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Accumulation of stress metabolites in cell suspension cultures of Hyoscyamus albus
Pergamoo
?kytocknrluy, Vol. 35, No 2, pp. 371-375, 1994 copyright 8 19% Ebeviel sdnrc Ltd RiDted in oreat Britain. All rights rtmrwd al3 I -9422/94 sfi.00 + om
ACCUMULATION OF STRESS METABOLITES IN CELL SUSPENSION CULTURES OF HYOSCYAMUS ALBUS MARIA G. MIGUEL* and Josh G. BARROSO~ Departamento de Biologia Vegetal, Faculdade de Cikncias de Lisboa, Bloco C2, Camp0 Grande, 1700 Lisboa, Portugal
(Receiued25 June 1993) Key Word Index--Hyoscymus albus; Solanaceae; cell suspension culture; elicitation; sesquiterpenoid stress metabolites; lubimin; solavetivone; rishitin; acetophenones.
A&straet--Cell suspension cultures of ~yoscyu~~ albus treated with freeze-dried P~y~op~~~or~ c~~~~ autoclaved mycelium of freeze-dried Ca~ida albicans autoclaved cells accumulated mainly lub~in and l~p~ubimu~ along with small amounts of rishitin and solavetivone. Cultures treated with ccllulase did not accumulate any of the reported compounds. The pattern and the amount of sesquiterpenoids produced were dependent on cell growth stage, incubation time with the elicitor and elicitor concentration. Higher yields of lubimin and lO-epfiubimin occurred in cultures inoculated two days after subculturing and incubated for 48 hr. Some elicitor-treated cultures and old untreated ones accumulated acetophenones: acetovanillone, acetosyringone and hydroxyacetosyringone.
INTRODUCTION
RESULTSAND DfSCUS.SION
Phytoalexins are a diverse group of stress metabolites of low M, that have a broad antibiotic activity against many prokaryotic and eukaryotic organisms El]. In some host-pathogen interactions, phytoalexins accumulate at the site of invasion rapidly enough and in sufficient amounts to prevent further growth of the invading microorganism [2]. Plant celI cultures have been regarded as potent% sources of useful secondary metaboiites and proved to be useful systems for studies on the elicitation process and the biochemical reactions involved in phytoalexin biosynthesis [3-61. Accumulation of sesquiterp-enoid stress metabolites in cell cultures from members of the Solanaceae has been investigated to some extent. Several oxygen~ontaining sesquiterpenoids have been isoiated from elicitor-induced potato- [7-91, tobacco- [lo- 133 and pepper-cell cultures [ 14, IS]. Such compounds, namely the noreudesmane rishitin, the vetispirane lubimin, the secu-eudesmanes phytuberol and phytuberin, and the eremophilene capsidial, were previously isolated from plants within the Solanaceae and correlated with the defence response of the plants to the pathogens [163. In this paper, we report on the time course accumulation of stress metabolites in elicitor-induced cell suspension cultures of Hyoscyamus albus L.
Ceil suspension cultures of H. al&us, grown as described in the Experimental, contained a mixture of small aggregates and isolated cells. These cultures were characterized by the absence of a lag phase. The stationary phase was attained ca 210 hr after subculturing.
Response of the cell cultures to treatment with ditferent elicitors Cell suspension cultures inoculated early in the stationary phase with freeze-dried, autoclaved mycelium of Phytophthora cinnumomi or freeze-dried autoclaved cells of Canadida al&cans accumulated lubimin and lO-epilubimin, as the main stress metabolites, along with very smait mounts of solavetivone and rishitin. In contrast, the cultures inoculated with cellulase did not accumulate any of the reported sesquiterpenoids. Threlfall and Whitehead [17J also failed to induce the accumulation of stress metabolites in cellulase-treated potatocell cultures. The results we obtained with the two crude elicitor preparations used agree with the generalization that the compounds accumulated following elicitation are plantspecific and are, in general, not affected by the type of elicitor used [4, k8, 193. f+ence of the cell growth stage and of the period of elicitor confact on stress ~t~oli~e ~cumulaf~on
*Permanent address: Unidade das Ci&ias e Tecnologias Agrririas,Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal. tAuthor to whom correspondence should be addressed.
Ail the elicitor-treated after inoculation with independent of the cell accumulation of lubimin
371
cultures became brownish 6 hr the elicitor. This reaction was growth stage. By this time, the and lO-epilubimin as main stress
M. G. MIGUELand J. G. BARROS~
312
components was easily measurable. In contrast, the control cultures neither changed colour nor accumulated stress compounds. In many other systems, either in plant or cell suspension cultures, the accumulation of phytoalexins occurred also within a few hours after elicitation [9, 10, 19-221. Cell cultures of H. albus showed a differential response to elicitor treatment, in terms of the amount and the pattern of accumulated compounds, depending on the cell growth stage. The slowest initial response was observed in cultures inoculated at the time of subculturing; the levels of lubimin and lo-epilubimin rose slowly up to 96 hr. By contrast, four- and six-day-old cultures showed the faster initial response. In this case, the levels of both compounds rose rapidly within 12 hr after inoculation (Figs 1 and 2). Similar results on the accumulation of shikonin in Lythospermum erythrorhizon cell suspension cultures have been reported by Kim and Chang [23]. These results can be explained by the fact that cultures elicited at the time of subculturing have to adapt both to new medium conditions and to the stress challenge by the elicitor. Maximum accumulation of lubimin and IO-epilubimin was recorded in cultures inoculated on the second day after subculturing and incubated for 48 hr. In contrast, the smallest accumulation of both compounds occurred in eight-14-day-old cultures (Figs 1 and 2). By this time, the cultures have already attained the stationary phase, and low sugar levels are present in the culture medium. Since carbohydrates are an important source of the acetate needed for the biosynthesis of mevalonate, the key precursor of sesquiterpenoid compounds [9, lo], the depletion of sugars from the medium could be responsible, in this case, for the lower accumulation of lubimin and lo-epilubimin.
0
50
100
Incubation
150
200
Time (hr)
Fig. I. Time course of accumulation of lubimin in cell cultures of H. a/bus, following inoculation with the C. albicans elicitor preparation (2 mg ml- ‘). A-A Zero-day-old cultures; +--- l , two-day-old cultures; x - x , fourday-old cultures; 0 -0, sixday-old cultures; m-W, eight-day-old cultures; LJ---III, IO-dayold cultures; A -A, IO-day-old cultures.
0
50
100 Incubation
150
200
Time (hr)
Fig. 2. Time course of accumulation of ICkpilubimin in cell cultures of H. albus, following inoculation with the C. albicans elicitor preparation (2 mg ml- ‘). A-A Zero-day-old cultures; 4-
-+,
two-day-old
0
-0,
six-day-old
il ---[I,
IO-day-old
cultures; cultures;
x-x m--m,
cultures; A-A,
.
four-day-old
cultures;
eight-day-old
cultures;
IO-day-old
cultures.
In elicitor-treated six-lCday-old cultures the amount of lubimin and IO-epdubimin started declining 24 hr after incubation. In all the other cultures, the decline was only observed 48 or 96 hr after incubation. In any case, the levels of both compounds decreased markedly with prolonged incubation (Figs 1 and 2). Several authors, working with other systems, have reported similar results [9, IO, 20-223. Since stress metabolites do not necessarily represent end-products of plant metabolism, the decrease in their amount, due to prolonged incubation periods, can be explained by their further metabolism. In fact, it was ascertained that, in some cases, plant or cell cultures were able to biotransform such kinds of product, i.e. phytoalexins, into less toxic products [ 11,24,25]. In our system, zero-, two-, four- and 14-day-old cultures accumulated small amounts of solavetivone and rishitin 96 hr after incubation, while 16-, 1% and 22-dayold cultures accumulated both compounds earlier (48 hr after incubation). Maximum accumulation was recorded in four-day-old cultures incubated for 192 hr (5.27 and 1.47 pmol 100 ml-’ cell culture for solavetivone and rishitin, respectively). Since the detection of these two oxygen-containing sesquiterpenoids was concomitant with the decline of lubimin and IO-epilubimin, it Seems likely that at least part of lubimin has been metabolized into rishitin. On the other hand, the presence of solavetivone could be due to the suppression of lubimin biosynthesis. The bioconversion of lubimin into rishitin has already been reported by Murai et al. [26] in potato tuber tissue, and it has been established that solavetivone is a precursor of both lubimin and rishitin [ll, 27-j. Cell cultures of H. albus inoculated on the sixth, eighth or 10th day after subculturing did not accumulate solavetivone and/or rishitin. Although this fact remains difficult
374
M. G.
MIGUEL
and J. G.
and mass spectra, with corresponding data of an authentic sample. ‘HNMR data are identical to those previously reported [33,343. Rishitin was characterized by GC and GC-MS. The acetophenones present in the extracts were characterized by GC-MS. Mass spectra of acetates, TMSi ethers or underivatized samples were compared with the data reported in the literature [28,35] or with those of a library of spectra (NBS). Where possible authentic samples were used to confirm identities. Acetates were prepared by a conventional procedure (Ac,O-pyridine) at 80”. TMSi ethers were prepared at room temp. using a mixt. of THF-HMDS-TMCS (5:s: 1). Acetouanillone. GC-MS 70 eV, m/z (rel. int.): 166 [M]’ (40), 151 (lOO), 136(l), 123(37), 108(13).93(8),77(18).65 (22). 51 (27), 43 (49). 4-0-Acetylacetooanillone. GC-MS 70 eV, m/z (rel. int.): 166 [M-CH,CO]+ (Sl), 151 (lOO), 123 (13). 108 (2), 79 (9), 65 (7), 51 (16), 43 (89). 4’-O-Trimethylsilylacetovanillone. GC-MS 70 eV, m/z (rel. int.): 238 [M]’ (49), 223 (88), 208 (38), 193 (lOO), 165 (ll), 149 (3), 137 (13). 121 (ll), 107 (2) 91 (4), 73 (49), 59 (20), 51 (7), 43 (78). Acetosyringone. GC-MS 70 eV, m/z (rel. int.): 196 [M] + (42). 181(100), 153(20), 137(l), 123(6), 110(7),93(11),79 (12), 65 (24), 53 (22), 43 (70). 4’-O-Acetylacetosyringone. GC-MS 70 eV, m/z (rel. int.): 196[M-CH,CO]+ (44), 181 (78), 153(11), 122(11), 109 (7). 93 (6) 79 (4), 65 (7), 53 (8), 43 (100). 4’-O-Trimethylsilylacetosyringone. GC-MS 70 eV, m/z (rel. int.): 268 CM]’ (51), 253 (89), 238 (53), 223 (lOO), 195 (7). 137 (13) 109 (6). 96 (4), 81 (3), 73 (68), 59 (22), 43 (91). Hydroxyacetosyringone. GC-MS 70 eV, m/z (rel. int.): 212[M]+ (ll), 181 (lOO), 153(18), 147(2), 138(4), 123(11), 108 (13), 93 (12), 79 (16), 67 (27). 53 (20), 41 (24). 2,4’-di-0-Acetylhydroxyacetosyringone. GC-MS 70 eV, m/z(rel. int.): 254 [M-CH,CO]+ (32), 181 (100) 153 (7), 123 (4). 109 (2), 79 (2), 65 (7). 53 (6), 43 (88). 2,4’-di-Trimethylsilylhydroxyacetosyringone. GC-MS 70 eV, m/z (rel. int.): 356 CM] + (lo), 341(20X 253 (lOO),223 (4), 103 (6) 73 (46), 59 (1 1), 45 (21). For analytical purposes, the cultures were sonicated for 3 min, at 20 kHz, prior to centrifugation for recovering the culture medium. Stress metabolites were isolated by a combined purification/concn method using C,s Sep-Pak cartridges. Culture medium (20ml) was applied to the cartridge followed by 10 ml distilled H,O. The adsorbed compounds were then eluted with 20ml MeGH. The extract was taken to dryness and the residue redissolved in CH,Cl,. Aliquots of this soln, containing methyl palmitate as standard, were assayed for GC analyses. Analytical conditions. GC analyses were performed using either a Perkin Elmer 8600 gas chromatograph equipped with a FID, a data handling system and a bonded phase DB-1 fused-silica column (30 m x 0.25 mm id., film thickness 0.25 pm) or a Perkin Elmer 8700 gas chromatograph equipped with two FIDs, a data handling system and a vaporizing injection port into which 2 columns of different polarities were installed: a bonded
BARROSO
phase DB-1 fused-silica column (30 m x 0.25 mm id., film thickness 0.25pm) and a bonded phase DB-Wax fused-silica column (30 m x 0.25 mm i.d., film thickness 0.25 pm). In both cases, the analyses were performed as follows: oven temp. was held at 170” for 5 min and then programmed to 220” at 2” min _ i; injection and detector temps, 220” and 240”. respectively; carrier gas, H,, adjusted to a linear velocity of 30 cm set- ‘. The samples were injected using the split sampling technique, ratio 1: 50. The GC-MS unit consisted of a Carlo Erba 6ooo Vega gas chromatograph, equipped with a bonded phase DB-1 fused-silica column (30 m x 25 mm i.d., film thickness 0.25 pm), and interfaced with a Finnigan MAT 800 Ion Trap Detector (ITD), operating in EI mode. Oven and injector temps were as above; transfer line temp., 280’, ion trap temp., 220”; carrier gas, He, adjusted to a linear velocity of 30 cm set - ‘; ionization energy, 70 eV, ionization current, 60 PA: scan range, 40-400 u; scan time, 1 set; splitting ratio, 1 : 40. authors wish to thank Dr Jan Schripsema (Leiden/Amsterdam Center for Drug Research, The Netherlands) for performing the ‘HNMR measurements, Dr Susane McCormick (National Center for Agricultural Utilization Research, U.S.A.) for a sample of lubimin and Junta National de InvestigaGo Cientifica e Technologica (JNICT) for financial support (Research contract no PMCT/B10/901/90). Acknowledgements-The
REFERENCES
1. Davis, K. R., Darvill, A. G., Albersheim, P. and Dell, A. (1986) Plant Physiol. 80, 568. 2. Mansfield, J. W. (1982) in Phytoalexins (Bayley, J. A. and Mansfield, J. W., eds), p. 253. Blackie, Glasgow. 3. Barz, W., Daniel, S., Hinderer, W., Jacques, U., Kessman, H., Koster, J. and Tiemann, K. (1988) in Plant Cell Biotechnology(Pais, M. S. S., Mavituna, F. and Novais, J. M., eds), p. 211. Springer, Berlin. 4. Barz, W., Bless, W., Daniel, S., Gunia, W., Hinderer, W., Jacques, U., Kessman, H., Tiemann, K. and Wittkampf, U. (1989) in Primary and Secondary Metabolism of Plant Cell Cultures (Kurz, W. G. W., ed.), p. 208. Springer, Berlin. 5. Eilert, U. (1987) in Cell Culture and Somatic Cell Genetics oj Plants (Vasil, I. K., ed.), Vol. 4, p. 153. Academic Press, New York. 6. Fowler, M. W. and Stafford, A. M. (1992) in Plant Biotechnology, Comprehensive Biotechnology, 2nd Suppl. (Fowler, M. W., Warren, G. S. and MooYoung, M., eds). Pergamon Press, Oxford. 7. Brindle, P. A., Kuhn, P. J. and Threlfall, D. R. (1983) Phytochemistry 22, 2719. 8. Brindle, P. A., Kuhn, P. J. and Threlfall, D. R. (1983) Biochem. Sot. Trans. 11, 5 16.
9. Brindle, P. A., Kuhn, P. J. and Threlfall, D. R. (1988) Phytochemistry 27, 133. 10. Chappell, J. and Nable, R. (1987) Plant Physiol. 85, 469.
Stress metabolites in Hyoscyamus cell cultures
11. Threlfall, D. R. and Whitehead, I. M. (1988) Phytochemistry 27, 2567. 12. Watson, D. G., Rycroft, D. S., Freer, I. M. and Brooks, C. J. W. (1985) Phytochemistry 24,2195. 13. Whitehead, I. M., Ewing, D. F. and Threlfall, D. R. (1988) Phytochcmistry 27, 1365. 14. Brooks, C. J. W., Watson, D. G. and Freer, I. M. (1985) Phytochemistry 25, 1089. 15. Whitehead, I. M., Threlfall, D. R. and Ewing, D. F. (1987) Phytochemistry 26, 1367. 16. Stoessl, A., Stothers, J. B. and Ward, E. W. B. (1976) Phytochemistry 15, 855. 17. Threlfall, D. R. and Whitehead, I. M. (1988) Biochem. Sot. Trans. 16, 71. 18. Lisker, N. and KCc, J. A. (1977) Phytoporhology 67, 1356. 19. Zook, M. N. and Kuc, J. A. (1987) Phytopathology 77, 1217. 20. Brodelius. P. (1988) in Plant Cell Biotechnology (Pais, M. S. S., Mavituna, F. and Novais, J. M., eds), p. 203. Springer, Berlin. 21. Edwards, R., Blount, J. W. and Dixon, R. A. (1991) Planra 184,403.
22. Marinehi, F., Gregorio, S. D. and Ronchi, V. N. (1991) Plant Science 77, 261. 23. Kim, D. J. and Chang, H. N. (1990) Biorechnol. Letters 12, 443. 24. Vanetten. H. D., Mattews, D. E. and Smith, D. A.
25. 26. 27. 28.
37s
(1982) in Phytoalexitrs (Bayley, J. A. and Mansfield, 1. W., eds), p. 181. Blaekie, Glasgow. Zaeeharius, R. M., Kalan, E. B. and Kimoto, W. I. (1985) Plant Cell Reports 4, 1. Murai, A., Sato, S., Osada, A., Katsui, N. and Masamune, T. (I 982) J. Chem. Sot. Chem. Common. 1,32. Whitehead, I. M., Atkinson, A. L. and Threlfah, D. R. (1990) Planta 182, 81. Spencer, P. A. and Towers, G. H. N. (1991) Phyto-
chemistry 30, 2933. 29. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15, 473. 30. Schripsema, J., Meijer, A., Van Iren, F., Hoopen, H. and Verpoorte, R. (1990) Plant Cell, Tissue and Organ Culture 22, 55. 31. Katsui, N., Matsunaga, A., Kitahara, H., Yagihashi, F., Murai, A., Musamune, T. and Sato, N. (1977) Bull. Chem. Sot. Jpn SO, 1217.
32. Ewing, D. F., Whitehead, I. M., Atkinson, A. L. and Threlfall, D. R. (1990) J. Chem Sot. Perkin Trans 2, 343. 33. Coxon, D. T., Price, K. R., Howard, B., Osman, S. F., Kalan, E. B. and Zacharius, R. M. (1974) Tetrahedron Letters 34, 2921. 34. Sannai, A., Fujimori, T. and Kato, K. (1982) Phytochemistry 12, 2986. 35. Stachel, S. E., Messens, E., Van Montagu, M. and Zambriski, P. (1985) Nature 318, 624.
?kytocknrluy, Vol. 35, No 2, pp. 371-375, 1994 copyright 8 19% Ebeviel sdnrc Ltd RiDted in oreat Britain. All rights rtmrwd al3 I -9422/94 sfi.00 + om
ACCUMULATION OF STRESS METABOLITES IN CELL SUSPENSION CULTURES OF HYOSCYAMUS ALBUS MARIA G. MIGUEL* and Josh G. BARROSO~ Departamento de Biologia Vegetal, Faculdade de Cikncias de Lisboa, Bloco C2, Camp0 Grande, 1700 Lisboa, Portugal
(Receiued25 June 1993) Key Word Index--Hyoscymus albus; Solanaceae; cell suspension culture; elicitation; sesquiterpenoid stress metabolites; lubimin; solavetivone; rishitin; acetophenones.
A&straet--Cell suspension cultures of ~yoscyu~~ albus treated with freeze-dried P~y~op~~~or~ c~~~~ autoclaved mycelium of freeze-dried Ca~ida albicans autoclaved cells accumulated mainly lub~in and l~p~ubimu~ along with small amounts of rishitin and solavetivone. Cultures treated with ccllulase did not accumulate any of the reported compounds. The pattern and the amount of sesquiterpenoids produced were dependent on cell growth stage, incubation time with the elicitor and elicitor concentration. Higher yields of lubimin and lO-epfiubimin occurred in cultures inoculated two days after subculturing and incubated for 48 hr. Some elicitor-treated cultures and old untreated ones accumulated acetophenones: acetovanillone, acetosyringone and hydroxyacetosyringone.
INTRODUCTION
RESULTSAND DfSCUS.SION
Phytoalexins are a diverse group of stress metabolites of low M, that have a broad antibiotic activity against many prokaryotic and eukaryotic organisms El]. In some host-pathogen interactions, phytoalexins accumulate at the site of invasion rapidly enough and in sufficient amounts to prevent further growth of the invading microorganism [2]. Plant celI cultures have been regarded as potent% sources of useful secondary metaboiites and proved to be useful systems for studies on the elicitation process and the biochemical reactions involved in phytoalexin biosynthesis [3-61. Accumulation of sesquiterp-enoid stress metabolites in cell cultures from members of the Solanaceae has been investigated to some extent. Several oxygen~ontaining sesquiterpenoids have been isoiated from elicitor-induced potato- [7-91, tobacco- [lo- 133 and pepper-cell cultures [ 14, IS]. Such compounds, namely the noreudesmane rishitin, the vetispirane lubimin, the secu-eudesmanes phytuberol and phytuberin, and the eremophilene capsidial, were previously isolated from plants within the Solanaceae and correlated with the defence response of the plants to the pathogens [163. In this paper, we report on the time course accumulation of stress metabolites in elicitor-induced cell suspension cultures of Hyoscyamus albus L.
Ceil suspension cultures of H. al&us, grown as described in the Experimental, contained a mixture of small aggregates and isolated cells. These cultures were characterized by the absence of a lag phase. The stationary phase was attained ca 210 hr after subculturing.
Response of the cell cultures to treatment with ditferent elicitors Cell suspension cultures inoculated early in the stationary phase with freeze-dried, autoclaved mycelium of Phytophthora cinnumomi or freeze-dried autoclaved cells of Canadida al&cans accumulated lubimin and lO-epilubimin, as the main stress metabolites, along with very smait mounts of solavetivone and rishitin. In contrast, the cultures inoculated with cellulase did not accumulate any of the reported sesquiterpenoids. Threlfall and Whitehead [17J also failed to induce the accumulation of stress metabolites in cellulase-treated potatocell cultures. The results we obtained with the two crude elicitor preparations used agree with the generalization that the compounds accumulated following elicitation are plantspecific and are, in general, not affected by the type of elicitor used [4, k8, 193. f+ence of the cell growth stage and of the period of elicitor confact on stress ~t~oli~e ~cumulaf~on
*Permanent address: Unidade das Ci&ias e Tecnologias Agrririas,Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal. tAuthor to whom correspondence should be addressed.
Ail the elicitor-treated after inoculation with independent of the cell accumulation of lubimin
371
cultures became brownish 6 hr the elicitor. This reaction was growth stage. By this time, the and lO-epilubimin as main stress
M. G. MIGUELand J. G. BARROS~
312
components was easily measurable. In contrast, the control cultures neither changed colour nor accumulated stress compounds. In many other systems, either in plant or cell suspension cultures, the accumulation of phytoalexins occurred also within a few hours after elicitation [9, 10, 19-221. Cell cultures of H. albus showed a differential response to elicitor treatment, in terms of the amount and the pattern of accumulated compounds, depending on the cell growth stage. The slowest initial response was observed in cultures inoculated at the time of subculturing; the levels of lubimin and lo-epilubimin rose slowly up to 96 hr. By contrast, four- and six-day-old cultures showed the faster initial response. In this case, the levels of both compounds rose rapidly within 12 hr after inoculation (Figs 1 and 2). Similar results on the accumulation of shikonin in Lythospermum erythrorhizon cell suspension cultures have been reported by Kim and Chang [23]. These results can be explained by the fact that cultures elicited at the time of subculturing have to adapt both to new medium conditions and to the stress challenge by the elicitor. Maximum accumulation of lubimin and IO-epilubimin was recorded in cultures inoculated on the second day after subculturing and incubated for 48 hr. In contrast, the smallest accumulation of both compounds occurred in eight-14-day-old cultures (Figs 1 and 2). By this time, the cultures have already attained the stationary phase, and low sugar levels are present in the culture medium. Since carbohydrates are an important source of the acetate needed for the biosynthesis of mevalonate, the key precursor of sesquiterpenoid compounds [9, lo], the depletion of sugars from the medium could be responsible, in this case, for the lower accumulation of lubimin and lo-epilubimin.
0
50
100
Incubation
150
200
Time (hr)
Fig. I. Time course of accumulation of lubimin in cell cultures of H. a/bus, following inoculation with the C. albicans elicitor preparation (2 mg ml- ‘). A-A Zero-day-old cultures; +--- l , two-day-old cultures; x - x , fourday-old cultures; 0 -0, sixday-old cultures; m-W, eight-day-old cultures; LJ---III, IO-dayold cultures; A -A, IO-day-old cultures.
0
50
100 Incubation
150
200
Time (hr)
Fig. 2. Time course of accumulation of ICkpilubimin in cell cultures of H. albus, following inoculation with the C. albicans elicitor preparation (2 mg ml- ‘). A-A Zero-day-old cultures; 4-
-+,
two-day-old
0
-0,
six-day-old
il ---[I,
IO-day-old
cultures; cultures;
x-x m--m,
cultures; A-A,
.
four-day-old
cultures;
eight-day-old
cultures;
IO-day-old
cultures.
In elicitor-treated six-lCday-old cultures the amount of lubimin and IO-epdubimin started declining 24 hr after incubation. In all the other cultures, the decline was only observed 48 or 96 hr after incubation. In any case, the levels of both compounds decreased markedly with prolonged incubation (Figs 1 and 2). Several authors, working with other systems, have reported similar results [9, IO, 20-223. Since stress metabolites do not necessarily represent end-products of plant metabolism, the decrease in their amount, due to prolonged incubation periods, can be explained by their further metabolism. In fact, it was ascertained that, in some cases, plant or cell cultures were able to biotransform such kinds of product, i.e. phytoalexins, into less toxic products [ 11,24,25]. In our system, zero-, two-, four- and 14-day-old cultures accumulated small amounts of solavetivone and rishitin 96 hr after incubation, while 16-, 1% and 22-dayold cultures accumulated both compounds earlier (48 hr after incubation). Maximum accumulation was recorded in four-day-old cultures incubated for 192 hr (5.27 and 1.47 pmol 100 ml-’ cell culture for solavetivone and rishitin, respectively). Since the detection of these two oxygen-containing sesquiterpenoids was concomitant with the decline of lubimin and IO-epilubimin, it Seems likely that at least part of lubimin has been metabolized into rishitin. On the other hand, the presence of solavetivone could be due to the suppression of lubimin biosynthesis. The bioconversion of lubimin into rishitin has already been reported by Murai et al. [26] in potato tuber tissue, and it has been established that solavetivone is a precursor of both lubimin and rishitin [ll, 27-j. Cell cultures of H. albus inoculated on the sixth, eighth or 10th day after subculturing did not accumulate solavetivone and/or rishitin. Although this fact remains difficult
374
M. G.
MIGUEL
and J. G.
and mass spectra, with corresponding data of an authentic sample. ‘HNMR data are identical to those previously reported [33,343. Rishitin was characterized by GC and GC-MS. The acetophenones present in the extracts were characterized by GC-MS. Mass spectra of acetates, TMSi ethers or underivatized samples were compared with the data reported in the literature [28,35] or with those of a library of spectra (NBS). Where possible authentic samples were used to confirm identities. Acetates were prepared by a conventional procedure (Ac,O-pyridine) at 80”. TMSi ethers were prepared at room temp. using a mixt. of THF-HMDS-TMCS (5:s: 1). Acetouanillone. GC-MS 70 eV, m/z (rel. int.): 166 [M]’ (40), 151 (lOO), 136(l), 123(37), 108(13).93(8),77(18).65 (22). 51 (27), 43 (49). 4-0-Acetylacetooanillone. GC-MS 70 eV, m/z (rel. int.): 166 [M-CH,CO]+ (Sl), 151 (lOO), 123 (13). 108 (2), 79 (9), 65 (7), 51 (16), 43 (89). 4’-O-Trimethylsilylacetovanillone. GC-MS 70 eV, m/z (rel. int.): 238 [M]’ (49), 223 (88), 208 (38), 193 (lOO), 165 (ll), 149 (3), 137 (13). 121 (ll), 107 (2) 91 (4), 73 (49), 59 (20), 51 (7), 43 (78). Acetosyringone. GC-MS 70 eV, m/z (rel. int.): 196 [M] + (42). 181(100), 153(20), 137(l), 123(6), 110(7),93(11),79 (12), 65 (24), 53 (22), 43 (70). 4’-O-Acetylacetosyringone. GC-MS 70 eV, m/z (rel. int.): 196[M-CH,CO]+ (44), 181 (78), 153(11), 122(11), 109 (7). 93 (6) 79 (4), 65 (7), 53 (8), 43 (100). 4’-O-Trimethylsilylacetosyringone. GC-MS 70 eV, m/z (rel. int.): 268 CM]’ (51), 253 (89), 238 (53), 223 (lOO), 195 (7). 137 (13) 109 (6). 96 (4), 81 (3), 73 (68), 59 (22), 43 (91). Hydroxyacetosyringone. GC-MS 70 eV, m/z (rel. int.): 212[M]+ (ll), 181 (lOO), 153(18), 147(2), 138(4), 123(11), 108 (13), 93 (12), 79 (16), 67 (27). 53 (20), 41 (24). 2,4’-di-0-Acetylhydroxyacetosyringone. GC-MS 70 eV, m/z(rel. int.): 254 [M-CH,CO]+ (32), 181 (100) 153 (7), 123 (4). 109 (2), 79 (2), 65 (7). 53 (6), 43 (88). 2,4’-di-Trimethylsilylhydroxyacetosyringone. GC-MS 70 eV, m/z (rel. int.): 356 CM] + (lo), 341(20X 253 (lOO),223 (4), 103 (6) 73 (46), 59 (1 1), 45 (21). For analytical purposes, the cultures were sonicated for 3 min, at 20 kHz, prior to centrifugation for recovering the culture medium. Stress metabolites were isolated by a combined purification/concn method using C,s Sep-Pak cartridges. Culture medium (20ml) was applied to the cartridge followed by 10 ml distilled H,O. The adsorbed compounds were then eluted with 20ml MeGH. The extract was taken to dryness and the residue redissolved in CH,Cl,. Aliquots of this soln, containing methyl palmitate as standard, were assayed for GC analyses. Analytical conditions. GC analyses were performed using either a Perkin Elmer 8600 gas chromatograph equipped with a FID, a data handling system and a bonded phase DB-1 fused-silica column (30 m x 0.25 mm id., film thickness 0.25 pm) or a Perkin Elmer 8700 gas chromatograph equipped with two FIDs, a data handling system and a vaporizing injection port into which 2 columns of different polarities were installed: a bonded
BARROSO
phase DB-1 fused-silica column (30 m x 0.25 mm id., film thickness 0.25pm) and a bonded phase DB-Wax fused-silica column (30 m x 0.25 mm i.d., film thickness 0.25 pm). In both cases, the analyses were performed as follows: oven temp. was held at 170” for 5 min and then programmed to 220” at 2” min _ i; injection and detector temps, 220” and 240”. respectively; carrier gas, H,, adjusted to a linear velocity of 30 cm set- ‘. The samples were injected using the split sampling technique, ratio 1: 50. The GC-MS unit consisted of a Carlo Erba 6ooo Vega gas chromatograph, equipped with a bonded phase DB-1 fused-silica column (30 m x 25 mm i.d., film thickness 0.25 pm), and interfaced with a Finnigan MAT 800 Ion Trap Detector (ITD), operating in EI mode. Oven and injector temps were as above; transfer line temp., 280’, ion trap temp., 220”; carrier gas, He, adjusted to a linear velocity of 30 cm set - ‘; ionization energy, 70 eV, ionization current, 60 PA: scan range, 40-400 u; scan time, 1 set; splitting ratio, 1 : 40. authors wish to thank Dr Jan Schripsema (Leiden/Amsterdam Center for Drug Research, The Netherlands) for performing the ‘HNMR measurements, Dr Susane McCormick (National Center for Agricultural Utilization Research, U.S.A.) for a sample of lubimin and Junta National de InvestigaGo Cientifica e Technologica (JNICT) for financial support (Research contract no PMCT/B10/901/90). Acknowledgements-The
REFERENCES
1. Davis, K. R., Darvill, A. G., Albersheim, P. and Dell, A. (1986) Plant Physiol. 80, 568. 2. Mansfield, J. W. (1982) in Phytoalexins (Bayley, J. A. and Mansfield, J. W., eds), p. 253. Blackie, Glasgow. 3. Barz, W., Daniel, S., Hinderer, W., Jacques, U., Kessman, H., Koster, J. and Tiemann, K. (1988) in Plant Cell Biotechnology(Pais, M. S. S., Mavituna, F. and Novais, J. M., eds), p. 211. Springer, Berlin. 4. Barz, W., Bless, W., Daniel, S., Gunia, W., Hinderer, W., Jacques, U., Kessman, H., Tiemann, K. and Wittkampf, U. (1989) in Primary and Secondary Metabolism of Plant Cell Cultures (Kurz, W. G. W., ed.), p. 208. Springer, Berlin. 5. Eilert, U. (1987) in Cell Culture and Somatic Cell Genetics oj Plants (Vasil, I. K., ed.), Vol. 4, p. 153. Academic Press, New York. 6. Fowler, M. W. and Stafford, A. M. (1992) in Plant Biotechnology, Comprehensive Biotechnology, 2nd Suppl. (Fowler, M. W., Warren, G. S. and MooYoung, M., eds). Pergamon Press, Oxford. 7. Brindle, P. A., Kuhn, P. J. and Threlfall, D. R. (1983) Phytochemistry 22, 2719. 8. Brindle, P. A., Kuhn, P. J. and Threlfall, D. R. (1983) Biochem. Sot. Trans. 11, 5 16.
9. Brindle, P. A., Kuhn, P. J. and Threlfall, D. R. (1988) Phytochemistry 27, 133. 10. Chappell, J. and Nable, R. (1987) Plant Physiol. 85, 469.
Stress metabolites in Hyoscyamus cell cultures
11. Threlfall, D. R. and Whitehead, I. M. (1988) Phytochemistry 27, 2567. 12. Watson, D. G., Rycroft, D. S., Freer, I. M. and Brooks, C. J. W. (1985) Phytochemistry 24,2195. 13. Whitehead, I. M., Ewing, D. F. and Threlfall, D. R. (1988) Phytochcmistry 27, 1365. 14. Brooks, C. J. W., Watson, D. G. and Freer, I. M. (1985) Phytochemistry 25, 1089. 15. Whitehead, I. M., Threlfall, D. R. and Ewing, D. F. (1987) Phytochemistry 26, 1367. 16. Stoessl, A., Stothers, J. B. and Ward, E. W. B. (1976) Phytochemistry 15, 855. 17. Threlfall, D. R. and Whitehead, I. M. (1988) Biochem. Sot. Trans. 16, 71. 18. Lisker, N. and KCc, J. A. (1977) Phytoporhology 67, 1356. 19. Zook, M. N. and Kuc, J. A. (1987) Phytopathology 77, 1217. 20. Brodelius. P. (1988) in Plant Cell Biotechnology (Pais, M. S. S., Mavituna, F. and Novais, J. M., eds), p. 203. Springer, Berlin. 21. Edwards, R., Blount, J. W. and Dixon, R. A. (1991) Planra 184,403.
22. Marinehi, F., Gregorio, S. D. and Ronchi, V. N. (1991) Plant Science 77, 261. 23. Kim, D. J. and Chang, H. N. (1990) Biorechnol. Letters 12, 443. 24. Vanetten. H. D., Mattews, D. E. and Smith, D. A.
25. 26. 27. 28.
37s
(1982) in Phytoalexitrs (Bayley, J. A. and Mansfield, 1. W., eds), p. 181. Blaekie, Glasgow. Zaeeharius, R. M., Kalan, E. B. and Kimoto, W. I. (1985) Plant Cell Reports 4, 1. Murai, A., Sato, S., Osada, A., Katsui, N. and Masamune, T. (I 982) J. Chem. Sot. Chem. Common. 1,32. Whitehead, I. M., Atkinson, A. L. and Threlfah, D. R. (1990) Planta 182, 81. Spencer, P. A. and Towers, G. H. N. (1991) Phyto-
chemistry 30, 2933. 29. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15, 473. 30. Schripsema, J., Meijer, A., Van Iren, F., Hoopen, H. and Verpoorte, R. (1990) Plant Cell, Tissue and Organ Culture 22, 55. 31. Katsui, N., Matsunaga, A., Kitahara, H., Yagihashi, F., Murai, A., Musamune, T. and Sato, N. (1977) Bull. Chem. Sot. Jpn SO, 1217.
32. Ewing, D. F., Whitehead, I. M., Atkinson, A. L. and Threlfall, D. R. (1990) J. Chem Sot. Perkin Trans 2, 343. 33. Coxon, D. T., Price, K. R., Howard, B., Osman, S. F., Kalan, E. B. and Zacharius, R. M. (1974) Tetrahedron Letters 34, 2921. 34. Sannai, A., Fujimori, T. and Kato, K. (1982) Phytochemistry 12, 2986. 35. Stachel, S. E., Messens, E., Van Montagu, M. and Zambriski, P. (1985) Nature 318, 624.