Differential accumulation of phenolic compounds and expression of related genes in black- and yellow-seeded Brassica napus.

Developing yellow-seeded Brassica napus (rapeseed) with improved qualities is a major breeding goal. The intermediate and final metabolites of the phenylpropanoid and flavonoid pathways affect not only oil quality but also seed coat colour of B. napus. Here, the accumulation of phenolic compounds was analysed in the seed coats of black-seeded (ZY821) and yellow-seeded (GH06) B. napus. Using toluidine blue O staining and liquid chromatography-mass spectrometry, histochemical and biochemical differences were identified in the accumulation of phenolic compounds between ZY821 and GH06. Two and 13 unique flavonol derivatives were detected in ZY821 and GH06, respectively. Quantitative real-time PCR analysis revealed significant differences between ZY821 and GH06 in the expression of common phenylpropanoid biosynthetic genes (BnPAL and BnC4H), common flavonoid biosynthetic genes (BnTT4 and BnTT6), anthocyanin- and proanthocyandin-specific genes (BnTT3 and BnTT18), proanthocyandin-specific genes (BnTT12, BnTT10, and BnUGT2) and three transcription factor genes (BnTTG1, BnTTG2, and BnTT8) that function in the flavonoid biosynthetic pathway. These data provide insight into pigment accumulation in B. napus, and serve as a useful resource for researchers analysing the formation of seed coat colour and the underlying regulatory mechanisms in B. napus.

Introduction

Brassica napus (2n=38, AACC) is one of the most important oilseed crops worldwide. Increasing oil yield is the foremost objective of B. napus breeding programmes. In B. napus, the oil yield is simultaneously determined by seed yield and oil content. Much effort has focused on identifying a germplasm and hybrid cross with higher seed oil content. A remarkable advancement was the development of yellow-seeded B. napus, which has a significantly thinner seed coat, lower husk proportion, higher oil content, and much lower crude oil pigment content than the black-seeded type (Olsson, 1960; Stringam ; Tang ; Meng ). However, the genetic mechanism underlying seed coat colour formation in B. napus is not well understood, and 1–4 genes are thought to be involved in this process (Somers ; Liu ; Xiao ; Zhang ).

The seed coat colour of B. napus is determined by the content of phenolic compounds cyanidin and procyanidins (Marles and Gruber, 2004; Akhov ; Nesi ). Flavonoids possess diverse chemical structures with a common C6-C3-C6 body. According to the linkage position of the aromatic ring, the saturation level, and the oxidation degree of the C-ring, flavonoids are classified into flavonols, anthocyanins, phlobaphenes, isoflavones, and proanthocyanidins (PAs) (Winkel-Shirley, 2001). Both cyanidin and procyanidins are phenolic compounds belonging to the PA class of flavonoids. In B. napus, the coats of the black-seeded variety have a higher flavonol and procyanidin content than do those of the yellow-seeded variety (Akhov ). In mature B. napus seeds, the coat is usually dark brown to black (Fu ). A previous study reported that the seed coats of B. napus accumulate high amounts of insoluble PAs (in-PAs), which are difficult to quantify. Auger further demonstrated that soluble PA starts to accumulate early in embryogenesis in the micropyle–chalaza area and continues to collect during mid to late embryogenesis in the seed body. The flavonols present in Brassica seeds are kaempferol derivatives, quercetin derivatives, isorhamnetin derivatives, and epicatechin glucoside, and flavonols were also detected in the vegetative parts and inflorescences of Brassica spp. (Romani ; Lin and Harnly, 2009, 2010).

The phenylpropanoid and flavonoid pathway synthesizes a large array of secondary metabolites and has now been well characterized by the identification and functional analysis of several Arabidopsis thaliana mutants (Winkel-Shirley, 2002; Bharti and Khurana, 2003; Gachon ). Research in Arabidopsis has shown that the pathway starts with the conversion of l-phenylalanine into trans-cinnamic acid by phenylalanine ammonia lyase (PAL) (Supplementary Fig. S1 available at JXB online). Trans-cinnamic acid is hydroxylated by cinnamate 4-hydroxylase (C4H) to form p-coumaric acid (Russell, 1971), and 4-coumarate:CoA ligase (4CL) then converts p-coumaric acid into its coenzyme-A ester, which is the precursor of various phenylpropanoid derivatives, including flavonoids, lignins, and isoflavonoids (Winkel-Shirley, 2002). The formation of tetrahydroxychalcone, the precursor of all flavonoids, is catalysed by chalcone synthase (CHS), which is encoded by TRANSPARENT TESTA 4 (TT4) (Burbulis and Winkel-Shirley, 1999). This is the first dedicated step of flavonoid biosynthesis in higher plants, and is thus critical for many important flavonoid-related characteristics, such as seed coat colour, flower colour, and pigmentation of the stem and leaf surface (Hoffmann ; Kasai ). Chalcone is converted into naringenin by chalcone isomerase (CHI), which is encoded by TT5. Subsequently, naringenin is converted into dihydrokaempferol and dihydroquercetin by flavone 3-hydroxylase (F3H; also known as TT6) and flavone 3′-hydroxylase (F3′H; also known as TT7) (Holton and Cornish, 1995; Pelletier and Shirley, 1996; Wisman ; Schoenbohm ; Grotewold, 2006). Then, the products are further metabolized via two biosynthetic branches (Supplementary Fig. S1). On one branch, dihydrokaempferol and dihydroquercetin are converted into leucocyanidin and leucopelargonidin, respectively, by DFR (TT3). Leucoanthocyanidin dioxygenase (LODX) then catalyses the conversion of leucocyanidin and leucopelargonidin into cyanidin and pelargonidin, respectively (Abrahams ). In the last step, cyanidin is converted into epicatechin by anthocyanidin reductase (ANR; also called BAN). On the other branch, dihydrokaempferol and dihydroquercetin are converted by flavonol synthase (FLS) to flavonols, such as kaempferol and quercetin. Quercetin can be converted into isorhamnetin by O-methyl transferase (OMT). These products are believed to be synthesized in the cytoplasm and transported to the vacuole by some transporters, such as TT12 (Marinova ; Zhao and Dixon, 2009). In addition, TT19 (Sun ) was shown to encode a GST (glutathione S-transferase) protein related to flavonoid transport to the vacuole. Kaempferol, quercetin, and isorhamnetin are colourless or pale yellow, while cyanidin and pelargonidin are pink-red in the cytoplasm and pink-purple in the vacuole. Epicatechin units are polymerized to procyanidin oligomers and then oxidized to form insoluble procyanidins by TT10 (Pourcel ; Zhao ). BAN encodes one anthocyanidin reductase and is involved in plant flavonoid biosynthesis (Albert ; Devic ; Xie ; Baudry ). The expression of the underlying biosynthetic genes is regulated by a set of transcription factors and regulatory genes, such as TT2, TT8, TT1, TTG1, and TTG2 (Nesi , 2001; Chiu ).

In this study, cDNAs for phenylpropanoid biosynthetic genes (PAL, C4HA, and C4HB), flavonoid biosynthetic genes (CHS, CHI, F3H, F3′H, DFR, LDOX, LAR, TT12, BAN, and TT10), and regulatory genes (TT2, TTG1, TTG2, and TT8) were isolated from the seeds of B. napus. Differential transcriptional profiles of these genes in the black-seeded coat parent ZY821 and the yellow-seeded coat parent GH06 were analysed by quantitative real-time PCR (qRT-PCR) at seven different seed developmental stages. In parallel, the spatiotemporal accumulation of polyphenol compounds in different development stages of ZY821 and GH06 was analysed using toluidine blue O (TBO). Moreover, the accumulation of flavonols, PAs, and in-PAs in the seed coat was also quantified by HPLC-DAD-MS (high-performance liquid chromatography with diode-array detection and mass spectrometry). The results suggest that the differential expression of flavonoid biosynthetic genes plays a crucial role in determining the seed coat pigmentation patterns of black- and yellow-seeded B. napus.

Materials and methods

Plant materials and growth conditions

Inbred lines of GH06 and ZY821 were used to represent typical yellow- and black-seeded genotypes of B. napus, respectively. ZY821 is a major variety of rapeseed that has been cultivated in China since the 1980s and has a 39.69% oil content, 29.81% protein content, and 19.75% fibre content (Supplementary Fig. S2A, C, E at JXB online). GH06 is a yellow-seeded rape that is used as one of the breeding parents in the Chongqing Rapeseed Technology Research Center (CRTRC) and has a 44.57% oil content, 33.65% protein content, and 14.57% fibre content (Supplementary Fig. S2B, D, F). The plants were grown under normal field conditions at the CRTRC in 2010. Field management essentially followed normal agronomic procedures. Seeds of the two parental lines (i.e. GH06 and ZY821) were harvested and used for total RNA isolation at seven developmental stages, namely 7, 14, 21, 28, 35, 42, and 49 days after pollination (DAP).

Tissue preparation and light microscopy observations

Brassica napus seeds were harvested at 7, 14, 21, 28, 35, 42, and 49 DAP and immediately fixed for 24h at 4 °C in a fixation solution containing 5% acetic acid, 5% formaldehyde, and 50% ethanol. Following fixation, seeds were dehydrated at 60min intervals through a 20% step-graded series of ethanol–water mixtures, ending at 100% ethanol. Then, the seeds were processed at 60min intevals through a 30% step-graded series of ethanol–TBA (tert-butyl alcohol) mixtures, ending at 100% TBA. Seeds were subsequently infiltrated over a 24h period with saturated paraffin–TBA mixtures, and then embedded over a 48h period in paraffin. Blocks were completely polymerized at 4 °C.

Semi-thin (5–8 μm thick) sections were cut with a microtome blade R-35 (Feather Safety Razor Co., Ltd Medical Division, Japan) and viewed under a stereo microscope (SZX12, Olympus, Japan). Three blocks were sectioned for each time point, and a minimum of 60 sections were collected for each block. Sections were stained with TBO and observed with a Nikon Eclipse E600 microscope (Nikon Instruments, Japan).

Reagents and standards

Liquid chromatography–mass spectrometry (LC-MS) solvents were from Fisher Scientific (Rockford, IL, USA); ultra-pure water was obtained using a model MilliQ Plus system from Millipore (Billerica, MA, USA). Flavonoid standards were from Indofine (Somerville, NJ, USA), Sigma-Aldrich (St Louis, MO, USA), and Chromax (Irvine, CA, USA).

Extraction of flavonoids from seed coats

Frozen fresh seed material (100mg fresh weight) was homogenized in 80% methanol (1ml), and the suspension was placed in an ultrasonic bath for 1h. The extract was centrifuged (13 000rpm, 20min) and the supernatant was filtered. The samples were immediately subjected to LC-UV-MS analysis. The in-PA measurement method was followed as previously described by Liang .

High-performance liquid chromatography analysis

Plant extracts were analysed with an Agilent 1100 HPLC system (Hewlett-Packard, Palo Alto, CA, USA) combined with an iron trap mass spectrometer and a Bruker Esquire 3000 (Bruker Daltonics, Bremen Germany). Instrument analyses were carried out using a Grace column (20×250mm, grain diameter 4.6 μm). UV spectra were obtained by scanning from 200nm to 600nm. The mobile phase consisted of (A) water containing 0.1% formic acid (v/v) and (B) acetonitrile, using the following binary gradient: 0–5min, isocratic 95% A and 5% B; 5–10min, isocratic 10% B; 10–17min, isocratic 17% B; 17–25min, isocratic 25% B; 25–30min, isocratic 30% B; 30–55min, isocratic 55% B; 55–65min, isocratic 70% B; 65–70min, isocratic 5% B; and 70–75min, isocratic 95% A and 5% B. The flow rate was 0.8ml min–1 and the temperature of the column was maintained at 25 °C. Negative-ion electrospray ionization (ESI) mass spectra was employed, using an ion source voltage of 3.5kV, a counter current nitrogen flow set at a pressure of 12 psi, and a capillary temperature of 350 °C. Mass spectra were recorded over the range 50–2200 m/z. The Bruker ion-trap mass spectrometer (ITMS) was operated using an ion current control (ICC) of ~10 000 with a maximum acquisition time of 100ms. Tandem mass spectra were obtained in manual mode for targeted masses using an isolation width of 2.0, fragmentation amplitude of 2.2, and threshold set at 6000.

RNA extraction

Total RNA was extracted using the Plant RNA Mini Kit (Watson Biotechnologies, Inc., China). RNA aliquots were treated with RNase-free DNase I (TaKaRa, Dalian, China) to remove any DNA contamination. The quality and concentration of total RNA samples were assessed by agarose gel electrophoresis and spectrophotometry.

Sequence analysis and primer design

Primers for amplifying partial sequences of the genes involved in the phenylpropanoid (PAL and C4H) and flavonoid biosynthesis pathways and of those encoding transcription factors involved in these pathways were designed from conserved nucleotide regions identified by multiple alignments of sequences (Supplementary file 1 at JXB online) from Arabidopsis and B. napus obtained from public databases at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/BLAST/) and the Arabidopsis Biological Resource Center (http://www.arabidopsis.org/). Gene sequences of B. rapa and B. oleracea were obtained from public databases at the Brassica database (BRAD; http://brassicadb.org/brad/) and The Brassica oleracea Genome Database (Bolbase; http://www.ocri-genomics.org/bolbase/). Primer sequences used for the qRT-PCR are listed in Supplementary Table S1.

Quantitative real-time PCR

First-strand cDNA was synthesized from 1 μg of each RNA sample in a 20 μl reaction volume with Oligo dT-Adaptor Primer using the RNA PCR Kit (AMV) Ver. 3.0 (TaKaRa). Brassica napus 26S rRNA was used as the internal control to monitor sample uniformity of initial RNA input and reverse transcription efficiency. The specific primers were F26S (5′-CACAATGATAGGAAGAGCCGAC-3′) and R26S (5′-CAAGGGAACGGGCTTGGCAGAATC-3′). The amplification protocol used for Bn26S was 94 °C for 2min, followed by 21 cycles of amplification (94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1min), and then 72 °C for 10min. Real-time PCR was conducted using SYBR® Premix Ex Taq™ II (Perfect Real Time) (TaKaRa) in a typical 20 μl PCR mixture that included 10 μl of SYBR® Premix Ex Taq™ II, 1–5 μl of template cDNA, and 0.4 μM of each PCR primer. Cycling conditions were 95 °C for 2min, followed by 40 cycles at 95 °C for 20 s, 56 °C for 1min, and 72°C for 30 s, and samples were run on the Stratagene Mx3000P Real-Time PCR System (Stratagene) as described previously (Fricker ).

Results

Spatiotemporal accumulation of polyphenol compounds in the seed coats of black- and yellow-seeded B. napus

To determine the cellular distribution of polyphenol compounds in developing seeds of black- (ZY821) and yellow-seeded (GH06) B. napus, a histochemical analysis of seeds harvested at different developmental stages was performed. TBO staining of transverse sections of developing seeds revealed that seed coats lacked polymeric phenolic compounds at 7 DAP (Fig. 1A, H), and that these compounds began to accumulate in the hilum of seeds at 14 DAP (Fig. 1B, I). At these early time points, staining was similar in ZY821 and GH06. At 21 DAP, ZY821 stained more intensely than GH06, indicating that more polymeric phenolic compounds had accumulated in ZY821 (Fig. 1C, J). The distribution of polymeric phenolic compounds was significantly different at 28 DAP. Whereas these compounds were mainly distributed to the palisade and pigment layers of the seed coat in ZY821 (Fig. 1D), they accumulated in the hilum of GH06 seeds (Fig. 1K). At 35 DAP, polymeric phenolic compound accumulation had increased more significantly in the palisade and pigment layers of ZY821 seed coats than in those of GH06 (Fig. 1E, L). However, the pigment layer of GH06 seeds accumulated more polymeric phenolic compounds near the hilum than did that of ZY821 (Fig. 1L). During late developmental stages (42 and 49 DAP), significant differences in polymeric phenolic compounds were particularly obvious in the palisade and pigment layers of the ZY821 and GH06 seed coats (Fig. 1F, G, M, N).

Fig. 1.

Changes of proanthocyanidins and phenolic compounds during caryopses development of yellow- and black-seeded B. napus. (A–G) Detection and localization of proanthocyanidin and phenolic compounds in seed coat development in ZY821. Bars=20 μm. (H–N) Detection and localization of proanthocyanidin and phenolic compounds in seed coat development in GH06. Bars=20 μm. Black arrows, accumulation site for polymeric phenolic compounds; nu, nucleus; v, vacuole; e, epidermis; s, subepidermis; pal, palisade layer; pig, pigment layer; hi, hilum; em, embryo; co, cotyledon.

These data show that polymeric phenolic compounds localize mainly to the palisade and pigment layers of the seed coat of black-seeded ZY821. In contrast, these compounds were mainly distributed near the hilum of the pigment layer of yellow-seeded GH06 seed coats (Fig. 1). PA and polyphenol compounds gradually increase during seed maturation and show significant differences in the seed coats of ZY821 and GH06. Moreover, the PA and polyphenol compounds were more abundant in the black-seeded than the yellow-seeded variety of B. napus (Fig. 1).

Flavonoid profiling and accumulation in black- and yellow-seeded B. napus

To determine which phenolic compounds accumulated in the seed coats of ZY821 and GH06, seed coats were isolated from seeds of different developmental stages and analysed using LC-UV-MS. Thirty-five phenolic compounds were detected in the flavonoid extracts from both ZY821 and GH06 seed coats. Interestingly, 13 and two compounds were unique to ZY821 and GH06, respectively (Table 1). Three types of flavonol derivatives, including quercetin, isorhamnetin, and kaempferol, and some epicatechin derivatives, were identified. Epicatechin derivatives were only detected in the seed coat extracts of ZY821 (Table 1, Fig. 2).

Table 1.

Main flavonols identified by LC-UV-ESI-MS2 in seed coat flavonoid extracts from B. napus (ZY821 and GH06).

No.	RT (min)a	[M-H]–b	Putative identityc	MS2 [M-H]– (m/z)b	UVd	ZY821	GH06
1	12.3	463	Quercetin-3-O-glucoside	301	64, 324
2	13.3	386	Putative epicatechin derivative	306, 275, 259, 241, 208	ND
3	19	447	Kaempferol-3-O-glucoside	285e	280, 326
4	22	787	Quercetin 3-O-diglucoside-7-O-glucoside	625, 463, 301e	256, 286sh, 358
5	22.6	423	Putative quercetin derivative	342, 301	266, 342
6	23.6	771	Kaempferol-3-O-diglucoside-7-O-glucoside	609, 447, 285	266, 348
7	24.8	801	Isorhametin-dihexoside-O-glucoside	639, 315	254, 284sh, 358
8	25.4	447	Quercetin-deoxyhex	301		ND
9	26.9	993	Quercetin 3-O-sinapoyldiglucoside-7-O-glucoside	831, 625, 301	250, 284sh, 348
10	28.3	577	[DP2]-B2*	425, 407, 289	222, 264sh, 328		ND
11	28.8	977	Kaempferol-sinapoyl-trihexoside	815, 653, 447	222, 270sh, 330
12	29.9	561	Unknown	505, 432, 292, 190	ND
13	30.1	609	Kaempferol-3-O-glucoside-7-O-glucoside	489, 447, 285	ND
14	30.5	289	Epicatechin	245, 205, 179e	280		ND
15	30.7	418	Putative quercetin derivative	301	ND	ND
16	30.7	801	Isorhametin-dihexoside-O-glucoside	639, 315	254, 286sh, 354
17	31.9	477	Isorhametin-3-O-glucoside	315	280, 360
18	32.8	865	[DP3]-	739, 695, 577, 407	280		ND
19	33.9	625	Quercetin-dihexoside	463, 301	280, 316
20	34.7	639	Isorhametin-dihexoside	477, 315	260, 314
21	35.5	545	Putative flavonol derivative	432, 292, 190	262, 310
22	36	609	Sophoraflavonoloside	447,429, 285	ND
23	36.1	765	Putative quercetin derivative	596, 471,301	284, 368
24	37.2	557	Isorhametin-hexoside-sulphate	477, 315	ND
25	37.3	577	[DP2]-B5*	451, 425, 407, 289	270, 338		ND
26	37.9	559	Unknown	432, 292, 190	262, 330		ND
27	38.5	591	Putative quercetin derivative	547, 439, 301	280, 360		ND
28	41.3	495	Putative epicatechin derivative	451, 289	ND		ND
29	41.5	753	Putative epicatechin derivative	529, 457, 429, 289	226, 266, 330		ND
30	44.3	434	Putative epicatechin derivative	289	ND		ND
31	44.8	591	Putative quercetin derivative	547, 439, 395, 301	ND		ND
32	45.6	591	Putative quercetin derivative	559, 439, 367, 301	238, 330
33	48.4	434	Putative epicatechin derivative	289	ND		ND
34	50.4	418	Putative epicatechin derivative	289	ND		ND
35	54.2	418	Putative epicatechin derivative	289	ND		ND

RT, retention time; measured with a C18 Grace column and a corresponding gradient profile (see the Materials and methods).

Obtained with an ion trap mass spectrometer.

Compounds were identified after comparison with standards (*) and references.

Obtained with a UV detector.

Standard identified.

ND, none detected; others were detected.

Fig. 2.

Flavonoid content in ZY821 and GH06 seed coat during different developmental stages by LC-ESI-MS. Values represent the average of three biological replicates. Isorhamnetin derivates, quercetin derivates, kaempferol derivates, and epicatechin derivates (refer to the colour key depicted on the figure).

The concentration of epicatechin, quercetin, isorhametin, and kaempferol derivatives in the coats of ZY821 and GH06 seeds was determined (Fig. 2). It was found that the amount of all four flavonol derivatives increased during seed maturation. The total amount of flavonol derivatives peaked at 42 DAP, and declined thereafter. Epicatechin derivatives, which are reported to be an important type of flavonol in the synthesis of the seed coat of Arabidopsis and Medicago (Marinova ; Zhao and Dixon, 2009), were not detected in the seed coats of GH06 at any stage of seed development.

Additionally, the levels of in-PAs were measured by subjecting the pellet remaining after solvent extraction directly to oxidative cleavage under hot acidic butanol. The seed coat began to accumulate in-PAs at 28 DAP (Fig. 3). A maximum level of 2.0±0.1 μg mg–1 of seed coat (fresh weight) was detected during late seed maturation (49 DAP) (Fig. 3). The seed coats of ZY821 exhibited a much higher in-PA content than did those of GH06 (Fig. 3).

Fig. 3.

In-PA content in ZY821 and GH06 B. napus seed coats during seed development using BuOH-HCl assays. (A) In-PA content of ZY821and GH06 in the extraction pellet after oxidative cleavage in hot acidic butanol. Values represent the average ±SD of three biological replicates. (B) Extraction of pellets of GH06 using hot acidic butanol. (C) Extraction of pellets of ZY821 using hot acidic butanol.

Sequence analysis of phenylpropanoid biosynthesis genes from B. napus

The phenylpropanoid biosynthesis pathway is one of the most important secondary metabolism pathways in higher plants. A variety of key phytochemicals, including lignins, stilbenes, coumarins, salicylates, sinapateesters, and flavonoids, are synthesized in this pathway. In Arabidopsis, several key genes in this pathway have been cloned and their functions identified (Albert ; Nesi , 2001; Baudry ). Homologous genes in B. napus have also been cloned and characterized to some extent (Chen ; Wei ; Xu ; Lu ; Akhov ; Auger ). To study the expression of flavonoid genes in the seed coats of the ZY821 and GH06 varieties of B. napus, expressed sequence tag (EST) sequences of genes involved in this biosynthetic pathway were searched for by blasting the EST sequences of the corresponding genes from Arabidopsis against the NCBI database. These gene sequences cloned from ZY821 and GH06 showed a high level of nucleotide similarity with phenylpropanoid and flavonoid genes from other Brassica species (Supplementary file 1 at JXB online).

The triplication of genes involved in the flavonoid pathway

All current Brassicaceae plants are derived from the same postulated Brassicaceae ancestry by triplicate genomes. In the B. rapa genome, the mesohexaploidy event started between 5 and 9 million years ago (Wang ). The Brassica mesohexaploidy provides a chance to study gene retention in triplicated genomes. In fact, the substantial gene loss is typical after polyploidy formation in eukaryotes. All copies of TT genes were identified in the B. rapa and B. oleracea genome (Supplementary Table S2 at JXB online) and each of the orthologous blocks corresponding to ancestral blocks was identified using collinearity between orthologues on the genome of B. rapa and B. oleracea and the A. thaliana genome (Table 2). Five copies of C4H were identified and distributed in LF, MF1, MF2, and non-genome triplication in the genome of B. rapa and B. oleracea, respectively. However, TT7, TT3, TT12, TT8, TTG1, and TT2 were only identified in one copy in the genome of B. rapa and B. oleracea, respectively. All copies per gene were conserved based on alignment of the DNA sequence (Suppplementary file 1).

Table 2.

The different copies of TT genes in the B. rapa and B. oleracea genome.

Gene	AGI ID	Triplication block	B. rapa					B. oleracea
			LFa	MF1b	MF2c	Non-genome triplicationd		LF	MF1	MF2	Non-genome triplication
PAL	AT2G37040	J	Bra005221	Bra017210				Bol025522	Bol037689
C4H	AT2G30490		Bra018311	Bra021636	Bra022802	Bra022803	Bra021637	Bol006704	Bol004610	Bol033349	Bol033347	Bol004608
TT4	AT5G13930	R	Bra008792	Bra023441	Bra006224	Bra020688	Bra036307	Bol043396	Bol004244	Bol034259
TT5	AT3G55120	N	Bra007142		Bra003209	Bra007145	Bra017728	Bol044343		Bol008652	Bol018696	Bol044344
TT6	AT3G51240	N	Bra036828		Bra012862			Bol010585		Bol002277
TT7	AT5G07990	R	Bra009312					Bol043829
TT3	AT5G42800		Bra027457					Bol035269
TT18	AT4G22880	U	Bra013652	Bra019350				Bol014986	Bol042059
TT12	AT3G59030	N			Bra003361					Bol023767
TT10	AT5G48100	V	Bra037510	Bra020720					Bol033117
UGT2	AT4G01070	O	Bra037386	Bra000942	Bra008540	Bra008535		Bol011466	Bol010781	Bol040647	Bol040645
TT8	AT4G09820	P	Bra037887					Bol004077
TTG1	AT5G24520	Q	Bra009770					Bol022420
TTG2	AT2G37260	J	Bra005210		Bra023112			Bol025535		Bol005555
TT2	AT5G35550	S			Bra035532					Bol014029

LF indicates the least fractionated blocks.

MF1 indicates the medium fractionated blocks.

MF2 indicated the most fractionated blocks.

Non-genome triplication indicates that these copies are not triplicated genome segments.

The expression of genes involved in the biosynthesis of phenolic compounds regulates flavonoid synthesis

BnPAL, which encodes a protein that connects primary and secondary metabolism and transfers phenylalanine to trans-cinnamic acid, a precursor of other phenolic compounds (Ohl ), is a key gene in phenylpropanoid synthesis in B. napus. It was found that the expression level of BnPAL was much higher in ZY821 than in GH06, and that expression peaked at 42 and 49 DAP, respectively (Fig. 4A). BnTT4 (BnCHS), which converts three molecules of malonyl-CoA and one molecule of 4-coumaroyl-CoA into nargingenin chalcone, catalyses the first committed step of the flavonoid biosynthesis pathway (Li ; Shirley, 1998). All flavonoids and isoflavonoids are derived from the nargingenin chalcone generated by this first enzyme. During both early (7, 14, and 21 DAP) and late (42 and 49 DAP) development stages, BnTT4 was expressed at the same level in the seed coats of ZY821 and GH06. Only during the middle development stages (28 and 35 DAP) was the expression of BnTT4 significantly higher in ZY821 than in GH06 (Fig. 4D, E). BnTT5 (BnCHI) stereospecifically directs and greatly accelerates the spontaneous cyclization of chalcones to form the flavonoid core. Since the activity of this enzyme is closely related to BnCHS activity, the gene encoding BnCHS exhibited an expression pattern similar to that of BnTT4 and BnTT5. However, the expression of BnTT5 peaked after that of BnTT4 (Fig. 4F). Moreover, BnTT6, BnTT7, BnTT3, and BnTT18 had expression patterns and levels similar to those of BnTT4 and BnTT5, but they peaked at different development stages. Whereas BnTT5, BnTT6, and BnTT7 expression peaked at 35 DAP (Fig. 4G, H), that of BnTT3 and BnTT18 peaked only at 42 DAP (Fig. 4I, J). Furthermore, these genes were expressed at higher levels in ZY821 than in GH06.

Fig. 4.

Analysis of the expression levels of genes involved in flavonoid biosynthesis during seed coat development of ZY821 and GH06 by qRT-PCR (A–O). Seed coat development was divided into seven developmental stages (7, 14, 21, 28, 35, 42, and 49 DAP). Values represent the average ±SD of three biological replicates with three technical replicates of each development stage. Relative gene expression levels were normalized according to the expression values of 7 in ZY821.

In addition, ZY821 and GH06 contain two copies of BnTT12, which peak at 28 DAP, and then decline. BnTT12 was expressed more strongly in ZY821 than in GH06 (Fig. 4K, L). TT10 encodes an enzyme that oxidizes procyanidins to yield PAs in the Arabidopsis seed coat (Pourcel ). In addition to its role in flavonoid biosynthesis, TT10 also regulates lignin biosynthesis (Liang ). The expression of BnTT10 peaks at 42 DAP (Fig. 4M). However, there was no significant difference in the expression of BnTT10 between ZY821 and GH06, except at 28 and 42 DAP. BnUGT2 encodes an epicatechin-specific glucosyltransferase that is involved in the production of epicatechin 3′-O-glucoside in the seed coat (Pang ). In both ZY821 and GH06 seed coats, BnUGT2 expression peaked at 35 DAP (Fig. 4N), suggesting that most anthocyanin precursors are synthesized at this stage. The visible function of anthocyanin is to promote colour formation in plant organs (Sparvoli ), which supports the hypothesis that anthocyanin accumulation plays an important role in the formation of seed coat colour in B. napus

The flavonoid pathway in Arabidopsis is known to be regulated not only by structural genes, but also by regulatory genes, such as TT1, TTG1, TT2, TTG2, and TT8 (Walker ; Nesi , 2001; Johnson ; Sagasser ). In this study, BnTTG1 and BnTT2 were found to possess a similar expression pattern and showed the highest levels of expression at 35 DAP in the seed coat of ZY821. However, the expression of BnTT2 peaked sooner in the seed coat of GH06 (Fig. 4P, Q). The expression of BnTT8 and BnTTG2 was enhanced at the early stages of seed development (21 and 21 DAP), decreased during the middle stages (35 and 28 DAP), and increased again at the late stages (49 and 42 DAP) (Fig. 4O, R). During most development stages, the expression of BnTTG1, BnTT8, and BnTTG2 was stronger in ZY821 than in GH06, and the expression of BnTT2 was higher in GH06 than in ZY821 at 21 and 28 DAP.

Discussion

Potential mechanisms underlying the histological and biochemical differences in seed coat colour in B. napus

Flavonoids are secondary metabolites that accumulate in vascular plants and, to a lesser extent, in mosses. They promote seed and pollen dispersal by contributing to colour formation in fruits and flowers. Previously, researchers showed that epicatechin derivatives (Marinova ; Zhao and Dixon, 2009) and a PA monomer (Holton and Cornish, 1995; Grotewold, 2006) are important flavonols in the synthesis of the seed coat of Arabidopsis and Medicago.

Recently, much research has focused on deciphering the genetic mechanisms underlying colour formation in B. napus seeds, and great progress has been made in this regard. However, there was no consensus on the inheritance model of the seed coat colour trait in B. napus. Theander reported that the main factor contributing to seed colour is soluble anthocyanin. Subsequent research showed that the colour of immature seeds is determined by anthocyanidin, polyphenols, and flavonoid, but that the colour of mature seeds is controlled by melanin (Wang and Liu, 1996). PA accumulation contributes to the differences in B. juncea (Sharma and Dixon, 2005). In Arabidopsis, PAs are synthesized as colourless polymers during the early developmental stages, and are then oxidized into brown complexes that cross-link within the cell during seed maturation. These modifications account for the typical brown colour of the Arabidopsis wild-type testa (Stafford, 1988; Kitamura ; Pourcel ). In B. napus, PAs accumulated strongly in the black-seeded line, but weakly in the yellow-seeded line. The flavonol content, however, did not differ much between these two lines (Akhov ). In agreement with these findings, Marles and Gruber (2004) found that PA was deposited over the entire seed coat of the dark-seeded germplasm and in patches in some yellow-seeded germplasms. However, the correlation between total flavonoid content and seed colour was comparatively weak (Lipsa ).

In this study, it was shown that flavonoid and its derivatives, including epicatechin, quercetin, isorhametin, and kaempferol, mainly accumulated in the black-seeded variety. In addition, 35 phenolic compounds were detected in the flavonoid extracts from both ZY821 and GH06 seed coats according to the retention time analysis of LC-UV-MS. Interestingly, 13 and two compounds could be detected only in ZY821 and GH06, respectively (Table 1). Moreover, epicatechin derivatives were not detected in the seed coat extracts of GH06 (Table 1, Fig. 2). Large differences were also found in the polyphenol composition of seed coats from the black- and yellow-seeded lines. The cytological analysis showed that polymeric phenol compounds began to accumulate in the hilum of seeds during the early developmental stages, and were mainly confined to the palisade and pigment layers of the seed coat during the middle to late stages of development. Moreover, PA and polyphenol content were higher in ZY821 than in GH06 seed coats (Fig. 1). In addition, the in-PAs were not detected during the early developmental stages of B. napus, but accumulated during the middle to late stages, and the in-PA content of the black-seeded variety was much higher than that of the yellow-seeded line. It was also found that in-PAs accumulated in the seed coats of GH06, but did not vary much during development (Fig. 3A). The differences in in-PA content may be due to differences in the synthesis of epicatechin, as epicatechin is thought to be the major precursor of in-PAs in Arabidopsis seed coats (Lepiniec ). These results suggest that the differences in seed coat colour observed in B. napus are largely attributable to differences in the content of epicatechin and its derivatives and of in-PAs.

The expression of genes involved in the flavonoid biosynthesis pathway in the seed coats of black- and yellow-seeded B. napus

Several key genes involved in flavonoid biosynthesis have been cloned and their functions identified by analysing various mutants in Arabidopsis (Albert ; Nesi , 2001; Baudry ; Arsovski ; Le ) and other species (Pang , 2008; Gallardo ; Jiang and Deyholos, 2010). Homologous genes in the B. napus pathway have also been cloned and characterized (Chen ; Wei ; Xu ; Lu ; Akhov ; Auger ). In B. napus, BnPAL and BnC4H are the key enzymes of the phenylpropanoid pathway, which synthesizes primary and secondary metabolites and converts phenylalanine into trans-cinnamic acid, which is a precursor of other phenolic compounds (Ohl ). Blount and co-workers reported that PAL activity was reduced by the antisense-mediated silencing of C4H, and concluded that C4H was involved in regulating the expression of PAL (Blount ). In a previous study, using three pairs of near isogenic lines of B. napus, it was found that the enzyme activity of BnPAL was much higher in the black-seeded lines than in the yellow-seeded lines, and differed significantly among the lines. Moreover, it was found that cinnamic acid accumulated robustly and p-coumaric acid weakly in the young seed coats of yellow-seeded lines, but conversely during the middle to late stages of seed development (Liang and Li, 2004).

In this study, the expression level of BnPAL in ZY821 was found to be much higher than in GH06, and expression in ZY821 was strongest at 42 DAP, which was also 7 d earlier than in GH06 (Fig. 4A). These results are consistent with the PAL activity and expression in B. napus, and confirm that the activity of BnPAL is closely related to the expression pattern of BnPAL. Furthermore, the expression level of BnC4H was much higher in ZY821 than in GH06 during the middle developmental stages, and the expression of BnC4H peaked 7 d before that of BnPAL (Fig. 4A–C). This result supports the hypothesis that flux into the phenylpropanoid pathway is controlled, at least in part, by feedback regulation of PAL sensed through the production of cinnamic acid (Blount ). In addition, it also confirmed the previous results that showed that cinnamic acid accumulated robustly and p-coumaric acid weakly in the young seed coats of yellow-seeded lines, but conversely during the middle to late stages of seed development (Liang and Li, 2004).

UGT2 encodes an epicatechin-specific glucosyltransferase involved in the production of epicatechin 3′-O-glucoside in the seed coat (Pang ). TT12 is an ABC transporter that is responsible for epicatechin 3′-O-glucoside and anthocyanin cyanidin-3-O-glucoside uptake in Medicago and Arabidopsis, respectively (Debeaujon ; Marinova ; Chai ; Zhao and Dixon, 2009). Moreover, ANR converts cyanidin into epicatechin. Epicatechin is one of the precursors for biosynthesis of PA (Albert ; Devic ; Xie ; Baudry ). In this study, the expression of BnUGT2 and BnTT12 was found to be higher in ZY821 than in GH06; however, both genes were expressed throughout the development of GH06 seed coats (Fig. 4N, K, L). This result also indicates that epicatechin, which was not detected at any stage of seed coat development in GH06, contributes to seed coat colour formation (Fig. 2).

It was shown here that the expression of genes involved in flavonoid biosynthesis varied throughout seed development, and that the genes had different expression patterns in the black- and yellow-seeded lines. The genes were classified according to the time at which their expression peaked. The expression of the first group of genes, including BnTT4, BnTT8, and BnTT12, peaked at the early stages of seed development in black-seeded ZY821. Another group of genes (BnC4H, BnTT5, BnTT6, BnTT7, BnUGT2 BnPAL, BnTT3, BnTT18, and BnTT10) showed the highest expression levels at 35 and 42 DAP, respectively. Thus, these genes could play important roles in flavonoid biosynthesis during the middle to late stages of seed coat development (Fig. 4). In addition, BnTT6, BnTT7, BnTT3, and BnTT18 exhibited similar expression patterns between the black- and yellow-seeded varieties of B. napus. In contrast, the expression of BnTT7, BnTT18, and BnTT10 peaked earlier in GH06 than in ZY821 (Fig. 4H, J, M). Furthermore, there were no significant differences in the expression level of BnPAL, BnC4H, BnTT4, and BnTT6 between the different stages of seed development in GH06. The differences in the expression patterns of genes revealed genetic differences in the synthesis of pigments between the black- and yellow-seeded varieties of B. napus. These results suggested that BnPAL, BnC4H, BnTT4, and BnTT6 are the key genes underlying differences in seed coat pigmentation in B. napus, and that the expression of these genes may be inhibited in the yellow-seeded line of B. napus.

Regulation mechanisms underlying the biosynthesis of seed coat pigment in B. napus

The inheritance of seed coat colour in B. napus is quite complicated, and the regulatory mechanisms underlying the biosynthesis of the relevant genes were hitherto unknown. In this study, the expression level of genes involved in pigment biosynthesis was found to vary greatly between the yellow- and black-seeded lines of B. napus. However, all of these genes were also expressed in the yellow-seeded lines. These data indicate that the absence of pigment synthesis in the yellow-seeded line of B. napus involves the down-regulation, but not the complete inactivation, of several key genes. In addition, the seed coat colour of B. napus was affected by environmental factors, such as temperature and light (Deynze ). Future research should aim to decipher the mechanisms that down-regulate the expression of genes involved in pigment synthesis in yellow-seeded B. napus.

For the reasons mentioned above, this study has almost certainly analysed the metabolic networks underlying pigment formation, investigated the laws of variation in the accumulation of flavonol and phenolic compounds using histochemical and LC-UV-MS methods, and monitored the differential expression of the main structural genes and transcription factors involved in the flavonoid biosynthesis pathway (Fig. 5). The findings illuminate the mechanisms underlying colour formation in B. napus seed coats.

Fig. 5.

Proposed model for the molecular mechanisms underlying seed coat colour in B. napus. BnPAL, l-phenylalanine ammonia-lyase; BnC4H, cinnamate 4-hydroxylase; Bn4CL, 4-coumarate: CoA ligase; BnCHS, chalcone synthase; BnCHI, chalcone isomerase; BnF3H, flavanone 3-hydroxylase; BnDFR, dihydroflavonol reductase; BnANS, anthocyanidin synthase; BnANR, anthocyanidin reductase.

Supplementary data

Supplementary data are available at JXB online.

Figure S1. The pathway of flavonoid biosynthesis, modification, and transport in Arabidopsis.

Figure S2. The phenotype of GH06 and ZY821.

Table S1. Specific primers used for qRT-PCR.

Table S2. The distribution of TT genes in the genome of B. rapa and B. oleracea.

Supplementary file 1. Multiple alignments of sequences obtained from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/BLAST/) and the Arabidopsis Biological Resource Center (http://www.arabidopsis.org/) database.