Identification and Characterization of Abiotic Stress Induced  novel UDP-Glucosyltransferase (UGT72L11) Gene from Glycyrrhiza glabra L.

Identification and Characterization of Abiotic Stress Induced novel UDP-Glucosyltransferase (UGT72L11) Gene from Glycyrrhiza glabra L. | 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 Identification and Characterization of Abiotic Stress Induced novel UDP-Glucosyltransferase (UGT72L11) Gene from Glycyrrhiza glabra L. Shahnawaz Hussain, Bhawna Verma, Malik Muzafar Manzoor, Pooja Goyal, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3981251/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The present study reports a unique broad spectrum UDP-glycosyltransferase from Glycyrrhiza glabra involved in multiple stress responses and abscisic acid mediated glycosylation. The identified UGT72L11 gene was cytoplasmic with ORF of 1425 bp encoding a 52.2 kDa protein of 474 amino acids. Phylogenetic analysis revealed maximum homology (73.3%) with epicatechin 3-glucosyltransferase (ACC38470) from Medicago truncatula exhibiting sequence uniqueness. The gene was differentially expressed in shoot tissues and significantly upregulated in abscisic acid treatment (122.3 folds) and under cold stress (36 folds) in planta . In-silico Structure-Activity-Relationship revealed GLU279, ARG386, PRO380 and TRP379 residues being involved in receptor-ligand interactions. The UGT72L11 protein was optimal between 10ºC to 30ºC preferring quercetin-UDPGlc ( K m 0.23) over kaempferol-UDPGlc ( K m 0.47). The purified recombinant protein showed multi-substrate O-glycosylation towards various classes of aglycones, abscisic acid, and also displayed C-glycosylation with colchicine as a foundation for the future medicinal applications. ABA enzymatic assay Flavonoid Glycyrrhiza glabra in-vitro stress UDP glycosyltransferase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Plant UDP dependent glycosyltransferases (UGTs) belongs to a large multi-gene superfamily of glucosyltransferases (GTs) that catalyses the transfer of UDP-activated sugars to diverse set of aglycons forming corresponding glycol-conjugates [ 1 ] as observed in secondary metabolites, hormones [ 2 ], xenobiotics [ 3 ] and several other small molecules. The glycosylation of secondary metabolites effectively regulate their properties and functions imparting altered characteristics [ 4 ]. Hence the chemical modification plays critical role in the response to plant growth and adaptation to biotic and abiotic stresses. Studies have highlighted role of plant UGTs in stress management. The rice UGT85E1 has been demonstrated to mediate plant response to drought and oxidative stresses [ 5 ]. Similarly, ectopic over expression of UGT76E11 from Arabidopsis thaliana showed enhanced tolerance to salinity and drought [ 6 ]. On the other hand, silencing of UGT75C1 from tomato played crucial role in ABA-mediated fruit ripening, seed germination, and drought responses [ 7 ]. Higher accumulation of glycosylated specialized metabolites modified by UGTs have shown to be involved in drought and salt stress tolerance and higher oxidative stress management in plants (Li et al., 2018c, 2018d; Gharibi et al., 2019). Chill induced expression of OsUGT90A1 from Oryza sativa imparted tolerance to low temperature through cell membrane integrity [ 8 ]. Glycosyltransferases have shown specialized metabolites modulations. The UGTs like CrUGT87A , identified from Carex rigescens exhibited increased flavonoid accumulation and salt stress resistance [ 9 ] and CsUGT78A15 from Camellia sinensis was shown to be involved in biosynthesis of eugenol glucoside under cold stress [ 10 ] as was also demonstrated in A. thaliana AtUGT87A2 associated with salinity, osmotic stress, drought and ABA [ 11 ]. Multiple stress inducible command further insight into the response mechanism in plants to understand the interplay between the UGT catalyzed glycosylation under the influence of stress, and specialized metabolite accumulation. The crystal structure of the di-C-glycosyltransferase from G.glabra catalyzing a two-step di-C-glycosylation of flopropione-containing substrates revealed the role of hydrogen-bond interactions of sugar hydroxyl groups in sugar donor selectivity and space-chemistry in di-C-glycosylation capability [ 12 ]. This finding has immense application in developing efficient biocatalysts to synthesize C-glycosides of medicinal potential. Understanding of the intricate interplay between UGTs in plants is needed to unlock the full potential of UGTs in plant adaptive behaviour under climate change scenario and also as a prospective catalytic tool. In recent years putative UGT genes have been identified from several plant species, but their functional characterization is equitably small, coming predominantly from studies conducted on A. thaliana [ 13 ]. Further, only a few members of the UGT72 subfamily have been characterized from Fabaceae as in the seed coat-specific UGT72L1 from M. truncatula [ 14 ] and UGT72L6 from Lotus. japonicas (KT895087). In the Glycyrrhiza genus, literature reports few UGTs identified and characterized from G.uralensis species for terpenoids [ 15 ], saponin [ 16 ] and flavonoid [ 17 ] biosyntheses. A recent study on metabolic profiling in G. uralensis identified few key differentially expressed UGT genes co-regulating flavonoid and saponin biosyntheses in licorice under salt stress [ 18 ]. The present study mined a novel UGT gene from the transcriptome resource of G. glabra species having wide-substrate acceptability and involvement in multiple stresses. The purified recombinant protein was enzymatically characterized and in-planta assessed. Additionally, the isolated recombinant protein was studied for various kinetic parameters and in-vitro evaluated for substrate promiscuity utilizing diverse class of aglycons. 2. Materials and methods 2.1 Plant material Plant material was collected from the experimental farm of the Council for Scientific and Industrial Research-Indian Institute of Integrative Medicine Jammu (32.73ºN and 74.87ºE) for RNA extraction and as explants for tissue culture. The regenerated plants were used throughout the experiments for various treatments. All tissues were frozen in liquid nitrogen (N) and stored at − 80°C until required. 2.2 Chemicals Reagents used in the study were purchased from Takara Bio-USA, Promega, Thermofisher and Thermoscientific. Substrates and Isopropyl ß-D-1-thiogalactopyranoside (IPTG) for enzyme assay were purchased from Sigma-Aldrich (Oakville, CA, USA). All the chemicals used in this study were of molecular/analytical/HPLC/MS grade. 2.3 Retrieval of UGTs from the transcriptomic data Transcriptome data (PRJNA664636) generated through Next Generation Sequencing (NGS) of G.glabra plant was used as resource for the identification of UGT gene from the species using the Hidden Markov Model (HMM) profile (Pfam family: PF00201). The local similarity search (tBLASTN) was performed for the mining of GgUGTs using the BioEdit with an E-value cut off of 10 − 3 . The obtained contigs were further filtered by ORF finder ( https://www.ncbi.nlm.nih.gov/orffinder/ ) tool to select full-length sequences and translated by ExPasy ( https://web.expasy.org/ ) tool. The molecular weight (MW), theoretical isoelectric point (pI), instability index, aliphatic index, grand average of hydropathicity (GRAVY) were predicted via the ProtParam ( http://web.expasy.org/protparam/ ). Additionally, sub-cellular localisation prediction was performed by an advanced protein sub-cellular localisation prediction tool CELLO2GO ( http://cello.life.nctu.edu.tw/cello2go/ ). 2.4 Sequence alignment and phylogenetic analysis The protein sequence of the identified UGTs were aligned with the sequences of the functionally characterized proteins from other plant species of the same subfamily, and proteins having mono- and disaccharide glycoside forming activities [ 19 ]. Multiple sequence alignment (MSA) of the deduced amino acid sequences was carried out using Clustal omega [ 20 ] and DNAMAN( https://dnaman.software.informer.com/7.0/ ). To construct the phylogenetic tree, amino acid sequences were aligned using the BLOSUM62 matrix with the ClustalW algorithm-based AlignX module from Mega MEGA (Ver7.0) to generate a neighbour joining tree with bootstrapping (1000 replicates) analysis and handling gaps with pair wise deletion. The mono/di-glucoside-based categorization was done by the Geneious alignment tool ( https://www.geneious.com/ ) to construct phylogenetic tree using neighbour joining method in default parameters of the Geneious program. The conserved motifs of the UGTs were predicted using the Multiple Expectation Maximization for Motif Elicitation (MEME: http://meme-suite.org/tools/meme ) tool having maximum ten motifs with minimum 6 and maximum 50 motif width. 2.5 In-silico 3D model construction The three-dimensional protein structure of UGT72L11 protein was predicted by I-TASSER ( https://zhanglab.ccmb.med.umich.edu/I-TASSER/ ) and validated by Ramachandran plot. Ligand binding site was predicted using 3DLigandSite [ 21 ]. The ligand structure of kaempferol was downloaded from PubChem Compound Database (National Centre for Biotechnology Information; https://pubchem.ncbi.nlm.nih.gov/).Fo r primary molecular docking, visualization and modifying receptor and ligand structures the DockThor-VS web online server ( https://dockthor.lncc.br/v2/ ) was used (Discovery Studio Biovia 2017; Dassault Systèmes, San Diego, California, USA). Discovery Studio Biovia 2017 was also used for post-docking analyses, like prediction of the size and location of binding site, hydrogen-bond interactions, hydrophobic interactions, and bonding distances. 2.6 Cellular localisation: Sub-cellular localisation of the gene was performed in onion cells using Agrobacterium mediated transformation having 35S: UGT72L11 -GFP construct in PCAMBIA1302 vector following the published protocol [ 22 ]. Briefly, healthy and fresh onion scales (1–1.5×1 cm) were placed on a petridish with re suspension of Agrobacterium solution (OD600) having sucrose, acetosyringone and Silwet-for 24 hours at 28 ◦ C. Subsequently, onion scales were co-cultivated with Agrobacterium containing ½ strength Murashige and Skoog basal medium. GFP imaging of transient expression was visualised under confocal fluorescence microscope (Olympus FLUOVIEW FV1000).For GFP detection, the excitation source was an argon-ion laser at 488 nm, and emission was observed between 510 and 530 nm. 2.7 RNA isolation, gene and promoter cloning Total RNA having purity between 1.9–2.1 (A260/280) was isolated using Pure Link RNA Isolation kit (Thermofisher). Extracted RNA (2 µg) was reverse-transcribed to cDNA using primeScriptIII First-Strand cDNA kit (Thermofisher). The full-length amplification of the UGT72L11 gene was performed using gene specific primers (Table S1 ). The amplified gene was cloned into pJET1.2 (ThermoScientific, USA) cloning vector, confirmed by Sanger sequencing and subsequently submitted to NCBI Data Bank. The corresponding amino acid sequence encoding the candidate UGT was protein-BLAST searched for homology using NCBI search tools [ 23 ]. For promoter cloning, the 5’ upstream region of the identified UGT was cloned using Genome Walker kit (TAKARA, Japan) following the instruction manual. The amplified promoter region was scanned for the presence of various cis-regulatory elements using Plant Cis-acting Regulatory DNA Elements database (PLACE, http://www.dna.affrc.go.jp/PLACE/ ). 2.8 Real-time expression analysis 2.7.1 Treatments The in-vitro regenerated plants grown under three different environmental conditions namely, tissue culture, hardening unit and field conditions were assessed for the expression of the identified gene. The G. glabra plants were also subjected to one biotic stress, eight abiotic stress treatments and three hormone based elicitations. The treatments were essentially performed following the standardized protocol published earlier [ 24 , 25 ]. Each treatment along with control was given to a set of three in-vitro plants (2 months old) for different time durations. Abiotic treatments like dark and carbon starvation were subjected for 48 hrs; cold (4°C) for 64 hrs; wounding for 8 hrs; UV-C for 30 min, salinity for 128 hrs and drought for 4 days. For the senescence treatment, green and yellow leaves of the in-vitro plants were used. For biotic elicitation 3 pathogenic isolates of Colletotrichum sp. and one Calonectria sp. were employed. The hormone treatment was subjected for various time duration; NAA was given for 2 hrs, GA 3 for 6 hrs and ABA for 16 hrs to the in-vitro grown plants. RNA extraction and cDNA synthesis from the tissues of the control and treated plants, and plants grown under three different conditions ( in-vitro , field and glass house) were performed as mentioned earlier. 2.8.2 Expression Analysis The expression of UGT72L11 transcripts in the aerial tissues of G. glabra plant were examined by quantitative reverse-transcription PCR following the thermal profile published earlier using β-actin gene as an internal control [ 26 ]. The primers for quantitative real-time (qRT)-PCR are listed in Table S1 . qRT-PCR was performed on StepOne™ (Applied Biosystems) using SYBR premix ExTaq II (Clontech) following instructions of the manufacturer. The relative expression level was calculated by the 2 -ΔΔct method [ 27 ]. For each gene, three independent experiments were performed, and three technical replicates were analyzed for each sample [ 26 ]. Statistical analysis was performed using Graph Pad prism software/EXCEL sheet. 2.9 Heterologous expression and protein purification The full-length gene in pJET1.2 (ThermoScientific, USA) cloning vector was amplified using unique NcoI and XhoI site specific forward and reverse primers, respectively. The amplified gene was digested with respective restriction enzymes and sub-cloned into previously digested pET28a(+) vector with the same set of enzymes. The resultant construct was transformed into Escherichia coli BL21 (DE3) cells and the transformed colonies were screened on the Kanamycin (50mg/ml) supplemented plates and confirmed by colony PCR. PCR amplification was performed using gene specific forward/reverse primers (supplementary tabl e1 ) and T7 promoter/terminator primer combination using the plasmid extracted from the positive colony for the conformity of the transformation. For the recombinant protein expression and purification, the confirmed transformant was inoculated into 5 ml Luria Bertani (LB) containing appropriate antibiotic and incubated at 37ºC overnight. Subsequently, the primary inoculum (1%) was added to 200 ml fresh LB media and incubated similarly, until its OD 600 reached a value of 0.6. The cloned gene was induced with the optimized conditions of IPTG concentration (0.8 mM), incubating temperature (22°C) and under shaking (180 rpm) for 20 hrs. The recombinant E. coli cells were harvested and the recombinant protein was extracted by cell lysis followed by centrifugation (6500 g for 10 min at 4°C).The crude protein (20 µl) was initially checked on SDS-PAGE (10%) to confirm protein induction. Subsequently the recombinant protein was purified using TALON single step column (Takara, Japan) essentially following the manufacturer’s protocol. The purified recombinant protein (20 µl) was confirmed on SDS-PAGE gel (10%) and quantified using Bradford’s method[ 28 ]. 2.10 Enzyme Kinetics Prior to the in-vitro assay using aglycon compound library, optimization of the reaction conditions for the recombinant protein was performed. The purified protein was optimized for parameters like incubation time (15–90 min), temperature (4ºC-70ºC) and pH in citrate (pH 3–6), phosphate (6-7.5) and Tris-HCl (7.5–10.5) buffers at a constant protein concentration (2.5 µg). The sugar acceptability using kaempferol and quercetin as substrates was assessed essentially following the UDP Glo™ assay protocol (Promega, USA) which detects UDP after UDP-sugar hydrolysis/ transfer by converting UDP to light in luciferase type reaction (measured in relative units). A standard curve of UDP concentration (0–25 µm) was generated, and linear range of detection was determined, where the luminescence is directly proportional to the UDP concentration. Following the protocol published earlier [ 29 ], each sugar nucleotide hydrolysis reaction was combined with the detection reagent in equal ratio and allowed to incubate at room temperature. After 1h of incubation, the luminescence was measured using FLUOstar Omega microplate reader (BMG Labtech).The amount of UDP released during the glycosylation reaction was recorded and the relative catalytic activity was calculated. Also, four substrates (kaempferol and quercetin) concentrations (3, 5,7 &9 µM) were examined and the released UDP amounts were quantified by relative luminescence for the enzyme activity. The enzyme kinetics parameter namely, Km, Vmax, kcat and kcat/Km were calculated for the two substrates. 2.11 In vitro enzyme assay Enzyme reaction (200 µl) was performed under the in-vitro conditions using the optimized parameters of citrate buffer (50 mM) pH 6.0, purified protein (5µg), UDP-Glc (5 mM) and substrate (0.9 mM) at 30°C incubation temperature for 15 min. The reaction was quenched by the addition of ethyl acetate (100 µl) thrice and partitioned with water. The organic part was concentrated and re-suspended in methanol for UP-HPLC/LCMS analysis. After successful glycosylation of kaempferol and quercetin, a panel of sixteen natural products belonging to various classes of compounds (flavones, terpenes, alkaloids, phenolics, vitamins, Phyto-hormone and carboxylic acid) and drug molecules were tested under similar conditions. 2.12 LC-MS Analysis Experiments were performed on an Agilent 1260 Infinity (Agilent, USA) HPLC system coupled with an Agilent 6410 (Agilent Technologies, USA) triple quadrupole MS/MS instrument equipped with an ESI ion source in the positive ion mode. Chromatographic separation was performed on a Chromolith high resolution RP18e column(100 x 4.6 mm) maintained at 300º C. Mobile phases of 0.1% (v/v) formic acid in water (eluent A) and acetonitrile (eluent B) were used at a flow rate of 600 µl/min to analyze each sample having injection volume of 10µl. A gradient programme used was: 0–7 min, 50%B followed by increment of 10% in eluent B after 8–12 min, 12–25 min, 25–30 min reaching upto80% for30–45 min and returning to 50% for 45–50 min. Data handling was performed using Mass Hunter workstation. The standards (kaempferol/quercetin/3-O-glycosylated kaempferol and quercetin) were analyzed under the optimized conditions before analysing the samples. 3. Results 3.1 Sequence and phylogenetic analysis The selected full-length gene encoding a UGT gene was mined from the in-house library (PRJNA664636) of G. glabra plant and submitted to the NCBI database (MN163014.1) bearing 52.16 kDa protein (QHW04706.1). The full-length GgUGT72L11 ORF was sequenced to be 1425bp encoding a cytoplasmic protein of 474 amino acid having instability index of 40.62, aliphatic index of 99.75, GRAVY value of 0.032 and a theoretical pI of 6.61. The MEME based motif analysis showed ten highly conserved fifty sequence length motifs (including the signature Plant Secondary Product Glycosyltransferase motif) common to all the studied protein homologs (Fig. 1 ). The MSA based homology of the UDPGT specific region ranged between 72.61% 85.90% to (supplementary Fig. 1). A 199 aa long GTB region (from 262 to 461 aa) was found to be highly variable (from 305 to 321aa); however, a 69 aa long region (from amino acid 322 to 391) in this region was highly conserved including the 44 aa long PSPG box (aa344 to aa388) at the C-terminal. Further, the protein-protein blast of the 58 aa long stretch was found to be highly specific to the proteins identified from Fabaceae family which was endorsed by the results of the MSA (Supplementary Fig. 1). Further, to predict the putative function of UGT72L11, twenty five functionally annotated UGT72 protein sequences reported from various plant species, were aligned (Fig. 1 ). The phylogeny classified the identified protein akin to epicatechin 3-glucosyltransferase (UGT72L1) from M. truncatula and flavonol 3-O-glucosyltransferases from Glycine max (UGT72Z3), G. uralensis (UGT72X5) and G. max . (UGT72X4) plants displaying affinity towards flavonoids. Additionally, phylogenetic tree (Fig. 2 ) based on thirty four proteins sequences having mono- and di-glycosylation propensity revealed the identified protein to be mono-glycosylated forming a distinct cluster with AtUGT71C1 and VvGT7A (XP_002276546) corroborated by mono-glucoside characteristic GSS consensus motif (563–566) as reported by Huang et al.[ 19 ]. Contrary to this finding, the proline amino acid at position 95 (Supplementary Fig. 2) which is the characteristic feature of disaccharides was also present in the GgUGT72L11 protein further highlighting the sequence uniqueness of the identified protein. 3.2 Promoter Analysis To further investigate the role of the identified gene, upstream region (1174 bp) of the selected gene was isolated by genome walking. In silico analysis of the promoter sequence revealed putative cis- acting regulatory elements and transcription factor-binding sites. The results demonstrated that the mapped cis -acting elements included promoter core elements (TATA-box and CAAT-box), phytochrome activated light-responsive element (5’GATAAGR3’), abscisic acid signalling/regulated gene expression motif (5’CACGTG/CANNTG3’). Stress responsive elements including dehydration (5’ACGT3’), cold induced (5’CAANTG3’) and ABRE-Related Sequences (MACGYGB) were also found. The promoter region also had carbon (5’AAAG3’) and sugar metabolism (5’TGACT3’) motifs, pathogenesis related elements (5’YTGTCWC3’) and wound (5’TGACY3’) perceptive elements (supplementary Fig. 3). 3.33-D Model Prediction In plants, the characteristic 44 aa long consensus sequence of the PSPG domain plays a crucial role during glycosylation [ 30 ]. The 10 aa highly conserved C-terminal domain binds to the donor sugar molecule, while the N-terminal is highly variable acceptor binding site potentially for accommodating wide variety of aglycons. Ramachandran plot analysis of protein under study, (supplementary Fig. 4a,b) showed maximum number of amino acids in favored region (97.40%) and allowed region (1.94%). A small fraction (0.65%) was found in the outliner region (supplementary Fig. 4b) also. The glycosylation can only be catalyzed if the acceptor molecule is positioned correctly in the substrate pocket such that the functional group of the sugar acceptor is in close proximity to the first carbon of the sugar for the formation of glycosidic bond [ 31 ]. Docking simulations of candidate ligand kaempferol with receptor UGT72L11 showed good binding affinity and stability of the complex as indicated by binding affinity of kaempferol (-6.140 kcal/mol), total energy (-31.247 kcal/mol), vdW energy (-0.438 kcal/mol) and electrostatic energy (-16.390 kcal/mol). 3.4 Subcellular localisation, recombinant Protein Expression and Purification Sub-cellular localization of UGT72L11 protein was found to be predominantly in plasma membrane as demonstrated by onion cells transiently transformed with 35S:ugt72l11-GFP construct (Fig. 4 ). The recombinant protein was expressed in E. coli (BL-21 cells) and optimized for optimal induction. The protein was found to be optimally induced (Fig. 5 ) in 20 hr under 0.8 mM IPTG concentration at 20°C. The recombinant protein with C-terminal HIS tag was column purified and employed for enzyme kinetic studies and in-vitro assays. 3.5 Enzyme Kinetics The purified recombinant protein activity was assessed in three different pH -acidic (citrate), neutral (phosphate) and basic (Tris HCl) buffer systems. The protein was found to be active in all the three buffers studied. However, optimum enzyme activity was found in citrate buffer (pH6.0) (Fig. 6 a). Further, the time duration of 15 min to 90 min assessed for the enzyme activity showed its optimal activity at 15 min (Fig. 6 b). The effect of temperature studied between 4°C to 70°C, revealed protein to be optimally active between 10°C to 30°C (Fig. 6 c). The optimized parameters were further used for the enzyme kinetic studies. The enzyme-substrate relationship was evaluated with two substrates (quercetin and kaempferol) at four concentrations (3, 5, 7 & 9 µM) employing UDP-glucose as the sugar donor (Fig. 6 d). The kinetic properties were determined for the selected substrates using previously optimized concentrations of 2.5µg protein and 15 µM UDP sugar. The reaction was kept for 15-min at 30°C. The Michaelis constant ( K m ), which signifies the concentration of the substrate at which half of the enzyme is saturated, was found to be more for quercetin-UDPGlc (0.23) than kaempferol-UDPGlc (0.47) and the catalytic number ( K cat )/ K m were calculated to be 0.66 and 0.44 m m − 1 s − 1 , respectively reflecting its preference for quercetin over kaempferol. 3.6 In-vitro enzyme activity and product identification The substrate affinity of the purified recombinant protein was established using in-vitro enzyme assay employing various classes of substrates as acceptors and UDP-glucose as the sugar donor following the optimized conditions of 30ºC for 15 min. The protein was initially assessed with quercetin and kaempferol as the two model substrates as they harbour several hydroxyl groups. The in-vitro enzyme activity and the position of glycosylation were confirmed by their respective 3-O-glucoside standards. The retention time (RT) corresponding to the substrates and respective glycosylated products were compared and confirmed by peaks and mass spectra. It was conclusively shown that the substrates, kaempferol (Fig. 7 a) and quercetin (Fig. 7 b) were glycosylated to quercetin-3-glucoside and kaempferol-3-glucoside, respectively. 3.7 In-vitro activity of GgUGT72L11 Enzyme (Substrate scope) The substrates investigated in the present study belonged to flavones, terpenes, carboxylic acids, vitamins, alkaloid, phenol classes of compounds and few commercial drugs (Fig. 8 ). Flavones, such as quercetin and catechin hydrate having polyhydroxyl groups, when subjected to glycosylation under optimized conditions showed the formation of respective glucosides as confirmed by LCMS ( supplementary Data 1; Table 1 ). Similarly, diosgenin having monohydroxy group and andrographolide with carboxylic groups were catalyzed for the addition of glucose moiety to form respective glycosides by the purified protein (supplementary Data 1). Isovanillin, (a phenolic), dihydrozeatin & abscisic acid (phytohormones) were also seen to form their respective glycosides catalyzed by GgUGT72L11. The alkaloidal substrates studied were colchicine, monocrotaline and quinine hydrochloride. The identified GgUGT72L11 catalyzed glycosylation in colchicine whereas monocrotaline and quinine hydrochloride underwent hydroxyl glycosylation forming respective glycosides. Structurally, colchicine possess no hydroxyl and/or carboxyl groups, contrary to monocrotaline and quinine hydrochloride that had -OH groups Table 1 List of glycosylated compounds (flavanols, terpenes, carboxylic acids, vitamins, alkaloid, phenolics and commercial drugs) with molecular formulae observed, and calculated molecular weight of the glycosides S.no Product Mass calculated for Calculated molecular weight Observed molecular weight 1 Kaempferol glucoside (M-H) (C 21 H 19 O 11 ) 448.10 448.02 2 Quercetin glucoside (M + H) (C 21 H 21 O 12 ) 465.10 464.65 3 Catechin hydrate glucoside (M-H) (C 21 H 25 O 12 ) 469.14 469.83 4 Diosgenin glucoside (M + H) (C 33 H 53 O 8 ) 577.37 577.35 5 Andrographolide glucoside (M-H) (C 26 H 39 0 10 ) 513.26 514.88 6 Citric acid glucoside (M-H) (C 12 H 17 O 12 ) 353.07 353.26 7 Nicotinic acid glucoside (M-H) (C 12 H 14 NO 7 ) 284.08 285,25 8 Riboflavin glucoside (M-H) (C 23 H 29 N 4 O 11 ) 537.18 537.32 9 Iso vanillin glucoside (M-H) (C 14 H 17 O 8 ) 313.09 313.51 10 Dihydro zeatin glucoside (M + H) (C 16 H 26 N 5 0 6 ) 384.19 383.8 11 Abscisic acid (M-H) (C20H29O9) 425.45 425.35 12 Colchicine glucoside (M + Na) C 28 H 35 NNaO 11 ) 585.21 585.67 13 Quinine hydrochloride glucoside (M-H) (C 26 H 34 ClN 2 O 7 ) 521.21 520.9 14 Monocrotaline glucoside (M + H) (C 22 H 34 NO 11 ) 488.21 488.18/486.6 15 Podophyllotoxin glucoside (M + H) (C 28 H 33 0 13 ) 577.19 576.94 16 Sofosbuvir glucoside (M-H) (C 28 H3 8 FN 3 O 14 P) 690.21 690.42 17 Ephedrine HCL glucoside (M-H) (C 16 H 25 ClNO 6 ) 362.14 361.99 13 Quinine hydrochloride glucoside (M-H) (C 26 H 34 ClN 2 O 7 ) 521.21 520.9 14 Monocrotaline glucoside (M + H) (C 22 H 34 NO 11 ) 488.21 488.18/486.6 15 Podophyllotoxin glucoside (M + H) (C 28 H 33 0 13 ) 577.19 576.94 16 Sofosbuvir glucoside (M-H) (C 28 H3 8 FN 3 O 14 P) 690.21 690.42 17 Ephedrine HCL glucoside (M-H) (C 16 H 25 ClNO 6 ) 362.14 361.99 3.8 Expression Dynamics of UGT72L11 3.8.1Growth Conditions qRT-PCR analysis was performed to analyze the expression profile and levels of the identified gene in different tissues. The real-time quantitative analysis of the transcripts was performed using the RNA extracted from the aerial and underground tissues on the plants grown under three different conditions namely, tissue culture, hardening unit and the experimental farms (Fig. 9 a). The identified gene displayed preferential expression under the three surroundings. The GgUGTL11 was found to be significantly up-regulated (2.41 folds) in the roots of the field grown plants, while it was significantly down regulated in the roots of the glass house stationed plants as compared to the respective shoot tissues (Fig. 9 a). The gene was found to be similarly expressed in the stem tissues under the three conditions. 3.8.2 Stress Total RNA extracted from the aerial tissues of the in-vitro grown plants was subjected to nine stressors and assessed for transcript accumulation. Eight abiotic treatments (Fig. 9 b) given to the plants were dark, carbon starvation, wounding, senescence, drought, salinity, cold and UV light. The GgUGT72L11 gene was found to be repressed under the influence of carbon starvation (0.6 fold), salinity (0.9 folds) and dark (0.3 fold) conditions. The gene transcripts were found to be maximally up regulated under cold (36 folds) conditions followed by drought (9 folds), senescence (1.7 folds), and UV light (1.2 folds). Further, the biotic stress subjected through co-cultivation of in-vitro grown G. glabra plants (individually) with four plant pathogens (3 isolates of Colletotrichum sp.( P1- Colletotrichum gloeosporioides , P2- Colletotrichum siamense sub sp. , P3- Colletotrichum siamense sub sp. and P4- one isolate of Calonectria cylindrospora) showed increased transcript levels with P2 (2.86 folds), while P1 was found to be suppressed (Fig. 9 c). The expression profile of the GgUGT72L11 gene in the in-vitro plants subjected to auxin, abscisic acid and GA hormone treatments showed significant reverberation. The gene was found to be highly up regulated under ABA (400 folds) treatment at 16 hrs (Fig. 9 d) and moderately up regulated in the presence of auxin (2 folds) between 30 to 60 minutes. At the same time, it was seen to be down regulated (0.4 to 0.8) under GA treatment between 1 to 6 hrs in the aerial tissues of the plant as compared to the untreated control plants. 3.8.3 Determination of expression of ROS scavenging genes and cold responsive genes The above results indicated higher accumulation of transcripts in the aerial tissues of the in-vitro grown plants under cold (4°C) and ABA stresses. Further, cold -specific COR genes ( COR47, ABI5, DREB1 ) and Reactive Oxygen Species scavenging genes catalase (CAT1) , super oxide dismutase ( SOD) , Glutathione Peroxidase (GPX ) were studied to understand the molecular mechanism (Fig. 10 A). The cold responsive genes were seen to be significantly up regulated- COR 47 (22.3 folds), DREB1 (13.8 folds) and ABI5 (10.2 folds) under cold stress. Also expression analysis of ROS scavenging enzymes and cold responsive genes was carried out in ABA treated plants (Fig. 10 B). Both the ROS scavenging genes -CAT1 ( 5.97 folds), SOD (18.6 folds) and GPX (16 folds), and cold responsive genes- DREB1 (2.9 folds) and ABI5 (14.6folds) were found to be up regulated in the plants subjected to ABA treatments suggesting the role of GgUGT72L11 under multiple stress. However, ROS scavenging genes ( CAT1, SOD, GPX ) were observed to be uninfluenced under the cold stress suggesting no involvement of ROS scavenging enzymes under cold stress. Discussion The pBLAST search (NCBI) displayed maximum homology (73.3%) homology with UGT72L1 (ACC38470) encoding epicatechin 3-glucosyltransferase from M. truncatula . The CAZy database divulged information on only two proteins from G. uralensis species (NCBI accession No. MK341791 and MK341793) which were involved in flavonoid biosynthesis [ 17 ]. The identified protein from G. glabra seems to be unique as it showed less than 40% homology with the two proteins reported from the G. uralensis . The uniqueness of the protein was further enhanced by having both mono- and disaccharide glycoside forming activities. The protein sequence possessed mono-glucoside characteristic GSS consensus motif (563–566), and also had proline amino acid at position 95 characteristic of disaccharides as reported by Huang et al.[ 19 ]. Understanding the glycosylation mechanism and its physiological significance in plants hold importance not only in-planta for channelizing metabolic pathways for producing the desired compound [ 32 ]; but also for valuable glycosides in synthetic medical biology. In our study, the in-silico mapping on receptor (UGT72L11) and ligand (kaempferol) showed that kaempferol formed five hydrogen bond interactions with UGT72L11protein specifically with four amino acid residues GLU279, ARG386, PRO380 and TRP379 (Fig. 3 ). On the other hand, in the well characterized structure of AtUGT72B1 protein the substrate was demonstrated to be enclosed in six hydrophobic residues (I-86, L-118, F-119, F-148, L-183, and L-197) to form catalytic site roofed by E83 covering the site[ 33 ]. Further, the ligand and receptor interaction was also stabilized by pi donor hydrogen bond and pi alkyl interactions involving SER277 and LEU409 amino acid residues respectively. Protein-ligand binding occurs spontaneously when the binding affinity/free energy change is negative [ 34 ] as these complexes are stable which is an essential characteristic of effective ligands. The present study further experimented to confirm the results of docking experiment with the in-vitro enzyme activity results using the substrates. In vitro assay using the purified protein demonstrated multi-substrate acceptability with wide range of aglycons and medicinally important drugs like ephedrine hydrochloride, podophyllotoxin, and sofosbuvir (Table 1and supplementary Data1). The GgUGT72L11 protein introduced glucose moiety to the mono hydroxyl group in the studied compounds forming their respective mono-glycosylated products as confirmed by LCMS analyses (supplementary Data 1). Previous in-vitro enzymatic studies in thirty reported recombinant UGT72 family proteins, across 12 different angiosperm species, have shown acceptance to varied substrates including flavonoids, monolignols, and their precursors/derivatives [ 35 ]. UGTs can glycosylate at O -position (3, 5, 7, 3',4', or 5'), and\or C -positions as seen in rice, maize and buckwheat (Du et al., 2010; Falcone Ferreyra et al., 2013;Nagatomo et al., 2014). Evidences suggest members of UGT72 family possess broad spectrum substrate recognition predominantly in flavanols with different glycosylation patterns forming 3- O -monoglycosides as in L. japonicus ( LjUGT72AH1, LjUGT72Z2, LjUGT72V3) [ 36 ], Glycine max (GmUGT72X4, GmUGT72Z3)[ 37 ] and M. truncatula (MtUGT72L1)[ 14 ], to multi-site glycosylation at 3, 4, and 7 positions by Clonorchis sinensis CsUGT72A1[ 38 ] and Hieracium pilosella HpUGT72B11 [ 39 ]. The identified GgUGT72L11 protein also catalyzed C -glycosylation in colchicine. The C -glycosylation pattern and its extent have a crucial role in forming potent drug-like molecule [ 12 ]. The conjugates of C -glycosides exhibit unique characteristics as these bonds are resistant to acid hydrolysis and glycosidase cleavage [ 40 ]. The UGTs catalyzing C -glycosylation has been reported from several plants including Fagopyrum esculentum (FeCGTa and FeCGTb) [ 41 ], Glycine max (UGT708D1), Dendrobium catenatum [ 40 ], Gentiana trifloral [ 42 ] and Citrus plants (UGT708G1, UGT708G2) [ 43 ]. Literature cites singular report of C -glycosyltransferase (GgCGT) from G. glabra catalysing two-step C -glycosylation of the drug flopropione [ 12 ]. The presence of light, hormonal and stress responsive motifs indicated involvement of gene in complex hormone regulatory network pertaining to stress and secondary metabolite biosyntheses. Overall, the cis -element analysis suggested gene responsive to different kinds of stresses. This was explored further to assess the involvement of the identified UGT under the influence of various stresses. The identified flavonoid glycosyl transferase displayed high expression under cold, senescence, drought and abscisic acid stress. Recent comprehensive review [ 44 ] highlighted buffering effects of UGTs in multiple biotic and abiotic stresses. A series of protective mechanisms are triggered when plants sense stress [ 45 ] underpinning the overlapping but not completely redundant biological functions of UGTs in mediating developmental and stress responses. In the current study, GgUGT72L11 gene was shown to be significantly up regulated under cold stress along with the downstream chill-induced COR and DREB genes. However, ROS scavenging genes were down regulated under similar conditions. Contrary to this studies have highlighted up regulation of ROS-scavenging genes such as superoxide dismutase (SOD) , catalase (CAT 1) and Glutathione Peroxidase (GPX) in detoxification particularly under stress[ 46 ] .One probable reason for down regulation could be the formation of glycosylated flavonoids catalyzed by the identified flavonol glycosyl transferase ( GgUGT72L11 ) imparting hydrophilic and cartable [ 47 ] properties to counter the oxidative burst more efficiently under stressed conditions [ 45 ]. Our findings also revealed the gene was significantly induced when subjected to ABA treatment and catalyses ABA to respective glycosylated conjugate (supplementary data) under in-vitro conditions. Hence, the ABA homeostasis was maintained thereby inhibiting ROS accumulation. Corroborating our findings, few studies have confirmed strong induction of UGTs under the cold stress simultaneously participating in flavonol biosyntheses [ 45 ] ,[ 48 ]. Studies have also confirmed involvement of several UGTs (AtUGT79B2, AtUGT79B3 and CsUGT78A14) in glycosylation of ABA regulating the dynamic state of cellular ABA/ABA-GE levels (ABA homeostasis). Conclusion Studies targeting association of flavonoids under cold stress and ROS scavenging ability will provide novel insights into the biological role of the UGTs and flavonoids in plant defence and stress biology. Further, investigation into the underlying molecular processes involving ABA homeostasis and ABA-mediated stresses (cold and drought in particular) will throw light on the interplay between stressors, glycosylated metabolites and signalling mechanisms for multiple stress tolerance. The recognition of a simple but unique glycosylation mechanism in the identified UGT offers physiological advantage to the plant and could be the foundation for numerous applications in medical biology expanding the magnitude to manipulate the biological and physicochemical properties of molecules. Further investigation of the underlying downstream processes will elucidate the regulatory mechanism of UGT72L11 in plant stress tolerance. Declarations Contribution: Malik Muzafar Manzoor, Shahnawaz Husain, Bhawna Verma, Suphla Gupta - Conceptualization, Malik Muzafar Manzoor, Shahnawaz Husain, Bhawna Verma ,Pooja Goyal, Ritu Devi, 4Fariha chowdhary- Methodology :, Ajai P Gupta ,Pooja Goyal, Bhawna Verma, Suphla Gupta, Bhawna Verma - Writing- Original draft preparation. Suphla Gupta,: Visualization, Investigation and Supervision.: Ajai P Gupta: Software, Validation.: Suphla Gupta, Bhawna Verma- Writing- Reviewing and Editing. Funding and Acknowledgements: Authors acknowledge the grant (SERB/SB/SO/PS/90/2013) from Science and Engineering Research Board, India for funding the study. MM and SH acknowledges Council for Scientific & Industrial Research for Fellowships; PG and BV acknowledges University Grant Commission for Fellowship; SH & BV acknowledges Council for Scientific & Industrial Research and Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Sector-19, Ghaziabad, U.P., India is also acknowledged. All the authors acknowledge director, CSIR-IIIM for providing all the necessary facilities. Data Statement: The article's supporting data and materials are in the public domain and can be accessed at NCBI SRA Data (PRJNA664636). Declaration of competing interest All the authors declare no competing interest. Declaration of generative AI in scientific writing We have not used any generative AI in scientific writing. References Bock KW (2016) The UDP-glycosyltransferase (UGT) superfamily expressed in humans, insects and plants: Animal-plant arms-race and co-evolution. Biochem Pharmacol 99:11–17 Gilbert MK, Bland JM, Shockey JM, Cao H, Hinchliffe DJ, Fang DD, Naoumkina M (2013) A transcript profiling approach reveals an abscisic acid-specific glycosyltransferase (UGT73C14) induced in developing fiber of Ligon lintless-2 mutant of cotton (Gossypium hirsutum L). PLoS ONE 8(9):e75268 Zhao Y, Chow TF, Puckrin RS, Alfred SE, Korir AK, Larive CK, Cutler SR (2007) Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated. Nat Chem Biol 3(11):716–721 Jarrar Y, Lee S-J (2021) The Functionality of UDP-Glucuronosyltransferase Genetic Variants and their Association with Drug Responses and Human Diseases. J Pers Med 11(6):554 Zheng S, Luo J, Zhu X, Gao X, Hua H, Cui J (2022) Transcriptomic analysis of salivary gland and proteomic analysis of oral secretion in Helicoverpa armigera under cotton plant leaves, gossypol, and tannin stresses. Genomics 114(2):110267 Li Q, Yu H-M, Meng X-F, Lin J-S, Li Y-J, Hou B-K (2018) Ectopic expression of glycosyltransferase UGT76E11 increases flavonoid accumulation and enhances abiotic stress tolerance in Arabidopsis. Plant Biol 20(1):10–19 Sun Y, Ji K, Liang B, Du Y, Jiang L, Wang J, Kai W, Zhang Y, Zhai X, Chen P et al (2017) Suppressing ABA uridine diphosphate glucosyltransferase (SlUGT75C1) alters fruit ripening and the stress response in tomato. Plant J 91(4):574–589 Shi Y, Phan H, Liu Y, Cao S, Zhang Z, Chu C, Schläppi MR (2020) Glycosyltransferase OsUGT90A1 helps protect the plasma membrane during chilling stress in rice. J Exp Bot 71(9):2723–2739 Zhang K, Sun Y, Li M, Long R (2021) CrUGT87A1, a UDP-sugar glycosyltransferases (UGTs) gene from Carex rigescens, increases salt tolerance by accumulating flavonoids for antioxidation in Arabidopsis thaliana. Plant Physiol Biochem 159:28–36 Zhao M, Cai B, Jin J, Zhang N, Jing T, Wang J, Pan Y, Zhou Z, Zhao Y, Feng Y et al (2020) Cold Stress-induced Glucosyltransferase CsUGT78A15 is Involved in the Formation of Eugenol Glucoside in Camellia sinensis. Hortic Plant J 6(6):439–449 Li P, Li Y-j, Wang B, Yu H-m, Li Q, Hou B (2017) -k: The Arabidopsis UGT87A2, a stress-inducible family 1 glycosyltransferase, is involved in the plant adaptation to abiotic stresses. Physiol Plant 159(4):416–432 Zhang M, Li FD, Li K, Wang ZL, Wang YX, He JB, Su HF, Zhang ZY, Chi CB, Shi XM et al (2020) Functional Characterization and Structural Basis of an Efficient Di-C-glycosyltransferase from Glycyrrhiza glabra. J Am Chem Soc 142(7):3506–3512 Caputi L, Malnoy M, Goremykin V, Nikiforova S, Martens S (2012) A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land. Plant J 69(6):1030–1042 Pang Y, Cheng X, Huhman DV, Ma J, Peel GJ, Yonekura-Sakakibara K, Saito K, Shen G, Sumner LW, Tang Y et al (2013) Medicago glucosyltransferase UGT72L1: potential roles in proanthocyanidin biosynthesis. Planta 238(1):139–154 Nomura Y, Seki H, Suzuki T, Ohyama K, Mizutani M, Kaku T, Tamura K, Ono E, Horikawa M, Sudo H et al (2019) Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin. Plant J 99(6):1127–1143 Chung SY, Seki H, Fujisawa Y, Shimoda Y, Hiraga S, Nomura Y, Saito K, Ishimoto M, Muranaka T (2020) A cellulose synthase-derived enzyme catalyses 3-O-glucuronosylation in saponin biosynthesis. Nat Commun 11(1):5664 Jiang D, Li P, Yin Y, Ren G, Liu C (2021) Molecular cloning and functional characterization of UGTs from Glycyrrhiza uralensis flavonoid pathway. Int J Biol Macromol 192:1108–1116 Wang C, Chen L, Cai Z, Chen C, Liu Z, Liu S, Zou L, Tan M, Chen J, Liu X et al (2021) Metabolite Profiling and Transcriptome Analysis Explains Difference in Accumulation of Bioactive Constituents in Licorice (Glycyrrhiza uralensis) Under Salt Stress. Front Plant Sci 12:727882 Huang F-C, Giri A, Daniilidis M, Sun G, Härtl K, Hoffmann T, Schwab W (2018) Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases. Plant Cell Physiol 59(4):862–875 McWilliam H, Li W, Uludag M, Squizzato S, Park YM, Buso N, Cowley AP, Lopez R (2013) Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res 41(W1):W597–W600 Wass MN, Kelley LA, Sternberg MJ 3DLigandSite: predicting ligand-binding sites using similar structures. Nucleic Acids Res 2010:gkq406 Sun W, Cao Z, Li Y, Zhao Y, Zhang H (2007) A simple and effective method for protein subcellular localization using Agrobacterium-mediated transformation of onion epidermal cells. Biologia 62(5):529–532 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410 Manzoor MM, Goyal P, Pandotra P, Dar MS, Dar MJ, Misra P, Gupta AP, Vishwakarma RA, Ahuja A, Dhar MK Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis. Protoplasma 2021:1–17 Goyal P, Muzafar Manzoor M, Gupta AP, Pandotra P, Gupta S (2022) : Molecular Dissection of Genes and Promoters involved in Glycyrrhizin Biosynthesis Revealed Phytohormone Induced Modulation in Glycyrrhiza glabra L. Gene :146682 Li Y, Liang X, Zhou X, Wu Z, Yuan L, Wang Y, Li Y (2020) : Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments. Plants (Basel Switzerland) 9(11) Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW (2000) Quantitative Reverse Transcriptionân-Polymerase Chain Reaction to Study mRNA Decay: Comparison of Endpoint and Real-Time Methods. Anal Biochem 285(2):194–204 Bradford M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7: 248–254. Find this article online 1976. McGraphery K, Schwab W (2020) : Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases. Int J Mol Sci 21(6) Louveau T, Leitao C, Green S, Hamiaux C, van der Rest B, Dechy-Cabaret O, Atkinson RG, Chervin C (2011) Predicting the substrate specificity of a glycosyltransferase implicated in the production of phenolic volatiles in tomato fruit. FEBS J 278(2):390–400 Breton C, Šnajdrová L, Jeanneau C, Koča J, Imberty A (2005) Structures and mechanisms of glycosyltransferases. Glycobiology 16(2):29R–37R Guo H, Wang H, Huo YX (2020) Engineering Critical Enzymes and Pathways for Improved Triterpenoid Biosynthesis in Yeast. ACS Synth Biol 9(9):2214–2227 Brazier-Hicks M, Offen WA, Gershater MC, Revett TJ, Lim E-K, Bowles DJ, Davies GJ, Edwards R (2007) : Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants. Proceedings of the National Academy of Sciences 104(51):20238–20243 Afriza D, Suriyah W, Ichwan S (2018) : In silico analysis of molecular interactions between the anti-apoptotic protein survivin and dentatin, nordentatin, and quercetin. In: Journal of Physics: Conference Series : : IOP Publishing; 2018: 032001 Speeckaert N, El Jaziri M, Baucher M, Behr M (2022) : UGT72, a Major Glycosyltransferase Family for Flavonoid and Monolignol Homeostasis in Plants. Biology (Basel) 11(3) Yin Q, Shen G, Chang Z, Tang Y, Gao H, Pang Y (2016) Involvement of three putative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis in Lotus japonicus seeds. J Exp Bot 68(3):597–612 Noguchi A, Saito A, Homma Y, Nakao M, Sasaki N, Nishino T, Takahashi S, Nakayama T (2007) A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis. J Biol Chem 282(32):23581–23590 Zhao X, Dai X, Gao L, Guo L, Zhuang J, Liu Y, Ma X, Wang R, Xia T, Wang Y (2017) Functional Analysis of an Uridine Diphosphate Glycosyltransferase Involved in the Biosynthesis of Polyphenolic Glucoside in Tea Plants (Camellia sinensis). J Agric Food Chem 65(50):10993–11001 Speeckaert N, Adamou NM, Hassane HA, Baldacci-Cresp F, Mol A, Goeminne G, Boerjan W, Duez P, Hawkins S, Neutelings G et al (2020) : Characterization of the UDP-glycosyltransferase UGT72 Family in Poplar and Identification of Genes Involved in the Glycosylation of Monolignols. Int J Mol Sci 21(14) Ren Z, Ji X, Jiao Z, Luo Y, Zhang G-Q, Tao S, Lei Z, Zhang J, Wang Y, Liu Z-J et al (2020) Functional analysis of a novel C-glycosyltransferase in the orchid Dendrobium catenatum. Hortic Res 7(1):111 Nagatomo Y, Usui S, Ito T, Kato A, Shimosaka M, Taguchi G (2014) Purification, molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon. Plant J 80(3):437–448 Sasaki N, Nishizaki Y, Yamada E, Tatsuzawa F, Nakatsuka T, Takahashi H, Nishihara M (2015) Identification of the glucosyltransferase that mediates direct flavone C-glucosylation in Gentiana triflora. FEBS Lett 589(1):182–187 Ito T, Fujimoto S, Suito F, Shimosaka M, Taguchi G (2017) C-Glycosyltransferases catalyzing the formation of di-C-glucosyl flavonoids in citrus plants. Plant J 91(2):187–198 Zhang W, Wang S, Yang J, Kang C, Huang L, Guo L (2022) Glycosylation of plant secondary metabolites: Regulating from chaos to harmony. Environ Exp Bot 194:104703 Zhao M, Jin J, Gao T, Zhang N, Jing T, Wang J, Ban Q, Schwab W, Song C (2019) : Glucosyltransferase CsUGT78A14 Regulates Flavonols Accumulation and Reactive Oxygen Species Scavenging in Response to Cold Stress in Camellia sinensis. Front Plant Sci 10 Suzuki N (2005) Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction. Physiol Plant 126(1):45–51 Song C, Gu L, Liu J, Zhao S, Hong X, Schulenburg K, Schwab W (2015) Functional Characterization and Substrate Promiscuity of UGT71 Glycosyltransferases from Strawberry (Fragaria x ananassa). Plant Cell Physiol 56(12):2478–2493 Chen X (2023) : BcMYB111 Responds to BcCBF2 and Induces Flavonol Biosynthesis to Enhance Tolerance under Cold Stress in Non-Heading Chinese Cabbage. Int J Mol Sci 24(Issue 10). Supplementary Files supplementaryfig1.pdf Supplementary Fig: Multiple sequence alignment of amino acid sequences of the identified Glycyrrrhiza glabra UGT, GgUGT72L11 (QHW04706.1), with 25 homologs from other plant species ACC38471.1 ( Medicago truncatula ), XP_039686479.1 ( Medicago truncatula ), KEH17970.1 ( Medicago truncatula ), XP_024629184.1 ( Medicago truncatula ), XP_013443945.2 ( Medicago truncatula ), AET01253.1 ( Medicago truncatula ), ( Glycyrrhiza glabra ), XP_013443944.2 ( Medicago truncatula ), XP_020230839.1 ( Cajanus cajan ), AGU14145.1 ( Cicer arietinum ), ACC38470.1 ( Medicago truncatula ), AKK25346.1 ( Lotus japonicas ), XP_003626779.1 ( Medicago truncatula ), XP_040873063.1 ( Glycine max ), XP_012567790.1 ( Cicer arietinum ), XP_003626784.2 ( Medicago truncatula ), QCE04782.1 ( Vigna unguiculata ), TKY68848.1 ( Spatholobus suberectus ), XP_003521043.1 ( Glycine max ), KYP40073.1 (Cajanus cajan), XP_006576739.1 ( Glycine max ), KAH1257819.1 ( Glycine max ), XP_014515709.1 ( Vigna radiata var. radiata ), XP_003532193.1 ( Glycine max ), TKY66296.1 ( Spatholobus suberectus ) and OAP00532.1 ( Arabidopsis thaliana ). supplementaryfig2.pdf Supplementary Fig2: Amino acid alignment of GgUGT72L11 biochemically characterized UGT from Glycyyrhiza glabra , UGT92G6 (XP_010650424.1), Vitis vinifera , caffeic acid 3-O-UGT from Arabidopsis thaliana (AtUGT71C1; AEC08300) and di/trisaccharide glycoside forming UGT from various plants. The GenBank accession numbers for the sequences are shown in parentheses: Monosaccharide glycoside forming GTs overdraw with orange colour: AcA3Gat (ADC34700); CsUGT85K11 (BAO51834); VvGt1 (gi|261260083); VvGT7a (XP_002276546); VvGT14 (XP_002285770); VvGT15 (XP_010650963 ); VvUGT85K14 (gi|225468660); VvUGT78A11 (CAN74919); VvUGT78A12 (BAI22847); VvgGT1 (gi|363805186); VvgGT2 (gi|363805188); VvgGT3 (gi|363805190); VlRSGT (gi|110932098) ;GmSap3Glu2‘‘Gat (D4Q9Z4); GmSap22A3’’’Gt (BAM29362); GmSap22A3‘‘Xt (BAM29363) Di/trisaccharide glycoside forming GTs overdraw with black colour AtF3GT2’’Xt (gi|75311632); AtF3G2’’GT (XP_002866013); BpA3G2’’Glt (Q5NTH0); CmF7G2’’Rt (gi|378405177); GmSap3Glu2’’Ga2’’’Rt (D4Q9Z5);; CrF3G6’’Gt (gi|242345159); CsiF7G6’’Rt (gi|75265643); CsTerG6’’Xt (BAO51835); GmF3G6’’Rt (BAN91401); GmF3G6’’GT (BAV56172); GmF3G2’’Gt (BAR88077); IpA3G2’’Gt (gi|62857206); PgGin3G2’’Gt (AKA44579); PhA3G6’’Rt (gi|397567); SiSes2G6’’Gt (BAF99027); SlPhe2G2’’X6’’’Gt (AGO03777). Gt, glucosyl-; Gat, galactosyl-; Rt, rhamnosyl-; Xt, xylosyl-; Glt, glucuronosyltransferase. Ac, Actinidia chinensis ; At, Arabidopsis thaliana ; Bp, Bellis perennis ; Cm, Citrus maxima ; Cr , Catharanthus roseus ; Cs, Camellia sinensis ; Csi, Citrus sinensis ; Gm, Glycine max , Ip, Ipomoea purpurea; Pg, Panax ginseng; Ph, Petunia hybrida ; Si, Sesamum indicum ; Sl, Solanum lycopersicum ; Vv, Vitis vinifera . entary supplementaryfig3.tif Supplementary Fig3: Predicted three dimensional model of UGT72L11 protein and Validation of 3D model. (a) 3-D structure of UGT72L11 (Ribbon model); (b) Ramachandran plot. Black, Dark Grey, Grey, Light Grey represents highly preferred conformations (Delta >=-2). White with Black Grid represents preferred conformations (-2> Delta >=-4). White with Grey Grid represents questionable conformations (Delta<-4). Highly preferred observations are shown in green crosses. Preferred observations are shown as brown triangles. Questionable observations are shown as red circles. Supplementaryfig.4.jpg Supplementary Fig4: Analysis of cis - regulatory elements of the promoter region in UGT72L11 Supplementary data1: LC-MS/HRMS chromatograms. Arrows indicate the excepted position of glycosylation by UGT72L11. Enzymatic reaction constitutes the 100Mm Tris HCL buffer pH 7.5, 0.5mM substrate, 5mM UDP glucose ,50 µg protein reaction incubated at 30 ºC for 1 hour . The reaction was stopped by the addition of 500 µl of ethyl acetate concentrated and analyzed using LCMS/HRMS. SUPTABLE.docx Supplementary Table1: Details of PCR primers SupportingData1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 27 Aug, 2024 Reviewers invited by journal 15 Mar, 2024 Editor assigned by journal 22 Feb, 2024 First submitted to journal 15 Feb, 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3981251","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280012591,"identity":"854d1928-c26f-44a1-9800-f272c3e60c01","order_by":0,"name":"Shahnawaz Hussain","email":"","orcid":"","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shahnawaz","middleName":"","lastName":"Hussain","suffix":""},{"id":280012592,"identity":"dde4cd6f-5f09-4434-a6cf-e168a127f4d4","order_by":1,"name":"Bhawna Verma","email":"","orcid":"","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bhawna","middleName":"","lastName":"Verma","suffix":""},{"id":280012593,"identity":"7ee0353b-5864-4ecc-a7b6-6f04bef60e4b","order_by":2,"name":"Malik Muzafar Manzoor","email":"","orcid":"","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Malik","middleName":"Muzafar","lastName":"Manzoor","suffix":""},{"id":280012594,"identity":"9a585bbc-9d1f-48bc-9c1b-09a0071f544f","order_by":3,"name":"Pooja Goyal","email":"","orcid":"","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pooja","middleName":"","lastName":"Goyal","suffix":""},{"id":280012595,"identity":"910f7c04-e2d8-468f-8f60-a0c768bc6e1a","order_by":4,"name":"Ritu Devi","email":"","orcid":"","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ritu","middleName":"","lastName":"Devi","suffix":""},{"id":280012596,"identity":"93212246-af87-402d-801c-e7291efd17fe","order_by":5,"name":"Ajai Prakash Gupta","email":"","orcid":"","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ajai","middleName":"Prakash","lastName":"Gupta","suffix":""},{"id":280012597,"identity":"2ff9c46f-1aef-4bd7-a62e-b6dcc3fe881e","order_by":6,"name":"Manoj kumar Dhar","email":"","orcid":"","institution":"University of Jammu","correspondingAuthor":false,"prefix":"","firstName":"Manoj","middleName":"kumar","lastName":"Dhar","suffix":""},{"id":280012598,"identity":"77a8987c-cacc-4ac4-ab41-e91473a26c37","order_by":7,"name":"Fariha chowdhary","email":"","orcid":"","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fariha","middleName":"","lastName":"chowdhary","suffix":""},{"id":280012599,"identity":"d7731582-73db-4379-8d03-df6d092e4143","order_by":8,"name":"Suphla Gupta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYPACGx42ZiiTjSgNBxjSSNdymAQXyfevMXv8oea8DB87dwJzRUUdA590A34tBjfemBscOHYb6DDeDYxnzhxmYJM5QECLxBkziQNsUC2NbQcY2CQSCDhsBkjLv3NQLf/qCGthON9jJnGw7QBUSwMzYS0GN9jKDc72JYO1HGw4dpiHsMP6D297UPHNzl6+/+zGhw01dXLyMwg5TCIBEXcHgJiHgHog4D9AXHSPglEwCkbBCAYAgrU7ffJ+lqEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6688-5575","institution":"CSIR-Indian Institute of Integrative Medicine: Council of Scientific \u0026 Industrial Research Indian Institute of Integrative Medicine","correspondingAuthor":true,"prefix":"","firstName":"Suphla","middleName":"","lastName":"Gupta","suffix":""}],"badges":[],"createdAt":"2024-02-23 09:01:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3981251/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3981251/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52956869,"identity":"1d2f71df-fcad-4ccf-b9c5-513b6e66eecb","added_by":"auto","created_at":"2024-03-19 05:32:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":328381,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree and MEME of deduced amino acid sequence of GgUGT72L11 (pink color) along with 25 UGT proteins of UGT72 subfamily identified from other plant species (green color) displaying homology with UGT72L1 (ACC38470) epicatechin 3-glucosyltransferase from Medicago truncatula. GenBank accession numbers for the sequences are shown in parentheses: GgUGT72L11 (QHW04706.1) Hp_UGT72B11(ACB56923.1); UGT728B2 (AAL06646.2); UGT727A5(BAD52007.1); UGT725D3 (BAG32255.1); UGT724B2 (AAK53551.1); UGT723F9 (ACM66950.1); UGT723A3 (BAD06874.1); UGT721B8 (Q8S3B6); UGT720D4 (ABA18631.1); UGT720A5 (CAA50376.1); UGT72Z3 (XP_003532192.3); UGT72X5( QDM38910.1); UGT72X4 (XP_003532193.1); UGT72L1 (ACC38470); UGT72G15 (NpUGT6); UGT72G4 (MK644229.1); UGT72E8 (IiUGT1); UGT72E2 (NP_201470.1); UGT72C1 (CAB16822.1); UGT72BD1 (QHB92369.1); UGT72B42 (QDM38905.1); UGT72B29 (5NLM); UGT72B17 (6JEM); UGT72B14 (ACD87062.1); UGT72B1( 2VCE); Pt_UGT72B29(AUO17143.1).\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/a832a623c8d083b0ac7c0698.png"},{"id":52957468,"identity":"0bb4e236-0497-4df3-a226-ae7c1cdd4369","added_by":"auto","created_at":"2024-03-19 05:40:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":166102,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of mono- and disaccharide glycoside producing UGTs with UGT72L11. The GenBank accession numbers for the sequences are shown in parentheses: \u003cstrong\u003eMonosaccharide glycoside\u003c/strong\u003e-forming glycosyltransferases (marked with blue colour): AcA3Gat (ADC34700);CsUGT85K11 (BAO51834); VvGt1 (gij261260083); VvGT7a (XP_002276546); VvGT14 (XP_002285770); VvGT15 (XP_010650963);VvUGT85K14 (gij225468660); VvUGT78A11 (CAN74919); VvUGT78A12 (BAI22847); VvgGT1 (gij363805186); VvgGT2 (gij363805188);VvgGT3 (gij363805190); VlRSGT (gij110932098); AtUGT71C1(AEC08300) and UGT92G6 (XP_010650424.1)GmSap3Glu2’’Gat (D4Q9Z4); GmSap22A3 Gt(BAM29362); GmSap22A3Xt (BAM29363); \u003cstrong\u003edi-/trisaccharide glycoside\u003c/strong\u003e-forming glycosyltransferases marked with pink colour: AtF3GT2’’Xt (gij75311632); AtF3G2’’GT (XP_002866013); BpA3G2’’Glt (Q5NTH0); CmF7G2’’Rt (gij378405177); GmSap3Glu2’’Ga2’’’Rt (D4Q9Z5); CrF3G6’’Gt (gij242345159); CsiF7G6’’Rt(gij75265643); CsTerG6’’Xt (BAO51835); GmF3G6’’Rt (BAN91401); GmF3G6’’Gt (BAV56172); GmF3G2’’Gt (BAR88077); IpA3G2’’Gt (gij62857206); PgGin3G2’’Gt (AKA44579); PhA3G6’’Rt (gij397567); SiSes2G6’’Gt (BAF99027); SlPhe2G2’’X6’’’Gt (AGO03777)\u003cem\u003e.\u003c/em\u003eGt, glucosyl-; Gat,galactosyl-; Rt, rhamnosyl-; Xt, xylosyl-; Glt, glucuronosyltransferase. Ac, \u003cem\u003eActinidia chinensis\u003c/em\u003e; At, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e; Bp, \u003cem\u003eBellis perennis\u003c/em\u003e; Cm, \u003cem\u003eCitrus maxima\u003c/em\u003e; Cr, \u003cem\u003eCatharanthus roseus\u003c/em\u003e; Cs, \u003cem\u003eCamellia sinensis\u003c/em\u003e; Csi, \u003cem\u003eCitrus sinensis\u003c/em\u003e; Gm, \u003cem\u003eGlycinemax\u003c/em\u003e, Ip, \u003cem\u003eIpomoea purpurea\u003c/em\u003e; Pg, \u003cem\u003ePanax ginseng\u003c/em\u003e; Ph, \u003cem\u003ePetunia hybrida\u003c/em\u003e; Si, \u003cem\u003eSesamum indicum\u003c/em\u003e; Sl, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e; Vv, \u003cem\u003eVitis vinifera\u003c/em\u003e. The bar represents 0.10 amino acid substitutions per site.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/c6288f881c35b58ab5558587.png"},{"id":52956871,"identity":"ffb33cc6-a6d6-4cd1-8fa6-1a6d3877b342","added_by":"auto","created_at":"2024-03-19 05:32:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":129693,"visible":true,"origin":"","legend":"\u003cp\u003eBinding conformation and interaction of substrate (kaempferol) with receptor protein (GgUGT72L11). (a) Ribbon 3D model showing binding orientation of ligand (Kaempferol) in the active site of receptor (GgUGT72L11); (b) Showing interactions of ligand (kaempferol) with amino acid residues of ligand GgUGT72L11.\u003c/p\u003e","description":"","filename":"FIG.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/e4391fe941cb5f348f165131.png"},{"id":52956875,"identity":"3e69d36c-022a-4db9-afeb-12c84d590387","added_by":"auto","created_at":"2024-03-19 05:32:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":327405,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization of transiently expressed UGT72L11 in onion epidermal cells using GFP tagging . The transformed onion cells were obtained after introduction of 35S:ugt72l11-GFP through the \u003cem\u003eAgrobacterium\u003c/em\u003e- mediated transformation. (a) Images of cells in white light (b) in fluorescence photographed by confocal microscopy.\u003c/p\u003e","description":"","filename":"FIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/dc0855c343c0c3d3d82ce49e.png"},{"id":52956879,"identity":"ef42ca00-ba89-4ffa-8081-08d0b8a02d62","added_by":"auto","created_at":"2024-03-19 05:32:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":432672,"visible":true,"origin":"","legend":"\u003cp\u003e(A): SDS-PAGE electrophoresis of prokaryotic expression of pET28a-UGT72L11. Isopropyl-β-D-thiogalactopyranoside (IPTG) of the gene in various timeline. Lane a-3hr; Lane b-6 hr; Lane c-12hr; Lane d-20hr ; Lane e- 24hr ; Lane f- protein marker; Lane g- control without isopropyl-β-D-thiogalactopyranoside; arrow indicates maximum induction of the fused protein at 20 hrs; (5B) SDS-PAGE electrophoresis of prokaryotic expression of eluted pET28a-UGT72L11; Lane5-8,flow through; lane10-lane14- eluted protein having approximately 52kDa of molecular weight\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/feb4762fc26c5e3105ff8ab3.png"},{"id":52956872,"identity":"bc7a6725-2097-432a-9549-feac04485290","added_by":"auto","created_at":"2024-03-19 05:32:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":124342,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of various parameters (X axis) with respect to UDP concentration (nm) using purified recombinant GgUGTL11 protein; (a) buffers/pH -C/3, C/4, C/5, C/6 in citrate buffer; P/6, P/7.5 in phosphate buffer; and T/7.5, T/8.5, T/9.5 and T/10.5 in tris-HCl buffer; (b) various time intervals (in minutes) 15,30,45,60 \u0026amp; 90; (c) different temperature (ºC) at 4,10, 22, 30, 37, 50 \u0026amp; 70; and (d) kaempferol substrate (in µm) at 3,5,7,9 concentrations.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/545681b8da39920a982d92fb.png"},{"id":52956873,"identity":"c62ff4dc-7391-47da-9918-5219e4b9b4e5","added_by":"auto","created_at":"2024-03-19 05:32:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":194804,"visible":true,"origin":"","legend":"\u003cp\u003e(1) HPLC/LCMS data of (a) standard kaempferol evidence peak with molecular weight 285 and RT value 53.4; (b) standard kaempferol 3-O-glucoside evidence peak with molecular weight 447 and retention time value 40.7; (c1) UGT72L11 enzymatic reaction peaks (c2): extraction of peak from (c)1 at molecular weight 447 confirmed peak with retention time value 40.9 of kaempferol 3-O-glucoside; (c3) extraction of peak from (c1) at molecular weight 285 confirmed peak with retention time value 53.7 of kaempferol as in standard. (2) HPLC LC-MS analysis of UGT72L11 enzymatic reaction using quercetin as a substrate. (a) total ion chromatograph (TIC) of the standards in positive/negative-ion mode showing peaks at RT of 12.5 and 9.4 mins for quercetin and quercetin 3-oglucoside respectively; (b) control reactions (without protein) showing the base peak intensity (BPI) chromatogram in positive/negative-ion mode at RT of 12.5 min; (c) \u003cem\u003ein-vitro\u003c/em\u003e enzymatic reaction with protein at the base peak intensity in positive/negative-ion mode showing peaks at retention times of 12.5 and 9.4 min; (d) peaks in the extracted ion chromatogram at a mass/charge ratio (m/z) of 300.8[M- H]− and 462.9 [M-H]− corresponding to quercetin and quercetin 3-0 glucoside, respectively\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/3baf0195d1d9d053aca338e9.png"},{"id":52957471,"identity":"06c48366-a527-4a3a-bdcd-29c5f6134f2e","added_by":"auto","created_at":"2024-03-19 05:40:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":98536,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of substrates used in the study (flavanols, terpenes, carboxylic acids, vitamins, alkaloid, phenolics and commercial drugs) for glycosylation.\u003c/p\u003e","description":"","filename":"fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/9d162b22450cba89f7c380db.png"},{"id":52956881,"identity":"660d3f9f-bf7a-4b3a-a54d-bdcdfdf87181","added_by":"auto","created_at":"2024-03-19 05:32:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":51649,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative expression level of\u0026nbsp;\u003cem\u003eGgUGT72L11\u003c/em\u003e by qRT-PCR. (a) tissue-specific expression under three conditions (tissue culture, hardening unit and field); (b) abiotic stress (NaCl , mannitol , UV, carbon starvation, dark, cold senescence and wounding); (c) pathogens(P1 -\u003cem\u003e Colletotrichum gloeosporioides\u003c/em\u003e , P2-\u003cem\u003e Colletotrichum siamense.subsp\u003c/em\u003e, P3-\u003cem\u003eColletotrichum siamense sub sp. and\u003c/em\u003e\u0026nbsp; P4- \u003cem\u003eCalonectria cylindrospora)\u003c/em\u003e (d) hormonal treatments (NAA, ABA \u0026amp; GA\u003csub\u003e3\u003c/sub\u003e).\u0026nbsp; The y – axis indicates relative expression value\u0026nbsp; and x-axis indicate the control and treated\u0026nbsp; shoot tissue under different conditions/stress,. Three biological replicates were used to calculate error bars using standard deviation. Asterisks indicate the statistical significance of observed values when compared with control [(*) p value ≤0.05, (**) p value≤ 0.01, (***) p value≤ 0.001] calculated by One -way ANNOVA analysis in Graph pad prism 8.0.\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/e8e7db8aae2ef6e7aff1780b.png"},{"id":52957472,"identity":"858a7050-07a0-4279-bd82-b63fb6f61163","added_by":"auto","created_at":"2024-03-19 05:40:29","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":33801,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative expression levels of Reactive Oxygen Species scavenging genes catalase\u003cem\u003e (CAT1), \u003c/em\u003esuper oxide dismutase (\u003cem\u003eSOD)\u003c/em\u003e, Glutathione Peroxidase\u003cem\u003e (GPX\u003c/em\u003e) and cold –responsive genes (\u003cem\u003eCOR47, ABI5, DREB1\u003c/em\u003e) \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026nbsp;by qRT-PCR. (A) 64hr cold treated shoot; (B) 32 hr ABA treated shoot. The y – axis indicates relative expression value and x-axis indicate the control and treated shoot tissue under stress. Three biological replicates were used to calculate error bars using standard deviation. Asterisks indicate the statistical significance of observed values [(*) p value ≤0.05, (**) p value≤ 0.01, (***) p value≤ 0.001] calculated by One way ANNOVA analysis in Graph pad prism 8.0\u003c/p\u003e","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/84d56d6c7b071d7cddf3a147.png"},{"id":52958247,"identity":"d99f2b7a-1e2d-410f-b6f6-061a4d39671f","added_by":"auto","created_at":"2024-03-19 05:48:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2751669,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/22519a00-e185-463c-be61-5f7dff44ac49.pdf"},{"id":52956882,"identity":"5563d736-28bd-4f74-8d40-01669c1d43fa","added_by":"auto","created_at":"2024-03-19 05:32:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3237602,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig: Multiple sequence alignment of amino acid sequences of the identified \u003cem\u003eGlycyrrrhiza glabra\u003c/em\u003e UGT, GgUGT72L11 (QHW04706.1), with 25 homologs from other plant species ACC38471.1 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), XP_039686479.1 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), KEH17970.1 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), XP_024629184.1 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), XP_013443945.2 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), AET01253.1 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), (\u003cem\u003eGlycyrrhiza glabra\u003c/em\u003e), XP_013443944.2 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), XP_020230839.1 (\u003cem\u003eCajanus cajan\u003c/em\u003e), AGU14145.1 (\u003cem\u003eCicer arietinum\u003c/em\u003e), ACC38470.1 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), AKK25346.1 (\u003cem\u003eLotus japonicas\u003c/em\u003e), XP_003626779.1 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), XP_040873063.1 (\u003cem\u003eGlycine max\u003c/em\u003e), XP_012567790.1 (\u003cem\u003eCicer arietinum\u003c/em\u003e), XP_003626784.2 (\u003cem\u003eMedicago truncatula\u003c/em\u003e), QCE04782.1 (\u003cem\u003eVigna unguiculata\u003c/em\u003e), TKY68848.1 (\u003cem\u003eSpatholobus suberectus\u003c/em\u003e), XP_003521043.1 (\u003cem\u003eGlycine max\u003c/em\u003e), KYP40073.1 (Cajanus cajan), XP_006576739.1 (\u003cem\u003eGlycine max\u003c/em\u003e), KAH1257819.1 (\u003cem\u003eGlycine max\u003c/em\u003e), XP_014515709.1 (\u003cem\u003eVigna radiata var. radiata\u003c/em\u003e), XP_003532193.1 (\u003cem\u003eGlycine max\u003c/em\u003e), TKY66296.1 (\u003cem\u003eSpatholobus suberectus\u003c/em\u003e) and OAP00532.1 (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"supplementaryfig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/ff6c12e6e63c0156f2b95098.pdf"},{"id":52957470,"identity":"735d8ec4-457d-414c-8ae5-169d3bea7a7f","added_by":"auto","created_at":"2024-03-19 05:40:29","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5115032,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig2: Amino acid alignment of GgUGT72L11 biochemically characterized UGT from \u003cem\u003eGlycyyrhiza glabra\u003c/em\u003e, UGT92G6 (XP_010650424.1), \u003cem\u003eVitis vinifera\u003c/em\u003e, caffeic acid 3-O-UGT from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AtUGT71C1; AEC08300) and di/trisaccharide glycoside forming UGT from various plants. The GenBank accession numbers for the sequences are shown in parentheses: Monosaccharide glycoside forming GTs overdraw with orange colour: AcA3Gat (ADC34700); CsUGT85K11 (BAO51834); VvGt1 (gi|261260083); VvGT7a (XP_002276546); VvGT14 (XP_002285770); VvGT15 (XP_010650963 ); VvUGT85K14 (gi|225468660); VvUGT78A11 (CAN74919); VvUGT78A12 (BAI22847); VvgGT1 (gi|363805186); VvgGT2 (gi|363805188); VvgGT3 (gi|363805190); VlRSGT (gi|110932098) ;GmSap3Glu2‘‘Gat (D4Q9Z4); GmSap22A3’’’Gt (BAM29362); GmSap22A3‘‘Xt (BAM29363) Di/trisaccharide glycoside forming GTs overdraw with black colour AtF3GT2’’Xt (gi|75311632); AtF3G2’’GT (XP_002866013); BpA3G2’’Glt (Q5NTH0); CmF7G2’’Rt (gi|378405177); GmSap3Glu2’’Ga2’’’Rt (D4Q9Z5);; CrF3G6’’Gt (gi|242345159); CsiF7G6’’Rt (gi|75265643); CsTerG6’’Xt (BAO51835); GmF3G6’’Rt (BAN91401); GmF3G6’’GT (BAV56172); GmF3G2’’Gt (BAR88077); IpA3G2’’Gt (gi|62857206); PgGin3G2’’Gt (AKA44579); PhA3G6’’Rt (gi|397567); SiSes2G6’’Gt (BAF99027); SlPhe2G2’’X6’’’Gt (AGO03777). Gt, glucosyl-; Gat, galactosyl-; Rt, rhamnosyl-; Xt, xylosyl-; Glt, glucuronosyltransferase. Ac, \u003cem\u003eActinidia chinensis\u003c/em\u003e; At, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e; Bp, \u003cem\u003eBellis perennis\u003c/em\u003e; Cm, \u003cem\u003eCitrus maxima\u003c/em\u003e; Cr\u003cem\u003e, Catharanthus roseus\u003c/em\u003e; Cs, \u003cem\u003eCamellia sinensis\u003c/em\u003e; Csi, \u003cem\u003eCitrus sinensis\u003c/em\u003e; Gm, \u003cem\u003eGlycine\u003c/em\u003e \u003cem\u003emax\u003c/em\u003e, Ip, Ipomoea purpurea; Pg, Panax ginseng; Ph, \u003cem\u003ePetunia hybrida\u003c/em\u003e; Si, \u003cem\u003eSesamum indicum\u003c/em\u003e; Sl, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e; Vv, \u003cem\u003eVitis vinifera\u003c/em\u003e. entary\u003c/p\u003e","description":"","filename":"supplementaryfig2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/242f0bda1ecd92e9f37d9e16.pdf"},{"id":52956877,"identity":"dbdd2c45-0e74-4acd-8db3-01dcaad2aa01","added_by":"auto","created_at":"2024-03-19 05:32:29","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":106760,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig3: Predicted three dimensional model of UGT72L11 protein and Validation of 3D model. (a) 3-D structure of UGT72L11 (Ribbon model); \u0026nbsp;(b) Ramachandran plot. Black, Dark Grey, Grey, Light Grey represents highly preferred conformations (Delta \u0026gt;=-2). White with Black Grid represents preferred conformations (-2\u0026gt; Delta \u0026gt;=-4). White with Grey Grid represents questionable conformations (Delta\u0026lt;-4). Highly preferred observations are shown in green crosses. Preferred observations are shown as brown triangles. Questionable observations are shown as red circles.\u003c/p\u003e","description":"","filename":"supplementaryfig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/202d3b4b9866c4cc5b6ea1ba.tif"},{"id":52956884,"identity":"6ff3ca31-1532-4e1a-9b10-de143091feb5","added_by":"auto","created_at":"2024-03-19 05:32:29","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":130248,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig4: Analysis of \u003cem\u003ecis\u003c/em\u003e- regulatory elements of the promoter region in UGT72L11\u003c/p\u003e\n\u003cp\u003eSupplementary data1: LC-MS/HRMS chromatograms. Arrows indicate the excepted position of glycosylation by UGT72L11. Enzymatic reaction constitutes the 100Mm Tris HCL buffer pH 7.5, 0.5mM substrate, 5mM UDP glucose ,50 µg protein reaction incubated at 30 ºC for 1 hour . The reaction was stopped by the addition of 500 µl of ethyl acetate concentrated and analyzed using LCMS/HRMS.\u003c/p\u003e","description":"","filename":"Supplementaryfig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/04a05b4325106adb23371e93.jpg"},{"id":52957469,"identity":"ee0ab6c7-63f8-478a-81f2-38dc1c2e95ed","added_by":"auto","created_at":"2024-03-19 05:40:28","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11995,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table1: Details of PCR primers\u003c/p\u003e","description":"","filename":"SUPTABLE.docx","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/74e5e603dea4b528ed3990a3.docx"},{"id":52956885,"identity":"ca6103b2-033e-448e-85d0-cf050b336c68","added_by":"auto","created_at":"2024-03-19 05:32:30","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2527564,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingData1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3981251/v1/7d6961704252f777c8836a6f.pdf"}],"financialInterests":"","formattedTitle":"Identification and Characterization of Abiotic Stress Induced novel UDP-Glucosyltransferase (UGT72L11) Gene from Glycyrrhiza glabra L.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlant UDP dependent glycosyltransferases (UGTs) belongs to a large multi-gene superfamily of glucosyltransferases (GTs) that catalyses the transfer of UDP-activated sugars to diverse set of aglycons forming corresponding glycol-conjugates [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] as observed in secondary metabolites, hormones [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], xenobiotics [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and several other small molecules. The glycosylation of secondary metabolites effectively regulate their properties and functions imparting altered characteristics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Hence the chemical modification plays critical role in the response to plant growth and adaptation to biotic and abiotic stresses. Studies have highlighted role of plant UGTs in stress management. The rice \u003cem\u003eUGT85E1\u003c/em\u003e has been demonstrated to mediate plant response to drought and oxidative stresses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, ectopic over expression of \u003cem\u003eUGT76E11\u003c/em\u003e from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e showed enhanced tolerance to salinity and drought [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. On the other hand, silencing of \u003cem\u003eUGT75C1\u003c/em\u003e from tomato played crucial role in ABA-mediated fruit ripening, seed germination, and drought responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Higher accumulation of glycosylated specialized metabolites modified by UGTs have shown to be involved in drought and salt stress tolerance and higher oxidative stress management in plants (Li et al., 2018c, 2018d; Gharibi et al., 2019). Chill induced expression of \u003cem\u003eOsUGT90A1\u003c/em\u003e from \u003cem\u003eOryza sativa\u003c/em\u003e imparted tolerance to low temperature through cell membrane integrity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Glycosyltransferases have shown specialized metabolites modulations. The UGTs like \u003cem\u003eCrUGT87A\u003c/em\u003e, identified from \u003cem\u003eCarex rigescens\u003c/em\u003e exhibited increased flavonoid accumulation and salt stress resistance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and \u003cem\u003eCsUGT78A15\u003c/em\u003efrom \u003cem\u003eCamellia sinensis\u003c/em\u003e was shown to be involved in biosynthesis of eugenol glucoside under cold stress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] as was also demonstrated in \u003cem\u003eA. thaliana AtUGT87A2\u003c/em\u003e associated with salinity, osmotic stress, drought and ABA [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Multiple stress inducible command further insight into the response mechanism in plants to understand the interplay between the UGT catalyzed glycosylation under the influence of stress, and specialized metabolite accumulation. The crystal structure of the di-C-glycosyltransferase from \u003cem\u003eG.glabra\u003c/em\u003e catalyzing a two-step di-C-glycosylation of flopropione-containing substrates revealed the role of hydrogen-bond interactions of sugar hydroxyl groups in sugar donor selectivity and space-chemistry in di-C-glycosylation capability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This finding has immense application in developing efficient biocatalysts to synthesize C-glycosides of medicinal potential. Understanding of the intricate interplay between UGTs in plants is needed to unlock the full potential of UGTs in plant adaptive behaviour under climate change scenario and also as a prospective catalytic tool.\u003c/p\u003e \u003cp\u003eIn recent years putative UGT genes have been identified from several plant species, but their functional characterization is equitably small, coming predominantly from studies conducted on \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Further, only a few members of the UGT72 subfamily have been characterized from Fabaceae as in the seed coat-specific UGT72L1 from \u003cem\u003eM. truncatula\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and UGT72L6 from \u003cem\u003eLotus. japonicas\u003c/em\u003e (KT895087). In the Glycyrrhiza genus, literature reports few UGTs identified and characterized from \u003cem\u003eG.uralensis\u003c/em\u003e species for terpenoids [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], saponin [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and flavonoid [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] biosyntheses. A recent study on metabolic profiling in \u003cem\u003eG. uralensis\u003c/em\u003e identified few key differentially expressed UGT genes co-regulating flavonoid and saponin biosyntheses in licorice under salt stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study mined a novel \u003cem\u003eUGT\u003c/em\u003e gene from the transcriptome resource of \u003cem\u003eG. glabra\u003c/em\u003e species having wide-substrate acceptability and involvement in multiple stresses. The purified recombinant protein was enzymatically characterized and \u003cem\u003ein-planta\u003c/em\u003e assessed. Additionally, the isolated recombinant protein was studied for various kinetic parameters and \u003cem\u003ein-vitro\u003c/em\u003e evaluated for substrate promiscuity utilizing diverse class of aglycons.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material\u003c/h2\u003e \u003cp\u003ePlant material was collected from the experimental farm of the Council for Scientific and Industrial Research-Indian Institute of Integrative Medicine Jammu (32.73\u0026ordm;N and 74.87\u0026ordm;E) for RNA extraction and as explants for tissue culture. The regenerated plants were used throughout the experiments for various treatments. All tissues were frozen in liquid nitrogen (N) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Chemicals\u003c/h2\u003e \u003cp\u003eReagents used in the study were purchased from Takara Bio-USA, Promega, Thermofisher and Thermoscientific. Substrates and Isopropyl \u0026szlig;-D-1-thiogalactopyranoside (IPTG) for enzyme assay were purchased from Sigma-Aldrich (Oakville, CA, USA). All the chemicals used in this study were of molecular/analytical/HPLC/MS grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Retrieval of UGTs from the transcriptomic data\u003c/h2\u003e \u003cp\u003eTranscriptome data (PRJNA664636) generated through Next Generation Sequencing (NGS) of \u003cem\u003eG.glabra\u003c/em\u003e plant was used as resource for the identification of UGT gene from the species using the Hidden Markov Model (HMM) profile (Pfam family: PF00201). The local similarity search (tBLASTN) was performed for the mining of \u003cem\u003eGgUGTs\u003c/em\u003e using the BioEdit with an E-value cut off of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. The obtained contigs were further filtered by ORF finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) tool to select full-length sequences and translated by ExPasy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) tool. The molecular weight (MW), theoretical isoelectric point (pI), instability index, aliphatic index, grand average of hydropathicity (GRAVY) were predicted \u003cem\u003evia\u003c/em\u003e the ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Additionally, sub-cellular localisation prediction was performed by an advanced protein sub-cellular localisation prediction tool CELLO2GO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/cello2go/\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw/cello2go/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Sequence alignment and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe protein sequence of the identified UGTs were aligned with the sequences of the functionally characterized proteins from other plant species of the same subfamily, and proteins having mono- and disaccharide glycoside forming activities [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Multiple sequence alignment (MSA) of the deduced amino acid sequences was carried out using Clustal omega [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and DNAMAN(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dnaman.software.informer.com/7.0/\u003c/span\u003e\u003cspan address=\"https://dnaman.software.informer.com/7.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To construct the phylogenetic tree, amino acid sequences were aligned using the BLOSUM62 matrix with the ClustalW algorithm-based AlignX module from Mega MEGA (Ver7.0) to generate a neighbour joining tree with bootstrapping (1000 replicates) analysis and handling gaps with pair wise deletion. The mono/di-glucoside-based categorization was done by the Geneious alignment tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneious.com/\u003c/span\u003e\u003cspan address=\"https://www.geneious.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to construct phylogenetic tree using neighbour joining method in default parameters of the Geneious program. The conserved motifs of the UGTs were predicted using the Multiple Expectation Maximization for Motif Elicitation (MEME:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) tool having maximum ten motifs with minimum 6 and maximum 50 motif width.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 In-silico 3D model construction\u003c/h2\u003e \u003cp\u003eThe three-dimensional protein structure of UGT72L11 protein was predicted by I-TASSER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zhanglab.ccmb.med.umich.edu/I-TASSER/\u003c/span\u003e\u003cspan address=\"https://zhanglab.ccmb.med.umich.edu/I-TASSER/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and validated by Ramachandran plot. Ligand binding site was predicted using 3DLigandSite [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The ligand structure of kaempferol was downloaded from PubChem Compound Database (National Centre for Biotechnology Information; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/).Fo\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/).Fo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003er primary molecular docking, visualization and modifying receptor and ligand structures the DockThor-VS web online server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dockthor.lncc.br/v2/\u003c/span\u003e\u003cspan address=\"https://dockthor.lncc.br/v2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used (Discovery Studio Biovia 2017; Dassault Syst\u0026egrave;mes, San Diego, California, USA). Discovery Studio Biovia 2017 was also used for post-docking analyses, like prediction of the size and location of binding site, hydrogen-bond interactions, hydrophobic interactions, and bonding distances.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cellular localisation:\u003c/h2\u003e \u003cp\u003eSub-cellular localisation of the gene was performed in onion cells using \u003cem\u003eAgrobacterium\u003c/em\u003e mediated transformation having 35S:\u003cem\u003eUGT72L11\u003c/em\u003e-GFP construct in PCAMBIA1302 vector following the published protocol [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, healthy and fresh onion scales (1\u0026ndash;1.5\u0026times;1 cm) were placed on a petridish with re suspension of \u003cem\u003eAgrobacterium\u003c/em\u003e solution (OD600) having sucrose, acetosyringone and Silwet-for 24 hours at 28\u003csup\u003e◦\u003c/sup\u003eC. Subsequently, onion scales were co-cultivated with \u003cem\u003eAgrobacterium\u003c/em\u003e containing \u0026frac12; strength Murashige and Skoog basal medium. GFP imaging of transient expression was visualised under confocal fluorescence microscope (Olympus FLUOVIEW FV1000).For GFP detection, the excitation source was an argon-ion laser at 488 nm, and emission was observed between 510 and 530 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA isolation, gene and promoter cloning\u003c/h2\u003e \u003cp\u003eTotal RNA having purity between 1.9\u0026ndash;2.1 (A260/280) was isolated using Pure Link RNA Isolation kit (Thermofisher). Extracted RNA (2 \u0026micro;g) was reverse-transcribed to cDNA using primeScriptIII First-Strand cDNA kit (Thermofisher). The full-length amplification of the \u003cem\u003eUGT72L11\u003c/em\u003egene was performed using gene specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The amplified gene was cloned into pJET1.2 (ThermoScientific, USA) cloning vector, confirmed by Sanger sequencing and subsequently submitted to NCBI Data Bank. The corresponding amino acid sequence encoding the candidate UGT was protein-BLAST searched for homology using NCBI search tools [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For promoter cloning, the 5\u0026rsquo; upstream region of the identified UGT was cloned using Genome Walker kit (TAKARA, Japan) following the instruction manual. The amplified promoter region was scanned for the presence of various cis-regulatory elements using Plant Cis-acting Regulatory DNA Elements database (PLACE, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.dna.affrc.go.jp/PLACE/\u003c/span\u003e\u003cspan address=\"http://www.dna.affrc.go.jp/PLACE/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Real-time expression analysis\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Treatments\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein-vitro\u003c/em\u003e regenerated plants grown under three different environmental conditions namely, tissue culture, hardening unit and field conditions were assessed for the expression of the identified gene. The \u003cem\u003eG. glabra\u003c/em\u003e plants were also subjected to one biotic stress, eight abiotic stress treatments and three hormone based elicitations. The treatments were essentially performed following the standardized protocol published earlier [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Each treatment along with control was given to a set of three \u003cem\u003ein-vitro\u003c/em\u003e plants (2 months old) for different time durations. Abiotic treatments like dark and carbon starvation were subjected for 48 hrs; cold (4\u0026deg;C) for 64 hrs; wounding for 8 hrs; UV-C for 30 min, salinity for 128 hrs and drought for 4 days. For the senescence treatment, green and yellow leaves of the \u003cem\u003ein-vitro\u003c/em\u003e plants were used. For biotic elicitation 3 pathogenic isolates of \u003cem\u003eColletotrichum\u003c/em\u003e sp. and one \u003cem\u003eCalonectria\u003c/em\u003e sp. were employed. The hormone treatment was subjected for various time duration; NAA was given for 2 hrs, GA\u003csub\u003e3\u003c/sub\u003e for 6 hrs and ABA for 16 hrs to the \u003cem\u003ein-vitro\u003c/em\u003e grown plants. RNA extraction and cDNA synthesis from the tissues of the control and treated plants, and plants grown under three different conditions (\u003cem\u003ein-vitro\u003c/em\u003e, field and glass house) were performed as mentioned earlier.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Expression Analysis\u003c/h2\u003e \u003cp\u003eThe expression of \u003cem\u003eUGT72L11\u003c/em\u003e transcripts in the aerial tissues of \u003cem\u003eG. glabra\u003c/em\u003e plant were examined by quantitative reverse-transcription PCR following the thermal profile published earlier using \u003cem\u003eβ-actin\u003c/em\u003e gene as an internal control [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The primers for quantitative real-time (qRT)-PCR are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. qRT-PCR was performed on StepOne\u0026trade; (Applied Biosystems) using SYBR premix ExTaq II (Clontech) following instructions of the manufacturer. The relative expression level was calculated by the 2\u003csup\u003e-ΔΔct\u003c/sup\u003e method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For each gene, three independent experiments were performed, and three technical replicates were analyzed for each sample [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Statistical analysis was performed using Graph Pad prism software/EXCEL sheet.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Heterologous expression and protein purification\u003c/h2\u003e \u003cp\u003eThe full-length gene in pJET1.2 (ThermoScientific, USA) cloning vector was amplified using unique \u003cem\u003eNcoI\u003c/em\u003e and \u003cem\u003eXhoI\u003c/em\u003e site specific forward and reverse primers, respectively. The amplified gene was digested with respective restriction enzymes and sub-cloned into previously digested pET28a(+) vector with the same set of enzymes. The resultant construct was transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) cells and the transformed colonies were screened on the Kanamycin (50mg/ml) supplemented plates and confirmed by colony PCR. PCR amplification was performed using gene specific forward/reverse primers (supplementary tabl\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003ee1\u003c/span\u003e) and T7 promoter/terminator primer combination using the plasmid extracted from the positive colony for the conformity of the transformation. For the recombinant protein expression and purification, the confirmed transformant was inoculated into 5 ml Luria Bertani (LB) containing appropriate antibiotic and incubated at 37\u0026ordm;C overnight. Subsequently, the primary inoculum (1%) was added to 200 ml fresh LB media and incubated similarly, until its OD\u003csub\u003e600\u003c/sub\u003e reached a value of 0.6. The cloned gene was induced with the optimized conditions of IPTG concentration (0.8 mM), incubating temperature (22\u0026deg;C) and under shaking (180 rpm) for 20 hrs. The recombinant \u003cem\u003eE. coli\u003c/em\u003e cells were harvested and the recombinant protein was extracted by cell lysis followed by centrifugation (6500 g for 10 min at 4\u0026deg;C).The crude protein (20 \u0026micro;l) was initially checked on SDS-PAGE (10%) to confirm protein induction. Subsequently the recombinant protein was purified using TALON single step column (Takara, Japan) essentially following the manufacturer\u0026rsquo;s protocol. The purified recombinant protein (20 \u0026micro;l) was confirmed on SDS-PAGE gel (10%) and quantified using Bradford\u0026rsquo;s method[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Enzyme Kinetics\u003c/h2\u003e \u003cp\u003ePrior to the \u003cem\u003ein-vitro\u003c/em\u003e assay using aglycon compound library, optimization of the reaction conditions for the recombinant protein was performed. The purified protein was optimized for parameters like incubation time (15\u0026ndash;90 min), temperature (4\u0026ordm;C-70\u0026ordm;C) and pH in citrate (pH 3\u0026ndash;6), phosphate (6-7.5) and Tris-HCl (7.5\u0026ndash;10.5) buffers at a constant protein concentration (2.5 \u0026micro;g). The sugar acceptability using kaempferol and quercetin as substrates was assessed essentially following the UDP Glo\u0026trade; assay protocol (Promega, USA) which detects UDP after UDP-sugar hydrolysis/ transfer by converting UDP to light in luciferase type reaction (measured in relative units). A standard curve of UDP concentration (0\u0026ndash;25 \u0026micro;m) was generated, and linear range of detection was determined, where the luminescence is directly proportional to the UDP concentration. Following the protocol published earlier [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], each sugar nucleotide hydrolysis reaction was combined with the detection reagent in equal ratio and allowed to incubate at room temperature. After 1h of incubation, the luminescence was measured using FLUOstar Omega microplate reader (BMG Labtech).The amount of UDP released during the glycosylation reaction was recorded and the relative catalytic activity was calculated. Also, four substrates (kaempferol and quercetin) concentrations (3, 5,7 \u0026amp;9 \u0026micro;M) were examined and the released UDP amounts were quantified by relative luminescence for the enzyme activity. The enzyme kinetics parameter namely, Km, Vmax, kcat and kcat/Km were calculated for the two substrates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.11 In vitro enzyme assay\u003c/h2\u003e \u003cp\u003eEnzyme reaction (200 \u0026micro;l) was performed under the \u003cem\u003ein-vitro\u003c/em\u003e conditions using the optimized parameters of citrate buffer (50 mM) pH 6.0, purified protein (5\u0026micro;g), UDP-Glc (5 mM) and substrate (0.9 mM) at 30\u0026deg;C incubation temperature for 15 min. The reaction was quenched by the addition of ethyl acetate (100 \u0026micro;l) thrice and partitioned with water. The organic part was concentrated and re-suspended in methanol for UP-HPLC/LCMS analysis. After successful glycosylation of kaempferol and quercetin, a panel of sixteen natural products belonging to various classes of compounds (flavones, terpenes, alkaloids, phenolics, vitamins, Phyto-hormone and carboxylic acid) and drug molecules were tested under similar conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.12 LC-MS Analysis\u003c/h2\u003e \u003cp\u003eExperiments were performed on an Agilent 1260 Infinity (Agilent, USA) HPLC system coupled with an Agilent 6410 (Agilent Technologies, USA) triple quadrupole MS/MS instrument equipped with an ESI ion source in the positive ion mode. Chromatographic separation was performed on a Chromolith high resolution RP18e column(100 x 4.6 mm) maintained at 300\u0026ordm; C. Mobile phases of 0.1% (v/v) formic acid in water (eluent A) and acetonitrile (eluent B) were used at a flow rate of 600 \u0026micro;l/min to analyze each sample having injection volume of 10\u0026micro;l. A gradient programme used was: 0\u0026ndash;7 min, 50%B followed by increment of 10% in eluent B after 8\u0026ndash;12 min, 12\u0026ndash;25 min, 25\u0026ndash;30 min reaching upto80% for30\u0026ndash;45 min and returning to 50% for 45\u0026ndash;50 min. Data handling was performed using Mass Hunter workstation. The standards (kaempferol/quercetin/3-O-glycosylated kaempferol and quercetin) were analyzed under the optimized conditions before analysing the samples.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sequence and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe selected full-length gene encoding a UGT gene was mined from the \u003cem\u003ein-house\u003c/em\u003e library (PRJNA664636) of \u003cem\u003eG. glabra\u003c/em\u003e plant and submitted to the NCBI database (MN163014.1) bearing 52.16 kDa protein (QHW04706.1). The full-length \u003cem\u003eGgUGT72L11\u003c/em\u003e ORF was sequenced to be 1425bp encoding a cytoplasmic protein of 474 amino acid having instability index of 40.62, aliphatic index of 99.75, GRAVY value of 0.032 and a theoretical pI of 6.61. The MEME based motif analysis showed ten highly conserved fifty sequence length motifs (including the signature Plant Secondary Product Glycosyltransferase motif) common to all the studied protein homologs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The MSA based homology of the UDPGT specific region ranged between 72.61% 85.90% to (supplementary Fig.\u0026nbsp;1). A 199 aa long GTB region (from 262 to 461 aa) was found to be highly variable (from 305 to 321aa); however, a 69 aa long region (from amino acid 322 to 391) in this region was highly conserved including the 44 aa long PSPG box (aa344 to aa388) at the C-terminal. Further, the protein-protein blast of the 58 aa long stretch was found to be highly specific to the proteins identified from Fabaceae family which was endorsed by the results of the MSA (Supplementary Fig.\u0026nbsp;1). Further, to predict the putative function of UGT72L11, twenty five functionally annotated UGT72 protein sequences reported from various plant species, were aligned (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The phylogeny classified the identified protein akin to epicatechin 3-glucosyltransferase (UGT72L1) from \u003cem\u003eM. truncatula\u003c/em\u003e and flavonol 3-O-glucosyltransferases from \u003cem\u003eGlycine max\u003c/em\u003e (UGT72Z3), \u003cem\u003eG. uralensis\u003c/em\u003e (UGT72X5) and \u003cem\u003eG. max\u003c/em\u003e. (UGT72X4) plants displaying affinity towards flavonoids.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) based on thirty four proteins sequences having mono- and di-glycosylation propensity revealed the identified protein to be mono-glycosylated forming a distinct cluster with AtUGT71C1 and VvGT7A (XP_002276546) corroborated by mono-glucoside characteristic GSS consensus motif (563–566) as reported by Huang et al.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Contrary to this finding, the proline amino acid at position 95 (Supplementary Fig.\u0026nbsp;2) which is the characteristic feature of disaccharides was also present in the GgUGT72L11 protein further highlighting the sequence uniqueness of the identified protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Promoter Analysis\u003c/h2\u003e \u003cp\u003eTo further investigate the role of the identified gene, upstream region (1174 bp) of the selected gene was isolated by genome walking. \u003cem\u003eIn silico\u003c/em\u003e analysis of the promoter sequence revealed putative \u003cem\u003ecis-\u003c/em\u003eacting regulatory elements and transcription factor-binding sites. The results demonstrated that the mapped \u003cem\u003ecis\u003c/em\u003e-acting elements included promoter core elements (TATA-box and CAAT-box), phytochrome activated light-responsive element (5’GATAAGR3’), abscisic acid signalling/regulated gene expression motif (5’CACGTG/CANNTG3’). Stress responsive elements including dehydration (5’ACGT3’), cold induced (5’CAANTG3’) and ABRE-Related Sequences (MACGYGB) were also found. The promoter region also had carbon (5’AAAG3’) and sugar metabolism (5’TGACT3’) motifs, pathogenesis related elements (5’YTGTCWC3’) and wound (5’TGACY3’) perceptive elements (supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.33-D Model Prediction\u003c/h2\u003e \u003cp\u003eIn plants, the characteristic 44 aa long consensus sequence of the PSPG domain plays a crucial role during glycosylation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The 10 aa highly conserved C-terminal domain binds to the donor sugar molecule, while the N-terminal is highly variable acceptor binding site potentially for accommodating wide variety of aglycons. Ramachandran plot analysis of protein under study, (supplementary Fig.\u0026nbsp;4a,b) showed maximum number of amino acids in favored region (97.40%) and allowed region (1.94%). A small fraction (0.65%) was found in the outliner region (supplementary Fig.\u0026nbsp;4b) also. The glycosylation can only be catalyzed if the acceptor molecule is positioned correctly in the substrate pocket such that the functional group of the sugar acceptor is in close proximity to the first carbon of the sugar for the formation of glycosidic bond [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Docking simulations of candidate ligand kaempferol with receptor UGT72L11 showed good binding affinity and stability of the complex as indicated by binding affinity of kaempferol (-6.140 kcal/mol), total energy (-31.247 kcal/mol), vdW energy (-0.438 kcal/mol) and electrostatic energy (-16.390 kcal/mol).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Subcellular localisation, recombinant Protein Expression and Purification\u003c/h2\u003e \u003cp\u003eSub-cellular localization of UGT72L11 protein was found to be predominantly in plasma membrane as demonstrated by onion cells transiently transformed with 35S:ugt72l11-GFP construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The recombinant protein was expressed in \u003cem\u003eE. coli\u003c/em\u003e (BL-21 cells) and optimized for optimal induction. The protein was found to be optimally induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) in 20 hr under 0.8 mM IPTG concentration at 20°C. The recombinant protein with C-terminal HIS tag was column purified and employed for enzyme kinetic studies and \u003cem\u003ein-vitro\u003c/em\u003e assays.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Enzyme Kinetics\u003c/h2\u003e \u003cp\u003eThe purified recombinant protein activity was assessed in three different pH -acidic (citrate), neutral (phosphate) and basic (Tris HCl) buffer systems. The protein was found to be active in all the three buffers studied. However, optimum enzyme activity was found in citrate buffer (pH6.0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Further, the time duration of 15 min to 90 min assessed for the enzyme activity showed its optimal activity at 15 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The effect of temperature studied between 4°C to 70°C, revealed protein to be optimally active between 10°C to 30°C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The optimized parameters were further used for the enzyme kinetic studies. The enzyme-substrate relationship was evaluated with two substrates (quercetin and kaempferol) at four concentrations (3, 5, 7 \u0026amp; 9 µM) employing UDP-glucose as the sugar donor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The kinetic properties were determined for the selected substrates using previously optimized concentrations of 2.5µg protein and 15 µM UDP sugar. The reaction was kept for 15-min at 30°C. The Michaelis constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e), which signifies the concentration of the substrate at which half of the enzyme is saturated, was found to be more for quercetin-UDPGlc (0.23) than kaempferol-UDPGlc (0.47) and the catalytic number (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e)/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e were calculated to be 0.66 and 0.44 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e\u003csup\u003e− 1\u003c/sup\u003e s\u003csup\u003e− 1\u003c/sup\u003e, respectively reflecting its preference for quercetin over kaempferol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 In-vitro enzyme activity and product identification\u003c/h2\u003e \u003cp\u003eThe substrate affinity of the purified recombinant protein was established using \u003cem\u003ein-vitro\u003c/em\u003e enzyme assay employing various classes of substrates as acceptors and UDP-glucose as the sugar donor following the optimized conditions of 30ºC for 15 min. The protein was initially assessed with quercetin and kaempferol as the two model substrates as they harbour several hydroxyl groups. The \u003cem\u003ein-vitro\u003c/em\u003e enzyme activity and the position of glycosylation were confirmed by their respective 3-O-glucoside standards. The retention time (RT) corresponding to the substrates and respective glycosylated products were compared and confirmed by peaks and mass spectra. It was conclusively shown that the substrates, kaempferol (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) and quercetin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) were glycosylated to quercetin-3-glucoside and kaempferol-3-glucoside, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7 In-vitro activity of GgUGT72L11 Enzyme (Substrate scope)\u003c/h2\u003e \u003cp\u003eThe substrates investigated in the present study belonged to flavones, terpenes, carboxylic acids, vitamins, alkaloid, phenol classes of compounds and few commercial drugs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Flavones, such as quercetin and catechin hydrate having polyhydroxyl groups, when subjected to glycosylation under optimized conditions showed the formation of respective glucosides as confirmed by LCMS ( supplementary Data 1; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similarly, diosgenin having monohydroxy group and andrographolide with carboxylic groups were catalyzed for the addition of glucose moiety to form respective glycosides by the purified protein (supplementary Data 1). Isovanillin, (a phenolic), dihydrozeatin \u0026amp; abscisic acid (phytohormones) were also seen to form their respective glycosides catalyzed by GgUGT72L11. The alkaloidal substrates studied were colchicine, monocrotaline and quinine hydrochloride. The identified GgUGT72L11 catalyzed glycosylation in colchicine whereas monocrotaline and quinine hydrochloride underwent hydroxyl glycosylation forming respective glycosides. Structurally, colchicine possess no hydroxyl and/or carboxyl groups, contrary to monocrotaline and quinine hydrochloride that had -OH groups\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of glycosylated compounds (flavanols, terpenes, carboxylic acids, vitamins, alkaloid, phenolics and commercial drugs) with molecular formulae observed, and calculated molecular weight of the glycosides\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.no\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMass calculated for\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCalculated molecular weight\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eObserved molecular weight\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKaempferol glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e448.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e448.02\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuercetin glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + H) (C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e465.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e464.65\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatechin hydrate glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e469.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e469.83\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiosgenin glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + H) (C\u003csub\u003e33\u003c/sub\u003eH\u003csub\u003e53\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e577.37\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e577.35\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAndrographolide glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e39\u003c/sub\u003e0\u003csub\u003e10\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e513.26\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e514.88\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCitric acid glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e353.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e353.26\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNicotinic acid glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eNO\u003csub\u003e7\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e284.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e285,25\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRiboflavin glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e29\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e537.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e537.32\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIso vanillin glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e313.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e313.51\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDihydro zeatin glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + H) (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003e0\u003csub\u003e6\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e384.19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e383.8\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbscisic acid\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C20H29O9)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e425.45\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e425.35\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColchicine glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + Na) C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e35\u003c/sub\u003eNNaO\u003csub\u003e11\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e585.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e585.67\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuinine hydrochloride glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e521.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e520.9\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocrotaline glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + H) (C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eNO\u003csub\u003e11\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e488.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e488.18/486.6\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePodophyllotoxin glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + H) (C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e33\u003c/sub\u003e0\u003csub\u003e13\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e577.19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e576.94\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSofosbuvir glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e28\u003c/sub\u003eH3\u003csub\u003e8\u003c/sub\u003eFN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003eP)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e690.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e690.42\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEphedrine HCL glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eClNO\u003csub\u003e6\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e362.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e361.99\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuinine hydrochloride glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e521.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e520.9\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocrotaline glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + H) (C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eNO\u003csub\u003e11\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e488.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e488.18/486.6\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePodophyllotoxin glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M + H) (C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e33\u003c/sub\u003e0\u003csub\u003e13\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e577.19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e576.94\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSofosbuvir glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e28\u003c/sub\u003eH3\u003csub\u003e8\u003c/sub\u003eFN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003eP)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e690.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e690.42\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEphedrine HCL glucoside\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(M-H) (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eClNO\u003csub\u003e6\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e362.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e361.99\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Expression Dynamics of UGT72L11\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.8.1Growth Conditions\u003c/h2\u003e \u003cp\u003eqRT-PCR analysis was performed to analyze the expression profile and levels of the identified gene in different tissues. The real-time quantitative analysis of the transcripts was performed using the RNA extracted from the aerial and underground tissues on the plants grown under three different conditions namely, tissue culture, hardening unit and the experimental farms (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). The identified gene displayed preferential expression under the three surroundings. The \u003cem\u003eGgUGTL11\u003c/em\u003e was found to be significantly up-regulated (2.41 folds) in the roots of the field grown plants, while it was significantly down regulated in the roots of the glass house stationed plants as compared to the respective shoot tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). The gene was found to be similarly expressed in the stem tissues under the three conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.8.2 Stress\u003c/h2\u003e \u003cp\u003eTotal RNA extracted from the aerial tissues of the \u003cem\u003ein-vitro\u003c/em\u003e grown plants was subjected to nine stressors and assessed for transcript accumulation. Eight abiotic treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb) given to the plants were dark, carbon starvation, wounding, senescence, drought, salinity, cold and UV light. The \u003cem\u003eGgUGT72L11\u003c/em\u003egene was found to be repressed under the influence of carbon starvation (0.6 fold), salinity (0.9 folds) and dark (0.3 fold) conditions. The gene transcripts were found to be maximally up regulated under cold (36 folds) conditions followed by drought (9 folds), senescence (1.7 folds), and UV light (1.2 folds). Further, the biotic stress subjected through co-cultivation of \u003cem\u003ein-vitro\u003c/em\u003e grown \u003cem\u003eG. glabra\u003c/em\u003e plants (individually) with four plant pathogens (3 isolates of \u003cem\u003eColletotrichum\u003c/em\u003e sp.( P1-\u003cem\u003eColletotrichum gloeosporioides\u003c/em\u003e, P2- \u003cem\u003eColletotrichum siamense sub sp.\u003c/em\u003e, P3- \u003cem\u003eColletotrichum siamense sub sp.\u003c/em\u003e and P4- one isolate of \u003cem\u003eCalonectria cylindrospora)\u003c/em\u003e showed increased transcript levels with P2 (2.86 folds), while P1 was found to be suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec). The expression profile of the \u003cem\u003eGgUGT72L11\u003c/em\u003e gene in the \u003cem\u003ein-vitro\u003c/em\u003e plants subjected to auxin, abscisic acid and GA hormone treatments showed significant reverberation. The gene was found to be highly up regulated under ABA (400 folds) treatment at 16 hrs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed) and moderately up regulated in the presence of auxin (2 folds) between 30 to 60 minutes. At the same time, it was seen to be down regulated (0.4 to 0.8) under GA treatment between 1 to 6 hrs in the aerial tissues of the plant as compared to the untreated control plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.8.3 Determination of expression of ROS scavenging genes and cold responsive genes\u003c/h2\u003e \u003cp\u003eThe above results indicated higher accumulation of transcripts in the aerial tissues of the \u003cem\u003ein-vitro\u003c/em\u003e grown plants under cold (4°C) and ABA stresses. Further, cold -specific COR genes (\u003cem\u003eCOR47, ABI5, DREB1\u003c/em\u003e) and Reactive Oxygen Species scavenging genes catalase \u003cem\u003e(CAT1)\u003c/em\u003e, super oxide dismutase (\u003cem\u003eSOD)\u003c/em\u003e, Glutathione Peroxidase \u003cem\u003e(GPX\u003c/em\u003e) were studied to understand the molecular mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). The cold responsive genes were seen to be significantly up regulated-\u003cem\u003eCOR 47\u003c/em\u003e (22.3 folds), \u003cem\u003eDREB1\u003c/em\u003e (13.8 folds) and \u003cem\u003eABI5\u003c/em\u003e (10.2 folds) under cold stress. Also expression analysis of ROS scavenging enzymes and cold responsive genes was carried out in ABA treated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Both the ROS scavenging genes -CAT1 ( 5.97 folds), \u003cem\u003eSOD\u003c/em\u003e (18.6 folds) and \u003cem\u003eGPX\u003c/em\u003e (16 folds), and cold responsive genes-\u003cem\u003eDREB1\u003c/em\u003e (2.9 folds) and \u003cem\u003eABI5\u003c/em\u003e (14.6folds) were found to be up regulated in the plants subjected to ABA treatments suggesting the role of \u003cem\u003eGgUGT72L11\u003c/em\u003e under multiple stress. However, ROS scavenging genes (\u003cem\u003eCAT1, SOD, GPX\u003c/em\u003e) were observed to be uninfluenced under the cold stress suggesting no involvement of ROS scavenging enzymes under cold stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pBLAST search (NCBI) displayed maximum homology (73.3%) homology with UGT72L1 (ACC38470) encoding epicatechin 3-glucosyltransferase from \u003cem\u003eM. truncatula\u003c/em\u003e. The CAZy database divulged information on only two proteins from \u003cem\u003eG. uralensis\u003c/em\u003e species (NCBI accession No. MK341791 and MK341793) which were involved in flavonoid biosynthesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The identified protein from \u003cem\u003eG. glabra\u003c/em\u003e seems to be unique as it showed less than 40% homology with the two proteins reported from the \u003cem\u003eG. uralensis\u003c/em\u003e. The uniqueness of the protein was further enhanced by having both mono- and disaccharide glycoside forming activities. The protein sequence possessed mono-glucoside characteristic GSS consensus motif (563–566), and also had proline amino acid at position 95 characteristic of disaccharides as reported by Huang et al.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUnderstanding the glycosylation mechanism and its physiological significance in plants hold importance not only \u003cem\u003ein-planta\u003c/em\u003e for channelizing metabolic pathways for producing the desired compound [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; but also for valuable glycosides in synthetic medical biology. In our study, the \u003cem\u003ein-silico\u003c/em\u003e mapping on receptor (UGT72L11) and ligand (kaempferol) showed that kaempferol formed five hydrogen bond interactions with UGT72L11protein specifically with four amino acid residues GLU279, ARG386, PRO380 and TRP379 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On the other hand, in the well characterized structure of AtUGT72B1 protein the substrate was demonstrated to be enclosed in six hydrophobic residues (I-86, L-118, F-119, F-148, L-183, and L-197) to form catalytic site roofed by E83 covering the site[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Further, the ligand and receptor interaction was also stabilized by pi donor hydrogen bond and pi alkyl interactions involving SER277 and LEU409 amino acid residues respectively. Protein-ligand binding occurs spontaneously when the binding affinity/free energy change is negative [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] as these complexes are stable which is an essential characteristic of effective ligands. The present study further experimented to confirm the results of docking experiment with the \u003cem\u003ein-vitro\u003c/em\u003e enzyme activity results using the substrates. \u003cem\u003eIn vitro\u003c/em\u003e assay using the purified protein demonstrated multi-substrate acceptability with wide range of aglycons and medicinally important drugs like ephedrine hydrochloride, podophyllotoxin, and sofosbuvir (Table\u0026nbsp;1and supplementary Data1). The GgUGT72L11 protein introduced glucose moiety to the mono hydroxyl group in the studied compounds forming their respective mono-glycosylated products as confirmed by LCMS analyses (supplementary Data 1). Previous \u003cem\u003ein-vitro\u003c/em\u003e enzymatic studies in thirty reported recombinant UGT72 family proteins, across 12 different angiosperm species, have shown acceptance to varied substrates including flavonoids, monolignols, and their precursors/derivatives [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. UGTs can glycosylate at \u003cem\u003eO\u003c/em\u003e-position (3, 5, 7, 3',4', or 5'), and\\or \u003cem\u003eC\u003c/em\u003e-positions as seen in rice, maize and buckwheat (Du et al., 2010; Falcone Ferreyra et al., 2013;Nagatomo et al., 2014). Evidences suggest members of UGT72 family possess broad spectrum substrate recognition predominantly in flavanols with different glycosylation patterns forming 3-\u003cem\u003eO\u003c/em\u003e-monoglycosides as in \u003cem\u003eL. japonicus\u003c/em\u003e ( LjUGT72AH1, LjUGT72Z2, LjUGT72V3) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], \u003cem\u003eGlycine max\u003c/em\u003e (GmUGT72X4, GmUGT72Z3)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and \u003cem\u003eM. truncatula\u003c/em\u003e (MtUGT72L1)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], to multi-site glycosylation at 3, 4, and 7 positions by \u003cem\u003eClonorchis sinensis\u003c/em\u003e CsUGT72A1[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and \u003cem\u003eHieracium pilosella\u003c/em\u003e HpUGT72B11 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe identified GgUGT72L11 protein also catalyzed \u003cem\u003eC\u003c/em\u003e-glycosylation in colchicine. The \u003cem\u003eC\u003c/em\u003e-glycosylation pattern and its extent have a crucial role in forming potent drug-like molecule [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The conjugates of \u003cem\u003eC\u003c/em\u003e-glycosides exhibit unique characteristics as these bonds are resistant to acid hydrolysis and glycosidase cleavage [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The UGTs catalyzing \u003cem\u003eC\u003c/em\u003e-glycosylation has been reported from several plants including \u003cem\u003eFagopyrum esculentum\u003c/em\u003e (FeCGTa and FeCGTb) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], \u003cem\u003eGlycine max\u003c/em\u003e (UGT708D1), \u003cem\u003eDendrobium catenatum\u003c/em\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], \u003cem\u003eGentiana trifloral\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and Citrus plants (UGT708G1, UGT708G2) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Literature cites singular report of \u003cem\u003eC\u003c/em\u003e-glycosyltransferase (GgCGT) from \u003cem\u003eG. glabra\u003c/em\u003e catalysing two-step \u003cem\u003eC\u003c/em\u003e-glycosylation of the drug flopropione [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe presence of light, hormonal and stress responsive motifs indicated involvement of gene in complex hormone regulatory network pertaining to stress and secondary metabolite biosyntheses. Overall, the \u003cem\u003ecis\u003c/em\u003e-element analysis suggested gene responsive to different kinds of stresses. This was explored further to assess the involvement of the identified UGT under the influence of various stresses. The identified flavonoid glycosyl transferase displayed high expression under cold, senescence, drought and abscisic acid stress. Recent comprehensive review [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] highlighted buffering effects of UGTs in multiple biotic and abiotic stresses. A series of protective mechanisms are triggered when plants sense stress [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] underpinning the overlapping but not completely redundant biological functions of UGTs in mediating developmental and stress responses.\u003c/p\u003e\u003cp\u003eIn the current study, \u003cem\u003eGgUGT72L11\u003c/em\u003e gene was shown to be significantly up regulated under cold stress along with the downstream chill-induced \u003cem\u003eCOR\u003c/em\u003e and \u003cem\u003eDREB\u003c/em\u003e genes. However, ROS scavenging genes were down regulated under similar conditions. Contrary to this studies have highlighted up regulation of ROS-scavenging genes such as superoxide dismutase \u003cem\u003e(SOD)\u003c/em\u003e, catalase \u003cem\u003e(CAT 1) and\u003c/em\u003e Glutathione Peroxidase \u003cem\u003e(GPX)\u003c/em\u003e in detoxification particularly under stress[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] .One probable reason for down regulation could be the formation of glycosylated flavonoids catalyzed by the identified \u003cem\u003eflavonol glycosyl transferase\u003c/em\u003e (\u003cem\u003eGgUGT72L11\u003c/em\u003e) imparting hydrophilic and cartable [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] properties to counter the oxidative burst more efficiently under stressed conditions [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our findings also revealed the gene was significantly induced when subjected to ABA treatment and catalyses ABA to respective glycosylated conjugate (supplementary data) under \u003cem\u003ein-vitro\u003c/em\u003e conditions. Hence, the ABA homeostasis was maintained thereby inhibiting ROS accumulation. Corroborating our findings, few studies have confirmed strong induction of UGTs under the cold stress simultaneously participating in flavonol biosyntheses [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] ,[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Studies have also confirmed involvement of several UGTs (AtUGT79B2, AtUGT79B3 and CsUGT78A14) in glycosylation of ABA regulating the dynamic state of cellular ABA/ABA-GE levels (ABA homeostasis).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eStudies targeting association of flavonoids under cold stress and ROS scavenging ability will provide novel insights into the biological role of the UGTs and flavonoids in plant defence and stress biology. Further, investigation into the underlying molecular processes involving ABA homeostasis and ABA-mediated stresses (cold and drought in particular) will throw light on the interplay between stressors, glycosylated metabolites and signalling mechanisms for multiple stress tolerance. The recognition of a simple but unique glycosylation mechanism in the identified UGT offers physiological advantage to the plant and could be the foundation for numerous applications in medical biology expanding the magnitude to manipulate the biological and physicochemical properties of molecules. Further investigation of the underlying downstream processes will elucidate the regulatory mechanism of \u003cem\u003eUGT72L11\u003c/em\u003e in plant stress tolerance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMalik Muzafar Manzoor, Shahnawaz Husain, Bhawna Verma, Suphla Gupta - Conceptualization, Malik Muzafar Manzoor, Shahnawaz Husain, Bhawna Verma ,Pooja Goyal, Ritu Devi, 4Fariha chowdhary- Methodology :, Ajai P Gupta ,Pooja Goyal, Bhawna Verma, \u0026nbsp;Suphla Gupta, Bhawna Verma - Writing- Original draft preparation. Suphla Gupta,: Visualization, Investigation and \u0026nbsp;Supervision.: Ajai P Gupta: Software, Validation.: Suphla Gupta, Bhawna Verma- Writing- Reviewing and Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;and Acknowledgements:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors acknowledge the grant (SERB/SB/SO/PS/90/2013) from Science and Engineering Research Board, India for funding the study. MM and SH acknowledges Council for Scientific \u0026amp; Industrial Research for Fellowships; PG and BV acknowledges University Grant Commission for Fellowship; SH \u0026amp; BV acknowledges Council for Scientific \u0026amp; Industrial Research and Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Sector-19, Ghaziabad, U.P., India is also acknowledged. All the authors acknowledge director, CSIR-IIIM for providing all the necessary facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Statement:\u0026nbsp;\u003c/strong\u003eThe article\u0026apos;s supporting data and materials are in the public domain and can be accessed at NCBI SRA Data (PRJNA664636).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI in scientific writing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have not used any generative AI in scientific writing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBock KW (2016) The UDP-glycosyltransferase (UGT) superfamily expressed in humans, insects and plants: Animal-plant arms-race and co-evolution. Biochem Pharmacol 99:11\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilbert MK, Bland JM, Shockey JM, Cao H, Hinchliffe DJ, Fang DD, Naoumkina M (2013) A transcript profiling approach reveals an abscisic acid-specific glycosyltransferase (UGT73C14) induced in developing fiber of Ligon lintless-2 mutant of cotton (Gossypium hirsutum L). PLoS ONE 8(9):e75268\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Chow TF, Puckrin RS, Alfred SE, Korir AK, Larive CK, Cutler SR (2007) Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated. Nat Chem Biol 3(11):716\u0026ndash;721\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJarrar Y, Lee S-J (2021) The Functionality of UDP-Glucuronosyltransferase Genetic Variants and their Association with Drug Responses and Human Diseases. J Pers Med 11(6):554\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng S, Luo J, Zhu X, Gao X, Hua H, Cui J (2022) Transcriptomic analysis of salivary gland and proteomic analysis of oral secretion in Helicoverpa armigera under cotton plant leaves, gossypol, and tannin stresses. Genomics 114(2):110267\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Yu H-M, Meng X-F, Lin J-S, Li Y-J, Hou B-K (2018) Ectopic expression of glycosyltransferase UGT76E11 increases flavonoid accumulation and enhances abiotic stress tolerance in Arabidopsis. Plant Biol 20(1):10\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Ji K, Liang B, Du Y, Jiang L, Wang J, Kai W, Zhang Y, Zhai X, Chen P et al (2017) Suppressing ABA uridine diphosphate glucosyltransferase (SlUGT75C1) alters fruit ripening and the stress response in tomato. Plant J 91(4):574\u0026ndash;589\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi Y, Phan H, Liu Y, Cao S, Zhang Z, Chu C, Schl\u0026auml;ppi MR (2020) Glycosyltransferase OsUGT90A1 helps protect the plasma membrane during chilling stress in rice. J Exp Bot 71(9):2723\u0026ndash;2739\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang K, Sun Y, Li M, Long R (2021) CrUGT87A1, a UDP-sugar glycosyltransferases (UGTs) gene from Carex rigescens, increases salt tolerance by accumulating flavonoids for antioxidation in Arabidopsis thaliana. Plant Physiol Biochem 159:28\u0026ndash;36\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Cai B, Jin J, Zhang N, Jing T, Wang J, Pan Y, Zhou Z, Zhao Y, Feng Y et al (2020) Cold Stress-induced Glucosyltransferase CsUGT78A15 is Involved in the Formation of Eugenol Glucoside in Camellia sinensis. Hortic Plant J 6(6):439\u0026ndash;449\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi P, Li Y-j, Wang B, Yu H-m, Li Q, Hou B (2017) -k: The Arabidopsis UGT87A2, a stress-inducible family 1 glycosyltransferase, is involved in the plant adaptation to abiotic stresses. Physiol Plant 159(4):416\u0026ndash;432\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang M, Li FD, Li K, Wang ZL, Wang YX, He JB, Su HF, Zhang ZY, Chi CB, Shi XM et al (2020) Functional Characterization and Structural Basis of an Efficient Di-C-glycosyltransferase from Glycyrrhiza glabra. J Am Chem Soc 142(7):3506\u0026ndash;3512\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaputi L, Malnoy M, Goremykin V, Nikiforova S, Martens S (2012) A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land. Plant J 69(6):1030\u0026ndash;1042\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang Y, Cheng X, Huhman DV, Ma J, Peel GJ, Yonekura-Sakakibara K, Saito K, Shen G, Sumner LW, Tang Y et al (2013) Medicago glucosyltransferase UGT72L1: potential roles in proanthocyanidin biosynthesis. Planta 238(1):139\u0026ndash;154\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNomura Y, Seki H, Suzuki T, Ohyama K, Mizutani M, Kaku T, Tamura K, Ono E, Horikawa M, Sudo H et al (2019) Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin. Plant J 99(6):1127\u0026ndash;1143\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung SY, Seki H, Fujisawa Y, Shimoda Y, Hiraga S, Nomura Y, Saito K, Ishimoto M, Muranaka T (2020) A cellulose synthase-derived enzyme catalyses 3-O-glucuronosylation in saponin biosynthesis. Nat Commun 11(1):5664\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang D, Li P, Yin Y, Ren G, Liu C (2021) Molecular cloning and functional characterization of UGTs from Glycyrrhiza uralensis flavonoid pathway. Int J Biol Macromol 192:1108\u0026ndash;1116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Chen L, Cai Z, Chen C, Liu Z, Liu S, Zou L, Tan M, Chen J, Liu X et al (2021) Metabolite Profiling and Transcriptome Analysis Explains Difference in Accumulation of Bioactive Constituents in Licorice (Glycyrrhiza uralensis) Under Salt Stress. Front Plant Sci 12:727882\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang F-C, Giri A, Daniilidis M, Sun G, H\u0026auml;rtl K, Hoffmann T, Schwab W (2018) Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases. Plant Cell Physiol 59(4):862\u0026ndash;875\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcWilliam H, Li W, Uludag M, Squizzato S, Park YM, Buso N, Cowley AP, Lopez R (2013) Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res 41(W1):W597\u0026ndash;W600\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWass MN, Kelley LA, Sternberg MJ 3DLigandSite: predicting ligand-binding sites using similar structures. Nucleic Acids Res 2010:gkq406\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun W, Cao Z, Li Y, Zhao Y, Zhang H (2007) A simple and effective method for protein subcellular localization using Agrobacterium-mediated transformation of onion epidermal cells. Biologia 62(5):529\u0026ndash;532\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403\u0026ndash;410\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManzoor MM, Goyal P, Pandotra P, Dar MS, Dar MJ, Misra P, Gupta AP, Vishwakarma RA, Ahuja A, Dhar MK Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis. Protoplasma 2021:1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoyal P, Muzafar Manzoor M, Gupta AP, Pandotra P, Gupta S (2022) : Molecular Dissection of Genes and Promoters involved in Glycyrrhizin Biosynthesis Revealed Phytohormone Induced Modulation in Glycyrrhiza glabra L. Gene :146682\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Liang X, Zhou X, Wu Z, Yuan L, Wang Y, Li Y (2020) : Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments. Plants (Basel Switzerland) 9(11)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW (2000) Quantitative Reverse Transcription\u0026acirc;n-Polymerase Chain Reaction to Study mRNA Decay: Comparison of Endpoint and Real-Time Methods. Anal Biochem 285(2):194\u0026ndash;204\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradford M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7: 248\u0026ndash;254. \u003cem\u003eFind this article online\u003c/em\u003e 1976.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcGraphery K, Schwab W (2020) : Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases. Int J Mol Sci 21(6)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLouveau T, Leitao C, Green S, Hamiaux C, van der Rest B, Dechy-Cabaret O, Atkinson RG, Chervin C (2011) Predicting the substrate specificity of a glycosyltransferase implicated in the production of phenolic volatiles in tomato fruit. FEBS J 278(2):390\u0026ndash;400\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreton C, Šnajdrov\u0026aacute; L, Jeanneau C, Koča J, Imberty A (2005) Structures and mechanisms of glycosyltransferases. Glycobiology 16(2):29R\u0026ndash;37R\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo H, Wang H, Huo YX (2020) Engineering Critical Enzymes and Pathways for Improved Triterpenoid Biosynthesis in Yeast. ACS Synth Biol 9(9):2214\u0026ndash;2227\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrazier-Hicks M, Offen WA, Gershater MC, Revett TJ, Lim E-K, Bowles DJ, Davies GJ, Edwards R (2007) : Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 104(51):20238\u0026ndash;20243\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfriza D, Suriyah W, Ichwan S (2018) : In silico analysis of molecular interactions between the anti-apoptotic protein survivin and dentatin, nordentatin, and quercetin. In: \u003cem\u003eJournal of Physics: Conference Series\u003c/em\u003e: : IOP Publishing; 2018: 032001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpeeckaert N, El Jaziri M, Baucher M, Behr M (2022) : UGT72, a Major Glycosyltransferase Family for Flavonoid and Monolignol Homeostasis in Plants. Biology (Basel) 11(3)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin Q, Shen G, Chang Z, Tang Y, Gao H, Pang Y (2016) Involvement of three putative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis in Lotus japonicus seeds. J Exp Bot 68(3):597\u0026ndash;612\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoguchi A, Saito A, Homma Y, Nakao M, Sasaki N, Nishino T, Takahashi S, Nakayama T (2007) A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis. J Biol Chem 282(32):23581\u0026ndash;23590\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Dai X, Gao L, Guo L, Zhuang J, Liu Y, Ma X, Wang R, Xia T, Wang Y (2017) Functional Analysis of an Uridine Diphosphate Glycosyltransferase Involved in the Biosynthesis of Polyphenolic Glucoside in Tea Plants (Camellia sinensis). J Agric Food Chem 65(50):10993\u0026ndash;11001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpeeckaert N, Adamou NM, Hassane HA, Baldacci-Cresp F, Mol A, Goeminne G, Boerjan W, Duez P, Hawkins S, Neutelings G et al (2020) : Characterization of the UDP-glycosyltransferase UGT72 Family in Poplar and Identification of Genes Involved in the Glycosylation of Monolignols. Int J Mol Sci 21(14)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen Z, Ji X, Jiao Z, Luo Y, Zhang G-Q, Tao S, Lei Z, Zhang J, Wang Y, Liu Z-J et al (2020) Functional analysis of a novel C-glycosyltransferase in the orchid Dendrobium catenatum. Hortic Res 7(1):111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagatomo Y, Usui S, Ito T, Kato A, Shimosaka M, Taguchi G (2014) Purification, molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon. Plant J 80(3):437\u0026ndash;448\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasaki N, Nishizaki Y, Yamada E, Tatsuzawa F, Nakatsuka T, Takahashi H, Nishihara M (2015) Identification of the glucosyltransferase that mediates direct flavone C-glucosylation in Gentiana triflora. FEBS Lett 589(1):182\u0026ndash;187\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIto T, Fujimoto S, Suito F, Shimosaka M, Taguchi G (2017) C-Glycosyltransferases catalyzing the formation of di-C-glucosyl flavonoids in citrus plants. Plant J 91(2):187\u0026ndash;198\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Wang S, Yang J, Kang C, Huang L, Guo L (2022) Glycosylation of plant secondary metabolites: Regulating from chaos to harmony. Environ Exp Bot 194:104703\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Jin J, Gao T, Zhang N, Jing T, Wang J, Ban Q, Schwab W, Song C (2019) : Glucosyltransferase CsUGT78A14 Regulates Flavonols Accumulation and Reactive Oxygen Species Scavenging in Response to Cold Stress in Camellia sinensis. Front Plant Sci 10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki N (2005) Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction. Physiol Plant 126(1):45\u0026ndash;51\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong C, Gu L, Liu J, Zhao S, Hong X, Schulenburg K, Schwab W (2015) Functional Characterization and Substrate Promiscuity of UGT71 Glycosyltransferases from Strawberry (Fragaria x ananassa). Plant Cell Physiol 56(12):2478\u0026ndash;2493\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X (2023) : BcMYB111 Responds to BcCBF2 and Induces Flavonol Biosynthesis to Enhance Tolerance under Cold Stress in Non-Heading Chinese Cabbage. Int J Mol Sci 24(Issue 10).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ABA, enzymatic assay, Flavonoid, Glycyrrhiza glabra, in-vitro stress, UDP glycosyltransferase","lastPublishedDoi":"10.21203/rs.3.rs-3981251/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3981251/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study reports a unique broad spectrum UDP-glycosyltransferase from \u003cem\u003eGlycyrrhiza glabra\u003c/em\u003e involved in multiple stress responses and abscisic acid mediated glycosylation. The identified \u003cem\u003eUGT72L11\u003c/em\u003egene was cytoplasmic with ORF of 1425 bp encoding a 52.2 kDa protein of 474 amino acids. Phylogenetic analysis revealed maximum homology (73.3%) with epicatechin 3-glucosyltransferase (ACC38470) from \u003cem\u003eMedicago truncatula\u003c/em\u003e exhibiting sequence uniqueness. The gene was differentially expressed in shoot tissues and significantly upregulated in abscisic acid treatment (122.3 folds) and under cold stress (36 folds) \u003cem\u003ein planta\u003c/em\u003e. \u003cem\u003eIn-silico\u003c/em\u003e Structure-Activity-Relationship revealed GLU279, ARG386, PRO380 and TRP379 residues being involved in receptor-ligand interactions. The UGT72L11 protein was optimal between 10\u0026ordm;C to 30\u0026ordm;C preferring quercetin-UDPGlc (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e 0.23) over kaempferol-UDPGlc (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e 0.47). The purified recombinant protein showed multi-substrate O-glycosylation towards various classes of aglycones, abscisic acid, and also displayed C-glycosylation with colchicine as a foundation for the future medicinal applications.\u003c/p\u003e","manuscriptTitle":"Identification and Characterization of Abiotic Stress Induced novel UDP-Glucosyltransferase (UGT72L11) Gene from Glycyrrhiza glabra L.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 05:32:23","doi":"10.21203/rs.3.rs-3981251/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-08-27T05:49:35+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-15T12:47:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-22T06:39:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Genetics","date":"2024-02-15T07:54:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5f9721a1-75f8-4396-a2b7-23f330a24e00","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-19T05:32:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-19 05:32:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3981251","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3981251","identity":"rs-3981251","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}