Vous êtes sur la page 1sur 4

Plant Cell

Reports

Plant Cell Reports (1997) 16:287-290

Springer-Verlag 1997

Effect of acetylsalicylic acid on secondary metabolism


of C a t h a r a n t h u s r o s e u s tumor suspension cultures
Gregorio Godoy-Hern~ndez and Victor M. Loyola-Vargas
Departamento de Biologia Experimental, Divisi6n de Biologia Vegetal, Centro de Investigacidn Cientifica de Yucat/m,
Apartado Postal 87, Cordemex, 97310, Yucatfin, M~xico
Received 17 May 1996/Revised version received 23 July 1996 - Communicated by F. Constabel

Summary. Addition of various concentrations (0.5-20


raM) of acetylsalicylie acid (ASA) to tumor lines of
Catharanthus roseus cultivated in vitro and requiring
corn starch as carbon source, produced remarkable
effects on secondary metabolite production. An
increase of 505% total alkaloids per culture (ceils plus
liquid medium), 1587% total phenolics (liquid
medium), 612%
total furanocoumarins (liquid
medium)~ and 1476% total anthocyanins (liquid
medium) was detected. 1 mM ASA in combination
with other elicitors, such as homogenates o f Aspergillus
fumigatus or trans-cinnamic acid, did not further
increase the metabolite content substantially. The
results suggest that ASA could act as a new biotic
elicitor of metabolite production in C. roseus cell
suspension culture.
Key words: Catharanthus roseus - Acetylsalicylic acid Aspirin - Alkaloids - suspension cultures - tumor

Introduction
Application of salicylic acid (SA) or acetylsalicylic acid
(ASA) to higher plants, algae and cultured plant cells
has been shown to influence diverse plant development
processes such as germination (Reynolds 1978), growth
of mesocotyls, coleoptiles, and roots (Kumar and
Nanda 1981), flowering induction (Kumar and Nanda
1981; Klmrana and Maheshwari 1983; Kaihara et al.
1981; Khurana and Maheshwari 1978; Watanabe and
Takimoto 1979), shoot induction in Nicotiana tabacum
tissue (Lee and Skoog 1996), vegetative reproduction
in Fucus spiralis (Fries 1984), inhibition of tumor tissue
growth in N. tabacurn (Saint-Pierre et al. 1984).
Likewise affected have been a wide variety of metabolic
processes. For a review see (Raskin 1992; Klessig and
Malamy 1994; Lee et al. 1995).

At present no information is available regarding the


effect of ASA on secondary metabolite production.
The aim of the present communication is to examine
the effect of ASA on secondary metabolism in a
Catharanthus roseus tumor cell line cultivated in vitro
in the absence of growth regulators and with corn
starch as a carbon source.

Materials and methods


Cell line LBA1 Cultures. Catharanthus roseu~fL~G. Don-transformed
tumor cultures were obtained from stems of plants grown at the
greenhouse, and which had been infected with the PC 2500 strain of
Agrobacterium tumefaciens (J. Schell-Gent strain). Tumors formed
were dissected into pieces of 0.5 cm and transferred to PC medium
(Phillips and Collins 1979) without growth regulators for callus
formation. Callus was subcultured in the same medium every 15 days
for two years. Thereafter 1 g callus was transferred to 50 ml of PC
medium in 250 ml Erlenmeyer flasks for the obtention of cell
suspension cultures. One year later cells were transferred to 50 ml of
PC medium with a supplement of 10 g/L of corn starch, without
sucrose, in 250 Erlenmeyer flasks and routinely subcultured under
continuos light, temperature of 28"C and 100 rpm. Finally, cell
suspensions (3 g fresh weight) were subcultured in 100 ml of PC
medium with corn starch (10 g/L) in 500 ml Erlenmeyer flasks and
routinely subcultured under the above conditins. All experiments
were performed with 10 day-old cell suspensions (12/100 ml), in
three replicates. Viability was measured using fluorescein diacetate.
Experiments were repeated twice in triplicate.
Acetylsalicylic acid preparation. ASA (Sigma) was prepared at
different concentrations (0.5 - 20 mM) in distilled water and the pH
was adjusted to 5.8 with KOH. Preparations were sterilized by
filtration (Millipore membrane 45/~m) prior to their addition to the
10oday-old suspension cultures. The cultures were sampled after 72
hours of incubation.

Fungal homogenate preparation. Aspergillus fumigatus (ATCC-10787)


homogenates were added to the 10~day-old cultures to render a final

Correspondence to: V. M. Loyola-Vargas

288
concentrationof 15 mg/ml of total sugar (glucose). The culture

3OO

300

300

conditions and homogenate preparation have been previously


250

described (Godoy-Hernfindez and Loyola-Vargas 1991),


Determination

of

total

sugars

determination of total sugars was


(1956).

~3oo.~o

(glucose). The method for


according to Dubois et al.

liquid

medium,

was

i
determinated

according

o
=o
z

150

'~ 150

100

=.o

150

50

50

150

100
3

Determination of total alkaloid. Total alkaloid concentration, in cells

and

200

to
0

Godoy-Hern~ndez and Loyola-Vargas (1991).

I
5

I
10

t
15

I
20

ACETYL SALICYLIC ACID (raM)

Determination of total phenolies. Total phenol concentration was


determinated according to Seitz et al. (1989). The culture medium
was separated from cells by filtration (Whatman No. 1) under
suction. Filtered medium (0.5 ml) was diluted in 6 ml of distilled
water and 0.5 ml of Folin reagent. After incubation for 3 min, 1 ml of
saturated sodium carbonate solution was added. The mixture was
diluted to a final volume of 10 ml with distilled water. The solution
was allowed to stand for 1 h and absorbance (OD) (~,=~x= 725 nm)
was determined. A calibration curve was constructed with caffeic
acid as standard.
Determination of total furanoconmarins. The method to
determinate total furanoeoumarin concentration was similar to the
one reported by Davis and Hahlbrock (1987), with slight
modifications. Culture medium (20 ml) was extracted three times
with 20 ml chloroform. The extracts were combined and evaporated
to dryness. The residue was resuspended in 1.0 ml of analytical grade
methanol and a 1:10 (v/v) dilution into methanol was prepared and
absorbance at 320 nm was determined. Molar furanocoumarin
content was calculated using an extinction coefficient of 12,000 L/mol
x cm. All measurements were done in triplicate.
Determination of total anthoeyanins. The method of total
anthocyanin concentration was similar to that reported by Do and
Cormier (1990), with some modifications. Total anthocyanins were
extracted from freeze-dried
media with ethanol-HC1 1%
(85:15 v/v). Optical density of extracts was measured at 535 nm.
Molar anthocyanin content was calculated using an extinction
coefficient of 98.2 l/mol x cm. All measurements were done in
triplicate.

Fig. 1. Total alkaloid content in cells and liquid medium as a


function of acetylsalicylic acid concentration in C. roseus
transformed cell suspension cultures (10-days old) after 72 h of
treatment.

ASA also affected other secondary metabolite


pathways. Phenolics, furanocumarins and anthocyanins
were increased in a dose-response manner (Fig. 2A -

2C).
At 20 mM of A S A there was a 1587% increase in
phenolics with respect to the control (Fig. 2A). While
furanocoumarin content increased 612% at 10 mM
ASA (Fig. 2B). This accumulation of phenolics and
furanocoumarins is correlated with the induction of
enzymes involved in general phenylpropanoid
metabolism, e.g. phenylalanine ammonia lyase (PAL)
and 4-coumarate:CoA ligase (4CL) (Kombrink and
Hahlbrock 1986) and in the furanocoumarin
biosynthetic pathway, eg. S-adenosyl-L-methionine:
anthotoxol O-methyltransferase (XMT) (Davis and
Hahlbrock 1987).
35

25
15O0
~o

|
=

Results and discussion

ASA produced a general increase in secondary


metabolite concentration in C. roseus cell suspension
cultures. Total alkaloids increased at each of the ASA
concentrations used (Fig. 1). Addition of 1 mM ASA
for 72 h increased the alkaloids 160% in the culture
medium, while in the cells the increase was 345% over
the control. This effect was higher than the one
obtained
with
1 mM
trans-cinnamic
acid
(Godoy-Hernfindez and Loyola-Vargas 1991). In
general, there was a 2 to 2.5-fold increase in the total
alkaloids per flask (Fig. 1).

5
1o
15
ACEllYL$AUCYBCACID(mbl)

2o

Figure 2. Total phenolics (2A), furanocoumarins (2B) and


anthocyanins (2C) in liquid medium as a function of acetylsalicylie
acid concentration in C. roseus transformed cell suspension cultures
(10-days old) after 72 h of treatment.

Not only the synthesis of phenolic compounds was


induced, but anthocyanins also (Fig. 2C). At 20 mM
ASA, there was an almost fifteen-fold increase

289
(1476%) in the content of these pigments. This
increase in anthocyanins with the ASA treatment was
1250% higher than that obtained with osmotic stress
(sucrose or mannitol) by Do and Cormier (1990) in
cells of Vitis vinifera; 1180% higher than that obtained
with high ammonium concentrations (Do and Cormier
1991) and 1050% higher than the treatment with low
nitrate and high sugar concentration (Do and Cormier
1991) with the same plant cell suspension. However, a
1000% increase in anthocyanin production has been
obtained by medium with low nitrate and high sucrose
concentration in a Vitis hybrid cell culture (Hirasuna et
al. 1991). Raising the sucrose concentration from 2 to
11% in C. roseus cell suspension caused an increase in
anthocyanin concentration of more than 300% in an
experiment by Knobloch et al. (1982), whereas
starvation of phosphate and nitrogen in MS medium
caused an increase of 1200%.

250
225
200

175
150

the other hand, the use of i mM of trans-cinnamic acid


produced an increase of 282% in the alkaloid content
(Fig. 3). Similar results have been obtained in C. roseus
cell suspension culture (line BAP-2) grown with
sucrose as a carbon source (Godoy-Hernfindez and
Loyola-Vargas 1991). When the trans-cinnamic acid
was used in the presence of ASA, the initial effect was
reverted (Fig. 3).
The addition of A. fumigatus homogenates did not
modify the phenol content of the cultures, but
decreased the effect of ASA. While the presence of
trans-cinnamic acid did not modify the ASA increase in
the phenol content (Fig. 4A).
The addition ofA. fumigatus homogenates decreased
the level of furanocoumarins compared to control and
ASA treatment. Trans-cinnamic acid did not enhance
the ASA effect on furanocoumarin production (Fig. 4B).
The addition of A. furnigatus homogenates increased
the anthocyanins more than 3-fold with respect to the
control. How ever, when it was added in combination
with ASA the effect diminished by almost 50%. The
addition of trans-cinnamic acid also increased the
anthocyanin content and did not affect the increase
produced by ASA (Fig. 4C).

125
100

IA
1000
1500
1250

75
5O

i:

25
0
C

AF AF+ASAASAASA+TC TC
CULTURECONDITIONS

Fig. 3. Total alkaloid content in cells (1~) and liquid medium (r---7)
due to the effect of different culture conditions in C. roseus
transformed cell suspension cultures (10-days old) after 72 h of
treatment. (C): control at day 13; (AF) Aspergillus fumigatus
homogenate (15 mg/ml glucose); (AF + ASA)Aspergillusfumigatus
homogenate (15 mg/ml glucose) plus 1.0 mM acetylsalicylic acid;
(ASA) 1.0 mM aeetylsalicylic acid; (ASA + TC) 1.0 mM
acetylsalicylic acid plus 1.0 mM trans-cinnamic acid; (TC) 1.0 mM
trans-cinnamic acid.

The response in the alkaloid content to some


compounds and treatments can be additive or
synergistic (Godoy-Hernfindez and Loyola-Vargas
1991). To test if this phenomenon had been happening
in the presence of ASA, the combination of
homogenates of Aspergillusfumigatus or trans-cinnamic
acid and ASA was tested.
The addition ofA. fumigatus homogenates did not alter
the alkaloid of the cells (Fig, 3). When the
homogenates were used in combination with ASA, they
reverted the increment induced by this compound. On

CULTURE
CONDITIONS
AF AF*ASAASAASATC TC

Fig. 4. Total phenolics (4A), furanocoumarins (4B) and


anthocyanins (4C) in liquid medium due to the effect of different
culture conditions in C. roseus transformed cell suspension cultures
(10-days old) after 72 h of treatment. (C): control at day 13; (AF)
Aspogillus fumigatus homogenate (15 mg/ml glucose); (AF + ASA)
Aspergillus fumigatus homogenate (15 mg/ml glucose) plus 1.0 mM
acetylsalicylic acid; (ASA) 1.0 mM acetylsalicylic acid; (ASA + TC)
1.0 mM acetylsalicylic acid plus 1.0 mM trans-cinnamic acid; (TC)
1.0 mM trans-cinnamic acid.

The role of ASA in plant cells is still unknown and is


under investigation. It has been suggested to act as
chelator (Watanabe and Takimoto 1979) to inhibit
ethylene synthesis in cultured plant cells by blocking
the action of the ethylene forming enzyme (Leslie and

290
Romani 1986). Other authors have hypothethized that
ASA could favor the enzyme production that degrades
or inactivates endogenous auxins or behaves like a
hormone, and may trigger other processes inside
plants.
Results suggest that ASA may act as modulator of
phenylpropanoid metabolism, because its effect on the
production of different secondary metabolites was
noted, e. g. alkaloids (505%), phenolics (1587%),
furanocoumarins (612%) and anthocyanins (1476%)
(Fig. 1 and 2A - 2C). ASA is a more powerful elicitor
than
Aspergillus
fumigatus
homogenate
or
trans-cinnamic acid (TCA) for stimulation of
secondary metabolism
(Fig. 3 and 4A - 4C).
The role of ASA on anthocyanin production in line
LBA1 of C. roseus transformed cell suspension cultures
is unknown, one may hypothesize that ASA could be
activating the key enzyme (Chalcone synthase) in the
anthoeyanin biosynthetic pathway.

Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956)


AnalyticalChemistry28:350-356
Fries L (1984) Canadian Journal of Botany 62:1616-1620
Godoy-HernfindezG, Loyola-VargasVM (1991) Plant Cell Reports
10:537-540
Hirasuna TJ, Shuler ML, Lackney VK, SpanswickRM (1991) Plant
Science 78:107-120
Kaihara S, Watanabe K, Takimoto A (1981) Plant Cell Physiology
22:819-825
Khurana JP, Maheshwari SC (1978) Plant Science Letters 12:
127-131
Khurana JP, Maheshwari SC (1983) Plant Cell Physiology 24:
907-912
KlessigDF, MalamyJ (1994) Plant Molecular Biology26:1439-1458
Knobloch K-H, Bast G, Berlin J (1982) Phytochemistry21:591-594
Kombrink E, Hahlbrock K (1986) Plant Physiology81:216-221
Kumar S, Nanda KK (1981) BiologiaPlantarum 23:321-327
Lee H, Le6n J, Raskin I (1995) Proceedings of the National
Academy of Science (USA) 92:4076-4079
Lee TF, Skoog F (1996) PhysiologiaPlantarum 18:386-402
Leslie CA, Romani RJ (1986) Plant Cell Reports 5:144-146
Phillips GC, CollinsGB (1979) Crop Science 19:59-64

References

Raskin I (1992) Annual Review of Plant Physiology and Plant


Molecular Biology43:439-463
Reynolds T (1978) Annals of Botany 42:419-427

Davis KR, Hahlbrock K (1987) Plant Physiology85:1286-1290


Do CB, Cormier F (1990) Plant Cell Reports 9:143-146
Do CB, Cormier F (1991) Plant Cell Tissue and Organ Culture 27:
169-174
Do CB, Cormier F (1991) Plant Cell Reports 9:500-504

Saint-Pierre B, MivilleL, Dion P (1984) Canadian Journal of Botany


62:729-734
Seitz HU, De Luca V, Kurz WGW (1989) Plant Cell Tissue and
Organ Culture 18:71-78
Watanabe K, Takimoto A (1979) Plant Cell Physiology20:874-850