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

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.