Molecular mechanisms of catechins regulation by EfMYB5b in Euryale ferox

Molecular mechanisms of catechins regulation by EfMYB5b in Euryale ferox | 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 Molecular mechanisms of catechins regulation by EfMYB5b in Euryale ferox Peng Wu, Chenyan Qu, Tianyu Wang, Wenjing Ling, Mengnan He, Yuerui Fang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8027490/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 Flavonoids are important secondary metabolites, which exist widely and play different roles in plants. Many kinds of flavonoid compounds have been identified in E. ferox seed kernels, but the main flavonoid components and biosynthesis mechanism of E. ferox are still unclear. In this study, UPLC-MS/MS was used to identify catechin as the main flavonoid compound in seed kernel of E. ferox . The 32 structural genes (2 EfDFRs and 30 EfANRs ) related to catechin synthesis were identified. Among them, key genes EfDFR-1 and EfANR-11 were screened by transcriptome, real-time fluorescence quantification and enzyme activity analysis. Further, in vitro enzyme activity assay demonstrated that EfANR-11 and EfDFR-1 could catalyze the formation of gallocatechin (GC) and epigallocatechin (EGC) from the substrate, respectively. Then, subcellular localization showed that EfDFR-1 and EfANR-11 were located in the Golgi apparatus. Transient overexpression of EfDFR-1 and EfANR-11 significantly increased catechin content in seed kernels of E. ferox . Subsequently, the EfMYB5b directly bind to TAACCA and ACCTAC in the EfDFR-1 and EfANR-11 promoter and promote their expression. Meanwhile, transientoverexpression of EfMYB5b showed that the expression of EfDFR-1 and EfANR-11 were significantly enhanced, and the content of catechin was increased in seed kernel E. ferox . Our findings clarified the molecular mechanism of transcription factor EfMYB5b regulating key genes EfDFR-1 and EfANR-11 in catechin synthesis pathway. It provides theoretical basis for improving the quality of E. ferox and breeding new varieties. Catechins EfANR-11 EfDFR-1 EfMYB5b E. ferox Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message EfMYB5b positively regulates the synthesis of catechins in the seed kernels of Euryale ferox by promoting the expression of EfDFR-1 and EfANR-11 . 1. Introduction Catechins, namely flavan-3-ols, are a class of polyphenolic compounds belonging to flavonoid compounds (Gadkari et al., 2015). It has the basic structure of 2-phenylbenzopyran (C6-C3-C6), comprising A-, C-, and B-rings. Catechins can be classified into two groups, one group of trans flavonoid 3 alcohols includes (+)-catechin (C) and (-)-gallocatechin (GC) (Liu et al., 2012 ). And another group of cis flavonoid 3 alcohols includes (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) (Zhang et al., 2012 ). The latter being the predominant type among the catechins (Wang et al., 2018 ). Catechins, recognized as vital defensive compounds that protect plants from ultraviolet(UV) radiation, pathogens, and pests (Petrussa et al., 2013 ), are also scientifically validated to provide diverse health benefits for humans. By comparing the different infection stages of disease-resistant tea and susceptible tea plants, it was found that the content of EC in disease-resistant tea plants was significantly higher than that in susceptible tea plants, and EGCG was the opposite (Punyasiri et al., 2005 ). In addition, many studies have confirmed that catechins can combat a wide range of diseases (Wolfram et al., 2006 ), such as anti-cancer, anti-obesity, anti-diabetic, neuroprotective and reducing the risk of coronary heart disease (Kapoor et al., 2022 ; Thomasset et al., 2006 ), moderate intake of catechins can also improve overall health (Isemura, 2019 ). Therefore, a deeper study of the regulatory mechanisms of catechins is of great research importance. The biosynthesis of catechins, through the phenylpropanoid and flavonoid biosynthetic pathways (Xiong et al., 2013 ), is a large and complex process involving the participation of multiple enzymes and genes. Firstly, in benzene propane way, L-Phenylalanine via phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumaroyl-CoA ligase (4CL) continuous catalytic, the resulting 4-coumaroyl-CoA. Subsequently, chalcone synthase (CHS) converts 4-coumaroyl-CoA into chalcone, which was further catalyzed by chalcone isomerase (CHI) to produce naringenin (Li et al., 2015 ). Then, naringenin undergoes hydroxylation by flavanone 3-hydroxylase (F3H) and flavonoid 3',5'-hydroxylase (F3'5'H), followed by dihydroflavonol 4-reductase (DFR) mediated reduction to leucoanthocyanidins (Zhou et al., 2016 ). Downstream of DFR, the pathway bifurcates into two distinct branches, one is leucoanthocyanidin reductase (LAR) directly converts leucoanthocyanidins to catechin (C) and gallocatechin (GC). Another is anthocyanidin synthase (ANS) oxidizes leucoanthocyanidins to cyanidin and delphinidin, which are subsequently reduced by anthocyanidin reductase (ANR) to produce epicatechin (EC) and epigallocatechin (EGC) (Zhang et al., 2019 ). Among them, DFR is a key enzyme in the synthesis of anthocyanins and proanthocyanidins. It can catalyze the synthesis of colorless anthocyanins from the precursor substance dihydroflavonol, which is the common precursor substance of biosynthesis such as catechin and proanthocyanidins. Catechin epimerization, primarily mediated by the enzymatic activities of LAR,ANS, and ANR, serves as a critical determinant in establishing the equilibrium between epicatechin derivatives (EGCG, ECG, EGC, EC) and their non-epi counterparts (GCG, CG, GC, C) (Pang et al., 2013 ; Punyasiri et al., 2004 ; Wu et al., 2014 ). Notably, ANR plays a dual catalytic role in this biochemical process, not only participating in the biosynthesis of flavan-3-ol monomers through the conversion of anthocyanidins to epicatechin (EC) and epigallocatechin (EGC), but also contributing to the epimerization regulation (Ashihara et al., 2010 ; Zhang et al., 2016 ). In plants, the biosynthesis of catechins is regulated by transcription factors. These transcription factors regulate the expression of one or more structural genes in this pathway by binding to cis-acting elements in structural gene promoters, thereby regulating catechin biosynthesis. Studies have demonstrated that the biosynthesis of catechins is regulated by multiple transcription factors, including NAC, MYB, bHLH, and others (Meraj et al., 2020 ). For example, overexpression of CsMYB5 was found to significantly increase the expression of CsANR , thereby promote catechin synthesis (Wang et al., 2022b ). Based on DAP-seq verification, it was found that CsMYB196 directly combines with CsANR to promote the synthesis of catechins (Shan et al., 2025 ). Additionally, CsMYBL2 can promote the expression of CsANR and thus promote the accumulation of catechins (Zhao et al., 2023 ). VviMYBC2-L1 and VviMYBC2-L2 negatively regulate the expression of VviDFR and inhibit the biosynthesis of proanthocyanidins and catechins in the green fruit stage of grape growth and development (Vale et al., 2024 ). Euryale ferox , is an annual floating-leaved macrophyte aquatic herb that belongs to the genus Euryale from the angiosperm basal plant family Nymphaeaceae, and is one of the important characteristic aquatic vegetables in China (Wu et al., 2022b ). The Chinese Pharmacopoeia records that E. ferox mainly has the effects of tonifying the spleen, stopping diarrhoea, benefiting the kidneys and fixing the sperm, etc (Jiang et al., 2023a ), and it is a typical representative of homology of medicine and food. The seed kernel of E. ferox is the main edible organ, is characterized by a rich nutritional profile comprising flavonoids, carbohydrates, proteins, and other bioactive compounds. Flavonoid compounds are the main efficacy components of E. ferox (Naik et al., 2022 ). Proteomic analysis of seed kernels at different periods showed that PAL, F3H, FLS, DFR, and ANS were key enzymes in E. ferox flavonoid biosynthesis (Wu et al., 2022a ). A total of 129 flavonoid substances were identified in E. ferox , and the content of flavonoid compounds reaches the highest level in seed kernels of E. ferox 30 days after flowering (DAF30) (Wu et al., 2021 ), but the main components of flavonoid compounds have not been determined. In this study, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology was used to identify the main flavonoids in seeds kernel. Subsequently, two key genes EfDFR-1 and EfANR-11 , were identified through integrated transcriptomic profiling, RT-qPCR validation, and enzyme activity assays. Their functional roles were further characterized via subcellular localization analysis and transient overexpression experiments. The transcriptional activation of key catechin biosynthetic genes by EfMYB5b was further validated through yeast one-hybrid (Y1H) assays, dual-luciferase reporter (LUC) systems, and electrophoretic mobility shift assays (EMSA). Finally, transient overexpression trials further established EfMYB5b's capacity to modulate catechin accumulation. Collectively, this systematic approach elucidates critical genes and regulatory networks governing catechin biosynthesis, providing a theoretical foundation for targeted quality improvement and cultivar development in E. ferox . 2. Materials and methods 2.1 Plant material and sample collection 'ZHSQ' was planted in the aquatic vegetable experimental base of Yangzhou University, under normal cultivation and management (Yangzhou, China, 2024). The seed kernels were collected at four periods of DAF10 (days after flowering), DAF20, DAF30, and DAF40, with three biological replicates for each period. All samples were snap frozen in liquid nitrogen immediately after collection and kept at -80°C. 2.2 Determination of flavonoid content The seed kernels of E. ferox DAF30 were quantitatively analysed for flavonoid compounds using UPLC-MS/MS. Ultra Performance Liquid Chromatography (UPLC) (ExionLC™ AD, https://sciex.com.cn/ ) and Tandem Mass Spectrometry (MS/MS) (QTRAP® 6500+, https://sciex.com.cn/ ) for data acquisition. A MWDB (Metware Database) database was constructed based on the standards to qualitatively analyse the data detected by mass spectrometry. Analyst 1.6.3 software was used to process the mass spectrometry data. The above data are shown in Table S1 . 2.3 Sequence identification of EfDFRs, EfANRs DFR genes from Camellia sinensis and Arabidopsis thaliana were utilized for Blast in the E. ferox genome. Then, pfam ( http://pfam.xfam.org ) and SMART ( http://smart.embl-heidelberg.de/ ) were performed to further identify the candidate genes for DFR and ANR. Molecular weight and isoelectric point were finally predicted by ExPASy tool ( https://www.expasy.org/ ). 2.4 Analysis of conserved motifs and domains of candidate genes Gene structure information was obtained from the GFF file of the E. ferox genome and conserved motifs were analyzed in the MEME suite ( https://meme-suite.org/meme/tools/meme ). Finally, Tbtools software was used for visualization (Chen et al., 2020 ). The relevant results are shown in Tables S2 . 2.5 Phylogenetic tree analysis of candidate genes Amino acid sequence comparison was performed using MEGA 11 software, followed by neighbour-joining method for the construction of phylogenetic tree, which was further refined using iTOL ( https://itol.embl.de/tree ). The related gene sequences are shown in Table S3 . 2.6 Quantitative real-time PCR analysis (RT-qPCR) Total RNA was extracted from E. ferox seed kernels at different developmental stages (DAF10-DAF40) and different E. ferox organs using an RNA extraction kit (Takara, Dalian, China). The extracted RNA was reverse transcribed into cDNA by applying HiScript® II Q RT SuperMix (Vazyme, Nanjing, China). EfUBQ5 (ID: EF11G001150) was used as internal reference genes (Wu et al., 2022c ). The CFX-96 real-time fluorescence quantitative PCR system (Bio-Rad) was used for amplification, and the amplification program was 95°C for 30s, 95°C for 10s, and 60°C for 30s, for a total of 40 cycles. qPCR concentration and amplification efficiency were also determined. The 2 −ΔΔCT method was used for the analysis of RT-qPCR data(Zhao et al., 2021 ). The relevant primers are shown in Table S4 . 2.7 Subcellular localisation Utilizing Plant-mPloc ( http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ ) predicting the specific location of EfDFR-1, EfANR-11 and EfMYB5b proteins within a cell. Tobacco transient expression system was applied to analyse the subcellular localisation of EfDFR-1, EfANR-11 and EfMYB5b. The CDS sequence of these genes were constructed through the method of homologous recombination into the pCAMBIA1300-35S-sGFP vector, resulting in p35S::EfDFR-1-GFP, p35S::EfANR-11-GFP and p35S::EfMYB5b-GFP. Using the freeze-thaw method, the recombinant plasmid construct was transferred into the Agrobacterium GV3101 strain. Subsequently, it was expressed in tobacco leaves through the Agrobacterium infection method. Bacterial cultures were resuspended to OD₆₀₀ = 0.8-1.0 in infiltration buffer (10 mM MgCl 2 , 0.5M MES, 100 mM acetosyringone, pH 5.8). Needleless injector was used to inject on the abaxial surface of the tobacco leaf until the entire leaf was submerged. The plants were cultured at room temperature in the dark for three days. The fluorescence signals were observed under an ultra-high resolution laser confocal microscope, with the unloaded state serving as the control. The above primers are listed in Table S5 . 2.8 Enzyme activity assay The CDS regions of EfDFR-1 , EfDFR-2 , EfANR-11 and EfANR-25 were ligated into the pCold-TF vector, and then the recombinant plasmids were transformed into E. coli BL21 for prokaryotic expression. Select a single colony, incubate it in 100 mL of LB liquid medium containing 50 mg/L Amp at 37°C, 200 rpm in a shaking incubator for 4–6 hours until the OD600 reaches approximately 0.5. Add 1 mM of IPTG (isopropyl-β-D-thiogalactopyranoside), and incubate it at 16°C, 100 rpm on a shaking incubator for 16 hours. Further, collect the bacterial suspension by centrifugation at 4°C, 4000 rpm for 20 minutes, discard the supernatant and add 5 mL of lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM β-mercaptoethanol, 10 mM imidazole) for suspension. Use low-temperature ultrasonic disruption for 20 minutes, centrifuge at 10000 rpm for 10 minutes, and elute the target protein using a resin elution column. The fused protein was subjected to Ni-Agarose column (CWBIO, Jiangsu, China) to elute the target protein with gradient imidazole concentration (100–500 mM). And single-purpose bands were obtained by SDS-PAGE electrophoresis, respectively. The reaction system was as follows (1 mL): 300 µg of recombinant protein, 100 mM phosphate buffer (pH 6.5), 20 mM NADPH, and 1 mM substrate. After incubation at 45℃ for 30 minutes, 1 mL of methanol was added to terminate the reaction. The mixture was then shaken evenly and centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected. The filtrate was filtered through a 0.22 µm filter membrane and analyzed using UPLC. The products of the reaction were analyzed by HPLC (Agilent 1260, USA). A C18 column (4.6 ×250 mm) was applied with 1% acetic acid in ultrapure water (v/v) as solvent A and 100% acetonitrile solvent B. The elution gradients were as follows: 20% B at 0 min, 95% B at 15 min, and 20% B at 15.01 min. A flow rate of 1.0 mL/min and a column temperature of 25°C were applied and the chromatogram was obtained at 280 nm. A list of the above primers is shown in Table S6 . 2.9 Transient overexpression assay The gene was constructed into pCAMBIA1300 vector. The correctly sequenced plasmid was transformed into Agrobacterium tumefaciens GV3101 (Miao et al., 2020 ). The successfully transformed Agrobacterium solution was mixed with an infection solution containing acetosyringone, and the OD 600 was adjusted to 0.8 and left in the dark for 2–3 hours. The E. ferox seed kernels of DAF30 were immersed in the aforementioned solution. (Wei et al., 2023 ). The materials used were fresh 30 days after flower opening seed kernels harvested from our experimental field. After cleaning it thoroughly, it was immediately disinfected with 75% ethanol in clean bench for 15 seconds, then disinfected with 3% hypochlorous acid for 10 minutes. The seeds were then placed in the infection solution containing positive Agrobacterium in the dark environment and infected for 30 minutes. Then, the kernels were washed three times with sterilized water for 1 minute each time. The surface moisture was then absorbed using sterilized filter paper. The kernels were then inoculated onto the MS medium and incubated in the dark for 3–4 days. The experiment was repeated three times, with 6 kernels being tested each time. The 6 kernels were mixed and ground into powder, and then the content of catechins and RNA extraction were carried out. The above primers are listed in Table S5 . 2.10 Relevance analysis Correlation analyses of EfMYB5b and EfDFR-1 and EfANR-11 were performed by Qmic studio ( https://www.omicstudio.cn/video ). 2.11 Yeast one-hybrid (Y1H) assay Utilizing PLANTCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) predicted binding sites of EfMYB5b to key genes. Sequences of EfMYB5b was inserted into the pGADT7 vector. EfDFR-1 and EfANR-11 were ligated into the pAbAi vector. Positive bacterial fluids were sent to the company for sequencing and plasmid extraction. The p EfDFR-1 -pAbAi, p EfANR-11 -pAbAi plasmid was linearised using Bbs I restriction endonuclease, and cultured in SD/-Leu + AbA 200 medium at 30°C for 3–5 days. The above primers are listed in Table S7 . 2.12 Dual-luciferase reporter assay (LUC) The coding sequences of EfDFR-1, EfANR-11 and EfMYB5b were cloned into the pGreenII-0800 and pGreenII-62-SK reporter vectors, respectively, after which the after which the reporter plasmid was transferred into Agrobacterium tumefaciens strain GV3101 and then diluted with infection solution (100mM acetosyringone, 0.5M MES, 10mM MgCl 2 , pH5.8); Agrobacterium containing pGreenPro- EfDFR-1 and pGreenPro- EfANR-11 was mixed with strains containing pGreenII62-SK-EfMYB5b at a ratio of 1:1 (v/v). Adjust the OD600 value of the bacterial solution to 0.8-1.0, leave it in the dark for 3 hours, and then inject it into the tobacco leaves. At the same time, the leaves were sprayed with 100 mM luciferin and incubated for 10 minutes. The LUC luminescence signal was detected using the GelView 6000ProⅡ multi-functional imaging workstation. The above primers are listed in Table S8 . 2.13 Electrophoretic Mobility Shift Assay (EMSA) The full-length EfMYB5b fragment was inserted into pCold-TF vector, and the prokaryotic expression vector pCold-TF-EFMYB5b was obtained, and the purified protein was obtained. Use the EMSA probe biotin labeling kit (Beyotime, China) and follow the instructions. The probe was synthesized by Sangon. SDS-PAGE electrophoresis detection was performed after the probe reaction. After the steps of coating, transfer and fixation, electrophoretic transfer was carried out, and then the gel was cross-linked in the purple diplomatic linkage instrument for chemiluminescence reaction and photographed for observation. The primers of the probe are shown in Table S9 . 2.14 Statistical analysis Three independent biological replicates were used for each sample in each experiment. Experimental data were analyzed and visualized using GraphPad Prism 8 software. Statistical significance was tested using multiple t tests (* P < 0.05, ** P < 0.01). 3. Results 3.1 Catechins are main flavonoid compounds in E. ferox seed kernels During the developmental process of E. ferox seed kernels, flavonoid compounds exhibit significant accumulation at DAF30 (Wu et al., 2021 ). Consequently, UPLC-MS/MS was employed to determine the types and contents of flavonoid compounds in DAF30 E. ferox seed kernels. The results identified 31 flavonoid compounds across six categories: flavones, flavonols, flavanols, dihydroflavonols, chalcones, and flavone C-glycosides (Fig. 1 ). Among them, the content of flavanols was the highest, mainly including EGCG (87.45ng/g DW), GC (54.48ng/g DW), EC (22.91ng/g DW), CG (12.90ng/g DW), GCG (7.08ng/g DW), C (5.23ng/g DW). Secondly, there are chalcones, mainly including PRO B2(Procyanidin B2) and MHYOG(Medicarpin), with their contents being 1.62 and 1.07 ng/g DW. The contents of flavones, flavonols, dihydroflavonols and flavone C-glycosides were relatively low. Their concentrations range from 0.002 to 0.91 ng/g DW ( Table S1 ). The comparative analysis demonstrated pronounced accumulation of flavanols in the seed kernels, with catechins constituting the highest proportion at 96.07% of the total detected flavonoids, and approximately 24 times the combined concentration of all other flavonoid subclasses (Fig. 1 ). These findings indicate that catechins are among the most critical bioactive components in E. ferox seed kernels. 3.2 Identification of DFRs and ANRs gene in E. ferox The 6 candidate DFR and 38 ANR genes were identified in E. ferox genome by blast. Then, 2 EfDFRs and 30 EfANRs were screened with pfam and SMART ( Table S2 ). The analysis of the physicochemical properties revealed that there was a wide variation in their molecular weights (Mw), ranging from 31.49 KDa to 64.05 KDa, with isoelectric points (pI) between 5.35 and 9.11 ( Table S10 ). Further, we used DFR proteins to construct phylogenetic tree (Fig. 2 a). The results showed that EfDFR-1 and EfDFR-2 clustered together with OsDFR1 , OsDFR2 , and OsDFR3 . It indicated that the EfDFRs has higher similarity with the OsDFRs sequence and functions. Then, the EfDFRs domains have typical PLN02650 structural features, which is a specific site of the DFR protein (Fig. 2 b). And motif analysis showed that EfDFRs contained motif1 ~ motif8, and the arrangement was consistent (Fig. 2 b). Meanwhile, the ANR phylogenetic tree was constructed by using the E. ferox , Arabidopsis thaliana , and Camellia sinensis ANR proteins. The 30 EfANRs genes can be divided into 5 subfamilies. The distribution proportions of ANR family members of the three species in each subfamily are different, and there are also differences in cluster analysis among different plants (Fig. 2 c). Further analysis revealed that the majority of EfANRs have a relatively high homology with AtANRs and CsANRs, which might indicate that the ANR of plants is evolutionarily conserved. Similarly, EfANRs conserved motifs and domains are visualized. Most of EfANRs contain motif1 ~ motif10 and are arranged relatively consistently in different subgroups, indicating that these motifs are highly conserved (Fig. 2 d). Besides, EfANR-5 and EfANR-12 contained 5 motifs, while EfANR-1, EfANR-8, EfANR-9, and EfANR-13 only contained 4 motifs. A total of 5 conserved domains were identified in EfANRs. 15 EfANRs contained NADB_Rossmann superfamily and 10 EfANRs contained FR_SDR_e specific domain. Other EfANRs contained Reticulon, PLN00198 superfamily and PLN02896 superfamily respectively. They all play an important role in the NADP-dependent reduction of flavonoids (Fig. 2 d). 3.3 EfDFR-1 and EfANR-11 are key candidate genes for catechins biosynthesis in E. ferox Transcriptome and RT-qPCR analysis showed that EfANR-1 , EfANR-11 , EfANR-15 , and EfDFR-1 were highly expressed in different organs of E. ferox and their expression levels were higher in seeds ( Fig. S1 a, Table S11 ). EfDFR-2 was almost not expressed, while EfANR-25 expression was relatively low, but significantly higher in seed than in other tissues. In seed kernels of E. ferox at different developmental stages (DAF10-DAF40), the expression of EfDFR-1 was 35.51 times that of EfDFR-2 , and the expression of EfANR-11 and EfANR-25 was significantly higher than that of other candidate genes ( Fig. S1 b, Table S12 ). Therefore, EfANR-11 , EfANR-25 and EfDFR-1 may be the important genes for catechin synthesis in seed kernels of E. ferox . In order to further verify the enzymatic activity of EfDFR-1, EfDFR-2, EfANR-11 and EfANR-25, their sequences were cloned into pCold vector, and single target bands were obtained by SDS-PAGE gel electrophoresis ( Fig. S1 c ). At the same time, enzyme activity was measured with the same amount of protein solution. The results showed that the enzyme activity of EfDFR-1 was 5.56 times that of EfDFR-2, and the enzyme activity of EfANR-11 was 2.41 times that of EfANR-25 ( Fig. S1 d ). Finally, centaurea was reacted with purified EfANR-11 protein by High-performance liquid chromatography (HPLC) with centaurea standard and inactivated enzyme protein as controls. The results showed that no substance was detected in the control reaction solution, while the reaction solution containing the prokaryotic recombinant protein EfANR-11 on the cyanidin substrate was detectable in the gallocatechin (Fig. 3 a). DFR and LAR are closely linked two-step catalytic enzymatic reaction and both depend on NADPH. Because of the high instability of leucoanthocyanidins, it is difficult to be detected and cannot be directly used as the substrate of enzymatic reaction to determine the activity of recombinant protease alone. We used dihydromyricetin as substrate to react with purified EfDFR-1 and EfLAR-1 protein solution, and found that epigallocatechin could be detected in the reaction solution of EfDFR-1 and EfLAR-1 (Fig. 3 b). To further determine the role of EfDFR-1 and EfANR-11 in catechins biosynthesis, transient overexpression experiments were performed. The expression changes of EfDFR-1 and EfANR-11 were analyzed by RT-qPCR, and the contents of catechin in overexpressed seed kernels were determined. The results showed that the expression of EfDFR-1 was significantly increased after overexpression and the catechin content of EfDFR-1 overexpressed seed kernels was 1.2 times higher than that of the control. The expression of EfANR-11 and the catechin content of EfANR-11 overexpressed seed kernels was significantly increased, too (Fig. 3 c). Ultimately, to determine the localization of EfDFR-1 and EfANR-11, recombinant vectors and empty vector were introduced into tobacco epidermal cells. The results showed that the fluorescence signals of p35S:EFDFR-1-GFP and p35S:EFANR-11-GFP were only observed in the Golgi apparatus, indicating that EfDFR-1 and EfANR-11 were located in the Golgi apparatus (Fig. 3 d). The results were consistent with those predicted by Plant-mPloc. 3.4 EfMYB5b positively regulates EfDFR-1 and EfANR-11 expression, and promotes catechins biosynthesis Transcriptome and qPCR analysis found that the expression trend of EfMYB5b in seeds of E. ferox at different periods was consistent with the expression trend of EfDFR-1 and EfANR-11 ( Fig. S2 ). And correlation analysis showed that EfMYB5b was positively correlated with EfDFR-1 and EfANR-11 ( Table. S13 ). In addition, in the PLANTCARE database, it was found that there are respectively the binding sites TAACCA and ACCTAC for EfMYB5b on the promoters of EfDFR-1 and EfANR-11 . Therefore, EfMYB5b may be an important transcription factor in the regulation of EfDFR-1 and EfANR-11 . The results of subcellular localization experiments showed that the fluorescence signal of p35S::EfMYB5B-GFP was only visible in the nucleus and overlapped with nuclear markers, suggesting that EfMYB5b is nuclear-localized transcription factors (Fig. 4 a). To further verify whether EfMYB5b protein could interact with EfDFR-1 , we conducted the Y1H experiment. The results showed that AD + EfDFR-1 promoter could not grow on SD-Leu 200 medium, while EfMYB5b-AD + EfDFR-1 promoter could grow on SD-Leu 200 medium, which indicated that EfMYB5b protein could directly bind to EfDFR-1 promoter and thus regulate its expression (Fig. 4 b). Subsequently, LUC assay was used to investigate the regulation of EfDFR-1 by EfMYB5b protein. When EfMYB5b and EfDFR-1 were co-expressed in tobacco leaves, LUC activity was significantly increased, and the relative expression of luciferase increased 3.3 times (Fig. 4 c). The Y1H results indicated that EfMYB5b can interact with the promoter of EfANR-11 (Fig. 5 a). Similarly, dual-luciferase assay was performed in tobacco leaves. The results showed that the co-expression of EfANR-11pro::LUC and 35S::EfMYB5b in tobacco leaves significantly increased the activity of LUC, and the relative expression of luciferase increased by 1.9 times (Fig. 5 b). This indicating that EfMYB5b could promote the expression of EfDFR-1 and EfANR-11 . To further verify whether the EfMYB5b protein can bind to the ‘TAACCA’ and ‘ACCTAC’ site, we designed biotin-labeled probes containing the binding site for the EMSA assays. The purified HIS- EfMYB5b fusion protein was confirmed by Western blot and successfully obtained (Fig. 4 d, Fig. 5 c). EMSA results showed that EfMYB5b binds to the labeled probe. When unlabeled probes (cold probes) were added, the binding band significantly weakened (Fig. 4 d, Fig. 5 c), indicating that EfMYB5b can specifically bind to the ‘TAACCA’ and ‘ACCTAC’ site. These results suggest that EfMYB5b can target multiple key enzyme genes EfDFR-1 and EfANR-11 involved in the synthesis of catechins. Finally, the CDS sequence of EfMYB5b was constructed onto the vector pCAMBIA1300, and the empty vector was used as the negative control for overexpression. The treated seed kernels of E. ferox was soaked in the bacterial solution an cultured on MS medium for 4 days. RT-qPCR showed that the expression of EfDFR-1 and EfANR-11 in overexpressed seed kernels of E. ferox was significantly increased (Fig. 4 e, Fig. 5 d). The content of catechins also significantly increased in the seed kernels after overexpression of EfMYB5b compared to CK (Fig. 4 e). These experimental results consistently indicated that EfMYB5b could promote the expression of EfDFR-1 and EfANR-11 , thus promoting the accumulation of catechins in seed kernels of E. ferox . 4. Discussion Flavonoids are polyphenolic compounds that are widely distributed in plants and are rich in functions (Nabavi et al., 2020 ; Shen et al., 2022 ). An increasing number of flavonoids in plants have been shown to be functional components and research on their biosynthesis has been intensified (Wu et al., 2020 ). The main component of flavonoid compounds in tea is catechin, and catechin is an important functional component of tea (Liu et al., 2012 ). A rich variety of flavonoid compounds have been identified in the seed kernels of E. ferox and significant accumulation of flavonoid compounds at DAF30 seed kernels. A total of 129 metabolites measured were identified, including favanones, dihydrofavanols, favanols, favones, isofavones, anthocyanins, favonols, favonoid carbonosides, chalcones and proan-thocyanidins (Wu et al., 2021 ). However, which substance is the main component of flavonoid compounds in E. ferox seed kernels has not yet been determined. In our study, it was found that the content of catechins was the highest among flavonoids, by using UPLC-MS/MS to analyze the types and contents of flavonoid compounds in DAF30 seed kernels of E. ferox . Therefore, we speculate that the high content of catechins may be an important functional component of E. ferox seed kernels. DFR and ANR are key enzymes in catechins biosynthesis. Studies have shown that DFR gene expression is significantly correlated with catechin accumulation during leaf development of different tea cultivars (Mamati et al., 2006 ). Overexpressed the CsDFR and CsANR genes increased catechin content and improvied its antioxidant capacity in tobacco (Kumar et al., 2013 ). Besides, three MdANRs were overexpressed, the contents of catechin and epicatechin were significantly increased in tobacco (Han et al., 2012 ). A total of 5 DFR genes and 21 ANR genes were identified in tea (Duan et al., 2023; Punyasiri et al., 2017 ), 12 DFR gene and 7 ANR genes were identified in Ginkgo biloba (Liu et al., 2022 ). In this study, 2 DFR genes and 30 ANR genes were identified in E. ferox , and the number of ANR genes was significantly higher than that of other species. We speculated that ANR plays a more important role in the accumulation of catechins in seed kernels of E. ferox . To study the evolution of the ANR gene family, we compared the number of ANRs in Camellia sinensis and Arabidopsis thaliana . E. ferox have a relatively large number of 30 ANR genes. The ANR gene family expand reason is that E. ferox had experienced a whole genome triplication (WGT) event(Wu et al., 2022b ). Furthermore, we identified that 90% of the duplication types of the ANR gene belong to whole genome duplication (WGD) ( Table S14 ). These results together suggest that ANR genes cloud play important roles in the adaptive to the aquatic environment of E. ferox . Furthermore, we screened and identified the key genes EfDFR-1 and EfANR-11 . Among them, EfANR-11 and CsANR are clustered on the same branch, have similar functions, and both play important roles in the synthesis of catechins. Although EfDFR-1 and CSDFR are not clustered in the same branch, both contain PLN02650 domains. The R2R3-MYB family is a class of transcription factors widely involved in the synthesis and regulation of plant secondary metabolites. Overexpression ZmMYB31 in Arabidopsis thaliana showed that the expression of DFR gene was up-regulated in transgenic plants, resulting in increased catechin accumulation (Fornalé et al., 2010 ). Overexpression of CsMYB5b , the expression of NtANR was up-regulated and catechins were accumulated in large quantities in tobacco leaves (Wang et al., 2018 ). Further, we screened the transcription factor EfMYB5b by transcriptome, qPCR and correlation analysis. Then we verified that it can interact with EfDFR-1 and EfANR-11 , promote their expression, and then positively regulate the synthesis of catechin. The biological functions of transcription factor MYB5 are diverse in different species. AtMYB5 mainly regulates the accumulation of tannin in the inner cortex of Arabidopsis seeds (Xu et al., 2015 ). FaMYB5 promotes the biosynthesis of anthocyanins and PA through F3’H and LAR genes in strawberry fruit (Jiang et al., 2023b). MtMYB5 activates the expression of LAR and ANR genes, thereby promoting the accumulation of PAs in seeds of Medicago truncatula (Liu et al., 2014 ), which is similar to EfMYB5b. Our research has for the first time demonstrated a new mechanism by which the MYB5b transcription factor, in synergy with DFR and ANR, regulates catechins in seed kernels of E. ferox . In addition, catechin biosynthesis is widely reported to be regulated by the conserved MYB-bHLH-WD40 (MBW) complex (Wang et al., 2022a ). CsMYB60 formed complexes with CsbHLH42/MYC1 and CsWD40 to promote CsANR expression and positively regulate catechin synthesis in tea (Li et al., 2020 ). VvMYBPA1 formed a trimer complex with VvMYC2 and VvWDR1, which promotes the accumulation of catechins in grape fruits (Liang et al., 2023 ). Based on the calculation of Pearson correlation coefficients (PCC) among EfDFR-1 , EfANR-11 , EfMYB5b, and bHLH/WD40 genes, we constructed a co-expression network. The results indicate that the bHLH/WD40 complex is closely correlated with EfDFR-1 , EfANR-11 , EfMYB5b (Fig. S3 ). Therefore, we hypothesize that the bHLH/WD40 complex may cooperate with EfMYB5b to coregulate EfDFR-1 and EfANR-11 , thereby influencing catechin synthesis. 5. Conclusion The seed kernels of E. ferox are rich in flavonoid compounds. In this study, catechins were found to be the mainly flavonoid compounds in E. ferox seed kernels. Further, 2 DFRs and 30 ANRs genes were identified from the E. ferox genome. And then we screened the key genes EfDFR-1 and EfANR-11 through RT-qPCR, enzyme activity analysis. In addition, the transcription factor EfMYB5b can respectively bind to EfDFR-1 and EfANR-11 and promote their expression, thereby increasing the content of catechins in the seed kernels of E. ferox . (Fig. 6 ). The present study provides new ideas on the mode of regulation of catechins biosynthesis in plants. Declarations Competing interests The authors declare that there are no competing interests. Acknowledgements This work was supported by, China Agriculture Research System (grant number: CARS-24), Jiangsu seed industry revitalization’Jie Bang Gua Shuai’project (grant number: JBGS [2021] 017), and Yangzhou University qinglan project. CRediT authorship contribution statement Peng Wu : Investigation, Visualization, Roles/Writing-original draft, Writing-review& editing. Chen-Yan Qu : Investigation, Visualization, Roles/Writing-original draft, Writing-review& editing. Tian-Yu Wang : Investigation, Visualization. Wen-Jing Ling : Validation, Data curation. Yue-Rui Fang : Software, Data curation. Yu-Da Guo : Software, Data curation. Meng-Nan He: Formal analysis, Methodology. Shu-Ping Zhao : Supervision. Kai Feng : Supervision. Liang-Jun Li : Conceptualization, Funding acquisition, Project administration, Supervision. 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Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant ( Camellia sinensis ). Int. J. Mol. Sci., 17 (2), 261. https://www.mdpi.com/1422-0067/17/2/261 Supplementary Files Fig.S1.jpg Figure S1 Screening of key genes EfDFR-1 and EfANR-11 for catechin synthesis in E. ferox . (a) The expression of EfDFRs , EfANRs in different organs of E. ferox . (b)The expression of EfDFRs , EfANRs during the development (DAF10-DAF40) in seed kernels of E. ferox . (c) SDS-PAGE electropherograms of purified recombinant EfDFR-1, EfDFR-2, EfANR-11, and EfANR-25. (d) Enzyme activity assays of purified recombinant EfPAL1 and EfPAL2. Values with different letters are significantly different from each other (P < 0.05). Error bars indicate three biological replicates. Fig.S2.jpg Figure S2 Expression of EfMYB5b during different organs and seed kernel development of E. ferox by qPCR and transcriptome together. (a) The expression of EfMYB5b in different organs of E. ferox . (b)The expression of EfMYB5b during the development (DAF10-DAF40) in seed kernels of E. ferox . Values with different letters are significantly different from each other (P < 0.05). Error bars indicate three biological replicates. Fig.S3.jpg Figure S3 The co-expression interaction network with EfDFR-1, EfANR-11, EfMYB5b and bHLH/WD40. SupplementaryFile.xlsx 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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NRGC: Naringenin Chalcone. The data are presented as the mean (n=3) + SD; a, b, c, d indicate significant differences (P\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/43e2e2d0b3193844dfbcc4a7.jpg"},{"id":96711449,"identity":"b27742b3-7ae2-486b-95fe-c610dd94b9bb","added_by":"auto","created_at":"2025-11-25 10:12:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3988736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMotif distribution, conserved domains and phylogenetic analysis. \u003c/strong\u003e(a) Phylogenetic tree of \u003cem\u003eEfDFRs\u003c/em\u003e. (b) Motif distribution, conserved domains for the 2 putative DFRs in \u003cem\u003eE. ferox\u003c/em\u003e. (c) Phylogenetic tree of \u003cem\u003eEfANRs\u003c/em\u003e. (d) Motif distribution, conserved domains for the 30 putative ANRs in \u003cem\u003eE. ferox\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/5df64ee7d9dd0b52027bf858.jpg"},{"id":96710813,"identity":"c1b7031c-8455-4e98-b72f-3c8fe42d8625","added_by":"auto","created_at":"2025-11-25 10:11:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2901424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening and functional identification of key genes for catechin synthesis. \u003c/strong\u003e(a-b) HPLC analysis of the product of enzyme assays using recombinant EfDFR-1 and EfANR-11. (c) Relative expression of itself and catechins content after transient overexpression of \u003cem\u003eEfDFR-1\u003c/em\u003e, \u003cem\u003eEfANR-11\u003c/em\u003e. (d) Subcellular localization of EfDFR-1 and EfANR-11 in tobacco. Bars=50 μm. The '*' or '**' above the histogram indicated the statistical significance at the level of 0.05 or 0.01(p \u0026lt; 0.05; p \u0026lt; 0.01). Error bars show SD from three biological replicates.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/ef7286ec6edf3be65edaf1f8.jpg"},{"id":96699685,"identity":"d7590d63-bce0-4353-a1d5-5642ffe9c30c","added_by":"auto","created_at":"2025-11-25 08:11:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4106352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfMYB5b binds to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEfDFR-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter and positively regulates catechin synthesis. \u003c/strong\u003e(a) Subcellular localization of EfMYB5b in tobacco. (b) Y1H assay used to identify the binding of EfMYB5b and the\u003cem\u003e EfDFR-1\u003c/em\u003e promoter. (c) LUC assay used to identify the regulation of EfMYB5b on the\u003cem\u003e EfDFR-1\u003c/em\u003e promoter. (d) EMSA used to identify the binding of EfMYB5b and the \u003cem\u003eEfDFR-1\u003c/em\u003e promoter. (e)Relative expression of \u003cem\u003eEfDFR-1\u003c/em\u003e and catechins content after transient overexpression of \u003cem\u003eEfMYB5b\u003c/em\u003e. Bars=50μm. SD/-Leu, SD medium lacking Leu; SD/-Leu+AbA\u003csup\u003e200\u003c/sup\u003e, SD medium with 200 ng/mL AbA lacking Leu; AbA, Aureobasidin A; Leu, leucine. The '*' above the histogram indicated the statistical significance at the level of 0.05 (p \u0026lt; 0.05). Error bars show SD from three biological replicates.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/8cf542682b224be2d8a573aa.jpg"},{"id":96699678,"identity":"3b1b2d27-5db7-402a-9798-6d4d67674ce3","added_by":"auto","created_at":"2025-11-25 08:11:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3365554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfMYB5b binds to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEfANR-11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter and positively regulates catechin synthesis. \u003c/strong\u003e(a) Y1H assay used to identify the binding of EfMYB5b and the\u003cem\u003e EfANR-11\u003c/em\u003e promoter. (b) LUC assay used to identify the regulation of EfMYB5b on the\u003cem\u003e EfANR-11\u003c/em\u003e promoter. (c) EMSA used to identify the binding of EfMYB5b and the \u003cem\u003eEfANR-11\u003c/em\u003e promoter. (d) Relative expression of \u003cem\u003eEfANR-11\u003c/em\u003e and catechins content after transient overexpression of \u003cem\u003eEfMYB5b\u003c/em\u003e. Bars=50μm. SD/-Leu, SD medium lacking Leu; SD/-Leu+AbA\u003csup\u003e100\u003c/sup\u003e, SD medium with 100 ng/mL AbA lacking Leu; AbA, Aureobasidin A; Leu, leucine. Values with different letters are significantly different from each other (P \u0026lt; 0.05). Error bars indicate three biological replicates.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/3c12c5c33c9cf9fb5b9a0a11.jpg"},{"id":96710892,"identity":"a12610ee-58a4-4d7a-b438-31eba8ddc808","added_by":"auto","created_at":"2025-11-25 10:11:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":769895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypothetical model of EfMYB5b mediating catechins accumulation through regulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEfDFR-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEfANR-11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/1f2ee4f3710c584956d93136.jpg"},{"id":99797607,"identity":"9292a1a4-6d62-4758-baa0-3bbc46d97636","added_by":"auto","created_at":"2026-01-08 13:46:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14312533,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/6923e0c6-6206-484f-b0b5-fa31ab52c8f4.pdf"},{"id":96699679,"identity":"fef16e9e-3b28-45f2-8de9-684812c08437","added_by":"auto","created_at":"2025-11-25 08:11:47","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5488742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1 Screening of key genes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEfDFR-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and EfANR-11 for catechin synthesis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. ferox\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(a) The expression of \u003cem\u003eEfDFRs\u003c/em\u003e, \u003cem\u003eEfANRs\u003c/em\u003e in different organs of \u003cem\u003eE. ferox\u003c/em\u003e. (b)The expression of \u003cem\u003eEfDFRs\u003c/em\u003e, \u003cem\u003eEfANRs\u003c/em\u003e during the development (DAF10-DAF40) in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e. (c) SDS-PAGE electropherograms of purified recombinant EfDFR-1, EfDFR-2, EfANR-11, and EfANR-25. (d) Enzyme activity assays of purified recombinant EfPAL1 and EfPAL2. Values with different letters are significantly different from each other (P \u0026lt; 0.05). Error bars indicate three biological replicates.\u003c/p\u003e","description":"","filename":"Fig.S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/dccd007d446bce7040c7e6e7.jpg"},{"id":96699677,"identity":"3f793b38-97f7-4c1e-a5ed-243458d6028a","added_by":"auto","created_at":"2025-11-25 08:11:47","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":801241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2 Expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEfMYB5b \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eduring different organs and seed kernel development of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. ferox\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by qPCR and transcriptome together.\u003c/strong\u003e (a) The expression of \u003cem\u003eEfMYB5b\u003c/em\u003e in different organs of \u003cem\u003eE. ferox\u003c/em\u003e. (b)The expression of \u003cem\u003eEfMYB5b\u003c/em\u003e during the development (DAF10-DAF40) in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e. Values with different letters are significantly different from each other (P \u0026lt; 0.05). Error bars indicate three biological replicates.\u003c/p\u003e","description":"","filename":"Fig.S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/32d03b478af762fc0d526d10.jpg"},{"id":96711458,"identity":"3a803cd2-7b0b-4cec-9e61-88cdb7bd90c3","added_by":"auto","created_at":"2025-11-25 10:12:02","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2860293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3 The co-expression interaction network with EfDFR-1, EfANR-11, EfMYB5b and bHLH/WD40.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.S3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/50d60dc45c35206bd941de8a.jpg"},{"id":96699676,"identity":"3a1a1059-3e73-4742-abcd-f98efa88d040","added_by":"auto","created_at":"2025-11-25 08:11:47","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":58688,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8027490/v1/6b17b058501bf6428e94436b.xlsx"}],"financialInterests":"","formattedTitle":"Molecular mechanisms of catechins regulation by EfMYB5b in Euryale ferox","fulltext":[{"header":"Key Message","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eEfMYB5b positively regulates the synthesis of catechins in the seed kernels of\u0026nbsp;\u003cem\u003eEuryale ferox\u003c/em\u003e by promoting the expression of\u0026nbsp;\u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eCatechins, namely flavan-3-ols, are a class of polyphenolic compounds belonging to flavonoid compounds (Gadkari et al., 2015). It has the basic structure of 2-phenylbenzopyran (C6-C3-C6), comprising A-, C-, and B-rings. Catechins can be classified into two groups, one group of trans flavonoid 3 alcohols includes (+)-catechin (C) and (-)-gallocatechin (GC) (Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). And another group of cis flavonoid 3 alcohols includes (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) (Zhang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The latter being the predominant type among the catechins (Wang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Catechins, recognized as vital defensive compounds that protect plants from ultraviolet(UV) radiation, pathogens, and pests (Petrussa et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), are also scientifically validated to provide diverse health benefits for humans. By comparing the different infection stages of disease-resistant tea and susceptible tea plants, it was found that the content of EC in disease-resistant tea plants was significantly higher than that in susceptible tea plants, and EGCG was the opposite (Punyasiri et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In addition, many studies have confirmed that catechins can combat a wide range of diseases (Wolfram et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), such as anti-cancer, anti-obesity, anti-diabetic, neuroprotective and reducing the risk of coronary heart disease (Kapoor et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Thomasset et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), moderate intake of catechins can also improve overall health (Isemura, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, a deeper study of the regulatory mechanisms of catechins is of great research importance.\u003c/p\u003e\u003cp\u003eThe biosynthesis of catechins, through the phenylpropanoid and flavonoid biosynthetic pathways (Xiong et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), is a large and complex process involving the participation of multiple enzymes and genes. Firstly, in benzene propane way, L-Phenylalanine via phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumaroyl-CoA ligase (4CL) continuous catalytic, the resulting 4-coumaroyl-CoA. Subsequently, chalcone synthase (CHS) converts 4-coumaroyl-CoA into chalcone, which was further catalyzed by chalcone isomerase (CHI) to produce naringenin (Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Then, naringenin undergoes hydroxylation by flavanone 3-hydroxylase (F3H) and flavonoid 3',5'-hydroxylase (F3'5'H), followed by dihydroflavonol 4-reductase (DFR) mediated reduction to leucoanthocyanidins (Zhou et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Downstream of DFR, the pathway bifurcates into two distinct branches, one is leucoanthocyanidin reductase (LAR) directly converts leucoanthocyanidins to catechin (C) and gallocatechin (GC). Another is anthocyanidin synthase (ANS) oxidizes leucoanthocyanidins to cyanidin and delphinidin, which are subsequently reduced by anthocyanidin reductase (ANR) to produce epicatechin (EC) and epigallocatechin (EGC) (Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among them, DFR is a key enzyme in the synthesis of anthocyanins and proanthocyanidins. It can catalyze the synthesis of colorless anthocyanins from the precursor substance dihydroflavonol, which is the common precursor substance of biosynthesis such as catechin and proanthocyanidins. Catechin epimerization, primarily mediated by the enzymatic activities of LAR,ANS, and ANR, serves as a critical determinant in establishing the equilibrium between epicatechin derivatives (EGCG, ECG, EGC, EC) and their non-epi counterparts (GCG, CG, GC, C) (Pang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Punyasiri et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Notably, ANR plays a dual catalytic role in this biochemical process, not only participating in the biosynthesis of flavan-3-ol monomers through the conversion of anthocyanidins to epicatechin (EC) and epigallocatechin (EGC), but also contributing to the epimerization regulation (Ashihara et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn plants, the biosynthesis of catechins is regulated by transcription factors. These transcription factors regulate the expression of one or more structural genes in this pathway by binding to cis-acting elements in structural gene promoters, thereby regulating catechin biosynthesis. Studies have demonstrated that the biosynthesis of catechins is regulated by multiple transcription factors, including NAC, MYB, bHLH, and others (Meraj et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, overexpression of \u003cem\u003eCsMYB5\u003c/em\u003e was found to significantly increase the expression of \u003cem\u003eCsANR\u003c/em\u003e, thereby promote catechin synthesis (Wang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Based on DAP-seq verification, it was found that CsMYB196 directly combines with \u003cem\u003eCsANR\u003c/em\u003e to promote the synthesis of catechins (Shan et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, CsMYBL2 can promote the expression of \u003cem\u003eCsANR\u003c/em\u003e and thus promote the accumulation of catechins (Zhao et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). VviMYBC2-L1 and VviMYBC2-L2 negatively regulate the expression of \u003cem\u003eVviDFR\u003c/em\u003e and inhibit the biosynthesis of proanthocyanidins and catechins in the green fruit stage of grape growth and development (Vale et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eEuryale ferox\u003c/em\u003e, is an annual floating-leaved macrophyte aquatic herb that belongs to the genus \u003cem\u003eEuryale\u003c/em\u003e from the angiosperm basal plant family Nymphaeaceae, and is one of the important characteristic aquatic vegetables in China (Wu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). The Chinese Pharmacopoeia records that \u003cem\u003eE. ferox\u003c/em\u003e mainly has the effects of tonifying the spleen, stopping diarrhoea, benefiting the kidneys and fixing the sperm, etc (Jiang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), and it is a typical representative of homology of medicine and food. The seed kernel of \u003cem\u003eE. ferox\u003c/em\u003e is the main edible organ, is characterized by a rich nutritional profile comprising flavonoids, carbohydrates, proteins, and other bioactive compounds. Flavonoid compounds are the main efficacy components of \u003cem\u003eE. ferox\u003c/em\u003e (Naik et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Proteomic analysis of seed kernels at different periods showed that PAL, F3H, FLS, DFR, and ANS were key enzymes in \u003cem\u003eE. ferox\u003c/em\u003e flavonoid biosynthesis (Wu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). A total of 129 flavonoid substances were identified in \u003cem\u003eE. ferox\u003c/em\u003e, and the content of flavonoid compounds reaches the highest level in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e 30 days after flowering (DAF30) (Wu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), but the main components of flavonoid compounds have not been determined.\u003c/p\u003e\u003cp\u003eIn this study, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology was used to identify the main flavonoids in seeds kernel. Subsequently, two key genes \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e, were identified through integrated transcriptomic profiling, RT-qPCR validation, and enzyme activity assays. Their functional roles were further characterized via subcellular localization analysis and transient overexpression experiments. The transcriptional activation of key catechin biosynthetic genes by EfMYB5b was further validated through yeast one-hybrid (Y1H) assays, dual-luciferase reporter (LUC) systems, and electrophoretic mobility shift assays (EMSA). Finally, transient overexpression trials further established EfMYB5b's capacity to modulate catechin accumulation. Collectively, this systematic approach elucidates critical genes and regulatory networks governing catechin biosynthesis, providing a theoretical foundation for targeted quality improvement and cultivar development in \u003cem\u003eE. ferox\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plant material and sample collection\u003c/h2\u003e\u003cp\u003e'ZHSQ' was planted in the aquatic vegetable experimental base of Yangzhou University, under normal cultivation and management (Yangzhou, China, 2024). The seed kernels were collected at four periods of DAF10 (days after flowering), DAF20, DAF30, and DAF40, with three biological replicates for each period. All samples were snap frozen in liquid nitrogen immediately after collection and kept at -80\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Determination of flavonoid content\u003c/h2\u003e\u003cp\u003eThe seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e DAF30 were quantitatively analysed for flavonoid compounds using UPLC-MS/MS. Ultra Performance Liquid Chromatography (UPLC) (ExionLC\u0026trade; AD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Tandem Mass Spectrometry (MS/MS) (QTRAP\u0026reg; 6500+, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for data acquisition. A MWDB (Metware Database) database was constructed based on the standards to qualitatively analyse the data detected by mass spectrometry. Analyst 1.6.3 software was used to process the mass spectrometry data. The above data are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Sequence identification of EfDFRs, EfANRs\u003c/h2\u003e\u003cp\u003eDFR genes from \u003cem\u003eCamellia sinensis\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were utilized for Blast in the \u003cem\u003eE. ferox\u003c/em\u003e genome. Then, pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were performed to further identify the candidate genes for DFR and ANR. Molecular weight and isoelectric point were finally predicted by ExPASy tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.expasy.org/\u003c/span\u003e\u003cspan address=\"https://www.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Analysis of conserved motifs and domains of candidate genes\u003c/h2\u003e\u003cp\u003eGene structure information was obtained from the GFF file of the \u003cem\u003eE. ferox\u003c/em\u003e genome and conserved motifs were analyzed in the MEME suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, Tbtools software was used for visualization (Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The relevant results are shown in \u003cb\u003eTables S2\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Phylogenetic tree analysis of candidate genes\u003c/h2\u003e\u003cp\u003eAmino acid sequence comparison was performed using MEGA 11 software, followed by neighbour-joining method for the construction of phylogenetic tree, which was further refined using iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/tree\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/tree\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The related gene sequences are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Quantitative real-time PCR analysis (RT-qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from \u003cem\u003eE. ferox\u003c/em\u003e seed kernels at different developmental stages (DAF10-DAF40) and different \u003cem\u003eE. ferox\u003c/em\u003e organs using an RNA extraction kit (Takara, Dalian, China). The extracted RNA was reverse transcribed into cDNA by applying HiScript\u0026reg; II Q RT SuperMix (Vazyme, Nanjing, China). \u003cem\u003eEfUBQ5\u003c/em\u003e (ID: EF11G001150) was used as internal reference genes (Wu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022c\u003c/span\u003e). The CFX-96 real-time fluorescence quantitative PCR system (Bio-Rad) was used for amplification, and the amplification program was 95\u0026deg;C for 30s, 95\u0026deg;C for 10s, and 60\u0026deg;C for 30s, for a total of 40 cycles. qPCR concentration and amplification efficiency were also determined. The 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method was used for the analysis of RT-qPCR data(Zhao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The relevant primers are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Subcellular localisation\u003c/h2\u003e\u003cp\u003eUtilizing Plant-mPloc (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) predicting the specific location of EfDFR-1, EfANR-11 and EfMYB5b proteins within a cell. Tobacco transient expression system was applied to analyse the subcellular localisation of EfDFR-1, EfANR-11 and EfMYB5b. The CDS sequence of these genes were constructed through the method of homologous recombination into the pCAMBIA1300-35S-sGFP vector, resulting in p35S::EfDFR-1-GFP, p35S::EfANR-11-GFP and p35S::EfMYB5b-GFP. Using the freeze-thaw method, the recombinant plasmid construct was transferred into the Agrobacterium GV3101 strain. Subsequently, it was expressed in tobacco leaves through the Agrobacterium infection method. Bacterial cultures were resuspended to OD₆₀₀ = 0.8-1.0 in infiltration buffer (10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5M MES, 100 mM acetosyringone, pH 5.8). Needleless injector was used to inject on the abaxial surface of the tobacco leaf until the entire leaf was submerged. The plants were cultured at room temperature in the dark for three days. The fluorescence signals were observed under an ultra-high resolution laser confocal microscope, with the unloaded state serving as the control. The above primers are listed in \u003cb\u003eTable S5\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Enzyme activity assay\u003c/h2\u003e\u003cp\u003eThe CDS regions of \u003cem\u003eEfDFR-1\u003c/em\u003e, \u003cem\u003eEfDFR-2\u003c/em\u003e, \u003cem\u003eEfANR-11\u003c/em\u003e and \u003cem\u003eEfANR-25\u003c/em\u003e were ligated into the pCold-TF vector, and then the recombinant plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 for prokaryotic expression. Select a single colony, incubate it in 100 mL of LB liquid medium containing 50 mg/L Amp at 37\u0026deg;C, 200 rpm in a shaking incubator for 4\u0026ndash;6 hours until the OD600 reaches approximately 0.5. Add 1 mM of IPTG (isopropyl-β-D-thiogalactopyranoside), and incubate it at 16\u0026deg;C, 100 rpm on a shaking incubator for 16 hours. Further, collect the bacterial suspension by centrifugation at 4\u0026deg;C, 4000 rpm for 20 minutes, discard the supernatant and add 5 mL of lysis buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, 10 mM β-mercaptoethanol, 10 mM imidazole) for suspension. Use low-temperature ultrasonic disruption for 20 minutes, centrifuge at 10000 rpm for 10 minutes, and elute the target protein using a resin elution column. The fused protein was subjected to Ni-Agarose column (CWBIO, Jiangsu, China) to elute the target protein with gradient imidazole concentration (100\u0026ndash;500 mM). And single-purpose bands were obtained by SDS-PAGE electrophoresis, respectively. The reaction system was as follows (1 mL): 300 \u0026micro;g of recombinant protein, 100 mM phosphate buffer (pH 6.5), 20 mM NADPH, and 1 mM substrate. After incubation at 45℃ for 30 minutes, 1 mL of methanol was added to terminate the reaction. The mixture was then shaken evenly and centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected. The filtrate was filtered through a 0.22 \u0026micro;m filter membrane and analyzed using UPLC. The products of the reaction were analyzed by HPLC (Agilent 1260, USA). A C18 column (4.6 \u0026times;250 mm) was applied with 1% acetic acid in ultrapure water (v/v) as solvent A and 100% acetonitrile solvent B. The elution gradients were as follows: 20% B at 0 min, 95% B at 15 min, and 20% B at 15.01 min. A flow rate of 1.0 mL/min and a column temperature of 25\u0026deg;C were applied and the chromatogram was obtained at 280 nm. A list of the above primers is shown in \u003cb\u003eTable S6\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Transient overexpression assay\u003c/h2\u003e\u003cp\u003eThe gene was constructed into pCAMBIA1300 vector. The correctly sequenced plasmid was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 (Miao et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The successfully transformed \u003cem\u003eAgrobacterium\u003c/em\u003e solution was mixed with an infection solution containing acetosyringone, and the OD\u003csub\u003e600\u003c/sub\u003e was adjusted to 0.8 and left in the dark for 2\u0026ndash;3 hours. The \u003cem\u003eE. ferox\u003c/em\u003e seed kernels of DAF30 were immersed in the aforementioned solution. (Wei et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The materials used were fresh 30 days after flower opening seed kernels harvested from our experimental field. After cleaning it thoroughly, it was immediately disinfected with 75% ethanol in clean bench for 15 seconds, then disinfected with 3% hypochlorous acid for 10 minutes. The seeds were then placed in the infection solution containing positive Agrobacterium in the dark environment and infected for 30 minutes. Then, the kernels were washed three times with sterilized water for 1 minute each time. The surface moisture was then absorbed using sterilized filter paper. The kernels were then inoculated onto the MS medium and incubated in the dark for 3\u0026ndash;4 days. The experiment was repeated three times, with 6 kernels being tested each time. The 6 kernels were mixed and ground into powder, and then the content of catechins and RNA extraction were carried out. The above primers are listed in \u003cb\u003eTable S5\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Relevance analysis\u003c/h2\u003e\u003cp\u003eCorrelation analyses of \u003cem\u003eEfMYB5b\u003c/em\u003e and \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e were performed by Qmic studio (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omicstudio.cn/video\u003c/span\u003e\u003cspan address=\"https://www.omicstudio.cn/video\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Yeast one-hybrid (Y1H) assay\u003c/h2\u003e\u003cp\u003eUtilizing PLANTCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) predicted binding sites of EfMYB5b to key genes. Sequences of \u003cem\u003eEfMYB5b\u003c/em\u003e was inserted into the pGADT7 vector. \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e were ligated into the pAbAi vector. Positive bacterial fluids were sent to the company for sequencing and plasmid extraction. The p\u003cem\u003eEfDFR-1\u003c/em\u003e-pAbAi, p\u003cem\u003eEfANR-11\u003c/em\u003e-pAbAi plasmid was linearised using \u003cem\u003eBbs\u003c/em\u003e I restriction endonuclease, and cultured in SD/-Leu\u0026thinsp;+\u0026thinsp;AbA\u003csup\u003e200\u003c/sup\u003e medium at 30\u0026deg;C for 3\u0026ndash;5 days. The above primers are listed in \u003cb\u003eTable S7\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Dual-luciferase reporter assay (LUC)\u003c/h2\u003e\u003cp\u003eThe coding sequences of EfDFR-1, EfANR-11 and EfMYB5b were cloned into the pGreenII-0800 and pGreenII-62-SK reporter vectors, respectively, after which the after which the reporter plasmid was transferred into Agrobacterium tumefaciens strain GV3101 and then diluted with infection solution (100mM acetosyringone, 0.5M MES, 10mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH5.8); Agrobacterium containing pGreenPro-\u003cem\u003eEfDFR-1\u003c/em\u003e and pGreenPro-\u003cem\u003eEfANR-11\u003c/em\u003e was mixed with strains containing pGreenII62-SK-EfMYB5b at a ratio of 1:1 (v/v). Adjust the OD600 value of the bacterial solution to 0.8-1.0, leave it in the dark for 3 hours, and then inject it into the tobacco leaves. At the same time, the leaves were sprayed with 100 mM luciferin and incubated for 10 minutes. The LUC luminescence signal was detected using the GelView 6000ProⅡ multi-functional imaging workstation. The above primers are listed in \u003cb\u003eTable S8\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Electrophoretic Mobility Shift Assay (EMSA)\u003c/h2\u003e\u003cp\u003eThe full-length EfMYB5b fragment was inserted into pCold-TF vector, and the prokaryotic expression vector pCold-TF-EFMYB5b was obtained, and the purified protein was obtained. Use the EMSA probe biotin labeling kit (Beyotime, China) and follow the instructions. The probe was synthesized by Sangon. SDS-PAGE electrophoresis detection was performed after the probe reaction. After the steps of coating, transfer and fixation, electrophoretic transfer was carried out, and then the gel was cross-linked in the purple diplomatic linkage instrument for chemiluminescence reaction and photographed for observation. The primers of the probe are shown in \u003cb\u003eTable S9\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Statistical analysis\u003c/h2\u003e\u003cp\u003eThree independent biological replicates were used for each sample in each experiment. Experimental data were analyzed and visualized using GraphPad Prism 8 software. Statistical significance was tested using multiple t tests (* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Catechins are main flavonoid compounds in \u003cem\u003eE. ferox\u003c/em\u003e seed kernels\u003c/h2\u003e\u003cp\u003eDuring the developmental process of \u003cem\u003eE. ferox\u003c/em\u003e seed kernels, flavonoid compounds exhibit significant accumulation at DAF30 (Wu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, UPLC-MS/MS was employed to determine the types and contents of flavonoid compounds in DAF30 \u003cem\u003eE. ferox\u003c/em\u003e seed kernels. The results identified 31 flavonoid compounds across six categories: flavones, flavonols, flavanols, dihydroflavonols, chalcones, and flavone C-glycosides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among them, the content of flavanols was the highest, mainly including EGCG (87.45ng/g DW), GC (54.48ng/g DW), EC (22.91ng/g DW), CG (12.90ng/g DW), GCG (7.08ng/g DW), C (5.23ng/g DW). Secondly, there are chalcones, mainly including PRO B2(Procyanidin B2) and MHYOG(Medicarpin), with their contents being 1.62 and 1.07 ng/g DW. The contents of flavones, flavonols, dihydroflavonols and flavone C-glycosides were relatively low. Their concentrations range from 0.002 to 0.91 ng/g DW (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe comparative analysis demonstrated pronounced accumulation of flavanols in the seed kernels, with catechins constituting the highest proportion at 96.07% of the total detected flavonoids, and approximately 24 times the combined concentration of all other flavonoid subclasses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings indicate that catechins are among the most critical bioactive components in \u003cem\u003eE. ferox\u003c/em\u003e seed kernels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Identification of DFRs and ANRs gene in \u003cem\u003eE. ferox\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe 6 candidate DFR and 38 ANR genes were identified in \u003cem\u003eE. ferox\u003c/em\u003e genome by blast. Then, 2 \u003cem\u003eEfDFRs\u003c/em\u003e and 30 \u003cem\u003eEfANRs\u003c/em\u003e were screened with pfam and SMART (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). The analysis of the physicochemical properties revealed that there was a wide variation in their molecular weights (Mw), ranging from 31.49 KDa to 64.05 KDa, with isoelectric points (pI) between 5.35 and 9.11 (\u003cb\u003eTable S10\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eFurther, we used DFR proteins to construct phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The results showed that \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfDFR-2\u003c/em\u003e clustered together with \u003cem\u003eOsDFR1\u003c/em\u003e, \u003cem\u003eOsDFR2\u003c/em\u003e, and \u003cem\u003eOsDFR3\u003c/em\u003e. It indicated that the \u003cem\u003eEfDFRs\u003c/em\u003e has higher similarity with the \u003cem\u003eOsDFRs\u003c/em\u003e sequence and functions. Then, the EfDFRs domains have typical PLN02650 structural features, which is a specific site of the DFR protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). And motif analysis showed that EfDFRs contained motif1\u0026thinsp;~\u0026thinsp;motif8, and the arrangement was consistent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Meanwhile, the ANR phylogenetic tree was constructed by using the \u003cem\u003eE. ferox\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and \u003cem\u003eCamellia sinensis\u003c/em\u003e ANR proteins. The 30 \u003cem\u003eEfANRs\u003c/em\u003e genes can be divided into 5 subfamilies. The distribution proportions of ANR family members of the three species in each subfamily are different, and there are also differences in cluster analysis among different plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Further analysis revealed that the majority of EfANRs have a relatively high homology with AtANRs and CsANRs, which might indicate that the ANR of plants is evolutionarily conserved. Similarly, EfANRs conserved motifs and domains are visualized. Most of EfANRs contain motif1\u0026thinsp;~\u0026thinsp;motif10 and are arranged relatively consistently in different subgroups, indicating that these motifs are highly conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Besides, EfANR-5 and EfANR-12 contained 5 motifs, while EfANR-1, EfANR-8, EfANR-9, and EfANR-13 only contained 4 motifs. A total of 5 conserved domains were identified in EfANRs. 15 EfANRs contained NADB_Rossmann superfamily and 10 EfANRs contained FR_SDR_e specific domain. Other EfANRs contained Reticulon, PLN00198 superfamily and PLN02896 superfamily respectively. They all play an important role in the NADP-dependent reduction of flavonoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3 \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e are key candidate genes for catechins biosynthesis in \u003cem\u003eE. ferox\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTranscriptome and RT-qPCR analysis showed that \u003cem\u003eEfANR-1\u003c/em\u003e, \u003cem\u003eEfANR-11\u003c/em\u003e, \u003cem\u003eEfANR-15\u003c/em\u003e, and \u003cem\u003eEfDFR-1\u003c/em\u003e were highly expressed in different organs of \u003cem\u003eE. ferox\u003c/em\u003e and their expression levels were higher in seeds (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, Table S11\u003c/b\u003e). \u003cem\u003eEfDFR-2\u003c/em\u003e was almost not expressed, while \u003cem\u003eEfANR-25\u003c/em\u003e expression was relatively low, but significantly higher in seed than in other tissues. In seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e at different developmental stages (DAF10-DAF40), the expression of \u003cem\u003eEfDFR-1\u003c/em\u003e was 35.51 times that of \u003cem\u003eEfDFR-2\u003c/em\u003e, and the expression of \u003cem\u003eEfANR-11\u003c/em\u003e and \u003cem\u003eEfANR-25\u003c/em\u003e was significantly higher than that of other candidate genes (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb, Table S12\u003c/b\u003e). Therefore, \u003cem\u003eEfANR-11\u003c/em\u003e, \u003cem\u003eEfANR-25\u003c/em\u003e and \u003cem\u003eEfDFR-1\u003c/em\u003e may be the important genes for catechin synthesis in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn order to further verify the enzymatic activity of EfDFR-1, EfDFR-2, EfANR-11 and EfANR-25, their sequences were cloned into pCold vector, and single target bands were obtained by SDS-PAGE gel electrophoresis (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec\u003c/b\u003e). At the same time, enzyme activity was measured with the same amount of protein solution. The results showed that the enzyme activity of EfDFR-1 was 5.56 times that of EfDFR-2, and the enzyme activity of EfANR-11 was 2.41 times that of EfANR-25 (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed\u003c/b\u003e). Finally, centaurea was reacted with purified EfANR-11 protein by High-performance liquid chromatography (HPLC) with centaurea standard and inactivated enzyme protein as controls. The results showed that no substance was detected in the control reaction solution, while the reaction solution containing the prokaryotic recombinant protein EfANR-11 on the cyanidin substrate was detectable in the gallocatechin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). DFR and LAR are closely linked two-step catalytic enzymatic reaction and both depend on NADPH. Because of the high instability of leucoanthocyanidins, it is difficult to be detected and cannot be directly used as the substrate of enzymatic reaction to determine the activity of recombinant protease alone. We used dihydromyricetin as substrate to react with purified EfDFR-1 and EfLAR-1 protein solution, and found that epigallocatechin could be detected in the reaction solution of EfDFR-1 and EfLAR-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). To further determine the role of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e in catechins biosynthesis, transient overexpression experiments were performed. The expression changes of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e were analyzed by RT-qPCR, and the contents of catechin in overexpressed seed kernels were determined. The results showed that the expression of \u003cem\u003eEfDFR-1\u003c/em\u003e was significantly increased after overexpression and the catechin content of \u003cem\u003eEfDFR-1\u003c/em\u003e overexpressed seed kernels was 1.2 times higher than that of the control. The expression of \u003cem\u003eEfANR-11\u003c/em\u003e and the catechin content of \u003cem\u003eEfANR-11\u003c/em\u003e overexpressed seed kernels was significantly increased, too (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUltimately, to determine the localization of EfDFR-1 and EfANR-11, recombinant vectors and empty vector were introduced into tobacco epidermal cells. The results showed that the fluorescence signals of p35S:EFDFR-1-GFP and p35S:EFANR-11-GFP were only observed in the Golgi apparatus, indicating that EfDFR-1 and EfANR-11 were located in the Golgi apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The results were consistent with those predicted by Plant-mPloc.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4 EfMYB5b positively regulates \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e expression, and promotes catechins biosynthesis\u003c/h2\u003e\u003cp\u003eTranscriptome and qPCR analysis found that the expression trend of \u003cem\u003eEfMYB5b\u003c/em\u003e in seeds of \u003cem\u003eE. ferox\u003c/em\u003e at different periods was consistent with the expression trend of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e(\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). And correlation analysis showed that \u003cem\u003eEfMYB5b\u003c/em\u003e was positively correlated with \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e (\u003cb\u003eTable. S13\u003c/b\u003e). In addition, in the PLANTCARE database, it was found that there are respectively the binding sites TAACCA and ACCTAC for EfMYB5b on the promoters of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e. Therefore, \u003cem\u003eEfMYB5b\u003c/em\u003e may be an important transcription factor in the regulation of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results of subcellular localization experiments showed that the fluorescence signal of p35S::EfMYB5B-GFP was only visible in the nucleus and overlapped with nuclear markers, suggesting that EfMYB5b is nuclear-localized transcription factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). To further verify whether EfMYB5b protein could interact with \u003cem\u003eEfDFR-1\u003c/em\u003e, we conducted the Y1H experiment. The results showed that AD\u0026thinsp;+\u0026thinsp;\u003cem\u003eEfDFR-1\u003c/em\u003epromoter could not grow on SD-Leu\u003csup\u003e200\u003c/sup\u003e medium, while EfMYB5b-AD\u0026thinsp;+\u0026thinsp;\u003cem\u003eEfDFR-1\u003c/em\u003epromoter could grow on SD-Leu\u003csup\u003e200\u003c/sup\u003e medium, which indicated that EfMYB5b protein could directly bind to \u003cem\u003eEfDFR-1\u003c/em\u003e promoter and thus regulate its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Subsequently, LUC assay was used to investigate the regulation of \u003cem\u003eEfDFR-1\u003c/em\u003e by EfMYB5b protein. When EfMYB5b and \u003cem\u003eEfDFR-1\u003c/em\u003e were co-expressed in tobacco leaves, LUC activity was significantly increased, and the relative expression of luciferase increased 3.3 times (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The Y1H results indicated that EfMYB5b can interact with the promoter of EfANR-11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Similarly, dual-luciferase assay was performed in tobacco leaves. The results showed that the co-expression of EfANR-11pro::LUC and 35S::EfMYB5b in tobacco leaves significantly increased the activity of LUC, and the relative expression of luciferase increased by 1.9 times (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This indicating that EfMYB5b could promote the expression of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further verify whether the EfMYB5b protein can bind to the \u0026lsquo;TAACCA\u0026rsquo; and \u0026lsquo;ACCTAC\u0026rsquo; site, we designed biotin-labeled probes containing the binding site for the EMSA assays. The purified HIS- EfMYB5b fusion protein was confirmed by Western blot and successfully obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). EMSA results showed that EfMYB5b binds to the labeled probe. When unlabeled probes (cold probes) were added, the binding band significantly weakened (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), indicating that EfMYB5b can specifically bind to the \u0026lsquo;TAACCA\u0026rsquo; and \u0026lsquo;ACCTAC\u0026rsquo; site. These results suggest that EfMYB5b can target multiple key enzyme genes \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e involved in the synthesis of catechins.\u003c/p\u003e\u003cp\u003eFinally, the CDS sequence of EfMYB5b was constructed onto the vector pCAMBIA1300, and the empty vector was used as the negative control for overexpression. The treated seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e was soaked in the bacterial solution an cultured on MS medium for 4 days. RT-qPCR showed that the expression of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e in overexpressed seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The content of catechins also significantly increased in the seed kernels after overexpression of \u003cem\u003eEfMYB5b\u003c/em\u003e compared to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). These experimental results consistently indicated that EfMYB5b could promote the expression of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e, thus promoting the accumulation of catechins in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eFlavonoids are polyphenolic compounds that are widely distributed in plants and are rich in functions (Nabavi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). An increasing number of flavonoids in plants have been shown to be functional components and research on their biosynthesis has been intensified (Wu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The main component of flavonoid compounds in tea is catechin, and catechin is an important functional component of tea (Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A rich variety of flavonoid compounds have been identified in the seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e and significant accumulation of flavonoid compounds at DAF30 seed kernels. A total of 129 metabolites measured were identified, including favanones, dihydrofavanols, favanols, favones, isofavones, anthocyanins, favonols, favonoid carbonosides, chalcones and proan-thocyanidins (Wu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, which substance is the main component of flavonoid compounds in \u003cem\u003eE. ferox\u003c/em\u003e seed kernels has not yet been determined. In our study, it was found that the content of catechins was the highest among flavonoids, by using UPLC-MS/MS to analyze the types and contents of flavonoid compounds in DAF30 seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e. Therefore, we speculate that the high content of catechins may be an important functional component of \u003cem\u003eE. ferox\u003c/em\u003e seed kernels.\u003c/p\u003e\u003cp\u003eDFR and ANR are key enzymes in catechins biosynthesis. Studies have shown that DFR gene expression is significantly correlated with catechin accumulation during leaf development of different tea cultivars (Mamati et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Overexpressed the \u003cem\u003eCsDFR\u003c/em\u003e and \u003cem\u003eCsANR\u003c/em\u003e genes increased catechin content and improvied its antioxidant capacity in tobacco (Kumar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Besides, three \u003cem\u003eMdANRs\u003c/em\u003e were overexpressed, the contents of catechin and epicatechin were significantly increased in tobacco (Han et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A total of 5 DFR genes and 21 ANR genes were identified in tea (Duan et al., 2023; Punyasiri et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), 12 DFR gene and 7 ANR genes were identified in \u003cem\u003eGinkgo biloba\u003c/em\u003e (Liu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, 2 DFR genes and 30 ANR genes were identified in \u003cem\u003eE. ferox\u003c/em\u003e, and the number of ANR genes was significantly higher than that of other species. We speculated that ANR plays a more important role in the accumulation of catechins in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e. To study the evolution of the ANR gene family, we compared the number of ANRs in \u003cem\u003eCamellia sinensis\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eE. ferox\u003c/em\u003e have a relatively large number of 30 ANR genes. The ANR gene family expand reason is that E. ferox had experienced a whole genome triplication (WGT) event(Wu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Furthermore, we identified that 90% of the duplication types of the ANR gene belong to whole genome duplication (WGD) (\u003cb\u003eTable S14\u003c/b\u003e). These results together suggest that ANR genes cloud play important roles in the adaptive to the aquatic environment of \u003cem\u003eE. ferox\u003c/em\u003e. Furthermore, we screened and identified the key genes \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e. Among them, \u003cem\u003eEfANR-11\u003c/em\u003e and \u003cem\u003eCsANR\u003c/em\u003e are clustered on the same branch, have similar functions, and both play important roles in the synthesis of catechins. Although \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eCSDFR\u003c/em\u003e are not clustered in the same branch, both contain PLN02650 domains.\u003c/p\u003e\u003cp\u003eThe R2R3-MYB family is a class of transcription factors widely involved in the synthesis and regulation of plant secondary metabolites. Overexpression \u003cem\u003eZmMYB31\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e showed that the expression of DFR gene was up-regulated in transgenic plants, resulting in increased catechin accumulation (Fornal\u0026eacute; et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Overexpression of \u003cem\u003eCsMYB5b\u003c/em\u003e, the expression of \u003cem\u003eNtANR\u003c/em\u003e was up-regulated and catechins were accumulated in large quantities in tobacco leaves (Wang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Further, we screened the transcription factor EfMYB5b by transcriptome, qPCR and correlation analysis. Then we verified that it can interact with \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e, promote their expression, and then positively regulate the synthesis of catechin. The biological functions of transcription factor MYB5 are diverse in different species. AtMYB5 mainly regulates the accumulation of tannin in the inner cortex of Arabidopsis seeds (Xu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). FaMYB5 promotes the biosynthesis of anthocyanins and PA through \u003cem\u003eF3\u0026rsquo;H\u003c/em\u003e and \u003cem\u003eLAR\u003c/em\u003e genes in strawberry fruit (Jiang et al., 2023b). MtMYB5 activates the expression of \u003cem\u003eLAR\u003c/em\u003e and \u003cem\u003eANR\u003c/em\u003e genes, thereby promoting the accumulation of PAs in seeds of \u003cem\u003eMedicago truncatula\u003c/em\u003e (Liu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which is similar to EfMYB5b. Our research has for the first time demonstrated a new mechanism by which the MYB5b transcription factor, in synergy with DFR and ANR, regulates catechins in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn addition, catechin biosynthesis is widely reported to be regulated by the conserved MYB-bHLH-WD40 (MBW) complex (Wang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). CsMYB60 formed complexes with CsbHLH42/MYC1 and CsWD40 to promote \u003cem\u003eCsANR\u003c/em\u003e expression and positively regulate catechin synthesis in tea (Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). VvMYBPA1 formed a trimer complex with VvMYC2 and VvWDR1, which promotes the accumulation of catechins in grape fruits (Liang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Based on the calculation of Pearson correlation coefficients (PCC) among \u003cem\u003eEfDFR-1\u003c/em\u003e, \u003cem\u003eEfANR-11\u003c/em\u003e, EfMYB5b, and bHLH/WD40 genes, we constructed a co-expression network. The results indicate that the bHLH/WD40 complex is closely correlated with \u003cem\u003eEfDFR-1\u003c/em\u003e, \u003cem\u003eEfANR-11\u003c/em\u003e, EfMYB5b (Fig.\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Therefore, we hypothesize that the bHLH/WD40 complex may cooperate with EfMYB5b to coregulate \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e, thereby influencing catechin synthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e are rich in flavonoid compounds. In this study, catechins were found to be the mainly flavonoid compounds in \u003cem\u003eE. ferox\u003c/em\u003e seed kernels. Further, 2 DFRs and 30 ANRs genes were identified from the \u003cem\u003eE. ferox\u003c/em\u003e genome. And then we screened the key genes \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e through RT-qPCR, enzyme activity analysis. In addition, the transcription factor EfMYB5b can respectively bind to EfDFR-1 and EfANR-11 and promote their expression, thereby increasing the content of catechins in the seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The present study provides new ideas on the mode of regulation of catechins biosynthesis in plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by, China Agriculture Research System (grant number: CARS-24), Jiangsu seed industry revitalization\u0026rsquo;Jie Bang Gua Shuai\u0026rsquo;project (grant number: JBGS [2021] 017), and Yangzhou University qinglan project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeng Wu\u003c/strong\u003e: Investigation, Visualization, Roles/Writing-original draft, Writing-review\u0026amp; editing.\u0026nbsp;\u003cstrong\u003eChen-Yan Qu\u003c/strong\u003e: Investigation, Visualization, Roles/Writing-original draft, Writing-review\u0026amp; editing.\u0026nbsp;\u003cstrong\u003eTian-Yu Wang\u003c/strong\u003e: Investigation, Visualization.\u003cstrong\u003e\u0026nbsp;Wen-Jing Ling\u003c/strong\u003e: Validation, Data curation.\u0026nbsp;\u003cstrong\u003eYue-Rui Fang\u003c/strong\u003e: Software, Data curation.\u0026nbsp;\u003cstrong\u003eYu-Da Guo\u003c/strong\u003e: Software, Data curation.\u0026nbsp;\u003cstrong\u003eMeng-Nan He:\u003c/strong\u003e Formal analysis, Methodology.\u0026nbsp;\u003cstrong\u003eShu-Ping Zhao\u003c/strong\u003e: Supervision.\u0026nbsp;\u003cstrong\u003eKai Feng\u003c/strong\u003e: Supervision.\u0026nbsp;\u003cstrong\u003eLiang-Jun Li\u003c/strong\u003e: Conceptualization, Funding acquisition, Project administration, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAshihara H, Deng W, Mullen W, Crozier A. 2010. 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Cloning and Characterization of a Flavonoid 3\u0026prime;-Hydroxylase Gene from Tea Plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e). Int. J. Mol. Sci.,\u003cem\u003e 17\u003c/em\u003e(2), 261. https://www.mdpi.com/1422-0067/17/2/261 \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":"Catechins, EfANR-11, EfDFR-1, EfMYB5b, E. ferox","lastPublishedDoi":"10.21203/rs.3.rs-8027490/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8027490/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlavonoids are important secondary metabolites, which exist widely and play different roles in plants. Many kinds of flavonoid compounds have been identified in\u003cem\u003e E. ferox\u003c/em\u003e seed kernels, but the main flavonoid components and biosynthesis mechanism of \u003cem\u003eE. ferox\u003c/em\u003e are still unclear. In this study, UPLC-MS/MS was used to identify catechin as the main flavonoid compound in seed kernel of \u003cem\u003eE. ferox\u003c/em\u003e. The 32 structural genes (2 \u003cem\u003eEfDFRs\u003c/em\u003e and 30 \u003cem\u003eEfANRs\u003c/em\u003e) related to catechin synthesis were identified. Among them, key genes EfDFR-1 and EfANR-11 were screened by transcriptome, real-time fluorescence quantification and enzyme activity analysis. Further, \u003cem\u003ein vitro\u003c/em\u003e enzyme activity assay demonstrated that EfANR-11 and EfDFR-1 could catalyze the formation of gallocatechin (GC) and epigallocatechin (EGC) from the substrate, respectively. Then, subcellular localization showed that \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003e were located in the Golgi apparatus. Transient overexpression of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003esignificantly increased catechin content in seed kernels of \u003cem\u003eE. ferox\u003c/em\u003e. Subsequently, the EfMYB5b directly bind to TAACCA and ACCTAC in the \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003epromoter and promote their expression. Meanwhile, transientoverexpression of EfMYB5b showed that the expression of \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003ewere significantly enhanced, and the content of catechin was increased in seed kernel\u003cem\u003e E. ferox\u003c/em\u003e. Our findings clarified the molecular mechanism of transcription factor EfMYB5b regulating key genes \u003cem\u003eEfDFR-1\u003c/em\u003e and \u003cem\u003eEfANR-11\u003c/em\u003ein catechin synthesis pathway. It provides theoretical basis for improving the quality of \u003cem\u003eE. ferox\u003c/em\u003e and breeding new varieties.\u003c/p\u003e","manuscriptTitle":"Molecular mechanisms of catechins regulation by EfMYB5b in Euryale ferox","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 08:11:41","doi":"10.21203/rs.3.rs-8027490/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":"d3537e6a-c4f6-493c-a2b5-6b11649fca6a","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T16:27:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 08:11:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8027490","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8027490","identity":"rs-8027490","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}