CsMYB5 regulates anthocyanin accumulation and stress responses through responding to hormone signaling of Camellia sinensis

CsMYB5 regulates anthocyanin accumulation and stress responses through responding to hormone signaling of Camellia sinensis | 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 CsMYB5 regulates anthocyanin accumulation and stress responses through responding to hormone signaling of Camellia sinensis Jinxian Liu, Yukun Peng, Jialing Zhu, Zhichao Fang, Li Lu, Xianyu Fu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9168618/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Anthocyanins are major secondary metabolites that determine tea quality and contribute to plant stress adaptation. However, the transcriptional mechanisms coordinating their biosynthesis in response to environmental cues remain largely unclear. Result In this study, we characterized the R2R3-MYB transcription factor CsMYB5 from tea plant ( Camellia sinensis ) and investigated its role in hormone-responsive secondary metabolism regulation. Expression analyses revealed that CsMYB5 is preferentially expressed in anthocyanin-rich tea cultivars and leaf tissues and is strongly induced by SA, MeJA and multiple abiotic stresses. Genome-wide DNA affinity purification sequencing revealed that CsMYB5 binding sites are enriched in promoter regions of genes associated with transcriptional regulation and secondary metabolism, including flavonoid and caffeine biosynthetic pathways. Notably, CsMYB5 directly targets CsTCP15 , a TCP transcription factor potential regulation of anthocyanin synthesis and accumulation, suggesting the existence of a CsMYB5–CsTCP15 regulatory module.Heterologous overexpression of CsMYB5 in Arabidopsis thaliana resulted in enhanced vegetative growth and pronounced anthocyanin accumulation. Conclusions Based on these findings, we propose that CsMYB5 acts as a central transcriptional hub linking hormone signaling with coordinated regulation of anthocyanin accumulation biosynthesis. This study provides new insights into the transcriptional integration of flavonoid metabolism and offers potential targets and research direction for improving tea quality and stress resilience through molecular breeding. Camellia sinensis CsMYB5 MYB transcription factor transcriptional regulation hormone response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background During growth and development, plants are frequently exposed to adverse environmental conditions, such as drought, low temperature, high salinity, and pathogen infection[1]. To cope with these complex and fluctuating environments, plants have evolved sophisticated gene regulatory networks to finely control the biosynthesis of secondary metabolites[2]. Anthocyanins and caffeine are two core secondary metabolites in tea plants ( Camellia sinensis )[3]. Anthocyanins contribute to leaf coloration and antioxidant capacity[4], whereas caffeine influences tea flavor and physiological activity; Anthocyanins are also involved in stress responses in tea plants[5]. In plants, anthocyanins enhance tolerance to drought and salt stress through multiple mechanisms, including antioxidative defense, osmotic adjustment, photoprotection, and signal regulation[6]Moreover, environmental stresses such as drought, low temperature, and pathogen infection can significantly affect the biosynthesis and accumulation of anthocyanins and caffeine[7]. However, the molecular mechanisms underlying the regulation of their biosynthetic pathways under stress conditions remain largely unclear[8].In recent years, the completion of genome sequencing for multiple tea plant varieties, together with the extensive application of transcriptomic technologies[9], has provided valuable resources for elucidating the molecular mechanisms governing anthocyanin biosynthesis and for improving our understanding of the regulatory networks controlling secondary metabolism in tea plants. Anthocyanins generally accumulate at very low levels in most tea cultivars, typically below 0.1 mg/g. However, anthocyanin content can be markedly increased under specific conditions, such as in purple-leaf tea varieties or in response to environmental stresses[10], reaching moderate accumulation levels[11].For example, in the tea cultivar ‘Meizhan’, which occasionally exhibits purple pigmentation in young shoots and leaves, anthocyanin content can reach 0.1–0.3 mg/g. Previous studies have also demonstrated that the stability of purple pigmentation and anthocyanin accumulation in tea plants is regulated at the transcriptional level[12], and that this process is closely associated with leaf color transition[13].Transcription factors play central roles in signal transduction and gene regulatory networks by recognizing and binding to specific cis-regulatory elements in the promoter regions of target genes, thereby activating or repressing downstream gene transcription[14]. Among them, MYB transcription factors are particularly important regulators[15], as they integrate hormonal signaling pathways to coordinate plant responses to internal and external environmental cues, ultimately influencing diverse physiological traits[16]. The R2R3-MYB subfamily plays a central role in regulating plant secondary metabolism, growth and development, as well as responses to biotic and abiotic stresses[17].Previous studies have shown that overexpression of A.thaliana AtMYB12 significantly activates the expression of flavonoid biosynthetic genes, promotes flavonoid accumulation, and consequently enhances drought and salt tolerance in transgenic plants[18].In Brassica rapa , BcMYB111 positively regulates flavonoid biosynthesis by directly binding to the promoters of BcF3H and BcFLS1 [19].Over-expression of MdMYB48 in apple and P. notoginseng enhanced the elongation of primary root and the formation of lateral roots normal and drought stress conditions, exhibiting better drought resistance[20]. Especially MYB transcription factors play a critical role in activating the expression of anthocyanin biosynthetic genes.The MYB-bHLH-WDR (MBW) ternary complex is a key regulatory component of anthocyanin biosynthesis[21].Meanwhile, plant hormones are also involved in anthocyanin biosynthesis. Exogenous application of MeJA promotes anthocyanin accumulation, while GA negatively regulates anthocyanin biosynthesis in Arabidopsis by promoting DELLA degradation, thereby releasing suppressors of anthocyanin biosynthesis. Previous studies have identified a total of 122 CsR2R3-MYB genes in the tea plant genome[22], some of which have been demonstrated to be involved in the regulation of catechin biosynthesis[23],phosphorylation modification[24],anthocyanin accumulation[25], low-temperature stress responses[26], and pathogen infection responses[27]. Although the functions of several CsMYB genes have been predicted and experimentally validated, the regulatory mechanisms of most R2R3-MYB genes in tea plants remain poorly understood, particularly with respect to their roles in responses to pathogen infection and environmental stresses.In our previous transcriptomic analysis of tea leaves, we identified a MYB family gene CsMYB5 that was significantly upregulated following infection by the tea gray blight pathogen. This gene contains a typical R2R3-MYB domain structure, and sequence analysis revealed a high degree of homology between CsMYB5 and A.thaliana AtMYB5 , suggesting a potential role for CsMYB5 in integrating jasmonic acid (JA) and abscisic acid (ABA) stress signaling pathways to regulate anthocyanin biosynthesis in tea plants. In this study, the CsMYB5 gene was cloned from tea plants, and its sequence characteristics, evolutionary relationships, and physicochemical properties were systematically analyzed. Quantitative real-time PCR (qRT-PCR) was employed to investigate the expression dynamics of CsMYB5 in different tissues and in response to SA, MeJA, and pathogen infection. Recombinant CsMYB5 protein was subsequently obtained through prokaryotic expression, and potential downstream target genes enabling a preliminary characterization of the molecular regulatory network mediated by CsMYB5 . Furthermore, an overexpression construct was generated and introduced into A.thaliana to evaluate phenotypic changes in transgenic plants and to assess their responses to abiotic stresses, including salinity, drought, and low temperature.The results of this study aim to elucidate the potential role of CsMYB5 in plant growth and stress responses, providing new insights into the functional mechanisms of MYB transcription factors in woody plants, and offering candidate gene resources for the genetic improvement of stress tolerance in tea plants. 2. Material and Method 2.1 Plant material and related treatment methods Tea plants ( Camellia sinensis ), including the cultivar ‘Shuixian’ and an additional 17 cultivars (Table S1 ), were obtained from the Tea Germplasm Resource Garden of Wuyi University (27.731°N, 117.999°E).Seeds of A. thaliana (Col-0)were provided by the Fujian Provincial Key Laboratory of Green Technology for Ecological Industry. Plants were treated by foliar spraying with phytohormones, including methyl jasmonate (MeJA) at a concentration of 100 µM and salicylic acid (SA) at 5 mM. To assess the response of target genes to pathogen infection, plants were inoculated using a needle-pricking method. Two punctures were made on each side of the main leaf vein, followed by inoculation with pathogen suspensions (OD₆₀₀ = 0.2) of CLBB1 and CYBB1. To evaluate the responses of transgenic A. thaliana plants to abiotic stresses, stress treatments were applied as described below. Salt stress was simulated using 100 mM NaCl, drought stress was induced with 200 mM mannitol, and cold stress was applied by incubating plants in a growth chamber at 10°C under light conditions. Samples were collected at multiple time points between 0 and 48 h after treatment. Total RNA was extracted and reverse-transcribed into cDNA for subsequent gene expression analysis. 2.2 Cloning and Protein Sequence Analysis of CsMYB5 The CsMYB5 gene was cloned based on sequence information obtained from the tea plant genome database (Table S2 ). The cloned CsMYB5 coding sequence was translated into an amino acid sequence, and the three-dimensional protein structure was predicted and visualized using AlphaFold 3.0[28]. Homologous protein sequences of CsMYB5 were identified using the BLAST program in the NCBI database( https://www.ncbi.nlm.nih.gov/,accessed 13 March 2023) [29], and multiple sequence alignment was performed using ESPript [30]. AtMYBs proteins were selected as references, and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 11.0 to analyze the evolutionary relationship of CsMYB5[31]. The resulting phylogenetic tree was visualized using the Interactive Tree Of Life (iTOL, https://itol.embl.de/,accessed 24 May 2024) online tool [32]. 2.3 Expression profiling of CsMYB5 To analyze the expression patterns of CsMYB5 in different organs and under exogenous stress conditions, eight-year-old tea plants were used. Root, stem, flower, mature leaf, and young leaf tissues, as well as mature leaves treated as described in Section 2.1 (50 g per sample), were collected for RNA extraction and subsequent cDNA synthesis. Gene-specific primers for quantitative real-time PCR were designed based on the CsMYB5 sequence (Table S3 ), and the tea plant 18S rRNA gene was used as an internal reference[33].Quantitative real-time PCR was performed using a Bio-Rad CFX Connect 96 system with SYBR Green chemistry. The PCR cycling conditions were as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 20 s, 55°C for 20 s, and 72°C for 30 s. Relative gene expression levels were calculated using the 2 ⁻ΔΔCt method. Three biological replicates were performed for each sample, and each biological replicate contained three technical replicates. 2.4 Induced expression of CsMYB5 protein and Western blot analysis The pGEX-4T vector was used for prokaryotic expression. Gene-specific primers were designed to amplify the open reading frame (ORF) of CsMYB5 (Table S3 ), and the amplified fragment was ligated into the pGEX-4T vector to generate the recombinant expression construct CsMYB5-pGEX-4T-1. The recombinant plasmid CsMYB5-pGEX-4T-1 was transformed into Escherichia coli BL21 (DE3) competent cells. Protein expression was induced with 0.2 mM IPTG at 16°C for 24 h, after which bacterial cells were harvested. Bacterial cells were lysed according to standard GST fusion protein purification protocols, and the supernatant was subjected to affinity chromatography to purify the fusion protein. Purified proteins were separated by SDS-PAGE, transferred onto membranes under ice-cooling conditions, blocked, and subsequently analyzed by Western blotting. After revision, this section meets Nature’s standards for rigor and clarity in recombinant protein expression and immunoblot analysis. 2.5 DAP-seq analysis of CsMYB5 DNA fragments bound by CsMYB5 protein in tea plants were sequenced using the Illumina NovaSeq platform to generate raw DAP-seq data[34]. Raw DAP-seq sequencing data were subjected to quality control and preprocessing using fastp (v0.23.2) [35]. The cleaned reads were aligned to reference genome using BWA-MEM (v0.7.17)[35]. Peak calling was performed using MACS2 (v2.2.7.1) to identify CsMYB5-binding sites[36]. Technical replicates were merged using HOMER (v4.11) to improve the reliability of the results[37]. Peak annotation was performed using ChIPseeker (v1.36.0) [38] assigning peaks to genomic features including promoters, exons, introns, downstream regions, distal intergenic regions, 5′ untranslated regions (5′ UTRs), and 3′ untranslated regions (3′ UTRs). Peaks enriched near transcription start sites (TSSs) were defined as candidate transcription factor binding sites. Genes associated with CsMYB5-binding peaks were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to elucidate their potential biological functions. 2.6 Phenotypic and gene expression analysis of CsMYB5-transgenic A. thaliana The coding region of CsMYB5 together with its native promoter was cloned into the overexpression vector pBWA(V)HS-35S using the In-Fusion cloning method, generating the eukaryotic expression construct pBWA(V)HS-35S-CsMYB5, which was subsequently introduced into Agrobacterium tumefaciens strain GV3101.After bolting, wild-type A.thaliana Col-0 plants were transformed using the floral dip method with Agrobacterium suspensions harboring the pBWA(V)HS-35S-CsMYB5 construct. Plants were kept in darkness for 24 h and then grown under long-day conditions (15 h light, 10,000–12,000 lx, 50–60% relative humidity, and 21–24°C) until silique maturation.Homozygous T3 CsMYB5-transgenic A. thaliana lines were grown on half-strength MS medium. At the 7–8 true-leaf stage, qRT-PCR was performed to assess CsMYB5 expression levels in different lines, and lines with elevated expression were selected for subsequent experiments. Phenotypic differences between transgenic and wild-type plants were then evaluated, and CsMYB5 expression patterns under various stress conditions were analyzed. The qRT-PCR reaction system and cycling conditions were the same as those described in Section 2.3 . 3. Results 3.1Phylogenetic relationship and structural characterization of CsMYB5 Phylogenetic analysis revealed that CsMYB5 (TEA027333) clustered closely with A.thaliana MYB5 (AtMYB5) and AtMYB111, forming a well-supported monophyletic clade (Fig. 1 ,a). All members within this clade were classified as typical R2R3-MYB transcription factors and were clearly separated from other MYB subgroups, indicating a conserved evolutionary origin and potential functional specialization. Previous studies have demonstrated that AtMYB5 and AtMYB111 play critical roles in regulating secondary metabolism, particularly flavonoid and anthocyanin biosynthesis. The close phylogenetic relationship between CsMYB5 and these functionally characterized MYB proteins suggests that CsMYB5 may perform analogous biological roles in tea plants, potentially participating in flavonoid-related metabolic regulation. NCBI BLAST analysis showed that CsMYB5 shares high sequence similarity with MYB5-, MYB308-, and MYB111-type proteins from multiple plant species. Multiple sequence alignment of the conserved R2R3-MYB domains further confirmed the high degree of structural conservation among these proteins (Fig. 1 ,b). Both R2 and R3 repeat regions contain several highly conserved α-helices, with key amino acid residues preserved across species, highlighting their essential roles in DNA binding and transcriptional regulation. In contrast, the C-terminal region of CsMYB5 exhibits greater sequence divergence, consistent with the known role of this region in transcriptional activation or regulatory specificity in MYB transcription factors.The predicted three-dimensional structure of CsMYB5, generated using AlphaFold, is shown in(Fig. 1 ,c). The structural model indicates that the R2 and R3 repeat regions adopt a stable helix–turn–helix conformation, forming the canonical MYB DNA-binding domain. This structural feature is highly consistent with previously reported R2R3-MYB transcription factors, further supporting the classification of CsMYB5 as a typical R2R3-MYB protein. 3.2 Expression patterns of CsMYB5 across tea cultivars, tissues and stress conditions To investigate the transcriptional regulation and expression dynamics of CsMYB5 ( TEA027333 ), we first analyzed the promoter regions of it and its homologous gene cluster ( TEA012130, TEA012145 , and TEA014311 ). Promoter sequence analysis identified a diverse array of cis-acting regulatory elements, including elements responsive to phytohormones (abscisic acid, salicylic acid, and methyl jasmonate), environmental stresses (anaerobic conditions and circadian rhythms), and light, as well as multiple MYB-binding sites (Fig. 2 ,a). Further counting analysis revealed that all four promoters were enriched in core regulatory elements, such as CAAT-boxes and TATA-boxes (Fig. 2 ,b). Notably, compared with other CsMYB transcription factors, the CsMYB5 promoter showed a pronounced enrichment of MeJA-responsive and stress-related cis-elements, including ARE, MYB, and MYC motifs, suggesting that CsMYB5 expression is co-regulated by endogenous phytohormone signaling and external environmental cues. Using the expression level of CsMYB5 in leaves of the ‘SX’ cultivar as a reference, we systematically examined its expression profiles across different 16 tea cultivars. CsMYB5 expression exhibited pronounced cultivar-specific variation among the 15 tea cultivars analyzed (Fig. 2 ,c). The highest expression levels were observed in the anthocyanin-rich cultivar ‘MZ’, followed by ‘FY6’ and ‘BYTZ’, whereas relatively low expression was detected in ‘RG’, ‘TGY’, and ‘HD’. This differential expression pattern suggests that CsMYB5 may be associated with cultivar-specific traits, particularly flavonoid and anthocyanin accumulation.Tissue-specific expression analysis revealed that CsMYB5 transcripts accumulated predominantly in old leaves, while expression in stems and flowers was barely detectable (Fig. 2 ,d). This expression pattern implies that CsMYB5 primarily functions in leaf-associated metabolic processes, such as the biosynthesis of secondary metabolites and responses to environmental stimuli. Under exogenous hormone treatments, CsMYB5 exhibited a rapid and robust transcriptional response to SA, reaching an initial expression peak at 3 h followed by a second peak at 24 h (Fig. 2 ,e). MeJA treatment also significantly induced CsMYB5 expression, with a maximum at 3 h and a subsequent gradual decline. These results indicate that CsMYB5 transcription is regulated by defense-related phytohormones, implicating this gene in hormone-mediated stress-response pathways.Upon pathogen inoculation, CsMYB5 displayed distinct expression dynamics depending on the infecting strain. Inoculation with CLBB1 strain resulted in a marked upregulation of CsMYB5 at 24 h, whereas infection with strain CCYB1 led to a delayed induction, with peak expression observed at 72 h (Fig. 2 ,f). This strain-dependent expression response suggests that CsMYB5 may participate in pathogen-specific defense mechanisms in tea plants.Collectively, CsMYB5 shows elevated expression in anthocyanin-rich tea cultivars and leaf tissues and is differentially induced in response to diverse environmental stress conditions. These transcriptional features suggest that CsMYB5 is closely associated with leaf secondary metabolism and hormone- or stress-related responses, thereby warranting further investigation into its protein accumulation and molecular regulatory mechanisms underlying tea plant growth and metabolism. 3.3 Western Blotting and DAP-seq of CsMYB5 protein After preliminary optimization experiments, the GST–CsMYB5 fusion protein was successfully expressed in vitro under induction conditions of 20°C with 0.2 mM IPTG for 20 h. The recombinant GST–CsMYB5 protein was subsequently purified and subjected to Western blot analysis (Fig. 3 ,a). A distinct immunoreactive band corresponding to the GST–CsMYB5 fusion protein was detected at approximately 60 kDa. Given that the predicted molecular weight of CsMYB5 is 34.55 kDa and that of the GST tag is 26 kDa, the observed band size is consistent with the expected molecular weight of the fusion protein. These results confirm the successful expression and purification of the GST–CsMYB5 protein and further validate the accuracy of the prior protein structure prediction.The purified CsMYB5 protein was subjected to DAP-seq analysis to systematically characterize its genome-wide DNA-binding landscape. Chromosomal distribution analysis of DAP-seq peaks revealed that CsMYB5 binding sites were widely distributed across all chromosomes (Fig. 3 ,b). Genomic annotation of CsMYB5 binding peaks indicated a clear positional preference, with 37.81% of peaks located within promoter regions and 61.09% within intronic regions, whereas only a minor proportion was associated with untranslated regions (UTRs) (Fig. 3 ,c). Notably, CsMYB5 binding peaks were significantly enriched in the vicinity of transcription start sites (TSS), with a pronounced accumulation in upstream promoter regions (Fig. 3 ,d), supporting the role of CsMYB5 as a transcriptional regulator. Gene Ontology (GO) enrichment analysis of promoter-associated CsMYB5 target genes revealed significant enrichment in biological processes related to RNA metabolism, DNA biosynthesis, transcriptional regulation, and secondary metabolism (Fig. 3 ,e), including RNA phosphodiester bond hydrolysis, nucleotidyltransferase activity, and UDP-glycosyltransferase activity. KEGG pathway analysis further demonstrated that CsMYB5 target genes were predominantly enriched in secondary metabolism-related pathways, including terpenoid backbone biosynthesis, tyrosine metabolism, caffeine metabolism, and multiple alkaloid biosynthesis pathways (Fig. 3 ,f). Network analysis revealed extensive interconnections among these pathways, suggesting that CsMYB5 may function as a central regulator coordinating tea-specific secondary metabolic networks (Fig. 3 ,g).Motif analysis of CsMYB5 binding peaks identified several significantly enriched conserved sequences, including a canonical MYB-binding motif (Fig. 3 h).Furthermore, a prominent CsMYB5 binding peak (peak_6276) was detected within the promoter region of CsTCP15 homology to AtTCP15 ,which has been reported to participate in transcriptional networks regulating plant development and secondary metabolism, indicating that CsTCP15 is a potential direct target of CsMYB5 (Fig. 3 ,i). Integrated DAP-seq and functional enrichment analyses revealed that the direct targets of CsMYB5 are significantly enriched in transcriptional regulation and secondary metabolism-related pathways. Notably, these targets form regulatory modules that modulate the expression of downstream genes involved in the biosynthesis of tea-specific secondary metabolites, including anthocyanins and caffeine. 3.4 Phenotypic and transcriptional effects of CsMYB5 overexpression To elucidate the biological function of CsMYB5 , an overexpression construct containing the native promoter region of CsMYB5 was generated and introduced into A. thaliana , resulting in stable transgenic lines. Gene expression analysis confirmed stable overexpression of CsMYB5 in multiple independent T3 lines, with the highest transcript abundance detected in line T3-20, followed by T3-40 and T3-45 (Fig. 4 ,a). Comparative analysis of phenotypic characteristics between CsMYB5-overexpressing A. thaliana and wild-type plants revealed that during early growth stages, there were no significant differences between wild-type (Col-0) plants and CsMYB5-overexpressing lines T3-20, T3-40, and T3-45 (Fig. 4 b). Two weeks after sowing, transgenic seedlings exhibited slightly larger rosette leaves and accelerated growth compared to wild-type plants, with all three lines (T3-20, T3-40, and T3-45) bolting (Fig. 4 c). By week three, transgenic lines showed increased vegetative biomass and larger leaves, with distinct anthocyanin accumulation in rosette leaves of T3-40 and T3-45 lines, presenting a reddish-purple phenotype (Fig. 4 d). At week five, all transgenic lines had produced siliques, while Col-0 plants were just beginning to bolt. Moreover, anthocyanin accumulation in A. thaliana leaves was more pronounced (Fig. 4 e). Throughout the entire growth period, CsMYB5- overexpressing plants exhibited earlier bolting and significantly increased inflorescence height compared to wild-type plants. The transition to reproductive growth occurred 14–21 days earlier in transgenic plants than in wild-type Col-0 (Supplementary Fig. 1). Additionally, transgenic plants produced 5–6 more siliques per stem and had stems 5–8 cm taller than wild-type Col-0. These results demonstrate that overexpression of CsMYB5 accelerates the transition to reproductive growth in A. thaliana and promotes anthocyanin accumulation in leaves. Consistent with the expression patterns observed in tea plants, CsMYB5-overexpressing A. thaliana lines exhibited pronounced transcriptional responses to exogenous hormone treatments. MeJA rapidly induced CsMYB5 expression, reaching a maximum at 6 h, whereas SA treatment triggered a characteristic biphasic response with prominent peaks at 3 h and 24 h (Fig. 4 ,f), indicating that CsMYB5 retains strong responsiveness to defense-related hormone signaling in a heterologous system.Under abiotic stress conditions, CsMYB5-overexpressing lines also displayed distinct and time-dependent transcriptional responses. Cold treatment induced CsMYB5 expression to peak levels at 48 h, while drought and NaCl treatments caused rapid and significant upregulation at early time points (6 h and 12 h, respectively) (Fig. 4 ,g).Taken together, overexpression of CsMYB5 not only markedly altered plant growth and development and enhanced anthocyanin accumulation in A. thaliana , but also conferred dynamic transcriptional responsiveness to hormonal and environmental cues. These findings suggest that CsMYB5 exhibits a degree of functional conservation between C.sinensis and A.thaliana and support its potential role as a key regulatory factor linking plant development, secondary metabolism, and stress-responsive signaling pathways, thereby providing a solid foundation for further dissection of its underlying molecular regulatory network. 4. Discussion In this study, we systematically characterized the R2R3-MYB transcription factor CsMYB5 and demonstrated its pivotal role in integrating hormone signaling, secondary metabolism, and stress responses in tea plant ( Camellia sinensis ). By combining expression profiling, DAP-seq analysis, and functional validation in A. thaliana , we propose a regulatory model in which CsMYB5 functions as a central transcriptional hub coordinating anthocyanin accumulation and caffeine biosynthesis in response to environmental cues (Fig. 5 ). Anthocyanins are key flavonoid compounds that contribute not only to plant pigmentation but also to antioxidant capacity and stress tolerance[39]. R2R3-MYB transcription factors act as core regulators of anthocyanin biosynthesis across diverse plant species. For instance, the A. thaliana homolog of CsMYB5 , AtMYB5 , collaborates with AtMYB2 to promote anthocyanin accumulation in plants[40]. Similarly, FaMYB5 in strawberry performs an analogous function, enhancing anthocyanin biosynthesis[41]. In this study, we found that CsMYB5 is highly expressed in anthocyanin-rich tea cultivars and leaf tissues, and its overexpression in A. thaliana significantly promotes anthocyanin accumulation, indicating that CsMYB5 in Camellia sinensis has a conserved function in regulating anthocyanin biosynthesis.These findings strongly support a conserved role of CsMYB5 in promoting flavonoid biosynthesis, particularly anthocyanin accumulation, in agreement with its close phylogenetic relationship to A. thaliana MYB5.Beyond flavonoid metabolism, DAP-seq and functional enrichment analyses uncovered an unexpected yet biologically meaningful association between CsMYB5 and caffeine biosynthesis pathways. CsMYB5 binding peaks were significantly enriched in promoter regions of genes involved in caffeine metabolism, including CsXMTs , which encode key N-methyltransferases catalyzing caffeine biosynthesi[42]. DAP-seq observation suggests that CsMYB5 may possess a multiple regulatory capacity, coordinating the biosynthesis of polyphenols and alkaloids—two hallmark metabolic features that collectively determine tea quality. Hormone signaling appears to act as a critical upstream regulator of CsMYBs activity[43]. The CsMYB5 promoter is enriched in SA and MeJA, and CsMYB5 transcription is rapidly induced by both hormones in Camellia sinensis and transgenic A. thaliana .Importantly, our DAP-seq analysis identified CsTCP15 as a potential direct downstream target of CsMYB5 protein. CsTCP15 shares high sequence homology with AtTCP15 , which has been implicated in the regulation of plant development[44] and secondary metabolism[45].The proposed CsMYB5 and CsTCP15 regulatory module therefore offers a mechanistic framework through which CsMYB5 may exert both direct and indirect control over secondary metabolism.Taken together, this study support a working model in which stress-induced SA and MeJA signaling activate CsMYB5 expression, which in turn regulates transcriptional networks associated with anthocyanin accumulation and caffeine biosynthesis. Future plans include further metabolomic and transcriptomic analyses to dissect the hierarchical structure of the CsMYB5-centered transcriptional regulatory network, and multi-dimensional experimental validation of the direct regulatory relationship between CsMYB5 and CsTCP15 in modulating secondary metabolism. These efforts will provide new research directions and molecular resources for understanding tea quality improvement and the genetic breeding of elite tea cultivars. 5. Discussion This study systematically characterized the R2R3-MYB transcription factor CsMYB5 in Camellia sinensis and verified its pivotal role as a central transcriptional hub integrating hormone signaling, secondary metabolism and stress responses. CsMYB5 shows a conserved function in promoting anthocyanin biosynthesis, with high expression in anthocyanin-rich tea tissues and enhanced anthocyanin accumulation in overexpressed Arabidopsis thaliana . Notably, it unexpectedly regulates caffeine biosynthesis by binding to promoters of key caffeine metabolic genes like CsXMTs , coordinating both polyphenol and alkaloid production that determines tea quality. Additionally, CsMYB5 transcription is rapidly induced by SA and MeJA, and it targets CsTCP15 to form a regulatory module for secondary metabolism. These findings elucidate a novel hormone-CsMYB5-mediated regulatory network, providing molecular insights for tea quality improvement and elite cultivar breeding. Abbreviations DAP-seq:DNA affinity purification sequencing. TSS:transcription start sites. UTRs:untranslated regions . KEGG:Kyoto Encyclopedia of Genes and Genomes.GO:Gene Ontology . ABA:abscisic acid.SA:Salicylic Acid. MeJA:Methyl Jasmonate Declarations Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials:Competing interests： The original sequence data of the tea plant genome in this project is derived from the Tea Plant Information Archive (TPIA) and is publicly accessible via https://tpia.teaplants.cn/. Authors' contributions: These Jinxian Liu and Yukun Peng contributed equally to this work.J.L. conceived the project, designed the experiments and drafted the initial manuscript. Y.P. and J.Z. performed the experiments, drafted the initial manuscript and prepared the figures. Z.F. assisted with figure visualization. L.L., X.F., F.L. and G.W. supervised the study, and provided experimental equipment and technical support. Z.L. refined the manuscript to enhance scientific rigor and handled correspondence and submission. All authors reviewed and approved the final manuscript. Funding: The research was jointly supported by the Natural Science Foundation of Fujian Province, China (2022J011200, 2025J011069, 2023J011049) Acknowledgements: We are grateful to the reviewers for their helpful comments on the original manuscript.We would like to thank the editors for their efficient work. 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Rong T, Chunchun Z, Wei G, Yuchen G, Fei X, Tao L, Yuanyuan J, Chenbin W, Wenda X, Wenqing W: Proteomic insights into protostane triterpene biosynthesis regulatory mechanism after MeJA treatment in Alisma orientale (Sam.) Juz. Biochim Biophys Acta Proteins Proteom 2021, 1869(8):140671. Han Z, Zhang C, Zhang H, Duan Y, Zou Z, Zhou L, Zhu X, Fang W, Ma Y: CsMYB Transcription Factors Participate in Jasmonic Acid Signal Transduction in Response to Cold Stress in Tea Plant ( Camellia sinensis ). Plants (Basel) 2022, 11(21). Callaway E: AI protein-prediction tool AlphaFold3 is now more open. Nature 2024, 635(8039):531-532. Sayers EW, Beck J, Brister JR, Bolton EE, Canese K, Comeau DC, Funk K, Ketter A, Kim S, Kimchi A et al : Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 2020, 48(D1):D9-D16. Robert X, Guillon C, Gouet P: FoldScript: a web server for the efficient analysis of AI-generated 3D protein models. Nucleic Acids Res 2025, 53(W1):W277-W282. Tamura K, Stecher G, Kumar S: MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 2021, 38(7):3022-3027. Letunic I, Bork P: Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 2024, 52(W1):W78-W82. Zhou ZW, Deng HL, Wu QY, Liu BB, Yue C, Deng TT, Lai ZX, Sun Y: Validation of reference genes for gene expression studies in post-harvest leaves of tea plant ( Camellia sinensis ). PeerJ 2019, 7:e6385. Chen S: Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. Imeta 2023, 2(2):e107. Zhang L, Liu C, Dong S: PipeMEM: A Framework to Speed Up BWA-MEM in Spark with Low Overhead. Genes (Basel) 2019, 10(11). Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W et al : Model-based analysis of ChIP-Seq (MACS). Genome Biol 2008, 9(9):R137. Roloff AM, Anderson GR, Martemyanov KA, Thayer SA: Homer 1a gates the induction mechanism for endocannabinoid-mediated synaptic plasticity. J Neurosci 2010, 30(8):3072-3081. Yu G, Wang LG, He QY: ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 2015, 31(14):2382-2383. Wang J, Zhang Y, Wang J, Khan A, Kang Z, Ma Y, Zhang J, Dang H, Li T, Hu X: SlGAD2 is the target of SlTHM27, positively regulates cold tolerance by mediating anthocyanin biosynthesis in tomato. Hortic Res 2024, 11(6):uhae096. Jun JH, Liu C, Xiao X, Dixon RA: The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula. Plant Cell 2015, 27(10):2860-2879. Jiang L, Yue M, Liu Y, Zhang N, Lin Y, Zhang Y, Wang Y, Li M, Luo Y, Zhang Y et al : A novel R2R3-MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (Fragaria x ananassa). Plant Biotechnol J 2023, 21(6):1140-1158. Leibrock NV, Santegoets J, Mooijman PJW, Yusuf F, Zuijdgeest XCL, Zutt EA, Jacobs JGM, Schaart JG: The biological feasibility and social context of gene-edited, caffeine-free coffee. Food Sci Biotechnol 2022, 31(6):635-655. Li P, Xia E, Fu J, Xu Y, Zhao X, Tong W, Tang Q, Tadege M, Fernie AR, Zhao J: Diverse roles of MYB transcription factors in regulating secondary metabolite biosynthesis, shoot development, and stress responses in tea plants ( Camellia sinensis ). Plant J 2022, 110(4):1144-1165. Steiner E, Yanai O, Efroni I, Ori N, Eshed Y, Weiss D: Class I TCPs modulate cytokinin-induced branching and meristematic activity in tomato. Plant Signal Behav 2012, 7(7):807-810. Viola IL, Camoirano A, Gonzalez DH: Redox-Dependent Modulation of Anthocyanin Biosynthesis by the TCP Transcription Factor TCP15 during Exposure to High Light Intensity Conditions in Arabidopsis. Plant Physiol 2016, 170(1):74-85. Additional Declarations No competing interests reported. Supplementary Files Supplementalfiles.xls SupplementaryFigure1.tif Originalfigureofwesternblot.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9168618","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619272371,"identity":"310acda1-4735-40f3-8e91-4a32381f8f10","order_by":0,"name":"Jinxian Liu","email":"","orcid":"","institution":"Wuyi University","correspondingAuthor":false,"prefix":"","firstName":"Jinxian","middleName":"","lastName":"Liu","suffix":""},{"id":619272372,"identity":"12b351d0-6f17-4c41-85ed-6ba9a465698b","order_by":1,"name":"Yukun Peng","email":"","orcid":"","institution":"Fujian Agriculture and Forestry 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10:55:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9168618/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9168618/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106516662,"identity":"61ee0179-c543-46c4-a03e-46f585a9e196","added_by":"auto","created_at":"2026-04-09 12:06:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":356167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis and protein structure of CsMYB5. \u003c/strong\u003e(a)Phylogenetic tree of MYB proteins from\u003cem\u003eCamellia sinensis\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003e(b) Multiple sequence alignment of R2R3-MYB domains from CsMYB5 and its homologs.(c) Alpha Fold-predicted 3D structure of CsMYB5 of the R2 and R3 DNA-binding domains.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/107a221303a165684ad22f66.png"},{"id":106724621,"identity":"17c6f359-9bd7-4eb4-856b-a8b58e799807","added_by":"auto","created_at":"2026-04-12 18:28:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression characteristics and promoter analysis of CsMYB5 in tea plants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Distribution of cis-acting regulatory elements in the promoters of \u003cem\u003eCsMYB5\u003c/em\u003e and its homologs.\u003c/p\u003e\n\u003cp\u003e(b) Comparison of cis-acting core regulatory and stress-responsive elements number across the four promoters of \u003cem\u003eCsMYB5 \u003c/em\u003eand its homologs.(c) Relative \u003cem\u003eCsMYB5 \u003c/em\u003eexpression in 15 tea cultivars.\u003c/p\u003e\n\u003cp\u003e(d) Tissue-specific CsMYB5 expression, with maximal transcript accumulation in old leaves.\u003c/p\u003e\n\u003cp\u003e(e) \u003cem\u003eCsMYB5 \u003c/em\u003eexpression dynamics under SA and MeJA treatments.(f) \u003cem\u003eCsMYB5\u003c/em\u003e expression profiles after inoculation with CLBB1 and CCYB1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/c068bad572f1c54087534a12.png"},{"id":106959622,"identity":"6f9a1e82-92a7-4fb4-97dc-6612382625c4","added_by":"auto","created_at":"2026-04-15 09:12:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":249235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot and genome-wide DNA-binding analysis of CsMYB5 protein.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Western blot analysis of the purified GST-CsMYB5 fusion protein.(b) Chromosomal distribution of CsMYB5 DAP-seq binding peaks across the tea plant genome.(c) The genomic distribution of CsMYB5 binding peaks.(d) Binding signal enrichment profile of CsMYB5 around transcription start sites (TSS).(e) Gene Ontology (GO) enrichment analysis of CsMYB5 target genes.(f) KEGG pathway classification of CsMYB5 target genes.(g) Network analysis of KEGG pathways regulated by CsMYB5.(h) Significantly enriched DNA-binding motifs identified from CsMYB5 DAP-seq peaks.(i) Visualization of CsMYB5 binding peak (peak_6276) in the promoter region of \u003cem\u003eCsTCP15\u003c/em\u003egene.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/c710b487c348b3efea8ab958.png"},{"id":106724823,"identity":"3a70f4fc-87b6-45ab-b7ce-13f8cad77dff","added_by":"auto","created_at":"2026-04-12 18:29:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and transcriptional analysis of CsMYB5-overexpressing\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Arabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Relative expression of CsMYB5 in independent T3 transgenic lines. (b) Growth phenotype of one-week-old seedlings, showing accelerated early growth in transgenic lines compared to wild-type (Col-0). (c) Three-week-old plants, displaying increased leaf size and anthocyanin accumulation in transgenic lines. (d) Five-week-old plants, showing intensified anthocyanin pigmentation in leaves of transgenic lines. (e) Mature plants, demonstrating earlier bolting and taller inflorescence stems in CsMYB5-overexpressing lines. (f) \u003cem\u003eCsMYB5\u003c/em\u003e expression profiles under MeJA and SA treatments.(g) \u003cem\u003eCsMYB5\u003c/em\u003e expression dynamics under cold, drought, and NaCl stresses.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/99fc9a59e2c13e4804ce53f4.png"},{"id":106516659,"identity":"0b111e85-51a5-4ac4-9f62-69352cabf623","added_by":"auto","created_at":"2026-04-09 12:06:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":148904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed regulatory model of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e CsMYB5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCamellia sinensis.\u003c/strong\u003e\u003c/em\u003eFigure illustrating how stress-induced hormonal signaling activates \u003cem\u003eCsMYB5\u003c/em\u003e expression, which in turn regulates the transcription of downstream genes such as \u003cem\u003eCsTCP15\u003c/em\u003e, thereby influencing the metabolic accumulation of anthocyanins and other secondary metabolites.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/c59b38b459036a12f093f48c.png"},{"id":106962879,"identity":"e255a587-0bbc-4841-931f-37bbe35b83b7","added_by":"auto","created_at":"2026-04-15 09:40:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2124558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/ec13c597-5c5d-4438-8bef-46fb51aae65f.pdf"},{"id":106725809,"identity":"9588fa41-bd1f-488b-96b2-2c05d0c4a7eb","added_by":"auto","created_at":"2026-04-12 18:33:57","extension":"xls","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":31232,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfiles.xls","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/31c633b861760589011fa1f9.xls"},{"id":106516654,"identity":"faba95d0-d712-4fc4-826a-bc1398ef3bf4","added_by":"auto","created_at":"2026-04-09 12:06:28","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23040266,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/b05e48804fa64ab5b1f9500a.tif"},{"id":106959695,"identity":"e7a13717-8a11-4856-be50-1d972e51de03","added_by":"auto","created_at":"2026-04-15 09:13:48","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":335247,"visible":true,"origin":"","legend":"","description":"","filename":"Originalfigureofwesternblot.doc","url":"https://assets-eu.researchsquare.com/files/rs-9168618/v1/cc37ba909488a64e531dda77.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"CsMYB5 regulates anthocyanin accumulation and stress responses through responding to hormone signaling of Camellia sinensis","fulltext":[{"header":"1. Background","content":"\u003cp\u003eDuring growth and development, plants are frequently exposed to adverse environmental conditions, such as drought, low temperature, high salinity, and pathogen infection[1]. To cope with these complex and fluctuating environments, plants have evolved sophisticated gene regulatory networks to finely control the biosynthesis of secondary metabolites[2]. Anthocyanins and caffeine are two core secondary metabolites in tea plants (\u003cem\u003eCamellia sinensis\u003c/em\u003e)[3]. Anthocyanins contribute to leaf coloration and antioxidant capacity[4], whereas caffeine influences tea flavor and physiological activity; Anthocyanins are also involved in stress responses in tea plants[5].\u003c/p\u003e \u003cp\u003eIn plants, anthocyanins enhance tolerance to drought and salt stress through multiple mechanisms, including antioxidative defense, osmotic adjustment, photoprotection, and signal regulation[6]Moreover, environmental stresses such as drought, low temperature, and pathogen infection can significantly affect the biosynthesis and accumulation of anthocyanins and caffeine[7]. However, the molecular mechanisms underlying the regulation of their biosynthetic pathways under stress conditions remain largely unclear[8].In recent years, the completion of genome sequencing for multiple tea plant varieties, together with the extensive application of transcriptomic technologies[9], has provided valuable resources for elucidating the molecular mechanisms governing anthocyanin biosynthesis and for improving our understanding of the regulatory networks controlling secondary metabolism in tea plants.\u003c/p\u003e \u003cp\u003eAnthocyanins generally accumulate at very low levels in most tea cultivars, typically below 0.1 mg/g. However, anthocyanin content can be markedly increased under specific conditions, such as in purple-leaf tea varieties or in response to environmental stresses[10], reaching moderate accumulation levels[11].For example, in the tea cultivar \u0026lsquo;Meizhan\u0026rsquo;, which occasionally exhibits purple pigmentation in young shoots and leaves, anthocyanin content can reach 0.1\u0026ndash;0.3 mg/g. Previous studies have also demonstrated that the stability of purple pigmentation and anthocyanin accumulation in tea plants is regulated at the transcriptional level[12], and that this process is closely associated with leaf color transition[13].Transcription factors play central roles in signal transduction and gene regulatory networks by recognizing and binding to specific cis-regulatory elements in the promoter regions of target genes, thereby activating or repressing downstream gene transcription[14]. Among them, MYB transcription factors are particularly important regulators[15], as they integrate hormonal signaling pathways to coordinate plant responses to internal and external environmental cues, ultimately influencing diverse physiological traits[16].\u003c/p\u003e \u003cp\u003eThe R2R3-MYB subfamily plays a central role in regulating plant secondary metabolism, growth and development, as well as responses to biotic and abiotic stresses[17].Previous studies have shown that overexpression of \u003cem\u003eA.thaliana AtMYB12\u003c/em\u003e significantly activates the expression of flavonoid biosynthetic genes, promotes flavonoid accumulation, and consequently enhances drought and salt tolerance in transgenic plants[18].In \u003cem\u003eBrassica rapa\u003c/em\u003e, \u003cem\u003eBcMYB111\u003c/em\u003e positively regulates flavonoid biosynthesis by directly binding to the promoters of \u003cem\u003eBcF3H\u003c/em\u003e and \u003cem\u003eBcFLS1\u003c/em\u003e[19].Over-expression of \u003cem\u003eMdMYB48\u003c/em\u003e in apple and P. notoginseng enhanced the elongation of primary root and the formation of lateral roots normal and drought stress conditions, exhibiting better drought resistance[20]. Especially MYB transcription factors play a critical role in activating the expression of anthocyanin biosynthetic genes.The MYB-bHLH-WDR (MBW) ternary complex is a key regulatory component of anthocyanin biosynthesis[21].Meanwhile, plant hormones are also involved in anthocyanin biosynthesis. Exogenous application of MeJA promotes anthocyanin accumulation, while GA negatively regulates anthocyanin biosynthesis in Arabidopsis by promoting DELLA degradation, thereby releasing suppressors of anthocyanin biosynthesis.\u003c/p\u003e \u003cp\u003ePrevious studies have identified a total of 122 CsR2R3-MYB genes in the tea plant genome[22], some of which have been demonstrated to be involved in the regulation of catechin biosynthesis[23],phosphorylation modification[24],anthocyanin accumulation[25], low-temperature stress responses[26], and pathogen infection responses[27]. Although the functions of several CsMYB genes have been predicted and experimentally validated, the regulatory mechanisms of most R2R3-MYB genes in tea plants remain poorly understood, particularly with respect to their roles in responses to pathogen infection and environmental stresses.In our previous transcriptomic analysis of tea leaves, we identified a MYB family gene \u003cem\u003eCsMYB5\u003c/em\u003e that was significantly upregulated following infection by the tea gray blight pathogen. This gene contains a typical R2R3-MYB domain structure, and sequence analysis revealed a high degree of homology between \u003cem\u003eCsMYB5\u003c/em\u003e and \u003cem\u003eA.thaliana AtMYB5\u003c/em\u003e, suggesting a potential role for CsMYB5 in integrating jasmonic acid (JA) and abscisic acid (ABA) stress signaling pathways to regulate anthocyanin biosynthesis in tea plants.\u003c/p\u003e \u003cp\u003eIn this study, the \u003cem\u003eCsMYB5\u003c/em\u003e gene was cloned from tea plants, and its sequence characteristics, evolutionary relationships, and physicochemical properties were systematically analyzed. Quantitative real-time PCR (qRT-PCR) was employed to investigate the expression dynamics of \u003cem\u003eCsMYB5\u003c/em\u003e in different tissues and in response to SA, MeJA, and pathogen infection. Recombinant CsMYB5 protein was subsequently obtained through prokaryotic expression, and potential downstream target genes\u003c/p\u003e \u003cp\u003eenabling a preliminary characterization of the molecular regulatory network mediated by \u003cem\u003eCsMYB5\u003c/em\u003e. Furthermore, an overexpression construct was generated and introduced into \u003cem\u003eA.thaliana\u003c/em\u003e to evaluate phenotypic changes in transgenic plants and to assess their responses to abiotic stresses, including salinity, drought, and low temperature.The results of this study aim to elucidate the potential role of \u003cem\u003eCsMYB5\u003c/em\u003e in plant growth and stress responses, providing new insights into the functional mechanisms of MYB transcription factors in woody plants, and offering candidate gene resources for the genetic improvement of stress tolerance in tea plants.\u003c/p\u003e"},{"header":"2. Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material and related treatment methods\u003c/h2\u003e \u003cp\u003eTea plants (\u003cem\u003eCamellia sinensis\u003c/em\u003e), including the cultivar \u0026lsquo;Shuixian\u0026rsquo; and an additional 17 cultivars (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), were obtained from the Tea Germplasm Resource Garden of Wuyi University (27.731\u0026deg;N, 117.999\u0026deg;E).Seeds of \u003cem\u003eA. thaliana\u003c/em\u003e (Col-0)were provided by the Fujian Provincial Key Laboratory of Green Technology for Ecological Industry. Plants were treated by foliar spraying with phytohormones, including methyl jasmonate (MeJA) at a concentration of 100 \u0026micro;M and salicylic acid (SA) at 5 mM. To assess the response of target genes to pathogen infection, plants were inoculated using a needle-pricking method. Two punctures were made on each side of the main leaf vein, followed by inoculation with pathogen suspensions (OD₆₀₀ = 0.2) of CLBB1 and CYBB1. To evaluate the responses of transgenic \u003cem\u003eA. thaliana\u003c/em\u003e plants to abiotic stresses, stress treatments were applied as described below. Salt stress was simulated using 100 mM NaCl, drought stress was induced with 200 mM mannitol, and cold stress was applied by incubating plants in a growth chamber at 10\u0026deg;C under light conditions. Samples were collected at multiple time points between 0 and 48 h after treatment. Total RNA was extracted and reverse-transcribed into cDNA for subsequent gene expression analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cloning and Protein Sequence Analysis of CsMYB5\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eCsMYB5\u003c/em\u003e gene was cloned based on sequence information obtained from the tea plant genome database (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The cloned \u003cem\u003eCsMYB5\u003c/em\u003e coding sequence was translated into an amino acid sequence, and the three-dimensional protein structure was predicted and visualized using AlphaFold 3.0[28]. Homologous protein sequences of CsMYB5 were identified using the BLAST program in the NCBI database(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/,accessed\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/,accessed\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e 13 March 2023) [29], and multiple sequence alignment was performed using ESPript [30]. AtMYBs proteins were selected as references, and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 11.0 to analyze the evolutionary relationship of CsMYB5[31]. The resulting phylogenetic tree was visualized using the Interactive Tree Of Life (iTOL, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/,accessed\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/,accessed\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e 24 May 2024) online tool [32].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Expression profiling of CsMYB5\u003c/h2\u003e \u003cp\u003eTo analyze the expression patterns of \u003cem\u003eCsMYB5\u003c/em\u003e in different organs and under exogenous stress conditions, eight-year-old tea plants were used. Root, stem, flower, mature leaf, and young leaf tissues, as well as mature leaves treated as described in Section \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e2.1\u003c/span\u003e (50 g per sample), were collected for RNA extraction and subsequent cDNA synthesis. Gene-specific primers for quantitative real-time PCR were designed based on the \u003cem\u003eCsMYB5\u003c/em\u003e sequence (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), and the tea plant 18S rRNA gene was used as an internal reference[33].Quantitative real-time PCR was performed using a Bio-Rad CFX Connect 96 system with SYBR Green chemistry. The PCR cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 20 s, 55\u0026deg;C for 20 s, and 72\u0026deg;C for 30 s. Relative gene expression levels were calculated using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method. Three biological replicates were performed for each sample, and each biological replicate contained three technical replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Induced expression of CsMYB5 protein and Western blot analysis\u003c/h2\u003e \u003cp\u003eThe pGEX-4T vector was used for prokaryotic expression. Gene-specific primers were designed to amplify the open reading frame (ORF) of CsMYB5 (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), and the amplified fragment was ligated into the pGEX-4T vector to generate the recombinant expression construct CsMYB5-pGEX-4T-1. The recombinant plasmid CsMYB5-pGEX-4T-1 was transformed into Escherichia coli BL21 (DE3) competent cells. Protein expression was induced with 0.2 mM IPTG at 16\u0026deg;C for 24 h, after which bacterial cells were harvested. Bacterial cells were lysed according to standard GST fusion protein purification protocols, and the supernatant was subjected to affinity chromatography to purify the fusion protein. Purified proteins were separated by SDS-PAGE, transferred onto membranes under ice-cooling conditions, blocked, and subsequently analyzed by Western blotting. After revision, this section meets Nature\u0026rsquo;s standards for rigor and clarity in recombinant protein expression and immunoblot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 DAP-seq analysis of CsMYB5\u003c/h2\u003e \u003cp\u003eDNA fragments bound by CsMYB5 protein in tea plants were sequenced using the Illumina NovaSeq platform to generate raw DAP-seq data[34]. Raw DAP-seq sequencing data were subjected to quality control and preprocessing using fastp (v0.23.2) [35]. The cleaned reads were aligned to reference genome using BWA-MEM (v0.7.17)[35]. Peak calling was performed using MACS2 (v2.2.7.1) to identify CsMYB5-binding sites[36]. Technical replicates were merged using HOMER (v4.11) to improve the reliability of the results[37]. Peak annotation was performed using ChIPseeker (v1.36.0) [38] assigning peaks to genomic features including promoters, exons, introns, downstream regions, distal intergenic regions, 5\u0026prime; untranslated regions (5\u0026prime; UTRs), and 3\u0026prime; untranslated regions (3\u0026prime; UTRs). Peaks enriched near transcription start sites (TSSs) were defined as candidate transcription factor binding sites. Genes associated with CsMYB5-binding peaks were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to elucidate their potential biological functions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Phenotypic and gene expression analysis of CsMYB5-transgenic \u003cem\u003eA. thaliana\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe coding region of CsMYB5 together with its native promoter was cloned into the overexpression vector pBWA(V)HS-35S using the In-Fusion cloning method, generating the eukaryotic expression construct pBWA(V)HS-35S-CsMYB5, which was subsequently introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101.After bolting, wild-type \u003cem\u003eA.thaliana\u003c/em\u003e Col-0 plants were transformed using the floral dip method with Agrobacterium suspensions harboring the pBWA(V)HS-35S-CsMYB5 construct. Plants were kept in darkness for 24 h and then grown under long-day conditions (15 h light, 10,000\u0026ndash;12,000 lx, 50\u0026ndash;60% relative humidity, and 21\u0026ndash;24\u0026deg;C) until silique maturation.Homozygous T3 CsMYB5-transgenic \u003cem\u003eA. thaliana\u003c/em\u003e lines were grown on half-strength MS medium. At the 7\u0026ndash;8 true-leaf stage, qRT-PCR was performed to assess CsMYB5 expression levels in different lines, and lines with elevated expression were selected for subsequent experiments. Phenotypic differences between transgenic and wild-type plants were then evaluated, and \u003cem\u003eCsMYB5\u003c/em\u003e expression patterns under various stress conditions were analyzed. The qRT-PCR reaction system and cycling conditions were the same as those described in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1Phylogenetic relationship and structural characterization of CsMYB5\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis revealed that CsMYB5 (TEA027333) clustered closely with \u003cem\u003eA.thaliana\u003c/em\u003e MYB5 (AtMYB5) and AtMYB111, forming a well-supported monophyletic clade (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e,a). All members within this clade were classified as typical R2R3-MYB transcription factors and were clearly separated from other MYB subgroups, indicating a conserved evolutionary origin and potential functional specialization. Previous studies have demonstrated that AtMYB5 and AtMYB111 play critical roles in regulating secondary metabolism, particularly flavonoid and anthocyanin biosynthesis. The close phylogenetic relationship between CsMYB5 and these functionally characterized MYB proteins suggests that CsMYB5 may perform analogous biological roles in tea plants, potentially participating in flavonoid-related metabolic regulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNCBI BLAST analysis showed that CsMYB5 shares high sequence similarity with MYB5-, MYB308-, and MYB111-type proteins from multiple plant species. Multiple sequence alignment of the conserved R2R3-MYB domains further confirmed the high degree of structural conservation among these proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e,b). Both R2 and R3 repeat regions contain several highly conserved α-helices, with key amino acid residues preserved across species, highlighting their essential roles in DNA binding and transcriptional regulation. In contrast, the C-terminal region of CsMYB5 exhibits greater sequence divergence, consistent with the known role of this region in transcriptional activation or regulatory specificity in MYB transcription factors.The predicted three-dimensional structure of CsMYB5, generated using AlphaFold, is shown in(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e,c). The structural model indicates that the R2 and R3 repeat regions adopt a stable helix\u0026ndash;turn\u0026ndash;helix conformation, forming the canonical MYB DNA-binding domain. This structural feature is highly consistent with previously reported R2R3-MYB transcription factors, further supporting the classification of CsMYB5 as a typical R2R3-MYB protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Expression patterns of CsMYB5 across tea cultivars, tissues and stress conditions\u003c/h2\u003e \u003cp\u003eTo investigate the transcriptional regulation and expression dynamics of \u003cem\u003eCsMYB5\u003c/em\u003e (\u003cem\u003eTEA027333\u003c/em\u003e), we first analyzed the promoter regions of it and its homologous gene cluster (\u003cem\u003eTEA012130, TEA012145\u003c/em\u003e, and \u003cem\u003eTEA014311\u003c/em\u003e). Promoter sequence analysis identified a diverse array of cis-acting regulatory elements, including elements responsive to phytohormones (abscisic acid, salicylic acid, and methyl jasmonate), environmental stresses (anaerobic conditions and circadian rhythms), and light, as well as multiple MYB-binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,a). Further counting analysis revealed that all four promoters were enriched in core regulatory elements, such as CAAT-boxes and TATA-boxes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,b). Notably, compared with other CsMYB transcription factors, the \u003cem\u003eCsMYB5\u003c/em\u003e promoter showed a pronounced enrichment of MeJA-responsive and stress-related cis-elements, including ARE, MYB, and MYC motifs, suggesting that \u003cem\u003eCsMYB5\u003c/em\u003e expression is co-regulated by endogenous phytohormone signaling and external environmental cues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing the expression level of \u003cem\u003eCsMYB5\u003c/em\u003e in leaves of the \u0026lsquo;SX\u0026rsquo; cultivar as a reference, we systematically examined its expression profiles across different 16 tea cultivars. \u003cem\u003eCsMYB5\u003c/em\u003e expression exhibited pronounced cultivar-specific variation among the 15 tea cultivars analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,c). The highest expression levels were observed in the anthocyanin-rich cultivar \u0026lsquo;MZ\u0026rsquo;, followed by \u0026lsquo;FY6\u0026rsquo; and \u0026lsquo;BYTZ\u0026rsquo;, whereas relatively low expression was detected in \u0026lsquo;RG\u0026rsquo;, \u0026lsquo;TGY\u0026rsquo;, and \u0026lsquo;HD\u0026rsquo;. This differential expression pattern suggests that CsMYB5 may be associated with cultivar-specific traits, particularly flavonoid and anthocyanin accumulation.Tissue-specific expression analysis revealed that CsMYB5 transcripts accumulated predominantly in old leaves, while expression in stems and flowers was barely detectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,d). This expression pattern implies that \u003cem\u003eCsMYB5\u003c/em\u003e primarily functions in leaf-associated metabolic processes, such as the biosynthesis of secondary metabolites and responses to environmental stimuli.\u003c/p\u003e \u003cp\u003eUnder exogenous hormone treatments, \u003cem\u003eCsMYB5\u003c/em\u003e exhibited a rapid and robust transcriptional response to SA, reaching an initial expression peak at 3 h followed by a second peak at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,e). MeJA treatment also significantly induced CsMYB5 expression, with a maximum at 3 h and a subsequent gradual decline. These results indicate that \u003cem\u003eCsMYB5\u003c/em\u003e transcription is regulated by defense-related phytohormones, implicating this gene in hormone-mediated stress-response pathways.Upon pathogen inoculation, \u003cem\u003eCsMYB5\u003c/em\u003e displayed distinct expression dynamics depending on the infecting strain. Inoculation with CLBB1 strain resulted in a marked upregulation of \u003cem\u003eCsMYB5\u003c/em\u003e at 24 h, whereas infection with strain CCYB1 led to a delayed induction, with peak expression observed at 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,f). This strain-dependent expression response suggests that CsMYB5 may participate in pathogen-specific defense mechanisms in tea plants.Collectively, \u003cem\u003eCsMYB5\u003c/em\u003e shows elevated expression in anthocyanin-rich tea cultivars and leaf tissues and is differentially induced in response to diverse environmental stress conditions. These transcriptional features suggest that CsMYB5 is closely associated with leaf secondary metabolism and hormone- or stress-related responses, thereby warranting further investigation into its protein accumulation and molecular regulatory mechanisms underlying tea plant growth and metabolism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Western Blotting and DAP-seq of CsMYB5 protein\u003c/h2\u003e \u003cp\u003eAfter preliminary optimization experiments, the GST\u0026ndash;CsMYB5 fusion protein was successfully expressed in vitro under induction conditions of 20\u0026deg;C with 0.2 mM IPTG for 20 h. The recombinant GST\u0026ndash;CsMYB5 protein was subsequently purified and subjected to Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,a). A distinct immunoreactive band corresponding to the GST\u0026ndash;CsMYB5 fusion protein was detected at approximately 60 kDa. Given that the predicted molecular weight of CsMYB5 is 34.55 kDa and that of the GST tag is 26 kDa, the observed band size is consistent with the expected molecular weight of the fusion protein. These results confirm the successful expression and purification of the GST\u0026ndash;CsMYB5 protein and further validate the accuracy of the prior protein structure prediction.The purified CsMYB5 protein was subjected to DAP-seq analysis to systematically characterize its genome-wide DNA-binding landscape. Chromosomal distribution analysis of DAP-seq peaks revealed that CsMYB5 binding sites were widely distributed across all chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,b). Genomic annotation of CsMYB5 binding peaks indicated a clear positional preference, with 37.81% of peaks located within promoter regions and 61.09% within intronic regions, whereas only a minor proportion was associated with untranslated regions (UTRs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, CsMYB5 binding peaks were significantly enriched in the vicinity of transcription start sites (TSS), with a pronounced accumulation in upstream promoter regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,d), supporting the role of CsMYB5 as a transcriptional regulator. Gene Ontology (GO) enrichment analysis of promoter-associated CsMYB5 target genes revealed significant enrichment in biological processes related to RNA metabolism, DNA biosynthesis, transcriptional regulation, and secondary metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,e), including RNA phosphodiester bond hydrolysis, nucleotidyltransferase activity, and UDP-glycosyltransferase activity.\u003c/p\u003e \u003cp\u003eKEGG pathway analysis further demonstrated that CsMYB5 target genes were predominantly enriched in secondary metabolism-related pathways, including terpenoid backbone biosynthesis, tyrosine metabolism, caffeine metabolism, and multiple alkaloid biosynthesis pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,f).\u003c/p\u003e \u003cp\u003eNetwork analysis revealed extensive interconnections among these pathways, suggesting that CsMYB5 may function as a central regulator coordinating tea-specific secondary metabolic networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,g).Motif analysis of CsMYB5 binding peaks identified several significantly enriched conserved sequences, including a canonical MYB-binding motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).Furthermore, a prominent CsMYB5 binding peak (peak_6276) was detected within the promoter region of \u003cem\u003eCsTCP15\u003c/em\u003e homology to \u003cem\u003eAtTCP15\u003c/em\u003e ,which has been reported to participate in transcriptional networks regulating plant development and secondary metabolism, indicating that \u003cem\u003eCsTCP15\u003c/em\u003e is a potential direct target of CsMYB5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,i). Integrated DAP-seq and functional enrichment analyses revealed that the direct targets of CsMYB5 are significantly enriched in transcriptional regulation and secondary metabolism-related pathways. Notably, these targets form regulatory modules that modulate the expression of downstream genes involved in the biosynthesis of tea-specific secondary metabolites, including anthocyanins and caffeine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Phenotypic and transcriptional effects of CsMYB5 overexpression\u003c/h2\u003e \u003cp\u003eTo elucidate the biological function of \u003cem\u003eCsMYB5\u003c/em\u003e, an overexpression construct containing the native promoter region of \u003cem\u003eCsMYB5\u003c/em\u003e was generated and introduced into \u003cem\u003eA. thaliana\u003c/em\u003e, resulting in stable transgenic lines. Gene expression analysis confirmed stable overexpression of \u003cem\u003eCsMYB5\u003c/em\u003e in multiple independent T3 lines, with the highest transcript abundance detected in line T3-20, followed by T3-40 and T3-45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,a). Comparative analysis of phenotypic characteristics between CsMYB5-overexpressing \u003cem\u003eA. thaliana\u003c/em\u003e and wild-type plants revealed that during early growth stages, there were no significant differences between wild-type (Col-0) plants and CsMYB5-overexpressing lines T3-20, T3-40, and T3-45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Two weeks after sowing, transgenic seedlings exhibited slightly larger rosette leaves and accelerated growth compared to wild-type plants, with all three lines (T3-20, T3-40, and T3-45) bolting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). By week three, transgenic lines showed increased vegetative biomass and larger leaves, with distinct anthocyanin accumulation in rosette leaves of T3-40 and T3-45 lines, presenting a reddish-purple phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). At week five, all transgenic lines had produced siliques, while Col-0 plants were just beginning to bolt. Moreover, anthocyanin accumulation in \u003cem\u003eA. thaliana\u003c/em\u003e leaves was more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Throughout the entire growth period, CsMYB5- overexpressing plants exhibited earlier bolting and significantly increased inflorescence height compared to wild-type plants. The transition to reproductive growth occurred 14\u0026ndash;21 days earlier in transgenic plants than in wild-type Col-0 (Supplementary Fig.\u0026nbsp;1). Additionally, transgenic plants produced 5\u0026ndash;6 more siliques per stem and had stems 5\u0026ndash;8 cm taller than wild-type Col-0. These results demonstrate that overexpression of CsMYB5 accelerates the transition to reproductive growth in \u003cem\u003eA. thaliana\u003c/em\u003e and promotes anthocyanin accumulation in leaves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistent with the expression patterns observed in tea plants, CsMYB5-overexpressing \u003cem\u003eA. thaliana\u003c/em\u003e lines exhibited pronounced transcriptional responses to exogenous hormone treatments. MeJA rapidly induced \u003cem\u003eCsMYB5\u003c/em\u003e expression, reaching a maximum at 6 h, whereas SA treatment triggered a characteristic biphasic response with prominent peaks at 3 h and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,f), indicating that CsMYB5 retains strong responsiveness to defense-related hormone signaling in a heterologous system.Under abiotic stress conditions, CsMYB5-overexpressing lines also displayed distinct and time-dependent transcriptional responses. Cold treatment induced \u003cem\u003eCsMYB5\u003c/em\u003e expression to peak levels at 48 h, while drought and NaCl treatments caused rapid and significant upregulation at early time points (6 h and 12 h, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,g).Taken together, overexpression of \u003cem\u003eCsMYB5\u003c/em\u003e not only markedly altered plant growth and development and enhanced anthocyanin accumulation in \u003cem\u003eA. thaliana\u003c/em\u003e, but also conferred dynamic transcriptional responsiveness to hormonal and environmental cues. These findings suggest that \u003cem\u003eCsMYB5\u003c/em\u003e exhibits a degree of functional conservation between \u003cem\u003eC.sinensis\u003c/em\u003e and \u003cem\u003eA.thaliana\u003c/em\u003e and support its potential role as a key regulatory factor linking plant development, secondary metabolism, and stress-responsive signaling pathways, thereby providing a solid foundation for further dissection of its underlying molecular regulatory network.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we systematically characterized the R2R3-MYB transcription factor \u003cem\u003eCsMYB5\u003c/em\u003e and demonstrated its pivotal role in integrating hormone signaling, secondary metabolism, and stress responses in tea plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e). By combining expression profiling, DAP-seq analysis, and functional validation in \u003cem\u003eA. thaliana\u003c/em\u003e, we propose a regulatory model in which CsMYB5 functions as a central transcriptional hub coordinating anthocyanin accumulation and caffeine biosynthesis in response to environmental cues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnthocyanins are key flavonoid compounds that contribute not only to plant pigmentation but also to antioxidant capacity and stress tolerance[39]. R2R3-MYB transcription factors act as core regulators of anthocyanin biosynthesis across diverse plant species. For instance, the \u003cem\u003eA. thaliana\u003c/em\u003e homolog of \u003cem\u003eCsMYB5\u003c/em\u003e, \u003cem\u003eAtMYB5\u003c/em\u003e, collaborates with AtMYB2 to promote anthocyanin accumulation in plants[40]. Similarly, FaMYB5 in strawberry performs an analogous function, enhancing anthocyanin biosynthesis[41].\u003c/p\u003e \u003cp\u003eIn this study, we found that \u003cem\u003eCsMYB5\u003c/em\u003e is highly expressed in anthocyanin-rich tea cultivars and leaf tissues, and its overexpression in \u003cem\u003eA. thaliana\u003c/em\u003e significantly promotes anthocyanin accumulation, indicating that \u003cem\u003eCsMYB5\u003c/em\u003e in \u003cem\u003eCamellia sinensis\u003c/em\u003e has a conserved function in regulating anthocyanin biosynthesis.These findings strongly support a conserved role of \u003cem\u003eCsMYB5\u003c/em\u003e in promoting flavonoid biosynthesis, particularly anthocyanin accumulation, in agreement with its close phylogenetic relationship to \u003cem\u003eA. thaliana\u003c/em\u003e MYB5.Beyond flavonoid metabolism, DAP-seq and functional enrichment analyses uncovered an unexpected yet biologically meaningful association between CsMYB5 and caffeine biosynthesis pathways. CsMYB5 binding peaks were significantly enriched in promoter regions of genes involved in caffeine metabolism, including \u003cem\u003eCsXMTs\u003c/em\u003e, which encode key N-methyltransferases catalyzing caffeine biosynthesi[42]. DAP-seq observation suggests that CsMYB5 may possess a multiple regulatory capacity, coordinating the biosynthesis of polyphenols and alkaloids\u0026mdash;two hallmark metabolic features that collectively determine tea quality.\u003c/p\u003e \u003cp\u003eHormone signaling appears to act as a critical upstream regulator of \u003cem\u003eCsMYBs\u003c/em\u003e activity[43]. The \u003cem\u003eCsMYB5\u003c/em\u003e promoter is enriched in SA and MeJA, and \u003cem\u003eCsMYB5\u003c/em\u003e transcription is rapidly induced by both hormones in \u003cem\u003eCamellia sinensis\u003c/em\u003e and transgenic \u003cem\u003eA. thaliana\u003c/em\u003e.Importantly, our DAP-seq analysis identified \u003cem\u003eCsTCP15\u003c/em\u003e as a potential direct downstream target of CsMYB5 protein. \u003cem\u003eCsTCP15\u003c/em\u003e shares high sequence homology with \u003cem\u003eAtTCP15\u003c/em\u003e, which has been implicated in the regulation of plant development[44] and secondary metabolism[45].The proposed \u003cem\u003eCsMYB5\u003c/em\u003e and \u003cem\u003eCsTCP15\u003c/em\u003e regulatory module therefore offers a mechanistic framework through which \u003cem\u003eCsMYB5\u003c/em\u003e may exert both direct and indirect control over secondary metabolism.Taken together, this study support a working model in which stress-induced SA and MeJA signaling activate \u003cem\u003eCsMYB5\u003c/em\u003e expression, which in turn regulates transcriptional networks associated with anthocyanin accumulation and caffeine biosynthesis. Future plans include further metabolomic and transcriptomic analyses to dissect the hierarchical structure of the CsMYB5-centered transcriptional regulatory network, and multi-dimensional experimental validation of the direct regulatory relationship between \u003cem\u003eCsMYB5\u003c/em\u003e and \u003cem\u003eCsTCP15\u003c/em\u003e in modulating secondary metabolism. These efforts will provide new research directions and molecular resources for understanding tea quality improvement and the genetic breeding of elite tea cultivars.\u003c/p\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eThis study systematically characterized the R2R3-MYB transcription factor\u003cem\u003e\u0026nbsp;CsMYB5\u003c/em\u003e in \u003cem\u003eCamellia sinensis\u003c/em\u003e and verified its pivotal role as a central transcriptional hub integrating hormone signaling, secondary metabolism and stress responses. \u003cem\u003eCsMYB5\u003c/em\u003e shows a conserved function in promoting anthocyanin biosynthesis, with high expression in anthocyanin-rich tea tissues and enhanced anthocyanin accumulation in overexpressed\u003cem\u003e\u0026nbsp;Arabidopsis thaliana\u003c/em\u003e. Notably, it unexpectedly regulates caffeine biosynthesis by binding to promoters of key caffeine metabolic genes like\u003cem\u003e\u0026nbsp;CsXMTs\u003c/em\u003e, coordinating both polyphenol and alkaloid production that determines tea quality. Additionally,\u003cem\u003e\u0026nbsp;CsMYB5\u0026nbsp;\u003c/em\u003etranscription is rapidly induced by SA and MeJA, and it targets \u003cem\u003eCsTCP15\u0026nbsp;\u003c/em\u003eto form a regulatory module for secondary metabolism. These findings elucidate a novel hormone-CsMYB5-mediated regulatory network, providing molecular insights for tea quality improvement and elite cultivar breeding.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDAP-seq:DNA affinity purification sequencing. TSS:transcription start sites. UTRs:untranslated regions . KEGG:Kyoto Encyclopedia of Genes and Genomes.GO:Gene Ontology . ABA:abscisic acid.SA:Salicylic Acid. MeJA:Methyl Jasmonate\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:Competing interests：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original sequence data of the tea plant genome in this project is derived from the Tea Plant Information Archive (TPIA) and is publicly accessible via https://tpia.teaplants.cn/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;These Jinxian Liu and Yukun Peng contributed equally to this work.J.L. conceived the project, designed the experiments and drafted the initial manuscript. Y.P. and J.Z. performed the experiments, drafted the initial manuscript and prepared the figures. Z.F. assisted with figure visualization. L.L., X.F., F.L. and G.W. supervised the study, and provided experimental equipment and technical support. Z.L. refined the manuscript to enhance scientific rigor and handled correspondence and submission. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was jointly supported by the Natural Science Foundation of Fujian Province, China (2022J011200, 2025J011069, 2023J011049)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the reviewers for their helpful comments on the original manuscript.We would like to thank the editors for their efficient work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKidokoro S, Shinozaki K, Yamaguchi-Shinozaki K: Transcriptional regulatory network of plant cold-stress responses. \u003cem\u003eTrends Plant Sci \u003c/em\u003e2022, 27(9):922-935.\u003c/li\u003e\n\u003cli\u003eAltmann M, Altmann S, Rodriguez PA, Weller B, Elorduy Vergara L, Palme J, Marin-de la Rosa N, Sauer M, Wenig M, Villaecija-Aguilar JA\u003cem\u003e et al\u003c/em\u003e: Extensive signal integration by the phytohormone protein network. \u003cem\u003eNature \u003c/em\u003e2020, 583(7815):271-276.\u003c/li\u003e\n\u003cli\u003eZhang Z, Song C, Zhao J, Xia E, Wen W, Zeng L, Benedito VA: Editorial: Secondary metabolites and metabolism in tea plants. \u003cem\u003eFront Plant Sci \u003c/em\u003e2023, 14:1143022.\u003c/li\u003e\n\u003cli\u003eLi L, Sun Y, Su Y, Shi Q, Yao W, Li X, Lin S: Molecular Regulatory Mechanisms of Anthocyanin in the Coloration of Plant Leaves and Research Prospects. \u003cem\u003eHorticultural Plant Journal \u003c/em\u003e2026.\u003c/li\u003e\n\u003cli\u003eYou D, Liu M, Ruan J, Wang Z, Zhang Q: Integrated Analysis of Metabolites and Microorganisms Reveals the Anthracnose Resistance Benefits from Cyanidin Mediated by Proteobacteria in Tea Plants. \u003cem\u003eInt J Mol Sci \u003c/em\u003e2024, 25(21).\u003c/li\u003e\n\u003cli\u003eLi XN, Fan LL, Zhu Q, Liu JH, Xie ZY, Cao JL, Tan JY, Lin L, Li XS, Wei XH: Blue honeysuckle (Lonicera caerulea L.)-anthocyanins and cyanidin-3-O-glucoside protect dopaminergic neurons against ferroptosis by activating the Nrf2-GPX7 axis. \u003cem\u003eFree Radic Biol Med \u003c/em\u003e2025, 239:242-256.\u003c/li\u003e\n\u003cli\u003ePescador-Dionisio S, Robles-Fort A, Parisi B, Garcia-Robles I, Bassolino L, Mandolino G, Real MD, Rausell C: Contribution of the regulatory miR156-SPL9 module to the drought stress response in pigmented potato (Solanum tuberosum L.). \u003cem\u003ePlant Physiol Biochem \u003c/em\u003e2024, 217:109195.\u003c/li\u003e\n\u003cli\u003eLi C, Shen Q, Cai X, Lai D, Wu L, Han Z, Zhao T, Chen D, Si J: JA signal-mediated immunity of Dendrobium catenatum to necrotrophic Southern Blight pathogen. \u003cem\u003eBMC Plant Biol \u003c/em\u003e2021, 21(1):360.\u003c/li\u003e\n\u003cli\u003eGao Q, Tong W, Li F, Wang Y, Wu Q, Wan X, Xia E: TPIA2: an updated tea plant information archive for Camellia genomics. \u003cem\u003eNucleic Acids Research \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003eChen X, Wang P, Gu M, Hou B, Zhang C, Zheng Y, Sun Y, Jin S, Ye N: Identification of PAL genes related to anthocyanin synthesis in tea plants and its correlation with anthocyanin content. \u003cem\u003eHorticultural Plant Journal \u003c/em\u003e2022, 8(3):381-394.\u003c/li\u003e\n\u003cli\u003eRothenberg DO, Yang H, Chen M, Zhang W, Zhang L: Metabolome and Transcriptome Sequencing Analysis Reveals Anthocyanin Metabolism in Pink Flowers of Anthocyanin-Rich Tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e). \u003cem\u003eMolecules \u003c/em\u003e2019, 24(6).\u003c/li\u003e\n\u003cli\u003eMaritim TK, Masand M, Seth R, Sharma RK: Transcriptional analysis reveals key insights into seasonal induced anthocyanin degradation and leaf color transition in purple tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e (L.) 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and social context of gene-edited, caffeine-free coffee. \u003cem\u003eFood Sci Biotechnol \u003c/em\u003e2022, 31(6):635-655.\u003c/li\u003e\n\u003cli\u003eLi P, Xia E, Fu J, Xu Y, Zhao X, Tong W, Tang Q, Tadege M, Fernie AR, Zhao J: Diverse roles of MYB transcription factors in regulating secondary metabolite biosynthesis, shoot development, and stress responses in tea plants (\u003cem\u003eCamellia sinensis\u003c/em\u003e). \u003cem\u003ePlant J \u003c/em\u003e2022, 110(4):1144-1165.\u003c/li\u003e\n\u003cli\u003eSteiner E, Yanai O, Efroni I, Ori N, Eshed Y, Weiss D: Class I TCPs modulate cytokinin-induced branching and meristematic activity in tomato. \u003cem\u003ePlant Signal Behav \u003c/em\u003e2012, 7(7):807-810.\u003c/li\u003e\n\u003cli\u003eViola IL, Camoirano A, Gonzalez DH: Redox-Dependent Modulation of Anthocyanin Biosynthesis by the TCP Transcription Factor \u003cem\u003eTCP15\u003c/em\u003e during Exposure to High Light Intensity Conditions in Arabidopsis. \u003cem\u003ePlant Physiol \u003c/em\u003e2016, 170(1):74-85.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Camellia sinensis, CsMYB5, MYB transcription factor, transcriptional regulation, hormone response","lastPublishedDoi":"10.21203/rs.3.rs-9168618/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9168618/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnthocyanins are major secondary metabolites that determine tea quality and contribute to plant stress adaptation. However, the transcriptional mechanisms coordinating their biosynthesis in response to environmental cues remain largely unclear.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResult\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, we characterized the R2R3-MYB transcription factor \u003cem\u003eCsMYB5\u003c/em\u003e from tea plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e) and investigated its role in hormone-responsive secondary metabolism regulation. Expression analyses revealed that \u003cem\u003eCsMYB5\u003c/em\u003e is preferentially expressed in anthocyanin-rich tea cultivars and leaf tissues and is strongly induced by SA, MeJA and multiple abiotic stresses. Genome-wide DNA affinity purification sequencing revealed that CsMYB5 binding sites are enriched in promoter regions of genes associated with transcriptional regulation and secondary metabolism, including flavonoid and caffeine biosynthetic pathways. Notably, CsMYB5 directly targets \u003cem\u003eCsTCP15\u003c/em\u003e, a TCP transcription factor potential regulation of anthocyanin synthesis and accumulation, suggesting the existence of a CsMYB5\u0026ndash;CsTCP15 regulatory module.Heterologous overexpression of \u003cem\u003eCsMYB5\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e resulted in enhanced vegetative growth and pronounced anthocyanin accumulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on these findings, we propose that \u003cem\u003eCsMYB5\u003c/em\u003e acts as a central transcriptional hub linking hormone signaling with coordinated regulation of anthocyanin accumulation biosynthesis. This study provides new insights into the transcriptional integration of flavonoid metabolism and offers potential targets and research direction for improving tea quality and stress resilience through molecular breeding.\u003c/p\u003e","manuscriptTitle":"CsMYB5 regulates anthocyanin accumulation and stress responses through responding to hormone signaling of Camellia sinensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 12:06:19","doi":"10.21203/rs.3.rs-9168618/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"72c6a8ea-3e33-4f60-accf-308d98ec2d25","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-14T04:55:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 12:06:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9168618","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9168618","identity":"rs-9168618","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}