pubs.acs.

org/JAFC Article

Targeted Knockout of BnTT2 Homologues for Yellow-Seeded

Brassica napus with Reduced Flavonoids and Improved Fatty Acid
Composition
Tao Xie, Xin Chen, Tuli Guo, Hao Rong, Ziyi Chen, Qinfu Sun, Jacqueline Batley, Jinjin Jiang,*
and Youping Wang*
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ABSTRACT: Brassica napus is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in
rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this
study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the
four alleles of two BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant
functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that the BnTT2 mutation
hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the BnTT2 mutation
inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the
complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of BnTT2 homologues increased oil content and
improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic
improvement of rapeseed. Overall, this research showed that the BnTT2 mutation can be used for yellow seed breeding and oil
improvement, which is of great significance in improving the economic value of rapeseeds.
KEYWORDS: Brassica napus, yellow seed, TRANSPARENT TESTA 2, CRISPR/Cas9, flavonoids, fatty acid

■ INTRODUCTION
Brassica napus L. (AACC, 2n = 38) is a natural allotetraploid
with great economical value in producing oil for the human
diet and industrial use as well as oil-seed-cake for animal
feeding.1,2 The yellow-seeded B. napus has been preferred for
decades, with prominent improvements in oil and protein
content compared with the black-seeded counterparts.3 The
improved quality of yellow-seeded B. napus is also related with
the thinner seed coat and reduced proanthocyanidin content.4
Hitherto, many yellow-seeded B. napus have been created
through interspecific crosses in Brassica5 and intergeneric
hybridization between B. napus and Sinapis alba or Descurainia
sophia.6,7
In Arabidopsis thaliana, the yellow seed character has been
clarified and all the related genes are named Transparent Testa
(TT) genes that are involved in the flavonoid biosynthetic
pathway.8 The major types of flavonoids are flavonols,
anthocyanins, isoflavonols, and proanthocyanidins (PAs), of
which only flavonols and PAs are deposited in Arabidopsis
seeds. Flavonols form copigments with anthocyanins to
contribute to seed color, and oxidation of PAs with seed
maturation forms oxidized tannins and a brown color.9
Kaempferol, quercetin, and isorhamnetin are the main
flavonols accumulated in Arabidopsis seeds, which could form
flavonol derivatives, anthocyanins, or tannins. Epicatechin and
catechin are two stereoisomers that form the polymers of PAs,
of which only epicatechin is synthesized in Arabidopsis.10 The
genes involved in the biosynthesis of the above chemicals are
classified into early biosynthesis genes (EBGs), later biosyn-
thesis genes (LBGs), and transcriptional regulatory factors.8
The EBGs include TT4/CHS, TT5/CHI, TT6/F3H, and TT7/
F3′H; the LBGs include TT3/DFR, TT18/LDOX/ANS, BAN/
ANR, TT12, TT19/GSTF12/GST26, and AHA10. The MYB-
bHLH-WD40 (MBW) complex is well-known as a regulator in
the flavonoid pathway, including TT2-TT8-TTG1, MYB5-
TT8-TTG1, TT2-EGL3-TTG1, and TT2-GL3-TTG1.11
PAP1/MYB75 and PAP2/MYB90 were also identified as
components of the MBW complex in Arabidopsis.12 The
molecular mechanism and cloning of TTs in Brassica was
reviewed in 2013, and the duplication history of TTs in 17
Brassicaceae species was analyzed in 2016.13,14 Due to the
duplication history and genome complexity15 and lack of
genetic mutants, gene functional analysis was greatly hindered
in B. napus. Hitherto, only BnTT10, BnTT1, and BnTT8 were
functionally confirmed in regulating PA metabolism, lignin
synthesis, seed coat pigmentation, and fatty acid (FA)
biosynthesis.16−18
As mentioned above, R2R3-MYB is an essential component
of the MBW complex. Different MBW complexes with
Received: February 19, 2020
Revised: April 29, 2020
Accepted: April 29, 2020
Published: May 12, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.jafc.0c01126

5676 J. Agric. Food Chem. 2020, 68, 5676−5690
Journal of Agricultural and Food Chemistry
different MYBs were reported as tissue-specific regulators
controlling anthocyanin and PA accumulation in Arabidopsis.
For example, PAP1/MYB75, PAP2/MYB90, MYB113, and
MYB114 regulate flavonoid biosynthesis in vegetative tissues;
TT2/MYB123 regulates PA biosynthesis in seeds, and MYB5
regulates PA accumulation with minor functions, which could
functionally replace TT2.11,19,20 Recently, MYB4 was reported
with dual roles in flavonoid biosynthesis in Arabidopsis, which
could repress phenylpropanoid metabolism together with
TT8/GL3/EGL3 and inhibit flavonoid biosynthesis by
repressing Arogenate Dehydratase 6 (ADT6). 21 MYBs
regulating anthocyanin and PA synthesis were also reported
in Rosa rugosa, Malus × domestica, Vitis vinifera, Malus sieversii,
and Marchantia polymorpha.22−28 Besides, B-box proteins, such
as BBX16 in pear and BBX22 in apple, positively regulate light-
induced flavonoid accumulation by activating a TT2
homologue (MYB10).22,29 In B. napus, BnTT2 homologues
were cloned30 and the allelic variation of BnTT2 was identified
on the C genome, which is associated with seed coat color and
FA biosynthesis.31 Hitherto, functional analysis on BnTT2
regulating anthocyanin and PA biosynthesis has not been
reported yet.
The duplication history of B. napus resulted in two or more
homologues for each gene, and it is quite challenging to
simultaneously regulate the expression of all the homologues.15
Genome editing with the CRISPR/Cas9 systems greatly
helped the gene functional analysis in plants, especially in
the polyploid species,32 and the newly developed Cas12 system
has been proven to be a promising tool for precise genome
editing in A. thaliana, Oryza sativa, Nicotiana benthamiana,
Gossypium hirsutum, and Citrus sinensis.33−36 Hitherto, several
genes were target mutated with CRISPR/Cas9 in B. napus,
including BnCLV3, BnFAD2, BnLIM1, BnSDG8, BnMAX1,
BnLPAT2/5, BnMLPK, BnTT8, BnIND, and BnALC.18,37−44 In
the present study, we targeted knockout two BnTT2
homologues in B. napus with the CRISPR/Cas9 system and
confirmed that the mutation of the BnTT2 homologues
significantly reduced the flavonoid content, especially the
epicatechin and isorhamnetin content, through the down-
regulation of genes involved in the flavonoid biosynthetic
pathway. Besides the yellow seed character in the BnTT2
knockout mutants, we found the oil content was increased and
the FA composition was improved with higher linoleic acid
(C18:2) and linolenic acid (C18:3). This is valuable in
providing improved rapeseed germplasm for breeding.
■
MATERIALS AND METHODS
Plant Materials. The A. thaliana ecotype Columbia (Col-0), tt2-5
mutant (CS2105593), and transgenic A. thaliana were grown in a
climate chamber under a photoperiod of 16 h light/8 h dark and a
temperature of 22 °C. The B. napus line J9712 was used for genetic
transformation. Both J9712 and the transgenic B. napus were grown in
the experimental field in Yangzhou University, Jiangsu, China. Three
replicates of 15 samples representing the major developmental tissues
and organs of the B. napus line J9712 were collected for RNA-seq
analysis, including leaf, cotyledon, hypocotyl, root, stem, shoot apical
meristem (SAM), 3 mm bud, 6 mm bud, endosperm, silique at 2
weeks after flowering (WAF), and five seed samples at 3 WAF, 4
WAF, 5 WAF, 6 WAF, and the mature stage. Three replicates of seed
samples at 3 to 7 WAF and the mature stage from two knockout lines
of BnTT2 homologues were collected for metabolism analysis and
RNA-seq analysis, using B. napus line J9712 as CK.
Sequence Analysis of BnTT2. The protein sequence of AtTT2
was used for protein-blast against the B. napus reference genome
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(http://www.genoscope.cns.fr/brassicanapus/). Two highly con-
s er ved s equenc es in B. n apus (BnaA08g299 30D and
BnaC08g07960D) were named BnaA.TT2 and BnaC.TT2. Multiple
sequence alignments of the protein sequences of TT2 genes were
performed using the clustalX program (http://www.clustal.org/
clustal2/) and edited by Jalview (http://www.jalview.org/). Phyloge-
netic trees were constructed using the Neighbor-Joining (NJ) method
with 1000 bootstrap replications, the p-distance, and pairwise deletion
in MEGA 6.0 (https://www.megasoftware.net/).
Genetic Complementation of BnTT2 in Arabidopsis tt2
Mutant. The coding sequences (CDSs) of BnaA.TT2 and BnaC.TT2
were cloned from B. napus cv. ‘Darmor-bzh’ and ligated into the
pMDC83 vector under the control of the 35S promoter.
35S::BnaA.TT2 and 35S::BnaC.TT2 were introduced into the
Agrobacterium tumefaciens strain GV3101 by electroporation and
then transformed into Arabidopsis tt2-5 mutants using the floral dip
method.45 The T1 seeds were screened on 1/2 MS medium
containing 50 mg/L hygromycin B2 (Hyg), and the putative
transformants were confirmed by PCR analysis. The T3 seeds were
used for homozygotes screening and phenotypic observation. All the
primers used in this study are listed in Table S1.
CRISPR/Cas9 Plasmid Construction, Genetic Transforma-
tion, and Identification of Target Mutated Transgenic Plants.
Two sgRNAs (small guide RNA) sequences were designed to target
two BnTT2 genes using CRISPR-P 2.0 (http://crispr.hzau.edu.cn/
CRISPR2/). The two target sequences were used to generate sgRNA
expression cassettes under the control of AtU6-1 and AtU6-29
promoters, respectively. Then, the expression cassettes were
integrated into the pYLCRISPR/Cas9-DH vector using a Golden
Gate clone method.46 The Cas9/sgRNA constructs were introduced
into A. tumefaciens GV3101 by electroporation. The A. tumefaciens-
mediated hypocotyl transformation in B. napus line J9712 was
proceeded as reported.38 All the regenerated plants under a selective
pressure of 25 mg/L Hyg were screened by PCR using plasmid
specific primers. To analyze the genotype of BnTT2 mutant lines,
sequencing was carried out on the PCR products using specific
primers spanning the Cas9/sgRNA target sequences (Tsingke
Biotech, China). To analyze the type and frequency of mutations,
the PCR products were cloned into pEASY-T1 Cloning Vector
(Transgene Biotech, China) and sequenced using M13F primer.
Seed Pigmentation Observation. As reported before,47 we used
vanillin and p-dimethylaminocinnamaldehyde (DMACA) for PA
staining in the seed coat of B. napus line J9712 and BnTT2 knockout
mutants. The DMACA assay was also used for the detection of
flavonoids in Arabidopsis seeds, including Col-0, tt2-5 mutant, and
transgenic plants. To know the deposition of flavonoids in the seed
structure, semithin sections were cut with mature rapeseeds and
viewed under a light microscope (BX53, Olympus, Japan).48
Flavonoid and FA Profiling. Flavonoids were extracted with 100
seeds from six developmental stages (3 WAF, 4 WAF, 5 WAF, 6
WAF, 7 WAF, and the mature stage) of B. napus line J9712 and
BnTT2 knockout mutants, and the total contents of soluble phenolics
and favonoids were measured as previously reported.49 The detailed
profiling of the polyphenolic components was analyzed using HPLC−
DAD−ESI/MS.50
The lipids were extracted from 100 mature seeds, which were
ground with isopropanol and incubated at 100 °C for 5 min and then
vortexed with CH2Cl2 for 30 min. The organic and aqueous phases
were separated with centrifugation, after adding CH2Cl2 and 1 M KCl
in 0.2 M H3PO4. The aqueous phase was washed twice with CH2Cl2,
and all the organic phases were concentrated under a nitrogen
evaporator.51 Transmethylation of total lipid was conducted to obtain
fatty acid methyl esters (FAMEs). The lipid extracts were mixed with
methanolic HCl (20 mL of acetyl chloride in 100 mL of cold
methanol) and incubated for 1 h at 80 °C. Then, 0.9% NaCl (2 mL)
was added to terminate the reaction. The FAMEs were extracted
twice with hexane; then, the combined organic phases were
concentrated for gas chromatography−mass spectrometry (GC−
MS) analysis.52 The extracts were filtered through a nylon membrane
(0.22 μm) and separated with a DB-WAX capillary column (30 m ×
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Figure 1. Two BnTT2 homologues in the rapeseed genome. (A) Alignment and structural domains of TT2 in B. napus, B. rapa, B. oleracea, and
A. thaliana. (B) The phylogenetic tree of TT2 in B. napus, B. rapa, B. oleracea, R. sativus, Vitis vinifera, Camelina sativa, Arachis hypogaea,
G. arboreum, Eucalyptus grandis, Glysine max, and A. thaliana. (C) The expression pattern of BnTT2 during B. napus development.
0.25 mm, 0.25 μm, Agilent Technologies, Waldbronn, Germany). The
chromatographic separation of FAMEs was performed on a Trace GC
DSQII (ThermoFinnigan, San Jose, CA, USA) with 1 mL/min helium
and an inlet temperature of 250 °C. The chemicals were detected
using a quadrupole MS with an iron source at 250 °C (both positive
and negative modes were applied); 70 eV spectrometric voltage; m/z
30 to 450 scanning range. The oven temperature was set at 50 °C for
2 min, increased at 4 °C/min to 200 °C for 5 min, and increased
again at 4 °C/min to 220 °C for 20 min. The FAs were identified in
comparison with the Supelco 37 Component FAME Mix (Sigma,
California, USA).
RNA-seq and qRT-PCR Analysis. Three replicates of seed
samples at 3 WAF, 4 WAF, 5 WAF, and 6 WAF from B. napus line
J9712 and two BnTT2 knockout lines were collected for RNA-seq
analysis. The total RNA was extracted using the RNAprep pure Plant
Kit (Tiangen Biotech, China). The RNA quality and concentration
were verified using agarose gel electrophoresis, Nanodrop, Qubit, and
Agilent 2100. cDNA library construction and sequencing, clean data
mapping, DEG screening, and annotation were performed as
reported.3 RNA-seq of 15 samples representing the major
developmental tissues and organs of B. napus line J9712 was carried
out.
Subsamples of RNA-seq were reverse transcribed into cDNA for
real-time qPCR validation using the Revert Aid First Strand cDNA
Synthesis Kit and SYBR Green Real-Time PCR Master Mixes
(Thermo, USA). qRT-PCR was performed on a fluorescence
quantitative system StepOnePlus (Applied Biosystems, USA).
B. napus Actin-7 (NC_027775.2) was used as an endogenous control.
Protein−Protein Interaction Assay. For the Y2H assay, the
CDSs of BnTT2, BnTT8, BnTTG1, BnMYBL2, BnCPC, BnLBD37,
BnLBD38, and BnLBD39 were cloned into yeast two-hybrid (Y2H)
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bait vector pGBKT7 and prey vector pGADT7 (Clontech, Japan).
Bait and prey constructs were cotransformed into Saccharomyces
cerevisiae strain AH109 according to the manufacturer’s instructions of
the Matchmaker Gold Yeast Two-Hybrid system (Clontech, Japan).
After being cultured in SD/-Leu/-Trp liquid medium, the positive
clones were diluted with sterile water and plated on the SD/-Ade/-
His/-Leu/-Trp plates to test the interaction between bait and prey.
The cotransformations of pGBKT7-53 and pGADT7-T were used as
positive controls; pGBKT7-Lam and pGADT7-T were negative
controls.
The bimolecular fluorescence complementation (BiFC) assay was
performed as described by Waadt et al. with some modifications.53
The CDSs of BnTT2, BnTT8, BnTTG1, BnMYBL2, BnCPC,
BnLBD37, BnLBD38, and BnLBD39 were cloned into pVYNE and
pVYCE to fuse with the N- or C-terminal fragments of yellow
fluorescent protein (YFP). The different combinations of the
constructs were cotransformed into 5-week-old N. benthamiana by
infiltration with the A. tumefaciens strain GV3101. The florescence
images were captured using a confocal laser-scanning microscope
(TCS SP8 STED, Leica, Germany).
Near-Infrared Spectroscopy Analysis of Rapeseeds. The FA
content, crude protein, and glucosinolate in mature seeds of J9712
and BnTT2 knockout lines were analyzed with near-infrared
spectroscopy (NIRS) (NIRS DS2500F, Foss, Denmark).
Statistical Analysis. The flavonoid and FA contents were
expressed as the means ± standard error from three independent
extractions, and p < 0.05 was considered significant.
https://dx.doi.org/10.1021/acs.jafc.0c01126
J. Agric. Food Chem. 2020, 68, 5676−5690
Journal of Agricultural and Food Chemistry
Figure 2. Overexpression of BnTT2 recovered yellow seed phenotype in the Arabidopsis tt2 mutant. (A) Expression level of BnTT2 in
complemented tt2 mutants. (B) Seed phenotype of Col, tt2 mutant, and BnTT2 complementation materials. (C) DMACA stained seeds. Bar = 1
mm.
■ RESULTS
Two TT2 Homologues in B. napus Recover Seed Color
of Arabidopsis tt2 Mutant. Two BnTT2 homologues
identified in the rapeseed genome were named BnaA.TT2
(BnaA08g29930D) and BnaC.TT2 (BnaC08g07960D).
BnaA.TT2 shared 72.93% identity and 78.95% similarity and
BnaC.TT2 shared 72.12% identity and 78.07% similarity with
AtTT2 at the amino acid level. BnaA.TT2 shared 96.93%
identity and 97.32% similarity with BnaC.TT2 at the amino
acid level. Similar to the orthologs in A. thaliana, B. rapa, and
B. oleracea, BnaA.TT2 and BnaC.TT2 have R2 and R3 domains
in the N-terminus and a TT2 domain (Figure 1A).
Phylogenetic analysis confirmed that BnaA.TT2 and BnaC.TT2
were clustered with TT2 in B. rapa, B. oleracea, and Raphanus
sativus, indicating the two BnTT2 homologues might have
conserved but redundant functions, which were originated
from B. rapa and B. oleracea, respectively (Figure 1B). RNA-
seq analysis revealed that both BnaA.TT2 and BnaC.TT2 were
highly expressed in endosperm, silique, and developing seeds
(Figure 1C). This agreed with a previous report that AtTT2
was specifically expressed in seeds.11 We also found that the
expressional pattern of BnTT2 homologues was similar,
maximized in seeds at 3 WAF, and then downregulated
afterward. To know if BnTT2 has conserved function with
AtTT2, the BnTT2 homologues were overexpressed in the
Arabidopsis tt2 mutant, respectively (Figure 2A). We found the
transparent seed color was recovered with overexpression of
either BnaA.TT2 or BnaC.TT2 and PA content was increased
with DMACA staining (Figure 2B,C). This indicated that both
BnaA.TT2 and BnaC.TT2 have conserved functions with
AtTT2.
Creation of Targeted Knockout Lines of BnTT2
Homologues. On the basis of the functional analysis of
BnTT2 in Arabidopsis tt2 mutants, we created the knockout
lines of BnTT2 homologues to obtain yellow-seeded B. napus.
Two sgRNAs (sgRNA-1 and sgRNA-2) both targeting two
BnTT2 homologues were designed with CRISPR-P 2.0
(http://crispr.hzau.edu.cn/CRISPR2/) (Figure 3A). The
expression cassettes with two sgRNAs were integrated into
the pYLCRISPR/Cas9-DH vector (Figure 3B) and then
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introduced into B. napus line J9712 using A. tumifaciens-
mediated hypocotyl transformation. A total of 81 T0 positive
transgenic plants were created, and four of them were
identified with mutations on the target site of sgRNA-2
(Figure 3C). The mutation on the target site of sgRNA-1 was
not identified. All the mutations on the A and C genome were
defined as six types: A1 (−8 bp), A2 (−31 bp), A3 (+1 bp),
C1 (+1 bp), C2 (−7 bp), and C3 (−1 bp). Two T0 plants
were identified with a mutation on BnaA.TT2 and BnaC.TT2,
respectively. Other two T0 plants were identified with
mutations on both A and C homologues, but no homozygous
mutation was obtained.
The four mutated plants mentioned above were further
identified by sanger sequencing, and homozygous mutations
on A genome and C genome were screened in T1 generation
(Table 1). Five out of 24 T1 plants of line 3 were identified
with homozygous mutations on BnaA.TT2, and 23 out of 24
T1 plants of line 33 were identified with homozygous
mutations on BnaC.TT2. Homozygous mutations on
BnaA.TT2 and BnaC.TT2 were identified in T1 plants of
lines 3 and 33, respectively, but the seed coat color was not
changed in mutants of BnaA.TT2 (named aaCC) or BnaC.TT2
(named AAcc) (Figure 4A). Homozygous mutations on both
BnaA.TT2 and BnaC.TT2 were identified in T1 plants of lines
4 and 27, both showing a visible yellow seed phenotype
(Figure 4A). The two yellow-seeded BnTT2 mutation lines,
named aacc-4 and aacc-27, were used for phenotypic analysis,
flavonoid and FA profiling, gene expression, and seed quality
analysis.
BnTT2 Mutation Reduced Flavonoid Accumulation in
Rapeseed. Using vanillin and DMACA staining to visualize
the PA accumulation in the seed coat of J9712 (CK) and
BnTT2 knockout mutants (aacc-4 and aacc-27), we found the
pigmentation of flavonoids was reduced in two BnTT2 mutants
compared with that of CK (Figure 4B,C). The semithin
section in mature seeds confirmed that the palisade layers of
aacc-4 and aacc-27 were significantly thinner than that of CK,
and the red-stained PA accumulation was absent in the
palisade layer of the seed coat in the BnTT2 knockout mutants
(Figure 4D,E).
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Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

Figure 3. Creation of BnTT2 knockout lines with CRISPR/Cas9. (A) Two target sites on the first two exons of the BnTT2 homologues. (B) Vector
structure of CRISPR/Cas9 hosting two sgRNA expression cassettes. (C) Sequences at the mutation sites of BnTT2 homologues in the T0
generation. A1 (−8 bp), A2 (−31 bp), A3 (+1 bp), C1 (+1 bp), C2 (−7 bp), and C3 (−1 bp) are six types of identified mutations. HZ and HM
represent heterozygous and homozygous mutations.

Table 1. Genotypic Analysis of BnTT2 Mutants in T1 Generation

line TT2 genotypes positive
#3 AACC AA1CC A1A1CC 17/24
9/24 10/24 5/24
#4 A2A2CC A2A2C1C1 A3A3CC1 A2A2CC1 A2A3C1C1 A2A3CC1 A3A3CC A3A3C1C1 AA3CC1 A2A3CC 16/23
1/23 3/23 4/23 3/23 3/23 2/23 2/23 2/23 1/23 2/23
#27 AA3C1C1 A3A3C2C2 AA3CC1 A3A3C1C2 AAC2C2 AA3C1C2 AA3C2C2 AAC1C2 A3A3CC2 A3A3C1C1 15/23
4/22 1/22 1/22 4/22 2/22 2/22 3/22 3/22 1/22 1/22
#33 AACC3 AAC3C3 19/24
1/24 23/24

Compared with J9712, we found both total phenolic and
flavonoid contents in BnTT2 knockout mutants were reduced
throughout seed development (Figure 5A,B). Detailed
profiling of phenolic compounds in BnTT2 mutants and
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J9712 using HPLC-DAD-ESI/MS analysis, and a total of 46
hydroxycinnamic acid derivatives and flavonoid compounds
were identified, including 18 hydroxycinnamic acid derivatives,
12 kaempferols (km), 6 isorhamnetins (is), 5 quercetins (qn),
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Figure 4. Seed phenotype of BnTT2 knockout mutants. (A) Mature seed phenotype of two BnTT2 knockout mutants. (B) Vanillin stained
developing seeds. (C) DMACA stained developing seeds. (D) Semithin section and red PA staining in mature seeds. (E) Thickness of palisade
layer. CK is J9712. aaCC and AAcc represent homozygous mutants on BnaA.TT2 and BnaC.TT2, respectively. aacc-4 and aacc-27 are two
homozygous mutants on both BnaA.TT2 and BnaC.TT2. WAF, weeks after flowering. Bar = 20 μm. The data represents the means ± SE (n = 10);
the Student’s t-test was used for statistical analysis between the knockout mutant and CK (**, p ≤ 0.01).

5 epicatechin and its derivatives (Table S2). We found several
hydroxycinnamic acid derivatives, including sinapoylhexose,
trans-sinapic acid, sinapoylgentiobiose, disinapoylgentiobiose,
sinapoylmalic acid, disinapoylglucoside, and sinapoylhydrox-
yferuloylgentioside were reduced in BnTT2 mutants compared
with J9712 (Figure S1). Three of the quercetins, such as qn-3-
O-sophoroside-7-O-glucoside and two qn-3-O-sinapoylso-
phoroside-7-O-glucoside, were less accumulated in BnTT2
mutants than in J9712 (Figure S2). Only five kaempferols,
including km-3-O-glucoside-7-O-glucoside, km-3-O-sophoro-
side-7-O-glucoside, two km-3-O-sinapoyldiglucoside-7-O-glu-
coside, and km-3-O-feruloylsophoroside-7-O-glucoside, were
reduced in BnTT2 mutants than in J9712 (Figure S3).
Interestingly, all the isorhamnetins and epicatechins were
significantly reduced throughout the seed development of the
BnTT2 mutants than that of J9712 (Figures S4 and S5). Since
isorhamnetins and epicatechins are two main groups of
flavonoid compounds in rapeseed, we may suspect that
BnTT2 plays important roles in regulating flavonol and PA
biosynthesis (Figure 5C). Since the flavonoid content was
significantly reduced in BnTT2 mutants, we found the
percentage of the total content of hydroxycinnamic acid
derivatives, as the main group of polyphenolic compounds in
rapeseed, was increased throughout the seed development of
BnTT2 mutants than that of J9712 (Figure 5D).
BnTT2 Mutation Improves the FA Composition in
Rapeseed. To know the effects of BnTT2 mutation on seed
quality, we used NIRS to analyze the fatty acid and protein
contents. We found the oil content was significantly increased
in BnTT2 mutants (45.04−47.63%) than in J9712 (40.11%),
which is of great value to rapeseed breeding as the seed oil is
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the main value embodiment (Table 2). GC-MS analysis was
carried out to further confirm and identify the FA changes in
BnTT2 mutants, and the results revealed that palmitic acid
(C16:0) was not significantly changed after BnTT2 mutation;
stearic acid (C18:0) and oleic acid (C18:1) were significantly
decreased in BnTT2 mutants, while linoleic acid (C18:2) and
linolenic acid (C18:3) were significantly increased in BnTT2
mutants (Table 2). Generally, the FA profiles in BnTT2
mutants were consistent with the altered FA composition, and
the nutritional value of the mutants was improved compared
with that of CK.
BnTT2 Regulates the Phenylpropanoid and Flavo-
noid Biosynthetic Pathway. On the basis of the RNA-seq
analysis of the developing seeds of two BnTT2 knockout lines
(aacc-4 and aacc-27) and J9712 (CK), we screened the
differentially expressed genes (DEGs) coexisting in the
comparisons of aacc-4 vs CK and aacc-27 vs CK. The three
biological replicates of RNA-seq data at each seed devel-
opmental stage were confirmed with personal correlation
coefficient values of R = 0.89−0.99 (Figure S6). Generally,
9194 DEGs (4250 upregulated and 4944 downregulated),
2948 DEGs (1520 upregulated and 1428 downregulated),
2305 DEGs (890 upregulated and 1415 downregulated), and
12554 DEGs (7328 upregulated and 5226 downregulated)
were identified at 3 WAF, 4 WAF, 5 WAF, and 6 WAF,
respectively (Figure 6A, Table S3). Only 89 upregulated and
460 downregulated DEGs between the BnTT2 mutant and
J9712 coexisted throughout seed development (Figure 6B,C).
Gene ontology (GO) enrichment analysis revealed that most
DEGs were involved in a cellular process, a metabolic process,
binding, catalytic activity, cells, cell parts, and organelles
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Figure 5. Phenolic and flavonoid content reduced in BnTT2 mutants. (A) Total phenolic content. (B) Total flavonoid content. (C) Content of
different flavonoid compounds. (D) Percentage of phenolic components. CK is J9712. aacc-4 and aacc-27 are mutants of BnTT2 alleles. WAF,
weeks after flowering. The data represents the means ± SE (n = 3).

Table 2. Oil and Protein Content and FA Composition in T2 Generation of BnTT2 Mutantsa
line fat (%) protein (%) C16:0 (%) C18:0 (%) C18:1 (%) C18:2 (%) C18:3 (%)
CK 40.11 ± 1.23 22.33 ± 0.26 9.547 ± 0.08 4.367 ± 0.30 62.41 ± 0.34 12.89 ± 0.04 10.77 ± 0.16
aacc-4-2 47.01 ± 0.91** 24.52 ± 0.48** 8.950 ± 0.23 3.149 ± 0.06** 60.15 ± 0.00** 14.91 ± 0.00** 12.83 ± 0.29**
aacc-4-6 45.04 ± 0.94** 23.79 ± 0.26** 9.048 ± 0.48 3.534 ± 0.13** 58.72 ± 0.28** 15.29 ± 0.18** 13.39 ± 0.45**
aacc-4-14 47.63 ± 0.8** 22.42 ± 0.33 9.634 ± 0.19 3.269 ± 0.07** 58.97 ± 0.29** 15.41 ± 0.11** 12.70 ± 0.29**
aacc-4-16 46.87 ± 0.2** 22.92 ± 0.12* 9.702 ± 0.11 3.641 ± 0.03** 60.70 ± 0.16** 14.23 ± 0.05** 11.70 ± 0.07**
aacc-27-2 47.13 ± 0.51** 23.96 ± 0.05** 8.898 ± 0.05 3.178 ± 0.07** 59.76 ± 0.00** 14.98 ± 0.02** 13.16 ± 0.04**
aacc-27-4 45.11 ± 0.38** 23.13 ± 0.12** 9.280 ± 0.00 3.675 ± 0.02** 62.34 ± 0.03 13.77 ± 0.05** 10.92 ± 0.05
aacc-27-23 46.32 ± 0.64** 24.12 ± 0.16** 8.972 ± 0.10 3.211 ± 0.08** 61.72 ± 0.03** 14.19 ± 0.02** 11.89 ± 0.08**
a
The data represents the means ± SE (n = 3); the Student’s t-test was used for statistical analysis between the knockout mutant and CK (*, p ≤
0.05; **, p ≤ 0.01).

(Figure S7 and Table S4). Compared with CK, the
downregulated DEGs in BnTT2 mutants were significantly
enriched in the flavonoid biosynthesis pathway according to
the Kyoto Encyclopedia of Genes and Genomes (KEGG)
enrichment analysis (Figure S8 and Table S5). We annotated
the DEGs to the phenylpropanoid and flavonoid biosynthetic
pathway (Table S6) and found all the genes involved in the
two pathways were significantly downregulated at 3 to 5 WAF
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of BnTT2 mutants compared to those of J9712 (Figure 7A,B).
For instance, PAL, C4H, and 4CL were downregulated, and
this agreed with the reduced sinapic acid content in BnTT2
mutants. Besides, CHS, CHI, F3H, F3′H, DFR, LDOX, ANR,
GSTF12, TT12, and AHA10 were significantly downregulated
(Figure 7B). These DEGs are responsible for the reduced PA
biosynthesis and yellow seed color. qRT-PCR data of the
DEGs in the flavonoid biosynthetic pathway were in
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Figure 6. DEGs after BnTT2 mutation. (A) Number of DEGs at each seed developmental stage. (B) Venn diagram of upregulated genes in BnTT2
mutants compared with CK. (C) Venn diagram of downregulated genes in BnTT2 mutants compared with CK. CK is J9712. aacc-4 and aacc-27 are
mutants of BnTT2 alleles. WAF, weeks after flowering.
accordance with the sequencing results throughout the seed
development of yellow- and black-seeded rapeseeds (Figure
S9).
BnTT2 Interacts with BnTT8 and BnTTG1 in Regulat-
ing PA Biosynthesis. As reported in Arabidopsis, AtTT2
forms an MBW complex with AtTT8 and AtTTG1 and
regulates the transcription levels of other genes in the flavonoid
biosynthetic pathway.12 Here, we confirmed that BnTT2
interacts with both BnTT8 and BnTTG1 using Y2H, and
BnTT8 also interacts with BnTTG1, indicating BnTT2,
BnTT8, and BnTTG1 formed an MBW complex (Figure
8A). BiFC analysis also confirmed the interaction among
BnTT2, BnTT8, and BnTTG1 (Figure 8B). On the basis of
the Y2H and BiFC analysis, we found BnTT2 interacts with
BnLBD37/38/39, BnCPC, and BnMYBL2 (Figure 9A,B).
These proteins have been reported as negative regulators of
anthocyanin biosynthesis in Arabidopsis.54−56 We found the
expression levels of BnCPC, BnMYBL2, BnTT2, BnTT8, and
BnTTG1 were downregulated in 3 WAF, 4 WAF, and 5 WAF
developing seeds of BnTT2 mutants compared with those of
J9712. BnLBD37/38/39 was downregulated in 3 WAF seeds
and upregulated in 6 WAF seeds of the BnTT2 mutants
(Figure 10A).
BnTT2 Mutation Affects the Gene Expression in the
FA Biosynthesis Pathway. As mentioned above, the oil
content and FA composition in BnTT2 knockout mutants were
changed compared with those in J9712. We analyzed the key
genes involved in the FA biosynthesis pathway, including
transcription factors LEC1, LEC2, FUS2, and FUS3, genes
involved in the initial steps of the FA biosynthesis (BCCP1,
BCCP2, MOD1, CAC2, and FAB1), and genes encoding other
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key enzymes (FAE1, FAD2, FAD3, FAB2, FatA, and CDS2)
(Table S7). Compared with J9712, the expression of FAD3
and FAB2 was significantly upregulated in 3 WAF seeds;
BCCP1 and LEC1 were upregulated in 6 WAF seeds, while
FAD2 (except for BnaA05g26900D), FAE1, and BCCP2
(except for BnaA01g28580D) were upregulated in 3 WAF
and 6 WAF seeds of the BnTT2 mutants (Figure 10B). The
upregulation of these enzyme genes or transcription factors
could contribute to variations in oil content and FA
composition, which were in accordance with the seed quality
analysis. Taken together, these results suggested that BnTT2
plays an important role in regulating oil content and FA
composition.
■ DISCUSSION
B. napus is the third-largest oil crop in the world and provides
∼55% of plant oil in China.57 Yellow-seeded B. napus has been
preferred by breeders for the improved oil and protein content
compared to black rapeseeds. However, yellow seed
germplasm does not exist in natural B. napus, and the
yellow-seeded B. napus bred from interspecific crosses were
susceptible to environmental changes. Similar to that reported
in Arabidopsis, seed color variation in B. napus has been proven
to be correlated to the expression or mutation of TT genes in
the flavonoid biosynthetic pathway.13 Hitherto, these genes
have been cloned, but their molecular mechanisms in
regulating flavonoid biosynthesis have not been clarified.14
Gene functional analysis was greatly hindered in B. napus due
to the multiple homologues resulting from its duplication
history. CRISPR/Cas9 mediated genome editing is an effective
method to knockout the homologous genes in B. napus, and
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Figure 7. DEGs related to flavonoid and phenylpropanoid pathways. (A) Heatmap of DEGs. (B) Annotation of DEGs in flavonoid and
phenylpropanoid pathways.

Figure 8. Protein interaction among BnTT2, BnTT8, and BnTTG1. (A) Y2H analysis of BnTT2, BnTT8, and BnTTG1 interactions. (B) BiFC
analysis of BnTT2, BnTT8, and BnTTG1 interactions.

many agronomic traits have been obtained by specific gene
mutation, such as BnLIM1 regulated leaf lobe formation,
BnSDG8 regulated early flowering, BnFAD2 regulated oleic
acid content, and BnTT8 regulated PA accumulation.18,39−41
Hence, we adopted CRISPR/Cas9 systems to knockout
BnTT2, aiming to clarify its regulatory role in flavonoid
biosynthesis and create yellow-seeded B. napus for breeding.
BnTT2 Homologues Regulate Flavonoid Accumula-
tion in B. napus. In Arabidopsis, TT2 together with TT8 and
TTG1 forms the MBW complex, which is necessary for
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temporal and spatial regulation of flavonoid biosynthesis in
seeds,9 and T-DNA insertion resulted in Arabidopsis tt2
mutants with a yellowish seed coat. In the present study, we
cloned two BnTT2 homologues and found they could recover
the yellow seed phenotype of Arabidopsis tt2 mutants,
indicating that BnTT2 has a similar function as AtTT2 in
regulating PA accumulation in seeds. The expression pattern of
the BnTT2 homologues during rapeseed development
resembled that of Arabidopsis, which were specifically ex-
pressed in seeds.11
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Figure 9. BnTT2 interacts with negative regulators of anthocyanin
biosynthesis. (A) Y2H analysis of BnTT2, BnCPC, and BnLBD37/
38/39 interactions. (B) BiFC analysis of BnTT2, BnCPC, BnMYBL2,
and BnLBD37/38/39 interactions.
To elaborate the molecular function of BnTT2 in B. napus,
we created the homozygous mutants of BnaA.TT2 and
BnaC.TT2 with genome editing, and the knockout mutants
exhibited a yellow seed color. Histology and chemical analysis
showed that phenolic and flavonoid content were reduced in
BnTT2 mutants, and the palisade layer of the BnTT2 mutant
was thinner than that of CK. This indicated BnTT2 mutation
affected flavonoid biosynthesis in rapeseed, reduced PA
accumulation, and resulted seed coat color variation. Chemical
staining on the developing seeds also revealed that PAs were
not obviously accumulated in the BnTT2 mutants, while the
red pigmentation was visible in CK after 3 WAF. Detailed
profiling of hydroxycinnamic acid derivative and flavonoid
compounds in the BnTT2 mutants revealed that a significant
reduction of isorhamnetins and epicatechins might be the main
reason for the yellow seed phenotype. Isorhamnetins are a
group of flavonols that could form flavonol derivatives,
anthocyanins, and tannins.9 PAs identified in Arabidopsis
seeds are polymers of epicatechin.10 Thus, it is reasonable to
speculate that BnTT2 regulated the biosynthesis of these
chemicals.
BnTT2 Mutation Represses Phenylpropanoid and
Flavonoid Biosynthetic Genes in B. napus. The
annotation of DEGs between developing seeds of the BnTT2
knockout mutant and CK revealed that genes in the flavonoid
biosynthetic pathway were downregulated, including CHS,
CHI, F3H, F3′H, DFR, LDOX, ANR, GSTF12, TT12, and
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AHA10. In Arabidopsis, CHS catalyzes the biosynthesis of
chalcones, and CHI catalyzes chalcones to form flavanones,
which could be catalyzed by F3H to form dihydroflavonols.
Dihydroflavonols are upstream substrates of flavonols.9 Thus,
downregulation of CHS, CHI, F3H, and F3′H in B. napus
would directly affect the biosynthesis of isorhamnetins,
kaempferols, and quercetins. Repression of DFR, LDOX, and
ANR would reduce the synthesis of flavan-3,4-diols,
anthocyanidins, and downstream epicatechins. Moreover,
ANR expression in Arabidopsis is specific in cells with PA
accumulation, which is mainly regulated by TT2.58 Hence, the
BnTT2 knockout mutation could cause the ANR repression.
Finally, expression changes of GSTF12, TT12, and AHA10 in
BnTT2 mutants influenced the flavonoid compartmenta-
tion.59,60 All these gene expressional changes in the BnTT2
mutants agreed with the reduced flavonoid components.
Interestingly, we found the expression of FLS, a key gene
involved in the synthesis of kaempferol and quercetin, was not
significantly changed in the BnTT2 mutant. This might be
helpful to explain why the content of kaempferol and quercetin
was not significantly changed in BnTT2 mutants.
Besides the expressional changes of structural genes and
genes encoding transfer proteins, we found BnTT8 was also
downregulated after BnTT2 mutation. As reported, TT2, TT8,
and TTG1 are the major regulators of PA biosynthesis in
Arabidopsis seeds.8 TT2 and TT8 can activate ANR expression
by binding to its promoter, only when TTG1 is expressed.58
ANR triggers the first enzymatic step of PA biosynthesis.9,61 In
the present study, the protein interaction among BnTT2,
BnTT8, and BnTTG1 was confirmed. Thus, it is reasonable
that downregulation of MBW members blocked the PA
synthesis in rapeseed. We found BnTT2 could interact with
BnLBD37/38/39, BnCPC, and BnMYBL2. In Arabidopsis,
these proteins have been reported as negative regulators of
anthocyanin biosynthesis.54−56 Anthocyanin and PA are two
downstream compounds in the flavonoid biosynthetic path-
way, both using anthocyanidin as a precursor. We found the
expression of BnCPC and BnMYBL2 was downregulated in
BnTT2 mutants, while that of BnLBD37/38/39 was down-
regulated in 3 WAF seeds and upregulated in 6 WAF seeds of
the BnTT2 mutants. Thus, repression of these negative
regulators of anthocyanin biosynthesis in BnTT2 mutants
might contribute to the decreased PA content.
Similar to BnTT8 knockout mutants, we found genes in the
phenylpropanoid pathway, such as PAL, C4H, and 4CL, were
downregulated at early seed developmental stages of BnTT2
mutants.18 These gene encoded enzymes participated in the
initial steps of the lignin biosynthesis pathway. Previously, a
transcriptome comparison between yellow- and black-seeded
rapeseed germplasms also revealed the repression of these
genes in yellow seeds, which was correlated with less lignin in
yellow seeds.3 This agreed with the reduced sinapic acid
content in the BnTT2 mutants, which is a major compound in
the phenylpropanoid pathway. Since the phenylpropanoid
pathway shares common substrates with the flavonoid
biosynthetic pathway, we may suspect that the mutation of
BnTT2 indirectly affected the gene expression in the lignin
biosynthesis.
BnTT2 Mutation Affected FA Composition in
B. napus. In B. napus, improved oil content in yellow-seeded
rapeseed has been correlated with the thinner seed coat and
lower husk proportion.62 We found the palisade layer of
BnTT2 mutants was significantly thinner than that of CK,
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Figure 10. Expression changes of regulators in PA and anthocyanin biosynthesis and genes in the FA biosynthetic pathway. (A) Expression pattern
of regulators in PA and anthocyanin biosynthesis. (B) Heatmap of genes in the FA biosynthetic pathway.
which may greatly contribute to the improved FA content
since positive correlation between seed coat thickness and oil
content has been previously reported in Brassicas.62,63 In
Arabidopsis, tt2 mutation significantly increases the seed FA
content and upregulates the FUS3 expression, which is mostly
expressed during seed filling and promotes oil deposition.64 As
reported, AtTT2 binds to the regulatory region of FUS3,
represses BCCP2, CAC2, MOD1, and KASII expression, and
finally inhibits FA accumulation in seeds.64,65 Here, we found a
BnTT2 mutation increased the oil content in rapeseeds and
BCCP1/2 was upregulated in 3 WAF and 6 WAF seeds,
although the expression of FUS3 was not significantly changed.
BCCP1/2 encodes subunits of acetyl-coenzyme A carboxylase,
which catalyze the first step of FA biosynthesis.66 GC-MS
analysis revealed the FA composition in the BnTT2 mutant
was improved with higher C18:2 and C18:3 and lower C18:0
and C18:1. As two unsaturated fatty acids, C18:2 and C18:3
are essential to the human body and good to human health. As
reported, linoleic acid can prevent or reduce the incidence of
cardiovascular disease. Linolenic acid is a main component
synthesized and metabolized in human cells, which can be
transformed into DHA and EPA.67,68 Wang et al. reported that
AtTT2 could regulate the expression of FAE1, FAD2, and
FAD3 in developing seeds.65 We found FAE1, FAD2 (except
for BnaA05g26900D), and FAD3, which are involved in FA
desaturation and elongation, were upregulated at early seed
developmental stages of the BnTT2 mutant. We found the
homologues of AtLEC1, encoding a key transcription factor in
FA biosynthesis and having unique but overlapped functions
with FUS3, were upregulated in the BnTT2 mutants.69 In
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Arabidopsis, AtTT8 binds to AtLEC1 promoter and represses
AtLEC1 expression and FA accumulation.70 Since TT8 and
TT2 are MBW components and BnTT8 expression was
downregulated in BnTT2 mutants, it is reasonable that the
BnTT2 mutation affected the MBW stability and may further
influence the expression of genes regulated by MBW members.
Hence, we may suspect that the BnTT2 mutation increased the
expression of FA biosynthetic related genes and finally affected
the oil content and FA composition.
■ CONCLUSIONS
Overall, we proved that the BnTT2 mutation repressed the
expression of PA biosynthetic and transport genes as well as
several regulators of anthocyanin biosynthesis. Meanwhile, the
BnTT2 mutation resulted in the upregulation of FA
biosynthetic genes. These results provided a putative function
of BnTT2 in regulating flavonoid content, oil content, and FA
composition in rapeseeds, and the BnTT2 mutant would be
interesting for yellow seed breeding in B. napus.
■
ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c01126.
Figure S1: Accumulation pattern of hydroxycinnamic
acid derivatives in BnTT2 mutants and CK; Figure S2:
Accumulation pattern of quercetins in BnTT2 mutants
and CK; Figure S3: Accumulation pattern of kaempfer-
ols in BnTT2 mutants and CK; Figure S4: Accumulation
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Journal of Agricultural and Food Chemistry
pattern of isorhamnetins in BnTT2 mutants and CK;
Figure S5: Accumulation pattern of epicatechin and
derivatives in BnTT2 mutants and CK; Figure S6:
Personal correlation coefficient analysis of three bio-
logical replicates of RNA-seq data; Figure S7: GO
enrichment of DEGs between BnTT2 mutant and CK;
Figure S8: KEGG enrichment of DEGs between BnTT2
mutant and CK; Figure S9: qPCR analysis of DEGs in
flavonoid biosynthetic pathway (PDF)
Table S1: Primers used in the present study (XLSX)
Table S2: Phenolic compounds identified in rapeseeds
(XLSX)
Table S3: RNA-seq data (FPKM value) of developing
seeds in BnTT2 double mutant and CK (XLSX)
Table S4: GO annotation of DEGs (XLSX)
Table S5: KEGG annotation of DEGs (XLSX)
Table S6: DEGs related to the phenylpropanoid and
flavonoid metabolic processes (XLSX)
Table S7: DEGs related to the FA biosynthetic pathway
(XLSX)
■
AUTHOR INFORMATION
Corresponding Authors
Jinjin Jiang − Jiangsu Provincial Key Laboratory of Crop
Genetics and Physiology, Yangzhou University, Yangzhou,
Jiangsu 225009, China; Phone: +86 514 87997303;
Email: jjjiang@yzu.edu.cn
Youping Wang − Jiangsu Provincial Key Laboratory of Crop
Genetics and Physiology, Yangzhou University, Yangzhou,
Jiangsu 225009, China; Joint International Research
Laboratory of Agriculture and Agri-Product Safety, The
Ministry of Education of China, Yangzhou, Jiangsu 225009,
China; orcid.org/0000-0001-6832-8561; Phone: +86 514
87997303; Email: wangyp@yzu.edu.cn
Authors
Tao Xie − Jiangsu Provincial Key Laboratory of Crop Genetics
and Physiology, Yangzhou University, Yangzhou, Jiangsu
225009, China
Xin Chen − Jiangsu Provincial Key Laboratory of Crop Genetics
and Physiology, Yangzhou University, Yangzhou, Jiangsu
225009, China
Tuli Guo − Jiangsu Provincial Key Laboratory of Crop Genetics
and Physiology, Yangzhou University, Yangzhou, Jiangsu
225009, China
Hao Rong − Jiangsu Provincial Key Laboratory of Crop Genetics
and Physiology, Yangzhou University, Yangzhou, Jiangsu
225009, China
Ziyi Chen − Jiangsu Provincial Key Laboratory of Crop Genetics
and Physiology, Yangzhou University, Yangzhou, Jiangsu
225009, China
Qinfu Sun − Jiangsu Provincial Key Laboratory of Crop Genetics
and Physiology, Yangzhou University, Yangzhou, Jiangsu
225009, China
Jacqueline Batley − School of Biological Sciences, University of
Western Australia, Perth, Western Australia 6009, Australia
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c01126
Author Contributions
Y.W. and J.J. designed the experiments. T.X., X.C., T.G., and
Q.S. performed the experiments. T.X. and H.R. analyzed the
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sequencing data. X.C. and Z.C. collected the seed samples. J.J.
and T.X. wrote the manuscript. J.B. and Y.W. revised the
manuscript. All the authors approved the final manuscript.
Funding
This study was supported by the National Key Research and
Development Program of China (2016YFD0102000,
2018YFE0108000), the National Science and Technology
Major Project (2018ZX08020001), the National Natural
Science Foundations (31771825, 31972963), the Natural
Science Foundation of Jiangsu Province (BK20180101,
BE2018356), the Undergraduate Training Program for
Innovation and Entrepreneurship (X20190712), the Priority
Academic Program Development of Jiangsu Higher Education
Institutions. We sincerely thank Prof. Yaoguang Liu (South
China Agricultural University) for providing the CRISPR/
Cas9 plant expression vectors, Prof. Yongming Zhou
(Huazhong Agricultural University) for providing the
B. napus line J9712, and the Test Center of Yangzhou
University for the HPLC and GC-MS facilities.
Notes
The authors declare no competing financial interest.
■ ABBREVIATIONS USED
TT, Transparent Testa; PA, proanthocyanidins; EBG, early
biosynthesis gene; LBG, later biosynthesis gene; MBW, MYB-
bHLH-WD40; FA, fatty acid; ADT6, Arogenate Dehydratase
6; SAM, shoot apical meristem; WAF, weeks after flowering;
NJ, Neighbor-Joining; CDS, coding sequence; Hyg, hygrom-
ycin B2; sgRNA, small guide RNA; DMACA, p-dimethylami-
nocinnamaldehyde; FAME, fatty acid methyl ester; GC-MS,
gas chromatography−mass spectrometer; Y2H, yeast two-
hybrid; BiFC, bimolecular fluorescence complementation;
YFP, yellow fluorescent protein; NIRS, near-infrared spectros-
copy; km, kaempferols; is, isorhamnetins; qn, quercetins; DEG,
differentially expressed gene; GO, gene ontology; KEGG,
Kyoto Encyclopedia of Genes and Genomes
■
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