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

Abstract

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.