Silencing of Dihydroflavonol 4-reductase in Chrysanthemum Ray Florets Enhances Flavonoid Biosynthesis and Antioxidant Capacity

Flavonoid biosynthesis requires the activities of several enzymes, which form weakly-bound, ordered protein complexes termed metabolons. To decipher flux regulation in the flavonoid biosynthetic pathway of chrysanthemum (Chrysanthemum morifolium Ramat), we suppressed the gene-encoding dihydroflavonol 4-reductase (DFR) through RNA interference (RNAi)-mediated post-transcriptional gene silencing under a floral-specific promoter. Transgenic CmDFR-RNAi chrysanthemum plants were obtained by Agrobacterium mediated transformation. Genomic PCR analysis of CmDFR RNAi chrysanthemums propagated by several rounds of stem cuttings verified stable transgene integration into the genome. CmDFR mRNA levels were reduced by 60-80% in CmDFR-RNAi lines compared to those in wild-type (WT) plants in ray florets, but not leaves. Additionally, transcript levels of flavonoid biosynthetic genes were highly upregulated in ray florets of CmDFR-RNAi chrysanthemum relative to those in WT plants, while transcript levels in leaves were similar to WT. Total flavonoid contents were high in ray florets of CmDFR-RNAi chrysanthemums, but flavonoid contents of leaves were similar to WT, consistent with transcript levels of flavonoid biosynthetic genes. Ray florets of CmDFR-RNAi chrysanthemums exhibited stronger antioxidant capacity than those of WT plants. We propose that post-transcriptional silencing of CmDFR in ray florets modifies metabolic flux, resulting in enhanced flavonoid content and antioxidant activity.