The taxonomic distribution of plastoquinone and tocopherolquinone and their intracellular distribution in leaves of Vicia faba L.

BIOCH1MICA ET BIOPHYSICA ACTA

BBA

19

45261

T H E TAXONOMIC D I S T R I B U T I O N OF P L A S T O Q U I N O N E AND T O C O P H E R O L Q U I N O N E AND T H E I R I N T R A C E L L U L A R D I S T R I B U T I O N IN LEAVES OF VICIA FABA L. C. BUCKE, R A C H E L M. LEECH, MARY H A L L A W A Y AND R. A. MORTON

Department of Biochemistry, University of Liverpool, Liverpool, and Department of Botany, Imperial College, London (Great Britain) (Received May 6th, 1965)

SUMMARY

I. ~-Tocopherolquinone and plastoquinone- 9 were detected in photosynthetic tissues from algae, bryophytes and angiosperms; the molar ratio of ~-TQ to PQ-9 varied from 0. 4 to 2. 2. The levels of TQ, PQ and chlorophyll in different subcellular fractions from young leaves of Vicia fabc~ L. were estimated. The fractions investigated included the material sedimented at 600 × g, IOOO × g, ioooo × g and the ioooo × g supernatant; chloroplasts free from contamination but without their outer m e m b r a n e ; structurally undamaged but slightly contaminated chloroplasts; and lamellar and stroma preparations from uncontaminated chloroplasts. 3. From the examination of the ratio of PQ and TQ to chlorophyll and to each other in the different fractions of V. faba leaves it was concluded that both TQ and PQ were localized exclusively in the chloroplasts and that not less than 95 % of both was in the lamellae. 4- Up to 6o % of the total TQ of young leaves of V. faba could be extracted in a reduced form, probably tocopherolquinol, which was readily converted to TQ b y mild oxidation of the extract. 5- It was concluded that the concentration, widespread occurrence and intracellular distribution of TQ indicate that, like PQ-9, it m a y be a component of the photosynthetic process in m a n y types of plant.

INTRODUCTION

Information on the occurrence and intracellular distribution of quinone derivatives is of interest in connexion with their postulated role as intermediates in electron transport associated with both photosynthesis and respiration l-a. The group includes the naphthoquinones, ubiquinones, plastoquinones and tocopherolquinones, and one or more members of each type have been detected in leaves ~ 10. In plants the naphthoquinones4, ~ and tocopherolquinones 1° have been found only in green tissues, Abbreviations: PQ-9, plastoquinone45; P Q - I I , plastoquinon%s; PQ-4, plastoquinone20; TQ, tocopherolquinone; TQH2, tocopherolquinol.

Biochim. Biophys. Acta, 112 (1966) 19-34

20

c. BUCKE et al,

and the level of PQ is much higher in green than in non-green tissues; however, there is less ubiquinone in leaves than in non-photosynthetic tissueG,L PQ, which was detected and characterized b y KOFLER et a/. s,ll accompanies the chloroplast fraction after differential centrifugation of cell-free preparations of leaves 12. If the chloroplast lipids are partially removed by extracting isolated chloroplasts with organic solvents, both the Hill reaction and photophosphorylation are inhibited; but both activities are restored by adding back purified PQ-9 (refs. 1 3 - 1 5 ) . It therefore seems likely that PQ is localized in the chloroplasts and is an intermediate in the photosynthetic process 1~. In addition to PQ-9 other plastoquinones are present in much smaller amounts in extracts from leaves. PQ-A, PQ-B, PQ-C and PQ-D have been distinguished by thin-layer chromatography and ultraviolet spectrophotometry 17 19. PQ-A is PQ-9, PQ-B is almost certainly a higher isoprenologue (perhaps P Q - I I ) , and PQ-C and PQ-D m a y be dimeric and trimeric forms of PQ-9. ECK AND TREBST2~ found in addition to PQ-9 a PQ-X difficult to distinguish from PQ-B ; they also found a lower isoprenologue (apparently PQ-4) and its dimeric form together with a dimeric form of PQ-9 akin to PQ-C. c~-TQ was first detected in chloroplast preparations by LICHTENTHALER AND PARK1° and b y DILLEY AND CRANE 21 and was characterized by BUCKE et al. z2. ]~-TQ, v-TQ and 3-TQ have also been identified in leaf preparations21, 2a. The effects of extracting and then replacing chloroplast lipids on the Hill reaction and on photophosphorylation catalysed by isolated chloroplasts, suggest that the tocopherolquinones m a y also be intermediates in photosynthetic electron transfer 2a-za. Although LESTER AND CRANE6 and EGGER26 have determined the occurrence of PQ in a variety of plants, nothing is known of the occurrence of TQ in different taxa. A study has therefore been made of the incidence of TQ in a number of plants including algae, liverworts, mosses and angiosperms; the PQ was also estimated at the same time. The quantitative distribution of the plastoquinones and tocopherolquinones among the subcellular organelles is unknown, and their location within the chloroplast has been little studied. Since this information is necessary in considering their function we have investigated the subcellular distribution of PQ and TQ in leaves of Viciafaba L. and have compared the molar ratio of TQ and of PQ to chlorophyll in whole leaves with the ratio in chloroplasts free from contamination. We have also examined the distribution of the quinones within purified chloroplasts. MATERIALS

Diethyl ether and light petroleum (b.p. 4o-6o °) were left to stand over Na wire and were decanted before distillation not more than 36 h before use. Spectroscopically pure ethanol was prepared b y refluxing absolute ethanol with Zn (IO g/l) and K O H (2o g/l) for 6 h and then collecting the ethanol by distillation. Activated alumina (Grade "O") was obtained from P. Spence and Co., Widnes ; silica gel G from E. Merck, A.G., Darmstadt ; and N a B H 4 from L. Light and Co., Ltd., Colnbrook. c~-TQ was prepared by oxidizing a-tocopherol (bought from Roche Products, Ltd., Welwyn Garden City) with AgNOa (ref. 27) and PQ-9 was isolated from the green tissues of various plants, chiefly holly (Ilex aquifolium L.), bracken (Pteridium Biochim. Bioph.ys. Acta, 112 (1966) 19-34

D I S T R I B U T I O N OF

PQ AND TQ

IN PLANTS

21

aquilinum L.) and beans (Phaseolus vulgctris), b y chromatographing acetone extracts of the tissues on alumina. Seeds of V. faba var. "The Sutton" were obtained from Sutton's Seeds, Reading. In a few experiments another variety "Masterpiece" was used. The plants were grown either at Imperial College under the same conditions as those employed before 2~ or at Liverpool out of doors during the summer. Leaves were harvested 14-28 days after planting and only leaves with two leaflets were used. Cultures of Anabaena variabilis (Ktitzing) and Chlorogloeafritschii (Mitra) were kindly supplied by Dr. N. G. CARR (Department of Biochemistry, Liverpool). Both organisms were grown at 32-34 ° on Medium C (ref. 29) supplemented with 0.05 % (w/v) NaHCOa, gassed with a mixture of air and COz (95:5, v/v), and illuminated b y 6o-W daylight strip lights at 9 inch distance. Other plant tissues were collected in the field (see Table III).

METHODS

Extraction of plastoquinones and tocopherolquinones The quinones were extracted from whole leaves and other intact tissues by macerating them thoroughly with acetone in the "Liquidizer" attachment of the Kenwood "Chef". The quinones were extracted from subcellular fractions b y stirring them with excess acetone. The acetone extracts were cleared b y centrifugation or filtration, diluted with water and then extracted with a large volume of diethyl ether and light petroleum (b.p. 40-60 °; 5 ° : 5o, v/v), using a gentle swirling motion rather than vigorous shaking. Only one extraction was usually necessary to remove all the chlorophyll into the epiphase. The e t h e r - p e t r o l e u m extracts were dehydrated by standing over anhydrous NaeSO 4 for not less than 15 rain and were then taken to dryness in a rotary evaporator. The residues were stored at 5 ° in a vacuum desiccator overnight pending chromatography.

Column chromatography The residues, dissolved in light petroleum, were chromatographed on columns of acid-washed alumina deactivated with water to Brockmann grade I I I . The lipids were eluted with increasingly polar mixtures of diethyl ether and light petroleum, usually 0% (v/v), 2 %, 6 %, 8%, I 2 % , I 5 % , 25%, lOO% ether in light petroleum. The eluates were evaporated to small volume on a water bath and dried in a current of N 2. The residues were dissolved in spectroscopically pure ethanol, and their absorption spectra over the range 19o to 400 m/, determined by means of a Unicam SP 800 recording spectrophotometer. The recoveries of purified PQ-9 and c~-TQ from alumina columns were 95.4 % =k 1.4 and lO2 % ~ 5.1, respectively.

Estimation of quinones and chlorophyll Plastoquinones and tocopherolquinones were assayed in ethanolic solution by measuring the decrease in absorbance at 254 mtz and 262 mtz respectively caused b y % employed reducing the quinone to the quinol with N a B H 4. The values for AE It om were 200 for PQ (ref. 30) and 400 for TQ (ref. 31). Total chlorophyll was determined b y the method of ARNONs2. Biochim. Biophys. Acta, 112 (1966) 1 9 - 3 4

22

C. BUCKE et al.

Estimatio~ of protein Samples of chloroplast preparations for protein determination were pipetted into cold trichloroacetic acid (final concentration 6 % (w/v) trichloroacetic acid) allowed to stand for Io rain and centrifuged. The sediment was washed once in 5 % (w/v) trichloroaeetic acid, then twice in ethanol-chloroform (3:~, v/v) and the final pale cream precipitate taken up in I N NaOH and stored in the deep-freeze. Protein was determined according to LOWRY et at. aa in samples containing 0-25 ° /~g protein in o.6 ml, using soluble casein (Hopkins and Williams) for the standard. The optimum dilution of Folin reagent was determined by the method of OYAMA AND EAGLE3~.

Thin-layer chromatography The eluates from the alumina columns were evaporated to dryness and then dissolved in sufficient benzene to give a concentration of about I ~g quinone per/~1. The compounds eluted by 2 % and 6 % ether in light petroleum were chromatographed on silica gel using 20 % (v/v) heptane in benzene as developing solvent. The compounds eluted by 15 % and 25 % ether in light petroleum were chromatographed on silica gel and developed with I5 % (v/v) ethyl acetate in benzene. After development the plates were sprayed with 0.2 % (w/v) fluoreseein in ethanol and the areas which absorbed ultraviolet light were marked. To identify tile quinones, the plates were sprayed first with o.I % (w/v) NaBH4 in ethanol to reduce the quinones to quinols. This was followed by 1% aqueous HC1, and finally with E m m e r i e - E n g e l reagent to detect the reducing compounds. The major quinone eluted by 2 % and 6 % ether in light petroleum was PQ-9 which accounted for over 95 % of the quinone content of the combined fractions. The / 15 O/o and 25 ,% ether in light petroleum eluted a more complex mixture of quinones. The predominant component was a-TQ (which accounted for usually over 9° % and always over 8o % of the total quinone content) but traces of/3-TQ and y-TQ and of PQ-C or PQ-D were occasionally present. The composition of the mixture varied with the age of tile leaves and with the time of the year, and a study of the changes in composition is being carried out. The tocophcrolquinones were estimated together by measuring the decrease in absorbance at 262 m/, on reduction with NaBH4, and correcting for the decrease in absorbance attributable to PQ-C in the following way: For a highly purified sample of ~-TQ, dA~54m/,/alA262m# ~ o.73 and for PQ-C, AA2a4mu/AA2a2m/~ = I. For any mixture of c~-TQ and PQ-C, AA2a2ml~- AA2a4ml~, 0.27 AA (TQ).

Possibili@ lhat the tocopherolquinones may be artifacts Tocopherols have been detected in leaves and since each can be oxidized to the corresponding quinone the TQ detected might be an artifact of the extraction procedure. However, this explanation of their presence is not easily tenable for two reasons. Firstly, the toeopberol content of young leaves is low and in fact we have been unable to detect any tocopherols in the bean leaves of I4-28 days used in these experiments. In leaves which have been darkened for 63 h a trace of ~-tocopherol was detectable, which is consistent with BOOTH'Sobservation 35that the tocopherol content of green tissues increased in the dark. Secondly, when samples of c~-tocopherol were subjected to the standard extraction and chromatography procedures in the presence

Biochim. Biophys. Acla, 112 (i966) I9-34

DISTRIBUTION OF PQ A~,'D TQ IN PLANTS

23

or absence of leaf tissue, 98.5 % was recovered as c~-tocopherol and no increase in a-TQ was detectable.

Isolation of "chloroplast" fractions IOOO x g fraction. A leaf brei was prepared by grinding batches of IO g of leaves in a pestle and mortar with 20 ml 67 mM Na2HPO4-KH~PO 4 buffer (pH 7.3) containing sucrose (0.3 M). The brei was filtered through a single layer of White Permanent organdie and the filtrate centrifuged at 200 x g for 2 rain. The supernatant from this spin was centrifuged at IOOO x g for 12 min, the pellet resuspended and termed "IOOO x g fraction". Stripped chloroplasts. These were prepared from a IOOO x g fraction b y the method of JAMES AND DAS36. This procedure involves the centrifugation of a IOOO × g fraction through a discontinuous gradient made up of layers ot 60 % and 25 % (w/v) glycerol in buffered sucrose (0.3 M). After gradient centrifugation the deep-green band at the junction of the two glycerol layers was removed, diluted with an equal volume of phosphate buffer, spun down at IOOO × g for 12 min and the resuspended pellet termed "stripped" chloroplasts. Intact chloroplasts. These were prepared by the method of LEECI-I~ using a sucrose density gradient.

Fractionation of the chloroplasts Either sonic or osmotic rupture was used for the preparation of lalnellae and stroma fractions from stripped chloroplasts and from intact chloroplasts. Before fractionation chloroplasts were suspended in 67 mM phosphate buffer (pH 7.3)Sonic rupture. The procedure described by JA~ES AND LEECH3s was employed. An MSE-Mullard ultrasonic disintegrator was used with a cooled titanium probe vibrating with a frequency of 20 kcycles/sec. Osmotic rupture. The chloroplasts were kept in melting ice and stirred continuously until microscopic examination showed that all the chloroplasts were completely ruptured. After sonic or osmotic rupture the chloroplast material was centrifuged at 105000 x g for 13 rain. The resuspended green sediment from this operation was termed "lamella fraction" and the supernatant "stroma fraction".

The appearance of the chloroplast preparations in the electron microscope zooo ~ g fraction. This fraction, prepared in buffered 0.3 M sucrose, consists mainly of chloroplasts stripped of their bounding membranes, chloroplast fragments and a few intact chloroplasts. In addition there is considerable contamination with other cellular constituents: in particular m a n y profiles of structurally well preserved mitochondria are present, in numbers about equal to the total (broken plus intact) profiles of chloroplasts. Unidentifiable cytoplasmic debris, cell-wall material, whole nuclei and nuclear fragments are also present. Stripped chloroplasts. The appearance of this kind of preparation has been illustrated elsewhere (ref. 38, Fig. 5). Mitochondria and other cytoplasmic constituents are completely absent from these preparations, but all the chloroplasts have lost their bounding envelopes and a considerable portion of their stroma. The fretwork of the lamella system remains intact. Biochim. Biophys. Acla, 112 (1966) I9--34

24

c. BUCKE el al.

Intact chloroplasts. The appearance of these chloroplasts has been illustrated earlier (ref. 37, Plate A). 9 ° % of the profiles are of chloroplasts with completely i n t a c t b o u n d i n g envelopes, 5 % of chloroplast fragments a n d 5 % of mitochondria. These observations are s u m m a r i z e d in Table I. After comparing the light- a n d electron-microscope images of the same prepar a t i o n it is possible from light-microscope observations alone to assess to some e x t e n t the degree of preservation of chloroplast structure a n d the degree of c o n t a m i n a t i o n of the preparations. All the p r e p a r a t i o n s were e x a m i n e d in the light microscope a n d o n l y those which h a d the appearance expected were used for q u i n o n e d e t e r m i n a t i o n . TABLE I CHARACTERISTICS

OF

Preparation

SUSPENSIONS

Contamination

OF

ISOLATED

CHLOROPLASTS

State of the chloroplasts Envelope

Stroma

Thylahoids

Considerable loss in ahnost all Considerable loss

No swelling

Loss in only io %

iooo × g fraction

Considerable

Stripped

None

Lost from more than 90 % Lost

Intact

Very slight

Intact

Lanlellar systems intact Unchanged

TABLE II TOTAL

PROTEIN

IN

CHLOROPLASTS

PREPARED

BY

DIFFERENT

METHODS

Preparative techniques described in the METHODSsection. The data are given as itg protein per t*g total chlorophyll. Expt.

IOOO X g fraction

Stripped Intact chloroplast chloroplasts

i 2 3 4 5

1.895 2.51 2.23 2.62 2.04

0.74 1.o4 1-13 ---

--3-18 3.66 3.2i

Mean Variance of mean

2.26

0.97

3.35

0.25

o.15

0.20

Protein content of the chloroplast preparations The protein c o n t e n t of each of the three types of chloroplast p r e p a r a t i o n is a n a d d i t i o n a l measure of their relative integrity, since stroraa protein is easily lost from d a m a g e d chloroplasts ag. I n Table I I the protein level relative to chlorophyll in five IOOO X g fractions is listed together with t h a t of either the stripped or the i n t a c t chloroplasts isolated from the same b a t c h of leaves; in E x p t . 3 b o t h stripped a n d i n t a c t chloroplasts were isolated from the same cell-free preparation. The i n t a c t chloroplasts c o n t a i n a b o u t three times more protein t h a n stripped chloroplasts a n d a b o u t 5 ° % more t h a n the I000 ~< g preparation, despite the fact t h a t the I000 >: g p r e p a r a t i o n is heavily c o n t a m i n a t e d with mitochondria. Biochim. Biophys. Acta, 112 (1966) 19-34

DISTRIBUTION- OF P Q

IN PLANTS

AND T Q

o

ooo

o

o

25

ooo

o

o

o + o

+ + +

o

o

o

~

6

5

~

+ + o +

o

6~6~ o

o

o

o

+ + + + + + +

o,,

5~666

5 5 5 5 6 ~

eo t.,i

~

to

555

o

6

~; I 5

o ~ 55

•~ ~ ~

~

=

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~-~

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®

~ 5 5 5

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0

0

= ' ~0 ~

p~

~ 5 ~

0

0

~ 0

~ 0

~ ;>

to

~

o--

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"~ •

o~

-.~ ~

~

+

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~ ~

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~-~

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0~-~

o

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'~

k

o~

.<

Biochim. Biophys. Acta, 112 (1966) 19_34

26

c. BUCKE et al.

RESULTS

The levels of TQ and PQ in photosynthetic tissues from different types of plant Both PQ and TQ were present in the photosynthetic tissues of every plant examined. The molar ratios of TQ to PQ-9 were between 0. 5 and 0.8 in green tissues from the angiosperms and rather higher in the green and blue-green algae, in the brown algae the ratio was nearer 2 and in the red algae it was close to 0. 5 (Table I I I ) . The leaves from which stripped and intact chloroplasts were isolated were always placed in the dark for 48 h before extraction in order to deplete the chloroplasts of starch, and it was possible that the extended dark period might modify the quinone levels. We therefore estimated the quinone content of leaves from plants kept in natural light and dark regime and from plants of the same sowing but which were darkened for 63 h before extraction (Table IV). The levels of both TQ and PQ were lower after the dark period but the decrease was not very striking (I 4 % and 20 % respectively) and the molar ratio of TQ to PQ remained near I.

The intracellular distribution of TQ and PQ in leaves of V. faba The evidence available supports the view that much of the TQ and PQ of the cell is localized within the chloroplast and probably in the lamellae (see INTRODUCTIOX). Assuming that, chlorophyll in vivo is restricted entirely to the chloroplast lamellae, and that the fractionation procedures release the quinones and chlorophyll from the lamellae to an identical degree, then, if TQ and PQ are present only in the lamellae, the ratio of TQ or PQ to chlorophyll in different subcellular fractions should be constant. Fig. I shows the distribution of TQ and PQ relative to chlorophyll and the ratio of TQ to PQ in four fractions obtained b y differential centrifugation of a cell-free preparation of bean leaves; the absolute amounts of TQ, PQ and chlorophyll in the fractions and the recoveries are listed in Table V. Although over 6o % of the quinones and over 5 ° % of the chlorophyll were recovered in the iooo x g sediment, TQ, PQ and chlorophyll were present in all fractions and the molar ratios of each of the quinones to chlorophyll and to each other varied considerably from one fraction to another. In addition the amount of TQ (but not of PQ) extracted from the homogenate (A) was less than that extracted b y macerating whole leaves (cf. Table V and Table I I I , bottom line), so that the value of T Q : P Q in the homogenate was only o.44 rather than about I. The high recovery of TQ (Table V) suggested an explanation for these variations. In vivo both TQ and T Q H 2 might be presenO °, and TABLE

IV

COMPARISON

OF THE

RATIO

OF QUINONE

TO CHLOROPHYLL

IN EXTRACTS

OF TOTAL

LIPIDS

FROM V. faba

Pretreatment

Total chlorophyll (i, moles)

TQ : chlorophyll PQ : chlorophyll (l, moles/mmole chlorophyll)

Molar ratio TQ : PQ

A* B *~

27.1 28-o5

36 31

o,9o o.97

4° 32

* From plants kept under natural diurnal light and dark regime. ** F r o m p l a n t s k e p t for 63 h i n t h e d a r k b e f o r e e x t r a c t i o n .

Biochim. Biophys. Acta, 112 (1966) 19.-34

OF LEAVES

DISTRIBUTION OF

PQ AND TQ

IN PLANTS

27

the quinol might be more readily oxidized to the quinone form during the vigorous maceration of the whole leaves than during the much gentler extraction of the leaf brei; oxidation of the quinol to quinone might also occur during further fractionation f.O

$6"" --'~ 110

E

1oo

~

1.o

/,,

~60

/;, ,

c° 20

o n.-

Q2

o TQ PQ A

// TQ PQ B

TQ PQ C

TQ t::'Q TQ PQ D E

o A

B

C

D

E

Fig. I. H i s t o g r a m s of (a) the ratio of T Q and P Q to chlorophyll (#moles q u i n o n e per mmole chlorophyll), and (b) the molar ratio of TQ to PQ, in four subcellular fractions of bean leaves. 5 ° g leaves were g r o u n d in 67 mM p h o s p h a t e buffer (pH 7.3) containing o. 3 M sucrose and the suspension strained to provide H o m o g e n a t e A. This h o m o g e n a t e was separated b y differential centrifugation into Fractions B - E . F r a c t i o n B sedimented in 2 rain at 6oo × g; F r a c t i o n C sedimented in IO min at iooo × g; F r a c t i o n D sedimented in 3 ° min at i o o o o × g; and F r a c t i o n E is the s u p e r n a t a n t from the i o o o o × g centrifugation. P r e p a r a t i o n s A - E were analysed for TQ, P Q and total chlorophyll.

b or'( J 120ia

1.2~1.1 b

100!

1.0

so

0.6

I

o

8 o

40

5o"

_

L

-

-

y

Q5

0.4 0.3

20

_

L__.

0.2 0.1

~o OTQ PQ TQ PQ TQ PQ TQ PQ 0 A B+C D E A rr

B*C

D

E

Fig. 2. H i s t o g r a m s of (a) the ratio of T Q and P Q to chlorophyll (ffmoles quinone per mmole chlorophyll) and (b) the molar ratio of T Q to P Q in three subcellular fractions of bean leaves, after oxidation of endogenous T Q H 2 to TQ. 75 g leaves were g r o u n d in 67 mM p h o s p h a t e buffer (pH 7.3) containing o. 3 ?4 sucrose and the suspension strained to provide H o m o g e n a t e A. This h o m o g e n a t e was separated b y differential centrifugation into Fractions B - E . Fractions B plus C sedimented in io min at IOOO × g; F r a c t i o n D sedimented in 30 min at IOOOO × g; a n d F r a c t i o n E is the s u p e r n a t a n t from the IOOOO × g centrifugation. P r e p a r a t i o n s A-t?; were analysed for TQ, P Q and chlorophyll after aerating the acetone e x t r a c t of the fractions to c o n v e r t the T Q H 2 to TQ.

Biochim. Biophys. Acla, 112 (i966) 19-34

28

C. BUCKE et al.

of the brei. Since only the q u i n o n e form is estimated, different proportions of T Q a n d T Q H ~ in the various fractions could account for the observed v a r i a t i o n s in T Q : P Q in F r a c t i o n s B - E (Fig. z) a n d for the difference between the T Q c o n t e n t of F r a c t i o n A a n d the extract of whole leaves. This was tested b y dividing the acetone e x t r a c t of a Iooo × g p r e p a r a t i o n in half a n d a e r a t i n g one sample for I5 m i n at room t e m p e r a t u r e before c h r o m a t o g r a p h i n g b o t h samples as usual. The results are shown in Table VI ; aeration h a d no effect on the level of P Q b u t increased t h a t of T Q b y n e a r l y 4 ° %. I n addition when the acetone extracts of Fractions A - E prepared from a n o t h e r sample of leaves were aerated before e s t i m a t i o n of the quinones, the a m o u n t of T Q increased to a level slightly above t h a t of P Q to give an almost c o n s t a n t value of T Q : P Q in all fractions (Fig. 2 a n d Table VII). We conclude from the c o n s t a n c y of the T Q : P Q ratio t h a t PQ, T Q a n d a reduced form of T Q (plausibly TQH2) occur together in the cell a n d t h a t , in V. f a b a leaves of the age used, the molar c o n c e n t r a t i o n of P Q is a p p r o x i m a t e l y equal to t h a t of T Q plus T Q H 2. The m a i n intracellular site for P Q a n d T Q plus T Q H 2 is in association with the chlorophyll, in the lamellae. However, the v a r i a t i o n in the ratios of P Q a n d TQ to chlorophyll in different subcellular fractions, even after mild oxidation, suggests t h a t a small a m o u n t of the quinones m a y be present elsewhere, either within the chloroplast or outside it. The location of this small fraction of P Q a n d T Q was n e x t investigated. TABLE V DISTRIBUTION

OF

CHLOROPHYLL,

TQ

AND

PQ

IN

FOUR

SUBCELLULAR

FRACTIONS

FROM

~'.faba

LEAVES

The preparation of the fractions is described in the legend to Fig. I.

A B C D E

Fraction

Chlorophyll (l~moles)

JPQ (pmoles)

TQ (l~moles)

Molar ratio TQ :PQ

Total homogenate 600 × g sediment Iooo × g sediment ioooo × g sediment Supernatant

53.4 5.95 28.8 i8.8 2.5°

1.83 o. I5 o.88 o.42 o..z2

o.8i 0.07 o.7i o.28 o. 23

0.44 0.48 o.So o.66 i. o3

Io4.I %

92 °/o

I59 ~o~

I~ecovery

* High recovery of TQ is discussed in the text. TABLE VI EFFECT

OF A E R A T I O N

ON

THE

LEVELS

OF

PQ

AND

TQ

IN

AN

ACETONE

EXTRACT

OF

I000

X g

PREPARATION

The iooo × g fraction from 25 g V, faba leaves was extracted with acetone. Half of the extract was aerated for 15 min, then TQ, PQ, and chlorophyll were estimated in the aerated and nonaerated solutions. Sample

PQ :chlorophyll TQ :chlorophyll (#moles/mmole chlorophyll)

Molar ratio TQ: PQ

Non-aerated (o.87/*mole chlorophyll per nil) Aerated (o.87/*mole chlorophyll per ml)

27 27

o.75 1.o4

Biochirn. Biophys. Acta, I12 (1966) i9-34

2o. 4 28

DISTRIBUTION

OF

PQ

AND

TQ

I~" P L A N T S

29

If the quinone to chlorophyll ratio in a IOOO x g fraction which contains mitochondria as well as whole and broken chloroplasts were significantly higher than in a preparation of intact chloroplasts, this could indicate an extrachloroplast site for the quinones in mitochondria. Similarly, if the quinone to chlorophyll ratio were higher in the 600 × g fraction (which contains cell walls and nuclear material in addition to chloroplasts) than in intact chloroplasts, this would suggest other possible extrachloroplast sites. However, in both IOOO x g preparations and 600 × g preparations the ratio of PQ:chlorophyll was no higher than in the intact chloroplasts (Table VIII) (the ratios of T Q : P Q and of TQ:chlorophyll varied, due almost certainly to differences in the relative amounts of TQ and T Q H 2 since, if the acetone extracts were oxidized before assay, the value of T Q : P Q approached I). There is thus no evidence for a non-chloroplast particulate location for PQ and TQ. Moreover, since the level of PQ and therefore of TQ plus T Q H 2 relative to chlorophyll in the intact chloroplasts is of the same order as in the whole leaf any major extrachloroplast location for the quinones seems to be exchlded. TABLE

VII

DISTRIBUTION OF CHLOROPHYLL,

TQ

AND

PQ

IN THREE

SUBCELLULAR FRACTIONS FROM V. faba

LEAVES The preparation of the fractions is described in the legend to Fig. 2. The subjected to mild oxidation.

A B + C D E

extracted

lipids were

Fraction

Chlorophyll (ffmoles)

PQ (ffmoles)

TQ (ffmoles)

Molar ratio TQ : JPQ

Total homogenate iooo × g sediment ioooo × g sediment ioooo × g supernatant

28. i o 17.9o 9.41 2.28

1.365 o.79o 0.308 O.lO9

1.588 o.835 o.343 o.116

i. 16 1.o6 i.ii 1.o6

Recovery

lO 5 %

89 %

82 %

The location of TQ ansl PQ within the chloroplast The evidence presented in the previous section supports the hypothesis that in young bean leaves TQ and PQ are found only in the chloroplasts and t h a t most of the T Q and PQ is closely associated with the chlorophyll, i.e. present in the lamellae. I t cannot be stated that the quinones are localized exclusively in the lamellae since, after differential centrifugation both are present in the ioooo x g supernatant as well as in the IOOOO x g sediment (which contains the larger fragments of chloroplasts) and in different amounts relative to chlorophyll in the two fractions (Figs. I and 2). Two explanations are possible : the quinones m a y be localized in the lamellae in vivo but during fractionation a proportion m a y be more readily removed from the lamellae than chlorophyll; and/or some TQ and PQ m a y occur in other parts of the chloroplast. These two possibilities were examined by comparing the quinone to chlorophyll ratio in intact chloroplasts with the ratio in chlorophyll-containing (lamellar) and chlorophyll-free (stroma) preparations from chloroplasts. The levels of PQ and TQ relative to chlorophyll are of the same order in stripped chloroplasts, in which the lamellar structure appears relatively intact as in the IOOO x g particles which include damaged and undamaged chloroplasts as well as Biochim. ]~iophys. Acla, 112 (1966) 1 9 - 3 4

c . BUCKE et al.

30

mitochondria (cf. Tables V I I I and IX). The levels of the quinones in these two preparations are slightly lower than in the intact chloroplasts or total lipids of the leaf. This provides additional evidence of some loss of quinones during preparation of stripped chloroplasts or Iooo x g particles, but does not give any indication of the site within the chloroplast from which quinones have been lost. TABLE

VIII

TQ

1DQ L E V E L S

AND

AND

RELATIVE

IOOO X g FRACTIONS

AND

TO CHLOROPHYLL IN INTACT

M e t h o d s of i s o l a t i o n a n d e s t i m a t i o n

Preparation

Whole leaf homogenate

6oo × g fraction

i 2 3 [4] *

IN WHOLE

CHLOROPLASTS

LEAF

HOMOGENATES,

PREPARED

FROM

AND

LEAVES

IN 600

OF

X g

V. faba

d e s c r i b e d i n t h e METHODS s e c t i o n .

Chlorophyll (#moles)

TO:chlorophyll PQ:chlorophyll (~moles/mmolechlorophyll)

Molar ratio T~ :PQ

75-9 26. 4 62.0 28-3

31 36 56 31

29 4° 49 32

1.o 7 o.9o 1.16 0.95

5 6

4.o4 6.74

25 27

42 4°

o.61 o.66

IOOO × g f r a c t i o n

7 8 9 IO ii iz 13 [14] *

48-4 17.8 1.73 28. 4 30.0 9.1 1.73 9.0

29 44 28 24 27 18 20 23

27 41 27 30 28 28 27 35

1.o8"* 1.o8"* 1.o4"* 0.8 0.97 0.64 0.75 0.65

Intact chloroplasts

[15] * [I6j *

2.36 2.65

16 15

42 36

0.39 0.42

* Leaves kept in the dark 24-48 h prior to chloroplast isolation. ** S a m p l e o x i d i z e d b e f o r e c h r o m a t o g r a p h y . TABLE

IX

T Q AND P Q LEVELS RELATIVE TO CHLOROPHYLL IN STRIPPED CHLOROPLASTS AND IN LAMELLAE FRACTIONS PREPARED FROM THRM P r e p a r a t i o n o f f r a c t i o n s d e s c r i b e d i n t h e METHODS s e c t i o n . N o n e of t h e p r e p a r a t i o n s before analysis.

Preparation

Stripped chloroplasts

Lamellae fraction

Chlorophyll (pmoles) I 2 L3~ * 4 5 6 7

was oxidized

TO_, :chlorophyll PQ:chlorophyll (ktmoles/mmolechlorophyll)

Molar ratio TQ :PQ

6.16 15. 5 5-55 5 .62

IO 15 18 16

29 43 29 25

0.34 o-36 o.63 0.65

18.18 9.28 5.59

12 9 12

22 27 33

o.56 o.34 o.36

* Plants kept in dark 7 2 h before experiment.

Biochim, Biophys. Acla, 112 (1966) 1 9 - 3 4

DISTRIBUTION OF P Q AND T Q IN PLANTS

31

The ratios of PQ and TQ to chlorophyll are of the same order in stripped chloroplasts as in the lamellar fraction prepared from them (Table IX), which suggests there is no significant amount of TQ or PQ in the stroma fraction of stripped chloroplasts. Furthermore when either stripped or intact chloroplasts were separated into stroma and lamellar fractions in every experiment but one, neither quinones nor chlorophyll were detected in the stroma fraction (Table X). An explanation of the anomalous result is provided by the results of recent experiments which have shown that prolonged ultrasonic disintegration of chloroplasts liberates a quinone-rich fraction from the lamellae which could account for the presence of PQ in the stroma fraction isolated from a preparation of stripped chloroplasts which had been ruptured sonically (Table X). Finally, washing a IOOO × g preparation with buffered sucrose removed small amounts of PQ and TQ but in an approximately constant ratio to chlorophyll (Table XI). All these findings suggest that TQ and PQ are present only in the lamellae. When, however, 17 g of leaves were homogenized in buffer without sucrose (so that most of the chloroplasts would be disrupted), and the homogenate TABLE

X

DISTRIBUTION

OF T Q

AND

PQ

AND

C H L O R O P H Y L L IN G R E E N A N D N O N - G R E E N

F R A C T I O N S FROM

LEAVES P r e p a r a t i o n o f l e a f f r a c t i o n s d e s c r i b e d i n t h e METHODS s e c t i o n ; c h l o r o p l a s t s i n l e a f b r e i a n d i n t a c t chloroplasts ruptured osmotically, and stripped chloroplasts broken by ultrasonic treatment.

Preparation

Chlorophyll (ffmoles)

TO, (#moles)

PO, (ffmoles)

Leaf hrei 1o5ooo × g sediment IO5OOO X g s u p e r n a t a n t

7.59 21.9 o

O.lO4 o.25o o.o12

o.138 o.354 O.OLO

Intact chloroplasts 20000 x g sediment 20000 × g supernatant

1.68 o

O.Ol 4 o

0.037 o

Stripped chloroplasts (I) 1 0 5 0 0 0 X g s e d i m e n t 105000 X g s u p e r n a t a n t (2) 1 0 5 0 0 0 x g s e d i m e n t 105000 X g supernatant

5.59 o 3.4 ° o

0.067 o 0.035 o

o.186 0.093 ---

TABLE

XI

REMOVAL OF P Q , T Q

AND CHLOROPHYLL FROM IOOO X g PREPARATION BY WASHING

IOOO X g p r e p a r a t i o n w a s h e d 0. 3 M s u c r o s e , b y s u s p e n d i n g

t h r e e t i m e s w i t h 6 7 m M p h o s p h a t e b u f f e r ( p H 7.5) c o n t a i n i n g s e d i m e n t i n m e d i u m a n d c e n t r i f u g i n g a t i o o o × g f o r lO m i n .

Sample

Chlorophyll (ffmoles)

TQ (ltmoles)

PQ (ffmoles)

TQ : chlorophyll PQ : chlorophyll (ffmoles/mmole chlorophyll)

iooo x g ist wash 2rid wasia 3rd wash

46.9o ii.oi 7.60 7.63

1.83 °-34 o.32 o. 17

2.76 o.37 0-33 o.29

39 31 43 23

Biochim. Biophys. Acta,

59 34 44 39

112 (1966) 1 9 - 3 4

c. BUCKE et al.

32

was then separated into a high-speed supernatant and sediment, a very small amount (less than 5 %) of the TQ and PQ was recovered in the chlorophyll-free supernatant (Table X). If this proportion of the quinones were present in the stroma fraction isolated from intact or stripped chloroplasts it would be below the concentration detectable by the methods used. We conclude that in the young bean leaves at least 95 % of the TQ and PQ is present in the chloroplast lamellae. The remaining 5 % is localized in the chloroplast in vivo, but the precise intrachloroplast site is uncertain. In this connexion it is important to note that the chloroplasts in the leaves we have used (V. faba var. "The Sutton") contain very few osmiophilic globules, since both GREENWOOD el al. 23 and BAILEY AND WIIYBORN41 detected PQ in globules isolated from chloroplasts of V. faba (var. "Giant Windsor"), Beta vulgaris and other species. DISCUSSION

~-TQ was present in the photosynthetic tissue of all the species examined which included representatives from algae bryophytes and angiosperms; the molar concentration of TQ was of the same order of magnitude as that of PQ-9. In young leaves of V. faba both quinones are restricted to the chloroplast and within the chloroplast are associated almost entirely with the lamellae. Up to 6o o/,.oof the total TQ extractable from the tissue arises from the oxidation of a reduced form, probably T Q H 2. It has been shown by DILLEY AND CRANE40 that the endogenous TQ and PQ of spinach chloroplasts exist in states of equilibrium between oxidized and reduced forms, and the position of the equilibrium is sensitive to light and dark. It seems probable from their wide-spread distribution, their relative concentration, intracellular localization, and redox changes, and their role in the Hill reaction (see INTRODUCTION')that both TQ and PQ m a y be components of the photosynthetic process in all types of plant. There are considerable differences in the levels of PQ-A, PQ-B and PQ-C found in spinach (Spinacia oleracea) chloroplasts by different investigators (the amount of TQ varies rather less) (Table X I I ) and the levels of PQ-A in spinach are in general higher than those in Table I I I . It is not possible to account precisely for these differences as several factors are probably involved. In the first place the level of PQ is known to increase with the age of tissue in some species2a,2% and is also affected TABLE

XI[

QUINONE LEVELS IN SPINACH CHLOROPLASTS

C h l o r o p l a s t s were s c d i m e n t e d t r o m cell-free p r e p a r a t i o n s of S p i n a c i a oleracea l e a v e s a t a b o u t Iooo × g. L e v e l s e x p r e s s e d a s / * m o l e s / n m l o l e c h l o r o p h y l l c a l c u l a t e d from t h e d a t a w h e r e ne c e s s a ry. A d a s h m e a n s no v a l u e r e p o r t e d . Reference

~-~Q

PQ

I~EGEL et al. 17

--

~6o (A), 8 (B) 3 I O - - 4 2 0 (A @ B )

D I L L E Y AND CRANE 43

8 20

HENNINGER et al. 28

IO

80 (A), 15 (B)

H E N N I N G E R AND CRANE 23 ~)ILLEY AND C R A N E 40 L I C H T E N T H A L E R AND CALVIN 42

I2

I I 6 (A), 2~ (B), 2I ( C + D )

10 20

115-I46

20

80 (A), 40 (B)

B i o c h i m . B i o p h y s . A c t a , ~I2 (I966) I9--34

(A - ' B )

DISTRIBUTION OF

PQ

AND T Q

IN PLANTS

33

by the degree of illumination of the tissue 26. Secondly, the level of PQ-9 varies with the species (Table III and ref. 26) and the relative amounts of the different plastoquinones may also differ between species ~7. Thirdly a variety of methods have been used by different workers for separating the quinones which may differ in efficiency; for example LICHTENTHALERAND CALVIN42 suggest that the solutions they prepared by elution of quinone zones from paper chromatograms might be purer than the fractions eluted from columns by KEGEL et a l Y . In addition the structures of all the quinones have not been elucidated, the relationship of PQ-4 and the dimers of PQ-9 and PQ-4 to PQ-B, PQ-C, and PQ-D is uncertain; ECK AND TREBST20 also point out that the level of the dimers increases on storage of the lipid-containing extracts at the expense of the monomers, so the possibility of such artifacts arising may also have to be taken into account. In considering the quantitative aspects of the possible role of the quinones in photosynthesis it is important to note that in vigorously growing leaves up to half the total PQ may be present in the osmiophilic globules 41, and the proportion increases with a g o 4. The PQ in the globules is probably not involved in photosynthesis. There is no report of the presence of TQ in chlorophyll-free globules, although LICHTENTHALER45 found a trace of TQ in a globule preparation which contained chlorophyll and which was isolated by a technique differing from those used earlier2S, 41. ACKNOWLEDGEMENTS

We should like to thank Professor W. 0. JAMES, F.R.S. for his encouragement of this work and Miss M. A. GEORGE and Miss V. FLYNN for their valuable technical assistance. C.B. holds a research studentship from the Department of Scientific and Industrial Research. REFERENCES I C. MARTILIS,in Ciba Found. Syrup. Quinones Electron Transport, •96o, Churchill, L o n d o n , 1961, p. 312. 2 E. R. REDFEARN, in Ciba Found. Syrup. Quinones Electron Transport, i96o , Churchill, London, 1961, p. 346. 3 N. I. BISHOP, in Ciba Found. Syrup. Quinones Electron Transport, I96o, Churchill, London, 1961, p. 385 • 4 M. DAM, J. GLAVIND AND E. K. GABRIELSEN, Aeta Physiol. Scand., 13 (1947) 9. 5 M. MCKENNA, M. S. HENNINGER AND F. L. CRANE, Nature, 203 (1964) 524. 6 R. L. LESTER AND F. L. CRANE, J. Biol. Chem., 234 (1959) 2169. 7 F. L. CRANE, Plant Physiol., 34 (1959) 128. 8 M. KOELER, Festschrift E. C. Barell, H o f f m a n - L a Roche, Basle, 1946, p. 199. 9 F. L. CRANE, Plant Physiol., 34 (1959) 546. IO H . K . LICHTENTHALER AND R . B. PARK, Nature, 198 (1963) lO7O. I I 3/[. KOFLER, i .

12 13 14 15 16

17 IS

19 20

LANGERMANN, R . R/JEGG, L. H . CHOPARD-DIT-JEAN, i .

R. AYROLID AND

ISLER, Helv. Chim. Acta, 42 (1959) 1283. 1. BISHOP, Proc. Natl. Acad. Sci. U.S., 44 (1958) 5 °11. BISHOP, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1696. R. FEDFEARN AND J. FRIEND, Biochem. J., 84 (1962) 34 P. W. KROGMANN, Biochem. Biophys. Res. Commun., 4 (1961) 275. F. I{. V~THATLEY AND A. A. MORTON, Acta Chem. Scand., 17 (1963) 14o. L. P. KE~EL, M. D. FIENNINGER AND F. L. CRANE, Biochem. Biophys. Res. Commun., 8 (1962) 294. L. P. I~EGEL AND M. D. HENNINGER, Plant Physiol., 38 (1963) xi. M. D. HENNINGER AND F. L. CRANE, Plant Physiol., 39 (1964) 598. H. ECK AND A. TREBST, Z. Naturforsch., i8b (1963) 446.

O. N. N. E. D.

Bioehim. Biophys. Acla, 112 (I966) 19-34

34 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45

c. BUCKE et

al.

R. A. DILLEY AND ]7. L. CRANE, Plant Physiol., 38 (1963) 452. C. BUCKE, M. HALLA\X,'AY AND 1~. A. MORTON, Biochem. J., 90 (1964) 4 I P . M. D. ]71ENNINGER AND F. L. CRANE, Biochemistry, 2 (1963) 1168. M. D. HENNINGER AND F. L. CRANE, Biochim. Biophys. Acta, 75 (1963) 144. ,]V[. D. HENNINGER, R. A. DILLEY AND F. L. CRANE, Biochem. Biophys. Res. Commun., i o (1963) 237K. EGGER, Planta, 64 (1965) 41. \V. JOHN, E . DIETZEL AND E. EMTE, Z. Physiol. Chem., 257 (1939) 173A. D. GREENWOOD, 1~. M. LEECH AND J. P. V~ILLIAMS, Biochim. Biophys. Acta, 78 (1963) 148. W . A. KRATZ AND J. MYERS, Am. J. Botany, 42 (1955) 282. J. STEVENSON, F. W . HEMMING AND R. A. MORTON, Biochem. J., 88 (1963) 52. R. A. DILLEY AND F. L. CRANE, Anal. Biochem., 5 (1963) 531. D. I. ARNON, Plant Physiol., 24 (1949) I. O. ]71. LOWRY, N. J. ROSEBOROUGH, A. L. FARR AND I~. J. RANDALL, J. Biol. Chem., 193 (1951 ) 265. V. [. OYAMA AND ]7I. EAGLE, Proc. Soc. Exptl. Biol. ~VIed., 91 (1956) 305 • V. H. BOOTH, Phytochemistry, 3 (1964) 273. \V. O. JAMES AND x¢~. S. R. DAS, New Phytologist, 56 (1957) 325 . R. M. LEECH, Biochim. Biophys. Acta, 79 (1964) 637. W . O. JAMES AND R. M. LEECH, Proc. Roy. Soc. London, Ser. B, 16o (1964) 13. G. J A c o n I AND E . PERNER, Flora Jena, 15o (1961) 209. R. A. DILLEY AND F. L. CRANE, Plant Physiol., 39 (1964) 33" J. L. BAILEY AND A. G. \¥HYBORN, Biochim. Biophys. Acta, 78 (1963) 163. H . K. LICHTENTHALER AND M. CALVIN, Biochim. Biophys. Acta, 79 (1964) 3 °. R. A. DILLEY AND F. L. CRANE, Biochim. Biophys. Acta, 75 (1963) 142. A. R. \¥ELLBURN AND F. \ ¥ . HEMMING, p e r s o n a l c o m m u n i c a t i o n . H. K. LICHTENTHALER, Ber. Deut. Botan. Ges., 77 (1965) 398.

Biochim. Biophys. Acta, 112 (1966) 1 9 - 3 4