Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

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 3 β-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.

Acknowledgements

This work was supported by the New Breeding Technologies Development Program [grant number PJ016545] of the Rural Development Administration, Republic of Korea.

Author contributions

S.P. performed gene cloning, enzyme assay, HPLC, LC-ESI-QTOF- MS analysis, and wrote the manuscript. H.L. performed gene cloning, HPLC analysis, Chinese cabbage breeding, and off-target analysis. J.H., J.S., C.J.L., and J.O. contributed to the Chinese cabbage breeding, cultivation, and genotyping. S.H.L. analyzed the LC-ESI- QTOF-MS data. S.B.L., J.L., and S.L. performed data analysis and review. J.A.K. was in charge of Chinese cabbage transformation. B.K. designed and supervised this research and revised the final version of the manuscript.

Data availability

All relevant data generated or analyzed are included in the manuscript or in supplementary materials.

Conflict of interest statement

No conflict of interest declared.

References

[1] Li Z, Lee HW, Liang X. et al. Profiling of phenolic compounds and antioxidant activity of 12 cruciferous vegetables. Molecules. 2018; 23: 1139

[2] Park CH, Yeo HJ, Park SY. et al. Comparative phytochemical analyses and metabolic profiling of different phenotypes of Chinese cabbage Brassica rapa ssp. pekinensis. Foods. 2019; 8: 587

[3] Owens DK, Alerding AB, Crosby KC. et al. Functional analysis of a predicted flavonol synthase gene family in Arabidopsis. Plant Physiol. 2008; 147: 1046-61

[4] Preuß A, Stracke R, Weisshaar B. et al. Arabidopsis thaliana expresses a second functional flavonol synthase. FEBS Lett. 2009; 583: 1981-6

[5] Schilbert HM, Schone M, Baier T. et al. Characterization of the Brassica napus flavonol synthase gene family reveals bifunctional flavonol synthases. Front Plant Sci. 2021; 12: 733762

[6] Cheng F, Wu J, Fang L. et al. Biased gene fractionation and dominant gene expression among the subgenomes of Brassica rapa. PLoS One. 2012; 7: e36442

[7] Guo N, Cheng F, Wu J. et al. Anthocyanin biosynthetic genes in Brassica rapa. BMC Genomics. 2014; 15: 426

[8] Wang X, Wang H, Wang J. et al. The genome of the mesopolyploid crop species Brassica rapa. Nat Genet. 2011; 43: 1035-9

[9] He Q, Lu Q, He Y. et al. Dynamic changes of the anthocyanin biosynthesis mechanism during the development of heading Chinese cabbage Brassica rapa L. and arabidopsis under the control of BrMYB2. Front Plant Sci. 2020a; 11: 593766

[10] Gao C . Genome engineering for crop improvement and future agriculture. Cell. 2021; 184: 1621-35

[11] Li J, Yu X, Zhang C. et al. The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives. aBIOTECH. 2022; 3: 146-61

[12] Hong JK, Suh EJ, Park SR. et al. Multiplex CRISPR/Cas9 mutagenesis of BrVRN1 delays flowering time in Chinese cabbage Brassica rapa L. ssp. pekinensis. Agriculture. 2021; 11: 1286

[13] Su T, Wang W, Li P. et al. Natural variations of BrHISN2 provide a genetic basis for growth-flavour trade-off in different Brassica rapa subspecies. New Phytol. 2021; 231: 2186-99

[14] Xiong X, Liu W, Jiang J. et al. Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system. Mol Gen Genomics. 2019; 294: 1251-61

[15] Shin NR, Shin YH, Kim HS. et al. Function analysis of the PR55/B gene related to self-incompatibility in Chinese cabbage using CRISPR/Cas9. Int J Mol Sci. 2022; 23: 5062

[16] Kim NS, Yu J, Bae S. et al. Identification and characterization of PSEUDO-RESPONSE REGULATOR (PRR) 1a and 1b genes by CRISPR/Cas9-targeted mutagenesis in Chinese cabbage Brassica rapa L. Int J Mol Sci. 2022; 23: 6963

[17] Wellmann F, Lukacin R, Moriguchi T. et al. Functional expression and mutational analysis of flavonol synthase from Ctrus unshiu. Eur J Biochem. 2002; 269: 4134-42

[18] Stracke R, de Vos RC, Bartelniewoehner L. et al. Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase. Planta. 2009; 229: 427-45

[19] Chua CS, Biermann D, Goo KS. et al. Elucidation of active site residues of Arabidopsis thaliana flavonol synthase provides a molecular platform for engineering flavonols. Phytochemistry. 2008; 69: 66-75

[20] Kim H, Kim ST, Ryu J. et al. A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system. J Integr Plant Biol. 2016; 58: 705-12

[21] Falcone Ferreyra ML, Rius S, Emiliani J. et al. Cloning and characterization of a UV-B-inducible maize flavonol synthase. Plant J. 2010; 62: 77-91

[22] He Q, Wu J, Xue Y. et al. The novel gene BrMYB2, located on chromosome A07, with a short intron 1 controls the purple-head trait of Chinese cabbage Brassica rapa L. Hortic Res. 2020b; 7: 97

[23] Yin R, Messner B, Faus-Kessler T. et al. Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation. J Exp Bot. 2012; 63: 2465-78

[24] Auger B, Marnet N, Gautier V. et al. A detailed survey of seed coat flavonoids in developing seeds of Brassica napus L. J Agric Food Chem. 2010; 58: 6246-56

[25] Hald C, Dawid C, Tressel R. et al. Kaempferol 3-O-2-O-Sinapoyl-beta-sophoroside causes the undesired bitter taste of canola/rapeseed protein isolates. J Agric Food Chem. 2019; 67: 372-8

[26] Wen J, Zhu L, Qi L. et al. Characterization of interploid hybrids from crosses between Brassica juncea and B. Oleracea and the production of yellow-seeded B. Napus. Theoretical and Applied Genetic. 2012; 125: 19-32

[27] Jiang J, Shao Y, Li A. et al. Phenolic composition analysis and gene expression in developing seeds of yellow- and black-seeded Brassica napus. J Integr Plant Biol. 2013; 55: 537-51

[28] Zhang XF, Hung TM, Phuong PT. et al. Anti-inflammatory activity of flavonoids from Populus davidiana. Arch Pharm Res. 2006; 29: 1102-8

[29] Zhang WY, Lee JJ, Kim IS. et al. Stimulation of glucose uptake and improvement of insulin resistance by aromadendrin. Pharmacology. 2011; 88: 266-74

[30] Zhang Y, Yan G, Sun C. et al. Apoptosis effects of dihydrokaempferol isolated from bauhinia championii on synoviocytes. Evid Based Complement Alternat Med. 2018; 2018: 9806160

[31] Zhang Y, Zhang D, Tang Y. et al. Aromadendrin: a dual amyloid promoter to accelerate fibrillization and reduce cytotoxicity of both amyloid-β and hIAPP. Materials Advances. 2020; 1: 1241-52

[32] Lee HS, Jeong GS . Aromadendrin inhibits T cell activation via regulation of calcium influx and NFAT activity. Molecules. 2020; 25: 4590

[33] Lee HS, Kim EN, Jeong GS . Aromadendrin protects neuronal cells from methamphetamine-induced neurotoxicity by regulating endoplasmic reticulum stress and PI3K/Akt/mTOR signaling pathway. Int J Mol Sci. 2021; 22: 2274

[34] Kim D, Pertea G, Trapnell C. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013; 14: R36

[35] Tarpnell C, Williams BA, Pertea G. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28: 511-5

[36] Park S, Lee H, Min MK. et al. Functional characterization of BrF3′H, which determines the typical flavonoid profile of purple Chinese cabbage. Front Plant Sci. 2021; 12: 793589

[37] Earley KW, Haag JR, Pontes O. et al. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 2006; 45: 616-29

[38] Holsters M, de Waele D, Depicker A. et al. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genomics. 1978; 163: 181-7

[39] Park S, Choi MJ, Lee JY. et al. Molecular and biochemical analysis of two rice flavonoid 3′-hydroxylase to evaluate their roles in flavonoid biosynthesis in rice grain. Int J Mol Sci. 2016; 17: 1549

[40] Bae S, Kweon J, Kim HS. et al. Microhomology-based choice of Cas9 nuclease target sites. Nat Methods. 2014; 11: 705-6

[41] Xie X, Ma X, Zhu Q. et al. CRISPR-GE: a convenient software toolkit for CRISPR-based genome editing. Mol Plant. 2017; 10: 1246-9

[42] Manolopoulou E, Varzakas TH, Petsalaki A . Chlorophyll determination in green pepper using two different extraction methods. Current research in nutrition and food science. 2016; 4: 52-60