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Article|14 Nov 2023|OPEN
Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage
Sangkyu Park1 ,† , Hyo Lee1 ,† , Jaeeun Song1 , Chan Ju Lim2 , Jinpyo Oh2 , Sang Hoon Lee3 , Saet Buyl Lee1 , Jong-Yeol Lee1 , Sunhyung Lim4 , Jin A. Kim1 and Beom-Gi Kim,1 ,
1Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju, 54874, South Korea
2Institute of Biotechnology and Breeding, Asiaseed Inc., Icheon, 17414, South Korea
3Food and Nutrition Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju, 55365, South Korea
4Department of Horticultural Biotechnology, School of Biotechnology, Hankyong National University, Anseong, 17579, South Korea
*Corresponding author. E-mail: bgkimpeace@gmail.com
Both authors contributed equally to the study.

Horticulture Research 11,
Article number: uhad239 (2024)
doi: https://doi.org/10.1093/hr/uhad239
Views: 44

Received: 11 Sep 2023
Accepted: 06 Nov 2023
Published online: 14 Nov 2023

Abstract

Flavonols are the major class of flavonoids of green Chinese cabbage (Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes (BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3β-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout (BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants, while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.