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BIOTROPIA No. 20,2003: 24 - 35
INFLUENCE OF MEVALONIC ACID AND LINALOOL ON LIMONENE
ACCUMULATION IN CALLUS TISSUES OF CITRUS GRANDIS OSBECK
NIK NORULAINI NIK AB RAHMAN', ZARINA ZAKARiA2 .AND MOHD OMAR ADD KADIRS
'School fur Distance Learning Education, Universiti Sains Malaysia,
11800 USM. Penang, Malaysia. 2 Faculty of Applied Sciences,
Universiti Teknologi MARA, Arau Campus,
02600 Arau, Perils, Malaysia.
3 School of Industrial Technology, Universiti Sains Malaysia,
11800 USM, Penang, Malaysia.
ABSTRACT
Effect on callus growth was studied for Citrus grandis cultured with feeding of exogenous mevalonic acid
(MVA) at concentrations of 0.04, 0.08, 0.38, 0.77, 1.15 and 1.54 mM. Similar effect with linalool ranging from 10 to 200
nl was studied under various incubation periods. The growth was proportional to the concentrations of precursors
used meaning that higher precursors concentrations influenced more growth on C. grandis callus culture. Mevalonic acid
and linalool showed quite similar precursor feeding effects on limonene accumulation of C. grandis callus cultures. It was
revealed that limonene production was triggered with the introduction of MVA and linalool even at low concentration.
Limonene accumulation was detected as early as week four and continued to increase at about 0.0030 and 0.0032 mg/g with
MVA and linalool, respectively, after the seventh week incubation. In comparison to the unfed cultures, no limonene was
detected from the callus up to eight weeks in incubation.
Keywords : C/rrusgrarafo/Limonene/Linalool/Mevalonic acid
Abbreviations: 2,4-D: 2,4 — Dichlorophenoxyacetic acid, ABA: Abscisic acid, MVA : Mevalonic acid
INTRODUCTION
Bioconversion using an exogenous supply of biosynthetic precursor is believed to improve the
accumulation of desired metabolites compound where the productivity is limited because of the
lack of particular precursors. Naturally occurring as well as related synthetic compounds may be
used as precursors. Many attempts have been made to produce valuable compounds by adding
precursors to several culture species. Often the precursors undergo more than one bioconversion
resulting in complex mixture of unknown products (Kawaguchi et al. 1988). Dorisse et al. (1988)
had studied the bioconversion of papaverine in Glycyrrhiza gabra cell suspension and found the
bioconversion rate into papaverinol (31.5%), papa-veraldine (4%) and demethylpapaverine
was rather slow. In other case, hydro-
quinone was glucosylated into arbutin after addition to cell suspension cultures of
Datura innoxia (Suzuki et al. 1987) in production rate of 2.5 gl-1 after of
incubation
BIOTROPIA No. 20, 2003
Feeding of 3 mM L-tyrosine or phenylalanine into Lithospermum flavum cultures
yielded a 2-3 fold increase in 5-methoxy podophyllotoxin accumulation
(Van Uden et al. 1990). The byconversion rates were low; 0.34 and 0.5 mg g d , respectively. It is
clear that there are significant differences in bioconversion rates. Several reasons include that;
the rate may be dependent on the solubility of precursors and/or the intracellularly present
amounts of enzyme. Furthermore, the precursors can be metabolized by other reactions and
even a specific transport system may be involved. Funk and Brodelius (1990) have used
precursor feeding and addition of metabolic inhibitors techniques as a tool to evaluate the
possibility that involved to the undetactable vanillin from the cultivated cells of Vanilla
plan/folia. They found that cinnamic acid, but not ferulic acid, is a precursor of vanillic acid in
these cultivated cells of V. plamfolia.
Extraction of Citrus grandis or pomelo peel contained almost exclusively limonene
(92.6%) and myrcena (1.9%) suggests that pomelo may be utilized as source material for the
production of limonene (Wong 1992). The precursors for the synthesis of monoterpenoids are
obtained from acetate-mevalonate pathway, which supply to mevalonic acid. Mevalonic acid is
the primary precursor of all the terpenoids biosynthesized by plant, including limonene.
Orange juice vesicles (Citrus sp., Rutaceae) contain an enzyme system which is capable of
phospho-rylating mevalonic acid and forming the final product, a monoterpene, linalool. Intact
fruits and leaves convert linalooi to limonene and other cyclic monoterpenoids. Linalool is
considered as an immediate precursor which is expected to readily converted to limonene under
favourable conditions.
There are numerous reports of experiments on mevalonic acid as a precursor. Moreno et al.
(1993) reported that feeding mevalonic acid did not increase alkaloid accumulation in cell
suspension cultures of Catharanthus roseus. Mevalonic acid
added to cultures at concentration of 3.3 mM was reported to saturate the 030 (steroid)
biosynthetic pathway in Nicotiana tabaccum suspension cultures (Threfall & Whitehead 1988).
With the aim of shortening the period for limonene production and enhancing its accumulation,
the effect of feeding primary and immediate precursors was investigated.
MATERIALS AND METHODS
Culture conditions
Young C. grandis fruits about five weeks after anthesis and 4-5 cm in diameter were obtained
from established plantation. Sterilization technique was established by immersing the whole fruit
into 20% Clorox® (5.2% sodium hypochloride) for 2 hours prior to peeling and cutting. The fruits
were rinsed with sterile distilled water three times and cut transversely into small segments.
Callus was initiated on MS (Murashige & Skoog 1962) basal medium supplemented with 30
mg/1 sucrose. 3.0 me/1 2.4-D 3.0 mg/1 kinetin and 0.2 mg/1 ABA and incubated in the dark 24
Influence of mevalonic acid and linalool on limonene accumulation - Nik Norulaini Nik Ab Rahman et al.
hours/day at room temperature. Callus were formed after 2-6 weeks with a success rate of 80-90%.
Callus were maintained on the media and subcultured to the same fresh media every 4 weeks
interval.
Feeding of precursors
Mevalonic acid lactone ( MVA) and linalool (Analytical grades) were purchased from
Fluka ( Japan). Aqueous solution of MVA was prepared by adding 50 ml distilled water to 1 g of
MVA powder. MVA was filter sterilized and aliquots were added to different MS media to get a
range of final MVA concentrations of 0.04, 0.08, 0.38, 0.77, 1.15 and 1.54 mM in the media.
Various volume of linalool (~ 97% purity), 10, 50, 100, 150 and 200 μj were added to 1 liter MS
media prior to autoclave. After 4 weeks, callus from maintenance media were cut into smaller
pieces and transferred by spreading over the same MS medium with varying added precursors.
Callus growth determination
Callus fresh weight was taken as final weight started from the fourth week followed by
fifth, sixth, seventh and eighth week of incubation period by direct measurement. To minimize
the differences in growth, the initial weight of the callus prior transferring to the experimental MS
media was ensured about equal according to each treatment. Callus was cleaned from attached
media and dried using tissue paper before weighing using analytical balance (Sartorius, Germany).
Several plates were sacrificed along the time course of the experiment, to measure the biomass
accumulate.
Limonene determination and analysis
Extraction of limonene was carried out from four to eight weeks old callus. Fresh callus
weighing 2.0 - 3.0 g were extracted with approximately 70 ml methanol using soxhlet apparatus
according to Morris et al. (1985). Identifications and quantification of limonene were made by
comparing with standard purchased from Fluka using Gas Chromatography (Shimadzu GC -
17A) equipped with FID detector. Column used was BP-20 (0.25 (im, 30m X 0.25mm) from
SGE. The temperature was programmed at 60 C, 4 min, 140 C, 8 min. Injector and detector
temperature was 250 C respectively. The experiments were performed in triplicates and data were
subjected to analysis of variance (ANOVA).
BIOTROPIA No. 20, 2003
RESULTS
Effect of various concentrations of MVA feeding and incubation period on callus growth
rate
Figure 1 shows the effect of various concentrations of MVA feeding on callus fresh weight.
Comparisons of callus fresh weights, taken as an indicator of cell proliferation were determined
within the eighth weeks period for all MVA feeding tested. Addition of exogenous MVA
enhanced the callus growth even at low level except for 0.04 mM compared to the control.
However, as the concentration of MVA feeding increased, the callus fresh weight increased
concomitantly as pointed out in the figure. Among the concentrations used, there was no significant
difference (P>0.05) between 0.38 and 0.77 mM and between 1.15 and 1.54 mM (Table 1).
Highest callus fresh weight was obtained with the feeding of 1.15 and 1.54 mM of MVA where
increase in fresh weight was about two times higher compared to control callus.
Influence of mevalonic acid and linalool on limonene accumulation - Nik Norulaini Nik Ab Rahman et al.
Table 1 : Effects of mevalonic acid concentrations on callus growth and limonene accumulation of Citrus grandis after
culture for 7 weeks.
MVA(mM) Fresh weight (g) Limonene (mg/g)
0.04 3.127a 0.0015c
0.08 3.900c 0.003 le
0.38 4.868d 0.0030e
0.77 4.807d 0.0020d
1.15 5.335e O.OOISc
1.54 5.574e 0.0013c
control 3.218b 0
Different alphabet for each data indicates a significant difference at a =
0.05 according to Tukey HSD test.
The figure also reveals that MVA feeding exerts positive effect on callus growth throughout
the eight-week culture duration. The most active growth occurred from fourth to sixth week in
incubation. Callus fresh weight was measured beginning the fourth week of incubation. First
measurements of fresh weight were done at week four and at this point all MVA feedings showed
a higher callus fresh weight than control except for 0.04 mM MVA feeding. However, subsequently
each feeding concentration displays similar callus growth. Higher concentrations of MVA feeding
illustrates greater callus growth from fourth to seventh week in culture. For all corcentrations, the
fresh weights achieved constant level after the seventh week.
Effect of various linalool feeding concentrations and incubation period on callus growth rate
All feeding concentrations of linalool enhanced callus growth rate, though to a different
degree. Figure 2 shows that only the addition of 10 to 100 jil/1 had resulted on distinctively
different (P<0.005) in callus fresh weight proportional to linalool concentrations. Feeding of
higher than 100 (j.1/1 showed slightly reduction in callus growth but still higher than control callus.
Callus showed fresh weight increment at the beginning of incubation period at the fourth week and
increased in fresh weight occurred after longer incubation period until the seventh week. After this
period, fresh weight remained about level indicating callus had slowed down or reached constant
growth. It was about twofold increment in callus fresh weight compared to control treatment when
added with 50 (il/l and above of linalool.
Effect of incubation period and various MVA feeding concentrations on limonene
accumulation
The combined effect of various concentrations of MVA feeding and the incubation period is
shown in Figure 3. Compared to cultures with no exogenous MVA added, other cultures with
MVA feeding showed limonene as early as four weeks in culture. This accumulation continues up
to the seventh week, whereby limonene accumulation displays a sudden decline. This phenomenon
was exhibited in all MVA concentrations used. However, cultures fed with 0.08 mM MVA shows a
more active limonene accumulation from fourth to seventh week and this is followed by 0.38 mM
MVA which after the fifth week, shows a drastic increase in limonene accumulation to match that
accumulated in cultures fed with 0.08 mM MVA. Other concentrations of MVA feeding profile
displayed an increase from fourth to seventh week and a decline thereafter in much lower
limonene accumulation.
Greatest effect of MVA concentrations was observed on limonene accumulation
using 0.08 and 0.38 mM, whereby there is no significant difference
Influence of mevalonic acid and linalool on limonene accumulation - Nik Norulaini Nik Ab Rahman et al.
between the two (Table 1). Outside these concentrations, the accumulation of limonene was
significantly lower (P>0.05). For cultures with no exogenous MVA added that is the control, no
limonene was detectable.
Effect of various linalool feeding concentrations and incubation period on limonene
accumulation
Results from Figure 4 shows that limonene accumulation was detected from feeding of
linalool at all concentrations studied. There was no particular pattern on limonene accumulation
with various linalool feeding concentrations. Limonene content seems to accumulate at a
significant amount when 10 and 50 μl/1 of linalool was added. This amount slightly decreased with
feeding of 100 μl/l of linalool and increased instantly with 150 μ.1/1 before dropped again at
concentration of 200 μ.1/1 of linalool.
The figure also demonstrated that limonene accumulation was highest at seventh
week of incubation period. However, limonene accumulation with 200 μl/1 exogenous linalool
addition gave maximum peak at week six. Limonene value dropped after the seventh week of
incubation regardless of linalool feeding concentrations. Most rapid increase of limonene
accumulation occurred between six to seven weeks of incubation and the feeding of 150 μl/1 of
linalool giving the highest accumulation (Table 2).
Influence of mevalonic acid and Imalool on limonene accumulation - Nik Norulaini Nik Ab Rahman et al.
DISCUSSION
Effect of precursor feeding on callus growth
Effect on callus growth was studied for C. grandis cultured with feeding of exogenous
MVA ( Figure 1) and linalool ( Figure 2) under various concentrations and incubation period.
The growth was proportional to the concentrations of precursors used meaning that higher
precursors concentrations influenced more growth on C. grandis callus culture except at very low
concentration, which is 0.04 mM of MVA. Obvious callus growth was observed from week five to
week six and began to reduce after week seven where the increase was about threefold by fresh
weight. During this time, callus turned brown indicated that the accumulation of secondary
metabolites in cells had caused toxicity and started to die.
Increase in callus growth showed that precursor supplied at the studied range did not inhibit
callus growth in fact improve growth development. Similar result by Moreno et al. (1993) stated that
feeding of the terpenoid precursors mevalonic acid, loganin, loganic acid or secologanin to
suspension cultures of Catharanthus roseus did not affect the culture growth, as determined by
dry weight concentrations measured at 72 and 120 hrs after feeding. These results demonstrated no
toxic effect for all precursors added to the cultures.
Decreased in callus fresh weight after the seventh week could coincide with the initiation of
secondary metabolites synthesize as cell growth approached stationary phase. Lindsey and
Yeoman (1983) concluded that there is indirect relationship between cells growth and
secondary metabolites accumulation. It is expected that secondary metabolites accumulation was
stimulated only when cells slow down their rate of growth i.e. at the end of the logarithmic phase
and the beginning of the stationary phase. Ketchum et al. (1999) showed that Taxus
suspension cultures accumulated maximum levels of taxoid at the end of the growth phase.
This conclusion could be related to the finding of this study. Interestingly, the rate of growth
and highest fresh weight value attained for both precursors did not differ significantly (P>0.05).
This concluded that both precursors have had same potential to serve as internal growth stimulator
acting to shoot up various aspects of cell metabolism.
It was suggested that rapid growth at the early stage of callus was due to synergistic effect
of feeding precursors and added growth factors. This phenomenon was demonstrated when 2,4-D
was reduced in immobilized cultures of Catharanthus roseus cells that rised up the production of
alkaloid (Tom et al 1991). Decrease in growth rate after week seven for fed cells was found a
little greater than nonfed cells. Reasonable explanation for this phenomenon is that the
accumulation of limonene and linalool themselves might have caused cells toxicity which led to
cell fatality (Brown et al 1987; Charlwood & Brown 1987).
BIOTROPIA No. 20,2003
Effect of precursors feeding on limonene accumulation
Mevalonic acid lactone and linalool showed quite similar precursor feeding effects on C.
grandis callus cultures. Illustrations in Figure 3 for MVA feeding and Figure 4 for linalool feeding
revealed that limonene production was triggered with the introduction of MVA and linalool at low
concentration. Limonene accumulation was detected as early as week four and continued to increase
until the seventh week. The highest limonene accumulation was 0.0030 mg/g and 0.0032 mg/g
obtained from cultures fed with 0.08 mM of MVA for the former and 150 uM of linalool for the
later on the seventh week of incubation (Tables 1 & 2).
The results proved that precursor feeding had triggered limonene production in C. grandis
cultures earlier than non fed cultures as suggested by Moreno et al. (1993). They stated that
exogenous supply of a biosynthetic precursor, secologanin, to the culture medium may improve
metabolites, such as strictosidine and ajmalicine accumulation where the productivity is limited by
lack of that particular precursor. It was showed when they added the terpenoid precursor during
the growth phase, about 11-fold increase in strictosidine occurred 72 hrs after feeding and decreased
to 5 fold after 120 hrs. Similar pattern was observed from Imbault et al. (1996) on MVA
feeding effects. They showed that the inhibition of endogenous MVA production by
pravastatin treatment blocked the alkaloid biosynthesis; supplying such cells with exogenous
MVA allowed accumulation of alkaloid, but did not result in incorporation of MVA. However,
Moreno et al. (1993) did not find any significant increase in alkaloid level after mevalonic
acid feeding, the basic precursor for all terpenoids, in Catharanthus roseus suspension cultures.
For both treatments, it is concluded that limonene production was not dependent on amount of
precursors added. It showed that only a small amount of MVA and linalool was needed as a
stimulator for limonene production.
Other possibility that contributes to this finding was the facts that MVA must be catalytically
phosphorylated by ATP and mevalonic kinase itself for the pathway to continue. Therefore, the
lack of those co-factor and enzyme had restricted limonene production eventhough with the
presence of high amount of precursor (Rohmer et al. 1996; Lichtenthaler et al. 1997). The strong
role of enzymes and its incorporation with feeding precursor in study of biosynthesis was briefly
described by Zenk (1991) in his review. Besides, for accumulation of a secondary compound to
occur in cultured cells, it is clearly necessary that the system retains the ability both to synthesize
and to store the product.
Trend in limonene accumulation shows that cell maturity has direct relation to secondary
metabolite accumulation with both precursor feeding. It was obvious that feeding of both precursors
accumulated highest peak of same amount of limonene at the same incubation period. Highest
limonene accumulation at the seventh week suggested that cells had slowed down their
division to prepare themselves for metabolite production. This result fulfilled the suggestion
from Lindsey and Yeoman (1983) that rapidly growing cultures accumulate cells and not
secondary products. A similar pattern of results was obtained from suspension culture of
Influence of mevalonic acid and linalool on limonene accumulation - Nik Norulaini Nik Ab Rahman et al.
Podophyllum hexandrum on the production of podophyllotoxin (Pras 1992). Both situations
demonstrated that the increase in metabolite compounds accumulation occurred only at the
mature-death stage of the growth curve.
Consideration also has to take account for limonene itself an intermediate product and
would be converted to other form of monoterpenoid. This situation is definitely uncontrollable
because biosynthesis is a continuous process and when and how the bioconversion occurred need
further and deeper study. Potential compounds synthesize from limonene are a-terpeniol,
perillic acid and carveon. Further experiments on identifying the accumulation of those
compounds in C. grandis cultures should be carried out to prove this statement. For future work
on limonene improvement, it can be suggested that study on the regulation of enzymes involved in
limonene biosynthesis could impose clearer picture of the biosynthetic pathway. The use of
suspension cultures for fast limonene accumulation is among the future work that can be
considered.
CONCLUSION
The period of limonene production in C. grandis callus culture was shortened and
accumulation was enhanced by precursor feeding with MVA and linalool. Callus growth rate
improved significantly in the presence of the precursors. This result also reconfirmed the
presence of MVA and linalool in limonene pathway.
ACKNOWLEDGEMENT
The authors would like to thank Universiti Sains Malaysia for the research grant and the
Ministry of Science, Technology & Environment (MOSTE) for National Science Fellowship
scheme.
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