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 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.

Figure 1

Comparative analysis of gene expression and amino acid sequences of BrFLS homologs. (A) Relative gene expression levels of BrFLS homologs based on mRNA-TPM (transcript per million) from the RNA-seq analysis of leaves from 9- and 42-day-old plants of 5546 and 5923 varieties. Error bars indicate ± SD from three replicates. (B) Alignment was conducted using the ClustalW program. Identical amino acids are indicated with black backgrounds. Amino acids that are >80% conserved are indicated with dark grey backgrounds, and those that are >60% conserved are indicated with light grey backgrounds. Three boxes represent FLS-specific motifs ‘PxxxIRxxxEQP’, ‘CPQ/RPxLAL’, and ‘SxxTxLVP’. Amino acid residues responsible for binding ferrous iron and 2-oxoglutarate are marked with black and grey arrows, respectively. Predicted residues involved in substrate binding are marked with black circles. Functional residues suggested to be involved in proper folding of the FLS polypeptide are marked with black squares. Amino acid variations of BrFLS1 and BrFLS3.1 that were cloned from variety 8045 are indicated by red rectangles.

Figure 2

Substrate-feeding assays of recombinant BrFLS homologs. (A) GST-fused BrFLS1, BrFLS2, BrFLS3.1, and BrFLS4.2 were expressed in E. coli by IPTG induction, which was verified by SDS-PAGE. VC, vector control. (B) IPTG-induced bacterial cultures were fed with DHK or Nar as a substrate. HPLC identified K production from DHK-fed reactant and DHK and K production from Nar-fed reactant. Nar and DHK were identified at 288 nm (grey chromatogram), and K was identified at 350 nm (black chromatogram). Small peaks of K and DHK are enlarged and shown in the insets. (C) Conversion rates were calculated based on the molar ratio between input and consumed substrates. The mean values were determined from three replicates.

Figure 3

Generation of transgene-free homozygous brfls1 plants. (A) Diagram of binary vector construction. Three sgRNAs (sg1, sg2, and sg3) were selected in exon 1 of the BrFLS1 gene and were introduced in the binary vector pHAtC carrying SpCas9, sgRNA scaffold, and the hygromycin resistance gene (Hyg-R). (B) T₂ progenies of brfls1 plants (NB62–180, −203, and −204) and control plants (8045 and T₂ progenies of NB61–99) grown in green house. (C) The regions encompassing the sg3 target site (dashed line) were amplified by genomic PCR and sequenced, which showed single T (red arrow) or A (green arrow) insertion 3 bp upstream of the PAM sequence (5′-CGG-3′) (shaded grey) in the target site of BrFLS1 in the brfls1 T₂ plants. (D) Deduced amino acid sequences of the brfls1 harboring the T or A insertion show early stops of BrFLS1 translation after 107^th amino acid caused by a frameshift. (E) Genomic PCR of four individuals (1–4) of the brfls1 T₂ progenies showing absence of transgene in their genome.

Figure 4

Investigation of changes in the flavonoid profile of brfls1 plants using LC-ESI-QTOF-MS analysis. LC-ESI-QTOF-MS analysis of flavonoid aglycones extracted from the leaves of four T₂ individuals generated from the NB61–99 (WT) and brfls1 (NB62–180, NB62–203, NB62–204) with negative ionization mode. (A) Representative XICs displaying mass spectra of flavonoid aglycones in the T₂ plants. XICs at m/z 285.23, m/z 287.25, m/z 301.23, m/z 303.25, m/z 315.26, and m/z 286.24 show deprotonated K, DHK, Q, DHQ, IR, and cyanidin Cya aglycones, respectively. (B) The average values of flavonoid content in the four individuals of each T₂ line were calculated based on the areas of corresponding standards. The mean values ± SD of four independent biological samples are shown. Significant differences were determined by Student’s t-tests. Asterisks indicate significant differences from the WT (^*P < 0.05, ^**P < 0.01, ^***P < 0.001). (C) Representative XICs of dihydroflavonol glycosides extracted from the brfls1 T₂ plants using 70% methanol (Hex, hexoside; diHex, dihexoside).

Figure 5

Analyses of changes in the expression of flavonoid biosynthetic genes and total phenolic contents in the brfls1 T₂ plants. qPCR analysis was performed and total phenolic content was measured in the four T₂ individuals generated from the NB61–99 and brfls1 (NB62–180, NB62–203, NB62–204). Expression values were normalized using B. rapa Actin7 (BrACT7) transcript. The total phenolic contents were calculated using gallic acid as a standard, and the mean values were expressed as milligrams of gallic acid equivalents per gram of fresh weight (mg GAE·g⁻¹ FW). The mean values ± SD of four independent biological samples are shown. Significant differences were determined by Student’s t-tests. Asterisks indicate significant differences from the WT (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Figure 6

Phenotypic analyses of the brfls1 T₂ plants. (A) Cross-sections of heads of two brfls1 T₂ plants and controls (8045 and NB61–99). (B) Indices related to agricultural traits of their heads (C) Root lengths of seedlings of brfls1 T₂ plants and control plants four (for control treatment) or six (for mannitol or NaCl treatment) days after osmolytes treatment. (D) Chlorophyll contents measured from aerial parts of the seedlings 4 days after mannitol treatment. The mean values ± SD of four independent biological samples are shown. Statistical significance was determined by Duncan’s Multiple Range Test using SAS (version 9.1) software. Significant differences between means (P < 0.05) are indicated by different lower case letters (a, b, and c).