Plant Cell Reports

Plant Cell Reports (1986) 3:195-198

© Springer-Verlag 1986

Photoautotrophic cell suspension cultures of periwinkle (Catharanthus roseus (L.) G. Don): Transition from heterotrophic to photoautotrophic growth R. T. Tyler, W. G. W. Kurz, and B. D. Panchuk Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Road, Saskatoon, Saskatchewan, Canada S7N OW9 Received February 11, 1986 / Revised version received April 17, 1986 - Communicated by F. Constabel

ABSTRACT Chlorophyllous, heterotrophic periwinkle (Catharanthus roseus (L.) G. Don) c e l l s were capable of sustained photoautotrophic growth in sugar-free B5 medium containing naphthaleneacet i c acid and k i n e t i n when provided with a COp-enriched atmosphere. An increase in c e l l fresh weight, f i r s t observed approximately 2 weeks a f t e r t r a n s f e r from heterotrophic to photoautotrophic conditions, coincided with the development of maximum chlorophyll content and photosynthetic a c t i v i t y . Electron micrographs revealed that chloroplasts of c e l l s cultured photoautotrophically in continuous l i g h t contained large starch granules and exhibited a less extensive thylakoid system than did periwinkle mesophyll chloroplasts. Photoautotrophic c e l l s did not accumulate v i n d o l i n e or dimeric a l k a l o i d s . Abbreviations: Chl - chlorophyll dry wt - dry weight f r wt - fresh weight 2,4-D - 2,4-dichlorophenoxyacetic acid NAA naphthaleneacetic acid -

INTRODUCTION Photoautotrophic cell cultures have been developed f o r a number of plant species. Accordingly, the c h a r a c t e r i s t i c s , manipulation, and potential biotechnological applications of such cultures have been well summarized CBarz and H~semann 1982; Horn and Dalton 1984; Horn and Widholm 1984; HUsemann 1984, 1985; Yamada and Sato 1978, 1983). Our i n t e r e s t in photoautotrophy stemmed from a long-term study of indole a l k a l o i d production in c e l l cultures of periwinkle (Catharanthus roseus CL.) G. Don). The dimeric a l k a l o i d s , v i n c r i s t i n e and v i n b l a s t ine, and a monomeric precursor, v i n d o l i n e , could be extracted from periwinkle leaves but were c o n s i s t e n t l y absent from heterotrophic cultures. This led us to speculate that c e l l cultures which contained well developed chloroplasts would simulate metabolism in leaves and might synthesize and accumulate these compounds.

Offprint requests to." R. T. Tyler

The development of photoautotrophic periwinkle cell cultures had not previously been reported. Preliminary i n v e s t i g a t i o n s revealed that of four chlorophyllous periwinkle c e l l l i n e s , only one, coded as 555G, demonstrated a propensity f o r sustained growth on sugar-free medium. Although 2,4-D did not i n h i b i t chlorophyll synthesis in heterotrophic suspensions of the four c e l l l i n e s , degreening occurred under photoautotrophic conditions unless 2,4-D was omitted and both NAA and a c y t o k i n i n were included in the c u l t u r e medium, and a COp-enriched ~tmosphere was provided. High lePels of NHa enhanced both the chlorophyll content and ~hotosynthetic a c t i v i t y of photoautotrophic periwinkle c e l l s . However, growth rates were markedly higher when NO3 was the primary source of nitrogen. This paper describes conditions suitable f o r the i n i t i a t i o n of photoautotrophic cultures from c e l l l i n e 555G and characterizes such cultures during t h e i r t r a n s i t i o n from heterotrophic to photoautotrophic growth. MATERIALS AND METHODS Photomixotrophic cultures of periwinkle (Catharanthus roseus (L.) G. Don cv. L i t t l e Delicata) c e l l l i n e 555G were maintained as heavy suspensions (approximately I00 ml ( s e t t l e d volume) of c e l l s in 150 ml of medium) in 500 ml Delong-type culture fl~sks u~der continuous i l l u m i n a t i o n 140 ~E m - s e c -~ at c u l t u r e l e v e l ) at 26°C in B5 medium (Gamborg et al _2968) containing 1 mg I - 2,4-D and 30 g i sucrose. Cultures were agitated on a gyrotory shaker (150 rpm) and subcultured at weekly i n t e r v a l s . T r a n s i t i o n to photoautotrophic growth was achieved by i n o c u l a t i n g 751mi of sugar-free B5 medium, containing I mg I - of each of NAA and k i n e t i n , with 5 g ( f r wt) of c e l l s collected by f i l t r a t i o n from photomixotrophic suspension cultures containing approximately 60 ~g Chl (g f r wt)- . The 250-mi Delong-type c u l t u r e flasks were equipped with gas i n l e t and o u t l e t por~s to permit continuous f l u s h i n g (I0 - 20 ml min- ) with humidified a i r containing 2% CO~. Photoautotrophic cultures were mai~t~ine~ under continuous i l l u m i n a t i o n (I00 wE m- secat

196 culture l e v e l ) at 26°C with agitation at 140 rpm on a gyrotory shaker. Replicate culture~ were harvested at weekly intervals f o r growth measurements and chemical analysis. Cells were collected for fresh weight measurements by vacuum f i l t r a t i o n and subsequently oven-dried {80°C, 24h) f o r determination of dry weight. Chlorophyll was determined spectrophotometrically in 80% acetone extracts using the equations of Arnon (1949). Photosynthetic Oo evolution was measured with a Rank oxygen electrode (Rank Brothers, Cambridge, 2 Engl~nd) under strong illumination (1500 ~E msec-~) at 26°C in 50 mM phosphate - I0 mM NaHCO3 buffer (pH 7.8), e s s e n t i a l l y as described by Hagimori et a l . (1984b). Starch in freeze-dried samples was assayed as described by Tyler (1984). Alkaloids were extracted and p u r i f i e d according to Kurz and Constabel {1982). The resulting alkaloid mixture was dissolved in 2 ml of ethyl acetate containing 4% methanol. A l i quots (30 - 200 ~I) were resolved by t h i n - l a y e r chromatography (Kurz and Constabel 1982) and individual components t e n t a t i v e l y i d e n t i f i e d on the basis of Rc value and chromogenic reaction with ceric ammbnium sulfate reagent (Farnsworth et a l . 1964). Specimens for transmission electron microscopy were fixed f o r 3 h in 4% glutaraldehyde/ 0 . I M Na cacodylate buffer (pH 7.3). After washing, c e l l s received additional f i x a t i o n f o r 2 h in 1% Os%/O.l M phosphate buffer (pH 7.3). The fixed c e l l s were then washed with water, dehydrated with increasing concentrations of ethanol, infused with propylene oxide, and embedded in Spurr's resin ( E i l e r t et a l . 1985). All processing steps up to infusion with propylene oxide were performed at 0 - 4°C. Sections were stained with uranyl acetate and lead c i t r a t e p r i o r to evaluation (EM 420 Transmission Electron Microscope, N.V. P h i l i p s , Eindhoven, The Netherlands). RESULTS AND DISCUSSION Following an i n i t i a l decline in fresh weight during the f i r s t two weeks in culture, the c e l l s exhibited slow, steady growth under photoautotrophic conditions, resulting in a total increase in fresh weight a f t e r 8 weeks of 156% (Fig. I ) . The 149% increase in fresh weight achieved during the logarithmic phase of growth (2 to 6 weeks) corresponded to a doubling time of approximately 19 days over t h i s period. After approximately 4 weeks, the chlorophyll content of the c e l l s (60 ~g (g f r wt)i n i t i a l l Y ) l h a d increased to approximately i00 ~g (g f r wt)- , a level maintained f o r the duration of the culture period (Fig. 2A). Growth rates and chlorophyll contents of photoautotrophic cultures have varied widely, with higher rates of growth generally associated with higher levels of chlorophyll. For example, Horn et al. (1983) reported, f o r soybean cultures, large ClO00 to 1400%)increases in cell weight over 14-day growth periods, and . chlorophyll levels (450 to 600 ~g (g f r w t ) - z ) , on a dry weight basis, 75 to 90% that of soybean leaves grown at equivalent l i g h t i n t e n s i t i e s . In contrast, the maximum chlorophyll level we observed with our r e l a t i v e l y §~ow growing cultures was 107 pg (g f r wt) (1669 ~g (g dry wt)- ), less than 20% that of young greenhousegrown periwinkle leaves (8760 ~g (g dry wt)- ). Among species, however, chlorophyll is not a

176t

5.2

5,1

160 I

IIF

5,0

4,9 //z

'-,E

E

~ I-

I

/

130

4.8

f"

/

"I"

4.7

120

-m ,.n,

~:

110

// /

4.6 S

/ ~

100

4,5

90

4.4

80

4.3

76

4.2

60

4,1

TIME IN CULTURE (Days)

Figure 1. Change in cell fresh weight during photoautotrophic growth of periwinkle cell suspensions. Bars represent standard errors (n = 2). Logarithmic curve is derived from fresh weight means Istandard errors not shown). r e l i a b l e indicator of photoautotrophic potential since r e l a t i v e l y rapid growth has been observed for cultures lower in chlorophyll {Berlyn and Zelitch 1975; H~semann and Barz 1977; Yamada and Sato 1978), and r e l a t i v e l y slow growth f o r cells higher in chlorophyll (Yasuda et a l . 1980). Yamada and Sato (1983) have concluded that a measure of photosynthetic a c t i v i t y is more useful than chlorophyll content for comparative purposes and in the selection of c e l l s with superior photoautotrophic potential. I t is apparent from our photosynthetic 09 evolution data (Fig. 2B) that for a particula~ culture, photosynthetic a c t i v i t y and chlorophyll content are closely related, as values f o r both parameters increased s t e a d i l y during the f i r s t 2 to 3 weeks and remained e s s e n t i a l l y constant thereafter. As expected, the maximum rate of increase in fresh weight was observed a f t e r t h i s i n i t i a l period of chloroplast development. Repeated selection of highly chlorophyllous c e l l s has been employed in the development of photoautotrophic cultures with improved growth rates (Horn et a l . 1983; H~semann and Barz 1977; Yamada and Sato 1978). However, we were unable to increase s i g n i f i c a n t l y either the chlorophyll content or the i n i t i a l photoautotrophic growth rate of the cell l i n e by such a procedure, as even the greenest c e l l s , when selected, gave rise to variegated cultures composed of c e l l s varying widely in chlorophyll content. The concentration of starch in the c e l l s (Fig. 2C) declined from 8.2% (dry weight basis)

197 11o

o

A

~-

o ~o ..r o

50

100

z o

~:

~-

=J::8 0

B

40

16 '.rCj

C

12

8

I

I

I

I

T I M E IN C U L T U R E ( D a y s )

Figure 2. Changes in chlorophyll content IA), photosynthetic Oo evaluation (B), and starch content ( t ) during photoautotrophic growth of periwinkle c e l l suspensions. Bars represent standard errors (n = 2). i n i t i a l l y to a low of 4.4% by day 7 as the c e l l s adapted to the sugar-free medium. As the chlorophyll content and photosynthetic a c t i v i t y of the c e l l s increased, however, there was again a net synthesis of starch such that by day 21 i t s concentration (9.9%) had surpassed the i n i t i a l value. Levels were e s s e n t i a l l y constant during the l a t t e r h a l f of the c u l t u r e period, with mean values ranging from I I to 13%. As expected from the r e l a t i v e l y high starch concentrations, chloroplasts in photoautotrophic c e l l s maintained under continuous i l l u m i n a t i o n f o r 6 weeks were heavily laden with starch (Fig. 3A). A reduction in the photosynthetic a c t i v i t y of chloroplasts may r e s u l t from physical d i s t o r t i o n of the chloroplast by starch grains IJensen 1980). This might account, at least in part, f o r the slow growth rates of photoautotrophic periwinkle cultures in continuous l i g h t . Chloroplasts in photoautotrophic c e l l s also exhibited a less extensive thylakoid system than did periwinkle mesophyll c e l l s (Fig. 3B) which would also contribute to low chlorophyll contents and slow growth. Photoautotrophic periwinkle c e l l s accumulated n e i t h e r v i n d o l i n e nor dimeric a l k a l o i d s . S i m i l a r l y , i t has been observed in cultures of other species that t r a n s i t i o n from heterotrophic to photoautotrophic conditions was not s u f f i c i e n t to induce the secondary metabolism c h a r a c t e r i s t i c of leaves, and that photoautotrophic cultures were generally i n f e r i o r to heterotrophic and

Figure 3. Electron micrographs of chloroplasts from periwinkle c e l l s cultured photoautotrophically in continuous l i g h t f o r 8 weeks (A) and from l e a f mesophyll c e l l s of greenhouse-grown periwinkle plants (B). photomixotrophic cultures with respect to the production of secondary metabolites (Barz and HUsemann 1982; Hagimori et a l . 1984a, 1984b). In such cases, d i f f e r e n t i a t i o n other than, or in addition to the presence of active chloroplasts may be required to confer upon c e l l s the a b i l i t y to produce certain secondary compounds. A l t e r n a t i v e l y , photoautotrophic cultures may lack e i t h e r the level of photosynthetic a c t i v i t y or the quantity of p a r t i c u l a r carbon skeletons required to support the production of secondary metabolites. V i n d o l i n i n e , 1 9 - e p i v i n d o l i n i n e , horhammerinine, horhammericine and l o c h n e r i c i n e , a l l Aspidosperma-type alkaloids (Kurz 1980), and s t r i c t o s i d i n e lactam were t e n t a t i v e l y i d e n t i f i e d in photoautotrophic periwinkle c u l t u r e s , a l b e i t in trace amounts. However, as these compounds were also detected in the photomixotrophic inoculum, and have been i d e n t i f i e d in t h i s and other periwinkle cell l i n e s under heterotrophic conditions (Kurz et al. 1985), t h e i r synthesis was not induced by the s h i f t to photoautotrophic conditions. Indeed, as a l l c e l l s in the photoautotrophic cultures were not necessarily photosynthetic and, in f a c t , did not appear to be so, the alkaloids detected may have been synthesized e n t i r e l y by heterotrophic c e l l s . In summary, photoautotrophic growth of a periwinkle cell l i n e has been demonstrated, and changes in chlorophyll content, photosynthetic a c t i v i t y , starch content, and a l k a l o i d pattern monitored as c e l l s adapted to photoautotrophic growth. Cultures have now been maintained photoautotrophically f o r approximately one year, with subculturing at 4- to 6-week i n t e r v a l s , without a s i g n i f i c a n t increase in growth rate over that reported in t h i s study. C u r r e n t l y , we are attempting to improve the growth rate of, and production of alkaloids by photoautotrophic periwinkle cultures by a l t e r a t i o n of some c u l t u r a l parameters.

198 ACKNOWLEDGEMENTS We thank Dr. U. E i l e r t for helpful advice and L.R. Nesbitt for excellent technical assistance in the preparation of electron micrographs. REFERENCES Arnon DI {1949) Plant Physiol. 24:1-15 Barz W, HUsemann W (1982) In: Fujiwara A (ed) Plant tissue culture 1982, Maruzen, Tokyo, pp 245-248 Berlyn MB, Zelitch I (1975) Plant Physiol. 56:752-756 E i l e r t U, Nesbitt LR, Constabel F (1985) Can. J. Bot. 63:1540-1546 Farnsworth NR, Blomster RN, Damratoski D, Meer WA, Cammarato LV {1964) Lloydia 27: 302-314 Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 50:151-158 Hagimori M, Matsumoto T, Mikami Y (1984a) Plant Cell Physiol. 25:947-953 Hagimori M, Matsumoto T, Mikami Y (1984b) Plant Cell Physiol. 25:1099-1102 Horn ME, Dalton CC (1984) Int. Assoc. Plant Tissue Culture Newsletter 43:2-7 Horn ME, Sherrard JH, Widholm JM (1983) Plant Physiol. 72:426-429 Horn ME, Widholm JM (1984) In: Collins GB, Petolino JG (eds) Applications of genetic engineering to crop improvement, Nijhoff-Junk, Dordrecht, pp 113-161

H~semann W (1984) In: Vasil IK (ed) Cell culture and somatic cell genetics of plants, vol I, Academic Press, Orlando, pp 182-191 HUsemann W (1985) In: Vasil IK (ed) Cell culture and somatic cell genetics of plants, vol 2, Academic Press, Orlando, pp 213-252 H~semann W, Barz W (1977) Physiol. Plant. 40: 77-81 Jensen RG (1980) In: Stumpf PK, Conn EE (eds) The biochemistry of plants, vol 1, Academic Press, New York, pp 273-313 Kurz WGW, Chatson KB, Constabel F (1985) In: Neumann K-H, Barz W, Reinhard E (eds) Primary and secondary metabolism of plant cell cultures, Springer-Verlag, Berlin, pp 143-153 Kurz, WGW, Chatson KB, Constabel F, Kutney JP, Choi LSL, Kolodziejczyk P, Sleigh SK, Stuart KL, Worth BR (1980) Phytochem. 19: 2583-2587 Kurz WGW, Constabel F (1982) In: Wetter LR, Constabel F (eds) Plant tissue culture methods, National Research Council of Canada, Saskatoon, pp 128-131 Tyler RT (1984) J. Food Sci. 49:925-930 Yamada Y, Sato F (1978) Plant Cell Physiol. 19: 691-699 Yamada Y, Sato F (1983) In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, vol I , Macmillan Publishing Co., New York, pp 489-500 Yasuda T, Hashimoto T, Sato F, Yamada Y 11980) Plant Cell Physiol. 21:929-932