Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol150.Issue1.2000:

(1)

Mevalonate kinase activity in

Catharanthus roseus

plants and

suspension cultured cells

Anna E. Schulte

_{, Eva M. Llamas Dura´n, Robert van der Heijden *, Robert Verpoorte}

Di6ision of Pharmacognosy,Leiden/Amsterdam Center for Drug Research,Gorlaeus Laboratories,P.O.Box9502, 2300RA Leiden,The Netherlands

Received 17 May 1999; received in revised form 10 August 1999; accepted 27 August 1999

Abstract

Mevalonate kinase is an early enzyme in plant isoprenoid biosynthesis. Its activity was studied in different parts ofCatharanthus roseus (L.) G. Don (Apocynaceae) plants and in C. roseus suspension cultured cells. In the plant specific mevalonate kinase activities were found to be relatively high in the fruits, stem, roots, flowers and buds, and relatively low in young and completely elongated leaves. In suspension cultured cells, the specific mevalonate kinase activity increased during the exponential phase of growth, reaching a maximum of 0.5 nkat/mg protein at 4 days after subculturing. After 6 days, which corresponded to the beginning of the stationary phase, the specific activity decreased. After transferring 14-day-oldC.roseuscells to a medium known to induce the production of terpenoid indole alkaloids, an increase in the specific mevalonate kinase activity was observed, reaching a maximum of 1.7 nkat/mg protein at 8 days after transfer. The alkaloid accumulation in the cultures was monitored, and ajmalicine was found to be the major product. Under standard conditions, mevalonate kinase activity showed a diurnal rhythm, with highest activities at 12:00 and 24:00. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Catharanthus roseus; Apocynaceae; Terpenoid biosynthesis; Mevalonate kinase; Mevalonate

www.elsevier.com/locate/plantsci

1. Introduction

Isoprenoid biosynthesis is one of the major pathways in plants leading to a huge number of compounds. Many of these play important roles in growth and development, such as abscisic acid, chlorophyll, ubiquinone, sterols and phytoalexins [1,2]. The common precursor of all isoprenoids is isopentenyl diphosphate (IPP). The demand and competition for this precursor must be extremely high, as a consequence of its unique position in this physiologically important pathway. At

present, two pathways are known that result in the biosynthesis of IPP, i.e., the mevalonate (MVA) pathway [1 – 3], and the 2-C-methyl-D-erythritol

4-phosphate (MEP) pathway (at the 4th European Symposium on Plant Isoprenoids (Barcelona, April 21 – 23 1999) it was agreed to use only the names ‘‘Rohmer pathway’’ (after its discoverer) or ‘‘MEP pathway’’ (after 2-C-methyl-D-erythritol

4-phosphate, the first committed precursor) for the recently discovered non-mevalonate pathway of IPP biosynthesis.) [4,5]. The MVA pathway is supposed to be exclusively cytosolic and in plants the MEP pathway has been shown to be localized in the chloroplasts.

As to the mevalonate pathway, it consists of a sequence of six enzyme reactions (Fig. 1). First, three molecules of acetyl-CoA are condensed to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is reduced to MVA by HMG-CoA reductase (HMGR; EC 1.1.1.34). MVA is

subse-* Corresponding author. Fax: +31-71-527-4511.

E-mail addresses: [email protected] (A.E. Schulte), [email protected] (R. van der Heijden)

1_{Present address: Section Enzymology, Department of} Biotechnol-ogy, Delft University of TechnolBiotechnol-ogy, Julianalaan 67, 2628 BC, Delft, The Netherlands.

(2)

quently phosphorylated to the diphosphate before it is decarboxylated to form IPP. In this sequence of enzyme reactions HMGR has been recognised as an important enzyme for the regulation of substrate flux into isoprenoid biosynthesis [1,2,6]. In addition, IPP isomerase (EC 5.3.3.2) [7], and MVA kinase (MK; EC 2.7.1.36) have been indi-cated to be regulators of the metabolic flux to-wards isoprenoids.

Considering plant MK, its regulatory role has been proposed due to the fact that in vitro the enzyme has been found to be inhibited by geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), two later intermediates of isoprenoid biosynthesis, in cotyledons of Phaseolus 6_ulgaris

and Cucumis melo, and in He6_ea _{latex [8]. This}

characteristic has also been shown in yeast, hu-man, and rat MK [9 – 11]. In addition, the rat liver MK has been found to be regulated by sterols on the protein [10] and mRNA [12] levels. Indeed, the presence of a sterol regulatory element, known to control gene expression of HMGR and HMG-CoA synthase, has been recognised in the pro-moter region of the human MK gene [13].

At present, the genes for MK have been cloned from yeast [9], rat [12], human [14] and Arabidop

-sis thaliana [15], and the sequences showed to be

highly homologous.Therefore, regulatory charac-teristics as found for mammalian MK might also hold for MK from a plant source. Consequently,

MK is expected to be an important enzyme in the regulation of isoprenoid biosynthesis in plants. Indeed, MK activity has been correlated with the accumulation of phytoalexins in potato tuber after wounding and elicitation [16], and with increased rubber production in Guayule plants after treat-ment with a bioregulator [17].

However, the importance of the MVA pathway in the biosynthesis of isoprenoids of plastidial origin has been questioned since the discovery of the presence of the MEP pathway in plant chloro-plasts. Certainly, the MVA pathway is predomi-nantly localized in the cytosol. Nevertheless, MK activity was detected in transforming maize etio-plasts [18], and labeling studies showed the pres-ence of the MVA pathway in the leucoplasts of peppermint glandular cells [19], and immature chloroplasts of young spinach plants [20].

In addition, numerous labeling studies have been performed on seedlings showing incorpora-tion of MVA into alkaloids (reviewed in [21]). On the other hand, labeling studies with photo-mixotrophic suspension cultures of Catharanthas

roseus have indicated that the plastidial

iso-prenoids were derived from the MEP pathway [22]. In addition, the ultimate precursor of the terpenoid moiety of terpenoid indole alkaloids, i.e. secologanin, has been shown to be derived from the MEP pathway in a specially selected, secolo-ganin-accumulating green cell-line of C. roseus in

Fig. 1. Biosynthesis of isopentenyl diphosphate according to the mevalonate pathway. AACT, acetoacetyl-CoA thiolase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; PMK, 5-phosphomevalonate kinase; PMDC, 5-diphosphomevalonate decarboxylase; and IPP, isopentenyl diphosphate.

(3)

our laboratory [23]. More studies will be necessary to determine the possibility of cross-talk and ex-change of intermediates between the MVA and MEP pathways in the formation of different ter-penoid classes, in particular in relation to the differentational and physiological state of the cell. Imbault et al. [24] showed that the inhibition of endogenous MVA production by pravastatin treatment blocked the alkaloid biosynthesis; sup-plying such cells with exogenous MVA allowed accumulation of alkaloids, but did not result in incorporation of MVA. Recently, it was suggested that isoprenylated proteins are involved in the regulation of secologanin biosynthesis. These proteins may thus form the link between the MVA and the MEP pathway in alkaloid biosynthesis [25]. The MVA pathway thus seems to play an essential role in the biosynthesis of terpenoid-in-dole alkaloids, either in a direct or an indirect way.

Therefore, in continuation of our studies on the early steps of isoprenoid biosynthesis [26] atten-tion was focused on MK. MK was studied in C.

roseus plants and suspension cultured cells grown

under standard conditions and on a medium known to induce the accumulation of terpenoid-indole alkaloids [27].

2. Materials and methods

2.1. Chemicals and enzymes

Polyvinylpolypyrrolidone (PVPP), phospho-enolpyruvate (PEP), bovine serum albumin (BSA) and the mixed solution of pyruvate kinase/lactate dehydrogenase (PK/LDH) rabbit muscle enzymes in ammonium sulphate or glycerol were obtained from Sigma. Sucrose, KCl, MgCl2·6H2O,

1,4-dithiothreitol (DTT), ATP and NADH were ob-tained from Merck. KF, Tris Ultrapure and mevalonolactone were obtained from J.T. Baker, GibcoBRL and Fluka, respectively. Mevalonolac-tone was hydrolyzed to the free acid according to Popja´k [28].

2.2. Biological materials

2.2.1. Plants

C. roseus roseus (L.) G. Don plants were grown

in the greenhouse. Growth conditions were:

tem-perature 24°C, humidity 60% and in summer nor-mal daylight.

2.2.2. Suspension cultured cells

Cell suspensions ofC.roseuswere grown on MS medium [29] without growth regulators and con-taining 3% sucrose. The cultures were kept under continuous light (ca. 1200 lx) at 100 rpm and 25°C, and were subcultured weekly by weighing 5 g cells fresh weight (fr. wt.) in 50 ml medium. C.

roseus cells grown for 14 days on MS medium

were transferred to MS medium or IM 2 medium [27] by weighing 20 g cells fr. wt in 500 ml medium. Due to practical constraints the experi-ment was performed in batches.

2.3. Sampling for MK acti6ity

2.3.1. Plants

Two plants were collected and subdivided in flowers and buds, young leaves up to 3 cm long, completely elongated leaves, stem, roots and fruits. The flowers and buds from each plant were pooled because of the small quantities. Extracts were prepared and the MK activity was deter-mined by means of the radiochemical assay (see below).

2.3.2. Cell suspensions: effects of media and inoculum size

At the days indicated in Fig. 2, cells from two flasks were harvested, frozen separately in liquid nitrogen and stored at −80°C until further use. This was done for the three growth conditions mentioned above. Determinations were made of accumulation of fr. wt. and dry wt. (after lyophili-sation) and MK activity in the acetone precipitate by means of spectrophotometrical assay (see be-low). In addition, the alkaloid accumulation was monitored in the samples collected at the days indicated in Fig. 3.

2.3.3. Variation during the day

For the determination of the variation of MK activity during the day, two flasks cultured under standard growth conditions were harvested every 4 h during the exponential phase of the growth cycle; the cells were frozen in liquid nitrogen and stored at −80°C until further use. Determinations were made of fr. wt. and dry wt. accumulation and MK activity in the crude extract by means of a radiochemical assay (see below).

(4)

2.4. Enzyme extraction

Crude extracts were prepared by grinding the frozen biomass in a precooled (−20°C) mortar in

the presence of sea-sand and

polyvinyl-polypyrrolidone (10% wt./fr. wt. each) and in the presence of homogenisation buffer (HB) in a ratio of 2 ml buffer to 1 g fr. wt. The HB consisted of 0.1 M Tris – HCl, pH 7.5, 0.1 M sucrose, 50 mM KCl and 10 mM 1,4-dithiothreitol. All manipula-tions were performed at 4°C and the materials, extracts and buffers were kept on ice. The crude extracts were centrifuged at 3500×gfor 30 min. at 4°C, and the supernatants were filtered over Mira-cloth to remove any floating particles. Where indi-cated, the proteins were precipitated with cold acetone (−20°C) to 60% v/v, and collected by centrifugation at 3500×g for 30 min. at 4°C. The protein precipitate was resuspended in 1:1 water diluted HB, and subsequently centrifuged at 13 000 g for 5 min. at 4°C. The protein concentrations were determined by the method of Peterson [30].

2.5. Enzyme assays

MK activity was measured by means of a spec-trophotometrical assay and a radiochemical assay, both principally according to Popja´k [28].

2.5.1. Spectrophotometrical assay

Enzyme solution was incubated at 30°C with reaction mixture to determine base-line activities, e.g. ATPases and NADH oxidases. Subsequently, MVA was added to start the measurement of MK activity. The decrease in absorption at 340 nm was monitored for both the base line and MK activities for at least 2 min, when stable. Final concentrations in a 0.5 ml final volume were: 3.3 mM MVA (R,S), 2.3 mM ATP, 3.5 mM MgCl2, 3.5 mM KF, 0.3 mM

phosphoenolpyruvate, 0.14 mM NADH, 5.75 U LDH, 4 U PK, and 3.5 mM 1,4-dithiothreitol in 34.5 mM Tris – HCl, pH 7.5.

2.5.2. Radiochemical assay

Enzyme solution (10 ml) was incubated at 30°C with MVA in a reaction mixture giving final con-centrations of 0.5 mM MVA (R,S) (59.24 MBq/ mmol [2-14_{C]MVA), 2 mM ATP, 2 mM MgCl}

2, 2

mM KF and 50 mM Tris – HCl, pH 7.5, in a total volume of 20 ml. The reaction was started by addition of MVA and stopped after 15 min

by addition of 5 ml 72% TCA. The samples were applied to Silica F254TLC plates after

centri-fugation at 16 000×g for 2 min. The plates were developed in ethanol – ammonia – water (80:12.5:7.5%). The radioactivity on the TLC plate was visualised and quantified using a beta-scope. MK activity was calculated from the sum of the radioactivity in the phosphorylated products. Rf

values were 0.79, 0.18, 0, and 0.05 for MVA, 5 - phosphomevalonate, 5 - diphosphomevalonate and IPP, respectively.

2.6. Alkaloid determination

The alkaloid extraction was performed as previ-ously described [31]. Freeze-dried cell material (50 mg) was extracted twice with 5 ml dichloromethane and the extracts were combined. To reach extrac-tion of tryptamine, 0.5 ml 1 M NaOH was added to the remaining biomass and the extraction was repeated with 5 ml dichloromethane. Medium, 2 ml, was extracted with 4 ml dichloromethane; 3 ml extract were collected. The solvent was evaporated and the residues were dissolved in 0.5 ml HPLC eluent (5.52 g NaH2PO4.H2O in 800 ml water, 150

ml acetonitrile, and 50 ml 2-methoxy-ethanol, pH set to 3.9 using concentrated phosphoric acid). The alkaloids were identified by HPLC analysis using photodiode-array detection [32]. Retention times: ajmalicine 21 min., vindolinine 8.3 min., catha-ranthine 17.8 min, and tabersonine 25.4 min.

2.7. Statistical analysis

The data for the determination of the variation of MK activity during the day, were grouped according to the time of harvesting the cells, yield-ing six groups with harvestyield-ing times at 4, 8, 12, 16, 20 and 24 h, respectively. The variances were determined to be equal according to the Bartlett’s test. The statistical significance between means was assessed by the Kruskal – Wallis test and the Dun-can’s multiple range test.

3. Results and discussion

3.1. Assaying MK acti6ity

MK activity can be assayed by spectrophotomet-rical or radiochemical methods [28].

(5)

The spectrophotometrical assay is a continuous, but indirect, enzyme-coupled assay; the ADP formed in the reaction catalysed by MK is coupled to the oxidation of NADH by the activities of pyruvate kinase (PK; EC 2.7.1.40) and lactate dehydrogenase (LDH; EC 1.1.1.28). Consequently, for the optimisation of the MK assay, the reaction conditions for the auxiliary enzymes PK and LDH have to be optimised.

In addition, it has been recognized that enzyme-coupled assays are susceptible to problems associated with contaminants in specific assay components, which may result in misleading interpretation of the measurements. Strategies for the identification of such artifacts have been reviewed in Ref. [33]. Our experience showed that a non-linear relationship between the protein concentration and enzyme activity was obtained when using the spectrophotometrical assay with crude extracts from the C. roseus plants and cells (results not shown); similar problems were encountered by others [34]. We expect that these effects were due to inhibitory effects on PK and LDH activity. These inhibitory effects were overcome by sample pre-treatment, e.g. gel filtration, ultrafiltration or acetone precipitation. We think that specific plant compounds present in crude extracts cause these inhibitory effects, as even high salt concentrations did not seem to influence the assay system.

The radiochemical assay has the advantage that it can be used directly on the crude extract. However, care has to be taken to inhibit phosphatase activities present in plant extracts. The values of MK activity obtained by means of the two assays differ, the spectrophotometrical assay giving on average six to seven times higher values than the radiochemical assay. Likely, this is due to interference of 5-phosphomevalonate kinase (PMK; EC 2.7.4.2) and 5-diphosphom-evalonate decarboxylase (PMDC; EC 4.1.1.33) in the spectrophotometrical assay. Nevertheless, the profile of MK activity in different cell extracts proved to be quite similar when using the spectrophotometrical assay or the radiochemical assay.

3.2. MK acti6_{ity in plants}

The MK activities in the different parts of the

C. roseus plants are presented in Table 1. The

mevalonate phosphorylating enzymes have been studied in extracts from different plant sources using an assay similar to the radiochemical assay applied here (Table 2). The specific MK activity in

C. roseus plants is relatively high (Table 1)

com-pared to the reported values for Nepeta [35],

Spinacia [35] and Parthenium [17] (Table 2). High

MK specific activities were recovered from the flowers and buds, fruit, stem and root compared to rather low specific activities from young and fully elongated leaves (Table 1). A similar distribu-tion has been found for the enzymes acetoacetyl-CoA thiolase (AACT; EC 2.3.1.9) and HMG-acetoacetyl-CoA synthase (HMGS; EC 4.1.3.5) in C. roseus plants [26].

Flowers and buds might have a high MK activ-ity because of their developmentally related, high metabolic activity. As in He6_ea _{the MK activity}

was extremely high in the latex [36] (Table 2), it might be that the MK activity in the fruit and stem is related to a high content of latex. Unfortu-nately, attempts to measure MK activity in some droplets of latex collected from the C. roseus

plants were unsuccessful. Stem and root had simi-lar MK activities, both as related to their protein content as well as related to their biomass. Fur-thermore, both young leaves and fully elongated leaves had low MK specific activities, due to their high protein content. Only in young leaves the MK activities showed to be quite high when re-lated to the amount of biomass. This is due to the lower dry weight content of young leaves (14.3%) compared to fully elongated leaves (20.5%).

3.3. MK acti6ity in suspension cultured cells

Biomass accumulation and MK activity were studied for C. roseus suspension cultured cells grown under standard conditions (Fig. 2A) and on an induction medium (IM 2) (Fig. 2C), known to enhance the accumulation of terpenoid indole al-kaloids in C. roseus cell suspensions [27]. As for the IM 2 medium, two parameters were changed at once, i.e. the age of the cells at the moment of inoculation (14 instead of 7 days), and the size of the inoculum (4% fr. wt./vol. instead of 10% fr. wt./vol.), the same conditions were applied for cells on the standard growth medium (Fig. 2B). The biomass accumulation in C. roseus suspen-sion cultured cells differed greatly dependent on a combination of the size of the inoculum and the age of the cells, and the medium type.

(6)

Subcultur-ing 7-day-old cells with a 10% inoculum (fr. wt./vol.) in standard medium resulted in a

sigmoidal growth curve (Fig. 2A). When

14-day-old cells were transferred to standard medium or IM 2 medium with a 4% inoculum (fr. wt./vol.), biomass accumulated in a linear way (Fig. 2B and C). The dry wt accumulation in IM 2 medium was about 1.5 times higher than in standard medium at the end of the experiment. The increase in dry wt. in this medium is mainly due to the accumulation of starch.

Fig. 2 also presents the time courses of MK activity for the C. roseus cells grown under different conditions. Under standard conditions (Fig. 2A) MK activity increased during the exponential phase reaching a maximum of 0.5 nkat/mg protein after 4 days. After 6 days, at the onset of the stationary phase, MK activity decreased. Only after 18 days did MK activity increase again to 0.85 nkat/mg protein. This increase in activity might be related to alkaloid production, as the ajmalicine accumulation increased concomitantly (Fig. 3A).

When 14-day-old cells were subcultured in standard medium MK activity reached values of 0.7 to 0.9 nkat/mg protein between 11 and 17 days after inoculation (Fig. 2B). When 14-day-old cells were transferred to IM 2 medium MK activity increased to about 1.5 nkat/mg protein after 8 and 9 days (Fig. 2C). Compared to the cells growing under standard conditions, this is a 2 – 3-fold increase in activity.

The accumulation of terpenoid indole alkaloids in the C. roseuscell suspensions was monitored at different times during the culturing period under the three culturing conditions mentioned. Alkaloids and tryptamine, the indole moiety of the terpenoid indole alkaloids, were recovered only from the biomass; no alkaloids or tryptamine were detected in the medium. In addition, no alkaloids were detected in the cells subcultured in MS medium after 14 days and at a high dilution (4% fr. wt./vol.); however, these cultures did accumulate tryptamine. The tryptamine levels in these cultures increased from 0.7 to 1.5 mg/l cell suspension from the 3rd to the 15th day after subculturing, and increased even further to 13.4 mg/l cell suspension after 20 days (results not shown). Fig. 3A and B present the ajmalicine and tryptamine accumulations in the cell cultures grown under standard conditions (10% fr. wt./vol.) and in the IM2 medium (4% fr. wt./vol.), respectively. The results clearly show the substrate/product relationship between tryptamine and ajmalicine; when alkaloid levels increase, tryptamine levels decrease.

Fig. 2. Biomass accumulation and mevalonate kinase activity in C. roseus suspension cultured cells grown under different conditions. (A) In standard medium at an inoculum of 10% (fr. wt./vol.), (B) in standard medium at an inoculum of 4% (fr. wt./vol.) and (C) in induction medium at an inoculum of 4% (fr. wt./vol). The crude extracts were prepared as indi-cated in Section 2 and the MK activity was determined by means of the spectrophotometrical assay using acetone pre-cipitates obtained from the crude extracts. Indicated are the averaged values; bars represent individual values. DW, dry wt.

(7)

Fig. 3. Ajmalicine and tryptamine accumulation (mg/l cell suspension) inC.roseussuspension cultured cells grown (A) in standard growth medium at an inoculum of 10% (fr. wt./vol.) and (B) in induction medium at an inoculum of 4% (fr. wt./vol.). Culture conditions and the alkaloid determinations are described in Section 2. The MK activities indicated are the averaged values from Fig. 2A and C at the corresponding culture ages.

In addition to ajmalicine, trace amounts of taber-sonine and vindolinine were detected in the cell biomass cultured under standard conditions, and trace amounts of vindolinine and catharanthine were recovered from the cells subcultured into the induction medium (results not shown). Further-more, Fig. 3 shows that, in contrast to expectation, the accumulation of ajmalicine in the induction medium is lower than in the standard growth medium, except at prolonged culturing periods. Additional results within our laboratory showed that the accumulation of alkaloids or tryptamine is

dependent on the inoculum size in the growth medium [37]. An increase of inoculum-size from 40 to 160 g fr. wt./l medium favored the accumulation of secologanin and ajmalicine; this is in agreement with the results presented here.

In addition, the MK activity profiles from Fig. 2A and C have been added to their corresponding Fig. 3A and B, respectively. Under standard conditions (Fig. 3A), the ajmalicine accumulation correlates well with the MK activity profile. However, after the cells have been transferred to induction medium, there does not seem to be any correlation

(8)

Table 1

MK activity in different parts of twoC.roseusplantsa

Plant part MK in pkat/mg protein MK in nkat/g dry wt.

Plant 1 Plant 2 Plant 1 Plant 2

63 2.4 1.5

Flowers/buds 112

n.d. n.d.

n.d.

Fruit 111

36 1.4

Young leaf 32 1.2

30 0.4

Leaf 25 0.5

119 89

Stem 0.9 1.1

103

Root 86 0.9 1.1

a_{MK activities are related to protein contents of the extracts (pkat/mg protein) and the amount of biomass used for the}

extractions (nkat/g dry wt.). Crude extracts were prepared as described in Section 2 and the MK activity was determined using the radiochemical assay. n.d., Not determined.

between alkaloid production and MK activity (Fig. 3B). As mentioned earlier, higher plants have two pathways for the biosynthesis of IPP [5]. The involvement of the MEP pathway in the biosyn-thesis of secologanin, a precursor of the terpenoid-indole alkaloids, in C. roseus is now well established [23]. However, these studies were per-formed on a specially selected, secologanin-accu-mulating, green cell line; presumably, labeling studies will also confirm the relationship between the MEP pathway and ajmalicine production in the yellowish/white C. roseus cell line used in this study. The results presented may indicate that under standard growth conditions (Fig. 3A) MK activity could be indirectly involved in the biosyn-thesis of terpenoid indole alkaloids, e.g. via regula-tion of indole-alkaloid biosynthesis by prenylated proteins [25].

As presented in Fig. 2, the MK activity showed a high variation in activity. Also, during some preliminary feeding experiments (unpublished re-sults) it was found that the MK activity varied considerably depending on the time of harvesting the suspension culturedC. roseuscells. It has been reported before that the activities of the MVA phosphorylating enzymes in plants show a

sea-sonal variation, e.g. for Pelargonium gra6eolens

[38] and Cymbopogon citratus[39]. For HMGR in

He6ea latex a diurnal variation has been reported [40]. Also, HMGS inHe6_ealatex showed a diurnal variation, with high activities at 10:00 and 22:00 [41].

A 12 h variation was found for MK inC.roseus

suspension cultured cells, with higher activities at 12:00 and 24:00 (Fig. 4). Statistical analyses of the means as calculated for the combined values at each harvesting time showed that the mean MK activity is significantly higher at the 95% level at 24 h than at 4, 8 and 16 h.

As the cells are grown under continuous light, the physiological importance of this phenomenon is not clear. One explanation might be that the variation in MK activity is related to plant cell growth. For mammalian cells it has already been proven that MVA-derivatives, especially preny-lated proteins, are essential for growth activation, e.g. initiation of DNA synthesis [42], and control cell morphology and cell duplication by their ac-tion on the cytoskeleton [43]. Prenylaac-tion of proteins has also been found in suspension cul-tured cells of tobacco, and was considered to be essential during the early stages of growth [44,45].

Table 2

Reported values for MK activities from different plant sources

Source MK activity (pkat/mg protein)

Plant Reference

[35]* 5.1

Nepeta cataria Leaf

2.5 [35]

N.cataria Callus

[35]* 2.5

Spinacea oleracea Leaf

Plant 12.2

Parthenium argentatum [17]

Latex serum 190

He6_{ea brasiliensis} _[36]

(9)

Fig. 4. Mevalonate kinase specific activity during the growth phase ofC.roseussuspension cultured cells grown under standard growth conditions. The crude extracts were prepared as indicated in Section 2. MK activity was determined by means of the radiochemical assay. Indicated are the mean values; bars represent individual values. The insert graph presents the averaged MK activities as a function of harvesting time; for harvesting at 24:00, 04:00, and 08:00n=4), and for harvesting at 12:00, 16:00, and 20:00 (n=6).

Nevertheless, now that it has also been shown that in suspension cultured cells variations in enzyme activities can occur, this should be accounted for. Certainly, strict control of the moments of subcul-turing and harvesting might reduce variation in enzyme activities.

During the growth phase of suspension cultured

C. roseus cells grown under standard conditions,

MK values of about 60 – 100 pkat/mg protein are obtained using the radiochemical assay on crude extracts (Fig. 4). These values are comparable to the MK activities found in the stem and roots of

the C. roseus plant. Taking into account that

biomass can be easily obtained with the fast

grow-ing C. roseus cell suspensions and that extracts of

the suspension cultured cells are cleaner, e.g. no chlorophylls, the cell suspensions are considered to be the best source for the purification of the enzyme MK, allowing further studies on the regu-lation of this enzyme (manuscript in preparation).

Acknowledgements

The research of RvdH has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. Financial support by the ‘Van Leersum fonds’ is gratefully acknowledged.

References

[1] J.C. Gray, Control of isoprenoid biosynthesis in higher plants, Adv. Bot. Res. 14 (1987) 25 – 90.

[2] T.J. Bach, Some new aspects of isoprenoid biosynthesis in plants — a review, Lipids 30 (1995) 191 – 202. [3] J.L. Goldstein, M.S. Brown, Regulation of the

meval-onate pathway, Nature 343 (1990) 425 – 430.

[4] M. Rohmer, M. Seemann, S. Horbach, S. Bringer-Meyer, H. Sahm, Glyceraldehyde 3-phosphate and pyru-vate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis, J. Am. Chem. Soc. 118 (1996) 2564 – 2566.

[5] H.K. Lichtenthaler, M. Rohmer, J. Schwender, Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants, Physiol. Plant. 101 (1997) 643 – 652.

[6] B.A. Stermer, G.M. Bianchini, K.L. Korth, Regulation of HMG-CoA reductase activity in plants: review, J. Lip. Res. 35 (1994) 1133 – 1140.

[7] A.C. Ramos-Valdivia, R. van der Heijden, R. Verpoorte, Isopentenyl diphosphate isomerase: a core enzyme in isoprenoid biosynthesis; a review of its biochemistry and function, Nat. Prod. Rep. 14 (1997) 591 – 604.

[8] J.C. Gray, R.G.O. Kekwick, The inhibition of plant mevalonate kinase preparations by prenyl pyrophos-phates, Biochim. Biophys. Acta 279 (1972) 290 – 296. [9] A. Oulmouden, F. Karst, Nucleotide sequence of the

ERG12 gene ofSaccaromyces cere6_isiaeencoding meval-onate kinase, Curr. Genet. 19 (1991) 9 – 14.

[10] R.D. Tanaka, B.L. Schafer, L.Y. Lee, J.S. Freuden-berger, S.T. Mosley, Purification and regulation of mevalonate kinase from rat liver, J. Biol. Chem. 265 (1990) 2391 – 2398.

(10)

[11] D. Potter, H.M. Miziorko, Identification of catalytic residues in human mevalonate kinase, J. Biol. Chem. 272 (1997) 25449 – 25454.

[12] R.D. Tanaka, L.Y. Lee, B.L. Schafer, et al., Molecular cloning of mevalonate kinase and regulation of its mRNA levels in rat liver, Proc. Natl. Acad. Sci. USA 87 (1990) 2872 – 2876.

[13] R.W. Bishop, K.L. Chambliss, G.F. Hoffmann, R.D. Tanaka, K.M. Gibson, Characterization of the meval-onate kinase 5%-untranslated region provides evidence for coordinate regulation of cholesterol biosynthesis, Biochem. Biophys. Res. Commun. 242 (1998) 518 – 524. [14] B.L. Schafer, R.W. Bishop, V.J. Kratunis, et al.,

Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic dis-ease mevalonic aciduria, J. Biol. Chem. 267 (1992) 13229 – 13238.

[15] C. Riou, Y. Tourte, F. Lacroute, F. Karst, Isolation and characterization of a cDNA encoding Arabidopsis thaliana mevalonate kinase by genetic complementation in yeast, Gene 148 (1994) 293 – 297.

[16] G.M. Bianchini, B.A. Stermer, N.L. Paiva, Induction of early mevalonate pathway enzymes and biosynthesis of end products in potato (Solanum tuberosum) by wounding and elicitation, Phytochemistry 42 (1996) 1563 – 1571.

[17] C.R. Benedict, P.H. Reibach, S. Madhavan, R.V. Sti-panovic, J.H. Keithly, H. Yokoyama, Effect of 2-(3,4-dichlorophenoxy)-triethylamine on the synthesis of

cis-polyisoprene in Guayule plants (Parthenium argen

-tatumGray), Plant Physiol. 72 (1983) 897 – 899.

[18] M. Albrecht, G. Sandmann, Light-stimulated biosyn-thesis during transformation of maize etioplasts is regu-lated by increased activity of isopentenyl pyrophosphate isomerase, Plant Physiol. 105 (1994) 529 – 534.

[19] D. McCaskill, R. Croteau, Monoterpene and sesquiter-pene biosynthesis in glandular trichomes of peppermint (Mentha×piperita) rely exclusively on plastid derived isopentenyl diphosphate, Planta 197 (1995) 49 – 56. [20] A. Heintze, A. Riedel, S. Aydogdu, G. Schultz,

Forma-tion of chloroplast isoprenoids from pyruvate and ac-etate by chloroplasts from young spinach plants; evidence for a mevalonate pathway in immature chloroplasts, Plant Physiol. Biochem. 32 (1994) 791 – 797.

[21] A. Contin, The biosynthesis of secologanin in Catha

-ranthus roseus cell suspension cultures, PhD Thesis, Leiden University, 1999.

[22] D. Arigoni, S. Sagner, C. Latzel, W. Eisenreich, A. Bacher, M.H. Zenk, Terpenoid biosynthesis from 1-de-oxy-D-xylulose in higher plants by intramolecular skele-tal rearrangement, Proc. Natl. Acad. Sci. USA 94 (1997) 10600 – 10605.

[23] A. Contin, R. van der Heijden, A.W.M. Lefeber, R. Verpoorte, The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a

Catharanthus roseus cell culture, FEBS Lett. 434 (1998) 413 – 416.

[24] N. Imbault, M. Thiersault, P. Dupe´ron, A. Benabdel-mouna, P. Doireau, Pravastatin: a tool for

investigat-ing the availability of mevalonate metabolites for primary and secondary metabolism in Catharanthus roseus cell suspensions, Physiol. Plant. 98 (1996) 803 – 809.

[25] N. Gigliolo-Guivarc’h, A.L. Tarroa, M. Thiersault, P Doireau, Involvement of isoprenylated proteins in the activation of alkaloid biosynthesis in cell suspensions of Catharanthus roseus, poster presentation, In Vitro Plant Cell Engineering, Amiens, 2 – 4 December 1998. [26] R. van der Heijden, V. de Boer-Hlupa´, R. Verpoorte,

J.A. Duine, Enzymes involved in the metabolism of 3-hydroxy-3-methylglutaryl Coenzyme A in Catha

-ranthus roseus, Plant Cell Tissue Org. Cult. 38 (1994) 345 – 349.

[27] K.H. Knobloch, J. Berlin, Influence of medium compo-sition on the formation of secondary compounds in cell suspension cultures of Catharanthus roseus (L.) G. Don, Z. Naturforsch 35c (1980) 551 – 556.

[28] G. Popja´k, Enzymes of sterol biosynthesis in liver and intermediates of sterol biosynthesis, Methods Enzymol. 15 (1969) 393 – 423.

[29] T. Murashige, F. Skoog, A revised medium for rapid growth and bio-assays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473 – 497.

[30] G.L. Peterson, A simplification of the protein assay method of Lowry et al. which is more generally appli-cable, Anal. Biochem. 83 (1977) 346 – 356.

[31] J. Schripsema, R. Verpoorte, Search for factors in-volved in indole alkaloid production in cell suspension cultures of Tabernaemontana di6_aricata, Planta Med. 58 (1992) 245 – 249.

[32] R. van der Heijden, P.J. Lamping, P.P. Out, R. Wi-jnsma, R. Verpoorte, High performance liquid chro-matographic determination of indole alkaloids in a cell suspension culture of Tabernaemontana di6_aricata_{, J.}

Chromatogr. 396 (1987) 287 – 295.

[33] N.J. Kruger, Errors and artifacts in coupled spec-trophotometrical assays of enzyme activity, Phytochem-istry 38 (1995) 1065 – 1071.

[34] G. Sandmann, M. Albrecht, Assays for three enzymes involved in mevalonic acid metabolism, Physiol. Plant. 92 (1994) 297 – 301.

[35] R.E. Arebalo, E.D. Mitchell Jr, Cellular distribution of 3-hydroxy-3-methylglutaryl Coenzyme A reductase and mevalonate kinase in leaves of Nepeta cataria, Phyto-chemistry 23 (1984) 13 – 18.

[36] D.N. Skilleter, R.G.O. Kekwick, The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate-5-phosphate) in the latex of He6_ea

brasiliensis, Biochem. J. 124 (1971) 407 – 417.

[37] A. Contin, R. Van der Heijden, H.J.G. ten Hoopen, R. Verpoorte, The inoculum size triggers tryptamine or secologanin biosynthesis in a Catharanthus roseus cell culture, Plant Science 139 (1998) 205 – 211.

[38] D.V. Banthorpe, D.R.S. Long, C.R. Pink, Biosynthesis of geraniol and related monoterpenes in Pelargonium gra6eolens, Phytochemistry 22 (1983) 2459 – 2463. [39] R. Lalitha, R. George, T. Ramasarma, Mevalonate

de-carboxylation in lemon grass leaves, Phytochemistry 24 (1985) 2569 – 2571.

(11)

[40] R. Wititsuwannakul, Diurnal variation of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase activity in latex of He6_{ea brasiliensis}_{and its relation to rubber content,}

Experientia 42 (1986) 44 – 45.

[41] W. Suvachittanont, R. Wititsuwannakul, 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase in He6ea brasiliensis, Phytochemistry 40 (1995) 757 – 761.

[42] M. Carlberg, M. Hjertman, J. Wejde, O. Larsson, Mevalonate is essential for growth activation of human fibroblasts: evidence for a critical role of protein glyco-sylation in the prereplicative period, Exp. Cell. Res. 212 (1994) 359 – 366.

[43] M. Bifulco, C. Laezza, S.M. Aloj, C. Garbi, Meval-onate controls cytoskeleton organization and cell mor-phology in thyroid epithelial cells, J. Cell. Physiol. 155 (1993) 340 – 348.

[44] S.K. Randall, M.S. Marshall, D.N. Crowell, Protein isoprenylation in suspension-cultured tobacco cells, Plant Cell 5 (1993) 433 – 442.

[45] T.A. Morehead, B.J. Biermann, D.N. Crowell, S.K. Randall, Changes in protein isoprenylation during the growth of suspension-cultured tobacco cells, Plant Physiol. 109 (1995) 277 – 284.

(1)

A.E.Schulte et al./Plant Science150 (2000) 59 – 69

ing 7-day-old cells with a 10% inoculum (fr.

wt.

/

vol.) in standard medium resulted in a

sigmoidal

growth

curve

(Fig.

2A).

When

14-day-old cells were transferred to standard

medium or IM 2 medium with a 4% inoculum (fr.

wt.

/

vol.), biomass accumulated in a linear way

(Fig. 2B and C). The dry wt accumulation in IM 2

medium was about 1.5 times higher than in

standard medium at the end of the experiment.

The increase in dry wt. in this medium is mainly

due to the accumulation of starch.

Fig. 2 also presents the time courses of MK

activity for the

C

.

roseus

cells grown under

different conditions. Under standard conditions

(Fig. 2A) MK activity increased during the

exponential phase reaching a maximum of 0.5

nkat

/

mg protein after 4 days. After 6 days, at the

onset of the stationary phase, MK activity

decreased. Only after 18 days did MK activity

increase again to 0.85 nkat

/

mg protein. This

increase in activity might be related to alkaloid

production,

as

the

ajmalicine

accumulation

increased concomitantly (Fig. 3A).

When 14-day-old cells were subcultured in

standard medium MK activity reached values of

0.7 to 0.9 nkat

/

mg protein between 11 and 17 days

after inoculation (Fig. 2B). When 14-day-old cells

were transferred to IM 2 medium MK activity

increased to about 1.5 nkat

/

mg protein after 8 and

9 days (Fig. 2C). Compared to the cells growing

under standard conditions, this is a 2 – 3-fold

increase in activity.

The accumulation of terpenoid indole alkaloids

in the

C

.

roseus

cell suspensions was monitored at

different times during the culturing period under

the three culturing conditions mentioned. Alkaloids

and tryptamine, the indole moiety of the terpenoid

indole alkaloids, were recovered only from the

biomass; no alkaloids or tryptamine were detected

in the medium. In addition, no alkaloids were

detected in the cells subcultured in MS medium

after 14 days and at a high dilution (4% fr. wt.

/

vol.);

however, these cultures did accumulate tryptamine.

The tryptamine levels in these cultures increased

from 0.7 to 1.5 mg

/

l cell suspension from the 3rd

to the 15th day after subculturing, and increased

even further to 13.4 mg

/

l cell suspension after 20

days (results not shown). Fig. 3A and B present the

ajmalicine and tryptamine accumulations in the cell

cultures grown under standard conditions (10% fr.

wt.

/

vol.) and in the IM2 medium (4% fr. wt.

/

vol.),

respectively.

The

results

clearly

show

the

substrate

/

product relationship between tryptamine

and ajmalicine; when alkaloid levels increase,

tryptamine levels decrease.

(2)

In addition to ajmalicine, trace amounts of

taber-sonine and vindolinine were detected in the cell

biomass cultured under standard conditions, and

trace amounts of vindolinine and catharanthine

were recovered from the cells subcultured into the

induction medium (results not shown).

Further-more, Fig. 3 shows that, in contrast to expectation,

the accumulation of ajmalicine in the induction

medium is lower than in the standard growth

medium, except at prolonged culturing periods.

Additional results within our laboratory showed

that the accumulation of alkaloids or tryptamine is

dependent on the inoculum size in the growth

medium [37]. An increase of inoculum-size from 40

to 160 g fr. wt.

/

l medium favored the accumulation

of secologanin and ajmalicine; this is in agreement

with the results presented here.

In addition, the MK activity profiles from Fig. 2A

and C have been added to their corresponding Fig.

3A and B, respectively. Under standard conditions

(Fig. 3A), the ajmalicine accumulation correlates

well with the MK activity profile. However, after the

cells have been transferred to induction medium,

there does not seem to be any correlation

(3)

A.E.Schulte et al./Plant Science150 (2000) 59 – 69

Table 1

MK activity in different parts of twoC.roseusplantsa

Plant part MK in pkat/mg protein MK in nkat/g dry wt.

Plant 1 Plant 2 Plant 1 Plant 2

63 2.4 1.5

Flowers/buds 112

n.d. n.d.

n.d.

Fruit 111

36 1.4

Young leaf 32 1.2

30 0.4

Leaf 25 0.5

119 89

Stem 0.9 1.1

103

Root 86 0.9 1.1

a_{MK activities are related to protein contents of the extracts (pkat/mg protein) and the amount of biomass used for the}

extractions (nkat/g dry wt.). Crude extracts were prepared as described in Section 2 and the MK activity was determined using the radiochemical assay. n.d., Not determined.

between alkaloid production and MK activity

(Fig. 3B). As mentioned earlier, higher plants have

two pathways for the biosynthesis of IPP [5]. The

involvement of the MEP pathway in the

biosyn-thesis of secologanin, a precursor of the

terpenoid-indole alkaloids, in

C

.

roseus

is now well

established [23]. However, these studies were

per-formed on a specially selected,

secologanin-accu-mulating, green cell line; presumably, labeling

studies will also confirm the relationship between

the MEP pathway and ajmalicine production in

the yellowish

/

white

C

.

roseus

cell line used in this

study. The results presented may indicate that

under standard growth conditions (Fig. 3A) MK

activity could be indirectly involved in the

biosyn-thesis of terpenoid indole alkaloids, e.g. via

regula-tion of indole-alkaloid biosynthesis by prenylated

proteins [25].

As presented in Fig. 2, the MK activity showed

a high variation in activity. Also, during some

preliminary feeding experiments (unpublished

re-sults) it was found that the MK activity varied

considerably depending on the time of harvesting

the suspension cultured

C

.

roseus

cells. It has been

reported before that the activities of the MVA

phosphorylating enzymes in plants show a

sea-sonal variation, e.g. for

Pelargonium gra

6 eolens

[38] and

Cymbopogon citratus

[39]. For HMGR in

He

6 ea

latex a diurnal variation has been reported

[40]. Also, HMGS in

He

6 _ea

latex showed a diurnal

variation, with high activities at 10:00 and 22:00

[41].

A 12 h variation was found for MK in

C

.

roseus

suspension cultured cells, with higher activities at

12:00 and 24:00 (Fig. 4). Statistical analyses of the

means as calculated for the combined values at

each harvesting time showed that the mean MK

activity is significantly higher at the 95% level at

24 h than at 4, 8 and 16 h.

As the cells are grown under continuous light,

the physiological importance of this phenomenon

is not clear. One explanation might be that the

variation in MK activity is related to plant cell

growth. For mammalian cells it has already been

proven that MVA-derivatives, especially

preny-lated proteins, are essential for growth activation,

e.g. initiation of DNA synthesis [42], and control

cell morphology and cell duplication by their

ac-tion on the cytoskeleton [43]. Prenylaac-tion of

proteins has also been found in suspension

cul-tured cells of tobacco, and was considered to be

essential during the early stages of growth [44,45].

Table 2

Reported values for MK activities from different plant sources

Source MK activity (pkat/mg protein)

Plant Reference

[35]* 5.1

Nepeta cataria Leaf

2.5 [35]

N.cataria Callus

[35]* 2.5

Spinacea oleracea Leaf

Plant 12.2

Parthenium argentatum [17]

Latex serum 190

He6_{ea brasiliensis} _[36]

(4)

Nevertheless, now that it has also been shown that

in suspension cultured cells variations in enzyme

activities can occur, this should be accounted for.

Certainly, strict control of the moments of

subcul-turing and harvesting might reduce variation in

enzyme activities.

During the growth phase of suspension cultured

C

.

roseus

cells grown under standard conditions,

MK values of about 60 – 100 pkat

/

mg protein are

obtained using the radiochemical assay on crude

extracts (Fig. 4). These values are comparable to

the MK activities found in the stem and roots of

the

C

.

roseus

plant. Taking into account that

biomass can be easily obtained with the fast

grow-ing

C

.

roseus

cell suspensions and that extracts of

the suspension cultured cells are cleaner, e.g. no

chlorophylls, the cell suspensions are considered to

be the best source for the purification of the

enzyme MK, allowing further studies on the

regu-lation of this enzyme (manuscript in preparation).

Acknowledgements

The research of RvdH has been made possible

by a fellowship of the Royal Netherlands Academy

of Arts and Sciences. Financial support by the

‘Van Leersum fonds’ is gratefully acknowledged.

References