Work

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

Year: 2025

Type: article

Abstract: Purple varieties of Chinese cabbage (Brassica rapa subsp. pekinensis) predominantly accumulate cyanidin-based anthocyanins. Dihydroflavonol 4-reductase (DFR) plays a key role in anthocyanin biosynthesis, but the DFR gene in B. rapa has only been tentatively identified, and the activity of its encoded protein remains unexplored. To characterize the candidate B. rapa DFR gene (Bra027457), we isolated its coding sequences (CDSs) from four B. rapa varieties exhibiting either the green or purple trait. Sequence analysis revealed that all CDSs were identical to the registered sequence. Bra027457 exhibited expression patterns correlated with anthocyanin accumulation in Chinese cabbage leaves. Enzyme assays demonstrated that Bra027457 is capable of reducing all three dihydroflavonol substrates of DFR. We generated BrDFR-knockout (BrDFR-KO) Chinese cabbage using CRISPR/Cas9 to target Bra027457 in the purple variety 8267. We successfully obtained transgene-free, homozygous plants exhibiting a green phenotype due to a complete loss of anthocyanins, confirming that Bra027457 is the authentic BrDFR gene responsible for anthocyanin accumulation in Chinese cabbage leaves. In BrDFR-KO T2 heads, quercetin (Q), isorhamnetin (IR), and dihydroquercetin (DHQ) levels were significantly elevated. Our analysis revealed five flavonol glycosides and one DHQ glycoside that accumulated in BrDFR-KO heads, with Q 3,7-di-O-glucoside and IR 3-O-(2′′′-O-feruloyl)sophoroside-7-O-glucoside representing the predominant flavonoids. These findings provide advanced insights into flavonoid biosynthesis in Brassica species and lay the foundation for developing valuable breeding resources for Chinese cabbage.

Source: Plant Physiology and Biochemistry

DOI: 10.1016/j.plaphy.2025.110534