CRISPR/Cas9-Mediated DFR Disruption Suggests Coordinated Changes in Flavonoid Flux and Development in Petunia × hybrida

CRISPR/Cas9-Mediated DFR Disruption Suggests Coordinated Changes in Flavonoid Flux and Development in Petunia × hybrida | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CRISPR/Cas9-Mediated DFR Disruption Suggests Coordinated Changes in Flavonoid Flux and Development in Petunia × hybrida Fangchen Liu, Tao Jiang, Sameena Ejaz Tanwir, Wisnu Handoyo Ardi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9227413/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Dihydroflavonol 4-reductase ( DFR ) occupies a critical branch point in flavonoid metabolism, channeling dihydroflavonol substrates toward anthocyanin biosynthesis in competition with flavonol synthase. While DFR's role in floral pigmentation is well established, the broader physiological and transcriptional consequences of its disruption remain poorly characterized, particularly in commercially important ornamental species. Here, we report the generation and comprehensive phenotyping of five independent CRISPR/Cas9-mediated DFR knockout alleles in the commercial Petunia × hybrida cultivar 'Carmine Velour'. The edited lines showed an allele-associated spectrum of loss of floral pigmentation that was broadly consistent with mutation severity, confirming DFR-A as the dominant isoform governing corolla anthocyanin accumulation. Beyond pigmentation, dfr mutants exhibited unexpected reductions in floral dimensions (20–40%), leaf biomass (30–50%), and plastidial pigment content, with chlorophyll and carotenoid levels declining 35–60% in petals despite unchanged leaf anthocyanins. Stem anatomy remained unaffected, revealing organ-specific pleiotropic effects. Transcriptional profiling uncovered feedback reprogramming within the flavonoid pathway: chalcone synthase A ( CHSA ) and chalcone isomerase A ( CHIA ) were downregulated while the competing branch enzyme flavonol synthase ( FLS ) was upregulated almost 2-fold, consistent with the possibility of altered flux partitioning toward flavonol biosynthesis. Strikingly, protochlorophyllide oxidoreductase A ( PORA ), encoding a key chlorophyll biosynthetic enzyme, was severely suppressedby 60–75%, suggesting a possible connection between flavonoid disruption and tetrapyrrole metabolism. Correlation analyses suggested coordinated variation, with floral anthocyanin content positively associated with leaf chlorophyll and carotenoid levels across genotypes. These findings support the view that DFR acts as a functionally important metabolic node whose disruption is associated with effects across pigment classes and organ types, with implications for precision trait engineering in floriculture. Petunia dihydroflavonol 4-reductase (DFR) genome-editing anthocyanin biosynthesis flavonoid metabolism pleiotropic effects Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message Loss of DFR function in petunia alters pigment metabolism and reduces organ size, revealing unexpected links between flavonoid biosynthesis, plastidial pigments, and development. Introduction Anthocyanins are water-soluble flavonoid pigments responsible for much of the red, purple, and blue coloration observed in flowers, fruits, and vegetative organs across the plant kingdom. Beyond aesthetics, these compounds serve protective functions: they attenuate photodamage under high-light conditions, scavenge reactive oxygen species during oxidative stress, and contribute to plant defense against both abiotic challenges, including drought, temperature extremes, and salinity, and biotic pressures such as herbivory and pathogen attack (Holton and Cornish 1995 ; Winkel-Shirley 2001 ). In reproductive structures, anthocyanins attract pollinators and seed dispersers, directly influencing reproductive fitness (Grotewold 2006 ). For ornamental crops, where flower color ranks among the most commercially decisive traits, anthocyanin content and composition largely determine market value. Unlike agronomic species prioritizing yield or nutritional output, ornamentals face intense selective pressure on visual appeal—color intensity, pattern stability, and novelty all drive consumer preference and breeding priorities (Tanaka et al. 2008 ; Zhao and Tao 2015 ). The anthocyanin biosynthetic pathway is well characterized at the molecular level. It initiates when chalcone synthase (CHS) condenses phenylalanine-derived precursors into naringenin chalcone, which chalcone isomerase (CHI) then cyclizes to form flavanones. Flavanone 3-hydroxylase (F3H) subsequently hydroxylates these intermediates to yield dihydroflavonols, such as dihydrokaempferol, dihydroquercetin, and dihydromyricetin, whose B-ring hydroxylation patterns can be further modified by flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). At this juncture, the pathway reaches a critical branch point: dihydroflavonols either enter flavonol biosynthesis via flavonol synthase (FLS) or proceed toward anthocyanin production through dihydroflavonol 4-reductase (DFR). DFR catalyzes the NADPH-dependent reduction of dihydroflavonols to leucoanthocyanidins, which anthocyanidin synthase (ANS) oxidizes to colored anthocyanidins; glycosylation by UDP-glucose: flavonoid 3- O -glucosyltransferase (UFGT) then stabilizes these products for vacuolar storage (Holton and Cornish 1995 ; Liu et al. 2021 ; Winkel-Shirley 2001 ). Transcriptional control of this pathway is largely mediated by the MBW complex, a ternary regulatory module comprising R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) proteins, and WD40-repeat proteins that together activate promoters of late biosynthetic genes, including DFR , ANS , and UFGT (Xu et al. 2015 ; Lloyd et al. 2017 ). In Antirrhinum majus , the bHLH component DELILA was among the first such regulators to be characterized (Goodrich et al. 1992 ), and subsequent work has continued to elucidate the functional roles of conserved bHLH factors in this species (Jiang et al. 2026 ). The combinatorial specificity of MBW components determines both the spatial patterning and intensity of anthocyanin accumulation across tissues, as different R2R3-MYB activators or repressors recruit distinct bHLH partners to fine-tune pathway output (Albert et al. 2014 ). DFR occupies a strategic position within flavonoid metabolism. Because it competes directly with FLS for dihydroflavonol substrates, the relative activities of these two enzymes dictate whether carbon flux proceeds toward colored anthocyanins or colorless flavonols (Choudhary and Pucker 2024 ; Luo et al. 2016 ). This competition has measurable phenotypic consequences. In Rubus chingii , biochemical analyses demonstrated that FLS exhibits higher affinity for dihydroflavonol substrates than DFR and that flavonols can inhibit DFR activity, establishing FLS as the dominant competitor under most physiological conditions (Lei et al. 2023 ). Comparative studies across species, including rose, petunia, carnation, azalea, and camellia, have consistently linked white-flowered phenotypes to elevated FLS expression relative to DFR , whereas red-flowered forms show the opposite pattern (Luo et al. 2016 ). Early transgenic work reinforced this view: constitutive DFR expression combined with antisense suppression of FLS maximized anthocyanin accumulation in petunia, positioning DFR as a metabolic gatekeeper whose manipulation can predictably redirect flavonoid flux (Davies et al. 2003 ). Functional evidence for DFR's role in anthocyanin production spans diverse experimental systems. Heterologous expression of OjDFR1 from Ophiorrhiza japonica in tobacco intensified floral pigmentation and upregulated endogenous ANS and UFGT transcripts, suggesting coordinated activation of downstream pathway components (Sun et al. 2021 ). Similarly, ectopic overexpression of GbDFR from Ginkgo biloba in tobacco elevated both DFR enzymatic activity and anthocyanin content while darkening flower color (Ni et al. 2020 ). In Hosta ventricosa , HvDFR overexpression in tobacco increased anthocyanin levels 1.7- to 2.4-fold and elevated total flavonoid content substantially (Qin et al. 2022 ). Studies in Arabidopsis tt3 mutants have been particularly informative: complementation with DFR homologs from sweet potato, freesia, and chrysanthemum restores both seed coat pigmentation and anthocyanin accumulation in vegetative tissues, confirming functional conservation across angiosperms (Li et al. 2017 ; Lim et al. 2020 ; Wang et al. 2013 ). Loss-of-function approaches have provided complementary insights. RNA interference targeting NtDFR1 and NtDFR2 in tobacco produced pale pink to white flowers and, unexpectedly, induced compensatory upregulation of other flavonoid biosynthetic genes, evidence that DFR perturbation triggers pathway-wide transcriptional feedback (Lim et al. 2016 ). RNAi-mediated downregulation of IbDFR in purple sweet potato dramatically reduced anthocyanin accumulation in leaves, stems, and storage roots while simultaneously increasing quercetin glycosides, directly demonstrating the metabolic trade-off between anthocyanin and flavonol branches (Wang et al. 2013 ). More recently, genome editing has enabled precise interrogation of DFR function. Watanabe et al. ( 2017 ) employed CRISPR/Cas9 to knock out DFR-B , one of three DFR genes in Japanese morning glory ( Ipomoea nil ); 75% of transgenic plants produced white flowers with biallelic mutations at the target locus, representing the first CRISPR-mediated flower color change reported in higher plants. This finding established that targeted DFR disruption can completely abolish anthocyanin biosynthesis without off-target effects on related members of the gene family. Despite substantial progress, several questions remain poorly understood. Most functional studies have concentrated on pigmentation phenotypes, with limited attention to broader developmental or physiological effects associated with altered DFR activity. The existing literature relies heavily on heterologous expression or gene suppression, approaches that demonstrate functional relevance but do not fully capture the consequences of precise loss-of-function mutations within native genetic and regulatory contexts. Whether DFR disruption affects traits beyond flower color has not been systematically examined, particularly in commercially valuable cultivars where growth performance and metabolic homeostasis are critical. The potential for pleiotropic effects, influences on chlorophyll or carotenoid metabolism, organ development, or stress physiology, warrants investigation, especially given emerging evidence that flavonoid pathway alterations can trigger unexpected transcriptional and metabolic responses. Petunia × hybrida offers distinct advantages for such investigations. As both a major ornamental crop and an established model for flower color research, petunia combines commercial relevance with extensive genetic and molecular resources. Petunia is amenable to stable Agrobacterium -mediated transformation and regenerates efficiently from tissue culture (Vandenbussche et al. 2016 ). Its flavonoid biosynthetic pathway has been characterized in detail, and the regulatory networks governing anthocyanin accumulation are well understood (Holton and Cornish 1995 ). Although CRISPR/Cas9 has been applied in petunia for purposes ranging from self-incompatibility studies to architectural modifications (Abdulla et al. 2024 ; Sun et al. 2018 ), targeted disruption of DFR in this species, with systematic characterization of the resulting phenotypes, has not been reported. Here we report the generation and characterization of CRISPR/Cas9-mediated DFR knockout lines in the commercial petunia cultivar 'Carmine Velour'. Five independently edited lines exhibiting varying degrees of loss of floral pigmentation were recovered, enabling the construction of an allelic series for phenotype–genotype correlation. By integrating molecular validation, comprehensive pigment quantification, morphometric analysis, and gene expression profiling, we addressed two primary objectives: first, to define how different levels of DFR inactivation reshapes floral anthocyanin accumulation and related pigment profiles; second, to assess whether DFR disruption affects traits beyond pigmentation, including organ development, chlorophyll and carotenoid metabolism, and transcriptional regulation of associated biosynthetic pathways. Our findings reveal that DFR knockout produces not only the expected anthocyanin depletion but also unanticipated effects on floral and vegetative physiology, demonstrating previously unrecognized connections between flavonoid metabolism and broader plant performance. These results advance understanding of DFR function and illustrate the utility of precise genome editing for both mechanistic inquiry and trait manipulation in ornamental crops. Materials and Methods Plant materials and growth conditions Petunia × hybrida 'Carmine Velour' (Wave® series) seeds were provided by Ball Horticultural Company. Seeds were surface-sterilized in 75% (v/v) ethanol for 1 min, followed by 15% (v/v) commercial bleach (6% sodium hypochlorite) for 10 min with gentle agitation, and rinsed six times with sterile distilled water. Sterilized seeds were germinated on Murashige and Skoog (MS) basal medium (Murashige and Skoog 1962 ) supplemented with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar (pH 5.8). Following acclimatization, transgenic and wild-type control plants were transferred to 10-cm pots containing a commercial soilless substrate (Premium Garden Soil Blend; Reliable Peat Company, Groveland, FL, USA) and cultivated in a controlled-environment growth chamber maintained at 25°C under a 16-h light/8-h dark photoperiod with approximately 60% relative humidity at the Mid-Florida Research and Education Center, University of Florida Institute of Food and Agricultural Sciences (Apopka, FL, USA) (Jiang et al. 2025 ). CRISPR/Cas9 vector construction and guide RNA design The CRISPR/Cas9 binary vector system was adapted from (Nguyen et al. 2021 ). The T-DNA cassette comprises an Arabidopsis -codon-optimized Streptococcus pyogenes Cas9 (HypaCas9) driven by the parsley ( Petroselinum crispum ) ubiquitin promoter ( PcUBI ), a single-guide RNA (sgRNA) expression module under the Arabidopsis U6-26 promoter utilizing a tRNA–sgRNA polycistronic architecture, and an eGFP–NPTII fusion selectable marker driven by the double-enhanced cassava vein mosaic virus (CsVMV) promoter for kanamycin selection and visual screening of transformants (Fig. S1 ). Guide RNA target sites were identified within conserved regions of the Petunia DFR coding sequence. Briefly, Petunia DFR homologs were retrieved from NCBI GenBank using the Arabidopsis thaliana DFR protein sequence (AT5G42800; TAIR) as query. Multiple Petunia DFR coding sequences from diverse cultivars and species were aligned using ClustalW (Thompson et al. 1994 , Fig. S2) to identify highly conserved exonic regions amenable to multiplex targeting. Candidate gRNAs were designed using CRISPOR (Concordet and Haeussler 2018 ) and CHOPCHOP (Labun et al. 2019 ), prioritizing guides with canonical NGG protospacer adjacent motif (PAM) sequences, high predicted on-target activity scores, and minimal predicted off-target activity as assessed by CRISPOR based on available petunia reference genomes. Three gRNAs (gRNA1–gRNA3) targeting exons 3 and 4 were selected for multiplex editing (Fig. 1 , Table S1 ). Synthetic oligonucleotides encoding the gRNA spacer sequences were synthesized (Gene Universal Inc., Newark, DE, USA) and cloned into the PHN-HypaCas9-4×BsaI-GFP backbone via Golden Gate assembly using BsaI restriction sites. All constructs were sequence-verified by Sanger sequencing and subsequently introduced into Agrobacterium tumefaciens strain EHA105 by freeze–thaw transformation. Agrobacterium-mediated transformation and plant regeneration Agrobacterium tumefaciens strain EHA105 harboring the CRISPR/Cas9 construct was cultured in Luria-Bertani (LB) medium supplemented with rifampicin (50 mg L − 1 ) and spectinomycin (100 mg L − 1 ) at 28°C with shaking (200 rpm) until reaching an optical density at 600 nm (OD600) of 0.3–0.5. Bacterial cells were harvested by centrifugation (4,000 × g , 10 min, 4°C), gently resuspended in liquid MS medium (pH 5.8) containing 100 µM acetosyringone, and incubated at room temperature for 1 h to induce virulence gene expression. Cotyledonary explants excised from 10-day-old in vitro -germinated seedlings were used for transformation. Prior to inoculation, explants were pre-cultured on MS basal medium containing 3% (w/v) sucrose and 0.7% (w/v) agar (pH 5.8) for 2 days in darkness to promote competence for transformation. Pre-cultured explants were immersed in the Agrobacterium suspension for 10 min with gentle agitation, blotted on sterile filter paper to remove excess inoculum, and transferred to co-cultivation medium (CCM) consisting of MS salts supplemented with 0.75 mg L − 1 6-benzylaminopurine (BAP) and 0.15 mg L − 1 1-naphthaleneacetic acid (NAA). Co-cultivation proceeded for 2 days at 25°C in darkness. Following co-cultivation, explants were transferred to callus induction medium (CIM) containing 0.75 mg L − 1 BAP, 0.15 mg L − 1 NAA, 100 mg L − 1 kanamycin for selection, and 100 mg L − 1 timentin to suppress Agrobacterium overgrowth. Cultures were maintained at 25°C under a 16-h light/8-h dark photoperiod. Regenerating calli were transferred to shoot induction medium (SIM) containing 0.5 mg L − 1 BAP and 0.01 mg L − 1 NAA. Elongated shoots were excised and transferred to root induction medium (RIM) supplemented with 0.5 mg L − 1 indole-3-butyric acid (IBA). All regeneration media contained identical antibiotic concentrations as CIM. Putative transformants were identified by kanamycin resistance and confirmed by GFP fluorescence visualization using a fluorescence stereomicroscope (Leica M205 FA; Leica Microsystems, Wetzlar, Germany). Only GFP-positive regenerants were retained for rooting and subsequent analyses. Mutant genotyping and validation Genomic DNA was isolated from young, fully expanded leaves using a cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle and Doyle 1987). The DFR target region spanning all three gRNA sites and selected candidate off-target loci were amplified by PCR using gene-specific primers (Table S1 ). Amplicons were purified using a commercial PCR purification kit (QIAGEN, Hilden, Germany) and subjected to Sanger sequencing. Editing outcomes and candidate off-target loci were determined by aligning sequence chromatograms to the wild-type reference sequence using SnapGene software (GSL Biotech LLC, San Diego, CA, USA). Mutant alleles were classified based on the nature and position of insertions, deletions, or substitutions relative to the predicted Cas9 cleavage sites. Morphological phenotyping Floral and vegetative organ dimensions were quantified from calibrated digital images using ImageJ software (Schneider et al. 2012 ). Flowers were harvested at anthesis (stage 13), and leaves were sampled from the third to fourth nodes below the shoot apex to ensure comparable physiological age across genotypes. Images containing a metric scale reference were converted to 8-bit grayscale, spatially calibrated, and analyzed using the freehand selection tool to delineate individual organ boundaries. Projected area was measured using the "Analyze > Measure" function. A minimum of ten biological replicates per genotype were analyzed for each trait. Fresh weight of excised leaves and flowers was determined using a precision analytical balance (MS105DU; Mettler Toledo, Columbus, OH, USA) with 0.01 mg readability. Protein sequence prediction and structural modeling Predicted protein sequences corresponding to wild-type and edited DFR alleles were derived by conceptual translation of inferred coding sequences using the ExPASy Translate tool ( https://web.expasy.org/translate/ ). Three-dimensional protein structure models were generated by homology modeling using the SWISS-MODEL server ( https://swissmodel.expasy.org/ ; Waterhouse et al. 2018 ) with default parameters. Template selection, model building, and quality estimation were performed automatically. Predicted structures were visualized and compared using PyMOL (Schrödinger LLC, New York, NY, USA) to illustrate the extent of truncation and loss of conserved structural domains in mutant DFR proteins. Anthocyanin extraction and quantification Anthocyanins were extracted and quantified following the acidified methanol protocol of Neff and Chory ( 1998 ) with modifications. Freshly harvested petal or leaf tissue (50–100 mg) was weighed, flash-frozen in liquid nitrogen, and homogenized to a fine powder. Samples were extracted in 300 µL acidified methanol [1% (v/v) HCl in methanol] by vortexing and incubating overnight at room temperature in complete darkness. Following extraction, 200 µL Milli-Q water and 500 µL chloroform were added to facilitate phase separation. Samples were vortexed vigorously and centrifuged (12,000 × g , 5 min, 4°C). A 350-µL aliquot of the upper aqueous-methanolic phase was transferred to a fresh tube and diluted to a final volume of 1.05 mL with 415.8 µL methanol, 4.2 µL concentrated HCl, and 280 µL Milli-Q water to maintain absorbance within the linear detection range. Absorbance was measured at 530 nm (anthocyanin peak) and 657 nm (chlorophyll interference) using a microplate reader (Synergy H1; BioTek Instruments, Winooski, VT, USA). Corrected anthocyanin absorbance was calculated as Acorr = A530 − 0.25 × A657. Anthocyanin content was expressed as milligrams of cyanidin-3-glucoside equivalents per gram fresh weight (mg C3G eq g − 1 FW) using the equation: Anthocyanin = (Acorr × V × MW × 1000) / (ε × l × FW), where V is the final extract volume (L), MW = 449.2 g mol − 1 (molecular weight of cyanidin-3-glucoside), ε = 33,000 L mol − 1 cm − 1 (molar extinction coefficient), l = 1 cm (path length), and FW is the fresh tissue weight (g). Blank extractions processed without tissue were included to correct for background absorbance. Chlorophyll and carotenoid extraction and quantification Chlorophylls and carotenoids were extracted following (Lichtenthaler 1987 ) with modifications. Approximately 50 mg of fresh petal or leaf tissue was incubated in 1.0 mL of 95% (v/v) ethanol for 16 h at 25°C in complete darkness to ensure exhaustive pigment solubilization. Extracts were clarified by centrifugation (12,000 × g , 5 min), and absorbance was measured at 470 nm, 649 nm, and 665 nm using a BioTek Synergy H1 microplate reader (Agilent Technologies, Santa Clara, CA, USA) with 95% ethanol as the reference blank. Pigment concentrations (µg mL − 1 ) were calculated using the spectrophotometric equations of Wellburn ( 1994 ) optimized for 95% ethanol: chlorophyll a = 13.95 × A665 − 6.88 × A649; chlorophyll b = 24.96 × A649 − 7.32 × A665; total carotenoids = (1000 × A470 − 2.13 × Chl a − 97.63 × Chl b ) / 209. Final pigment contents were expressed as mg g − 1 FW by multiplying concentrations by the extraction volume and normalizing to fresh tissue mass. RNA isolation and quantitative real-time PCR Total RNA was extracted from 80–100 mg of flash-frozen petal tissue using RNAzol RT reagent (Molecular Research Center, Cincinnati, OH, USA) following the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA); samples with A260/A280 ratios between 1.9 and 2.1 were retained for downstream applications. Genomic DNA contamination was eliminated by on-column DNase I digestion (RNase-Free DNase Set; QIAGEN). First-strand cDNA was synthesized from 1 µg of DNase-treated RNA using the QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer's instructions. The resulting cDNA was diluted 20- to 25-fold in nuclease-free water for use as qRT-PCR template. Quantitative real-time PCR (qRT-PCR) was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iTaq Universal SYBR Green Supermix (Bio-Rad). Each 10-µL reaction contained 5 µL 2× SYBR Green master mix, 4 µL diluted cDNA template, and 0.5 µL of a 10 µM forward and reverse primer mixture. Gene-specific primers were designed using Primer-BLAST (NCBI) to span exon–exon junctions where possible and to generate amplicons of 100–150 bp with melting temperatures of 58–62°C. Primer sequences for all target genes are provided in Table S1 . Target genes included structural genes of the anthocyanin biosynthetic pathway ( CHSA , CHIA , F3′H , F3′5′H , ANS ), the flavonol branch pathway ( FLS ), and chlorophyll biosynthesis ( PORA , CHLH ). Thermal cycling conditions consisted of an initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Melt curve analysis was performed after amplification to verify primer specificity. Relative gene expression was calculated using the 2 −ΔΔCt method (Livak and Schmittgen 2001 ), with EF1α serving as the internal reference gene based on its stable expression across all experimental samples. Three biological replicates, each comprising three technical replicates, were analyzed per genotype. Statistical analysis All statistical analyses were performed using R software (version 4.3.1; R Core Team, 2023 ). Biochemical quantifications (anthocyanin, chlorophyll, carotenoid) and gene expression analyses were conducted using a minimum of three independent biological replicates, with three technical replicates per biological sample for qRT-PCR assays. Morphological traits were quantified from at least ten biological replicates per genotype. Differences among genotypes were evaluated by one-way analysis of variance (ANOVA) following verification of normality (Shapiro–Wilk test) and homogeneity of variances (Levene's test). Pairwise comparisons were performed using Tukey's honestly significant difference (HSD) post hoc test. Compact letter displays indicating statistically distinct groups ( P < 0.05) were generated using the multcompView package and are displayed above corresponding data points in all figures. Pearson correlation coefficients and associated P -values were calculated using the cor.test function to assess relationships between phenotypic traits. All data visualizations were generated using the ggplot2 package (Wickham 2016 ). Box plots display the median, interquartile range (IQR; 25th–75th percentiles), and whiskers extending to 1.5× IQR; individual data points are overlaid to show the full distribution of biological replicates. Bar plots display mean values with error bars representing standard deviation (SD) unless otherwise specified. Phylogenetic analysis and sequence alignment The amino acid sequence of Petunia × hybrida DFR-A (PhDFRa) was retrieved from NCBI based on the published DFR-A sequence (Beld et al. 1989 ). Additional DFR protein sequences from Solanaceae species were selected according to the DFR family analysis reported by Li et al. ( 2023 ). Full-length amino acid sequences were aligned in MEGA (version 11.0.13) using default parameters, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replicates. Bootstrap values were used to assess branch support, and the tree was visualized using iTOL v6 (Letunic and Bork., 2024). Conserved motifs in DFR proteins were identified using the MEME Suite with the following settings: maximum number of motifs = 10 and motif width = 6–50 amino acids. The predicted three-dimensional structure of PhDFRa was generated using SWISS-MODEL. Sequence similarity among DFR proteins was evaluated by BLASTP using the PhDFRa amino acid sequence as the query. Comparative DFR protein sequences from other Solanaceae species, selected from Li et al. ( 2023 ), were aligned against PhDFRa, and the resulting similarity statistics, including Max score, Total score, Query cover, E-value, Percent identity, and protein length, were summarized. Results CRISPR/Cas9-mediated disruption of DFR produces an allele-associated spectrum of pigment loss in petunia To dissect the functional role of dihydroflavonol 4-reductase (DFR) in anthocyanin biosynthesis, we employed a multiplex CRISPR/Cas9 strategy targeting the canonical floral DFR gene in Petunia × hybrida 'Carmine Velour'. Three guide RNAs (gRNA1–gRNA3) were designed to target conserved coding sequences within exons 3 and 4, with inter-target distances of 122 bp (gRNA1–gRNA2) and 244 bp (gRNA2–gRNA3) (Fig. 1 ). Although P. hybrida harbors multiple DFR paralogs, previous biochemical and genetic evidence indicates that DFR-A is the principal enzyme channeling dihydroflavonols toward anthocyanidin biosynthesis in corolla tissue (Huits et al. 1994 ); accordingly, all genotyping and phenotypic analyses focused on this locus. This target choice was further supported by phylogenetic, structural, and sequence-comparison analyses, which placed the selected petunia DFR with functionally characterized anthocyanin-associated DFR proteins and indicated that all queried petunia DFR versions showed the highest similarity to the same PhDFR homolog (Fig. S2). These results support the conclusion that this locus represents the major DFR copy associated with anthocyanin biosynthesis in P. hybrida . Potential off-target sites for each guide RNA were predicted using CRISPOR, and the highest-ranking candidate loci were subsequently amplified and examined by Sanger sequencing (Fig. S3). No mutations were detected at these predicted off-target sites in the edited plants analyzed, supporting the specificity of the CRISPR/Cas9 system used in this study. Five independent edited lines ( dfr-1 to dfr-5 ) were recovered and cultivated under controlled greenhouse conditions. These lines displayed a striking, overall reduction in floral pigmentation relative to the deeply magenta wild-type (WT) control (Fig. 1 A). Lines dfr-1 and dfr-2 retained partial magenta pigmentation with characteristic variegated patterning, dfr-3 exhibited markedly attenuated coloration, and dfr-4 and dfr-5 produced flowers with near-complete absence of visible anthocyanin accumulation, yielding pale cream to yellow-green corollas. Sanger sequencing of PCR amplicons spanning the three CRISPR target sites revealed a diverse spectrum of editing outcomes (Fig. 1 B). The detected lesions included: a 3-bp in-frame deletion at the gRNA1 site coupled with a single-nucleotide insertion at gRNA2 in dfr-1 (net Δ = −3/+1); single-base insertions at the gRNA2 locus in dfr-2 and dfr-3 (Δ = +1); a 76-bp deletion spanning the gRNA2 region in dfr-4 (Δ = −76); and a large 304-bp deletion extending across the gRNA1–gRNA3 interval in dfr-5 (Δ = −304). All identified mutations introduced frameshifts relative to the WT open reading frame, resulting in premature translation termination. Computational protein modeling corroborated these predictions (Fig. 1 C). Whereas the WT DFR enzyme comprises 380 amino acids with characteristic Rossmann-fold architecture, the edited alleles encode severely truncated polypeptides: 155 aa ( dfr-1 ), 165 aa ( dfr-2 and dfr-3 ), 142 aa ( dfr-4 ), and 114 aa ( dfr-5 ). Structural predictions indicate extensive loss of conserved secondary and tertiary elements (Fig. 1 C), including the catalytic domain and NADPH-binding site, consistent with strong loss of function. Together, these mutations generate DFR variants predicted to cause differing degrees of functional impairment, enabling analysis of how differing degrees of DFR disruption influence pigmentation and broader physiological consequences. DFR perturbation depletes floral anthocyanins and alters pigment composition alongside organ morphology Biochemical and morphometric characterization revealed a broad phenotypic spectrum across all edited genotypes (Fig. 2 , Fig. S4). Methanolic extracts from wild-type (WT) petals were intensely magenta. dfr-1 and dfr-2 yielded diluted pink solutions; dfr-3 extracts appeared markedly paler; those from dfr-4 and dfr-5 were nearly colorless (Fig. 2 A). This gradient was broadly consistent with the intact flower phenotypes. WT corollas were uniformly pigmented across the limb, while dfr-1 and dfr-2 showed variegated patterning instead, with pigmented sectors interspersed among unpigmented tissue. This mosaic appearance likely reflects chimeric editing or mosaicism within primary regenerants. dfr-3 flowers were uniformly pale pink. dfr-4 and dfr-5 lacked visible anthocyanin altogether, their cream-to-yellowish hue attributable to underlying carotenoids and chlorophyll (Fig. 2 B). Transgene integration was verified by fluorescence imaging of dissected corolla tubes (Fig. 2 C). WT flowers exhibited minimal autofluorescence confined to anther tissue, with no signal in filaments. All dfr lines, by contrast, displayed strong GFP fluorescence throughout filaments and anthers, confirming successful incorporation of the CRISPR/Cas9 T-DNA cassette harboring the eGFP reporter. Spectrophotometric quantification supported the visual observations (Fig. 2 D). WT flowers accumulated 0.47 mg g − 1 fresh weight (FW) anthocyanin. dfr-1 retained roughly 80% of this value (0.37 mg g − 1 ). dfr-2 and dfr-3 showed reductions of approximately 40% and 80%, respectively (Fig. 2 D). The most severely edited lines, dfr-4 and dfr-5 , contained only trace amounts (≤ 0.05 mg g − 1 ), representing greater than 90% depletion relative to WT. The overall decline across the allelic series closely corresponded to predicted protein truncation severity. Floral dimensions declined in parallel with anthocyanin content (Fig. 2 E, F). WT corolla area averaged 31.2 ± 1.8 cm 2 . dfr-1 through dfr-3 were 20–30% smaller; dfr-4 and dfr-5 approached 40% reduction, averaging 18–19 cm 2 . Fresh weight followed a similar though less pronounced trend: WT corollas averaged 0.32 ± 0.02 g, while mutant values ranged from 0.23 to 0.28 g (10–30% decreases). The coupling between pigment loss and reduced organ size was consistent across independent lines, suggesting an association rather than coincidental variation. The morphological phenotypes prompted examination of non-anthocyanin pigments in petal tissue (Fig. 3 ). Interestingly, total carotenoid content declined significantly across all mutant lines (Fig. 3 B), with dfr mutant petals containing 0.015 to 0.025 mg g − 1 compared to 0.041 mg g − 1 FW in WT, representing 35–60% reductions. Unlike anthocyanins, carotenoid levels showed no clear relationship with the apparent anthocyanin gradient: dfr-2 (165 aa truncation) and dfr-5 (114 aa truncation) exhibited comparable reductions despite different predicted degrees of DFR disruption. This pattern argues against direct enzymatic involvement and instead implicates indirect regulatory or metabolic feedback mechanisms. Chlorophyll reductions were more pronounced (Fig. 3 C–E). WT petals contained 0.133 ± 0.012 mg g − 1 FW total chlorophyll, whereas dfr mutant lines ranged from 37–55% of this level. Critically, both chlorophyll a and b declined coordinately rather than selectively, indicating general suppression of tetrapyrrole biosynthesis rather than pathway-specific effects. The reduction ranges from dfr-2 (~ 55% of WT) to dfr-4 (~ 37%) did not show a clear quantitative correspondence to anthocyanin loss, consistent with the carotenoid pattern. The coordinated decline of both chlorophylls and carotenoids across all five independent alleles reveals that DFR disruption broadly perturbs plastidial pigment metabolism, extending well beyond anthocyanin biosynthesis in the cytosol. The consistency of this phenotype across multiple independently edited alleles with distinct molecular lesions argues against somaclonal variation or T-DNA positional effects, supporting plastid pigment reduction as a likely pleiotropic consequence of DFR loss. Vegetative tissues show organ-specific responses: leaf physiology altered, stem anatomy unaffected To determine whether DFR-A governs anthocyanin biosynthesis and development beyond flowers, we examined leaf pigmentation, morphology, and stem anatomy across all genotypes (Figs. 4 and 5 ). Leaf anthocyanin accumulation is DFR-independent. In striking contrast to the floral phenotypes, anthocyanin extracts from leaves of WT and all dfr mutants were uniformly pale and visually indistinguishable (Fig. 4 A). Quantitative analysis confirmed this observation: mean leaf anthocyanin content ranged from 0.013 to 0.022 mg g − 1 FW across genotypes, with no statistically significant differences detected between WT and any mutant lines (Fig. 4 C). These findings indicate that DFR-A is dispensable for basal anthocyanin accumulation in petunia leaves, potentially reflecting functional redundancy with other DFR paralogs or tissue-specific regulatory differences. Despite unchanged anthocyanin levels, leaf development is compromised. Leaf area showed modest reductions across mutants, with only dfr-5 reaching significance ( P < 0.05) compared to WT (0.95 ± 0.08 cm²) (Fig. 4 B, D). Leaf biomass, however, declined substantially: WT leaves averaged 0.028 ± 0.003 g, whereas all dfr mutant lines ranged from 0.014 to 0.020 g, representing decreases of 30–50% (Fig. 4 E). Paralleling the petal phenotype, leaf chlorophyll and carotenoids also decreased by 20–45% (Fig. S5), with dfr-4 and dfr-5 showing the most severe depletion. Despite unchanged anthocyanin content, the coordinated reductions of biomass and plastidial pigments suggest that DFR disruption is associated with altered leaf physiology through anthocyanin-independent mechanisms. In contrast to the leaf phenotypes, stem morphology and anatomy were unaffected by DFR disruption. Flowering shoots exhibited the expected floral pigmentation gradient but normal overall architecture, with internode length, phyllotaxis, and branching comparable between WT and all dfr lines (Fig. S6). Transverse sections of young stems revealed no discernible anatomical abnormalities in any mutant genotype (Fig. 5 B). Vascular organization, cortical thickness, epidermal cell morphology, and central pith structure showed no abnormalities, hypertrophy, or irregular patterning in any mutant. Collectively, these observations establish that DFR-A function is essential for floral pigmentation and associated with changes in leaf biomass and plastidial pigment accumulation, but dispensable for leaf anthocyanin accumulation and stem morphogenesis. The reduction in leaf biomass and plastidial pigments, occurring independently of anthocyanin deficiency, suggests that DFR perturbation influences vegetative traits through mechanisms extending beyond its canonical role in floral anthocyanin biosynthesis. Floral anthocyanin content correlates with morphological and pigment traits across genotypes To examine relationships between anthocyanin accumulation and other traits, we performed Pearson correlation analyses using genotype-level mean values (Fig. 5 ). Within floral organs, anthocyanin content showed positive associations with morphological traits. Flower area exhibited a strong positive correlation with anthocyanin levels (R = 0.78, P = 0.069), and flower fresh weight followed a similar trend (R = 0.56, P = 0.25), though neither reached statistical significance. Positive correlations were also detected between floral anthocyanin content and other pigment classes: carotenoid content (R = 0.65, P = 0.16) and total chlorophyll (R = 0.71, P = 0.11). Notably, the strongest correlations were observed between floral anthocyanin content and vegetative traits, with several relationships reaching statistical significance. Leaf area was significantly correlated with floral anthocyanin levels (R = 0.84, P = 0.037), indicating that genotypes with higher floral pigmentation tend to produce larger leaves. Even more robust associations were observed for leaf pigment contents: total leaf chlorophyll (R = 0.89, P = 0.018) and leaf carotenoid content (R = 0.93, P = 0.0075) both showed highly significant positive correlations with floral anthocyanin accumulation. Leaf fresh weight exhibited a positive but non-significant relationship (R = 0.56, P = 0.24). The tight correlations between floral anthocyanin and leaf plastidial pigments suggest an association between floral and vegetative pigment traits across genotypes. This pattern is consistent with but does not by itself demonstrate broader coordination of pigment-related processes across organs. Shared regulatory networks or whole-plant resource allocation constraints could contribute to these relationships, although the current analysis does not distinguish among these possibilities. Overall, these data indicate that variation in floral anthocyanin content is accompanied by corresponding shifts in several vegetative traits following DFR perturbation. DFR dysfunction triggers coordinate transcriptional changes in flavonoid and chlorophyll biosynthesis To assess transcriptional responses to DFR loss of function, we quantified gene expression in corolla tissue from WT, dfr-3 , and dfr-5 across flavonoid biosynthesis and chlorophyll metabolism pathways (Fig. 6 ). Upstream anthocyanin genes show coordinate downregulation. CHSA (chalcone synthase A), encoding the entry-point enzyme of flavonoid biosynthesis, showed moderate decline to ~ 75% of WT in dfr-3 (not significant) and ~ 60% in dfr-5 ( P < 0.05). CHIA (chalcone isomerase A) exhibited a more pronounced and consistent response: transcript levels declined to ~ 55% of WT in dfr-3 ( P < 0.001) and 70% in dfr-5 ( P < 0.01). The upstream transcriptional downregulation triggered by DFR disruption suggests feedback sensing of the metabolic blockage, potentially triggered by reduced flux or intermediate accumulation. Unlike the upstream anthocyanin genes, genes involved in parallel or downstream branches of flavonoid metabolism showed divergent responses. FLS (flavonol synthase), which competes with DFR for dihydroflavonol substrates, increased significantly: 1.9-fold in dfr-3 and 1.4-fold in dfr-5 relative to WT (both P < 0.05). This upregulation is consistent with the possibility of substrate redirection toward flavonol production when DFR is inactive. In contrast, F3′H , F3′5′H , and ANS showed no significant differences in transcript abundance between WT and either mutant line, with expression values remaining within ± 30% of WT levels. Notably, genes involved in chlorophyll metabolism exhibited marked transcriptional alterations. PORA (protochlorophyllide oxidoreductase A), encoding a rate-limiting enzyme in chlorophyll biosynthesis, was severely downregulated: ~25% of WT in dfr-3 ( P < 0.001) and 40% of WT in dfr-5 ( P < 0.01). By contrast, CHLH , encoding the H subunit of magnesium chelatase, showed no significant changes, with transcript levels remaining comparable to WT in both mutant backgrounds. The selective downregulation of PORA may contribute to the reduced chlorophyll accumulation observed in dfr mutant flowers and suggests a possible link between cytosolic flavonoid and plastidial tetrapyrrole pathways. Discussion Targeted DFR knockout in P. hybrida eliminated floral anthocyanin in an allele-dependent fashion, with the five edited lines spanning partial to near-complete depigmentation (Fig. 1 ). Overall, the severity of depigmentation was generally consistent with mutation type and predicted protein truncation, supporting the conclusion that DFR-A is the principal isoform governing corolla pigmentation in petunia (Fig. S2). Earlier biochemical work had pointed in this direction (Holton and Cornish 1995 ), but precise loss-of-function alleles had not been available to test the inference directly. Beyond pigmentation, the mutants displayed reduced floral dimensions, diminished leaf biomass, and coordinated declines in chlorophyll and carotenoid content across both reproductive and vegetative tissues, while stem anatomy remained normal (Figs. 2 – 4 , Figs. S4-6). Multiple independent alleles converging on similar phenotypes strengthen confidence that these traits represent genuine consequences of DFR loss rather than transformation artifacts or background variation. This consideration is especially important in commercial hybrid backgrounds, where genetic heterogeneity can obscure single-allele effects. The following sections address the metabolic function of DFR in flavonoid biosynthesis and the pleiotropic consequences of its disruption, but first the interpretation of pigmentation phenotypes in these primary regenerants warrants qualification. The pigmentation phenotypes in T₀ plants should be interpreted with caution. Several edited lines, particularly dfr-2, dfr-3, and dfr-4, display sectoral pigmentation patterns characterized by white stripes or unpigmented patches interspersed with residual anthocyanin-containing tissue, rather than uniformly reduced coloration across the corolla. Such patterns are consistent with somatic chimerism, in which independently edited and unedited cell lineages coexist within a single regenerant. Chimeric tissues are a well-recognized outcome of Agrobacterium -mediated transformation and CRISPR/Cas9 editing in T₀ plants, because mutagenesis can occur at different developmental stages during callus proliferation and shoot regeneration (Fauser et al. 2014 ; Feng et al. 2014 ). The sectoral distribution of pigment loss in these lines, therefore, may reflect mosaic editing across floral cell layers, rather than solely a simple graded, allele-dependent reduction in DFR activity. Because individual flower sectors were not isolated and sequenced separately in this study, the relative contribution of chimerism versus residual enzymatic function to the observed variation in pigmentation cannot be resolved from the current data. Future work should include genotyping of micro-dissected pigmented and unpigmented sectors from the same corolla, as well as analysis of T₁ segregants, to distinguish true hypomorphic alleles from chimeric mosaics and to establish stable genotype-phenotype relationships in non-chimeric backgrounds. DFR functions in flavonoid biosynthesis and metabolic flux partitioning DFR and FLS occupy the same metabolic branch point, competing for dihydroflavonol substrates (Choudhary and Pucker 2024 ; Luo et al. 2016 ). Eliminating DFR activity would be expected to favor the accumulation of dihydroflavonols and increase the potential for flux to be redirected toward flavonol biosynthesis. The significant upregulation of FLS transcripts in dfr-3 and dfr-5 corollas supports this prediction (Fig. 6 ). Wang et al. ( 2013 ) observed exactly this pattern when suppressing IbDFR by RNAi in purple sweet potato: anthocyanins declined while quercetin glycosides rose. Direct quantification of flavonols was not performed in the present study; therefore, a shift in metabolic flux cannot be demonstrated from the current data. Nevertheless, the transcriptional data, together with the well-documented substrate competition between DFR and FLS , are consistent with partial flux redirection toward flavonol biosynthesis. Prior DFR loss-of-function studies across diverse species provide useful context for evaluating these findings. Watanabe et al. ( 2017 ) used CRISPR/Cas9 to knock out DFR-B in Japanese morning glory ( Ipomoea nil ); 75% of regenerants bore white flowers, confirming that DFR loss abolishes anthocyanin accumulation in Convolvulaceae. That study focused on pigmentation and did not examine developmental or physiological consequences. In black rice ( Oryza sativa ), Jung et al. ( 2019 ) applied CRISPR/Cas9 to target OsF3′H , OsDFR , and OsLDOX simultaneously; the resulting mutants showed altered seed coloration and reduced anthocyanin content, with the mutations stably inherited into subsequent generations. RNAi knockdown of NtDFR1 and NtDFR2 in tobacco produced pale flowers and triggered compensatory upregulation of other flavonoid genes (Lim et al. 2016 ). A similar transcriptional feedback response was observed here, though the magnitude differed. Functional characterization of DFR homologs in ornamental species has expanded considerably. Qin et al. ( 2022 ) isolated HvDFR from Hosta ventricosa and demonstrated that ectopic expression in Arabidopsis increased anthocyanin accumulation 1.7- to 2.4-fold, confirming the enzyme's conserved role across monocots and dicots. In chrysanthemum, Lim et al. ( 2020 ) showed that C-terminal sequence variation in CmDFR between white- and red-flowered cultivars affects substrate specificity for dihydrokaempferol, contributing to flower color differences. The Arabidopsis tt3 mutant accumulates elevated flavonols, exhibits altered auxin transport and displays developmental anomalies (Kuhn et al. 2011 ; Peer et al. 2004 ). These phenotypes align broadly with ours. What sets the present work apart is the systematic characterization of pigment, morphological, and transcriptional phenotypes across multiple organs using multiple independent alleles in a commercially relevant cultivar. Most earlier DFR studies examined pigmentation alone or relied on single mutant alleles. The allelic series strategy enabled the establishment of an overall relationship between mutation severity and phenotypic intensity, as well as the distinction between genuine DFR-dependent effects and background noise. Including leaf and stem phenotyping revealed organ-specific consequences: leaves exhibited reduced biomass and plastidial pigments despite unchanged anthocyanin content, whereas stems remained anatomically normal (Figs. 4 , Figs. S5-6). This pattern has not been documented in previous DFR mutant analyses. Recent genome-wide surveys have identified DFR gene families in strawberry (Chen et al. 2024 ), rapeseed (Qian et al. 2023 ), and Solanaceae species (Li et al. 2023 ), underscoring the complexity of DFR regulation. Our findings add a layer by demonstrating that even knockout of a single dominant isoform can propagate effects across connected pathways. Pleiotropic consequences of DFR disruption and anthocyanin depletion A possible increase in flavonol levels could have functional consequences that help explain the observed growth phenotypes. A recent comprehensive review by Daryanavard et al. ( 2023 ) summarized the current understanding of flavonol action in plant development, emphasizing two primary mechanisms: inhibition of polar auxin transport and modulation of reactive oxygen species homeostasis. Flavonols negatively regulate auxin transport by modulating PIN-dependent auxin distribution and auxin efflux processes (Buer and Muday 2004 ; Kuhn et al. 2011 ; Peer et al. 2004 ). In Arabidopsis , flavonoid accumulation caused by repression of lignin biosynthesis (HCT silencing) correlated directly with reduced auxin transport and growth inhibition (Besseau et al. 2007 ). Arabidopsis tt3 mutants lack functional DFR and accumulate excess kaempferol and quercetin; these plants display altered auxin distribution together with modified root and shoot architecture (Peer et al. 2004 ). The reduced flower size and leaf biomass in our dfr lines (Figs. 2 and 4 ) are consistent with auxin-mediated growth limitation, although confirmation would require direct measurement of auxin gradients in petunia using DR5 reporter lines or targeted IAA quantification. Flavonols also act as potent antioxidants. Quercetin derivatives in particular scavenge reactive oxygen species and modulate cellular redox balance (Agati et al. 2012 ; Sharma et al. 2012 ). ROS serve dual roles as signaling molecules and as agents of oxidative damage, so shifts in flavonol content can alter developmental programs through redox-sensitive pathways (Gayomba et al. 2017 ). Kurepa et al. ( 2023 ) recently argued that flavonoids act as central mediators of the auxin-ROS-flavonol feedback loop, linking environmental sensing to developmental adjustment. Whether growth reductions in dfr mutants stem from perturbed auxin transport, altered ROS homeostasis, or both cannot be resolved with current data. Support for the redox hypothesis also comes from purple sweet potato, where RNAi suppression of IbDFR reduced anthocyanin accumulation and led to greater oxidative damage under cold stress, including increased H₂O₂ accumulation and electrolyte leakage, consistent with a protective role for anthocyanins in ROS homeostasis (Wang et al. 2013 ). The two mechanisms are not mutually exclusive. Histochemical ROS staining or genetically encoded biosensors would help clarify the contribution of redox perturbation. One of the less anticipated outcomes of this work is the coordinated decline of chlorophyll and carotenoid content in dfr mutant flowers (Fig. 3 ). Chlorophyll a and b fell in parallel, as did total carotenoids, indicating general suppression of plastidial pigment biosynthesis rather than selective loss of a single branch. Severe downregulation of PORA in mutant corollas may provide a plausible mechanism (Fig. 6 ). PORA encodes protochlorophyllide oxidoreductase A, which catalyzes a light-dependent step in chlorophyll synthesis; its suppression would predictably limit chlorophyll accumulation. CHLH , encoding the H subunit of magnesium chelatase, remained unchanged, indicating that the perturbation is specific to PORA rather than global across the tetrapyrrole pathway. Consistent with this possibility, transcriptome analysis of CRISPR/Cas9 NtDFR1/NtDFR2 knockout tobacco plants revealed significant downregulation of photosynthesis-related pathways, including genes encoding light-harvesting complex proteins, suggesting that disruption of DFR can influence chloroplast-associated metabolic processes beyond flavonoid biosynthesis (Jiang et al. 2023 ). How flavonoid disruption impinges on chlorophyll biosynthesis is not immediately obvious. Several possibilities warrant consideration. Redox status influences chloroplast development and gene expression, so altered flavonol-mediated ROS scavenging could affect plastidial processes secondarily. Shared transcriptional regulators offer another route. HY5, a bZIP-type transcription factor, has emerged as a master regulator coordinating light signaling with flavonoid biosynthesis and photomorphogenic chlorophyll accumulation (Xiao et al. 2022 ). HY5 directly activates MYB12 and multiple anthocyanin biosynthetic genes, and in roots it also modulates auxin transporter abundance (van Gelderen et al. 2017 ; Xiao et al. 2022 ). In apple, MbHY5 has been implicated in regulation of both iron transport and chlorophyll synthesis under iron deficiency (Sun et al. 2022 ). The strong positive correlation between floral anthocyanin and leaf chlorophyll across genotypes ( R = 0.89, P = 0.018; Fig. 5 ) is consistent with coordinated variation between these traits across organs. The underlying mechanism remains to be identified, but the data suggest that flavonoid and tetrapyrrole metabolism may be more closely linked in petunia than is currently appreciated. This coupling may be especially pronounced. CRISPR/Cas9-based modification of flavonoid pathway genes is increasingly applied in ornamental breeding. Nitarska et al. ( 2021 ) targeted F3′H in poinsettia to shift bract color from red to reddish-orange through reduced cyanidin levels. Nishihara et al. ( 2018 ) edited F3H into Torenia to produce white-flowered lines with high efficiency. In chrysanthemum, genome editing faces additional challenges due to hexaploidy, though progress has been made using multicopy transgenes as editing targets (Kishi-Kaboshi et al. 2017 ). Our results add to this literature by showing that DFR knockout in a commercial petunia cultivar produces a range of pigment phenotypes accompanied by growth phenotypes that breeders must consider. The edited lines collectively suggest that a variety of phenotypes may be achievable, not merely binary on/off states, expanding the toolkit for rational trait engineering in ornamentals. Breeding implications, limitations, and future directions From a breeding perspective, these findings carry practical weight. Targeted DFR editing provides a direct route to modulate flower color intensity in petunia and related ornamentals. The edited lines suggest that a spectrum of pigmentation phenotypes may be achievable, not merely binary on/off states. Although the edited lines collectively showed an allele-associated spectrum of floral depigmentation, the striped and sectorial patterns observed in some lines suggest that petal color may also be influenced by somatic mosaicism or chimerism in primary regenerants. Therefore, these visible differences should be interpreted with caution, and validation in fixed later-generation lines or in separately genotyped petal sectors will be needed to confirm stable genotype–phenotype relationships. This expands the palette available to breeders pursuing novel color variants. At the same time, the associated reductions in flower size and biomass warrant attention. Breeders aiming for lighter flower color may need to screen against growth penalties or pair DFR edits with compensatory modifications elsewhere in the pathway. The pleiotropic effects documented here also hint at opportunities beyond color engineering. Manipulation of the flavonoid pathway could modulate stress tolerance by altering flavonol-mediated ROS scavenging. Meyer et al. ( 1987 ) first demonstrated heterologous DFR expression in petunia to redirect pigment biosynthesis. Subsequent work across species has linked flavonoid accumulation to enhanced tolerance of oxidative and drought stress (Nakabayashi et al. 2014 ). Consistent with this broader view, transgenic tobacco overexpressing tea DFR or ANR accumulated more flavonoids, showed enhanced antioxidant capacity, flowered earlier, and displayed improved resistance to herbivory, indicating that manipulation of downstream flavonoid flux can influence both development and stress-related traits (Kumar et al. 2013 ). Whether DFR knockout, together with a possible shift in flavonoid composition, improves or compromises stress resilience in petunia remains untested. The outcome likely depends on which flavonoid classes predominate and how effectively they counteract particular stressors. Several limitations of this work point toward productive follow-up. Flavonol accumulation was inferred from transcriptional data and the known biochemistry of the DFR/FLS branch point; direct quantification was not performed. Targeted metabolomics, for example, LC-MS profiling of kaempferol, quercetin, and their glycosides, would confirm the inference and identify which specific flavonol species accumulate. Such data would also clarify whether developmental phenotypes correlate more tightly with total flavonol content or with particular derivatives. The mechanistic underpinnings of the growth phenotypes require further investigation. Measurements of auxin distribution using DR5 reporters or direct quantification of IAA would test the auxin transport hypothesis. Parallel assessment of ROS levels would evaluate the redox hypothesis. The connection between DFR disruption and PORA downregulation is particularly intriguing. ChIP-seq to identify shared transcription factor binding sites between flavonoid and tetrapyrrole pathway genes could illuminate this possible link. Reproductive fitness was not systematically examined here. Pollen viability, seed set, and germination rate all merit attention, given the documented roles of flavonols in pollen tube growth and fertilization (Mo et al. 1992 ; Taylor and Grotewold 2005 ). DFR disruption could affect these processes. Finally, combinatorial editing offers a promising avenue. Targeting DFR together with FLS , F3′H , or F3′5′H would permit finer control over metabolic flux partitioning and may help disentangle the relative contributions of anthocyanin loss versus flavonol gain to the phenotypes observed in this study. Conclusions CRISPR/Cas9-mediated knockout of DFR in Petunia confirms that this enzyme is essential for floral anthocyanin biosynthesis and reveals unanticipated pleiotropic consequences extending to organ size, biomass, and plastidial pigment metabolism. The availability of multiple edited alleles strengthens inference about genotype–phenotype relationships, although some pigmentation patterns in T₀ plants likely reflect mosaicism. The transcriptional and correlational data suggest that DFR functions not merely as a biosynthetic enzyme but as a functionally important metabolic node whose disruption is associated with effects across connected pathways. These findings expand current understanding of flavonoid pathway integration and provide a foundation for rational engineering of pigmentation and related traits in ornamental crops. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests Declare any potential competing interests. Funding Information This project was funded by the USDA-NIFA grant 2019-67013-29236 and the USDA HATCH program FLA-MFC-006387, awarded to H.H. Author Contribution FL and TJ conceived and designed the study. FL and SET performed the experiments. FL and TJ collected and analyzed the data and wrote the manuscript. WA provided technical assistance. HH and TJ supervised the research, secured resources, and revised the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors gratefully acknowledge PanAmerican Seed (Ball Horticultural Company) for providing the Petunia × hybrida ‘Carmine Velour’ cultivar for this research. The authors sincerely thank Dr. Jianping Ren for coordinating seed shipment and for valuable guidance and helpful discussions regarding petunia materials and cultivation. 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Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 05 May, 2026 Reviews received at journal 22 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers agreed at journal 28 Mar, 2026 Reviewers invited by journal 28 Mar, 2026 Editor assigned by journal 26 Mar, 2026 Submission checks completed at journal 26 Mar, 2026 First submitted to journal 25 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-9227413","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":615502774,"identity":"5d4debde-f354-46db-976f-ffa9bc9a1d65","order_by":0,"name":"Fangchen Liu","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Fangchen","middleName":"","lastName":"Liu","suffix":""},{"id":615502779,"identity":"893e85f5-c43b-4465-88a9-5a3fa9cc6b44","order_by":1,"name":"Tao Jiang","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Jiang","suffix":""},{"id":615502785,"identity":"c105c5e4-4c69-44bd-ac8b-bc581d8feb6b","order_by":2,"name":"Sameena Ejaz Tanwir","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Sameena","middleName":"Ejaz","lastName":"Tanwir","suffix":""},{"id":615502789,"identity":"93aea4da-ba6f-4959-9bea-0c68acbeaa86","order_by":3,"name":"Wisnu Handoyo Ardi","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Wisnu","middleName":"Handoyo","lastName":"Ardi","suffix":""},{"id":615502793,"identity":"94e686d7-d293-4c5a-b044-587f1400d273","order_by":4,"name":"Heqiang Huo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAkElEQVRIiWNgGAWjYBACAwkg8YFBTgbEJl4L4wwGYx7StDDzkKTFXLrH8LFtmwEPA3vzNgmitFjOOWNsnAvSwnOsjDgtBjdyzKRz2/7wMEjkmJGgxRJki/wbUrQwgrRI8BCtJa3YsOecAQ8bT1qxBZFakjc++FFmIMfPfnjjDaK0wAEbacpHwSgYBaNgFOAFAK2JJCgPtvQoAAAAAElFTkSuQmCC","orcid":"","institution":"University of Florida","correspondingAuthor":true,"prefix":"","firstName":"Heqiang","middleName":"","lastName":"Huo","suffix":""}],"badges":[],"createdAt":"2026-03-25 22:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9227413/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9227413/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106094092,"identity":"225f9620-7e95-4db8-8f28-3d8ae689b1b6","added_by":"auto","created_at":"2026-04-03 11:40:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":544688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRISPR/Cas9-mediated disruption of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDFR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e locus in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePetunia × hybrida\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ‘Carmine Velour’ produces an allele-associated spectrum of floral pigmentation. (A)\u003c/strong\u003eWhole-plant phenotypes of wild type (WT) and five independent CRISPR-edited lines (\u003cem\u003edfr-1 \u003c/em\u003eto \u003cem\u003edfr-5\u003c/em\u003e) grown under greenhouse conditions. WT plants display deep magenta corollas, whereas edited lines exhibit varying degrees of reduced anthocyanin pigmentation, ranging from partial pigmentation (\u003cem\u003edfr-1\u003c/em\u003e, \u003cem\u003edfr-2\u003c/em\u003e) to markedly reduced coloration (\u003cem\u003edfr-3\u003c/em\u003e) and near-complete loss of visible pigment (\u003cem\u003edfr-4\u003c/em\u003e, \u003cem\u003edfr-5\u003c/em\u003e). Scale bars = 10 cm. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic of the \u003cem\u003eDFR\u003c/em\u003e gene structure showing exon organization and positions of three CRISPR guide RNAs (gRNA1–gRNA3, red) within exons 3 and 4. Distances between adjacent target sites are indicated (122 bp between gRNA1–gRNA2 and 244 bp between gRNA2- gRNA3). Representative sequence alignments of WT and mutant alleles are shown below, with PAM sequences highlighted in blue. The net size change relative to WT is summarized in the Δ column. Detected lesions include small local indels near gRNA1 (\u003cem\u003edfr-1\u003c/em\u003e), single-base insertions at the gRNA2 site (\u003cem\u003edfr-1\u003c/em\u003e, \u003cem\u003edfr-2\u003c/em\u003e, \u003cem\u003edfr-3\u003c/em\u003e), a 76-bp deletion spanning the gRNA2 region (\u003cem\u003edfr-4\u003c/em\u003e), and a 304-bp deletion extending across the gRNA2–gRNA3 interval (\u003cem\u003edfr-5\u003c/em\u003e). Insertions are highlighted in orange, and deletions are indicated by gray shading. \u003cstrong\u003e(C)\u003c/strong\u003ePredicted protein structures derived from WT and edited \u003cem\u003eDFR\u003c/em\u003e coding sequences. \u003cem\u003eDFR\u003c/em\u003e in WT encodes a 380-amino-acid protein, whereas all edited alleles produce truncated polypeptides of reduced length (\u003cem\u003edfr-1\u003c/em\u003e, 155 aa; \u003cem\u003edfr-2\u003c/em\u003e, 165 aa; \u003cem\u003edfr-3\u003c/em\u003e, 165 aa; \u003cem\u003edfr-4\u003c/em\u003e, 142 aa; \u003cem\u003edfr-5\u003c/em\u003e, 114 aa), with frameshift-induced premature termination and loss of functional domains.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/6f694894dc8edf4fea8c6953.png"},{"id":106402119,"identity":"5411d551-f17c-4f13-bc2d-30a503d0feab","added_by":"auto","created_at":"2026-04-08 09:11:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFloral pigmentation and morphological traits of petunia WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edfr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutants.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Anthocyanin extracts from WT and \u003cem\u003edfr\u003c/em\u003e flowers showing varying extents of anthocyanin pigmentation loss; \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative corolla images illustrating variation in floral pigmentation: uniformly magenta WT flowers; partially pigmented \u003cem\u003edfr-1\u003c/em\u003e and \u003cem\u003edfr-2\u003c/em\u003e; pale-pink \u003cem\u003edfr-3\u003c/em\u003e; and anthocyanin-deficient \u003cem\u003edfr-4\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e. Scale bars, 1 cm. \u003cstrong\u003e(C)\u003c/strong\u003eDissected corolla tubes imaged under bright field and GFP fluorescence. WT shows autofluorescence in anthers, whereas all \u003cem\u003edfr\u003c/em\u003e mutants exhibit stronger fluorescence, including in filaments. Scale bars, 5 mm. \u003cstrong\u003e(D)\u003c/strong\u003eQuantification of floral anthocyanin content (mg g⁻¹ FW). \u003cstrong\u003e(E)\u003c/strong\u003e Flower area (cm²). \u0026nbsp;\u003cstrong\u003e(F)\u003c/strong\u003e Flower fresh weight (g). For panel (D), values represent at least three biological replicates, each measured with three technical repeats. For panels (E–F), values represent at least ten biological replicates. Box plots show the median, interquartile range, and full data range. Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD test; different letters indicate significant differences between genotypes (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/2b00844e0c95bc879ceab43e.png"},{"id":106046743,"identity":"79d58f67-62b5-476e-a720-9632995e5455","added_by":"auto","created_at":"2026-04-02 19:54:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCarotenoid and chlorophyll content in petunia WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edfr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant flowers\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Representative pigment extracts from flower corollas of WT and \u003cem\u003edfr\u003c/em\u003emutants (\u003cem\u003edfr-1 to dfr-5\u003c/em\u003e), showing lighter coloration in the mutant lines. \u003cstrong\u003e(B)\u003c/strong\u003e Total carotenoid content (mg g⁻¹ FW). \u003cstrong\u003e(C) \u003c/strong\u003eChlorophyll \u003cem\u003ea \u003c/em\u003econtent (mg g⁻¹ FW). \u003cstrong\u003e(D) \u003c/strong\u003eChlorophyll\u003cem\u003e b\u003c/em\u003e content (mg g⁻¹ FW). \u003cstrong\u003e(E)\u003c/strong\u003eTotal chlorophyll content (mg g⁻¹ FW). For panels (B–E), each point represents one replicate, derived from four biological replicates per genotype, with three technical measurements per biological replicate. Box plots display the median, interquartile range, and full distribution of the plotted values. Statistical differences among genotypes were determined using one-way ANOVA followed by Tukey’s HSD test; different letters denote significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/ea0407466a66f1ff667a0e64.png"},{"id":106046741,"identity":"ea74bbaf-1936-47be-bf83-bb1b0355287b","added_by":"auto","created_at":"2026-04-02 19:54:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":244509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLeaf anthocyanin content and morphological traits in WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edfr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutant petunia lines.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Anthocyanin extracts from leaves of wild-type (WT) and \u003cem\u003edfr\u003c/em\u003emutants (\u003cem\u003edfr-1\u003c/em\u003e to \u003cem\u003edfr-5\u003c/em\u003e) showing pale coloration with no visible differences among genotypes. \u003cstrong\u003e(B)\u003c/strong\u003e Representative leaf images displaying comparable shape and coloration across WT and mutant lines. Scale bars, 1 cm. \u003cstrong\u003e(C)\u003c/strong\u003e Leaf anthocyanin content (mg g⁻¹ FW). Consistent with the visual extracts, no significant differences were detected between WT and any of the \u003cem\u003edfr\u003c/em\u003e mutants. \u003cstrong\u003e(D)\u003c/strong\u003e Leaf area (cm²). All \u003cem\u003edfr\u003c/em\u003emutants displayed reduced mean leaf area compared to WT, though only \u003cem\u003edfr-5\u003c/em\u003ereached statistical significance. \u003cstrong\u003e(E)\u003c/strong\u003e Leaf fresh weight (g). WT leaves were significantly heavier than all \u003cem\u003edfr\u003c/em\u003emutant lines; no significant differences were detected among the mutants. For panel (C), values represent at least three biological replicates, each measured with three technical repeats. For panels (D–E), values represent at least ten biological replicates. Statistical significance was assessed using one-way ANOVA followed by Tukey's HSD test; different letters indicate significant differences among genotypes (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Scale bar, 1 cm in B.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/cc14f12823d9766afa595709.png"},{"id":106046748,"identity":"9a0c622d-fd99-4fc0-9142-de042fead76b","added_by":"auto","created_at":"2026-04-02 19:54:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":349109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analyses of anthocyanin content with morphological and pigment traits in petunia WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003edfr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant lines. \u003c/strong\u003ePearson correlation analyses were performed using genotype-level mean values to examine associations between floral anthocyanin content and morphological or pigment-related traits. Scatter plots show relationships between floral anthocyanin content and \u003cstrong\u003e(A)\u003c/strong\u003e flower area, \u003cstrong\u003e(B)\u003c/strong\u003e floral fresh weight, \u003cstrong\u003e(C)\u003c/strong\u003e floral carotenoid content, \u003cstrong\u003e(D)\u003c/strong\u003e floral chlorophyll content, \u003cstrong\u003e(E)\u003c/strong\u003eleaf area, \u003cstrong\u003e(F)\u003c/strong\u003e leaf fresh weight, \u003cstrong\u003e(G)\u003c/strong\u003e leaf total chlorophyll content, and \u003cstrong\u003e(H)\u003c/strong\u003e leaf carotenoid content. Each point represents the mean value for one genotype (WT and \u003cem\u003edfr-1\u003c/em\u003e to \u003cem\u003edfr-5\u003c/em\u003e), based on at least 10 biological replicates per genotype. Solid blue lines indicate linear regression fits, and shaded areas represent 95% confidence intervals. Pearson correlation coefficients (R) and associated \u003cem\u003eP\u003c/em\u003e-values are indicated in each panel.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/bd9865aa0258c6fa4e635068.png"},{"id":106046750,"identity":"1c4fc9a6-694e-4c52-9a89-aa2c25f996ec","added_by":"auto","created_at":"2026-04-02 19:54:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":191786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression analysis of flavonoid and chlorophyll pathway genes in WT and dfr mutant petals.\u003c/strong\u003e Relative transcript abundance of \u003cstrong\u003e(A)\u003c/strong\u003eanthocyanin biosynthetic genes (\u003cem\u003eCHSA\u003c/em\u003e, \u003cem\u003eCHIA\u003c/em\u003e, \u003cem\u003eF3’H\u003c/em\u003e, \u003cem\u003eF3′5′H\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e), \u003cstrong\u003e(B)\u003c/strong\u003e the competing branch gene (\u003cem\u003eFLS\u003c/em\u003e),\u003cstrong\u003e (C) \u003c/strong\u003echlorophyll metabolism–associated genes (\u003cem\u003ePORA\u003c/em\u003e, \u003cem\u003eCHLH\u003c/em\u003e) in fully expanded flower corollas. Expression levels were measured by qRT-PCR in wild-type (WT), \u003cem\u003edfr\u003c/em\u003e-3, and \u003cem\u003edfr\u003c/em\u003e-5 lines, normalized to an internal reference gene EF1α and are presented relative to WT (set to 1.0). Bars indicate mean ± s.d. of three biological replicates. WT, \u003cem\u003edfr\u003c/em\u003e-3, and \u003cem\u003edfr\u003c/em\u003e-5 are shown in dark, medium, and light purple, respectively. Statistical significance versus WT was determined by one-way ANOVA followed by Tukey’s HSD test; ns, not significant; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/aafadf313a7cc8665e847621.png"},{"id":106405592,"identity":"03051b76-5e7a-4eb3-b899-450687a2263a","added_by":"auto","created_at":"2026-04-08 09:27:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3335612,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/b44fffa3-5add-4857-bf5f-0e6406ee6139.pdf"},{"id":106046742,"identity":"1869512d-b123-4971-94e1-4f4319c99fc8","added_by":"auto","created_at":"2026-04-02 19:54:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2740018,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9227413/v1/e417aa77140462a4a894bdaa.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CRISPR/Cas9-Mediated DFR Disruption Suggests Coordinated Changes in Flavonoid Flux and Development in Petunia × hybrida","fulltext":[{"header":"Key Message","content":"\u003cp\u003eLoss of DFR function in petunia alters pigment metabolism and reduces organ size, revealing unexpected links between flavonoid biosynthesis, plastidial pigments, and development.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAnthocyanins are water-soluble flavonoid pigments responsible for much of the red, purple, and blue coloration observed in flowers, fruits, and vegetative organs across the plant kingdom. Beyond aesthetics, these compounds serve protective functions: they attenuate photodamage under high-light conditions, scavenge reactive oxygen species during oxidative stress, and contribute to plant defense against both abiotic challenges, including drought, temperature extremes, and salinity, and biotic pressures such as herbivory and pathogen attack (Holton and Cornish \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Winkel-Shirley \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In reproductive structures, anthocyanins attract pollinators and seed dispersers, directly influencing reproductive fitness (Grotewold \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For ornamental crops, where flower color ranks among the most commercially decisive traits, anthocyanin content and composition largely determine market value. Unlike agronomic species prioritizing yield or nutritional output, ornamentals face intense selective pressure on visual appeal\u0026mdash;color intensity, pattern stability, and novelty all drive consumer preference and breeding priorities (Tanaka et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhao and Tao \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe anthocyanin biosynthetic pathway is well characterized at the molecular level. It initiates when chalcone synthase (CHS) condenses phenylalanine-derived precursors into naringenin chalcone, which chalcone isomerase (CHI) then cyclizes to form flavanones. Flavanone 3-hydroxylase (F3H) subsequently hydroxylates these intermediates to yield dihydroflavonols, such as dihydrokaempferol, dihydroquercetin, and dihydromyricetin, whose B-ring hydroxylation patterns can be further modified by flavonoid 3\u0026prime;-hydroxylase (F3\u0026prime;H) and flavonoid 3\u0026prime;,5\u0026prime;-hydroxylase (F3\u0026prime;5\u0026prime;H). At this juncture, the pathway reaches a critical branch point: dihydroflavonols either enter flavonol biosynthesis via flavonol synthase (FLS) or proceed toward anthocyanin production through dihydroflavonol 4-reductase (DFR). DFR catalyzes the NADPH-dependent reduction of dihydroflavonols to leucoanthocyanidins, which anthocyanidin synthase (ANS) oxidizes to colored anthocyanidins; glycosylation by UDP-glucose: flavonoid 3-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase (UFGT) then stabilizes these products for vacuolar storage (Holton and Cornish \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Winkel-Shirley \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Transcriptional control of this pathway is largely mediated by the MBW complex, a ternary regulatory module comprising R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) proteins, and WD40-repeat proteins that together activate promoters of late biosynthetic genes, including \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e, and \u003cem\u003eUFGT\u003c/em\u003e (Xu et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lloyd et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In \u003cem\u003eAntirrhinum majus\u003c/em\u003e, the bHLH component DELILA was among the first such regulators to be characterized (Goodrich et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), and subsequent work has continued to elucidate the functional roles of conserved bHLH factors in this species (Jiang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). The combinatorial specificity of MBW components determines both the spatial patterning and intensity of anthocyanin accumulation across tissues, as different R2R3-MYB activators or repressors recruit distinct bHLH partners to fine-tune pathway output (Albert et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDFR occupies a strategic position within flavonoid metabolism. Because it competes directly with FLS for dihydroflavonol substrates, the relative activities of these two enzymes dictate whether carbon flux proceeds toward colored anthocyanins or colorless flavonols (Choudhary and Pucker \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This competition has measurable phenotypic consequences. In \u003cem\u003eRubus chingii\u003c/em\u003e, biochemical analyses demonstrated that FLS exhibits higher affinity for dihydroflavonol substrates than DFR and that flavonols can inhibit DFR activity, establishing FLS as the dominant competitor under most physiological conditions (Lei et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Comparative studies across species, including rose, petunia, carnation, azalea, and camellia, have consistently linked white-flowered phenotypes to elevated \u003cem\u003eFLS\u003c/em\u003e expression relative to \u003cem\u003eDFR\u003c/em\u003e, whereas red-flowered forms show the opposite pattern (Luo et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Early transgenic work reinforced this view: constitutive \u003cem\u003eDFR\u003c/em\u003e expression combined with antisense suppression of \u003cem\u003eFLS\u003c/em\u003e maximized anthocyanin accumulation in petunia, positioning DFR as a metabolic gatekeeper whose manipulation can predictably redirect flavonoid flux (Davies et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFunctional evidence for DFR's role in anthocyanin production spans diverse experimental systems. Heterologous expression of \u003cem\u003eOjDFR1\u003c/em\u003e from \u003cem\u003eOphiorrhiza japonica\u003c/em\u003e in tobacco intensified floral pigmentation and upregulated endogenous \u003cem\u003eANS\u003c/em\u003e and \u003cem\u003eUFGT\u003c/em\u003e transcripts, suggesting coordinated activation of downstream pathway components (Sun et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, ectopic overexpression of \u003cem\u003eGbDFR\u003c/em\u003e from \u003cem\u003eGinkgo biloba\u003c/em\u003e in tobacco elevated both DFR enzymatic activity and anthocyanin content while darkening flower color (Ni et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In \u003cem\u003eHosta ventricosa\u003c/em\u003e, \u003cem\u003eHvDFR\u003c/em\u003e overexpression in tobacco increased anthocyanin levels 1.7- to 2.4-fold and elevated total flavonoid content substantially (Qin et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies in \u003cem\u003eArabidopsis tt3\u003c/em\u003e mutants have been particularly informative: complementation with \u003cem\u003eDFR\u003c/em\u003e homologs from sweet potato, freesia, and chrysanthemum restores both seed coat pigmentation and anthocyanin accumulation in vegetative tissues, confirming functional conservation across angiosperms (Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLoss-of-function approaches have provided complementary insights. RNA interference targeting \u003cem\u003eNtDFR1\u003c/em\u003e and \u003cem\u003eNtDFR2\u003c/em\u003e in tobacco produced pale pink to white flowers and, unexpectedly, induced compensatory upregulation of other flavonoid biosynthetic genes, evidence that DFR perturbation triggers pathway-wide transcriptional feedback (Lim et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). RNAi-mediated downregulation of \u003cem\u003eIbDFR\u003c/em\u003e in purple sweet potato dramatically reduced anthocyanin accumulation in leaves, stems, and storage roots while simultaneously increasing quercetin glycosides, directly demonstrating the metabolic trade-off between anthocyanin and flavonol branches (Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). More recently, genome editing has enabled precise interrogation of \u003cem\u003eDFR\u003c/em\u003e function. Watanabe et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) employed CRISPR/Cas9 to knock out \u003cem\u003eDFR-B\u003c/em\u003e, one of three DFR genes in Japanese morning glory (\u003cem\u003eIpomoea nil\u003c/em\u003e); 75% of transgenic plants produced white flowers with biallelic mutations at the target locus, representing the first CRISPR-mediated flower color change reported in higher plants. This finding established that targeted \u003cem\u003eDFR\u003c/em\u003e disruption can completely abolish anthocyanin biosynthesis without off-target effects on related members of the gene family.\u003c/p\u003e \u003cp\u003eDespite substantial progress, several questions remain poorly understood. Most functional studies have concentrated on pigmentation phenotypes, with limited attention to broader developmental or physiological effects associated with altered DFR activity. The existing literature relies heavily on heterologous expression or gene suppression, approaches that demonstrate functional relevance but do not fully capture the consequences of precise loss-of-function mutations within native genetic and regulatory contexts. Whether \u003cem\u003eDFR\u003c/em\u003e disruption affects traits beyond flower color has not been systematically examined, particularly in commercially valuable cultivars where growth performance and metabolic homeostasis are critical. The potential for pleiotropic effects, influences on chlorophyll or carotenoid metabolism, organ development, or stress physiology, warrants investigation, especially given emerging evidence that flavonoid pathway alterations can trigger unexpected transcriptional and metabolic responses.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePetunia \u0026times; hybrida\u003c/em\u003e offers distinct advantages for such investigations. As both a major ornamental crop and an established model for flower color research, petunia combines commercial relevance with extensive genetic and molecular resources. Petunia is amenable to stable \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation and regenerates efficiently from tissue culture (Vandenbussche et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Its flavonoid biosynthetic pathway has been characterized in detail, and the regulatory networks governing anthocyanin accumulation are well understood (Holton and Cornish \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Although CRISPR/Cas9 has been applied in petunia for purposes ranging from self-incompatibility studies to architectural modifications (Abdulla et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), targeted disruption of \u003cem\u003eDFR\u003c/em\u003e in this species, with systematic characterization of the resulting phenotypes, has not been reported.\u003c/p\u003e \u003cp\u003eHere we report the generation and characterization of CRISPR/Cas9-mediated \u003cem\u003eDFR\u003c/em\u003e knockout lines in the commercial petunia cultivar 'Carmine Velour'. Five independently edited lines exhibiting varying degrees of loss of floral pigmentation were recovered, enabling the construction of an allelic series for phenotype\u0026ndash;genotype correlation. By integrating molecular validation, comprehensive pigment quantification, morphometric analysis, and gene expression profiling, we addressed two primary objectives: first, to define how different levels of DFR inactivation reshapes floral anthocyanin accumulation and related pigment profiles; second, to assess whether \u003cem\u003eDFR\u003c/em\u003e disruption affects traits beyond pigmentation, including organ development, chlorophyll and carotenoid metabolism, and transcriptional regulation of associated biosynthetic pathways. Our findings reveal that \u003cem\u003eDFR\u003c/em\u003e knockout produces not only the expected anthocyanin depletion but also unanticipated effects on floral and vegetative physiology, demonstrating previously unrecognized connections between flavonoid metabolism and broader plant performance. These results advance understanding of DFR function and illustrate the utility of precise genome editing for both mechanistic inquiry and trait manipulation in ornamental crops.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePlant materials and growth conditions\u003c/p\u003e \u003cp\u003e \u003cem\u003ePetunia \u0026times; hybrida\u003c/em\u003e 'Carmine Velour' (Wave\u0026reg; series) seeds were provided by Ball Horticultural Company. Seeds were surface-sterilized in 75% (v/v) ethanol for 1 min, followed by 15% (v/v) commercial bleach (6% sodium hypochlorite) for 10 min with gentle agitation, and rinsed six times with sterile distilled water. Sterilized seeds were germinated on Murashige and Skoog (MS) basal medium (Murashige and Skoog \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) supplemented with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar (pH 5.8). Following acclimatization, transgenic and wild-type control plants were transferred to 10-cm pots containing a commercial soilless substrate (Premium Garden Soil Blend; Reliable Peat Company, Groveland, FL, USA) and cultivated in a controlled-environment growth chamber maintained at 25\u0026deg;C under a 16-h light/8-h dark photoperiod with approximately 60% relative humidity at the Mid-Florida Research and Education Center, University of Florida Institute of Food and Agricultural Sciences (Apopka, FL, USA) (Jiang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCRISPR/Cas9 vector construction and guide RNA design\u003c/p\u003e \u003cp\u003eThe CRISPR/Cas9 binary vector system was adapted from (Nguyen et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The T-DNA cassette comprises an \u003cem\u003eArabidopsis\u003c/em\u003e-codon-optimized \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e Cas9 (HypaCas9) driven by the parsley (\u003cem\u003ePetroselinum crispum\u003c/em\u003e) ubiquitin promoter (\u003cem\u003ePcUBI\u003c/em\u003e), a single-guide RNA (sgRNA) expression module under the \u003cem\u003eArabidopsis U6-26\u003c/em\u003e promoter utilizing a tRNA\u0026ndash;sgRNA polycistronic architecture, and an \u003cem\u003eeGFP\u0026ndash;NPTII\u003c/em\u003e fusion selectable marker driven by the double-enhanced cassava vein mosaic virus (CsVMV) promoter for kanamycin selection and visual screening of transformants (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGuide RNA target sites were identified within conserved regions of the \u003cem\u003ePetunia DFR\u003c/em\u003e coding sequence. Briefly, \u003cem\u003ePetunia DFR\u003c/em\u003e homologs were retrieved from NCBI GenBank using the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e DFR protein sequence (AT5G42800; TAIR) as query. Multiple \u003cem\u003ePetunia DFR\u003c/em\u003e coding sequences from diverse cultivars and species were aligned using ClustalW (Thompson et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Fig. S2) to identify highly conserved exonic regions amenable to multiplex targeting. Candidate gRNAs were designed using CRISPOR (Concordet and Haeussler \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and CHOPCHOP (Labun et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), prioritizing guides with canonical NGG protospacer adjacent motif (PAM) sequences, high predicted on-target activity scores, and minimal predicted off-target activity as assessed by CRISPOR based on available petunia reference genomes. Three gRNAs (gRNA1\u0026ndash;gRNA3) targeting exons 3 and 4 were selected for multiplex editing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSynthetic oligonucleotides encoding the gRNA spacer sequences were synthesized (Gene Universal Inc., Newark, DE, USA) and cloned into the PHN-HypaCas9-4\u0026times;BsaI-GFP backbone via Golden Gate assembly using BsaI restriction sites. All constructs were sequence-verified by Sanger sequencing and subsequently introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 by freeze\u0026ndash;thaw transformation.\u003c/p\u003e \u003cp\u003eAgrobacterium-mediated transformation and plant regeneration\u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 harboring the CRISPR/Cas9 construct was cultured in Luria-Bertani (LB) medium supplemented with rifampicin (50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and spectinomycin (100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 28\u0026deg;C with shaking (200 rpm) until reaching an optical density at 600 nm (OD600) of 0.3\u0026ndash;0.5. Bacterial cells were harvested by centrifugation (4,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 10 min, 4\u0026deg;C), gently resuspended in liquid MS medium (pH 5.8) containing 100 \u0026micro;M acetosyringone, and incubated at room temperature for 1 h to induce virulence gene expression.\u003c/p\u003e \u003cp\u003eCotyledonary explants excised from 10-day-old \u003cem\u003ein vitro\u003c/em\u003e-germinated seedlings were used for transformation. Prior to inoculation, explants were pre-cultured on MS basal medium containing 3% (w/v) sucrose and 0.7% (w/v) agar (pH 5.8) for 2 days in darkness to promote competence for transformation. Pre-cultured explants were immersed in the \u003cem\u003eAgrobacterium\u003c/em\u003e suspension for 10 min with gentle agitation, blotted on sterile filter paper to remove excess inoculum, and transferred to co-cultivation medium (CCM) consisting of MS salts supplemented with 0.75 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 6-benzylaminopurine (BAP) and 0.15 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 1-naphthaleneacetic acid (NAA). Co-cultivation proceeded for 2 days at 25\u0026deg;C in darkness.\u003c/p\u003e \u003cp\u003eFollowing co-cultivation, explants were transferred to callus induction medium (CIM) containing 0.75 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP, 0.15 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA, 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin for selection, and 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e timentin to suppress \u003cem\u003eAgrobacterium\u003c/em\u003e overgrowth. Cultures were maintained at 25\u0026deg;C under a 16-h light/8-h dark photoperiod. Regenerating calli were transferred to shoot induction medium (SIM) containing 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP and 0.01 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA. Elongated shoots were excised and transferred to root induction medium (RIM) supplemented with 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indole-3-butyric acid (IBA). All regeneration media contained identical antibiotic concentrations as CIM. Putative transformants were identified by kanamycin resistance and confirmed by GFP fluorescence visualization using a fluorescence stereomicroscope (Leica M205 FA; Leica Microsystems, Wetzlar, Germany). Only GFP-positive regenerants were retained for rooting and subsequent analyses.\u003c/p\u003e \u003cp\u003eMutant genotyping and validation\u003c/p\u003e \u003cp\u003eGenomic DNA was isolated from young, fully expanded leaves using a cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle and Doyle 1987). The \u003cem\u003eDFR\u003c/em\u003e target region spanning all three gRNA sites and selected candidate off-target loci were amplified by PCR using gene-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Amplicons were purified using a commercial PCR purification kit (QIAGEN, Hilden, Germany) and subjected to Sanger sequencing. Editing outcomes and candidate off-target loci were determined by aligning sequence chromatograms to the wild-type reference sequence using SnapGene software (GSL Biotech LLC, San Diego, CA, USA). Mutant alleles were classified based on the nature and position of insertions, deletions, or substitutions relative to the predicted Cas9 cleavage sites.\u003c/p\u003e \u003cp\u003eMorphological phenotyping\u003c/p\u003e \u003cp\u003eFloral and vegetative organ dimensions were quantified from calibrated digital images using ImageJ software (Schneider et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Flowers were harvested at anthesis (stage 13), and leaves were sampled from the third to fourth nodes below the shoot apex to ensure comparable physiological age across genotypes. Images containing a metric scale reference were converted to 8-bit grayscale, spatially calibrated, and analyzed using the freehand selection tool to delineate individual organ boundaries. Projected area was measured using the \"Analyze\u0026thinsp;\u0026gt;\u0026thinsp;Measure\" function. A minimum of ten biological replicates per genotype were analyzed for each trait. Fresh weight of excised leaves and flowers was determined using a precision analytical balance (MS105DU; Mettler Toledo, Columbus, OH, USA) with 0.01 mg readability.\u003c/p\u003e \u003cp\u003eProtein sequence prediction and structural modeling\u003c/p\u003e \u003cp\u003ePredicted protein sequences corresponding to wild-type and edited \u003cem\u003eDFR\u003c/em\u003e alleles were derived by conceptual translation of inferred coding sequences using the ExPASy Translate tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/translate/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/translate/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Three-dimensional protein structure models were generated by homology modeling using the SWISS-MODEL server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Waterhouse et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with default parameters. Template selection, model building, and quality estimation were performed automatically. Predicted structures were visualized and compared using PyMOL (Schr\u0026ouml;dinger LLC, New York, NY, USA) to illustrate the extent of truncation and loss of conserved structural domains in mutant DFR proteins.\u003c/p\u003e \u003cp\u003eAnthocyanin extraction and quantification\u003c/p\u003e \u003cp\u003eAnthocyanins were extracted and quantified following the acidified methanol protocol of Neff and Chory (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) with modifications. Freshly harvested petal or leaf tissue (50\u0026ndash;100 mg) was weighed, flash-frozen in liquid nitrogen, and homogenized to a fine powder. Samples were extracted in 300 \u0026micro;L acidified methanol [1% (v/v) HCl in methanol] by vortexing and incubating overnight at room temperature in complete darkness. Following extraction, 200 \u0026micro;L Milli-Q water and 500 \u0026micro;L chloroform were added to facilitate phase separation. Samples were vortexed vigorously and centrifuged (12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 5 min, 4\u0026deg;C). A 350-\u0026micro;L aliquot of the upper aqueous-methanolic phase was transferred to a fresh tube and diluted to a final volume of 1.05 mL with 415.8 \u0026micro;L methanol, 4.2 \u0026micro;L concentrated HCl, and 280 \u0026micro;L Milli-Q water to maintain absorbance within the linear detection range.\u003c/p\u003e \u003cp\u003eAbsorbance was measured at 530 nm (anthocyanin peak) and 657 nm (chlorophyll interference) using a microplate reader (Synergy H1; BioTek Instruments, Winooski, VT, USA). Corrected anthocyanin absorbance was calculated as Acorr\u0026thinsp;=\u0026thinsp;A530\u0026thinsp;\u0026minus;\u0026thinsp;0.25 \u0026times; A657. Anthocyanin content was expressed as milligrams of cyanidin-3-glucoside equivalents per gram fresh weight (mg C3G eq g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) using the equation: Anthocyanin = (Acorr \u0026times; V \u0026times; MW \u0026times; 1000) / (ε\u0026thinsp;\u0026times;\u0026thinsp;\u003cem\u003el\u003c/em\u003e \u0026times; FW), where V is the final extract volume (L), MW\u0026thinsp;=\u0026thinsp;449.2 g mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (molecular weight of cyanidin-3-glucoside), ε\u0026thinsp;=\u0026thinsp;33,000 L mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (molar extinction coefficient), \u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1 cm (path length), and FW is the fresh tissue weight (g). Blank extractions processed without tissue were included to correct for background absorbance.\u003c/p\u003e \u003cp\u003eChlorophyll and carotenoid extraction and quantification\u003c/p\u003e \u003cp\u003eChlorophylls and carotenoids were extracted following (Lichtenthaler \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) with modifications. Approximately 50 mg of fresh petal or leaf tissue was incubated in 1.0 mL of 95% (v/v) ethanol for 16 h at 25\u0026deg;C in complete darkness to ensure exhaustive pigment solubilization. Extracts were clarified by centrifugation (12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 5 min), and absorbance was measured at 470 nm, 649 nm, and 665 nm using a BioTek Synergy H1 microplate reader (Agilent Technologies, Santa Clara, CA, USA) with 95% ethanol as the reference blank.\u003c/p\u003e \u003cp\u003ePigment concentrations (\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were calculated using the spectrophotometric equations of Wellburn (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) optimized for 95% ethanol: chlorophyll \u003cem\u003ea\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.95 \u0026times; A665\u0026thinsp;\u0026minus;\u0026thinsp;6.88 \u0026times; A649; chlorophyll \u003cem\u003eb\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24.96 \u0026times; A649\u0026thinsp;\u0026minus;\u0026thinsp;7.32 \u0026times; A665; total carotenoids = (1000 \u0026times; A470\u0026thinsp;\u0026minus;\u0026thinsp;2.13 \u0026times; Chl \u003cem\u003ea\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;97.63 \u0026times; Chl \u003cem\u003eb\u003c/em\u003e) / 209. Final pigment contents were expressed as mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW by multiplying concentrations by the extraction volume and normalizing to fresh tissue mass.\u003c/p\u003e \u003cp\u003eRNA isolation and quantitative real-time PCR\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from 80\u0026ndash;100 mg of flash-frozen petal tissue using RNAzol RT reagent (Molecular Research Center, Cincinnati, OH, USA) following the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA); samples with A260/A280 ratios between 1.9 and 2.1 were retained for downstream applications. Genomic DNA contamination was eliminated by on-column DNase I digestion (RNase-Free DNase Set; QIAGEN). First-strand cDNA was synthesized from 1 \u0026micro;g of DNase-treated RNA using the QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer's instructions. The resulting cDNA was diluted 20- to 25-fold in nuclease-free water for use as qRT-PCR template.\u003c/p\u003e \u003cp\u003eQuantitative real-time PCR (qRT-PCR) was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iTaq Universal SYBR Green Supermix (Bio-Rad). Each 10-\u0026micro;L reaction contained 5 \u0026micro;L 2\u0026times; SYBR Green master mix, 4 \u0026micro;L diluted cDNA template, and 0.5 \u0026micro;L of a 10 \u0026micro;M forward and reverse primer mixture. Gene-specific primers were designed using Primer-BLAST (NCBI) to span exon\u0026ndash;exon junctions where possible and to generate amplicons of 100\u0026ndash;150 bp with melting temperatures of 58\u0026ndash;62\u0026deg;C. Primer sequences for all target genes are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Target genes included structural genes of the anthocyanin biosynthetic pathway (\u003cem\u003eCHSA\u003c/em\u003e, \u003cem\u003eCHIA\u003c/em\u003e, \u003cem\u003eF3\u0026prime;H\u003c/em\u003e, \u003cem\u003eF3\u0026prime;5\u0026prime;H\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e), the flavonol branch pathway (\u003cem\u003eFLS\u003c/em\u003e), and chlorophyll biosynthesis (\u003cem\u003ePORA\u003c/em\u003e, \u003cem\u003eCHLH\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThermal cycling conditions consisted of an initial denaturation at 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 10 s and 60\u0026deg;C for 30 s. Melt curve analysis was performed after amplification to verify primer specificity. Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method (Livak and Schmittgen \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), with \u003cem\u003eEF1α\u003c/em\u003e serving as the internal reference gene based on its stable expression across all experimental samples. Three biological replicates, each comprising three technical replicates, were analyzed per genotype.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using R software (version 4.3.1; R Core Team, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Biochemical quantifications (anthocyanin, chlorophyll, carotenoid) and gene expression analyses were conducted using a minimum of three independent biological replicates, with three technical replicates per biological sample for qRT-PCR assays. Morphological traits were quantified from at least ten biological replicates per genotype.\u003c/p\u003e \u003cp\u003eDifferences among genotypes were evaluated by one-way analysis of variance (ANOVA) following verification of normality (Shapiro\u0026ndash;Wilk test) and homogeneity of variances (Levene's test). Pairwise comparisons were performed using Tukey's honestly significant difference (HSD) \u003cem\u003epost hoc\u003c/em\u003e test. Compact letter displays indicating statistically distinct groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were generated using the \u003cem\u003emultcompView\u003c/em\u003e package and are displayed above corresponding data points in all figures. Pearson correlation coefficients and associated \u003cem\u003eP\u003c/em\u003e-values were calculated using the \u003cem\u003ecor.test\u003c/em\u003e function to assess relationships between phenotypic traits.\u003c/p\u003e \u003cp\u003eAll data visualizations were generated using the \u003cem\u003eggplot2\u003c/em\u003e package (Wickham \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Box plots display the median, interquartile range (IQR; 25th\u0026ndash;75th percentiles), and whiskers extending to 1.5\u0026times; IQR; individual data points are overlaid to show the full distribution of biological replicates. Bar plots display mean values with error bars representing standard deviation (SD) unless otherwise specified.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis and sequence alignment\u003c/p\u003e \u003cp\u003eThe amino acid sequence of \u003cem\u003ePetunia \u0026times; hybrida\u003c/em\u003e DFR-A (PhDFRa) was retrieved from NCBI based on the published DFR-A sequence (Beld et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Additional DFR protein sequences from Solanaceae species were selected according to the DFR family analysis reported by Li et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Full-length amino acid sequences were aligned in MEGA (version 11.0.13) using default parameters, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replicates. Bootstrap values were used to assess branch support, and the tree was visualized using iTOL v6 (Letunic and Bork., 2024). Conserved motifs in DFR proteins were identified using the MEME Suite with the following settings: maximum number of motifs\u0026thinsp;=\u0026thinsp;10 and motif width\u0026thinsp;=\u0026thinsp;6\u0026ndash;50 amino acids.\u003c/p\u003e \u003cp\u003eThe predicted three-dimensional structure of PhDFRa was generated using SWISS-MODEL. Sequence similarity among DFR proteins was evaluated by BLASTP using the PhDFRa amino acid sequence as the query. Comparative DFR protein sequences from other Solanaceae species, selected from Li et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), were aligned against PhDFRa, and the resulting similarity statistics, including Max score, Total score, Query cover, E-value, Percent identity, and protein length, were summarized.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCRISPR/Cas9-mediated disruption of\u003c/b\u003e \u003cb\u003eDFR\u003c/b\u003e \u003cb\u003eproduces an allele-associated spectrum of pigment loss in petunia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo dissect the functional role of dihydroflavonol 4-reductase (DFR) in anthocyanin biosynthesis, we employed a multiplex CRISPR/Cas9 strategy targeting the canonical floral \u003cem\u003eDFR\u003c/em\u003e gene in \u003cem\u003ePetunia \u0026times; hybrida\u003c/em\u003e 'Carmine Velour'. Three guide RNAs (gRNA1\u0026ndash;gRNA3) were designed to target conserved coding sequences within exons 3 and 4, with inter-target distances of 122 bp (gRNA1\u0026ndash;gRNA2) and 244 bp (gRNA2\u0026ndash;gRNA3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although \u003cem\u003eP. hybrida\u003c/em\u003e harbors multiple \u003cem\u003eDFR\u003c/em\u003e paralogs, previous biochemical and genetic evidence indicates that DFR-A is the principal enzyme channeling dihydroflavonols toward anthocyanidin biosynthesis in corolla tissue (Huits et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1994\u003c/span\u003e); accordingly, all genotyping and phenotypic analyses focused on this locus. This target choice was further supported by phylogenetic, structural, and sequence-comparison analyses, which placed the selected petunia DFR with functionally characterized anthocyanin-associated DFR proteins and indicated that all queried petunia DFR versions showed the highest similarity to the same PhDFR homolog (Fig. S2). These results support the conclusion that this locus represents the major DFR copy associated with anthocyanin biosynthesis in \u003cem\u003eP. hybrida\u003c/em\u003e. Potential off-target sites for each guide RNA were predicted using CRISPOR, and the highest-ranking candidate loci were subsequently amplified and examined by Sanger sequencing (Fig. S3). No mutations were detected at these predicted off-target sites in the edited plants analyzed, supporting the specificity of the CRISPR/Cas9 system used in this study. Five independent edited lines (\u003cem\u003edfr-1\u003c/em\u003e to \u003cem\u003edfr-5\u003c/em\u003e) were recovered and cultivated under controlled greenhouse conditions. These lines displayed a striking, overall reduction in floral pigmentation relative to the deeply magenta wild-type (WT) control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Lines \u003cem\u003edfr-1\u003c/em\u003e and \u003cem\u003edfr-2\u003c/em\u003e retained partial magenta pigmentation with characteristic variegated patterning, \u003cem\u003edfr-3\u003c/em\u003e exhibited markedly attenuated coloration, and \u003cem\u003edfr-4\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e produced flowers with near-complete absence of visible anthocyanin accumulation, yielding pale cream to yellow-green corollas.\u003c/p\u003e \u003cp\u003eSanger sequencing of PCR amplicons spanning the three CRISPR target sites revealed a diverse spectrum of editing outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The detected lesions included: a 3-bp in-frame deletion at the gRNA1 site coupled with a single-nucleotide insertion at gRNA2 in \u003cem\u003edfr-1\u003c/em\u003e (net Δ = \u0026minus;3/+1); single-base insertions at the gRNA2 locus in \u003cem\u003edfr-2\u003c/em\u003e and \u003cem\u003edfr-3\u003c/em\u003e (Δ = +1); a 76-bp deletion spanning the gRNA2 region in \u003cem\u003edfr-4\u003c/em\u003e (Δ = \u0026minus;76); and a large 304-bp deletion extending across the gRNA1\u0026ndash;gRNA3 interval in \u003cem\u003edfr-5\u003c/em\u003e (Δ = \u0026minus;304). All identified mutations introduced frameshifts relative to the WT open reading frame, resulting in premature translation termination. Computational protein modeling corroborated these predictions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Whereas the WT DFR enzyme comprises 380 amino acids with characteristic Rossmann-fold architecture, the edited alleles encode severely truncated polypeptides: 155 aa (\u003cem\u003edfr-1\u003c/em\u003e), 165 aa (\u003cem\u003edfr-2\u003c/em\u003e and \u003cem\u003edfr-3\u003c/em\u003e), 142 aa (\u003cem\u003edfr-4\u003c/em\u003e), and 114 aa (\u003cem\u003edfr-5\u003c/em\u003e). Structural predictions indicate extensive loss of conserved secondary and tertiary elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), including the catalytic domain and NADPH-binding site, consistent with strong loss of function. Together, these mutations generate DFR variants predicted to cause differing degrees of functional impairment, enabling analysis of how differing degrees of DFR disruption influence pigmentation and broader physiological consequences.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDFR\u003c/b\u003e \u003cb\u003eperturbation depletes floral anthocyanins and alters pigment composition alongside organ morphology\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBiochemical and morphometric characterization revealed a broad phenotypic spectrum across all edited genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. S4). Methanolic extracts from wild-type (WT) petals were intensely magenta. \u003cem\u003edfr-1\u003c/em\u003e and \u003cem\u003edfr-2\u003c/em\u003e yielded diluted pink solutions; \u003cem\u003edfr-3\u003c/em\u003e extracts appeared markedly paler; those from \u003cem\u003edfr-4\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e were nearly colorless (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This gradient was broadly consistent with the intact flower phenotypes. WT corollas were uniformly pigmented across the limb, while \u003cem\u003edfr-1\u003c/em\u003e and \u003cem\u003edfr-2\u003c/em\u003e showed variegated patterning instead, with pigmented sectors interspersed among unpigmented tissue. This mosaic appearance likely reflects chimeric editing or mosaicism within primary regenerants. \u003cem\u003edfr-3\u003c/em\u003e flowers were uniformly pale pink. \u003cem\u003edfr-4\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e lacked visible anthocyanin altogether, their cream-to-yellowish hue attributable to underlying carotenoids and chlorophyll (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTransgene integration was verified by fluorescence imaging of dissected corolla tubes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). WT flowers exhibited minimal autofluorescence confined to anther tissue, with no signal in filaments. All \u003cem\u003edfr\u003c/em\u003e lines, by contrast, displayed strong GFP fluorescence throughout filaments and anthers, confirming successful incorporation of the CRISPR/Cas9 T-DNA cassette harboring the eGFP reporter. Spectrophotometric quantification supported the visual observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). WT flowers accumulated 0.47 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight (FW) anthocyanin. \u003cem\u003edfr-1\u003c/em\u003e retained roughly 80% of this value (0.37 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003edfr-2\u003c/em\u003e and \u003cem\u003edfr-3\u003c/em\u003e showed reductions of approximately 40% and 80%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The most severely edited lines, \u003cem\u003edfr-4\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e, contained only trace amounts (\u0026le;\u0026thinsp;0.05 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), representing greater than 90% depletion relative to WT. The overall decline across the allelic series closely corresponded to predicted protein truncation severity. Floral dimensions declined in parallel with anthocyanin content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). WT corolla area averaged 31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 cm\u003csup\u003e2\u003c/sup\u003e. \u003cem\u003edfr-1\u003c/em\u003e through \u003cem\u003edfr-3\u003c/em\u003e were 20\u0026ndash;30% smaller; \u003cem\u003edfr-4\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e approached 40% reduction, averaging 18\u0026ndash;19 cm\u003csup\u003e2\u003c/sup\u003e. Fresh weight followed a similar though less pronounced trend: WT corollas averaged 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g, while mutant values ranged from 0.23 to 0.28 g (10\u0026ndash;30% decreases). The coupling between pigment loss and reduced organ size was consistent across independent lines, suggesting an association rather than coincidental variation.\u003c/p\u003e \u003cp\u003eThe morphological phenotypes prompted examination of non-anthocyanin pigments in petal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Interestingly, total carotenoid content declined significantly across all mutant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), with \u003cem\u003edfr\u003c/em\u003e mutant petals containing 0.015 to 0.025 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e compared to 0.041 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW in WT, representing 35\u0026ndash;60% reductions. Unlike anthocyanins, carotenoid levels showed no clear relationship with the apparent anthocyanin gradient: \u003cem\u003edfr-2\u003c/em\u003e (165 aa truncation) and \u003cem\u003edfr-5\u003c/em\u003e (114 aa truncation) exhibited comparable reductions despite different predicted degrees of DFR disruption. This pattern argues against direct enzymatic involvement and instead implicates indirect regulatory or metabolic feedback mechanisms. Chlorophyll reductions were more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;E). WT petals contained 0.133\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW total chlorophyll, whereas \u003cem\u003edfr\u003c/em\u003e mutant lines ranged from 37\u0026ndash;55% of this level. Critically, both chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e declined coordinately rather than selectively, indicating general suppression of tetrapyrrole biosynthesis rather than pathway-specific effects. The reduction ranges from \u003cem\u003edfr-2\u003c/em\u003e (~\u0026thinsp;55% of WT) to dfr-4 (~\u0026thinsp;37%) did not show a clear quantitative correspondence to anthocyanin loss, consistent with the carotenoid pattern. The coordinated decline of both chlorophylls and carotenoids across all five independent alleles reveals that DFR disruption broadly perturbs plastidial pigment metabolism, extending well beyond anthocyanin biosynthesis in the cytosol. The consistency of this phenotype across multiple independently edited alleles with distinct molecular lesions argues against somaclonal variation or T-DNA positional effects, supporting plastid pigment reduction as a likely pleiotropic consequence of \u003cem\u003eDFR\u003c/em\u003e loss.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eVegetative tissues show organ-specific responses: leaf physiology altered, stem anatomy unaffected\u003c/h3\u003e\n\u003cp\u003eTo determine whether DFR-A governs anthocyanin biosynthesis and development beyond flowers, we examined leaf pigmentation, morphology, and stem anatomy across all genotypes (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeaf anthocyanin accumulation is DFR-independent. In striking contrast to the floral phenotypes, anthocyanin extracts from leaves of WT and all \u003cem\u003edfr\u003c/em\u003e mutants were uniformly pale and visually indistinguishable (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Quantitative analysis confirmed this observation: mean leaf anthocyanin content ranged from 0.013 to 0.022 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW across genotypes, with no statistically significant differences detected between WT and any mutant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings indicate that DFR-A is dispensable for basal anthocyanin accumulation in petunia leaves, potentially reflecting functional redundancy with other \u003cem\u003eDFR\u003c/em\u003e paralogs or tissue-specific regulatory differences.\u003c/p\u003e \u003cp\u003eDespite unchanged anthocyanin levels, leaf development is compromised. Leaf area showed modest reductions across mutants, with only \u003cem\u003edfr-5\u003c/em\u003e reaching significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to WT (0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 cm\u0026sup2;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D). Leaf biomass, however, declined substantially: WT leaves averaged 0.028\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 g, whereas all \u003cem\u003edfr\u003c/em\u003e mutant lines ranged from 0.014 to 0.020 g, representing decreases of 30\u0026ndash;50% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Paralleling the petal phenotype, leaf chlorophyll and carotenoids also decreased by 20\u0026ndash;45% (Fig. S5), with \u003cem\u003edfr-4\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e showing the most severe depletion. Despite unchanged anthocyanin content, the coordinated reductions of biomass and plastidial pigments suggest that \u003cem\u003eDFR\u003c/em\u003e disruption is associated with altered leaf physiology through anthocyanin-independent mechanisms.\u003c/p\u003e \u003cp\u003eIn contrast to the leaf phenotypes, stem morphology and anatomy were unaffected by \u003cem\u003eDFR\u003c/em\u003e disruption. Flowering shoots exhibited the expected floral pigmentation gradient but normal overall architecture, with internode length, phyllotaxis, and branching comparable between WT and all \u003cem\u003edfr\u003c/em\u003e lines (Fig. S6). Transverse sections of young stems revealed no discernible anatomical abnormalities in any mutant genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Vascular organization, cortical thickness, epidermal cell morphology, and central pith structure showed no abnormalities, hypertrophy, or irregular patterning in any mutant. Collectively, these observations establish that DFR-A function is essential for floral pigmentation and associated with changes in leaf biomass and plastidial pigment accumulation, but dispensable for leaf anthocyanin accumulation and stem morphogenesis. The reduction in leaf biomass and plastidial pigments, occurring independently of anthocyanin deficiency, suggests that DFR perturbation influences vegetative traits through mechanisms extending beyond its canonical role in floral anthocyanin biosynthesis.\u003c/p\u003e\n\u003ch3\u003eFloral anthocyanin content correlates with morphological and pigment traits across genotypes\u003c/h3\u003e\n\u003cp\u003eTo examine relationships between anthocyanin accumulation and other traits, we performed Pearson correlation analyses using genotype-level mean values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Within floral organs, anthocyanin content showed positive associations with morphological traits. Flower area exhibited a strong positive correlation with anthocyanin levels (R\u0026thinsp;=\u0026thinsp;0.78, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.069), and flower fresh weight followed a similar trend (R\u0026thinsp;=\u0026thinsp;0.56, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.25), though neither reached statistical significance. Positive correlations were also detected between floral anthocyanin content and other pigment classes: carotenoid content (R\u0026thinsp;=\u0026thinsp;0.65, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.16) and total chlorophyll (R\u0026thinsp;=\u0026thinsp;0.71, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.11).\u003c/p\u003e \u003cp\u003eNotably, the strongest correlations were observed between floral anthocyanin content and vegetative traits, with several relationships reaching statistical significance. Leaf area was significantly correlated with floral anthocyanin levels (R\u0026thinsp;=\u0026thinsp;0.84, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037), indicating that genotypes with higher floral pigmentation tend to produce larger leaves. Even more robust associations were observed for leaf pigment contents: total leaf chlorophyll (R\u0026thinsp;=\u0026thinsp;0.89, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) and leaf carotenoid content (R\u0026thinsp;=\u0026thinsp;0.93, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0075) both showed highly significant positive correlations with floral anthocyanin accumulation. Leaf fresh weight exhibited a positive but non-significant relationship (R\u0026thinsp;=\u0026thinsp;0.56, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.24).\u003c/p\u003e \u003cp\u003eThe tight correlations between floral anthocyanin and leaf plastidial pigments suggest an association between floral and vegetative pigment traits across genotypes. This pattern is consistent with but does not by itself demonstrate broader coordination of pigment-related processes across organs. Shared regulatory networks or whole-plant resource allocation constraints could contribute to these relationships, although the current analysis does not distinguish among these possibilities. Overall, these data indicate that variation in floral anthocyanin content is accompanied by corresponding shifts in several vegetative traits following DFR perturbation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDFR\u003c/b\u003e \u003cb\u003edysfunction triggers coordinate transcriptional changes in flavonoid and chlorophyll biosynthesis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess transcriptional responses to \u003cem\u003eDFR\u003c/em\u003e loss of function, we quantified gene expression in corolla tissue from WT, \u003cem\u003edfr-3\u003c/em\u003e, and \u003cem\u003edfr-5\u003c/em\u003e across flavonoid biosynthesis and chlorophyll metabolism pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpstream anthocyanin genes show coordinate downregulation. \u003cem\u003eCHSA\u003c/em\u003e (chalcone synthase A), encoding the entry-point enzyme of flavonoid biosynthesis, showed moderate decline to ~\u0026thinsp;75% of WT in \u003cem\u003edfr-3\u003c/em\u003e (not significant) and ~\u0026thinsp;60% in \u003cem\u003edfr-5\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eCHIA\u003c/em\u003e (chalcone isomerase A) exhibited a more pronounced and consistent response: transcript levels declined to ~\u0026thinsp;55% of WT in \u003cem\u003edfr-3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 70% in \u003cem\u003edfr-5\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The upstream transcriptional downregulation triggered by \u003cem\u003eDFR\u003c/em\u003e disruption suggests feedback sensing of the metabolic blockage, potentially triggered by reduced flux or intermediate accumulation.\u003c/p\u003e \u003cp\u003eUnlike the upstream anthocyanin genes, genes involved in parallel or downstream branches of flavonoid metabolism showed divergent responses. \u003cem\u003eFLS\u003c/em\u003e (flavonol synthase), which competes with \u003cem\u003eDFR\u003c/em\u003e for dihydroflavonol substrates, increased significantly: 1.9-fold in \u003cem\u003edfr-3\u003c/em\u003e and 1.4-fold in \u003cem\u003edfr-5\u003c/em\u003e relative to WT (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This upregulation is consistent with the possibility of substrate redirection toward flavonol production when \u003cem\u003eDFR\u003c/em\u003e is inactive. In contrast, \u003cem\u003eF3\u0026prime;H\u003c/em\u003e, \u003cem\u003eF3\u0026prime;5\u0026prime;H\u003c/em\u003e, and \u003cem\u003eANS\u003c/em\u003e showed no significant differences in transcript abundance between WT and either mutant line, with expression values remaining within \u0026plusmn;\u0026thinsp;30% of WT levels.\u003c/p\u003e \u003cp\u003eNotably, genes involved in chlorophyll metabolism exhibited marked transcriptional alterations. \u003cem\u003ePORA\u003c/em\u003e (protochlorophyllide oxidoreductase A), encoding a rate-limiting enzyme in chlorophyll biosynthesis, was severely downregulated: ~25% of WT in \u003cem\u003edfr-3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 40% of WT in \u003cem\u003edfr-5\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). By contrast, \u003cem\u003eCHLH\u003c/em\u003e, encoding the H subunit of magnesium chelatase, showed no significant changes, with transcript levels remaining comparable to WT in both mutant backgrounds. The selective downregulation of PORA may contribute to the reduced chlorophyll accumulation observed in \u003cem\u003edfr\u003c/em\u003e mutant flowers and suggests a possible link between cytosolic flavonoid and plastidial tetrapyrrole pathways.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTargeted \u003cem\u003eDFR\u003c/em\u003e knockout in \u003cem\u003eP. hybrida\u003c/em\u003e eliminated floral anthocyanin in an allele-dependent fashion, with the five edited lines spanning partial to near-complete depigmentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, the severity of depigmentation was generally consistent with mutation type and predicted protein truncation, supporting the conclusion that \u003cem\u003eDFR-A\u003c/em\u003e is the principal isoform governing corolla pigmentation in petunia (Fig. S2). Earlier biochemical work had pointed in this direction (Holton and Cornish \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), but precise loss-of-function alleles had not been available to test the inference directly. Beyond pigmentation, the mutants displayed reduced floral dimensions, diminished leaf biomass, and coordinated declines in chlorophyll and carotenoid content across both reproductive and vegetative tissues, while stem anatomy remained normal (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Figs. S4-6). Multiple independent alleles converging on similar phenotypes strengthen confidence that these traits represent genuine consequences of \u003cem\u003eDFR\u003c/em\u003e loss rather than transformation artifacts or background variation. This consideration is especially important in commercial hybrid backgrounds, where genetic heterogeneity can obscure single-allele effects. The following sections address the metabolic function of \u003cem\u003eDFR\u003c/em\u003e in flavonoid biosynthesis and the pleiotropic consequences of its disruption, but first the interpretation of pigmentation phenotypes in these primary regenerants warrants qualification.\u003c/p\u003e \u003cp\u003eThe pigmentation phenotypes in T₀ plants should be interpreted with caution. Several edited lines, particularly dfr-2, dfr-3, and dfr-4, display sectoral pigmentation patterns characterized by white stripes or unpigmented patches interspersed with residual anthocyanin-containing tissue, rather than uniformly reduced coloration across the corolla. Such patterns are consistent with somatic chimerism, in which independently edited and unedited cell lineages coexist within a single regenerant. Chimeric tissues are a well-recognized outcome of \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation and CRISPR/Cas9 editing in T₀ plants, because mutagenesis can occur at different developmental stages during callus proliferation and shoot regeneration (Fauser et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The sectoral distribution of pigment loss in these lines, therefore, may reflect mosaic editing across floral cell layers, rather than solely a simple graded, allele-dependent reduction in \u003cem\u003eDFR\u003c/em\u003e activity. Because individual flower sectors were not isolated and sequenced separately in this study, the relative contribution of chimerism versus residual enzymatic function to the observed variation in pigmentation cannot be resolved from the current data. Future work should include genotyping of micro-dissected pigmented and unpigmented sectors from the same corolla, as well as analysis of T₁ segregants, to distinguish true hypomorphic alleles from chimeric mosaics and to establish stable genotype-phenotype relationships in non-chimeric backgrounds.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDFR functions in flavonoid biosynthesis and metabolic flux partitioning\u003c/h2\u003e \u003cp\u003eDFR and FLS occupy the same metabolic branch point, competing for dihydroflavonol substrates (Choudhary and Pucker \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Eliminating \u003cem\u003eDFR\u003c/em\u003e activity would be expected to favor the accumulation of dihydroflavonols and increase the potential for flux to be redirected toward flavonol biosynthesis. The significant upregulation of \u003cem\u003eFLS\u003c/em\u003e transcripts in \u003cem\u003edfr-3\u003c/em\u003e and \u003cem\u003edfr-5\u003c/em\u003e corollas supports this prediction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Wang et al. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) observed exactly this pattern when suppressing \u003cem\u003eIbDFR\u003c/em\u003e by RNAi in purple sweet potato: anthocyanins declined while quercetin glycosides rose. Direct quantification of flavonols was not performed in the present study; therefore, a shift in metabolic flux cannot be demonstrated from the current data. Nevertheless, the transcriptional data, together with the well-documented substrate competition between \u003cem\u003eDFR\u003c/em\u003e and \u003cem\u003eFLS\u003c/em\u003e, are consistent with partial flux redirection toward flavonol biosynthesis.\u003c/p\u003e \u003cp\u003ePrior DFR loss-of-function studies across diverse species provide useful context for evaluating these findings. Watanabe et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) used CRISPR/Cas9 to knock out \u003cem\u003eDFR-B\u003c/em\u003e in Japanese morning glory (\u003cem\u003eIpomoea nil\u003c/em\u003e); 75% of regenerants bore white flowers, confirming that DFR loss abolishes anthocyanin accumulation in Convolvulaceae. That study focused on pigmentation and did not examine developmental or physiological consequences. In black rice (\u003cem\u003eOryza sativa\u003c/em\u003e), Jung et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) applied CRISPR/Cas9 to target \u003cem\u003eOsF3\u0026prime;H\u003c/em\u003e, \u003cem\u003eOsDFR\u003c/em\u003e, and \u003cem\u003eOsLDOX\u003c/em\u003e simultaneously; the resulting mutants showed altered seed coloration and reduced anthocyanin content, with the mutations stably inherited into subsequent generations. RNAi knockdown of \u003cem\u003eNtDFR1\u003c/em\u003e and \u003cem\u003eNtDFR2\u003c/em\u003e in tobacco produced pale flowers and triggered compensatory upregulation of other flavonoid genes (Lim et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A similar transcriptional feedback response was observed here, though the magnitude differed.\u003c/p\u003e \u003cp\u003eFunctional characterization of DFR homologs in ornamental species has expanded considerably. Qin et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) isolated \u003cem\u003eHvDFR\u003c/em\u003e from \u003cem\u003eHosta ventricosa\u003c/em\u003e and demonstrated that ectopic expression in \u003cem\u003eArabidopsis\u003c/em\u003e increased anthocyanin accumulation 1.7- to 2.4-fold, confirming the enzyme's conserved role across monocots and dicots. In chrysanthemum, Lim et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) showed that C-terminal sequence variation in \u003cem\u003eCmDFR\u003c/em\u003e between white- and red-flowered cultivars affects substrate specificity for dihydrokaempferol, contributing to flower color differences. The \u003cem\u003eArabidopsis tt3\u003c/em\u003e mutant accumulates elevated flavonols, exhibits altered auxin transport and displays developmental anomalies (Kuhn et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Peer et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These phenotypes align broadly with ours.\u003c/p\u003e \u003cp\u003eWhat sets the present work apart is the systematic characterization of pigment, morphological, and transcriptional phenotypes across multiple organs using multiple independent alleles in a commercially relevant cultivar. Most earlier DFR studies examined pigmentation alone or relied on single mutant alleles. The allelic series strategy enabled the establishment of an overall relationship between mutation severity and phenotypic intensity, as well as the distinction between genuine DFR-dependent effects and background noise. Including leaf and stem phenotyping revealed organ-specific consequences: leaves exhibited reduced biomass and plastidial pigments despite unchanged anthocyanin content, whereas stems remained anatomically normal (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Figs. S5-6). This pattern has not been documented in previous DFR mutant analyses. Recent genome-wide surveys have identified DFR gene families in strawberry (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), rapeseed (Qian et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and Solanaceae species (Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), underscoring the complexity of DFR regulation. Our findings add a layer by demonstrating that even knockout of a single dominant isoform can propagate effects across connected pathways.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePleiotropic consequences of\u003c/b\u003e \u003cb\u003eDFR\u003c/b\u003e \u003cb\u003edisruption and anthocyanin depletion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA possible increase in flavonol levels could have functional consequences that help explain the observed growth phenotypes. A recent comprehensive review by Daryanavard et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) summarized the current understanding of flavonol action in plant development, emphasizing two primary mechanisms: inhibition of polar auxin transport and modulation of reactive oxygen species homeostasis. Flavonols negatively regulate auxin transport by modulating PIN-dependent auxin distribution and auxin efflux processes (Buer and Muday \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kuhn et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Peer et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, flavonoid accumulation caused by repression of lignin biosynthesis (HCT silencing) correlated directly with reduced auxin transport and growth inhibition (Besseau et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). \u003cem\u003eArabidopsis tt3\u003c/em\u003e mutants lack functional DFR and accumulate excess kaempferol and quercetin; these plants display altered auxin distribution together with modified root and shoot architecture (Peer et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The reduced flower size and leaf biomass in our \u003cem\u003edfr\u003c/em\u003e lines (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) are consistent with auxin-mediated growth limitation, although confirmation would require direct measurement of auxin gradients in petunia using DR5 reporter lines or targeted IAA quantification.\u003c/p\u003e \u003cp\u003eFlavonols also act as potent antioxidants. Quercetin derivatives in particular scavenge reactive oxygen species and modulate cellular redox balance (Agati et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). ROS serve dual roles as signaling molecules and as agents of oxidative damage, so shifts in flavonol content can alter developmental programs through redox-sensitive pathways (Gayomba et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Kurepa et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) recently argued that flavonoids act as central mediators of the auxin-ROS-flavonol feedback loop, linking environmental sensing to developmental adjustment. Whether growth reductions in \u003cem\u003edfr\u003c/em\u003e mutants stem from perturbed auxin transport, altered ROS homeostasis, or both cannot be resolved with current data. Support for the redox hypothesis also comes from purple sweet potato, where RNAi suppression of IbDFR reduced anthocyanin accumulation and led to greater oxidative damage under cold stress, including increased H₂O₂ accumulation and electrolyte leakage, consistent with a protective role for anthocyanins in ROS homeostasis (Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The two mechanisms are not mutually exclusive. Histochemical ROS staining or genetically encoded biosensors would help clarify the contribution of redox perturbation.\u003c/p\u003e \u003cp\u003eOne of the less anticipated outcomes of this work is the coordinated decline of chlorophyll and carotenoid content in \u003cem\u003edfr\u003c/em\u003e mutant flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e fell in parallel, as did total carotenoids, indicating general suppression of plastidial pigment biosynthesis rather than selective loss of a single branch. Severe downregulation of \u003cem\u003ePORA\u003c/em\u003e in mutant corollas may provide a plausible mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). PORA encodes protochlorophyllide oxidoreductase A, which catalyzes a light-dependent step in chlorophyll synthesis; its suppression would predictably limit chlorophyll accumulation. \u003cem\u003eCHLH\u003c/em\u003e, encoding the H subunit of magnesium chelatase, remained unchanged, indicating that the perturbation is specific to \u003cem\u003ePORA\u003c/em\u003e rather than global across the tetrapyrrole pathway. Consistent with this possibility, transcriptome analysis of CRISPR/Cas9 NtDFR1/NtDFR2 knockout tobacco plants revealed significant downregulation of photosynthesis-related pathways, including genes encoding light-harvesting complex proteins, suggesting that disruption of DFR can influence chloroplast-associated metabolic processes beyond flavonoid biosynthesis (Jiang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHow flavonoid disruption impinges on chlorophyll biosynthesis is not immediately obvious. Several possibilities warrant consideration. Redox status influences chloroplast development and gene expression, so altered flavonol-mediated ROS scavenging could affect plastidial processes secondarily. Shared transcriptional regulators offer another route. HY5, a bZIP-type transcription factor, has emerged as a master regulator coordinating light signaling with flavonoid biosynthesis and photomorphogenic chlorophyll accumulation (Xiao et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). HY5 directly activates MYB12 and multiple anthocyanin biosynthetic genes, and in roots it also modulates auxin transporter abundance (van Gelderen et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xiao et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In apple, MbHY5 has been implicated in regulation of both iron transport and chlorophyll synthesis under iron deficiency (Sun et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The strong positive correlation between floral anthocyanin and leaf chlorophyll across genotypes (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.89, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) is consistent with coordinated variation between these traits across organs. The underlying mechanism remains to be identified, but the data suggest that flavonoid and tetrapyrrole metabolism may be more closely linked in petunia than is currently appreciated. This coupling may be especially pronounced.\u003c/p\u003e \u003cp\u003eCRISPR/Cas9-based modification of flavonoid pathway genes is increasingly applied in ornamental breeding. Nitarska et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) targeted \u003cem\u003eF3\u0026prime;H\u003c/em\u003e in poinsettia to shift bract color from red to reddish-orange through reduced cyanidin levels. Nishihara et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) edited \u003cem\u003eF3H\u003c/em\u003e into Torenia to produce white-flowered lines with high efficiency. In chrysanthemum, genome editing faces additional challenges due to hexaploidy, though progress has been made using multicopy transgenes as editing targets (Kishi-Kaboshi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our results add to this literature by showing that \u003cem\u003eDFR\u003c/em\u003e knockout in a commercial petunia cultivar produces a range of pigment phenotypes accompanied by growth phenotypes that breeders must consider. The edited lines collectively suggest that a variety of phenotypes may be achievable, not merely binary on/off states, expanding the toolkit for rational trait engineering in ornamentals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBreeding implications, limitations, and future directions\u003c/h3\u003e\n\u003cp\u003eFrom a breeding perspective, these findings carry practical weight. Targeted \u003cem\u003eDFR\u003c/em\u003e editing provides a direct route to modulate flower color intensity in petunia and related ornamentals. The edited lines suggest that a spectrum of pigmentation phenotypes may be achievable, not merely binary on/off states. Although the edited lines collectively showed an allele-associated spectrum of floral depigmentation, the striped and sectorial patterns observed in some lines suggest that petal color may also be influenced by somatic mosaicism or chimerism in primary regenerants. Therefore, these visible differences should be interpreted with caution, and validation in fixed later-generation lines or in separately genotyped petal sectors will be needed to confirm stable genotype\u0026ndash;phenotype relationships. This expands the palette available to breeders pursuing novel color variants. At the same time, the associated reductions in flower size and biomass warrant attention. Breeders aiming for lighter flower color may need to screen against growth penalties or pair \u003cem\u003eDFR\u003c/em\u003e edits with compensatory modifications elsewhere in the pathway.\u003c/p\u003e \u003cp\u003eThe pleiotropic effects documented here also hint at opportunities beyond color engineering. Manipulation of the flavonoid pathway could modulate stress tolerance by altering flavonol-mediated ROS scavenging. Meyer et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) first demonstrated heterologous \u003cem\u003eDFR\u003c/em\u003e expression in petunia to redirect pigment biosynthesis. Subsequent work across species has linked flavonoid accumulation to enhanced tolerance of oxidative and drought stress (Nakabayashi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Consistent with this broader view, transgenic tobacco overexpressing tea DFR or ANR accumulated more flavonoids, showed enhanced antioxidant capacity, flowered earlier, and displayed improved resistance to herbivory, indicating that manipulation of downstream flavonoid flux can influence both development and stress-related traits (Kumar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Whether DFR knockout, together with a possible shift in flavonoid composition, improves or compromises stress resilience in petunia remains untested. The outcome likely depends on which flavonoid classes predominate and how effectively they counteract particular stressors.\u003c/p\u003e \u003cp\u003eSeveral limitations of this work point toward productive follow-up. Flavonol accumulation was inferred from transcriptional data and the known biochemistry of the DFR/FLS branch point; direct quantification was not performed. Targeted metabolomics, for example, LC-MS profiling of kaempferol, quercetin, and their glycosides, would confirm the inference and identify which specific flavonol species accumulate. Such data would also clarify whether developmental phenotypes correlate more tightly with total flavonol content or with particular derivatives.\u003c/p\u003e \u003cp\u003eThe mechanistic underpinnings of the growth phenotypes require further investigation. Measurements of auxin distribution using DR5 reporters or direct quantification of IAA would test the auxin transport hypothesis. Parallel assessment of ROS levels would evaluate the redox hypothesis. The connection between \u003cem\u003eDFR\u003c/em\u003e disruption and \u003cem\u003ePORA\u003c/em\u003e downregulation is particularly intriguing. ChIP-seq to identify shared transcription factor binding sites between flavonoid and tetrapyrrole pathway genes could illuminate this possible link.\u003c/p\u003e \u003cp\u003eReproductive fitness was not systematically examined here. Pollen viability, seed set, and germination rate all merit attention, given the documented roles of flavonols in pollen tube growth and fertilization (Mo et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Taylor and Grotewold \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003eDFR\u003c/em\u003e disruption could affect these processes. Finally, combinatorial editing offers a promising avenue. Targeting \u003cem\u003eDFR\u003c/em\u003e together with \u003cem\u003eFLS\u003c/em\u003e, \u003cem\u003eF3\u0026prime;H\u003c/em\u003e, or \u003cem\u003eF3\u0026prime;5\u0026prime;H\u003c/em\u003e would permit finer control over metabolic flux partitioning and may help disentangle the relative contributions of anthocyanin loss versus flavonol gain to the phenotypes observed in this study.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCRISPR/Cas9-mediated knockout of \u003cem\u003eDFR\u003c/em\u003e in Petunia confirms that this enzyme is essential for floral anthocyanin biosynthesis and reveals unanticipated pleiotropic consequences extending to organ size, biomass, and plastidial pigment metabolism. The availability of multiple edited alleles strengthens inference about genotype\u0026ndash;phenotype relationships, although some pigmentation patterns in T₀ plants likely reflect mosaicism. The transcriptional and correlational data suggest that DFR functions not merely as a biosynthetic enzyme but as a functionally important metabolic node whose disruption is associated with effects across connected pathways. These findings expand current understanding of flavonoid pathway integration and provide a foundation for rational engineering of pigmentation and related traits in ornamental crops.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeclare any potential competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding Information\u003c/h2\u003e\n\u003cp\u003eThis project was funded by the USDA-NIFA grant 2019-67013-29236 and the USDA HATCH program FLA-MFC-006387, awarded to H.H.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eFL and TJ conceived and designed the study. FL and SET performed the experiments. FL and TJ collected and analyzed the data and wrote the manuscript. WA provided technical assistance. HH and TJ supervised the research, secured resources, and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors gratefully acknowledge PanAmerican Seed (Ball Horticultural Company) for providing the Petunia \u0026times; hybrida \u0026lsquo;Carmine Velour\u0026rsquo; cultivar for this research. The authors sincerely thank Dr. Jianping Ren for coordinating seed shipment and for valuable guidance and helpful discussions regarding petunia materials and cultivation. The authors also thank Matthew Creech for support in procuring chemicals, maintaining seed stocks, and managing equipment and facilities.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eThe data supporting the findings of this study are available upon reasonable request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdulla MF, Mostafa K, Kavas M (2024) CRISPR/Cas9-mediated mutagenesis of FT/TFL1 in petunia improves plant architecture and early flowering. 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Front Plant Sci 6:261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2015.00261\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2015.00261\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"Petunia, dihydroflavonol 4-reductase (DFR), genome-editing, anthocyanin biosynthesis, flavonoid metabolism, pleiotropic effects","lastPublishedDoi":"10.21203/rs.3.rs-9227413/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9227413/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDihydroflavonol 4-reductase (\u003cem\u003eDFR\u003c/em\u003e) occupies a critical branch point in flavonoid metabolism, channeling dihydroflavonol substrates toward anthocyanin biosynthesis in competition with flavonol synthase. While DFR's role in floral pigmentation is well established, the broader physiological and transcriptional consequences of its disruption remain poorly characterized, particularly in commercially important ornamental species. Here, we report the generation and comprehensive phenotyping of five independent CRISPR/Cas9-mediated DFR knockout alleles in the commercial \u003cem\u003ePetunia × hybrida\u003c/em\u003e cultivar 'Carmine Velour'. The edited lines showed an allele-associated spectrum of loss of floral pigmentation that was broadly consistent with mutation severity, confirming \u003cem\u003eDFR-A\u003c/em\u003e as the dominant isoform governing corolla anthocyanin accumulation. Beyond pigmentation, \u003cem\u003edfr\u003c/em\u003e mutants exhibited unexpected reductions in floral dimensions (20–40%), leaf biomass (30–50%), and plastidial pigment content, with chlorophyll and carotenoid levels declining 35–60% in petals despite unchanged leaf anthocyanins. Stem anatomy remained unaffected, revealing organ-specific pleiotropic effects. Transcriptional profiling uncovered feedback reprogramming within the flavonoid pathway: chalcone synthase A (\u003cem\u003eCHSA\u003c/em\u003e) and chalcone isomerase A (\u003cem\u003eCHIA\u003c/em\u003e) were downregulated while the competing branch enzyme flavonol synthase (\u003cem\u003eFLS\u003c/em\u003e) was upregulated almost 2-fold, consistent with the possibility of altered flux partitioning toward flavonol biosynthesis. Strikingly, protochlorophyllide oxidoreductase A\u003cem\u003e \u003c/em\u003e(\u003cem\u003ePORA\u003c/em\u003e), encoding a key chlorophyll biosynthetic enzyme, was severely suppressedby 60–75%, suggesting a possible connection between flavonoid disruption and tetrapyrrole metabolism. Correlation analyses suggested coordinated variation, with floral anthocyanin content positively associated with leaf chlorophyll and carotenoid levels across genotypes. These findings support the view that DFR acts as a functionally important metabolic node whose disruption is associated with effects across pigment classes and organ types, with implications for precision trait engineering in floriculture.\u003c/p\u003e","manuscriptTitle":"CRISPR/Cas9-Mediated DFR Disruption Suggests Coordinated Changes in Flavonoid Flux and Development in Petunia × hybrida","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-02 19:54:34","doi":"10.21203/rs.3.rs-9227413/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T02:31:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T16:44:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T03:52:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258391482045984554533301931991704887277","date":"2026-04-02T20:33:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267147790203807482402204905492348547768","date":"2026-03-29T03:34:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T00:28:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-26T14:35:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T14:34:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-03-25T22:47:05+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":"7fbcc186-fa21-4f54-89ef-30305f67eba9","owner":[],"postedDate":"April 2nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T02:31:01+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T02:38:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-02 19:54:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9227413","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9227413","identity":"rs-9227413","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}