaMYB13-1 , was demonstrated to activate the promoter functions of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT) in transient transactivation experiments, thereby increasing fructan levels in both leaves and stems[ 14 ]. The accumulation of bioactive constituents in medicinal plants is influenced by the special ecological environment factors of the genuine production areas, and moderate environmental stress can promote the biosynthesis of the bioactive constituents[ 16 ]. Lu Dangshen, a genuine medicinal material of CR from Shanxi Province, contains higher levels of CPPs compared with materials from other producing areas. It is primarily cultivated in high-altitude regions characterized by a cold and semiarid climate and iron-rich soils. In plants, transcription factors play a central role in abiotic stress responses, where they can either activate or repress the expression of downstream target genes [ 17 ]. Under stress conditions, plants activate internal signaling cascades to modulate gene expression, thereby adjusting the production of secondary metabolites and enhancing adaptation to environmental challenges[ 18 , 19 ]. However, the transcriptional regulatory mechanism of the key enzyme gene Cp1-SST for ITFs synthesis remains unclear in C. pilosula . Here, the CpMYB14 gene was characterized based on the cis-element of the Cp1-SST promoter to investigate its function by gene expression analysis, subcellular localization, virus-induced gene silencing (VIGS), and overexpression genetic transformation. The interaction between CpMYB14 and the Cp1-SST gene promoter was confirmed in vivo and in vitro via yeast one-hybrid (Y1H) and chromatin immunoprecipitation (ChIP) technologies. The primary objective of this research is to elucidate the regulatory role of the transcription factor CpMYB14 in ITFs synthesis in C. pilosula , thereby establishing a theoretical basis for understanding the genetic mechanisms underlying the genuineness of Lu Dangshen. 2. Materials and methods 2.1 Plant materials and growth conditions C. pilosula plants were grown in a field in Lingchuan, Shanxi, China (1,522 m altitude, 35°47′ N, 113°24′ E) for two years under good agricultural practices. Different tissues of the plants and the roots at different growth stages were used for gene expression analysis. The flower buds, stems, leaves, and roots were collected at the bloom stage and the roots were collected at the seedling, bloom, fruiting and harvest stages for gene expression analysis. The samples were immediately frozen in liquid nitrogen and stored at -80°C for library preparation. To analyze gene expression under stress, the seeds of C. pilosula were germinated in petri dishes and subsequently grown in plastic trays containing steam-sterilized growing medium at 25℃ with a 16 h light/8 h dark cycle. Four weeks after germination, the seedlings were transferred to individual pots containing an equal amount of dried soil and maintained under the same climatic conditions for 3 d. Thereafter, drought stress was applied by irrigation with 12% PEG6000, while cold stress was imposed by transferring plants to 4℃. Root samples were collected at 0, 4, 8, 12, 24 and 48 h after each treatment. Plants irrigated with water and grown at 25℃ were used as the controls for drought and cold stress, respectively. For large day-and-night temperature-difference stress, plants were subjected to 22℃/4℃ (day/night) and the roots were collected at 0, 6, 12, 18, and 24 d after treatment. Plants cultured at 22℃/16℃ (day/night) were used as the controls. Iron deficiency and iron overload stress were applied by culturing seedlings in liquid 1/2MS medium with 0 µM EDTA-Fe and 150 µM EDTA-Fe, respectively. Plants treated with 50 µM EDTA-Fe were used as the iron-sufficient control. All collected root samples were stored at -80℃ for further analysis. 2.2 Gene Expression analysis RNA extraction and real-time polymerase chain reaction (RT-PCR) were performed as described by Ji et al. [ 12 ]. The primer sequences are shown in Table 1 . 2.3 Cloning and analysis of Cp1-SST promoter and GUS staining The upstream region of Cp1-SST was amplified via Tail-PCR as described by Ji et al. [ 20 ]. To generate the Cp1-SST promoter-GUS construct, the 5´-flanking DNA of the Cp1-SST coding region was amplified with p 1-SST F and p 1-SST R. The 750-bp PCR fragment was cloned into the pCAMBIA1381 vector. The construct was introduced into tobacco leaves via transient transformation. Histochemical staining for GUS activity in transgenic plants was performed using the GUS stain kit (Takara, Japan). Plants transformed with pCAMBIA1381 were used as a parallel negative control. 2.4 Subcellular Localization The open reading frame (ORF) of CpMYB14 was recombined into the pCAMBIA3301-eGFP vector using seamless cloning (TransGen, Beijing, China) to generate plasmid pCAMBIA3301:: CpMYB14 - eGFP . The plasmid was used for transient transformation of tobacco ( N. benthamiana ) leaf epidermal cells. A confocal microscope was used to visualize green fluorescent protein (GFP) in transformed protoplasts after 48 h of incubation. 2.5 Virus-induced gene silencing of CpMYB14 in C. pilosula The 213-bp fragment of the CpMYB14 cDNA (vCpMYB14) was cloned from C. pilosula and inserted into the pTRV2 vector to yield pTRV2:: CpMYB14 for CpMYB14 gene silencing. The pTRV1 and the pTRV2 vectors with or without VCpMYB14 were transformed into the Agrobacterium strain, GV3101. The C. pilosula plants inoculated with pTRV2 empty vector were used as vector control (VCK). Subsequently, the Agrobacterium transformants were co-injected into the C. polosula roots as described by Ji et al. [ 12 ]. 2.6 Overexpression of CpMYB14 in C. pilosula The entire coding sequence of CpMYB14 was ligated into pCAMBIA1381-35S vector by seamless cloning (TransGen, Beijing, China) to construct 35S:: CpMYB14 . Recombinant vector was used to transform C. pilosula with the stem as the explant as described Ji et al. [ 12 ]. The transgenic calli inoculated with the pCAMBIA1381-35S empty vector were used as the controls (35S::). 2.7 Yeast one-hybrid assay The binding assay utilizing yeast one-hybrid system (Clontech) was performed according to the manual provided by the manufacture. The ORF sequence of CpMYB14 were constructed into the pGADT7 vector. The Cp1-SST promoter sequences (from − 567bp to -750bp, relative to translation start) were inserted into a pAbAi plasmid. Subsequently, the recombinant plasmids were co-introduced into yeast strain Y1H. The transformants were cultured and selected on SD/-Ura/-Leu solid medium supplemented with 700 ng/µL AbA for 72 h. The empty vectors pAbAi and pGADT7 were used as negative controls. 2.8 Chromatin immunoprecipitation (ChIP)-qPCR Assay The transgenic C. pilosula calli 35S:: CpMYB14 -eGFP were applied to ChIP analysis. Chromatin immunoprecipitation was conducted according to Pierce Magnetic ChIP Kit (Thermo Scientific, Waltham, USA). An anti-GFP antibody (Engibody, Shanghai, China) was used for ChIP qPCR. The immunoprecipitated samples were used as template for qPCR analysis. The primers were designed according to the sequence containing MYB recognition sites in the Cp1-SST promoter (Table 1 ). The samples without anti-GFP were used as the control. 2.9 High Performance Gel Permeation Chromatography (HPGPC) Analysis of ITFs content The ITFs content was measured by high-performance gel-permeation chromatography (HPGPC) analysis [ 21 ]. 3. Results 3.1 Cis-acting elements analysis of the Cp1-SST promoter in C. pilosula Cp1-SST is the key enzyme gene involved in the synthesis of ITFs in C. pilosula [ 12 ]. In our previous study, the expression of Cp1-SST was found to be induced by cold stress [ 12 ]. To explain why its expression is induced by cold stress and identify the transcription factors interacting with Cp1-SST , the promoter region of Cp1-SST was analyzed via Tail-PCR. The Cp1-SST promoter was found to contain various cis-elements, including three MYB binding sites (Fig. 1 A), three light responsive elements, one MeJA-responsiveness element, one dehydration-responsive element, one gibberellin-responsive element, one low-temperature response element and one auxin-responsive element (Table S1 ). To examine if the transcription activity of the Cp1-SST promoter is induced by cold stress, a GUS reporter gene was fused downstream from the promoter. The resulting p 1-SST ::GUS construct was then genetically transformed into the tobacco leaves. GUS staining demonstrated that cold stress treatment noticeably decreased the GUS activity (Fig. 1 B), which indicated that p Cp1-SST had transcription activation activity and negatively responded to cold stress. 3.2 Isolation and characterization of CpMYB14 To screen for transcription factors interacting with Cp1-SST based on cis-acting elements in its promoter, three MYB transcription factors were obtained from the C. pilosula transcriptome database [ 22 ]. Gene expression pattern analysis revealed that the gene expression level of MYB32 was significantly higher at the seedling, bloom, and fruiting stages compared to the harvest stage, while PHL11 gene expression showed no significant differences at different developmental stages. The expression of MYB14 was extremely significantly higher at the seedling stage than at other stages. Notably, the expression trends of MYB32 and MYB14 were opposite to the changes in Codonopsis polysaccharides (CPPs) content at different developmental stages (Fig. 2 ). Tissue-specific expression analysis indicated that the expression of MYB14 in C. pilosula roots was significantly higher than in stems, leaves, and flowers, consistent with the significantly higher CPPs content observed in roots compared to other tissues. Gene sequence analysis revealed that CpMYB14 contained an 879bp ORF encoding 292 amino acids. The results of the BLAST-Protein (BLASTP) online ( http://www.ncbi.nlm.gov/blast ) showed that CpMYB14 contained two characteristic SANT (SWI3, ADA2, N-CoR and TFIIIB) domains, confirming its classification within the R2R3-MYB transcription factor family. Phylogenetic tree analysis indicated that CpMYB14 had the closest evolutionary relationships to CiMYB14 (Fig. 3 ). 3.3 Expression patterns under different environmental stress of CpMYB14 The accumulation of secondary metabolites in medicinal plants is influenced by the specific ecological factors in their genuine producing regions, and moderate environmental stress can enhance this process [ 16 ]. To investigate the stress-responsive behavior of CpMYB14 , the expression pattern of CpMYB14 was analyzed in the roots of C. pilosula seedlings subjected to different stress treatments. Under drought stress, expression of the CpMYB14 gene increased sharply after 24 h, whereas it remained relatively stable in the control group. Interestingly, under cold stress, the expression pattern of the CpMYB14 gene was opposite to that of the control, suggesting its potential involvement in cold stress response. Furthermore, temperature difference stress also triggered upregulation of CpMYB14 expression in C. pilosula roots. Notably, the overall expression level of CpMYB14 under temperature difference stress was significantly higher compared with the control. Under iron deficiency stress, the expression of CpMYB14 in both the control and treatment group showed a fluctuating trend, characterized by an initial increase, followed by a decline, and then a subsequent rise. Notably, at 48 h after treatment, the expression level of CpMYB14 in the treated plants was significantly higher than that in the control group. Under iron overload stress, the CpMYB14 expression sharply increased and peaked at 8 h and 48 h after treatment, while the CK maintained a consistently low and stable expression level throughout the experimental period (Fig. 4 A). All these finding indicated that the expression of CpMYB14 could respond to cold, temperature difference and iron overload stress, especially the cold stress. 3.4 Subcellular localization of CpMYB14 To identify the subcellular location of CpMYB14 in vivo , the coding sequence of CpMYB14 was fused to the 5' end of the GFP reporter gene driven by the cauliflower mosaic virus (CaMV) 35S promoter. The CpMYB14-eGFP fused expression vector was used to carry out a transient expression assay in A. thaliana protoplasts. CpMYB14 was identified to be localized to the nucleus (Fig. 4 B), whereas GFP controls were distributed evenly throughout the cell. 3.5 CpMYB14 Gene silencing promotes Cp1-SST gene expression, plant growth and ITFs accumulation in C. pilosula To further investigate the function of CpMYB14 , the virus-induced transient gene silencing vector pTRV2- CpMYB14 was constructed for infection of the C. pilosula roots. Expression levels of CpMYB14 were significantly reduced in the gene silenced plants (VCpMYB14) compared to the non-silenced plants (VCK), confirming the effectiveness of CpMYB14 silencing (Fig. 5 C). Comparative morphological analysis revealed striking phenotypic changes in the CpMYB14 -silenced plants (Fig. 5 A). Their plant height was significantly greater than that of VCK. Consistently, the root length was also markedly increased following CpMYB14 gene silencing (Fig. 5 B). To further investigate the effect of CpMYB14 on inulin-fructan biosynthesis in C. pilosula , the expression levels of the genes related to ITFs biosynthesis were detected using qRT-PCR. The results showed that the expression of Cp1-SST was markedly elevated in the CpMYB14 -silenced plants. However, a significant down-regulation was observed in the expression of Cp1-FEH (Fructan 1-Exohydrolase) and Cp6-FEH (Fructan 6-Exohydrolase), the key enzyme genes involved in ITFs hydrolysis. Similarly, the expression of CpNI (Neutral invertase) related to sucrose hydrolysis significantly decreased (Fig. 5 C). These inverse expression patterns indicated that CpMYB14 gene silencing inhibited the expression of ITFs hydrolysis-related genes in C. pilosula . Notably, the ITFs content (the peak at 31 min) in the CpMYB14 -silenced roots was significantly upregulated (Fig. 5 D). Collectively, the results implied that CpMYB14 might negatively regulated the expression of Cp1-SST and ITFs accumulation. 3.6 Overexpression of CpMYB14 inhibits the expression of Cp1-SST and promotes the expression of the genes related to ITFs hydrolysis in C. pilosula To further confirm the effect of CpMYB14 on ITFs biosynthesis, we also conducted CpMYB14 -overexpression vector for induction of callus lines of C. pilosula . The positive transgenic lines were detected with genomic PCR. We further used GUS staining by GUS stain kit (Solarbio, China) to confirm the positive transgenic lines which could be stained blue (Fig. 6 A). qRT-PCR results also showed that CpMYB14 gene was successfully overexpressed in C. pilosula calli. Overexpression of CpMYB14 resulted in the significantly reduction of Cp1-SST expression. In addition, the expression level of CpSuSy and the key enzyme genes involved in inulin-fructan hydrolysis, Cp1-FEH and Cp6-FEH , significantly increased in CpMYB14 -overexpressed C. pilosula calli (Fig. 6 A). The results were consistently with CpMYB14 gene silencing and indicated that expression of CpMYB14 negatively regulated the expression of Cp1-SST , but positively regulated the expression of the genes related to inulin-fructan hydrolysis. HPGPC was used to analyze the CPPs profile characteristics and detect ITFs concentrations in the CpMYB14 -overexpressed calli. The results showed that the content of the large-molecular-weight polysaccharides (the peak at 17–29 min) in the overexpressed calli was significantly lower than that in the control. No ITFs (the peak at 31 min) was detected (Fig. 6 B), likely because the calli lack chloroplasts, the organelle where Cp1-SST is localized [ 12 ]. Therefore, CpMYB14 is a key transcription factor in negatively regulating CPPs accumulation probably through regulating the expression of Cp1-SST . 3.7 CpMYB14 binds to the promoter of Cp1-SST in C. pilosula To determine whether CpMYB14 actually binds to the Cp1-SST promoter to activate the gene transcription, yeast one-hybrid (Y1H) and chromatin immunoprecipitation (ChIP-PCR) assays were performed. For the Y1H assays, the Cp1-SST promoter sequence containing MYB elements was fused to the pAbAi vector, and the full sequence of CpMYB14 was fused to the activation domain AD. When fused p 1-SST -pAbAi was co-expressed with CpMYB14-pGADT7 in yeast, the strain was able to grow on an SD/-Leu/AbA700 plate. No growth was observed in the negative control expression in which the CpMYB14 transcription factor was lacked (Fig. 7 A). These results provided in vitro evidence for the specific binding of CpMYB14 to the Cp1-SST promoter. To verify the specific in vivo binding of CpMYB14 to Cp1-SST promoter, ChIP-PCR assays were conducted using pCAMBIA3301:: CpMYB14 -eGFP and pCAMBIA3301::eGFP transgenic C. pilosula calli, respectively. The MYB element-containing promoter regions of Cp1-SST were enriched by ChIP in the pCAMBIA3301:: CpMYB14 -eGFP transgenic calli compared to the pCAMBIA3301::eGFP control (Fig. 7 B). It showed that CpMYB14 specifically bound to the MYB element on the promoter of Cp1-SST . 4. Discussion Inulin-type fructans (ITFs), the primary constituents of Codonopsis polysaccharides (CPPs), serve as the key bioactive compounds mediating the intestinal protective and immunomodulatory effects of CR [ 23 ]. The expression of Cp1-SST , a crucial enzyme gene in ITFs biosynthesis in C. pilosula , is known to be induced by cold stress, yet the underlying regulatory mechanism remains unclear. In this study, we analyzed the promoter cis-elements of Cp1-SST and, based on the presence of MYB-binding motifs, identified the transcription factor CpMYB14 . Functional characterization revealed that CpMYB14 responds to abiotic stresses such as low temperature, interacts with Cp1-SST , and acts as a negative regulator of ITF accumulation in C. pilosula . Recent studies have confirmed that various soluble polysaccharides, including ITFs, play significant roles in plant stress responses[ 24 ]. Previous work has shown that overexpression of Cp1-SST in tobacco plants which naturally lack fructans effectively promotes fructan synthesis and enhances cold stress tolerance [ 12 ]. In the present study, transgenic tobacco plants carrying the p 1-SST ::GUS plasmid were subjected to cold stress and analyzed using GUS staining. The results revealed a marked reduction in GUS activity over the course of cold treatment, indicating that the Cp1-SST promoter responds negatively to low temperature by downregulating downstream gene expression. This observation further corroborates earlier findings on the stress-responsive regulation of the Cp1-SST gene. The MYB transcription factors family is well recognized for its critical role in regulating plant growth, development, and abiotic stress responses [ 25 ]. MYB proteins are classified into four groups according to the number of conserved MYB repeats in their sequences: 1R-MYB (containing one repeat), R2R3-MYB (two repeats), 3R-MYB (three repeats), and 4R-MYB (four repeats) [ 26 ]. Notably, R2R3-MYB proteins, the largest subgroup within the MYB family, have been shown to play a key role in mediating plant adaptation to drought and low-temperature stress [ 27 ]. CpMYB14 with two SANT domains, was characterized as an R2R3-MYB transcription factor in this study [ 28 , 29 ]. Subcellular localization confirmed that CpMYB14 localizes to the nucleus, which aligns with reports on other R2R3-MYB transcription factors such as SmMYB88 in eggplant ( Solanum melongena ) and VvMYB14 in grapevine ( Vitis vinifera ) [ 30 , 31 ]. Gene expression patterns under multiple stress conditions indicated that CpMYB14 is primarily induced by cold and iron overload stress. Previous studies have demonstrated that overexpression of the strawberry FvMYB44 gene in Arabidopsis thaliana improves cold tolerance in transgenic plants [ 32 ]. Similarly, overexpression of VaMyb14 in A. thaliana upregulated antioxidant enzyme activity and enhanced cold resistance compared with wild-type plants [ 33 ]. While iron is an essential micronutrient for plants, the role of MYB transcription factors in iron overload stress responses remains largely unexplored. In the present study, we observed a pronounced upregulation of CpMYB14 under high-iron treatment (150 µM EDTA-Fe), in contrast to the control, which showed no significant change. These findings suggest that CpMYB14 is capable of actively responding to iron overload stress. Previous studies have reported that in radish ( Raphanus sativus ) taproots, the transcription factor RsMYB90 is cold-inducible, directly binds to low-temperature-responsive elements within the RsCOR78 promoter, and activates its expression to enhance cold tolerance [ 34 ]. Here, we observed that the Cp1-SST promoter was down-regulated under low-temperature stress, consistent with the presence of low-temperature-responsive cis -element. Furthermore, the expression pattern of CpMYB14 under cold stress was inversely correlated with that of Cp1-SST , and the Cp1-SST promoter harbors multiple MYB-binding sites [ 12 ]. CiMYB17 binds to the consensus DNA-motif DTTHGGT and activate 1-SST transcription in Cichorium intybus [ 13 ]. TaMYB13 binds to a (A/G/T)TT(A/T/C)GGT core sequence in the promoters of wheat Ta1-SST and markedly enhance the expression of 1-SST and 6-SFT promoter-driven reporter genes in wheat [ 35 ]. The interaction between CpMYB14 and p Cp1-SST was verified in vitro and in vivo through Y1H and CHIP experiments, respectively. Unlike other MYB transcription factors that positively regulate the expression of 1-SST, gene silencing and overexpression analysis demonstrated that CpMYB14 negatively regulates the expression of Cp1-SST . After silencing and overexpressing the CpMYB14 gene, the expression of the key enzyme gene Cp1-SST related to ITFs synthesis in C. polosula was negatively correlated with the expression of CpMYB14 , and also negatively correlated with the expression of the fructan hydrolysis-related gene CpFEHs and the sucrose hydrolysis-related genes sucrose synthase and acid invertase. These results indicated that the Cp1-SST gene expression was negatively correlated with the expression of CpFEHs , CpSuSy and CpNI , which is consistent with the previous research results [ 12 ]. In this study, overexpression of CpMYB14 in C. pilosula significantly downregulated the Cp1-SST expression, yet no ITFs were detected via HPGPC analysis. This is likely attributable to the chloroplast localization of the Cp1-SST protein, coupled with the extremely low chloroplast content in callus, which together impede fructan accumulation[ 12 ]. Nonetheless, the diminished Cp1-SST activity still impaired the accumulation of other polysaccharides in C. pilosula . Conversely, silencing of CpMYB14 markedly increased the Cp1-SST expression and ITFs content, consistent with prior observations that Cp1-SST catalyze the biosynthesis of fructooligosaccharides with different degrees of polymerization[ 12 ]. However, the silenced lines displayed significantly enhanced root length and plant height, a phenotype opposite to the stunted growth previously reported in tobacco overexpression of Cp1-SST . This phenotypic divergence may arise from the high Cp1-SST expression in silenced lines, which could deplete its substrate, sucrose, while concurrently reducing the activity of sucrose-hydrolyzing enzymes such as NI and SuSy. Consequently, the typical pathway for carbon skeleton provision via sucrose hydrolysis may be disrupted. Given the complexity of plant carbon metabolism, alternative compensatory pathways might be activated [ 36 – 38 ]. Additionally, some MYB transcription factors are recognized as growth repressors in plants. The binding of MYB73/77 to UVR8 inhibits shoot elongation and lateral root growth by reducing expression of IAA19 in Arabidopsis [ 39 ]. Plants overexpression of OsMYB91 showed reduced plant growth [ 40 ]. Overexpression of StMYB66 resulted in shorter plant height[ 41 ]. Therefore, it can be inferred that the CpMYB14 transcription factor may be a core growth inhibitory factor. Its down-regulation directly relieves the inhibitory effect on plant growth-promoting genes. Conclusions In summary, the Cp1-SST promoter contained three MYB recognition sites and negatively regulated downstream gene expression under cold stress. CpMYB14 could respond to cold, temperature difference and iron overload stress. Express patterns indicated that CpMYB14 was located to the nucleus and shared the closest evolutionary relationships with CiMYB14. Functionally analysis revealed that CpMYB14 interacted with the Cp1-SST promoter, negatively regulating the Cp1-SST gene expression while positively regulating the genes related to fructan hydrolysis including FEHs and SuSy , thereby negatively regulating ITFs accumulation. Meanwhile, CpMYB14 expression could affect the plant height and root length. These findings provide a foundation for elucidating the regulatory mechanism underlying CR quality formation mediated by ITFs, with potential economic value. Declarations Funding : This work was supported by National Natural Science Foundation of China [grant number 82204569], Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources [grant number 2060302], the Natural Science Foundation of Shanxi Province [grant number 202103021224225], Applied Basic Research Project of Shanxi Province [grant number 202203021221180], Key R&D Projects in Forestry and Grassland of Shanxi Province [grant number JLYF-2026-40], Shanxi Medical University Doctoral Fund project [grant number No.XD2033]. Authors' Contributions: J.J desiganed, investigated, revised, wrote and revised the manuscript. J.G. and J.L. designed, instructed, investigated, and revised the manuscript. L.Z. and X.G. wrote the manuscript. L.Z., X.G., T.Z., Z.L.,Y.W.,X.L.and J.X. investigated and validated the experiment .J.J. and T.Z. prepared figures and tables. J.X., M.S. and J.J founded the manuscript. All authors reviewed the manuscript. Data Availability Statement: All data supporting the findings of this study are available within the paper and its Supplementary Information. Ethics approval and consent to participate: Not applicable. Conflicts of Interest : The authors declare no conflict of interest. Clinical trial number: Not applicable. Consent for publication : Not applicable. References Zou YF, Zhang YY, Paulsen BS, Fu YP, Huang C, Feng B, Li XL, Chen XF, Ji RY, Song X, He CL, Yin LZ, Ye G, Liang XX, Lv C, Yin ZQ. Prospects of Codonopsis pilosula polysaccharides: Structural features and bioactivities diversity. Trends Food Sci Tech. 2020; 103: 1-11. Luan F, Ji Y, Peng L, Liu Q, Cao H, Yang Y, He X, Zeng N. Extraction, purification, structural characteristics and biological properties of the polysaccharides from Codonopsis pilosula : A review. Carbohyd Polym. 2021; 261: 117863. Chen Q, Lin H, Huang X, Yu W. 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Wei H, Bausewein A, Greiner S, Dauchot N, Harms K, Rausch T. CiMYB17 , a stress-induced chicory R2R3-MYB transcription factor, activates promoters of genes involved in fructan synthesis and degradation. New Phytol. 2017; 215(1): 281-298. Kooiker M, Drenth J, Glassop D, McIntyre CL, Xue GP. TaMYB13-1, a R2R3 MYB transcription factor, regulates the fructan synthetic pathway and contributes to enhanced fructan accumulation in bread wheat. J Exp Bot. 2013; 64(14): 4349-4360. Mare CJ, Chalmers KJ, Henry RJ. The DOF transcription factor family in barley: identification, characterization and expression analysis. Funct Integr Genomic. 2004; 4(3): 149-161. Yuan Y, Zheng H, Huang LQ. Review of contributing factors and research model of excellent shape, high quality, and superior effect of Dao-di herbs. China J Chinese Mater Medi. 2024; 49(15)：3977-3985. Sun C, Wang CX, Liang Y, Li CM, Zhang ZY, Jiao XJ, Han CM. Research progress on plant WRKY transcription factors. 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MYB proteins: Versatile regulators of plant development, stress responses, and secondary metabolite biosynthetic pathways. Int J Biol Macromol. 2025; 288: 138588. Touqeer A, Yuanbo H, Li M, Wu S. Regulatory roles of MYB transcription factors in root barrier under abiotic stress. Plants. 2026; 15(2): 275. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis . Trends Plant Sci. 2010; 15(10): 573-81. Fu S, Liu F, Li Y, Lu C, Liu T, Tian S, Jin W, Guo P, Chen L, Geng X. SlMYB2: an R2R3-MYB transcription factor enhancing drought, salt, and cadmium tolerance in Solanum lycopersicum . Plant Cell Rep. 2026; 45: 24. Lan Y, Gong F, Dong R. A R2R3-MYB transcription factor SmMYB88 of Solanum melongena regulates cold tolerance by activating antioxidant system. Plant Cell Rep. 2026; 45(2): 28. Janine H, Alessandro V, Stefan C, Claudio D, Amanda R, Walker, Thomas R, Margherita L, Paul K, Boss IB, Dry JB. The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera . Plant Cell. 2013; 25(10): 4135-4149. Li W, Wei Y, Zhang L, Wang Y, Song P, Li X, Han D. FvMYB44 , a strawberryR2R3-MYB transcription factor, improved salt and cold stress tolerance in transgenic Arabidopsis . Agronomy. 2023; 13(4): 1051. Fang L, Wang Z, Su L, Gong L, Xin H. Vitis Myb14 confer cold and drought tolerance by activating Lipid transfer protein genes expression and Reactive oxygen species scavenge. Gene. 2023; 890: 147792-147792. Qin T, Zhang M, Yi X. RsMYB90 , a R2R3-MYB transcription factor, plays a positive role in regulating low temperature stress in radish . Plant Biology. 2025; 28(1):69-78. Xue GP, Kooiker M, Drenth J, McIntyre CL. TaMYB13 is a transcriptional activator of fructosyltransferase genes involved in beta -2,6-linked fructan synthesis in wheat. Plant J. 2011; 68(5): 857-870. Brendan M. Playing with Pyr: alternate sources of mitochondrial pyruvate fuel plant respiration. Plant Cell. 2021; 33(8): 2519-2520. Yang LY, Wang ZX, Zhang AQ, Bhawal R, Li CL, Zhang S, Cheng LL, Hua J. Reduction of the canonical function of a glycolytic enzyme enolase triggers immune responses that further affect metabolism and growth in Arabidopsis . Plant Cell. 2021; 34(5): 1745-1767. Dong WK, Zhang YJ, Ma HL. Research advances in the role of central carbon metabolic pathways in plant growth, development and stress responses. Pratacultural Sci. 2025; 43: 1-13. Xu Z, Zhu ZQ. Spotlight UV-B response: when UVR8 meets MYBs. Trends Plant Sci. 2020; 25(6): 515-517. Zhu N, Cheng S, Liu X. The R2R3-type MYB gene OsMYB91 has a function in coordinating plant growth and salt stress tolerance in rice. Plant Sci. 2025; 236: 146-156. Yu T, Wang D, Fu X, Liu Z. StMYB66, a R2R3-myb transcription factor, regulates plant growth and anthocyanin accumulation in potato ( Solanum tuberosum L.). J Am Soc Hortic Sci. 2025; 150(3): 147-158. Table 1 Table 1 is available in the supplementary files section Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Apr, 2026 Reviews received at journal 06 Apr, 2026 Reviews received at journal 27 Mar, 2026 Reviewers agreed at journal 25 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers invited by journal 15 Mar, 2026 Editor invited by journal 09 Mar, 2026 Editor assigned by journal 09 Mar, 2026 Submission checks completed at journal 09 Mar, 2026 First submitted to journal 04 Mar, 2026 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. 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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-9035409","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":606490142,"identity":"46592601-e9fd-484c-bb31-cec8015ce5c4","order_by":0,"name":"Lifang Zhang","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lifang","middleName":"","lastName":"Zhang","suffix":""},{"id":606490154,"identity":"2ad9f886-2bcb-4225-b1bb-da007322902f","order_by":1,"name":"Xiao Guo","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Guo","suffix":""},{"id":606490157,"identity":"02caaa61-850d-44f9-9bf5-4d3eda0b2854","order_by":2,"name":"Tingting Zhang","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Zhang","suffix":""},{"id":606490160,"identity":"cf772b79-2f27-4a08-aeef-3d386d865a8e","order_by":3,"name":"Hao Niu","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Niu","suffix":""},{"id":606490164,"identity":"e31ebf29-f16b-425e-9d60-560d3f2ec9d3","order_by":4,"name":"Zheyu Liu","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zheyu","middleName":"","lastName":"Liu","suffix":""},{"id":606490166,"identity":"4f42522a-fbed-4377-a02b-02ba09f15b91","order_by":5,"name":"Yarong Wang","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yarong","middleName":"","lastName":"Wang","suffix":""},{"id":606490170,"identity":"f2a49c41-6cd3-49d6-8b6b-fe386eb8ae9b","order_by":6,"name":"Xing Liu","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"Liu","suffix":""},{"id":606490179,"identity":"8f2a2e1b-14ea-4701-a764-e079566783f1","order_by":7,"name":"Jianping Gao","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Gao","suffix":""},{"id":606490181,"identity":"abafc4a3-979f-4b9a-b6a3-655db75abd70","order_by":8,"name":"Jinfang Xu","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinfang","middleName":"","lastName":"Xu","suffix":""},{"id":606490183,"identity":"444dd02a-5abe-4938-9210-5c473e60c736","order_by":9,"name":"Mingyue Shen","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingyue","middleName":"","lastName":"Shen","suffix":""},{"id":606490192,"identity":"cb1deb17-1be3-4bfe-96d8-3666c95fdde8","order_by":10,"name":"Jiankuan Li","email":"","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiankuan","middleName":"","lastName":"Li","suffix":""},{"id":606490193,"identity":"a266bca8-c9d3-483e-b257-3efa9dc989d9","order_by":11,"name":"Jiaojiao Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACxmaGhAMMDDZy8uyNjQ8/kKAlzdiw53CzsQQJlh1OZLiR3ibAQ4xa5naGhwd+VBxOYJz5sI1BgsFOTreBCIcd7DmTnscundj2oIAh2djsABFaDvC2WRczzk5sN5BgOJC4jRgtB/+2MSc23DzYJsFDrJbDvG3OiQ03GEnRInMGFMiJwEA2IMIvhv1nkj++qQBF5fGHDz9U2MkR1tLAk4DENSCgHATkGdgJmToKRsEoGAUjHgAARBFG0DXWTXUAAAAASUVORK5CYII=","orcid":"","institution":"Shanxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jiaojiao","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2026-03-05 03:39:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9035409/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9035409/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104783401,"identity":"1ab1533b-b952-481f-b785-7221dad26e0a","added_by":"auto","created_at":"2026-03-17 07:58:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":403661,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram and transcriptional activate activity of the \u003cem\u003eCp1-SST\u003c/em\u003e promoter. (A) Schematic diagram showing the cis-element of the \u003cem\u003eCp1-SST\u003c/em\u003epromoter (PlantCARE; \u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html\u003c/a\u003e). Red box indicated MYB recognizition sites in the \u003cem\u003eCp1-SST\u003c/em\u003e promoter. (B) β-Glucuronidase (GUS) staining of transgenic tobacco leaves transformed with the p\u003cem\u003e1-SST\u003c/em\u003e::\u003cem\u003eGUS\u003c/em\u003e plasmid at different time of treatment with 4℃ cold stress.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/3b0cf58a67748b9cffe4857b.png"},{"id":104782784,"identity":"b87f601b-f196-4885-95c8-90817fb1014e","added_by":"auto","created_at":"2026-03-17 07:57:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121626,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial and temporal expression profile of \u003cem\u003eMYBs\u003c/em\u003etranscription factor and CPPs content in \u003cem\u003eC. pilosula\u003c/em\u003e. Statistical significance was assessed with Student’s t test (**: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *: \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/76cb628d06b738a1d8209cf8.png"},{"id":104783405,"identity":"d086f89b-7e41-4eef-a4ff-4dcd207b44a4","added_by":"auto","created_at":"2026-03-17 07:58:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215970,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of MYB14 in different species. The tree was constructed using Neighbor-joining method based on the complete amino acid sequences by MEGA 7.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/7e0982026d65a8594c883f46.png"},{"id":104783441,"identity":"bb83cc40-e48a-4df0-9c3e-631c5b39b14e","added_by":"auto","created_at":"2026-03-17 07:58:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":233137,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns under different environmental stress and subcellular localization of CpMYB14. (A) Gene expression of \u003cem\u003eCpMYB14\u003c/em\u003e in the roots of \u003cem\u003eC. pilosula\u003c/em\u003esubjected to different environmental stress. Statistical significance was assessed with Student’s t test (**: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). (B) Nuclear localization of CpMYB14 proteins in tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) leaves. Tobacco leaf cells were transiently transformed with constructs containing \u003cem\u003eCpMYB14\u003c/em\u003e-eGFP, or eGFP (control) under the control of the 35S promoter.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/e58a47c76856e08ef25e846b.png"},{"id":104774987,"identity":"4b2eeee6-9078-427d-a635-39b3f758329a","added_by":"auto","created_at":"2026-03-17 06:31:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230967,"visible":true,"origin":"","legend":"\u003cp\u003eGene silencing of \u003cem\u003eCpMYB14\u003c/em\u003eby VIGS in \u003cem\u003eC. pilosula\u003c/em\u003e. (A) Seedling phenotype of pTRV2::\u003cem\u003eCpMYB14 \u003c/em\u003e(VMYB14) and pTRV2:: (VCK) transgenic \u003cem\u003eC. pilosula\u003c/em\u003e plants. (B) Morphological analysis of the transgenic plants. (C) Expression analysis of the genes related to ITFs metabolism in the transgenic plants. (D) HPGPC analysis of CPPs for the determination of ITFs content in the transgenic plants. Statistical significance was assessed with Student’s t test (**: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *: \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/a04a1e0fd19b48457b375a79.png"},{"id":104774991,"identity":"64a866e3-514d-4729-aa54-6a99f7e2fe01","added_by":"auto","created_at":"2026-03-17 06:31:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72525,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eCpMYB14\u003c/em\u003e in \u003cem\u003eC. pilosula \u003c/em\u003ecalli. (A) Expression analysis of the genes related to ITFs metabolism in the 35S::\u003cem\u003eCpMYB14 \u003c/em\u003e-GUS (OE) and 35S::GUS (CK) transgenic \u003cem\u003eC. pilosula\u003c/em\u003e calli. (B) HPGPC analysis of CPPs for the determination of ITFs content in the transgenic calli. Statistical significance was assessed with Student’s t test (**: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/b2a9d39b481977102f3fa997.png"},{"id":104774994,"identity":"8e57d190-59b9-43ba-851d-49a0a4f261a2","added_by":"auto","created_at":"2026-03-17 06:31:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":68289,"visible":true,"origin":"","legend":"\u003cp\u003eThe transcription factor CpMYB14 binds to the MYB element of the \u003cem\u003eCp1-SST\u003c/em\u003epromoter. (A) Yeast one-hybrid assay showed that the promoter of \u003cem\u003eCp1-SST\u003c/em\u003ewas fused to the pAbAi vector, and CpMYB14 was fused to activation domain AD. (B) ChIP-qPCR showed the DNA specific enrichment with GFP-antibody or without GFP antibody in 35S::\u003cem\u003eCpMYB14\u003c/em\u003e-eGFP (OE) and 35S::-eGFP (CK) transgenic \u003cem\u003eC. pilosul \u003c/em\u003ecalli. (C) ChIP-qPCR showed the DNA specific enrichment in 35S::\u003cem\u003eCpMYB14\u003c/em\u003e-eGFP (OE) and 35S::-eGFP (CK) transgenic \u003cem\u003eC. pilosul \u003c/em\u003ecalli. Statistical significance was assessed with Student’s t test (**: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *: \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/77500c0a5c84fd51332c6d86.png"},{"id":104808713,"identity":"b238b157-a4ee-4cf1-b330-ddebe02d16ed","added_by":"auto","created_at":"2026-03-17 12:39:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2352057,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/0d0b11f4-6e34-4f1f-a535-44ca221aceaa.pdf"},{"id":104774989,"identity":"60f6ec31-71bd-499f-bec4-3a1745ae03d9","added_by":"auto","created_at":"2026-03-17 06:31:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19029,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9035409/v1/30bb19839186a82078b27fcd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CpMYB14, a novel R2R3-MYB transcription factor, interacts with Cp1-SST to negatively regulate inulin-type fructan accumulation and modulates plant growth in Codonopsis pilosula","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eCodonopsis pilosula\u003c/em\u003e (Franch.) Nannf. is a perennial herb whose root is utilized both as the traditional Chinese medicinal material known as Codonopsis Radix (CR) and as a functional food ingredient. The main bioactive constituents of CR are Codonopsis polysaccharides (CPPs), which exhibit a range of notable biological activities including immunomodulation, anti-tumor, prebiotic, antifatigue, antiviral, and antioxidative effects[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recent studies further shows that CPPs can alleviate diet-induced metabolic disorders; for instance, they help reduce hepatic lipid accumulation by modulating related metabolic pathways [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Interestingly, most CPPs belong to the inulin-type fructans (ITFs) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. ITFs have long been used as sweeteners in diabetic foods and as dietary fiber sources [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Specifically, ITFs derived from CR have been shown to promote gastrointestinal functions, such as anti-gastric, immunoregulation, and intestinal microbial regulation activities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Within the plant itself, fructans play a dual role: they act not only as reserve carbohydrates but also as important regulatory molecules that aid in adapting to environmental stress [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eITFs metabolism involves several key enzymes. In plants, sucrose 1-fructosyltransferase (1-SST) acts as the gateway enzyme for ITFs biosynthesis, catalyzing the transfer of a fructosyl unit between sucrose molecules to yield 1-kestose, the foundational building block of ITFs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Plant fructan exohydrolases (FEHs) are evolutionarily derived from ancestral cell wall invertases (CW-INVs) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Various types of plant FEHs have been cloned and identified from species such as chicory, wheat, and perennial ryegrass. Among them, fructan 1-exohydrolase (1-FEH) specifically hydrolyzes the β-(2\u0026rarr;1)-linked fructosyl group at the terminal of ITFs or mixed-type fructans [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while fructan 6-exohydrolase (6-FEH) cleaves the β-(2\u0026rarr;6) fructosyl group at the terminal of levan or mixed-type fructans[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous study has demonstrated that Cp1-SST which synthesizes fructooligosaccharides with different degrees of polymerization is the key enzyme for ITFs biosynthesis in \u003cem\u003eC. pilosula\u003c/em\u003e. Furthermore, its overexpression enhances cold tolerance by modulating fructan accumulation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranscription factors (TFs), which play an important role in plant stress response and metabolic engineering, can coordinately regulate the expression of genes across multiple biosynthetic pathways. In the Asteraceae and Poaceae families, several TFs, primarily from the MYB and DOF families, have been identified as regulators of 1-SST expression [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Notably, CiMYB17, a stress-induced R2R3-MYB transcription factor in \u003cem\u003eCichorium intybus\u003c/em\u003e, directly activates 1-SST transcription by binding to the conserved DTT HGGt motif in the 1-SST promoter [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Beyond 1-SST, CiMYB17 also coregulates the downstream genes involved in fructan synthesis (\u003cem\u003e1-FFT\u003c/em\u003e) and hydrolysis (\u003cem\u003e1-FEH\u003c/em\u003e), establishing a bidirectional \"synthesis-degradation\" network. Genomic analysis further reveals that fructan-active enzymes (FAZYs) such as 1-SST and 1-FFT are spatially clustered with transcription factor genes like \u003cem\u003eMYB17\u003c/em\u003e, \u003cem\u003eMYB3\u003c/em\u003e, and \u003cem\u003eMYB5\u003c/em\u003e in chicory, providing a structural basis for their coordinated regulation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In barley, Overexpression of a sucrose-induced wheat MYB transcription factor, \u003cem\u003eTaMYB13-1\u003c/em\u003e, was demonstrated to activate the promoter functions of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT) in transient transactivation experiments, thereby increasing fructan levels in both leaves and stems[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe accumulation of bioactive constituents in medicinal plants is influenced by the special ecological environment factors of the genuine production areas, and moderate environmental stress can promote the biosynthesis of the bioactive constituents[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Lu Dangshen, a genuine medicinal material of CR from Shanxi Province, contains higher levels of CPPs compared with materials from other producing areas. It is primarily cultivated in high-altitude regions characterized by a cold and semiarid climate and iron-rich soils. In plants, transcription factors play a central role in abiotic stress responses, where they can either activate or repress the expression of downstream target genes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Under stress conditions, plants activate internal signaling cascades to modulate gene expression, thereby adjusting the production of secondary metabolites and enhancing adaptation to environmental challenges[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the transcriptional regulatory mechanism of the key enzyme gene \u003cem\u003eCp1-SST\u003c/em\u003e for ITFs synthesis remains unclear in \u003cem\u003eC. pilosula\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHere, the \u003cem\u003eCpMYB14\u003c/em\u003e gene was characterized based on the cis-element of the \u003cem\u003eCp1-SST\u003c/em\u003e promoter to investigate its function by gene expression analysis, subcellular localization, virus-induced gene silencing (VIGS), and overexpression genetic transformation. The interaction between CpMYB14 and the \u003cem\u003eCp1-SST\u003c/em\u003e gene promoter was confirmed in vivo and in vitro via yeast one-hybrid (Y1H) and chromatin immunoprecipitation (ChIP) technologies. The primary objective of this research is to elucidate the regulatory role of the transcription factor CpMYB14 in ITFs synthesis in \u003cem\u003eC. pilosula\u003c/em\u003e, thereby establishing a theoretical basis for understanding the genetic mechanisms underlying the genuineness of Lu Dangshen.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Plant materials and growth conditions\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eC. pilosula\u003c/em\u003e plants were grown in a field in Lingchuan, Shanxi, China (1,522 m altitude, 35\u0026deg;47\u0026prime; N, 113\u0026deg;24\u0026prime; E) for two years under good agricultural practices. Different tissues of the plants and the roots at different growth stages were used for gene expression analysis. The flower buds, stems, leaves, and roots were collected at the bloom stage and the roots were collected at the seedling, bloom, fruiting and harvest stages for gene expression analysis. The samples were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C for library preparation.\u003c/p\u003e\n \u003cp\u003eTo analyze gene expression under stress, the seeds of \u003cem\u003eC. pilosula\u003c/em\u003e were germinated in petri dishes and subsequently grown in plastic trays containing steam-sterilized growing medium at 25℃ with a 16 h light/8 h dark cycle. Four weeks after germination, the seedlings were transferred to individual pots containing an equal amount of dried soil and maintained under the same climatic conditions for 3 d. Thereafter, drought stress was applied by irrigation with 12% PEG6000, while cold stress was imposed by transferring plants to 4℃. Root samples were collected at 0, 4, 8, 12, 24 and 48 h after each treatment. Plants irrigated with water and grown at 25℃ were used as the controls for drought and cold stress, respectively. For large day-and-night temperature-difference stress, plants were subjected to 22℃/4℃ (day/night) and the roots were collected at 0, 6, 12, 18, and 24 d after treatment. Plants cultured at 22℃/16℃ (day/night) were used as the controls. Iron deficiency and iron overload stress were applied by culturing seedlings in liquid 1/2MS medium with 0 \u0026micro;M EDTA-Fe and 150 \u0026micro;M EDTA-Fe, respectively. Plants treated with 50 \u0026micro;M EDTA-Fe were used as the iron-sufficient control. All collected root samples were stored at -80℃ for further analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Gene \u003cem\u003eExpression analysis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eRNA extraction and real-time polymerase chain reaction (RT-PCR) were performed as described by Ji et al. [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. The primer sequences are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Cloning and analysis of Cp1-SST promoter and GUS staining\u003c/h2\u003e\n \u003cp\u003eThe upstream region of \u003cem\u003eCp1-SST\u003c/em\u003e was amplified via Tail-PCR as described by Ji et al. [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. To generate the \u003cem\u003eCp1-SST\u003c/em\u003e promoter-GUS construct, the 5\u0026acute;-flanking DNA of the Cp1-SST coding region was amplified with p\u003cem\u003e1-SST\u003c/em\u003e F and p\u003cem\u003e1-SST\u003c/em\u003e R. The 750-bp PCR fragment was cloned into the pCAMBIA1381 vector. The construct was introduced into tobacco leaves via transient transformation. Histochemical staining for GUS activity in transgenic plants was performed using the GUS stain kit (Takara, Japan). Plants transformed with pCAMBIA1381 were used as a parallel negative control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Subcellular Localization\u003c/h2\u003e\n \u003cp\u003eThe open reading frame (ORF) of \u003cem\u003eCpMYB14\u003c/em\u003e was recombined into the pCAMBIA3301-eGFP vector using seamless cloning (TransGen, Beijing, China) to generate plasmid pCAMBIA3301::\u003cem\u003eCpMYB14\u003c/em\u003e-\u003cem\u003eeGFP\u003c/em\u003e. The plasmid was used for transient transformation of tobacco (\u003cem\u003eN. benthamiana\u003c/em\u003e) leaf epidermal cells. A confocal microscope was used to visualize green fluorescent protein (GFP) in transformed protoplasts after 48 h of incubation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Virus-induced gene silencing of CpMYB14 in C. pilosula\u003c/h2\u003e\n \u003cp\u003eThe 213-bp fragment of the \u003cem\u003eCpMYB14\u003c/em\u003e cDNA (vCpMYB14) was cloned from \u003cem\u003eC. pilosula\u003c/em\u003e and inserted into the pTRV2 vector to yield pTRV2::\u003cem\u003eCpMYB14\u003c/em\u003e for \u003cem\u003eCpMYB14\u003c/em\u003e gene silencing. The pTRV1 and the pTRV2 vectors with or without VCpMYB14 were transformed into the \u003cem\u003eAgrobacterium\u003c/em\u003e strain, GV3101. The \u003cem\u003eC. pilosula\u003c/em\u003e plants inoculated with pTRV2 empty vector were used as vector control (VCK). Subsequently, the Agrobacterium transformants were co-injected into the \u003cem\u003eC. polosula\u003c/em\u003e roots as described by Ji et al. [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Overexpression of CpMYB14 in C. pilosula\u003c/h2\u003e\n \u003cp\u003eThe entire coding sequence of \u003cem\u003eCpMYB14\u003c/em\u003e was ligated into pCAMBIA1381-35S vector by seamless cloning (TransGen, Beijing, China) to construct 35S::\u003cem\u003eCpMYB14\u003c/em\u003e. Recombinant vector was used to transform \u003cem\u003eC. pilosula\u003c/em\u003e with the stem as the explant as described Ji et al. [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. The transgenic calli inoculated with the pCAMBIA1381-35S empty vector were used as the controls (35S::).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Yeast one-hybrid assay\u003c/h2\u003e\n \u003cp\u003eThe binding assay utilizing yeast one-hybrid system (Clontech) was performed according to the manual provided by the manufacture. The ORF sequence of \u003cem\u003eCpMYB14\u003c/em\u003e were constructed into the pGADT7 vector. The \u003cem\u003eCp1-SST\u003c/em\u003e promoter sequences (from \u0026minus;\u0026thinsp;567bp to -750bp, relative to translation start) were inserted into a pAbAi plasmid. Subsequently, the recombinant plasmids were co-introduced into yeast strain Y1H. The transformants were cultured and selected on SD/-Ura/-Leu solid medium supplemented with 700 ng/\u0026micro;L AbA for 72 h. The empty vectors pAbAi and pGADT7 were used as negative controls.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Chromatin immunoprecipitation (ChIP)-qPCR Assay\u003c/h2\u003e\n \u003cp\u003eThe transgenic \u003cem\u003eC. pilosula\u003c/em\u003e calli 35S::\u003cem\u003eCpMYB14\u003c/em\u003e-eGFP were applied to ChIP analysis. Chromatin immunoprecipitation was conducted according to Pierce Magnetic ChIP Kit (Thermo Scientific, Waltham, USA). An anti-GFP antibody (Engibody, Shanghai, China) was used for ChIP qPCR. The immunoprecipitated samples were used as template for qPCR analysis. The primers were designed according to the sequence containing MYB recognition sites in the \u003cem\u003eCp1-SST\u003c/em\u003e promoter (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The samples without anti-GFP were used as the control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 High Performance Gel Permeation Chromatography (HPGPC) Analysis of ITFs content\u003c/h2\u003e\n \u003cp\u003eThe ITFs content was measured by high-performance gel-permeation chromatography (HPGPC) analysis [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cis-acting elements analysis of the Cp1-SST promoter in C. pilosula\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCp1-SST\u003c/em\u003e is the key enzyme gene involved in the synthesis of ITFs in \u003cem\u003eC. pilosula\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In our previous study, the expression of \u003cem\u003eCp1-SST\u003c/em\u003e was found to be induced by cold stress [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To explain why its expression is induced by cold stress and identify the transcription factors interacting with \u003cem\u003eCp1-SST\u003c/em\u003e, the promoter region of \u003cem\u003eCp1-SST\u003c/em\u003e was analyzed via Tail-PCR. The \u003cem\u003eCp1-SST\u003c/em\u003e promoter was found to contain various cis-elements, including three MYB binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), three light responsive elements, one MeJA-responsiveness element, one dehydration-responsive element, one gibberellin-responsive element, one low-temperature response element and one auxin-responsive element (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo examine if the transcription activity of the \u003cem\u003eCp1-SST\u003c/em\u003e promoter is induced by cold stress, a GUS reporter gene was fused downstream from the promoter. The resulting p\u003cem\u003e1-SST\u003c/em\u003e::GUS construct was then genetically transformed into the tobacco leaves. GUS staining demonstrated that cold stress treatment noticeably decreased the GUS activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which indicated that p\u003cem\u003eCp1-SST\u003c/em\u003e had transcription activation activity and negatively responded to cold stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Isolation and characterization of CpMYB14\u003c/h2\u003e \u003cp\u003eTo screen for transcription factors interacting with \u003cem\u003eCp1-SST\u003c/em\u003e based on cis-acting elements in its promoter, three MYB transcription factors were obtained from the \u003cem\u003eC. pilosula\u003c/em\u003e transcriptome database [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Gene expression pattern analysis revealed that the gene expression level of \u003cem\u003eMYB32\u003c/em\u003e was significantly higher at the seedling, bloom, and fruiting stages compared to the harvest stage, while \u003cem\u003ePHL11\u003c/em\u003e gene expression showed no significant differences at different developmental stages. The expression of \u003cem\u003eMYB14\u003c/em\u003e was extremely significantly higher at the seedling stage than at other stages. Notably, the expression trends of \u003cem\u003eMYB32\u003c/em\u003e and \u003cem\u003eMYB14\u003c/em\u003e were opposite to the changes in Codonopsis polysaccharides (CPPs) content at different developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Tissue-specific expression analysis indicated that the expression of \u003cem\u003eMYB14\u003c/em\u003e in \u003cem\u003eC. pilosula\u003c/em\u003e roots was significantly higher than in stems, leaves, and flowers, consistent with the significantly higher CPPs content observed in roots compared to other tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene sequence analysis revealed that CpMYB14 contained an 879bp ORF encoding 292 amino acids. The results of the BLAST-Protein (BLASTP) online (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.gov/blast\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.gov/blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) showed that CpMYB14 contained two characteristic SANT (SWI3, ADA2, N-CoR and TFIIIB) domains, confirming its classification within the R2R3-MYB transcription factor family. Phylogenetic tree analysis indicated that CpMYB14 had the closest evolutionary relationships to CiMYB14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Expression patterns under different environmental stress of CpMYB14\u003c/h2\u003e \u003cp\u003eThe accumulation of secondary metabolites in medicinal plants is influenced by the specific ecological factors in their genuine producing regions, and moderate environmental stress can enhance this process [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To investigate the stress-responsive behavior of \u003cem\u003eCpMYB14\u003c/em\u003e, the expression pattern of \u003cem\u003eCpMYB14\u003c/em\u003e was analyzed in the roots of \u003cem\u003eC. pilosula\u003c/em\u003e seedlings subjected to different stress treatments.\u003c/p\u003e \u003cp\u003eUnder drought stress, expression of the \u003cem\u003eCpMYB14\u003c/em\u003e gene increased sharply after 24 h, whereas it remained relatively stable in the control group. Interestingly, under cold stress, the expression pattern of the \u003cem\u003eCpMYB14\u003c/em\u003e gene was opposite to that of the control, suggesting its potential involvement in cold stress response. Furthermore, temperature difference stress also triggered upregulation of \u003cem\u003eCpMYB14\u003c/em\u003e expression in \u003cem\u003eC. pilosula\u003c/em\u003e roots. Notably, the overall expression level of \u003cem\u003eCpMYB14\u003c/em\u003e under temperature difference stress was significantly higher compared with the control. Under iron deficiency stress, the expression of \u003cem\u003eCpMYB14\u003c/em\u003e in both the control and treatment group showed a fluctuating trend, characterized by an initial increase, followed by a decline, and then a subsequent rise. Notably, at 48 h after treatment, the expression level of \u003cem\u003eCpMYB14\u003c/em\u003e in the treated plants was significantly higher than that in the control group. Under iron overload stress, the \u003cem\u003eCpMYB14\u003c/em\u003e expression sharply increased and peaked at 8 h and 48 h after treatment, while the CK maintained a consistently low and stable expression level throughout the experimental period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). All these finding indicated that the expression of CpMYB14 could respond to cold, temperature difference and iron overload stress, especially the cold stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Subcellular localization of CpMYB14\u003c/h2\u003e \u003cp\u003eTo identify the subcellular location of CpMYB14 \u003cem\u003ein vivo\u003c/em\u003e, the coding sequence of \u003cem\u003eCpMYB14\u003c/em\u003e was fused to the 5' end of the GFP reporter gene driven by the cauliflower mosaic virus (CaMV) 35S promoter. The \u003cem\u003eCpMYB14-eGFP\u003c/em\u003e fused expression vector was used to carry out a transient expression assay in \u003cem\u003eA. thaliana\u003c/em\u003e protoplasts. CpMYB14 was identified to be localized to the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), whereas GFP controls were distributed evenly throughout the cell.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.5 CpMYB14 Gene silencing promotes Cp1-SST gene expression, plant growth and ITFs accumulation in C. pilosula\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo further investigate the function of \u003cem\u003eCpMYB14\u003c/em\u003e, the virus-induced transient gene silencing vector pTRV2-\u003cem\u003eCpMYB14\u003c/em\u003e was constructed for infection of the \u003cem\u003eC. pilosula\u003c/em\u003e roots. Expression levels of \u003cem\u003eCpMYB14\u003c/em\u003e were significantly reduced in the gene silenced plants (VCpMYB14) compared to the non-silenced plants (VCK), confirming the effectiveness of \u003cem\u003eCpMYB14\u003c/em\u003e silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eComparative morphological analysis revealed striking phenotypic changes in the \u003cem\u003eCpMYB14\u003c/em\u003e-silenced plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Their plant height was significantly greater than that of VCK. Consistently, the root length was also markedly increased following \u003cem\u003eCpMYB14\u003c/em\u003e gene silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo further investigate the effect of \u003cem\u003eCpMYB14\u003c/em\u003e on inulin-fructan biosynthesis in \u003cem\u003eC. pilosula\u003c/em\u003e, the expression levels of the genes related to ITFs biosynthesis were detected using qRT-PCR. The results showed that the expression of \u003cem\u003eCp1-SST\u003c/em\u003e was markedly elevated in the \u003cem\u003eCpMYB14\u003c/em\u003e-silenced plants. However, a significant down-regulation was observed in the expression of \u003cem\u003eCp1-FEH\u003c/em\u003e (Fructan 1-Exohydrolase) and \u003cem\u003eCp6-FEH\u003c/em\u003e (Fructan 6-Exohydrolase), the key enzyme genes involved in ITFs hydrolysis. Similarly, the expression of \u003cem\u003eCpNI\u003c/em\u003e (Neutral invertase) related to sucrose hydrolysis significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These inverse expression patterns indicated that \u003cem\u003eCpMYB14\u003c/em\u003e gene silencing inhibited the expression of ITFs hydrolysis-related genes in \u003cem\u003eC. pilosula\u003c/em\u003e. Notably, the ITFs content (the peak at 31 min) in the \u003cem\u003eCpMYB14\u003c/em\u003e-silenced roots was significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Collectively, the results implied that \u003cem\u003eCpMYB14\u003c/em\u003e might negatively regulated the expression of \u003cem\u003eCp1-SST\u003c/em\u003e and ITFs accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 Overexpression of CpMYB14 inhibits the expression of Cp1-SST and promotes the expression of the genes related to ITFs hydrolysis in C. pilosula\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further confirm the effect of CpMYB14 on ITFs biosynthesis, we also conducted \u003cem\u003eCpMYB14\u003c/em\u003e-overexpression vector for induction of callus lines of \u003cem\u003eC. pilosula\u003c/em\u003e. The positive transgenic lines were detected with genomic PCR. We further used GUS staining by GUS stain kit (Solarbio, China) to confirm the positive transgenic lines which could be stained blue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). qRT-PCR results also showed that \u003cem\u003eCpMYB14\u003c/em\u003e gene was successfully overexpressed in \u003cem\u003eC. pilosula\u003c/em\u003e calli.\u003c/p\u003e \u003cp\u003eOverexpression of \u003cem\u003eCpMYB14\u003c/em\u003e resulted in the significantly reduction of \u003cem\u003eCp1-SST\u003c/em\u003e expression. In addition, the expression level of \u003cem\u003eCpSuSy\u003c/em\u003e and the key enzyme genes involved in inulin-fructan hydrolysis, \u003cem\u003eCp1-FEH\u003c/em\u003e and \u003cem\u003eCp6-FEH\u003c/em\u003e, significantly increased in \u003cem\u003eCpMYB14\u003c/em\u003e-overexpressed \u003cem\u003eC. pilosula\u003c/em\u003e calli (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The results were consistently with \u003cem\u003eCpMYB14\u003c/em\u003e gene silencing and indicated that expression of \u003cem\u003eCpMYB14\u003c/em\u003e negatively regulated the expression of \u003cem\u003eCp1-SST\u003c/em\u003e, but positively regulated the expression of the genes related to inulin-fructan hydrolysis.\u003c/p\u003e \u003cp\u003eHPGPC was used to analyze the CPPs profile characteristics and detect ITFs concentrations in the \u003cem\u003eCpMYB14\u003c/em\u003e-overexpressed calli. The results showed that the content of the large-molecular-weight polysaccharides (the peak at 17\u0026ndash;29 min) in the overexpressed calli was significantly lower than that in the control. No ITFs (the peak at 31 min) was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), likely because the calli lack chloroplasts, the organelle where Cp1-SST is localized [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, CpMYB14 is a key transcription factor in negatively regulating CPPs accumulation probably through regulating the expression of \u003cem\u003eCp1-SST\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 CpMYB14 binds to the promoter of Cp1-SST in C. pilosula\u003c/h2\u003e \u003cp\u003eTo determine whether CpMYB14 actually binds to the \u003cem\u003eCp1-SST\u003c/em\u003e promoter to activate the gene transcription, yeast one-hybrid (Y1H) and chromatin immunoprecipitation (ChIP-PCR) assays were performed. For the Y1H assays, the \u003cem\u003eCp1-SST\u003c/em\u003e promoter sequence containing MYB elements was fused to the pAbAi vector, and the full sequence of \u003cem\u003eCpMYB14\u003c/em\u003e was fused to the activation domain AD. When fused p\u003cem\u003e1-SST\u003c/em\u003e-pAbAi was co-expressed with CpMYB14-pGADT7 in yeast, the strain was able to grow on an SD/-Leu/AbA700 plate. No growth was observed in the negative control expression in which the CpMYB14 transcription factor was lacked (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). These results provided \u003cem\u003ein vitro\u003c/em\u003e evidence for the specific binding of CpMYB14 to the \u003cem\u003eCp1-SST\u003c/em\u003e promoter.\u003c/p\u003e \u003cp\u003eTo verify the specific \u003cem\u003ein vivo\u003c/em\u003e binding of CpMYB14 to \u003cem\u003eCp1-SST\u003c/em\u003e promoter, ChIP-PCR assays were conducted using pCAMBIA3301::\u003cem\u003eCpMYB14\u003c/em\u003e-eGFP and pCAMBIA3301::eGFP transgenic \u003cem\u003eC. pilosula\u003c/em\u003e calli, respectively. The MYB element-containing promoter regions of \u003cem\u003eCp1-SST\u003c/em\u003e were enriched by ChIP in the pCAMBIA3301::\u003cem\u003eCpMYB14\u003c/em\u003e-eGFP transgenic calli compared to the pCAMBIA3301::eGFP control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). It showed that CpMYB14 specifically bound to the MYB element on the promoter of \u003cem\u003eCp1-SST\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eInulin-type fructans (ITFs), the primary constituents of \u003cem\u003eCodonopsis\u003c/em\u003e polysaccharides (CPPs), serve as the key bioactive compounds mediating the intestinal protective and immunomodulatory effects of CR [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The expression of \u003cem\u003eCp1-SST\u003c/em\u003e, a crucial enzyme gene in ITFs biosynthesis in \u003cem\u003eC. pilosula\u003c/em\u003e, is known to be induced by cold stress, yet the underlying regulatory mechanism remains unclear. In this study, we analyzed the promoter cis-elements of \u003cem\u003eCp1-SST\u003c/em\u003e and, based on the presence of MYB-binding motifs, identified the transcription factor \u003cem\u003eCpMYB14\u003c/em\u003e. Functional characterization revealed that CpMYB14 responds to abiotic stresses such as low temperature, interacts with \u003cem\u003eCp1-SST\u003c/em\u003e, and acts as a negative regulator of ITF accumulation in \u003cem\u003eC. pilosula\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRecent studies have confirmed that various soluble polysaccharides, including ITFs, play significant roles in plant stress responses[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Previous work has shown that overexpression of \u003cem\u003eCp1-SST\u003c/em\u003e in tobacco plants which naturally lack fructans effectively promotes fructan synthesis and enhances cold stress tolerance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the present study, transgenic tobacco plants carrying the p\u003cem\u003e1-SST\u003c/em\u003e::GUS plasmid were subjected to cold stress and analyzed using GUS staining. The results revealed a marked reduction in GUS activity over the course of cold treatment, indicating that the \u003cem\u003eCp1-SST\u003c/em\u003e promoter responds negatively to low temperature by downregulating downstream gene expression. This observation further corroborates earlier findings on the stress-responsive regulation of the \u003cem\u003eCp1-SST\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eThe MYB transcription factors family is well recognized for its critical role in regulating plant growth, development, and abiotic stress responses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. MYB proteins are classified into four groups according to the number of conserved MYB repeats in their sequences: 1R-MYB (containing one repeat), R2R3-MYB (two repeats), 3R-MYB (three repeats), and 4R-MYB (four repeats) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Notably, R2R3-MYB proteins, the largest subgroup within the MYB family, have been shown to play a key role in mediating plant adaptation to drought and low-temperature stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. CpMYB14 with two SANT domains, was characterized as an R2R3-MYB transcription factor in this study [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Subcellular localization confirmed that CpMYB14 localizes to the nucleus, which aligns with reports on other R2R3-MYB transcription factors such as SmMYB88 in eggplant (\u003cem\u003eSolanum melongena\u003c/em\u003e) and VvMYB14 in grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Gene expression patterns under multiple stress conditions indicated that \u003cem\u003eCpMYB14\u003c/em\u003e is primarily induced by cold and iron overload stress. Previous studies have demonstrated that overexpression of the strawberry \u003cem\u003eFvMYB44\u003c/em\u003e gene in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e improves cold tolerance in transgenic plants [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, overexpression of \u003cem\u003eVaMyb14\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e upregulated antioxidant enzyme activity and enhanced cold resistance compared with wild-type plants [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. While iron is an essential micronutrient for plants, the role of MYB transcription factors in iron overload stress responses remains largely unexplored. In the present study, we observed a pronounced upregulation of \u003cem\u003eCpMYB14\u003c/em\u003e under high-iron treatment (150 \u0026micro;M EDTA-Fe), in contrast to the control, which showed no significant change. These findings suggest that CpMYB14 is capable of actively responding to iron overload stress.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that in radish (\u003cem\u003eRaphanus sativus\u003c/em\u003e) taproots, the transcription factor RsMYB90 is cold-inducible, directly binds to low-temperature-responsive elements within the \u003cem\u003eRsCOR78\u003c/em\u003e promoter, and activates its expression to enhance cold tolerance [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Here, we observed that the \u003cem\u003eCp1-SST\u003c/em\u003e promoter was down-regulated under low-temperature stress, consistent with the presence of low-temperature-responsive \u003cem\u003ecis\u003c/em\u003e-element. Furthermore, the expression pattern of \u003cem\u003eCpMYB14\u003c/em\u003e under cold stress was inversely correlated with that of \u003cem\u003eCp1-SST\u003c/em\u003e, and the \u003cem\u003eCp1-SST\u003c/em\u003e promoter harbors multiple MYB-binding sites [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. CiMYB17 binds to the consensus DNA-motif DTTHGGT and activate 1-SST transcription in Cichorium intybus [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. TaMYB13 binds to a (A/G/T)TT(A/T/C)GGT core sequence in the promoters of wheat Ta1-SST and markedly enhance the expression of 1-SST and 6-SFT promoter-driven reporter genes in wheat [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The interaction between CpMYB14 and p\u003cem\u003eCp1-SST\u003c/em\u003e was verified in vitro and in vivo through Y1H and CHIP experiments, respectively. Unlike other MYB transcription factors that positively regulate the expression of 1-SST, gene silencing and overexpression analysis demonstrated that CpMYB14 negatively regulates the expression of \u003cem\u003eCp1-SST\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAfter silencing and overexpressing the \u003cem\u003eCpMYB14\u003c/em\u003e gene, the expression of the key enzyme gene \u003cem\u003eCp1-SST\u003c/em\u003e related to ITFs synthesis in \u003cem\u003eC. polosula\u003c/em\u003e was negatively correlated with the expression of \u003cem\u003eCpMYB14\u003c/em\u003e, and also negatively correlated with the expression of the fructan hydrolysis-related gene \u003cem\u003eCpFEHs\u003c/em\u003e and the sucrose hydrolysis-related genes sucrose synthase and acid invertase. These results indicated that the \u003cem\u003eCp1-SST\u003c/em\u003e gene expression was negatively correlated with the expression of \u003cem\u003eCpFEHs\u003c/em\u003e, \u003cem\u003eCpSuSy\u003c/em\u003e and \u003cem\u003eCpNI\u003c/em\u003e, which is consistent with the previous research results [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, overexpression of \u003cem\u003eCpMYB14\u003c/em\u003e in \u003cem\u003eC. pilosula\u003c/em\u003e significantly downregulated the \u003cem\u003eCp1-SST\u003c/em\u003e expression, yet no ITFs were detected via HPGPC analysis. This is likely attributable to the chloroplast localization of the Cp1-SST protein, coupled with the extremely low chloroplast content in callus, which together impede fructan accumulation[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nonetheless, the diminished Cp1-SST activity still impaired the accumulation of other polysaccharides in \u003cem\u003eC. pilosula\u003c/em\u003e. Conversely, silencing of \u003cem\u003eCpMYB14\u003c/em\u003e markedly increased the \u003cem\u003eCp1-SST\u003c/em\u003e expression and ITFs content, consistent with prior observations that Cp1-SST catalyze the biosynthesis of fructooligosaccharides with different degrees of polymerization[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the silenced lines displayed significantly enhanced root length and plant height, a phenotype opposite to the stunted growth previously reported in tobacco overexpression of \u003cem\u003eCp1-SST\u003c/em\u003e. This phenotypic divergence may arise from the high \u003cem\u003eCp1-SST\u003c/em\u003e expression in silenced lines, which could deplete its substrate, sucrose, while concurrently reducing the activity of sucrose-hydrolyzing enzymes such as NI and SuSy. Consequently, the typical pathway for carbon skeleton provision via sucrose hydrolysis may be disrupted. Given the complexity of plant carbon metabolism, alternative compensatory pathways might be activated [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additionally, some MYB transcription factors are recognized as growth repressors in plants. The binding of MYB73/77 to UVR8 inhibits shoot elongation and lateral root growth by reducing expression of IAA19 in Arabidopsis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Plants overexpression of \u003cem\u003eOsMYB91\u003c/em\u003e showed reduced plant growth [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Overexpression of \u003cem\u003eStMYB66\u003c/em\u003e resulted in shorter plant height[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, it can be inferred that the CpMYB14 transcription factor may be a core growth inhibitory factor. Its down-regulation directly relieves the inhibitory effect on plant growth-promoting genes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the \u003cem\u003eCp1-SST\u003c/em\u003e promoter contained three MYB recognition sites and negatively regulated downstream gene expression under cold stress. \u003cem\u003eCpMYB14\u003c/em\u003e could respond to cold, temperature difference and iron overload stress. Express patterns indicated that CpMYB14 was located to the nucleus and shared the closest evolutionary relationships with CiMYB14. Functionally analysis revealed that CpMYB14 interacted with the \u003cem\u003eCp1-SST\u003c/em\u003e promoter, negatively regulating the \u003cem\u003eCp1-SST\u003c/em\u003e gene expression while positively regulating the genes related to fructan hydrolysis including \u003cem\u003eFEHs\u003c/em\u003e and \u003cem\u003eSuSy\u003c/em\u003e, thereby negatively regulating ITFs accumulation. Meanwhile, CpMYB14 expression could affect the plant height and root length. These findings provide a foundation for elucidating the regulatory mechanism underlying CR quality formation mediated by ITFs, with potential economic value.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e This work was supported by National Natural Science Foundation of China [grant number 82204569], Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources [grant number 2060302], the Natural Science Foundation of Shanxi Province [grant number 202103021224225], Applied Basic Research Project of Shanxi Province [grant number 202203021221180], Key R\u0026amp;D Projects in Forestry and Grassland of Shanxi Province [grant number JLYF-2026-40], Shanxi Medical University Doctoral Fund project [grant number No.XD2033].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions:\u003c/strong\u003eJ.J desiganed, investigated, revised, wrote and revised the manuscript. J.G. and J.L. designed, instructed, investigated, and revised the manuscript. L.Z. and X.G. wrote the manuscript. L.Z., X.G., T.Z., Z.L.,Y.W.,X.L.and J.X. investigated and validated the experiment .J.J. and T.Z. prepared figures and tables. J.X., M.S. and J.J founded the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZou YF, Zhang YY, Paulsen BS, Fu YP, Huang C, Feng B, Li XL, Chen XF, Ji RY, Song X, He CL, Yin LZ, Ye G, Liang XX, Lv C, Yin ZQ. 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Int J Biol Macromol. 2025; 288: \u0026nbsp;138588.\u003c/li\u003e\n \u003cli\u003eTouqeer A, Yuanbo H, Li M, Wu S. Regulatory roles of MYB transcription factors in root barrier under abiotic stress. Plants. 2026; 15(2): 275.\u003c/li\u003e\n \u003cli\u003eDubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in \u003cem\u003eArabidopsis\u003c/em\u003e. Trends Plant Sci. 2010; 15(10): 573-81.\u003c/li\u003e\n \u003cli\u003eFu S, Liu F, Li Y, Lu C, Liu T, Tian S, Jin W, Guo P, Chen L, Geng X. SlMYB2: an R2R3-MYB transcription factor enhancing drought, salt, and cadmium tolerance in \u003cem\u003eSolanum lycopersicum\u003c/em\u003e. Plant Cell Rep. 2026; 45: 24.\u003c/li\u003e\n \u003cli\u003eLan Y, Gong F, Dong R. A R2R3-MYB transcription factor \u003cem\u003eSmMYB88\u003c/em\u003e of \u003cem\u003eSolanum melongena\u003c/em\u003e regulates cold tolerance by activating antioxidant system. 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Reduction of the canonical function of a glycolytic enzyme enolase triggers immune responses that further affect metabolism and growth in\u003cem\u003e\u0026nbsp;Arabidopsis\u003c/em\u003e. Plant Cell. 2021; 34(5): 1745-1767.\u003c/li\u003e\n \u003cli\u003eDong WK, Zhang YJ, Ma HL. Research advances in the role of central carbon metabolic pathways in plant growth, development and stress responses. Pratacultural Sci. 2025; 43: 1-13.\u003c/li\u003e\n \u003cli\u003eXu Z, Zhu ZQ. Spotlight UV-B response: when UVR8 meets MYBs. Trends Plant Sci. 2020; 25(6): 515-517.\u003c/li\u003e\n \u003cli\u003eZhu N, Cheng S, Liu X. The R2R3-type MYB gene \u003cem\u003eOsMYB91\u003c/em\u003e has a function in coordinating plant growth and salt stress tolerance in rice. Plant Sci. 2025; 236: 146-156.\u003c/li\u003e\n \u003cli\u003eYu T, Wang D, Fu X, Liu Z. StMYB66, a R2R3-myb transcription factor, regulates plant growth and anthocyanin accumulation in potato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L.). J Am Soc Hortic Sci. 2025; 150(3): \u0026nbsp;147-158.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the supplementary files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Codonopsis pilosula, CpMYB14, Cp1-SST, inulin-type fructan biosynthesis, transcription regulation","lastPublishedDoi":"10.21203/rs.3.rs-9035409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9035409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCodonopsis polysaccharides (CPPs), the primary bioactive constituents of the edible Chinese medicinal plant \u003cem\u003eCodonopsis pilosula\u003c/em\u003e (Franch.) Nannf., are mainly composed of inulin-type fructans. The biosynthesis of these fructans is catalyzed by Cp1-SST (sucrose:sucrose 1-fructosyltransferase), a key enzyme whose gene expression can be regulated by MYB transcription factors. However, the regulatory mechanism of \u003cem\u003eCp1-SST\u003c/em\u003e remained unclear. Here, two MYB cis-elements were found in \u003cem\u003eCp1-SST\u003c/em\u003e promoter which exhibited transcriptional activation activity and responded to cold stress. subsequently a R2R3-MYB transcription factor, CpMYB14, was isolated and functionally characterized. CpMYB14 was most responsive to cold, temperature difference, iron overload stress and was localized in the nucleus. Silencing of \u003cem\u003eCpMYB14\u003c/em\u003e resulted in a significant upregulation of \u003cem\u003eCp1-SST\u003c/em\u003e expression and inulin-type fructan content but a decreased expression of the genes associated with CPPs hydrolysis metabolism, including \u003cem\u003eCp1-FEH\u003c/em\u003e, \u003cem\u003eCp6-FEH\u003c/em\u003e, \u003cem\u003eCpSuSy\u003c/em\u003e and \u003cem\u003eCpNI\u003c/em\u003e. Concurrently, the plant stem height and root length were markedly increased in \u003cem\u003eCpMYB14\u003c/em\u003e gene-silenced lines. Oppositely, overexpression of \u003cem\u003eCpMYB14\u003c/em\u003e significantly decreased the \u003cem\u003eCp1-SST\u003c/em\u003e expression in \u003cem\u003eC. pilosula\u003c/em\u003e but with significantly increased \u003cem\u003eCp1-FEH\u003c/em\u003e, \u003cem\u003eCp6-FEH\u003c/em\u003e and \u003cem\u003eCpSuSy\u003c/em\u003e expression. Yeast one-hybrid and chromatin immunoprecipitation assays confirmed that CpMYB14 directly binds to the promoter of \u003cem\u003eCp1-SST\u003c/em\u003e. Therefore, CpMYB14 is a negative regulator of \u003cem\u003eCp1-SST\u003c/em\u003e and inulin-type fructan biosynthesis. These findings provide a theoretical foundation for the molecular breeding aimed at developing high-quality varieties of \u003cem\u003eC. pilosula\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"CpMYB14, a novel R2R3-MYB transcription factor, interacts with Cp1-SST to negatively regulate inulin-type fructan accumulation and modulates plant growth in Codonopsis pilosula","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 06:31:21","doi":"10.21203/rs.3.rs-9035409/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-10T03:36:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T14:55:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-27T19:46:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94825214376347076497359719047028998091","date":"2026-03-25T07:31:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273845567595805343883902668037923342641","date":"2026-03-23T13:25:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206650952185426556204590226926434882406","date":"2026-03-23T12:25:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-15T23:55:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-09T09:41:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-09T07:37:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-09T07:37:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-03-05T03:28:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"04005643-9858-4cdb-8ea6-7e5857cd19ab","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T08:23:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 06:31:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9035409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9035409","identity":"rs-9035409","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}