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

Michae 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

In supension cultured tobacco cells only blue light induces and maintains chlorophyll synthesis if the li- quid nutrient medium is supplemented with sucrose. The yield per gram fresh weight is closely correlated with

the energy fluence rate of blue light, but not with the initial amount of sucrose added to the medium (3-12

g/l). The uptake of sucrose by the cells proceeds with a constant rate over the growth period independently of the initial amount leading within 10-25 days to suc- rose-free media. Under these conditions the cells

continue to synthesize chlorophyll for about iO days.

This limitation is overcome by adding sucrose to the

medium at equal time intervals thus establishing a constant sugar level beyond the growth period. In con- trast, glucose as carbon source cannot adequately re- place sucrose in inducing and maintaining blue light- induced chlorophyll synthesis. Depending on the initial amount (3-10 g/l)this sugar is rapidly disappearing from the mediu/n within i-5 days after inoculation of

the cells. It apparently serves as a preferential source of energy and carbon skeletons thus suppressing chlorophyll synthesis. On the other hand, glucose

combined with sucrose in the medium brings about the characteristic induction and accumulation of chloro-

phyll in blue light which is observed with sucrose as the sole carbon source.

Introduction

Cell suspensions from callus tissue of Nicotiana taba- cum vat. "Samsun"j when exposed to blue or white light~

start to form chlorophyll, and plastids are transfor- med to functional chloroplasts (Bergmann and Berger 1966; Richter et al. 1980). These events are observed with either CO 2 or sucrose as carbon source; but with

the latter, i.e. under mixotrophic conditions, chloro- phyll production and cell growth are manifoldly in- creased (Bergmann 1967). While the direct influence of light quality on chloroplast differentiation in cul- tured tobacco cells is well documentedtthe role of sucrose as another significant parameter has not yet received much attention. The same holds true for glu- cose which in several instances successfully served as carbon source in maintaining growth and greening of cultured cells (De Klerk-Kiebert et al. 1982). There- fore it seemed worthwhile to follow the uptake and the consumption of these two sugars and to study their effect on blue light-induced chlorophyll synthesis respective chloroplast development in suspension cul- tured tobacco cells.

Materials and Methods

Cell culture. The source of the suspension cell cul- tures used in this study were callus cultures from Ni~ cotiana tabacum vat. Samsun (Bergmann 1960); they were

grown under sterile conditions in liquid medium con- taining the salts, vitamins and inositol of Murashige and Skoog (1962), Fe as EDTA-complex (21 mg/l FeSO4 • 7 H20 + 28.5 mg/l Na2EDTA), 0.6 mg/1 naphthalene ace- tic 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 sus-

pensions were maintained in 500-ml cotton-stoppered Erlenmeyer flasks containing 200 ml of medium. They were agitated on a gyratory shaker (120 rev/min) at 26oc. The setup for illumination of these cultures with white, blue or red light has been described pre- viously (Hundrieser and Richter 1982). Additionally, the blue fluorescent tubes (Philips TL 40/18) were combined with the plexiglas filter 627/3 mm (RShm and Haas) in order to filter out the undesired region beyond 520 nm of the spectrum. The energy fluence rate (EFR; W - m 22) was measured with a digital photometer

(Tektronix, Model J-16 with J 6502 irradiance probe; spectral response flat within ~ 7% from 420-950 nm).

Determination of Chlorophyll. Chlorophylls were extract- ed from O.5-g cell portions with about i ml of 80% ace-

ton + O.1% NH 3 in a Potter-Elvehjem type teflon-glass homogenizer. The hcmogenate was centrifuged (20 min at 6000 - g), the supernatant removed, and the pellet

washed with the aceton/NH 3 solvent; after centrifuga- tion the supernatant and the one of the first separa- tion were combined, and the volume brought to 4 ml. The absorbance of the extract was measured from 300 to 700 nm in a Pye Unicam (SP 18000) double beam spectro- meter. Total chlorophyll was calculated from its absorbanee at 649 and 665 nm according to Ziegler and Egle (1965).

Sugar analyses. Glucose or sucrose in the culture me- dium was determined by the GOD-Perid method (Glucotest combination of Boehringer) as recommended by the manu- factures; sucrose was hydrolyzed (O.i M HCI at 90°C for 30 min) prior to the assay.

Results and Discussion

When cell suspension cultures of tobacco were transfer- red to the modified MS-medium with 12 g/l sucrose and exposed to blue light of different energy fluence rate (EFR), chlorophyll became detectable from day 2 onwards (Fig. i). A close relationship between the rate of

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289

(W.m-2)

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F i g . 1, T ime c o u r s e o f c h l o r o p h y l l a c c u m u l a t i o n i n su - s p e n s i o n c u l t u r e d t o b a c c o c e l l s i r r a d i a t e d w i t h b l u e l i g h t o f d i f f e r e n t e n e r g y f l u e n c e r a t e (EFR; W - m -2 ) e x p r e s s e d as mg - 10 - 3 t o t a l c h l o r o p h y l l p e r gram o f f r e s h w e i g h t (FW). Fo 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 ) has been added f o r EFR o f 15 W • m - 2 .

s y n t h e s i s and t h e EFR a p p l i e d was r e g i s t e r e d : r a i s i n g t h e e n e r g y i m p u t l e d t o a s i g n i f i c a n t enhancemen 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 f a s h i o n 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 . S i m i l a r re - - s u i t s were o b t a i n e d w i t h w h i t e l i g h t o f e q u a l EFR v a l u e s . F o r r e d l i g h t , h o w e v e r , we f a i l e d t o d e t e c t a s i m i l a r p r o n o u n c e d i n d u c t i o n o f c h l o r o p h y l l s y n t h e s i s even a t h i g h v a l u e s o f EFR. A f t e r 12 days 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 b l u e l i g h t t r e a t e d c e l l s . The i n d u c e d c h l o r o p h y l l s y n - t 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 w i t h c e l l p r o l i f e r a - t i o n , i . e . w i t h t h e l o g a r i t h m i c phase o f t h e c u l t u r e c y c l e . T h i s does n o t mean t h a t c e l l d i v i s i o n i s a p r e - r e q u i s i t e o f c h l o r o p h y l l s y n t h e s i s - 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 o f t h e s t a t i o n a r y phase (see b e l o w ) .

I n o r d e r t o a n a l y z e t h e i n f l u e n c e o f s u c r o s e on b l u e l i g h t - i n d u c e d c h l o r o p h y l l s y n t h e s i s f u r t h e r we have f o l l o w e d i t s f a t e o v e r t h e g r o w t h p e r i o d o f t h e t o - bacco c e l l c u l t u r e . F i g . 2 shows t h a t d i f f e r e n t amounts 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 d i s a p p e a r . 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 depends upon t h e amount o f s u c r o s e a t t h e s t a r t . I n r e s p e c t t o c h l o r o p h y l l f o r m a t i o n n e i t h e r t h e i n i t i a l s u g a r c o n - c e n t r a t i o n n o r t h e d i f f e r e n t r a t e s o f i t s d i s a p p e a - r ance i s r e f l e c t e d i n t h e d a i l y s y n t h e s i s r a t e . On day 20 no s i g n i f i c a n t d i f f e r e n c e s i n t h e c h l o r o p h y l l y i e l d o f t h e t h r e e t e s t s u s p e n s i o n s we re d e t e c t a b l e . I n h i b i - t i o n o f c h l o r o p h y l l s y n t h e s i s b y t h e i n c r e a s e s i n e x - t r a c e l l u l a r s u c r o s e (Pamp l i n and Chapman 1975) was n o t o b s e r v e d . These r e s u l t s i n d i c a t e t h a t a c e r t a i n l e v e l 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 a l s o t o m a i n t a i n c h l o r o p h y l l s y n t h e s i s i n t h e b l u e and w h i t e l i g h t i r r a d i a t e d t o b a c c o c e l l s .

S t a r t i n g w i t h 15 g / 1 s u c r o s e and 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 s u c r o s e a f t e r 32 days ( d a t a n o t shown) . N e v e r t h e l e s s , t h e c e l l s s y n t h e s i z e d c h l o r o p h y l l up t o day 43 w i t h a c o n s t a n t d a i l y r a t e a m o u n t i n g t o a b o u t 330 pg c h l o r o - p h y l l / g FW ( c o r r e s p o n d i n g t o one t h i r d o f t h e c h l o r o - p h y l l c o n t e n t o f young t o b a c c o l e a v e s on FW b a s i s ) .

200

6

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%

_

1 ~ % o _~

. . . . . ~ . ~ . . . . . . . . . ~ ~

o ,/ I 7.~o ~ ," ~ 8

50 /' ~ 5.8 ~ m

. 6

3 . ~

5 10 15 20 25 days

F i g . 2. C h l o r o p h y l l s y n t h e s i s and suga r u p t a k e by b l u e

l i g h t - i r r a d i a t e d t o b a c c o c e l l s (12 W • m -2 ) i n i t i a l l y s u p p l i e d w i t h t h r e e d i f f e r e n t c o n c e n t r a t i o n s o f s u - c r o s e : 3, 6 and ~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 measured f o r ~2 g / 1 s u c r o s e .

Generally, cells in sucrose-depleted media tend to preserve chlorophyll synthesis up to iO days. This time limit was abolished by adding sucrose periodically to the otherwise unaltered medium (Fig. 3).

When the tobacco cells were grown with glucose, surpri- singly in blue light the different amounts initially added to the medium were taken up by the cells within 1 - 5 days with a rate of about 1.5 g per 1 and day (Fig. 4). On the other hand, the initial supply of

glucose turned out as the crucial parameter for the yield and the maintainance of chlorophyll synthesis. Only with relatively high concentrations (I0 g/l)

chlorophyll formation was approximately correlated with

400

350. /

~ ~oo- /~ -

~2~0- / ~

~ 1501 /

~ 100

50

10 -e 0 10 20 30 40 50

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.6

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Fig. 3. Chlorophyll synthesis and sugar uptake by blue light-irradiated tobacco cells (12 W - m -2) in a medium periodically supplemented with equal amounts of sucrose yielding about 5 g/l medium.

290

cell growth (expressed as dry weight increase) for

about 10 days, then it dropped rapidly.

~100J

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Fig. 4, As described in Fig. 2, except: cells were ini-

tially supplied with three different concentrations of

glucose: 3, 6 and ]0 g/l medium; cell mass was deter-

mined in the suspension with 10 g/l glucose.

AS compared with sucrosefglucose does not only disap ~

pear more rapidly from the culture medium but is also

rather inefficient in promoting chlorophyll synthesis.

Moreover, the cells produce insoluble fibrous material

in relatively large quantities which tend to form

massive aggregates and thus interfer with the growth

of cells: They stop dividing, become brown and even-

tually die. Because of these shortcomings glucose is

unfit to adequately replace sucrose in light-induced

chlorophyll synthesis of cultured tobacco cells. When

the tobacco cells were inoculated into a medium with

both sucrose and glucose each sugar disappeared with

about the same rate as measured in media supplemented

exclusively with sucrose or glucose alone (Fig. 5). In

respect to the induction and yield of chlorophyll syn-

thesis as well as to cell growth no differences to

sucrose-supplied cells were registered.

150- / x

×

,, / ~ O

% / * b 100" , , * ~

E / x

- I k

lO o ~ : 8 2 50;

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5 10 15 20 d eys

Fig. 5. As described in Fig. 2, except: cells were

initially supplied with 7 g/l sucrose and 8 g/l

glucose.

The results obtained demonstrate the dependency on suc-

rose of blue light-induced chlorophyll synthesis and

its superiority to glucose. One explanation for the

discrepancy in net disappearance of the two sugars

would be a different capacity in uptake and utiliza-

tion. Apparently the monosugar glucose is readily im-

ported by the cells and metabolized thus establishing

conditions which in turn prevent an efficient produc-

tion of chlorophyll. On the other hand, the sucrose

due to a retarded uptake is less available as primary

source for energy and carbon skeletons thus creating

a situation in metabolism most favourable for chloro-

phyll synthesis in blue light. This may include a

light-dependent loading of sucrose into the cell vacu-

ole (Lawyer et al. 1981). Indications are that in

plant callus cultures added sucrose is primarily hy-

drolyzed by invertase action (Fowler 1978). It seems

likely that the enzyme is bound to the cell wall and

responsible for cleaving extracellular sucrose. This

view is supported by our finding that in a few in-

stances immediately after the inoculation of callus

cells into a sucrose-containing medium increasing

amounts of glucose became detectable which were in-

versely correlated with the sucrose ones. Greening and

growth proceeded as in glucose-supplied cells.

Acknowledgement

The authors are grateful to Prof. L. Bergmann for

supplying callus cultures. They thank Mrs. E.

Scharfenorth for the skillfull technical assistance.

The investigation was supported by Deutsche For-

schungsgemeinschaft (Ri 73/22).

Abbreviation: EFR = energy fluence rate, FW = fresh

weight, MS-medium = Murashige-Skoog medium (Murashige

and Skoog 1962).

References

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Bergmann L (1967) Planta 74: 243 - 249

Bergmann L, Berger Ch (1966) Planta 69: 58 - 69

De Klerk-Kiebert YM, Kneppers TJA, Bakker PAHM,

Schale BH (1982) Z Pflanzenphysiol 105:445 - 456

Fowler MW (1975) in: Thorpe TA (ed) Frontiers in plant

tissue culture, Univ. Calgary, Calgary, pp 443 - 451

Hundrieser J, Richter G (1982) Plant Cell Reports i:

115 - 118

Lawyer AL, Grady KLiBassham JA (1981) Plant Physiol

68: 857 - 864

Murashige T, Skoog F (1962) Physiol Plant 15:473 - 497

Pamplin EJ, Chapman JM (1975) J Exp Bot 26: 212 - 220

Richter G, Reihl W, Wietoska B, Beckmann J (1980) in:

Senger H (ed) The blue light syndrome, Springer,

Berlin Heidelberg New York, pp 465 - 472

Ziegler R, Egle K (1965) Beitr Biol Pflanz 41: 11-37