Synthesis of plastoquinone-9 and phytylplastoquinone from homogentisate in lettuce chloroplasts

630

Biochimica et Biophysica Acta, 6 3 2 ( 1 9 8 0 ) 6 3 0 - - 6 4 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 29404

SYNTHESIS OF PLASTOQUINONE-9 AND P H Y T Y L P L A S T O Q U I N O N E FROM HOMOGENTISATE IN LETTUCE C H L O R O P L A S T S

K E N N E T H G. H U T S O N a n d D A V I D R. T H R E L F A L L *

Department of Plan t Biology, University of Hull, Hull HU6 7RX (U.K.) ( R e c e i v e d M a r c h 3rd, 1 9 8 0 )

Key words: Homogentisate; Plastoquinone synthesis; Phytylplastoquinone; (Lettuce chloroplast)

Summary Chloroform-soluble extracts of unpurified chloroplast preparations of lettuce, pea and spinach and of class I lettuce chloroplasts that have been incubated in the light with [methylene-3H]homogentisate contain 3H-labelled plastoquinones-9 and -8 (minor homologue), 2-demethylplastoquinones-9 and -8 (minor homologue), pytylplastoquinone and 2-demethylphytylplastoquinone. The absence of demethylquinols, the presumed precursors of the dimethylquinones, from the extracts to the fact that no precautions were taken in the extraction procedure to present their oxidation to the corresponding quinones.

In unpurified lettuce chloroplasts the synthesis of these c o m p o u n d s from Mg2+-dependent and it is stimulated by light. The addition of isopentenyl pyrophosphate to the incubation mixtures increases the amounts of both groups of quinones (polyprenyl quinones and phytyl quinones) synthesised in the light and the amounts of polyprenyl quinones synthesised in the dark. Replacement of isopentenyl pyrophosphate with a source of preformed polyprenyl pyrophosphates brings a b o u t a marked rise in the amounts of polyprenyl quinones synthesized. This rise in polyprenyl quinone synthesis is further increased if the chloroplasts are subjected to osmotic shock. The presence of S-adenosylmethionine increases the amounts of dimethylquinones synthesized at the expense of the demethylquinones. The implied precursor-product relationships between 2-demethylphytylplastoquinone (quinol?) and phytylplastoquinone and between the 2-demethylplastoquinones (quinols) and plastoquinones were verified in a pulse-labelling experiment. Confirmation that these quinones, or their corresponding quinols, axe synthesized

[methylene-3H]homogentisate is

* To whom correspondence should be addressed.. Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane sulphonic acid.

631 in the chloroplast is provided by the fact that they are made in class I lettuce chloroplasts. In none of the many incubations carried out in the course of the study were any [3H]tocopherols produced.

Introduction Plastoquinone-9 and its metabolites (plastoquinone-Bs, -Cs and -Zs and the plastochromanols), a-tocopherol, 7-tocopherol and a-tocopherolquinone are normal constituents of the photosynthetic tissues of higher plants. In addition, some tissues contain 5-tocopherol, fi-tocopherol or non<~-tocopherolquinones. The quinones, plastochromanols and the greater part of the a-tocopherol are located in the chloroplasts [1,2]. The non-a-tocopherols appear to be localized in the chloroplasts to some extent and perhaps also in the endoplasmic reticulum [2]. The results of feeding experiments designed to elucidate the natures of the intermediates involved in the biosynthesis of these compounds in shoots and leaves [1,2] and, more recently, chloroplasts [3,4] have indicated that the chloroplast is able to carry out all of the reactions involved in the synthesis of the chloroplast quinones and chromanols from COz, apart from the formation of homogentisate from 4-hydroxyphenylpyruvate (a reaction which probably takes place in the peroxisomes [4]), and that there are other sites in the cell for the syntheses of the extrachloroplastid tocopherols [ 2]. It is generally believed that the key reactions in the biosyntheses of plastoquinone-9 and the tocopherols are the formation of a nonaprenyltoluquinol and either a phytyltoluquinol or a geranylgeranyltoluquinol, respectively, from homogentisate and the appropriate terpenyl pyrophosphate [1,2] (Fig. 1). In the biosynthesis of plastoquinone-9, the product of this reaction would be either 2
~

~

OH

R'

OH

R'

3- Demet hylpnyt ylplastoqulnol

CH 3

OH

2 Demethyl0hytylplastoqumol

~H 3

OH

OH

OH

/

OH

~

CH3

/V=4

R'

7-MethyltOcoI

6-Tocopherol

CH3

~H 3~ A ~

CH3"'~7

HO

R

Plastoqulnol N

~H 3

~N 3

R'

R"

~ / ~ 1 ~

~N=4

t~ : 9 or 4

HO

R

CH3

# - Tocot rlenol

Tocopherol

CH3

~H3

#-Tocopnerol

~

CH3

O Plastoqulnone-N

EH 3

O

R"

............

S

sAM

,1

Tocopherc!

CH3

R

o< -Tocopherclqulnorle

CH3

•

Tocotmenol

Fig. 1. Possible pathways for the biosynthesis of plastoquinone-9 and tocopherols from homogentisate [1,2]. The labelling patterns of the 7- and 8-methyl groups of 7" and ~-tocopherol will be reversed if these tocopherols are formed from 7-methyltocol.

~ ,

CH2CO2 H

Homogentlslc actd

OH

OH

OH

2 Demethylplastoquind

~H 3

OH

05 bo

633 a-tocopherol (Fig. 1). The evidence offered in support of the intermediacy of 2-demethylterpenyltoluquinols was that radioactivity from DL-[1,2-14C]shiki mate is incorporated in the predicted manner into T-tocopherol and a-tocopherol in maize shoots [6] and that chloroplast preparations of sugar beet and E. gracilis are able to synthesize from homogentisate and phytyl pyrophosphate small amounts of a compound with the thin-layer chromatographic properties expected of a phytyltoluquinol [7]. In 1976, however, the intermediacy of 2-demethylterpenyltoluquinols in the synthesis of tocopherols other than 6-tocopherol was brought into question with the findings that on careful analysis of photosynthetic tissues, it is possible to detect small amounts of the other positional isomers of phytyltoluquinol [2] and that radioactivity from tritiated species of each of the positional isomers of phytyltoluquinone and monomethyltocol, is incorporated into a-tocopherol by leaf tissue [2]. On the basis of the quantitative data obtained in the latter studies, it was suggested that in the chloroplast a major route to a-tocopherol is: homogentisate-+ 3-demethylphytylplastoquinol-+ 7-methyltocol (a positional isomer of 5-tocopherol, 8-methyltocol)-~ 7,8-dimethyltocol (5-tocopherol)-+ a-tocopherol {Fig. 1). A major criticism which can be levelled against this work is that the assumption was made that the side-chain is formed from phytyl pyrophosphate, as a consequence of which no experiments were performed to test for the possible involvement of geranylgeranyltoluquinols or tocotrienols (unsaturated forms of tocopherols) in the biosynthesis of tocopherols. In the present paper we report the results of experiments designed to establish the identities of the first and subsequent intermediates in the pathway leading from homogentisate to a-tocopherol and plastoquinone-9 in higher plant chloroplasts. Materials and Methods Radiochemical. [ m e t h y l e n e - 3 H ] H o m o g e n t i s i c acid (2.5-dihydroxyphenyl acetic acid) (0.88 Ci. mmo1-1) was prepared from the unlabelled acid by catalytic exchange in solution by tritium gas (Method TR. 7) at the Radiochemical Centre, Amersham, U.K. It was purified before use by thin-layer chromatography on 0.5 mm silica gel HF254+366 (500 #Ci/plate) developed with benzene/methanol/acetic acid ( 4 5 : 8 : 4 , v/v) (RE = 0.25). The acid, which accounted for some 95% of the radioactivity on the plate, was located by examination of the developed plate under ultraviolet radiation (254 nm) and eluted off the gel with peroxide-free diethyl ether. The ether extract was taken to dryness under a stream of N2 gas and the residual homogentisic acid taken up in 4 ml ethanolic 0.01 M ascorbate. The ethanolic solution was adjusted to 100 pCi • m1-1 by the addition of ethanol and stored at --20°C until required. Triammonium [1-14C]isopentenyl pyrophosphate (56 mCi. mmol -~) was purchased from the Radiochemical Centre, Amersham, U.K. Chemicals. Plastoquinone-10, -9, -4 and -3, phytylplastoquinone, crude mixtures of phytyltoluquinones and monomethyltocols, fl-tocopherol and 5.7-dimethyltocol were obtained as generous gifts (see Acknowledgements). 2-Demethylplastoquinone-9 (contaminated with a small amount of 2-demethylplastoquinone-8) was isolated from bulbs of Iris hollandica var. Golden Harvest [8].

634 2- and 3-Demethylphytylplastoquinone (i.e. 3- and 4-phytyltoluquinone) were synthesized by condensation of phytol with toluquinol in dioxane in the presence of boron trifluoride etherate [2]. The demethylphytylplastoquinones were separated from the other reaction products by elution with 1% and 2% (v/v) diethyl ether in light petroleum ether (b.p. 40--60°C) from a column of Brockman grade III acid-washed alumina and then the mixture of 2'-cis and 2'-trans (major components) quinones subjected to quantitative thin-layer chromatography oa 0.5 mm silica gel HF254+366 developed with diethyl ether/light petroleum (b.p. 40--60°C) (2 : 23 v/v) (2'-trans-2-demethyl isomer, RE = 0.25; 2'-trans-3
635 the Botanic Gardens of the University of Hull. Spray dried cells of Micrococcus luteus ATCC 4698 were purchased from Miles Lab. Inc., Slough, U.K. Isolation o f lettuce chloroplasts. The suspension buffer used for the isolation of chloroplasts was based on one used for the isolation of barley etiochloroplasts [12] and consisted of 30 mM Hepes/0.5 M sucrose/5 mM cysteine/1 mM MgC12/1 mM EDTA/0.2% (w/v} bovine serum albumin fraction V, adjusted to pH 7.6 with KOH. Undamaged leaves (80--120 g) were removed from the lettuce plant, washed with distilled water and homogenized with ice-cold suspension buffer (30 ml/10 g leaf tissue) in an MSE top drive blender for 20 s (2 X 10 s). The homogenate was filtered through four layers of cheesecloth and the filtrate centrifuged at 500 X g for 90 s. The 500 X g supernatant was then centrifuged at 1500 X g for 5 min and the resultant pellet was washed by resuspension in 40 ml of suspension buffer followed by resedimentation at 1500 X g for 5 min before resuspension in 2 ml of suspension buffer. The concentration of chlorophyll in the preparation was a b o u t 1.0 m g . m1-1. This unpurified chloroplast suspension contained 50--60% intact plastids as judged by phase contrast microscopy and ferricyanide reduction [ 13]. In some experiments, the chloroplasts were further purified by centrifugation in a biphasic Dextran-poly(ethylene glycol) system [14]. The crude chloroplast pellet was resuspended in 10 ml of top phase, mixed with 5 ml of b o t t o m phase and centrifuged in a swing-out bucket at 1000 X g for 10 min. The top phase containing ruptured chloroplasts, a small proportion of intact chloroplasts and cellular contaminants was discarded. Pelleted chloroplasts and chloroplasts in the b o t t o m phase were washed by dilution in 50 ml of a 1 : 4 mixture of t o p phase and suspension buffer followed by resedimentation at 1500 X g for 5 min before resuspension in 2 ml of suspension buffer. The preparation (Class I chloroplasts) contained in excess of 95% intact plastids and was free of other cellular material (electron microscopy). Isolation o f spinach chloroplasts and pea chloroplasts. These were prepared by the method just described for the preparation of unpurified lettuce chloroplasts. Preincubation o f M. luteus extracts with trilithium isopentenyl pyrophosphate. Cell-free extracts of M. luteus prepared by the m e t h o d of Hutson and Threlfall [15] were incubated with trilithium isopentenyl pyrophosphate (10 p m o l . ml -~ extract} for 30 min at 30°C. This preparation was used as source of preformed polyprenyl pyrophosphates. Assay of the formation o f quinones, quinols and tocopherols by chloroplasts. The standard assay mixture (total volume 3 ml) was made up of: 2.3 ml suspension buffer, pH 7.6; 0.3 ml chloroplast preparation (approx. 0.3 mg chlorophyll}; 0.1 ml 10 mM trilithium isopentenyl pyrophosphate; 0.1 ml 5 mM S-adenosylmethionine; 0.1 ml 100 mM MgCI~ and 20 t~l of ethanolic [methylene-3H]homogentisate (2 pCi; 0.88 Ci • mmol-1). In incubations requiring ruptured chloroplasts the suspension buffer was replaced by 30 mM HEPES buffer, pH 7.6. In assays requiring the presence of an exogenous supply of preformed polyprenyl pyrophosphates the incubation mixture (total volume 3 ml) was made up of: 1.8 ml suspension buffer, pH 7.6; 0.3 ml chloroplast preparation (approx. 0.3 mg chlorophyll}; 0.5 ml M. luteus extract that had been preincu-

636 bated with trilithium isopentenyl pyrophosphate (see previous section); 0.1 ml 5 mM S-adenosylmethionine; 0.1 M MgC12 and 20 #l of ethanolic [3H]homogentisate. The assay mixtures were incubated in 25 ml conical flasks with constant shaking (120 X 3 cm stroke • min -1) at 30°C and 5000 lux (150 W reflector spotlight) for 60 min. At the end of the incubation period the reactions were terminated by the addition to each flask of 12 ml chloroform/methanol (1 : 2, v/v). The resultant mixtures were left in the dark at 4°C for 30 min and then worked up for. chloroform-soluble lipids [ 15 ]. The lipid extracts were assayed for 3H and then subjected to quantitative thin-layer chromatography on 0.5 mm silica gel HF254+366 developed with benzene. In those analyses in which it was known that the only products would be plastoquinone-9 (and -8), 2-demethylplastoquinone-9 ( a n d - 8 ) , phytylplastoquinone and 2-demethylphytylplastoquinone the area of gel (zones A and B in Fig. 2) containing the 3H was eluted with diethyl ether and the recovered compounds along with suitable marker quinones were subjected to high performance liquid chromatography on LiChrosorb S1 60 5/~ (Merck) developed with dioxane/iso-octane (see below). In all of the assays reported in this paper, 90-95% of the 3H-activity applied to the column was distributed between the fractions corresponding to the mass peaks of plastoquinone-9 (and-8), 2-demethylplastoquinone-9 (and -8), phytylplastoquinone and 2-demethylphytylplastoquinone. In those analyses where the natures of the reaction products had not been established, the developed thin-layer chromatogram was scanned to locate the 3H-labelled compounds which were then eluted with diethyl ether and characterized by the procedures described in Results. High performance liquid chromatography. This was performed in a Jobling System 1 high performance liquid chromatograph. Adsorptive chromatography of quinones was carried out on either a 200 X 2 mm column of LiChrosorb $1 60 5t~ (Merck) developed with 0.15% dioxane/iso-octane (0.3 ml • min -1) or a 100 X 5 mm column of LiChrosorb S1 60 developed with 0.1% dioxane-isooctane (1.8 ml • min-~). Adsorptive chromatography of tocopherols was carried out on the 200 X 2 mm column developed with 2% dioxane/iso-octane (0.3 ml • min-1). The samples and marker compounds (0.5--2 pg of each quinone; 5--10 #g of each tocopherol) in 5--10 #l of cyclohexane were injected directly onto the columns. The column effluents were monitored at 254 nm for the presence of quinones and at 280 nm for the presence of tocochromanols. The effluent from the ultraviolet detector was collected as a series of fractions which corresponded to the mass peaks of the marker compounds and to the periods between the mass peaks. In routine analytical work the fractions were collected in scintillation vials and assayed for 3H. If the compounds were to be the subjects of further characterization studies, then only aliquots of the fractions were assayed for 3H. The retention times of the quinones on the 2 mm (internal diameter) and 5 mm (internal diameter) columns are given in Fig. 3. The retention times (min) of the tocopherols on the 2 m m (internal diameter) column were: a-tocopherol, 12; 5,7-dimethyltocol, 14.5; /3-tocopherol, 22.5; 7-tocopherol, 24.5; 7-methyltocol, 31; 5-methyltocol, 33, and 5-tocopherol, 40.5. Reversed-phase chromatography was performed on a 200 X 2 mm {internal

637 diameter) column of ODS-Hypersil 5# (Shandon) developed with methanol. With a flow rate of 0.28 ml • min -1 the retention times (min) were: phytyltoluquinones, 5; phytylplastoquinones, 6; demethylplastoquinone-8, 18; plastoquinone-8, 22.5; demethylplastoquinone-9, 29, and plastoquinone-9, 38. If aqueous 95% methanol was used then the phytyltoluquinones and phytylplastoquinones were eluted after 7.5 and 10 min, respectively. Chemical conversion of phytylquinones to mono- and dimethyltocols. Two methods were used. In the first m e t h o d , phytyl-[3H]quinones recovered from high performance liquid chromatography were mixed with 5--10 mg of carrier quinone and refluxed in pyridine to form the chromenol which was then catalytically hydrogenated to the chromanol [16]. In the second method, the mixture of 3H-labelled and unlabelled quinone was treated with KHSO3 and glacial acetic acid [2]. The products were purified by thin-layer chromatography on 0.5 mm silica gel HF254+366 developed twice with chloroform (for RF values, see above). The 3H-chromanol was eluted off the gel with diethyl ether and its specific radioactivity measured. A sample was then subjected to high performance liquid chromatography and its 3H-elution and mass-elution profiles determined. In addition, the specific radioactivity of the eluted chromanol was measured. Chemical reduction of quinones to form quinols. The quinone was taken up in a small volume of aqueous 95% ethanol and treated with Zn dust and a drop of conc. HC1. The ethanolic phase was then subjected to thin-layer chromatography on silica gel HF2~4+366 developed with benzene (for RE values, see Fig. 2). Spectrophotometric estimation of quinones and chromanols. Quinones and chromanols were assayed in cyclohexane by using the appropriate absorbance factors (see above and Ref. 16). Chlorophyll determination. Chlorophyll was measured by the method of Arnon [18]. Radioassay. Distribution of 3H-activity on thin-layer chromatograms was determined by using a Panax Radio-TLC Scanner System. All other samples were dissolved in scintillation fluid [7] and assayed for radioactivity in a Intertechnique SL 4000 liquid scintillation counter. The machine efficiency for 3H was 64%. Results and Discussion

Identification of 3H-compounds formed from [methylene-3H]homogentisate To try and obtain more direct evidence as to the natures of the first and subsequent intermediates on the pathways leading from homogentisate to plastoquinone-9 and a-tocopherol, chloroplasts of lettuce, pea and spinach were tested for their abilities to synthesize chloroform-soluble 3H-labelled quinones, quinols and chromanols, when incubated in the light with [methylene-3H] homogentisate and what were considered to be appropriate supplements (Table I). It was found that, with the exception of the incubations which contained mature spinach chloroplasts, all the incubation mixtures tested incorporated radioactivity into chloroform-soluble c o m p o u n d s (Table I). Radioadsorptive thin-layer chromatography demonstrated that most of the

638 TABLE I SYNTHESIS OF 3 H - L A B E L L E D Q U I N O N E S FROM [ M E T H Y L E N E - 3 H ] H O M O G E N T I S A T E CHLOROPLAST PREPARATIONS FROM HIGHER PLANTS

BY

T h e c h l o r o p h y l l c o n t e n t o f t h e i n c u b a t i o n m i x t u r e s c o n t a i n i n g c h l o r o p l a s t s f r o m l e t t u c e , pea, 1 4 - d a y - o l d

spinach and m a t u r e spinach were 0.34, 0.35, 0.37 and 0.42 mg, respectively. The incubation period was 90 min. Source of preparation and nature of incubation mixture

R a d i o a c t i v i t y ( 1 0 -3 × d p m ) Chloroformsoluble compounds

Lettuce (obtained f r o m local m a r k e t gardens) Standard --S-adenosylmethionine 65 Standard 179.4 M. l u t e u s s y s t e m --S-adenosylmethionine 127.7 M. l u t e u s s y s t e m 146.7 Pea l e a v e s ( 1 2 - d a y -old) Standard --isopentenyl pyrophosphate and --S-adenosylmethionine Standard --S-adenosylmethionine M. luteu$ s y s t e m --S-adenosylmethionine S p i n a c h leaves ( 1 4 - d a y - o l d ) Standard --isopentenyl pyrophosphate and --S-adenosylmethionine Standard --S-adenosylmethionine Standard M. l u t e u s s y s t e m --S-adenosyhnethinnine S p i n a c h leaves ( m a t u r e ) A s i n c u b a t i o n s 8, 9 a n d 1 0

2-Demethylphytylplastoquinone

34.4 10.6

Phytylplastoquinone

2-Demethylplastoquinone-9 *

Plastoquinone-9 *

12.2 126.1

13.3 1.1

4.8 39.8

0.3 0.9

70.2 2

53 139.5

0 0

16.6

1.3

13.7

0

0.5

30.8

6.4

19.7

0.3

2.7

172.3

0.8

11.3

28.6

131.6

10.2

6.9

1.3

0.5

1.1

18.8 22.3

7.8 1.1

0.9 10.3

3.8 0.6

4.1 9.4

5.9

134.2

103.9

260.5

2

No i n c o ~ o r a t i o n of radioactivity

* S t a n d a r d i n c u b a t i o n s : 5 - - 8 % o f r a d i o a c t i v i t y a s s o c i a t e d w i t h t h e o c t a p r e n y l h o m o l o g u e . M. luteus systems: 10--15% of radioactivity associated with the o c t a p r e n y l h o m o l o g u e .

radioactivity in the chloroform extracts of the incubations which were either unsupplemented or supplemented with isopentenyl pyrophosphate -+ S-adenosylmethionine was distributed between c o m p o u n d s which co-chromatographed with the plastoquinones, 2-demethylplastoquinone-9, the phytylplastoquinones and the demethylphytylplastoquinones and that little, if any, was associated with quinols and tocopherols (e.g. Fig. 2). In the extracts from the incubations supplemented with polyprenyl pyrophosphates, the radioactivity was present mainly in c o m p o u n d s which co-chromatographed with plastoquinone-9 and demethylplastoquinone-9. The presence of S-adenosylmethionine led to a marked increase in the radioactivity which co-chromatographed with the dimethylquinones, at the apparent expense of the radioactive compounds which co-chromatographed with the demethylquinones (e.g. Fig. 2). The characterizations of the 3H-labelled compounds in the chloroform

639 C

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Fig. 2. R a d i o a d s o r P t i v e t h i n - l a y e r c h r o m a t o g r a p h y t r a c e s o f t h e p r o d u c t s o b t a i n e d o n i n c u b a t i o n o f u n p u r i f i e d l e t t u c e c h l o r o p l a s t s w i t h [methylene-3H]homogentisate. S c a n (a), c h l o r o f o r m - s o l u b l e p r o d u c t s f r o m S - a d e n o s y l m e t h i o n i n e - d e f i c i e n t s t a n d a r d i n c u b a t i o n m i x t u r e (Table I, i n c u b a t i o n 1.) S c a n (b), c h l o r o f o r m - s o l u b l e p r o d u c t s f r o m s t a n d a r d i n c u b a t i o n m i x t u r e ( T a b l e I, i n c u b a t i o n 2). M a r k e r c o m p o u n d s (2'-trans i s o m e r s o f q u i n o n e s ) : P Q - 1 0 , -9, -4 a n d -3; p l a s t o q u i n o n e - 1 0 , -9, -4 a n d -3; 2 - D P Q - 9 , 2 - d e m e t h y l plastoquinone-9; PPQ, phytylplastoquinonc; 2,6-PPQ and 3,6-PPQ, 2,6-and 3,6-dimethyl isomers of phytylplastoquinone; 6-PTQ, 6-phytyltoluquinone; 2-DPPQ(3-PTQ), 2-demethylphytylplastoquinone (3-phytyltoluquinone); 3-DPPQ (4-PTQ), 3-demethylphytylplastoquinone (4-phytyltoluquinone); GGTQs, g e r a n y l g e r a n y l t o l u q u i n o n e s ; P Q H 2 - 9 a n d P T Q H s , q u i n o l s o f P Q - 9 a n d P T Q s ; a - , 3'- a n d 5 - T , ~-, 3'- a n d 5 -tocopherol.

extracts of the incubations which were either unsupplemented or supplemented with isopentenyl pyrophosphate with or w i t h o u t S-adenosylmethionine were carried out as follows. The radioactivity on the thin-layer chromatography plates was eluted off the areas of gel corresponding to either the zones shown as A (upper zone), B (mid zone) and C (lower zone) in Fig. 2 or the individual peaks on the radiochromatograph trace, and subjected to analytical and preparative radio high-performance liquid chromatography on adsorptive columns. In the case of each extract, all the radioactivity from zone A was eluted coincident with the mass peaks of plastoquinone-9 and phytylplastoquinone whilst most of that from zone B was eluted coincident with the mass peaks of demethylplastoquinone-9 and 2-demethylphytylplastoquinone (e.g. Fig. 3). The small amounts of radioactivity from the zone Cs was not eluted coincident with any of the tocopherols. The elution patterns for the radioactivity eluted off the areas of gel from under the individual peaks gave a complementary set of results. The quinone nature of each of the 3H-labelled compounds recovered from the columns was established by the demonstration that it could be reduced chemically (Zn/HC1) to give a 3H-labelled c o m p o u n d which migrated with the same R F as t h a t of the appropriate marker quinol (For Re values, see Materials and

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C 25

0 Timelmin)

Fig. 3. R a d i o a d s o r p t i v e h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y t r a c e s o f t h e c o m p o u n d s r e c o v e r e d f r o m t h i n - l a y e r c h z o m a t o g r a m s . Scan a, c o m p o u n d s f r o m s t a n d a r d i n c u b a t i o n m i x t u r e ( T a b l e II, i n c u b a t i o n 7 ) r e c o v e r e d f r o m z o n e A o f t h e t h i n - l a y e r c h r o m a t o g r a m (see Fig. 2) a n d a p p r o p r i a t e m a r k e r c o m p o u n d s (PPQ, 2,6-PPQ, 3,6-PPQ, PQ-9, P Q - 1 0 a n d 2 - D P Q - 9 ) c h r o m a t o g r a p h e d o n 1 0 0 X 2 m m o f L i C h r o sorb $1 6 0 d e v e l o p e d w i t h 0 . 1 5 % d i o x a n e / i s o - o c t a n e . Scan b, c o m p o u n d s f r o m S - a d e n o s y l m e t h i o n i n e d e f i c i e n t s t a n d a r d i n c u b a t i o n m i x t u r e ( T a b l e II, i n c u b a t i o n 5) r e c o v e r e d f r o m z o n e B o f t h e t h i n - l a y e r c h r o m a t o g r a m (see Fig. 2) a n d a p p r o p r i a t e m a r k e r c o m p o u n d s (PPQ, 3-DPPQ, 2-DPPQ, PQ-9 a n d 2 D P Q - 9 ) c h r o m a t o g r a p h e d u n d e r t h e s a m e c o n d i t i o n s as (a). S c a n c, m a r k e r c o m p o u n d s c h r o m a t o g r a p h e d o n 1 0 0 X 5 m m L i C h r o s o r b S1 60 d e v e l o p e d w i t h 0.1% d i o x a n e ] i s o - o c t a n e . T h e a b b r e v i a t i o n s (see Fig. 2 for k e y ) are a l i g n e d w i t h t h e 2'-trans-isomers o f t h e q u i n o n e s . T h e 2'-cis-isomers w h i c h are f o r m e d o n s t o r a g e o f t h e m a r k e r c o m p o u n d s are t h e s m a l l a d j a c e n t p e a k s w i t h s h o r t e r e l u t i o n t i m e s . T h e t i m e s o f e l u t i o n o f PQ*10 (scan a), P Q - 3 , PQ-4, G G T Q s (Scan b), 7 - T a n d (x-T ( S c a n c) arc s h o w n b y a r r o w s .

Methods). Radio-reversed-phase high-performance liquid chromatography of the polyprenyl [3H] quinones recovered from the adsorptive columns confirmed the identities of the [3H]plastoquinone-9 and [3H]demethylplastoquinone-9 and showed that small amounts of the octaprenyl homologues (or more unlikely, positional isomers} of these two compounds had also been produced. The phytyl[3H]quinone samples were eluted coincident with the appropriate mixtures of positional isomers (reversed-phase chromatography does not separate positional isomers). Additional confirmation of the identities of the 3H-labelled plastoquinone-9 and phytylplastoquinone and proof that the compound which behaved as 2-demethylphytylplastoquinone was 2-demethylphytylquinone and not the 3-demethyl isomer that we had been unable to prepare (see Materials and Methods) was provided by the demonstrations: that the specific radioactivity of a mixture of 20 000 cpm [3H]plastoquinone-9 and 10 mg plastoquinone-9 remained unaltered through five recrystallizations from ethanol; and that mixtures of the 2-demethylphytyl-[3H]plastoquinone and 2-demethylphytylplastoquinone and of phytyl-[3H]plastoquinone and phytylplastoquinone could be cyclised by chemical means to form 5-[3H]tocopherol and 7-[3H]tocopherol,

641 respectively, with the same specific radioactivities as the starting mixtures and which gave coincident radioactive elution and mass-elution profiles On high performance liquid chromatography. More absolute p r o o f of the identities of 2-demethylplastoquinone-9, 2-demethylplastoquinone-8 and plastoquinone-8 was n o t obtained in this study. It seemed fairly certain, however, that the [3H]quinones are of the same substitution pattern as the marker quinones with which they are co-eluted on adsorptive high performance liquid chromatography. Indirect support for this belief was provided by the facts that the positional isomers of p h y t y l t o l u q u i n o n e and of d i m e t h y l p h y t y l b e n z o q u i n o n e are well separated from each other on adsorptive high performance liquid chromatography (Fig. 3). Analysis of the chloroform-soluble c o m p o u n d s from the incubations supplemented with polyprenyl pyrophosphates showed that they contained mainly 3H-labelled plastoquinone-9 and -8 and demethylplastoquinone-9 and -8 (Table I). The apparent absence of the quinols of the [3H]quinones isolated was due to the fact that no precautions were taken to prevent their oxidation to quinones in the course of the extraction procedure. The oxidations were allowed to proceed unhindered because the presence of quinols in the extracts interferes with the isolation and identification of the tocopherols (see Fig. 2). In the rest of this paper, it will be assumed for the sake of simplicity of presentation that the quinones are the biosynthetic products of the chloroplasts. The above results differed from those of Bickel, et al. [4], who reported that both class II and class I spinach chloroplasts (prepared from 6-week-old plants) were unable to synthesize 14C-labelled plastoquinone-9 and tocopherols from [~-14C]homogentisate, even though illuminated class II chloroplasts were able T A B L E II IN V I V O S Y N T H E S I S O F Q U I N O N E S A N D T O C O P H E R O L S BY L E T T U C E C H L O R O P L A S T S L e t t u c e leaves ( 2 3 g w e t w t . ) w e r e c o a t e d w i t h 2 m l o f 0 . 0 5 M pho~'phate b u f f e r , p H 7.0, c o n t a i n i n g 5 0 ~Ci o f [methylene-3H]homogentisate, a n d i n c u b a t e d o n m o i s t filter p a p e r f o r 4 h at r o o m t e m p e r a t u r e w i t h c o n t i n u o u s i l l u m i n a t i o n . T h e leaves w e r e t h e n h o m o g e n i z e d in 3 0 m M H e p e s / 0 . 5 M s u c r o s e b u f f e r , p H 7.6, a n d t h e r e s u l t a n t h o m o g e n a t e filtered a n d t h e n c e n t r i f u g e d at 5 0 0 X g f o r 9 0 s. ( T h e b u f f e r v o l u m e s u s e d , t i m e o f h o m o g e n i z a t i o n etc. w e r e i d e n t i c a l t o t h o s e u s e d f o r t h e p r e p a r a t i o n o f c h l o r o plasts f o r in v i t r o studies.) T h e 5 0 0 X g s u p e r n a t a n t w a s a d j u s t e d t o 1 0 0 m l w i t h b u f f e r a n d d i v i d e d i n t o a 25 m l f r a c t i o n ( h o m o g e n a t e ) , w h i c h w a s i m m e d i a t e l y a n a l y s e d (see b e l o w ) a n d a 75 m l f r a c t i o n w h i c h was c e n t r i f u g e d a t 1 5 0 0 × g f o r 5 rain. T h e h o m o g e n a t e f r a c t i o n , 1 5 0 0 X g c h l o r o p l a s t p e l l e t a n d 25 m l o f the 1500 X g s u p e r n a t a n t were analysed for chlorophyll c o n t e n t and 3H-labelled quinones and tocop h e r o l s . T h e q u i n o n e s a n d c h r o m a n o l s w e r e e x t r a c t e d w i t h a c e t o n e a n d p a r t i o n e d i n t o light p e t r o l e u m e t h e r ( b . p . 40°---60°C). T h e y w e r e t h a n s e p a r a t e d f r o m e a c h o t h e r b y t h i n - l a y e r c h r o m a t o g r a p h y a n d a n a l y s e d b y high p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y . T h e r e s u l t s f r o m t h e a n a l y s e s o f t h e t h r e e f r a c t i o n s w e r e u s e d t o c o n s t r u c t t h e f o l l o w i n g t a b l e . V a l u e s e x p r e s s e d as p e r 1 0 0 m l o f h o m o g e n a t e . Compound

Chloroplast (~g)

Chlorophyll Plastoquinone-9 Phytylplastoquinone 7-Tocopherol ~-Tocopherol

2840 * 72 9.7 25.7

Outside chloroplast ( 1 0 -3 X d p m )

168 111 13 51

* All c h l o r o p h y l l a s s u m e d t o be p r e s e n t in c h l o r o p l a s t .

(/.Lg)

0 43.7 8.3

(10 -3 X d p m )

0 101 320 16

642 to synthesize 14C-labelled plastoquinone-9 and tocopherols from ~4CO2, D-[1,614C]shikimate or DL-[fl-14C]tyrosine. The reason for the failure of these workers to obtain any incorporation of radioactivity fom homogentisate into plastoquinone-9 and the tocopherols is not easily explained. It was decided to use lettuce chloroplasts for the rest of this study, since of the chloroplasts tested they incorporated most radioactivity into chloroformsoluble quinones when incubated with [methylene-3H]homogentisate and isopentenyl pyrophosphate + S-adenosylmethionine (Table I). In vivo biosynthetic capabilities of lettuce chloroplasts. In a previous study, it was shown that lettuce leaves are able to incorporate radioactivity from [UJ4C]homogentisate into plastoquinone-9, a-tocopherol, T-tocopherol and a-tocopherolquinone [19]. The results of an experiment designed to measure the biosynthetic potential of isolated lettuce chloroplasts, indicated that in vivo the lettuce chloroplasts used in this study were capable of the synthesis of plastoquinone-9, phytylplastoquinone, a-tocopherol and 7-tocopherol and that there are additional sites in the cell for the synthesis of the phytylplastoquinone, a-tocopherol and 7-tocopherol present outside the chloroplasts (Table II).

Some properties of the lettuce chloroplast system Time course of quinone production. The amount of each [3H]quinone in the S-adenosylmethionine-deficient standard incubation mixture and of each [3H]dimethylquinone in the standard incubation mixture rose over the first 90 min of the incubation period and then declined (Fig. 4). In contrast, the amounts of [3H]demethylquinones in the latter mixture increased for only some 30 min before they declined (Fig. 4). The disappearance of the [3H]quinones was not accompanied by the appearance of new 3H-labelled compounds in the chloroform-soluble extracts of the incubation mixtures. Effect of Mg z÷, light and isopentenyl pyrophosphate. The synthesis of [3H]quinones from [methylene-3H]homogentisate by the unpurified chloroplast preparations is MgZ+-dependent and is stimulated by light (Table III). The addi-

30

60 [hi +SAM

/

SAM

Y

t ~o

o 20 b

ru 30 l

//!i '

"

o

o i

10

i

60 120 Time(min)

60 120 Time(rain)

Fig. 4. Time courses of [3H]quinone production by unpurified lettuce chloroplasts in a standard incubat i o n m i x t u r e -+ S - a d e n o s y l m e t h i o n i n e . The two time courses were determined on different days and with different preparations. Each point represents the analysis of one incubation mixture containing 0.4 mg chlorophyll, o o, 2-demethylphytylplastoquinone; ,~ -~ 2 - d e m e t h y l p l a s t o q u i n o n e s ; • •, phytylplastoquinone; • •, plastoquinones.

Isopentenyl pyrophosphate and } { S-adenosylmethionine Isopentenyl pyrophosphate Isopentenyl pyrophosphate S-Adenosylmethionine S-Adenosylmethionine None None

Omissions f r o m standard incubation mixture

5.5 26.1 38.4

40.2 8.8 43.9 S a m p l e lost 108.1 13.6 101.4 16.5

Chloroformsoluble compounds

7.2 2.5 65 5.8

63.4 4.2 2.7 0.6

2.2 5.9 9.8

11.3 2.3 39.5

22.2 5.9 0.9

0.6 2.3 1.3

Phytylplastoquinone

2-Demethylphytylplastoquinone

Radioactivity (10 -3 × d p m )

* 5--8% of the radioactivitywas associated with the octaprenyl homologue.

Osmotically shocked chloroplast + Isopentenyl pyrophosphate and S-adenosylmethionine + S*A d e n o s y l m e t h l o n i n e + None

+ + -+ -+ --

Presence or absence of illumination

E a c h i n c u b a t i o n m i x t u r e c o n t a i n e d 0.3 m g o f c h l o r o p h y l l . T h e i n c u b a t i o n p e r i o d w a s 6 0 rain.

E F F E C T O F L I G H T A N D O S M O T I C S H O C K ON T H E S Y N T H E S I S O F 3 H - L A B E L L E D Q U I N O N E S F R O M PURIFIED LETTUCE CHLOROPLASTS

TABLE III

2.2 4.2 0.5

18.4 2 0 0.2

0.5 0.2 0.2

2-Demethylplastoquinone-9 *

2 11.4 25.3

6.4 4.1 27.3 8.8

4.1 0.6 1.1

Plastoquinone-9 *

[METHYLENE-3H]HOMOGENTISATEBY

UN-

644 TABLE

IV

INCORPORATION OF RADIOACTIVITY [1-14C]ISOPENTENYL PYROPHOSPHATE PLASTS

FROM [METHYLENE-3H]HOMOGENTISATE A N D INTO QUINONES BY UNPURIFIED LETTUCE CHLORO-

T h e i n c u b a t i o n m i x t u r e c o n t a i n e d 0 . 4 m g c h l o r o p h y l l a n d h a d t h e s a m e c o m p o s i t i o n as t h e s t a n d a r d i n c u bation mixture, with the exception that the isopentenyl pyrophosphate w a s r e p l a c e d b y 2 . 5 ~zCi o f [ l_l 4 C ] i s o p e n t e n y l p y r o p h o s p h a t e ( 5 6 m C i - m m o F 1). Quin one

2-Demethylplastoquinone-9 Plastoquinone-9 2-Demethylphytylplastoquinone Phytylplastoquinone

R adioactivity

(10-3

× dp m )

3H

14 C

1,059 2.097 45.4 47.6

0,509 0,702 0 0

A t o m i c 3 H : 14 C r a t i o

1:8 1:5

tion of isopentenyl pyrophosphate, a precursor of the side chains of the quinones, leads to a marked increase in the amounts of polyprenyl quinones synthesized in the light and the dark and in the amounts of phytylquinones synthesized in the light (Table III). Chloroplasts incubated in the light with [methylene-3H]homogentisate and [1-14C]isopentenyl pyrophosphate synthesized [14C, 3H]plastoquinone-9 and [~4C, 3H]demethylplastoquinone-9 with ~4C : 3H ratios consistent with the added isopentenyl pyrophosphate acting as a major contributor of Cs units to their nonaprenyl side chains (Table IV). The absence of 14C from the phytylquinones indicated that the chloroplast must contain a relatively large endogenous pool of a C20 pyrophosphate (see below) which was not added to significantly by the [1-14C]isopentenyl pyrophosphate. The stimulatory effect of light on polyprenyl quinone synthesis is difficult to explain, since it seems to entail more than the provision of extra substrates for side-chain synthesis and regeneration of S-adenosylmethionine by photosynthetic activity. Thus light still has a marked stimulatory effect even in the presence of added isopentenyl pyrophosphate (Table III). The observations that the stimulation of p h y t y l q u i n o n e formation is light dependent (Table III), that radioactivity from [1-~4C]isopentenyl pyrophosphate was n o t incorporated into phytylquinones, and that geranylgeranyl quinones were not produced in any of the incubations performed in this study, suggests that the synthesis of phytylquinones requires p h y t y l p y r o p h o s p h a t e and that light is needed to provide the reducing equivalents for the formation of this c o m p o u n d from an endogenous pool of geranylgeranyl pyrophosphate present in isolated chloroplasts and from geranylgeranyl pyrophosphate newly synthesized from added isopentenyl pyrophosphate. Stability of preparations. If the preparation of the chloroplasts is protracted or if the chloroplasts are stored at 0°C before use then there is a progressive loss in the amounts of quinones synthesized (Fig. 5). In addition, the stimulatory effect of isopentenyl pyrophosphate is reduced in the cases of the polyprenylquinones and is completely lost in the cases of the phytylquinones (Fig. 5). Effect of osmotic shock. Incubation mixtures which contain chloroplasts that have been ruptured by osmotic shock are still able to carry o u t the synthe-

645 ~5C

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Time for which chtoroptast preporotior" wc~s held at O*C prior to incubotion(h) F i g . 5. E f f e c t o f s t o r a g e o n [ 3 H ] q u i n o n e p r o d u c t i o n b y u n p u r i f i e d l e t t u c e c h l o r o p l a s t s . (a) E a c h p o i n t represents the analysis of one incubation mixture from which S-adenosylmethionine and isopentenyl pyrophosphate h a d b e e n o m i t t e d . ( b ) A s ( a ) e x c e p t t h a t i s o p e n t e n y l p y r o p h o s p h a t e w a s i n c l u d e d in t h e incubation mixtures. Each incubation c o n t a i n e d 0 . 3 5 m g c h l o r o p h y l l . ~A polyprenylquinones © o, phytylquinones.

ses of both groups of quinones from homogentisate and isopentenyl pyrophosphate (Table III). However, the quantitative data from such incubations does n o t give rise to a consistent pattern. Thus, in some incubations the amounts of phytylquinones and of polyprenylquinones synthesized in ruptured chloroplasts compared to the controls remain the same whereas in others they either increase or decline. A further complication is that the changes in the amounts of the two groups of quinones are independent of each other. These disparities in the amounts of quinones synthesized b y ruptured chloroplasts may reflect physiological differences in the leaves. This suggestion is supported by the observation that the most marked reductions in p h y t y l q u i n o n e synthesis are only observed in chloroplasts of lettuces harvested in late summer. Effect of polyprenyl pyrophosphates. If the isopentenyl p y r o p h o s p h a t e is replaced by a source of preformed polyprenyl pyrophosphates then greater amounts of polyprenyl[3H]quinones are produced (Tables II, III and V). This synthesis, in agreement with previous work [7] is independent of light, and the amounts of quinones produced can be increased further b y exposure of the chloroplasts to osmotic shock (Tables III and V). This latter effect is probably a reflection of the inability of the added protein b o u n d polyprenyl pyrophosphates to pass through the envelopes of the intact chloroplasts. Effect of S-adenosylmethionine. The ratio of 2-demethylplastoquinones: plastoquinones and of 2-demethylphytylplastoquinones : phytylplastoquinone in the incubations unsupplemented with S-adenosylmethionine varied from preparation to preparation and presumably reflected the endogenous levels of this cofactor (cf. Tables I, III and V and Figs. 4 and 6). The addition of S-adenosylmethionine in the incubation mixtures usually had little effect on the a m o u n t of radioactivity incorporated into the t w o groups of quinones, although its presence did bring a b o u t a quantitative transfer of radioactivity from the 2-demethylplastoquinones to the plastoquinones and from 2-demethylphytylplast o q u i n o n e to phytylplastoquinone (Table I and III and Fig. 4). It was sometimes found that the presence of S-adenosylmethionine in the incubation mixture resulted in an increase in the a m o u n t of radioactivity incorporated into the

646 30

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60 9'0 Time (mln)

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60 90 Time (rain}

Fig. 6. S y n t h e s i s o f p h y t y l p l a s t o q u i n o n e (o --o) from 2-demethylphytylplastoquinone (e e ) or its q u i n o l a n d o f p l a s t o q u i n o n e - 9 ( a n d -S) (~ /~) f r o m 2 - d e m c t h y l p l a s t o q u i n o n c - 9 ( a n d -8) (A A) or its q u i n o l in u n p u r i f i e d l e t t u c e c h l o r o p l a s t s . 9 m l o f S - a d e n o s y l m e t h i o n i n e - d e f i c i e n t standard i n c u b a t i o n m i x t u r e ( 1 . 5 m g c h l o r o p h y l l ) w a s i n c u b a t e d for 6 0 m i n . A t t h e e n d o f this p e r i o d t h e i n c u b a t i o n m i x t u r e w a s r e s u s p e n d e d in 1 0 vols. s u s p e n s i o n b u f f e r a n d t h e c h l o r o p l a s t s i s o l a t e d b y c c n t r i f u g a t i o n at 1 5 0 0 X g f o r 1 0 rain. T h e c h l o r o p l a s t s w e r e t h e n r e s u s p e n d e d in 9 m l o f s u s p e n s i o n b u f f e r . O n e - t h i r d o f t h e c h l o r o p l a s t s u s p e n s i o n w a s a n a l y s e d for [ 3 H ] q u i n o n e s . T h e r e m a i n i n g t w o - t h i r d s w a s divided into two equal volumes which, after supplementation of one of the volumes with 1 pmol of S-adenosylmethionine, w e r e i n c u b a t e d for 3 0 m i n a n d t h e n a n a l y s e d . - . . . . . , c h a n g e s in r a d i o a c t i v i t y c o n t e n t o f q u i n o n e s in i n c u b a t i o n w h i c h w a s u n s u p p l e m e n t e d w i t h S - a d e n o s y l m e t h i o n i n e .

dimethylquinones that was well in excess of that lost from the demethylquinones (e.g. Table I). The addition of S-adenosylhomocysteine, a potent inhibitor of methyl transferases, caused the radioactivity to accumulate in the demethylquinones. Confirmation of the implied precursor-product relationship between the 2-demethylplastoquinones and plastoquinones (or, as is more likely, 2-demethylplastoquinols and plastoquinols) and between 2-demethylphytylplastoquinone and phytylplastoquinone (or their corresponding quinols) was established with the demonstration that when chloroplasts that had been allowed to accumulate [3H]quinones were incubated with S-adenosylmethionine there was a transfer of radioactivity from demethylquinones (or demethylquinols) to dimethylquinones (or dimethylquinols) (Fig. 6). The radioactivity lost from the demethylplastoquinones was entirely accounted for by the increase in the amount of 3H associated with the plastoquinones, whereas only 58% of the 3H lost from 2-demethylphytylplastoquinone was accounted for by the increase in the amount of 3H associated with phytylplastoquinone (Fig. 6).

Properties of class I lettuce chloroplasts Class I chloroplasts (i.e. chloroplasts essentially free from contamination by other cellular material) had the same biosynthetic capabilities as the chloroplast preparation used in the rest of this study (Table V). The only real difference between t h e two types of preparation was that the stimulatory effect of isopentenyl pyrophosphate on the synthesis of the quinones, particularly the polyprenyl quinones, was much reduced in the incubations which contained purified chloroplasts (Table V). This reduction was not unexpected in view of the longer period of time and additional manipulations needed to prepare class I chloroplasts (Table V cf. Fig. 5).

Class I Class I

Class I

60 60

Experiment 2 60

/

( | ~ | \

Isopentenyl pyrophosphate and S-adenosylmethionine replaced by M. luteus p r e p a r a t i o n

[ Isopentenyl pyro~ phosphate and [ S-adenosylmethionine S-adenosylmethionine S-adenosylmethionine Isopentenyl pyrophosphate and S-adenosylmethionine S-adenosylmethionine None

Omissions from standard incubation mixture

736.4

14.1

19.8

5.3 15.6 1.3

12.2 25.6 23.1 214.5

3.6 53.1 24.1

3.3

2-Demethylphytylplastoquinone

10.9 92.2 40.5

13.9

Chloroformsoluble compounds

R a d i o a c t i v i t y ( 1 0 -3 X d p m )

7

21.7

5.3 6.3 18.1

5.8 10.3 7

8.9

Phytylplastoquinone

444.7

80.5

0.2 0.9 0.5

0.3 13 2.5

0.3

2-Demethylplastoquinone-9 *

177

61.9

0.5 1.6 1.1

0.5 10.8 4.5

0.5

Plastoquinone-9 *

* S t a n d a r d i n c u b a t i o n s : 5 - - 8 % o f r a d i o a c t i v i t y a s s o c i a t e d w i t h t h e o c t a p r e n y l h o m o l o g u e . M. luteus s y s t e m s : 1 0 - - 1 5 % o f r a d i o a c t i v i t y a s s o c i a t e d w i t h t h e o c t a prenyl homologue.

Osmotically shocked Class I

Unpurified Unpurified Unpurified Class I

60 0 60 6O

60

Unpurified

Type of preparation

Expe~ment 1 O

Time elapsed from preparation of unpurified chloroplasts to start of incubation (rain)

I n e x p e r i m e n t 1, t h e i n c u b a t i o n m i x t u r e s w e r e s c a l e d d o w n b y o n e - t h i r d w i t h r e s p e c t t o all c o m p o n e n t s , a p a r t f r o m t h e r a d i o - s u b s t r a t e w h i c h w a s a d d e d i n t h e s a m e a m o u n t as in t h e s t a n d a r d i n c u b a t i o n m i x t u r e . E a c h o f t h e i n c u b a t i o n s in e x p e 1 ~ n e n t 1, c o n t a i n e d 0 . 1 5 m g o f c h l o r o p h y l l w h i l s t t h o s e in e x p e r i m e n t 2, c o n tained 0.32 mg of chlorophyll. The incubation period was 60 min.

SYNTHESIS OF 3H-LABELLED QUINONES FROM [METHYLENE-3H]HOMOGENTISATE BY CLASS I LETTUCE CHLOROPLASTS

TABLE V

648

Conclusions When the results of the above experiments are considered in conjunction with those obtained in previous studies, they are consistent with the proposals that the plastoquinone-9 and the small amounts of phytylplastoquinone found in the chloroplasts of higher plants are synthesized from homogentisate by the following pathways: homogentisate + nonaprenyl pyrophosphate -* 2-demethylplastoquinol-9 -* plastoquinol-9 -~ plastoquinone-9; homogentisate + phytyl pyrophosphate -~ 2-demethylphytylplastoquinol -~ phytylplastoquinol -~ phytylplastoquinone. They do not, however, provide any direct evidence as to the identities of the intermediates involved in the biosynthesis of tocopherols from homogentisate. Indeed, they cast some d o u b t over the recent reports that in spinach chloroplasts the synthesis of a-tocopherol from homogentisate involves the intermediacy of 2-demethylphytylplastoquinol and phytylplastoquinol (Fig. 1) [20--22].

Acknowledgements This work was supported by a grant from the Science Research Council. We thank Professor O. Isler (Hoffmann-La Roche and Company) and Dr. J.F. Pennock (University of Liverpool) for generous gifts of quinones and tocopherols, Professor G. Schultz (University of H a n n o v e r ) f o r making his manuscripts available to us prior to publication and Mrs. Susan Swetez for expert technical assistance.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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