PgF3H gene enhances drought tolerance in transgenic Arabidopsis by regulating flavonoid biosynthesis and stress response

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Flavonoids, crucial secondary metabolites, aid in plant development and stress responses. Pearl millet, a drought-tolerant crop, produces high levels of secondary metabolites like flavonoids and anthocyanins via the phenylpropanoid pathway. Research indicates that flavonoid-encoding genes are prevalent in drought-tolerant pearl millet variants, hinting at their role in drought response, though their exact functions are not fully understood. This study highlights the essential role of pearl millet flavanone 3-hydroxylase ( PgF3H ) in flavonoid biosynthesis. Overexpressing PgF3H in Arabidopsis enhances flavonol and anthocyanin content, improving tolerance to water-deficit stress without affecting antioxidant gene expression. Supporting evidence includes increased flavanone 3-hydroxylase activity in the Atf3h mutant and variable anthocyanin levels in Atans and Atanr mutants. In silico analysis of the PgF3H promoter revealed stress-responsive elements, and ProPgF3H::GUS expressing lines showed increased GUS expression with higher PEG concentrations. The in silico structure of PgF3H revealed a 2OG-Fe(II) oxygenase domain, crucial in the flavonoid biosynthetic pathway. In conclusion, PgF3H overexpression enhances drought tolerance in Arabidopsis , suggesting a potential strategy for improving crop drought resistance by manipulating flavonoid biosynthesis. Pennisetum glaucum flavanone 3-hydroxylase overexpression Arabidopsis thailiana water stress flavonoids Atf3h Atans Atanr PgF3H promoter protein structure ROS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key message Water stress stimulates plants to regulate flavonoid biosynthesis. Overexpression of the PgF3H gene increases flavonoid levels and drought tolerance in Arabidopsis , with stress-responsive elements in the PgF3H promoter indicating its role in drought response. Introduction Drought resistance is a multifaceted trait governed by an intricate network of numerous genes. It entails complex interactions between these genes and environmental signals, influencing diverse morphological and metabolic pathways (Tardieu and Tuberosa, 2010). The genes that are triggered by drought stress produce proteins that have a diverse function, including signal transduction, gene expression, damage control from stress, and treatment (Valliyodan and Nguyen, 2006 ). Different secondary metabolites that are produced by the phenylpropanoid metabolic pathway have robust antioxidant action under abiotic stress conditions. Plants have very potent chemicals called phenolic compounds, particularly flavonoids, which can provide resistance to a number of biotic and abiotic stressors (Saini et al., 2024). Flavonoids are phenolic compounds composed of two aromatic rings connected by a three-carbon bridge. Despite their diverse structures and functions, they are primarily synthesized through the phenylpropanoid pathway, a secondary metabolic pathway. Numerous researchers have been interested in flavonoids over the years. The biosynthetic process for flavonoids has been extensively studied (Liu et al., 2013 ). According to altered seed coat color, a number of mutant lines in Arabidopsis have been found to exhibit flavonoid biosynthesis defects (Winkel-Shirley, 2001). Most of the pathway's enzymes have been found in a different plant species (Hassan and Mathesius, 2012 ; Yu et al., 2012). The flavonoid biosynthesis pathway is initiated by chalcone synthase (CHS), a key enzyme that catalyzes the condensation of malonyl-CoA and 4-coumaroyl-CoA to form chalcone, marking the entry point of this metabolic route (Niu et al., 2021). Chalcone then undergoes a series of enzymatic transformations, leading to the production of various flavonoid compounds. Chalcone isomerase (CHI) facilitates the isomerization of chalcone into flavanone, which serves as a precursor for multiple flavonoid subclasses. Flavanone 3-hydroxylase (F3H) further hydroxylates flavanone, contributing to the biosynthesis of flavonols and anthocyanins. Dihydroflavonol 4-reductase (DFR) plays a crucial role in converting dihydroflavonols into leucoanthocyanidins, which are subsequently processed by flavonol synthase (FLS) to generate flavonols or by anthocyanin synthase (ANS) to produce anthocyanins. These enzymes collectively regulate the structural diversity of flavonoids, which are essential for plant pigmentation, defense, and environmental adaptation (Zha et al., 2019). The Flavanone 3-hydroxylase (F3H) gene belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) family and functions as the third key enzyme in the core flavonoid biosynthetic pathway. It plays a crucial role in catalyzing the hydroxylation of flavanones, facilitating the biosynthesis of flavonols, anthocyanins, and other essential flavonoid derivatives involved in plant defense and pigmentation (Jiang et al., 2016 ). F3H is crucial for flavonoid biosynthesis, where it hydroxylates flavanones like naringenin to produce 3-hydroxy flavonol. This compound is a key precursor for the formation of anthocyanins, flavanols, and proanthocyanidins (Pervaiz et al., 2017 ; Liu et al., 2018 ). Recent studies have increasingly concentrated on the protective role of flavonoids in stress responses. These compounds are particularly noted for their involvement in responding to abiotic signals, including temperature variations, nutrient scarcity, and water deficiency. Flavonoids have been shown to play a crucial role in helping plants adapt to and withstand these challenging environmental conditions. Research has demonstrated that flavonoids rapidly accumulate in plants exposed to drought conditions, providing a protective function (Winkel-Shirley, 2002 ; Li et al., 2016 ). Furthermore, flavonoids may help mitigate oxidative stress triggered by environmental challenges (Pietta, 2000 ). Observations have shown that genes related to the flavonoid pathway are up-regulated in response to stress in various plants, including potatoes, birch, and rice (André et al., 2009 ). These studies suggest that the flavonoid pathway plays a significant role in stress responses. However, the specific roles of key enzymes and the regulatory mechanisms involved in the flavonoid biosynthetic pathway under abiotic stress conditions are not yet fully understood. Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a significant C4 Panicoid crop cultivated for its grain, forage, and stover, particularly in the arid and semi-arid regions of Africa and Asia (Shivhare et al., 2024). Ranked as the sixth most important cereal after rice, wheat, maize, barley, and sorghum, it is highly valued for its rich nutritional profile and is also recognized as a promising feedstock for biofuel production (Lata et al., 2013 ; Shivhare and Lata, 2016 ; 2017 ). Pearl millet thrives in regions with low rainfall and demonstrates exceptional adaptability to different abiotic stresses, including high temperatures, osmotic stress, and drought, whether these challenges arise individually or in combination. Its resilience under harsh environmental conditions makes it an ideal model for studying the molecular basis of abiotic stress tolerance. Furthermore, it serves as a crucial resource for functional genomics research focused on improving stress resistance in crops (Shivhare and Lata 2024 ). Genome sequencing of pearl millet has identified an abundance of gene families associated with secondary metabolite regulatory pathways, including those involved in flavonoid, anthocyanin, terpene, and wax biosynthesis, which may contribute to its drought tolerance (Varshney et al., 2017 ). Additionally, transcriptomic studies under drought stress have shown an upregulation of genes related to the phenylpropanoid pathway, further highlighting its adaptive mechanisms (Shivhare et al., 2020a ; Shivhare et al., 2020b ). In this study, the flavanone 3-hydroxylase gene ( PgF3H ) from the flavonoid biosynthetic pathway was isolated from pearl millet and successfully expressed in Arabidopsis thaliana wild type (Col0) and specific mutants designed to disrupt the flavonoid biosynthetic pathway, aiming to explore the stress tolerance mechanisms mediated by flavonoids under drought conditions. These Arabidopsis mutants, including the AtSALK_113904; tt6 mutant with a crucial loss of function in flavanone 3-hydroxylase (F3H) necessary for flavonol synthesis, resulting in undetectable kaempferol or quercetin levels, were employed alongside mutants AtSALK_073183; tt11-11 and ANR, and AtSALK_040250; ban4, with disruptions in the anthocyanidin synthase and reductase genes, respectively, affecting anthocyanin content and seed coat pigmentation. Overexpressing PgF3H in these lines, we investigated the gene's expression levels, enzyme activity, total flavonoid and anthocyanin contents, and the antioxidant capacity of flavonoids. This comprehensive approach allowed us to assess the contribution of the PgF3H gene to enhancing drought stress tolerance through flavonoid-mediated pathways in both the wild-type Col0 and the mutant Arabidopsis plants, providing insights into the underlying stress response mechanisms in pearl millet. Material and methods Plant Materials, RNA Isolation PRLT2/89 − 33 seeds were grown in 3 kg pots with composite soil inside a glasshouse under controlled conditions: 32 ± 2°C (day), 28 ± 2°C (night), 40–70% humidity, and natural sunlight. Drought stress was imposed at the full panicle emergence stage (40 days after sowing) using a dry-down protocol. Two groups—well-watered (WW) and water-stressed (WS)—were maintained, each with three replicates. WW plants had an NTR of 1, while WS plants were stressed until NTR ≤ 0.2. Leaf samples were collected, frozen in liquid nitrogen, and stored at -80°C to preserve RNA integrity, ensuring reliable molecular analysis of drought stress responses (Shivhare et al., 2020a ). The total RNA was isolated from flag leaf using RNAiso plus Reagent (TaKaRa, Japan). RNA samples with OD260/OD230 (≥ 2.0) and OD260/OD280 nm absorption ratio (1.98–2.01) and were used for cDNA synthesis. About 5 µg of total RNA underwent DNase treatment to ensure thorough removal of any DNA contamination. This process utilized the RNase-free DNase set from Qiagen, Germany, or TURBO DNAse from Ambion, effectively eliminating potential DNA residues from the RNA samples. First-strand complementary DNA (cDNA) was synthesized using either the SuperScript™ III First-Strand Synthesis System (Invitrogen). The reaction was carried out with 1 µg of DNA-free total RNA as the template, utilizing oligo(dT) primers to ensure selective reverse transcription of polyadenylated messenger RNA (mRNA). The total reaction volume was adjusted to 20 µl, following the respective manufacturer’s protocols for optimal efficiency. After synthesis, the resulting cDNA was stored at -20°C to preserve its integrity for subsequent gene expression and molecular analyses. Selection of terminal drought-responsive gene from pearl millet Pearl millet terminal drought-responsive gene, flavanone 3-hydroxylase gene ( PgF3H ) was selected from previous data, have the higher expression under drought stress for cloning and functional characterization (Shivhare et al., 2020). Complete open reading frame (ORF) of PgF3H (1020 bp) was amplified from water stressed RNA sample of pearl millet drought tolerant genotype, PRLT2/89 − 33 using gene-specific primers (Supplementary Table 1) by PCR using high fidelity Platinum™ Taq DNA Polymerase (Invitrogen). Sequence homology and phylogenetic analysis Multiple sequence alignment was conducted using ClustalOmega to identify conserved regions and assess the functional homology of the PgF3H gene with selected orthologous genes from various plant species. The analysis was performed through the ClustalOmega web server ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ) to ensure accurate sequence comparison. A phylogenetic tree was constructed using the neighbor-joining method with a bootstrap value of 1000 for statistical reliability, employing MEGA6 software. Additionally, all protein sequences were retrieved and analyzed through the NCBI database using the web-based BLAST tool ( http://ncbi.nlm.nih.gov/blast ) to determine sequence similarity and evolutionary relationships with other plant species. In silico analysis of promoter, structure, and homology modeling of the PgF3H Protein The promoter sequence of PgF3H gene was analyzed using PlantCARE (Lescot et al., 2002) and PlantPAN (Chang et al., 2008) databases as well as motifs taken from the literature for putative cis-acting elements. The PgF3H molecular model was created using the web-based homology modelling server SWISS-MODEL automated protein homology modelling server ( http:/swissmodel.expasy.org/workspace ) to create homology models of protein structure using as guide information on the crystal structure of the recombinant proteins. The molecular model of PgF3H was visualized using the PyMOL software ( http://pymol.sourceforge.net/ ). To predict the secondary structure of the protein, the PSIPRED server ( http://bioinf.cs.ucl.ac.uk/psipred/ ) was utilized, following the methodology described by Jones (2002). Isolation of the 5ˊ-flanking region of PgF3H The 5′ flanking sequence of PgF3H (Pgl_GLEAN_10000809) was retrieved from the IPMGSC database ( https://cegsb.icrisat.org/ipmgsc/ ). Forward and reverse primers were meticulously designed based on the PgF3H sequence along with its 5′ flanking region (Supplementary Table 2). A 1109-base pair fragment located upstream of the translation start codon “ATG” of the PgF3H gene was identified and subsequently amplified from the genomic DNA of the pearl millet genotype PRLT2/89 − 33. This amplification was performed using polymerase chain reaction (PCR) with primers specific to the PgF3H promoter. The amplified fragment was then verified through sequencing to ensure accuracy. Construction of transformation vectors and generation of transgenic plants The promoter region of PgF3H , spanning from − 1109 base pairs to − 1 base pair, was isolated through PCR amplification using specific oligonucleotide primers (Supplementary Table 2). For the purpose of constructing PgF3H promoter::GUS vectors, the PCR products were inserted into the pBI101.2 plasmid (Cambia, Australia) at the HindIII and BamHI restriction sites. This insertion was subsequently verified by sequencing. The confirmed plasmids were then utilized to transform A. thaliana . A. thaliana wild type and three Arabidopsis mutants from phenylpropanoid biosynthesis pathways in the Col0 background were employed to develop lines overexpressing PgF3H gene. The AtSALK_113904; tt6 mutant, characterized by the loss of function of flavanone 3-hydroxylase (F3H), crucial for flavonol synthesis, exhibited undetectable levels of kaempferol or quercetin, the primary flavonol components in Arabidopsis . Additionally, the mutants AtSALK_073183; tt11-11 and ANR, along with AtSALK_040250; ban4, had insertions in the ANS gene encoding anthocyanidin synthase and the ANR gene encoding anthocyanidin reductase, respectively. The tt11-11 mutants lacked red pigmentation due to the absence of anthocyanins, while ban-4 mutants displayed a red seed coat in immature seeds and a darker black seed coat in fully desiccated seeds (Bowerman et al., 2012). The full-length cDNA of the PgF3H gene was cloned into the pCAMBIA1304 vector (Cambia, Australia) under the regulation of the CaMV35S promoter. To achieve this, the complete PgF3H gene was PCR amplified using gene-specific primers. The cDNA template for this amplification was derived from the flag leaf of the pearl millet genotype PRLT2/89 − 33 (Supplementary Table 2) using Platinum II Taq Hot-Start DNA Polymerase (Thermo Fisher Scientific, United States). The amplicon was cloned into pCAMBIA1304 plant binary vector using the NcoI and BstII restriction sites and transformed into Agrobacterium GV3101. A. thaliana Col-0 and mutant lines were transformed using the floral dip method facilitated by Agrobacterium tumefaciens . After transformation, T0 seeds were harvested from treated plants and sown on 1/2 Murashige and Skoog (MS) medium supplemented with 30 mg/L hygromycin (Sigma-Aldrich, USA) for selection of transformants. Hygromycin-resistant seedlings were identified after eight days of germination and subsequently transferred to soil for further growth in a controlled environment. To serve as controls, plants transformed with an empty pCAMBIA1304 vector were also generated for comparative analysis. Histochemical GUS staining GUS staining was carried out following a modified protocol from Sharma et al. ( 2020 ). Transgenic seedlings harboring the ProPgF3H::GUS construct were submerged in a staining solution composed of 100 mM sodium phosphate buffer (pH 7.2), 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide, 0.1% Triton X-100, 10 mM EDTA, and 1 mg/mL of the chromogenic substrate X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide). The samples were incubated at 37°C for four hours to facilitate the enzymatic reaction, leading to the formation of a distinct blue precipitate in regions exhibiting GUS activity. To enhance visibility and eliminate chlorophyll, the stained seedlings were washed several times with 70% ethanol. The cleared samples were then examined under a Leica microscope (LAS version 4.12.0, Leica Microsystems) to assess GUS expression patterns, providing valuable insights into the spatial regulation and activity of the PgF3H promoter in plant tissues. Expression analysis qRT-PCR was conducted using SYBR Green chemistry on a 7500 Fast Real-Time PCR System (Applied Biosystems). Each 10 µl reaction contained 1 µl of diluted cDNA (50 ng RNA equivalent), 10 µM gene-specific primers, and 5 µl of 2X Power SYBR Green PCR Master Mix. The cycling conditions included 50 ºC for 20 s, 95 ºC for 10 min, followed by 40 cycles of 95 ºC for 15 s and 60 ºC for 1 min. Melt curve analysis (60 ºC–90 ºC) was used to confirm amplification specificity (Supplementary Table 1). Assessment of drought tolerance in transgenic A. thaliana plants The drought stress tolerance of PgF3H -overexpressing transgenic A. thaliana plants, along with the mutants Atf3h (AtSALK_113904), Atans (AtSALK_073183), and Atanr (AtSALK_040250), was assessed using the three best homozygous lines. At the germination stage, T3 seeds of transgenic lines, wild-type (Col-0), and empty vector (EV) controls were surface sterilized and stratified in darkness at 4°C for three days. The stratified seeds were then placed on ½ MS medium supplemented with PEG 6000 (0, 1.5, 3, and 6%) or mannitol (0, 100, 200, and 300 mM). Germination percentage was recorded on the ninth day to evaluate stress tolerance. Seeds of transgenic lines, WT and EV were grown on composite soil in well-watered conditions supplemented with nutrient media for four weeks in order to evaluate the drought tolerance behavior under the influence of physiological drought conditions. There were two treatments: control (WW) and water stressed (WS) plants. WW plants were watered regularly and WS plants were exposed to water stress by withdrawing the water for next 14 days. At 15 days, leaf and root tissue of both the treatments were harvested for physiological and biochemical assays and for RNA isolation leaf tissue were frozen in liquid nitrogen and stored in -80 ºC. Water loss was assessed in one-month-old plants subjected to 14 days of drought stress. Fresh leaves from control and stressed plants were harvested, weighed immediately, and dried on filter paper at 25°C (50–60% RH). Leaves were reweighed at 0 h, 1 h, 2 h, and 4 h to estimate water loss based on initial fresh weight. Electrolyte leakage (EL) was measured following Dionisio-Sese and Tobita (1998). Root tissues (100 mg) from control and stressed plants were immersed in 20 ml deionized water and shaken at 100 rpm for 2 h. Initial conductivity (C1) was recorded, followed by boiling for 30 min to release electrolytes. After cooling, final conductivity (C2) was measured, and EL was calculated as (C1/C2) × 100. Leaf relative water content (RWC) was measured following Slatyer (1967). Fully expanded leaves were excised, and their fresh weight (LFW) was recorded. Leaves were then submerged in distilled water for 4 hours at room temperature, blotted dry, and weighed for turgid weight (LTW). After drying at 80°C for 24 hours, dry weight (LDW) was recorded. RWC (%) was calculated using Barrs and Weatherley’s (1962) formula: RWC (%) = [(LFW – LDW) / (LTW – LDW)] × 100. Total chlorophyll content was estimated by suspending 100 mg of leaf tissue in 10 mL of 80% acetone, homogenizing, and incubating in darkness at 4°C overnight. The extract was centrifuged at 5000 rpm, and absorbance was recorded at 663 and 645 nm using a spectrophotometer. Chlorophyll content was calculated following Arnon ( 1949 ). Biochemical and enzymatic assays Leaf tissues were ground in liquid nitrogen and homogenized in bicarbonate buffer to extract total soluble proteins, which were then quantified using the Bradford assay (Bradford, 1976). The supernatant obtained after centrifugation was mixed with Bradford reagent, and absorbance was measured at 595 nm using a BSA standard curve. Proline content was determined following the method of Bates et al. ( 1973 ), where leaf tissues were homogenized in sulfosalicylic acid, reacted with ninhydrin and acetic acid, and heated. The proline-chromophore complex was then extracted with toluene, and its absorbance was recorded at 520 nm. Lipid peroxidation levels were assessed by estimating malondialdehyde (MDA) content through the TBARS assay. Leaf extracts were treated with thiobarbituric acid (TBA), heated, and absorbance was measured at 532 nm. Antioxidant enzyme activities were evaluated by different spectrophotometric assays: superoxide dismutase (SOD) activity was analyzed based on its inhibition of nitroblue tetrazolium (NBT) reduction at 560 nm (Beauchamp and Fridovich, 1971 ); catalase (CAT) activity was assessed by monitoring the decomposition of H₂O₂ at 240 nm (Aebi, 1974 ); and ascorbate peroxidase (APX) activity was measured by tracking ascorbate oxidation at 290 nm (Nakano and Asada, 1981 ). Guaiacol peroxidase (GPX) activity was analyzed by tracking guaiacol oxidation at 470 nm (Hemeda and Klein, 1990 ). Chlorophyll content was determined using acetone extraction, with absorbance measured at 663 nm and 645 nm (Arnon, 1949 ). Reactive oxygen species (ROS) accumulation was evaluated via NBT and DAB staining. Superoxide anions were visualized as blue formazan deposits from NBT staining, while H 2 O 2 accumulation was identified by brown precipitates from DAB staining. Quantitative densitometry of the stained tissues was performed using ImageJ software(version 1.46 for Windows 8), providing a comparative analysis of ROS levels across treatments (Schneider et al., 2012 ). This comprehensive approach highlights biochemical and physiological changes under various plant treatments. Total anthocyanin quantification Total anthocyanin was measured as previously recommended method (Li et al., 2016 ). In this procedure, leaf tissue of control and stressed plants (100 mg) were crushed in liquid N2 and transmitted to extraction buffer solution (propanol: HCl: H 2 O; 18:1:81). Samples solutions were boiled for 3 min at 95ºC then incubated in the dark condition for 2 h at room temperature. Samples were centrifuged and supernatant was collected in fresh tube. Absorbance of the supernatant solution was taken at 535 and 650 nm. Anthocyanin content was calculated as (A535–2.2A650)/g • FW. Total flavonol quantification Flavonoid content was measured using a modified aluminum chloride colorimetric assay based on Loyola (2016). Leaf tissues from control and drought-stressed plants were extracted in 1 ml of 80% ethanol at 4°C for 2 hours with continuous shaking. The extracts were centrifuged at 12,000×g for 12 minutes, and the supernatant was collected. For quantification, 5 ml of the supernatant was diluted with methanol to a final volume of 2 ml and mixed with 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate, and 2.8 ml of Milli-Q water. After 30 minutes of incubation, absorbance was recorded at 415 nm. Flavonoid concentration was determined using a quercetin standard curve. Extraction and quantification of flavonols To extract flavonols, 500 mg of seedlings were ground in liquid nitrogen and then extracted in 80% methanol overnight at room temperature. The extracts were subsequently hydrolyzed with an equal volume of 6 N HCl at 70°C for 40 minutes. To prevent the precipitation of aglycones, an equal volume of methanol was added after hydrolysis. The resulting extracts were filtered through 0.2 µm filters (Millipore) prior to metabolite analysis. The analysis was performed using High-Performance Liquid Chromatography with Photodiode Array detection (HPLC-PDA), employing a Waters 1525 Binary HPLC Pump system equipped with a PDA detector. The method followed was developed by Niranjan et al. ( 2011 ). Quantification of the various metabolites was achieved using Breeze 2 software (Waters). Statistical Analysis The results are expressed as the mean ± standard deviation (SD) from three independent experiments conducted under identical environmental conditions. Statistical significance between control and drought-stressed samples was assessed using one-way analysis of variance (ANOVA). Further comparison of mean values was performed using Duncan’s multiple range test in SPSS software (version 16.0, SPSS Inc./IBM Corp., Chicago, USA). Graphical representation of the experimental data was generated using GraphPad Prism software (version 5.03, San Diego, California, USA). Results Validation, characterization, cloning sequence analysis of the PgF3H gene Pearl millet terminal drought-responsive gene, PgF3H (Pgl_GLEAN_10000809) was identified from pearl millet exposed to drought stress. To confirm the increased expression of PgF3H in a drought-tolerant pearl millet genotype, a quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis have been performed. The results revealed a significant up-regulation of PgF3H , showing a 7-fold increase in expression under drought stress conditions. The PgF3H is located on pearl millet chromosome number 3 and has 1020 bp open reading frame (ORF), was amplified using RT-PCR and cloned in to plant expression vector, pCAMBIA1304 and then in to Agrobacterium strain GV3101 confirmed with colony PCR (Supplementary Figure S1 ). Multiple sequence alignment of PgF3H exhibit high resemblance with other plant Flavonone 3-hydroxygenase encoding genes (Supplementary Figure S2a). High sequence similarity in the 2OG-Fe(II) oxygenase domain suggested the importance of conservation of F3H genes among gramineae crops. In order to further establish the closeness of PgF3H to the other 2OG-Fe(II) oxygenase proteins, a systematic phylogenetic study focused on the similarity of 2OG-Fe(II) oxygenase domains in proteins isolated from Arabidopsis , cotton, capsicum, maize, rice, sorghum, barley, cannabis and wheat using MEGA 6.0 by NJ method was performed (Supplementary Figure S2b). Structure of the full length PgF3H protein The deduced PgF3H protein contained 339 amino acids with a molecular mass estimated to be 38.75 kDa and a theoretical pI of 5.67. Recently full-length F6H proteins from Arabidopsis containing a family protein domain of 2OG-Fe(II) oxygenase have been discovered which are found to play a key role in flavonoid biosynthetic pathway (Sun et al., 2015 ). At the primary amino acid sequence level, PgF3H and AtF6H proteins shared 39 percent similarity. Using the homology-modeling server SWISS-MODEL, Arabidopsis F6H protein (PDB ID: 4XAE) crystal structure was chosen as a template for PgF3H model development. The PyMOL software ( http:/pymol.sourceforge.net/ ) was used to generate PgF3H molecular model (Supplementary Figure S3a). Generally, PgF3H overall architecture was close to that of AtF6H , and the Arabidopsis protein's structural findings were also relevant to the PgF3H protein. A protein domain study using the CDD database showed the existence of highly conserved PLN02639 domain [2OG-Fe(II) oxygenase family protein)], a single member of superfamily cl31913 with secondary metabolite activity, transport and catabolism ( https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml ). The secondary structure analysis of the PgF3H amino acid sequence using PSIPRED revealed the presence of three alpha-helical structures near the N-terminus and two alpha-helical structures near the C-terminus (Supplementary Figure S3b). Further structural modeling using SWISS-MODEL software confirmed these findings, showing that the protein structure of PgF3H comprises 12 β-strands and 16 α-helices. This structural data aligns with the secondary structure prediction made by PSIPRED. Additionally, hydropathy analysis indicated that the predicted PgF3H protein is hydrophilic, with the highest level of hydrophilicity observed near the N-terminus. Cloning and sequence analysis of the PgF3H promoter region The PgF3H promoter region ( PgF3H pro) was isolated from pearl millet genomic DNA with PgF3H pro specific primers and confirmed by sequencing. PgF3Hpro sequence was analyzed in silico using the PlantCARE database which showed that PgF3Hpro harbors cis -acting elements, including ABRE (involved in abscisic acid (ABA) response), Box 4, G-box, GT1-motif (light responsive component), two copies of AuxRR-core, TGA-element (involved in auxin response), MBS (MYB binding site involved in drought-inducibility), TGACG-motif (MeJA-responsivenes), and LTR (Low-temperature response (Supplementary Figure S4a; Supplementary Table 2). Selection and assessment of PgF3H promoter A.thailiana line under water deficit condition To analyze the activity of PgF3H promoter ( PgF3Hpro ) in response to stress condition, A. thaliana promoter–reporter lines (ProPgF3H::GUS) were generated and positive lines were selected on kanamycin + ½ MS selection plate harboring the PgF3Hpro sequence. Homozygous PgF3Hpro A. thailiana transgenic lines were analyzed for water deficit response. The promoter lines were grown on ½ MS media with or without supplementation with PEG (0, 1.5, 3 and 6%). A histochemical GUS assay showed progressive increase in GUS activity in ProPgF3H::GUS seedlings with the increase of PEG concentration (Supplementary Figure S4b). Modulation of water stress response in PgF3H overexpressing A.thailiana line under water deficit condition Transgenic Arabidopsis plants with resistance to hygromycin were developed by incorporating the PgF3H gene. To confirm the successful integration of the PgF3H transgene into the genome of the selected Arabidopsis thaliana lines, PCR analysis was performed. The results of the PCR analysis revealed the amplification of a 1020-bp fragment corresponding to PgF3H, thus verifying the presence of the transgene in these genetically modified lines. Best three homozygous T3 transgenic lines with higher expression of PgF3H were selected for further analysis (Supplementary Figure S5 a-f). These three PgF3H overexpressing A. thaliana lines were further used for water stress assessment assays. PgF3H overexpressing A. thailiana lines were analysed for water stress tolerance capacity at germination level and in four week old plants. Germination percentage (G%) of WT, EV and PgF3H overexpressing lines (OE1, OE2 and OE3) were normal and nearly 100% in ½ MS media with no treatment. In response to different concentrations of mannitol and PEG treatment, G% was found to vary in PgF3H overexpressing lines compared to WT and EV. Under simulated osmotic stress conditions induced by PEG, the germination rate of the transgenic lines showed significant improvements compared to the wild-type (WT). By day 9, the average germination rates for transgenic lines were 83.5%, 62.2%, and 42% under 1.5%, 3%, and 6% PEG concentrations, respectively. In contrast, the WT plants exhibited germination rates of 64.6%, 27.3%, and 16% under the same conditions. The empty vector (EV) plants showed germination rates of 64%, 26%, and 15.3% under the respective PEG concentrations. At 100, 200 and 300 mM mannitol treatment, the average G % of transgenic seeds were 76, 60 and 42.6% compared to 48, 20.6 and 12% for WT and 44.6, 20 and 14% for EV seeds on the 9th day of treatment (Supplementary Fig. 6). To assess the water deficit resistance of PgF3H- overexpressing transgenic A. thaliana plants, they were cultivated in soilrite under controlled conditions for 4 weeks in a growth chamber. Following this period, water stress was induced by withholding water for the subsequent 14 days (see Fig. 1 a). The growth of the PgF3H overexpressing lines, WT and EV plants was similar under well-watered conditions. However, only 50% of the WT and 48.7% of EV plants survived under water-deficit conditions, while the PgF3H overexpressing plants showed a survival rate of about 81% (Fig. 1 c). Further, dry weight of drought stresses plants were calculated and result showed significant higher dry weight of PgF3H overexpressing Arabidopsis plants compared to WT and EV plants (Fig. 1 b). The RWC, water loss and electrolytic leakage were also calculated in control and stressed plants of PgF3H overexpressing lines, WT and EV (Fig. 1 d). Average RWC for PgF3H overexpressing Arabidopsis plants were 86% whereas the same for WT and EV were 84, and 86% in controlled plants treatment. Under drought stress, average RWC of PgF3H overexpressing plants was 65% whereas in WT and EV was 42%. The average water loss after 1, 2, and 3 h was 29.6, 54.7, and 66.84% respectively in PgF3H overexpressing plants compared to 30, 53, and 65% in the WT and 29.9, 54.6 and 68% in EV plants under well- watered conditions whereas 40.7, 66.5 and 84.5% in PgF3H overexpressing plants compared to 50, 80 and 94% in WT and 52, 78.3 and 95% in EV plants under water-deficit condition (Fig. 1 e). Average EL of PgF3H overexpressing plants was 13.6% compared to 14% in WT and 14.3% in EV plants in control conditions and 24.6% for transgenic lines compared to 37% for WT and 38% for EV plants under drought stress condition (Fig. 1 f). Proline, MDA and chlorophyll content were also calculated in order to see the effect of water stress on various biochemical parameters under drought stress. Average 2-fold higher accumulation in proline content was recorded in PgF3H overexpressing lines while 1.3-fold increase was observed in WT (2.4-fold), and 1.4-fold in EV (2.8-fold) in water stressed tissues compared control plants (Supplementary Figure S7a). An estimation of MDA content showed 1.3-fold average increase in PgF3H overexpressing lines, lowered to WT and EV have more than 3-fold increase under water deficit condition (Supplementary Figure S7b). In response to drought stress, chlorophyll content decreases in WT, EV as well as in transgenic lines, however no significant decrease was recorded in PgF3H overexpressing Arabidopsis lines (Supplementary Figure S7c). To evaluate the impact of drought stress on the antioxidant levels of transgenic lines relative to the wild type (WT), various enzymatic assays including APX, CAT, and SOD were conducted under both control and water stress conditions. The results of the enzymatic assays showed no notable differences among the transgenic lines, WT, and EV plants under normal conditions. However, their activities significantly increased in both WT and transgenic plants under water-deficit conditions. Specifically, in response to drought stress, the average APX and CAT activities were 1.4 and 1.6 times higher in transgenic plants compared to WT plants, respectively, whereas there was no significant difference in SOD activity among PgF3H overexpressing lines, WT, and EV plants (Supplementary Figure S8a-c). These findings were further supported by the gene expression analysis of APX, CAT, and SOD genes using qRT-PCR (Supplementary Figure S8d-f). Change in total flavonols and anthocyanin content in PgF3H overexpressing plants Total flavonols and anthocyanin content were estimated in WT, EV and transgenic plants under control and drought stress conditions to assess the role of PgF3H in the synthesis and accumulation of these metabolites (Fig. 2 a; Fig. 3 a-b). Results showed in controlled as well as in drought stressed leaf samples of WT, EV and transgenic plants have significant difference in accumulation of these metabolites with higher content in PgF3H overexpressed lines. Average accumulation of total flavonols and anthocyanin were about 1.5 and 2.5-fold higher in PgF3H overexpressing plants as compared to WT under the water deficit environment (Fig. 2 a; Fig. 3 a). It was also confirmed by the up regulation of expression of genes involved in flavonols ( AtFLS1 , AtFLS3 , and AtUGT78D2 ) and anthocyanin biosynthesis ( AtDFR, and AtLDOX ) in transgenic plants compared to WT and EV in response to drought stress (Fig. 2 b-f; Fig. 3 c-d). However, AtUGT78D3 did not show any major change in its expression under drought stress in WT as well as in transgenic lines. HPLC analysis results also revealed that control and drought stressed leaf samples of WT, EV and transgenic lines for flavonol metabolites including kaempferol, quercetin and rutin also have significantly enhanced expression in transgenic lines compared to its control as well as WT under water deficit conditions (Supplementary Fig. 9). In response to drought stress, average accumulation of kaempferol, quercetin and rutin were 1.8-, 2.10- and 2.11- fold respectively, higher in comparison to WT. Development and analysis of PgF3H overexpressing mutants To further confirm the function of PgF3H gene involved in flavonoid biosynthesis, PgF3H was transformed in Atf3h mutant (AtSALK_113904; tt6) have the loss of function of flavanone 3-hydroxylase (F3H) involved in flavonols formation, Atans mutant (AtSALK_073183; tt11-11) and Atanr mutant (AtSALK_040250; ban4) having the insertion in anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) genes respectively involved in anthocyanin biosynthesisin Col0 background. Transgenic seeds for each mutant ( Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H ) having the insertion of PgF3H gene were screened on ½ MS + hygromycin plate. T3 homozygous seeds of the overexpressing or transgenics lines (OE1, OE2 and OE3) of each mutant harboring the PgF3H gene were generated and the PCR results showed the amplification of a 1020-bp fragment of PgF3H confirming the integration of the transgene in the genome of Atf3h, Atans and Atanr mutants (Supplementary Fig. 10). Best three homozygous T3 transgenic lines of mutants with higher expression of PgF3H were selected for further analysis (Supplementary Fig. 10) and were further used for water stress assessment assays. Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H overexpressing A. thailiana lines (OE1, OE2 and OE3) were analysed for water stress tolerance capacity in four week old plants. To analyze the water stress tolerance capacity of mutants PgF3H transgenics, were grown for four weeks than subjected to drought stress by withdrawing water for the next 14 days (Fig. 4 ). The growth of the Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H transgenics and all the mutants plants were similar under WW conditions. However all Atf3h mutant seeds were grown in WW condition but the rosette size were smaller than Atf3h + PgF3H plants. Under the water deficit condition PgF3H overexpressing transgenic lines of all the three mutants have significant higher survival percentage as well dry weight compared to their respective mutant plants (Fig. 4 ). Further Atans + PgF3H , and Atanr + PgF3H transgenics have almost similar survival percentage and dry weight as was showed by PgF3H Col0 transgenic plants while Atf3h + PgF3H transgenics have lower survival percentage and dry weight than PgF3H Col0 transgenic plants but significant higher than Atf3h mutant plants under WS condition (Fig. 4 ). Gene expression of antioxidant enzymes including AtAPX, AtCAT and AtSOD showed significantly higher expression in all the mutants and Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H transgenic plants in response to WS compared to WW condition but almost all the transgenics have lower expression for these antioxidant enzymes compared to respective mutant under WS. While PgF3H overexpressing, mutant transgenic plants have the lower expression of AtAPX, AtCAT and AtSOD genes compared to their respective mutant plants under WS condition (Supplementary Fig. 11). Change in total flavonols and anthocyanin content in PgF3H overexpressing mutants plants To evaluate the role of PgF3H gene involved in flavonoid biosynthesis, Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H transgenics lines (OE1, OE2 and OE3) were analyzed for flavonone and anthocyanin contents. Results showed Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H overexpressing plants have enhanced flavonone content under water deficit condition (Fig. 5 ; Fig. 6 ). Atans + PgF3H , and Atanr + PgF3H have flavonone content comparable with PgF3H overexpressing Col0 plants whereas, At f 3h + PgF3H has higher flavone content compared to Atf3h mutant plants but lower than PgF3H overexpressing Col0 plants. These results were also supported by the gene expression analysis of flavonone biosynthesis genes ( AtFLS1 and AtFLS3) which showed higher expression in all the mutant transgenic plants in response to WS. Anthocyanin content was found to be higher in Atf3h + PgF3H , and Atanr + PgF3H whereas there was no increase in anthocyanin content in Atans + PgF3H transgenic plants. Gene expression analysis of anthocyanin biosynthesis genes including AtDFR, AtLDOX, AtUGT78D2 , and AtUGT78D3 also have the increased expression in Atf3h + PgF3H , and Atanr + PgF3H transgenic plants while Atans + PgF3H plants have no significant change in expression of these genes in response to WS condition. Genes involved in lignin biosynthesis ( AtCCR1 and AtCAD6) exhibited significantly higher expression in Atf3h + PgF3H , Atans + PgF3H plants except AtSND1 under WS whereas Atanr + PgF3H follow the similar expression pattern of these genes as found in PgF3H Col0 transgenic plants. Surprisingly gene expression of AtHCT involved in flavonoids accumulation was significantly higher in Atans + PgF3H , and Atanr + PgF3H transgenic plants compared to their mutants while Atf3h + PgF3H showed decline in expression of AtHCT gene as present in PgF3H Col0 transgenic plants under WS. Flavonol metabolites including kaempferol, quercetin and rutin were also evaluated in Atf3h + PgF3H and Atf3h mutant plants using HPLC. Atf3h + PgF3H transgenic plants have significant higher accumulation of these flavones compared to Atf3h mutant plants under water stress and the average accumulation of kaempferol, quercetin and rutin were 1.57-, 4.75- and 1.9- fold higher respectively, in comparison to control Atf3h + PgF3H transgenic plants (Supplementary Fig. 12). Change in lignin biosynthesis gene expression in PgF3H overexpressing Col0 and mutants plants To determine the impact of PgF3H gene overexpression on the lignin production pathway, expression analyses of the genes ( AtSND1, AtCCR1 , and AtCAD6) were carried out in PgF3H overexpressing Col0 and mutants plants. Expression profile of lignin biosynthesis genes were either down regulated or having basal expression in both the WT and PgF3H overexpressing Col0 and mutants lines (Supplementary Fig. 13). PgF3H overexpressing Col0, Atans + PgF3H and Atanr + PgF3H transgenic plants showed the similar expression of lignin biosynthesis genes with lower expression in response to drought stress (Supplementary Fig. 14). While during drought stress, AtSND1, AtCCR1 , and AtCAD6 genes are expressed more in Atf3h mutant and Atf3h + PgF3H transgenic plants than in their respective controls, although Atf3h mutant drought plants higher expression of these genes compared to Atf3h + PgF3H transgenic plants. Effect of PgF3H gene in ROS accumulation in PgF3H overexpressing Col0 and mutants plants Plants exposed to drought stress can cause ROS accumulation. The level of endogenous O2¯ in leaves was measured using NBT staining, and of H 2 O 2 was measured using DAB staining. Under well-watered condition, all the WT, mutants, PgF3H overexpressing Col0 and mutants OE1, OE2 and OE3 transgenic lines showed similar basal levels of superoxide anion radicals and H 2 O 2 except Atf3h plants. However, elevated NBT staining, representing higher O2¯ levels was observed in WT and mutant plants compared to PgF3H overexpressing Col0 and mutants transgenic plants under drought conditions. Likewise, enhanced DAB staining was observed in WT and mutant plants, representing higher levels of H 2 O 2 , relative to that in PgF3H , overexpressing transgenic lines under drought conditions (Supplementary Fig. 15). Quantification of both the staining was done using ImageJ software which also confirmed higher accumulation of ROS in WT as compared to transgenic lines under drought stress (Supplementary Fig. 15). These findings show that overexpression of PgF3H can improve the tolerance of plants to drought stress in Arabidopsis . Discussion Flavonoids are a group of natural compounds present in vegetables and fruits, serving as significant antioxidants in the human diet while also playing essential roles in various fundamental plant functions (Song et al., 2016 ; Wang et al., 2010 ). Flavonoids derived from a phenylpropanoid pathway where F3H is essential for the formation of intermediate shared biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins (Pervaiz et al., 2017 ; Song et al., 2016 ). In our previous transciptome study, PgF3H gene responsible for flavonoids biosynthesis exhibited higher expression in pearl millet drought tolerant genotype under terminal drought stress (Shivhare et al., 2020a ; Shivhare et al., 2020b ). In this work, PgF3H was overexpressed in Arabidopsis to facilitate detailed functional analysis, focusing on screening transgenic plants for their response to drought stress. The coding sequence of PgF3H underwent bioinformatic characterization, revealing significant sequence similarity with F3H proteins from various plant species, particularly those within the Gramineae family. Five conserved motifs, which include residues binding 2-oxoglutarate and ferrous ions within the 2OG-Fe(II) oxygenase domains of F3H proteins, show a high degree of similarity across various plant species (Song et al., 2016 ; Lukacin and Britsch, 1997 ; Britsch et al., 1992 ). To evaluate the evolution relationship of PgF3H and other orthologs in plants, a phlyogenetic tree was built and the results showed that PgF3H is substantially identical to XP 004976589.1, XP 025825120.1, XP 002446984.2, THU69177.1, XP 030501918.1, PHT87608.1, and XP 016738006.11, this is why these proteins are in the same phylogenetic research subgroup. Through in silico sequence analysis of approximately 1 Kb of the PgF3H gene promoter, various putative cis-acting regulatory elements were identified. These include GTAC motifs associated with anoxic stress response, LTR elements linked to low temperature responsiveness, G-box motifs indicative of light responsiveness, ARE elements for anaerobic induction, as well as core sequences of ABRE elements involved in ABA responsiveness, and MBS motifs associated with drought inducibility. This indicates that not only the F3H gene is activated during drought stress, but its promoter is also stimulated to protect the plants during environmental stresses and it was confirmed by gradual increase in GUS expression in Pro PgF3H ::GUS Arabidopsis seedlings by increasing the PEG6000 concentration. However, in combination with the corresponding transcription factors, the presence of various cis -regulatory elements and their coordinated action can controls PgF3H transcript expression in response to different climatic conditions and also in plant’s growth conditions (Shinde et al., 2018 ; Singh et al., 2015 ; Reddy et al., 2012 ). Although flavonols and anthocyanin secondary metabolites were reported for their higher accumulation in plants in response to biotic and abiotic stress induction, such as drought, salt, UV light stimulus, cold and abscisic acid (ABA) stress responses (Zhang et al., 2014 ; Jeong et al., 2004 ; Winkel-Shirley et al., 1995). In this study, PgF3H was cloned in the binary vector pCAMBIA1304 under the control of the promoter CaMV35S to observe its water stress tolerance capacity in transgenic A. thaliana. Up regulation of the PgF3H gene was seen in the qRT-PCR analysis of the produced transgenic lines. To characterize the mechanisms involved, PEG 6000 and mannitol were used to simulate water deficit conditions. For the stress tolerance assay, three homozygous (T3) transgenic lines of A. thaliana were used. These transgenic Arabidopsis plants exhibited a higher rate of germination under stress caused by PEG and mannitol. The process of maintaining the water potential in plant cells to align the osmoticum under osmotic stress with the external environment is called osmotic adjustment (Shivhare and Lata, 2019 ; Kumar et al., 2019 ). Under water deficit conditions, the aggregation of solutes results in a reduction in the cell's osmotic potential, which draws water molecules into the cells and helps to retain turgor (Lata and Prasad, 2011 ; Lata et al., 2010 ). PEG and mannitol are commonly used for the artificial induction of osmotic stress and these solutes lower the osmotic potential (Khakwani et al., 2011 ; Rauf et al., 2007 ; Bohnert et al., 1999 ). Previous studies have also shown that under mannitol, PEG and NaCl stress, higher germination rate, root length, and biomass was recorded in transgenic Arabidopsis plants that overexpress proteins related to stress response (Kumar et al., 2019 ; Yu et al., 2016 ; Xu et al., 2015 ; Huang et al., 2015 ). In recent researches, transgenic Arabidopsis and tobacco plants overexpressing regulatory proteins with flavonoids showed higher survival rates under water deficit conditions relative to WT plants (Li et al., 2017 ; Song et al., 2016 ; Mahajan and Yadav et al., 2014; Liu et al., 2013 ). Similar to other stress-responsive proteins, the constitutive overexpression of the PgF3H gene in A. thaliana plants resulted in enhanced drought tolerance. This was evidenced by reduced water loss rates and higher relative water content (RWC) in the transgenic plants (Kumar et al., 2019 ; Lu et al., 2013 ). In order to determine the water balance of plants, the RWC is seen as a significant marker (Lata et al., 2011). In other side, EL is inversely linked to the integrity of the cell membrane, and abiotic stress tolerance has generally been correlated with the ability to avoid or restore membrane damage (Lata et al., 2011). RWC and EL of both the PgF3H overexpressing lines, WT and EV were similar at WW condition while WT and EV have higher decrease in RWC and excess EL compared to transgenic lines under WS condition. However overexpressing transgenic Arabidopsis plants with PgF3H led to improved conservation of plant water status and integrity of the membrane, as confirmed by previous works (Butt et al., 2017 ; Lu et al., 2013 ). An increase in malondialdehyde (MDA) content serves as an indicator of stress-induced lipid peroxidation (LP) in cellular membranes and is considered a marker of elevated oxidative damage. These findings align with previous research which demonstrated that LP in drought-stressed Arabidopsis plants is linked to membrane integrity. Additionally, LP, along with electrolyte leakage (EL), has been recognized as a direct indicator of a plant's tolerance to dehydration stress (Dong et al., 2003 ). The reduced MDA and H 2 O 2 content in LcF3H overexpressed transgenics Arabidopsis plants under stressed and unstressed conditions also have shown correlation with our results (Song et al., 2016 ). Compatible osmolyte accumulation such as proline, by maintaining osmotic turgor, helps plants to withstand drought stress (Grover et al., 2013 ). During stress conditions, their synthesis and aggregation has been documented to enhance multiplicity (Lata et al., 2015 ). Accordingly, this study also records a rise in proline content during drought in transgenic plants. Similarly, Under water-deficit conditions, transgenic plants exhibited higher chlorophyll content compared to wild-type (WT) plants. This increased chlorophyll content was associated with enhanced net photosynthesis, suggesting that transgenic plants were able to sustain more efficient photosynthetic activity during drought stress. This observation is supported by findings from Do et al. ( 2004 ), which reported that transgenic plants maintain better photosynthetic performance under drought conditions. Drought stress effect the molecular and physiological properties of plants stemmed in to oxidative damage through the formation of toxic ROS that needs to be foraged by low molecular weight antioxidative enzymes. Plants can regulate reactive oxygen species (ROS) levels through the activity of antioxidant enzymes such as ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD). These enzymes play a crucial role in scavenging ROS molecules, thereby enhancing the plant's resistance to drought stress (Yao et al., 2017 ; Kumar et al., 2016 ; Cai et al., 2015 ; Hameed et al., 2012 ). In cell stroma, thalakoids, cytosol, and mitochondria, APX is present and serves as an electron donor for H 2 O 2 decomposition. SOD is a protection enzyme that catalyses the hydrolysis of superoxides (O 2 ¯) into less reactive molecules, such as H 2 O 2 or molecular O 2 . Finally, the H 2 O 2 thus produced is broken down into water and oxygen without the need to reduce power through the action of CAT (Shivhare and Lata, 2019 ; Lata et al., 2011). Similarly in this study, results also showed that antioxidants enzymetic activity increased in both the PgF3H transgenic lines and in WT on exposure to drought stress but transgenic plants showed lower activity of APX, CAT, GPX and SOD antioxidative enzymes compared to WT in response to drought stress. Gene expression analysis of AtAPX, AtCAT and AtSOD also showed increase expression in WT, EV, mutants, and PgF3H overexpressing Col0 and mutant transgenic plants in comparison to their control but transgenics plants have lower expression of these genes compared to WT and mutant plants in under drought stress. So these results suggested that overexpression of PgF3H gene did not induced the enzymatic pathway rather than it activate the non-enzymatic pathway via inducing the flavonone biosynthesis to overcome and minimize the oxidative stress response in PgF3H overexpressing Col0 and mutant transgenic plants and increases its survival rate compared to WT plants (Jan et al., 2022 ). F3H genes are known for their role in flavonols and anthocyanins biosynthesis, and their participation in plant responses to abiotic stresses have also been well described (Page et al., 2012 ; Quina et al., 2009 ). In response to drought stress, total flavonols and anthocyanin content was quantified in PgF3H tansgenics and WT plants. Transgenic lines have significant higher accumulation of flavonols as well as anthocyanin pigments compared to WT. It was also confirmed by higher accumulation of flavonones and anthocyanins content in PgF3H overexpressing Arabidopsis mutants ( Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H ) under drought stress. Quantification of kaempferol, quercetin, and rutin in PgF3H overexpressing Col0 and Atf3h + PgF3H transgenic plants using HPLC also showed the positive correlation with these results. All the results revealed that these secondary metabolites participate in stress responsive regulatory pathways and protect the plants from deleterious effects of drought stress (Goswami et al., 2018 ; Song et al., 2016 ; Onkokesung et al., 2014 ). Expression profiles of downstream genes of F3H including FLS1, FLS3, DFR, LDOX, UGT78D2, UGT78D3 and AUGT89A also exhibited differential expression pattern in PgF3H overexpressing Col0 and mutants transgenic plants under water deficit condition. Genes responsible for flavonol biosynthesis including FLS1 and FLS3 were highly expressed in PgF3H overexpressing transgenic lines of Col0 and mutants with more than four – tenfold higher expression for FLS3 compared to WT and mutant plants under drought stress. Along with flavonol biosynthesis, genes for anthocyanin biosynthesis including DFR, LDOX, UGT78D2 , and UGT78D3 also showed enhanced expression except UGT78D3 in transgenic lines of Col0 and mutants in response to drought stress. AtHCT involved in flavonoids accumulation was significantly higher in Atans + PgF3H , and Atanr + PgF3H transgenic plants compared to their mutants while Atf3h + PgF3H have down expression of AtHCT gene as present in PgF3H Col0 transgenic plants under WS. Whereas, under drought stress, expression of gene involved in lignin biosynthesis including SND1, CCR1 and CAD6 were either have basal expression or down regulated in PgF3H overexpressing Col0 plants as well as in WT. The downregulation of these lignin biosynthesis genes is closely associated with the observed decrease in lignin content in the genetically edited plants. This correlation suggests that the reduced expression of these specific genes directly contributes to the lower lignin levels. Whereas, Atf3h + PgF3H , Atans + PgF3H , and Atanr + PgF3H transgenic plants have up-regulation of lignin biosynthesis genes but lower than their respective mutant plants under drought stress. Overall, these findings indicate that the increased accumulation of flavonoids and anthocyanins in PgF3H overexpressing Col0 plants can be attributed to the redirection of substrate flow towards flavonoid biosynthesis, occurring at the expense of lignin synthesis. This shift in metabolic flux suggests that the overexpression of PgF3H prioritizes the production of flavonoids and anthocyanins, thereby reducing the resources available for lignin formation. These findings indicate that entire pathway possibilities are redirected to the development of flavonols and anthocyanin (Song et al., 2016 ; Onkokesung et al., 2014 ; Mahajan and Yadav, 2014 ). Therefore, we propose that the up-regulation of downstream genes within the DFR and ANS pathways in the flavonoid biosynthetic process might be responsible for the heightened anthocyanin accumulation observed in the transgenic plants. This hypothesis suggests that the increased expression of these specific genes enhances the production of anthocyanins, contributing to their elevated levels in the genetically modified plants (Survey et al., 2011). Further, flavonols and anthocyanin secondary metabolites were also reported for scavenging ROS species including, superoxide radicals O 2 ¯, hydroxyl radicals •OH and hydrogen peroxide H 2 O 2 (Di Ferdinando et al., 2012). Anthocyanins, which generally accumulate in the vacuoles, prevent oxidative damage in plants and are important for exhibiting oxidative and drought tolerance (Hernandez et al., 2004). In our study, results of NBT and DAB staining of stressed leaves of transgenic lines and WT also showed the minimum accumulation of ROS in PgF3H transgenic plants of Col0 and mutants as compared to WT and their respective mutants. In conclusion, the overexpression of flavanone 3-hydroxylase ( PgF3H ) gene in Arabidopsis helps it to better tolerate drought stress by modulating various morpho-physiological, biochemical, and molecular parameters via inducing the flavonoid biosynthesis pathway (Fig. 7 ). This research has yielded valuable insights into the roles of PgF3H in enhancing plant resistance to abiotic stress. By elucidating the functions of PgF3H , the study advances our understanding of how this gene contributes to the plant's ability to withstand challenging environmental conditions, such as drought and temperature extremes. Finally, we conclude that in improving drought stress tolerance in crops, PgF3H may be a valuable candidate gene. Declarations Data availability Data will be made available on request. Acknowledgements Dr. Charu Lata acknowledges the INSPIRE Faculty Award [IFA-11LSPA-01] from Department of Science & Technology (DST), GoI, New Delhi. The authors are thankful Dr. Rakesh Srivastava, International Crops Research Institute for the Semi- Arid Tropics, Patancheru, India for providing pearl millet seed materials and Arabidopsis Biological Resource Center, The Ohio State University for providing Arabidopsis mutants. Dr. Radha Shivhare acknowledges CSIR for the SRF fellowship (File No: 31/08(0348)/2018-EMR-I) awarded to her. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6030840","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":428177856,"identity":"6f9f0568-ece6-4e93-ace8-9c3c21e340fc","order_by":0,"name":"Radha Shivhare","email":"","orcid":"","institution":"National Botanical Research Institute CSIR","correspondingAuthor":false,"prefix":"","firstName":"Radha","middleName":"","lastName":"Shivhare","suffix":""},{"id":428177857,"identity":"b510c1f2-303d-47d3-8454-775a4fae1b43","order_by":1,"name":"Priyamvada Mishra","email":"","orcid":"","institution":"CSIR-National Botanical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Priyamvada","middleName":"","lastName":"Mishra","suffix":""},{"id":428177858,"identity":"fed58da9-9b5c-41b0-9324-72e359c83280","order_by":2,"name":"Poorwa Kamal Badola","email":"","orcid":"","institution":"National Botanical Research Institute CSIR","correspondingAuthor":false,"prefix":"","firstName":"Poorwa","middleName":"Kamal","lastName":"Badola","suffix":""},{"id":428177859,"identity":"2d31decf-46e7-434b-a7d9-5c1624bdf69e","order_by":3,"name":"Puneet Singh Chauhan","email":"","orcid":"","institution":"CSIR-National Botanical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Puneet","middleName":"Singh","lastName":"Chauhan","suffix":""},{"id":428177860,"identity":"fbbb81d0-e15f-42e2-847c-1231ac4c8e90","order_by":4,"name":"Charu Lata","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACCRBRAMTsDQwHEMIGhLQYACmeAyRrkUgg0l38s5uffeYxsKvjn/n24MEfNXfyGaQPH/7AUHAHtyV3jhnP5jFIlpC4nZdwmOfYM8sGvrQ0CQaDZzi1GEgkGDPzGDBLMNzOMTjM2HDYgIGHxwwofhiPlvTPQC31EvI3zxgc/AnWwv/5A34tOSBbDksY3OAxOMALsQUYGHi0SNzIKWacY3BccuMZoMOAfjFg42Ezk0jAo4V/RvpmhjcV1fxyx88YfwSGmAE/D/PjDx/+4NaCDg4wsIGoBKI1MCAngFEwCkbBKBgFUAAAJtVNugt+ycQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2840-6999","institution":"National Institute of Science Communication: CSIR-National Institute of Science Communication \u0026 Policy Research","correspondingAuthor":true,"prefix":"","firstName":"Charu","middleName":"","lastName":"Lata","suffix":""}],"badges":[],"createdAt":"2025-02-14 13:06:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6030840/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6030840/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-025-03524-8","type":"published","date":"2025-06-20T15:57:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78660156,"identity":"b74d024e-fbbb-4d52-abd5-9c96c990491b","added_by":"auto","created_at":"2025-03-17 10:02:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":703550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrought stress tolerance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpressing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thailiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eplants.\u003c/strong\u003e Transgenic lines, WT and EV plants under control and drought conditions \u003cstrong\u003e(a)\u003c/strong\u003e, dry weight \u003cstrong\u003e(b)\u003c/strong\u003e, survival rates \u003cstrong\u003e(c)\u003c/strong\u003e, electrolytic leakage \u003cstrong\u003e(d)\u003c/strong\u003e, relative water content (RWC) \u003cstrong\u003e(e)\u003c/strong\u003e, and water loss \u003cstrong\u003e(f)\u003c/strong\u003e of the transgenic, WT and EV plants under water-deficit conditions. Values are means ± SD (n = 3) of three independent experiments. Letters over the bars revealed a significant difference (p \u0026lt; 0.05) as evaluated by Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/3a1f10dff02aeafb93b606fb.png"},{"id":78660154,"identity":"c2aee19c-a743-435a-9111-9abab750c583","added_by":"auto","created_at":"2025-03-17 10:02:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":511528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e affects flavonol accumulation and altered expression of flavonoid biosynthesis genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thailiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transgenic lines, WT and EV plants under control and drought conditions.\u003c/strong\u003eQuantification of total flavonols content \u003cstrong\u003e(a)\u003c/strong\u003e, gene expression of \u003cem\u003eAtFLS1\u003c/em\u003e \u003cstrong\u003e(b)\u003c/strong\u003e, \u003cem\u003eAtFLS3\u003c/em\u003e \u003cstrong\u003e(c)\u003c/strong\u003e, \u003cem\u003eAtUGT78D2\u003c/em\u003e \u003cstrong\u003e(d)\u003c/strong\u003e, \u003cem\u003eAtUGT78D3\u003c/em\u003e \u003cstrong\u003e(e)\u003c/strong\u003e, and \u003cem\u003eAtHCT\u003c/em\u003e \u003cstrong\u003e(f)\u003c/strong\u003e in WT, EV and transgenic plants under control and drought stress. Values are means ± SD (n = 3) of three independent experiments. Letters over the bars revealed a significant difference (p \u0026lt; 0.05) as evaluated by Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/88819166293169223bf2cede.png"},{"id":78661124,"identity":"fe06f80a-832a-4808-ba26-4b50de8e5d0b","added_by":"auto","created_at":"2025-03-17 10:10:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":395965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e affects anthocyanin accumulation and altered expression of anthocyanin biosynthesis genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thailiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transgenic lines, WT and EV plants under control and drought conditions.\u003c/strong\u003eQuantification of anthocyanin content \u003cstrong\u003e(a-b)\u003c/strong\u003e, gene expression of \u003cem\u003eAtDFR\u003c/em\u003e \u003cstrong\u003e(c)\u003c/strong\u003e, and \u003cem\u003eAtLDOX\u003c/em\u003e \u003cstrong\u003e(d) \u003c/strong\u003ein WT, EV and transgenic plants under control and drought stress. Values are means ± SD (n = 3) of three independent experiments. Letters over the bars revealed a significant difference (p \u0026lt; 0.05) as evaluated by Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/0455fac1b4482776e34988d7.png"},{"id":78660158,"identity":"afbe1fa0-f7ae-49e6-9c59-349cac3e8175","added_by":"auto","created_at":"2025-03-17 10:02:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3746175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrought stress tolerance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thailiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutants (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtf3h, Atans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtanr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) and respective \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e overexpressing transgenic (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtf3h+ PgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtans + PgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtanr + PgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eplants.\u003c/strong\u003e \u003cem\u003eAtf3h\u003c/em\u003e mutant and \u003cem\u003eAtf3h+PgF3H\u003c/em\u003e transgenic plants under control and drought conditions \u003cstrong\u003e(a)\u003c/strong\u003e, dry weight and survival rates \u003cem\u003eAtf3h\u003c/em\u003e mutant and \u003cem\u003eAtf3h+PgF3H\u003c/em\u003e transgenic plants (\u003cstrong\u003eb-c)\u003c/strong\u003e. \u003cem\u003eAtans\u003c/em\u003e mutant and \u003cem\u003eAtans+PgF3H\u003c/em\u003e transgenic plants under control and drought conditions \u003cstrong\u003e(d)\u003c/strong\u003e, dry weight and survival rates \u003cem\u003eAtans\u003c/em\u003e mutant and \u003cem\u003eAtans+PgF3H\u003c/em\u003e transgenic plants \u003cstrong\u003e(e-f)\u003c/strong\u003e. \u003cem\u003eAtanr\u003c/em\u003e mutant and \u003cem\u003eAtanr+PgF3H\u003c/em\u003e transgenic plants under control and drought conditions \u003cstrong\u003e(g)\u003c/strong\u003e, dry weight and survival rates \u003cem\u003eAtanr\u003c/em\u003e mutant and \u003cem\u003eAtanrs+PgF3H\u003c/em\u003e transgenic plants \u003cstrong\u003e(h-i)\u003c/strong\u003e. Values are means ± SD (n = 3) of three independent experiments. Letters over the bars revealed a significant difference (p \u0026lt; 0.05) as evaluated by Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/9566c9240c0df7c9868e13ea.png"},{"id":78661477,"identity":"0c3acbf9-f871-4d33-85d3-11764cc38329","added_by":"auto","created_at":"2025-03-17 10:18:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":813307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e affects flavonol accumulation and altered expression of flavonoid biosynthesis genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thailiana mutants (Atf3h, Atans and Atanr) and respective PgF3H overexpressing transgenic (Atf3h+ PgF3H, Atans + PgF3H, and Atanr + PgF3H) plants \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder control and drought conditions.\u003c/strong\u003eQuantification of total flavonols content \u003cstrong\u003e(a)\u003c/strong\u003e, gene expression of \u003cem\u003eAtFLS1\u003c/em\u003e \u003cstrong\u003e(b)\u003c/strong\u003e, \u003cem\u003eAtFLS3\u003c/em\u003e \u003cstrong\u003e(c)\u003c/strong\u003e, \u003cem\u003eAtUGT78D2\u003c/em\u003e \u003cstrong\u003e(d)\u003c/strong\u003e, \u003cem\u003eAtUGT78D3\u003c/em\u003e \u003cstrong\u003e(e)\u003c/strong\u003e, and \u003cem\u003eAtHCT\u003c/em\u003e \u003cstrong\u003e(f)\u003c/strong\u003e in \u003cem\u003eAtf3h \u003c/em\u003emutant and \u003cem\u003eAtf3h+PgF3H\u003c/em\u003e under control and drought stress. Quantification of total flavonols content \u003cstrong\u003e(g)\u003c/strong\u003e, gene expression of \u003cem\u003eAtFLS1\u003c/em\u003e \u003cstrong\u003e(h)\u003c/strong\u003e, \u003cem\u003eAtFLS3\u003c/em\u003e \u003cstrong\u003e(i)\u003c/strong\u003e, \u003cem\u003eAtUGT78D2\u003c/em\u003e \u003cstrong\u003e(j)\u003c/strong\u003e, \u003cem\u003eAtUGT78D3\u003c/em\u003e \u003cstrong\u003e(k)\u003c/strong\u003e, and \u003cem\u003eAtHCT\u003c/em\u003e \u003cstrong\u003e(l)\u003c/strong\u003e in \u003cem\u003eAtans \u003c/em\u003emutant and \u003cem\u003eAtans+PgF3H\u003c/em\u003e under control and drought stress. Quantification of total flavonols content \u003cstrong\u003e(m)\u003c/strong\u003e, gene expression of \u003cem\u003eAtFLS1\u003c/em\u003e \u003cstrong\u003e(n)\u003c/strong\u003e, \u003cem\u003eAtFLS3\u003c/em\u003e \u003cstrong\u003e(o)\u003c/strong\u003e, \u003cem\u003eAtUGT78D2\u003c/em\u003e \u003cstrong\u003e(p)\u003c/strong\u003e, \u003cem\u003eAtUGT78D3\u003c/em\u003e \u003cstrong\u003e(q)\u003c/strong\u003e, and \u003cem\u003eAtHCT\u003c/em\u003e \u003cstrong\u003e(r)\u003c/strong\u003e in \u003cem\u003eAtanr \u003c/em\u003emutant and \u003cem\u003eAtanr+PgF3H\u003c/em\u003e under control and drought stress. Values are means ± SD (n = 3) of three independent experiments. Letters over the bars revealed a significant difference (p \u0026lt; 0.05) as evaluated by Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/530b5957b546a979f1c32195.png"},{"id":78660157,"identity":"cb226873-2518-432c-9884-a80009a15e26","added_by":"auto","created_at":"2025-03-17 10:02:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":735110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e affects anthocyanin accumulation and altered expression of anthocyanin biosynthesis genes in in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thailiana mutants (Atf3h, Atans and Atanr) and respective PgF3H overexpressing transgenic (Atf3h+ PgF3H, Atans + PgF3H, and Atanr + PgF3H) plants \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder control and drought conditions.\u003c/strong\u003eQuantification of anthocyanin content \u003cstrong\u003e(a-b)\u003c/strong\u003e, gene expression of \u003cem\u003eAtDFR\u003c/em\u003e \u003cstrong\u003e(c)\u003c/strong\u003e, and \u003cem\u003eAtLDOX\u003c/em\u003e \u003cstrong\u003e(d) \u003c/strong\u003ein \u003cem\u003eAtf3h \u003c/em\u003emutant and \u003cem\u003eAtf3h+PgF3H\u003c/em\u003e under control and drought stress. Quantification of anthocyanin content \u003cstrong\u003e(e-f)\u003c/strong\u003e, gene expression of \u003cem\u003eAtDFR\u003c/em\u003e \u003cstrong\u003e(g)\u003c/strong\u003e, and \u003cem\u003eAtLDOX\u003c/em\u003e \u003cstrong\u003e(h) \u003c/strong\u003ein \u003cem\u003eAtans \u003c/em\u003emutant and \u003cem\u003eAtans+PgF3H\u003c/em\u003e under control and drought stress. Quantification of anthocyanin content \u003cstrong\u003e(i-j)\u003c/strong\u003e, gene expression of \u003cem\u003eAtDFR\u003c/em\u003e \u003cstrong\u003e(k)\u003c/strong\u003e, and \u003cem\u003eAtLDOX\u003c/em\u003e \u003cstrong\u003e(l) \u003c/strong\u003ein \u003cem\u003eAtanr \u003c/em\u003emutant and \u003cem\u003eAtanr+PgF3H\u003c/em\u003e under control and drought stress. Values are means ± SD (n = 3) of three independent experiments. Letters over the bars revealed a significant difference (p \u0026lt; 0.05) as evaluated by Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/f2662fba9b36d11a5ece53aa.png"},{"id":78661125,"identity":"74541c3a-61f3-4e7f-91ce-c9d5246f9e3d","added_by":"auto","created_at":"2025-03-17 10:10:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":130011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model for the regulatory network of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgF3H\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eresponse under water-deficit condition. \u003c/strong\u003eFlavonol and anthocyanin have antioxidant activities and in response to abiotic stress in plants, biosynthesis and accumulation of flavonoids takes part in reactive oxygen species (ROS) scavenging in different plant cell organelles and reduces oxidative damage in plant cells.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/8af384be1caeea0b7dc9abe8.png"},{"id":85231624,"identity":"bd533e32-6be7-4919-82d9-cc7c217e68d7","added_by":"auto","created_at":"2025-06-23 16:09:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9789388,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/98341670-cfa4-4143-9f71-8d083fa8575a.pdf"},{"id":78660152,"identity":"31958802-1796-435c-899d-25f8bc34439c","added_by":"auto","created_at":"2025-03-17 10:02:49","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31097,"visible":true,"origin":"","legend":"","description":"","filename":"LegendstoSupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/3764d0de8778ffe548c01454.docx"},{"id":78661127,"identity":"cc5fdbde-e146-4fa8-b015-558a4a4a27b7","added_by":"auto","created_at":"2025-03-17 10:10:49","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3326711,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6030840/v1/91574411ea1d6a8dedd64d84.pdf"}],"financialInterests":"","formattedTitle":"PgF3H gene enhances drought tolerance in transgenic Arabidopsis by regulating flavonoid biosynthesis and stress response","fulltext":[{"header":"Key message","content":"\u003cp\u003eWater stress stimulates plants to regulate flavonoid biosynthesis. Overexpression of the \u003cem\u003ePgF3H\u003c/em\u003e gene increases flavonoid levels and drought tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e, with stress-responsive elements in the \u003cem\u003ePgF3H\u003c/em\u003e promoter indicating its role in drought response.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eDrought resistance is a multifaceted trait governed by an intricate network of numerous genes. It entails complex interactions between these genes and environmental signals, influencing diverse morphological and metabolic pathways (Tardieu and Tuberosa, 2010). The genes that are triggered by drought stress produce proteins that have a diverse function, including signal transduction, gene expression, damage control from stress, and treatment (Valliyodan and Nguyen, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Different secondary metabolites that are produced by the phenylpropanoid metabolic pathway have robust antioxidant action under abiotic stress conditions. Plants have very potent chemicals called phenolic compounds, particularly flavonoids, which can provide resistance to a number of biotic and abiotic stressors (Saini et al., 2024). Flavonoids are phenolic compounds composed of two aromatic rings connected by a three-carbon bridge. Despite their diverse structures and functions, they are primarily synthesized through the phenylpropanoid pathway, a secondary metabolic pathway. Numerous researchers have been interested in flavonoids over the years. The biosynthetic process for flavonoids has been extensively studied (Liu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). According to altered seed coat color, a number of mutant lines in \u003cem\u003eArabidopsis\u003c/em\u003e have been found to exhibit flavonoid biosynthesis defects (Winkel-Shirley, 2001). Most of the pathway's enzymes have been found in a different plant species (Hassan and Mathesius, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yu et al., 2012). The flavonoid biosynthesis pathway is initiated by chalcone synthase (CHS), a key enzyme that catalyzes the condensation of malonyl-CoA and 4-coumaroyl-CoA to form chalcone, marking the entry point of this metabolic route (Niu et al., 2021). Chalcone then undergoes a series of enzymatic transformations, leading to the production of various flavonoid compounds. Chalcone isomerase (CHI) facilitates the isomerization of chalcone into flavanone, which serves as a precursor for multiple flavonoid subclasses. Flavanone 3-hydroxylase (F3H) further hydroxylates flavanone, contributing to the biosynthesis of flavonols and anthocyanins. Dihydroflavonol 4-reductase (DFR) plays a crucial role in converting dihydroflavonols into leucoanthocyanidins, which are subsequently processed by flavonol synthase (FLS) to generate flavonols or by anthocyanin synthase (ANS) to produce anthocyanins. These enzymes collectively regulate the structural diversity of flavonoids, which are essential for plant pigmentation, defense, and environmental adaptation (Zha et al., 2019).\u003c/p\u003e \u003cp\u003eThe Flavanone 3-hydroxylase (F3H) gene belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) family and functions as the third key enzyme in the core flavonoid biosynthetic pathway. It plays a crucial role in catalyzing the hydroxylation of flavanones, facilitating the biosynthesis of flavonols, anthocyanins, and other essential flavonoid derivatives involved in plant defense and pigmentation (Jiang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). F3H is crucial for flavonoid biosynthesis, where it hydroxylates flavanones like naringenin to produce 3-hydroxy flavonol. This compound is a key precursor for the formation of anthocyanins, flavanols, and proanthocyanidins (Pervaiz et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Recent studies have increasingly concentrated on the protective role of flavonoids in stress responses. These compounds are particularly noted for their involvement in responding to abiotic signals, including temperature variations, nutrient scarcity, and water deficiency. Flavonoids have been shown to play a crucial role in helping plants adapt to and withstand these challenging environmental conditions. Research has demonstrated that flavonoids rapidly accumulate in plants exposed to drought conditions, providing a protective function (Winkel-Shirley, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, flavonoids may help mitigate oxidative stress triggered by environmental challenges (Pietta, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Observations have shown that genes related to the flavonoid pathway are up-regulated in response to stress in various plants, including potatoes, birch, and rice (Andr\u0026eacute; et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These studies suggest that the flavonoid pathway plays a significant role in stress responses. However, the specific roles of key enzymes and the regulatory mechanisms involved in the flavonoid biosynthetic pathway under abiotic stress conditions are not yet fully understood.\u003c/p\u003e \u003cp\u003ePearl millet [\u003cem\u003ePennisetum glaucum\u003c/em\u003e (L.) R. Br.] is a significant C4 Panicoid crop cultivated for its grain, forage, and stover, particularly in the arid and semi-arid regions of Africa and Asia (Shivhare et al., 2024). Ranked as the sixth most important cereal after rice, wheat, maize, barley, and sorghum, it is highly valued for its rich nutritional profile and is also recognized as a promising feedstock for biofuel production (Lata et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shivhare and Lata, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Pearl millet thrives in regions with low rainfall and demonstrates exceptional adaptability to different abiotic stresses, including high temperatures, osmotic stress, and drought, whether these challenges arise individually or in combination. Its resilience under harsh environmental conditions makes it an ideal model for studying the molecular basis of abiotic stress tolerance. Furthermore, it serves as a crucial resource for functional genomics research focused on improving stress resistance in crops (Shivhare and Lata \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Genome sequencing of pearl millet has identified an abundance of gene families associated with secondary metabolite regulatory pathways, including those involved in flavonoid, anthocyanin, terpene, and wax biosynthesis, which may contribute to its drought tolerance (Varshney et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, transcriptomic studies under drought stress have shown an upregulation of genes related to the phenylpropanoid pathway, further highlighting its adaptive mechanisms (Shivhare et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Shivhare et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). In this study, the flavanone 3-hydroxylase gene (\u003cem\u003ePgF3H\u003c/em\u003e) from the flavonoid biosynthetic pathway was isolated from pearl millet and successfully expressed in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e wild type (Col0) and specific mutants designed to disrupt the flavonoid biosynthetic pathway, aiming to explore the stress tolerance mechanisms mediated by flavonoids under drought conditions. These \u003cem\u003eArabidopsis\u003c/em\u003e mutants, including the AtSALK_113904; tt6 mutant with a crucial loss of function in flavanone 3-hydroxylase (F3H) necessary for flavonol synthesis, resulting in undetectable kaempferol or quercetin levels, were employed alongside mutants AtSALK_073183; tt11-11 and ANR, and AtSALK_040250; ban4, with disruptions in the anthocyanidin synthase and reductase genes, respectively, affecting anthocyanin content and seed coat pigmentation. Overexpressing \u003cem\u003ePgF3H\u003c/em\u003e in these lines, we investigated the gene's expression levels, enzyme activity, total flavonoid and anthocyanin contents, and the antioxidant capacity of flavonoids. This comprehensive approach allowed us to assess the contribution of the \u003cem\u003ePgF3H\u003c/em\u003e gene to enhancing drought stress tolerance through flavonoid-mediated pathways in both the wild-type Col0 and the mutant \u003cem\u003eArabidopsis\u003c/em\u003e plants, providing insights into the underlying stress response mechanisms in pearl millet.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials, RNA Isolation\u003c/h2\u003e \u003cp\u003ePRLT2/89\u0026thinsp;\u0026minus;\u0026thinsp;33 seeds were grown in 3 kg pots with composite soil inside a glasshouse under controlled conditions: 32\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C (day), 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C (night), 40\u0026ndash;70% humidity, and natural sunlight. Drought stress was imposed at the full panicle emergence stage (40 days after sowing) using a dry-down protocol. Two groups\u0026mdash;well-watered (WW) and water-stressed (WS)\u0026mdash;were maintained, each with three replicates. WW plants had an NTR of 1, while WS plants were stressed until NTR\u0026thinsp;\u0026le;\u0026thinsp;0.2. Leaf samples were collected, frozen in liquid nitrogen, and stored at -80\u0026deg;C to preserve RNA integrity, ensuring reliable molecular analysis of drought stress responses (Shivhare et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe total RNA was isolated from flag leaf using RNAiso plus Reagent (TaKaRa, Japan). RNA samples with OD260/OD230 (\u0026ge;\u0026thinsp;2.0) and OD260/OD280 nm absorption ratio (1.98\u0026ndash;2.01) and were used for cDNA synthesis. About 5 \u0026micro;g of total RNA underwent DNase treatment to ensure thorough removal of any DNA contamination. This process utilized the RNase-free DNase set from Qiagen, Germany, or TURBO DNAse from Ambion, effectively eliminating potential DNA residues from the RNA samples. First-strand complementary DNA (cDNA) was synthesized using either the SuperScript\u0026trade; III First-Strand Synthesis System (Invitrogen). The reaction was carried out with 1 \u0026micro;g of DNA-free total RNA as the template, utilizing oligo(dT) primers to ensure selective reverse transcription of polyadenylated messenger RNA (mRNA). The total reaction volume was adjusted to 20 \u0026micro;l, following the respective manufacturer\u0026rsquo;s protocols for optimal efficiency. After synthesis, the resulting cDNA was stored at -20\u0026deg;C to preserve its integrity for subsequent gene expression and molecular analyses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSelection of terminal drought-responsive gene from pearl millet\u003c/h3\u003e\n\u003cp\u003ePearl millet terminal drought-responsive gene, flavanone 3-hydroxylase gene (\u003cem\u003ePgF3H\u003c/em\u003e) was selected from previous data, have the higher expression under drought stress for cloning and functional characterization (Shivhare et al., 2020). Complete open reading frame (ORF) of \u003cem\u003ePgF3H\u003c/em\u003e (1020 bp) was amplified from water stressed RNA sample of pearl millet drought tolerant genotype, PRLT2/89\u0026thinsp;\u0026minus;\u0026thinsp;33 using gene-specific primers (Supplementary Table\u0026nbsp;1) by PCR using high fidelity Platinum\u0026trade; Taq DNA Polymerase (Invitrogen).\u003c/p\u003e\n\u003ch3\u003eSequence homology and phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eMultiple sequence alignment was conducted using ClustalOmega to identify conserved regions and assess the functional homology of the PgF3H gene with selected orthologous genes from various plant species. The analysis was performed through the ClustalOmega web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/msa/clustalo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to ensure accurate sequence comparison. A phylogenetic tree was constructed using the neighbor-joining method with a bootstrap value of 1000 for statistical reliability, employing MEGA6 software. Additionally, all protein sequences were retrieved and analyzed through the NCBI database using the web-based BLAST tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ncbi.nlm.nih.gov/blast\u003c/span\u003e\u003cspan address=\"http://ncbi.nlm.nih.gov/blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to determine sequence similarity and evolutionary relationships with other plant species.\u003c/p\u003e\n\u003ch3\u003eIn silico analysis of promoter, structure, and homology modeling of the PgF3H Protein\u003c/h3\u003e\n\u003cp\u003eThe promoter sequence of \u003cem\u003ePgF3H\u003c/em\u003e gene was analyzed using PlantCARE (Lescot et al., 2002) and PlantPAN (Chang et al., 2008) databases as well as motifs taken from the literature for putative cis-acting elements. The \u003cem\u003ePgF3H\u003c/em\u003e molecular model was created using the web-based homology modelling server SWISS-MODEL automated protein homology modelling server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp:/swissmodel.expasy.org/workspace\u003c/span\u003e\u003cspan address=\"http://swissmodel.expasy.org/workspace\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to create homology models of protein structure using as guide information on the crystal structure of the recombinant proteins. The molecular model of \u003cem\u003ePgF3H\u003c/em\u003e was visualized using the PyMOL software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pymol.sourceforge.net/\u003c/span\u003e\u003cspan address=\"http://pymol.sourceforge.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To predict the secondary structure of the protein, the PSIPRED server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.cs.ucl.ac.uk/psipred/\u003c/span\u003e\u003cspan address=\"http://bioinf.cs.ucl.ac.uk/psipred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized, following the methodology described by Jones (2002).\u003c/p\u003e\n\u003ch3\u003eIsolation of the 5ˊ-flanking region of PgF3H\u003c/h3\u003e\n\u003cp\u003eThe 5\u0026prime; flanking sequence of \u003cem\u003ePgF3H\u003c/em\u003e (Pgl_GLEAN_10000809) was retrieved from the IPMGSC database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cegsb.icrisat.org/ipmgsc/\u003c/span\u003e\u003cspan address=\"https://cegsb.icrisat.org/ipmgsc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Forward and reverse primers were meticulously designed based on the \u003cem\u003ePgF3H\u003c/em\u003e sequence along with its 5\u0026prime; flanking region (Supplementary Table\u0026nbsp;2). A 1109-base pair fragment located upstream of the translation start codon \u0026ldquo;ATG\u0026rdquo; of the PgF3H gene was identified and subsequently amplified from the genomic DNA of the pearl millet genotype PRLT2/89\u0026thinsp;\u0026minus;\u0026thinsp;33. This amplification was performed using polymerase chain reaction (PCR) with primers specific to the \u003cem\u003ePgF3H\u003c/em\u003e promoter. The amplified fragment was then verified through sequencing to ensure accuracy.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of transformation vectors and generation of transgenic plants\u003c/h2\u003e \u003cp\u003eThe promoter region of \u003cem\u003ePgF3H\u003c/em\u003e, spanning from \u0026minus;\u0026thinsp;1109 base pairs to \u0026minus;\u0026thinsp;1 base pair, was isolated through PCR amplification using specific oligonucleotide primers (Supplementary Table\u0026nbsp;2). For the purpose of constructing \u003cem\u003ePgF3H\u003c/em\u003e promoter::GUS vectors, the PCR products were inserted into the pBI101.2 plasmid (Cambia, Australia) at the HindIII and BamHI restriction sites. This insertion was subsequently verified by sequencing. The confirmed plasmids were then utilized to transform \u003cem\u003eA. thaliana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. thaliana\u003c/em\u003e wild type and three \u003cem\u003eArabidopsis\u003c/em\u003e mutants from phenylpropanoid biosynthesis pathways in the Col0 background were employed to develop lines overexpressing \u003cem\u003ePgF3H\u003c/em\u003e gene. The AtSALK_113904; tt6 mutant, characterized by the loss of function of flavanone 3-hydroxylase (F3H), crucial for flavonol synthesis, exhibited undetectable levels of kaempferol or quercetin, the primary flavonol components in \u003cem\u003eArabidopsis\u003c/em\u003e. Additionally, the mutants AtSALK_073183; tt11-11 and ANR, along with AtSALK_040250; ban4, had insertions in the ANS gene encoding anthocyanidin synthase and the ANR gene encoding anthocyanidin reductase, respectively. The tt11-11 mutants lacked red pigmentation due to the absence of anthocyanins, while ban-4 mutants displayed a red seed coat in immature seeds and a darker black seed coat in fully desiccated seeds (Bowerman et al., 2012). The full-length cDNA of the \u003cem\u003ePgF3H\u003c/em\u003e gene was cloned into the pCAMBIA1304 vector (Cambia, Australia) under the regulation of the CaMV35S promoter. To achieve this, the complete PgF3H gene was PCR amplified using gene-specific primers. The cDNA template for this amplification was derived from the flag leaf of the pearl millet genotype PRLT2/89\u0026thinsp;\u0026minus;\u0026thinsp;33 (Supplementary Table\u0026nbsp;2) using Platinum II Taq Hot-Start DNA Polymerase (Thermo Fisher Scientific, United States). The amplicon was cloned into pCAMBIA1304 plant binary vector using the NcoI and BstII restriction sites and transformed into \u003cem\u003eAgrobacterium\u003c/em\u003e GV3101. \u003cem\u003eA. thaliana\u003c/em\u003e Col-0 and mutant lines were transformed using the floral dip method facilitated by \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. After transformation, T0 seeds were harvested from treated plants and sown on 1/2 Murashige and Skoog (MS) medium supplemented with 30 mg/L hygromycin (Sigma-Aldrich, USA) for selection of transformants. Hygromycin-resistant seedlings were identified after eight days of germination and subsequently transferred to soil for further growth in a controlled environment. To serve as controls, plants transformed with an empty pCAMBIA1304 vector were also generated for comparative analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistochemical GUS staining\u003c/h3\u003e\n\u003cp\u003eGUS staining was carried out following a modified protocol from Sharma et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Transgenic seedlings harboring the ProPgF3H::GUS construct were submerged in a staining solution composed of 100 mM sodium phosphate buffer (pH 7.2), 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide, 0.1% Triton X-100, 10 mM EDTA, and 1 mg/mL of the chromogenic substrate X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide). The samples were incubated at 37\u0026deg;C for four hours to facilitate the enzymatic reaction, leading to the formation of a distinct blue precipitate in regions exhibiting GUS activity. To enhance visibility and eliminate chlorophyll, the stained seedlings were washed several times with 70% ethanol. The cleared samples were then examined under a Leica microscope (LAS version 4.12.0, Leica Microsystems) to assess GUS expression patterns, providing valuable insights into the spatial regulation and activity of the PgF3H promoter in plant tissues.\u003c/p\u003e\n\u003ch3\u003eExpression analysis\u003c/h3\u003e\n\u003cp\u003eqRT-PCR was conducted using SYBR Green chemistry on a 7500 Fast Real-Time PCR System (Applied Biosystems). Each 10 \u0026micro;l reaction contained 1 \u0026micro;l of diluted cDNA (50 ng RNA equivalent), 10 \u0026micro;M gene-specific primers, and 5 \u0026micro;l of 2X Power SYBR Green PCR Master Mix. The cycling conditions included 50 \u0026ordm;C for 20 s, 95 \u0026ordm;C for 10 min, followed by 40 cycles of 95 \u0026ordm;C for 15 s and 60 \u0026ordm;C for 1 min. Melt curve analysis (60 \u0026ordm;C\u0026ndash;90 \u0026ordm;C) was used to confirm amplification specificity (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of drought tolerance in transgenic A. thaliana plants\u003c/h2\u003e \u003cp\u003eThe drought stress tolerance of \u003cem\u003ePgF3H\u003c/em\u003e-overexpressing transgenic \u003cem\u003eA. thaliana\u003c/em\u003e plants, along with the mutants \u003cem\u003eAtf3h\u003c/em\u003e (AtSALK_113904), \u003cem\u003eAtans\u003c/em\u003e (AtSALK_073183), and \u003cem\u003eAtanr\u003c/em\u003e (AtSALK_040250), was assessed using the three best homozygous lines. At the germination stage, T3 seeds of transgenic lines, wild-type (Col-0), and empty vector (EV) controls were surface sterilized and stratified in darkness at 4\u0026deg;C for three days. The stratified seeds were then placed on \u0026frac12; MS medium supplemented with PEG 6000 (0, 1.5, 3, and 6%) or mannitol (0, 100, 200, and 300 mM). Germination percentage was recorded on the ninth day to evaluate stress tolerance.\u003c/p\u003e \u003cp\u003eSeeds of transgenic lines, WT and EV were grown on composite soil in well-watered conditions supplemented with nutrient media for four weeks in order to evaluate the drought tolerance behavior under the influence of physiological drought conditions. There were two treatments: control (WW) and water stressed (WS) plants. WW plants were watered regularly and WS plants were exposed to water stress by withdrawing the water for next 14 days. At 15 days, leaf and root tissue of both the treatments were harvested for physiological and biochemical assays and for RNA isolation leaf tissue were frozen in liquid nitrogen and stored in -80 \u0026ordm;C.\u003c/p\u003e \u003cp\u003eWater loss was assessed in one-month-old plants subjected to 14 days of drought stress. Fresh leaves from control and stressed plants were harvested, weighed immediately, and dried on filter paper at 25\u0026deg;C (50\u0026ndash;60% RH). Leaves were reweighed at 0 h, 1 h, 2 h, and 4 h to estimate water loss based on initial fresh weight. Electrolyte leakage (EL) was measured following Dionisio-Sese and Tobita (1998). Root tissues (100 mg) from control and stressed plants were immersed in 20 ml deionized water and shaken at 100 rpm for 2 h. Initial conductivity (C1) was recorded, followed by boiling for 30 min to release electrolytes. After cooling, final conductivity (C2) was measured, and EL was calculated as (C1/C2) \u0026times; 100. Leaf relative water content (RWC) was measured following Slatyer (1967). Fully expanded leaves were excised, and their fresh weight (LFW) was recorded. Leaves were then submerged in distilled water for 4 hours at room temperature, blotted dry, and weighed for turgid weight (LTW). After drying at 80\u0026deg;C for 24 hours, dry weight (LDW) was recorded. RWC (%) was calculated using Barrs and Weatherley\u0026rsquo;s (1962) formula: RWC (%) = [(LFW \u0026ndash; LDW) / (LTW \u0026ndash; LDW)] \u0026times; 100. Total chlorophyll content was estimated by suspending 100 mg of leaf tissue in 10 mL of 80% acetone, homogenizing, and incubating in darkness at 4\u0026deg;C overnight. The extract was centrifuged at 5000 rpm, and absorbance was recorded at 663 and 645 nm using a spectrophotometer. Chlorophyll content was calculated following Arnon (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1949\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical and enzymatic assays\u003c/h2\u003e \u003cp\u003eLeaf tissues were ground in liquid nitrogen and homogenized in bicarbonate buffer to extract total soluble proteins, which were then quantified using the Bradford assay (Bradford, 1976). The supernatant obtained after centrifugation was mixed with Bradford reagent, and absorbance was measured at 595 nm using a BSA standard curve. Proline content was determined following the method of Bates et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1973\u003c/span\u003e), where leaf tissues were homogenized in sulfosalicylic acid, reacted with ninhydrin and acetic acid, and heated. The proline-chromophore complex was then extracted with toluene, and its absorbance was recorded at 520 nm. Lipid peroxidation levels were assessed by estimating malondialdehyde (MDA) content through the TBARS assay. Leaf extracts were treated with thiobarbituric acid (TBA), heated, and absorbance was measured at 532 nm. Antioxidant enzyme activities were evaluated by different spectrophotometric assays: superoxide dismutase (SOD) activity was analyzed based on its inhibition of nitroblue tetrazolium (NBT) reduction at 560 nm (Beauchamp and Fridovich, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1971\u003c/span\u003e); catalase (CAT) activity was assessed by monitoring the decomposition of H₂O₂ at 240 nm (Aebi, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1974\u003c/span\u003e); and ascorbate peroxidase (APX) activity was measured by tracking ascorbate oxidation at 290 nm (Nakano and Asada, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Guaiacol peroxidase (GPX) activity was analyzed by tracking guaiacol oxidation at 470 nm (Hemeda and Klein, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Chlorophyll content was determined using acetone extraction, with absorbance measured at 663 nm and 645 nm (Arnon, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1949\u003c/span\u003e). Reactive oxygen species (ROS) accumulation was evaluated via NBT and DAB staining. Superoxide anions were visualized as blue formazan deposits from NBT staining, while H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation was identified by brown precipitates from DAB staining. Quantitative densitometry of the stained tissues was performed using ImageJ software(version 1.46 for Windows 8), providing a comparative analysis of ROS levels across treatments (Schneider et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This comprehensive approach highlights biochemical and physiological changes under various plant treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTotal anthocyanin quantification\u003c/h2\u003e \u003cp\u003eTotal anthocyanin was measured as previously recommended method (Li et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this procedure, leaf tissue of control and stressed plants (100 mg) were crushed in liquid N2 and transmitted to extraction buffer solution (propanol: HCl: H\u003csub\u003e2\u003c/sub\u003eO; 18:1:81). Samples solutions were boiled for 3 min at 95\u0026ordm;C then incubated in the dark condition for 2 h at room temperature. Samples were centrifuged and supernatant was collected in fresh tube. Absorbance of the supernatant solution was taken at 535 and 650 nm. Anthocyanin content was calculated as (A535\u0026ndash;2.2A650)/g \u0026bull; FW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTotal flavonol quantification\u003c/h2\u003e \u003cp\u003eFlavonoid content was measured using a modified aluminum chloride colorimetric assay based on Loyola (2016). Leaf tissues from control and drought-stressed plants were extracted in 1 ml of 80% ethanol at 4\u0026deg;C for 2 hours with continuous shaking. The extracts were centrifuged at 12,000\u0026times;g for 12 minutes, and the supernatant was collected. For quantification, 5 ml of the supernatant was diluted with methanol to a final volume of 2 ml and mixed with 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate, and 2.8 ml of Milli-Q water. After 30 minutes of incubation, absorbance was recorded at 415 nm. Flavonoid concentration was determined using a quercetin standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eExtraction and quantification of flavonols\u003c/h2\u003e \u003cp\u003eTo extract flavonols, 500 mg of seedlings were ground in liquid nitrogen and then extracted in 80% methanol overnight at room temperature. The extracts were subsequently hydrolyzed with an equal volume of 6 N HCl at 70\u0026deg;C for 40 minutes. To prevent the precipitation of aglycones, an equal volume of methanol was added after hydrolysis. The resulting extracts were filtered through 0.2 \u0026micro;m filters (Millipore) prior to metabolite analysis. The analysis was performed using High-Performance Liquid Chromatography with Photodiode Array detection (HPLC-PDA), employing a Waters 1525 Binary HPLC Pump system equipped with a PDA detector. The method followed was developed by Niranjan et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Quantification of the various metabolites was achieved using Breeze 2 software (Waters).\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent experiments conducted under identical environmental conditions. Statistical significance between control and drought-stressed samples was assessed using one-way analysis of variance (ANOVA). Further comparison of mean values was performed using Duncan\u0026rsquo;s multiple range test in SPSS software (version 16.0, SPSS Inc./IBM Corp., Chicago, USA). Graphical representation of the experimental data was generated using GraphPad Prism software (version 5.03, San Diego, California, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eValidation, characterization, cloning sequence analysis of the PgF3H gene\u003c/b\u003e\u003c/h2\u003e \u003cp\u003ePearl millet terminal drought-responsive gene, \u003cem\u003ePgF3H\u003c/em\u003e (Pgl_GLEAN_10000809) was identified from pearl millet exposed to drought stress. To confirm the increased expression of \u003cem\u003ePgF3H\u003c/em\u003e in a drought-tolerant pearl millet genotype, a quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis have been performed. The results revealed a significant up-regulation of \u003cem\u003ePgF3H\u003c/em\u003e, showing a 7-fold increase in expression under drought stress conditions. The \u003cem\u003ePgF3H\u003c/em\u003e is located on pearl millet chromosome number 3 and has 1020 bp open reading frame (ORF), was amplified using RT-PCR and cloned in to plant expression vector, pCAMBIA1304 and then in to \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101 confirmed with colony PCR (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Multiple sequence alignment of \u003cem\u003ePgF3H\u003c/em\u003e exhibit high resemblance with other plant Flavonone 3-hydroxygenase encoding genes (Supplementary Figure S2a). High sequence similarity in the 2OG-Fe(II) oxygenase domain suggested the importance of conservation of F3H genes among gramineae crops. In order to further establish the closeness of \u003cem\u003ePgF3H\u003c/em\u003e to the other 2OG-Fe(II) oxygenase proteins, a systematic phylogenetic study focused on the similarity of 2OG-Fe(II) oxygenase domains in proteins isolated from \u003cem\u003eArabidopsis\u003c/em\u003e, cotton, capsicum, maize, rice, sorghum, barley, cannabis and wheat using MEGA 6.0 by NJ method was performed (Supplementary Figure S2b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStructure of the full length PgF3H protein\u003c/h2\u003e \u003cp\u003eThe deduced \u003cem\u003ePgF3H\u003c/em\u003e protein contained 339 amino acids with a molecular mass estimated to be 38.75 kDa and a theoretical pI of 5.67. Recently full-length \u003cem\u003eF6H\u003c/em\u003e proteins from \u003cem\u003eArabidopsis\u003c/em\u003e containing a family protein domain of 2OG-Fe(II) oxygenase have been discovered which are found to play a key role in flavonoid biosynthetic pathway (Sun et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). At the primary amino acid sequence level, \u003cem\u003ePgF3H\u003c/em\u003e and \u003cem\u003eAtF6H\u003c/em\u003e proteins shared 39 percent similarity. Using the homology-modeling server SWISS-MODEL, \u003cem\u003eArabidopsis F6H\u003c/em\u003e protein (PDB ID: 4XAE) crystal structure was chosen as a template for \u003cem\u003ePgF3H\u003c/em\u003e model development. The PyMOL software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp:/pymol.sourceforge.net/\u003c/span\u003e\u003cspan address=\"http://pymol.sourceforge.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to generate \u003cem\u003ePgF3H\u003c/em\u003e molecular model (Supplementary Figure S3a). Generally, \u003cem\u003ePgF3H\u003c/em\u003e overall architecture was close to that of \u003cem\u003eAtF6H\u003c/em\u003e, and the \u003cem\u003eArabidopsis\u003c/em\u003e protein's structural findings were also relevant to the \u003cem\u003ePgF3H\u003c/em\u003e protein. A protein domain study using the CDD database showed the existence of highly conserved PLN02639 domain [2OG-Fe(II) oxygenase family protein)], a single member of superfamily cl31913 with secondary metabolite activity, transport and catabolism (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The secondary structure analysis of the \u003cem\u003ePgF3H\u003c/em\u003e amino acid sequence using PSIPRED revealed the presence of three alpha-helical structures near the N-terminus and two alpha-helical structures near the C-terminus (Supplementary Figure S3b). Further structural modeling using SWISS-MODEL software confirmed these findings, showing that the protein structure of \u003cem\u003ePgF3H\u003c/em\u003e comprises 12 β-strands and 16 α-helices. This structural data aligns with the secondary structure prediction made by PSIPRED. Additionally, hydropathy analysis indicated that the predicted \u003cem\u003ePgF3H\u003c/em\u003e protein is hydrophilic, with the highest level of hydrophilicity observed near the N-terminus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCloning and sequence analysis of the PgF3H promoter region\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ePgF3H\u003c/em\u003e promoter region (\u003cem\u003ePgF3H\u003c/em\u003epro) was isolated from pearl millet genomic DNA with \u003cem\u003ePgF3H\u003c/em\u003epro specific primers and confirmed by sequencing. \u003cem\u003ePgF3Hpro\u003c/em\u003e sequence was analyzed \u003cem\u003ein silico\u003c/em\u003e using the PlantCARE database which showed that \u003cem\u003ePgF3Hpro\u003c/em\u003e harbors \u003cem\u003ecis\u003c/em\u003e-acting elements, including ABRE (involved in abscisic acid (ABA) response), Box 4, G-box, GT1-motif (light responsive component), two copies of AuxRR-core, TGA-element (involved in auxin response), MBS (MYB binding site involved in drought-inducibility), TGACG-motif (MeJA-responsivenes), and LTR (Low-temperature response (Supplementary Figure S4a; Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSelection and assessment of PgF3H promoter A.thailiana line under water deficit condition\u003c/h2\u003e \u003cp\u003eTo analyze the activity of \u003cem\u003ePgF3H\u003c/em\u003e promoter (\u003cem\u003ePgF3Hpro\u003c/em\u003e) in response to stress condition, \u003cem\u003eA. thaliana\u003c/em\u003e promoter\u0026ndash;reporter lines (ProPgF3H::GUS) were generated and positive lines were selected on kanamycin + \u0026frac12; MS selection plate harboring the \u003cem\u003ePgF3Hpro\u003c/em\u003e sequence. Homozygous \u003cem\u003ePgF3Hpro A. thailiana\u003c/em\u003e transgenic lines were analyzed for water deficit response. The promoter lines were grown on \u0026frac12; MS media with or without supplementation with PEG (0, 1.5, 3 and 6%). A histochemical GUS assay showed progressive increase in GUS activity in ProPgF3H::GUS seedlings with the increase of PEG concentration (Supplementary Figure S4b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eModulation of water stress response in PgF3H overexpressing A.thailiana line under water deficit condition\u003c/h2\u003e \u003cp\u003eTransgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants with resistance to hygromycin were developed by incorporating the \u003cem\u003ePgF3H\u003c/em\u003e gene. To confirm the successful integration of the \u003cem\u003ePgF3H\u003c/em\u003e transgene into the genome of the selected \u003cem\u003eArabidopsis thaliana\u003c/em\u003e lines, PCR analysis was performed. The results of the PCR analysis revealed the amplification of a 1020-bp fragment corresponding to PgF3H, thus verifying the presence of the transgene in these genetically modified lines. Best three homozygous T3 transgenic lines with higher expression of \u003cem\u003ePgF3H\u003c/em\u003e were selected for further analysis (Supplementary Figure S5 a-f). These three \u003cem\u003ePgF3H\u003c/em\u003e overexpressing \u003cem\u003eA. thaliana\u003c/em\u003e lines were further used for water stress assessment assays. \u003cem\u003ePgF3H\u003c/em\u003e overexpressing \u003cem\u003eA. thailiana\u003c/em\u003e lines were analysed for water stress tolerance capacity at germination level and in four week old plants.\u003c/p\u003e \u003cp\u003eGermination percentage (G%) of WT, EV and \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines (OE1, OE2 and OE3) were normal and nearly 100% in \u0026frac12; MS media with no treatment. In response to different concentrations of mannitol and PEG treatment, G% was found to vary in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines compared to WT and EV. Under simulated osmotic stress conditions induced by PEG, the germination rate of the transgenic lines showed significant improvements compared to the wild-type (WT). By day 9, the average germination rates for transgenic lines were 83.5%, 62.2%, and 42% under 1.5%, 3%, and 6% PEG concentrations, respectively. In contrast, the WT plants exhibited germination rates of 64.6%, 27.3%, and 16% under the same conditions. The empty vector (EV) plants showed germination rates of 64%, 26%, and 15.3% under the respective PEG concentrations. At 100, 200 and 300 mM mannitol treatment, the average G % of transgenic seeds were 76, 60 and 42.6% compared to 48, 20.6 and 12% for WT and 44.6, 20 and 14% for EV seeds on the 9th day of treatment (Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eTo assess the water deficit resistance of \u003cem\u003ePgF3H-\u003c/em\u003eoverexpressing transgenic \u003cem\u003eA. thaliana\u003c/em\u003e plants, they were cultivated in soilrite under controlled conditions for 4 weeks in a growth chamber. Following this period, water stress was induced by withholding water for the subsequent 14 days (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The growth of the \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines, WT and EV plants was similar under well-watered conditions. However, only 50% of the WT and 48.7% of EV plants survived under water-deficit conditions, while the \u003cem\u003ePgF3H\u003c/em\u003e overexpressing plants showed a survival rate of about 81% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Further, dry weight of drought stresses plants were calculated and result showed significant higher dry weight of \u003cem\u003ePgF3H\u003c/em\u003e overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e plants compared to WT and EV plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The RWC, water loss and electrolytic leakage were also calculated in control and stressed plants of \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines, WT and EV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Average RWC for \u003cem\u003ePgF3H\u003c/em\u003e overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e plants were 86% whereas the same for WT and EV were 84, and 86% in controlled plants treatment. Under drought stress, average RWC of \u003cem\u003ePgF3H\u003c/em\u003e overexpressing plants was 65% whereas in WT and EV was 42%. The average water loss after 1, 2, and 3 h was 29.6, 54.7, and 66.84% respectively in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing plants compared to 30, 53, and 65% in the WT and 29.9, 54.6 and 68% in EV plants under well- watered conditions whereas 40.7, 66.5 and 84.5% in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing plants compared to 50, 80 and 94% in WT and 52, 78.3 and 95% in EV plants under water-deficit condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Average EL of \u003cem\u003ePgF3H\u003c/em\u003e overexpressing plants was 13.6% compared to 14% in WT and 14.3% in EV plants in control conditions and 24.6% for transgenic lines compared to 37% for WT and 38% for EV plants under drought stress condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eProline, MDA and chlorophyll content were also calculated in order to see the effect of water stress on various biochemical parameters under drought stress. Average 2-fold higher accumulation in proline content was recorded in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines while 1.3-fold increase was observed in WT (2.4-fold), and 1.4-fold in EV (2.8-fold) in water stressed tissues compared control plants (Supplementary Figure S7a). An estimation of MDA content showed 1.3-fold average increase in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines, lowered to WT and EV have more than 3-fold increase under water deficit condition (Supplementary Figure S7b). In response to drought stress, chlorophyll content decreases in WT, EV as well as in transgenic lines, however no significant decrease was recorded in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e lines (Supplementary Figure S7c). To evaluate the impact of drought stress on the antioxidant levels of transgenic lines relative to the wild type (WT), various enzymatic assays including APX, CAT, and SOD were conducted under both control and water stress conditions. The results of the enzymatic assays showed no notable differences among the transgenic lines, WT, and EV plants under normal conditions. However, their activities significantly increased in both WT and transgenic plants under water-deficit conditions. Specifically, in response to drought stress, the average APX and CAT activities were 1.4 and 1.6 times higher in transgenic plants compared to WT plants, respectively, whereas there was no significant difference in SOD activity among \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines, WT, and EV plants (Supplementary Figure S8a-c). These findings were further supported by the gene expression analysis of APX, CAT, and SOD genes using qRT-PCR (Supplementary Figure S8d-f).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eChange in total flavonols and anthocyanin content in PgF3H overexpressing plants\u003c/h2\u003e \u003cp\u003eTotal flavonols and anthocyanin content were estimated in WT, EV and transgenic plants under control and drought stress conditions to assess the role of \u003cem\u003ePgF3H\u003c/em\u003e in the synthesis and accumulation of these metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Results showed in controlled as well as in drought stressed leaf samples of WT, EV and transgenic plants have significant difference in accumulation of these metabolites with higher content in \u003cem\u003ePgF3H\u003c/em\u003e overexpressed lines. Average accumulation of total flavonols and anthocyanin were about 1.5 and 2.5-fold higher in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing plants as compared to WT under the water deficit environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). It was also confirmed by the up regulation of expression of genes involved in flavonols (\u003cem\u003eAtFLS1\u003c/em\u003e, \u003cem\u003eAtFLS3\u003c/em\u003e, and \u003cem\u003eAtUGT78D2\u003c/em\u003e) and anthocyanin biosynthesis (\u003cem\u003eAtDFR, and AtLDOX\u003c/em\u003e) in transgenic plants compared to WT and EV in response to drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-f; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). However, \u003cem\u003eAtUGT78D3\u003c/em\u003e did not show any major change in its expression under drought stress in WT as well as in transgenic lines.\u003c/p\u003e \u003cp\u003eHPLC analysis results also revealed that control and drought stressed leaf samples of WT, EV and transgenic lines for flavonol metabolites including kaempferol, quercetin and rutin also have significantly enhanced expression in transgenic lines compared to its control as well as WT under water deficit conditions (Supplementary Fig.\u0026nbsp;9). In response to drought stress, average accumulation of kaempferol, quercetin and rutin were 1.8-, 2.10- and 2.11- fold respectively, higher in comparison to WT.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment and analysis of PgF3H overexpressing mutants\u003c/h2\u003e \u003cp\u003eTo further confirm the function of \u003cem\u003ePgF3H\u003c/em\u003e gene involved in flavonoid biosynthesis, \u003cem\u003ePgF3H\u003c/em\u003e was transformed in \u003cem\u003eAtf3h\u003c/em\u003e mutant (AtSALK_113904; tt6) have the loss of function of flavanone 3-hydroxylase (F3H) involved in flavonols formation, \u003cem\u003eAtans\u003c/em\u003e mutant (AtSALK_073183; tt11-11) and \u003cem\u003eAtanr\u003c/em\u003e mutant (AtSALK_040250; ban4) having the insertion in anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) genes respectively involved in anthocyanin biosynthesisin Col0 background. Transgenic seeds for each mutant (\u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e) having the insertion of \u003cem\u003ePgF3H\u003c/em\u003e gene were screened on \u0026frac12; MS\u0026thinsp;+\u0026thinsp;hygromycin plate. T3 homozygous seeds of the overexpressing or transgenics lines (OE1, OE2 and OE3) of each mutant harboring the \u003cem\u003ePgF3H\u003c/em\u003e gene were generated and the PCR results showed the amplification of a 1020-bp fragment of \u003cem\u003ePgF3H\u003c/em\u003e confirming the integration of the transgene in the genome of \u003cem\u003eAtf3h, Atans\u003c/em\u003e and \u003cem\u003eAtanr\u003c/em\u003e mutants (Supplementary Fig.\u0026nbsp;10). Best three homozygous T3 transgenic lines of mutants with higher expression of \u003cem\u003ePgF3H\u003c/em\u003e were selected for further analysis (Supplementary Fig.\u0026nbsp;10) and were further used for water stress assessment assays. \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e overexpressing \u003cem\u003eA. thailiana\u003c/em\u003e lines (OE1, OE2 and OE3) were analysed for water stress tolerance capacity in four week old plants.\u003c/p\u003e \u003cp\u003eTo analyze the water stress tolerance capacity of mutants \u003cem\u003ePgF3H\u003c/em\u003e transgenics, were grown for four weeks than subjected to drought stress by withdrawing water for the next 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The growth of the \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenics and all the mutants plants were similar under WW conditions. However all \u003cem\u003eAtf3h\u003c/em\u003e mutant seeds were grown in WW condition but the rosette size were smaller than \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e plants. Under the water deficit condition \u003cem\u003ePgF3H\u003c/em\u003e overexpressing transgenic lines of all the three mutants have significant higher survival percentage as well dry weight compared to their respective mutant plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Further \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenics have almost similar survival percentage and dry weight as was showed by \u003cem\u003ePgF3H\u003c/em\u003e Col0 transgenic plants while \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e transgenics have lower survival percentage and dry weight than \u003cem\u003ePgF3H\u003c/em\u003e Col0 transgenic plants but significant higher than \u003cem\u003eAtf3h\u003c/em\u003e mutant plants under WS condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Gene expression of antioxidant enzymes including \u003cem\u003eAtAPX, AtCAT\u003c/em\u003e and \u003cem\u003eAtSOD\u003c/em\u003e showed significantly higher expression in all the mutants and \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenic plants in response to WS compared to WW condition but almost all the transgenics have lower expression for these antioxidant enzymes compared to respective mutant under WS. While \u003cem\u003ePgF3H\u003c/em\u003e overexpressing, mutant transgenic plants have the lower expression of \u003cem\u003eAtAPX, AtCAT\u003c/em\u003e and \u003cem\u003eAtSOD\u003c/em\u003e genes compared to their respective mutant plants under WS condition (Supplementary Fig.\u0026nbsp;11).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eChange in total flavonols and anthocyanin content in PgF3H overexpressing mutants plants\u003c/h2\u003e \u003cp\u003eTo evaluate the role of \u003cem\u003ePgF3H\u003c/em\u003e gene involved in flavonoid biosynthesis, \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenics lines (OE1, OE2 and OE3) were analyzed for flavonone and anthocyanin contents. Results showed \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e overexpressing plants have enhanced flavonone content under water deficit condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e have flavonone content comparable with \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 plants whereas, \u003cem\u003eAt\u003c/em\u003ef\u003cem\u003e3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e has higher flavone content compared to \u003cem\u003eAtf3h\u003c/em\u003e mutant plants but lower than \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 plants. These results were also supported by the gene expression analysis of flavonone biosynthesis genes (\u003cem\u003eAtFLS1 and AtFLS3)\u003c/em\u003e which showed higher expression in all the mutant transgenic plants in response to WS. Anthocyanin content was found to be higher in \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e whereas there was no increase in anthocyanin content in \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenic plants. Gene expression analysis of anthocyanin biosynthesis genes including \u003cem\u003eAtDFR, AtLDOX, AtUGT78D2\u003c/em\u003e, and \u003cem\u003eAtUGT78D3\u003c/em\u003e also have the increased expression in \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenic plants while \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e plants have no significant change in expression of these genes in response to WS condition. Genes involved in lignin biosynthesis (\u003cem\u003eAtCCR1 and AtCAD6)\u003c/em\u003e exhibited significantly higher expression in \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e plants except \u003cem\u003eAtSND1\u003c/em\u003e under WS whereas \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e follow the similar expression pattern of these genes as found in \u003cem\u003ePgF3H\u003c/em\u003e Col0 transgenic plants. Surprisingly gene expression of \u003cem\u003eAtHCT\u003c/em\u003e involved in flavonoids accumulation was significantly higher in \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenic plants compared to their mutants while \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e showed decline in expression of \u003cem\u003eAtHCT\u003c/em\u003e gene as present in \u003cem\u003ePgF3H\u003c/em\u003e Col0 transgenic plants under WS. Flavonol metabolites including kaempferol, quercetin and rutin were also evaluated in \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e and \u003cem\u003eAtf3h\u003c/em\u003e mutant plants using HPLC. \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e transgenic plants have significant higher accumulation of these flavones compared to \u003cem\u003eAtf3h\u003c/em\u003e mutant plants under water stress and the average accumulation of kaempferol, quercetin and rutin were 1.57-, 4.75- and 1.9- fold higher respectively, in comparison to control \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e transgenic plants (Supplementary Fig.\u0026nbsp;12).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eChange in lignin biosynthesis gene expression in PgF3H overexpressing Col0 and mutants plants\u003c/h2\u003e \u003cp\u003eTo determine the impact of \u003cem\u003ePgF3H\u003c/em\u003e gene overexpression on the lignin production pathway, expression analyses of the genes (\u003cem\u003eAtSND1, AtCCR1\u003c/em\u003e, and \u003cem\u003eAtCAD6)\u003c/em\u003e were carried out in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and mutants plants. Expression profile of lignin biosynthesis genes were either down regulated or having basal expression in both the WT and \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and mutants lines (Supplementary Fig.\u0026nbsp;13). \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0, \u003cem\u003eAtans\u0026thinsp;+\u0026thinsp;PgF3H and Atanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenic plants showed the similar expression of lignin biosynthesis genes with lower expression in response to drought stress (Supplementary Fig.\u0026nbsp;14). While during drought stress, \u003cem\u003eAtSND1, AtCCR1\u003c/em\u003e, and \u003cem\u003eAtCAD6\u003c/em\u003e genes are expressed more in \u003cem\u003eAtf3h\u003c/em\u003e mutant and \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e transgenic plants than in their respective controls, although \u003cem\u003eAtf3h\u003c/em\u003e mutant drought plants higher expression of these genes compared to \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e transgenic plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eEffect of PgF3H gene in ROS accumulation in PgF3H overexpressing Col0 and mutants plants\u003c/h2\u003e \u003cp\u003ePlants exposed to drought stress can cause ROS accumulation. The level of endogenous O2\u0026macr; in leaves was measured using NBT staining, and of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was measured using DAB staining. Under well-watered condition, all the WT, mutants, \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and mutants OE1, OE2 and OE3 transgenic lines showed similar basal levels of superoxide anion radicals and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e except \u003cem\u003eAtf3h\u003c/em\u003e plants. However, elevated NBT staining, representing higher O2\u0026macr; levels was observed in WT and mutant plants compared to \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and mutants transgenic plants under drought conditions. Likewise, enhanced DAB staining was observed in WT and mutant plants, representing higher levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, relative to that in \u003cem\u003ePgF3H\u003c/em\u003e, overexpressing transgenic lines under drought conditions (Supplementary Fig.\u0026nbsp;15). Quantification of both the staining was done using ImageJ software which also confirmed higher accumulation of ROS in WT as compared to transgenic lines under drought stress (Supplementary Fig.\u0026nbsp;15). These findings show that overexpression of \u003cem\u003ePgF3H\u003c/em\u003e can improve the tolerance of plants to drought stress in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFlavonoids are a group of natural compounds present in vegetables and fruits, serving as significant antioxidants in the human diet while also playing essential roles in various fundamental plant functions (Song et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Flavonoids derived from a phenylpropanoid pathway where \u003cem\u003eF3H\u003c/em\u003e is essential for the formation of intermediate shared biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins (Pervaiz et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In our previous transciptome study, \u003cem\u003ePgF3H\u003c/em\u003e gene responsible for flavonoids biosynthesis exhibited higher expression in pearl millet drought tolerant genotype under terminal drought stress (Shivhare et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Shivhare et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). In this work, \u003cem\u003ePgF3H\u003c/em\u003e was overexpressed in \u003cem\u003eArabidopsis\u003c/em\u003e to facilitate detailed functional analysis, focusing on screening transgenic plants for their response to drought stress. The coding sequence of \u003cem\u003ePgF3H\u003c/em\u003e underwent bioinformatic characterization, revealing significant sequence similarity with \u003cem\u003eF3H\u003c/em\u003e proteins from various plant species, particularly those within the Gramineae family. Five conserved motifs, which include residues binding 2-oxoglutarate and ferrous ions within the 2OG-Fe(II) oxygenase domains of \u003cem\u003eF3H\u003c/em\u003e proteins, show a high degree of similarity across various plant species (Song et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lukacin and Britsch, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Britsch et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). To evaluate the evolution relationship of \u003cem\u003ePgF3H\u003c/em\u003e and other orthologs in plants, a phlyogenetic tree was built and the results showed that \u003cem\u003ePgF3H\u003c/em\u003e is substantially identical to XP 004976589.1, XP 025825120.1, XP 002446984.2, THU69177.1, XP 030501918.1, PHT87608.1, and XP 016738006.11, this is why these proteins are in the same phylogenetic research subgroup. Through \u003cem\u003ein silico\u003c/em\u003e sequence analysis of approximately 1 Kb of the \u003cem\u003ePgF3H\u003c/em\u003e gene promoter, various putative cis-acting regulatory elements were identified. These include GTAC motifs associated with anoxic stress response, LTR elements linked to low temperature responsiveness, G-box motifs indicative of light responsiveness, ARE elements for anaerobic induction, as well as core sequences of ABRE elements involved in ABA responsiveness, and MBS motifs associated with drought inducibility. This indicates that not only the \u003cem\u003eF3H\u003c/em\u003e gene is activated during drought stress, but its promoter is also stimulated to protect the plants during environmental stresses and it was confirmed by gradual increase in GUS expression in Pro\u003cem\u003ePgF3H\u003c/em\u003e::GUS \u003cem\u003eArabidopsis\u003c/em\u003e seedlings by increasing the PEG6000 concentration. However, in combination with the corresponding transcription factors, the presence of various \u003cem\u003ecis\u003c/em\u003e-regulatory elements and their coordinated action can controls \u003cem\u003ePgF3H\u003c/em\u003e transcript expression in response to different climatic conditions and also in plant\u0026rsquo;s growth conditions (Shinde et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Reddy et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough flavonols and anthocyanin secondary metabolites were reported for their higher accumulation in plants in response to biotic and abiotic stress induction, such as drought, salt, UV light stimulus, cold and abscisic acid (ABA) stress responses (Zhang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jeong et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Winkel-Shirley et al., 1995). In this study, \u003cem\u003ePgF3H\u003c/em\u003e was cloned in the binary vector pCAMBIA1304 under the control of the promoter CaMV35S to observe its water stress tolerance capacity in transgenic \u003cem\u003eA. thaliana.\u003c/em\u003e Up regulation of the \u003cem\u003ePgF3H\u003c/em\u003e gene was seen in the qRT-PCR analysis of the produced transgenic lines. To characterize the mechanisms involved, PEG 6000 and mannitol were used to simulate water deficit conditions. For the stress tolerance assay, three homozygous (T3) transgenic lines of \u003cem\u003eA. thaliana\u003c/em\u003e were used. These transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants exhibited a higher rate of germination under stress caused by PEG and mannitol. The process of maintaining the water potential in plant cells to align the osmoticum under osmotic stress with the external environment is called osmotic adjustment (Shivhare and Lata, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Under water deficit conditions, the aggregation of solutes results in a reduction in the cell's osmotic potential, which draws water molecules into the cells and helps to retain turgor (Lata and Prasad, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lata et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). PEG and mannitol are commonly used for the artificial induction of osmotic stress and these solutes lower the osmotic potential (Khakwani et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rauf et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Bohnert et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Previous studies have also shown that under mannitol, PEG and NaCl stress, higher germination rate, root length, and biomass was recorded in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants that overexpress proteins related to stress response (Kumar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent researches, transgenic \u003cem\u003eArabidopsis\u003c/em\u003e and tobacco plants overexpressing regulatory proteins with flavonoids showed higher survival rates under water deficit conditions relative to WT plants (Li et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mahajan and Yadav et al., 2014; Liu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similar to other stress-responsive proteins, the constitutive overexpression of the \u003cem\u003ePgF3H\u003c/em\u003e gene in \u003cem\u003eA. thaliana\u003c/em\u003e plants resulted in enhanced drought tolerance. This was evidenced by reduced water loss rates and higher relative water content (RWC) in the transgenic plants (Kumar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In order to determine the water balance of plants, the RWC is seen as a significant marker (Lata et al., 2011). In other side, EL is inversely linked to the integrity of the cell membrane, and abiotic stress tolerance has generally been correlated with the ability to avoid or restore membrane damage (Lata et al., 2011). RWC and EL of both the \u003cem\u003ePgF3H\u003c/em\u003e overexpressing lines, WT and EV were similar at WW condition while WT and EV have higher decrease in RWC and excess EL compared to transgenic lines under WS condition. However overexpressing transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants with \u003cem\u003ePgF3H\u003c/em\u003e led to improved conservation of plant water status and integrity of the membrane, as confirmed by previous works (Butt et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). An increase in malondialdehyde (MDA) content serves as an indicator of stress-induced lipid peroxidation (LP) in cellular membranes and is considered a marker of elevated oxidative damage. These findings align with previous research which demonstrated that LP in drought-stressed \u003cem\u003eArabidopsis\u003c/em\u003e plants is linked to membrane integrity. Additionally, LP, along with electrolyte leakage (EL), has been recognized as a direct indicator of a plant's tolerance to dehydration stress (Dong et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The reduced MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in \u003cem\u003eLcF3H\u003c/em\u003e overexpressed transgenics \u003cem\u003eArabidopsis\u003c/em\u003e plants under stressed and unstressed conditions also have shown correlation with our results (Song et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Compatible osmolyte accumulation such as proline, by maintaining osmotic turgor, helps plants to withstand drought stress (Grover et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). During stress conditions, their synthesis and aggregation has been documented to enhance multiplicity (Lata et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Accordingly, this study also records a rise in proline content during drought in transgenic plants. Similarly, Under water-deficit conditions, transgenic plants exhibited higher chlorophyll content compared to wild-type (WT) plants. This increased chlorophyll content was associated with enhanced net photosynthesis, suggesting that transgenic plants were able to sustain more efficient photosynthetic activity during drought stress. This observation is supported by findings from Do et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), which reported that transgenic plants maintain better photosynthetic performance under drought conditions.\u003c/p\u003e \u003cp\u003eDrought stress effect the molecular and physiological properties of plants stemmed in to oxidative damage through the formation of toxic ROS that needs to be foraged by low molecular weight antioxidative enzymes. Plants can regulate reactive oxygen species (ROS) levels through the activity of antioxidant enzymes such as ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD). These enzymes play a crucial role in scavenging ROS molecules, thereby enhancing the plant's resistance to drought stress (Yao et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cai et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hameed et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In cell stroma, thalakoids, cytosol, and mitochondria, APX is present and serves as an electron donor for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition. SOD is a protection enzyme that catalyses the hydrolysis of superoxides (O\u003csub\u003e2\u003c/sub\u003e\u0026macr;) into less reactive molecules, such as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or molecular O\u003csub\u003e2\u003c/sub\u003e. Finally, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e thus produced is broken down into water and oxygen without the need to reduce power through the action of CAT (Shivhare and Lata, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lata et al., 2011). Similarly in this study, results also showed that antioxidants enzymetic activity increased in both the \u003cem\u003ePgF3H\u003c/em\u003e transgenic lines and in WT on exposure to drought stress but transgenic plants showed lower activity of APX, CAT, GPX and SOD antioxidative enzymes compared to WT in response to drought stress. Gene expression analysis of \u003cem\u003eAtAPX, AtCAT\u003c/em\u003e and \u003cem\u003eAtSOD\u003c/em\u003e also showed increase expression in WT, EV, mutants, and \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and mutant transgenic plants in comparison to their control but transgenics plants have lower expression of these genes compared to WT and mutant plants in under drought stress. So these results suggested that overexpression of \u003cem\u003ePgF3H\u003c/em\u003e gene did not induced the enzymatic pathway rather than it activate the non-enzymatic pathway via inducing the flavonone biosynthesis to overcome and minimize the oxidative stress response in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and mutant transgenic plants and increases its survival rate compared to WT plants (Jan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eF3H\u003c/em\u003e genes are known for their role in flavonols and anthocyanins biosynthesis, and their participation in plant responses to abiotic stresses have also been well described (Page et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Quina et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In response to drought stress, total flavonols and anthocyanin content was quantified in \u003cem\u003ePgF3H\u003c/em\u003e tansgenics and WT plants. Transgenic lines have significant higher accumulation of flavonols as well as anthocyanin pigments compared to WT. It was also confirmed by higher accumulation of flavonones and anthocyanins content in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e mutants (\u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e) under drought stress. Quantification of kaempferol, quercetin, and rutin in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e transgenic plants using HPLC also showed the positive correlation with these results. All the results revealed that these secondary metabolites participate in stress responsive regulatory pathways and protect the plants from deleterious effects of drought stress (Goswami et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Onkokesung et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Expression profiles of downstream genes of \u003cem\u003eF3H\u003c/em\u003e including \u003cem\u003eFLS1, FLS3, DFR, LDOX, UGT78D2, UGT78D3\u003c/em\u003e and \u003cem\u003eAUGT89A\u003c/em\u003e also exhibited differential expression pattern in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 and mutants transgenic plants under water deficit condition. Genes responsible for flavonol biosynthesis including \u003cem\u003eFLS1\u003c/em\u003e and \u003cem\u003eFLS3\u003c/em\u003e were highly expressed in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing transgenic lines of Col0 and mutants with more than four \u0026ndash; tenfold higher expression for \u003cem\u003eFLS3\u003c/em\u003e compared to WT and mutant plants under drought stress. Along with flavonol biosynthesis, genes for anthocyanin biosynthesis including \u003cem\u003eDFR, LDOX, UGT78D2\u003c/em\u003e, and \u003cem\u003eUGT78D3\u003c/em\u003e also showed enhanced expression except \u003cem\u003eUGT78D3\u003c/em\u003e in transgenic lines of Col0 and mutants in response to drought stress. \u003cem\u003eAtHCT\u003c/em\u003e involved in flavonoids accumulation was significantly higher in \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenic plants compared to their mutants while \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e have down expression of \u003cem\u003eAtHCT\u003c/em\u003e gene as present in \u003cem\u003ePgF3H\u003c/em\u003e Col0 transgenic plants under WS. Whereas, under drought stress, expression of gene involved in lignin biosynthesis including \u003cem\u003eSND1, CCR1\u003c/em\u003e and \u003cem\u003eCAD6\u003c/em\u003e were either have basal expression or down regulated in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 plants as well as in WT. The downregulation of these lignin biosynthesis genes is closely associated with the observed decrease in lignin content in the genetically edited plants. This correlation suggests that the reduced expression of these specific genes directly contributes to the lower lignin levels. Whereas, \u003cem\u003eAtf3h\u0026thinsp;+\u0026thinsp;PgF3H\u003c/em\u003e, \u003cem\u003eAtans\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e, and \u003cem\u003eAtanr\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePgF3H\u003c/em\u003e transgenic plants have up-regulation of lignin biosynthesis genes but lower than their respective mutant plants under drought stress. Overall, these findings indicate that the increased accumulation of flavonoids and anthocyanins in \u003cem\u003ePgF3H\u003c/em\u003e overexpressing Col0 plants can be attributed to the redirection of substrate flow towards flavonoid biosynthesis, occurring at the expense of lignin synthesis. This shift in metabolic flux suggests that the overexpression of \u003cem\u003ePgF3H\u003c/em\u003e prioritizes the production of flavonoids and anthocyanins, thereby reducing the resources available for lignin formation. These findings indicate that entire pathway possibilities are redirected to the development of flavonols and anthocyanin (Song et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Onkokesung et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mahajan and Yadav, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, we propose that the up-regulation of downstream genes within the DFR and ANS pathways in the flavonoid biosynthetic process might be responsible for the heightened anthocyanin accumulation observed in the transgenic plants. This hypothesis suggests that the increased expression of these specific genes enhances the production of anthocyanins, contributing to their elevated levels in the genetically modified plants (Survey et al., 2011). Further, flavonols and anthocyanin secondary metabolites were also reported for scavenging ROS species including, superoxide radicals O\u003csub\u003e2\u003c/sub\u003e\u0026macr;, hydroxyl radicals \u0026bull;OH and hydrogen peroxide H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Di Ferdinando et al., 2012). Anthocyanins, which generally accumulate in the vacuoles, prevent oxidative damage in plants and are important for exhibiting oxidative and drought tolerance (Hernandez et al., 2004). In our study, results of NBT and DAB staining of stressed leaves of transgenic lines and WT also showed the minimum accumulation of ROS in \u003cem\u003ePgF3H\u003c/em\u003e transgenic plants of Col0 and mutants as compared to WT and their respective mutants.\u003c/p\u003e \u003cp\u003eIn conclusion, the overexpression of flavanone 3-hydroxylase (\u003cem\u003ePgF3H\u003c/em\u003e) gene in \u003cem\u003eArabidopsis\u003c/em\u003e helps it to better tolerate drought stress by modulating various morpho-physiological, biochemical, and molecular parameters via inducing the flavonoid biosynthesis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This research has yielded valuable insights into the roles of \u003cem\u003ePgF3H\u003c/em\u003e in enhancing plant resistance to abiotic stress. By elucidating the functions of \u003cem\u003ePgF3H\u003c/em\u003e, the study advances our understanding of how this gene contributes to the plant's ability to withstand challenging environmental conditions, such as drought and temperature extremes. Finally, we conclude that in improving drought stress tolerance in crops, \u003cem\u003ePgF3H\u003c/em\u003e may be a valuable candidate gene.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Charu Lata acknowledges the INSPIRE Faculty Award [IFA-11LSPA-01] from Department of Science \u0026amp; Technology (DST), GoI, New Delhi. The authors are thankful Dr. Rakesh Srivastava, International Crops Research Institute for the Semi- Arid Tropics, Patancheru, India for providing pearl millet seed materials and \u003cem\u003eArabidopsis\u003c/em\u003e Biological Resource Center, The Ohio State University for providing \u003cem\u003eArabidopsis\u003c/em\u003e mutants. Dr. Radha Shivhare acknowledges CSIR for the SRF fellowship (File No: 31/08(0348)/2018-EMR-I) awarded to her.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.L. conceived and designed the experiment. R.S. and P.M. conducted the experiments. \u0026nbsp;P.K.B. performed the HPLC experiment. P.S.C. arranged the \u003cem\u003eArabidopsis\u003c/em\u003e mutants and lab facilities. R.S. analyzed the data. R.S. and C.L. wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAebi, H. (1974). \u0026ldquo;Catalase,\u0026rdquo; in Methods of Enzymatic Analysis, Vol. 2, ed. H. U. Bergmeyer (New York, NY: Academic Press Inc.), 673\u0026ndash;685.\u003c/li\u003e\n\u003cli\u003eAndr\u0026eacute;, C.M., Schafleitner, R., Legay, S., Lef\u0026egrave;vre, I., Aliaga, C.A., Nomberto, G., Hoffmann, L., Hausman, J.F., Larondelle, Y., Evers, D. 2009. Gene expression changes related to the production of phenolic compounds in potato tubers grown under drought stress. Phytochem. 70:1107\u0026ndash;1116.\u003c/li\u003e\n\u003cli\u003eArnon, D. I. (1949). Copper enzyme polyphenoloxides in isolated chloroplast in Beta vulgaris. 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Plant. 36:1221\u0026ndash;1229.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pennisetum glaucum, flavanone 3-hydroxylase, overexpression, Arabidopsis thailiana, water stress, flavonoids, Atf3h, Atans, Atanr, PgF3H promoter, protein structure, ROS","lastPublishedDoi":"10.21203/rs.3.rs-6030840/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6030840/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWater stress significantly impairs plant growth and yield, but plants combat this through various strategies, including flavonoid biosynthesis regulation. Flavonoids, crucial secondary metabolites, aid in plant development and stress responses. Pearl millet, a drought-tolerant crop, produces high levels of secondary metabolites like flavonoids and anthocyanins via the phenylpropanoid pathway. Research indicates that flavonoid-encoding genes are prevalent in drought-tolerant pearl millet variants, hinting at their role in drought response, though their exact functions are not fully understood. This study highlights the essential role of pearl millet flavanone 3-hydroxylase (\u003cem\u003ePgF3H\u003c/em\u003e) in flavonoid biosynthesis. Overexpressing \u003cem\u003ePgF3H\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e enhances flavonol and anthocyanin content, improving tolerance to water-deficit stress without affecting antioxidant gene expression. Supporting evidence includes increased flavanone 3-hydroxylase activity in the \u003cem\u003eAtf3h\u003c/em\u003e mutant and variable anthocyanin levels in \u003cem\u003eAtans\u003c/em\u003e and \u003cem\u003eAtanr\u003c/em\u003e mutants. In silico analysis of the \u003cem\u003ePgF3H\u003c/em\u003e promoter revealed stress-responsive elements, and ProPgF3H::GUS expressing lines showed increased GUS expression with higher PEG concentrations. The in silico structure of \u003cem\u003ePgF3H\u003c/em\u003e revealed a 2OG-Fe(II) oxygenase domain, crucial in the flavonoid biosynthetic pathway. In conclusion, \u003cem\u003ePgF3H\u003c/em\u003e overexpression enhances drought tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e, suggesting a potential strategy for improving crop drought resistance by manipulating flavonoid biosynthesis.\u003c/p\u003e","manuscriptTitle":"PgF3H gene enhances drought tolerance in transgenic Arabidopsis by regulating flavonoid biosynthesis and stress response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 10:02:44","doi":"10.21203/rs.3.rs-6030840/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-13T07:02:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-13T06:57:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-05T18:42:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-03-05T04:36:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a38f6b21-33b2-4713-aa49-92947dc97d0a","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T16:06:45+00:00","versionOfRecord":{"articleIdentity":"rs-6030840","link":"https://doi.org/10.1007/s00299-025-03524-8","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2025-06-20 15:57:30","publishedOnDateReadable":"June 20th, 2025"},"versionCreatedAt":"2025-03-17 10:02:44","video":"","vorDoi":"10.1007/s00299-025-03524-8","vorDoiUrl":"https://doi.org/10.1007/s00299-025-03524-8","workflowStages":[]},"version":"v1","identity":"rs-6030840","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6030840","identity":"rs-6030840","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}