Les roténoïdes, composés dérivés de la voie du métabolisme phénylpropanoïde, sont en grande partie accumulés dans les feuilles de "Tephrosia vogelii$. Des cultures de suspensions cellulaires hétérotrophes et photomixotrophes de cette plante tropicale ont été obtenues. Les lignées cellulaires sont toutes deux capables de produire des roténoïdes, mais une production spéciale est observée dans chaque type de culture cellulaire. La lignée cellulaire photomixotrophe accumulait de la roténone et de la dégueline, et la lignée cellulaire hétérotrophe produisait essentiellement de la dégueline et de la téphrosine. (Résumé d'auteur

Campa, Claudine

Chrestin, Hervé

Lambert, N.

Trouslot, Marie-France

Name not available

Production of rotenoids by heterotrophic and photomixotrophic cell cultures of Tephrosia vogelii

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Phytochemistry, VOL 34, No. 6, pp. 1515-1520, 1993 
Printed in Great Britain. 
0031-9422/93 96.00+0.00 
8 1993 Pcrgamon F'ress Ltd 
PRODUCTION OF ROTENOIDS BY HETEROTROPHIC AND 
PHOTOMIXOTROPHIC CELL CULTURES OF TEPHROSIA VOGELII 
NADINE LAMBERT, MARIE-FRANCE TROUSLOT,* CLAUDINE NEF-CAMPA* and ~ E R V É  CHRESTIN* 
Département de Physiologie VCg&& et Eoosystème, Centre d'étude de Cadarache, 13108 St Paul La Durance, France; I 
*Laboratoire de Physiologie et.Biotechnologie Végétales, Adiopodoumé, ORSTOM, BP V 51 Abidjan République de Côte d'Ivoire I 
(Received 13 May 1993) 
Key Word Index-Tephrosiu vogelii; Papilionaceae; heterotrophic; photomixotrophic cell culture; 
rotenoids; HPLC. 
Abstract-Rotenoid compounds provided from the phenylpropanoid pathway are largely accumulated in the leaves of 
Tephrosiu uogelii. Heterotrophic and photomixotrophic cell suspension cultures of this tropical plant have been 
obtained. Both cell lines are able to produce rotenoids, but a specific production is observed in each cell culture. The 
photomixotrophic cell line accumulated rotenone and deguelin in Tephrosia leaves and the heterotrophic cell line 
.produced essentially deguelin and tephrosin. 
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INTRODUCTION 
Rotenone and its derivatives with insecticidal properties 
are essentially extracted from Derris, a tropical plant 
originally from South America. These compounds are 
also accumulated in the leaves of Tephrosiu uogelii Hook 
f. Up to now, this tropical plant, which is obtained from 
West Africa, has been traditionally used for its ichtyo- 
toxic, insecticidal and food parasiticidal activities. In fact, 
a great variety of rotenoids are accumulated in the leaves 
of this plant where the total rotenoid content can reach 
4% of the dry matter. Rotenone and deguelin are the 
major compounds present [l]. 
The rotenoids (1-4) are composed of an isoflavone 
nucleus C&&s (ring ACB) with an isoprene moiety 
attached at C-8 of ring A. "'hese compounds are classified 
. in the isoflavonoid family, the end product of the phenyl- 
propanoid pathway [2]. The precursor for all phenylpro- 
panoids is L-phenylalanine provided from the shikimate 
pathway. Phenylalanine is converted into 4-coumaroyl 
COA by the concerted action of phenylalanine ammonia- 
lyase (PAL), cinnamate-4-hydroxylase (C4H) and 
4coumarate COA ligase (4CL). PAL, which catalyses the 
conversion of L-phenylalanine to truns~nnamic acid is 
subjected to a highly complicated pattern of control 
comprising endogenous as well as exogenous factors [3, 
41. One of these exogenous factors is light which dramat- 
ically increases the enzyme activity after a lag period of a 
few hours [a. The subcellular localization of the bio- 
synthetic pathway of the rotenoids is not well known. 
However, some steps can take place in the chloroplast; 
phenylpropanoids and/or flavonoids and the enzymes of 
the phenolic metabolism [3, 6, ?'I are located in this 
organelle of many plants. 
In this paper, we studied rotenoid production in chlor- 
ophyll-containing and heterstrophic cell lines of 
T. uogelii. Both callus and suspension cultures were first 
characterized with re8.d to fresh and dry weight and 
chlorophyll accumulation., 
RESULTS AND DISCUSSION 
Heterotrophic and photomixotrophic callus cultures 
were both obtained from T. vogelii hypocotyls. Growth 
kinetics were established after 15 months of subculture. 
The fresh and dry weight curves of heterotrophic callus 
cultures demonstrated a continuous growth throughout 
the cell culture cycle (Fig. 1A). In chlorophyll-containing 
cultures intensive growth was only observed between day 
10 and day 25 (Fig. 1B). Growth increases were similar in 
both cultures ( x 10 (expressed in dry wt) and 11, respect- 
ively, for heterotrophic and photomixotrophic cells). The 
estimation of the chlorophyll content of green callus 
cultures (Fig. 1C) demonstrated a loss of chlorophyll 
material during the first 10 days of culture and the 
'stationary phase of growth; synthesis occurred only dur- 
ing the intense growth phase. 
Identical growth patterns were observed when the 
culture of both cell lines was performed in liquid medium. 
The phase of growth started from the first day of sub- 
culture in the heterotrophic cell suspensions (Fig. 2A). 
For the photomixotrophic culture (Fig. 2B), the growth 
phase took place after two days of lag phase. The growth 
rates, expressed in dry weight, are relatively similar, the 
dry weight being 2.5-fold higher at the end of both 
cultures. In the chlorophyll-containing cell suspensions, 
the lag phase was accompanied by a decrease in the 
chlorophyll content as observed for the callus cultures 
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CH3 
\ 
ObH3 
3 
N. LAMBERT et al. 
CH3 
CH3 
O 
dCH3 
2 
CH3 
CH3 
OkH3 
4 
0.0 
o 10 20 30 40 
Time (days) 
no - 0.0 
Time (days) 
Fig. 1. Growth kinetics of heterotrophic (A) or chlorophyll-containing (B) callus of T. uogelii and evolution of 
chlorophyll content (C). 
(Fig. 2C). A synthesis of the chlorophyll occurred only 
during the intense growth phase and its final level 
(expressed in pg mg-' dry wt) raised the value 
observed at the subculturing. During the transfer to the 
fresh medium, cells were in the presence of a higher 
concentration of sucrose which inhibited the chlorophyl- 
logenese CS]. 
- Analysis of the rotenoid content of leaf extracts of 
T. vogelii showed that 1-4 are the main rotenoids pro- 
duced (Fig. 3). The amount of the rotenoid produced in 
the in vitro material was determined by HPLC analysis 
with reference to standard solutions of rotenolone (l), 
tephrosin (2), rotenone (3) and deguelin (4) (Fig. 3). 
In the heterotrophic callus cultures, it seemed that 4 
was the principal rotenoid produced (Fig.4A). Com- 
pound 1 was not detected and 2 and 3 were poorly 
synthesized (always <25pgg-' dry wt). The chloro- 
phyll-containing callus cultures produced the same roten- 
Fig. 2. Growth kinetics of heterotrophic (A) or photomixotrophic (B) cell suspension culturcs of T. vogelii and 
evolution of chlorophyll content (C). 
- 
6 
E! 
w 
E 
8 
f 
P 
I I 
10 20 
The (min) 
t 
10 20 
"e (min) 
Fig. 3. HPLC analysis of leafextract (left chromatogram) and callus cultures (right chromatogram) from T. wgelii.  
Each peak number corresponds to its compound number. 
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oids present in the leaves (Fig. 3). However, 3 appeared to 
be the main rotenoid throughout the culture cycle 
(Fig. 4B). Its production rate was approximately 
four-six-fold higher than that of the other rotenoid 
compounds. Its highest concentration was observed after 
20 days of culture (570 pg g- dry wt). Compounds 1 and 
2 were produced in lower amounts, their concentration 
being always lower than 50 pgg-' dry wt. 
In the heterotrophic cell suspension cultures (Fig. 5A), 
4 was the major rotenoid produced as in the heterotro- 
phic callus cultures. However, 2 was also found in large 
amounts, its concentration being equivalent to 4. Here 
also, 1 was not detected, although 1 was the major 
rotenoid produced by the photomixotrophic cell suspen- 
sion culture (Fig. 5B). Compound 3 was also actively 
synthesized by these cells. For both cell lines grown in 
liquid medium, rotenoid synthesis was increased during 
the growth stage with maximum concentrations of the 
compounds appearing at the end of the phase of growth. 
The rotenoid content was also determined in the culture 
e Tephrosin(2) v Rotenone(3) 
o 10 20 30 40 
%e (days) l'"e (days) 
Fig. 4. Rotenoid production in heterotrophic (A) and chlorophylltontaining callus (B) cultures. 
O Tephrosin (2) 
Rotenone (3) 
- -  
O 2 4 6 8 1 0 1 2  
Tlme (days) (days) 
, -  
Fig. 5. Rotenoid production in heterotrophic (A) and photomixotrophic (B) cell suspension cultures. 
- I   - i__ 
I 
. - , . I - . , . - _. - . . 
. .  . . _ _  
Production of rotenoids 1519 
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medium of the two cell lines (data not shown). The 
rotenoids were poorly released and only 4 was found in 
large amounts. Finally, the modification of some culture 
conditions (absence of light and different concentration of 
sucrose) during callus culture allowed the two cell lines to 
be differentiated by their ability to synthesize chlorophyll. 
The rotenoid production was markedly different in these 
two cell lines. Moreover, the rotenoid profile of the 
chlorophyll-containing cultures was compared to the leaf 
production. Although T. uogelii leaves accumulated 3,4 
and their derivatives, heterotrophic cell cultures were able 
to produce only 4 and 2, while photomixotrophic cell 
cultures accumulated preferentially compounds like 3. It 
seems that the biosynthesis of 3 and 1 is stimulated by 
photomixotrophic conditions. It is an open question 
whether light or photosynthesis plays a role in directing 
the biosynthetic pathway towards 3 or 1, rather than 
towards 2 and 4. Up to now we know that the common 
precursor to 4 and 3 is rot-2'enonic acid [9, lo]. Two 
different enzymes are responsible for the cycFation 
process which produces 3 and 4. Compound 1 is an 
oxidation product of natural rotenone (3) while 2 is 
derived from 4. It is possible that the relative importance 
of the second pathway may vary with culture conditions. 
There is another possibility that the lack of accumulation 
of 2 and 4 in chlorophyll-containing cells could be 
attributed to the catabolism of the products or inactiv- 
ation of some enzymes participating in this metabolic 
pathway. Detailed studies on the enzymes and inter- 
mediate compounds of rotenoid biosynthesis and their 
localization are needed to elucidate the site, as well as the 
control mechanism, of the rotenoid biosynthesis in 
T. uogelii cells. 
In this study, the establishment of the cell culture 
system allowed the demonstration that the photomixo- 
trophic condition of the culture (light essentially) pro- 
motes the production of rotenoids which are of eco- 
nomical interest. The present investigation also provides 
another example where the spectra of secondary consti- 
tuents of photomixotrophic and heterotrophic cell cul- 
tures are significantly different [ll, 12). Therefore, de- 
pending on the type of compound required, a specific 
form of cell culture (heterotrophic or photomixotrophic) 
could be selected for product accumulation and relevant 
biochemical investigations. 
EXPERIMENTAL ,. 
Establishment of the cell lines. Chlorophyll-containing 
callus cultures were initiated from hypocotyl explants of 
T. Yogelii seedlings, grown on a modified MS [13] 
medium [14], supplemented with 5.37 pM l-naphthalene 
acetic acid, 4.4 pM benzylaminopurine, sucrose 3% (w/v) 
and 0.6% (w/v) agar with the pH adjusted to 5.7. These 
cultures were maintained at 28" under diffuse light (18 hr 
light-6 hr dark, 100 pmol m-, sec-'). Heterotrophic cul- 
tures were obtained by transferring part of the chloro- 
phyll-containing culture to the dark (28"). For each cell 
line, subculturing was carried out every 3 weeks. 
After 8 months of callus subculture, cell suspension 
cultures were initiated from each callus cell line. Cultures 
were performed in 300-ml Erlenmeyer flasks containing 
80ml of liquid medium (modified MS medium supple- 
mented with 1.5% (w/v) or 2% (w/v) sucrose for photo- 
mixotrophic and heterotrophic cultures, respectively). 
The photomixotrophic cell line had to be filtered during 
the first subculture in order to remove cell clumps. Cell 
suspensions were routinely subcultured (every 1 or 2 
weeks, respectively for heterotrophic and photomixotro- 
phic cells) and maintained at 28" on a gyratory shaker 
(80 rpm) under diffuse light (18 hr light-6 hr dark, 
100 pmol m-, sec-') or in the dark. 
Estimation of growth. Cells and the medium were sepd 
by Htration under partial vacuum through a glass filter. 
Growth was estimated by measuring the total fresh 
weight and the dry weight (a 500-mg aliquot of fresh cells 
was dried at 60" for 48 hr). Data reported are the average 
of the values from triplicated experiments. 
Determination of chlorophyll (a+ b) content. Cultured 
cells collected on Miracloth by filtration (1 g fr. wt) were 
extracted with Me,CO (4 ml) in the dark and at 4". After 
centrifugation, the supernatant was adjusted to a final 
vol. of 15 ml with Me,CO-H20 (4: 1). The quantitative 
estimation of chlorophyll was by spectrophotometrically 
using the equation of Lichtenthaler and Wellburn [ 151. 
Extraction of rotenoids. Freeze-dried cells were ex- 
tracted twice with MeOH-H,O (9: 1) and oxalic acid 
(1 mgml-') [la at 30" and the extract was evapd to 
dryness (at 30"). The remaining residue was dissolved in 
CH,Cl, and partitioned (3 x ) against 0.25 M NaCI. The 
CH,CI, phase was reduced to dryness under vacuum and 
the final extract taken up in 1 ml of Me,CO-H,O (1 : 1). 
After centrifugation (27000 g, 15 min;\ the supernatant 
was decanted and stored at -20". 
Rotenoid analysis. The major rotenoids (1-4) were 
characterized qualitatively by TLC (silica gel 60 F 254 
plates, Merck) with CHC1,-Me,CO-HOAc (169: 3 : 1) 
and quantitatively by HPLC on a Beckman 332, using a 
Lichrospher 100CH 8/II column R.P., 25x0.4cm 
(5 pm), at a flow rate of 0.8 ml min-', the mobile phase 
MeOH-H20 (65:35), detection at 280 nm. 
Acknowledgements-Tephrosin and deguelin were a gift 
of Dr Delle Monache, Roma, Italy and rotenolone was 
provided by Dr Fujita, Kyoto, Japan. 
REFERENCES 
I. Irvine, J. E, and Freyre, H. (1959) J .  Agric. Food 
Chem 7, 106. 
2. Hahlbrock, K. and Grisebach, H. (1975) in The 
Flavonoids (Mabry, T. J. and Mabry, H., eds), 
pp. 866-915. Chapman and Hall, London. 
3. Hanson, K. R. and Havir, E. A. (1981) in The Bio- 
chemistry of Plants. A Comprehensive Treatise 
(Stumpf, P. K. and Conn, E. E., eds), Vol. 7, 
pp. 557-625. Academic Press, New York. 
4. Jones, D. H. (1984) Phytochemistry 23, 1349. 
1520 N. LAMBERT et al. 
5. Knypl, J. S., Janas, K. M. and Wolska, K. M. (1986) 
Physiol. Plant. 86, 543. 
6. Weissenbock, G. and Schneider, V. (1974) Z. Pflan- 
zenphysiol. 12, 23. 
7. Wiermann, R. (1981) in The Biochemistry ofPlants. A 
Comprehensive Treatise (Stumpf, P. K. and Conn, 
E. E., eds), Vol. 7, pp. 86-1 12. Academic Press, New 
York. 
8. Yamada, Y. and Sato, F. (1978) Plant Cell Physiol. 19, 
691. 
9. Crombie, L., Rossiter, J. and Whiting, D. A. (1986) 
J .  Chem. Soc. Chem. Comm. 352. 
10. Crombie, L., Holden, I., Kilbee, G.  W. and Whiting, 
D. A. (1982) J .  Chem. Soc. Perkin Trans I 789. 
1 1 .  Hüsemann, W., Fischer, K., Mittelbach, I., Hübner, - 
S., Richter, G. and Ban, W. (1989) in Primary and 
Secondary Metabolism of Plant Cell Cultures I I  
(Kurz, W. G. W., ed.), pp. 35-46. Springer, Berlin. 
12. Ikemeyer, D. and Ban, W. (1989) Plant Cell Reports 
13. Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15, 
14. Hardy, T., Chaumont, D., Brunel, L. and Gudin, C. 
15. Lichtenthaler, H. K. and Wellbum, A. R. (1983) 
16. a h n ,  R. S., Phipers, R. F. and Brodaty, E. (1945) 
8, 479. 
473. 
(1987) J .  Plant Physiol. 128, 11. 
Biochem. Soc. Trans. 603,591. 
J .  Soc. Chem Ind. London 64,33. 
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Production of rotenoids by heterotrophic and photomixotrophic cell cultures of Tephrosia vogelii

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