Isolated pea chloroplasts were treated with secondary metabolites acetylcholine, dopamine, adrenaline, noradrenaline, serotonin known as animal neurotransmitters or mediators and found in plant cell as well. There were no effects of all compounds on the NADP-photoreduction. Low concentrations (< 10-5 M)) of these, except serotonin, stimulated noncyclic photophosphorylation in thylakoids. Acetylcholine induced Na+, K+ output from intact chloroplasts, but had no effect on Ca2+, Mg2+ efflux. Dopamine, adrenaline, noradrenaline and serotonin had an opposite effect. A possible regulatory and mediatory role of the substances in chloroplasts and in cell is discussed.Izolirani kloroplasti tretirani su sekundarnim metabolitima acetilholinom, dopaminom, adrenalinom, noradrenalinom i serotoninom, koji su prisutni u biljnim stanicama. U životinja također dolaze ovi spojevi i imaju ulogu neurotransmitera ili medijatora. Svi osim serotonina u niskim koncentracijama (10-5 M) potiču necikličku fotofosforilaciju u tilakoidama. Acetilholin pospješuje izlaženje K‘ i Na+ ijona iz intaktnih kloro- plasta, ali ne utječe tako i na Ca2" i Ma2". Dopamin, adrenalin, noradrenalin i serotonin imaju suprotan učinak

Victoriya V. Roshchina

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Acta Bot. Croat. 49, 29— 35, 1990. CODEN: ABCRA2  Y U  ISSN 0365— 0588UDC 581.192.2:582.844.2 =  20 Original scientific paperR E G U L A T I O N  O F  C H L O R O P L A S T  R E A C T I O N S  B Y  S E C O N D A R Y  M E T A B O L I T E S  A C E T Y L C H O L I N E  A N D  B I O G E N I C  A M I N E SVICTORIYA  V. ROSHCHINA(Institute o f  Soil Science and Photosynthesis, U.S.S.R. A cadem y o f  Sciences, Pushchino,M oscow  Region)R eceived  Septem ber 5, 1989Isolated pea chloroplasts were treated with secondary metabolites acetylcholine, dopamine, adrenaline, noradrena­line, serotonin known as animal neurotransmitters or m e­diators and found in plant cell as well. There were no effects of all compounds on the NADP-photoreduction. Low  concentrations ( <  10-s  M)) of these, except serotonin, sti­mulated noncyclic photophosphorylation in thylakoids. A ce­tylcholine induced N a + , K +  output from  intact chloroplasts, but had no effect on Ca2+ , M g2+ efflux. Dopamine, adre­naline, noradrenaline and serotonin had an opposite effect. A  possible regulatory and mediatory role of the substances in chloroplasts and in cell is discussed.(Report at the 7th FEPPS Congress in Split, September 5— 9, 1988)A C T A  B O T . C R O A T . V O L . 49, 1990. 29V JC T O R IY A  V . K O SH C H IN AI n t r o d u c t i o nAccording to Frey-Wyssling hypothesis, membranes of chloroplasts can originate from plasmalemma which is sensitive to external chemicals. There is an evolutionary likeness between both types of membranes as chemical sensors. Plasmalemma can receive signals of environment directly from external media while chloroplast — indirectly from internal cell media possibly by means of mediators as has been observed between animal cells in synapse ( R o s h c h i n a  1989). The role of neuromediators are characteristic of cholinic ester acetylcholine and biogenic amines dopa­mine, adrenaline, noradrenaline and serotonin, forming cholinergic, dopaminergic, adrenergic, serotoninergic systems of regulation. Such substances as acetylcholine ( F l u c h  and J a f f e  1976), dopamine, noradrenaline ( U d e n f r i e n d  et al. 1959), adrenaline ( A s k a r  et al. 1972) serotonin ( R e g u l a  1986) were found in plant cells as well. Acetyl­choline is a part of the cholinergic regulation system in animals, including cholinoreceptor, enzyme of the acetylcholine synthesis (choline acetyl- transferase) and hydrolysis (cholinesterase). Acetylcholine, choline acetyl- transferase and cholinesterase were found in plants (F 1 u c k and J a f f e  1976, H a r t m a n n  and G u p t a  1989). Acetylcholine, cholinesterase, adrenaline and noradrenaline were also observed in isolated intact chloro­plasts ( R o s h c h i n a  1988, 1989).We do not excatly know the function of the substances in plastids. Since acetylcholine and biogenic amines were found in multicellular plants and animals without nervous system, their intracellular role is assumed ( B u z n i k o v  1987) to be regulators of development or (and) energy and metabolism. It is possible that they are involved in signalling between plasmatic membranes and organelles and between organelles ( R o s h c h i n a  1989). Intracellular mediation and regulation by them could be a more ancient mechanism than the known role of intercellular mediators in synapse between cells of animals with nervous system ( B u ­z n i k o v  1987). This possibility needs experimental and theoretical ana­lysis. A  model of the study could be isolated chloroplasts as a test for the action of the exogenous acetylcholine and biogenic amines.The aim of the paper was to analyze the regulatory function of the substances in chloroplasts.M e t h o d sSubjects of the study were chloroplasts isolated from the leaves of 12-day-old seedlings of pea (Pisum sativum L., cv. Prevoskhodnii), bean (Phaseolus aureus L.), sting nettle (Urtica dioica L.). Intact type A chlo­roplasts were isolated according to R o b i n s o n  et al. (1979) and type B chloroplasts according to R o s h c h i n a  and M u k h i n  (1985). The NADP+ - photoreduction and coupled photophosphorylation were measured in type B chloroplasts as described earlier ( R o s h c h i n a  and M u k h i n  1985), and the Na+, K+, Ca2+, Mg2+ output from intact plastids with ion-se­lective electrodes ( R o s h c h i n a  1987). Isolation and purification of plasto- cyanin, ferredoxin, ferredoxin-NADP-reductase from pea leaves and cyto­chrome f from Chlorella cells were done as described earlier (R o s h c h i- n a 1989), Acetylcholine in leaves and intact chloroplasts was determined by paper chromatography ( R o s h c h i n a  1989). Cholinesterase activity was measured according to E l l  m a n  et al. (1961).30 A C T A  B O T . C R O A T . V O L . 49, 1990.R E G U L A T IO N  O F C H L O R O P L A S T  R E A C T IO N SRes ul t sTable 1 shows sensitivity of intact chloroplasts and type B chloro- plasts without envelope to exogenous acetylcholine and biogenic amines measured as concentration affecting ion permeability and photochemical activity. Low concentration of acetylcholine stimulated Na+, K+ efflux from intact chloroplasts, but there was no effect on Ca2+, Mg-' output thro­ugh envelope. Noradrenaline, adrenaline and serotonin had an opposite action. Response reactions of thylakoids to these substances were estima­ted as changes in photochemical activity. The compounds studied were not sensitive to rates of the NADP+-photoreduction. Low concentrations of exogenous acetylcholine, noradrenaline, adrenaline stimulated non-cyclic photophosphorylation ~2-fold, but dopamine did so only to 30°/o of control ( R o s h c h i n a  1989). Serotonin did not act on this reaction. It could be supposed that due to phenolic or indolic groups, biogenic amines are capa­ble of redox reactions. High concentrations of catecholamines interacted with individual electron carries (Table 2), reducing cytochrome f and pla- stocyanin. Serotonin had no such effect. Thus catecholamines can take part in redox reactions with metal-containing proteins of the electron transport chain.T able 1. S 50 (M ), concentrations of acetylcholine and biogenic amines stimulated the pea chloroplasts reactions on 50% of control (0, no effect; — , no data)SubstancesIntact chloroplasts ThylakoidsN a+Ion effluxK  + Ca2 + M g 2 +NADP + photo- reductionATPsynthesisAcetylcholine i o - 7 1 0 "7 0 0 0 5 X  1 0 -9Dopamine — — — — 0 5 X  1 0 - 9Noradrenaline 0 0 1 0 '9 1 0 -9 0 7 X  IO’ 9Adrenaline 0 0 1 0 "9 1 0 -9 0 i o - 8Serotonin 0 0 1 0 '9 2 X  I O '9 0 0Table 2. Redox interaction of high concentrations (10" 6— 10 ~ 3 M ) of biogenic amines with isolated electron carries (0, no effect)Substances Cytochrome f Plastocyanin FerredoxinFerredoxin-NADP-reductaseDopamine reduced reduced 0 0Noradrenaline reduced reduced 0 0Adrenaline reduced reduced 0 0Serotonin 0 0 0 0A C T A  B O T . C R O A T . V O L . 49, 1990. 31V IC T O R IY A  V . R O S H C H IN AT able 3. The acetylcholine content and the cholinesterase activity in isolated i ntac chloroplasts of higher plants (traces, SE 15%)PlantAcetylcholine x 10 " 6 (mol kg-1  of fresh leaf mass)Hydrolysis of acetylthiocholine x 10" 6 (mol s -1  kg-1  of fresh leaf mass)Pisum sativum L. 0.2 39Phaseolus aureus L. 49 1.1Urtica dioica L. traces 29The phenomena observed can take place in vivo and be dependent on the rates of synthesis and katabolism of acetylcholine and biogenic amines. For example, the acetylcholine content and cholinesterase activity were compared for some species (Table 3). Both parameters were estima­ted by the amount of chloroplasts isolated from 1 kg of fresh leaf mass. The lowest accumulation of choline ester in chloroplasts of pea and sting nettle correlated with the highest activity of the enzyme hydrolyzing it. The opposite correlation was observed for bean plastids.D i s c u s s i o nOur results show that in low concentrations acetylcholine and cate­cholamines can be regulators of ion permeability and photophosphoryla­tion in chloroplasts. As for serotonin, it seems to reguate only Ca2* and Mg2+ permeability but to be non-effective on photophosphorylation. These readtions can be estimated as replies of chloroplasts to the intracellular chemical signalling by acetylcholine and biogenic amines. This possibility becomes more real because acetylcholine and catecholamines were found not only in cytoplasm of plant cell, but also in chloroplasts. Other argu­ments should be sought in evolution.Photosyntetic organelle chloroplas'ts were an independent organism in ancient times which then during evolution was possibly included into eukaryotic cell by endocytosis. Symbiont preserved the envelope formed by a double membrane layer and thylakoids as internal structures. The envelope can receive a chemical signal from cytoplasm and thylakoids from stroma as it happened with plasmalemma.Molecular targets of the acetylcholine and catecholamines action in chloroplasts can be specific receptors, conformational changes of which in response to the binding of the active substance induce shifts in rates of photophosphorylation and Na+, K+, Ca2+, Mg2+ output. The reactions using agonists (muscarine and arecoline) and antagonists (atropine and d-tubocurarine) of acetylcholine were shown in earlier papers ( R o s h c h i -  n a 1987, 1989). The functional analog of cholinoreceptor which binds with chemical agents and is excited (or broken) by them was supposed to re­gulate the opening of ion channel. The presence of adrenoreceptors in chloroplasts is proposed as well, based on the sensitivity of plastids to catecholamines. In animals cholino- and adrenoreceptors regulate not only ion permeability but also the triggering of adenylate cyclase activity.32 A C T A  B O T . C R O A T . V O L . 49, 1990.R E G U L A T IO N  O F C H L O R O P L A S T  R E A C T IO N SScheme 1The envelope membraneAcetylcholine o f cytoplasmAdrenaline, noradrenaline (serotonin?) of cytoplasmt=t>4>proposed cholino- - receptorconformational ■ changes ----------adenylatecyclasephosphorylated--------proteins ATP — v-̂-cAMP"̂^proposed adreno- -  receptorN a+/K +-perme- ability (opening of ion chammels)-►physiologicaleffectsuadenylatecyclaseIconformational changes-----------CaJ+/M g 2+-perme--►  ability (opening of ion channels)Acetylcholine of chloroplast (stroma)Adrenaline, noradrenaline of chloroplast (stroma)The thylakoid membrane>proposed cholino- -  receptorconformational 1 changesadenylatecyclasephosphorylated ------proteins ATP^------c A M P -* 'proposed adreno- -  receptoradenylatecyclase!conformationalchanges• physiological effectsphotochemicalactivityThis enzyme catalyzes the synthesis of secondary messenger cyclic AMP (cAMP) ( B r o w n  and N e w t o n  1981). cAMP phosphorylâtes membra­ne proteins. Phosphorylation of proteins is a common regulatory mecha­nism in physiological processes. Similar ways of regulation are proposed for chloroplasts (Scheme 1) based on the presence of adenylate cyclaseA C T A  B O T . C R O A T . V O L . 49, 1990. 33V IC T O R IY A  V . R O S H C H IN A( B r o w n  and N e w t o n  1981) and protein kinase ( S o i l  1988) in pla- stids, the protein phosphorylation in chloroplasts ( S u s s  1981), stimula­tion of ATP synthesis in thylakoids by acetylcholine and biogenic amines (ATP can be a substrate for cAMP synthesis), changes in ion permeabi­lity of envelope of plastids by acetylcholine and catecholamines.Intracellular functions of the substances are more ancient than their specialized role as intercellular neuromediators ( B u z n i k o v  1987). Pos­sible reasons for this conclusion are common distribution of acetylcholine and catecholamines in both kingdoms (animals and plants), the presence of the substances, their receptors and cholinesterase in unicellular and multicellular organisms, common membrane reactions on biomediators (changes in ion permeability and activation of adenylate cyclase). Ace­tylcholine and catecholamines can play the role of regulators of energetic and transport processes as well as of chemical signals or mediators in the information transmission from cytoplasm to plastids or other organel­les.R e f e r e n c e sAskar, A., K. Rubacli, J. Schormuller, 1972: Dunnschichtchromatographische Trennung der in Banenen vorkommenden Amin-Fraktion. Chem. M ikro- biol. Technol. Lebensm. 1, 187— 190.Brown, E. G., R. P. Newton, 1981: Cyclic A M P  and higher plants. Phyto­chemistry, 20, 2453— 2463.Buznikov, G. A., 1987: Neurotransmittery v  embriogeneze. Moskva, Nauka.Ellman, G. L„ K. D. Courtney, V. Andres Jr., R. M. Featherstone, 1961: A  new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88— 95.Fluck, R. A., M. J. Jaffe, 1976: The acetylcholine system in plants. In Com­mentaries in Plant Science (H. Smith, ed.), Oxford: Pergamon Press, pp. 119— 136.Hartmann, E., R. Gupta, 1989: Acetylcholine as a signalling system in plants. In Second Messengers in Plant Growth and Development (Boss, W . F. and D. I. Morve, eds). N ew  York: Allan R. Liss, pp. 257— 287.Regula, I., 1986: The presence of serotonin in embryo of black walnut (Juglans nigra). Acta Bot. Croat 45, 91— 95.Robinson, S. P., G. E. Edwards, D. A. Walker, 1979: Established methods for the isolation of intact chloroplasts. In Plant Organelles (Reid, E., ed.), Chichester et al.: J. W iley &  Sons, pp. 13— 24.Roshchina, V. V., E. N. Mukhin, 1985: Acetylcholinesterase activity in chlo­roplasts and acetylcholine effects on the photochemical reactions. Photo- synthetica 19, 164— 171.Roshchina, V. V., 1987: The action of the acetylcholine agonists and antago­nists on the reactions of photosynthetic membranes. Photosynthetica 21, 296— 300.Roshchina, V. V., 1988: Characterization of pea chloroplast cholinesterase: ef­fect of inhibitors of animal enzymes. Photosynthetica 22, 20— 26.Roshchina, V. V., 1989: Biomediators in chloroplasts of higher plants. 1. The interaction with photosynthetic membranes as chemical sensors. Photo­synthetica 23, 197— 206.Soil, J., 1988: Purification and characterization of a chloroplast outer-enve­lope-bound, ATP-dependent protein kinase. Plant Physiol. 87, 898— 903.Suss, K. H., 1981: Identification of chloroplast thylakoid phosphoproteins. Evidence for the absence of phosphorylpolypeptide intermediates in the ATPase complex. Biochem. Biophys. Res. Commun. 102, 724— 729.Udenfriend, S., W. Lovenberg, A. Sjoerdsma, 1959: Physiologically active amines in common fruits and vegetable. Arch. Biochem. Biophys. 85, 487—490.34 A C T A  B O T . C R O A T . V O L . 49, 1990.R E G U L A T IO N  O F C H L O R O P L A S T  R E A C T IO N SS A Ž E T A KREGULACIJA REAKCIJA KLOROPLASTA SEKUNDARNIM M ETABOLITIM A ACETILHOLINOM  I BIOGENIM AM INIM AViktoriya V. Roshchina(Institut za p edolog iju  i fotosintezu A kadem ije znanosti SSSR, M oskva)Izolirani kloroplasti tretirani su sekundarnim metabolitima acetilho- linom, dopaminom, adrenalinom, noradrenalinom i serotoninom, koji su prisutni u biljnim stanicama. U životinja također dolaze ovi spojevi i imaju ulogu neurotransmitera ili medijatora. Svi osim serotonina u niskim koncentracijama (<  10-;! M) potiču necikličku fotofosforilaciju u tilako- idama. Acetilholin pospješuje izlaženje K ‘ i Na+ ijona iz intaktnih kloro- plasta, ali ne utječe tako i na Ca2" i Ma2". Dopamin, adrenalin, noradre- nalin i serotonin imaju suprotan učinak.Victoriya V. Roshchina Institute o f  Soil Science and Photosynthesis,U.S.S.R. A cadem y o f  Sciences SU-142292 Pushchino, M oscow  RegionA C T A  E O T . C R O A T . V O L . 49, 1990. 35

Regulacija reakcija kloroplasta sekundarnim metabolitima acetilholinom i biogenim aminima

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