Characterization of the 2-ODD DOXC Family and its Members Involved in Flavonoid Biosynthesis in Scutellaria baicalensis

Characterization of the 2-ODD DOXC Family and its Members Involved in Flavonoid Biosynthesis in Scutellaria baicalensis | 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 Characterization of the 2-ODD DOXC Family and its Members Involved in Flavonoid Biosynthesis in Scutellaria baicalensis Sanming Zhu, Mengying Cui, Qing Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3877996/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Aug, 2024 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version Abstract Background 2-oxoglutarate-dependent dioxygenase (2ODD) superfamily is the second largest enzyme family in the plant genome and plays diverse roles in secondary metabolic pathways. The medicinal plant Scutellaria baicalensis Georgi contains various flavonoids, which have the potential to treat coronavirus disease 2019 (COVID-19), such as baicalein and myricetin. Flavone synthase I (FNSI) and flavanone 3-hydroxylase (F3H) from the DOXC class of the 2ODD family have been reported to participate in flavonoid biosynthesis. It is certainly interesting to study the 2ODD members involved in the biosynthesis of flavonoids in S. baicalensis . Results We provided a genome-wide analysis of the 2ODD family from DOXC class in S. baicalensis genome, a total of 88 2ODD genes were identified, 82 of which were grouped into 25 distinct clades based on phylogenetic analysis of At2ODDs. We then performed a functional analysis of Sb2ODDs involved in the biosynthesis of flavones and dihydroflavonols. Sb2ODD1 and Sb2ODD2 from DOXC38 clade exhibit the activity of FNSI (Flavone synthase I), which exclusively converts pinocembrin to chrysin. Sb2ODD1 has significantly higher transcription levels in the root. While Sb2ODD7 from DOXC28 clade exhibits high expression in flowers, it encodes a F3H (flavanone 3-hydroxylase). This enzyme is responsible for catalyzing the conversion of both naringenin and pinocembrin into dihydrokaempferol and pinobanksin, kinetic analysis showed that Sb2ODD7 had high catalytic efficiency to naringenin. Conclusions Our experiment suggests that Sb2ODD1 may serve as a supplementary factor to SbFNSII-2 and play a role in flavone biosynthesis specifically in the roots of S. baicalensis . Sb2ODD7 is mainly responsible for dihydrokaempferol biosynthesis in flowers, which can be further directed into the metabolic pathways of flavonols and anthocyanins. Scutellaria baicalensis Flavonoid Flavone synthase I Flavanone 3-hydroxylase Biosynthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Scutellaria baicalensis Georgi is a perennial herb belonging to Scutellaria genus in the Labiaceae family. It is cultivated worldwide due to its remarkable therapeutic properties. S. baicalensis contains two types of flavones, the 4′-hydroxyflavones like scutellarein and scutellarin accumulate in aerial tissues, and 4′-deoxyflavones such as baicalein, baicalin, wogonin and wogonoside rich in the roots [ 1 ]. In addition, S. baicalensis also contains various flavonoids like naringenin, pinocembrin, dihydrokaempferol, kaempferol and so on [ 2 , 3 ]. These flavonoids exhibit anti-bacterial, anti-inflammatory, anti-cancer and anti-viral effects [ 2 ]. The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to human health, and the 3C-like protease (3CL pro ) of SARS-CoV-2 is a primary target for the developing broad-spectrum antiviral drugs [ 4 ]. Baicalein and myricetin were found from S. baicalensis , and have been identified as inhibitors of the SARS-CoV-2 3CL pro , which have demonstrated inhibition of viral replication in Vero cells [ 5 ]. It has been reported that a total of 100 flavonoids were found in S. baicalensis , with flavones being the major compounds [ 3 ]. Recent studies have provided the genomic and transcriptome data for S. baicalensis , which serves as a robust foundation for the analysis of flavonoid biosynthesis in the medicinal plant[ 6 ]. Flavonoids are a class of secondary metabolites widely distributed in plants. They possess a common C6-C3-C6 skeleton with two aromatic rings connected by a three-carbon chain, typically arranged in a phenylchromane configuration [ 7 ]. Based on the degree of unsaturation and the substitution pattern, the structures of flavonoids are diverse. The major types of flavonoids include flavones, flavonols, flavanones, flavanols, dihydroflavonols, anthocyanins, isoflavones and chalcones [ 8 ]. In addition to serving as the primary source of plant pigments, flavonoids play a crucial role in influencing the colors of flowers and fruits, thus contributing to the process of plant reproduction [ 9 ]. Furthermore, flavonoids offer protection against various external stresses, such as drought [ 10 ], low temperature [ 11 ], diseases [ 12 ] and UV-B radiation [ 13 ]. The classic flavonoid biosynthesis pathway starts with L-phenylalanine, which is catalyzed by phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI), resulting in the formation of naringenin (4′-hydroxyflavanone), a crucial intermediate compound in the pathway (Fig. 1 ) [ 1 ]. Naringenin can subsequently serve as a substrate for various enzymes, leading to the production of different types of flavonoids, such as isoflavones, flavones, dihydroflavonols and so on [ 14 ]. However, S. baicalensis has evolved a specific 4′-deoxyflavones pathway for the biosynthesis of baicalein and wogonin (Fig. 1 ). In this pathway, L-phenylalanine is converted into 4′-deoxyflavanone pinocembrin by SbPAL, cinnamate CoA ligase (SbCLL-7), SbCHS-2 and SbCHI. Pinocembrin is also an important intermediate product, which is further converted into 4'-deoxyflavones through flavone synthase, flavone hydroxylases and methyl-transferases. The 2ODD superfamily is the second largest enzyme family in the plant genome, after the cytochrome P450 superfamily (CYP450). Members of the 2ODD family catalyze various oxidative reactions in plants, such as hydroxylations, demethylations, desaturations, epimerization, rearrangement, halogenation ring closure and ring cleavage [ 15 ]. The plant 2ODD family can be categorized into three distinct evolutionary classes based on amino acid sequence similarity: DOXA, DOXB and DOXC [ 16 ]. Plant homologs of Escherichia coli AlkB is a DNA repair protein [ 17 ], and it is classified into the DOXA class. The DOXB class includes Prolyl 4-hydroxylases (P4Hs), which participate in the synthesis of cell wall proteins in plants [ 18 ]. The DOXC class is involved in secondary metabolisms, such as alkaloids and flavonoids [ 19 ]. The 2ODDs involved in flavonoid biosynthesis are further classified into two clades: DOXC28 and DOXC47 [ 16 ]. DOXC28 clade comprises the classic FNSI and F3H enzymes, while the DOXC47 clade consists of flavonol synthase (FLS) and anthocyanidin synthase (ANS), which function downstream of F3H in the biosynthesis of flavonoids. Early studies of classic FNSI enzymes primarily focused on Apiaceae and monocots [ 20 , 21 ]. It has been reported that PcFNSI and PcF3H have a high level of sequence identity, and PcFNSI may have evolved from PcF3H by gene duplication [ 22 ]. However, there is another type of 2ODD subfamily was reported to have FNSI (AtDMR6) activity, which belongs to DOXC38 clade in Arabidopsis thaliana . AtDMR6 is involved not only in salicylic acid catabolism but also in apigenin (4′-hydroxyflavone) biosynthesis [ 23 , 24 ]. S. baicalensis is rich in various flavonoids. However, it is still unclear whether any member of the 2ODD family is involved in the biosynthesis of flavones in this plant. Furthermore, the role of F3H, an important enzyme involved in the flavonoid pathway, has not been studied in S. baicalensis . Here, we carried out a genome-wide study of 2ODD family members from DOXC class of S. baicalensis by phylogenetic analysis and expression profiles. Then we identified and characterized the Sb2ODD1-Sb2ODD7 of DOXC28 and DOXC38 clades, and performed enzyme assays. Our results elucidated the relationship between the Sb2ODDs and flavonoids accumulation patterns in S. baicalensis . Materials and methods Genome‑wide identification, sequence alignment and phylogenetic analysis of Sb2ODD genes The HMM profile of 2ODD domain (PF14226 and PF03171) from Pfam database ( https://www.ebi.ac.uk/interpro ) was used to extract full-length 2ODD candidates from the S. baicalensis genome by the HMM algorithm (HMMER) [ 25 ]. Multiple sequence alignments and phylogenetic analysis were performed using MEGA 11 [ 26 ]. The neighbor-joining tree was constructed under the default parameters with Sb2ODD candidates and At2ODD sequences. The maximum-likelihood tree was constructed under the default parameters with Sb2ODD sequences and reported F3H and FNSⅠ. The bootstrap statistics were calculated with 1,000 replications. Gene location visualization The location of the Sb2ODD genes on the chromosome was determined by TBtools [ 27 ]. Gene cloning and expression vector construction Based on the transcriptome data of different organs and the genome of S. baicalensis [ 6 ], we amplified the full-length coding regions of Sb2ODD1 using specific primers (Table S2 ). Sb2ODD2 , Sb2ODD3 , Sb2ODD4 , Sb2ODD5 , Sb2ODD6 and Sb2ODD7 were obtained by de novo synthesis (GenScript, Nanjing, China). According to the manufacturer’s instructions, fragments were cloned into the entry vector pDONR207 using the Gateway BP Clonase II Enzyme Kit (Invitrogen, MA, USA). These fragments were then cloned into the yeast expression vector pYesdest52 and prokaryotic expression vector pYesdest17 using the Gateway LR Clonase II Enzyme Kit, respectively. In vivo yeast enzyme assays S. cerevisiae WAT11 was used as the host strain for in vivo enzyme assays. The pYesdest52 empty vector or constructs were transformed into the yeast using the Yeast Transformation II Kit (ZYMO, CA, USA). Transformants were selected on synthetic drop-out medium without uracil (SD-Ura) containing 20 g/L glucose and grown at 28°C for 48 h. The recombinant strains were initially grown in SD-Ura liquid medium with 20 g/L glucose at 28°C for 24 h until the OD 600 reached 2–3. Yeast cells were centrifuged at 4000 rpm for 10 min, then resuspended in the SD-Ura liquid medium with 20 g/L galactose to induce expression of the target proteins. Different substrates and α-ketoglutaric acid were supplemented in the medium. After fermentation for 48 h, yeast cells were harvested by centrifugation, extracted with 1 mL of 70% MeOH (pH 5.0) for metabolite analysis. Enzyme assays and kinetic studies The empty vector or constructed prokaryotic expression vector was transformed into E. coli Rosetta (DE3). Transformants were initially grown in 10 ml of LB liquid medium with 100 µg/ml ampicillin at 37°C for 12 h and then transferred to 300 ml of LB liquid medium until the OD 600 reached 0.6–0.8. Recombinant protein expression was then induced with 1 mM Isopropyl β-D-thiogalactopyranoside (IPTG) at 16°C for 16 h. After harvesting the E. coli cells, high-pressure cell disruption equipment (Constant Systems, Northants, UK) was used to crush the cells. The crude protein lysate was centrifuged and purified by affinity chromatography with Ni-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Germany). The protein concentration was determined using the Bradford method and analyzed by SDS-polyacrylamide gel electrophoresis. The target protein concentration was further determined by Image J software. The enzyme assays in vitro was performed according to a previously described protocol with some modifications [ 23 ]. The reaction mixture contained 100mM NaH 2 PO 4 (pH 6.8), 2 mM DTT, 1 mM α-ketoglutaric acid, 2 mM ascorbic acid, 1 mM ATP, 0.25 mM ferrous sulfate, 50 µM substrate and 1 µg of recombinant purified protein in a final volume of 100 µL. Enzyme assay was performed at 37°C for 1 h in open tubes with shaking. The reaction was initiated by the addition of the enzyme and terminated by adding methanol. After centrifugation at the top speed for 10 min, the supernatant was analyzed by HPLC. For kinetics measurements, naringenin or pinocembrin were used at concentrations ranging from 1 to 250 µM. The reaction time was reduced to 10 min. K m and V max values were obtained by using GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com ). Standard compounds Naringenin and pinocembrin were purchased from Sigma-Aldrich (MO, USA), dihydrokaempferol, pinobanksin, salicylic acid and 2, 5-dihydroxybenzoic acid were purchased from Yuanye-Biotech (Shanghai, China). All of the above standard compounds were dissolved in dimethyl sulfoxide (DMSO). Metabolite analysis An Agilent 1260 Infinity II HPLC system was used for metabolite analysis. Flavones were detected at 280 nm. Separation was carried out on a 100 × 2 mm, 3 µ Luna C18 (2) column. The column was maintained at 35°C. The flow rate of the mobile phase consisting of 0.1% (v/v) formic acid in water (A) and 1:1 MeOH/Acetonitrile + 0.1% formic acid (B) was set to 0.26 ml/min. The gradient program was as follows: 0–3 min, 20% B; 20 min, 50% B; 20–30 min, 50% B; 36 min, 70% B; 37 min, 20% B and 37–43 min, 20% B. Salicylic acid and 2, 5-dihydroxybenzoic acid were detected at 300 nm. Separation was carried out on a 250 × 4.6 mm, 5 µm Eclipse XDB-C18 column. The column was maintained at 35°C. The flow rate of the mobile phase consisting of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in Acetonitrile (B) was set to 0.8 ml/min. The gradient program was as follows: 0–5 min, 10% B; 25 min, 40% B; 25–30 min, 40% B; 36 min, 60% B; 37 min, 10% B and 37–43 min, 10% B. Based on the retention time of standard substances and standard curves, metabolites were confirmed and measured. Results Gene identification of Sb2ODDs in S. baicalensis It has been reported that S. baicalensis is rich in not only flavones, but also in flavonols and anthocyanins [ 3 , 28 , 29 ]. Considering the structures of these compounds (Fig. 1 ), it can be inferred that S. baicalensis harbors Sb2ODDs involved in their biosynthetic pathway. Based on a HMMER search of the S. baicalensis genome, a total of 88 protein sequences were identified as members of the 2ODD enzyme family. By conducting a phylogenetic analysis of Sb2ODDs and At2ODDs, 82 candidates were grouped into 25 clades while 6 candidates remained unclassified (Fig. 2 ). We annotated these clades based on those annotation of A. thaliana enzymes, including gibberellin biosynthesis (DOXC3, DOXC7 and DOXC22), gibberellin catabolism (DOXC12 and DOXC13), auxin metabolism (DOXC15), glucosinolate metabolism (DOXC20 and DOXC31), alkaloid metabolism (DOXC31, DOXC41 and DOXC52), salicylic acid catabolism (DOXC38), coumarin biosynthesis (DOXC30), flavonoid biosynthesis (DOXC28 and DOXC47), ethylene biosynthesis (DOXC53) [ 16 ]. However, further research is needed to determine the functional classification of other clades (DOXC14, DOXC17, DOXC19, DOXC21, DOXC23, DOXC24, DOXC27, DOXC37, DOXC45, DOXC46, DOXC54 and DOXC55). Chromosomal location of Sb2ODDs in S. baicalensis We conducted chromosomal localization mapping of Sb2ODDs using gene annotation files and found that they were distributed unevenly across the S. baicalensis genome (Fig. 3 ). Chromosomes 01–09 contained 22, 9, 15, 8, 9, 6, 5, 4 and 7 Sb2ODDs , respectively. Additionally, 3 Sb2ODDs were not anchored onto any specific chromosomes. Some Sb2ODDs were located in close proximity to specific regions on the chromosomes, indicating possible tandem gene duplication events. A gene cluster is defined as the existence of two neighboring Sb2ODD genes on a chromosome with a distance of less than 50 kb [ 16 ]. According to the annotation of DOXC, these gene clusters located on chromosomes 01, 05, 08 and 09 were found to be involved in flavonoid biosynthesis, glucosinolate metabolism and alkaloid metabolism, respectively. Tissue‑specific expression patterns of Sb2ODDs in S. baicalensis The expression patterns of the Sb2ODDs were determined based on FPKM values from the transcriptome of flowers, flower buds, leaves, stems, roots and MeJA-treated roots of S. baicalensis [ 6 ]. These Sb2ODDs were classified into five groups based on their tissue-specific expression patterns (Fig. 4 and Table S1 ). Group A consisted of only 1 Sb2ODD , which exhibited high expression levels across all the tissues. Group B showed relatively high expression levels specifically in flowers and flower buds. Notably, DOXC47, which includes anthocyanidin synthase (ANS), was clustered in Group B, suggesting a potential association between these genes and anthocyanidin biosynthesis in S. baicalensis flowers. Groups C and D displayed low transcript levels across all the tissues, with Group D showing relatively higher levels compared to Group C. Group E showed predominant expression in roots and their transcripts could be induced by MeJA treatment, indicating its potential involvement in the accumulation of specific flavones in the roots of S. baicalensis , as we previously showed that MeJA treatment leads to increased flavones in S. baicalensis roots [ 1 ]. Gene isolation of Sb2ODDs The DOXC28 clade includes the classic FNSI and F3H, which are involved in the biosynthesis of flavones and dihydroflavonols, respectively [ 16 ]. It has been reported that AtDMR6 from the DOXC38 clade can also be involved in the biosynthesis of flavone and the catabolism of salicylic acid [ 23 , 24 ]. To analyze the phylogenetic relationship of the Sb2ODDs in DOXC38 and DOXC28, a phylogenetic tree was constructed with previously studied enzymes from other species (Fig. 5 A). The results showed that 4 putative Sb2ODDs (Sb2ODD1-Sb2ODD4) were clustered with AtDMR6 (AtFNSⅠ/S5H) on the DOXC38 clade, while Sb2ODD5-Sb2ODD7 clustered with AtF3H and PcFNSI on the DOXC28 clade (Fig. 5 A). Sequence alignment of Sb2ODDs and other homologous proteins revealed that they all had ferrous iron binding domain (HxDxnH) and 2-oxoglutarate binding domain (RxS, Fig. S1 ). Expression heat-map based on FPKM values showed that Sb2ODD1 was highly expressed in roots and JA-treated roots, while Sb2ODD3 and Sb2ODD4 had relatively higher transcripts in root tissues, Sb2ODD2 had very low expression levels in all the tissues analyzed. The expression of Sb2ODD5 , Sb2ODD6 and Sb2ODD7 were higher in flowers and flower buds than in other tissues. Therefore, Sb2ODD1, 3, 4 and Sb2ODD5-7 might be involved in the biosynthesis of flavones and anthocyanidins in roots and flowers, respectively. We isolated the ORFs of the 7 Sb2ODDs by RT-PCR (Table S2 ). Functional characterization of Sb2ODDs To explore the activities of the Sb2ODDs, we expressed coding regions of the 7 enzymes in yeast. As AtDMR6 in DOXC38 can converse naringenin and salicylic acid to apigenin and 2, 5-dihydroxybenzoic acid (2, 5-DHBA) [ 23 ]. We assayed the enzymes of Sb2ODD1-4 by feeding naringenin, salicylic acid and pinocembrin as substrates. The yeast strains expressing Sb2ODD1 and Sb2ODD2 fermented with pinocembrin produced new compound (Peak Ⅰ) with the same retention time as the chrysin standard (Fig. 6 A). We then expressed the proteins in E. coli and purified them from the strains, the enzyme activities were also confirmed through vitro enzyme assay. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that peaks Ⅰ had the same mass charge ratio (m/z) and MS/MS patterns as the chrysin standard (Fig. S2 A, B). Sb2ODD1 and Sb2ODD2 could specifically catalyze the conversion of pinocembrin to chrysin, but no new products were detected when naringenin was used as substrate (Fig. 6 B and Fig. S3A, B). The FNSⅠ’s activity (catalyzed 4′-deoxyflavanone) of Sb2ODD1 and Sb2ODD2 in S. baicalensis were different from the classic FNSⅠs (catalyzed 4′-hydroxyflavanone) in other species, as the previously reported FNSⅠs use naringenin as natural substrate. When we fed yeasts with salicylic acid as a substrate, no new products were detected by high-performance liquid chromatography (HPLC), but a very tiny peak was detected in the yeast extracts of Sb2ODD1 and Sb2ODD2 in mass spectrometry (MS), which had the same MS patterns as 2, 5-DHBA standard (Fig. S3C, D, E). Compared with the empty vector (EV) control, the transformed yeast cells expressing Sb2ODD3 and Sb2ODD4 showed no activity to the 3 substrates (Fig. 6 A and Fig. S3A, C). We then supplemented flavanones naringenin and pinocembrin into the yeasts transformed with Sb2ODD5-7, respectively, and two new products (Peak II and Peak III) were detected in the strains expressing Sb2ODD7, which had the same retention time and MS/MS patterns as dihydrokaempferol and pinobanksin standards (Fig. 6 C, E), showing it is a F3H. The results were also verified through in vitro enzyme activity experiments, (Fig. S2 C, D, E, F). However, Sb2ODD5 and Sb2ODD6 had no activity to naringenin and pinocembrin substrates (Fig. 6 C, E). Therefore, Sb2ODD7 was involved in the biosynthesis of dihydroflavonols, which not only catalyzed naringenin to dihydrokaempferol, but also catalyzed pinocembrin to pinobanksin (Fig. 6 D, F). Kinetic analysis of Sb2ODD7 We then conducted a study to explore the kinetic parameters of Sb2ODD7 to naringenin and pinocembrin (Fig. S4). The results demonstrated that the apparent Michaelis constant ( K m) values of Sb2ODD7 were 57.48 µM and 36.55 µM for naringenin and pinocembrin, respectively. Additionally, the apparent maximal velocity ( V max) values were 2032 pkat mg protein − 1 and 114.1 pkat mg protein − 1 for naringenin and pinocembrin, respectively (Fig. 6 G and Fig. S5). While Sb2ODD7 exhibited a lower K m for pinocembrin, it displayed significantly higher V max for naringenin, resulting in an 11.33-fold higher V max/ K m ratio for naringenin compared to pinocembrin. So Sb2ODD7 had a higher catalytic efficiency to naringenin. Moreover, the expression level of Sb2ODD7 was found to be higher in flowers than in roots, so this enzyme is involved in the conversion of naringenin in flowers of S. baicalensis , subsequently entering the biosynthesis pathway of anthocyanidins and flavonols. Discussion S. baicalensis is known to contain a variety of flavonoids, including classic flavonoids (4′-hydroxyflavonoids) and root-specific flavonoids (4′-deoxyflavonoids) [ 1 ]. Flavonoids have significant pharmacological activity and have been used to treat various diseases. Recently, baicalein and myricetin were reported to have anti-COVID-19 properties [ 5 ]. The DOXC class of 2ODD family is involved in the biosynthesis of the flavonoids that are of particular interest to us. The number of 2ODD genes varies in each plant genome, for example, there are 7, 49, 56, and 99 2ODDs DOXC class identified in Chlamydomonas reinhardtii , Physcomitrella patens , Selaginella moellendorffii and Arabidopsis thaliana respectively [ 16 ]. In our study, 88 2ODDs from DOXC class were identified by searching the genome of S. baicalensis . Compared to A. thaliana , S. baicalensis lacks the DOXC21 and DOXC24 clades (Fig. 2 ). Most members of the DOXC class are involved in specialized metabolites, such as flavonoids and phytohormones [ 16 ]. This suggests that there has been a large-scale duplication of 2ODD genes related to specific metabolism from green algae to higher plants. In S. baicalensis , we found Sb2ODD gene tandem duplications on chromosomes 01, 05, 08, and 09, which are annotated as being associated with flavonoid biosynthesis, glucosinolate metabolism, and alkaloid metabolism, respectively (Fig. 3 ). The Sb2ODD gene tandem duplication (Sb05g11050) on chromosome 05 shows relatively high expression levels in flowers and flower buds, it was annotated as FLS belings to DOXC47 clade, which most likely to be involved in flavonol biosynthesis (Fig. 4 ). Pinocembrin and naringenin are central intermediates in the biosynthesis of root-specific flavonoids and classic flavonoids, respectively [ 1 , 30 ]. There are two types of FNSs in plants that convert flavanones to flavones, namely FNSⅠ (2ODD) and FNSⅡ (CYP450). In S. baicalensis , SbFNSⅡ-1 primarily catalyzes the conversion of naringenin into apigenin aerial parts, respectively, while SbFNSⅡ-2 can specifically catalyze the conversion of pinocembrin into chrysin in the roots [ 1 ]. FNSI was first characterized in parsley, PcFNSI also catalyzed the desaturation reaction of naringenin to form apigenin and belongs to DOXC28 clade [ 21 ]. Interestingly, most DOXC28 members are F3H enzymes. In addition, AtDMR6 from the DOXC38 clade was found to exhibit FNSI activity. However, in A. thaliana , AtDMR6 preferred to hydroxylate salicylic acid and was involved in the catabolism of plant hormones [ 23 , 24 ]. The DOXC28 clade includes the classic FNSI and F3H, therefore we analyzed the the Sb2ODDs enzymes in DOXC38 and DOXC28. Phyogenetic analysis showed that four Sb2ODDs formed a cluster with AtDMR6 in the DOXC38 clade, while, three Sb2ODDs were grouped with AtF3H and PcFNSI in the DOXC28 clade (Fig. 5 A). Notably, Sb2ODD1 and Sb2ODD7 exhibited high expression levels in the roots and flowers, respectively, suggesting their different function in the biosynthesis of flavonoids in the different organs (Fig. 5 B). We isolated the Sb2ODDs that belong to DOXC38 clade, they encode proteins homologous to AtDMR6 protein (Table S2 ). Interestingly, Sb2ODD1 and Sb2ODD2 were involved in the conversion of pinocembrin to chrysin, but they did show any catalytic activity on naringenin (Fig. 6 A, B and Fig. S3A). Unlike AtDMR6, which mainly takes salicylic acid as the substrate, Sb2ODD1 and Sb2ODD2 can only produce a tiny amount of 2, 5-DHBA when salicylic acid is supplemented (Fig. S3C, D, E). Our previous studies have shown that SbFNSII-2 from CYP450 family is mainly responsible for the biosynthesis of chrysin in the S. baicalensis roots. Now, our results showed that Sb2ODD1 and Sb2ODD2 also have flavone synthase activity (FNSI). As Sb2ODD1 is highly expressed in the roots, while Sb2ODD2 has very low transcripts in all the tissues studied, therefore, Sb2ODD1 may act as supplements to participate in the root-specific flavones (4′-deoxyflavones) biosynthesis in S. baicalensis . FNSI might have undergone multiple evolutions to resist biotic or abiotic stress during land plant colonization and radiation. Then we isolated 3 candidate Sb2ODDs of the DOXC28 clade in S. baicalensis (Table S2 ). Sb2ODD7 could catalyze the conversion of naringenin (4′-hydroxyflavanone) and pinocembrin (4′-deoxyflavanone) into dihydrokaempferol and pinobanksin, respectively, showing that the enzyme is a typical F3H (Fig. 6 C, E). Kinetic analysis showed that Sb2ODD7 had high catalytic efficiency to naringenin (Fig. 6 G). Dihydrokaempferol was the precursor of anthocyanidin and myricetin. Sb2ODD7 was highly expressed in flowers and flower buds, therefore these tissues accumulate multiple cyanidin, delphinidin and myricetin (Fig. 1 , 5 B). Conclusions Our works provide a genome-wide analysis of the 2ODD gene family from DOXC class in S. baicalensis genome. We identified 88 2ODD DOXc genes. We also performed functional analysis of Sb2ODDs involved in the biosynthesis of flavones, dihydorflanonols. These works complement the biosynthesis pathway of flavonoids in S. baicalensis , providing genetic resources for the modification of biological chassis and large-scale production of medicinal active ingredients in synthetic biology. Declarations Supplementary Information Additional file 1: Table S1. The list of gene locus and thier groups from DOXC clade in S. baicalensis . Table S2. The list of enzyme names, gene locus and primers used for cloning of predicted Sb2ODD in S. baicalensis . Additional file 2: Fig. S1. Sb2ODDs and other species FNSI/F3H sequences alignment. Fig. S2. In vitro enzyme assays of Sb2ODDs. Fig. S3. In vivo yeast enzyme assays of Sb2ODDs. Fig. S4. SDS PAGE analysis of purification of Sb2ODD1 and Sb2ODD7 proteins. Fig. S5. Enzymatic kinetic curve of Sb2ODD7. Ethics approval and consent to participate All methods were carried out in accordance with local and national guidelines and regulations. Consent for publication Not applicable. Availability of data and materials The DNA and the protein sequences from S. baicalensis are provided in Table S2. Protein sequences from A. thaliana are available with the link of http://www.plants.ensembl.org/index.html. RNA sequencing data are available in the Sequence Read Archive (SRA) database with the link of www.ncbi.nlm.nih.gov/sra, under the accession number SRP156996. The genome of S. baicalensis is available in the National Genomics Data Center (https://bigd.big.ac.cn/gwh) with accession number GWHAOTC00000000. Competing interests The authors declare that they have no competing interests. Funding This work is sponsored by Natural Science Foundation of Shanghai (22ZR1479500), Special Fund for Scientific Research of Shanghai Landscaping & City Appearance Administrative Bureau (G212401), Ministry of Science and Technology of China (YDZX20223100001003) and Youth Innovation Promotion Association of Chinese Academy of Sciences. QZ is also supported by the Shanghai Youth Talent Support Program and SANOFI-SIBS scholarship. Authors’ contributions Q.Z. conceptualized and designed the project. S.M.Z. and M.Y.C. performed the experiments. All the authors analyzed and interpreted the data. S.M.Z. wrote the manuscript. M.Y.C. and Q.Z. revised the manuscript. All authors read and approved the final manuscript. Acknowledgments We greatly appreciate the experimental facilities and services provided by the office of Chenshan Plant Science Research Center. We also thank Xingguo Li from National Key Laboratory of Wheat Breeding, College of Life Sciences, Shandong Agricultural University for his suggestions on the manuscript. References Zhao Q, Zhang Y, Wang G, Hill L, Weng JK, Chen XY, et al. A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis . Sci Adv. 2016;2(4):1501780. Shang X, He X, He X, Li M, Zhang R, Fan P, et al. The genus Scutellaria an ethnopharmacological and phytochemical review. J Ethnopharmacol. 2010;128(2):279-313. Wang ZL, Wang S, Kuang Y, Hu ZM, Qiao X, Ye M. A comprehensive review on phytochemistry, pharmacology, and flavonoid biosynthesis of Scutellaria baicalensis . Pharm Biol. 2018;56(1):465-84. Liu H, Ye F, Sun Q, Liang H, Li C, Li S, et al. Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro . J Enzyme Inhib Med Chem. 2021;36(1):497-503. Su H, Yao S, Zhao W, Zhang Y, Liu J, Shao Q, et al. Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease. Nat Commun. 2021;12(1):3623. Zhao Q, Yang J, Cui MY, Liu J, Fang YM, Yan MX, et al. The reference genome sequence of Scutellaria baicalensis provides insights into the evolution of wogonin biosynthesis. Mol Plant. 2019;12(7):935-50. Wen W, Alseekh S, Fernie AR. Conservation and diversification of flavonoid metabolism in the plant kingdom. Curr Opin Plant Biol. 2020;55:100-8. Santos-Buelga C, Feliciano AS. Flavonoids: from structure to health issues. Mol. 2017;22(3):477. Sheehan H, Moser M, Klahre U, Esfeld K, Dell'Olivo A, Mandel T, et al. MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation. Nat Genet. 2016;48(2):159-66. Tian F, Jia T, Yu B. Physiological regulation of seed soaking with soybean isoflavones on drought tolerance of Glycine max and Glycine soja . Plant Growth Regul. 2014;74(3):229-37. Bhatia C, Pandey A, Gaddam SR, Hoecker U, Trivedi PK. Low temperature-enhanced flavonol synthesis requires light-associated regulatory components in Arabidopsis thaliana . Plant Cell Physiol. 2018;59(10):2099-112. Malhotra B, Onyilagha JC, Bohm BA, Towers GHN, James D, Harborne JB, et al. Inhibition of tomato ringspot virus by flavonoids. Phytochemistry. 1996;43(6):1271-6. Bieza K, Lois R. An Arabidopsis mutant tolerant to lethal ultraviolet-B levels shows constitutively elevated accumulation of flavonoids and other phenolics. Plant Physiol. 2001;126(3):1105-15. Liu W, Feng Y, Yu S, Fan Z, Li X, Li J, et al. The flavonoid biosynthesis network in plants. Int J Mol Sci. 2021;22(23). Farrow SC, Facchini PJ. Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism. Front Plant Sci. 2014;5:524. Kawai Y, Ono E, Mizutani M. Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants. Plant J. 2014;78(2):328-43. Kataoka H, Yamamoto Y, Sekiguchi M. A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate. J Bacteriol. 1983;153(3):1301-7. Keskiaho K, Hieta R, Sormunen R, Myllyharju J. Chlamydomonas reinhardtii has multiple prolyl 4-hydroxylases, one of which is essential for proper cell wall assembly. Plant Cell. 2007;19(1):256-69. Araujo WL, Martins AO, Fernie AR, Tohge T. 2-oxoglutarate: linking TCA cycle function with amino acid, glucosinolate, flavonoid, alkaloid, and gibberellin biosynthesis. Front Plant Sci. 2014;5:552. Lee YJ, Kim JH, Kim BG, Lim Y, Ahn JH. Characterization of flavone synthase I from rice. BMB Rep. 2008;41(1):68-71. Martens S, Forkmann G, Matern U, Lukacin R. Cloning of parsley flavone synthase I. Phytochemistry. 2001;58(1):43-6. Gebhardt YH, Witte S, Steuber H, Matern U, Martens S. Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis. Plant Physiol. 2007;144(3):1442-54. Ferreyra MLF, Emiliani J, Rodriguez EJ, Campos-Bermudez VA, Grotewold E, Casati P. The identification of maize and Arabidopsis type I flavone synthases links flavones with hormones and biotic interactions. Plant Physiol. 2015;169(2):1090-107. Zhang YJ, Zhao L, Zhao JZ, Li YJ, Wang JB, Guo R, et al. S5H/DMR6 encodes a salicylic acid 5-hydroxylase that fine-tunes salicylic acid homeostasis. Plant Physiol. 2017;175(3):1082-93. Eddy SR. Profile hidden Markov models. Bioinformatics. 1998;14(9):755-763. Tamura K, Stecher G, Kumar S, Battistuzzi FU. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022-7. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194-202. Cui MY, Lu AR, Li JX, Liu J, Fang YM, Pei TL, et al. Two types of O-methyltransferase are involved in biosynthesis of anticancer methoxylated 4'-deoxyflavones in Scutellaria baicalensis Georgi. Plant Biotechnol J. 2022;20(1):129-42. Wang D, Wang J, Wang Y, Yao D, Niu Y. Metabolomic and transcriptomic profiling uncover the underlying mechanism of color differentiation in Scutellaria baicalensis Georgi. flowers. Front Plant Sci. 2022;13:884957. Martens S, Mithofer A. Flavones and flavone synthases. Phytochemistry. 2005;66(20):2399-407. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Additional file 1: Table S1. The list of gene locus and thier groups from DOXC clade in S. baicalensis . Table S2. The list of enzyme names, gene locus and primers used for cloning of predicted Sb2ODD in S. baicalensis . Additionalfile2.docx Additional file 2: Fig. S1. Sb2ODDs and other species FNSI/F3H sequences alignment. Fig. S2. In vitro enzyme assays of Sb2ODDs. Fig. S3. In vivo yeast enzyme assays of Sb2ODDs. Fig. S4. SDS PAGE analysis of purification of Sb2ODD1 and Sb2ODD7 proteins. Fig. S5. Enzymatic kinetic curve of Sb2ODD7. Additionalfile3.docx Cite Share Download PDF Status: Published Journal Publication published 26 Aug, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 27 Jun, 2024 Reviews received at journal 25 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviews received at journal 20 Feb, 2024 Reviewers agreed at journal 30 Jan, 2024 Reviewers agreed at journal 30 Jan, 2024 Reviewers invited by journal 29 Jan, 2024 Editor assigned by journal 25 Jan, 2024 Submission checks completed at journal 25 Jan, 2024 First submitted to journal 19 Jan, 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-3877996","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269212638,"identity":"2ddceb42-c104-4d09-a572-0cd5d9dd995f","order_by":0,"name":"Sanming Zhu","email":"","orcid":"","institution":"Shanghai Chenshan Botanical Garden","correspondingAuthor":false,"prefix":"","firstName":"Sanming","middleName":"","lastName":"Zhu","suffix":""},{"id":269212639,"identity":"7470994f-64df-443f-a75e-16c2fce3c561","order_by":1,"name":"Mengying Cui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYFACxgYwxQZkPQDSMmAmsVqYDRgYDHiI0IIAbBJEaeGfkdy64eOO2sQ+6fZrFR/b/vDws7clMPyo2IZTi8SNxLabM88cT2yTOVN2c2abAY9kz7EDjD1nbuPUYiCR2Habt+1YYptEThqQYcBjcCO9gZmxjYCWv1AtxcRrYWyrAWpJP8YM0ZJ2AK8WiTMP2272th0wBtrCLDnjnDHILwkH8fmFvz392Y2fbXWy82ekP/zwoUxODhhihg9+VODWAgWHHRsYeAzg3AOE1ANBnT0DA/sDIhSOglEwCkbBSAQA7pRawBYx7yYAAAAASUVORK5CYII=","orcid":"","institution":"Shanghai Chenshan Botanical Garden","correspondingAuthor":true,"prefix":"","firstName":"Mengying","middleName":"","lastName":"Cui","suffix":""},{"id":269212641,"identity":"1bd4cc99-9e02-41f2-8c4c-1e2be8090b27","order_by":2,"name":"Qing Zhao","email":"","orcid":"","institution":"Shanghai Chenshan Botanical Garden","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-01-19 07:29:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3877996/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3877996/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05519-1","type":"published","date":"2024-08-26T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50311965,"identity":"0d043982-7f9e-4736-8977-aefa0c5ac8e8","added_by":"auto","created_at":"2024-01-29 14:53:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":741403,"visible":true,"origin":"","legend":"\u003cp\u003eTwo Pathways for biosynthesis of Flavonoids in \u003cem\u003eS. baicalensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFNSⅡ, flavone synthase II; FNSⅠ, flavone synthase I; F3H, flavanone 3-hydroxylase; F6H, flavone 6-hydroxylase; F8H, flavone 8-hydroxylase; OMT, 8-\u003cem\u003eO\u003c/em\u003e-methyl transferase; FLS, flavonol synthase; F3’5’H, flavonoid 3’5’-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase. The enzymes highlighted in red are still under investigation.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/079d77b0cab24552c3b09c49.png"},{"id":50311333,"identity":"ebca19d3-ef2f-4a0f-b983-416a80d06861","added_by":"auto","created_at":"2024-01-29 14:45:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1602929,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of 2ODDs proteins.\u003c/p\u003e\n\u003cp\u003eClades were annotated with 2ODDs of AtDOXC. The neighbor joining method was used to construct the tree with bootstrap (n=1000). A gray circle in the middle of each branch indicated bootstrap values greater than 0.5. Red asterisks indicated the DOXC28 and DOXC38 clades for further study.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/4891aa2e74915cf610ba2c83.png"},{"id":50311966,"identity":"46756a7e-31a2-476f-ba1d-e5fa710ec61d","added_by":"auto","created_at":"2024-01-29 14:53:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1426663,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution of \u003cem\u003eSb2ODDs\u003c/em\u003e in \u003cem\u003eS. baicalensis.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eYellow bars represent pseudochromosomes, blue rectangles represent that the distance between \u003cem\u003eSb2ODD\u003c/em\u003e genes in a gene cluster on the chromosome is less than 50kb.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/32f7671fc7e7e6ed74351757.png"},{"id":50311337,"identity":"039bd05d-f15f-4433-b358-5861f61cab78","added_by":"auto","created_at":"2024-01-29 14:45:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1940334,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression heatmap of \u003cem\u003eSb2ODDs\u003c/em\u003e in \u003cem\u003eS. baicalensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe color scale on the right represents the FPKM values normalized with log10. F, flower; FB, flower bud; L, leaf; S, stem; RJ, MeJA-treated root; R, root; the numbers behind indicated biological replicates. Colored rectangles represent genes clustered in different groups (group A-E) based on their expression patterns.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/326ae43c5d53897b77bd583a.png"},{"id":50311338,"identity":"a3fe9cc9-f51f-4975-9110-c63748bcc3f1","added_by":"auto","created_at":"2024-01-29 14:45:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":624712,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis and expression patterns of \u003cem\u003eSbDOXC28s \u003c/em\u003eand \u003cem\u003eSbDOXC38s\u003c/em\u003ein \u003cem\u003eS. baicalensis\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Phylogenetic tree of SbDOXC28 and SbDOXC38 proteins. The maximum likelihood method was used to construct the tree with bootstrap (n=1000). AtAOP1 from At2ODD was used as an outgroup. At, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e; Zm, \u003cem\u003eZea mays\u003c/em\u003e; Pc, \u003cem\u003ePetroselinum crispum\u003c/em\u003e. Accession numbers are as follows: AtDMR6, NP_197841; AtDLO1, NP_192788; AtDLO2, NP_192787; ZmFNSⅠ, NP_001151167; PcFNSⅠ, AAX21541; AtF3H, NP_190692; ZmF3H, NP_001130275; PcF3H, AAP57394; AtAOP1 (NM_116541); Sb2ODD1, Sb06g22120; Sb2ODD2, Sb02g38930; Sb2ODD3, Sb01g51050; Sb2ODD4, Sb01g51220; Sb2ODD5, Sb05g01401; Sb2ODD6, Sb05g01404; Sb2ODD7, Sb05g01631. B. Tissue-specific expression heatmap of \u003cem\u003eSbDOXC28s\u003c/em\u003e and \u003cem\u003eSbDOXC38s\u003c/em\u003e. The color scale on the right represents the FPKM values normalized with log10. F, flower; FB, flower bud; L, leaf; S, stem; RJ, MeJA-treated root; R, root; the numbers behind indicated biological replicates.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/0f6d8746aa389c73df6c095f.png"},{"id":50311335,"identity":"3f7741d4-e185-4eea-8e01-c7a9470f0040","added_by":"auto","created_at":"2024-01-29 14:45:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1149016,"visible":true,"origin":"","legend":"\u003cp\u003eEnzyme assays of Sb2ODDs.\u003c/p\u003e\n\u003cp\u003eA. HPLC analysis of Sb2ODD1-4 from DOXC38 using pinocembrin as a substrate \u003cem\u003ein vivo\u003c/em\u003e yeast enzyme assays. Top, chrysin standard; EV, empty vector control; Sb2ODD1-4, assays with corresponding Sb2ODD proteins. B. The reaction was catalyzed by Sb2ODD1-2 from DOXC38 using pinocembrin as a substrate. C. HPLC analysis of Sb2ODD5-7 from DOXC28 using naringenin as a substrate \u003cem\u003ein vivo\u003c/em\u003e yeast enzyme assays. Top, dihydrokaempferol standard; EV, empty vector control; Sb2ODD5-7, assays with corresponding Sb2ODD proteins. D. The reaction was catalyzed by Sb2ODD7 from DOXC28 using naringenin as a substrate. E. HPLC analysis of Sb2ODD5-7 from DOXC28 using pinocembrin as a substrate \u003cem\u003ein vivo\u003c/em\u003eyeast enzyme assays. Top, pinobanksin standard; EV, empty vector control; Sb2ODD5-7, assays with corresponding Sb2ODD proteins. F. The reaction was catalyzed by Sb2ODD7 from DOXC28 using pinocembrin as a substrate. G. Kinetic analysis of Sb2ODD7 to naringenin and pinocembrin.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/49fb810cbb3b67d8342aaff6.png"},{"id":63820803,"identity":"603995d0-d8a3-413b-a4d3-ea07ff028646","added_by":"auto","created_at":"2024-09-02 16:08:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8118492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/fd11e7c6-88fc-4e32-b657-0af9ce4bc88b.pdf"},{"id":50311332,"identity":"4bbb72cf-9d5b-4608-865e-d3d3d6ed2541","added_by":"auto","created_at":"2024-01-29 14:45:10","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1. \u003c/strong\u003eThe list of gene locus and thier groups from DOXC clade in \u003cem\u003eS. baicalensis\u003c/em\u003e. \u003cstrong\u003eTable S2. \u003c/strong\u003eThe list of enzyme names, gene locus and primers used for cloning of predicted Sb2ODD in \u003cem\u003eS. baicalensis\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/d9fee7011d78313999c6039c.xlsx"},{"id":50311341,"identity":"eba9736d-8885-467c-884d-325debf3159e","added_by":"auto","created_at":"2024-01-29 14:45:11","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14117480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Fig. S1. \u003c/strong\u003eSb2ODDs and other species FNSI/F3H sequences alignment. \u003cstrong\u003eFig. S2. \u003c/strong\u003e\u003cem\u003eIn vitro \u003c/em\u003eenzyme assays of Sb2ODDs. \u003cstrong\u003eFig. S3. \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e yeast enzyme assays of Sb2ODDs. \u003cstrong\u003eFig. S4. \u003c/strong\u003eSDS PAGE analysis of purification of Sb2ODD1 and Sb2ODD7 proteins. \u003cstrong\u003eFig. S5. \u003c/strong\u003eEnzymatic kinetic curve of Sb2ODD7.\u003c/p\u003e","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/34e4a5e064be581e900445b2.docx"},{"id":50311339,"identity":"67027702-7eff-4a8d-ae1c-67d5ba239fdd","added_by":"auto","created_at":"2024-01-29 14:45:10","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":227200,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3877996/v1/bfa546b417274840e5ee7fe5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of the 2-ODD DOXC Family and its Members Involved in Flavonoid Biosynthesis in Scutellaria baicalensis","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eScutellaria baicalensis\u003c/em\u003e Georgi is a perennial herb belonging to \u003cem\u003eScutellaria\u003c/em\u003e genus in the Labiaceae family. It is cultivated worldwide due to its remarkable therapeutic properties. \u003cem\u003eS. baicalensis\u003c/em\u003e contains two types of flavones, the 4\u0026prime;-hydroxyflavones like scutellarein and scutellarin accumulate in aerial tissues, and 4\u0026prime;-deoxyflavones such as baicalein, baicalin, wogonin and wogonoside rich in the roots [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition, \u003cem\u003eS. baicalensis\u003c/em\u003e also contains various flavonoids like naringenin, pinocembrin, dihydrokaempferol, kaempferol and so on [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These flavonoids exhibit anti-bacterial, anti-inflammatory, anti-cancer and anti-viral effects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to human health, and the 3C-like protease (3CL\u003csup\u003epro\u003c/sup\u003e) of SARS-CoV-2 is a primary target for the developing broad-spectrum antiviral drugs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Baicalein and myricetin were found from \u003cem\u003eS. baicalensis\u003c/em\u003e, and have been identified as inhibitors of the SARS-CoV-2 3CL\u003csup\u003epro\u003c/sup\u003e, which have demonstrated inhibition of viral replication in Vero cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It has been reported that a total of 100 flavonoids were found in \u003cem\u003eS. baicalensis\u003c/em\u003e, with flavones being the major compounds [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recent studies have provided the genomic and transcriptome data for \u003cem\u003eS. baicalensis\u003c/em\u003e, which serves as a robust foundation for the analysis of flavonoid biosynthesis in the medicinal plant[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFlavonoids are a class of secondary metabolites widely distributed in plants. They possess a common C6-C3-C6 skeleton with two aromatic rings connected by a three-carbon chain, typically arranged in a phenylchromane configuration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Based on the degree of unsaturation and the substitution pattern, the structures of flavonoids are diverse. The major types of flavonoids include flavones, flavonols, flavanones, flavanols, dihydroflavonols, anthocyanins, isoflavones and chalcones [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition to serving as the primary source of plant pigments, flavonoids play a crucial role in influencing the colors of flowers and fruits, thus contributing to the process of plant reproduction [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, flavonoids offer protection against various external stresses, such as drought [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], low temperature [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and UV-B radiation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe classic flavonoid biosynthesis pathway starts with L-phenylalanine, which is catalyzed by phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI), resulting in the formation of naringenin (4\u0026prime;-hydroxyflavanone), a crucial intermediate compound in the pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Naringenin can subsequently serve as a substrate for various enzymes, leading to the production of different types of flavonoids, such as isoflavones, flavones, dihydroflavonols and so on [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, \u003cem\u003eS. baicalensis\u003c/em\u003e has evolved a specific 4\u0026prime;-deoxyflavones pathway for the biosynthesis of baicalein and wogonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In this pathway, L-phenylalanine is converted into 4\u0026prime;-deoxyflavanone pinocembrin by SbPAL, cinnamate CoA ligase (SbCLL-7), SbCHS-2 and SbCHI. Pinocembrin is also an important intermediate product, which is further converted into 4'-deoxyflavones through flavone synthase, flavone hydroxylases and methyl-transferases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 2ODD superfamily is the second largest enzyme family in the plant genome, after the cytochrome P450 superfamily (CYP450). Members of the 2ODD family catalyze various oxidative reactions in plants, such as hydroxylations, demethylations, desaturations, epimerization, rearrangement, halogenation ring closure and ring cleavage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The plant 2ODD family can be categorized into three distinct evolutionary classes based on amino acid sequence similarity: DOXA, DOXB and DOXC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Plant homologs of \u003cem\u003eEscherichia coli\u003c/em\u003e AlkB is a DNA repair protein [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and it is classified into the DOXA class. The DOXB class includes Prolyl 4-hydroxylases (P4Hs), which participate in the synthesis of cell wall proteins in plants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The DOXC class is involved in secondary metabolisms, such as alkaloids and flavonoids [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The 2ODDs involved in flavonoid biosynthesis are further classified into two clades: DOXC28 and DOXC47 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. DOXC28 clade comprises the classic FNSI and F3H enzymes, while the DOXC47 clade consists of flavonol synthase (FLS) and anthocyanidin synthase (ANS), which function downstream of F3H in the biosynthesis of flavonoids. Early studies of classic FNSI enzymes primarily focused on Apiaceae and monocots [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has been reported that PcFNSI and PcF3H have a high level of sequence identity, and PcFNSI may have evolved from PcF3H by gene duplication [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, there is another type of 2ODD subfamily was reported to have FNSI (AtDMR6) activity, which belongs to DOXC38 clade in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. AtDMR6 is involved not only in salicylic acid catabolism but also in apigenin (4\u0026prime;-hydroxyflavone) biosynthesis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eS. baicalensis\u003c/em\u003e is rich in various flavonoids. However, it is still unclear whether any member of the 2ODD family is involved in the biosynthesis of flavones in this plant. Furthermore, the role of F3H, an important enzyme involved in the flavonoid pathway, has not been studied in \u003cem\u003eS. baicalensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHere, we carried out a genome-wide study of 2ODD family members from DOXC class of \u003cem\u003eS. baicalensis\u003c/em\u003e by phylogenetic analysis and expression profiles. Then we identified and characterized the Sb2ODD1-Sb2ODD7 of DOXC28 and DOXC38 clades, and performed enzyme assays. Our results elucidated the relationship between the Sb2ODDs and flavonoids accumulation patterns in \u003cem\u003eS. baicalensis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eGenome‑wide identification, sequence alignment and phylogenetic analysis of\u003c/strong\u003e \u003cstrong\u003eSb2ODD\u003c/strong\u003e \u003cstrong\u003egenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HMM profile of 2ODD domain (PF14226 and PF03171) from Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro\u003c/span\u003e\u003c/span\u003e) was used to extract full-length \u003cem\u003e2ODD\u003c/em\u003e candidates from the \u003cem\u003eS. baicalensis\u003c/em\u003e genome by the HMM algorithm (HMMER) [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Multiple sequence alignments and phylogenetic analysis were performed using MEGA 11 [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. The neighbor-joining tree was constructed under the default parameters with Sb2ODD candidates and At2ODD sequences. The maximum-likelihood tree was constructed under the default parameters with Sb2ODD sequences and reported F3H and FNSⅠ. The bootstrap statistics were calculated with 1,000 replications.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eGene location visualization\u003c/h2\u003e\n \u003cp\u003eThe location of the \u003cem\u003eSb2ODD\u003c/em\u003e genes on the chromosome was determined by TBtools [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eGene cloning and expression vector construction\u003c/h2\u003e\n \u003cp\u003eBased on the transcriptome data of different organs and the genome of \u003cem\u003eS. baicalensis\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], we amplified the full-length coding regions of \u003cem\u003eSb2ODD1\u003c/em\u003e using specific primers (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). \u003cem\u003eSb2ODD2\u003c/em\u003e, \u003cem\u003eSb2ODD3\u003c/em\u003e, \u003cem\u003eSb2ODD4\u003c/em\u003e, \u003cem\u003eSb2ODD5\u003c/em\u003e, \u003cem\u003eSb2ODD6\u003c/em\u003e and \u003cem\u003eSb2ODD7\u003c/em\u003e were obtained by \u003cem\u003ede novo\u003c/em\u003e synthesis (GenScript, Nanjing, China). According to the manufacturer\u0026rsquo;s instructions, fragments were cloned into the entry vector pDONR207 using the Gateway BP Clonase II Enzyme Kit (Invitrogen, MA, USA). These fragments were then cloned into the yeast expression vector pYesdest52 and prokaryotic expression vector pYesdest17 using the Gateway LR Clonase II Enzyme Kit, respectively.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eyeast enzyme assays\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e WAT11 was used as the host strain for \u003cem\u003ein vivo\u003c/em\u003e enzyme assays. The pYesdest52 empty vector or constructs were transformed into the yeast using the Yeast Transformation II Kit (ZYMO, CA, USA). Transformants were selected on synthetic drop-out medium without uracil (SD-Ura) containing 20 g/L glucose and grown at 28\u0026deg;C for 48 h. The recombinant strains were initially grown in SD-Ura liquid medium with 20 g/L glucose at 28\u0026deg;C for 24 h until the OD\u003csub\u003e600\u003c/sub\u003e reached 2\u0026ndash;3. Yeast cells were centrifuged at 4000 rpm for 10 min, then resuspended in the SD-Ura liquid medium with 20 g/L galactose to induce expression of the target proteins. Different substrates and \u0026alpha;-ketoglutaric acid were supplemented in the medium. After fermentation for 48 h, yeast cells were harvested by centrifugation, extracted with 1 mL of 70% MeOH (pH 5.0) for metabolite analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eEnzyme assays and kinetic studies\u003c/h2\u003e\n \u003cp\u003eThe empty vector or constructed prokaryotic expression vector was transformed into \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3). Transformants were initially grown in 10 ml of LB liquid medium with 100 \u0026micro;g/ml ampicillin at 37\u0026deg;C for 12 h and then transferred to 300 ml of LB liquid medium until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6\u0026ndash;0.8. Recombinant protein expression was then induced with 1 mM Isopropyl \u0026beta;-D-thiogalactopyranoside (IPTG) at 16\u0026deg;C for 16 h. After harvesting the \u003cem\u003eE. coli\u003c/em\u003e cells, high-pressure cell disruption equipment (Constant Systems, Northants, UK) was used to crush the cells. The crude protein lysate was centrifuged and purified by affinity chromatography with Ni-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Germany). The protein concentration was determined using the Bradford method and analyzed by SDS-polyacrylamide gel electrophoresis. The target protein concentration was further determined by Image J software.\u003c/p\u003e\n \u003cp\u003eThe enzyme assays \u003cem\u003ein vitro\u003c/em\u003e was performed according to a previously described protocol with some modifications [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The reaction mixture contained 100mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (pH 6.8), 2 mM DTT, 1 mM \u0026alpha;-ketoglutaric acid, 2 mM ascorbic acid, 1 mM ATP, 0.25 mM ferrous sulfate, 50 \u0026micro;M substrate and 1 \u0026micro;g of recombinant purified protein in a final volume of 100 \u0026micro;L. Enzyme assay was performed at 37\u0026deg;C for 1 h in open tubes with shaking. The reaction was initiated by the addition of the enzyme and terminated by adding methanol. After centrifugation at the top speed for 10 min, the supernatant was analyzed by HPLC.\u003c/p\u003e\n \u003cp\u003eFor kinetics measurements, naringenin or pinocembrin were used at concentrations ranging from 1 to 250 \u0026micro;M. The reaction time was reduced to 10 min. \u003cem\u003eK\u003c/em\u003em and \u003cem\u003eV\u003c/em\u003emax values were obtained by using GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, California USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eStandard compounds\u003c/h2\u003e\n \u003cp\u003eNaringenin and pinocembrin were purchased from Sigma-Aldrich (MO, USA), dihydrokaempferol, pinobanksin, salicylic acid and 2, 5-dihydroxybenzoic acid were purchased from Yuanye-Biotech (Shanghai, China). All of the above standard compounds were dissolved in dimethyl sulfoxide (DMSO).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eMetabolite analysis\u003c/h2\u003e\n \u003cp\u003eAn Agilent 1260 Infinity II HPLC system was used for metabolite analysis. Flavones were detected at 280 nm. Separation was carried out on a 100 \u0026times; 2 mm, 3 \u0026micro; Luna C18 (2) column. The column was maintained at 35\u0026deg;C. The flow rate of the mobile phase consisting of 0.1% (v/v) formic acid in water (A) and 1:1 MeOH/Acetonitrile\u0026thinsp;+\u0026thinsp;0.1% formic acid (B) was set to 0.26 ml/min. The gradient program was as follows: 0\u0026ndash;3 min, 20% B; 20 min, 50% B; 20\u0026ndash;30 min, 50% B; 36 min, 70% B; 37 min, 20% B and 37\u0026ndash;43 min, 20% B.\u003c/p\u003e\n \u003cp\u003eSalicylic acid and 2, 5-dihydroxybenzoic acid were detected at 300 nm. Separation was carried out on a 250 \u0026times; 4.6 mm, 5 \u0026micro;m Eclipse XDB-C18 column. The column was maintained at 35\u0026deg;C. The flow rate of the mobile phase consisting of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in Acetonitrile (B) was set to 0.8 ml/min. The gradient program was as follows: 0\u0026ndash;5 min, 10% B; 25 min, 40% B; 25\u0026ndash;30 min, 40% B; 36 min, 60% B; 37 min, 10% B and 37\u0026ndash;43 min, 10% B.\u003c/p\u003e\n \u003cp\u003eBased on the retention time of standard substances and standard curves, metabolites were confirmed and measured.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGene identification of\u003c/b\u003e \u003cb\u003eSb2ODDs\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eS. baicalensis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIt has been reported that \u003cem\u003eS. baicalensis\u003c/em\u003e is rich in not only flavones, but also in flavonols and anthocyanins [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Considering the structures of these compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), it can be inferred that \u003cem\u003eS. baicalensis\u003c/em\u003e harbors Sb2ODDs involved in their biosynthetic pathway. Based on a HMMER search of the \u003cem\u003eS. baicalensis\u003c/em\u003e genome, a total of 88 protein sequences were identified as members of the 2ODD enzyme family. By conducting a phylogenetic analysis of Sb2ODDs and At2ODDs, 82 candidates were grouped into 25 clades while 6 candidates remained unclassified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We annotated these clades based on those annotation of \u003cem\u003eA. thaliana\u003c/em\u003e enzymes, including gibberellin biosynthesis (DOXC3, DOXC7 and DOXC22), gibberellin catabolism (DOXC12 and DOXC13), auxin metabolism (DOXC15), glucosinolate metabolism (DOXC20 and DOXC31), alkaloid metabolism (DOXC31, DOXC41 and DOXC52), salicylic acid catabolism (DOXC38), coumarin biosynthesis (DOXC30), flavonoid biosynthesis (DOXC28 and DOXC47), ethylene biosynthesis (DOXC53) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, further research is needed to determine the functional classification of other clades (DOXC14, DOXC17, DOXC19, DOXC21, DOXC23, DOXC24, DOXC27, DOXC37, DOXC45, DOXC46, DOXC54 and DOXC55).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosomal location of\u003c/b\u003e \u003cb\u003eSb2ODDs\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eS. baicalensis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe conducted chromosomal localization mapping of \u003cem\u003eSb2ODDs\u003c/em\u003e using gene annotation files and found that they were distributed unevenly across the \u003cem\u003eS. baicalensis\u003c/em\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Chromosomes 01\u0026ndash;09 contained 22, 9, 15, 8, 9, 6, 5, 4 and 7 \u003cem\u003eSb2ODDs\u003c/em\u003e, respectively. Additionally, 3 \u003cem\u003eSb2ODDs\u003c/em\u003e were not anchored onto any specific chromosomes. Some \u003cem\u003eSb2ODDs\u003c/em\u003e were located in close proximity to specific regions on the chromosomes, indicating possible tandem gene duplication events. A gene cluster is defined as the existence of two neighboring \u003cem\u003eSb2ODD\u003c/em\u003e genes on a chromosome with a distance of less than 50 kb [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. According to the annotation of DOXC, these gene clusters located on chromosomes 01, 05, 08 and 09 were found to be involved in flavonoid biosynthesis, glucosinolate metabolism and alkaloid metabolism, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue‑specific expression patterns of\u003c/b\u003e \u003cb\u003eSb2ODDs\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eS. baicalensis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe expression patterns of the \u003cem\u003eSb2ODDs\u003c/em\u003e were determined based on FPKM values from the transcriptome of flowers, flower buds, leaves, stems, roots and MeJA-treated roots of \u003cem\u003eS. baicalensis\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These \u003cem\u003eSb2ODDs\u003c/em\u003e were classified into five groups based on their tissue-specific expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Group A consisted of only 1 \u003cem\u003eSb2ODD\u003c/em\u003e, which exhibited high expression levels across all the tissues. Group B showed relatively high expression levels specifically in flowers and flower buds. Notably, DOXC47, which includes anthocyanidin synthase (ANS), was clustered in Group B, suggesting a potential association between these genes and anthocyanidin biosynthesis in \u003cem\u003eS. baicalensis\u003c/em\u003e flowers. Groups C and D displayed low transcript levels across all the tissues, with Group D showing relatively higher levels compared to Group C. Group E showed predominant expression in roots and their transcripts could be induced by MeJA treatment, indicating its potential involvement in the accumulation of specific flavones in the roots of \u003cem\u003eS. baicalensis\u003c/em\u003e, as we previously showed that MeJA treatment leads to increased flavones in \u003cem\u003eS. baicalensis\u003c/em\u003e roots [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene isolation of\u003c/b\u003e \u003cb\u003eSb2ODDs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe DOXC28 clade includes the classic FNSI and F3H, which are involved in the biosynthesis of flavones and dihydroflavonols, respectively [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has been reported that AtDMR6 from the DOXC38 clade can also be involved in the biosynthesis of flavone and the catabolism of salicylic acid [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To analyze the phylogenetic relationship of the Sb2ODDs in DOXC38 and DOXC28, a phylogenetic tree was constructed with previously studied enzymes from other species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The results showed that 4 putative Sb2ODDs (Sb2ODD1-Sb2ODD4) were clustered with AtDMR6 (AtFNSⅠ/S5H) on the DOXC38 clade, while Sb2ODD5-Sb2ODD7 clustered with AtF3H and PcFNSI on the DOXC28 clade (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSequence alignment of Sb2ODDs and other homologous proteins revealed that they all had ferrous iron binding domain (HxDxnH) and 2-oxoglutarate binding domain (RxS, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Expression heat-map based on FPKM values showed that \u003cem\u003eSb2ODD1\u003c/em\u003e was highly expressed in roots and JA-treated roots, while Sb2ODD3 and \u003cem\u003eSb2ODD4\u003c/em\u003e had relatively higher transcripts in root tissues, Sb2ODD2 had very low expression levels in all the tissues analyzed. The expression of \u003cem\u003eSb2ODD5\u003c/em\u003e, \u003cem\u003eSb2ODD6\u003c/em\u003e and \u003cem\u003eSb2ODD7\u003c/em\u003e were higher in flowers and flower buds than in other tissues. Therefore, Sb2ODD1, 3, 4 and Sb2ODD5-7 might be involved in the biosynthesis of flavones and anthocyanidins in roots and flowers, respectively. We isolated the ORFs of the 7 \u003cem\u003eSb2ODDs\u003c/em\u003e by RT-PCR (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFunctional characterization of Sb2ODDs\u003c/h2\u003e \u003cp\u003eTo explore the activities of the Sb2ODDs, we expressed coding regions of the 7 enzymes in yeast. As AtDMR6 in DOXC38 can converse naringenin and salicylic acid to apigenin and 2, 5-dihydroxybenzoic acid (2, 5-DHBA) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We assayed the enzymes of Sb2ODD1-4 by feeding naringenin, salicylic acid and pinocembrin as substrates. The yeast strains expressing Sb2ODD1 and Sb2ODD2 fermented with pinocembrin produced new compound (Peak Ⅰ) with the same retention time as the chrysin standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We then expressed the proteins in \u003cem\u003eE. coli\u003c/em\u003e and purified them from the strains, the enzyme activities were also confirmed through vitro enzyme assay. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that peaks Ⅰ had the same mass charge ratio (m/z) and MS/MS patterns as the chrysin standard (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, B). Sb2ODD1 and Sb2ODD2 could specifically catalyze the conversion of pinocembrin to chrysin, but no new products were detected when naringenin was used as substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and Fig. S3A, B). The FNSⅠ\u0026rsquo;s activity (catalyzed 4\u0026prime;-deoxyflavanone) of Sb2ODD1 and Sb2ODD2 in \u003cem\u003eS. baicalensis\u003c/em\u003e were different from the classic FNSⅠs (catalyzed 4\u0026prime;-hydroxyflavanone) in other species, as the previously reported FNSⅠs use naringenin as natural substrate. When we fed yeasts with salicylic acid as a substrate, no new products were detected by high-performance liquid chromatography (HPLC), but a very tiny peak was detected in the yeast extracts of Sb2ODD1 and Sb2ODD2 in mass spectrometry (MS), which had the same MS patterns as 2, 5-DHBA standard (Fig. S3C, D, E). Compared with the empty vector (EV) control, the transformed yeast cells expressing Sb2ODD3 and Sb2ODD4 showed no activity to the 3 substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Fig. S3A, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then supplemented flavanones naringenin and pinocembrin into the yeasts transformed with Sb2ODD5-7, respectively, and two new products (Peak II and Peak III) were detected in the strains expressing Sb2ODD7, which had the same retention time and MS/MS patterns as dihydrokaempferol and pinobanksin standards (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, E), showing it is a F3H. The results were also verified through in vitro enzyme activity experiments, (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC, D, E, F). However, Sb2ODD5 and Sb2ODD6 had no activity to naringenin and pinocembrin substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, E). Therefore, Sb2ODD7 was involved in the biosynthesis of dihydroflavonols, which not only catalyzed naringenin to dihydrokaempferol, but also catalyzed pinocembrin to pinobanksin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, F).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eKinetic analysis of Sb2ODD7\u003c/h2\u003e \u003cp\u003eWe then conducted a study to explore the kinetic parameters of Sb2ODD7 to naringenin and pinocembrin (Fig. S4). The results demonstrated that the apparent Michaelis constant (\u003cem\u003eK\u003c/em\u003em) values of Sb2ODD7 were 57.48 \u0026micro;M and 36.55 \u0026micro;M for naringenin and pinocembrin, respectively. Additionally, the apparent maximal velocity (\u003cem\u003eV\u003c/em\u003emax) values were 2032 pkat mg protein\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 114.1 pkat mg protein\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for naringenin and pinocembrin, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and Fig. S5). While Sb2ODD7 exhibited a lower \u003cem\u003eK\u003c/em\u003em for pinocembrin, it displayed significantly higher \u003cem\u003eV\u003c/em\u003emax for naringenin, resulting in an 11.33-fold higher \u003cem\u003eV\u003c/em\u003emax/\u003cem\u003eK\u003c/em\u003em ratio for naringenin compared to pinocembrin. So Sb2ODD7 had a higher catalytic efficiency to naringenin. Moreover, the expression level of Sb2ODD7 was found to be higher in flowers than in roots, so this enzyme is involved in the conversion of naringenin in flowers of \u003cem\u003eS. baicalensis\u003c/em\u003e, subsequently entering the biosynthesis pathway of anthocyanidins and flavonols.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eS. baicalensis\u003c/em\u003e is known to contain a variety of flavonoids, including classic flavonoids (4\u0026prime;-hydroxyflavonoids) and root-specific flavonoids (4\u0026prime;-deoxyflavonoids) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Flavonoids have significant pharmacological activity and have been used to treat various diseases. Recently, baicalein and myricetin were reported to have anti-COVID-19 properties [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The DOXC class of 2ODD family is involved in the biosynthesis of the flavonoids that are of particular interest to us. The number of \u003cem\u003e2ODD\u003c/em\u003e genes varies in each plant genome, for example, there are 7, 49, 56, and 99 \u003cem\u003e2ODDs\u003c/em\u003e DOXC class identified in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e, \u003cem\u003ePhyscomitrella patens\u003c/em\u003e, \u003cem\u003eSelaginella moellendorffii\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e respectively [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In our study, 88 \u003cem\u003e2ODDs\u003c/em\u003e from DOXC class were identified by searching the genome of \u003cem\u003eS. baicalensis\u003c/em\u003e. Compared to \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eS. baicalensis\u003c/em\u003e lacks the DOXC21 and DOXC24 clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Most members of the DOXC class are involved in specialized metabolites, such as flavonoids and phytohormones [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This suggests that there has been a large-scale duplication of \u003cem\u003e2ODD\u003c/em\u003e genes related to specific metabolism from green algae to higher plants. In \u003cem\u003eS. baicalensis\u003c/em\u003e, we found \u003cem\u003eSb2ODD\u003c/em\u003e gene tandem duplications on chromosomes 01, 05, 08, and 09, which are annotated as being associated with flavonoid biosynthesis, glucosinolate metabolism, and alkaloid metabolism, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The \u003cem\u003eSb2ODD\u003c/em\u003e gene tandem duplication (Sb05g11050) on chromosome 05 shows relatively high expression levels in flowers and flower buds, it was annotated as FLS belings to DOXC47 clade, which most likely to be involved in flavonol biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePinocembrin and naringenin are central intermediates in the biosynthesis of root-specific flavonoids and classic flavonoids, respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. There are two types of FNSs in plants that convert flavanones to flavones, namely FNSⅠ (2ODD) and FNSⅡ (CYP450). In \u003cem\u003eS. baicalensis\u003c/em\u003e, SbFNSⅡ-1 primarily catalyzes the conversion of naringenin into apigenin aerial parts, respectively, while SbFNSⅡ-2 can specifically catalyze the conversion of pinocembrin into chrysin in the roots [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. FNSI was first characterized in parsley, PcFNSI also catalyzed the desaturation reaction of naringenin to form apigenin and belongs to DOXC28 clade [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Interestingly, most DOXC28 members are F3H enzymes. In addition, AtDMR6 from the DOXC38 clade was found to exhibit FNSI activity. However, in \u003cem\u003eA. thaliana\u003c/em\u003e, AtDMR6 preferred to hydroxylate salicylic acid and was involved in the catabolism of plant hormones [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The DOXC28 clade includes the classic FNSI and F3H, therefore we analyzed the the Sb2ODDs enzymes in DOXC38 and DOXC28. Phyogenetic analysis showed that four Sb2ODDs formed a cluster with AtDMR6 in the DOXC38 clade, while, three Sb2ODDs were grouped with AtF3H and PcFNSI in the DOXC28 clade (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Notably, \u003cem\u003eSb2ODD1\u003c/em\u003e and \u003cem\u003eSb2ODD7\u003c/em\u003e exhibited high expression levels in the roots and flowers, respectively, suggesting their different function in the biosynthesis of flavonoids in the different organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWe isolated the \u003cem\u003eSb2ODDs\u003c/em\u003e that belong to DOXC38 clade, they encode proteins homologous to AtDMR6 protein (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Interestingly, Sb2ODD1 and Sb2ODD2 were involved in the conversion of pinocembrin to chrysin, but they did show any catalytic activity on naringenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B and Fig. S3A). Unlike AtDMR6, which mainly takes salicylic acid as the substrate, Sb2ODD1 and Sb2ODD2 can only produce a tiny amount of 2, 5-DHBA when salicylic acid is supplemented (Fig. S3C, D, E). Our previous studies have shown that SbFNSII-2 from CYP450 family is mainly responsible for the biosynthesis of chrysin in the \u003cem\u003eS. baicalensis\u003c/em\u003e roots. Now, our results showed that Sb2ODD1 and Sb2ODD2 also have flavone synthase activity (FNSI). As \u003cem\u003eSb2ODD1\u003c/em\u003e is highly expressed in the roots, while Sb2ODD2 has very low transcripts in all the tissues studied, therefore, Sb2ODD1 may act as supplements to participate in the root-specific flavones (4\u0026prime;-deoxyflavones) biosynthesis in \u003cem\u003eS. baicalensis\u003c/em\u003e. FNSI might have undergone multiple evolutions to resist biotic or abiotic stress during land plant colonization and radiation.\u003c/p\u003e \u003cp\u003eThen we isolated 3 candidate \u003cem\u003eSb2ODDs\u003c/em\u003e of the DOXC28 clade in \u003cem\u003eS. baicalensis\u003c/em\u003e (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Sb2ODD7 could catalyze the conversion of naringenin (4\u0026prime;-hydroxyflavanone) and pinocembrin (4\u0026prime;-deoxyflavanone) into dihydrokaempferol and pinobanksin, respectively, showing that the enzyme is a typical F3H (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, E). Kinetic analysis showed that Sb2ODD7 had high catalytic efficiency to naringenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Dihydrokaempferol was the precursor of anthocyanidin and myricetin. Sb2ODD7 was highly expressed in flowers and flower buds, therefore these tissues accumulate multiple cyanidin, delphinidin and myricetin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur works provide a genome-wide analysis of the \u003cem\u003e2ODD\u003c/em\u003e gene family from DOXC class in \u003cem\u003eS. baicalensis\u003c/em\u003e genome. We identified 88 \u003cem\u003e2ODD\u003c/em\u003e DOXc genes. We also performed functional analysis of Sb2ODDs involved in the biosynthesis of flavones, dihydorflanonols. These works complement the biosynthesis pathway of flavonoids in \u003cem\u003eS. baicalensis\u003c/em\u003e, providing genetic resources for the modification of biological chassis and large-scale production of medicinal active ingredients in synthetic biology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1.\u0026nbsp;\u003c/strong\u003eThe list of gene locus and thier groups from DOXC clade in \u003cem\u003eS. baicalensis\u003c/em\u003e. \u003cstrong\u003eTable S2.\u0026nbsp;\u003c/strong\u003eThe list of enzyme names, gene locus and primers used for cloning of predicted Sb2ODD in \u003cem\u003eS. baicalensis\u003c/em\u003e\u003cstrong\u003e. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Fig. S1.\u0026nbsp;\u003c/strong\u003eSb2ODDs and other species FNSI/F3H sequences alignment. \u003cstrong\u003eFig. S2.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003eenzyme assays of Sb2ODDs. \u003cstrong\u003eFig. S3.\u0026nbsp;\u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e yeast enzyme assays of Sb2ODDs. \u003cstrong\u003eFig. S4.\u0026nbsp;\u003c/strong\u003eSDS PAGE analysis of purification of Sb2ODD1 and Sb2ODD7 proteins. \u003cstrong\u003eFig. S5.\u0026nbsp;\u003c/strong\u003eEnzymatic kinetic curve of Sb2ODD7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods were carried out in accordance with local and national guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA and the protein sequences from \u003cem\u003eS. baicalensis\u003c/em\u003e are provided in Table S2. Protein sequences from \u003cem\u003eA. thaliana\u003c/em\u003e are available with the link of http://www.plants.ensembl.org/index.html. RNA sequencing data are available in the Sequence Read Archive (SRA) database with the link of www.ncbi.nlm.nih.gov/sra, under the accession number SRP156996. The genome of \u003cem\u003eS. baicalensis\u003c/em\u003e is available in the National Genomics Data Center (https://bigd.big.ac.cn/gwh) with accession number GWHAOTC00000000.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is sponsored by Natural Science Foundation of Shanghai (22ZR1479500), Special Fund for Scientific Research of Shanghai Landscaping \u0026amp; City Appearance Administrative Bureau (G212401), Ministry of Science and Technology of China (YDZX20223100001003) and Youth Innovation Promotion Association of Chinese Academy of Sciences. QZ is also supported by the Shanghai Youth Talent Support Program and SANOFI-SIBS scholarship.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.Z. conceptualized and designed the project. S.M.Z. and M.Y.C. performed the experiments. All the authors analyzed and interpreted the data. S.M.Z. wrote the manuscript. M.Y.C. and Q.Z. revised the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe greatly appreciate the experimental facilities and services provided by the office of Chenshan Plant Science Research Center. We also thank Xingguo Li from National Key Laboratory of Wheat Breeding, College of Life Sciences, Shandong Agricultural University for his suggestions on the manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhao Q, Zhang Y, Wang G, Hill L, Weng JK, Chen XY, et al. A specialized flavone biosynthetic pathway has evolved in the medicinal plant, \u003cem\u003eScutellaria baicalensis\u003c/em\u003e. Sci Adv.\u003cem\u003e \u003c/em\u003e2016;2(4):1501780.\u003c/li\u003e\n\u003cli\u003eShang X, He X, He X, Li M, Zhang R, Fan P, et al. The genus \u003cem\u003eScutellaria\u003c/em\u003e an ethnopharmacological and phytochemical review. J Ethnopharmacol.\u003cem\u003e \u003c/em\u003e2010;128(2):279-313.\u003c/li\u003e\n\u003cli\u003eWang ZL, Wang S, Kuang Y, Hu ZM, Qiao X, Ye M. A comprehensive review on phytochemistry, pharmacology, and flavonoid biosynthesis of \u003cem\u003eScutellaria baicalensis\u003c/em\u003e. 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Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism. Front Plant Sci.\u003cem\u003e \u003c/em\u003e2014;5:524.\u003c/li\u003e\n\u003cli\u003eKawai Y, Ono E, Mizutani M. Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants. Plant J.\u003cem\u003e \u003c/em\u003e2014;78(2):328-43.\u003c/li\u003e\n\u003cli\u003eKataoka H, Yamamoto Y, Sekiguchi M. A new gene (alkB) of \u003cem\u003eEscherichia coli\u003c/em\u003e that controls sensitivity to methyl methane sulfonate. J Bacteriol.\u003cem\u003e \u003c/em\u003e1983;153(3):1301-7.\u003c/li\u003e\n\u003cli\u003eKeskiaho K, Hieta R, Sormunen R, Myllyharju J. Chlamydomonas reinhardtii has multiple prolyl 4-hydroxylases, one of which is essential for proper cell wall assembly. 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Flavones and flavone synthases. Phytochemistry.\u003cem\u003e \u003c/em\u003e2005;66(20):2399-407.\u003c/li\u003e\n\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Scutellaria baicalensis, Flavonoid, Flavone synthase I, Flavanone 3-hydroxylase, Biosynthesis","lastPublishedDoi":"10.21203/rs.3.rs-3877996/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3877996/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e2-oxoglutarate-dependent dioxygenase (2ODD) superfamily is the second largest enzyme family in the plant genome and plays diverse roles in secondary metabolic pathways. The medicinal plant \u003cem\u003eScutellaria baicalensis\u003c/em\u003e Georgi contains various flavonoids, which have the potential to treat coronavirus disease 2019 (COVID-19), such as baicalein and myricetin. Flavone synthase I (FNSI) and flavanone 3-hydroxylase (F3H) from the DOXC class of the 2ODD family have been reported to participate in flavonoid biosynthesis. It is certainly interesting to study the 2ODD members involved in the biosynthesis of flavonoids in \u003cem\u003eS. baicalensis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe provided a genome-wide analysis of the \u003cem\u003e2ODD\u003c/em\u003e family from DOXC class in \u003cem\u003eS. baicalensis\u003c/em\u003e genome, a total of 88 \u003cem\u003e2ODD\u003c/em\u003e genes were identified, 82 of which were grouped into 25 distinct clades based on phylogenetic analysis of At2ODDs. We then performed a functional analysis of Sb2ODDs involved in the biosynthesis of flavones and dihydroflavonols. Sb2ODD1 and Sb2ODD2 from DOXC38 clade exhibit the activity of FNSI (Flavone synthase I), which exclusively converts pinocembrin to chrysin. \u003cem\u003eSb2ODD1\u003c/em\u003e has significantly higher transcription levels in the root. While Sb2ODD7 from DOXC28 clade exhibits high expression in flowers, it encodes a F3H (flavanone 3-hydroxylase). This enzyme is responsible for catalyzing the conversion of both naringenin and pinocembrin into dihydrokaempferol and pinobanksin, kinetic analysis showed that Sb2ODD7 had high catalytic efficiency to naringenin.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur experiment suggests that Sb2ODD1 may serve as a supplementary factor to SbFNSII-2 and play a role in flavone biosynthesis specifically in the roots of \u003cem\u003eS. baicalensis\u003c/em\u003e. Sb2ODD7 is mainly responsible for dihydrokaempferol biosynthesis in flowers, which can be further directed into the metabolic pathways of flavonols and anthocyanins.\u003c/p\u003e","manuscriptTitle":"Characterization of the 2-ODD DOXC Family and its Members Involved in Flavonoid Biosynthesis in Scutellaria baicalensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-29 14:45:05","doi":"10.21203/rs.3.rs-3877996/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-27T12:22:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-25T18:12:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326985681027458000881210207244247972686","date":"2024-06-10T06:28:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-21T02:32:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ffe37e12-9599-4d3e-815b-a12456064aa8","date":"2024-01-31T00:57:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31bfca3f-cc7a-49af-88ee-d2011380364a","date":"2024-01-30T13:12:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-30T02:07:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-25T10:11:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-25T10:09:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-01-19T07:25:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"482791eb-bebb-4aa3-800a-b0a3b18bcd99","owner":[],"postedDate":"January 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T15:59:27+00:00","versionOfRecord":{"articleIdentity":"rs-3877996","link":"https://doi.org/10.1186/s12870-024-05519-1","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-08-26 15:57:02","publishedOnDateReadable":"August 26th, 2024"},"versionCreatedAt":"2024-01-29 14:45:05","video":"","vorDoi":"10.1186/s12870-024-05519-1","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05519-1","workflowStages":[]},"version":"v1","identity":"rs-3877996","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3877996","identity":"rs-3877996","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}