Metabolite Profiling of Chalcones and Flavanones in Tomato Fruit

Home , Eriodictyol, Homoeriodictyol, Naringenin chalcone

Yoko Iijima, Kunihiro Suda, Tatsuya Suzuki, Koh Aoki* and Daisuke Shibata

Kazusa DNA Research Institute, Kazusa-Kamatari, Kisarazu 292–0818, Japan

Tomato (Solanum lycopersicum) is one of a group of plants that accumulate chalcones and flavanones. However, the molecular diversity of chalcones, flavanones, and their conjugate metabolites has not been investigated intensively. Here, we report the profiling of chalcones and flavanones in fruits of the dwarf tomato cultivar Micro- Tom using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR- MS). We identified eriodictyol chalcone and eriodictyol aglycones, along with naringenin chalcone and naringenin aglycones. To our knowledge, this is the first report that demonstrates the presence of eriodictyol chalcone and eriodictyol in tomato. We detected 26 conjugate metabolites of chalcones and flavanones. Chemical information obtained simultaneously by LC-FTICR-MS, including m/z values, MS/MS spectra, UV absorption spectra, and retention times, facilitated the elucidation of molecular formulas and conjugate structures of the metabolites. Eriodictyol chalcone and eriodictyol conjugates had the same modification patterns seen in naringenin chalcone and naringenin conjugates. Chalcones and flavanones were much more abundant in tomato fruit peel than flesh. Accumulation profiles during ripening were classified into three groups. The first group included metabolites that showed the highest accumulation levels at the breaker stage, and then decreased during ripening. The second group included metabolites that accumulated to the highest levels at the turning stage, and then decreased at the red stage. The third group included metabolites that accumulated gradually during ripening, and showed the highest accumulation levels at the red stage. These accumulation profiles were mapped onto a putative modification pathway deduced from conjugate structures. Mapping revealed that the conjugate metabolites upstream of this pathway accumulated earlier, and those downstream accumulated later during ripening. This result demonstrated that chalcones and flavanones undergo sequential modification as ripening progresses.

Key Words: chalcone, eriodictyol, flavanone, LC-FTICR-MS, Solanum lycopersicum.

chalcone and flavanone starts with the conversion of Introduction cinnamic acid to p-coumaric acid by a monooxygenase, Flavonoids form a large group of secondary cinnamate 4-hydroxylase (C4H). p-Coumaric acid is then metabolites that have several ecological and physiolog- activated to p-coumaroyl-coenzyme A (CoA) by 4- ical functions in plants. The functions of flavonoids coumaroyl:CoA ligase (4CL). To p-coumaroyl-CoA, include pigmentation, signaling, protection against biotic three molecules of malonyl-CoA are sequentially added and abiotic stresses, and fertility (Weisshaar and Jenkins, by the action of chalcone synthase (CHS), yielding 1998; Winkel-Shirley, 2001) and nodulation in legumes naringenin chalcone. Finally, chalcones are converted to (Wasson et al., 2006). Additionally, increasing evidence flavanones by chalcone isomerase (CHI). The upstream suggests that flavonoids potentially have a range of part of this linear pathway has been revised to a grid- biological activity in animal cells as a result of their like pathway, in which cinnamic acid, caffeic acid, antioxidant capacity (Dugas et al., 2000; Sugihara et al., ferulic acid, and their respective CoA esters can be 1999). precursors of the flavanone biosynthetic pathway as well Chalcones and flavanones are precursors of the as p-coumaric acid (Dixon et al., 2001; Humphreys and majority of flavonoid compounds. Biosynthesis of Chapple, 2002; Nair et al., 2002). Consequently, different forms of flavanones, pinocembrin, naringenin, eriodictyol, and homoeriodictyol, can be synthesized by Received; July 10, 2007. Accepted; August 20, 2007. This work was supported in part by the Research and Development the same pathway (Fig. 1). Program for New Bio-Industry Initiatives. Tomato (Solanum lycopersicum) accumulates fla- * Corresponding author (E-mail: [email protected]). vonoids in the peel of the fruit, which contains naringenin

94 J. Japan. Soc. Hort. Sci. 77 (1): 94–102. 2008. 95

activity and antioxidant activity (Nguyen et al., 2004; Yao et al., 2006). However, the molecular diversity of chalcones and flavanones and their accumulation profiles in tomato fruit have not previously been investigated. In the present study, we profiled chalcones, flavanones, and their glycosides in the fruits of a dwarf tomato cultivar Micro-Tom, which has been used as a model of solanaceae plants (Shibata, 2005), using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) (Suzuki et al., 2007). We identified eriodictyol chalcone and eriodictyol for the first time in tomato plants. In addition, 26 conjugates of chalcones and flavanones were detected from tomato fruit. The data obtained by LC-FTICR-MS allowed us to predict the conjugate structures of the metabolites. Profiling of conjugates of naringenin chalcone, naringenin, eriodictyol chalcone, and eriodictyol revealed that these metabolites accumulated predominantly in peel. Accumulation profiles during ripening were classified into three distinct patterns. Superimposing the profiles onto the putative modification pathway suggests that sequential conjugation of chalcones and flavanones occurs during ripening. Materials and Methods Plant materials Seeds of cultivated tomato (Solanum lycopersicum ‘Micro-Tom’) were sown in pots (500 mL) filled with a mixture of vermiculite and Powersoil (1 to 1 mixing ratio, Kureha Chemical Ind., Tokyo, Japan, and Kanto Hiryou Ind., Saitama, Japan, respectively) and kept in a controlled room at 25℃. After 4 days of germination, they were grown with a photoperiod of 16 h light (7000 lux) and 8 h dark. Hyponex (Hyponex Ltd., Osaka, Fig. 1. Schematic representation of the biosynthetic pathway of Japan) at 1000-fold dilution was applied to plants once chalcones and flavanones. NGC, naringenin chalcone; NG naringenin, EDC, eriodictyol chalcone; ED, eriodictyol; PAL, a week as a nutrient. Fruits at mature green phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; (approximately 30 days after anthesis), breaker C3H, coumarate 3-hydroxylase; 4CL, 4-coumarate-CoA ligase; (approximately 35 days after anthesis), turning (approx- CHS, chalcone synthase; CHI, chalcone isomerase; F3'H, imately 38–40 days after anthesis) and red (approxi- flavonoid 3'-hydroxylase. mately 45–48 days after anthesis) stages were harvested. chalcone, naringenin, and flavonol glycosides (Bovy et Sample preparation for MS al., 2002; Moco et al., 2006; Muir et al., 2001; Verhoeyen The peel and flesh of tomato fruits were separated et al., 2002). In particular, the accumulation level of with a razor blade. Samples were sliced and immediately naringenin chalcone is approximately 10-fold higher than frozen in liquid nitrogen, and then ground to a powder that of rutin (quercetin 3-O-rutinoside) at the turning by a homogenizer, Shake Master (Biomedical Science, stage (Muir et al., 2001). This result showed that tomato Tokyo, Japan). The powdered samples were stored in a fruit is a member of a group of foods that accumulate −80℃ freezer for no longer than 3 months before chalcones and flavanones, including celery, citrus, and extraction of metabolites. We confirmed that powdered prunes (Ross and Kasum, 2002). Chalcones, flavanones, samples stored at −80℃ for 6 months showed the same and their glycosides are potential health-protecting metabolite profiles as those of freshly prepared samples. compounds (Ross and Kasum, 2002). For example, Powdered samples (50–70 mg) were extracted with naringenin chalcone showed anti-allergic activity which three volumes of methanol containing formononetin was not shown by other flavonoid compounds, including (20 mg·L−1) as an internal standard. After homogenizing rutin, quercetin, and kaempferol (Yamamoto et al., twice with Mixer Mill MM 300 (QIAGEN, Hilden, 2004). Additionally, different conjugates showed Germany) at 27 Hz for 2 min, homogenates were different activities, such as xanthine oxidase inhibition centrifuged (12000 × g, 10 min, 4℃). The supernatant 96 Y. Iijima, K. Suda, T. Suzuki, K. Aoki and D. Shibata was filtered through a 0.2-µm PVDF membrane Chemicals (Whatman, Brentford, UK), and the filtrate was used for Authentic eriodictyol, eriodictyol 7-O-glucoside, and LC-FTICR-MS analysis. naringenin 7-O-glucoside were purchased from Funa- koshi Co. Ltd. (Tokyo, Japan; catalog numbers 0056, LC-FTICR-MS 1112S, and 1090, respectively). Authentic eriodictyol An Agilent 1100 system (Agilent, Palo Alto, CA, chalcone (2',4',6',3,4-pentahydroxychalcone) was pur- USA) coupled with a mass spectrometer, Finnigan LTQ- chased from EXTRASYNTHESE (Genay, France; FT (Thermo Fisher, Waltham, MA, USA) was used for catalog number 1282B). Authentic naringenin chalcone LC-FTICR-MS analysis. Data were acquired with compound was generously provided by the Kikkoman Xcalibur 2.0 software (Thermo Fisher). Corporation (Noda, Japan). A methanol extract was applied to a TSKgel ODS- Results 100V column (4.6 × 250 mm, 5 µm; TOSOH, Tokyo, Japan). Water (HPLC grade; solvent A) and acetonitrile Identification of eriodictyol and eriodictyol chalcone (HPLC grade; solvent B) were used as the mobile phase We detected eight metabolites (peak 1, 7, 8, 9, 14, under gradient elution conditions. Formic acid was added 19, 23, and 24) that had an m/z 289 ([M + H]+) fragment to both solvents at a concentration of 0.1% (v/v). The in the MS/MS spectra (Table 1). The m/z value 289 gradient program was as follows: 10% B to 50% B matched [M + H]+ ions of a number of metabolites, in- (50 min), 50% to 90% B (20 min), 90% B (5 min), and cluding dihydrokaempferol, eriodictyol, and eriodictyol 10% B (10 min). The flow rate was set to 0.5 mL·min−1, chalcone. To identify the fragment, we performed MS3 and the column oven temperature was set to 40℃. analysis targeting the m/z 289 ion. MS3 spectra consisted Twenty microliters of each sample were injected. of fragments m/z 271, 179, 163, and 153 (Fig. 2A). This To monitor HPLC elution, a photodiode array detector fragmentation pattern was different from that reported was used in the wavelength range between 200 and for dihydrokaempferol (Le Gall et al., 2003). The MS3 650 nm. MS data were obtained in positive-ion fragmentation pattern was compared with that of electrospray ionization (ESI) mode. The settings were authentic eriodictyol. The MS/MS fragmentation pattern spray voltage 4.0 kV and capillary temperature 300℃. of authentic eriodictyol matched the MS3 fragmentation λ Nitrogen sheath gas and auxiliary gas were set at 40 and pattern of the observed m/z 289 ion (Fig. 2A). The max 15 arbitrary units, respectively. A full MS scan with of the UV absorption spectra of these peaks also matched internal standards was performed in the range of 100– that of authentic eriodictyol (Table 1). Thus, we 1500 (m/z) at resolution 100000 (at m/z 400). concluded that aglycones of peaks 1, 7, 8, 9, and 14 A mixture of internal calibration standards dissolved were eriodictyol. MS3 fragment ions were schematically in 50% (v/v) acetonitrile was introduced by a post- assigned to eriodictyol, as in Figure 2B. Peaks 19, 23, column method at a flow rate of 20 µL·min−1. The and 24 had m/z 289 ions, and the fragmentation patterns concentration of each standard in the mixture was as of the ions were identical to those for m/z 289 ions of µ + + µ λ follows: 10 M lidocaine (m/z 235.1805 [M H] ), 5 M peaks 1, 7, 8, 9, and 14. However, max (ranging from prochloraz, (m/z 376.0381 [M + H]+), 1.2 µM reserpine, 340 to 370 nm, Table 1) of the UV absorption spectra (m/z 609.2807 [M + H]+), 0.8 µM bombesin (m/z of peaks 19, 23, and 24 was different from that of 810.4148 [M + H]+), 0.4 µM aureobasidin A, (m/z authentic eriodictyol. By analyzing authentic eriodictyol + + µ λ 1123.6778 [M Na] ), and 22 M vancomycin (m/z chalcone, we confirmed that max of peaks 19, 23, and 1448.4375 [M + H]+). MS/MS and MS/MS/MS (MS3) 24 (364 nm) matched that of eriodictyol chalcone. MS3 fragmentation were carried out at a normalized collision fragment ions were schematically assigned to eriodictyol energy of 35.0% and isolation width 4.0 (m/z) in ion chalcone, as shown in Figure 2C. This is the first trap mode. Mass Frontier software version 4.0 (Thermo experimental evidence, as far as we know, demonstrating Fisher) was used to predict the fragmentation pattern. the presence of eriodictyol and eriodictyol chalcone in tomato. Analysis Eriodictyol chalcone, naringenin chalcone, eriodictyol Putative conjugate structures of chalcones and flavanone 7-O-glucoside, and naringenin 7-O-glucoside were derivatives quantified using standard calibration curves of authentic We assigned molecular formulas and conjugate compounds. structures of 8 naringenin chalcone conjugates, 9 Accumulation profiles were estimated from the area naringenin conjugates, 3 eriodictyol chalcone conju- ratio of the quasi-molecular ion to that of the internal gates, and 5 eriodictyol conjugates (Table 1). Peaks with standard, formononetin (m/z 269.0808 [M + H]+). The the same m/z and different retention time indicated the area ratios of metabolites were normalized over eight presence of isomers. Accurate m/z measurement with samples (peel and flesh from mature green, breaker, LC-FTICR-MS, with average ∆ppm of 0.44, facilitated turning, and red stages) so that the average value across the prediction of a small number of molecular formulas λ samples equaled 1.0. for each metabolite. The max of UV absorption spectra J. Japan. Soc. Hort. Sci. 77 (1): 94–102. 2008. 97

Table 1. Chalcones and flavanones detected in tomato (S. lycopersicum ‘Micro-Tom’) fruit.

Rt Obs. massa Molecular Theor. massa λ ∆ppm max Description MS/MSa (m/z, (rel. int.)) (min) (m/z) formula (m/z) (nm)

1 19.97 572.1430 C24H29O13NS 572.1432 −0.40 292 ED-hex, -121 289 (100), 290 (14), 451 (12) 2 20.22 556.1479 C24H29O12NS 556.1483 −0.76 286 NG-hex, -121 273 (100), 315 (2), 394 (2), 435 (55) 3 20.67 556.1479 C24H29O12NS 556.1483 −0.76 286 NG-hex, -121 273 (100), 284 (9), 297 (1), 394 (49), 399 (1), 417 (2), 435 (14) 4 20.96 556.1479 C24H29O12NS 556.1483 −0.76 328 NGC-hex, -121 273 (100), 274 (11), 315 (2), 394 (3), 435 (85) 5 21.26 556.1480 C24H29O12NS 556.1483 −0.53 286 NG-hex, -121 273 (100), 315 (1), 394 (2), 417 (2), 435 (7) 6 23.30 614.2074 C27H35O15N 614.2079 −0.84 286 NG-hex, -hex, -17 273 (100), 435 (66), 597 (7) 7 24.02 410.0903 C18H20O8NS 410.0904 −0.16 284 ED-121 289 (100), 375 (13) 8 24.48 410.0904 C18H20O8NS 410.0904 −0.13 284 ED-121 289 (100), 375 (13) 9 24.56 630.2025 C27H35O16N 630.2029 −0.54 284 ED-hex, -hex, -17 289 (49), 451 (100), 452 (3), 586 (5), 612 (8), 613 (29) 10 24.61 642.1481 C27H31O15NS 642.1487 −0.99 284 NG-121, -248 273 (57), 315 (6), 381 (3), 485 (4), 503 (8), 521 (100) 11 26.36 597.1813 C27H32O15 597.1814 −0.25 ND NG/NGC-hex, -hex 273 (100), 435 (27) 12 26.56 394.0954 C18H19O7NS 394.0955 −0.33 290 NG-121 273 (100) 13 27.06 394.0953 C18H19O7NS 394.0955 −0.58 290 NG-121 273 (100) b 14 29.31 451.1235 C21H22O11 451.1235 −0.08 284 ED-7-O-gluc 289 (100) 15 33.00 581.1861 C27H32O14 581.1865 −0.66 370 NGC-hex, -dhex 273 (100), 419 (13), 435 (24) 16 33.12 567.1706 C26H30O14 567.1708 −0.44 366 NGC-hex, -pen 273 (100), 315 (4), 405 (5), 417 (11), 435 (19) b 17 33.90 435.1283 C21H22O10 435.1286 −0.54 284 NG-7-O-gluc 273 (100) 18 34.86 435.1283 C21H22O10 435.1286 −0.72 362 NGC-hex 273 (100) 19 35.81 451.1232 C21H22O11 451.1235 −0.73 368 EDC-hex 289 (100) 20 37.19 435.1283 C21H22O10 435.1286 −0.53 368 NGC-hex 273 (100) 21 37.65 435.1283 C21H22O10 435.1286 −0.72 368 NGC-hex 273 (100) 22 37.78 955.2508 C45H46O23 955.2503 0.56 366 NGC-hex, -malonylhex, 273 (24), 435 (100), 521 (29) -272 23 40.29 451.1233 C21H22O11 451.1235 −0.51 370 EDC-hex 289 (100) b 24 44.14 289.0707 C15H12O6 289.0707 0.26 364 EDC 163 (100), 153 (29), 179 (24), 271 (18), 164 (9), 145 (7), 253 (6) 25 46.78 627.2073 C32H34O13 627.2072 0.07 330 NGC-354 273 (100), 315 (19), 339 (15), 381 (21), 399 (13), 488 (13), 591 (13), 609 (9) b 26 50.18 273.0756 C15H12O5 273.0757 −0.51 362 NGC 153 (100), 147 (76), 148 (7), 154 (6), 231 (4), 189 (4), 179 (4)

Abbreviations: Rt, retention time; Obs. mass, observed mass; Theor. mass, theoretical mass; rel. int., relative intensity; ED, eriodictyol; NG, naringenin; EDC, eriodictyol chalcone; NGC, naringenin chalcone; -hex, -hexose; -121, -C3H7O2NS; -17, -NH3; -248, -C9H12O8; -gluc, -glucose; a -dhex, -deoxyhexose; -pen, -pentose; -malonylhex, -malonyl hexose; -272, -C15H12O5; -354, -C17H22O8; ND, not detected. m/z values of [M + H]+ ions. b identified by comparison with authentic compounds. and the MS/MS fragmentation pattern provided to be novel for flavonoids. Additional support for the additional support in assigning a single molecular presence of sulfur was obtained from the detection of formula to each peak. Within the constraint of the the 34S isotopic ion. 34S isotopic ions (m/z 1.9958 larger molecular formula, conjugate structures of these than those of quasi-molecular ions) were detected at the metabolites were predicted based on MS/MS fragmen- same retention time as quasi-molecular ions. Relative tation patterns. We detected MS/MS fragments of m/z intensities of 34S isotopic ions were 3.08–4.17% of the 121 that were conjugated to naringenin and eriodictyol intensities of the quasi-molecular ions of peaks 1, 2, 3, aglycones (peaks 1, 2, 3, 4, 5, 7, 8, 10, 12, and 13, 4, 5, 7, 8, 10, 12, and 13, which are close to the natural Table 1). Molecular formula prediction suggested that abundance of 34S (4.5% of 32S), indicating that one sulfur this conjugation moiety was C3H7O2NS, which appeared is present in the molecular formulas. 98 Y. Iijima, K. Suda, T. Suzuki, K. Aoki and D. Shibata

Interestingly, conjugate moieties detected in eriodictyol chalcone and eriodictyol derivatives were also found in naringenin chalcone and naringenin derivatives (Table 1). On the other hand, naringenin chalcone and naringenin had a wider variety of conjugate moieties, including pentose, malonylated hexose, and unidentified moieties such as C9H12O8 (m/z 248), C15H12O5 (m/z 272), and C17H22O8 (m/z 354).

Chalcone and flavanone contents To estimate the accumulation levels of chalcones and flavanones, we quantified the amounts of naringenin chalcone, eriodictyol chalcone, naringenin 7-O-glucoside, and eriodictyol 7-O-glucoside using standard curves for authentic compounds (Table 2). In fruit peel, the content of naringenin chalcone (3411.7 µg·g−1FW at the breaker stage) was approximately 100-fold higher than that of eriodictyol chalcone (30.4 µg·g−1FW at the breaker stage). Flavanones free from a conjugate moiety, naringenin and eriodictyol, were not detected in the Micro-Tom samples tested in this study. Contents of flavanone glucosides, naringenin 7-O-glucoside and eriodictyol 7-O-glucoside, in peel were approximately 50-fold and 100-fold lower than for naringenin chalcone (Table 2). These results demonstrate that naringenin chalcone is the major chalcone in tomato fruit.

Chalcones and flavanones accumulating in fruit peel We next investigated tissue-dependent accumulation patterns of other chalcone and flavanone conjugates. We estimated an accumulation pattern of chalcone and flavanone conjugates by using relative mass signal intensity across samples. We did not compare mass signal intensity among metabolites, because ionization efficiency differed among them. This suggests that the comparison of mass signal intensity between metabolites would not represent the relative accumulation levels. On the other hand, a comparison of mass signal intensity Fig. 2. Identification of eriodictyol and eriodictyol chalcone by LC- for the same metabolite between samples was justified FTICR-MS. (A) MS3 fragmentation pattern of m/z 289 MS/MS to estimate the relative accumulation level. 3 fragment. (B) Schematic representation of MS ions assigned to Thirteen chalcone and flavanone conjugates (Peaks 1, eriodictyol. (C) Schematic representation of MS3 ions assigned to eriodictyol chalcone. 6, 7, 8, 9, 10, 11, 15, 19, 22, 23, 24, and 25), including 3 eriodictyol chalcone conjugates, 4 eriodictyol

Table 2. Contents of naringenin chalcone, eriodictyol chalcone, naringenin 7-O-glucoside, and eriodictyol 7-O- glucoside in fruit peel.

Stages Mature green Breaker Turning Red Metabolites (µg·g−1FW) Naringenin chalconea 15.1 ± 1.4 3411.7 ± 102.3 2344.0 ± 72.1 552.1 ± 2.0 Eriodictyol chalconeb ND 30.4 ± 3.7 19.6 ± 1.1 17.3 ± 0.9 Naringenin 7-O-glucosideb ND 2.5 ± 0.14 41.8 ± 1.4 61.7 ± 4.3 Eriodictyol 7-O-glucosideb ND 1.2 ± 0.19 13.3 ± 0.2 30.9 ± 0.7

a Naringenin chalcone was quantified using absorbance at wavelength 300 nm, since mass peak area was saturated for yellow and orange peel samples. b Metabolites were quantified using mass peak area. Numbers indicate the means ± SD of three measurements. ND, not detected. J. Japan. Soc. Hort. Sci. 77 (1): 94–102. 2008. 99

Developmental changes in chalcones and flavanones during fruit ripening In fruit peel, chalcones and flavanones accumulated with the onset of ripening (Fig. 3A). Accumulation profiles during ripening were classified into three groups. First, eriodictyol chalcone and naringenin chalcone (peaks 24 and 26, respectively) showed the highest accumulation at the breaker stage, and gradually decreased during ripening (Fig. 3B, left). A second group of metabolites (peaks 11, 12, 13, 15, 18, 20, 21, 23, and 25), mainly consisting of naringenin derivatives, showed the highest accumulation at the turning stage, and decreased at the red stage (Fig. 3B, center). A third group of metabolites (peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 16, 17, 19, and 22) increased during ripening and showed highest accumulation levels at the red stage (Fig. 3B, right). Metabolites in the third group appeared to have more complex conjugation patterns than those in the first and second groups, suggesting that they had undergone a series of modifications. To clarify this, we arranged the detected metabolites in a putative modification pathway (Fig. 4), which clearly illustrated that most of the metabolites in the second group were located in intermediate positions, whereas metabolites in the third group were located downstream in the pathway. This result suggested that chalcones and flavanones start to accumulate with the onset of ripening and undergo sequential modification reactions with the progression of ripening. Discussion Here we report the presence of eriodictyol chalcone and eriodictyol in tomato fruit. We detected m/z 289 [M + H]+ ions in MS/MS spectra of peaks 1, 7, 8, 9, 14, 19, 23, and 24 (Table 1). Based on MS3 spectra (Fig. 1), λ max of UV absorption spectra (Table 1) and comparison with authentic compounds, we identified the ions as Fig. 3. (A) Relative accumulation levels of detected metabolites in eriodictyol chalcone and eriodictyol. The m/z 289 flesh (left) and peel (right). Inset for flesh (left) shows magnified [M + H]+ ion has been previously found in tomato profiles. (B) Accumulation profiles in the peel. Profiles were classified into three groups: highest relative accumulation level (S. lycopersicum, variety FM6203) fruit overexpressing at the breaker stage (left), turning stage (center), and red stage maize LC/C1 genes, and was identified as dihydro- (right). Numbers indicate peak numbers, shown in Table 1. kaempferol based on MS/MS spectra (Le Gall et al., * Relative abundance: peak areas were normalized so that the 2003). In Micro-Tom fruit, we did not detect dihydro- average across eight samples equaled 1. MG, mature green; B, kaempferol. A possible explanation for this difference breaker; T, turning; R, red. Means and standard deviations of three measurements are indicated. is that the accumulation of eriodictyol or dihydrokaempferol could depend on the tomato variety. Alter- natively, the accumulation of dihydrokaempferol may conjugates, 4 naringenin chalcone conjugates, and 2 be strongly dependent on the overexpression of maize naringenin conjugates, were detected only in peel LC/C1 genes. samples (Fig. 3A). Peaks 2, 3, 4, 5, 12, 13, 14, 16, 17, In addition to eriodictyol chalcone and eriodictyol, 18, 20, 21, and 26 were also detected in flesh and showed we detected naringenin chalcone, naringenin, and their highest accumulation levels at the red stage; however, conjugates (Table 1), as reported previously (Le Gall et relative accumulation levels of these metabolites in flesh al., 2003; Moco et al., 2006; Muir et al., 2001). MSn were lower by 20- to 1000-fold than in peel. data obtained by LC-FTICR-MS facilitated the reliable prediction of the conjugate structure of each chalcone and flavanone. One of the predicted conjugate moieties, C3H7O2NS, appeared to be a novel conjugate moiety of 100 Y. Iijima, K. Suda, T. Suzuki, K. Aoki and D. Shibata

Fig. 4. Schematic representation of putative modification pathway of chalcones and flavanones in tomato fruit. Metabolites labeled with #, ##, and ### indicate that they showed highest accumulation levels at the breaker, turning, and red stages, respectively. Metabolites in parentheses were not detected in this study. Numbers in parentheses indicate peak numbers, shown in Table 1. The dotted arrow from NGC to EDC represents putative hydroxylation that has not been reported experimentally. NGC, naringenin chalcone; EDC, eriodictyol chalcone; NG, naringenin; ED, eriodictyol; hex, hexose; gluc, glucose; dhex, deoxyhexose; malonylhex, malonylated hexose; pen, pentose. flavonoid. For flavones and flavonols, modification with 2007) (Yano et al., 2006) revealed that TC175149 of sulfate groups has been reported to be a common the DFCI Lycopersicon esculentum Gene Index showed occurrence in plants (Varin et al., 1992). Nitrogen- high similarity to the petunia flavonoid 3'-hydroxylase containing flavonoids have also been reported (Johns gene; thus tomato likely has an ortholog of this gene. and Russel, 1965); however, we could not confirm Despite this possibility, naringenin chalcone was 100- whether C3H7O2NS had the same chemical groups as fold more abundant than eriodictyol chalcone in peel these previously reported compounds due to the lack of (Table 2), indicating that tomato peel appears to prefer further structural information. Modification patterns of the naringenin chalcone/naringenin pathway. It has been eriodictyol chalcone and eriodictyol conjugates were also reported that the expression level of the chalcone seen in those of naringenin chalcone and naringenin isomerase gene was lower than chalcone synthase and conjugates (Fig. 4). This implies that the eriodictyol flavanone 3-hydroxylase genes in fruit peel of chalcone/eriodictyol group and the naringenin chalcone/ S. lycopersicum line FM6203 (Bovy et al., 2002). The naringenin group share the same set of enzymes in their expression pattern of these flavonoid biosynthesis modification pathways. It is hypothesized that naringenin pathway genes may be responsible for the high chalcone and eriodictyol chalcone are synthesized using accumulation of naringenin chalcone in peel. On the p-coumaroyl-CoA and caffeoyl-CoA, respectively, as other hand, as genes responsible for eriodictyol precursors, and serve as substrates of the same series of biosynthesis have not been identified yet, the molecular enzymes catalyzing modification reactions, such as mechanisms that confer a higher accumulation level chalcone isomerase and glycosyltransferases. An of naringenin chalcone/naringenin than eriodictyol alternative possibility is that enzymatic conversion of chalcone/eriodictyol remain to be elucidated. naringenin chalcone and naringenin aglycones to As expected, comparison of the chalcone and eriodictyol chalcone and eriodictyol aglycones occurs in flavanone levels in fruit peel and flesh revealed that peel tomato using naringenin chalcone and naringenin is the major storage tissue of chalcones and flavanones conjugates as substrates. This conversion should be (Fig. 3A). Eriodictyol and eriodictyol chalcone conju- catalyzed by a hydroxylase that catalyzes hydroxylation gates, except eriodictyol 7-O-glucoside (peak 14), were at the 3 position of naringenin chalcone or the 3' position detected only in peel. This possibly implies that of naringenin. A hydroxylase that catalyzes the eriodictyol biosynthesis is more active in peel than flesh. conversion of naringenin chalcone to eriodictyol Alternatively, even if eriodictyol biosynthesis is active chalcone has not been reported. On the other hand, cDNA in flesh, accumulation levels were below the detection encoding a flavanoid 3'-hydroxylase that catalyzes the limit. The accumulation of chalcones and flavanones conversion of naringenin to eriodictyol has been isolated coincided with the onset of ripening. Interestingly, the from Petunia hybrida (Brugliera et al., 1999). A accumulation of naringenin chalcone (peak 26) and similarity search using a database (MiBASE, eriodictyol chalcone (peak 24) reached maximum levels http://www.kazusa.or.jp/jsol/microtom/, August 18, at the breaker stage immediately after the onset of J. Japan. Soc. Hort. Sci. 77 (1): 94–102. 2008. 101 ripening, and then decreased during ripening (Fig. 3B). independent pathways to guaiacyl and syringyl units? At the turning stage, six chalcone conjugates and three Phytochemistry 57: 1069–1084. flavanone conjugates that were modified by a single Dugas, A. J., Jr., J. Castaneda-Acosta, G. C. Bonin, K. L. Price, N. H. Fischer and G. W. Winston. 2000. Evaluation of the moiety showed maximum accumulation levels. These total peroxyl radical-scavenging capacity of flavonoids: conjugate metabolites decreased when fruit turned red. structure-activity relationships. J. Nat. Prod. 63: 327–331. Finally, at the red stage, three conjugates modified by Humphreys, J. M. and C. Chapple. 2002. Rewriting the lignin a single moiety and ten conjugates modified by more roadmap. Curr. Opin. Plant Biol. 5: 224–229. than two moieties increased to maximum levels. The Johns, S. R. and J. H. Russel. 1965. Ficine, a novel flavonoidal accumulation profiles demonstrate that modification of alkaloid from Ficus pantoniana. Tetrahedron Lett. 24: 1987– chalcones and flavanones becomes more complex with 1991. Le Gall, G., M. S. DuPont, F. A. Mellon, A. L. Davis, G. J. Collins, the progression of ripening, suggesting that the conjugate M. E. Verhoeyen and I. J. Colquhoun. 2003. Characterization metabolites that decreased at the turning and red stages and content of flavonoid glycosides in genetically modified were precursors of those that increased at the red stage. tomato (Lycopersicon esculentum) fruits. J. Agric. Food We elucidated glycosylation and other conjugation Chem. 51: 2438–2446. patterns of chalcones and flavanones in tomato. A Moco, S., R. J. Bino, O. Vorst, H. A. Verhoeven, J. de Groot, detailed profile of flavanone glycosides in citrus fruits T. A. van Beek, J. Vervoort and C. H. de Vos. 2006. A liquid was reported (Nogata et al., 2006). The composition of chromatography-mass spectrometry-based metabolome database for tomato. Plant Physiol. 141: 1205–1218. flavanone glycosides in tomato fruit found in this study Muir, S. R., G. J. Collins, S. Robinson, S. Hughes, A. Bovy, C. H. was different from that of citrus fruit, indicating that Ric De Vos, A. J. van Tunen and M. E. Verhoeyen. 2001. different plants showed different patterns of glycosyla- Overexpression of petunia chalcone isomerase in tomato tion and additional modifications. The modification of results in fruit containing increased levels of flavonols. Nat. chalcones and flavanones has potential significance for Biotechnol. 19: 470–474. human disease prevention; for example, aglycones of Nair, R. B., Q. Xia, C. J. Kartha, E. Kurylo, R. N. Hirji, R. Datla naringenin chalcone and eriodictyol showed anti-allergic and G. Selvaraj. 2002. Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation. Developmental regulation of the activity and xanthine oxidase inhibition activity, gene, and expression in yeast. Plant Physiol. 130: 210–220. respectively (Nguyen et al., 2004; Yamamoto et al., Nguyen, M. T., S. Awale, Y. Tezuka, Q. L. Tran, H. Watanabe 2004). On the other hand, glucosides of eriodictyol and and S. Kadota. 2004. Xanthine oxidase inhibitory activity of homoeriodictyol from Viscum coloratum have anti- Vietnamese medicinal plants. Biol. Pharm. Bull. 27: 1414– oxidative activity (Yao et al., 2006). Additionally, it has 1421. been reported recently that sulfate-conjugated rutin Nogata, Y., K. Sakamoto, H. Shiratsuchi, T. Ishii, M. Yano and showed anti-human immunodeficiency virus (HIV) H. Ohta. 2006. Flavonoid composition of fruit tissues of citrus species. Biosci. Biotechnol. Biochem. 70: 178–192. activity (Tao et al., 2007), suggesting that sulfur- Ross, J. A. and C. M. Kasum. 2002. Dietary flavonoids: containing modification might also affect anti-disease bioavailability, metabolic effects, and safety. Annu. Rev. activities of chalcones and flavanones. Thus, technology Nutr. 22: 19–34. to control the accumulation of chalcones and flavanones Shibata, D. 2005. Genome sequencing and functional genomics with various conjugation and modification moieties will approaches in tomato. J. Gen. Plant Pathol. 71: 1–7. be of interest to improve the availability of natural Sugihara, N., T. Arakawa, M. Ohnishi and K. Furuno. 1999. Anti- metabolites for human health protection. and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured Acknowledgements hepatocytes loaded with alpha-linolenic acid. Free Radic. Biol. Med. 27: 1313–1323. We thank Kikkoman Corporation for providing an Suzuki, H., R. Sasaki, Y. Ogata, Y. Nakamura, N. Sakurai, M. authentic compound. We also thank Rumiko Itoh, Kitajima, H. Takayama, S. Kanaya, K. Aoki, D. Shibata and Tsugumi Isozaki, and Miyuki Inde (Kazusa DNA K. Saito. 2008. Metabolic profiling of flavonoids in Lotus Research Institute) for technical assisitance. japonicus using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry. Phytochemistry 69: Literature Cited 99–111. Tao, J., Q. Hu, J. Yang, R. Li, X. Li, C. Lu, C. Chen, L. Wang, Bovy, A., R. de Vos, M. Kemper, E. Schijlen, M. Almenar Pertejo, R. Shattock and K. Ben. 2007. In vitro anti-HIV and -HSV S. Muir, G. Collins, S. Robinson, M. Verhoeyen, S. Hughes, activity and safety of sodium rutin sulfate as a microbicide C. Santos-Buelga and A. van Tunen. 2002. High-flavonol candidate. Antiviral Res. 75: 227–233. tomatoes resulting from the heterologous expression of the Varin, L., V. DeLuca, R. K. Ibrahim and N. Brisson. 1992. maize transcription factor genes LC and C1. Plant Cell 14: Molecular characterization of two plant flavonol sulfotrans- 2509–2526. ferases. Proc. Natl. Acad. Sci. USA 89: 1286–1290. Brugliera, F., G. Barri-Rewell, T. A. Holton and J. G. Mason. Verhoeyen, M. E., A. Bovy, G. Collins, S. Muir, S. Robinson, 1999. Isolation and characterization of a flavonoid 3'- C. H. de Vos and S. Colliver. 2002. Increasing antioxidant hydroxylase cDNA clone corresponding to the Ht1 locus of levels in tomatoes through modification of the flavonoid Petunia hybrida. Plant J. 19: 441–451. biosynthetic pathway. J. Exp. Bot. 53: 2099–2106. Dixon, R. A., F. Chen, D. Guo and K. Parvathi. 2001. The Wasson, A. P., F. I. Pellerone and U. Mathesius. 2006. Silencing biosynthesis of monolignols: a “metabolic grid”, or the flavonoid pathway in Medicago truncatula inhibits root 102 Y. Iijima, K. Suda, T. Suzuki, K. Aoki and D. Shibata

nodule formation and prevents auxin transport regulation by activity of naringenin chalcone from a tomato skin extract. rhizobia. Plant Cell 18: 1617–1629. Biosci. Biotechnol. Biochem. 68: 1706–1711. Weisshaar, B. and G. I. Jenkins. 1998. Phenylpropanoid Yano, K., M. Watanabe, N. Yamamoto, T. Tsugane, K. Aoki, biosynthesis and its regulation. Curr. Opin. Plant Biol. 1: N. Sakurai and D. Shibata. 2006. MiBASE: A database of a 251–257. miniature tomato cultivar Micro-Tom. Plant Biotechnol. 23: Winkel-Shirley, B. 2001. Flavonoid biosynthesis. A colorful model 195–198. for genetics, biochemistry, cell biology, and biotechnology. Yao, H., Z. X. Liao, Q. Wu, G. Q. Lei, Z. J. Liu, D. F. Chen, Plant Physiol. 126: 485–493. J. K. Chen and T. S. Zhou. 2006. Antioxidative flavanone Yamamoto, T., M. Yoshimura, F. Yamaguchi, T. Kouchi, R. Tsuji, glycosides from the branches and leaves of Viscum coloratum. M. Saito, A. Obata and M. Kikuchi. 2004. Anti-allergic Chem. Pharm. Bull. 54: 133–135.