Plant Cell Tissue Organ Culture 2 : 2 3 9 - 2 5 1 (1983). © Martinus Ni]hoff/Dr W. Junk Publishers, The Hague. Printed in the Netherlands

E f f e c t o f c a r b o n a n d n i t r o g e n g r o w t h l i m i t a t i o n u p o n n u t r i e n t

u p t a k e a n d m e t a b o l i s m in b a t c h c u l t u r e s o f C a t h a r a n t h u s r o s e u s

(L) G. D o n

ANGELA STAFFORD and '~J tCHAEL W. FOWLER

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

(Received 23 January 1983; in revised form 13 April 1983)

Key words: Catharanthus roseus, enzyme activities, growth limitation, plant cell cultures

Abstract. Cell suspension cultures of the Madagascan Periwinkle, Catharanthus roseus (L). G. Don were grown as batch cultures in two different types of media; in one medium the limiting nutrient was inorganic nitrogen, and in the other it was carbon. The response of the cells to these growth-limiting conditions was monitored by measuring cellular fresh weight, dry weight and protein accumulation, cell viability, medium sugar and nitrate levels, and the activities of certain intracellular enzymes throughout growth in batch culture. The enzymes investigated were glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), hexokinase (EC 2.7.1.40), phosphofructokinase (EC 2.7.1.11), nitrate reductase (EC 1.6.6.1), glutamate dehydrogenase (EC 1.4.1.2) and glutamine synthetase (EC 6.3.1.2). The effect of culturing the cells under different nutritional regimes was apparent in all aspects of growth; only some enzyme activities were unaffected. Cell viability remained at a high level for several days after growth limitation in both types of culture. The possibility that protein degradation in nitrogen-limited batch cultures is under very stringent control is discussed.

Introduction

An extensive literature now exists regarding the accumulation of indole alkaloids in cultured cells of Catharanthus roseus (Zenk, 1978; Barz and Ellis, 1981), the ultimate objective of much of this work being the develop- ment of an industrial process for indole alkaloids of commercial significance (Fowler, 1982). Major progress towards this goal may be seen in the work on the pathways for, and hormonal regulation of the biosynthesis of serpentine and ajmalicine (Zenk, 1978; Pfitzner and Stockigt, 1982). In addition, cultural variation in the chemical nature and level of the indole alkaloids has been explored by Kurz and co-workers (1980). A notable omission in these studies has been work on the primary metabolism of C. roseus

cells. This is important in two respects; firstly, there are indications that the nature and level of carbon and nitrogen supplied as substrate may influ- ence secondary metabolism not only in cell cultures of C. roseus (Zenk, 1977) but also in cultures of other species (Schuler, 1981). Secondly, the impor- tance of growth rate and biomass accumulation to the efficiency of an industrial process implies that the mode of conversion of primary substrates into essential cell material requires detailed attention. This paper reports

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on initial studies directed towards furthering our understanding of primary metabolism in C. roseus.

The operation of the classical pathways of glycolysis, pentose phosphate pathway and citric acid cycle have been previously demonstrated in plant cell cultures (Fowler, 1978; Wilson, 1971), as well as the incorporation of nitrate and ammonia via nitrate reductase, glutamine synthetase and gluta- mate synthase (Dougall, 1977). In this work, we aimed to manipulate the concentration of substrates supplied to C. roseus in such a way that culture growth was either carbon- or nitrogen-limited. The effects of such limitations on parameters such as growth, nutrient uptake and key assimilatory enzyme activities were then monitored for the duration of one culture cycle. The effects of varying primary substrates upon secondary metabolism will be reported in a later paper.

Materials and methods

Stock cultures and experimental material. Batch suspension cultures were initiated and routinely maintained on Gamborg's B5 medium (Gamborg et al 1968) as previously fully described (Morris and Fowler 1980). Samples from 13 day old suspension cultures were filtered under gentle suction through lO0/a nylon mesh, and inoculated into either (i) Gamborg's B5 medium containing 2mMNH4 ÷ 30mMNO3- and 2% (w/v) sucrose (the stan- dard B5 medium, designated N30) or (ii) B5 medium with 2mM NH4 ÷ and the concentrations of NO3- and sucrose adjusted to 5mM and 3% (w/v), respectively (designated N5).

Chemicals and enzymes. All chemicals used were of 'AR' grade where pos- sible, and were purchased from Fisons or BDH. Enzyme cofactors and purified enzymes were purchased from Boehringer (Mannheim, W. Germany) Ltd., or Sigma Ltd., U.K.

Measurement o f growth and viability. Fresh and dry weights were determined by filtering 3 ml of culture through a Whatman No. 1 filter paper supported on a stainless steel mesh, directly weighing for fresh weight and then drying overnight at 60°C prior to dry weight measurement. Protein estimations were made on extracts prepared for enzyme assays using the Folin method (Layne 1957), with bovine serum albumin as a standard. Viability was estimated using fluorescein diacetate according to the method of Widholm (1972).

Analysis o f medium sugars and nitrate. Culture medium was separated from cells by filtering suspensions under gentle suction through Whatman No. 1 filter paper. Medium was stored at --20°C and subsequently analysed for sucrose, fructose, glucose and nitrate. Sucrose and fructose were determined

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using the anthrone method (Van Handel 1967, 1968), while glucose was estimated using an enzymatic method (Boehringer Mannheim test kit, cata- logue No. 166391). Nitrate was measured using an Orion Nitrate Ion Elec- trode model 92-07.

Determination of enzyme activities. Cells were harvested from the cultures in a Buchner funnel using gentle suction, rinsed with ice-cold extraction buffer (0.1 M sodium phosphate pH 7.5 containing 5 x 10 -4 M EDTA), and resus- pended in 10 volumes of the same buffer also ice-cold. Cells were then disrupted for 30 s on ice using a Branson-Dawe sonicator, giving greater than 90% cell breakage as determined microscopically. The sonicated suspensions were centrifuged at 800g for 5 min at room temperature to remove large cell debris, and part of the supernatant (the crude extract) was reserved for the assay of protein and hexokinase (Fowler and Clifton 1975). The remain- ing crude extract was further fractionated by centrifugation at 37,000g for 30 min at 4°C in a Beckman L2-50 centrifuge. The resulting supernatant was assayed for protein and all enzyme activities listed below except for hexokinase. All enzyme assays were performed on fresh extracts, as storage at -- 20°C had previously been found to affect the stability of nitrate reduc- tase, glutamine synthetase and glutamate dehydrogenase in crude extracts. The glycolytic and pentose phosphate pathway enzymes assayed were glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), hexokinase (EC2.7.1.1), pyruvate kinase (EC 2.7.1.40), and phosphofructokinase (EC 2.7.1.11). With minor modifi- cations these assays were performed as described by Fowler and Clifton (1974). All activities were measured at 25°C by following the oxidation or reduction of NAD(P)÷/H at 340nm with a Gilford 250 recording spectro- photometer. Enzymes of nitrogen metabolism examined were nitrate reduc- tase (EC 1.6.6.1), glutamate dehydrogenase (EC 1.4.1.2) and glutamine synthetase (EC 6.3.1.2). The aminating activity of NAD-linked glutamate dehydrogenase and 7-glutamyl transferase activity of glutamine synthetase were assayed as described by Young (1973). The nitrate reductase assay was performed according to the method of Hageman and Hucklesby (1971).

Results

Effect of varying medium upon culture growth

At the time of inoculation into the two types of medium, cell cultures were more than 90% viable as demonstrated by fluorescence of live cells in the presence of fluorescein diacetate. In cultures grown on N30 medium, the proportion of viable cells only fell below 90% on day 22 (Figure 1). However, cells cultured in N5 medium were only 60% viable by day 12, and by day 18 the cell viability had fallen further to 25% of the total population. There were also marked differences in the profiles of fresh and dry weight accumulation

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300

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A A A A A N5 VIABILITY 90*•, 90% 60"1o 60% 25"/,

A A A A N30 VIABILITY 90% 90"/* 30% 30%

Figure 1. Fresh weight accumulation and cell viability in C. roseus suspension cultures grown under carbon-limited and nitrogen-limited conditions. Data are expressed as the mean of 3 separate samples taken from different cultures. Bars represent _+ SE where these exceed the span of the points.

N5 ~ N30 e

exhibited by the two culture regimes (Figure 1 and Figure 2). The dry weight data indicates that N5 medium became growth limiting on day 6, while the growth o f N30 cultures only became limited on day 11. As previously observed for growth o f C. roseus cells on Wood and Braun medium (MacCarthy et al., 1980) our cultures grown on both N5 and N30 media continued to accumulate fresh weight for a further 2 days after achieving maximum dry weight/ml culture.

In N5-cultures, net synthesis o f protein/ml culture ceased by day 4 (Figure 3), though some dry matter continued to accumulate beyond this time. This result suggests that on day 4, N5-grown cells contained the enzymic machinery to allow cell-wall synthesis for a further 2 4 - 4 8 h. The fact that the dry weight of these cultures did not decrease significantly after day 6 implies that either few storage reserves were laid down in N5 cultures, or else that after this time the cells had lost the enzymic or cofactor capability for the remobilisation of such reserves, for example starch grains. The small

15

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Days in culture

Figure 2. Dry weight accumulation in cell suspensions of C. roseus cultured under carbon-limited and nitrogen-limited growth conditions. Data are expressed as the mean of 3 separate samples taken from different cultures. Bars represents -+ SE where these exceed the span of the points.

N5 ~ N30 o

decrease in dry weight/ml in the N5 culture observed in the later growth stages was probably due to cell lysis; staining with fluorescein diacetate indicated a gradual loss o f membrane integrity. Cells grown on N30 medium achieved their highest protein and dry matter expressed per ml culture between day 11 and day 12. N5 and N30 cultures differed in their maximum content o f protein/ml by a factor of 5, which compares well with the differ- ence observed in the amount o f nitrate removed from the medium by the two cultures.

Relationship o f medium sugar and nitrate levels to growth

Medium sucrose as determined by a positive anthrone reaction was no longer detectable by day 4 (Figure 4a), while the concentrat ion o f glucose and fructose rose steadily up to this point in both N5 and N30 cultures (Figure 4a and 4b). After day 4, a slow uptake o f glucose and fructose was indicated in N5 cultures, while in N30 cultures the medium was rapidly depleted o f the monosaccharides until day 12 when virtually all medium sugar was exhausted (as determined by estimating total carbohydrate; data not shown). The

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Figure 3. (a) Cellular protein accumulation in suspension cultures of C. roseus grown under carbon-limited and nitrogen-limited conditions. (b) Semi-logarithmic plot of medium nitrate levels in media sampled from C. roseus cell suspensions cultured under carbon-limited and nitrogen-limited conditions. Data are expressed as the mean of 3 separate samples taken from different cultures. Bars represent + SE where these exceed the span of the points.

N5 N3o

difference in rate of monosaccharide depletion in the two types of media was reflected in the rates of dry weight accumulation in the two cultures, N30 cultures continuing to grow until all sugar had disappeared, while the dry weight increase in N5 cultures was evidently limited by some other factor.

That this factor was likely to be nitrate is suggested by the data in Figure 3. In N5 cultures medium nitrate fell to its minimum by day 4; at which point net protein synthesis ceased. In N30 cultures medium nitrate never fell below 4raM; in fact after reaching this minimum at day 18 the nitrate level in the medium rose again to 7raM; this rise coincided with a decrease in cellular fresh weight and protein, and also a loss of cell viability in these cultures. High concentrations of other anions, particularly chloride, are known to interfere with the electrode measurement o f nitrate; however, the low initial concentration o f chloride in N5 medium rules out the possibility that anion interference is the reason for the detection of residual nitrate after the point of growth limitation. It is quite obvious from these results that growth in

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Figure 4. (a) Levels of glucose and (b) Levels of fructose present in the media sampled from C. roseus cell suspensions cultured under carbon-limited and nitrogen-limited conditions. Data are expressed as the mean of 3 separate samples taken from different cultures. Bars represent ± SE where these exceed the span of the points. The point at which medium sucrose is exhausted in both N5 and N30 cultures is indicated by a vertical dashed line.

N5 A N30 o

N5 cultures was severely l imi ted b y nitrate avai labi l i ty , whi le in N 3 0 cultures

it appeared to be carbon avai labi l i ty w h i c h determined the final b iomass o f

the cells. In b o t h cul tures b e t w e e n 6 0 and 90% o f the cells remained viable for

several days after the point o f g r o w t h l imi ta t ion , indicat ing that while g r o w t h

246

was not possible in these cultures, the factors necessary for the continuation of basic cellular metabolism were available. Between day 12 and day 22 N30 cultures remobilised approximately 50% of their dry matter acquired since inoculation (Figure 2) and continued to remove nitrate from the medium, so the maintenance of high viability in these cultures was perhaps not sur- prising. However between day 4 and 12 in N5 cultures only a very low level of metabolism was evident from the data on monosaccharide uptake; appar- ently just sufficient to keep the cells intact and functional.

Relationship o f enzyme activities to culture growth

As the cell line used for this work had been maintained under the same conditions for 30 subcultures and was stable in terms of growth charac- teristics and culture morphology, it was assumed that day 0 cells would possess the same enzyme activities as N30 day 14 cultures (age of inoculum). Day 0 enzyme activities have been included in Table 1 for ease of comparison. Hexokinase and pyruvate kinase mark the entry and exit points respectively for both glycolysis and the pentose phosphate pathway; phosphofructokinase is exclusively a glycolytic enzyme; ghicose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are pentose phosphate pathway enzymes.

N5 and N30 displayed very similar profiles for hexokinase; low and steadily declining activity was observed throughout culture. However, nitrate limitation had a marked effect upon the levels of phosphofructokinase and pyruvate kinase activity in N5 cultures, the maximum activities of both these enzymes in N5 cultures coinciding with the cessation of dry matter accumulation.

With respect to the pentose phosphate pathway enzymes, the profile of gtucose-6-phosphate dehydrogenase was similar in N5 and N30 cultures though in the former the enzyme activity was consistently lower throughout culture, indicating a stimulation of glucose-6-phosphate dehydrogenase activity in the presence of high nitrate. A co-ordinate regulation of glucose-6- phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity in N5 cultures is suggested by the data; such a phenomenon has been observed previously in cultured sycamore cells (Fowler, 1971). However this postulated co-ordination of enzyme activity did not appear to be true of the N30 cul- tures, in which 6-phosphogluconate dehydrogenase maintained a constant activity until day 18.

Relative differences in the activities of enzymes involved in nitrogen metabolism were also detected. An apparent induction of nitrate reductase was observed in both N5 and N30 cultures; the maximum activity for both treatments occurred on day 1, at which time there was a fourfold greater enzyme activity in N30 cultures, reflecting the difference in initial nitrate concentration in the two types of media. It is interesting to note that in contrast to the results of Young (1973), the levels of nitrate reductase in the present studies did not correspond to the medium nitrate concentration;

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in N5-grown cells nitrate reductase activity was detectable (albeit at a low level) long after medium nitrate had been depleted (Figure 3), while the enzyme could no longer be detected in N30-grown cells on day 12, at which point nitrate was stilt present in the medium. The possibility that nitrate reductase activity and protein synthesis were regulated to some extent by the availability of carbon supply to N30 cultures is suggested by the fact that zero nitrate reductase and maximum protein/ml culture on day 12 coincided with the final depletion of medium monosaccharides.

Glutamine synthetase was assayed by virtue of its 7-glutamyl transferase reaction; activities in both cultures appeared very high as was also noted by Young (1973) in chemostat cultures of Acer pseudoplatanus. The profiles of enzyme activity were almost identical in N5 and N30 cultures under the conditions used in this experiment,indicating that the regulation of glutamine synthetase was not dependent upon medium nitrate availability. This result agrees with the observation that little change in the activity of glutamine synthetase in Paul's Scarlet Rose callus cultures could be induced by varying the medium nitrate level between 10-100 mM (Caldas and Caldas, 1976).

Higher plants appear to possess the enzymic potential for two routes of ammonium assimilation (Dougall 1977), one route involving glutamate dehydrogenase, the other involving glutamate synthase (GOGAT). In these studies only one route was investigated by measuring the aminating activity of NAD-linked glutamate dehydrogenase. Distinct differences in the levels of this enzyme were observed in N5 and N30 cultures after day 6; in N5 cells the peak in activity which occurred on day 11 could be the result of an age-related deinhibition of glutamate dehydrogenase rather than a mani- festation of assimilatory activity, as no net protein synthesis was occurring in N5-grown cells at that time (Figure 3). A further possibility is that the measured aminating activity of glutamate dehydrogenase was proportional to the enzyme's deaminating activity, and that the peak observed at day 11 was indicative of increased glutamate degradation with release of ammonia.

In N30 cells, the drop in enzyme activity at day 11 coincided with the cessation of net protein accumulation. Thus while in N5 cultures an associ- ation between glutamate dehydrogenase activity and biosynthetic ammonium assimilation was not evident, there was some suggestion that the enzyme played a significant assimilatory role in the carbon-limited N30 cultures.

Discussion

The ability of cultured cells of Catharanthus roseus to hydrolyse medium sucrose prior to uptake has been described previously (Morris and Fowler 1980; Fowler 1982) and the phenomenon also occurs in cell cultures of other species, for example, Saccharurn spp. (Maretzki et al 1974) and Daucus carota (Ueda et al 1974). The rate of sucrose hydrolysis was apparently not affected by the amount of nitrogen available to the cells; however, cellular

249

uptake of the monosaccharides was severely limited in N5 cultures after the point of nitrogen-limitation.

Certain of the enzymes assayed displayed significantly different profiles in the two types of culture; particularly phosphofructokinase, pyruvate kinase, 6-phosphogluconate dehydrogenase and glutamate dehydrogenase.

Nitrate assimilation necessitates the provision of both NADH and NADPH for the functioning of the biosynthetic route from nitrate reduction to glutamine synthesis. Therefore, it might be expected that the induction of nitrate assimilation would place demands upon both the pentose phosphate pathway and glycolysis. Certainly our data indicated an early stimulation of enzymes of the pentose phosphate pathway, glycolysis and nitrate assimi- lation. An increase in the activity of the pentose phosphate pathway relative to glycolysis during the induction of nitrate assimilation has been suggested by other workers (Jessup and Fowler 1977). In the present experiment, the peaks in glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and 6 phosphogluconate dehydrogenase activity occurring in day 11 N5 cultures could be interpreted as a demand fo r NADPH by glutamate dehydrogenase being met by the supply of reduced pyrimidine nucleotide via the pentose phosphate pathway. However, a similar peak of activity for this enzyme was not evident in N30 cultures, despite the higher nitrate concentration, and the role of glutamate dehydrogenase at this particular stage Of the N5 culture cycle is obscure.

The activity of hexokinase was the lowest of all the carbohydrate-assimi- lating enzymes assayed. This observation agrees with the findings of Fowler and Clifton with Acerpseudoplatanus (1974) and Sarkissian and Fowler with pea roots (1974), and again demonstrates the probable role of hexokinase as a key rate-limiting enzyme for the pathways of carbohydrate oxidation. This enzyme maintained approximately the same activity in both nitrate- limited and carbohydrate limited cultures.

One factor that has not yet been considered is the quality of nitrogen supplied to the cultures. While much of the data presented here is consistent with that of other workers in the area, some of the observed differences between the enzyme levels in N5 and N30 cultures may have been produced by differing nitrate:ammonium ion ratios and consequent pH effects in the media.

Cell viability was maintained long after the point of growth limitation on both N5 and N30 media. General deterioration of the integrity of the cell membrane only occurred 7 - 9 days after medium carbon depletion in N30 cultures, and 10-12 days after nitrogen limitation in N5 cultures. After the point of growth limitation the pattern of events was quite different in the two types of culture. N30 cells were capable of remobilising their stored carbohydrate reserves in the absence of extracellular sugar in order to provide the energy required for general metabolism. Once remobilisation was initiated, medium nitrate removal continued for a further 6 days, after which

250

point protein degradat ion began and viabili ty fell, presumably as no fur ther

source was available to the cells. Af te r g rowth l imi ta t ion in N5 cultures,

some prote in degradat ion occurred , but did not coincide wi th an immedia te

loss o f viabi l i ty; in contras t wi th the s i tuat ion in N30 cultures, cell protein

mobi l i sa t ion was associated with con t inued metabol ic act ivi ty as indicated

by the slow wi thdrawal o f monosacchar ides f rom the external med ium.

The phase o f 'prote in degradat ion ' in carbon- l imi ted and ni t rogen- l imited

batch cultures would be wor thy o f fur ther investigation, our data suggesting

that in the lat ter system, ut i l izat ion o f intracellular n i t rogen is a relatively

much more eff icient process and is l ikely to be subject to very str ingent

control .

Acknowledgements

'The authors wish to express their thanks to A E R E Harwell for support o f

this work which is part o f a Depar tment o f Indus t ry sponsored project on

immobi l ized plant cells, and to Dr G.S. Sarkissian for his helpful c o m m e n t s

on the manuscr ip t . '

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