Redirecting flavonoid flux in purple Chinese cabbage via Cas9-mediated BrDFR knockout

Purple varieties of Chinese cabbage (Brassica rapa subsp. pekinensis) predominantly accumulate cyanidin-based anthocyanins. Although dihydroflavonol 4-reductase (DFR) is a key enzyme in anthocyanin biosynthesis, the function of the putative B. rapa DFR gene (Bra027457) remained unverified. We isolated and sequenced the coding region of Bra027457 from four B. rapa inbred lines with either green or purple phenotypes and detected no sequence variation. Bra027457 expression correlated with anthocyanin accumulation, and in vitro assays confirmed its ability to reduce all three dihydroflavonol substrates. Using CRISPR/Cas9, we knocked out Bra027457 in the purple line 8267 and obtained transgene-free, homozygous BrDFR-KO plants. These exhibited a green phenotype due to complete anthocyanin loss, verifying Bra027457 as the authentic BrDFR gene. Metabolite profiling of BrDFR-KO heads revealed significant increases in quercetin (Q), isorhamnetin (IR), and dihydroquercetin (DHQ). LC/MS analysis further identified five flavonol glycosides and one DHQ glycoside, of whch Q 3,7-di-O-glucoside and IR 3-O-(2‴-O-feruloyl)sophoroside-7-O-glucoside were predominant. These findings advance our understanding of flavonoid biosynthesis in Brassica species and provide valuable genetic resources for Chinese cabbage improvement.