Plant Cell Reports (1982) 1:288-290

Plant Cell Reports © Springer-Verlag 1982

Influence of Sugars on Blue Light-Induced Synthesis of Chlorophyll in Cultured Plant Ceils M i c h a e l G r o g and Gerhard Richter Institut ftir Botanik, Universit/it Hannover, Herrenh/iuser Strage 2, D-3000 Hannover, Federal Republic of Germany Received September 8, 1982/November 5, 1982

Abstract

M a t e r i a l s and M e t h o d s

In supension cultured tobacco cells only blue light induces and m a i n t a i n s chlorophyll synthesis if the liquid n u t r i e n t m e d i u m is supplemented with sucrose. The y i e l d per g r a m fresh w e i g h t is closely c o r r e l a t e d with the energy fluence rate of blue light, but not with the initial amount of sucrose added to the m e d i u m (3-12 g/l). The uptake of sucrose b y the cells p r o c e e d s w i t h a c o n s t a n t rate over the g r o w t h p e r i o d i n d e p e n d e n t l y of the initial amount leading within 10-25 days to sucrose-free media. Under these conditions the cells continue to synthesize chlorophyll for a b o u t iO days. This limitation is overcome by adding sucrose to the m e d i u m at equal time intervals thus e s t a b l i s h i n g a constant sugar level b e y o n d the growth period. In contrast, glucose as carbon source cannot a d e q u a t e l y rep l a c e sucrose in inducing and m a i n t a i n i n g blue lightinduced chlorophyll synthesis. D e p e n d i n g on the initial amount (3-10 g / l ) t h i s sugar is r a p i d l y d i s a p p e a r i n g from the mediu/n w i t h i n i-5 days after i n o c u l a t i o n of the cells. It a p p a r e n t l y serves as a p r e f e r e n t i a l source of energy and carbon skeletons thus suppressing chlorophyll synthesis. On the other hand, glucose c o m b i n e d with sucrose in the m e d i u m brings about the c h a r a c t e r i s t i c induction and a c c u m u l a t i o n of chlorophyll in blue light w h i c h is o b s e r v e d with sucrose as the sole carbon source.

Cell culture. The source of the suspension cell cultures u s e d in this study were callus cultures from N i ~ cotiana t a b a c u m vat. Samsun (Bergmann 1960); they were grown under sterile conditions in liquid m e d i u m containing the salts, v i t a m i n s and inositol of M u r a s h i g e and Skoog (1962), Fe as E D T A - c o m p l e x (21 mg/l FeSO4 • 7 H20 + 28.5 mg/l Na2EDTA), 0.6 mg/1 naphthalene acetic acid, 0.3 mg/1 kinetin, 0.2 mg/1 glycine as the sole amino acid, sucrose and/or glucose in various amounts (see experiments) at pH 5.6. The cell susp e n s i o n s were m a i n t a i n e d in 500-ml c o t t o n - s t o p p e r e d E r l e n m e y e r flasks containing 200 ml of medium. T h e y were a g i t a t e d on a g y r a t o r y shaker (120 rev/min) at 26oc. The setup for i l l u m i n a t i o n of these cultures with white, blue or red light has been d e s c r i b e d prev i o u s l y (Hundrieser and R i c h t e r 1982). Additionally, the blue f l u o r e s c e n t tubes (Philips T L 40/18) were c o m b i n e d w i t h the plexiglas filter 627/3 m m (RShm and Haas) in order to filter out the u n d e s i r e d region b e y o n d 520 nm of the spectrum. The energy fluence rate (EFR; W - m 22) was m e a s u r e d w i t h a digital p h o t o m e t e r (Tektronix, Model J-16 w i t h J 6502 irradiance probe; spectral response flat w i t h i n ~ 7% from 4 2 0 - 9 5 0 nm).

Introduction Cell suspensions from callus tissue of N i c o t i a n a tabacum vat. "Samsun"j when exposed to blue or white light~ start to form chlorophyll, and p l a s t i d s are transformed to functional chloroplasts (Bergmann and Berger 1966; Richter et al. 1980). T h e s e events are o b s e r v e d w i t h either CO 2 or sucrose as carbon source; but w i t h the latter, i.e. under m i x o t r o p h i c conditions, chlorophyll p r o d u c t i o n and cell g r o w t h are m a n i f o l d l y increased (Bergmann 1967). W h i l e the direct influence of light q u a l i t y on c h l o r o p l a s t d i f f e r e n t i a t i o n in cultured tobacco cells is well d o c u m e n t e d t t h e role of sucrose as another s i g n i f i c a n t p a r a m e t e r has not yet r e c e i v e d m u c h attention. The same holds true for glucose w h i c h in several instances successfully served as carbon source in m a i n t a i n i n g growth and g r e e n i n g of c u l t u r e d cells (De K l e r k - K i e b e r t et al. 1982). Therefore it seemed w o r t h w h i l e to follow the uptake and the c o n s u m p t i o n of these two sugars and to study their effect on blue l i g h t - i n d u c e d chlorophyll synthesis r e s p e c t i v e c h l o r o p l a s t d e v e l o p m e n t in suspension cultured tobacco cells.

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D e t e r m i n a t i o n of Chlorophyll. C h l o r o p h y l l s were extracted from O . 5 - g cell p o r t i o n s w i t h a b o u t i ml of 80% aceton + O.1% N H 3 in a P o t t e r - E l v e h j e m type teflon-glass homogenizer. The h c m o g e n a t e was c e n t r i f u g e d (20 min at 6000 - g), the supernatant removed, and the p e l l e t w a s h e d w i t h the a c e t o n / N H 3 solvent; after centrifugation the s u p e r n a t a n t and the one of the first separation were combined, and the volume b r o u g h t to 4 ml. The a b s o r b a n c e of the extract was m e a s u r e d from 300 to 700 nm in a Pye U n i c a m (SP 18000) double b e a m spectrometer. Total chlorophyll was c a l c u l a t e d from its absorbanee at 649 and 665 nm according to Ziegler and Egle (1965). Sugar analyses. G l u c o s e or sucrose in the culture med i u m was d e t e r m i n e d b y the G O D - P e r i d m e t h o d (Glucotest c o m b i n a t i o n o f Boehringer) as r e c o m m e n d e d b y the m a n u factures; sucrose was h y d r o l y z e d (O.i M HCI at 90°C for 30 min) p r i o r to the assay. Results and D i s c u s s i o n When cell suspension cultures of tobacco were transferred to the m o d i f i e d M S - m e d i u m w i t h 12 g/l sucrose and e x p o s e d to blue light of d i f f e r e n t energy fluence rate (EFR), chlorophyll became d e t e c t a b l e from day 2 o n w a r d s (Fig. i). A close r e l a t i o n s h i p b e t w e e n the rate of

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F i g . 1, Time c o u r s e o f c h l o r o p h y l l accumulation in suspension cultured tobacco cells irradiated with blue light of different energy fluence rate (EFR; W - m- 2 ) e x p r e s s e d as mg - 10 - 3 t o t a l chlorophyll p e r gram o f fresh weight (FW). F o r c o m p a r i s o n t h e i n c r e a s e o f c e l l mass (as d r y w e i g h t ) h a s b e e n a d d e d f o r EFR o f 15 W • m- 2 . synthesis a n d t h e EFR a p p l i e d was r e g i s t e r e d : raising the energy imput led to a significant e n h a n c e m e n t , The y i e l d p e r g FW i n c r e a s e d i n an a b o u t l i n e a r fashion o v e r 12 d a y s f o r a l l v a l u e s o f EFR t e s t e d . Similar re-suits were obtained with white light o f e q u a l EFR values. For red light, h o w e v e r , we f a i l e d to detect a similar pronounced induction of chlorophyll synthesis e v e n a t h i g h v a l u e s o f EFR. A f t e r 12 d a y s o f c u l t u r e t h e y i e l d was i n t h e r a n g e o f 5 - 9% o f t h a t f o u n d i n blue light treated cells. The i n d u c e d c h l o r o p h y l l synt h e s i s a p p e a r s t o be c o r r e l a t e d with cell proliferation, i.e. with the logarithmic phase of the culture c y c l e . T h i s d o e s n o t mean t h a t c e l l d i v i s i o n is a prerequisite of chlorophyll synthesis - on t h e c o n t r a r y : i t may w e l l c o n t i n u e i n c e l l s of the stationary phase (see b e l o w ) . In order to analyze the influence o f s u c r o s e on b l u e light-induced chlorophyll synthesis further we h a v e followed its fate over the growth period of the tobacco cell culture. F i g . 2 shows t h a t d i f f e r e n t a m o u n t s o f s u c r o s e i n t h e medium d r o p a t a r a t e o f 0 . 2 - 0 . 5 g p e r 1 and d a y , and f i n a l l y disappear. The p o i n t o f t i m e when t h e t e s t becomes n . e g a t i v e d e p e n d s upon the amount of sucrose at the start. In respect to chlorophyll formation neither the initial sugar concentration nor the different rates of its disappearance is reflected in the daily synthesis rate. On d a y 20 no s i g n i f i c a n t differences in the chlorophyll yield of the three test suspensions were detectable. Inhibition of chlorophyll synthesis by the increases in extracellular s u c r o s e ( P a m p l i n and Chapman 1975) was n o t observed. These results indicate that a certain level o f s u c r o s e as l o w as 3 g / 1 i s m a n d a t o r y t o s t a r t and also to maintain chlorophyll synthesis i n t h e b l u e and white light irradiated tobacco cells. Starting w i t h 15 g / 1 s u c r o s e a n d a p p l y i n g b l u e l i g h t o f e q u a l EFR t h e medium was d e p l e t e d f r o m sucrose after 32 d a y s ( d a t a n o t s h o w n ) . N e v e r t h e l e s s , the cells synthesized chlorophyll up t o d a y 43 w i t h a constant daily r a t e a m o u n t i n g t o a b o u t 330 p g c h l o r o phyll/g FW ( c o r r e s p o n d i n g t o one t h i r d of the chlorophyll c o n t e n t o f y o u n g t o b a c c o l e a v e s on FW b a s i s ) .

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light-irradiated tobacco cells (12 W • m- 2 ) i n i t i a l l y supplied with three different concentrations of suc r o s e : 3, 6 a n d ~2 g / 1 m e d i ~ . The i n c r e a s e o f c e l l mass (as d r y w e i g h t ) was m e a s u r e d f o r ~2 g / 1 s u c r o s e . G e n e r a l l y , cells in s u c r o s e - d e p l e t e d m e d i a tend to p r e s e r v e c h l o r o p h y l l synthesis up to iO days. T h i s time limit w a s a b o l i s h e d b y adding sucrose p e r i o d i c a l l y to the o t h e r w i s e u n a l t e r e d m e d i u m (Fig. 3). W h e n the tobacco cells w e r e grown w i t h glucose, surprisingly in blue light the d i f f e r e n t a m o u n t s i n i t i a l l y added to the m e d i u m w e r e taken up by the cells w i t h i n 1 - 5 days w i t h a rate of about 1.5 g per 1 and day (Fig. 4). O n the other hand, the initial supply of g l u c o s e turned out as the crucial p a r a m e t e r for the y i e l d and the m a i n t a i n a n c e of c h l o r o p h y l l synthesis. O n l y w i t h r e l a t i v e l y h i g h c o n c e n t r a t i o n s (I0 g/l) c h l o r o p h y l l formation was a p p r o x i m a t e l y c o r r e l a t e d w i t h

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Fig. 4, As d e s c r i b e d in Fig. 2, except: cells w e r e initially s u p p l i e d w i t h three d i f f e r e n t c o n c e n t r a t i o n s of glucose: 3, 6 a n d ]0 g/l medium; cell m a s s was determ i n e d in the suspension w i t h 10 g/l glucose. AS c o m p a r e d w i t h s u c r o s e f g l u c o s e does n o t o n l y d i s a p ~ p e a r m o r e r a p i d l y from the culture m e d i u m b u t is also rather i n e f f i c i e n t in p r o m o t i n g c h l o r o p h y l l synthesis. Moreover, the cells p r o d u c e i n s o l u b l e fibrous m a t e r i a l in r e l a t i v e l y large q u a n t i t i e s w h i c h t e n d to form m a s s i v e a g g r e g a t e s and thus i n t e r f e r w i t h the g r o w t h of cells: T h e y stop dividing, b e c o m e b r o w n and event u a l l y die. B e c a u s e of these s h o r t c o m i n g s glucose is unfit to a d e q u a t e l y r e p l a c e sucrose in l i g h t - i n d u c e d c h l o r o p h y l l synthesis of c u l t u r e d tobacco cells. W h e n the tobacco cells were i n o c u l a t e d into a m e d i u m w i t h b o t h sucrose and g l u c o s e each sugar d i s a p p e a r e d w i t h about the same rate as m e a s u r e d in m e d i a s u p p l e m e n t e d e x c l u s i v e l y w i t h sucrose or glucose alone (Fig. 5). In r e s p e c t to the i n d u c t i o n a n d y i e l d of c h l o r o p h y l l synthesis as well as to cell g r o w t h no d i f f e r e n c e s to s u c r o s e - s u p p l i e d cells were registered.

The r e s u l t s o b t a i n e d d e m o n s t r a t e the d e p e n d e n c y on sucrose of blue l i g h t - i n d u c e d c h l o r o p h y l l synthesis and its s u p e r i o r i t y to glucose. One e x p l a n a t i o n for the d i s c r e p a n c y in net d i s a p p e a r a n c e of the two sugars w o u l d be a d i f f e r e n t c a p a c i t y in uptake and utilization. A p p a r e n t l y the m o n o s u g a r glucose is r e a d i l y imp o r t e d by the cells a n d m e t a b o l i z e d thus e s t a b l i s h i n g c o n d i t i o n s w h i c h in t u r n p r e v e n t an e f f i c i e n t p r o d u c tion of chlorophyll. On the other hand, the sucrose due to a r e t a r d e d uptake is less a v a i l a b l e as p r i m a r y source for e n e r g y a n d carbon skeletons thus c r e a t i n g a s i t u a t i o n in m e t a b o l i s m m o s t favourable for chlorophyll synthesis in b l u e light. This m a y include a l i g h t - d e p e n d e n t l o a d i n g of sucrose into the cell vacuole (Lawyer et al. 1981). I n d i c a t i o n s are that in p l a n t callus c u l t u r e s added sucrose is p r i m a r i l y hyd r o l y z e d b y i n v e r t a s e a c t i o n (Fowler 1978). It seems l i k e l y t h a t the enzyme is b o u n d to the cell wall and r e s p o n s i b l e for c l e a v i n g e x t r a c e l l u l a r sucrose. This v i e w is s u p p o r t e d by our f i n d i n g that in a few instances i m m e d i a t e l y after the i n o c u l a t i o n of callus cells into a s u c r o s e - c o n t a i n i n g m e d i u m i n c r e a s i n g amounts of g l u c o s e became d e t e c t a b l e w h i c h w e r e inv e r s e l y c o r r e l a t e d w i t h the sucrose ones. G r e e n i n g and g r o w t h p r o c e e d e d as in g l u c o s e - s u p p l i e d cells. Acknowledgement The authors are g r a t e f u l to Prof. L. B e r g m a n n for s u p p l y i n g callus cultures. T h e y thank Mrs. E. S c h a r f e n o r t h for the skillfull t e c h n i c a l assistance. The i n v e s t i g a t i o n was s u p p o r t e d b y D e u t s c h e Forschungsgemeinschaft (Ri 73/22). A b b r e v i a t i o n : E F R = e n e r g y fluence rate, FW = fresh weight, M S - m e d i u m = M u r a s h i g e - S k o o g m e d i u m (Murashige and Skoog 1962).

References Bergmann

L

(1960)

J Gen P h y s i o l

Bergmann

L

(1967)

Planta

Bergmann

L, B e r g e r

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Fig. 5. As d e s c r i b e d in Fig. 2, except: cells w e r e i n i t i a l l y s u p p l i e d w i t h 7 g/l sucrose and 8 g/l glucose.

74: 243 - 249

(1966)

P l a n t a 69:

58 - 69

in: T h o r p e T A (ed) F r o n t i e r s in p l a n t Univ. Calgary, Calgary, pp 443 - 451

H u n d r i e s e r J, R i c h t e r 115 - 118

/

- 851

De K l e r k - K i e b e r t YM, K n e p p e r s TJA, B a k k e r PAHM, Schale BH (1982) Z P f l a n z e n p h y s i o l 1 0 5 : 4 4 5 - 456 F o w l e r M W (1975) tissue culture,

150-

Ch

43:841

G

(1982)

P l a n t Cell R e p o r t s

Lawyer AL, G r a d y K L i B a s s h a m 68: 857 - 864

JA

Murashige

Physiol

T, Skoog F

P a m p l i n EJ,

(1962)

Chapman JM

(1975)

(1981)

Plant Physiol

Plant

15:473

J E x p B o t 26:

R, E g l e K

(1965)

B e i t r Biol Pflanz

- 497

212 - 220

R i c h t e r G, R e i h l W, W i e t o s k a B, B e c k m a n n J (1980) Senger H (ed) The b l u e light syndrome, Springer, B e r l i n H e i d e l b e r g N e w York, pp 465 - 472 Ziegler

i:

41:

in:

11-37