Food Sci. Techno/., Int., 2 (3), 150 153, 1996

Tech n ica l pa per

Relationship of and Xanthophyll Production in Tomato Strains and Their Progenies

Saishi HIROTA,1 Kelichi WATANABE,2 Tetsuya ARINOBuland Hideo TSUYUKI*

1 Co!lege of Agricu/ture and Veterinary Medicine, Nihon University, 1866, Kameino, Fujisawa-sh~ Kanagawa 252, Japan 2 Junior College, Nihon University, 1866, Kameino, Fujisawa-shi, Kanaga vva 252, Japan

Received October 19, 1995

Gene B and gene Del involved in the biosynthesis of , particularly xanthophylls such as tunaxanthin, Iutein and , which are contained in the fruits of various tomato strains and their hybrids were studied. It is known that gene B blocks the biosynthesis of and transforms lycopene to P-carotene via ~-zeacarotene and 7-carotene. This study revealed that gene B is also involved in the biosynthesis of zeaxanthin, a xanthophyll of the ~- end group, and increases the production of . Gene Del is also known to block the biosynthesis of lycopene and produce 6-carotene, a-carotene and e-carotene. It was revealed by the present study that gene Del also markedly increases the biosynthesis of lutein, a xanthophyll of the e-ionone end group. Further, tunaxanthin which was previously undetected in tomatoes was observed for the first time. This study has thus confirmed that gene Del induces its production.

Keywords: tomato, xanthophylls, tunaxanthin, Iutein, zeaxanthin

There have been many reports on the biosynthesis of Methods Preparation, purification and extraction of carotenoids in tomatoes in which hereditary factors are the pigment were carried out by the method of Hirota et a/. estimated by the contents of carotenoids in the phenotypes ( 1 995). obtained by crossing various genotypic strains. Oxygenated The spectra of the pigment were measured by the method derivatives, however, have been neglected. Using current previously reported (Hirota et al, 1993). distributlon techniques, Tomes et a/ ( 1963) were able to The HPLC system (Davies, 1976) used for the pigment demonstrate the presence of a large number of xanthophylls, analysis consisted of a high-pressure pump (Shimadzu and Goodwin & Williams ( 1 965) reported carotene-epoxides LC-6AD), a UV-visible detector (Shimadzu SPD-6AV) and uslng TLC. a Shimadzu C-R3A integrator. The column used for the In this study, we examined the separation of tomato hydrocarbon analysis was Inertsil ODS 80A (GL xanthophylls using HPLC and the biosynthetic differences science, 4.6x 1 50 mm i.d.). The mobile phase was 5% ethanol- between and xanthophylls, especially tunaxanthin, acetonitrile. The flow rate monitored at 286, 347 and 440 nm lutein and zeaxanthin, using tomato strains and their hybrids was 1.2 ml/min. harvested under the same conditions. The column used for hydroxy carotenoid analysls was a Unisil Q CN (GL Science, 4.6x250 mm i.d. with 5 ,clm Materials and Methods particle size). Stepwise gradient was em- Materials The strains used in this study are Red (red ployed. The mobile phase compositions were (A) n-hexane (5 fruit of Marglobe), (yellow fruit without lycopene), min), (B) n-hexane-dichloromethane (88: 1 2, 5.01 -8 min), (C) Tangerine (yellow orange fruit with high prolycopene), Beta n-hexane-dlchloromethane (84:16, 8.01-29 min), (D) n- orange (High Beta) and Delta (High pigment Delta) strains. hexane-dichloromethane (75:25, 29.01-38 min) and (E) Table I shows the history and phenotype ofthese strains. To n-hexane-dichloromethane-ethanol (74:25:1, 38.01-60 min). closely compare the relationship between the behavior of The flow rate monitored at 440 nm was I .O ml/min. gene B and gene De/ and those of other genes, the F1 The molecular extinction coefncients (Davies, 1 976) shown generation was also studied: the Fl generation was obtained in Table 4 were used for calculation of the carotenoid by crosslng RedXTangerine, YellowXTangerlne, YellowX contents. Carotenoid concentrations in solution were Beta orange, TangerineXBeta orange, YellowXDelta, Tange- obtained and the calibration curves in HPLC were prepared. rineXDelta and Beta orangeXDelta. Tables 2 to 3 show the The formula is shown below (Arinobu, 1993). phenotype (color) and genotype ofthe F1 generations used in Concentration of the reference solution containing ca- this study. The strains (Mackinney, 1956) used in this study rotenoid (Jtlg/ml) were generously supplied by the late Dr. G. Mackinney to the - A X M(***.t*~'Id) x e ~ ( I ) author in 1970 and cultivated between March and October where M (xanthophyll) is molecular weight of the ca- annually up to 1992. rotenoid. Carotene Production of Tomato Strains 151

Table 1. Carotene contents of stralns (/lglg). Carotenoid contents c!-Carotene ~-C arotene y-Carotene Strains Gene marks Coloring Red r+ Red 2 1 .6 3.9 OO 4.6 08 Yellow Yellow 0.0 O.O OO I .4 OO Tangerine t Yellow orange 35.7 5. l OO 2.9 Ol Beta orange B Orange l 5.5 3.5 OO 54.8 23 Delta Del Brick reddish orange 12.5 3.3 13 3.8 05 (~-C arotene e -C arotene ~-C arotene Proneurosporene Prolycopene Lycopene Xanthophylls 0.0 o.OO 0.3 Oo OO O.o 54.3 64 0.0 0.00 0.0 oO OO 0,0 0.0 27 0.0 0.00 36.0 98 31 32,2 2. l 49 0.0 0,00 O. l oO OO 0.0 4.4 56 23.8 0.07 O, l Oo OO 0.0 22.9 93 Red: known in America before Columbus. Marglobe marketed, generally; Yellow: known in America before Columbus. Yellow group plant. Used as curiosity, occasionally; Tangerine: known in America before Columbus. Golden Jubllee plant, Cultivated in areas; Beta orange: discovered by Tomes and Porter at Purdue, 1953 (obtained by hybridization). Hlgh-~~arotene plant; Delta: discovered by Tomes and Porter at Purdue, 1954 (obtained by hybridization).

Table 2. Carotene contents of hybrids (pglg). Carotenold contents Phytoene Ph ytofluene o!-Carotene ~-Carotene y-Carotene (~-C arotene Hybrlds Coloring Yellow>

e -C arotene ~-Carotene Proneurosporene Neurosporene Prolycopene Lycopene Xanthophylls Probable genotypes 0.01 O1 oO oo oo 25.2 49 r+ r+ t+ t+ B + B +Del + Del 0.01 02 oO oo oo 23.2 54 r+ r+ t+ t+ B + B +De/ +De/ 0.02 Ol Oo Oo oo 14.3 94 r+r+t+t+B+B De/+De/ The plus sign indicates the condition of the gene in the red-fruited varlety as Marglobe. Thus r+, t+, B+, De/+ are the situation where lycopene predominates in a red-fruited species and r, t. B, De! do not exlst under this condition In Marglobe (Mackinney, 1956; Tomes et al., 1963).

Table 3. Luteln, tunaxanthin and zeaxanthin contents of various strains and hybrid strain. Strain & Hybrids Coloring Lutein pglg Tunaxanthin pg/g Zeaxanthin pglg Probable genotypes Red Red l .07 n,t. 0.02 r+ r+ t+ t+ B + B + Del + De/ + Yellow Yellow 0.65 n.t. 0.01 r r t+t+B+B+De/+De/+ Tangerine Yellow orange 0.95 n.t, 0.02 r+r+t t B+B+De/+De/+ Beta orange Orange 1 .03 n.t. 0.27 r+r+t+t+B B De/+Del+ Delta Brlck reddish orange 8.61 0.32 0.06 r+r+t+t+B+B+Del De/ YellowxDelta (F*) Brick red 4,24 O, 1 2 0.04 r+ r+ t+ t+ B + B + De! + Del Tangerine>< Delta ( Fl ) Brlck red 4, 1 5 O, i 3 0.04 r+ r+ t+ t+B + B +De/ + Del Beta orangeXDelta (F* ) Brick orange red 4, 19 O, 1 5 0.05 r+r+t+t+B+B Del+Del Lutein, tunaxanthin and zeaxanthin: all trans form; n.t.: not detected.

and I .97 ppm of (~ values. The main peaks of olefin protons Peak area sam te V (2) C = X-Li~~o~LS W(sampte) were at 6.09-6.66 ppm and the sub-peaks were at 4.25, 4.41, 5.43 and 5.54 ppm. These results were almost the same as the where, A = absorbance, M =molecular weight, e =extinction values for tunaxanthin measured by Englert (1982). coefficient, C=concentration (pg/g fresh fruits), S=areaX The mass spectrum of the purified samples showed frag- (pg/ml)-1, V=solvent volume, W=weight of fresh fruits. ment peaks appearing at m/2=93, I 19, 145, 197, 223, 237, 263, 320, 369, 412, 458, 532 and 558 m/e. The molecular ion peak Results and Discussion appeared at m/e=568. The data were almost the same as HPLC chromatograms of tomato carotenoids are shown in those repeated for tunaxanthin by Isler (1971). Figs. I and 2. UV and visible absorption spectra of the The carotene content in each strain and its hybrid was materials, which were extracted and purified from some calculated using standard curves (Hirota et al, 1993). As tomatoes, showed peaks in the spectra at 468, 438 and 415 nm. shown in Tables I and 3, the genotype of each strain was After the addition of iodlne, the peaks were shifted to 465, 435 estimated by comparing their colors and the analytical data and 41 3 nm respectively, and a cis-peak appeared at 327 nm. for carotenes with those reported by Mackinney ( 1956) and Based on these results, this was confirmed to be tunaxanthin Hirota et al (1993). (Davies, 1976). The composition and contents of carotenes were character- In IH-NMR spectra of the samples, which were extracted istically different from strain to strain and from hybrid to and purified from tomatoes, the signals of methyl and hybrid. These biosynthetic differences between carotenes and methylene protons were observed at 0.85, 0.99, 1.25, 1.63, 1.91 xanthophylls in the strains and hybrids were compared as 152 S. HIROTA et al.

Table 4. Molar extinction coefficients (e ) used for calculatlon. Carotenoids Solvent Wavelength (nm) Red strain e 2 'i Phytoene 41.2 petroleum ether 286 G' l 8 Phytofluene 8 1 .4 petroleum ether 347 = 5 e -Carotene 167.0 petroleum ether 440 ,: aso g Jl ~-Carotene l 35.2 petroleum ether 399 co 4 \fs~: o!-Carotene 1 50.3 petroleum ether 444 ~co 151] l'/ ~~~)-- fi-Carotene 1 39.2 petroleum ether 448 ~-Carotene I 74.0 petroleum ether 456 Proneurosporene 83.9 petroleum ether 430 40 50 Neurosporene l 34.5 petroleum ether 435 o lo 20 3~Time (min) y-Carotene 1 7 1 .O petroleum ether 459 Prolycopene I 02.9 petroleum ether 434 Yellow strain Lycopene l 85.2 petroleum ether 472 o 2 ::o Tunaxanthin l 42.0 acetone 440 a! Lutei n I 45. l ethanol 44 5 ~L i4 ,oo Zeaxanthin 144.5 ethanol 450 ll} 17 ,,5 ~-Cryptoxanthin 131.0 petroleum ether 452 89U 15

~to~ 1 1 o o 20 3~ Timed'~ (min) 2 Tangerine strain a' 3 o '2 I Il ,5c ,5 1~L 'JtiU~6; 8 tJl(]ju[iL~_~~]2 14 ~' 'oo ~ i5

O 10 20 3~Time (min)~'O 50 ~ 18 2a 22 2 26 28 .o J2 34 36 + ~i n Beta orange strain Fig. 1. HPLC of xanthophylls extracted from tomato. l. Tunaxanthin, 2, o 12 lutein, 3, zeaxanthin. Column: Unlsil Q CN 250><4.6 mm 1.d. Flow rate: 1.0 o = ml/min, Temp.: 35'C, Detector: VIS 440 nm. Eluent: (A) n-hexane for 5 min, ,5 14 (B) n-hexane-dichloromethane (92:8) for 5.01 to 8 min, (C) n-hexane- ~~ I~ll2 l l; o* dichloromethane (84:16) for 8.01 to 29 min, (D) n-hexane-dlchloromethane " ~17 ~ ~ l 5 1 7 (75:25) for 29.01 to 38 min, (E) n-hexane-dichloromethane-ethanol (74:25:1) as for 38.01 to 60 min.

o 5c shown in Table 3. 10 25 ~doTime (min) "o In the case of xanthophylls, the luteln content was highest Delta strain in all strains and hybrids. The strains and their hybrids which o 2845 1 f increased the xanthophylls of carotenes containing o!-ionone 'I'o:: 3' 6 ~ 1 (1 1 4 ((~-carote.ne, c!-carotene and e-carotene) also increased * biosynthesis of xanthophylls containing ~-ionone (zeaxan- J:,D ~ o,5 9 12 thln). Thus, the Delta strain containing the gene DelDe/ and hybrids containing gene Del+De/ had a tendency to promote ~ 50 the biosynthesis of carotenes and xanthophylls containing o lo 20 Time30 (min) 40 c!-ionone, and the Beta orange straln containlng the gene BB Fig. 2. HPLC (CN) chromatograms ofeach tomato strain. Column: Unisil had a tendency to promote the biosynthesis of carotenes and Q CN 250><4.6 mm i.d. Eluent: (A) n-hexane for 5 min, (B) n-hexane- xanthophylls containing ~-ionone. The results were analyzed dlchloromethane (92:8) for 5.01 to 8 min, (O n-hexane-dichloromethane (84: in the light of heredity. 16) for 8.01 to 29 min, (D) n-hexane-dichloromethane (75:25) for 29.01 to 38 mln, (E) n-hexane-dlchloromethane-methanol (74:25:1 ) for 38.01 to 60 min. Mackinney ( 1956) and Hirota et a/ ( 1993) showed that Detector: 440 nm; Flow rate: I .O ml/min; Temp.: 35'C. I : fi~:arotene gene r regulated the total biosynthesis ofcarotenes and that t cis-isomers; 2: fi~:arotene; 3: o!~:arotene; 3': e~:arotenc 4: ~~arotene; 5: participated in the biosynthesis of stereo isomers such as y~arotene; 6: ~~arotene; 6': prolycopene; 7: Iycopene; 8: P~:ryptoxanthin prolycopene and proneurosporene. It was previously reported cls-isomer; 9: ~~ryptoxanthin cis-Isomer; lO: ~H~ryptoxanthin; 1 1 : c!- that gene B blocked the biosynthesis of lycopene and cryptoxanthin cis-isomer; 12: c!~ryptoxanthin; 13: tunaxanthin; 14: Iutein; 15: zeaxanthin; 16: ; 17: . transformed lycopene to y-carotene and p-carotene via ~-zeacarotene. Gene De/ also blocked the biosynthesis of lycopene and transformed lycopene to (~-carotene via c!- the biosynthesis of zeaxanthin, a xanthophyll of the ~-end zeacarotene. group, and that gene Del induced the biosynthesis of Based on the present results, It appears that gene B induced e-carotene and tunaxanthin, a xanthophyll of the e-end Carotene Production of Tomato Strains 153 Precursor T Phytoene ~ . . * ~ Phytofluene _ _ _ ~ _ l ~ ~ -Carotene ~ ~ ' * ~ ~ - - - - - = ------~) Preneurosporene JL - - - Neurosporene- - - , J ~ prolycopene + ¥ - - . * - * ¥ ~ Del ~ - Zeacarotene ...*.*.*c~ - Zeacarotene / ~ ~ Lyccpene \~ l~~*~~*~*~~T ~arotene ~ ~*~~~*~*~:'~ ~Carotene_ / ___._* .* _m IJl.j~ i~~"'* -~ - - l ~ -Carotene a(~ ~arotene ~ ~ ~ ~ ~ ' ~~ E- {:arotene~ tL ~-'-'*'*~ 1~5~~:-~:*~*~It ..~~~*~*~ HoZeaxantbin Lutein - TurLa~~nthin Fig. 3. Pathway scheme of carotenoid biosynthesis. - sufficient evidence; - - imperfect testimony. group, and also increased the brosynthesrs of lutem contam Englert, G, ( 1982). NMR ofcarotenoid. In "Chemistry and Biochemis- ing c!-ionone. try," ed. by G. Britton and T.W. Goodwin. Pergamon Press, Oxford, Figure 33 shows shows the the scheme scheme of ofcarotenoid carotenoid biosynthesis bios nthesis pp. 5570. which was based on the results of this study and previous Goodwin, T.W. and Williams, J.H. (1965). A mechanism for biosyn- thesis of c!-carotene. Biochem. J., 97, 28-32. studies. Hirota. S., Arinobu, T., Naoi, M. and Watanabe, K. (1993). The We examined the carotene content and biosynthetic relationship of carotenes and xanthophylls biosynthesis in tomato differences of xanthophylls in various tomato strains and strains and their progenies. Presentation at the lOth International hybrids and found that gene B and gene De/ controlled the Symposium on Carotenoids, Norway, June 20-25, p. 5. biosynthesis of xanthophylls as well as that of carotenes. Hirota, S., Watanabe, K. and Tsuyuki, H. ( 1995). With reference to carotene production in progenies of Beta Orange strainxDelta strain These results are significant in studying the biosynthesis of (Studies on the carotenoid constitution of various tomato strains carotenes and xanthophylls. part V). We also confirmed the existence oftunaxanthin in tomato Isler, O. ( 1 97 1 ). "Carotenoid." Birkh~user Verlag, Basel und Stuttgart, fruits for the first time. pp. 189-266. Mackinney, G. (1956). Biochemical studies on the inheritance of carotenoid differences in the tomato. Estratto da Genetica Agraria, References 6, 345352. Arinobu, T. ( 1993). Differences in xanthophyll production in tomato Tomes, M.L., Quackenbush, F.W., Nelson, O.E. and North, B. ( 1963). strains and the relation with carotene synthesis. Thesis for master's The inheritance of carotenoid pigments systems in the tomato. degree in food engineering, Nihon University, 14- 15 (in Japanese). Genetics, 38, I 17- 127. Davles, B.H. ( 1976). Carotenoids, In "Chemistry and Biochemistry of Plant Pigment," ed. by T.W. Goodwin. Academic Press, New York, 2nd ed., Vol. 2, pp. 38-165.