Identification and Characterization of Major Constituents in Different

Article

Cite This: J. Agric. Food Chem. 2019, 67, 11053−11065 pubs.acs.org/JAFC

Identiﬁcation and Characterization of Major Constituents in Diﬀerent-Colored Rapeseed Petals by UPLC−HESI-MS/MS † ‡ ∥ † ‡ ∥ † ‡ ∥ † ‡ † ‡ Neng-Wen Yin, , , Shu-Xian Wang, , , Le-Dong Jia, , , Mei-Chen Zhu, , Jing Yang, , § † ‡ † ‡ † ‡ † ‡ † ‡ Bao-Jin Zhou, Jia-Ming Yin, , Kun Lu, , Rui Wang, , Jia-Na Li,*, , and Cun-Min Qu*, , † ‡ Chongqing Engineering Research Center for Rapeseed, College of Agronomy and Biotechnology, and Academy of Agricultural Sciences, Southwest University, Chongqing 400716, China § Deepxomics-Shenzhen, Guangdong 518083, China

*S Supporting Information

ABSTRACT: Oilseed rape (Brassica napus L.) is the second highest yielding oil crop worldwide. In addition to being used as an edible oil and a feed for livestock, rapeseed has high ornamental value. In this study, we identified and characterized the main floral major constituents, including phenolic acids and flavonoids components, in rapeseed accessions with different-colored petals. A total of 144 constituents were identified using ultrahigh-performance liquid chromatography−HESI-mass spectrometry (UPLC−HESI-MS/MS), 57 of which were confirmed and quantified using known standards and mainly contained phenolic acids, flavonoids, and glucosinolates compounds. Most of the epicatechin, quercetin, and isorhamnetin derivates were found in red and pink petals of B. napus, while kaempferol derivates were in yellow and pale white petals. Moreover, petal-specific compounds, including a putative hydroxycinnamic acid derivative, sinapoyl malate, 1-O-sinapoyl-β-D-glucose, feruloyl glucose, naringenin-7-O-glucoside, cyanidin-3-glucoside, cyanidin-3,5-di-O-glucoside, petunidin-3-O-β-glucopyranoside, isorhamnetin-3- O-glucoside, kaempferol-3-O-glucoside-7-O-glucoside, quercetin-3,4′-O-di-β-glucopyranoside, quercetin-3-O-glucoside, and delphinidin-3-O-glucoside, might contribute to a variety of petal colors in B. napus. In addition, bound phenolics were tentatively identified and contained three abundant compounds (p-coumaric acid, ferulic acid, and 8-O-4′-diferulic acid). These results provide insight into the molecular mechanisms underlying petal color and suggest strategies for breeding rapeseed with a specific petal color in the future. KEYWORDS: Brassica napus L., phenolic acids, flavonoids, petal color, UPLC−HESI-MS/MS

■ INTRODUCTION nins and proanthocyanidins).7 Numerous compounds are found ff In nature, colors are important cues that signal warnings (e.g., in di erent petals, such as the diacylated delphinidin (gentiodelphin) in gentian flowers, cyanidin 3-(6′-malonyl or predator deterrence) or rewards (e.g., pollinator attraction). 8 ff fl 3′,6′-dimalonyl)glucoside in chrysanthemum petals, lutein in Di erences in ower color are a conspicuous and prized feature fl 6 fl fl 9 of ornamental plants. Rapeseed (Brassica napus L., AACC, 2n = marigold owers, and avones and avonols in carnation and 8 fi 38) is an allopolyploid species derived from hybridization of the chrysanthemum, respectively. However, the speci c pigments diploid Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, and genes underlying petal color in B. napus are unclear. 1 fl Downloaded via UNIV OF NEW ENGLAND on October 23, 2019 at 01:02:36 (UTC). 2n = 18). It is not only an economically important source of Numerous avonoids and hydroxycinnamic acid derivatives have been characterized in Brassica vegetables, including 30 vegetable oil, biodiesel, and protein-rich meal for animal feed 10−12 2 fl fi See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. worldwide but also has high ornamental value. For instance, avonoids with de ned chemical structures. Correspond- fl cultivars of rapeseed exhibit petals that range in color from ingly, 130 avonol glycosides and 50 hydroxycinnamic acids have been putatively identified in over 30 Brassica vegeta- yellow and pale white to orange, golden-yellow, and milky white. 13−19 Novel flower color variations (e.g., pink, red, and purple) have bles, and about 50 anthocyanins are known among the ff 20−23 24 fi been produced recently in B. napus by genetic transformation di erent-colored Brassica vegetables. Lin et al. identi ed fl techniques.3,4 However, the mechanisms regulating petal color 67 anthocyanins, 102 avonol glycosides, and 40 hydroxycin- of oilseeds are poorly understood. namic acid derivatives in red mustard greens (Brassica juncea Three biosynthetic pathways (carotenoid, anthocyanin, and variety CosS). However, the constituents of different-colored B. betalain biosynthesis) produce secondary metabolites that napus petals have not been characterized. contribute to the attractive natural display of flower colors.5,6 Ultrahigh-performance liquid chromatography−HESI-mass Yellow and red are attributed to the carotenoid and betalain spectrometry (UPLC−HESI-MS/MS) is a precise and sensitive pathways, whereas the anthocyanin pathway produces a diverse technique that has been widely used to identify flavonoids and range of colors, including orange, yellow, blue, and red.6 The anthocyanin pathway is the best understood in terms of its Received: August 10, 2019 structure and regulation and is involved in the biosynthesis of Revised: September 17, 2019 various flavonoid derivatives, including both colorless com- Accepted: September 17, 2019 pounds (e.g., flavonols) and colored pigments (e.g., anthocya- Published: September 17, 2019

© 2019 American Chemical Society 11053 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article hydroxycinnamic acid derivatives in Brassica seeds, including the and stored at −80 °C. The precipitate was extracted again using the main flavonoids (kaempferol, isorhamnetin, quercetin, and same conditions in a sonicator. The supernatants from both extractions were pooled to obtain crude extracts, which were filtered (0.22-μm epicatechin/catechin and their derivatives) and the main fi − hydroxycinnamic acids (sinapic, ferulic, and p-coumaric nylon lter) and analyzed by UPLC HESI-MS/MS. 12,24−28 Bound Phenolics Extraction. Extraction of bound phenolics was acids). These constituents were used as reference performed according to the method described by Krygier et al.29 with compounds in this study. To comprehensively assess the fi ff ff slight modi cation. The residue after removal of the crude extract was di erences between di erent-colored rapeseed petals, we hydrolyzed for bound phenolics by resuspending in 2 mL of 4 M NaOH characterized the multiple constituents from typical yellow and vortexing for 2 h at 30 °C in order to liberate the bound phenolics. and white petals and from novel lines with red and pink petals. Subsequently, the samples were acidified to pH 2.0 with 6 M HCl, and To our knowledge, this is the first comparative study on the the lipids were removed with 2 mL of hexanes three separate times by differences in constituents among different-colored rapeseed vortex. Finally, extraction of bound phenolics was carried out three − petals. times with diethyl ether ethyl acetate (1:1, v/v), and the mixtures were evaporated to dryness and dissolved in 500 μL of 70% methanol and fi μ fi ■ MATERIALS AND METHODS Phenex-NY syringe ltered (0.22- m nylon lter) prior to further analysis. Plant Materials and Sample Collection. Four rapeseed varieties UPLC−HESI-MS/MS Analysis. Identities of phenolic compounds ff with di erent-colored petals (Figure 1), yellow, pale white, pink, and were determined using a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific) controlled by Xcalibur 3.1 software and an Aquity UPLC BEH C18 column (2.1 i.d. × 150 mm, 1.7 μm particle size) (Waters, Ireland) protected by a guard column (Aquity UPLC BEH C18 1.7 μm VanGuard Pre-Column 2.1 × 5 mm, Waters). The mobile phase included solutions A (0.1%, v/v, formic acid in H2O) and B (0.1%, v/v, formic acid in acetonitrile) with the following gradient: 0−2 min, 5−10% B; 2−18 min, 10−95% B; 18−20 min, 95% B; 20− 20.5 min, 95−5% B; and 20.5−24 min, 5% B. The flow rate was set at 0.300 mL/min, with a sample injection volume of 10 μL and column temperature of 30 °C. Mass spectrometry was performed on a Thermo ScientificQ- Exactive System equipped with a S-Lens ionizer source (Thermo Scientific) in negative mode, and data were acquired in the mass range from m/z 100 to 1500. Operation parameters were as follows: spray voltage, 3.5 kV; sheath gas, 35 (arbitrary units); auxiliary gas, 10 (arbitrary units); sweep gas, 0 (arbitrary units); and capillary temperature, 350 °C. All raw UPLC−HESI-MS/MS data were analyzed using Xcalibur 3.1 software. Integrated peak areas from the inbuilt Analyst Quantitation Wizard were manually corrected. Compounds were identified by comparing their retention times and accurate MS and MS/MS spectral data with commercial standards and information previously reported in − the literature.12,24 28 Compounds were quantified relative to the corresponding external standards using calibration curves with eight points generated from spiked samples covering a range from 0.001 to 2 mg L−1. The stock solution of standards was used to prepare a mixed standard solution with a concentration of 5 mg L−1, and then the prepared mixture was diluted to eight concentration gradients (0.001, Figure 1. Flower color in B. napus lines: (A) pale white, (B) yellow, (C) − 0.005, 0.01, 0.05, 0.20, 0.50, 1.0, and 2.0 mg L 1) for the calibration pink, and (D) red. Bars: 1 cm. curve construction. Statistical Analysis. All analyses were conducted in triplicate, and ± red, were grown in the 2016−2017 growing seasons under normal field data were expressed as the mean standard deviation (SD) of three conditions in Beibei (Chongqing, China). Each line was planted in technical replicates. Statistical evaluation was performed using SPSS ’ three rows with 10 plants per row. All the flower petals among flowers in 15.0 (one-way ANOVA with Tukey s assay; SPSS Inc., Chicago, IL), ff fi full bloom were picked and weighed individually, immediately frozen in and di erences were considered signi cant at the p < 0.05 level. liquid nitrogen, and stored at −80 °C for further analysis. Reagents and Standards. HPLC-grade acetonitrile, methanol, ■ RESULTS and formic acid (Sigma, St. Louis, MO) were used for UPLC−HESI- ff UPLC−HESI-MS/MS Analysis of Constituents in Differ- MS/MS analysis. Standards of ca eic acid, ferulic acid, epicatechin B. napus fl (ep), kaempferol (km), isorhamnetin (is), p-coumaric acid, quercetin ent-Colored Petals of . B. napus owers are normally (qn), sinapic acid, and sinigrin (HPLC grade, purity >99%) were yellow or pale white (Figure 1), but may be other colors, such as purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). pink and red (Figure 1). To assess the relationship between petal Ultrapure water was obtained from a Milli-Q water purification system color and multiple constituents, we analyzed crude petal extracts (Millipore, Bedford, MA). Stock solutions of standards were dissolved from each line using UPLC−HESI-MS/MS in negative mode. − ° in 80% (v/v) methanol and stored at 20 C. Base peak chromatograms were identified from pale white, Extraction of Metabolites from Petals. Flower samples (200 yellow, pink, and red rapeseed petals, respectively (Figure 2). mg) freeze-dried and stored at −80 °C were ground by hand into a fine powder using a mortar and pestle, aliquoted into 2 mL plastic We detected 144 readily distinguished constituents among the different rapeseed petals on the basis of their retention times, microtubes (Eppendorf), homogenized in an aqueous solution of 2 formic acid (0.1%, v/v) and aqueous methanol (1 mL; 80%, v/v), and exacting mass, and MS fragmentation characteristics. We also extracted in a sonicator (KQ-100E, Kunshan) at 4 °C for 1 h. The observed the elution order tendency of the compounds supernatant was collected after centrifugation (9000g, 30 min at 4 °C) according to their chemical structure class (Figure 3). The

11054 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article

Figure 2. Base peak chromatograms in negative mode for diﬀerent-colored petals of B. napus.

Figure 3. Distribution of constituents identiﬁed in B. napus petals based on their retention time (RT) and m/z.

144 constituents could be categorized into five main groups: 15 standard compounds are displayed in Figure 4. A total of 57 phenolic acids, 28 flavonoids, 16 glucosinolates, 16 fatty acyls compounds could be unambiguously identified and quantified as and lipids, and 69 other polar compounds. Their corresponding phenolic acids, flavonoids, and glucosinolates. Of these, three retention times (RTs), fragment patterns, and ion masses are identifications were supported using commercially available shown in Table S1 of the Supporting Information (SI). ff fl To investigate the relationship between petal color and ca eic acid; 26 avonoids were determined using epicatechin, flavonoid composition, the contents of these phenolic isorhamnetin, kaempferol, and quercetin; and 2, 3, 7, and 16 compounds in different B. napus petals were quantified using compounds were confirmed using ferulic acid, p-coumaric acid, standard compounds. RT and fragmentation information for the sinapic acid, and sinigrin, respectively (Table 1).

11055 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article

Figure 4. Base peak chromatogram (A) and MS/MS fragmentation (B) scheme proposed for standard compounds.

Identification of Phenolic Acids. On the basis of the base compounds (putative hydroxycinnamic acide derivative and peak chromatograms of phenolic acids standards (caffeic acid, disinapoylgentiobiose) were present in relatively high levels in ferulic acid, p-coumaric acid, and sinapic acid) in negative mode the different B. napus petals, but a putative hydroxycinnamic acid (Figure 4A,B), we quantified 15 peaks with different contents in derivative was only present in trace amounts in red petals (Table different-colored petals (Figure 5A, Table 1). Of these, two 1). In addition, two isomers of 1-O-sinapoyl-β-D-glucose (RT =

11056 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 a Chemistry Food and Agricultural of Journal Table 1. Quantiﬁed Constituents That Were Diﬀerentially Accumulated in the Pale White, Yellow, Pink and Red Petals of B. napus

RT code average m/z adduct ion (min) MS2 compd name pale white yellow pink red standard 103 353.0887 [M − H]− 4.56 191.05, 179.03, 135.04, 3-caffeoylquinic acid 0.174 ± 0.019 0.100 ± 0.011 0.263 ± 0.008 0.127 ± 0.017 caffeic acid 353.09, 161.02, 192.05 111 341.0883 [M − H]− 4.95 179.03, 135.04, 114.05, caffeic acid hexoside 0.311 ± 0.050 0.165 ± 0.010 0.140 ± 0.009 0.043 ± 0.019 caffeic acid 226.12, 89.02, 59.01 130 353.0887 [M − H]− 5.59 191.05, 130.06, trans-5-O-caffeoylquinic acid 0.024 ± 0.002 0.015 ± 0.001 0.008 ± 0.001 0.004 ± 0.000 caffeic acid 192.05,298.50, 161.02, 267.88 116 609.1478 [M − H]− 4.90 447.09, 285.04, 284.03, luteolin-3′,7-di-O-glucoside 0.274 ± 0.022 0.146 ± 0.009 0.154 ± 0.002 0.047 ± 0.018 epicatechin 283.03, 446.08, 96.95 − − ± ± ± ± 126 465.1040 [M 2H + H2O] 5.40 285.04, 241.05, 125.02, cyanidin-3-glucoside 0.008 0.001 c 0.057 0.009 c 0.564 0.032 b 2.052 0.020 a epicatechin 149.02, 329.09, 199.04 169 609.1489 [M − 2H]− 6.56 283.03, 609.14, 446.08, cyanidin-3,5-di-O-glucoside 0.047 ± 0.003 b 0.042 ± 0.003 b 0.075 ± 0.005 b 1.344 ± 0.024 a epicatechin 285.04, 120.01, 255.03 174 609.1489 [M − 2H]− 6.95 284.03, 285.04, 609.15, cyanidin-3-O-(2″-O-β- 0.036 ± 0.002 b 0.024 ± 0.001 b 0.043 ± 0.002 b 0.111 ± 0.002 a epicatechin 255.03, 227.03, 151.00 glucopyranosyl-β- glucopyranoside) 299 463.08963 [M − 2H]− 7.39 300.03, 301.00, 463.00, delphinidin 3-O-glucoside 2.885 ± 0.306 a 5.818 ± 0.784 a 12.753 ± 0.781 b 28.658 ± 0.815 b epicatechin 271.00, 151.00, 143.00 300 448.0979 [M − H]− 7.83 284.03, 285.00, 255.00, cyanidin 3-galactoside 1.250 ± 0.107 1.328 ± 0.143 2.044 ± 0.001 0.773 ± 0.012 epicatechin 447.00, 227.00, 256.00 302 477.10504 [M − 2H]− 7.90 314.04, 477.10, 271.00, petunidin-3-O-β- 5.774 ± 0.695 a 15.256 ± 1.301 b 15.609 ± 0.303 b 48.988 ± 0.756 c epicatechin

11057 243.00, 285.00, 315.00 glucopyranoside − 209 433.1156 [M − H] 8.29 271.06, 151.00, 61.99, naringenin-7-O-glucoside 0.832 ± 0.059 a 1.044 ± 0.114 a 1.503 ± 0.181 a 0.018 ± 0.011 b epicatechin 119.05, 272.06, 93.03 232 271.0621 [M − H]− 10.30 151.00, 271.06, 119.05, naringenin 0.168 ± 0.027 0.109 ± 0.005 0.040 ± 0.001 0.062 ± 0.009 epicatechin 107.01, 93.03, 177.02 150 355.1041 [M − H]− 6.12 175.04, 193.05, 160.02, ferulic acid hexoside 6.258 ± 0.415 a 4.243 ± 0.223 a 4.558 ± 0.162 a 1.021 ± 0.001 b ferulic acid 178.03, 134.04, 355.19 159 355.1043 [M − H]− 6.35 175.04, 160.02, 294.13, feruloyl glucose 0.883 ± 0.105 a 0.134 ± 0.014 b 0.211 ± 0.001 b 0.001 ± 0.000 c ferulic acid 279.11, 193.05, 134.04 145 477.1057 [M − H]− 5.99 314.04, 477.11, 271.02, is-3-O-glucoside 0.024 ± 0.002 0.014 ± 0.002 0.017 ± 0.001 0.078 ± 0.001 isorhamnetin 243.03, 285.04, 315.05 172 639.1579 [M − H]− 6.69 313.04, 315.05, 477.10, is-3-O-diglucoside 0.629 ± 0.060 b 0.424 ± 0.028 c 1.335 ± 0.027 b 2.733 ± 0.078 a isorhamnetin 112.98, 639.15, 314.04 206 477.1056 [M − H]− 7.90 314.04, 477.11, 271.03, is-3-O-glucoside 0.003 ± 0.001 b 0.009 ± 0.001 b 0.007 ± 0.000 b 15.612 ± 0.311 a isorhamnetin 243.03, 285.04, 315.05 240 315.0521 [M − H]− 10.64 300.03, 315.05, 151.00, isorhamnetin 0.765 ± 0.149 0.178 ± 0.015 0.095 ± 0.002 0.130 ± 0.033 isorhamnetin

.Arc odChem. Food Agric. J. 301.03, 164.01, 271.02 135 1139.3113 [M − H]− 5.74 284.03, 977.26, 285.04, km-3-O- 0.023 ± 0.002 0.020 ± 0.001 0.019 ± 0.003 0.005 ± 0.000 kaempferol 771.20, 283.03, 255.03 sinapoylsophorotrioside-7- O-glucoside DOI: 137 771.2006 [M − H]− 5.82 285.04, 283.03, 609.15, km-3-O-sophoroside-7-O- 4.178 ± 0.204 b 4.547 ± 0.214 b 6.526 ± 0.023 a 0.799 ± 0.017 c kaempferol

10.1021/acs.jafc.9b05046 284.03, 446.09, 771.20 glucoside 09 7 11053 67, 2019, 139 609.1478 [M − 2H]− 5.86 285.04, 283.02, 447.09, km-3-O-glucoside-7-O- 14.109 ± 0.571 b 11.718 ± 0.453 b 19.791 ± 0.586 a 2.185 ± 0.119 c kaempferol 446.08, 284.03 glucoside 143 977.2587 [M − H]− 5.92 285.04, 447.09, 284.03, km-3-O-sinapoylsophoroside- 0.065 ± 0.006 a 0.042 ± 0.003 a 0.005 ± 0.008 b nd c kaempferol 283.02, 609.14, 815.19 7-O-glucoside Article

− − 11065 202 609.1474 [M − H] 7.65 285.04, 284.03, 609.15, km-O-sophoroside 0.474 ± 0.037 b 0.526 ± 0.036 b 1.126 ± 0.056 a 0.253 ± 0.020 b kaempferol 255.03, 227.03, 257.04 ora fArclua n odChemistry Food and Agricultural of Journal Table 1. continued

RT code average m/z adduct ion (min) MS2 compd name pale white yellow pink red standard 205 447.095 [M − H]− 7.82 284.03, 255.03, 285.04, km-3-O-glucoside 0.015 ± 0.001 b 0.015 ± 0.001 b 0.006 ± 0.001 b 0.946 ± 0.024 a kaempferol 447.10, 227.03, 256.04 234 285.0412 [M − H]− 10.55 285.04, 151.00, 229.05, kaempferol 0.187 ± 0.020 0.138 ± 0.014 0.115 ± 0.009 0.031 ± 0.001 kaempferol 107.01, 169.06, 213.05 − 87 164.0709 [M − H] 3.11 147.04, 164.07, 72.01, L-(−)-phenylalanine 0.457 ± 0.070 0.567 ± 0.077 1.88 ± 0.010 0.129 ± 0.001 p-coumaric 148.05, 91.05, 103.05 acid 106 163.039 [M − H]− 4.55 119.05, 162.84, 163.04, m-hydroxycinnamic acid 0.517 ± 0.055 0.720 ± 0.032 1.032 ± 0.044 0.410 ± 0.013 p-coumaric 162.89, 120.05, 59.01 acid 179 163.0392 [M − H]− 7.20 119.05, 163.04, 149.01, p-coumaric acid 2.109 ± 0.204 a 3.299 ± 0.194 a 1.982 ± 0.071 a 0.722 ± 0.245 b p-coumaric 120.05 acid 110 625.1426 [M − H]− 4.80 301.03, 463.09, 299.02, qn-3,4′-O-di-β- 0.010 ± 0.001 0.011 ± 0.001 0.040 ± 0.003 0.029 ± 0.001 quercetin 462.08, 300.02, 78.35 glucopyranoside 128 625.1433 [M − H]− 5.54 299.02, 301.04, 462.08, qn-3,4′-O-di-β- 0.047 ± 0.003b 0.076 ± 0.006b 0.097 ± 0.014b 31.374 ± 1.006a quercetin 625.14, 463.09, 300.03 glucopyranoside 166 625.1433 [M − H]− 6.52 301.03, 300.03, 463.09, qn-3,4′-O-di-β- 0.079 ± 0.010 b 0.074 ± 0.004 b 0.063 ± 0.001 b 3.840 ± 0.106 a quercetin 151.00, 178.10, 299.02 glucopyranoside 188 463.0893 [M − H]− 7.36 300.03, 301.04, 463.09, qn-3-O-glucoside 2.759 ± 0.239b 3.717 ± 0.424b 22.316 ± 1.496a 16.335 ± 0.959a quercetin 271.02, 151.00, 143.04 197 505.1006 [M − H]− 7.62 300.03, 301.04, 505.10, qn-3-(6-O-acetyl-β-glucoside) nd b nd b 0.013 ± 0.002 a 0.023 ± 0.001 a quercetin 271.02, 143.04, 255.03 226 301.0358 [M − H]− 9.51 151.00, 301.04, 178.10, quercetin 0.515 ± 0.109 0.123 ± 0.017 0.336 ± 0.021 0.124 ± 0.001 quercetin 11058 211.13, 301.20, 121.03

− 154 385.1155 [M − H] 6.13 205.05, 190.03, 223.06, 1-O-sinapoyl-β-D-glucose 0.106 ± 0.013 b 0.050 ± 0.007 b 0.054 ± 0.004 b 10.755 ± 0.506 a sinapic acid 59.01, 164.05, 89.02 153 223.0609 [M − H]− 6.24 149.02, 164.05, 223.06, sinapic acid 0.012 ± 0.004 a 0.030 ± 0.003 0.076 ± 0.004 a nd b sinapic acid 208.04, 193.01, 147.04 − 160 385.1155 [M − H] 6.32 205.05, 190.03, 223.06, 1-O-sinapoyl-β-D-glucose 0.125 ± 0.011 b 0.049 ± 0.006 b 0.149 ± 0.006 b 1.463 ± 0.010 a sinapic acid 164.05, 59.01, 89.02 161 431.1930 [M − H]− 6.36 153.09, 71.01, 385.19, putative hydroxycinnamic 26.550 ± 0.918 a 8.354 ± 0.484 b 22.183 ± 1.646 a 0.893 ± 0.003 c sinapic acid 89.02, 152.08, 59.01 acid derivative 198 339.0734 [M − H]− 7.63 223.06, 164.05, 71.01, sinapoyl malate 0.030 ± 0.004 0.016 ± 0.005 0.078 ± 0.011 0.828 ± 0.023 sinapic acid 149.02, 171.10, 115.00 192 223.061 [M − H]− 7.64 208.04, 164.05, 149.02 sinapic acid 0.520 ± 0.033 b 0.341 ± 0.026 b 1.430 ± 0.044 a 0.308 ± 0.025 b sinapic acid 193.01, 223.06, 179.07 210 753.2263 [M − H]− 8.07 205.05, 223.06, 190.03, disinapoylgentiobiose 3.686 ± 0.302 b 10.955 ± 0.537 a 12.912 ± 0.441 a 9.765 ± 0.317 a sinapic acid 164.05, 529.16, 59.01 31 388.0381 [M − H]− 1.29 96.96, 74.99, 95.95, R-2-hydroxy-3-butenyl GSL 65.583 ± 4.013 76.704 ± 6.099 101.049 ± 3.182 55.625 ± 2.110 sinigrin .Arc odChem. Food Agric. J. 388.04, 79.96, 135.97 55 436.0422 [M − H]− 1.65 96.96, 95.95, 74.99, 4-methylsulfinylbutyl GSL 2.436 ± 0.219 b 6.267 ± 0.335 b 13.982 ± 1.314 a 3.158 ± 0.155 b sinigrin 178.01, 436.03, 372.04 − − ± ± ± ±

DOI: 59 450.0579 [M H] 1.72 96.96, 95.95, 74.99, glucoalyssin 32.966 1.538 63.154 3.598 99.70 3.891 27.15 0.5261 sinigrin 79.96, 450.06, 192.03

10.1021/acs.jafc.9b05046 − 09 7 11053 67, 2019, 80 372.0435 [M − H] 2.45 96.96, 74.99, 95.95, 3-butenyl GSL 134.722 ± 13.049 a 70.624 ± 3.292 b 98.08 ± 1.725 b 37.237 ± 0.813 c sinigrin 372.04, 79.96, 130.03 85 479.0443 [M − H]− 3.39 96.96, 95.95, 74.99, 3-benzoyloxypropyl GSL 0.691 ± 0.055 b 0.937 ± 0.058 b 2.389 ± 0.055 a 0.341 ± 0.003 b sinigrin

160.04, 79.96, 185.03 Article − − ± ± ± ± − 84 463.0497 [M H] 3.40 96.96, 95.95, 74.99, 4-hydroxyindol-3-ylmethyl 6.196 0.410 b 5.842 0.233 b 15.707 0.038 a 3.558 0.124 b sinigrin 11065 169.04, 267.01, 96.97 GSL ora fArclua n odChemistry Food and Agricultural of Journal Table 1. continued

RT code average m/z adduct ion (min) MS2 compd name pale white yellow pink red standard 98 386.0592 [M − H]− 4.22 96.96, 74.99, 95.95, 4-pentenyl GSL 165.485 ± 8.321 b 112.913 ± 2.905 a 227.032 ± 10.377 b 81.09 ± 2.847 a sinigrin 79.96, 144.05, 85.03 104 408.0442 [M − H]− 4.64 96.96, 95.95, 74.98, benzyl GSL 0.033 ± 0.004 0.102 ± 0.018 0.009 ± 0.001 0.055 ± 0.004 sinigrin 408.00, 79.95, 63.96 105 420.0476 [M − H]− 4.64 96.96, 95.95, 74.98, 4-methylthiobutyl GSL 0.015 ± 0.002 0.016 ± 0.003 0.020 ± 0.001 0.008 ± 0.001 sinigrin 420.00, 79.95, 226.10 112 203.0822 [M − H]− 4.91 116.04, 203.00, 74.02, tryptophan 2.314 ± 0.247 a 1.591 ± 0.128 b 4.093 ± 0.168 a 0.210 ± 0.005 b sinigrin 142.00, 159.00, 72.00 119 447.054 [M − H]− 5.40 96.96, 95.95, 74.98, indolylmethyl GSL 11.749 ± 0.399 8.356 ± 0.258 35.646 ± 2.383 16.684 ± 0.857 sinigrin 79.95, 447.00, 205.00 142 434.0625 [M − H]− 6.02 96.96, 95.95, 74.99, 5-methylthiopentyl GSL 0.129 ± 0.015 a 0.175 ± 0.019 a 0.263 ± 0.010 a nd b sinigrin 434.06, 79.96, 223.30 138 422.0595 [M − H]− 6.06 96.96, 95.95, 74.98, gluconasturtiin 4.900 ± 0.4182 2.487 ± 0.165 7.603 ± 0.102 3.087 ± 0.099 sinigrin 422.00, 79.95, 259.00 155 477.0661 [M − H]− 6.30 96.96, 95.95, 74.98, 4-methoxy-3-indolylmethyl 0.030 ± 0.003 b 0.045 ± 0.004 b 0.051 ± 0.007 b 3.102 ± 0.003 a sinigrin 477.00, 79.95, 259.00 GSL 214 144.0444 [M − H]− 8.38 144.04, 143.00, 144.00, indole-3-carboxyaldehyde 0.187 ± 0.017 0.204 ± 0.007 0.402 ± 0.011 0.401 ± 0.025 sinigrin 71.01, 85.02, 87.04 217 160.0396 [M − H]− 8.81 85.03, 59.00, 132.00, 1H-indole-3-carboxylic acid 0.277 ± 0.025 0.103 ± 0.011 0.241 ± 0.000 0.032 ± 0.020 sinigrin 131.00, 73.00, 71.00 a μ ± ﬀ ﬁ

11059 Note: The results are expressed as g/g of fresh weight (FW) petals. Each value is presented as the mean standard deviation (n = 3), and values in each rows with di erent letters are signi cantly ﬀ

di erent (p < 0.05). Abbreviations: ep, epicatechin; km, kaempferol; is, isorhamnetin; qn, quercetin; GSL, glucosinolate; nd, not detected. .Arc odChem. Food Agric. J. DOI: 10.1021/acs.jafc.9b05046 09 7 11053 67, 2019, Article − 11065 Journal of Agricultural and Food Chemistry Article

Figure 5. Distribution of constituents in diﬀerent-colored petals of B. napus: (A) phenolic acid compounds, (B) ﬂavonoids, (C) glucosinolate compounds, and (D) lipid compounds. The horizontal and vertical coordinates represent the peak area and RT of the corresponding constituents, respectively.

6.13 and 6.32 min) were present in high levels only in red petals, glucoside), which was not detected in pale white or yellow petals and the contents of L-(−)-phenylalanine, m-hydroxycinnamic (Table 1). acid, and sinapic acid were markedly higher in pale white petals Notably, some constituents accumulated to higher concen- than in those of other colors (Table 1). The remaining trations in red petals than in those of other colors, such as β compounds were present in trace or medium amounts in the delphinidin 3-O-glucoside, petunidin-3-O- -glucopyranoside, different B. napus petals. an isomer of is-3-O-glucoside (RT = 7.90 min), and two ′ β Identification of Flavonoids. We detected 28 flavonoid isomers of qn-3,4 -O-di- -glucopyranoside (RT = 5.54 and 6.52 compounds from the different-colored B. napus petals, of which min), whereas km-3-O-sinapoylsophoroside-7-O-glucoside was 26 were quantified using epicatechin, isorhamnetin, kaempferol, not detected in red petals (Table 1). In addition, we found that and quercetin as standards [Table S1 (SI), Figure 5B]. Of these, naringenin-7-O-glucoside (less than 3% in red petals compared with other petals) and km-3-O-glucoside-7-O-glucoside (less nine and four compounds were quantitated using epicatechin than 20% in red petals compared with other petals) were and isorhamnetin, respectively, and seven and six compounds substantially lower in red petals compared with other petals were identified using kaempferol and quercetin, respectively (Table 1). These findings suggest that primary constituents (Table 1). Two and three isomers of is-3-O-glucoside and qn- ff ff ′ β fi di ered substantially among the di erent-colored petals of B. 3,4 -O-di- -glucopyranoside, respectively, were identi ed si- napus. fl multaneously. Most of the avonoid compounds were not Identification of Glucosinolate Compounds. Glucosi- ff markedly di erent between yellow and pale white petals, but the nolates (GSL) are sulfur-containing plant secondary metabolites mean amounts in pink and red petals were substantially found in Brassica. We characterized these compounds by different. For example, contents of cyanidin-3-glucoside, is-3- identifying the parental molecular ions (adduct ions), the O-diglucoside, qn-3-O-glucoside, and qn-3-(6-O-acetyl-β-gluco- fragmentation patterns of the daughter ions (m/z), and the RTs side) were relatively higher in pink and red petals compared with of these molecules and their standards.30,31 Sixteen compounds pale white and yellow petals, especially for qn-3-(6-O-acetyl-β- were identified, which were quantified using sinigrin as a

11060 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article

Figure 6. Changes in quantified constituents of different-colored petals in B. napus: (A) total phenolic acids and (B) total flavonoids. Error bars represent the standard deviations from three biological replicates and ** represent significant differences at P < 0.01. (C) Amounts (μg/g FW) of quantified constituents in different B. napus petals. Different petal colors are shown at the bottom of the heatmap. Numbers on the left indicate the code of mass signals. The scale bar represents the contents of identified constituents (μg/g FW), calculated as the mean of three replicates using a range of standard signal values. standard (Table 1, Figure 5C). Five of these, R-2-hydroxy-3- tin, ferulic acid, p-coumaric acid, sinapic acid, and sinigrin butenyl GSL, glucoalyssin, 3-butenyl GSL, 4-pentenyl GSL, and (Figure 6C, Table 1). For example, the major constituents indolylmethyl GSL, accumulated to high levels in B. napus petals cyanidin-3-glucoside and cyanidin-3,5-di-O-glucoside mainly (Table 1). Some of them also showed remarkable differences in accumulated in red petals, at concentrations of 2.052 and 1.344 the composition and abundance of these compounds among the μg/g FW, respectively (Figure 7A,B, Table 1); is-3-O-glucoside different lines. also accumulated to much higher levels in red petals than in Identification of Lipids and Other Polar Compounds. other petals, up to 15.61 μg/g FW (Figure 7C, Table 1). As an important worldwide source of vegetable oil, the physical, Additionally, two isomers of qn-3,4′-O-di-β-glucopyranoside chemical, and nutritional qualities of rapeseed oil mainly depend (RT = 5.54 min, Figure 7D, and RT = 6.52 min) also showed on its lipid composition. We identified 16 compounds according notable accumulation in red petals (Table 1), whereas − − to the parental molecular ions [M + FA − H] and [M − H] , delphinidin 3-O-glucoside (Figure 7E), qn-3-O-glucoside, and MS2 spectral data, and the RTs of these molecules, which were qn-3-(6-O-acetyl-β-glucoside) accumulated less in yellow and lipid precursors and classified as nine fatty acyls and seven pale white petals than in pink and red petals (Table 1). We found glycerophospholipids (Figure 5D, Table S1, SI). Additionally, that most kaempferol compounds, such as kaempferol, km-3-O- 69 polar compounds were also successfully identified from glucoside-7-O-glucoside (Figure 7E), and km-3-O-sophoroside- different-colored petals, including chlorogenic acid, carboxylic 7-O-glucoside, were present at lower levels in red petals than in acid, organooxygen compounds, and pyrimidine nucleotides the other petals, and km-3-O-sinapoylsophoroside-7-O-gluco- (Table S1, SI). Although these compounds were identified in side was not detected in red petals (Table 1). These results are this study, there are few reports of using UPLC−HESI-MS/MS useful for further elucidating the molecular mechanisms in rapeseed. Therefore, the variation of these constituents underlying flavonoid metabolic fluxes in different-colored petals should be quantified in future studies using pure standards. of B. napus. A total of 57 constituents could be quantified using standards Characterization of Bound Phenolics. In the residue, the for caffeic acid, epicatechin, isorhamnetin, kaempferol, querce- bound phenolics were insoluble and not extracted by 80%

11061 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article

Figure 8. Base peak chromatograms of bound phenolic fractions in negative mode for diﬀerent-colored petals of B. napus.

them, p-coumaric acid was the most abundant bound phenolic in petals, but it had lower levels in the pale white; ferulic acid and 8- O-4′-diferulic acid was the second most abundant bound phenolic (Table 2). Thus, the levels of total bound phenolic concentrations in pale white petals were lower than these in other petals, perhaps mainly due to the higher p-coumaric acid, ferulic acid, and 8-O-4′-diferulic acid (Table 2, Figure 9). In this study, the insoluble bound phenolics were identified for the first time in the petals of rapeseed, and further studies may also be required in the future. ■ DISCUSSION Colorful flowers affect the value of many ornamental flowering plants and are one of the most important breeding targets in horticultural plants.32 Tourists are attracted to the predom- inantly yellow flowers of B. napus, a globally distributed crop grown widely in China, where it is honored culturally and associated with nobility and prosperity.3,33 Flower colors in rapeseed also include less white, orange, and milky white. Recently, other colors, such as pink, red, and purple, have been reported for B. napus,3,34 which might improve its ornamental value. In Brassicaceae, previous studies have highlighted the − major chemical constituents of seeds,25 27,35 leaves,14,36 and − different-colored Brassica vegetables,14 16,18,19 whereas only a few recent works have considered the chemical constituents of petal color in Brassica plants. Petal color is mainly associated with the chemical structures of Figure 7. UPLC−HESI-MS/MS chromatograms of the significant different anthocyanins accumulating in flowers.37,38 Corre- constituents found in petals of B. napus: (A) cyanidin-3-glucoside, (B) 36 ′ β spondingly, Zhao et al. investigated the chemical constituents cyanidin-3,5-di-O-glucoside, (C) is-3-O-glucoside, (D) qn-3,4 -O-di- - fl glucopyranoside, (E) delphinidin 3-O-glucoside, and (F) km-3,7- of B. rapa owers, mainly focusing on phenolic glycosides and diglucoside. their bioactivity, and isolated two novel phenolic glycoside compounds and 10 known analogues from B. rapa flowers. In addition, more anthocyanins were abundant in red petals than MeOH, but the residue could be hydrolyzed with alkaline yellow petals, such as 12 anthocyanins, including cyanidin and followed by acid and the released phenolics back-extracted with delphinidin, which were identified and contributed to the red diethyl ether−ethyl acetate. As shown in the chromatogram, color, but only two types of anthocyanins were identified from numerous peak chromatograms could be discerned in this study yellow petals.3 Furthermore, these corresponding biosynthetic (Figure 8). On the basis of matching retention time and mass pathways are well-understood in higher plants such as spectrometric data with authentic standards and quantifing Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and − using standard curves generated for each specific compound by petunia (Petunia hybrida).5 7 Therefore, in this study, we UPLC−HESI-MS/MS, seven tentatively identified insoluble explored the relationship between petal color and flavonoid bound phenolics in four different color petals are presented in composition in B. napus, and we found that different-colored Table 2, p-coumaric acid, sinapic acid, ferulic acid, epicatechin, petals in B. napus had different chemical constituents. Flavonoid 8-O-4′-diferulic acid, 8,8′-disinapic acid, and naringenin. Among compositions showed obvious differences, with the highest level

11062 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article < 0.05). p -coumaric acid -coumaric acid -coumaric acid p p p erent ( ﬀ 13.632 c 0.296 a0.094 ferulic1.811 acid d 0.060 sinapic acid 0.0120.126 b epicatechin epicatechin cantly di ± ﬁ ± ± ± ± ± ±

Figure 9. Changes in quantiﬁed bound phenolics of diﬀerent-colored

9.069 c 88.772 μ ± 1.161 a0.056 2.814 0.168 c0.133 0.807 2.462 0.0210.152 0.457 b 0.049 0.870 petals of B. napus. Bar data are expressed as g/g FW petals standard ±

± ± ± ± ± ± deviation (n = 3). Shared letters indicate no signiﬁcant diﬀerences erent letters are signi

ﬀ between bound phenolics within the same fraction (p < 0.05).

ﬂ

7.352 b 92.125 of total avonoids detected in red petals, followed by pink, ± 0.759 b0.0570.229 2.825 b0.086 0.734 0.019 1.141 0.010 b 0.555 0.056 0.432 yellow, and pale white (Figure 6B), whereas phenolic acids also ± ± ± ± ± ± showed marked differences among petal colors (Figure 6A). a Among these compounds, we quantified 15 phenolic acids and 26 flavonoids from pale white, yellow, pink, and red petals of B. napus (Table 1), with marked differences in the total flavonoids B. napus in different-colored rapeseed petals (Figure 6A,B). We analyzed 0.779 a0.878 a 113.985 0.164 5.529 0.038 a 0.888 0.057 1.850 0.011 0.294 0.029 a 0.057 0.442 fl

± ± ± ± ± ± ± the avonoid components and found that most of the = 3), and values in each rows with di isorhamnetin- and quercetin-derived compounds and cyanidin n derivatives (cyanidin-3-glucoside and delphinidin-3-O-glucoside) had higher levels in pink and red petals compared with pale white and yellow petals. The contents of cyanidin-3-glucoside was up to 40- and 10-fold higher in red and pink petals, respectively, than in yellow and white petals (Figure 7A, Table -diferulic acid 0.686 ′ 1), in accordance with results that color change is associated -4 -disinapic acid 0.282 compd name pale white yellow pink red standard standard deviation ( ′ 39 3 O

-coumaric acid 2.282 with the biosynthesis of cyanidin in strawberry, B. napus, and ± p Oenothera.40 Some compounds, such as is-3-O-glucoside, isomers of 1-O-sinapoyl-β-D-glucose, and qn-3,4′-O-di-β- glucopyranoside, accumulated to much higher concentrations in red petals than in other-colored petals of B. napus (Figure 7, Table 1), indicating that they might be speciﬁc to red petals in B. napus. Additionally, a previous study revealed that delphinidin- 3-O-glucoside is an important component of ﬂower petal 2 coloration in many plants, including Oncidium “Gower Ramsey”,41 soybean (Glycine max),42 Yard-long beans (Vigna unguiculata ssp. sesquipedalis L.),43 and evergreen azalea (subgenus Tsutsusi).44 Here, we observed similar results showing that delphinidin-3-O-glucoside accumulated to higher levels in pink-red petals than yellow-white petals (Table 1), indicating that this compound might be responsible for pink-red petals in rapeseed. However, we found that kaempferol

g/g FW petals. Each value is present as the mean derivatives, including km-3-O-glucoside-7-O-glucoside, km-3- RT μ 7.207.68 162.84, 149.01,7.64 120.05, 119.05, 59.01 178.03, 157.57,8.08 149.06, 137.02, 134.04 208.04, 164.05,10.75 149.02 193.01, 223.06, 179.07 267.07, 282.09,8.84 297.11, 341.1, 121.03, sinapic 326.08, 225.05, acid 323.09 357.1, 181.06,10.38 401.09, ferulic 173.01 acid 8- 245.08, 221.08, 205.05, 203.07, 8,8 179.03, 299.02, 109.03 314.04, 329.07, 329.23, 328.22, 252.20 epicatechin naringenin 0.454 2.782 0.019 0.173 (min) MS O-sophoroside-7-O-glucoside, and km-3-O-sinapoylsophoroside-7-O-glucoside, had lower levels in red petals than in − − − − − − − other-colored petals (Table 1). The presence of kaempferol H] H] H] H] H] H] H] − − − − − − − derivatives is rarely reported to be involved in the formation of different flower colors in other plants, but these compounds adduct ion ff cation of Main Bound Compounds in the Pale White, Yellow, Pink, and Red Petals of displayed markedly di erent levels between yellow- and black- fi seeded rapeseed,27,35 suggesting that the flavonoids constituents z

/ of rapeseed petals are diﬀerent from those of ﬂoricultural plants. m

average Hence, more evidence is needed to confirm the mechanism of flower color formation, because the materials used here might have a different genetic background from those used in prior 179 163.0392 [M 303 193.0496 [M 192304 223.0610305 385.0928 [M 158 445.1140 [M 233 289.0723 [M 329.0673 [M [M Note: The results are expressed as code

Table 2. Identi a studies.

11063 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article

In addition, studies have been conducted that reported that is, isorhamnetin; qn, quercetin; GSL, glucosinolate; FW, fresh − bound phenolics are predominant phenolic acids in plants.45 49 weight; SD, standard deviation In the present study, the comparative analyses of the phenolic acids in different petals have also revealed higher amounts of ■ REFERENCES bound phenolics compared with the free forms (Figures 6A and (1) Chalhoub, B.; Denoeud, F.; Liu, S.; Parkin, I. A.; Tang, H.; Wang, 9), but the levels of bound phenolics in pale white were ́ fi X.; Chiquet, J.; Belcram, H.; Tong, C.; Samans, B.; Correa, M.; Da Silva, signi cantly lower than that in others (Figures 6A and 9), C.; Just, J.; Falentin, C.; Koh, C. S.; Le Clainche, I.; Bernard, M.; Bento, suggesting that free forms might be the predominant phenolic P.; Noel, B.; Labadie, K.; Alberti, A.; Charles, M.; Arnaud, D.; Guo, H.; acids in the pale white rapeseed petals. 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11064 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065 Journal of Agricultural and Food Chemistry Article

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11065 DOI: 10.1021/acs.jafc.9b05046 J. Agric. Food Chem. 2019, 67, 11053−11065