Planta (Berl.) 94, 124--133 (1970) 9 by Springer-Verlag 1970

Effects of Some Phenoloxidase Inhibitors on Chloroplasts and Carboxylating Enzymes of Sugar Cane and Spinach C. W . BALDRY, C. BUOKE a n d J. CooM~s Tare and Lyle Ltd., Research Centre, Keston, Kent, England Received June 18, 1970

Summary. A number of additives have been tested for their effects on o-diphenol : 02 oxidoreductase activity of cane leaves. The most inhibitory compounds were thioglycollate, fl-mercaptoethanol, polyethylene glycol and bovine serum albumin. Sulphydryl (SH) compounds did not affect rates of photosynthetic CO2 assimilation when used at concentrations below 10-2 M. However, in the presence of Mn++ ions they contributed to an 03 consumption which masked photosynthetic 03 evolution. Addition of SH compounds or of polymers to the grinding media increased rates of enzymic C03 assimilation in crude enzyme preparations from cane leaves, but did not affect rates of CO3 assimilation ii~ similar spinach preparations. Strong reducing agents, copper chelators, low 03 tension and high pH were effective in reducing phenoloxidase activity, but presented problems in the isolation and assay of chloroplasts. The results are discussed in relation to (a) design of suitable media for preparation of active cane chloroplasts and (b) comparative studies of enzyme levels in plants of various genera. L a c k of p h o t o c h e m i c a l a c t i v i t y in cane chloroplasts i s o l a t e d using c o n v e n t i o n a l techniques such as described b y C o c k b u r n et. al, 1968 m a y be a s s o c i a t e d with high levels of chlorogenie acid a n d p h e n o l o x i d a s e (o-diphenol: 02 o x i d o r e d u c t a s e , E. C. 1.10.3.1) in t h e cane leaves ( B a l d r y et al., 1970). T h e effects of p l a n t o-diphenols, t a n n i n s a n d t h e i r o x i d a t i o n p r o d u c t s d u r i n g isolation of p l a n t enzymes, m i t o e h o n d r i a a n d viruses, are well k n o w n (Loomis a n d Bataillc, 1966; Anderson, 1968). These a u t h o r s h a v e also discussed m e t h o d s of o v e r c o m i n g their i n h i b i t o r y action. Less i n f o r m a t i o n is a v a i l a b l e concerning m e t h o d s of o v e r c o m i n g similar p r o b l e m s when isolating chloroplasts from leaves of higher plants. P i e r p o i n t (1966) has suggested t h a t o x i d a t i o n of ehlorogenie a c i d b y o - d i p h e n o l o x i d a s e results in t h e f o r m a t i o n of t h e o-diquinone. The d i q u i n o n e m a y r e a c t with S t I groups of p r o t e i n s to i n h i b i t e n z y m e a c t i v i t y . Such a m e c h a n i s m would be consistent with o b s e r v a t i o n s ( B a l d r y et al., 1970) t h a t t h e c a r b o x y l a t i o n r e a c t i o n of t h e p h o t o s y n t h e t i c c a r b o n r e d u c t i o n cycle is the m o s t sensitive to phenol inhibition.

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Inhibition of enzymes b y o-diphenols m a y be prevented or reduced b y removing the substrates (02 and phenolics), inhibiting the phenoloxidase, or reducing the products (quinones) back to phenols. We have therefore investigated the potential of these methods for improving the photochemical activity of cane chloroplasts. Materials and Methods Materials and methods used were essentially as described previously (Baldry et al., 1970). Chloroplasts were prepared from leaf laminae of Saccharum, Spinacea oleracea or Pisum sativum by the method of Cockburn et al., 1968. Leaf brei was obtained by grinding 30 g leaf in 200 ml phosphate buffer, 0.1 M, pit 6.5, and filtering through muslin. Enzyme assays were on aliquots from 5 g leaf blended in 30 ml 0.1 M tris buffer, pH 8.3 containing 0.01 M MgCl2. Photosynthetic C02 fixation by chloroplasts and dark enzymic carboxylation reactions with ribose5-phosphate and ATP or with phosphoenol pyruvate as substrates were determined by measuring incorporation of 14C02 into acid-stable products. Oxygen evolution and o-diphenoloxidase-catalysed 02 consumption were measured polarographically using Clark type Oe electrodes. Reducing compounds (sodium iso-ascorbate, dithionite, and metabisulphite) copper chelators (diethyldithiocarbamic acid and 8-hydroxyquinoline) polymeric substances (bovine serum albumin, BSA; polyvinylpyrrolidone, PVP; polyethyleneglycol 4,000, carbowax) and sulphydryl reagents (thioglycollate, fl-mercapto~thanol, dithioerythritol, glutathione and cysteine) were added to the grinding or assay media as described in the text. Results

A. Sulphydryl ( S H ) Reagents and Polymers Effects on Cane Phenoloyddase Activity Inhibition of cane o-diphenoloxidase b y SH reagents and polymeric compounds was determined as follows: leaf brei (5 ml) was incubated with varying concentrations of each compound for five minutes at 25 ~. Three ml of each suspension was introduced into the 02 electrode chamber, 1 ~mole o-diphenol added as substrate, and the rate of 0 2 consumption determined (Fig. 1 a). The rate of 02 consumption at each concentration of additive was plotted as a percentage of the control rates (Fig. 2). These were in the order of 100 ~moles 0 2 consumed per g fresh weight of tissue per hour. The time taken for each c o m p o u n d to inhibit the phenoloxidase activity was investigated b y sequential addition of leaf brei, o-diphenol and inhibitor to the electrode chamber (Fig. 1 b). Similar results were obtained with chlorogenic acid, caffeic acid or catechol as substrate. Thioglycollate was the most effective inhibitor of cane phenoloxidase. A mM solution caused almost immediate inhibition of enzyme activity. A similar degree of inhibition was observed after a five minute preincubation period with fl-mercaptoethanol. However, onset of inhibition

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Fig. 3 Fig. 4 Fig. 3. Effects of SH reagents and polymers on photosynthetic COs-dependent 0~ evolution (1 to 5 and 8) and photosynthetic C0~ fixation (6 to 8) by isolated spinach chloroplasts. I and 8 eysteine; 2 and 7 dithioerythritol; 3 and 8 fl-mercaptoethanol; 4 and 8 glutathione; 5 and 6 thioglycollate; 8 carbowax, BSA and PVP Fig. 4. Traces from oxygen electrodes showing effects of SH reagents on 02 consumption by isolated pea chloroplasts. Traces A, B and C, electrode chamber contained clfloroplasts (100 Fg ctdorophyll) in 3 ml HEPES (N-2-hydroxyethylpiperazine-N'-2-eth~nesutphonic acid) buffered resuspending medium containing Mn++ and ~g++ ions. On/off refers to illumination. LqH addition of dithioerythritol (B) or thioglyeollate (C, D) to a final concentration of 10-a M. DCMU added to 10-~ M. Catalase addition of 10 ~1 commercial preparation. Mn addition of M~CI~ to a finsl concentration of 10-a 1Y[

Effects on Active Chloroplast Preparations Polymeric substances at concentrations up to 2 % had no effect on rates of photosynthetic 02 evolution, or C02 fixation b y spinach chloroplasts (Fig. 3). I n these experiments the control rates were a b o u t 20 ~moles 02 evolved or 15 ~zmoles CO 2 fixed per mg chlorophyll per hour. H i g h concentrations of thioglyco]late and dithioerythritol inhibited CO s fixation. All S H compounds inhibited 02 evolution at low concentrations and stimulated 03 consumption at higher concentrations. The mechanism whereby S H compounds stimulated 03 consumption b y active pea chloroplasts was investigated polarographically. Tracings from these experiments are shown in Fig. 4. Trace A, from a control sample, shows the expected changes in O~, gas exchange on illumination or addition of DCMU [3-(3-4-dichlorophenyl)-l-l-dimethyl urea]. Trace B shows the response of similar chloroplast suspensions on the addition 9

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Fig. 5. Effects of SH reagents (a and c) and polymers (b and d) on dark enzymic COS fixation by sugar cane leaf brei with ribose-5-phosphate and ATP (a and b) or phosphoenol pyruvate (c and d) as snbstrates. 9 p-mereapf~ethanol; o thioglycollate; 9 glutathione; o cysteine; - dithioerythritol; 9 BSA; a PVP; 9 carbowax

of dithioery~hritol (10 -a M). An immediate uptake of 0~ occurred which was slightly reduced on addition of DCMU. Further investigations of DCMU-inhibited chloroplasts (trace C) confirmed the increased rate of 0~ consumption in the light. Addition of eatalase reduced this uptake to a rate comparable with the dark raft. A similar dark rate of 03 consumption was observed if SH reagents were added to the buffered medium containing Mn++ ions but no chloroplasts (trace D). Further controls were run which established that neither the Good's buffers, the osmoticum or Mg++ ions contributed to the non-photochemical 0~ consumption. Effects on Enzymic Carboxylation Reactions Effects of SH reagents and polymeric substances on CO~ fixation, in the dark, by cane leaf preparations are shown in Fig. 5. I n these experiments the additive was included in the grinding media at the required concentration. Most SH reagents, when used at concentrations

Phenoloxidase Inhibitors and CO2 Fixation

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between 10-3 and 10-4 M, stimulated CO s assimilation b y cane preparations when ribose-5-phosphate and A T P were supplied as substrates. Increases of up to 6 times the control rate, which was in the order of 0.3 ~moles CO s fixed per mg protein per hour, were observed. I~igher concentrations of SH reagents reduced the observed activity below the control rate. Carbowax and BSA significantly increased the rate of CO s assimilation but P V P had little effect. Rates of enzymic CO s assimilation b y cane preparations with phosphoenolpyruvate as substrate were much higher t h a n those with ribose5-phosphate and A T P as substrates. About 180 ~moles CO s were fixed per mg protein per hour. The control rate was stimulated when SH compounds were incorporated in the grinding media. Increased activity was observed with increasing concentration of SH compound used up to 10-s M. This was found with all SH compounds investigated except thioglycollate, which was inhibitory at this concentration. Polymeric substances had little effect on the P E P carboxylase activity. Cane carboxylation reactions were stimulated only ff the reagents were added to the grinding media. When leaf tissue was ground in buffer alone, and the reagents added to the assay mixtures, enzyme activities did not differ significantly from those in control samples. Rates of ribose-5-phosphate/ATP-dependent CO s fixation observed with similar spinach preparations were much higher (over 18 ~moles CO s fixed per mg protein per hour) than in cane preparations. On the other hand, rates of P E P carboxylase were negligible (less t h a n 1 ~mole CO s fixed per mg protein per hour). Addition of SH reagents or polymers to either grinding media or assay mixtures did not significantly alter the observed activities from these control rates.

B. Other Methods o/Reducing Inhibition by Phenolics Concentrations of the second substrate of o-diphenoloxidase (molecular 03) in the leaf brei can be reduced b y bubbling the media with inert gas, or b y addition of strong reducing agents. The reducing agents m a y also reduce o-diquinones to o-diphcnols and inhibit the phenoloxidase. However, lowering the 02 concentration of the media b y gassing with Iq2 during the complex operations involved in chloroplast preparation was not possible without substantially increasing the total time taken. The use of iso-ascorbate, dithionite and metabisulphite at concentrations of up to 10-3 M was investigated. Polarographie measurements indicated t h a t low O s concentrations, in the medium, were not maintained unless the leaves were blended under an atmosphere of I ~ . Furthermore, non-enzymic 0 s consumption in the presence of these compounds interfered with polarographie assays. This difficulty could 9*

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be overcome b y adopting an elaborate washing procedure to prevent reducing compounds being carried over from the grinding media. Diethyldithiocarbamic acid and 8-hydroxyquinoline were effective inhibitors of cane o-diphenoloxidase at concentrations between 10-s and 10-4 M. However, both compounds inhibited photosynthetic reactions of active spinach and pea chloroplasts. This could be due both to chelating copper in plastoeyanin, and to less specific reactions. As shown in Fig. 6, increasing the p H of the grinding medium caused a significant decrease in the rate of endogenous phenoloxidase activity in cane leaf brei. A similar decrease was observed in 02 consumption stimulated b y addition of either chlorogenic acid or caffeic acid. Borate buffer, p i t 9.2, was particularly effective in reducing phenoloxidase activity, due to complexing of o-diphenols b y the borate ions.

Discussion

Failure to detect high enzymic activities in sugar cane preparations, or to prepare active chloroplasts from sugar cane leaves was correlated with the presence of high levels of endogenous o-diphenols and o-diphenoloxidase in the tissues (Baldry et al., 1970). Anderson, 1968, in reviewing the literature, concluded t h a t in order to predict the effectiveness of a n y of the suggested methods for increasing activity of a particular enzyme from a specific tissue the following information was essential. 1. The o-diphenoloxidase activity of the tissue; 2. The subcellular location of the o-diphenoloxidase; 3. The nature and amount

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of the phenolics and tannins in the tissue; 4. The mechanism of action of the diphenoloxidase(s) and which of the phenols is substrate for the oxidase; 5. The mechanism of action of the various compounds for preventing accumulation of the products of o-diphenoloxidase and 6. The susceptibility of the enzyme being extracted to products of o-diphenol oxidase. Such information is now available in respect to sugar cane leaf tissue. We have established that cane leaves contain high levels of chlorogenic acid, and an active cytoplasmic o-diphenoloxidase for which chlorogenic acid is the preferred substrate. Furthermore, we have shown that most partial photosynthetic reactions are inhibited by mM concentrations of o-diphenols. The most sensitive reactions, CO sdependent O~ evolution and carboxylation reactions of the photosynthetic carbon reduction cycle, are inhibited by concentrations as low as 10-5 M (Balch'y et al., 1970). The mechanism of oxidation of chlorogenic acid, and the reactions which products may undergo, have been investigated in detail by Pierpoint, 1966. The mechanisms whereby the various protective techniques reverse inhibition have been discussed by Loomis and Bataflle, 1966 and Anderson, 1968. Hence most of the required information is available for design of effective media for isolation of chloroplasts from cane leaves. The present observations indicate that many of the available methods are effective in reducing activity of cane phenoloxidase, but are impracticable when applied to isolation of chloroplasts. The action of SH reagents on chloroplast reactions was inconsistent. Photosynthetic C02 fixation was insensitive except to high concentrations of SIt compounds. However, COs-dependent 0.2 evolution was completely inhibited at relatively low concentrations. These conflicting observations may be rationalized in terms of structural integrity of the plastids. Most chloroplast preparations consist of a mixture of intact, membrane-bound plasrids, chloroplasts which have lost their outer envelope but retain some stroma and chloroplasts which have lost both envelope and stroma. Rapid rates of CO 2 fixation can only be obtained with preparations which contain a high proportion of intact chloroplasts. Rates of electron transport with exogenous electron donors or acceptors arc more rapid in broken chloroplasts (Walker, 1965; 1967). This is due both to uncoupling of photophosphorylation from electron transport when the chloroplasts are disrupted and to loss of the selectively permeable envelope. This envelope restricts access of exogenous compounds to the photochemical apparatus. Hence the observed rates of CO e fixation could reflect the number of intact chloroplasts. Photosynthetic 03 evolution by these plastid s could be overshadowed by 02 consumption due in part to photochemical oxidations catalyzed by broken chloroplasts and

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C.W. Baldry, C. Bucke and J. Coombs:

in p a r t to non-enzymic oxidations. I n this case use of SH compounds would not impede studies of photosynthetic 14C0~ fixation b y intact chloroplasts. Furthermore their use in grinding media does not preclude polarographie studies, if the SH reagents are completely removed by repeated resuspension of the chloroplasts in media of lower SH concentration after the technique of Stokes et al., 1968. The fact t h a t effects of SH reagents and polymers, on CO 2 fixation, were only observed with cane preparations when the reagents were added to the grinding media is consistent with these compounds reducing the deleterious effects of o-diphenol oxidation products in the brei. However, since the response of the two enzyme systems to varying concentrations of the reagents differed, it must be concluded t h a t this was not the only effect. I n particular it would appear t h a t high concentrations of SH reagents inhibited the ribose-5-phosphate/ATP dependent earboxylatiou. Obviously there are still m a n y problems to be overcome before ideal methods of preparing enzymes and chloroplasts from cane leaves can be formulated. However, b y addition of 10-a M thioglycollate and 1% carbowax to the grinding medium, chloroplasts were isolated from cane leaves and were shown to carry out a light-dependent carboxylation of phosphoenolpyruvate (Baldry et al., 1969). I t is also suggested t h a t accurate comparison of enzyme levels in plants of different genera require a detailed investigation of concentrations of o-diphenols in the tissues. Further investigations are also required of the sensitivity of enzymes to phenolics and to compounds introduced into grinding media. We are grateful for the technical assistance of B. F. J. Bowler and J. E. Brown.

References Anderson, J.W.: Extraction of enzymes and subcellular organelles from plant tissues. Phytochem. 27, 1973--1988 (1968). Baldry, C.W., Bucke, C., Coombs, J. : Light/phosphoenol pyruvate dependent carbon dioxide fixation by isolated sugar cane chloroplasts. Bioehem. biophys. Res. Commun. 87, 828---832 (1969). - - Gross, D. : Phenols, phenoloxidase and photosynthetic activity of chloroplasts isolated from sugar cane and spinach. Planta (Berl.) 94, 107--123 (1970). Cockburn, W., Walker, D. A., Baldry, C. W. : The isolation of spinach chloroplasts in pyrophosphate media. Plant Physiol. 48, 1415--1418 (1968). Loomis, W. D., Bataille, g.: Plant phenolic compounds and the isolation of plant enzymes. Phytochem. 5, 423--438 (1966). Pierpoint, W. S. : The enzymatic oxidation of chlorogenic acid and some reactions of the quinone produced. Biochem. J. 98, 567--580 (1966).

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Stokes, D. M., Anderson, J. W., Rowan, K. S. : The isolation of mitochondria from potato tuber tissue using sodium metabisulphite for preventing damage by phenolic compounds during extraction. Phytoehem. 7, 1509--1512 (1968). Walker, D. A. : Correlation between photosynthetic activity and membrane integrity in isolated pea chloroplasts. Plant Physiol. 40, 1157--1161 (1965). - - P h o t o s y n t h e t i c activity of isolated pea chloroplasts. In: Biochemistry of chloroplasts, ed. T. W. Goodwin, p. 53--70. New York: Academic Press 1961. Dr. J. Coombs Tare and Lyle Ltd., Westerham Road Keston, Kent, BR 2 6 HJ, England