J Plant PhysioL VtJI. 150. pp.

325-330 (1997)

Cell Division Versus Secondary Metabolite Production in

Morinda citrifolia Cell Suspensions

MARc J. M. HAGENDOORN, DlAAN C. L. JAMAR, BARBARA MEYKAMP, and

LINUS H. W. VAN DER PLAS
Dept. of Plant Physiology, Wageningen Agricultural University, Arboreturnlaan 4, 6703 BD, Wageningen, The
Netherlands

Received July 24, 1996 . Accepted September 1, 1996

Summary

Morinda citrifolia cell suspension cultures are capable of accumulating high levels of anthraquinones,
but appear to display an inverse relationship between the rate of cell division and anthraquinone produc-
tion: low auxin concentrations lead to a high anthraquinone production and a low growth rate while at
high auxin concentrations the reverse was observed. NAA and 2,4-D both showed this effect although
there was a difference with respect to the optimal concentration, 2,4-D being active at approximately a
hundred-fold lower concentration than NAA.
We studied the inverse relation between cell division and anthraquinone accumulation in two ways.
First, batch cultures of Morinda citrifolia were treated with hydroxyurea, which almost completely inhib-
ited cell division. The 2,4-D cultures still did not accumulate anthraquinones. Second, we compared con-
tinuous cultures grown in the presence of NAA or 2,4-D at the same cell division rates. Only the NAA
culture accumulated anthraquinones. The overall high sugar content of the cells under the various cultiva-
tion conditions indicated that sufficient energy and carbon substrates were always present. The possible
direct effect of auxins on the induction and activity of the pathways leading to anthraquinone production
is discussed.

Key words: Morinda citrifolia, anthraquinones, auxin, ceO division, ceO suspension, continuous culture,
hydroxyurea, secondary metabolites.

Abbreviations: AQ = anthraquinones; D = dilution rate; DW = dry weight; HU = hydroxyurea.

Introduction A prerequisite for a study of the competition between pri-

In plant cells the primary metabolism is of vital impor-
tance for the secondary metabolism because it supplies the
energy, the precursors and the machinery to synthesize sec-
ondary metabolites. Many precursors of secondary metabo-
lites, as well as ATp, are also necessary for the synthesis of cell
constituents. Therefore, there is often competition for precur-
sors and energy between growth and secondary metabolism.
For cell suspension cultures, this might result in a secondary
metabolite production that only occurs when growth is slow
or absent (e.g. anthocyanin production in the stationary
growth phase of Vitis cells, Hall and Yeoman, 1986).
mary and secondary metabolism in cell suspensions is that a
sufficient amount of secondary metabolites must be pro-
duced. Otherwise, the impact of the synthesis of these prod-
ucts on the physiology of the cells is probably small and
therefore difficult to study. There are only a few systems that
meet this condition, e.g. rosmarinic acid production in Coleus
blumei (Ulbrich et al., 1985) and anthraquinone (AQ)
accumulation in Morinda citrifolia (Zenk et al., 1975; Hagen-
doorn et al., 1994; Van der Plas et al., 1995). In the latter sys-
tem, anthraquinones can make up as much as 20 % of the dry
weight (DW), making these cells very suitable for studies on
the interaction between primary and secondary metabolism.

© 1997 by Gustav Fischer Verlag, Jena

326 MARc J. M. HAGENDOORN, DIAAN C. L. JAMAR, BARBARA MEYKAMP, and LINUS H. W. VAN DER PLAS
Previous experiments showed that in the presence of
0.5 mg· L -\ 2,4-0 Morinda cells do not produce anthraqui-
nones. When the cells are transferred to medium containing
0.5 mg . L -\ NM, they produce considerable amounts of
anthraquinones. This production can be blocked by addition
of an equal amount of 2,4-0 (Hagendoorn et al., 1994; Van
der Plas et al., 1995). In this paper we further analyse the
(background of the) differences between the synthetic auxins
NM and 2,4-0. Furthermore, we examine the relation be-
tween cell division and secondary metabolite production in
Morinda citrifolia.
Materials and Methods
Tissue culture
The Morinda citrifolia cultures (Zenk et al., 1975) were main-
tained in 250-mL Erlenmeyer flasks containing 60 mL of culture
medium (B5, Gamborg et al., 1968) supplemented with sucrose
(40 g. L-i), kinetin (0.2 mg . L-i) and auxin (different concentra-
tions, depending on the type of experiment). The cells were subcul-
tured every 14 days (10 mL of culture transferred to 50 mL of fresh
medium). In the hydroxyurea experiments (5 mmol/L hydroxyurea),
at each subculturing the inoculum density of the treated cultures
was the same as that of control cultures. The cultures were grown at
25 ·C with a 16-h day period, an 8-h night period, and an irradiance
oflO-20Wm- 2 (Philips no. 84).
For continuous cultures, cells were cultured in a standard 2-L
bioreactor with a round bottom vessel, stirred with a ADI 1012 mo-
tor controller (Applikon) at 200 rpm. Air was continuously supplied
at a rate of 20 Uh. For the aeration, air was filtered through 0.2 ~m
filters (Millipore). Cells were grown under phosphate limitation (at
0.2 mmol/L phosphate) in B5 medium supplemented with 10 g. L-i
sucrose and 0.2 mg· L-i kinetin, at a dilution rate of 0.1 day-i.
Auxin type and concentration depended on the experiment.
Wtights and ctll numb"
Fresh weight was determined after harvesting the cells on a
Buchner funnel with a paper filter. For determination of the dry
weight the cells were dried at 60 ·C for 24 h. For the determination
of the cell number, two samples of the culture were, after an appro-
priate dilution, counted in a haemocytometer (Fuchs-Rosenthal).
The-cells in 16 squares were counted and the mean was determined.
Dtttrmination ofcell viability
One volume of cell suspension was mixed with one volume of
50 mglL fluorescein diacetate. The latter was freshly prepared by di-
luting a 5 giL stock in growth medium. The percentage of viability
was calculated as the number of cells that showed green fluorescence
under UV illumination, divided by the total number of cells
(counted under white light).
Anthraquinone dtttrmination
The anthraquinone content of the cell suspension was deter-
mined spectrophotometricalIy as described earlier (Hagendoorn et
al., 1994). A millimolar extinction coefficient of 5.5 was used (Zenk
et al., 1975; Koblitz, 1988). For further calculations a mean molec-
ular weight value of 534 for the glycosylated AQ was used (based on
a sugar moiety of glucose and xylose, Inoue et al., 1981).
Dettrmination ofcarbohydrates
Carbohydrate concentrations were determined with a Dionex
HPLC system (Dionex corporation, Sunnyvale, USA) as described
earlier (Hagendoorn et al., 1994).
Results
Effects of2,4-D and NAA
Morinda citrifllia cells can be grown in the presence of
different synthetic auxins. We tested the effect of a range of
concentrations of 2,4-0 and NM on cell division. In Fig. 1
the cell numbers after three consecutive subculturings in
media with various auxin concentrations are depicted. NM
has a significant, positive effect on growth at a concentra-
tion of 10- 6 mol/L and higher, while similar effects of 2,4-0
are already visible at a hundred-fold lower concentration
(10- 8 moIlL).
Both synthetic auxins show a negative effect on anthraqui-
none accumulation (Fig. 2). When the auxin concentration
increased, the accumulation of anthraquinones decreased. In
the presence of 2,4-0 a steep drop in the AQ content occur-
red between 10- 8 and 10-7 M. A similar drop in AQ content
occurred in the case of NM, however, only when concentra-
tions exceeded 10- 6 M.
To check whether the effects of NM and 2,4-0 are not
fundamentally different, we determined the AQ production
by cells grown in the presence of mixtures of NM and 2,4-
O. Such auxin combinations resulted in a cumulative effect,
when a hundred-fold difference in effectiveness is taken into
account (see also discussion). This also indicates that NM
and 2,4-0 do not interact directly.
9
8
.-
:::i'
0
C
7
I
:::I
c:
5
i 4
3
2
1
0 10-11 10" 10-7 10"
auxin concentfllion (M)
Fig. 1: Effect of NM (closed circles) and 2,4-D (open circles) on
the cell number in Morinda citrifolia cell suspensions after three sub-
culturings, measured at the stationary phase (Hagendoorn et al.,
1994). Means are depicted ± standard deviations; n= 12. Data were
fitted according to a sigmoid function.
 Cell division vs. secondary metabolism in Morinda cells 327
200

~150
• 0
.-.
..J
6

~ S
0
ob
:::. 5
! ...
CD
:100 .a 4
c E;:,
0
c c 3
.
'5
•
I::r
,c
'ii
u
C 50 2
•
1
+ HU

o V~--~~--~~----"~~QQ~"""
o 1 0"
auxin concentr8llon (M)
Fig.2: Effect of NM (closed circles) and 2,4-0 (open circles) on
anthraquinone accumulation in Morinda citrifolia suspension cells
after three subculturings, measured at the stationary phase (Hagen-
doom et al., 1994). Data points represent means of duplicate experi-
ments. Data were fitted according to a sigmoid function.
o
NAA 2,4-D
Fig. 3: Increase in cell number during the second subculturing (dif-
ference in cell number at day 31 and day 15) in Morinda citrifolia
batch cultures treated with 5 mM hydroxyurea (+ HU), in the pres-
ence of NM or 2,4-0, compared with the cell number increase of
the controls. Error bars represent standard deviation (n= 12).

i'
c 80
Effects ofhydroxyurea ~
c
To study the correlation between cell number and AQ E
content, we tried to inhibit cell division without affecting .,
"-

CD
60
cell viability by application of 5· 10- 3 mol/L hydroxyurea C
0
(HU; Kihlman et al., 1966). There was no significant de- c 40
';
crease of cell viability in NM cultures after 14 days incuba-

-
•...
CT
tion in the presence of HU (cell viabilities all ranging from .r:
100-95 %). The viability of 2,4-0 cells decreased in the 20
c
same period from 100 % (control cells) to 70 % (cells treated •
with HU). After 30 days in the presence of 2,4-0 or NM, 0
the viability decreased to approximately 60 % in both cul-
tures. 0 10 20 30 40 50
Figure 3 shows the effect of HU on cell number between days after HU addition
day 15 and day 31. Both in 2,4-0- and in NM-cultures, the Fig. 4: Time course of anthraquinone accumulation in Morinda
increase in cell number is dramatically reduced in comparison citrifolia cells during three subsecutive subculturings, grown in the
with the controls. When the AQ accumulation of HU-treated presence of NM (closed symbols) or 2,4-0 (open symbols). Com-
cells is analysed there is no significant difference between parison of cells treated with 5 mM hydroxyurea (triangles) and con-
these cells and the control cells (see Fig. 4). In 2,4-0 cultures trol cells (circles). Subculturing took place after 15 and 30 days.
the AQ accumulation is still negligible; in NM cultures the Duplicate measurements are depicted.
AQ content reaches a maximal level of ca. 60 mg . g-I . dry
weight.
NM and at 0.5 mg· L-I 2,4-0. Figure 5 shows the growth
data for these two continuous cultures. It appeared that the
Continuous cultures
cell numbers were almost identical (Fig. 5A), while the mean
Another method to acquire low cell division rates in the dry weight content was somewhat higher in NM cells than
presence of high concentrations of auxin (i.e. 2,4-0) is lim- in 2,4-0 cells (4.2 ± 0.18 and 3.4 ± 0.07g.L-I, respectively,
itation of the cell division rate by growing cells in continuous see Fig. 5 B). The AQ content of both types of cells is shown
cultures. Under these circumstances, the dilution rate (D) de- in Fig. 6. Although the cell division rate in the two cultures
termines the cell division rate. By application of the same di- was identical, there is still a clear difference in the AQ con-
lution rate (i.e. 0.1 day-I), it was possible to establish identi- tent: 2.1 ± 0.4 mg· gOW- I in 2,4-0 cultures and 17.6 ±
cal cell division rates in cell cultures growing at 0.5 mg. L- I 1.6 mg· gOW- I in NM cultures.
328 MARc J. M. HAGENDOORN, DIAAN C. L. JAMAR, BARBARA MEYKAMP, and LINUS H. W. VAN DER PLAS

2 5

1.5 • • - • •••• n
4

.• i'
-'
• •
0
!! ~o o oo~~ n 3 Q 400
'8 -'
a. ~
••
1 'v

• 2
.§.. 300
0.5 f
IV
aI
A B il 200
o o
o 10 20 30 40 o 10 20 30 40 .!
J:I
days :s
days '0 100
10

Fig. 5: Cell number (A) and dry weight content (B) of continuous
cultures of Morinda citrifolia cell suspensions. Cultures were grown o
in the presence of 2,4-D (open circles) or NAA (closed circles), at a cont. batch +HU
dilution rate of 0.1 day-I. For dry weight, duplicate measurements
are depicted; for cell number the mearJs of 12 determinations are Fig.7: Soluble sugar content (sucrose+glucose+frucrose) of Mo-
shown. rinda citrifolia suspension cells, grown under different conditions.
COnt., continuous cultures, grown at a dilution rate of 0.1 day-I;
batch, untreated batch cultures; + HU, batch cultures treated with
2 5 fl 5 mM hydroxyurea. Open bars represent 2,4-D cultures, hatched
bars represent NAA cultures. Error bars represent standard devia-
i' tions (n=6).
• • ••
020
Ii
• •• • •
01
C!
§. 1 5 • • •
II)
4D
C
•
g 1 0 o o
:i
go
~ 150
o

-
I! ~
.c 5 .§..
C
IV (") n 0 r
I 0 v v : 100
tl v
o &
c
o 1 0 2 0 30 40 5 0 'S
[
days
~ 50
Fig. 6: Anthraquinone content of continuous cultures of Morinda •
citrifolia. Cultures were grown in the presence of 2,4-D (oren cir-
cles) or NAA (closed circles), at a dilution rate of 0.1 day- . Points
are mearJs of duplicate measurements.
o 10.11 10" 1006
auxin equivalents (1[2,4-DJ=110[NAA))

To check if the availability of energy or carbon substrates

was limiting AQ production, we compared the sugar contents
of the cells grown under different culture conditions. Figure 7
shows the sum of the contents of the most important soluble
sugars (sucrose, glucose and fructose) in Morinda cells from
batch cultures (with and without hydroxyurea) and continu-
ous cultures. In NM-cultures the sugar content is somewhat
higher than in the corresponding 2,4-D-cultures. The differ-
ences between the different groups are, however, not very
large. In all cultures there is a large excess of mono- and di-
saccharides: they make up 28 to 46 % of the cell contents on
a dry weight basis.
Discussion
The regulation of secondary metabolite accumulation dif-
fers for different classes of secondary compounds. In cell sus-
pensions of Phytolacca americana, the secondary metabolite
Fig.8: Effect of NAA (open circles), 2,4-D (open diamonds) and
combinations of NAA and 2,4-D (closed circles) on anthraquinone
accumulation in Morinda citrifolia suspension cells after three sub-
culturings, measured at the stationary phase. Data points represent
means of duplicate experiments. Auxin analogue concentrations are
depicted as <auxin equivalents>, where 1[2,4-D] = 1l0[NAA]. Data
were fitted according to a sigmoid function.
betacyanin is accumulated during the logarithmic phase of
growth (Sakuta et al., 1986). This is, however, an exception;
many secondary metabolites are chiefly accumulated during
the stationary phase (e.g. anthocyanins in Vitis cultures, Hall
and Yeoman, 1986). In Morinda citrifolia cell suspension cul-
tures the anthraquinone production continues through all
phases of growth and therefore cannot be assigned to one of
these modes/types of production.
The accumulation of anthraquinones seemed to be deter-
mined by the type of auxin analogue in the culture medium

(Zenk et al., 1975). Our results show that both NAA and 2,4-
o are able to stimulate cell division in Morinda citrifolia cell
cultures. At low concentrations, both auxins allow anthraqui-
none accumulation. The greatest difference between the two
synthetic auxins is the effectiveness. When the data from
Figs. 1 and 2 were fitted with a sigmoidal curve, comparison
of the inflection points showed a difference in effectiveness
between these two analogues of approximately a factor of
110. Analysis of the anthraquinone accumulation leads to the
same difference in effectiveness between NAA and 2,4-0: in-
hibition of this accumulation by 2,4-0 occurred at a concen-
tration that was about 110 times lower. When combinations
of NAA and 2,4-0 were used, the rates of cell division and
AQ accumulation could be predicted from the equation [2,4-
0] = llO*[NAA]. In Fig. 8 the effect of auxin combinations
on AQ production is depicted by expressing the concentra-
tions as «2,4-0-equivalents», calculated according to this for-
mula. The auxin effect, indeed, seemed to be determined by
the sum of the concentrations of NAA and 2,4-0 calculated
in this way. This approach was applicable both for AQ pro-
duction and for cell division (data not shown).
Figures 1 and 2 suggest that there is a close negative relation
between cell division and AQ production. Experiments were
therefore designed to investigate whether this was just a
statistical correlation or a causal one. When Morinda cells were
treated with hydroxyurea, the cell division was largely inhib-
ited (Kihlman et al., 1966; Fig. 3), while the majority of cells
remained viable. So a situation was created where 2,4-0 was
present and the cell division rate was low. This, however, did
not result in a significant AQ accumulation, indicating that
there is no direct causal relation between cell division and AQ
accumulation. In contrast with this observation, anthocyanin
accumulation in Vitis cells could be evoked by cessation of cell
division, due to inhibition of DNA synthesis (Sakuta et al.,
1994) or phosphate starvation (Kakegawa et al., 1995).
To corroborate these results with artificial growth inhib-
itors like HU, we grew Morinda cells in fermentors under
growth limiting conditions. In this way we wanted to estab-
lish the same cell division rate in 2,4-0-cultures as in NAA- GAMBORG, O. L., R. A. MILLER, and V. OJIMA: Nutrient require-
cultures. A continuous culture was started using growth me-
dium with a reduced phosphate concentration, thus creating
a (low) cell division rate that was independent of the type of HAGEN DOORN, M. J. M., L. H. W. VAN DER Pus, and G. J. SEGERS:
auxin analogue used (Fig. 5 A). The cell division rate in these
continuous cultures was the same as in batch cultures at that
cell density (compare Fig. 5 A and Van der Plas et al., 1995,
Fig. 1). Again, such a low cell division rate did not lead to sig- HALL, R. D. and M. M. YEOMAN: Temporal and spatial heterogene-
nificant AQ accumulation in 2,4-0 cultures (Fig. 6), while
NAA-cultures at this cell division rate showed a significant
production.
The AQ content of cells from a continuous culture using
NAA as auxin was significantly lower than that of batch-
grown cells, and ultimately reached a stable level, whereas in KAKEGAWA, K., J. SUDA, M. SUGIYAMA, and A. KOMAMINE: Regula-
batch cells the AQ accumulation increased until the cells died
(Van der Plas et al., 1995). A possible explanation for the
higher AQ production rate in batch cultures might be the
amount of auxin present per amount of cell volume. Due to
the higher cell densities in the later phases of the batch
growth, the medium auxin is shared by a larger number of KOBLITZ, H.: Anthraquinones. In: CONSTABEL, F. and I. K. VASIL
cells, possibly leading to a lower intracellular concentration.
The relatively high availability of auxin for cells grown in
Cell division vs. secondary metabolism in Morinda cells 329
continuous culture under our conditions, might lead to a
higher intracellular auxin concentration and, as a conse-
quence, a lower AQ production. In this way the negative ef-
fect of anthraquinone accumulation on cell viability, as was
seen previously in batch cultures, was avoided. Whether the
limited availability of phosphate in phosphate-limited contin-
uous cultures has any direct or indirect effect on the produc-
tion of anthraquinones is not known.
The sugar content of the different types of cell suspensions
of Morinda citrifolia was determined to examine the possibil-
ity of an insufficient availability of energy and precursors, as a
cause of the lack of AQ accumulation in 2,4-0 cells. Figure 7
shows that the differences between 2,4-0 and NAA cultures
are not always significant. All of the cultures studied have a
very high sugar content, suggesting that at least endogenous
substrate is not limiting AQ production. These data suggest
that the <down regulation) of the AQ production by auxins
seems to work more directly on the specific pathways leading
to anthraquinone synthesis.
Summarising, we conclude that the high cell division rate
in batch cultures growing in the presence of 2,4-0 is not the
direct cause of the lack of anthraquinone accumulation in
these cultures. In our Morinda citrifolia system, the produc-
tion and accumulation of secondary metabolites is not simply
regulated by a so-called overflow mechanism. It is possible
that auxins have a co-ordinated effect on cell division and
secondary metabolism (positive and negative, respectively).
Auxins might be able to inhibit one or more steps in the sec-
ondary metabolism. Being at a cross-roads in the pathways
leading to anthraquinone synthesis, chorismic acid turnover
may be a feasible regulation point. In the near future, the reg-
ulation of the transition of chorismic acid into isochorismic
acid, the first committed step towards anthraquinone synthe-
sis, will be examined.
References
ments of suspension cultures of soybean root cells. Exp. Cell. Res.
50, 151-158 (1968).
Accumulation' of anthraquinones in Morinda citrifolia cell sus-
pensions. A model system for the study of the interaction be-
tween secondary and primary metabolism. Plant CellTIss. Org.
Cult. 38, 227-234 (1994).
ity in the accumulation of anthocyanin in cell cultures of Catha-
ranthus roseus (L.) G. Don. J. Exp.. Bot. 145, 1055-1065 (1986).
INOUE, K., H. NAYESHIRO, H. INOUYE, and M. H. ZENK: Anthra-
quinones in cell suspension cultures of Morinda citrifolia. Phyto-
chemistry 12, 1693-1700 (1981).
tion of anthocyanin biosynthesis in cell suspension cultures of
Vitis in relation to cell division. Physiol. Plant. 94, 661-666
(1995).
KiHLMAN, B. A., T. ERIKSON, and G. ODMARK: Effects ofhydroxyu-
rea on chromosomes, cell divison and nucleic acid synthesis in
Viciafoba. Hereditas 55, 386-397 (1966).
(eds.): Cell culture and Somatic Cell Genetics of Plants, Vol. 5,
pp. 113-139. Academic Press, Orlando (1988).
330 MARcJ. M. HAGENDOORN, DIAAN C. L. JAMAR, BARBARA MEYKAMP, and LINUS H. W. VAN DER PLAS
SAKUTA, M., T. TAKAGI, and A. KOMAMINE: Growth related accumu-
lation of betacyanin in suspension cultures of Phytolacca ameri-
cana. Plant Physiol. 125,337-343 (1986).
SAKUTA, M., H. HIRANO, K. KAKEGAWA, J. SUDA, M. HIROSE, R.
W. JOY IV, M. SUGIYAMA, and A. KOMAMINE: Regulatory mecha-
nisms of biosynthesis of betacyanin and anthocyanin in relation
to cell division activity in suspension cultures. Plant Cell Tiss.
Org. Cult. 38, 167-169 (1994).
ULBRICH, 8., w. WIESNER, and H. ARENS: Large-scale production
of rosmarinic acid from plant cell cultures of Coleus blumei
Benth. In: NEUMANN, K.-H., W. BARZ, and E. REINHARD
(eds.): Primary and Secondary Metabolism of Plant Cell Cul-
tures (pp. 293-303). Springer-Verlag, Berlin, Heidelberg
(1985).
VAN DER PLAS, L. H. w., C. EI]KELBOOM, and M. J. M. HAGEN-
DOORN: Relation between primary and secondary metabolism in
plant cell suspensions: competition between secondarymetabolite
production and growth in a model system (Morinda citrifolia).
Plant Cell Tiss. Org. Cult. 43, 111-116 (1995).
ZENK, M. H., U. SCHULTE, and H. EL SHAGI: Anthraquinone pro-
duction by cell suspension cultures of Morinda citrifolia. Planta
Med., Supp!. pp. 79-101 (1975).