Inhibition of chloroplast differentiation by the thymidine analogue, 5-bromo-2′deoxyuridine in cultured tobacco cells

CellD~fferentiation, 6 (~9~7) 65--74 © Elsevier/North-Holland Scientific Publishers Ltd.

65

i

I N H I B I T I O N OF CHLOROPLAST D I F F E R E N T I A T I O N BY TH E T ~ M I D I N E ANALOGUE, 5-BROMO-2'DEOXYURIDINE IN CUL'I~JRED TOBACCO CELLS

P. SEYER and A.M. LE~3CURE Laborc~toire d~ Biochimie Fonctionnelte des Plantes, Centre Universitaire de Luminy, 13288 Marseille Cedex 2, France Accepted 22 December, 1976

When tobacco cell suspension~ were grown during one to ~hree ceil generations in the pre~nce of micromolar concentrations of 5-bromodeoxyuridine (BrdU), chloroplast differentiation was specifically and reversibly inhibited. Kinetics of the ir~hibition and of its reversion suggested a DNA-linked phenotypic effect of the analogue. DNA analysis by caesium chloride density gradient showed that 2-[14C]BrdU was not incorporated at random into total DNA, but that a particular fraction of DNA was more heavily loaded by the analogue. The possibility that this fraction is chloroplast DNA is di,~cussed..

INTRODUCTION It has been previously reported (Lescure, 1973) that growth of tobacco tissue calluses was n o t inhibited by moderate concentrations of the thymidhle analogue, 5-bromo-2'deoxyuridine (BrdU). There was no other visible impact o f the analogue upon the culture than the lack of chlorophyll, related to a failure to develop thylakoids in the plastids. When transferred ont o a BrdU-free medium, these bleached calluses reve~ed to the green state after a short period o f growth. Our purpose is now to understand the molecular mechanism of the impact o f BrdU on the chloroplast differentiation. From the described experiments, it was n o t clear whether this effect was of genetic or of phenotypic or;,gin. It was first necessary to study the kinetics o f the inhibition and the reversion with tobacco cells grown in shake liquid medium. W e also attempted to see if the inhibition was correlated with an incorporation of the drug into D N A , possibly into chloroplast D N A . MATERIAL AND METHODS Culture conditions and growth measurements

The AG14 cell line of N i c o t m n a t a b a c u m c.v. Wisconsin 38, used in the following e~periments was shown to contain normal chloroplasts when

66

grown in a medium containing a cytokinin.Culture conditionsand pl~ti,d evolution during the culture of cellsin s ~ e n liquid medium have b ~ n described (Seyeret al.,1975). Cell growth was m ~ u r e d by freshor dry matter weight or by ~ k e d ceU volume. When necessary, ceilswere separated and counted according to Henshaw et al. (1966). ChlomphyU contents were calculated from absorbance of the acetone extracts according to Vernon (1960).

Purifica~,ion of chloroplasts Chloroplasts were purified by flotation in heavy sucrose medium, as described by Hendriks (1973), with t w o modifications: 1~, Dextran T40 concentration in the homogenisation buffer was reduced to 25 g per liter. 2) Flotation of chloroplasts was carried o u t in the following buffer: 25 mM TrisCl, 100 mM potassium chloride, 650 g per liter sucrose pH 7.8.

Preparation of chloroplast acetone powder The purified chloroplasts were resuspended in 4 ml water. Four volumes of pure acetone at --15°C were added. The suspension was kept 15 rain at --15°C, with intermittent shaking and centrifuged 5 rain at 480 ×g.' The pellet was submitted to t w o additional acetone treatments, dried under vacuum and stored a t - - 2 0 ° C.

DNA isolation and analysis DNA was extracted from lyophilized cells or from acetone powde~ of chloroplasts b y the Sarkosyl-pronase method and purified on a Sepharose 4B column, according to Heyn et at. (1974). Ultracentrifugation of DNA was carried out according to Flamm et al. (1966}, in a Beckman L3 50 centrifuge (rotor Ti 50, 36,000 rpm, 15°C, 60 h), in a caesium chloride cleasity gradient of initial density 1.700 g per ml. Fractions of 0.15 ml were collected and made up to 0.5 ml with 15 mM so,:lium citrate, pH 7.0. The absorption at 260 nm was measured. An aliquot of each fraction was counted in a vial filled with Instagel solution (Packard Instrument, USA), using a scintillation spectrometer (Intertechnique $L 40, France)°

Chemicals Nucleosides, BrdU, Pancreatic DNase I, venom phosphodiesterase II and alkaline phosphatase III were purchased from Sigma Chemical C~).; Caesium chloride (suprapur) was obtained from Merck Inc.; Radioactive nucleosides, 2-[14C]thymidine and 2.[14C]BrdU, were purchased from New England

:

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Nuclear Chemicals. Dextran T 40 was obtained from Pharmacia, Uppsala. As BrdU is heat sensitive, this compound was sterilized by filtration before addition to the cold autoclaved medium.

RESULTS

Ce!l growth and chlorophyll synthesis in the presence of BrdU When cellswere grown under white light (1000 lux), the cellgrowth rate w ~ not affected by the presence of BrdU 5 pM, at least during three cell geaerations (Fig. I, a and b). Chlorophyll contents of the BrdU culture followed the control drop during the growth phase, but the synthetic phase was abolished (Fig. 1, a' and b'). W h e n these cells were transferred to an analogue free medium in order to check the reversibilityof the inhibition,a low viabilitywas ~!cund in the subcultures. This lethal effect m a y be due to near visible wavelergths which are known to induce lethal damage in BrdU substituted cells (J~,nes et aL, 1972, Carlson, 1970). In order to avoid this lethal effect of light, cultures were grown under an orange filter(Rhodoid 1201) which elimi:mtes wavelengths under 550 nm. Fig. 2 A shows that under these light conditions chlorophyll synthesis occurred in the control at a low rate and again no synthesis was observed in BrdU culture. Fig. 2B shows that when t~e BrdU cells were transferred in the absence of the analogue, no lethali~:;yoccurred under these lastconditions. W h e n white light was then restored at the onset of stationary phase, chlorophyll synthesis occurred in both cultures with the same kinetms. These kinetics support the

c~

Tim~ ~days)

Fig. 1. Growth and chlorophyll ~yntihe~is in the absence (curves a and a') or in the pre~ence (curv~ b and b') of BrdU. t ~ and o ~ : mg of dry matter per 20 m] of suspension, A ~ - - ~ t and ~ - ~ : chlorophyll(~g per m] of packed ce]]~).

68

•

o

i

White light(~OOlux)

- 15

fs i % 5~

e u

Time (days)

. . . . . . .

Time (-days)

Fig. 2. Viability and chlorophyll synthesis under o~nge lightconditions,A) Growth and chlorophyll syntl~esisin the absence (curves a and a r)or in the pre~ence (curves b and b') of I/~M BrdU. B) Growth and chlorophyll synthesis after the firsttransferto a BrdU free medium (a and at: control; b and bt: cells grown in the presence of BrdU during culture A). ®------~ and o'-----o: cell number per ml or suspension. A ~ - - ~ , and A--------A; chlorophyll(pg per 106 cells).

hypothesis tha~ the inhibition of chloroplast differentiation is due to a phenotypic effect of the analogue.

Prevention of the inhibitory effect of BrdU by nucleosides Relevant nucleosides were tested for their ability to prevent the inhibition of chloroplast differentiation by BrdU (Table I). Thymidine was found to prevent completely the inhibitory activity. Deoxycyfidine and deoxyuridine were h:ss effective. Uridine and cytidine had vo effect.

Effect of BrdU added at the onset of stationary phase tinder our culture conditions, plastids underwent dedifferentiation during log phase of cell growth, differentiation of the lamellar system occurred only during stationary phase (Seyer et al., 1975). W e tried t0find if the inhibition by BrdU occurred at the same time as the differentiation of the laradlaz system or if the inhibition required the presence of the analogue during the cell multiplication period, i.e. before any differentiation wa~ observed in the control. Table II shows that when BrdU was added atthe onset; of stationary

69 TABLE I Effect of natural pyrimidine°nuc]eosMes c,n the inhibition of chlorophyll synthesis by 4 p ~ BrdU. C o m p o u n d tested

Chlorophyll contents ** (% control)

BrdU alone BvdU + Thymidine * BrdU + Deoxycytidine BrdU + Deoxyurldlne BrdU + Cytidlne BrdU + Uridine

35 100 86 86 27 30

* Nueleoside concentration: 10 p2V/ ** Chlorophyll measured after 17 days of culture (7 days after the onset of stationary phase). phase (culture nr 4), c h l o r p h y l l c o n c e n t r a t i o n was n o t affected. T h e inhibit i o n was observed o n l y w h e n t h e analogue w ~ present during t h e ceR g r o w t h phase {culture nr 3). F u r t h e r m o r e , t o p r e v e n t t h e i n h i b i t o r y e f f e c t o f t h e analogue, t h y m i d i n e was effective o n l y w h e n a d d e d at t h e beginning o f t h e c u l t u r e period (nr 5 and 6).

Incorporation of 2-[14C]thymidine and 2-[14C]BrdU into DNA T h e r e q u i r e m e n t o f a cell division period in t h e presence o f t h e analogue t o observe t h e inhibition o f chloroplast d i f f e r e n t i a t i o n and t h e efficiency o f t h y m i d i n e in o v e r c o m i n g this inhibition, suggested t h a t t h e BrdU e f f e c t was m e d i a t e d b y its incorporate.on i n t o DNA. T h e r e f o r e tests were p e r f o r m e d t o c h e c k w h e t h e r such an i n c o r p o r a t i o n actually occurred. T w o 100 ml lots o f TABLE I! Sensitivity to the inhibitory effect of BrdU at different steps of the culture. Culture nr.

C o m p o u n d added

Chlorophyll % of control

(at 'the time of transfer)

(at the onset of stationary phase)

1

none

none

100

2

Thd 10/~M

none

115

3

BrdU

none

4

none BrdD l p M

1 p2VI

5 6

~Thd 10~M BrdU 1/~M

40

BrdU 1 pM

100

none

100

Thd 10pM

40

79 TABLE III Radioactivit7 and absorbance of DNA purified on Sepharose 4B column. Radiolabeled nucleoside

.426e units

cpm

2-[l+C]thymidine 2-[14C]BrdU

1.96 2.12

3.0 x lO s 1.3 x 10 s

In both experiments the DNA was extracted from 160 mg ot lyophilized cells.

cells were grown for two cell generatiovs in the presence of 1/~M 2-[~+C] thymidine (5 ~Ci) or of 1 #M 2-[1+C]BrdU (5/~Ci)i, Total cellular D N ~ was extracted from each lot au~, p~.~.rified by chromatoi~raphy on a Sepharol~ 4B column, (cf. Methods). A peak containing optical density and radioactivity eluted together with the void volume; DNA was contaln~-'d in this fraction. The radioactivities and optical densities a~ 260 nm, measured for each peak, are listed in Table IlL i For .each DNA sample, radioactive nucleoside~ were fractionated as a

~ ~ A Br~L/Td~,.,~,~.,,..,,,,..,.~,. 110 1'5" Di,Jstancefronorigin(cm) l

Fig. 3. Radioactive traces t n th~n layer chromatograms of aucleosides obtained after enzymatic digestion of the total DNA. A) DNA from cells grown in the presence of 2-[:|4C] thymidine. B) DNA from cells grown in the presence of 2-[14C]BrdU. One A250 unit of e~q.'h DNA sample was ~ubmitted to enzymatic digestion. After the dig~tion, proteins were precipitated by ethanol 70% and eliminated by eentr|fugation, 39 000 × g, 10 rain. Supernatant~ were concentrated and spotted on a cellulose plate. Chromatograms were developed by: n-butanol/methanol/ammonie/H20:60: 20: 2 0 : 1 (v/v). Radioactive traces were obtained by use of a 4 ~ scanner Traeerlab flow counter.

71 probe that these nucleosides were incorporated into DNA without previous molecular degradation. DNA preparations were hydrolysed by the sequential action o f DNase I (EC 3.1.4.5), venom phosphodiesterase (EC 3.1.4.18) and alkaline pho~:l~hatase (EC 3.1.3.1), according to B i c k e t al. (1974). The resulting nucleoside mixtures were analysed by TLC. No randomization was observed in the ease of the 2-[x~C]thymidine-labeled DNA (Fig. 3). In the case of the 2-[ ~C] BrdU-labeled DNA, a m i n m radioactive contaminant was observed on the chromatograrn with the Rf of thymidine although the analogue was essentially incorporated into DNA without prior molecular degradation.

° /l

ot, lxt.

o.1o

f ~.~.~.~. ~

o

-

v

2

O.05 o e~

0.20

7 x

® 0.10

25

30

35 40 fractiuns

45

50

25

30

35

40

45

50

fractions

F~g."4. Analysis of total and chloroplast DNA by ultraeentrifugation. 14 in CsCl density gradients. A) Total DNA extracted i#om call8 labeled wzth 2-[ 1:?]thymidlne. B) DNA extracted from the chloroplast fraction of cells labeled with 2-[ C]thymidine. C) Total DNA extracted from cells labeled w|~h ~:-[14C]BcdU. D) DNA extracted from the chloro~l~t fraction of cells labeled with 2-[ 14C]BrdU. u ~ radioacti~dty,o----==o A260. ~ specific radioactivity (cpmIA2so). The arrow indicates the po6itions of marker DNA from Mterocoecus lysodaikticu$ ( d - 1.731). Fraction8 were collected from the bottom of the tube.

u

72 TABLE IV

Incorporation of 2-[~4C ]thymidine and of 2-[14C ]BrdU intototaland chloroplastDNA. Type of DNA

Nucleoside

Specific radioactivity cpmlA260

nmoles of nucleosides per A26o unit

Total DNA Claloroplast DNA Total DNA Chloroplast DNA

thymidine thymidine BrdU BrdU

21,700 24,000 5,060 6,837

0.638 0.700 0.240 0.325

DNA analysis by caesium chloride density gradient centrifugation This experiment was performed in order to know if thymidine and its analogue were incorporated at random into D N A or if these nucleosidas were incorporated specifically into a particular fraction of D N A . The assumption was that this fraction might be chloroplast D N A . T w o cultures were grown either in the presence of I ~ M 2-[14C]thymidine (14 Ci/mole) or in the presence of I/~M 2-[14C]BrdU (11 Ci/mole). After two cell generations, each culture was divided into two lots: the first,(10 g flesh matter) was lyophilised and its total D N A was extracted. From the second lot (200 g) chloroplast D N A was extracted from the chloroplast acetone powder. These crude preparations of D N A were filtered on Sepharose 4B column and the eluted D N A s were then subjected t~#.caesium chloride gradient centrifugations. The absorbances at 260 nm" and the racUoactivities of the collected fractions were measured (Fig. 4). In the case of the 2-[ ~4C]thymidine labeled D N A the m a x i m u m of the absorbance curves were coincident (Fig. 4 A and B). However the specific radioactivity of the heavy fractions was systematically higher than the average. When DNAs were extracted from the 2-[14C] BrdU labered culture (Fig. 4C and D) the situation was different: whereas the, max hnum o f the absorbance curves were found at the same density v~ that of the 2.~a4C]thymidine labeled DNA, the maximum o f the radioactive pea!~s were shifted to a denser region.

Y ~ e n the specific radioactivitieswere calculated for the total area of each peak (Table IV), it was found that this specific radioactivity was 10% higher for the chloroplast D N A than for the total D N A , in the case of the 2-[~4C]thymidine labeled cells. It was 4 0 % higher in the case of the 2-[14C]BrdU labeled cells. DISCUSSION When tobacco cells were grown under filtered light, kinetics clearly

showed that the recovery of chlorophyll synthesis ability in the absence of

73 BrdU could n o t result from a selection of r e s i s ~ t cells. It might be due to the multiplication of resist~mt plastids selected during the three cell generations in the p~sence of the analogue. However chlorophyll synthes~.skinetics at the fn~t transfer in the BrdU-free medium were very shnflar to t!~atof the control and these Idndtics were consistent with reversible phenotypic effect of the analogue rather than with a recovery of the greening ability by a selective mechanism. A phenotypic effect of this type has been described in a variety of animal cell cultures treated by the drug. It was demonstrated that the mlalogue affected specifically and reversibly the expression of a number of differentiated cell functions, while exerting little influence on the proliferation rate (Rutter et al.,1973). T w o alternative hypotheses have been proposed to explain the phenotypic effect of BrdU: according to the former (Walther et al., 1974), 'the substitution of thymidine by B r d U into D N A would modify the binding of regulatory proteins to D N A and cause a block in trsnscription at cert~n sites'. Alternatively, Schube~ eta!. (1970), described ~ morphogenic effect of the drug on the differentiation of neuroblastomas under conditions where D N A synthesis was blocked. They suggested tha'~the activity of the drug was unrelated with D N A and m a y result of an al~eration in the synthasis of polysaccharides associated with the membranes. Our experiments, as described Table II, showed that chlor~p!last d'ifferentiaticn which normally occurred during the stationary phase, was not inhibited when the drug was added only at the onset of this phase: a cell division period in the presence of the analogue was essential to observe the inhibitory effect of BrdU. W e also showed that thymidine specifically prevented the inhibition. These results and the fact that the kinetics of the inhibition and of its reversal were symmetrical argued for a mechanism of the BrdU action associated with its incorporation into DNA. As the consequence of the presence of BrdU was the inhibition of chloroplast differentiation, the question was raised whether a preferential incorporation of the analogue occurred into chloroplast DNA. Such a,~#:uation would result of a compartmentation of the thymidine kinase ac#~i~ty, as described for some strains of Chlamydomonas (Chiang et al., ]972). A difficulty to check this hypothesis with tobacco cells was that chlc,roplast DNA and nuclear DNA have the same b u o y a n t density in caesium chlol~de gradients (1.967 g per ml). It is known that a substantial substitt:tion of thymidine by BrdU produces an increased buoyant density (Davids(,n et al., 1974). Therefore, if the incorporation of BrdU occurred specifically into a particular fraction of DNA, we should expect a shift of this DNA.banding to a denser region of the gradient. Gradient patterns of total DNA showed that the absorbance peaks banded at the stone density whether DNA was extracted from thymJdine cells or from BrdU cells: this means that thymidine substitution by the analogue into total DNA was not sufficient to produce a measurable increase of density. However, it can be observed on Fig. 4A, that in the case of the 2-['~C]~hymidine labeled DNA~ the specific

74

radioacfivit3 thi,~ indicat~ DNA. In the active peak tion has be l a b d e d DN., specific radioactivi~y was increased o f 40%, c o m p ~ with t h a t 0 f t o t a l DNA. This result i.,~in favor o f a p ~ f e r e n t i a l h l c ~ b r a t i o n o f t h e an~A0gue i n t o c h l o r o p l a s t DNA; h o w e v e r we k n o w f r o m eXl~eriments :not d e ~ e d m this p a p e r t h a t t h e IJNA e x t r a c t e d ~ O m t h ~ chloropiast .~vacfion was still highly c o n t a m i n a t e d b y nuclear DNA. Higher p ~ f i e a f i o n is still necessary t o k n o w w h e t h e r o r n o t t h e c h l o r o p l ~ t D N A is s p e c i f i c ~ l y l o a d e d b y BrdU.

Such a specific fraction of Soybean DNA more heavily labeled by BrdU, has been mentiored by Ohyama (1974); not been identified. In animal cell systems, it has been shown that BrdU is incorporated predominantly within moderately repetitive sequences of DNA (Schwartz et al., 1974) which might be involved in the control of specialized cell functions. BrdU substitution w o u l d lead t o a m o d i f i c a t i o n o f r e g u l a t o r y p r o t e i n binding t o these s~tes. F u r t h e r e x p e r i m e n t s will t ~ y t o clarify w h e t h e r t h e b l o c k o f plastid d i f f e r e n t i a t i o n is c o r r e l a t e d t o a specific l o a d o f c h l o r o p l a s t D N A o r w h e t h e r it is m e d i a t e d t h r o u g h a m o d i f i c a t i o n o f t h e expression o f t h e nuclear g e n o m e . ACKNOWLEDGEMENTS We thank Dr. Pdaud-Leno~l for fruitful critical discussions during this work. The competent technical help of M.C. Durand is gratefully acknowledged. We are indebted to the 'D~l~gation G~n~rale ~ la Recherche Scienfifique et Technique' for a financial support to the laboratory and the grant of a fellowship to one of us (P. Seyer). REFERBI~:CBS Bick, M.D. and R.L. Davidson: Proc. Natl. Acad. Sci. U.S.A. 71, 2082'2086 (1974). Carlson, P.S.: Science 168,487--469 (1970). Chiang, K.S., E. Eves and D. Swintom Develop. Biol. 42, 53--63 (1975). Davidson, R.L. and M.D. Bick: Proc. Nail. Aead. Sci. U.S.A. 70, 138--142 (1973). Flamm, W.G., H.E. Bond and H.B. Bun: Biochim. Biophy~. Acta 129, 310-319 (1966). Hendricks, A.W.: Studies on plant organe]les. PhD Thesis, Leiden (1973). Henshaw, G.G., K.K. Jha, A.R. Metha, D.J. Shake,haft and H.E. Street: J. Expfl. Bot. 17, 362--377 (1966). Heyn, R.F., A.K. Herman and R.A. Schilperoort: Plant Science Letters 2, 73--78 (1974). Jones, T.C. and W.F. Dove: J. Mol. Biol. 64,409--416 (1972). Lescure, A.M.: Plant Sci. Left. 1,375--383 (1973). Ohyama, K.: Expfl. Cell Res. 89, 31--38 (1974). Rutter, W.J., R.L. Pietet and P.W. Morris: Ann. Rev. Biochem. 42,614--619 (1973). Schubert, D. and F. Jacob: Proc. Natl. Acad. Sel. U.S.A. 67,247--254 (1970). Schwartz, S.~. and W.H. Kirstel~: Franc. Haft. Acad. SCi. U.S.A. 71, 3570--3574 (1974). Seyer, P., D. Marry, A.M. Lescure and C. P~aud-Leno~l: Cell Differentiation 4,187--197 (~975). Vernon, L.P.: Analyt. Chem. 32, 114--125 (1960). Walther, B.T., R.L. Pictet, J.D. David and W.J. Rutter: J. Biol. Chem. 48, 1953--1964 (1974).