Planta (198l)152:54 58

P l ~ P l - ~ 9 Springer-Verlag 1981

Chlorophyll biosynthesis by mesophyll protoplasts and plastids from etiolated oat (Arena sativa L.) leaves Jfirgen Benz 1, Rtidiger Hampp 2, and Wolfhart Rtidiger 1 t Botanisches Institut der Universitfit Mfinchen, Menzinger Stral3e 67, D-8000 Mtinchen 19, and 2 Institut ftir Botanik, Technische Universitfit Miinchen, Arcisstrage 21, D-8000 Mtinchen 2, Federal Republic of Germany

Abstract. The uptake of [1-3H]geranylgeranyl diphosphate (GGPP) into protoplasts and intact etioplasts and the metabolic interconversion therein was studied after a 2 min pulse of white light. The chlorophyll synthetase reaction, Chlide+GGPP~ChI~G, was taken as a natural probe for the etioplast compartment. This reaction yields labeled ChLa~ and, by hydrogenation, labeled Chip, when [1-3H]GGPP receives access to the etioplast stroma. It was found that penetration across the plastid envelope was, rapid and that penetration across the plasma membrane of protoplasts, however, was slow. A cellular pool of soluble GGPP was detected. This pool was lost, in part, during preparation of the protoplasts and almost completely during preparation of the etioplasts. The membrane-bound phytol pool of etioplasts could not be replaced by exogenous [3H]GG. The endogenous GG and phytol pools of protoplasts, which were larger than those of etioplasts, could be replaced in part by exogenous [3H]GGPP. That part of this pool exists as soluble GGPP or as a direct precursor in the cytoplasm is discussed. Key words: A r e n a - Chlorophyll biosynthesis - Etioplasts - Geranylgeranyldiphosphate - Protoplasts.

Introduction

The final steps of chlorophyll biosynthesis include the introduction of the phytyl residue, which is responsible for the highly lipophilic properties of the pigment. The phytylation is at least in greening etioplasts - a multistep reaction sequence in which geranylgerauyl chlorophyllide (Chloe) is formed first. Abbreviations." GGPP=geranylgeranyldiphosphate; Chl~c=geranylgeranyl chlorophyllide a, Chlp = phytyl chlorophyllide a; IPP = isopentenyl diphosphate; Chlide=chlorophyllide a

0032-0935/8 l/0 152/0054/$01.00

The Chl~a is then hydrogenated via 2 intermediates to phytyl chlorophyllide (Chlp) as the final product. This reaction sequence was derived from kinetic measurements after illumination of intact etiolated oat and bean seedlings (Schoch et al. 1977, Schoch 1978), inhibition of the hydrogenation in intact oat seedlings by aminotriazol (Rtidiger and Benz 1979) or anaerobiosis (Schoch etal., 1980), and from studies of the reaction in vitro (Rfidiger et al. 1977, 1980; Benz et al. 1980). The latter studies demonstrated that geranylgeranyl diphosphate (GGPP) is the best substrate for the esterifying enzyme for which the name "chlorophyll synthetase" has been proposed (Rfidiger et al. 1980). The highest rates of hydrogenation of Chl~G to Chip are obtained in the presence of NADPH. However, phytyl diphosphate is also a suitable substrate for chlorophyll synthetase (Riidiger et al. 1980). Further in vitro studies (Benz 1980) revealed that small amounts of an endogenous phytyl precursor which directly yields Chlp are present in etioplast membranes; exogenous GGPP or phytyl diphosphate could not replace this phytyl precursor, but only mobilize its endogenous pool. Because in vitro investigations so far largely made use of membrane fractions of broken etioplasts, only membrane-bound endogenous precursors were present in the assays, whereas water-soluble compounds had been lost during the preparation. It was therefore the aim of this paper to determine a possible pool of water-soluble precursors by making use of protoplasts prepared from the primary leaves of etiolated oat seedlings (Hampp and Ziegler 1980) and etioplasts derived from these protoplasts by a rapid procedure. Penetration into these compartments was investigated by application of exogenous [1-3H]GGPP. The [13H]GGPP should be incorporated into ChlcG as soon as it arrives at the protylakoid membranes which, after irradiation of protoplasts or etioplasts, contained Chlide.

J. Benz et al, : Chlorophyll biosynthesis by protoplasts and plastids

55

I

100-

0.6 +GGPP

d

o

80"

g,

~X

~z 6o,

~s

~X

8 u~ .2

o

0.4

+ GGPP

~6 ,j

g

~x

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x-

2 0 - -GGPP -GGPP

.-o

CL 0.2 (3_ 0 I

t

1'5

3' 0 dark

/.,~5

6' 0

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Fig. 1. Esterification of endogenous Chlide in intact etioplasts (o 9 and protoplasts (x x). Etioplasts and protoplasts were prepared in the dark or under dim-green safetylight. Endogenous Protochlide was phototransformed into Chlide. Subsequent incubation in the presence or absence of [1AH]GGPP (dark, 24 ~ C). Pigments were extracted into acetone/ethyl acetate and transferred into diethylether. Magnesium was then removed from Chl(ide) by acidification (HC1). Subsequent extraction with diluted NH3 removed pheophorbide (unesterified), whereas pheophytin (esterifled) remained in the organic layer. Esterified pigment is given as % of total Chlide present before incubation. Values are means of 4 independent determinations. Standard deviation _+3% at 15 min, +2% at 30 and 60 min

Materials and methods Protoplasts and etioplasts were prepared from 7-d-old etiolated oat seedlings (Arena sativa L), as previously described (Hampp and Ziegler 1980). All procedures were carried out under dim-green safetylight in order to avoid phototransformation of Protochlide to Chlide. Protoplasts were resuspended in 0.5 M sorbitol, 0.05 M HepesKOH (pH 7.6), and 0.002 M CaCI> If etioplasts were required, protoplasts were broken by sucking them twice through a 20 pm nylon net. The resulting homogenate was centrifuged for 60 s at 600 g, and the pellet was resuspended in 0.35 M sorbitol, containing 0.05 M Hepes-KOH (pH 7.6), 0.2% (W/V) BSA, 0.001 M MgCI> and 0.001 M inorganic phosphate. In some cases, etioplasts were separated from protoplasts, within 15 s, by a method of integrated homogenation and fractionation (Hampp, in press). To aliquots containing 10-30nmol of Protochlide, 100 200 nmol [IAH]GGPP (specific activity 1.26 TBq/mol, Rfidiger et al. 1980) were added. The suspension was irradiated for 2 rain with white light (87 Win-2) for optimum photoconversion of Protochlide to Chlide and then incubated for 15, 30, or 60 rain in the dark at 24 ~ C. Pigments were extracted, isolated, and analyzed in the form of pheophytins, as previously described (Riidiger et al. 1980). In some experiments, broken etiopiasts (RiJdiger et al. 1980) were used instead of intact etioplasts or protopIasts. These were incubated with a 3,000 g or a 100,000 g supernatant of etiolated oat laminae which had been homogenized in 0.05 M phosphate buffer (pH 7.t). The ratio in the amount of oat laminae used either for the preparation of broken etioplasts or supernatant varied from l:i to 1:10; it was 1:7 in most experiments. Time-dependent uptake of [3H]GGPP by isolated etioplasts was measured by silicone oil filtration as described earlier (Hampp

~b

zb

Period of incubation (rain)

Fig. 2. Uptake of [3H]GGPP by isolated protoplasts and plastids from etiolated mesophyll tissue. Uptake was initiated by the addition of GGPP (0.1 raM; specific activity 1.26 TBq/mol) and terminated after the times indicated by centrifugal silicone oil filtration. Incubation was carried out at 20 ~ C. Values are means of two independent experiments with four parallels each. Standard deviation +5% for 0 10min, +3% for 10-30min. e - e etioplasts; o - o protoplasts

and Schmidt 1976). When uptake by protoplasts was measured, centrifugation was carried out at room temperature (10 s, 10,000 g) and silicone AR 140 (Wacker-Chemie, Mfinchen, FRG) was used instead of AR 150. The rates of uptake were referred to the osmotic spaces, which were calculated by subtracting the [3H]sorbitol space from the tritiated water space (preincubation for 5 rain at 23 ~ C).

Results and discussion T h e e s t e r i f i c a t i o n in i n t a c t p l a s t i d s (Fig. 1) is s i m i l a r to t h a t in b r o k e n e t i o p l a s t s : A f t e r 60 m i n i n c u b a t i o n , t h e e s t e r i f i c a t i o n is a b o u t 2 0 % o f C h l i d e without a n d 9 0 % o f C h l i d e with s a t u r a t i n g c o n c e n t r a t i o n s o f e x o g e n o u s G G P P ( R f i d i g e r et al. 1980). A t s h o r t e r i n c u b a t i o n times, the e s t e r i f i c a t i o n is s o m e w h a t l o w e r ( 1 3 % at 15 m i n ) w i t h o u t e x o g e n o u s G G P P , b u t n o differe n c e is f o u n d at s h o r t e r t i m e s w i t h e x o g e n o u s G G P P . T h e l a r g e d i f f e r e n c e in e s t e r i f i c a t i o n at 15 m i n d u e to G G P P ( 1 3 % w i t h o u t a n d 9 0 % w i t h G G P P ) suggests t h a t G G P P m u s t h a v e a l r e a d y p e n e t r a t e d t h e i n t a c t p l a s t i d e n v e l o p e e v e n at this e a r l y stage. S u p p o r t f o r this a s s u m p t i o n c o m e s f r o m Fig. 2. T i m e - d e p e n d e n t u p t a k e o f label f r o m [ 3 H ] G G P P is q u i t e fast w i t h s h o r t i n c u b a t i o n t i m e s ( a b o u t 0.3 to 0.6 n m o l btl X m i n - 1) a n d r e a c h e s s a t u r a t i o n b e t w e e n 10 a n d 30 rain at a level t h a t is n e a r l y 6 t i m e s t h a t o f the e x t e r n a l o n e (0.6 n m o l ~tl-1 : 0.6 m M c o m p a r e d

56

J. Benz et al. : Chlorophyll biosynthesis by protoplasts and plastids

to 0.1 mM). As uptake should be due to simple diffusion of this semilipophilic substrate, the stromal accumulation of label must be a consequence of the metabolization of GGPP. Protoplasts, however, on a volume basis, show a much slower rate of inward diffusion of [3H]GGPP (Fig. 2). This could be due to a higher reflection coefficient of the plasma membrane toward G G P P in comparison to the plastid envelope, but more probably could be a surface effect, as at the same volume. The surface of a protoplast is much smaller than that of plastids. Obviously, as a consequence of the lower uptake of label by protoplasts, the difference in esterification between experiments in the presence or absence of exogenous G G P P is not as large as in broken plastids. The values without exogenous G G P P are higher (38% at 60 min) and the values with exogenous G G P P lower (70% at 60 min) than the corresponding values with plastids (Fig. 1). A slower rate of uptake of G G P P across the plasmalemma in comparison to the rapid permeation through the plastid envelope can also be envisaged from the results of esterification in protoplasts without exogenous G G P P : Because the value (38%) is higher than in isolated plastids (20%), there must be some G G P P in the protoplast which later has been lost during the isolation of the plastids. Due to the technique used (fractionation of protoplasts in less than 15 s; Hampp in press), this amount of G G P P was possibly localized in the cytoplasm. On the other hand, the esterification of Chl in intact oat plants under otherwise the same conditions is about 95% with 60 min incubation (Schoch et al. 1977). This implies that during the isolation of protoplasts a considerable amount of G G P P has been lost. It is obvious from the foregoing results that diffusion of this substrate from the cytoplasm into the isolation medium should take place, if G G P P was localized in soluble form in the cytoplasm before. The same result would be expected if G G P P pools inside the plastids and in the cytoplasm were in equilibrium. The easy penetration of

G G P P through the plastid membranes suggests such a situation, but also raises the question of where G G P P is formed within the cell. In a recent paper Block et al. (1980) showed that recombined chloroplast fractions (stroma+thylakoids+envelopes), in contrast to the separated fractions, were capable of forming GG, GGPP, and Chlca from IPP. Combined stromal and envelope fractions synthesized G G P P and GG, while stroma itself lacked the enzymic capacity. F r o m these observations the authors suggested an association of the respective enzymes, at least in part, with the envelope membranes. In this respect the high permeability of G G P P toward the etioplast envelope, together with the cytoplasmic pool of GGPP, could indicate an export of G G P P formed inside the plastid (or at least at the inner envelope membrane) to other cellular sites. Incubation of broken plastids with G G P P leads to a specific rise in Chla~ and a smaller rise in Chln2c~, whereas the control values of Chln,G~ and Chlp are not changed (Rtidiger et al. 1980). A considerably higher amount of Chln4GG and some increase of Chlp is only observed in the presence of a large excess of N A D P H (Benz et al. 1980). No N A D P H or other reducing agent was applied in the present experiments. Nevertheless, the enhanced esterification in intact plastids in the presence of exogenous G G P P did not only imply ChlGa but also the other Chl species (Table 1), resulting in a remarkable increase of Chlp. This was never found with broken plastids. We assume that a soluble cofactor or a structural element necessary for the formation of Chip from ChlGc was lost during lysis of the plastids. Thus, we take the high percentage of Chlp as a criterion for the high degree of integrity of plastids derived from protoplasts. Interestingly, incubation of protoplasts with G G P P leads to a higher increase in Chlca and ChlH2aa than in Chlmao and Chlp, contrary to the results with intact plastids. If the reaction sequence Chlide~Chl~G-+Chlp were obligatory, this result

Table I. Incorporation of [1-3H]GGPPinto endogenous Chlide of etiolated protoplasts and intact plastids isolated therefrom. All values are means of 4 to 6 determinations the incubation time being 30 or 60 min. Standard deviations in brackets. Analysis by HPLC after transformation of chlorophylls into pheophytins GGPP

Protoplast Plastids

+ +

Amounts of pigment (% of Chl+Chlide)

Derived from [1-3H]GGPP according to radioactivity (% of Chl+Chlide)

Chloa

ChlH~GG

ChlmGG

Chlp

ChlGG

Chlp

10 (+ 1) 28 (_+2) 9 ( _+2) 38 (+_2)

3 (_+2) 10 (_+3) 2 (_+1) 10 (_+3)

3 6 2 4

19 (+1) 21 (_+2) 9 (_2) 36 (_+1)

23 (_+2) -25 (_+2)

9 (_+I) -23 (_+2)

('-1) (__+2) (_+1) (+_1)

J. Benz et al. : Chlorophyll biosynthesis by protoplasts and plastids

57

Table 2. Time course of esterification and hydrogenation. Chlee and Chlp are given as percent of Chl+Chlide. All values are means of 2 independent determinations. Standard deviations in brackets. Analysis by HPLC after transformation of chlorophylls into pheophytins

GGPP

Protoplasts Plastids

+ +

15 rain

30 min

60 min

ChlGe

Chlp

Chlee

Chip

Chloe

Chip

9 (+1) n.d. 6 (-+1) 60 (_+2)

7 (+1) n.d. 5 (_+1) i7 (_+1)

8 26 9 43

19 20 8 37

i0 32 9 40

25 23 I1 48

would reflect a slower overall reaction in protoplasts than in plastids due to the slow permeation of G G P P . However, the determination of radioactivity of the isolated chlorophylls reveals a more complicated situation. F r o m the specific radioactivity of isolated chlorophylls, the respective amount of chlorophyll derived from the applied [3H]GGPP was determined (Table 1). In accordance with results from broken plastids (Benz 1980), the small endogenous pools of G G and P precursors contained in intact plastids could not be replaced by exogenous [3H]GGPP and should be regarded as membrane-associated. The inrease in the amount of Chlao and of Chlp theretbre roughly corresponds to the amount derived from [3H]GGPP. However, the endogenous pools of G G and P precursors in protoplasts (which are larger than those in plastids) were partly replaced by exogenous [3H]GGPP. This observation suggests that part of the endogenous precursor pool of protoplasts exists as soluble G G P P possibly located in the cytoplasm and in some equilibrium with the plastid stroma. The lower value for [3H]GGPP-derived Chlp in protoplasts (9%) as compared to plastids (23%) again supports the view of a slower hydrogenation in the former, possibly due to the slow esterification. In another series of experiments, the time course of esterification and hydrogenation was investigated (Table 2). It is evident that not only the esterification but also the hydrogenation was more rapid in plastids than in protoplasts. Interestingly, some hydrogenation was also observed without exogenous G G P P . This demonstrates that at least part of the endogenous precursor must be G G P P (or a related G G compound) and not a phytol derivative. Furthermore, the presence of soluble G G P P (or its direct precursor) in laminae of etiolated oat seedlings was demonstrated by means of the chlorophyll synthetase reaction. The soluble fraction o f a homogenate from etiolated oat laminae was incubated with an illuminated etioplast m e m b r a n e fraction which contained Chlide and chlorophyll synthetase. The esterification was increased, e.g., f r o m 20% to 65%, if the soluble fraction was prepared from 7 parts oat laminae, the m e m b r a n e fraction from one part. High-

(_+1) (_+1) (_+1) (_+2)

(_+1) (_+1) (_+1) (_+2)

~D

B

o) j=

(_+1) (_+2) (_+1) (_+2)

(_+1) (_+1) (_+1) (+2)

r a~ .~

O.. ill

f f{ { Fig. 3A, B. HPLC analysis of pheophytin prepared from broken etioplast membrane fraction after esterification for 45 min. A Incubation of the etioplast membrane fractions in phosphate buffer (control). The main pigment is Phee. B Incubation of the etioplast membrane fraction (prepared from t part oat laminae) with the 3,000 g supernatant of a homogenate in phosphate buffer (prepared from 7 parts of oat laminae). The main pigment is Pheoo. Because the esterification was higher in B than in A, a lower magnification was chosen for detection of pigments after HPLC separation, The absolute amounts of Phep were about the same in A and B. The increase of PheeG was also observed with a 100,000 g supernatant

er ratios of soluble fraction to m e m b r a n e fraction yielded higher rates of esterfication (up to 90%), lower ratios a corresponding reduction in esterification. These results indicate that a substrate for chlorophyll synthetase must be present in the supernatant. H P L C and analysis of the esterified chlorophyll formed by the incubation revealed a specific increase

58 o f ChlGa a n d a smaller increase o f ChlH2GG (Fig. 3). N o increase o f Chlp c o u l d be detected. This is t y p i c a l for i n c u b a t i o n o f i l l u m i n a t e d m e m b r a n e s with G G P P (Rfidiger et al. 1980) a n d thus proves the p r e s e n c e o f G G P P (or a p r e c u r s o r thereof) in the s u p e r n a t a n t a n d the absence o f p h y t y l d i p h o s p h a t e . P h y t y l d i p h o s p h a t e s h o u l d have been i n c o r p o r a t e d into Chlide with the f o r m a t i o n o f Chip u n d e r these c o n d i t i o n s (Rfidiger et al. 1980). O u r results c a n n o t , however, be t a k e n as direct evidence for the presence o f G G P P in the s u p e r n a t a n t . The finding o f B l o c k et al. (1980), t h a t r e c o m b i n e d fractions o f fully d e v e l o p e d c h l o r o p l a s t s ( s t r o m a + t h y l a k o i d s + envelopes) c a t a l y z e the f o r m a tion o f G G P P f r o m IPP, suggests t h a t this r e a c t i o n also t a k e s place in o u r b r o k e n e t i o p l a s t p r e p a r a t i o n . A m o d e r a t e esterification o f Chlide was d e t e c t e d after i n c u b a t i o n o f b r o k e n e t i o p l a s t s with IPP, I P P + gerany l d i p h o s p h a t e o r I P P + f a r n e s y l d i p h o s p h a t e (Benz 1980). These p r e c u r s o r s were specifically i n c o r p o r a t e d into ChlGG, n o t into Chlp. E x p e r i m e n t s a r e u n d e r w a y to d e t e r m i n e w h e t h e r G G P P o r one o f these p r e c u r s o r s is present in the s u p e r n a t a n t . We thank the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, for support of this work.

J. Benz et al. : Chlorophyll biosynthesis by protoplasts and plastids Benz, J., Wolf, C., Rfidiger, W. (1980) Chlorophyll biosynthesis: hydrogenation of geranylgeraniol. Plant Sci. Lett. 19, 225 230 Block, M.A., Joyard, J., Douce, R. (1980) Site of synthesis of geranylgeranioI derivatives in intact spinach chloroplasts. Biochim. Biophys. Acta 631, 210-219 Hampp, R. (198 l) Rapid separation of the plastid, mitochondrial, and cytoplasmic fractions from intact leaf protoplasts of Avena. Planta (in press) Hampp, R., Schmidt, H.-W. (1976) Changes in envelope permeability during chloroplast development. Planta 129, 69-73 Hampp, R., Ziegler, H. (1980) On the use of Arena protoplasts to study chloroplast development. Planta 147, 485494 Riidiger, W., Hedden, P., K6st, H.-P., Chapman, D.J. (1977) Esterification of chlorophyllide by geranylgeranyl pyrophosphate in a cell-free system from Maize shoots. Biochem. Biophys. Res. Comm. 74, 1268-1272 Rfidiger, W., Benz, J. (1979) Influence of aminotriazol on the biosynthesis of chlorophyll and phytol. Z. Naturforsch. 34c, 1055 1057 Rfidiger, W., Benz, J., Guthoff, C. (1980) Detection and partial characterization of activity of chlorophyll synthetase in etioplast membranes. Eur. J. Biochem. 109, 193 200 Schoch, S., Lempert, U., Rfidiger, W. (1977) lJber die letzten Stufen der Chlorophyll-Biosynthese. Zwischenprodukte zwischen Chlorophyllid und phytolhaltigem Chlorophyll. Z. Pflanzenphysiol. 83, 427436 Schoch, S. (1978) The esterification of chlorophyllide a in greening bean leaves. Z. Naturforsch. 33e, 712 714 Schoch, S., Hehlein, C., Rt~diger, W. (1980) Influence of anaerobiosis on chlorophyll biosynthesis in greening oat seedlings (Arena sativa L.) Plant Physiol. 66, 576 579

References Benz, J. (1980) Untersuchungen zur Chlorophyll-Biosynthese in etiolierten Haferkeimliugen: Veresterung yon Chlorophyllid in vitro. PhD thesis, UniversitS.t Mfinchen

Received 14 November 1980; accepted 18 January 1981