PHOTOPUOSPHORYLATION IN CHTOROPLASTS AI{D IN DIGITONIN SUBCHLOROPLAST PARTICLES A thesis submitÈed to the University of Adelaide as a requirement for the degree of DOCTOR OF PHILOSOPHY by THAN-\TYIINT, B. Sc. (ttons) Departrnent of Botany ïJniuersity of Adelaide Jarunny L973 ADDENDUM InexperímentsdescribedinFig.Il,ChapterlllandTable t'r, IV, Chapter VIII , equivalent to 31,000 and 41,000 cPm re- spectively I^Iere added to Lhe reaction mixtures ' CONTENTS SUMMARY DECLARATTON ACIAVO',ILEDGEMENTS ABBREVIATIONS CHAETER T - GENERAL IXITRODUCTTON 1_- 20 A. GENERAL BACKGROUND 1- B, THE PHOTOSYNTHETIC ELECTRON ELOW CHATN 2 , f. Cornponents of the photosyrtthetic electron fLou chain = (a) 'q' 4 (b) PLastoquinone E (e) b-type cytoehromes o (Ð CytoeVwome f 7 (e) PLastocyanín I (f) tzl o U " Photosynthetì,c phosphorg Latì.on 1-0 (a) CyeLíc photophosphorglation 1-L (b ) Non-eyeLíe photophosphoz,yLatì,on LL ( e ) Ps eudoey elíc photophosphonyLatíon LL C. THE ENERGY CONSERVATTON HYPOTHESES 73 T. Ihe chenícaL hypothesis t¿12 n. The confovnationaL hypothesis L5 ilr. Ihe chemiosmotic hypothesis t¿ I il. l|i L Lí atn s hy p o th e s i s L7 D, THE PRESENY STUDY 79 CHAPTER II MATERIALS AND METHODS áJ- ¿O A PLANT MATERIAL 2L B ISOLATION OE CHLOROPLASTS 21 L. Uru¡aehed ehLonopLasts 21- 2. Sonicati.on of ehLoropLasts 2L 3. Washed ehLoropLasts 22 4, )smoticaLly shoeked ehLonopLast and chlonopLast ¿¿ fnagments C. PREPARATTON OE DIGTTONIN SUBCHLOROPLAST PARTTCLES ¿¿ o2 D. OXYGEN EVOLUTTON TßASUREMENTS aÐ o? E. PH MEASUREMENTS F. 32pí twcoapoRAlrov 24 G. REDUCED MTNUS DIFEERENCE SPECTRA OE CYTACHROMES ÎN ISOLATED CHLOROPLASTS AND DTGTTOIITN SUB- 24 CHLOROPLAST PARTTCLES H. LIGHT_INÐUCED REDOX CHANGES OF CYTOCHROMES 'C I, ASSAYS ¿o J. REAGENTS ¿o auyIE_E_!rI N1N-CyCt rc, PsEUD)CyCLrc AND cycfic 27 -45 PHOTOPHOSPHORYLATTONS TN TSOLATED PEA CHLOROPLASTS, ONE OR ThlO STTES 0E PH2SPH)RYITAII)N 0N IHE N)N-CYCLIC ELECTRON ELOW PATHI¿AY INTRODUCTTON 27 RESULTS 29 A. FECN AS THE ELECTRON ACCEPTOR Ðo B. MV AS THE ELECTRON ACCEPTOR SITES OE FECN REDI]CTION AND LOCATTON OF THE T,IO STTES 32 OE PHOSPHORYLATTON (ENERGY TRANSDUCTTON) ON THE NON-CYCLIC ELECTRON FLOT,,I PATHT¡AY D PMS - CATALY ZED PHOTOPHO SPHORY LAT TON S AND L T GHT- IN DU CED 35 PROTON UPTAT<E ?o DTSCUSSTON 70 A. NON-CYCLIC PHOTOPHOSPHORYLATION B. PSEUDO-CYCLTC AND CYCLIC PHO?OPHOSPHORYLATTON 43 CIIAPTER IV EFEECY OF UNCOWNERS ON THE PHOTOCHEMTCAL 46-64 ACTTVTTTES OF TSOLATND CHLOROPLASTS TNTRODUCTTON 46 A. NH .CL AND VALTNOMYCTN 46 4 RESULTS 46 DTSCUSSTON s0 co B. CCCP AND VALTNOMYCTN ua RESULTS 52 DTSCUSSTON 56 C. ARSENATE 59 RESUMS tro DTSCUSSTON 62 CHAPTER V EEEECT OF ELECTRON ELOW TNTTTBTTORS ON 65-80 YHE PHOTOCHEI,TTCAT ACYTVTNES OP ßOLATED CHLOROPLASTS AND DTGTTONTN SUBCHLORO- PLAST PARTTCLES TNTRODUCTION 65 A. ANTIMYCTN A 65 RESUUS 65 DTSCUSSTON 69 B. 2-ALKY-4-HYDRü{YQUTN0LLNE-N-0XTDE (H08N0) 7L RESUMS 71 DISCUSSNil 73 C, 2, 5-DLBE0M0-3-METHY[J-6-IS0PR0PYL-P-BENZ08UIN0NE (DBMIB ) 75 RESUffiS 75 DTSCUSSÏON 78 CHAPTER VÏ PHOTOPHOSPHORYLATTON IN DIGITONTN BL-95 SUBCHL1R)PLAST PARIICLES - LPH ot' AE? RELATIONSHTP OF THE 51.8-52OWN ABSORBANCE CHANGE TO PHOTOPHOSPHORY- LATTON IN CHLOROPLASTS AND PITOTOSYSTEM I SUBCHLOROPLAST PARTTCLES o1 INTRODUCTION at RESULTS B4 A ABSENCE OE THE LIGHT-TNDUCED PROTON UPTAI{E AND ó4 PRESENCE OE CYCLrc PHOTOPHOSPHORYLATTON TN DIGITONIN PHOTOSYSTEM I SUBCHLOROPLAST PARTICLES B EEFECT OF T¡NCOT¡PLERS ON PHOTOPHOSPHORYLATTON ÏN B5 DIGITONIN PHOTOSYSTEM T SUBCHLOROPLAST PARTICLES THE 51B-520wn ABS1RBANCE CHANGE IN CHL)R)PLASTS B7 AND DTGITONTN PHOTOSYSTEM T SUBCHLORAPLAST PARTTCLES DISCUSSION 89 CHAPTER VII CYTOCHROMES OE ISOLATED CHLOROPLASTS AND 96-1 23 DIGITONIN SUBCHLOROPLAST PARTICLES TNTRODUCTTON 96 RESULTS L00 A. CYTOCHROMES OE ISOLATED CHLOROPLASTS 100 L" Effect of CCCP and pCCp on the redoæ state of L03 cytochnomn b in isolated chLoropLasts rrn B. CYTOCHROMES OF DTGITONIN SUBCHLOROPLAST PARTICLES 1"04 FROM SPINACH AND PEA L. L0,000 æg particLes (PSH Papticles) 104 (d Effeet of CCCP on the redos state .of 106 cytochno.mn in 1-0r000 rg pa'r'tícLes ?rrn from spinach--- 2. 1441000 æg panticLes from pea chLoz'opLasts 1-07 (PSI PartíeLes) C" LIGHT-INDUCED REDOX CHANGES OE CYTOCHROMES L0B L. ChLoroplasts 1AB 2o 70,000 æg patticLes LLL 3, 1441000 æg partì.cLes 1_L3 DTSCUSSTON 11.3 A" LTGHT-TNDUCED REDOX CHANGES LL5 L, ChLoropLaets 115 2. L01000 ry partíeLes L2L 3. L441000 æg pantieLes L23 CI]APTER VTfi - ELECTRON ELOTI FROM DCPTPH^ TO MV IN L24-L3ø TNTACT CHLOROPLASTS AND NfiCNONM PHOTOSYSTEM T SUBCHLOROPUST PARIICI'ES, TNYROÐUCTÏAN 124 RESULTS L25 DTSCUSSTON 128 CHAPTER TX - GENERAL DTSCUSSTON L32-1-36 REFERENCES L37-L55 SUMMARY l. chloroplasÈs r^rere isolated from pea (Píswrt satiuum var. Green Feast) and spinadn (spinacea, oLay.acea L.> leaves. The chloroplasts exhibited phoÈosyntheÈic eontrol with ferri- cyanide (FeCN) and nethyl víologen (1"1V) as electron accePtors' giving control raÈios ranging from 3'0-6'0' ADP/O raLios were also determined, and ranged froro l-.3-l-.9 wíth FeCN and MV. 2. Ibing the quinone analog, 2r5rdibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB) as an inhibitor of non-cyclic electron flow, íÈ was possible to determine the'sites of FeCN reducËion on the photosynthetic electron flow chain. Two sites of FecN reductÍon are postulated, one phosphoryl-ating and the other non-phosphorylating. From this, it was deduced Ëhat the energy transducing sites are locaÈed between the t\^Io sítes of FeCN reduction. 3" Phenazine methosulphaÈe (PMS) is generally accepted as being a cyclic elect.ron acceptor. However, it was found to catalyze a light-Índuced oxygen uptake under red light illumination which was DCMU sensitive . It also cataLyzes aerobic phosphorylation, under red light illurnination, and this was courpletely inhibited by DCMU. These observations indicate Èhat PMS was cataLyzing pseudo-cyclic electron flow" True cyclic phosphorylaÈion could be observed in the presence of DCMU' when an exogenous reducÈant (e. g. ascorbate or NADH) \¡ras Present to reduce PMS non- enzymically. It was suggested that PMS can catalyze both cyclic and pseudocyclic phosphorylaÈion simultaneously, as P/2e- determinations, gave values above 2.0 4. The effect of some inhibitors of electron flor,rr and uncouplers of photophosphorylation on the various photochemical activities of chloroplasts and digitonin subchloroplast particles \^tere studied, and Èheir sites of acËíon determined as much as possible. 5. Subchloroplast particles r¡tere prePared by Èreatment rrrith digitonin. Heavy particles (10,000 and 30,000 xg) which contained only PhoÈosystem II and light particles (50'000 and 14r000 xg) wíth Photosystem I actívity only were obtained" The Photosystem I particles, although exhíbiÈing no lighÈ-induced proton uptake ê actívity, \¡rere capable of eatalyzing cyclic photophosphorylation with PMS, at rates comparable to whole chloroplasÈ,s. The effect of uncouplers on cyclic photophosphorylation in these particles was studied and the relevance of these results to the chemiosmotic hypothesis is discussed, 6. An attempt \./as made Ëo characterise and study the light-induced redox changes of the cytochromes present in chloroplasÈs, Photo- system II and Photosystem I subchloroplast part,icles. 7. There is controversy concerning phosphorylaÈÍon accompanying electr,on flow from DCPIPH2 Èo l"lV or NADP in the presence of DCMU. This arises from the effects of uncouplers and cyclic photophosphorylation whieh may accomPany the non-cyclic electron flow. Thus; elecÈron flow from DCPIPH2 to ÌfV in chloroplasts and Photosystem I particles has been reinvestigated" Although there is no doubt that non-cyclic electron flow ís stimulated by uncouplers any ATP forned nay be a result of simultaneous cyclic phosphorylation. A possible explanation of the effects of uncouplers on non-cyclic flow is proposed; :--Jè _ -_ - DECLAR,ITTON The invesËigations describe<i in this thesis r'¡ere carríed out i.n the Departnent of BoËany, university of Adelai<ie, from NIay L969 to SepteDbex 1-972. One paPer I^las wriLten and ttiree more are in PreParaÈíon: 1. Light-indueed redox changes of eytochrome br-n: bY J.t"1. Anderson, Than-NyunË and N.K. Boardman (in press, Arch. Biochem. Biophys. ) 2. Phenazine uethosulphaËe as a pseudo-cyclic and cyclíc electron acceptor: by Than-ÌiyunÈ and J.T. I{iskich (manuscript in preparation). 3. Phosphorylaring and non-phosphorylation eites of ferri- cyanide reduction by isolaÊed pea chloroplasts: by Than-IJyunt and J.T. I^Iískich (uanuscript in Preparatíon). 4. Studies on the effecËs af 2,5-dibrono-3-nnethyl-6-isopropyl- p-benzoquinone on ísolated pea chloroplasts: by Than-NyunÈ and J.T. I{iskich (manuscript ín preparation). To Ëhe auÈhorrs belief anC knoi^rledge, this Ëhesis contains no material- previously subuíÈted fot a degree in any University by the author or by a:ry other person' except r,rhere due reference is made in tire Ëext. + Than-Nyunt ACI{]VOWLEDGEMENTS I am greatly indebted to Dr. J.T. I¡triskich for his supervision, guidance and encouragemenË Èhroughout the course of this work. I would also like Êo Èhank Dr. N.K. Boardman, C.S.I¡R.O., Division of Plant Industry, Canberra, for advice and helpful discussion. Thanks are also due Ëo Drs. K"R. trùest, J.M. Rungie and J.M. Anderson for the helpful discussions I had with Èhem during the course of mY studY. I would also like to thank Professors Sir R.N" Robertson and P.G. Martin for Èhe use of laboratory facilities. Thanks are also due Èo Dr. N.K" Boardman for kindty letÈing me use Èhe Aminco-Chance dual wavelength spectrophotometer and the Cary 14R specÈrophotometer, thus making it. possible to caxty out the studies on cytochromes and Ëhe light-induced absorbance change at Sl8nm" Acknowledgements are made to Miss Bronwyn Cooper for typing the thesis and Mrs.