CRISPR/Cas9-mediated DFR disruption suggests coordinated changes in flavonoid flux and development in Petunia × hybrida.

key messageLoss of DFR function in petunia alters pigment metabolism and reduces organ size, suggesting previously underexplored associations among flavonoid biosynthesis, plastidial pigments, and development. 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-edited lines in the commercial Petunia × hybrida cultivar 'Carmine Velour'. The edited lines showed a spectrum of floral pigmentation loss that was broadly consistent with the representative editing patterns inferred from Sanger sequencing, supporting the major contribution of DFR-A to 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, indicating organ-specific associations between DFR disruption and growth-related traits. 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 twofold, consistent with the possibility of altered flux partitioning toward flavonol biosynthesis. Strikingly, protochlorophyllide oxidoreductase A (PORA), encoding a key chlorophyll biosynthetic enzyme, was severely suppressed by 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.