Insight into the Rutin Biosynthesis in the Unique Flavonol Synthesis Pathway of Tartary Buckwheat Based on the Enzymatic Functions of FLSs

Insight into the Rutin Biosynthesis in the Unique Flavonol Synthesis Pathway of Tartary Buckwheat Based on the Enzymatic Functions of FLSs | 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 Insight into the Rutin Biosynthesis in the Unique Flavonol Synthesis Pathway of Tartary Buckwheat Based on the Enzymatic Functions of FLSs Chenglei Li, Jiayi Sun, Guanlan Shi, Xuerong Zhao, Jun Gu, Jiaqi Shi, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4548454/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Theoretical and Applied Genetics → Version 1 posted 4 You are reading this latest preprint version Abstract The flavonol biosynthesis branch generates the main flavonoids in Tartary buckwheat (TB), with rutin serving as a representative flavonol compound. Flavonol synthase (FLS) is a vital enzyme involved in this metabolic pathway. Out of the five known FLS genes in the TB genome, FtFLS1 is the only gene identified, while information about the remaining four genes is limited. In this study, we cloned the five FtFLS genes from TB and performed molecular identification. The results showed that FtFLS1-3 exhibit high homology and similar molecular characteristics, categorizing them as FLS-like enzymes, while FtFLS4 and FtFLS5 show a certain degree of similarity to other 2-oxoglutarate-dependent dioxygenases. Further investigation revealed a significant correlation between expression of FtFLS1 and the rutin content during the flowering stage of TB ( p < 0.05). The promoter sequences of FtFLS1-3 ( P FtFLS1-3 ) displayed distinctive cis-elements, transcriptional activities, and expression patterns, exhibiting different sensitivities to cold, UV-B, and drought stresses. The overexpression of FtFLS1-3 in Arabidopsis led to a significant elevation in total flavonoid and rutin levels, providing evidence for the FLS activity of FtFLS1-3 in plants. The enzymatic analysis showed that the recombinant FtFLS1-3 were capable of catalyzing the formation of their respective products from dihydroflavanols. FtFLS1 exhibited a superior specific activity, V max and affinity for dihydroquercetin (DQ) in terms of enzyme catalytic characteristics compared to FtFLS2 and FtFLS3. In summary, our study establishes the FLS activity of FtFLS1-3 and suggests that the metabolic flow of the flavonol biosynthesis branch in TB involves the conversion from dihydrokaempferol (DK) to DQ and subsequently to quercetin (Q), ultimately glycosylated to rutin. In this process, FtFLS1 plays a predominant role. Tatary buckwheat Flavonol synthase Characterization Activity analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Tartary buckwheat ( Fagopyrum tataricum Gaertn., abbreviated as TB) originated in the southwestern region of China[ 1 ]. This pseudo-cereal crop holds significant importance for countries surrounding the Himalayan regions[ 2 ]. The primary bioactive components in TB are flavonoids, which offer diverse benefits for human health, including antioxidation, anti-tumor, anti-hypertension, and so on[ 3 ]. To date, over 100 different types of flavonoid compounds have been identified in TB, falling into categories such as flavonols, flavones, isoflavones, flavanones, flavan-3-ols, anthocyanins, fagopyrins, proanthocyanidins, flavonolignans, and others[ 3 ]. Notably, rutin (quercetin 3- O -rutinoside) is the predominant flavonoid compound, accounting for 70–85% of the total flavonoid content[ 4 ]. As such, flavonol compounds, primarily composed of rutin, kaempferol (K), myricetin (M), and quercetin (Q), emerge as the most critical and essential bioactive constituents in TB[ 5 ]. The metabolic pathway of plant flavonoid biosynthesis constitutes a significant component of the phenylpropanoid metabolic pathway and has undergone extensive investigation[ 6 ]. Within this pathway, the flavonol biosynthesis branch is primarily governed by five enzymes: flavonone 3 β -hydroxylase (F3H), flavonoid 3′-hydroxylase (F3'H), flavonoid 3', 5'-hydroxylase (F3'5'H), flavonol synthase (FLS), and flavonoid 3- O -glucosyltransferase (UFGT)[ 7 ]. Of these enzymes, flavonol synthase (FLS, EC: 1.14.20.6) stands out as a pivotal catalyst in the flavonol synthesis pathway, occupying a central and crucial role[ 6 , 7 ]. FLS belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily and operates with 2-oxoglutarate as a cosubstrate, in conjunction with Fe 2+ serving as a cofactor[ 8 ]. In plants, the first detection of flavonol synthase FLS activity occurred in a culture of parsley cells in 1981[ 9 ]. Subsequently, the initial FLS cDNA was isolated from Petunia hybrida and characterized through yeast expression in 1993, while the first genomic FLS sequence was identified from Arabidopsis thaliana in 1997[ 10 , 11 ]. Presently, the NCBI database contains annotations for approximately 2340 FLS sequences across flowering plants. In reality, the presence of FLS varies in copy numbers across the genomes of different species. The genome of A. thaliana harbors 6 FLS genes, with AtFLS1 and AtFLS3 being the sole contributors to FLS activity, while AtFLS2 , AtFLS4 , and AtFLS6 are categorized as pseudogenes[ 12 ]. Similarly, Brassica napus exhibits 13 identified sequences of homologous FLS , displaying diverse expression intensities and tissue specificities at the transcriptional level[ 13 ]. Noteworthy are BnaFLS1-1 and BnaFLS1-2 , both identified in Escherichia coli as having simultaneous FLS and F3H activities. Jiang et al. heterologously expressed 3 CsFLS genes from Camellia sinensis , all of which demonstrated catalytic activity in in vitro experiments, enhancing kaempferol synthesis in Arabidopsis [ 14 ]. Thus, the comprehensive identification of diverse FLS copies in plants is imperative for a thorough comprehension of the metabolic pathway governing flavonol synthesis. From our previous studies, we have annotated five flavonol branch related genes based on the TB genome and transcript data[ 15 , 16 ]. Among these, FtF3'H1 , FtFLS1 , FtUFGT1-3 , FtUGT73BE5 , and FtUGT79A15 have been identified with confirmed biological functions, either in vivo or in vitro [ 17 – 22 ]. The catalytic products of the FLS encompass kaempferol, quercetin, and myricetin. Notably, these products could potentially serve as substrates for the uridine diphosphate UFGT, ultimately leading to the formation of rutin (Fig. 1 )[ 7 ]. Of utmost significance, the TB genome harbors five homologous sequences of FLS genes, among which FtFLS1 has been previously identified in our earlier work[ 19 ]. Nevertheless, the precise molecular characteristics of the remaining FtFLS genes remain undisclosed. Their catalytic properties on different substrates and their biological activities await confirmation. Consequently, further analysis is warranted to illuminate their regulatory role in the material flow within the biochemical process of rutin synthesis. In this investigation, we conducted an extensive molecular identification of the five FtFLS genes present in TB. Notably, two novel FLS genes, designated as FtFLS2 and FtFLS3 , were successfully discerned. Additionally, we performed a comparative analysis of promoter characteristics, recombinant protein activity, and biological impacts associated with FtFLS1-3 , utilizing transgenic tobacco , a prokaryotic expression system, and transgenic Arabidopsis . In summary, our study furnishes essential foundational data, contributing to an enhanced comprehension of the interplay between molecular attributes and functions exhibited by FtFLS genes in TB. Notably, it serves as a valuable point of reference for dissecting the metabolic flux within TB's flavonol synthesis pathway. Moreover, our findings offer comprehensive evidence substantiating the rutin biosynthesis process. 2. MATERIALS AND METHODS 2.1 Plant materials. The cultivated TB variety, known as "Xiqiao No. 2," was cultivated in Leying Township, Tianquan County, Ya'an city, located in Sichuan province, China (coordinates: longitude 102°36′E, latitude 29°79′N, with an elevation of approximately 800 m). After approximately 60 days of germination, TB entered the initial flowering stage, at which point the roots, stems, leaves, flowers, and seeds were meticulously collected to serve as experimental specimens. All collected samples were promptly frozen in liquid nitrogen, ensuring their preservation for subsequent processes such as DNA extraction, RNA extraction, flavonoid content analysis, and qRT-PCR experiments. Nicotiana tabacum (NC89) was cultivated under controlled conditions comprising 12 hours of light followed by 12 hours of darkness, maintaining a temperature of 23 ± 2°C, and providing a light intensity of approximately 100 µmol/(m²·s). For A. thaliana (Columbia-0), cultivation took place within an artificial climate chamber with parameters set to 16 hours of light and 8 hours of darkness, a constant temperature of 23 ± 2°C, and a light intensity ranging between 120 and 150 µmol/(m²·s). 2.2 Chemical reagents, Microbial strains and Plasmids. The chemical reagents and flavonoid standard substances used in this study were purchased from Vanke (China), Tiandz (China), GE Healthcare (United States), and Sigma (United States). The plant genomic DNA extraction kit, plant total RNA extraction kit, plasmid extraction kit, restriction endonucleases, Taq DNA polymerase, and qRT-PCR reagent kit were obtained from Takara (Japan). The E. coli strains DH5a and BL21(DE3), as well as the Agrobacterium tumefaciens strain GV3101 used in this experiment, were obtained from our laboratory's collection. The pMD19-T plasmid was used for T-cloning of the target gene, pET-30b(+) was used for prokaryotic expression, and pBI-101 and pCAMBIA1301 were used for heterologous expression of the target gene in Arabidopsis and tobacco . 2.3 Cloning and Characterization of FtFLSs . Based on our preceding research, we have successfully annotated and designated five sequences within the TB genome as FLS genes, identified as FtFLS1 through FtFLS5 [ 16 ]. To isolate these FtFLSs , we extracted genomic DNA and total RNA from diverse tissues of TB. Subsequent to this, we conducted PCR amplification, followed by cloning and sequencing of the resultant products. The obtained DNA and cDNA sequences of FtFLSs were compared using the Gene Structure Display Server 2.0 ( http://gsds.gao-lab.org ). The deduced amino acid sequence of FtFLSs protein were analyzed using the DNAMAN software (Version 9.0) and BLAST ( http://www.ncbi.nlm.nih.gov/BLAST ) programs in NCBI. The multiple sequence alignment was performed with Clustal X ( http://www.clustal.org ), and the phylogenetic tree was drawn using the MEGA 11 ( https://www.megasoftware.net ) with the maximum likelihood method. The three-dimensional modeling of the proteins was conducted on the Swiss-model website ( https://swissmodel.expasy.org ). The genome data of TB is sourced from the MBK database ( https://www.mbkbase.org/Pinku1 ), and the transcriptome is obtained from the SRA database ( https://www.ncbi.nlm.nih.gov/sra , Accession No. GSE111937). The primer sequences are shown in Supplementary Table 1. 2.4 Expression analysis of FtFLS1-3 and measurement of flavonol content. In order to accurately analyze gene expression in TB, we established a real-time reverse transcription PCR (qRT-PCR) method in our previous work, identified the housekeeping gene FtH3 (Accession NO. HM628903) as the optimal internal reference gene, and applied it to the current study[ 23 ]. Then, qRT-PCR was performed following the instructions provided by the SYBR® Premix Ex Taq ™ (Takara, Japan) kit and the operational manual of the CFX96 Real-Time PCR Machine (Bio-Rad, USA). The data were analyzed using the 2 −ΔΔCT method. The primer sequences are shown in Supplementary Table 1. The method described by Yao et al. was employed to extract flavonoid compounds from different tissues, which were then subjected to high-performance liquid chromatography (HPLC) analysis[ 24 ]. The creation of standard curves involved the preparation of quercetin, kaempferol, myricetin, and rutin standard solutions in methanol. The concentrations of these solutions were 0.12 mg/mL, 0.12 mg/mL, 0.12 mg/mL, and 0.16 mg/mL, respectively. For each solution, injections of 5 µL, 10 µL, 15 µL, 20 µL, and 25 µL were made, each executed in triplicate. The subsequent analytical analysis was carried out using HPLC with a C18 column (250 × 4.6 mm, 5 µm internal diameter, RStech, Korea). The mobile phase, composed of a mixture of methanol and water/acetic acid (98:2, v/v), was consistently maintained at 30°C. Each sample, introduced through a 10 µL injection, was subsequently eluted at a controlled flow rate of 1.0 mL/min. Detection occurred at a wavelength of 330 nm, facilitating the quantification of flavonol concentrations using a derived standard curve. Correlation analysis was performed using the statistical software SPSS (Version 20), where the Pearson method was applied. Additionally, the Influence Degree (ID) was employed to evaluate the association between gene expression levels and flavonol contents. 2.5 Cloning and identification of the promoters of FtFLS1-3 . To examine the expression profiles of FtFLS1-3 , we cloned the promoter sequences of these three genes using PCR. The acquired P FtFLS1 , P FtFLS2 , and P FtFLS3 sequences underwent analysis via the PlantCARE online database ( http://sphinx.rug.ac.be:8080/PlantCARE ) and the GSDS tool ( http://gsds.cbi.pku.edu.cn ). To identify P FtFLS1−3 , distinct plasmids were generated: pBI101- P FtFLS1 -GUS, pBI101- P FtFLS2 -GUS, pBI101- P FtFLS3 -GUS, pBI101-35S-GUS (positive control), and pBI101-GUS (Negative control, lacking the 35S promoter). These plasmids were employed for separate transient transformation assays in tobacco leaves, as well as for establishing stable expression in Arabidopsis . Following the protocols outlined by Zhang et al. , the 5 plasmids were introduced into tobacco leaves through transient transfection using Agrobacterium strain GV3101[ 25 ]. Subsequently, β -glucuronidase (GUS) activity was visualized via histochemical staining after a 48-hour incubation period. Similarly, employing the floral dip method as detailed by Yao et al. , we introduced the aforementioned 5 plasmids into Arabidopsis [ 24 ]. Kanamycin-resistant seedlings were transferred to pots, validated through PCR analysis, and subsequently, the transgenic T3 generation of Arabidopsis lines were confirmed and utilized for subsequent experiments. To identify the tissue-specific expression pattern of P FtFLS1−3 , GUS staining and qRT-PCR analysis were performed on the Arabidopsis lines at the 20-days old and flowering stages, respectively. To distinguish the responses of P FtFLS1−3 to various environmental factors, Arabidopsis lines at the flowering stage were subjected to treatments of cold (4℃), UV-B (302nm), and drought (30% PEG-6000) using the method outlined by Luo et al. [ 26 ]. The control group (Con) comprised of untreated Arabidopsis lines, and qRT-PCR was conducted following the previously established procedure, using AtActin (Accession No. AF149413) as the reference gene. The primer sequences are shown in Supplementary Table 1. 2.6 Expression of FtFLS1-3 in Arabidopsis . To determine the biological functions of FtFLS1-3 in plants, we generated transgenic Arabidopsis lines with overexpressed FtFLS1-3 genes, individually. The recombinant plasmids, namely pCAMBIA-1301- FtFLS1 , pCAMBIA-1301- FtFLS2 , and pCAMBIA-1301- FtFLS3 , were engineered and introduced into Agrobacterium strain GV3101. The transgenic manipulation of Arabidopsis was performed using the previously mentioned floral dipping method[ 24 ]. Following this, the seedlings resistant to hygromycin were transplanted into pots and verified as transgenic through PCR analysis. Subsequently, the transgenic T3 generation of Arabidopsis lines were confirmed via PCR. The transgenic Arabidopsis lines were cultivated until reaching the flowering stage, at which point fresh samples were harvested and promptly cryopreserved in liquid nitrogen. This procedure was carried out for the determination of both total flavonoid content and rutin content[ 24 ]. 2.7 Functional expression of FLS1-3 in E. coli . To gain a more comprehensive understanding of the biological impacts of FtFLS1-3 , 3 recombinant plasmids (pET-30(b)- FtFLS1 , pET-30(b)- FtFLS2 , and pET-30(b)- FtFLS3 ) were constructed and transformed into E. coli strain BL21(DE3) for prokaryotic expression[ 19 ]. The recombinant FtFLSs were tagged with (His)6-tags at the N- and C-termini to facilitate protein purification. Isolated clones harboring pET-30(b)- FtFLS1-3 were meticulously selected and cultivated in Luria-Bertani medium supplemented with kanamycin (50 µg/mL). The cultures were agitated at 180 rpm until the optical density at 600 nm ( OD 600) reached 0.5. For the production of soluble FtFLS1-3 proteins, the conclusive expression conditions were achieved by introducing isopropyl- β -D-thiogalactopyranoside (IPTG) to attain a final concentration of 1 mM. Subsequently, the culture was incubated at 25°C for a duration of 6 hours. Crude enzyme solutions encompassing the recombinant FtFLS1-3 were obtained through ultrasonic disruption. The contents of all samples were subjected to analysis through 12.5% SDS-PAGE. The crude enzyme solutions were utilized to determine the FLS enzyme activity using DQ as the substrate[ 19 ]. The reaction mixture, comprising 1 mL, included 100 µL of crude enzyme solution, 100 µM DQ, 111 mM sodium acetate, 83 µM 2-oxoglutaric acid, 42 µM ferrous sulfate, and 2.5 mM vitamin C. The reaction was carried out at 37°C and pH 5.0 for a duration of 30 minutes. After the reaction, the mixtures were extracted twice with an equivalent volume of ethyl acetate, followed by separation using thin-layer chromatography (TLC) on silica gel G (toluene/acetic ether/formic acid, 5:2:2). Internal standards included DQ, quercetin (Q), and a mixture of DQ-Q. The identification of reaction products was accomplished through standard sample comparisons or by determining their retention factor ( R f ) values. 2.8 Catalytic characteristics of recombinant FtFLS1-3 in vitro . After 10 hours of induction, following the approach by Li et al. (with minor modifications)[ 19 ], the soluble protein fractions of recombinant FtFLS1-3 were purified using a HiTrap FF column from GE Healthcare (USA). The samples underwent sequential washing with 2 mL volumes of 50 mM, 100 mM, 150 mM, and 200 mM imidazole buffer (containing 0.5 M NaCl and 20 mM sodium phosphate at pH 7.4) before undergoing assessment via 12.5% SDS-PAGE. To remove imidazole from the samples, we conducted a protein ultrafiltration experiment[ 27 ]. A 2 mL portion of the diluted sample was added to an Amicon @ Ultra-15 10K NMWL (Nominal Molecular Weight Limit) Centrifugal Filter device (Merk, Germany) and centrifuged at 4000 g for 30 minutes at 4ºC. The ultrafiltered samples were adjusted to a volume of 500 µL using sterile water, and protein content was determined using the Coomassie Brilliant Blue G-250 method[ 28 ]. To individually assess the catalytic activity of the three recombinant FtFLSs towards dihydroflavonols (DQ, DK, and DM), we established standard curves for quantifying the concentrations of the resultant reaction products[ 19 ]. Different concentrations of kaempferol, quercetin, and myricetin (0.5, 1, 2, 3, 5, 7, 9, 12, and 15 µg/mL) were dissolved in the enzyme reaction solution (composed of 111 mM sodium acetate, 83 µM 2-oxoglutaric acid, 42 µM ferrous sulfate, 2.5 mM vitamin C, pH 5.0). A spectrophotometer (Shimadzu, Japan) with a 0.5 cm path length was used to perform a full spectrum scan in order to obtain characteristic absorption peaks, standard curves, regression equations, and correlation coefficient ( R 2 ). We employed the optimal wavelength of 365.5 nm for measuring kaempferol in the enzyme assays. The corresponding calibration curve was characterized by the regression equation A = 0.0228C (A represents absorbance and C denotes concentration in µg/mL), yielding an R 2 value of 0.9973. Similarly, we utilized the optimal wavelength of 365.5 nm to measure quercetin in the enzyme assays. The calibration curve was determined based on the regression equation A = 0.0223C, resulting in an R 2 value of 0.9975. For the measurement of myricetin within the enzyme assays, the optimal wavelength of 426.5 nm was employed. The calibration curve for myricetin was determined based on the regression equation A = 0.0169C, resulting in an R 2 value of 0.9957. The data is presented in Supplementary Fig. 1. Following the procedure outlined by Wellmann et al., FLS activity was determined using 12.5 µg of purified FtFLS1-3 and 100 µM substrates (with a molar ratio of enzyme to substrate set at 180:1) at a temperature of 37°C and a pH of 5.0[ 29 ]. The determination of the initial velocity of the enzyme-catalyzed reaction was conducted at 37°C, with continuous measurements being recorded over a period of 20 minutes. Absorbance values were collected at 2-minute intervals to investigate the correlation between the product generated through catalysis by FtFLS1, FtFLS2, and FtFLS3. Subsequently, the kinetic parameters ( K m ) and maximum velocity ( V max ) values for each FtFLS enzyme were determined using three dihydroflavonols (20, 30, 40, 50, 60, 70, and 80 µmol/L) as substrates within the enzymes' linear range of product generation. The K m and V max values were calculated using the Lineweaver-Burk plot method[ 30 ]. A single unit (IU) was defined as the quantity of FLS catalyzing the generation of 1 µmol of product from the substrates per minute at 37°C (µmol/min). The specific activity was denoted as IU/mg[ 31 ]. 3. Results 3.1 Molecular Characterization of FtFLSs form TB. The DNA and cDNA sequences of the five FtFLSs ( FtFLS1 to FtFLS5 ) were successfully obtained through PCR. Alignment of these five FtFLSs with the TB genome and transcriptome data revealed their consistency with the sequences stored in the database. The outcomes indicated that FtFLS1 and FtFLS2 were positioned on chromosome 7 of TB and represented tandem repeat sequences. FtFLS3 was found on chromosome 2, FtFLS4 on chromosome 3, and FtFLS5 on chromosome 1. The coding DNA sequences (CDS) of the five FtFLSs demonstrated a similarity of 75.7%, while their deduced protein sequences exhibited a similarity of 43.5%. Supplementary Table 2 and Supplementary Fig. 2 were provided to offer supplementary details regarding the five FtFLSs . In order to dissect the molecular attributes and potential roles of the predicted FtFLS proteins, we selected protein sequences from three 2-ODD enzymes (F3H, FLS, and ANS) associated with rutin synthesis in F. tartaricum , A. thaliana , and N. tabacum . The phylogenetic analysis illuminated the evolutionary tree, which divided into two distinct branches termed Cluster I and Cluster II (Fig. 2 A). Within Cluster I, a close phylogenetic relationship was evident between FtFLS1, FtFLS3, 6 AtFLSs from Arabidopsis , and 4 NtFLSs from tobacco , as they were collectively placed into Group A. On the other hand, FtFLS2 was assigned to Group B, joined by AtANS from F. tartaricum and 4 NtANSs from tobacco . As for Cluster II, FtFLS4 occupied an independent position in Group C, while FtFLS5 exhibited marked similarity to AtANS. Additionally, all F3Hs from the three species were found to cluster together in Group E. These results collectively indicate that among the five FtFLS proteins, FtFLS1, FtFLS2, and FtFLS3 are likely to share comparable biological functions, whereas FtFLS4 and FtFLS5 might serve as multifunctional enzymes within the rutin synthesis metabolic pathway of TB[ 8 ]. The multiple sequence alignment revealed that all 31 selected 2-ODD sequences encompass the conserved functional regions recognized as Conserved domain A and Conserved domain[ 12 ]. It is noteworthy that the major divergences were localized within the variable region comprised of the initial 70 amino acids at the N-terminus (Fig. 2 B). Further insights into the impact of the N-terminal variable region of FtFLSs on the configuration of the enzyme-substrate pocket structure were obtained through additional 3D modeling results (Fig. 2 C and 2 D). Of significant importance, FtFLS4 and FtFLS5 exhibited a stronger similarity to the advanced structure of ANS (pdb No. A0A5J5AX16) as opposed to FLS (pdb No. F5BR59.1). By leveraging our previously documented transcriptome data of flowering TB, we generated an updated expression heatmap for FtFLS1-5 . The outcomes underscored the presence of tissue specificity and variations in the expression levels of FtFLS1-5 . Importantly, FtFLS1 displayed the highest average expression level, followed by FtFLS2 , while FtFLS3 exhibited the lowest expression level (Supplementary Fig. 3). Consequently, FtFLS1-3 were selected as prospective candidate genes for subsequent investigations. 3.2 Analysis of the expression of FtFLSs and flavonoid contents in TB. To validate the expression levels of FtFLS1-3 as indicated by transcriptome data and explore their potential correlation with flavonoid contents, qRT-PCR was employed to quantify gene expression, while flavonoid contents were measured in various tissues of TB during the flowering stage. The qRT-PCR analysis revealed that FtFLS1 displayed elevated expression levels in the stems, leaves, and flowers of flowering buckwheat, with a moderate level of expression in the roots. In contrast, both FtFLS2 and FtFLS3 shared a comparable expression pattern, marked by low expression in the roots and flowers, and negligible expression in other tissues (Fig. 3 A). The provided data strongly reinforces the observed expression patterns of FtFLS1-3 in our previous transcriptome analysis of buckwheat[ 15 ]. HPLC analysis unveiled rutin as the predominant flavonol component in TB (dry weight, DW), present in relatively substantial quantities across various tissues (114.96 mg/g in flowers, 107.53 mg/g in leaves, 8.32 mg/g in stems, and 1.62 mg/g in roots, DW). Quercetin, being the principal precursor of rutin, exhibited the highest content in buckwheat flowers (9.25 mg/g, DW), while kaempferol and myricetin were detected at low levels in all tissues (less than 1.5 mg/g, DW) (Fig. 3 B). The correlation between the expression levels of FtFLS1-3 and the content of four different flavonols was analyzed using Pearson's correlation coefficient method with an ID of 0.75, indicating strong positive correlation. The results demonstrated that the expression of FtFLS1 displayed a robust positive correlation with quercetin content (0.80) and rutin content (0.75), a pronounced negative correlation with kaempferol content (-0.79), while no significant correlation was observed with myricetin. Additionally, a correlation coefficient of 0.68 existed between the expression of FtFLS2 and kaempferol content, which did not meet the established threshold. Notably, no significant correlation was identified between the expression of FtFLS3 and the content of the four flavonols. (Table 1 ). Table 1 Correlation analysis of the expression levels of FtFLS1-3 and the content of 4 different flavonols in flowering Taratary buckwheat Names Kaempferol Quercetin Myricetin Rutin FtFLS1 -0.79* 0.80* 0.20 0.75* FtFLS2 0.68 -0.51 -0.03 -0.45 FtFLS3 0.50 -0.38 -0.14 -0.38 (*) denotes that the absolute value of A exceeds 0.5. Based on the preceding findings, it is plausible to infer that FtFLS1 is likely a predominant player in the quercetin biosynthesis across diverse tissues of flowering buckwheat. This role could involve enhancing rutin accumulation via quercetin synthesis. Conversely, FtFLS2 and FtFLS3 might participate in distinct flavonol synthesis pathways, possibly contributing to the synthesis of various flavonol types through alternative routes. 3.3 Cloning and identification of the promoters of FtFLS1-3 . In order to gain a better understanding of the expression patterns for FtFLS1-3 , we cloned the promoter sequences located approximately 2000 bp upstream of their respective start codons (ATG). These sequences have been designated as P FtFLS1 (2479 bp), P FtFLS2 (2574 bp) and P FtFL S3 (2046 bp). These sequences have a high AT content (over 60%), which is consistent with the characteristics of plant promoters. The web tools PlantCARE and GSDS were utilized to analyze the DNA sequences of P FtFLS1−3 , and the resulting details are presented in Supplementary Table 3. Our results showed that P FtFLS1−3 encompassed a wide range of cis -acting elements, exhibiting varying quantities. Additionally, among the core promoter elements (TATA-box and conserved CAAT-box), prominent elements comprised light-responsive components like G-box, Box-I, and Box-4. These outcomes propose the potential responsiveness of P FtFLS1−3 to various environmental stimuli. Consequently, we proceeded with the functional analysis of P FtFLS1−3 , employing transgenic tobacco and Arabidopsis methodologies. In the experiment involving agrobacterium-mediated transformation of tobacco leaves, all P FtFLS1−3 successfully initiated the expression of the downstream reporter gene GUS , resulting in confined blue coloration within the tobacco leaves. However, their transcriptional activities exhibited relatively lower strength in comparison to the positive control 35S promoter (Fig. 4 A). To further differentiate the functions of the three P FtFLS1−3 , we generated T3 Arabidopsis lines expressing P FtFLS1 , P FtFLS2 , and P FtFLS3 individually and identified positive transgenic lines. During the seedling stage of transgenic Arabidopsis , histochemical staining indicated predominant expression of P FtFLS2 in the roots, while no detectable GUS activity accumulation was observed for P FtFLS1 and P FtFLS3 (Fig. 4 B). Subsequent qRT-PCR analysis revealed varying expression levels of all three genes in Arabidopsis , with P FtFLS2 displaying the highest expression level, followed by P FtFLS1 and P FtFLS3 (Fig. 4 C). We further explored the expression-driving potential of the P FtFLS1−3 promoters in transgenic Arabidopsis lines during the flowering stage. Histochemical staining demonstrated that all three promoters could activate GUS expression in different organs of Arabidopsis , encompassing roots, stems, leaves, and flowers, resulting in noticeable coloration (Fig. 5 A). However, the qRT-PCR results unveiled diversity in the expression levels of the three promoters across various tissues of Arabidopsis . P FtFLS2 demonstrated a pattern akin to flowering TB, while P FtFLS1 and P FtFLS3 exhibited unique expression patterns (Fig. 5 B). Furthermore, to investigate the response of P FtFLS1−3 to environmental stress, including cold, UV-B, and drought, flowering Arabidopsis plants were exposed to these stressors. The qRT-PCR results indicated that all three environmental factors led to an elevation in the expression of P FtFLS1 and P FtFLS3 , displaying significant differences from the wild type ( p 0.05), exhibiting upregulation solely under drought conditions, which was highly significant compared to the wild type ( p < 0.01) (Fig. 5 D). The aforementioned results elucidated the differences among the P FtFLS1−3 in terms of cis -acting element composition, spatial and temporal expression specificity, and responsiveness to environmental factors. These findings provide crucial insights to advance our understanding of the regulatory mechanisms that control the expression of the three FtFLS genes. 3.4 Expression of FtFLS1-3 in Arabidopsis . After hygromycin screening and PCR identification, we successfully generated T3 generation transgenic Arabidopsis lines overexpressing FtFLS1-3 , respectively (Fig. 6 A). The qRT-PCR analysis revealed varying expression levels of the FtFLS1-3 in Arabidopsis lines under the control of the 35S promoter, with relative expression levels ranked as FtFLS2 > FtFLS3 > FtFLS1 (Fig. 6 B). Total flavonoid content analyses exhibited a significant increase in all transgenic Arabidopsis lines compared to the wild-type control ( p < 0.01) (Fig. 6 C). Moreover, subsequent analysis of rutin content yielded similar results (Fig. 6 D). Furthermore, we conducted an assessment of the levels of kaempferol, quercetin, and myricetin in the samples. The findings indicated that these three compounds were present in extremely low concentrations in Arabidopsis lines, each accounting for less than 1% of the total flavonoid and rutin contents. This phenomenon could be attributed to the efficient conversion of flavanols into the end product rutin in Arabidopsis [ 32 ]. Overall, these results conclusively demonstrate the functional role of FtFLS1-3 as flavonol synthases within the plant system. 3.5 Functional expression of FLS1-3 in E. coli . After 6 hours of IPTG induction, SDS-PAGE analysis revealed successful expression of recombinant FtFLS1, FtFLS2, and FtFLS3 as the primary protein products within the E. coli cultures (Fig. 7 A). These proteins exhibited molecular weights of approximately 40 kDa, consistent with the values in Supplementary Table 2. TLC analysis demonstrated the effective separation of DQ, Q, and a DQ-Q mixture standard substances, with corresponding R f values of 0.33, 0.45, and 0.33–0.45. The TLC analysis results showed effective separation of DQ, Q, and DQ-Q mixture standard substances, with their respective R f values being 0.33, 0.45, and 033-0.45. In the activity assay using crude enzyme solutions, partially reacted substrate DQ and newly produced product quercetin were observed. The R f values of these spots matched those of the DQ and quercetin standards (Fig. 7 B). These results indicate that all three recombinant proteins, FtFLS1-3, exhibited the capability to catalyze the substrate DQ into Q, demonstrating fundamental FLS activity. 3.6 Catalytic characteristics of recombinant FtFLS1-3 in vitro . After a 10-hour induction period, soluble expression of FtFLS1-3 was observed through SDS-PAGE analysis (Fig. 8 A, lanes 1 and 2). Subsequent elution was achieved effectively using a 200 mM imidazole buffer (Fig. 8 A, lane 7). Following ultrafiltration purification (Fig. 8 A, lane 8), the concentrations of recombinant proteins FtFLS1-3 were determined to be 1.35 g/L, 1.29 g/L, and 1.45 g/L (500 µL), respectively. Enzyme-catalyzed reactions were carried out using DK, DQ, and DM as substrates. For FtFLS1, the observed total activities were 8.88 × 10 − 3 IU, 22.33 × 10 − 3 IU, and 4.70 × 10 − 3 IU for each substrate, with specific activities of 6.58 × 10 − 3 IU/mg, 16.54 × 10 − 3 IU/mg, and 3.48 × 10 − 3 IU/mg, respectively. This outcome closely aligns with our earlier research on FtFLS1[ 19 ]. Regarding FtFLS2, the recorded total activities were 5.01 × 10 − 3 IU, 5.17 × 10 − 3 IU, and 3.26 × 10 − 3 IU for each substrate, along with specific activities of 3.88 × 10 − 3 IU/mg, 4.01 × 10 − 3 IU/mg, and 2.53 × 10 − 3 IU/mg, respectively. Concerning FtFLS3, the measured total activities were 10.48 × 10 − 3 IU, 10.32 × 10 − 3 IU, and 7.42 × 10 − 3 IU for each substrate, accompanied by specific activities of 7.23 × 10 − 3 IU/mg, 17.12 × 10 − 3 IU/mg, and 5.12 × 10 − 3 IU/mg, respectively. Continuous measurements were taken for a duration of 20 minutes to quantify the production of quercetin resulting from the catalytic activity of FtFLS1-3 enzymes on DQ (Fig. 8 B). The results indicated that as the enzyme-catalyzed reaction proceeds, the product content continually increased. Approximately within the time span of 8 to 18 minutes from the start of the reaction, there was a linear relationship between the product and time. At this point, the slope of the line represented the initial velocity of the enzyme-catalyzed reaction[ 33 ]. Therefore, the initial velocities of the enzyme-catalyzed reactions of recombinant FtFLS1, FtFLS2, and FtFLS3 with DQ as substrates were 22.30 ×10 − 3 µM/min, 5.16×10 − 3 µM/min and 10.32×10 − 3 µM/min, respectively. Accordingly, we chose a time point of 15 minutes following the initiation of the enzyme-catalyzed reaction to determine the K m and V max values of FtFLS1, FtFLS2, and FtFLS3, utilizing three dihydroflavonols as substrates. The calculated results were presented in Table 2 , and the double reciprocal plots (Lineweaver-Burk plots) were available in Supplementary Fig. 4. The data revealed that FtFLS1-3 exhibit diverse catalytic abilities and substrate preferences for the three dihydroflavonols. FtFLS1 exhibited a preference for DQ as a substrate, characterized by a low K m value (594.17 µmol/L) and the highest V max value (42.55 µmol/L·min). However, FtFLS2 displayed the lowest catalytic capacity and affinity for all three substrates on average, despite its preference for DQ ( K m = 8411.27 µmol/L). In contrast, FtFLS3 showed a preference for DK as a substrate ( K m = 717.93 µmol/L), which is the highest among the three FtFLS enzymes. Notably, all the FtFLS1-3 enzymes showed lower V max and higher K m values for DM. Taken together, these results demonstrate that FtFLS1 exhibited a stronger specific activity, a higher V max , and a better affinity for DQ in terms of enzyme catalytic characteristics compared to FtFLS2 and FtFLS3. Table 2 K m and V max values of FtFLS1-3 for different substrates Name Dihydrokaempferol Dihydroquercetin Dihydromyricetin FtFLS1 V max = 25.53 µmol/L·min K m = 1257.18 µmol/L V max = 42.55 µmol/L·min K m = 594.17 µmol/L V max = 17.76 µmol/L·min K m = 1516.34 µmol/L FtFLS2 V max = 13.59 µmol/L·min K m = 23646.74 µmol/L V max = 10.25 µmol/L·min K m = 8411.27 µmol/L V max = 12.15 µmol/L·min K m = 21583.23 µmol/L FtFLS3 V max = 24.04µmol/L·min K m = 717.93 µmol/L V max = 13.97 µmol/L·min K m = 6478.21 µmol/L V max = 17.82 µmol/L·min K m = 1609.21 µmol/L 4. Discussion In the plant flavonoid biosynthesis pathway, four 2-ODD members participate: F3H, FNS Ⅰ (Flavone synthase Ⅰ), FLS, and ANS (Anthocyanidin synthase)/leucoanthocyanidin dioxygenase (LDOX)[ 8 ]. These 4 enzymes catalyze oxidation reactions that involve hydroxylation and desaturation of the flavonoid "C ring". However, each enzyme has distinct substrates, leading to their involvement in different branches of the flavonoid biosynthesis pathway[ 34 ]. In TB, FLS catalyzes dihydroflavonols into flavonols, contributing to the flavonol biosynthesis branch, while ANS converts leucoanthocyanidins into anthocyanins, functioning in the anthocyanin biosynthesis branch (Fig. 1 ). F3H, on the other hand, transforms naringenin into DK, providing a common substrate for both branches[ 16 ]. Notably, information about FNS Ⅰ in this species is currently unavailable. Consequently, this study focused on comparing and analyzing FtFLS1-5 with FLSs , ANSs , and F3Hs from Arabidopsis and tobacco . Our findings indicated that deduced FtFLS1-5 and the selected 2-ODDs share a relatively conserved primary protein structure and common features in the Conserved domain A and B. The most significant difference between them lies in the variable N-terminal region (Fig. 2 B). Through a comparison of the 3D models of FtFLS1-5, it becomes evident that the variable region of this amino acid sequence is notably enriched with α -helices and random coils, resulting in the formation of the substrate-binding pocket within FLS proteins (Fig. 2 C)[ 35 ]. The distinct structures formed by these variable regions may play a role in determining the substrate selectivity and affinity of FtFLS1-5[ 36 ]. Furthermore, studies have indicated that FLS can exhibit broader substrate catalysis capabilities beyond dihydroflavonols, showcasing its versatile functionality[ 37 ]. For instance, recombinant OsFLS ( Oryza sativa FLS) demonstrated both FLS and F3H activities; while the recombinant AtFLS1 ( A. thaliana AtFLS1) and CuFLS ( Citrus unshiu FLS) showed both FLS and ANS/LDOX activities[ 38 – 40 ]. The analysis of the phylogenetic tree revealed that FtFLS4 and FtFLS5 belonged to distinct evolutionary branches, implying their potential as multifunctional 2-ODDs. Meanwhile, FtFLS1 and FtFLS3 exhibited FLS activity, and FtFLS2 might possess both FLS and ANS activities (Fig. 2 C). Taking into account the expression levels and tissue specificity of FtFLS1-5 during the flowering stage of TB (Supplementary Fig. 3), this study focused on FtFLS1-3 as the primary candidate genes for further investigation. Numerous studies have highlighted a strong association between upregulated FLS expression and the accumulation of flavonols in TB[ 1 , 18 , 41 ]. Both our previous research and the current study underscore a noteworthy positive correlation between the expression levels of FtFLS1 and the total flavonoid content throughout the flowering stage of TB[ 19 ]. Also, higher transcript levels of FtFLS1 and FtFLS2 were observed in the TB cultivars ‘Hokkai T10’ and ‘Hokkai T8’, along with an increased amount of flavonols, despite their varying responses to exogenous effects[ 42 ]. In a study involving the co-treatment of microwave irradiation and L -phenylalanine during the germination of TB seeds, a notable outcome was the upregulation of FtFLS1 expression along with an increase in specific activity[ 41 ]. This upregulation exhibited a significant positive correlation with the quercetin content. Yao et al. reported that FtFLS1 could be upregulated by a SG7-MYB transcription factor, FtMYB6, which was found to be positively correlated with rutin content in TB seeds[ 24 ]. Furthermore, a wealth of research has shown that environmental factors such as UV, drought, high salt, cold, along with transcription factors like WB40, MYB, bHLH, as well as various plant hormones, often enhance the rutin content in TB by directly or indirectly influencing FtFLS1-2 [ 43 – 45 ]. Therefore, to gain a more comprehensive understanding of the expression patterns of FtFLS1-3 , we cloned their promoter sequences and performed both bioinformatics analysis (Supplementary Table 3) and functional characterization in plants (Figs. 4 and 5 ). The results showed that P FtFLS1−3 exhibited distinct basal transcription activities and tissue specificities in tobacco and Arabidopsis , and also demonstrated differential responsiveness to cold, UV-B, and drought. Likewise, 12-day-old TB seedlings underwent cold, UV-B, and drought treatments, leading to distinct response patterns for FtFLS1-3 . Although the expression pattern of FtFLS1 aligned with that in Arabidopsis , FtFLS2 and FtFLS3 displayed differing expression profiles compared to their Arabidopsis counterparts (Supplementary Fig. 5). This divergence could be attributed to the distinct genetic backgrounds of TB and Arabidopsis , potentially involving different transcription factors in the regulation of specific gene expression during responses to environmental stimuli[ 6 , 24 , 46 ]. Consequently, considering the distinct expression patterns observed for FtFLS1-3 , it is plausible to infer that they might contribute to FLS activities during various developmental stages or in response to unfavorable conditions within the context of TB[ 42 , 44 ]. In the context of in vitro activity assays for recombinant FLS, three dihydroflavanols (DK, DQ, and DM) were employed as substrates to distinguish the catalytic attributes of different FLS enzymes[ 8 ]. Existing literature consistently showcases FLS's heightened affinity for substrates DQ and DK, a phenomenon evident in plants such as C amellia sinensis [ 14 ], Rubus chingii [ 47 ], Ornithogalum caudatum [ 48 ], and others[ 12 , 29 ]. In contrast, myricetin and DM are relatively rare compounds in plants, leading to limited studies on their utilization in assessing FLS enzyme activity[ 49 ]. Notably, FLS exhibits a lower preference for DM when compared to DK and DQ, as observed in examples like Solanum lycopersicum , Solanum tuberosum , and Crocosmia × crocosmiiflora [ 50 – 52 ]. In a recent study, Xing et al . reported on two MrFLSs from Morella rubra [ 53 ]. Their findings further emphasize FLSs' heightened preference for DQ and DK, highlighting that DM is less favorable as a substrate. This study employed both prokaryotic expression and transgenic Arabidopsis approaches to confirm the in vitro and in vivo biological roles of FtFLS1-3 . The findings indicated that recombinant FtFLS1-3 exhibit diverse catalytic abilities and substrate preferences for the three dihydroflavonols. Both FtFLS1 and FtFLS2 exhibited a preference for DQ as substrates, but FtFLS1 displayed significantly higher catalytic activity compared to FtFLS2. Furthermore, FtFLS3 showed a preference for DK, with its catalytic activity falling in between. Although DM is not the preferred substrate for any of the three enzymes, the FtFLSs still exhibit a certain degree of catalytic activity towards it. This phenomenon suggests that FtFLS1-3 have low catalytic ability and affinity towards DM, which is consistent with the typically lower content of myricetin compared to other flavonols in TB[ 54 , 55 ]. In addition, we performed assays to evaluate the activity of recombinant FtFLS4 and FtFLS5 towards dihydroflavanols (Data not shown). Both enzymes displayed remarkably low catalytic efficiency towards DQ (0.06×10 − 3 IU and 0.27×10 − 3 IU, respectively) and demonstrated no detectable activity towards DK and DM. Further investigations revealed that overexpression of FtFLS1-3 in Arabidopsis significantly enhances the levels of total flavonoids and rutin, demonstrating FLS activity (Fig. 8 ). Our findings suggest that the flavonol biosynthetic pathway in TB is primarily characterized by the sequential conversion of DK to DQ (probably by F3'H) and subsequently to quercetin, leading to the ultimate synthesis of rutin[ 5 ]. Notably, FtFLS1 exhibited heightened catalytic activity and substrate affinity, highlighting its pivotal role in orchestrating this process. Moreover, considering the tendency of FLS to exhibit activities resembling other 2-ODDs, our future experiments will delve into exploring the enzymatic characteristics of FtFLS1-5 using diverse substrate types. This approach aims to further advance our comprehension of the intricate regulation underlying TB flavonoid synthesis and metabolism. Conclusion In conclusion, this study cloned and charactered the molecular features of 5 FtFLSs from TB genome. The promoter functions, expression patterns, and biological activities of FtFLS1-3 were elucidated by in vitro and in vivo experiments. Our results indicate that FtFLS1-3 possess varying expression patterns and in vitro enzymatic features, yet share the same biological functions in plants, making them FLS enzymes participating in the flavonol biosynthetic pathway and contributing to rutin synthesis in TB. Declarations AUTHOR INFORMATION Corresponding Author * College of Life Science, Sichuan Agricultural University, Ya’an, Sichuan 625014, China; orcid.org/ 0000-0001-7864-906X; Phone: (+86)-835-2886126; Email: [email protected] AUTHOR CONTRIBUTIONS Chenglei Li performed most of the experiments and wrote the draft of the paper; Jiayi Sun, Guanlan Shi, and Xuerong Zhao participated in the transgenic experiments of Arabidopsis and tobacco ; Jiaqi Shi, Jun Gu, Qihan Ma, Daoping Zeng, Tao Wang, Zizhong Tang and Tongliang Bu carried out part of the material collection; Hui Chen guided biochemical experiment technology; An’hu Wang provided the buckwheat seeds for this study; Qi Wu and Haixia Zhao conceived and designed the studies. All authors have read and approved the final manuscript. Acknowledgements We thank An’hu Wang at the Xichang College, Xichang, China and Dabing Xiang at the Chengdu University, Sichuan, China, for the gift of Tartary buckwheat seeds. FUNDING This work was supported by the National Natural Science Foundation of China (31871698) and the Sichuan Science and Technology Program (2024YFHZ0202). CONFLICTS OF INTEREST The authors also declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this manuscript. References Zhang K, He M, Fan Y, Zhao H, Gao B, Yang K, Li F, Tang Y, Gao Q, Lin T, Quinet M, Janovska D, Meglic V, Kwiatkowski J, Romanova O, Chrungoo N, Suzuki T, Luthar Z, Germ M, Woo SH, Georgiev MI, Zhou M (2021) Resequencing of global Tartary buckwheat accessions reveals multiple domestication events and key loci associated with agronomic traits. Genome Biol 22(1):23 Tak AM, Hami A, Bhat B, Bhat SA, Masoodi KZ, Bhat MA, Shah M, Khan MK, Zargar SM (2022) Unravelling rutin content of tartary buckwheat of north western Himalayas and insights into nucleotide polymorphisms in PAL gene to infer the associations with rutin biosynthesis. 3 Biotech 12(8):156 Huda MN, Lu S, Jahan T, Ding M, Jha R, Zhang K, Zhang W, Georgiev MI, Park SU, Zhou M (2021) Treasure from garden: Bioactive compounds of buckwheat. Food Chem 335:127653 Fabjan N, Rode J, Košir IJ, Wang Z, Zhang Z, Kreft I (2003) Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin. J Agric Food Chem 51(22):6452–6455 Li H, Lv Q, Liu A, Wang J, Sun X, Deng J, Chen Q, Wu Q (2022) Comparative metabolomics study of Tartary (Fagopyrum tataricum (L.) Gaertn) and common (Fagopyrum esculentum Moench) buckwheat seeds. Food Chem 371:131125 Liu W, Feng Y, Yu S, Fan Z, Li X, Li J, Yin H (2021) The Flavonoid Biosynthesis Network in Plants. Int J Mol Sci 22(23):12824 Choi M, Sathasivam R, Nguyen BV, Park NI, Woo SH, Park SU (2021) Expression Analysis of Phenylpropanoid Pathway Genes and Metabolomic Analysis of Phenylpropanoid Compounds in Adventitious, Hairy, and Seedling Roots of Tartary Buckwheat. Plants (Basel) 11(1):90 Wang Y, Shi Y, Li K, Yang D, Liu N, Zhang L, Zhao L, Zhang X, Liu Y, Gao L, Xia T, Wang P (2021) Roles of the 2-Oxoglutarate-Dependent Dioxygenase Superfamily in the Flavonoid Pathway: A Review of the Functional Diversity of F3H, FNS I, FLS, and LDOX/ANS. Molecules 26(21) Britsch L, Heller W, Grisebach H (1981) Conversion of flavanone to flavone, dihydroflavonol and flavonol with an enzyme system from cell cultures of parsley. Z für Naturforschung C 36(9–10):742–750 Holton TA, Brugliera F, Tanaka Y (1993) Cloning and expression of flavonol synthase from Petunia hybrida. Plant J 4(6):1003–1010 Pelletier MK, Murrell JR, Shirley BW (1997) Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis (Further evidence for differential regulation of early and late genes). Plant Physiol 113(4):1437–1445 Owens DK, Alerding AB, Crosby KC, Bandara AB, Westwood JH, Winkel BS (2008) Functional analysis of a predicted flavonol synthase gene family in Arabidopsis. Plant Physiol 147(3):1046–1061 Schilbert HM, Schone M, Baier T, Busche M, Viehover P, Weisshaar B, Holtgrawe D (2021) Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases. Front Plant Sci 12:733762 Jiang X, Shi Y, Fu Z, Li WW, Lai S, Wu Y, Wang Y, Liu Y, Gao L, Xia T (2020) Functional characterization of three flavonol synthase genes from Camellia sinensis: Roles in flavonol accumulation. Plant Sci 300:110632 Liu M, Ma Z, Zheng T, Sun W, Zhang Y, Jin W, Zhan J, Cai Y, Tang Y, Wu Q, Tang Z, Bu T, Li C, Chen H (2018) Insights into the correlation between Physiological changes in and seed development of tartary buckwheat (Fagopyrum tataricum Gaertn). BMC Genomics 19(1):648 Yao Y, Sun L, Wu W, Wang S, Xiao X, Hu M, Li C, Zhao H, Chen H, Wu Q (2021) Genome-Wide Investigation of Major Enzyme-Encoding Genes in the Flavonoid Metabolic Pathway in Tartary Buckwheat (Fagopyrum tataricum). J Mol Evol 89(4–5):269–286 Li H, Lv Q, Ma C, Qu J, Cai F, Deng J, Huang J, Ran P, Shi T, Chen Q (2019) Metabolite profiling and transcriptome analyses provide insights into the flavonoid biosynthesis in the developing seed of tartary buckwheat (Fagopyrum tataricum). J Agric Food Chem 67(40):11262–11276 Li C, Yang K, Yang J, Wu H, Chen H, Wu Q, Zhao H (2022) Tartary buckwheat FtF3′ H1 as a metabolic branch switch to increase anthocyanin content in transgenic plant. Front Plant Sci 13:959698 Li C, Bai Y, Li S, Chen H, Han X, Zhao H, Shao J, Park SU, Wu Q (2012) Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in tartary buckwheat. J Agric Food Chem 60(20):5161–5168 Jing Z, Cheng-Lei L, Fei G, Xiao-Peng L, Qing-Qing L, Hai-Xia Z, Hui-Peng Y, Hui C, An-Hu W (2016) W. Qi, Characterization of Three Glucosyltransferase Genes in Tartary Buckwheat. and Their Expression after Cold Stress Yin Q, Han X, Han Z, Chen Q, Shi Y, Gao H, Zhang T, Dong G, Xiong C, Song C, Sun W, Chen S (2020) Genome-wide analyses reveals a glucosyltransferase involved in rutin and emodin glucoside biosynthesis in tartary buckwheat. Food Chem 318:126478 Xu H, Jiang Z, Lin Z, Yu Q, Song R, Wang B (2022) FtUGT79A15 is responsible for rutinosylation in flavonoid diglycoside biosynthesis in Fagopyrum tataricum. Plant Physiol Biochem 181:33–41 Li C, Zhao H, Li M, Yao P, Li Q, Zhao X, Wang A, Chen H, Tang Z, Bu T, Wu Q (2019) Validation of reference genes for gene expression studies in tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR. PeerJ 7:e6522 Yao P, Huang Y, Dong Q, Wan M, Wang A, Chen Y, Li C, Wu Q, Chen H, Zhao H (2020) FtMYB6, a Light-Induced SG7 R2R3-MYB Transcription Factor, Promotes Flavonol Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum). J Agric Food Chem 68(47):13685–13696 Zhang L, Yang T, Li X, Hao H, Xu S, Cheng W, Sun Y, Wang C (2014) Cloning and characterization of a novel Athspr promoter specifically active in vascular tissue. Plant Physiol Biochem 78:88–96 Luo X-P, Zhao H-X, Xue J, Li C-L, Chen H, Park S-U, Wu Q (2016) Cloning of two basic helix-loop-helix transcription factor genes from Tartary buckwheat (Fagopyrum tataricum) and their expression under abiotic stress. Turkish J Biology 40(6):1192–1201 Rosa FF, Pires CF, Zimmermannova O, Pereira CF (2020) Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors. Bio Protoc 10(10):e3619 Sedmak JJ, Grossberg SE (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem 79(1–2):544–552 Wellmann F, Lukacin R, Moriguchi T, Britsch L, Schiltz E, Matern U (2002) Functional expression and mutational analysis of flavonol synthase from Citrus unshiu. Eur J Biochem 269(16):4134–4142 Rodriguez J-MG, Hux NP, Philips SJ, Towns MH (2019) Michaelis–Menten graphs, Lineweaver–Burk plots, and reaction schemes: Investigating introductory biochemistry students’ conceptions of representations in enzyme kinetics. J Chem Educ 96(9):1833–1845 Prescott AG, John P (1996) Molecular Structure and Role in Plant Metabolism. Annu Rev Plant Physiol Plant Mol Biol 47(1):245–271 Ishihara H, Tohge T, Viehöver P, Fernie AR, Weisshaar B, Stracke R (2016) Natural variation in flavonol accumulation in Arabidopsis is determined by the flavonol glucosyltransferase BGLU6. J Exp Bot 67(5):1505–1517 Harris TK, Keshwani MM (2009) Measurement of enzyme activity. Methods Enzymol 463:57–71 Gebhardt Y, Witte S, Forkmann G, Lukacin R, Matern U, Martens S (2005) Molecular evolution of flavonoid dioxygenases in the family Apiaceae. Phytochemistry 66(11):1273–1284 Preuss A, Stracke R, Weisshaar B, Hillebrecht A, Matern U, Martens S (2009) Arabidopsis thaliana expresses a second functional flavonol synthase. FEBS Lett 583(12):1981–1986 Welford RW, Clifton IJ, Turnbull JJ, Wilson SC, Schofield CJ (2005) Structural and mechanistic studies on anthocyanidin synthase catalysed oxidation of flavanone substrates: the effect of C-2 stereochemistry on product selectivity and mechanism. Org Biomol Chem 3(17):3117–3126 Turnbull JJ, Nakajima J, Welford RW, Yamazaki M, Saito K, Schofield CJ (2004) Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase. J Biol Chem 279(2):1206–1216 Park S, Kim DH, Park BR, Lee JY, Lim SH (2019) Molecular and Functional Characterization of Oryza sativa Flavonol Synthase (OsFLS), a Bifunctional Dioxygenase. J Agric Food Chem 67(26):7399–7409 Martens S, Preuss A, Matern U (2010) Multifunctional flavonoid dioxygenases: flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L. Phytochemistry 71(10):1040–1049 Lukačin R, Wellmann F, Britsch L, Martens S, Matern U (2003) Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase. Phytochemistry 62(3):287–292 Peng W, Wang N, Wang S, Wang J, Bian Z (2023) Effect of co-treatment of microwave and exogenous l-phenylalanine on the enrichment of flavonoids in Tartary buckwheat sprouts. J Sci Food Agric 103(4):2014–2022 Li X, Kim YB, Kim Y, Zhao S, Kim HH, Chung E, Lee JH, Park SU (2013) Differential stress-response expression of two flavonol synthase genes and accumulation of flavonols in tartary buckwheat. J Plant Physiol 170(18):1630–1636 Goyal J, Verma PK (2023) An Overview of Biosynthetic Pathway and Therapeutic Potential of Rutin. Mini Rev Med Chem 23(14):1451–1460 Zargar SM, Hami A, Manzoor M, Mir RA, Mahajan R, Bhat KA, Gani U, Sofi NR, Sofi PA, Masi A (2023) Buckwheat OMICS: present status and future prospects. Crit Rev Biotechnol 1–18 Dong Y, Wang N, Wang S, Wang J, Peng W (2023) A review: The nutrition components, active substances and flavonoid accumulation of Tartary buckwheat sprouts and innovative physical technology for seeds germinating. Front Nutr 10:1168361 Wen W, Alseekh S, Fernie AR (2020) Conservation and diversification of flavonoid metabolism in the plant kingdom. Curr Opin Plant Biol 55:100–108 Lei T, Huang J, Ruan H, Qian W, Fang Z, Gu C, Zhang N, Liang Y, Wang Z, Gao L (2023) Competition between FLS and DFR regulates the distribution of flavonols and proanthocyanidins in Rubus chingii Hu. Front Plant Sci 14:1134993 Sun YJ, He JM, Kong JQ (2019) Characterization of two flavonol synthases with iron-independent flavanone 3-hydroxylase activity from Ornithogalum caudatum Jacq. BMC Plant Biol 19(1):195 Tohge T, Watanabe M, Hoefgen R, Fernie AR (2013) The evolution of phenylpropanoid metabolism in the green lineage. Crit Rev Biochem Mol Biol 48(2):123–152 Bovy A, de Vos R, Kemper M, Schijlen E, Almenar Pertejo M, Muir S, Collins G, Robinson S, Verhoeyen M, Hughes S, Santos-Buelga C, van Tunen A (2002) High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell 14(10):2509–2526 Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54(4):733–749 Irmisch S, Ruebsam H, Jancsik S, Man Saint Yuen M, Madilao LL, Bohlmann J (2019) Flavonol biosynthesis genes and their use in engineering the plant antidiabetic metabolite montbretin A. Plant Physiol 180(3):1277–1290 Xing M, Cao Y, Ren C, Liu Y, Li J, Grierson D, Martin C, Sun C, Chen K, Xu C, Li X (2021) Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae. Plant J 108(2):411–425 Zou L, Wu D, Ren G, Hu Y, Peng L, Zhao J, Garcia-Perez P, Carpena M, Prieto MA, Cao H, Cheng KW, Wang M, Simal-Gandara J, John OD, Rengasamy KRR, Zhao G, Xiao J (2023) Bioactive compounds, health benefits, and industrial applications of Tartary buckwheat (Fagopyrum tataricum). Crit Rev Food Sci Nutr 63(5):657–673 Luthar Z, Golob A, Germ M, Vombergar B, Kreft I (2021) Tartary Buckwheat Hum Nutr Plants (Basel) 10(4):700 Supplementary Files GraphicalAbstract.jpg Graphical abstract Supplementaryfigures.pdf Supplementarytables.pdf Cite Share Download PDF Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Theoretical and Applied Genetics → Version 1 posted Reviewers agreed at journal 24 Jul, 2024 Reviewers invited by journal 23 Jul, 2024 Editor assigned by journal 08 Jun, 2024 First submitted to journal 07 Jun, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4548454","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330930904,"identity":"ae17f76c-4501-4829-a6c0-ace810f2b78c","order_by":0,"name":"Chenglei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBACPmYGNhAtw8DMfIAhASJogFcLG1QLDwMzWwKRWhhgWhh44CoJaGFnfvbg445aHn52ns8fHtTYJTawN2+TYKi5g8dhbOaGM88c55Fs5t0mkXAsObGB51iZBMOxZ3i08LBJ87Yd4zE4zLuNIbGBObFBIsdMgrHhMH4tf4Fa7A/zPP6Q2FCf2CD/hggtjG01PAbMPAwSiQ2HgbbwENLCZibZ23aAR+IwmxnQL8eN23jSii0SjuHWws9/+JnEz7Y6Of7+w48//qiplu1nP7zxxoca3FqgAEkBOJoSCGlgYKgjrGQUjIJRMApGLgAAq5tITqWo1XMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6825-4304","institution":"Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Chenglei","middleName":"","lastName":"Li","suffix":""},{"id":330930905,"identity":"4a3a37ac-7da8-4720-b123-7c8bfe5dbfe8","order_by":1,"name":"Jiayi Sun","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Sun","suffix":""},{"id":330930906,"identity":"7b637916-d18d-47ac-858d-e98f4e929603","order_by":2,"name":"Guanlan Shi","email":"","orcid":"","institution":"HC science Limited Company","correspondingAuthor":false,"prefix":"","firstName":"Guanlan","middleName":"","lastName":"Shi","suffix":""},{"id":330930907,"identity":"a0ed3115-2895-4a5f-9ffe-248b26dc03dc","order_by":3,"name":"Xuerong Zhao","email":"","orcid":"","institution":"Guangxi Kingmed Diagnostics","correspondingAuthor":false,"prefix":"","firstName":"Xuerong","middleName":"","lastName":"Zhao","suffix":""},{"id":330930908,"identity":"bbd2668f-e8fb-4785-9516-ad8650bbfe1d","order_by":4,"name":"Jun Gu","email":"","orcid":"","institution":"Sichuan Agricultural 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University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Wu","suffix":""},{"id":330930918,"identity":"cb9bd4bd-c558-42f4-be18-45e8cf85072d","order_by":14,"name":"Haixia Zhao","email":"","orcid":"https://orcid.org/0000-0001-7864-906X","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haixia","middleName":"","lastName":"Zhao","suffix":""},{"id":330930919,"identity":"8835fe98-0ba6-45db-a20e-6b912b161cdf","order_by":15,"name":"An’hu Wang","email":"","orcid":"","institution":"Xichang University","correspondingAuthor":false,"prefix":"","firstName":"An’hu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-08 01:45:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4548454/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4548454/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00122-025-04997-7","type":"published","date":"2025-08-11T15:56:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62842730,"identity":"600c9977-eedc-4b7c-858f-f2f1a039f99f","added_by":"auto","created_at":"2024-08-20 06:51:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRutin biosynthetic pathway in Tartary buckwheat. \u003c/strong\u003eThe flavonol biosynthesis branch is indicated by blue boxes, and enzymes participating in it are marked in red, the anthocyanin biosynthesis branch is represented by red boxes, and asterisks denote the 2-ODD enzymes. PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumaric acid: CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavonone 3\u003cem\u003eβ\u003c/em\u003e-hydroxylase; F3'H, flavonol 3'-hydroxylase; F3'5'H, flavonoid 3',5'-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-Reductase; ANS, anthocyanidin synthase; UFGT, flavonoid 3-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase. The pathway scheme is adapted and modified from Choi \u003cem\u003eet al.\u003c/em\u003e[7].\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/420f32d885a0a7f84dcfa2e4.jpeg"},{"id":62843639,"identity":"2ae85147-5765-485c-a1f1-acc520ba4c02","added_by":"auto","created_at":"2024-08-20 06:59:30","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":240284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular Characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFtFLSs\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phylogenetic tree of FtFLSs and related sequences. The bars represent evolutionary distance, FtFLS1-3 are indicated by red dots, and FtFLS4-5 are represented by black squares. The following protein sequences were analyzed: AtFLS1 (NM_001203337), AtFLS2 (NM_125753), AtFLS3 (BT003134), AtFLS4 (NM_148158), AtFLS5 (AY114035), AtFLS6 (NM_148083), AtF3H (NP_190692), AtANS1 (Q96323), AtANS2 (NP_181359), NtFLS1 (MF445061), NtFLS2 (MF445062), NtFLS3 (MF445063), NtFLS4 (MF445064), NtANS1 (MF445065), NtANS2 (MF445066), NtANS3 (MF445067), NtANS4 (MF445068), NtF3H1 (MF445069), NtF3H2 (MF445070), NtF3H3 (MF445071), NtF3H4 (MF445072), NtF3H5 (MF445073), NtF3H6 (MF445074), FtFLS1 (FtPinG0006907100), FtFLS2 (FtPinG0006907000), FtFLS3 (FtPinG0008448800), FtFLS4 (FtPinG0004192700), FtFLS5 (FtPinG0002444200), FtANS (FtPinG0006306200), FtF3H1 (FtPinG0006662600), FtF3H2 (FtPinG0008251700). (B) Sequence multi-alignment of FtFLSs protein with other 2-ODDs. The variable region, conservative domain A, and conservative region B of the 2-ODDs are indicated by dashed lines within boxes. (C) The three-dimensional structure of FtFLS1 constructed based on template F5BR59.1. The 70 amino acid residues at the N-terminus are labeled in red, with the conservative motif A in blue and the conservative motif B in yellow. (D) The merged 3D structures of five FtFLSs. The color bar on the right side displays the level of confidence among the amino acid sequences of 5 FtFLS proteins.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/ee22fda5953a8842ccdd7059.jpeg"},{"id":62842735,"identity":"e574f2b6-ac3a-48d7-b16b-6caac43e78c3","added_by":"auto","created_at":"2024-08-20 06:51:31","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFtFLS1-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and flavonol content in different organs during florescence of Tartary buckwheat\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Relative expression analysis of \u003cem\u003eFtFLS1-3\u003c/em\u003e in the roots, stems, leaves, and flowers of TB. (B) Detection of quercetin, kaempferol, myricetin, and rutin content in different tissues of buckwheat during the flowering period. Data are indicated as mean ± SD according to 3 replicates.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/7140a0faa8828270dc98826f.jpeg"},{"id":62842741,"identity":"f5c09acb-e67a-4ef4-b389-3f47a0ac0d16","added_by":"auto","created_at":"2024-08-20 06:51:31","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":189910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe\u0026nbsp;activities\u0026nbsp;of\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003eFtFLS1-3\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;analysis\u0026nbsp;in\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etobacco\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;leaves and\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e seedlings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GUS staining of \u003cem\u003etobacco\u003c/em\u003e leaves, a-e: pBI101-35S-\u003cem\u003eGUS\u003c/em\u003e (positive control), pBI101-\u003cem\u003eGU\u003c/em\u003eS (Negative control, lacking the 35S promoter), pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003eGUS\u003c/em\u003e, pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-GUS\u003c/em\u003e, pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-GUS\u003c/em\u003e. (B) Whole-plant GUS staining of \u003cem\u003eArabidopsis\u003c/em\u003e seedlings, f-h: pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003eGUS\u003c/em\u003e, pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-GUS\u003c/em\u003e, pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-GUS\u003c/em\u003e. (C) The expression levels of \u003cem\u003eGUS\u003c/em\u003e genes promoted by \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e. \u003c/sub\u003eData represent mean ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/db3313a351fa56d31bd3573e.jpeg"},{"id":62843641,"identity":"22465955-e9e0-40f1-b57b-cfb358c53b84","added_by":"auto","created_at":"2024-08-20 06:59:31","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":228268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the expression patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003eFtFLSs\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cstrong\u003e in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Histochemical staining analysis of GUS in T3 transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines during the flowering stage. (B) The expression levels of \u003cem\u003eGUS\u003c/em\u003e genes promoted by \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e \u003c/sub\u003ein different organs. (C-D) The expression levels of \u003cem\u003eGUS\u003c/em\u003e genes promoted by \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e under cold, UV-B, and drought treatments. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/f53a918ed3809bf5afc36a6c.jpeg"},{"id":62842739,"identity":"846b6de0-135f-4139-a925-aed488c2564b","added_by":"auto","created_at":"2024-08-20 06:51:31","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":491414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFtFLS1-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines overexpressing \u003cem\u003eFtFLS1-3\u003c/em\u003eat the flowering stage. Two transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines were chosen from each transformed material and named as FtFLS1-L1, FtFLS1-L2, FtFLS2-L1, FtFLS2-L2, FtFLS3-L1, FtFLS3-L2. (B) Expression of\u003cem\u003e FtFLS1-3\u003c/em\u003e in transgenic \u003cem\u003eArabidopsis\u003c/em\u003elines. The \u003cem\u003eβ\u003c/em\u003e-actin gene of \u003cem\u003eArabidopsis\u003c/em\u003e was used as an internal reference, with its expression level set to \"1\". (C) Total flavonoid content in the transgenic \u003cem\u003eArabidopsis\u003c/em\u003elines. (D) Rutin content in the transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines. Data represent mean ± SD of three biological replicates. Asterisks indicate significant differences (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/c85ae4be6665e484322dfa59.jpeg"},{"id":62842740,"identity":"e92a3544-5532-4c53-bfb3-b64398e930fd","added_by":"auto","created_at":"2024-08-20 06:51:31","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":154936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProkaryotic expression and activity detection of recombinant FtFLS1-3 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) SDS-PAGE analysis of the recombinant FtFLS1, FtFLS2, and FtFLS3. M represents protein standard molecular weight. Lane 1 corresponds to the pre-induction culture, while Lanes 2-7 correspond to the culture fluids after induction for 1 to 6 hours, respectively. The recombinant protein is indicated by the red arrow. (B) TLC of reaction products using dihydroquercetin (D) as substrate. D (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e=\u003c/sub\u003e 0.33) stands for dihydroquercetin standard, Q (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e=\u003c/sub\u003e 0.45) stands for quercetin standard, and DQ (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e=\u003c/sub\u003e 0.33-0.45) represents a mixture of dihydroquercetin and quercetin standards. The reaction products of the three recombinant proteins are indicated by black arrows. The \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e \u003c/em\u003evalues of the spots in the figure were as follows: a2= 0.33, a1= 0.33, a3= 0.33, b2= 0.44, b1= 0.45 and b3= 0.44.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/cefb0bb5224ef06b75fa3228.jpeg"},{"id":62842736,"identity":"1e02a5ab-6dac-40a4-b20c-5a02cc0c61cf","added_by":"auto","created_at":"2024-08-20 06:51:31","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":146883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSDS-PAGE analysis and the initial velocity of recombinant FtFLS1-3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) SDS-PAGE analysis of recombinant FtFLS1-3 in the purification process. M: protein standard molecular weight; Lanes: 1, sample of soluble fraction (5 mL); 2, sample of insoluble fraction; 3, sample collected after His binding process (5 mL); 4−8, samples collected after gradient rinsing with 50 mM (2 mL), 100 mM (2 mL), 150 mM (2 mL), and 200 mM (2 mL) imidazole buffers; 8, sample of purified FtFLS1-3 (500 µL, indicated by the red arrow). (B) The initial velocity of the enzyme-catalyzed reaction.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/7f9965f4282d812ba16ae562.jpeg"},{"id":89310822,"identity":"61c73d12-1772-437c-a578-7fb57d157097","added_by":"auto","created_at":"2025-08-18 16:10:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3281486,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/f5b12c09-ea82-445f-b6b6-a6eabe0aa3e7.pdf"},{"id":62842731,"identity":"75b99113-126a-4f7d-aee3-5c27591c83e3","added_by":"auto","created_at":"2024-08-20 06:51:30","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":278975,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/5359efbd435ce1b26596221c.jpg"},{"id":62842733,"identity":"1bd1c2e8-3f97-467b-87d6-ffb8d47798ee","added_by":"auto","created_at":"2024-08-20 06:51:30","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":523270,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/e389a925d4d8858ce0e4df11.pdf"},{"id":62844096,"identity":"eced59a5-bd44-4c5e-b6c1-aec40952dd18","added_by":"auto","created_at":"2024-08-20 07:07:30","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":98733,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4548454/v1/166770c89726dbfa0dfa40af.pdf"}],"financialInterests":"","formattedTitle":"Insight into the Rutin Biosynthesis in the Unique Flavonol Synthesis Pathway of Tartary Buckwheat Based on the Enzymatic Functions of FLSs","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTartary buckwheat (\u003cem\u003eFagopyrum tataricum\u003c/em\u003e Gaertn., abbreviated as TB) originated in the southwestern region of China[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This pseudo-cereal crop holds significant importance for countries surrounding the Himalayan regions[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The primary bioactive components in TB are flavonoids, which offer diverse benefits for human health, including antioxidation, anti-tumor, anti-hypertension, and so on[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To date, over 100 different types of flavonoid compounds have been identified in TB, falling into categories such as flavonols, flavones, isoflavones, flavanones, flavan-3-ols, anthocyanins, fagopyrins, proanthocyanidins, flavonolignans, and others[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, rutin (quercetin 3-\u003cem\u003eO\u003c/em\u003e-rutinoside) is the predominant flavonoid compound, accounting for 70\u0026ndash;85% of the total flavonoid content[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As such, flavonol compounds, primarily composed of rutin, kaempferol (K), myricetin (M), and quercetin (Q), emerge as the most critical and essential bioactive constituents in TB[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe metabolic pathway of plant flavonoid biosynthesis constitutes a significant component of the phenylpropanoid metabolic pathway and has undergone extensive investigation[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Within this pathway, the flavonol biosynthesis branch is primarily governed by five enzymes: flavonone 3\u003cem\u003eβ\u003c/em\u003e-hydroxylase (F3H), flavonoid 3\u0026prime;-hydroxylase (F3'H), flavonoid 3', 5'-hydroxylase (F3'5'H), flavonol synthase (FLS), and flavonoid 3-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase (UFGT)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Of these enzymes, flavonol synthase (FLS, EC: 1.14.20.6) stands out as a pivotal catalyst in the flavonol synthesis pathway, occupying a central and crucial role[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. FLS belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily and operates with 2-oxoglutarate as a cosubstrate, in conjunction with Fe\u003csup\u003e2+\u003c/sup\u003e serving as a cofactor[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In plants, the first detection of flavonol synthase FLS activity occurred in a culture of parsley cells in 1981[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Subsequently, the initial \u003cem\u003eFLS\u003c/em\u003e cDNA was isolated from \u003cem\u003ePetunia hybrida\u003c/em\u003e and characterized through yeast expression in 1993, while the first genomic \u003cem\u003eFLS\u003c/em\u003e sequence was identified from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e in 1997[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Presently, the NCBI database contains annotations for approximately 2340 \u003cem\u003eFLS\u003c/em\u003e sequences across flowering plants. In reality, the presence of \u003cem\u003eFLS\u003c/em\u003e varies in copy numbers across the genomes of different species. The genome of \u003cem\u003eA. thaliana\u003c/em\u003e harbors 6 \u003cem\u003eFLS\u003c/em\u003e genes, with \u003cem\u003eAtFLS1\u003c/em\u003e and \u003cem\u003eAtFLS3\u003c/em\u003e being the sole contributors to FLS activity, while \u003cem\u003eAtFLS2\u003c/em\u003e, \u003cem\u003eAtFLS4\u003c/em\u003e, and \u003cem\u003eAtFLS6\u003c/em\u003e are categorized as pseudogenes[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similarly, \u003cem\u003eBrassica napus\u003c/em\u003e exhibits 13 identified sequences of homologous \u003cem\u003eFLS\u003c/em\u003e, displaying diverse expression intensities and tissue specificities at the transcriptional level[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Noteworthy are \u003cem\u003eBnaFLS1-1\u003c/em\u003e and \u003cem\u003eBnaFLS1-2\u003c/em\u003e, both identified in \u003cem\u003eEscherichia coli\u003c/em\u003e as having simultaneous FLS and F3H activities. Jiang \u003cem\u003eet al.\u003c/em\u003e heterologously expressed 3 \u003cem\u003eCsFLS\u003c/em\u003e genes from \u003cem\u003eCamellia sinensis\u003c/em\u003e, all of which demonstrated catalytic activity in \u003cem\u003ein vitro\u003c/em\u003e experiments, enhancing kaempferol synthesis in \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, the comprehensive identification of diverse \u003cem\u003eFLS\u003c/em\u003e copies in plants is imperative for a thorough comprehension of the metabolic pathway governing flavonol synthesis.\u003c/p\u003e \u003cp\u003eFrom our previous studies, we have annotated five flavonol branch related genes based on the TB genome and transcript data[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Among these, \u003cem\u003eFtF3'H1\u003c/em\u003e, \u003cem\u003eFtFLS1\u003c/em\u003e, \u003cem\u003eFtUFGT1-3\u003c/em\u003e, \u003cem\u003eFtUGT73BE5\u003c/em\u003e, and \u003cem\u003eFtUGT79A15\u003c/em\u003e have been identified with confirmed biological functions, either \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e[\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The catalytic products of the FLS encompass kaempferol, quercetin, and myricetin. Notably, these products could potentially serve as substrates for the uridine diphosphate UFGT, ultimately leading to the formation of rutin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Of utmost significance, the TB genome harbors five homologous sequences of \u003cem\u003eFLS\u003c/em\u003e genes, among which \u003cem\u003eFtFLS1\u003c/em\u003e has been previously identified in our earlier work[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nevertheless, the precise molecular characteristics of the remaining \u003cem\u003eFtFLS\u003c/em\u003e genes remain undisclosed. Their catalytic properties on different substrates and their biological activities await confirmation. Consequently, further analysis is warranted to illuminate their regulatory role in the material flow within the biochemical process of rutin synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this investigation, we conducted an extensive molecular identification of the five \u003cem\u003eFtFLS\u003c/em\u003e genes present in TB. Notably, two novel \u003cem\u003eFLS\u003c/em\u003e genes, designated as \u003cem\u003eFtFLS2\u003c/em\u003e and \u003cem\u003eFtFLS3\u003c/em\u003e, were successfully discerned. Additionally, we performed a comparative analysis of promoter characteristics, recombinant protein activity, and biological impacts associated with \u003cem\u003eFtFLS1-3\u003c/em\u003e, utilizing transgenic \u003cem\u003etobacco\u003c/em\u003e, a prokaryotic expression system, and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. In summary, our study furnishes essential foundational data, contributing to an enhanced comprehension of the interplay between molecular attributes and functions exhibited by \u003cem\u003eFtFLS\u003c/em\u003e genes in TB. Notably, it serves as a valuable point of reference for dissecting the metabolic flux within TB's flavonol synthesis pathway. Moreover, our findings offer comprehensive evidence substantiating the rutin biosynthesis process.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials.\u003c/h2\u003e \u003cp\u003eThe cultivated TB variety, known as \"Xiqiao No. 2,\" was cultivated in Leying Township, Tianquan County, Ya'an city, located in Sichuan province, China (coordinates: longitude 102\u0026deg;36\u0026prime;E, latitude 29\u0026deg;79\u0026prime;N, with an elevation of approximately 800 m). After approximately 60 days of germination, TB entered the initial flowering stage, at which point the roots, stems, leaves, flowers, and seeds were meticulously collected to serve as experimental specimens. All collected samples were promptly frozen in liquid nitrogen, ensuring their preservation for subsequent processes such as DNA extraction, RNA extraction, flavonoid content analysis, and qRT-PCR experiments. \u003cem\u003eNicotiana tabacum\u003c/em\u003e (NC89) was cultivated under controlled conditions comprising 12 hours of light followed by 12 hours of darkness, maintaining a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and providing a light intensity of approximately 100 \u0026micro;mol/(m\u0026sup2;\u0026middot;s). For \u003cem\u003eA. thaliana\u003c/em\u003e (Columbia-0), cultivation took place within an artificial climate chamber with parameters set to 16 hours of light and 8 hours of darkness, a constant temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and a light intensity ranging between 120 and 150 \u0026micro;mol/(m\u0026sup2;\u0026middot;s).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Chemical reagents, Microbial strains and Plasmids.\u003c/h2\u003e \u003cp\u003eThe chemical reagents and flavonoid standard substances used in this study were purchased from Vanke (China), Tiandz (China), GE Healthcare (United States), and Sigma (United States). The plant genomic DNA extraction kit, plant total RNA extraction kit, plasmid extraction kit, restriction endonucleases, \u003cem\u003eTaq\u003c/em\u003e DNA polymerase, and qRT-PCR reagent kit were obtained from Takara (Japan). The \u003cem\u003eE. coli\u003c/em\u003e strains DH5a and BL21(DE3), as well as the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 used in this experiment, were obtained from our laboratory's collection. The pMD19-T plasmid was used for T-cloning of the target gene, pET-30b(+) was used for prokaryotic expression, and pBI-101 and pCAMBIA1301 were used for heterologous expression of the target gene in \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003etobacco\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cloning and Characterization of \u003cem\u003eFtFLSs\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eBased on our preceding research, we have successfully annotated and designated five sequences within the TB genome as \u003cem\u003eFLS\u003c/em\u003e genes, identified as \u003cem\u003eFtFLS1\u003c/em\u003e through \u003cem\u003eFtFLS5\u003c/em\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To isolate these \u003cem\u003eFtFLSs\u003c/em\u003e, we extracted genomic DNA and total RNA from diverse tissues of TB. Subsequent to this, we conducted PCR amplification, followed by cloning and sequencing of the resultant products. The obtained DNA and cDNA sequences of \u003cem\u003eFtFLSs\u003c/em\u003e were compared using the Gene Structure Display Server 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The deduced amino acid sequence of FtFLSs protein were analyzed using the DNAMAN software (Version 9.0) and BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/BLAST\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/BLAST\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) programs in NCBI. The multiple sequence alignment was performed with Clustal X (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.clustal.org\u003c/span\u003e\u003cspan address=\"http://www.clustal.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the phylogenetic tree was drawn using the MEGA 11 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the maximum likelihood method. The three-dimensional modeling of the proteins was conducted on the Swiss-model website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The genome data of TB is sourced from the MBK database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mbkbase.org/Pinku1\u003c/span\u003e\u003cspan address=\"https://www.mbkbase.org/Pinku1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the transcriptome is obtained from the SRA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/sra\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Accession No. GSE111937). The primer sequences are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Expression analysis of \u003cem\u003eFtFLS1-3\u003c/em\u003e and measurement of flavonol content.\u003c/h2\u003e \u003cp\u003eIn order to accurately analyze gene expression in TB, we established a real-time reverse transcription PCR (qRT-PCR) method in our previous work, identified the housekeeping gene \u003cem\u003eFtH3\u003c/em\u003e (Accession NO. HM628903) as the optimal internal reference gene, and applied it to the current study[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Then, qRT-PCR was performed following the instructions provided by the SYBR\u0026reg; Premix Ex\u003cem\u003eTaq\u003c/em\u003e\u0026trade; (Takara, Japan) kit and the operational manual of the CFX96 Real-Time PCR Machine (Bio-Rad, USA). The data were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primer sequences are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eThe method described by Yao \u003cem\u003eet al.\u003c/em\u003e was employed to extract flavonoid compounds from different tissues, which were then subjected to high-performance liquid chromatography (HPLC) analysis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The creation of standard curves involved the preparation of quercetin, kaempferol, myricetin, and rutin standard solutions in methanol. The concentrations of these solutions were 0.12 mg/mL, 0.12 mg/mL, 0.12 mg/mL, and 0.16 mg/mL, respectively. For each solution, injections of 5 \u0026micro;L, 10 \u0026micro;L, 15 \u0026micro;L, 20 \u0026micro;L, and 25 \u0026micro;L were made, each executed in triplicate. The subsequent analytical analysis was carried out using HPLC with a C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m internal diameter, RStech, Korea). The mobile phase, composed of a mixture of methanol and water/acetic acid (98:2, v/v), was consistently maintained at 30\u0026deg;C. Each sample, introduced through a 10 \u0026micro;L injection, was subsequently eluted at a controlled flow rate of 1.0 mL/min. Detection occurred at a wavelength of 330 nm, facilitating the quantification of flavonol concentrations using a derived standard curve. Correlation analysis was performed using the statistical software SPSS (Version 20), where the Pearson method was applied. Additionally, the Influence Degree (ID) was employed to evaluate the association between gene expression levels and flavonol contents.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cloning and identification of the promoters of \u003cem\u003eFtFLS1-3\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eTo examine the expression profiles of \u003cem\u003eFtFLS1-3\u003c/em\u003e, we cloned the promoter sequences of these three genes using PCR. The acquired \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e sequences underwent analysis \u003cem\u003evia\u003c/em\u003e the PlantCARE online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sphinx.rug.ac.be:8080/PlantCARE\u003c/span\u003e\u003cspan address=\"http://sphinx.rug.ac.be:8080/PlantCARE\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the GSDS tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To identify \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e, distinct plasmids were generated: pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e-GUS, pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e-GUS, pBI101-\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e-GUS, pBI101-35S-GUS (positive control), and pBI101-GUS (Negative control, lacking the 35S promoter). These plasmids were employed for separate transient transformation assays in \u003cem\u003etobacco\u003c/em\u003e leaves, as well as for establishing stable expression in \u003cem\u003eArabidopsis\u003c/em\u003e. Following the protocols outlined by Zhang \u003cem\u003eet al.\u003c/em\u003e, the 5 plasmids were introduced into tobacco leaves through transient transfection using \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Subsequently, \u003cem\u003eβ\u003c/em\u003e-glucuronidase (GUS) activity was visualized \u003cem\u003evia\u003c/em\u003e histochemical staining after a 48-hour incubation period. Similarly, employing the floral dip method as detailed by Yao \u003cem\u003eet al.\u003c/em\u003e, we introduced the aforementioned 5 plasmids into \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Kanamycin-resistant seedlings were transferred to pots, validated through PCR analysis, and subsequently, the transgenic T3 generation of \u003cem\u003eArabidopsis\u003c/em\u003e lines were confirmed and utilized for subsequent experiments. To identify the tissue-specific expression pattern of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e, GUS staining and qRT-PCR analysis were performed on the \u003cem\u003eArabidopsis\u003c/em\u003e lines at the 20-days old and flowering stages, respectively. To distinguish the responses of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e to various environmental factors, \u003cem\u003eArabidopsis\u003c/em\u003e lines at the flowering stage were subjected to treatments of cold (4℃), UV-B (302nm), and drought (30% PEG-6000) using the method outlined by Luo \u003cem\u003eet al.\u003c/em\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The control group (Con) comprised of untreated \u003cem\u003eArabidopsis\u003c/em\u003e lines, and qRT-PCR was conducted following the previously established procedure, using \u003cem\u003eAtActin\u003c/em\u003e (Accession No. AF149413) as the reference gene. The primer sequences are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Expression of \u003cem\u003eFtFLS1-3\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eTo determine the biological functions of \u003cem\u003eFtFLS1-3\u003c/em\u003e in plants, we generated transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines with overexpressed \u003cem\u003eFtFLS1-3\u003c/em\u003e genes, individually. The recombinant plasmids, namely pCAMBIA-1301-\u003cem\u003eFtFLS1\u003c/em\u003e, pCAMBIA-1301-\u003cem\u003eFtFLS2\u003c/em\u003e, and pCAMBIA-1301-\u003cem\u003eFtFLS3\u003c/em\u003e, were engineered and introduced into \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101. The transgenic manipulation of \u003cem\u003eArabidopsis\u003c/em\u003e was performed using the previously mentioned floral dipping method[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Following this, the seedlings resistant to hygromycin were transplanted into pots and verified as transgenic through PCR analysis. Subsequently, the transgenic T3 generation of \u003cem\u003eArabidopsis\u003c/em\u003e lines were confirmed \u003cem\u003evia\u003c/em\u003e PCR. The transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines were cultivated until reaching the flowering stage, at which point fresh samples were harvested and promptly cryopreserved in liquid nitrogen. This procedure was carried out for the determination of both total flavonoid content and rutin content[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Functional expression of \u003cem\u003eFLS1-3\u003c/em\u003e in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eTo gain a more comprehensive understanding of the biological impacts of \u003cem\u003eFtFLS1-3\u003c/em\u003e, 3 recombinant plasmids (pET-30(b)-\u003cem\u003eFtFLS1\u003c/em\u003e, pET-30(b)-\u003cem\u003eFtFLS2\u003c/em\u003e, and pET-30(b)-\u003cem\u003eFtFLS3\u003c/em\u003e) were constructed and transformed into \u003cem\u003eE. coli\u003c/em\u003e strain BL21(DE3) for prokaryotic expression[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The recombinant FtFLSs were tagged with (His)6-tags at the N- and C-termini to facilitate protein purification. Isolated clones harboring pET-30(b)-\u003cem\u003eFtFLS1-3\u003c/em\u003e were meticulously selected and cultivated in Luria-Bertani medium supplemented with kanamycin (50 \u0026micro;g/mL). The cultures were agitated at 180 rpm until the optical density at 600 nm (\u003cem\u003eOD\u003c/em\u003e600) reached 0.5. For the production of soluble FtFLS1-3 proteins, the conclusive expression conditions were achieved by introducing isopropyl-\u003cem\u003eβ\u003c/em\u003e-D-thiogalactopyranoside (IPTG) to attain a final concentration of 1 mM. Subsequently, the culture was incubated at 25\u0026deg;C for a duration of 6 hours. Crude enzyme solutions encompassing the recombinant FtFLS1-3 were obtained through ultrasonic disruption. The contents of all samples were subjected to analysis through 12.5% SDS-PAGE.\u003c/p\u003e \u003cp\u003eThe crude enzyme solutions were utilized to determine the FLS enzyme activity using DQ as the substrate[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The reaction mixture, comprising 1 mL, included 100 \u0026micro;L of crude enzyme solution, 100 \u0026micro;M DQ, 111 mM sodium acetate, 83 \u0026micro;M 2-oxoglutaric acid, 42 \u0026micro;M ferrous sulfate, and 2.5 mM vitamin C. The reaction was carried out at 37\u0026deg;C and pH 5.0 for a duration of 30 minutes. After the reaction, the mixtures were extracted twice with an equivalent volume of ethyl acetate, followed by separation using thin-layer chromatography (TLC) on silica gel G (toluene/acetic ether/formic acid, 5:2:2). Internal standards included DQ, quercetin (Q), and a mixture of DQ-Q. The identification of reaction products was accomplished through standard sample comparisons or by determining their retention factor (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e) values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Catalytic characteristics of recombinant FtFLS1-3 \u003cem\u003ein vitro\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eAfter 10 hours of induction, following the approach by Li \u003cem\u003eet al.\u003c/em\u003e (with minor modifications)[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the soluble protein fractions of recombinant FtFLS1-3 were purified using a HiTrap FF column from GE Healthcare (USA). The samples underwent sequential washing with 2 mL volumes of 50 mM, 100 mM, 150 mM, and 200 mM imidazole buffer (containing 0.5 M NaCl and 20 mM sodium phosphate at pH 7.4) before undergoing assessment \u003cem\u003evia\u003c/em\u003e 12.5% SDS-PAGE. To remove imidazole from the samples, we conducted a protein ultrafiltration experiment[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A 2 mL portion of the diluted sample was added to an Amicon\u003csup\u003e@\u003c/sup\u003e Ultra-15 10K NMWL (Nominal Molecular Weight Limit) Centrifugal Filter device (Merk, Germany) and centrifuged at 4000 g for 30 minutes at 4\u0026ordm;C. The ultrafiltered samples were adjusted to a volume of 500 \u0026micro;L using sterile water, and protein content was determined using the Coomassie Brilliant Blue G-250 method[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo individually assess the catalytic activity of the three recombinant FtFLSs towards dihydroflavonols (DQ, DK, and DM), we established standard curves for quantifying the concentrations of the resultant reaction products[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Different concentrations of kaempferol, quercetin, and myricetin (0.5, 1, 2, 3, 5, 7, 9, 12, and 15 \u0026micro;g/mL) were dissolved in the enzyme reaction solution (composed of 111 mM sodium acetate, 83 \u0026micro;M 2-oxoglutaric acid, 42 \u0026micro;M ferrous sulfate, 2.5 mM vitamin C, pH 5.0). A spectrophotometer (Shimadzu, Japan) with a 0.5 cm path length was used to perform a full spectrum scan in order to obtain characteristic absorption peaks, standard curves, regression equations, and correlation coefficient (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e). We employed the optimal wavelength of 365.5 nm for measuring kaempferol in the enzyme assays. The corresponding calibration curve was characterized by the regression equation A\u0026thinsp;=\u0026thinsp;0.0228C (A represents absorbance and C denotes concentration in \u0026micro;g/mL), yielding an \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e value of 0.9973. Similarly, we utilized the optimal wavelength of 365.5 nm to measure quercetin in the enzyme assays. The calibration curve was determined based on the regression equation A\u0026thinsp;=\u0026thinsp;0.0223C, resulting in an \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e value of 0.9975. For the measurement of myricetin within the enzyme assays, the optimal wavelength of 426.5 nm was employed. The calibration curve for myricetin was determined based on the regression equation A\u0026thinsp;=\u0026thinsp;0.0169C, resulting in an \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e value of 0.9957. The data is presented in Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eFollowing the procedure outlined by Wellmann et al., FLS activity was determined using 12.5 \u0026micro;g of purified FtFLS1-3 and 100 \u0026micro;M substrates (with a molar ratio of enzyme to substrate set at 180:1) at a temperature of 37\u0026deg;C and a pH of 5.0[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The determination of the initial velocity of the enzyme-catalyzed reaction was conducted at 37\u0026deg;C, with continuous measurements being recorded over a period of 20 minutes. Absorbance values were collected at 2-minute intervals to investigate the correlation between the product generated through catalysis by FtFLS1, FtFLS2, and FtFLS3. Subsequently, the kinetic parameters (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) and maximum velocity (\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) values for each FtFLS enzyme were determined using three dihydroflavonols (20, 30, 40, 50, 60, 70, and 80 \u0026micro;mol/L) as substrates within the enzymes' linear range of product generation. The \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e values were calculated using the Lineweaver-Burk plot method[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A single unit (IU) was defined as the quantity of FLS catalyzing the generation of 1 \u0026micro;mol of product from the substrates per minute at 37\u0026deg;C (\u0026micro;mol/min). The specific activity was denoted as IU/mg[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Molecular Characterization of \u003cem\u003eFtFLSs\u003c/em\u003e form TB.\u003c/h2\u003e \u003cp\u003eThe DNA and cDNA sequences of the five \u003cem\u003eFtFLSs\u003c/em\u003e (\u003cem\u003eFtFLS1\u003c/em\u003e to \u003cem\u003eFtFLS5\u003c/em\u003e) were successfully obtained through PCR. Alignment of these five \u003cem\u003eFtFLSs\u003c/em\u003e with the TB genome and transcriptome data revealed their consistency with the sequences stored in the database. The outcomes indicated that \u003cem\u003eFtFLS1\u003c/em\u003e and \u003cem\u003eFtFLS2\u003c/em\u003e were positioned on chromosome 7 of TB and represented tandem repeat sequences. \u003cem\u003eFtFLS3\u003c/em\u003e was found on chromosome 2, \u003cem\u003eFtFLS4\u003c/em\u003e on chromosome 3, and \u003cem\u003eFtFLS5\u003c/em\u003e on chromosome 1. The coding DNA sequences (CDS) of the five \u003cem\u003eFtFLSs\u003c/em\u003e demonstrated a similarity of 75.7%, while their deduced protein sequences exhibited a similarity of 43.5%. Supplementary Table\u0026nbsp;2 and Supplementary Fig.\u0026nbsp;2 were provided to offer supplementary details regarding the five \u003cem\u003eFtFLSs\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn order to dissect the molecular attributes and potential roles of the predicted FtFLS proteins, we selected protein sequences from three 2-ODD enzymes (F3H, FLS, and ANS) associated with rutin synthesis in \u003cem\u003eF. tartaricum\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, and \u003cem\u003eN. tabacum\u003c/em\u003e. The phylogenetic analysis illuminated the evolutionary tree, which divided into two distinct branches termed Cluster I and Cluster II (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Within Cluster I, a close phylogenetic relationship was evident between FtFLS1, FtFLS3, 6 AtFLSs from \u003cem\u003eArabidopsis\u003c/em\u003e, and 4 NtFLSs from \u003cem\u003etobacco\u003c/em\u003e, as they were collectively placed into Group A. On the other hand, FtFLS2 was assigned to Group B, joined by AtANS from \u003cem\u003eF. tartaricum\u003c/em\u003e and 4 NtANSs from \u003cem\u003etobacco\u003c/em\u003e. As for Cluster II, FtFLS4 occupied an independent position in Group C, while FtFLS5 exhibited marked similarity to AtANS. Additionally, all F3Hs from the three species were found to cluster together in Group E. These results collectively indicate that among the five FtFLS proteins, FtFLS1, FtFLS2, and FtFLS3 are likely to share comparable biological functions, whereas FtFLS4 and FtFLS5 might serve as multifunctional enzymes within the rutin synthesis metabolic pathway of TB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe multiple sequence alignment revealed that all 31 selected 2-ODD sequences encompass the conserved functional regions recognized as Conserved domain A and Conserved domain[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is noteworthy that the major divergences were localized within the variable region comprised of the initial 70 amino acids at the N-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Further insights into the impact of the N-terminal variable region of FtFLSs on the configuration of the enzyme-substrate pocket structure were obtained through additional 3D modeling results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Of significant importance, FtFLS4 and FtFLS5 exhibited a stronger similarity to the advanced structure of ANS (pdb No. A0A5J5AX16) as opposed to FLS (pdb No. F5BR59.1). By leveraging our previously documented transcriptome data of flowering TB, we generated an updated expression heatmap for \u003cem\u003eFtFLS1-5\u003c/em\u003e. The outcomes underscored the presence of tissue specificity and variations in the expression levels of \u003cem\u003eFtFLS1-5\u003c/em\u003e. Importantly, \u003cem\u003eFtFLS1\u003c/em\u003e displayed the highest average expression level, followed by \u003cem\u003eFtFLS2\u003c/em\u003e, while \u003cem\u003eFtFLS3\u003c/em\u003e exhibited the lowest expression level (Supplementary Fig.\u0026nbsp;3). Consequently, \u003cem\u003eFtFLS1-3\u003c/em\u003e were selected as prospective candidate genes for subsequent investigations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Analysis of the expression of \u003cem\u003eFtFLSs\u003c/em\u003e and flavonoid contents in TB.\u003c/h2\u003e \u003cp\u003eTo validate the expression levels of \u003cem\u003eFtFLS1-3\u003c/em\u003e as indicated by transcriptome data and explore their potential correlation with flavonoid contents, qRT-PCR was employed to quantify gene expression, while flavonoid contents were measured in various tissues of TB during the flowering stage.\u003c/p\u003e \u003cp\u003eThe qRT-PCR analysis revealed that \u003cem\u003eFtFLS1\u003c/em\u003e displayed elevated expression levels in the stems, leaves, and flowers of flowering buckwheat, with a moderate level of expression in the roots. In contrast, both \u003cem\u003eFtFLS2\u003c/em\u003e and \u003cem\u003eFtFLS3\u003c/em\u003e shared a comparable expression pattern, marked by low expression in the roots and flowers, and negligible expression in other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The provided data strongly reinforces the observed expression patterns of \u003cem\u003eFtFLS1-3\u003c/em\u003e in our previous transcriptome analysis of buckwheat[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. HPLC analysis unveiled rutin as the predominant flavonol component in TB (dry weight, DW), present in relatively substantial quantities across various tissues (114.96 mg/g in flowers, 107.53 mg/g in leaves, 8.32 mg/g in stems, and 1.62 mg/g in roots, DW). Quercetin, being the principal precursor of rutin, exhibited the highest content in buckwheat flowers (9.25 mg/g, DW), while kaempferol and myricetin were detected at low levels in all tissues (less than 1.5 mg/g, DW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The correlation between the expression levels of \u003cem\u003eFtFLS1-3\u003c/em\u003e and the content of four different flavonols was analyzed using Pearson's correlation coefficient method with an ID of 0.75, indicating strong positive correlation. The results demonstrated that the expression of \u003cem\u003eFtFLS1\u003c/em\u003e displayed a robust positive correlation with quercetin content (0.80) and rutin content (0.75), a pronounced negative correlation with kaempferol content (-0.79), while no significant correlation was observed with myricetin. Additionally, a correlation coefficient of 0.68 existed between the expression of \u003cem\u003eFtFLS2\u003c/em\u003e and kaempferol content, which did not meet the established threshold. Notably, no significant correlation was identified between the expression of \u003cem\u003eFtFLS3\u003c/em\u003e and the content of the four flavonols. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation analysis of the expression levels of \u003cem\u003eFtFLS1-3\u003c/em\u003e and the content of 4 different flavonols in flowering Taratary buckwheat\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNames\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKaempferol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMyricetin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRutin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.79*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.80*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.75*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e(*) denotes that the absolute value of A exceeds 0.5.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on the preceding findings, it is plausible to infer that \u003cem\u003eFtFLS1\u003c/em\u003e is likely a predominant player in the quercetin biosynthesis across diverse tissues of flowering buckwheat. This role could involve enhancing rutin accumulation \u003cem\u003evia\u003c/em\u003e quercetin synthesis. Conversely, \u003cem\u003eFtFLS2\u003c/em\u003e and \u003cem\u003eFtFLS3\u003c/em\u003e might participate in distinct flavonol synthesis pathways, possibly contributing to the synthesis of various flavonol types through alternative routes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Cloning and identification of the promoters of \u003cem\u003eFtFLS1-3\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eIn order to gain a better understanding of the expression patterns for \u003cem\u003eFtFLS1-3\u003c/em\u003e, we cloned the promoter sequences located approximately 2000 bp upstream of their respective start codons (ATG). These sequences have been designated as \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e (2479 bp), \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e (2574 bp) and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFL\u003c/em\u003eS3\u003c/sub\u003e(2046 bp). These sequences have a high AT content (over 60%), which is consistent with the characteristics of plant promoters. The web tools PlantCARE and GSDS were utilized to analyze the DNA sequences of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e, and the resulting details are presented in Supplementary Table\u0026nbsp;3. Our results showed that \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e encompassed a wide range of \u003cem\u003ecis\u003c/em\u003e-acting elements, exhibiting varying quantities. Additionally, among the core promoter elements (TATA-box and conserved CAAT-box), prominent elements comprised light-responsive components like G-box, Box-I, and Box-4. These outcomes propose the potential responsiveness of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e to various environmental stimuli. Consequently, we proceeded with the functional analysis of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e, employing transgenic \u003cem\u003etobacco\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e methodologies.\u003c/p\u003e \u003cp\u003eIn the experiment involving agrobacterium-mediated transformation of \u003cem\u003etobacco\u003c/em\u003e leaves, all \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e successfully initiated the expression of the downstream reporter gene \u003cem\u003eGUS\u003c/em\u003e, resulting in confined blue coloration within the \u003cem\u003etobacco\u003c/em\u003e leaves. However, their transcriptional activities exhibited relatively lower strength in comparison to the positive control 35S promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further differentiate the functions of the three \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e, we generated T3 \u003cem\u003eArabidopsis\u003c/em\u003e lines expressing \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e individually and identified positive transgenic lines. During the seedling stage of transgenic \u003cem\u003eArabidopsis\u003c/em\u003e, histochemical staining indicated predominant expression of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e in the roots, while no detectable GUS activity accumulation was observed for \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Subsequent qRT-PCR analysis revealed varying expression levels of all three genes in \u003cem\u003eArabidopsis\u003c/em\u003e, with \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e displaying the highest expression level, followed by \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further explored the expression-driving potential of the \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e promoters in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines during the flowering stage. Histochemical staining demonstrated that all three promoters could activate GUS expression in different organs of \u003cem\u003eArabidopsis\u003c/em\u003e, encompassing roots, stems, leaves, and flowers, resulting in noticeable coloration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, the qRT-PCR results unveiled diversity in the expression levels of the three promoters across various tissues of \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e demonstrated a pattern akin to flowering TB, while \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e exhibited unique expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, to investigate the response of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e to environmental stress, including cold, UV-B, and drought, flowering \u003cem\u003eArabidopsis\u003c/em\u003e plants were exposed to these stressors. The qRT-PCR results indicated that all three environmental factors led to an elevation in the expression of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS3\u003c/em\u003e\u003c/sub\u003e, displaying significant differences from the wild type (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS2\u003c/em\u003e\u003c/sub\u003e demonstrated insensitivity to cold and UV treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), exhibiting upregulation solely under drought conditions, which was highly significant compared to the wild type (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe aforementioned results elucidated the differences among the \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1\u0026minus;3\u003c/em\u003e\u003c/sub\u003e in terms of \u003cem\u003ecis\u003c/em\u003e-acting element composition, spatial and temporal expression specificity, and responsiveness to environmental factors. These findings provide crucial insights to advance our understanding of the regulatory mechanisms that control the expression of the three \u003cem\u003eFtFLS\u003c/em\u003e genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Expression of \u003cem\u003eFtFLS1-3\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eAfter hygromycin screening and PCR identification, we successfully generated T3 generation transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines overexpressing \u003cem\u003eFtFLS1-3\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The qRT-PCR analysis revealed varying expression levels of the \u003cem\u003eFtFLS1-3\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e lines under the control of the 35S promoter, with relative expression levels ranked as \u003cem\u003eFtFLS2\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eFtFLS3\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eFtFLS1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Total flavonoid content analyses exhibited a significant increase in all transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines compared to the wild-type control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Moreover, subsequent analysis of rutin content yielded similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, we conducted an assessment of the levels of kaempferol, quercetin, and myricetin in the samples. The findings indicated that these three compounds were present in extremely low concentrations in \u003cem\u003eArabidopsis\u003c/em\u003e lines, each accounting for less than 1% of the total flavonoid and rutin contents. This phenomenon could be attributed to the efficient conversion of flavanols into the end product rutin in \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Overall, these results conclusively demonstrate the functional role of \u003cem\u003eFtFLS1-3\u003c/em\u003e as flavonol synthases within the plant system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Functional expression of FLS1-3 in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eAfter 6 hours of IPTG induction, SDS-PAGE analysis revealed successful expression of recombinant FtFLS1, FtFLS2, and FtFLS3 as the primary protein products within the \u003cem\u003eE. coli\u003c/em\u003e cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). These proteins exhibited molecular weights of approximately 40 kDa, consistent with the values in Supplementary Table\u0026nbsp;2. TLC analysis demonstrated the effective separation of DQ, Q, and a DQ-Q mixture standard substances, with corresponding \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e values of 0.33, 0.45, and 0.33\u0026ndash;0.45. The TLC analysis results showed effective separation of DQ, Q, and DQ-Q mixture standard substances, with their respective \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e values being 0.33, 0.45, and 033-0.45. In the activity assay using crude enzyme solutions, partially reacted substrate DQ and newly produced product quercetin were observed. The \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e values of these spots matched those of the DQ and quercetin standards (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These results indicate that all three recombinant proteins, FtFLS1-3, exhibited the capability to catalyze the substrate DQ into Q, demonstrating fundamental FLS activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Catalytic characteristics of recombinant FtFLS1-3 \u003cem\u003ein vitro\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eAfter a 10-hour induction period, soluble expression of FtFLS1-3 was observed through SDS-PAGE analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lanes 1 and 2). Subsequent elution was achieved effectively using a 200 mM imidazole buffer (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lane 7). Following ultrafiltration purification (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lane 8), the concentrations of recombinant proteins FtFLS1-3 were determined to be 1.35 g/L, 1.29 g/L, and 1.45 g/L (500 \u0026micro;L), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEnzyme-catalyzed reactions were carried out using DK, DQ, and DM as substrates. For FtFLS1, the observed total activities were 8.88 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU, 22.33 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU, and 4.70 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU for each substrate, with specific activities of 6.58 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, 16.54 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, and 3.48 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, respectively. This outcome closely aligns with our earlier research on FtFLS1[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Regarding FtFLS2, the recorded total activities were 5.01 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU, 5.17 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU, and 3.26 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU for each substrate, along with specific activities of 3.88 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, 4.01 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, and 2.53 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, respectively. Concerning FtFLS3, the measured total activities were 10.48 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU, 10.32 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU, and 7.42 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU for each substrate, accompanied by specific activities of 7.23 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, 17.12 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, and 5.12 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e IU/mg, respectively.\u003c/p\u003e \u003cp\u003eContinuous measurements were taken for a duration of 20 minutes to quantify the production of quercetin resulting from the catalytic activity of FtFLS1-3 enzymes on DQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The results indicated that as the enzyme-catalyzed reaction proceeds, the product content continually increased. Approximately within the time span of 8 to 18 minutes from the start of the reaction, there was a linear relationship between the product and time. At this point, the slope of the line represented the initial velocity of the enzyme-catalyzed reaction[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, the initial velocities of the enzyme-catalyzed reactions of recombinant FtFLS1, FtFLS2, and FtFLS3 with DQ as substrates were 22.30 \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;M/min, 5.16\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;M/min and 10.32\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;M/min, respectively. Accordingly, we chose a time point of 15 minutes following the initiation of the enzyme-catalyzed reaction to determine the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e values of FtFLS1, FtFLS2, and FtFLS3, utilizing three dihydroflavonols as substrates. The calculated results were presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and the double reciprocal plots (Lineweaver-Burk plots) were available in Supplementary Fig.\u0026nbsp;4. The data revealed that FtFLS1-3 exhibit diverse catalytic abilities and substrate preferences for the three dihydroflavonols. FtFLS1 exhibited a preference for DQ as a substrate, characterized by a low \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value (594.17 \u0026micro;mol/L) and the highest \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e value (42.55 \u0026micro;mol/L\u0026middot;min). However, FtFLS2 displayed the lowest catalytic capacity and affinity for all three substrates on average, despite its preference for DQ (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e= 8411.27 \u0026micro;mol/L). In contrast, FtFLS3 showed a preference for DK as a substrate (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e= 717.93 \u0026micro;mol/L), which is the highest among the three FtFLS enzymes. Notably, all the FtFLS1-3 enzymes showed lower \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values for DM. Taken together, these results demonstrate that FtFLS1 exhibited a stronger specific activity, a higher \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e, and a better affinity for DQ in terms of enzyme catalytic characteristics compared to FtFLS2 and FtFLS3.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e values of FtFLS1-3 for different substrates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDihydrokaempferol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDihydroquercetin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDihydromyricetin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFtFLS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 25.53 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e= 1257.18 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 42.55 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 594.17 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 17.76 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 1516.34 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFtFLS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 13.59 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e= 23646.74 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 10.25 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 8411.27 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 12.15 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 21583.23 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFtFLS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 24.04\u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e= 717.93 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 13.97 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 6478.21 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e= 17.82 \u0026micro;mol/L\u0026middot;min\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 1609.21 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the plant flavonoid biosynthesis pathway, four 2-ODD members participate: F3H, FNS Ⅰ (Flavone synthase Ⅰ), FLS, and ANS (Anthocyanidin synthase)/leucoanthocyanidin dioxygenase (LDOX)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These 4 enzymes catalyze oxidation reactions that involve hydroxylation and desaturation of the flavonoid \"C ring\". However, each enzyme has distinct substrates, leading to their involvement in different branches of the flavonoid biosynthesis pathway[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn TB, FLS catalyzes dihydroflavonols into flavonols, contributing to the flavonol biosynthesis branch, while ANS converts leucoanthocyanidins into anthocyanins, functioning in the anthocyanin biosynthesis branch (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). F3H, on the other hand, transforms naringenin into DK, providing a common substrate for both branches[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, information about FNS Ⅰ in this species is currently unavailable. Consequently, this study focused on comparing and analyzing \u003cem\u003eFtFLS1-5\u003c/em\u003e with \u003cem\u003eFLSs\u003c/em\u003e, \u003cem\u003eANSs\u003c/em\u003e, and \u003cem\u003eF3Hs\u003c/em\u003e from \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003etobacco\u003c/em\u003e. Our findings indicated that deduced FtFLS1-5 and the selected 2-ODDs share a relatively conserved primary protein structure and common features in the Conserved domain A and B. The most significant difference between them lies in the variable N-terminal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Through a comparison of the 3D models of FtFLS1-5, it becomes evident that the variable region of this amino acid sequence is notably enriched with \u003cem\u003eα\u003c/em\u003e-helices and random coils, resulting in the formation of the substrate-binding pocket within FLS proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The distinct structures formed by these variable regions may play a role in determining the substrate selectivity and affinity of FtFLS1-5[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, studies have indicated that FLS can exhibit broader substrate catalysis capabilities beyond dihydroflavonols, showcasing its versatile functionality[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For instance, recombinant OsFLS (\u003cem\u003eOryza sativa\u003c/em\u003e FLS) demonstrated both FLS and F3H activities; while the recombinant AtFLS1 (\u003cem\u003eA. thaliana\u003c/em\u003e AtFLS1) and CuFLS (\u003cem\u003eCitrus unshiu\u003c/em\u003e FLS) showed both FLS and ANS/LDOX activities[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e–\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The analysis of the phylogenetic tree revealed that FtFLS4 and FtFLS5 belonged to distinct evolutionary branches, implying their potential as multifunctional 2-ODDs. Meanwhile, FtFLS1 and FtFLS3 exhibited FLS activity, and FtFLS2 might possess both FLS and ANS activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Taking into account the expression levels and tissue specificity of \u003cem\u003eFtFLS1-5\u003c/em\u003e during the flowering stage of TB (Supplementary Fig.\u0026nbsp;3), this study focused on \u003cem\u003eFtFLS1-3\u003c/em\u003e as the primary candidate genes for further investigation.\u003c/p\u003e \u003cp\u003eNumerous studies have highlighted a strong association between upregulated FLS expression and the accumulation of flavonols in TB[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Both our previous research and the current study underscore a noteworthy positive correlation between the expression levels of \u003cem\u003eFtFLS1\u003c/em\u003e and the total flavonoid content throughout the flowering stage of TB[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Also, higher transcript levels of \u003cem\u003eFtFLS1\u003c/em\u003e and \u003cem\u003eFtFLS2\u003c/em\u003e were observed in the TB cultivars ‘Hokkai T10’ and ‘Hokkai T8’, along with an increased amount of flavonols, despite their varying responses to exogenous effects[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In a study involving the co-treatment of microwave irradiation and \u003cem\u003eL\u003c/em\u003e-phenylalanine during the germination of TB seeds, a notable outcome was the upregulation of \u003cem\u003eFtFLS1\u003c/em\u003e expression along with an increase in specific activity[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This upregulation exhibited a significant positive correlation with the quercetin content. Yao \u003cem\u003eet al.\u003c/em\u003e reported that \u003cem\u003eFtFLS1\u003c/em\u003e could be upregulated by a SG7-MYB transcription factor, FtMYB6, which was found to be positively correlated with rutin content in TB seeds[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, a wealth of research has shown that environmental factors such as UV, drought, high salt, cold, along with transcription factors like WB40, MYB, bHLH, as well as various plant hormones, often enhance the rutin content in TB by directly or indirectly influencing \u003cem\u003eFtFLS1-2\u003c/em\u003e[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e–\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Therefore, to gain a more comprehensive understanding of the expression patterns of \u003cem\u003eFtFLS1-3\u003c/em\u003e, we cloned their promoter sequences and performed both bioinformatics analysis (Supplementary Table\u0026nbsp;3) and functional characterization in plants (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results showed that \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1−3\u003c/em\u003e\u003c/sub\u003e exhibited distinct basal transcription activities and tissue specificities in \u003cem\u003etobacco\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e, and also demonstrated differential responsiveness to cold, UV-B, and drought. Likewise, 12-day-old TB seedlings underwent cold, UV-B, and drought treatments, leading to distinct response patterns for \u003cem\u003eFtFLS1-3\u003c/em\u003e. Although the expression pattern of \u003cem\u003eFtFLS1\u003c/em\u003e aligned with that in \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eFtFLS2\u003c/em\u003e and \u003cem\u003eFtFLS3\u003c/em\u003e displayed differing expression profiles compared to their \u003cem\u003eArabidopsis\u003c/em\u003e counterparts (Supplementary Fig.\u0026nbsp;5). This divergence could be attributed to the distinct genetic backgrounds of TB and \u003cem\u003eArabidopsis\u003c/em\u003e, potentially involving different transcription factors in the regulation of specific gene expression during responses to environmental stimuli[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Consequently, considering the distinct expression patterns observed for \u003cem\u003eFtFLS1-3\u003c/em\u003e, it is plausible to infer that they might contribute to FLS activities during various developmental stages or in response to unfavorable conditions within the context of TB[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the context of \u003cem\u003ein vitro\u003c/em\u003e activity assays for recombinant FLS, three dihydroflavanols (DK, DQ, and DM) were employed as substrates to distinguish the catalytic attributes of different FLS enzymes[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Existing literature consistently showcases FLS's heightened affinity for substrates DQ and DK, a phenomenon evident in plants such as C\u003cem\u003eamellia sinensis\u003c/em\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], \u003cem\u003eRubus chingii\u003c/em\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], \u003cem\u003eOrnithogalum caudatum\u003c/em\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and others[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In contrast, myricetin and DM are relatively rare compounds in plants, leading to limited studies on their utilization in assessing FLS enzyme activity[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Notably, FLS exhibits a lower preference for DM when compared to DK and DQ, as observed in examples like \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eSolanum tuberosum\u003c/em\u003e, and \u003cem\u003eCrocosmia × crocosmiiflora\u003c/em\u003e[\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e–\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In a recent study, Xing \u003cem\u003eet al\u003c/em\u003e. reported on two \u003cem\u003eMrFLSs\u003c/em\u003e from \u003cem\u003eMorella rubra\u003c/em\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Their findings further emphasize FLSs' heightened preference for DQ and DK, highlighting that DM is less favorable as a substrate. This study employed both prokaryotic expression and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e approaches to confirm the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e biological roles of \u003cem\u003eFtFLS1-3\u003c/em\u003e. The findings indicated that recombinant FtFLS1-3 exhibit diverse catalytic abilities and substrate preferences for the three dihydroflavonols. Both FtFLS1 and FtFLS2 exhibited a preference for DQ as substrates, but FtFLS1 displayed significantly higher catalytic activity compared to FtFLS2. Furthermore, FtFLS3 showed a preference for DK, with its catalytic activity falling in between. Although DM is not the preferred substrate for any of the three enzymes, the FtFLSs still exhibit a certain degree of catalytic activity towards it. This phenomenon suggests that FtFLS1-3 have low catalytic ability and affinity towards DM, which is consistent with the typically lower content of myricetin compared to other flavonols in TB[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In addition, we performed assays to evaluate the activity of recombinant FtFLS4 and FtFLS5 towards dihydroflavanols (Data not shown). Both enzymes displayed remarkably low catalytic efficiency towards DQ (0.06×10\u003csup\u003e− 3\u003c/sup\u003e IU and 0.27×10\u003csup\u003e− 3\u003c/sup\u003e IU, respectively) and demonstrated no detectable activity towards DK and DM. Further investigations revealed that overexpression of \u003cem\u003eFtFLS1-3\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e significantly enhances the levels of total flavonoids and rutin, demonstrating FLS activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur findings suggest that the flavonol biosynthetic pathway in TB is primarily characterized by the sequential conversion of DK to DQ (probably by F3'H) and subsequently to quercetin, leading to the ultimate synthesis of rutin[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, FtFLS1 exhibited heightened catalytic activity and substrate affinity, highlighting its pivotal role in orchestrating this process. Moreover, considering the tendency of FLS to exhibit activities resembling other 2-ODDs, our future experiments will delve into exploring the enzymatic characteristics of FtFLS1-5 using diverse substrate types. This approach aims to further advance our comprehension of the intricate regulation underlying TB flavonoid synthesis and metabolism.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study cloned and charactered the molecular features of 5 \u003cem\u003eFtFLSs\u003c/em\u003e from TB genome. The promoter functions, expression patterns, and biological activities of \u003cem\u003eFtFLS1-3\u003c/em\u003e were elucidated by \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. Our results indicate that \u003cem\u003eFtFLS1-3\u003c/em\u003e possess varying expression patterns and \u003cem\u003ein vitro\u003c/em\u003e enzymatic features, yet share the same biological functions in plants, making them FLS enzymes participating in the flavonol biosynthetic pathway and contributing to rutin synthesis in TB.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e* College of Life Science, Sichuan Agricultural University, Ya\u0026rsquo;an, Sichuan 625014, China; orcid.org/ 0000-0001-7864-906X;\u003c/p\u003e\n\u003cp\u003ePhone: (+86)-835-2886126;\u003c/p\u003e\n\u003cp\u003eEmail: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChenglei Li performed most of the experiments and wrote the draft of the paper;\u0026nbsp;Jiayi Sun, Guanlan Shi, and Xuerong Zhao\u0026nbsp;participated in the transgenic experiments of \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003etobacco\u003c/em\u003e;\u0026nbsp;Jiaqi Shi, Jun Gu, Qihan Ma, Daoping Zeng, Tao Wang, Zizhong Tang and Tongliang Bu\u0026nbsp;carried out part of the material collection; Hui Chen guided biochemical experiment technology;\u0026nbsp;An\u0026rsquo;hu Wang provided the buckwheat seeds for this study;\u0026nbsp;Qi Wu and Haixia Zhao conceived and designed the studies. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eWe thank An\u0026rsquo;hu Wang at the Xichang College, Xichang, China and Dabing Xiang at the Chengdu University, Sichuan, China, for the gift of Tartary buckwheat seeds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31871698)\u0026nbsp;and\u0026nbsp;the\u0026nbsp;Sichuan\u0026nbsp;Science\u0026nbsp;and\u0026nbsp;Technology\u0026nbsp;Program (2024YFHZ0202).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors also declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang K, He M, Fan Y, Zhao H, Gao B, Yang K, Li F, Tang Y, Gao Q, Lin T, Quinet M, Janovska D, Meglic V, Kwiatkowski J, Romanova O, Chrungoo N, Suzuki T, Luthar Z, Germ M, Woo SH, Georgiev MI, Zhou M (2021) Resequencing of global Tartary buckwheat accessions reveals multiple domestication events and key loci associated with agronomic traits. Genome Biol 22(1):23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTak AM, Hami A, Bhat B, Bhat SA, Masoodi KZ, Bhat MA, Shah M, Khan MK, Zargar SM (2022) Unravelling rutin content of tartary buckwheat of north western Himalayas and insights into nucleotide polymorphisms in PAL gene to infer the associations with rutin biosynthesis. 3 Biotech 12(8):156\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuda MN, Lu S, Jahan T, Ding M, Jha R, Zhang K, Zhang W, Georgiev MI, Park SU, Zhou M (2021) Treasure from garden: Bioactive compounds of buckwheat. Food Chem 335:127653\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFabjan N, Rode J, Košir IJ, Wang Z, Zhang Z, Kreft I (2003) Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin. J Agric Food Chem 51(22):6452\u0026ndash;6455\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Lv Q, Liu A, Wang J, Sun X, Deng J, Chen Q, Wu Q (2022) Comparative metabolomics study of Tartary (Fagopyrum tataricum (L.) Gaertn) and common (Fagopyrum esculentum Moench) buckwheat seeds. Food Chem 371:131125\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu W, Feng Y, Yu S, Fan Z, Li X, Li J, Yin H (2021) The Flavonoid Biosynthesis Network in Plants. Int J Mol Sci 22(23):12824\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi M, Sathasivam R, Nguyen BV, Park NI, Woo SH, Park SU (2021) Expression Analysis of Phenylpropanoid Pathway Genes and Metabolomic Analysis of Phenylpropanoid Compounds in Adventitious, Hairy, and Seedling Roots of Tartary Buckwheat. Plants (Basel) 11(1):90\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Shi Y, Li K, Yang D, Liu N, Zhang L, Zhao L, Zhang X, Liu Y, Gao L, Xia T, Wang P (2021) Roles of the 2-Oxoglutarate-Dependent Dioxygenase Superfamily in the Flavonoid Pathway: A Review of the Functional Diversity of F3H, FNS I, FLS, and LDOX/ANS. Molecules 26(21)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBritsch L, Heller W, Grisebach H (1981) Conversion of flavanone to flavone, dihydroflavonol and flavonol with an enzyme system from cell cultures of parsley. Z f\u0026uuml;r Naturforschung C 36(9\u0026ndash;10):742\u0026ndash;750\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolton TA, Brugliera F, Tanaka Y (1993) Cloning and expression of flavonol synthase from Petunia hybrida. Plant J 4(6):1003\u0026ndash;1010\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePelletier MK, Murrell JR, Shirley BW (1997) Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis (Further evidence for differential regulation of early and late genes). Plant Physiol 113(4):1437\u0026ndash;1445\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOwens DK, Alerding AB, Crosby KC, Bandara AB, Westwood JH, Winkel BS (2008) Functional analysis of a predicted flavonol synthase gene family in Arabidopsis. Plant Physiol 147(3):1046\u0026ndash;1061\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchilbert HM, Schone M, Baier T, Busche M, Viehover P, Weisshaar B, Holtgrawe D (2021) Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases. Front Plant Sci 12:733762\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang X, Shi Y, Fu Z, Li WW, Lai S, Wu Y, Wang Y, Liu Y, Gao L, Xia T (2020) Functional characterization of three flavonol synthase genes from Camellia sinensis: Roles in flavonol accumulation. Plant Sci 300:110632\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Ma Z, Zheng T, Sun W, Zhang Y, Jin W, Zhan J, Cai Y, Tang Y, Wu Q, Tang Z, Bu T, Li C, Chen H (2018) Insights into the correlation between Physiological changes in and seed development of tartary buckwheat (Fagopyrum tataricum Gaertn). BMC Genomics 19(1):648\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Y, Sun L, Wu W, Wang S, Xiao X, Hu M, Li C, Zhao H, Chen H, Wu Q (2021) Genome-Wide Investigation of Major Enzyme-Encoding Genes in the Flavonoid Metabolic Pathway in Tartary Buckwheat (Fagopyrum tataricum). J Mol Evol 89(4\u0026ndash;5):269\u0026ndash;286\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Lv Q, Ma C, Qu J, Cai F, Deng J, Huang J, Ran P, Shi T, Chen Q (2019) Metabolite profiling and transcriptome analyses provide insights into the flavonoid biosynthesis in the developing seed of tartary buckwheat (Fagopyrum tataricum). J Agric Food Chem 67(40):11262\u0026ndash;11276\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Yang K, Yang J, Wu H, Chen H, Wu Q, Zhao H (2022) Tartary buckwheat FtF3\u0026prime; H1 as a metabolic branch switch to increase anthocyanin content in transgenic plant. Front Plant Sci 13:959698\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Bai Y, Li S, Chen H, Han X, Zhao H, Shao J, Park SU, Wu Q (2012) Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in tartary buckwheat. J Agric Food Chem 60(20):5161\u0026ndash;5168\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing Z, Cheng-Lei L, Fei G, Xiao-Peng L, Qing-Qing L, Hai-Xia Z, Hui-Peng Y, Hui C, An-Hu W (2016) W. Qi, Characterization of Three Glucosyltransferase Genes in Tartary Buckwheat. and Their Expression after Cold Stress\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin Q, Han X, Han Z, Chen Q, Shi Y, Gao H, Zhang T, Dong G, Xiong C, Song C, Sun W, Chen S (2020) Genome-wide analyses reveals a glucosyltransferase involved in rutin and emodin glucoside biosynthesis in tartary buckwheat. Food Chem 318:126478\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu H, Jiang Z, Lin Z, Yu Q, Song R, Wang B (2022) FtUGT79A15 is responsible for rutinosylation in flavonoid diglycoside biosynthesis in Fagopyrum tataricum. Plant Physiol Biochem 181:33\u0026ndash;41\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Zhao H, Li M, Yao P, Li Q, Zhao X, Wang A, Chen H, Tang Z, Bu T, Wu Q (2019) Validation of reference genes for gene expression studies in tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR. PeerJ 7:e6522\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao P, Huang Y, Dong Q, Wan M, Wang A, Chen Y, Li C, Wu Q, Chen H, Zhao H (2020) FtMYB6, a Light-Induced SG7 R2R3-MYB Transcription Factor, Promotes Flavonol Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum). J Agric Food Chem 68(47):13685\u0026ndash;13696\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Yang T, Li X, Hao H, Xu S, Cheng W, Sun Y, Wang C (2014) Cloning and characterization of a novel Athspr promoter specifically active in vascular tissue. Plant Physiol Biochem 78:88\u0026ndash;96\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo X-P, Zhao H-X, Xue J, Li C-L, Chen H, Park S-U, Wu Q (2016) Cloning of two basic helix-loop-helix transcription factor genes from Tartary buckwheat (Fagopyrum tataricum) and their expression under abiotic stress. Turkish J Biology 40(6):1192\u0026ndash;1201\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosa FF, Pires CF, Zimmermannova O, Pereira CF (2020) Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors. Bio Protoc 10(10):e3619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSedmak JJ, Grossberg SE (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem 79(1\u0026ndash;2):544\u0026ndash;552\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWellmann F, Lukacin R, Moriguchi T, Britsch L, Schiltz E, Matern U (2002) Functional expression and mutational analysis of flavonol synthase from Citrus unshiu. Eur J Biochem 269(16):4134\u0026ndash;4142\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez J-MG, Hux NP, Philips SJ, Towns MH (2019) Michaelis\u0026ndash;Menten graphs, Lineweaver\u0026ndash;Burk plots, and reaction schemes: Investigating introductory biochemistry students\u0026rsquo; conceptions of representations in enzyme kinetics. J Chem Educ 96(9):1833\u0026ndash;1845\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrescott AG, John P (1996) Molecular Structure and Role in Plant Metabolism. Annu Rev Plant Physiol Plant Mol Biol 47(1):245\u0026ndash;271\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshihara H, Tohge T, Vieh\u0026ouml;ver P, Fernie AR, Weisshaar B, Stracke R (2016) Natural variation in flavonol accumulation in Arabidopsis is determined by the flavonol glucosyltransferase BGLU6. J Exp Bot 67(5):1505\u0026ndash;1517\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris TK, Keshwani MM (2009) Measurement of enzyme activity. Methods Enzymol 463:57\u0026ndash;71\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGebhardt Y, Witte S, Forkmann G, Lukacin R, Matern U, Martens S (2005) Molecular evolution of flavonoid dioxygenases in the family Apiaceae. Phytochemistry 66(11):1273\u0026ndash;1284\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePreuss A, Stracke R, Weisshaar B, Hillebrecht A, Matern U, Martens S (2009) Arabidopsis thaliana expresses a second functional flavonol synthase. FEBS Lett 583(12):1981\u0026ndash;1986\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWelford RW, Clifton IJ, Turnbull JJ, Wilson SC, Schofield CJ (2005) Structural and mechanistic studies on anthocyanidin synthase catalysed oxidation of flavanone substrates: the effect of C-2 stereochemistry on product selectivity and mechanism. Org Biomol Chem 3(17):3117\u0026ndash;3126\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurnbull JJ, Nakajima J, Welford RW, Yamazaki M, Saito K, Schofield CJ (2004) Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase. J Biol Chem 279(2):1206\u0026ndash;1216\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark S, Kim DH, Park BR, Lee JY, Lim SH (2019) Molecular and Functional Characterization of Oryza sativa Flavonol Synthase (OsFLS), a Bifunctional Dioxygenase. J Agric Food Chem 67(26):7399\u0026ndash;7409\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartens S, Preuss A, Matern U (2010) Multifunctional flavonoid dioxygenases: flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L. Phytochemistry 71(10):1040\u0026ndash;1049\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLukačin R, Wellmann F, Britsch L, Martens S, Matern U (2003) Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase. Phytochemistry 62(3):287\u0026ndash;292\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng W, Wang N, Wang S, Wang J, Bian Z (2023) Effect of co-treatment of microwave and exogenous l-phenylalanine on the enrichment of flavonoids in Tartary buckwheat sprouts. J Sci Food Agric 103(4):2014\u0026ndash;2022\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Kim YB, Kim Y, Zhao S, Kim HH, Chung E, Lee JH, Park SU (2013) Differential stress-response expression of two flavonol synthase genes and accumulation of flavonols in tartary buckwheat. J Plant Physiol 170(18):1630\u0026ndash;1636\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoyal J, Verma PK (2023) An Overview of Biosynthetic Pathway and Therapeutic Potential of Rutin. Mini Rev Med Chem 23(14):1451\u0026ndash;1460\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZargar SM, Hami A, Manzoor M, Mir RA, Mahajan R, Bhat KA, Gani U, Sofi NR, Sofi PA, Masi A (2023) Buckwheat OMICS: present status and future prospects. Crit Rev Biotechnol 1\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong Y, Wang N, Wang S, Wang J, Peng W (2023) A review: The nutrition components, active substances and flavonoid accumulation of Tartary buckwheat sprouts and innovative physical technology for seeds germinating. Front Nutr 10:1168361\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen W, Alseekh S, Fernie AR (2020) Conservation and diversification of flavonoid metabolism in the plant kingdom. Curr Opin Plant Biol 55:100\u0026ndash;108\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei T, Huang J, Ruan H, Qian W, Fang Z, Gu C, Zhang N, Liang Y, Wang Z, Gao L (2023) Competition between FLS and DFR regulates the distribution of flavonols and proanthocyanidins in Rubus chingii Hu. Front Plant Sci 14:1134993\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun YJ, He JM, Kong JQ (2019) Characterization of two flavonol synthases with iron-independent flavanone 3-hydroxylase activity from Ornithogalum caudatum Jacq. BMC Plant Biol 19(1):195\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTohge T, Watanabe M, Hoefgen R, Fernie AR (2013) The evolution of phenylpropanoid metabolism in the green lineage. Crit Rev Biochem Mol Biol 48(2):123\u0026ndash;152\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBovy A, de Vos R, Kemper M, Schijlen E, Almenar Pertejo M, Muir S, Collins G, Robinson S, Verhoeyen M, Hughes S, Santos-Buelga C, van Tunen A (2002) High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell 14(10):2509\u0026ndash;2526\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54(4):733\u0026ndash;749\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIrmisch S, Ruebsam H, Jancsik S, Man Saint Yuen M, Madilao LL, Bohlmann J (2019) Flavonol biosynthesis genes and their use in engineering the plant antidiabetic metabolite montbretin A. Plant Physiol 180(3):1277\u0026ndash;1290\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXing M, Cao Y, Ren C, Liu Y, Li J, Grierson D, Martin C, Sun C, Chen K, Xu C, Li X (2021) Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae. Plant J 108(2):411\u0026ndash;425\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou L, Wu D, Ren G, Hu Y, Peng L, Zhao J, Garcia-Perez P, Carpena M, Prieto MA, Cao H, Cheng KW, Wang M, Simal-Gandara J, John OD, Rengasamy KRR, Zhao G, Xiao J (2023) Bioactive compounds, health benefits, and industrial applications of Tartary buckwheat (Fagopyrum tataricum). Crit Rev Food Sci Nutr 63(5):657\u0026ndash;673\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuthar Z, Golob A, Germ M, Vombergar B, Kreft I (2021) Tartary Buckwheat Hum Nutr Plants (Basel) 10(4):700\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Tatary buckwheat, Flavonol synthase, Characterization, Activity analysis","lastPublishedDoi":"10.21203/rs.3.rs-4548454/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4548454/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe flavonol biosynthesis branch generates the main flavonoids in Tartary buckwheat (TB), with rutin serving as a representative flavonol compound. Flavonol synthase (FLS) is a vital enzyme involved in this metabolic pathway. Out of the five known \u003cem\u003eFLS\u003c/em\u003e genes in the TB genome, \u003cem\u003eFtFLS1\u003c/em\u003e is the only gene identified, while information about the remaining four genes is limited. In this study, we cloned the five FtFLS genes from TB and performed molecular identification. The results showed that \u003cem\u003eFtFLS1-3\u003c/em\u003e exhibit high homology and similar molecular characteristics, categorizing them as FLS-like enzymes, while \u003cem\u003eFtFLS4\u003c/em\u003e and \u003cem\u003eFtFLS5\u003c/em\u003e show a certain degree of similarity to other 2-oxoglutarate-dependent dioxygenases. Further investigation revealed a significant correlation between expression of \u003cem\u003eFtFLS1\u003c/em\u003e and the rutin content during the flowering stage of TB (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The promoter sequences of \u003cem\u003eFtFLS1-3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eFtFLS1-3\u003c/em\u003e\u003c/sub\u003e) displayed distinctive cis-elements, transcriptional activities, and expression patterns, exhibiting different sensitivities to cold, UV-B, and drought stresses. The overexpression of \u003cem\u003eFtFLS1-3\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e led to a significant elevation in total flavonoid and rutin levels, providing evidence for the FLS activity of \u003cem\u003eFtFLS1-3\u003c/em\u003e in plants. The enzymatic analysis showed that the recombinant FtFLS1-3 were capable of catalyzing the formation of their respective products from dihydroflavanols. FtFLS1 exhibited a superior specific activity, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and affinity for dihydroquercetin (DQ) in terms of enzyme catalytic characteristics compared to FtFLS2 and FtFLS3. In summary, our study establishes the FLS activity of FtFLS1-3 and suggests that the metabolic flow of the flavonol biosynthesis branch in TB involves the conversion from dihydrokaempferol (DK) to DQ and subsequently to quercetin (Q), ultimately glycosylated to rutin. In this process, FtFLS1 plays a predominant role.\u003c/p\u003e","manuscriptTitle":"Insight into the Rutin Biosynthesis in the Unique Flavonol Synthesis Pathway of Tartary Buckwheat Based on the Enzymatic Functions of FLSs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-20 06:51:26","doi":"10.21203/rs.3.rs-4548454/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-25T00:46:49+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-23T22:34:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-08T13:48:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Theoretical and Applied Genetics","date":"2024-06-07T21:45:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9834b3a5-704f-4f5e-a0a7-2c4d41d6d8a3","owner":[],"postedDate":"August 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-18T16:06:59+00:00","versionOfRecord":{"articleIdentity":"rs-4548454","link":"https://doi.org/10.1007/s00122-025-04997-7","journal":{"identity":"theoretical-and-applied-genetics","isVorOnly":false,"title":"Theoretical and Applied Genetics"},"publishedOn":"2025-08-11 15:56:50","publishedOnDateReadable":"August 11th, 2025"},"versionCreatedAt":"2024-08-20 06:51:26","video":"","vorDoi":"10.1007/s00122-025-04997-7","vorDoiUrl":"https://doi.org/10.1007/s00122-025-04997-7","workflowStages":[]},"version":"v1","identity":"rs-4548454","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4548454","identity":"rs-4548454","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}