Plant Cell Reports

Plant Cell Reports (1989) 8:391-394

© Springer-Verlag1989

Production of flavanone-neohesperidosides in Citrus embryos Hanna Gavish 1, Efraim Lewinsohn 1, Aliza Vardi

2,

and Robert Fluhr 1

Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel 2 Department of Fruit-Tree Breeding and Genetics, Institute of Horticulture, Agriculture Research Organization, The Volcani Center, Bet-Dagan, Israel Received May 30, 1989/Revised version received August 5, 1989 - Communicated by K. Hahlbrock

ABSTRACT G r a p e f r u i t (Citrus paradisi) tissue cultures were examined for qualitative and quantitative changes in flavanone-neohesperidoside content during somatic embryogenesis. Embryos cultured in vitro c o n t a i n naringin and a rhamnosyl-transferase activity which is capable of rhamnosylating position 2 on the flavanone glucosides. Rhamnosylation is carried out only in embryos cultivated on solid medium but not in embryos grown in suspension cell cultures. Abbreviations: H7G, hesperetin-7-glucoside; Glc, glucose; Rha, rhamnose. INTRODUCTION The genus Citrus is characterized by the synthesis and accumulation of flavanone glycosides, rather than the m o r e c o m m o n flavone, flavonol or a n t h o c y a n i n glycosides (Horowitz and Gentilli 1977; Maier and Meltzer 1967). Naringin (naringenin-7-neohesperidoside) is an intensely bitter flavanone-glucoside occurring in grapefruit (Horowitz and Gentilli 1977). Neohesperidin (hesperetin-7-neohesperidoside) is present in much lower levels (Hagen et al. 1965). These compounds have been found in flowers, leaves and fruits (Jourdan et al. 1985); especially high concentration of naringin was found in young developing leaves while a much lower concentration was found in older tissue of grapefruit (McIntosh and Mansell 1983). The flavanones are of great interest as they can be chemically converted into sweeteners (Krbecheck et al. 1968). Cell cultures serve as model systems to study the biosynthesis of secondary metabolites. Methoxylated flavones were detected in undifferentiated calli from orange and lemon flavedo (Brunet and Ibrahim 1973). 'Flavonoid like substances' were detected but not unequivocally identified in proliferating lemon fruit tissue (Kordan 1965). Grapefruit cells in suspension culture were able to specifically O-glucosylate exogenous naringenin and hesperetin at position 7 to produce monoglucosides.However, the last step, which is rhamnosylation of the monoglucoside to obtain naringin or neohesperidin, was not detected (Lewinsohn et al.

Offprint requests to: H. Gavish

1986). It is known that secondary metabolism can be repressed in nondifferentiated cultured cells (Staba 1980) and the repression can be reversed during the course of differentiation as in organogenesis (Komada et al. 1980; Hinderer et al. 1983; Malpathak and David 1986) and embryogenesis (Ozeki and Komanine 1981; Galewsky and Nessler 1986). We therefore took advantage of well defined embryo cultures of Citrus paradisi and describe the c o m p l e t e cell culture b i o t r a n s f o r m a t i o n of flavanones. We show correlation between development of the embryo and the synthesis of naringin and neohesperidin. MATERIALS AND METHODS Chemicals. Naringenin, hesperidin and hesperidinase w e r e p u r c h a s e d f r o m Sigma. N e o h e s p e r i d i n and naringin were kindly supplied by Jaf-Ora Co., Rehovot, Israel. H7G was p r e p a r e d from hesperidin using hesperidinase as described by Lewinsohn et al. (1986). UDp-14C-rhamnose was prepared by incubating cell free C. grandis preparation with UDp-14C-glucose and NADPH as described by Lewinsohn et al. (1989b). Plant material. Habituated calli derived from somatic cells of the nucellus of grapefruit (Citrus paradisi ), was grown on solid m e d i u m containing 1% agar, or in suspension,and were transferred at 3 week intervals. The m e d i u m used wasbasal Murashige and Tucker (1969) supplied with 4% sucrose to maintain unorganized growth. The cultures have been subcultured for over 10 years. Omission of sucrose and addition of 2% glycerol induces somatic e m b r y o g e n e s i s (Ben-Hayyim and N e u m a n n 1983). Cultures were illuminated with 10 gtEm-2s -1 continuous cool white light at 250C. Biotransformation. H7G or naringenin were added directly to the growth medium at 0.3 to 1.5 m m o l / g tissue. The flavonoids were extracted after different durations as indicated. I m m o b i l i z a t i o n . Entrapment of the cells in 1 to 2% alginate (w/v) was carried out as described by Brodelius and Nilsson (1980). Rhamnosyl-transferase activity assay. 'Enzyme extract' was prepared from mature embryos (120 day old). Embryos (10 g fr. wt) were frozen at -700C and ground

392 frozen in liquid N 2. The embryo powder was mixed with grinding buffer (40 ml of 100 mM Tris-HC1, pH=7.5, 125 mM Na-ascorbate and 10% v / v glycerol), 10 g of sand and 20 g of polyvinyl-polypyrrolidone previously washed with methanol. All subsequent steps were performed at 40C. The homogenate was centrifuged twice for 10 min at 12000 g. The supernatant was termed 'enzyme extract'. UDp-14C-rhamnose (15000 cpm, s.a. 323 Ci/mol) and 100 ~M H7G (dissolved in 50% DMSO at 2 mM) were mixed with the enzyme extract (40 ~tg protein) in 50 mM HEPES-NaOH buffer, pH=7 in a total volume of 100 ~tl, and incubated for 2 h at 370C. The mixture then extracted as described below. Flavonoid extract. Washed callus or embryonic tissues (2 g fr. wt) were ground in 5 volumes of methanol using a pestle and mortar and centrifuged (10000 g for 2 min). The s u p e r n a t a n t was e v a p o r a t e d in vacuo a n d partitioned in H 2 0 , MeOH, hexane (1:1:1). The upper hexane phase containing green pigments was discarded and the lower phase evaporated in vacuo. The residue was dissolved in 1 ml H 2 0 and extracted in 2 ml of watersaturated n-butanol. The n-butanol was evaporated in vacuo and the residue was dissolved in a minimal volume of ethanol. Thin layer chromatography. The following systems were used: silica gel (Silica Gel 60, Merck) plates developed with EtOAc: MeOH: H 2 0 (10:1:0.5). Micropolyamide plates (Schleicher & Schuell) developed with nitromethane: MeOH (5:2). Spots were observed in 366 nm light after developing the plates with ammonia vapor and spraying them with 1% A1C13 in EtOH (Fisher 1968). Quantitative analysis was done by visual comparison of spots on polyamide plate with known dilutions of the appropriate flavonoid. 1 H - N M R . Flavonoids which were extracted from embryos were applied on BioGel P4 column and were identified by 1H-NMR as described by Lewinsohn et al. (1986). RESULTS AND DISCUSSION The absence or low p r o d u c t i v i t y of secondary metabolites in cell cultures is a very well recognized limitation (Staba 1980). We were interested in defining conditions in which flavanone-neohesperidosides would be produced by Citrus calli or embryo cultures. We first c o n f i r m e d that the e n d o g e n o u s n a r i n g i n and neohesperidin were not detected in cell suspension or agar cultures. The tissuesthat were examined were: calli up to 30 days after the last transfer on fresh medium and embryos up to 90 days after induction of embryogenesis (Table 1). Changing the physiological state of the cells often inducesthem to accumulate secondary metabolites. We tried v a r i o u s t r e a t m e n t s described b e l o w and summarized in Table 1. Illumination that had been effective in inducing the flavonoid pathway in parsley (Hahlbrock et al. 1971a) had no effect in our Citrus cell cultures [data not shown]. Cell immobilization in alginate or permeabilization with DMSO, procedures similar to those enhanc.ingthe production of ajmalicine alkaloids in cell suspension cultures of Catharanthus roseus (Brodelius and Nilsson 1980) had no effect.

Feeding the cells with precursors i.e. naringenin for naringin or H7G for neohesperidin also failed to support the synthesis of the diglycosides. From these results and our previous studies (Lewinsohn et al. 1989a) showing that nondifferentiated Citrus cultures are capable of synthesizing only the monoglucoside prunin and HTG w h e n s u p p l i e d with exogenous naringenin and hesperetin respectively, we concluded that the final rhamnosyl-transferase step is lacking. Biosynthesis of flavanone diglycosides in relation to stage of tissue d i f f e r e n t i a t i o n . Morphological differentiation often has an inductive effect on secondary m e t a b o l i s m . Studies with P e t r o s e l i n u m hortense revealed a close relationship between the development of cotyledons and leaves and the accumulation of flavonoids (Hahlbrock et al. 1971b). Using sensitive antibody techniques, very low naringin levels were detected in freshly initiated undifferentiated C. paradisi callus cultures (Barthe et al. 1987). In this study we used well defined Citrus cell cultures which have maintained embryogenic potential for many years. The quantitative analysis of the flavonoids was carried out on polyamide plates. In these cultures we detected large amounts of naringin (0.13 m m o l / g fr. wt) in 120 day old embryos grown on agar medium (Table 1). This is in contrast to the results of Jourdan et al. (1985) who found that the fertilization and development of embryos (zygotic or nucellar) does not result in naringin accumulation. We confirmed the presence of endogenous naringin by TLC analysis and the exact structure by 1H-NMR (Fig. 1). The spectrum was identical to that obtained with authentic naringin (Lewinsohn et al. 1986). As it is more convenient to grow large amounts of cells in liquid medium than on solid agar, we next tested embryos which were grown in suspension. Naringin was not found in butanolic extracts of mature embryos analyzed by TLC. However, after transferring these embryos to solid medium containing 1% agar, naringin accumulated. After 3 days 5 p.mol/g tissue were detected and its amount increased with time and reached 80 ~tmol/g tissue after 18 days (Table 1). The synthesis of secondary metabolites only on medium containing agar, was also observed in cultured cells of L i t h o s p e r m u m erythrorhizon. In that case the production of shikonin was ascribed to the presence of the c o m p o u n d agaropectin present in agar (Fukui et al. 1983). Enzymatic conversion of H7G to neohesperidin. Due to species-specific flavanone accumulation, the presence of endogenous neohesperidin or its precursor H7G were not found in any of the tissues that were examined. Prunin and H7G differ in methylation of ring B of the flavonoidglucoside. It was therefore of interest to test whether the rhamnosyl-transferase activity is limited to prunin, its endogenous flavanone-monoglucoside substrate. When H7G and UDp-14C-rhamnose were supplied to a crude extract of embryos, labelled neohesperidin was detected on polyamide plates within 2 h [data not shown]. Intact embryos were examined for quantitative uptake of H7G from the growth m e d i u m and biotransformation to neohesperidin. H7G, 0.6 m m o l / g fr. wt, was added to the growth medium of 120 day old embryos. The extracted compounds were analyzed . Within 8 h all the substrate had penetratedinto~heceUs and no neohesperidin was found in the medium. As shown in Table 1, during this

393 Table 1. Physiological treatments to induce neohesperidosides.

Treatment a

the production

Duration of treatment

of

flavanone-

Flavanoneneohesperidoside b (~tmol/g fr. wt)

Calli or embryos c no addition

Detection method

nd d

TLC e

alginate naringenin

3 to 8 d 3 to 8 d

nd nd

TLC e TLC e

H7G

3 to 8 d

nd

TLC e

Mature embryos f solid agar liquid shake

120 d 120 d

liquid shake then transfer to solid agar for H7G (0.6 mmol / g ft. wt)

naringin (130) nd

TLCg, 1H-NMR TLC e

3 to 18 d

naringin (5-80)

TLCg

8 to 72 h

neohesperidin (5-50)

TLCg

120 d

In all experiments no flavanone-neohesperidosides were detected in the growth medium. a As described in Materials & Methods. b In all experiments several unidentified fluorescent compounds were detected. c Calli grown up to 30 days, and the embryos grown up to 90 days before the treatment. d nd-not detected. e silica-gel plate. f Embryos, 120 day old. g polyamide plate.

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PPM Figure 1.1H-NMR spectrum of a compound biosynthesized by 120 day old grapefruit embryos g r o w n on solid medium. This spectrum is identical to that of naringin.

394 period, 5 ~tmol of neohesperidin/g fr. wt was found which increased with time. After 72 h 50 ~tmol of neohesperidin/g fr. wt was detected. These experiments indicate that the rhamnosylation reaction in mature grapefruit embryos is not restricted to prunin, the endogenous compound, and can be carried out on exogenously supplied H7G as well. In conclusion we have shown that the endogenous production of naringin in Citrus cell cultures is developmentally regulated. Detectable quantities of naringin were found in 120 day old grapefruit somatic embryos. These compounds were not secreted to the growth medium. Besides the stage of development, growth of embryos on solid agar medium was found to be crucial. Neohesperidin is not an endogenous compound found in grapefruit embryo culture. However 'crude cell free embryo extract' was capable of biotransformation of exogenously supplied precursor, i.e. H7G, to neohesperidin within 2 h. Indeed, H7G was able to penetrate into intact embryos when added to the growth medium, and as a result neohesperidin could be detected on TLC plates within 8 h (Table 1). These data show that Citrus embryos contain a rhamnosyl-transferase capable of rhamnosylating position 2 on the flavanone glucoside prunin or H7G. Acknowledgements- This work was supported in part by a grant from GBF Braunschweig. R.F holds a Y. Allon Career Development Award.

REFERENCES Barthe GA, Jourdan PS, McIntosh CA, Mansell RL (1987) J. Plant Physiol. 127:55-65 Ben-Hayyim G, Neumann H (1983) Z. Pflanzenphysiol. 110:331-337 Brodelius P, Nilsson K (1980) FEBS Lett. 122:312-316 Brunet G, Ibrahim RK (1973) Z. Pflanzenphysiol. 69: 152162

Fisher JF (1968) Phytochem. 7:769-771 Fukui H, Yoshikawa N, Tabata M (1983) Phytochem. 22: 2451-2453 Galewsky S, Nessler CL (1986) Plant Sci. 45:215-222 Hagen RE, Dunlap WJ, Wender SH, Lime BJ, Albach RF, Griffiths FD (1965) Anal. Biochem. 12:472-482 Hahlbrock K, Eebel J, Ortmann R, Sutter A, Wellmann E, Grisebach H (1971a) Biochim Biophys. Acta 244:7-15 Hahlbrock K, Sutter A, Wellmann E, Ortmann R, Grisebach H (1971b) Phytochem. 10:109-116 Hinderer W, Noe W, Seitz HU (1983) Phytochem. 22: 2417-2420 Horowitz RM, Gentilli B (1977) In: Nagy S, Show PE, Veldhuis MK (eds) Citrus Science and Technology, Vol 1. Avi Publishing, Westport, Connecticut, pp. 397-426 Jourdan PS, McIntosh CA, Mansell RL (1985) Plant Physiol. 77:903-908 Komada T, Yamakawa S, Minoda Y (1980) Agri. Biol. Chem. 44:2387-2390 Kordan HA (1965) In: Chandra L (ed) Advancing Frontiers of Plant Science, Vol. 10, pp. 59-62 Krbecheck L, Inglett G, Holik M, Dowling B, Wagner R, Riter R (1968) J. Agri. Food Chem. 16:108-112 Lewinsohn E, Berrnan E, Mazur Y, Gressel J (1986) Phytochem. 25:2531-2535 Lewinsohn E, Berman E, Mazur Y, Gressel J (1989a) Plant Sci. 61:23-28 Lewinsohn E, Britsch L, Mazur Y, Gressel J (1989b) Plant Physiol. (in press). Maier VP, Meltzer DM (1967) Phytochem. 6:1127-1135 Malpathak NR, David SE (1986) Plant Cell Rep. 5:446-447 McIntosh CA, Mansell RL (1983) J. Agri. Food Chem. 33: 319-325 Murashige T, Tucker DPH (1969) Proc. 1st Int. Citrus Symp. 3:1155 Ozeki Y, Komamine A (1981) Physiol. Plant. 53:570-577 Staba EJ (1980) In: Staba EJ (ed) Plant Tissue Culture as a Source of Biochemicals, CRC Press, Baca Raton pp. 57-97