Plant Cell Tissue Organ Culture 4 (1985) 83-94. © 1985 Martinus Ni]hoff/Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands.

R e g u l a t i o n o f p r o d u c t syn thes i s in cell c u l t u r e s o f

C a t h a r a n t h u s roseus (L) G. D o n Growth-related indole alkaloid accumulation in batch cultures

ANGELA STAFFORD, LAURA SMITH and MICHAEL W. FOWLER

Wolfson Institute of Biotechnology, The University, Sheffield S 10 2TN, U.K.

(Received 1 7 July, 1984; in revised form and accepted 23 October 1984)

Key words: Catharanthus roseus, plant cell cultures, alkaloid production, growth rate, mitotic activity

Abstract. Ceil suspension cultures of the Madagascan Periwinkle Catharanthus roseus (L) G. Don were maintained on Gamborg's B5 medium and their growth monitored by measuring cellular fresh and dry weight, ceil number and mitotic activity. Samples of cells of different ages and physiological states were subcultured onto an alkaloid pro- duction medium and their rates of growth and alkaloid accumulation measured over a period of 30-45 days. In two experiments the rate of biomass accumulation was directly related to the rate of cellular serpentine accumulation. Possible mechanisms underlying this phenomenon are discussed in relation to the properties of cells comprising the inocula.

Introduction

In microbial systems, growth and secondary product synthesis are often mutually exclusive processes [4]. A similar situation appears to occur in plant cell systems, where in numerous cases, secondary product accumulation has been observed to lag behind culture growth eg. in Gallium mollugo (Wilson and Marron, 1978), Morinda citrifolia [20] and Catharanthus roseus grown in an airlift fermenter [20]. Furthermore it has been found that medium conditions which tend to restrict growth will sustain an increase in the level of secondary compounds [7]. A concept of competition for mutual pre- cursors has been proposed by Phillips and Henshaw [15], in relation to this phenomenon in Acer pseudoplatanus. Lindsey and Yeoman [11] working on the production o f tropane alkaloids by tissue cultures ofvariousSolanaceae species, obtained evidence for an inverse relationship between the culture growth phase and alkaloid content. These authors also suggested that a level of cell differentiation was a prerequisite for secondary product formation.

The above results and impressions were all gained from experimental procedure in which inocula o f similar physiological states were subjected to a variety of treatments. A somewhat different perspective is gained if this procedure is reversed ie. a number of physiologically different inocula are subject to the same treatment. Using this experimental approach we were

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able to demonstrate that the rate of biomass accumulation in our cultures was directly related to the rate of cellular serpentine accumulation.

Materials and methods

Stock suspensions and experimental material

Batch suspension cultures of a single leaf-derived cell line of Catharanthus roseus (L) G. Don were initiated and maintained on Gamborg's B5 medium [6] or on a modified B5 (minus kinetin; designated HBS). In experiment (1), after routine subculture on B5 at 14 day intervals, cells were transferred to an alkaloid induction medium MZ [20] at various times after subculture. In experiment (2), cultures derived from the same source culture but which had been maintained on a 7 day (line AV7) or 14 day (line AV6) cycle on modified B5 for 9 subcultures were transferred to the alkaloid induction medium MZ, 7 or 14 days respectively after subculture.

All transfers were accomplished by filtering the cultures through 100#m nylon mesh using gentle suction, washing and then resuspending cells in fresh MZ medium. Culture conditions were as previously described by Morris and Fowler [13].

Measurement of growth and nutrient uptake

Cell biomass, protein, viability and medium sugars and nitrate were measured as described previously [17]. Cell numbers and mitotic indices were deter- mined using the methods of Brown and Rickless [1] and Marks [12], respectively.

Measurement of cellular alkaloids

Cells were filtered through Whatman No. 1 filter paper, washed with distilled water and lyophilised prior to extraction. 100 mg of freeze-dried cells were extracted with methanol in a soxhlet apparatus for 2h; the extract dried down on a rotary evaporator and taken up in HPLC grade methanol. Analysis by HPLC was performed using a # Bondapak Cla column with MeOH:H20: nHS at a flow rate of 1.5 ml/min. Mass spectrometry and UV spectral data confirmed the authenticity of serpentine in a pooled sample of cultured cell extract collected by preparative HPLC. The identity of serpentine in sub- sequent cell extracts was accepted on the basis of a 254:280nm absorbance ratio and retention time comparable to that of a serpentine hydrogen tartrate standard (methods of P. Morris and H. Woodhead, at this laboratory).

Results

Experiment 1

The aim of this experiment was to determined the effect of the age and physiological state of cells maintained on a growth medium upon their

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subsequent biomass and serpentine yield when transferred to an alkaloid production medium. Certain key points of growth in 100 ml B5 batch cultures and the times of transfer of cells to MZ medium are indicated in Figure 1.

q'2CO

2 t~

E 0'~ ~ IC0, .~n

q" - ~

~ E E

, ~ . ~ v v v .100 A A

o, t t . ! ! I 0 5 10 15 25 25

,50

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0 30

Figure 1. Growth cycle of 100 ml batch culture of C. roseus cell suspensions growing on B5 'maintenance' medium. Samples were removed on days 4.7.11.15 and 22 (arrowed) and transferred to MZ 'production' medium. Each point represents the mean of 3 separate determinations. Standard errors on fresh and dry weight measurements were less than 5% of the mean.

Although the inoculum dry mass was very similar for all transfers to MZ, the growth rates showed a tendency to vary inversely with the age of the inoculum (Figure 2). As the cells were carefully washed and filtered prior to transfer to MZ medium these growth rate differences can only reflect cellular variation over the period of the growth cycle on B5 medium.

The inocula with the two highest growth rates contained dividing cells from day 4 and day 7 cultures, in which carbon and nitrogen were still available in the medium. The remaining inocula contained no or very few dividing cells, though the third sample was taken on day 11 while the culture was accumulating fresh weight and a small amount of cell expansion was still occurring. C. roseus cells grown on B5 medium contain a large number of starch grains while carbon is available in the culture medium [17], and the dry weight loss apparent after day 7 in Figure 1 indicates

86

30.

o= zD.

t )

b "12

lb ~ 30 ~b clays in production medium

Figure 2. Dry weight accumulation in cells grown on production medium MZ after transfer at different times from B5; 4 days (o), 7 days (z~), 11 days (o), 15 days (e), 22 days (A). All points are the mean value of 3 separate determinations. Bars indicate standard errors exceeding 5% of the mean.

remobilisation of these carbohydrate reserves; the final three inocula were removed during this period. Shortly after reaching maximum fresh weight/ml, cell viability began to fall, and the final sample to be transferred to MZ medium had an estimated viability o f about 80%. It is clear that the 5 samples which were removed to alkaloid production medium contained cells of distinct and transient physiological characteristics.

The ability of the BS-grown cells to accumulate dry weight when transferred to MZ-medium appeared to be directly related to their capacity for serpentine accumulation (Figure 3 and 4). While in these figures the alkaloid data is plotted in terms of serpentine/ml culture, the same trends emerge if the data is plotted as serpentine/g, dry weight cells. This data contradicts that of Courtois and Guern [2] which inferred an inverse relationship between alkaloid formation and growth rate in C. roseus cultured under different temperature regimes. However, Kutney e t al. [8] have observed that their fermentations yield optimum alkaloid content if the inocula have a high mitotic index, a finding whch agrees with our data.

Obviously a large number of variables could be invoked in attempting to explain these results; including mitotic activity, nutritional status, intraceUular metabolite pools, genetic regulation, and so on. In order to examine the

60

87

93

~ 4 0 -

-j

O. 10 20 30 ~ 50

days in production medium

Figure 3. Serpentine accumulation in cells grown on productio~l medium MZ after transfer at different times from B5; 4 days (o), 7 days (z~), 11 days (n), 15 days (o), 22 days (A). All points are the mean value of 3 separate determinations. Bars indicate standard errors around the mean.

phenomenon more closely a second type of experiment was performed in which cells maintained on a 7 day or 14 day cycle in HB5 medium were transferred on their routine subculture days to MZ.

Experiment 2

The growth characteristics of the 7 and 14 day cycle cells (lines AV7 and AV6 respectively) in 100ml batch cultures are shown in Figure 5a and 5b. The cell dry weight profiles were quite comparable for the 2 cultures, and the calculated specific growth rates were almost identical (u(days-1) = 0.36 (AV6) and 0.37 (AV7)). However the composition of the culture dry mass in terms of cell type was very different; throughout culture the average dry weight/cell was significantly lower for AV7 than for AV6 (Figure 5c). Micro- scopic examination of the cultures indicated differences in general morphology, the 7 day cell cultures comprising an extremely homogeneous cell population through the first 9 days of growth - these cells were mostly highly clumped, small, spherical and full of starch grains, conforming to a 'meristematic' cell type. AV6 cultures were more heterogeneous with respect to cell size, shape and degree of clumping throughout the growth period, containing a high proportion of expanded vacuolated cells. The peak of mitotic activity was

88

..., 50-

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0

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Figure 4. Serpentine accumulation versus growth rate in cells grown on production medium MZ over a 14 day period. Each point represents the mean value of 3 deter- minations for cultures transferred at different times from B5. The slope of the line was calculated by the least squares fit of data for linear functions. [b = -- 15.82; rn = 3.696].

higher and earlier for AV7 than for AV6, suggesting that the 7-day cycle cultures possessed a greater physiological homogeneity.

Upon transferring AV6 and AV7 cells to medium MZ at day 14 and day 7 respectively, large differences in their growth characteristics were again noted (Figure 6a). The specific growth rates (u) were calculated as 0.164 days -1 (AV6) and 0.280 days -1 (AV7). This difference could be explained almost entirely in terms of the number of ceils present in the inocula; on day 0, the MZ cultures contained 5.4 x l0 S AV7 cells/ml, or 3.5 x l0 S AV6ceUs/ml. Comparison of the mitotic index data presented in Figure 5 and 6a clearly illustrates the fact that MZ medium only supports a low level of mitotic activity of C roseus cells transferred from B5; this has also been noted by other workers [10]. The lack of significant cell division on MZ indicated that the observed biomass increase mainly resulted from cell expansion and deposition of cell wall or storage materials. Microscopic observations con- finned this; as the culture period progressed some cell enlargement occurred, but the main features noted were cell wall thickenings and the accumulation of many large starch granules.

Only very low levels of serpentine or any other alkaloid were detected in these cell lines when grown on HB5 medium. On MZ medium an increase in serpentine/ml culture could be detected by day 3, and by day 8 differences between cultures in the cellular serpentine content could be recognized. The

89

30

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0 0

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(a)oeLL line AV6 (14 day subculture)

viability(day 14) =98'/,

(b)cell line AV7 (7 day subculture) -5

viability (day 15) --99% ~"

• / O g lb 15

number days on medium HB5

o

e

lb 15 20 number days" on medium HB5

Figure 5. Growth characteristics of cells growing in HB5 medium on either a 14 day (line AV6) or 7 day (line AV7) subculture. All points are the mean of 3 separate determin- ations. Standard errors on dry weight and cell number data are less than 5%.

divergence between the two lines in both growth rate and the rate of intra- cellular serpentine accumulation continued until about day 15; at this point there was a four fold difference in serpentine concentration/ml culture between AV6 and AV7 (See Table 1). By day 30 on MZ, the cell viability of both lines was declining and was estimated at 70%, and though at this point culture biomass was similar, line AV6 was still about 30% lower than AV7 with respect to serpentine/g, dry weight ceils.

It is obvious from the data presented in Figure 5b that the enhanced ability of the AV7 cell line for accumulating serpentine in MZ was not purely a function of high mitotic activity at the time of inoculation, as might have been suggested by the results of Experiment (1). At the point of transfer from growth medium, the mitotic index of these ceils was estimated as only

90

5,

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.~10.

" u 5 .

y:

"13

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( a ) ~ I I tJ~

, ~ _ ~ Vlabllity at(Jay 30 : 80*/, i / ' - :K / ' - ~ ~ for both cell lines

1() -1~5 2b 213 3b days in production medium

(b)

a

S ID 15 20 25 313 clays in production medium

Figure 6. Growth and serpentine accumulation in cultures of cell lines AV6 and AV7 growing on production medium MZ. Dry weight mg/ml eulture AV6 (o), AV7 (*);mitotic index AV6 (zx); AV7 (A); serpentine mg/g.dry.weight AV6 (o), AV7 (*). All points are the mean of 3 separate determinations. Bars indicate standard errors around the mean where these exceed 5%.

0.20%. Neither could this difference be explained by poor viability o f AV6 cells; at the time o f inoculation onto MZ, cell viability in both lines was very high and similar, around 98%.

The marked differences between these cultures in their capacity for serpentine accumulation may be rationalised if each cell has a maximum and similar sink size for serpentine regardless of the cell's size or origin; therefore

91

Table 1. Serpentine ug/ml culture in AV6 and AV7 cell lines grown on medium MZ (mean of 3 separate determinations + SE)

Cell line Day 6 Day 14-15 Day 27-28

AV6 5.92 -+ 0.25 30.04 -+ 0.36 113.8 *- 13.77 AV7 9.87 -+ 1.75 128.13 +- 9.04 133.17 +- 6.32

the AV7 culture on MZ with its greater initial cell number would have a greater potential for alkaloid accumulation. In fact this concept is supported by the data; the inoculum cell number ratio AV7/AV6 (1.54) is almost identical to the AV7/AV6 ratio for serpentine/g, dry weight at day 30 (1.50).

Discussion

Because of the way in which we conducted our experiments, any differences between our cultures with respect to growth rate and product synthesis reflected the properties of the cells when removed from growth medium, rather than the effects of different cultural conditions in the alkaloid production medium. Courtois and Guern [2] presented preliminary data indicating that the total alkaloid content in C roseus cell suspensions was increased when the rate of cell division was depressed, an effect obtained by lowering the growth temperature. In our cultures, mitotic activity on alkaloid production medium was not a major contributing factor to growth of the cells. While the relative infrequence of cell division in cultures growing in production medium may be a primary condition for their ability to produce serpentine, it cannot explain the observed differences in rates of serpentine accumulation between different inocula. In fact, in the second experiment, a higher mitotic index was found in the cell line accumulating biomass and serpentine most rapidly.

From our data and the observations of Kutney e t al. [8] it is apparent that cultures maintained on a 2,4-D medium and containing a high proportion of young and/or rapidly dividing cells are particularly well 'primed' for both growth and alkaloid formation when placed in a suitable environment. The 3-day post inoculation lag in serpentine accumulation observed in Experiment (2) suggests that cells transferred from maintenance to production medium undergo an induction process. Bearing in mind that the nutritional and hormonal regime was being altered substantially, it is likely that some radical adjustments to cellular metabolism and regulatory processes were made upon transfer to alkaloid production medium. While we have already discussed the possibility that the number of cells comprising the inoculum could be a major factor affecting serpentine yield, another contributing factor could be the proportion of cells in the culture endowed with 'competence' for alkaloid synthesis and/or accumulation. In our experience most C. roseus cultures growing on a production medium of any composition contain a maximum

92

of 50% alkaloid-accumulating cells. These estimates have been obtained using the method described by Deus and Zenk [3] which exploits the fluorescent properties of serpentine; the alkaloid can be visualised in the cell vacuole using fluorescence microscopy. Rapidly-dividing relatively undifferentiated cells in a maintenance medium are likely to be 'plastic' with respect to their ability to differentiate into alkaloid accumulating cells when presented with the appropriate stimuli. Taking this concept further, cultures removed from maintenance medium at progressively later points during the growth cycle would contain fewer and fewer 'plastic' cells, and would yield decreasing levels of alkaloids, a situation compatable with the present data.

Neuman et al. [14] and Renaudin and Guern [16] have suggested that a low vacuole pH is associated with alkaloid accumulation in C roseus. Vacuole pH may have been one of the factors contributing to the differential ability for alkaloid accumulation of cultures taken at various stages of the growth cycle on maintenance medium, and could be one of the features of cell 'plasticity' as discussed above. While the proportion of accumulating cells in any culture may ultimately restrict the product yield, it does not necessarily reflect the synthetic capacity of that culture. It has been noted that the auxin 2,4-D suppresses product formation in many plant cell culture systems [15, 5, 20] and Kurkdjian et al. [9] found an auxin-associated increase in pH of the cell sap in A c e r pseudoplatanus cultures. We have found that the addition of 2,4-D to cells growing on an alkaloid production medium results in a burst of mitotic activity and a concomitant decrease in the rate of alkaloid accumulation (manuscript in prep.). Data presented here (Figure 6a and b) also suggest an association between cell division and the suppression of alkaloid accumulation. These two events are not necessarily directly related; it is quite possible that 2,4-D exerts its effect upon product formation independently of its cytokinetic effect.

It is also possible that intracellular 2,4-D transferred from maintenance medium to production medium could produce a suppression in alkaloid formation. The lag in serpentine production noted in Figure 6b may be an example of this type of effect. However, differential carry-over of 2,4-D would not explain the trends shown in Figure 3, unless progressively older cells were capable of storing the hormone in an available form to a greater extent than younger cells. Alternatively, the physiology of the culture from which the inoculum was taken could determine the capacity of the cells for alkaloid synthesis. Ageing cells in stationary phase cultures may lack certain cofactors which they are unable to produce in MZ medium, or may be irreversibly blocked in a portion of the secondary biosynthetic pathway at a genomic or a post-transcriptional level.

In this presentation we have been unable to propose definite mechanisms for our results, but have demonstrated that under certain conditions a direct relationship between growth rate and product formation can be obtained.

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The inference for a b io technologica l process in qui te clear; for cultures

of some species at least it is possible to predict a dramatic improvemen t in

the rate o f biomass and produc t accumula t ion through considering the pro-

perties o f the inoculum.

The ques t ion o f whe ther or no t cell division can comprise a significant p ropor t ion o f growth in cultures undergoing secondary p roduc t synthesis will

be addressed in a fur ther communica t ion .

Acknowledgements

The authors wish to express their thanks to A E R E Harwell for suppor t o f this

work which was part o f a Depa r tmen t o f Indust ry sponsored project on

immobi l i sed plant cells, and to Dr. P. Morris and Mrs. H. Woodhead for their

help wi th the alkaloid analysis.

References

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3. Deus B, Zenk MH (1982) Exploitation of plant cells for the production of natural compounds. Biotech Bioeng 24:1965-1974

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13. Morris P, Fowler MW (1980) Sucrose utilisation by suspension cultures of Catharan- thus roseus. G. Don. Biochem Soc Trans 8:638-639

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15. Phillips R, Henshaw, GG (1977) The regulation of synthesis of phenolics in station- ary phase cell cultures o f A c e r pseudoplatanus L. J Exp Bot 28:785-794

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16. Renaudin J-P, Guern J (1982) Compartmentation mechanisms of indole alkaloids in cell suspension cultures of Catharanthus roseus. Physiol Veg 20:533-547

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20. Zenk MH, E1-Shagi H, Arens H, Stockigt J, Weiler EW, Deus B (1977) Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catha- ranthus roseus. In Barz W, Reinhard E, Zenk MH (eds.) Plant tissue culture and its biotechnological application. Springer-Verlag pp 27-43