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Developmental and immunological studies on chlorophyll proteins White, Michael J. 1987

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D E V E L O P M E N T A L AND IMMUNOLOGICAL STUDIES ON CHLOROPHYLL PROTEINS by MICHAEL JOHN WHITE B.Sc, McMaster University, 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 4 May 1987 © Michael John White, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for Financial gain shall not be allowed without my written permission. Department of Botany The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: 5 June 1987 A B S T R A C T All the chlorophyll in higher plants is bound to specific proteins. These chlorophyll-proteins absorb light energy which is used by the electron transport processes of the photosynthetic apparatus. The synthesis and assembly of the photosynthetic apparatus and chlorophyll proteins is regulated by light and other factors which are incompletely understood. Antibodies were used to investigate the accumulation of chlorophyll proteins in plant systems which were completely lacking one or more of the factors involved in chlorophyll protein synthesis or degradation. Immunological cross-reactions among the chlorophyll a + b apoproteins were observed and characterized, and theoretical work was undertaken to predict the probability that immunological cross-reactions may arise due to chance arrangement of amino acids. Chlorophyll a+b protein complexes associated with photosystem I were isolated and characterized. These included a light-harvesting antenna complex (LHCI), which was shown to contain Chi a, some Chi b, and four polypeptides of 21-24 kDa. This complex was isolated from CP Ia complexes, which also contain the P700 chlorophyll a protein, CP I. Antibodies were raised against the chlorophyll a + b protein complexes of both the photosystem I antenna (LHCI) and the photosystem II antenna (CP II and CP 29), and against the chlorophyll a protein containing the photosystem I reaction center (CP I). Antibodies to LHCI were obtained by using highly purified CP Ia as an antigen. When antisera to Chi a + b proteins were reacted with the entire ii electrophoretic spectrum of denatured thylakoid proteins, they cross-reacted with the polypeptides belonging to other Chi a+b proteins. The strongest cross-reactions were observed between antibodies to CP Ia and the polypeptides of the photosystem II complexes, CP II and CP 29. These cross-reactions observed with anti-CP Ia were shown not to be due to antibodies to CP I, but rather to antibodies directed against the photosystem I antenna complex, LHCI. Affinity-purification of the anti-CP Ia antibodies did not eliminate these cross-reactions. The data suggest there are amino acid sequence similarities among the chlorophyll a/6-binding polypeptides. Theoretical work was undertaken to determine if these cross-reactions were expected due to chance alone or not. Attempts to address this question led to the synthesis of a mathematical model to predict the probability that antibodies will react with proteins other than the antigen they were raised against. This theoretical work was done as a collaboration with Peter Sibbald of our lab, with input from Dr. Michael Waterman, one of the mathematicians whose work this model is based on. The sequence similarities predicted to exist among the chlorophyll a+b proteins appear to be more extensive than those which would be expected to arise due to chance alone. It is postulated that the Chi 0+6 proteins may have arisen from a common ancestral gene. At least some polypeptides belonging to each of the three Chi a + b proteins, including those of CP 29 and LHCI, were found in the Chi Mess barley (Hordeum vulgare L.) mutant chlorina f2. Similar experiments were performed on barley grown under intermittent light, which also completely lacks iii Chi b. Only two polypeptides belonging to the Chi a+b proteins were found under these conditions. Taken together with published studies in the literature, these results provide evidence for the existence of differing posttranscriptional controls among the various chlorophyll proteins, and between these two systems. Antibodies were also used to determine the kinetics of synthesis of the chlorophyll-binding polypeptides during the light-induced greening of dark-grown barley. The chlorophyll a + b antenna polypeptides of both photosystems were absent in the dark. They were first detected after 1-2 hours of illumination, and then increased in amount exponentially. The kinetics of accumulation are almost identical for all the polypeptides belonging to the chlorophyll a+b protein complexes, suggesting their synthesis may be regulated by the same mechanism despite the existence of the differing posttranscriptional controls mentioned above. CP 43, a chlorophyll a antenna protein of photosystem II, appeared to follow the same time course. In contrast to some reports in the literature, small amounts of the photosystem I P700 chlorophyll o protein were detected in etiolated plants. This protein also shows a light-induced increase, showing that light plays an important role in its synthesis or degradation, even if it is not an absolute requirement for transcription and translation. iv T A B L E OF CONTENTS Abstract ii List of Figures viii List of Abbreviations ix Acknowledgements x I. INTRODUCTION 1 1. Photosynthesis and electron transport 1 2. Chi proteins 8 a. Discovery of the first Chi proteins: CP I and CP II 8 b. Discovery of the other Chl proteins 10 c. Further detail on the Chl a proteins 14 d. Further detail on the Chl a + b proteins 17 3. Factors affecting Chl protein synthesis 23 a. Experimental systems for studying Chl protein synthesis 23 b. The Chl b-less barley mutant chlorina fZ 24 c. Plants grown under intermittent light 26 d. The etioplast to chloroplast conversion 26 e. Transcriptional control of protein synthesis 28 f. Posttranscriptional control of protein synthesis 31 4. Molecular techniques for studying Chl proteins and their synthesis 33 5. Properties of antibodies and antigens: Immunological specificity and cross-reactivity 37 II. MATERIALS AND METHODS 42 1. Plant material and growth conditions 42 2. Preparation and Detergent Solubilization of Thylakoid Membranes 42 3. Polyacrylamide Gel Electrophoresis 43 4. Preparation of Chlorophyll Protein Complexes 45 5. Spectra and Chlorophyll a/b ratios of Purified Chlorophyll Protein Complexes 47 6. Preparation of antisera 47 7. Western Blotting and Immunoenzymatic Detection of Chlorophyll Proteins 48 8. Affinity purification of anti-CP Ia 49 9. Developmental Experiments 50 a. Preparation of Whole Cell Protein Extracts 50 b. Electrophoresis and Immunoblotting 51 c. Quantitation of Immunoblots 51 III. RESULTS 53 A. PREPARATION AND CHARACTERIZATION OF A PS I CHLOROPHYLL A+B PROTEIN COMPLEX 53 v 1. Isolation and characterization of the two CP Ia bands and the PSI antenna complex 53 2. Effect of detergents on the dissociation of CP Ia complexes . 59 3. CP Ia complexes can be obtained directly from intact chloroplasts 60 . B. IMMUNOLOGICAL CROSS-REACTION AMONG CHLOROPHYLL A + B-BINDING POLYPEPTIDES 61 1. Preparation of antibodies to CP Ia 61 2. Antibodies to CP Ia cross-react with LHCII and CP 29 polypeptides 65 3. Specificity of cross-reactions observed with anti-CP Ia 66 4. Preparation of Antibodies to CP D and CP 29 69 5. Antibodies to CP II and CP 29 also cross-react 71 C. STUDIES ON A BARLEY MUTANT LACKING CHLOROPHYLL B ... 83 1. Chlorina f2 is depleted in the major LHCII polypeptides 83 2. Chlorina f2 contains the CP 29 polypeptides 84 3. Chlorina f2 contains the PSI antenna (LHCI) polypeptides .... 87 4. CP la-like complexes in Chi . b-less barley 89 D. THE SYNTHESIS OF CHLOROPHYLL-BINDING POLYPEPTIDES DURING GREENING OF ETIOLATED BARLEY 93 1. Rationale and Experimental Objectives 93 2. Controls 94 3. Synthesis of polypeptides of the three Chi a+b proteins 98 4. Synthesis of the CP 43 apoprotein 98 5. Synthesis of the CP I apoprotein ; 102 6. Effect of Sample Preparation 103 E. CHLOROPHYLL-BINDING POLYPEPTIDES IN BARLEY GROWN UNDER INTERMITTENT LIGHT 109 1. Plants grown under intermittent light have many similarities to Chi b-less mutants 109 2. Intermittent light barley lacks LHCI and most LHCII polypeptides 110 3. Intermittent light barley contains the CP 29 polypeptides ... I l l F. MATHEMATICAL MODELING OF IMMUNOLOGICAL CROSS-REACTION 115 1. A null hypothesis for immunological cross-reaction: Some cross-reaction is due to chance alone 115 2. Dimensions of the antibody combining site 116 3. Comparing two polypeptides for matching amino acids is analogous to flipping a row of coins 118 4. Determining the probability of shared antibody binding sites due to chance alone : 121 IV. DISCUSSION 129 1. Characterization of Chi a+b proteins 129 2. Immunological relatedness of the Chi a+b proteins 133 3. Modelling immunological cross-reaction 148 vi 4. Regulation of Chi a+b apoprotein synthesis: Chlorina f2 and intermittent light barley 157 5. Greening of etiolated barley 162 a. Chi a+b proteins 162 b. Chi aproteins 168 V. REFERENCES 172 Appendix 1: List of Addresses 190 vii List of Figures Fig. 1. The four major macromolecular complexes of thylakoid membranes 3 Fig. 2. Unstained gel of thylakoids solubilized in 300 mM octylglucoside 12 Fig. 3. CP Ia contains CP I and the PSI antenna CP .55 Fig. 4. Polypeptide composition of CP Ia and the PSI antenna CP 56 Fig. 5. Visible absorption spectra of CP Ia, CP I and the PSI antenna CP. ..58 Fig. 6. Anti-CP Ia cross-reacts with polypeptides of LHCII and CP 29 62 Fig. 7. Dot blots reacted with anti-CP II and anti-CP Ia 64 Fig. 8. Cross-reaction is due to antibodies to the PSI antenna CP 67 Fig. 9. Affinity-purified anti-CP Ia also cross-reacts 70 Fig. 10. Purified CP II and CP 29 used as antigens 72 Fig. 11. Dot blots reacted with anti-CP II and anti-CP 29 73 Fig. 12. Immunoblot reacted with anti-CP II 75 Fig. 13. Polypeptide composition of LHCII vs. CP II 76 Fig. 14. Immunoblot reacted with anti-CP 29 77 Fig. 15. Summary figure of immunological cross-reactions 79 Fig. 16. Chl Mess barley vs. anti-CP II 85 Fig. 17. Chl 6-less barley vs. anti-CP 29 86 Fig. 18. Chl 6-less barley vs. anti-CP Ia 88 Fig. 19. Chl 6-less barley: CP la-like bands 90 Fig. 20. Chl 6-less barley: CP la-like bands lack PSI antenna polypeptides 91 Fig. 21. Etiolated barley: Total Chl and Chl alb ratio vs. time in light 95 Fig. 22. Etiolated barley: Immunoblot reacted with anti-CP 1 96 Fig. 23. Etiolated barley: Control proteins vs. time in light 97 Fig. 24. Etiolated barley: Chl a+b apoproteins vs. time in light 99 Fig. 25. Etiolated barley: CP 43 apoprotein vs. time in light 101 Fig. 26. Etiolated barley: CP I apoprotein in normal and extreme dark 104 Fig. 27. Etiolated barley processed as in Kreuz et al vs. anti-CP 1 105 Fig. 28. Etiolated barley processed as in Takabe et al. vs. anti-CP 1 107 Fig. 29. Intermittent light barley: immunoblots of whole cell extracts 112 Fig. 30. Intermittent light barley: immunoblots of thylakoids 113 Fig. 31. Probability of cross-reaction as a function of protein lengths 128 viii LIST OF ABBREVIATIONS Chi = chlorophyll CP = chlorophyll protein (if used as a prefix or suffix) EDTA = disodium ethylenediaminetetraacetate LHC = light-harvesting complex (used as a prefix) i.i.d. = independently and identically distributed PSI = photosystem I PSII = photosystem II SDS = sodium docecyl sulfate ix ACKNOWLEDGEMENTS I would especially like to thank my wife Adrian for her continued support and for technical assistance in the preparation of figures. Special thanks also go to my supervisor Dr. Beverley Green for many hours spent editing papers and this thesis, to Dr. Edith Caram for teaching me numerous lab techniques, to Peter Sibbald for many useful discussions and lab samples, and to all of the above for an endless list of ideas and edits. Murray Webb and Gopal Subramaniam of our lab are both thanked for many interesting discussions. The theoretical work on immunological cross-reaction would not have been possible without the collaboration of Peter Sibbald and the contributions of Drs. Michael Waterman and Joe Watkins. Drs. R. Martin . and R. Stace-Smith were both extremely helpful in teaching me to prepare antisera. Thanks go to Drs. Andrew Staehelin, Klaus Apel and Winslow Briggs for useful ideas and discussions. I am indebted to Drs. Elaine Tobin, Nam-Hai Chua, Lee Mcintosh, Terry Bricker and Elaine Moase for their antibodies to CP II, Chlamydomonas proteins 5 and 6, DI, CP 47 and CF1 respectively. The technical assistance of Danny Chui and Don Krawciw is greatly appreciated. Finally, I thank all the people in the department or at U.B.C. who taught me courses or assisted with lab materials, equipment, and many other favours. x I. I N T R O D U C T I O N A primary goal of this research was to study chlorophyll (Chl) protein synthesis, using antibodies as sensitive and specific tools for the detection of the Chl-binding polypeptides. This included studying those factors which regulate Chl protein synthesis, and their effects on the different kinds of Chl proteins. Thus a major focus of this work is the synthesis of these light-harvesting proteins during photoregulated plant development. This thesis does not attempt to answer questions regarding the function or mechanism of the photosynthetic apparatus, although some findings may be related to the light-harvesting or other functions of the Chl proteins. Immunological cross-reactions among antibodies to the Chl a + b proteins were observed and characterized, and the investigation of immunological cross-reaction became an important part of this work. Theory was developed to explain the causes of this cross-reaction. These antibodies and others were used to investigate the accumulation of Chl proteins in systems lacking Chl b only, lacking Chl b and continuous light, or lacking both Chls a and b and continuous light. 1. Photosynthesis and electron transport Photosynthesis is an essential process for plant growth and for all the ecological food chains dependent on plants. Carbon, nitrogen and sulfur arrive at the plastid in the form of C 0 2 , N 0 2 " and S O , 2 - [Salisbury and Ross 1978]. 1 2 CO 2 is attached to ribulose bisphosphate by ribulose 1,5 bisphosphate carboxylase and processed through the Calvin cycle to become carbohydrates. These carbohydrates are then used as an energy source and as a carbon source for the general metabolism of the cell. NO 3 " is reduced to NO 2 " in the cytoplasm, and N 0 2 " is reduced to N H | , + by nitrite reductase in plastids [Oaks 1985]. Both nitrogen and sulfur are ultimately incorporated into amino acids, nucleotides and many other compounds. The energy and reducing power for these processes is supplied by ATP and NADPH, direct products of photosynthesis. Light energy harvested by chlorophyll (Chi) proteins is used to split water into oxygen (0 2), protons and electrons. In the currently accepted Z scheme of photosynthesis, photosystem II (PSII) provides the energy needed to split water, and photosystem I (PSI) provides the electrons with the additional energy needed to reduce NADP* to NADPH. The source of this additional energy is light harvested by Chi proteins, in this case those associated with PSI. A diagram outlining the positions of the four major macromolecular complexes of thylakoids is shown in Fig. 1. The splitting of water is mediated by a peripheral membrane protein complex associated with PSII. The prosthetic group catalyzing this reaction is a cluster of four Mn atoms, the exact structure of which is not yet known. Also involved in this process are three extrinsic membrane polypeptides of approximately 33-34, 23-24 and 16-18 kDa [reviewed in Amesz 1983, Govindjee 1984 and Govindjee et al. 1985]. These hydrophilic polypeptides are located on the inner thylakoid surface [Staehelin et al. 1987] and are encoded in nuclear 3 Fig. 1. Diagram depicting the four major macromolecular complexes of thylakoid membranes in mature higher plant chloroplasts. The four complexes are PSI, PSII-water splitting complex, cytochrome fb 6 and coupling factor. This figure is adapted from Anderson [1986] 4 genes [Westhoff et al. 1985]. A number of PSII components are involved in transferring electrons from P680 to plastoquinone. Following absorption of light by P680, an electron is transferred to pheophytin [Klimov et al. 1977, Klimov et al. 1980, Murata et al. 1986], and eventually stabilized on Q^, the primary quinone acceptor of PSII. is believed to be bound to one or more of the Chi a protein CP 47 (also known as CPa-1), the Qg protein (also known as the 32 kDa herbicide-binding protein or DI), or a homologous protein of unknown function, D2. is likely associated with iron, as is the quinone in purple photosynthetic bacteria such as Rhodopseudomonas [Okamura et al. 1975, Shopes and Wraight 1985]. One or two electrons are then passed to the quinone Qg, located on a binding site on the Qg protein. This produces the semiquinone anion Qg" or the quinol Qg 2 ", one or both of which exchange electrons with the plastoquinone pool [Kyle 1985]. Cytochrome b559 is a two subunit, two heme cytochrome associated with most PSII preparations, and is seen as bands of about 10 kDa and 6 kDa on SDS polyacrylamide gels. A number of functions have been proposed for this cytochrome, but it does not appear to be linked to electron transport in PSII while the water-splitting enzyme is operational [Cramer et al. 1986]. For this reason, and because cytochrome b559 is readily photooxidized by PSII when water splitting is inhibited, it has been proposed that cytochrome b559 acts to remove oxidants produced at the PSII reaction center during chloroplast development or in thylakoids damaged by heat, chilling or water stress [Cramer et al. 1986]. 5 In addition to the two protons released from each water molecule, two other protons are believed to be deposited inside the thylakoid lumen for each plastoquinone molecule which crosses the membrane [Mitchell 1976, Govindjee 1984]. This occurs when a plastoquinone molecule accepts an electron pair from a quinone associated with PSII, thereby becoming negatively charged. It cannot cross the membrane until this charge is neutralized by the uptake of two protons from the stroma. The electron pair carried by the plastoquinone is transferred to the cytochrome fb 6 complex and the proton pair released into the thylakoid lumen. This net deposition of protons within the thylakoid lumen creates a transmembrane proton gradient. The electrochemical energy stored in this gradient is then used to synthesize ATP from ADP and inorganic phosphate [Mitchell 1961, Jagendorf 1967]. This reaction is catalyzed by coupling factor, one of the four major macromolecular complexes in thylakoid membranes. In addition to PSI, the PSII-water splitting complex and coupling factor, cytochrome fb 6 complex is the fourth major macromolecular complex in thylakoid membranes. It contains the 34 kDa polypeptide of cytochrome f, the 23 kDa polypeptide with two cytochrome bg hemes, the high potential Rieske-FeS protein of 20 kDa, polypeptides of 17 and 5 kDa, and bound plastoquinol [Haehnel 1984]. Studies with antibodies have indicated that cytochrome f and the soluble portion of the Rieske-FeS protein containing the F e 2 S 2 cluster are located on the lumenal surface of the thylakoid membrane [Hauska et al. 1983]. Thus the reduction of the Rieske-FeS protein by plastoquinol and the oxidation of cytochrome f by plastocyanin, a blue copper protein, occur at the inner membrane surface [Haehnel 1984]. 6 However, there are still unanswered questions as to the roles of cytochrome f and plastocyanin in the reduction of P700, and as to the function of cytochrome b„ in the Q cycle [Cramer and Whitmarsh 1977, Crofts and b Wraight 1983, Haehnel 1984]. Following reduction of the plastoquinone pool, cytochrome f is reduced with a half-time of about 10-20 milliseconds [Cramer and Whitmarsh 1977, Crofts and Wraight 1983]. Due to the lateral segregation of PSII and PSI into granal and stromal lamellae respectively [Armond and Arntzen 1977, Anderson and Melis 1983], plastoquinone would have to move from the granal to the stromal lamellae in the 10-20 milliseconds. However, the measured diffusion coefficient for quinones (D = 10"9 cm2/s) does not appear to be sufficiently large to allow plastoquinone to move this quickly [Crofts and Wraight 1983, Haehnel 1984]. It has therefore been suggested that a molecule such as plastocyanin (D = 3-4 x 10" 9 cm2/s) is also involved in this processs [Crofts and Wraight 1983, Haehnel 1984]. Nevertheless, after cytochrome f has been reduced there appears to be a 5-15 millisecond lag in the reduction of plastocyanin, prior to the reduction of P700 in less than 1 millisecond [reviewed in Cramer and Whitmarsh, 1977]. It has therefore been suggested that the diffusion coefficient of plastoquinone may be as large as 10" 7 cm2/s, and thus be consistent with long range electron transfer [Haehnel 1984]. P700 is the primary donor of PSI and is located on the P700-CW o-protein also known as CP I [Malkin 1982], (CP I and the other Chi proteins are discussed in detail in the following section.) P700 was initially believed to be a special pair of Chi a molecules based on its electron paramagnetic resonance signal. This interpretation is further supported by spectroscopic data and the 7 isolation of spinach (Spinacia oleracea L.) chloroplasts of two CIO epimers of Chl a per P700 molecule [Watanabe et al. 1985, Setif and Mathis 1986]. However, a newly discovered Chl, Chl RCI, occurs in a 1:1 molar ratio with both P700 measurements and P700 reaction centers [Senger et al. 1987]. It is found in all photosynthetic organisms studied to date, but never in PSII particles. Its absorption maxima are shifted to longer wavelengths than Chl a by 3-4 nm in the blue and 8 nm in the red, and it is chlorinated at the 6-carbon of the porphyrin ring (the carbon adjacent to carbon 1) [Senger et al. 1987]. Thus the chemical identity of P700 remains undetermined. Absorption of light by P700 causes electrons to be transferred through a series of acceptors designated as A 0 , A 1 ( X, B and A. After illumination of PSI particles with a 1.5 picosecond laser flash, P700*A" is formed in 13.7 ± 0.8 picoseconds [Wasielewski et al. 1987]. P700, A 0 and A , occur in a 1:1:1 molar ratio. It has been suggested that A 0 and A^ may be monomeric Chl a anions [Haehnel 1984], but recent evidence suggests that A^ is a menaquinone [Bender et al. 1987]. X, also known as A 2 , is likely an iron sulfur cluster located on CP I [Sakurai and San Pietro 1985, Golbeck and Cornelius 1986, Warden and Golbeck 1986]. A and B are likely closely interacting Fe 4 S|, centers attached to ferredoxin molecules. It has been suggested that one or both play a role in the reduction of soluble chloroplast ferredoxin, leading to the reduction of N A D P + , or that one functions in cyclic electron transport around PSI [Malkin 1982, Haehnel 1984]. 8 2. Chi proteins o. Discovery of the first Chi proteins: CP I and CP II All the Chi in higher plants is bound to specific thylakoid proteins, along with carotenoids, as Chi proteins (also called Chi protein complexes) [Thornber 1979, Markwell et al. 1979]. These Chi proteins are associated with photosystems I and II and are the primary mediators of the light-harvesting process. Light absorbed by these Chi proteins is transferred to the reaction center Chi molecules, P680 and P700, of photosystems I and II respectively [Haehnel 1984]. CP I, the P700 Chi o protein [Thornber 1975, Malkin 1982] and CP II (LHCII), the major Chi a + b antenna complex associated mostly with PSII [Thornber and Highkin 1974, Thornber 1975], were the first Chi proteins to be isolated from higher plants. In these initial experiments plant material was treated with SDS at room temperature, and the SDS extracts electrophoresed on polyacrylamide gels containing detergent [Thornber 1975]. Three chlorophyll-containing zones were resolved on these gels and termed Component or Complex I, H and III in order of increasing electrophoretic mobility. Zones I and II were shown to be Chi proteins while zone III contained only free pigment but no protein [Thornber 1975]. A number of other Chi proteins have since been discovered. Since the Chi, carotenoids and other prosthetic groups associated with Chi proteins are not covalently attached, most Chi proteins readily dissociate unless special precautions are taken to keep them intact. Thus under the conditions described above the Chi proteins other than CP I and CP II 9 dissociated into their constituent polypeptides and prosthetic groups, and therefore were not present as green bands on these gels. However, CP I and CP II are not only the most abundant Chl proteins in higher plants, but appear to be more resistant to heat denaturation. This is especially true for CP I, which can be heated at 65 °C for 15 min or longer without being totally denatured [M.J. White, unpublished results]. The combination of these two factors explains how CP I and CP II were detected under conditions where the other Chl proteins were not. If the SDS extraction of thylakoids is performed at 4°C, oligomers of CP II can also be obtained [Hiller et al. 1974, Anderson and Levine 1974, Hayden and Hopkins 1976, Thornber 1979]. CP II is also known as LHCII, LHCP or LHC depending on the method of preparation or on arbitrary usage. The terms LHCP and LHC are now less appropriate due to the discovery of LHCI (discussed below), and are not used hereafter. CP II/LHCII contains at least two polypeptides of approximately 26 and 27 kDa, although differences in molecular weight or polypeptide composition occur between species [e.g. Thaler and Jay 1985]. The method of purification can also affect polypeptide composition. Barley LHCII prepared by cation precipitation contains an additional minor 25 kDa polypeptide not found in barley CP H prepared by gel electrophoresis [Results]. Similar results have been obtained with some other plant species [Machold 1981]. 10 b. Discovery of the other Chi proteins Hayden and Hopkins [1977] also obtained a Chi a protein migrating between CP I and CP II, just ahead of one of the CP II oligomers. This Chi a protein band was named complex IV, but is now known as CPa. Since then the use of detergents other than SDS has led to the discovery of still more Chi proteins. By using zwitterionic detergents such as Deriphat 160 or nonionic detergents such as octyl-/3-D-glucopyranoside (octylglucoside) instead of the anionic detergent SDS it is possible to recover almost all of the Chi as Chi proteins, greatly reducing the amount of Chi in the free pigment zone [Markwell et al. 1979, Camm and Green 1980]. Other detergents such as deoxycholate and digitonin have also been used successfully to obtain intact Chi proteins [Waldron and Anderson 1979, Interschick-Niebler and Lichtenthaler 1981]. Camm and Green [1980] extracted thylakoids at 4°C with 30 mM octylglucoside to solubilize preferentially the Chi proteins associated with PSII. The CPa band was resolved into two distinct Chi a proteins, CP 47 and CP 43. An additional Chi a+b protein, CP 29, was resolved which migrates just behind the monomeric CP II band. The numbers following the CP designation in CP 47, CP 43 and CP 29 are the apparent molecular weights of these complexes when electrophoresed on 10 % polyacrylamide gels [Camm and Green 1980]. CP 47 and CP 43 have since been resolved in other gel systems and are also known as CPa-1 and CPa-2. In these experiments of Camm and Green [1980] the PSII Chi proteins 11 were isolated from a high speed supernatant while most of the CP I remained in the pellet. This could be demonstrated by solubilizing the pellet with SDS and electrophoresing it under nondenaturing conditions to obtain a green CP I complex. The use of 300 mM octylglucoside, instead of 30 mM, completely solubilizes thylakoid membranes including the Chl proteins associated with PSI. Under these conditions two additional Chl proteins with lower electrophoretic mobility than CP I, designated CP Ia top and bottom are detected [White and Green 1987a, Results]. All these Chl proteins are shown in Fig. 2. These CP la complexes contain both CP I and its associated antenna [Results]. The initial experiments performed with SDS at room temperature indicated that approximately 10 % of the total Chl was in CP I. Since then the use of different detergents for solubilizing thylakoids and the use of improved electrophoretic techniques suggest that approximately 30 % of the total Chl is associated with PSI [Malkin 1982], but approximately 50 % of this Chl occurs in an associated Chl a + b antenna complex, LHCI [Ortiz et al. 1984]. Up to 50 % of the total Chl in thylakoids and most of the Chl b is accounted for by LHCII (CP II and its oligomers) [Thornber 1975]. The remaining Chl is associated with CP 47, CP 43 and CP 29. All the Chl a + b proteins bind both Chls a and b, but it has not been demonstrated that every polypeptide in these proteins binds both Chls. Throughout this thesis the term Chl a/fe-binding polypeptide(s) is therefore used to refer to the polypeptides rather than the complete Chl a+b protein. The term Chl a+fe-binding polypeptide may also be used provided that this is kept in mind. Fig. 2. Unstained polyacrylamide gels of thylakoids solubilized in 300 mM octylglucoside. Electrophoresis is on 7.5 % (a), 10 % (b) and 15 % (c) polyacrylamide gels containing 0.1 % SDS. In addition to the Chi proteins I, II 47, 43, 29 and II are two CP Ia bands, designated CP Ia top (t) and CP la bottom (b). CP II* is an oligomer of CP II (LHCII). 13 Some of the polypeptides associated with these proteins may not bind any Chl at all. A minor Chl a+b protein, CP 24, which migrates just ahead of CP II on polyacrylamide gels has recently been reported [Dunahay and Staehelin 1986]. A green band running in this position, probably CP 24, can be seen in lane c of Fig. 2. It accounts for less than 2 % of the total Chl in PSII preparations, and less than 0.5 % of the total Chl loaded on preparative gels [Dunahay and Staehelin 1986]. Its absorption and fluorescence emission spectra, the molecular weight of its polypeptides and its antigenicity with certain monoclonal antibodies suggest that it may be part of LHCI [David Simpson, pers. comm.]. However, its association with PSII particles and the immunological cross-reaction of antibodies to Chl a+b proteins (discussed later) leave the exact identity and function of CP 24 unresolved at present. The same Chl proteins appear to be present in all green algae, bryophytes and vascular plants studied to date. Thylakoids of the green alga Acetabularia, mosses, Psilotum, and angiosperms produce approximately the same electrophoretic profile of Chl proteins when solubilized with octylglucoside, although distinct bands corresponding to CP II and CP 29 are not resolved in all species [Camm and Green 1981]. CP I has been found in all organisms known to contain P700, including all eukaryotic and prokaryotic algae and Euglena, and may be ubiquitous in green plants [Thornber 1975]. Antibodies to CP I of Swiss Chard (Beta vulgaris L.) react with the CP I apoprotein from spinach, from the unicellular green alga Chlamydomonas and even from the thermophilic 14 cyanobacterium Mastigocladus laminosus, although the intensity of reaction with the latter is considerably reduced [Nechushtai et al. 1983]. Antibodies to a lower molecular weight polypeptide, subunit II of PSI, also reacted with the corresponding polypeptide among all these species [Nechushtai et al. 1983]. The Chl a+b proteins are less evolutionarily conserved than the Chl o proteins, and appear to be restricted to vascular plants, bryophytes and green algae with a few exceptions such as Euglena [Lee et al. 1985] and certain cyanobacteria-like organisms such as Prochloron [Giddings et al. 1980, Hiller and Larkum 1985]. Nevertheless, LHCII shows a substantial degree of immunological relatedness across a large variety of species. Monoclonal antibodies to pea (Pisum sativum L.) LHCII react with the corresponding polypeptide(s) in thylakoids of angiosperms, the fern Nephrolepsis and Chlamydomonas reinhardii [Thaler and Jay 1985]. However, polyclonal antibodies to spinach LHCII did not react with any polypeptides in Prochloron [Hiller and Larkum 1985]. c. Further detail on the Chl a proteins In addition to Chl a, CP I also contains smaller amounts of j3-carotene [Rawyler et al. 1980]. and phylloquinone (vitamin K , ) [Interschick-Niebler and Lichtenthaler 1981]. If CP I is totally heat-denatured to remove all prosthetic groups and electrophoresed on polyacrylamide gels, at least two bands are seen in the 62-66 kDa range. These polypeptides are likely encoded by the chloroplast genes psIAl and psIA2, although it is still possible that both bands are just different forms of a single polypeptide obtained from one of these genes [Fish et al. 1985, Lehmbeck et al. 1986]. The psIAl and psIA2 genes code for 15 polypeptides which are 45 % homologous in maize (Zea mays L.) [Fish et al. 1985] and these polypeptides are 89 % and 95 % homologous respectively to the polypeptides encoded by the corresponding genes in pea [Lehmbeck et al. 1986]. CP 47 and CP 43 are also similar to CP I in that they contain Chi a and /3-carotene [Yamada et al. 1985] and are coded for by chloroplast genes [Morris and Herrmann 1984, Alt et al. 1984]. When heat-denatured and subjected to SDS polyacrylamide gel electrophoresis, their apoproteins have molecular weights similar to those of the green complexes. These apoproteins contain primary polypeptides of approximately 51 and 44 kDa for CP 47 and CP 43 respectively, but other polypeptides of slightly lower molecular weight can be seen [Green 1982, Morris and Herrmann 1984]. These lower molecular weight polypeptides are readily detected if more sensitive techniques, such as silver-staining or immunoblotting, are used [Morris and Herrmann 1984, M.J. White unpublished results]. They may be different forms or conformations of the primary polypeptides. The predicted amino acid sequences of the 51 kDa Chi aapoprotein of CP 47 and the 44 kDa Chi aapoprotein of CP 43 from spinach possess some limited sequence homology [Alt et al. 1984] and immunological cross-reaction of antibodies to these proteins has also been observed [Liveanu et al. 1986]. However, polyclonal antibodies to the corresponding proteins 5 and 6 of Chlamydomonas PSII were monospecific [Chua and Blomberg 1979]. The sequence of 20 amino acid residues of a proteolytic fragment of barley CP 47 purified from a PSII preparation was recently obtained [Hinz 1985]. Of these 20 residues, 18 were identical to those in the same position of the amino acid sequence predicted for spinach, suggesting that the CP 47 sequence is conserved 16 between monocots and dicots. It is of interest that pairs of sequence homology exist between the predicted amino acid sequences of the CP I genes psIAl and psIA2, between CP 47 and CP 43 and between the DI and D2 polypeptides of PSII [Rochaix et al. 1984, Rasmussen et al. 1984, Alt et al. 1984], all of which are chloroplast genes. All three protein pairs also possess conserved, characteristically spaced amino acid residues including histidine and cysteine. It has therefore been suggested that each of these gene pairs arose by a chloroplast gene duplication event [Alt et al. 1984]. The DI and D2 polypeptides exhibit weak homology to the L and M polypeptides of the photosystem in purple photosynthetic bacteria [Michel and Deisenhofer 1987]. The L and M polypeptides bind the reaction center bacteriochlorophyll between them, along with other bacteriochlorophylls, bacteriopheophytin and quinones. Faint Chi binding bands have been detected in the D l and D2 region of polyacrylamide gels using a sensitive electrophoretic procedure in which C a 2 * is added to increase the stability of these bands [Irrgang et al. 1986]. However, it was not conclusively demonstrated that D l and D2 actually bind chlorophyll. Nanba and Satoh [1986] recently isolated a pigment-protein complex, consisting of D l , D2 and cytochrome b-559, which was capable of reversible accumulation of reduced pheophytin. It contained five Chi a, two pheophytin a and one or two hemes (molar ratio). This strongly suggests that CP 47 and CP 43 need not participate in binding either P680 or the pheophytin associated with the PSII reaction center, but that this function is served by D l and D2 instead. 17 d. Further detail on the Chl a + b proteins The Chl a + b proteins comprise a distinct group, with many different properties from the Chl a proteins. The Chl a + b proteins each contain two or more polypeptides with molecular weights in the 20-30 kDa range, lower than those of the Chl a apoproteins. In addition to ^-carotene, LHCII contains the xanthophylls lutein, neoxanthin and violaxanthin. [Rawyler et al. 1980, Landis, et al. 1983]. The carotenoid composition of the other Chl a+b proteins has not been investigated. A major part of my work is directed towards demonstrating that the Chl a+b proteins also comprise an immunologically related group, as indicated by cross-reaction of antibodies to these polypeptides. While this work was being published, similar results were obtained by Evans and Anderson [1986] and by Sylvia Darr [Darr et al. 1986]. This immunological relatedness is discussed in detail in the last section of this Introduction and in the second chapter of the Results, and has been published by White and Green [1987a, 1987b, 1987c]. The polypeptides of the Chl a + b proteins are encoded in nuclear genes and are synthesized on cytoplasmic ribosomes. Their synthesis is inhibited by cycloheximide but not by chloramphenicol [Mullet et al. 1982, Machold 1983]. By translating polyadenylated mRNA in vitro it is then possible to immunoprecipitate LHCII polypeptides in the form of soluble precursors several kDa longer than the mature polypeptide. These precursors can then be taken up by intact chloroplasts in vitro, or even by thylakoids • supplied with the necessary factors, and incorporated into thylakoids [Schmidt et al. 1981, Kline 1986]. Chl a, Chl b and 18 carotenoids are then attached and a 4-5 kDa transit peptide is removed [Schmidt et al. 1981, Chitnis et al. 1986, Kohorn et al. 1986]. Similar studies have shown that the PSI antenna (LHCI) polypeptides are also synthesized as slightly higher molecular weight precursors in the cytoplasm [Bellemare et al. 1982, Mullet et al. 1982]. Although this has not been demonstrated directly for the CP 29 polypeptides, the protein synthesis inhibitor data combined with its absence among the translation products of intact chloroplasts [Green 1982], strongly suggest that the CP 29 apoprotein is encoded by nuclear genes too. The polypeptides of LHCII are encoded by a large nuclear multigene family. For example, 16 such genes have been reported for petunia (Petunia  hybrida Vilm.) [Dunsmuir et al. 1983]. Since these genes have been isolated by immunological or probe hybridization techniques, it is possible that not all of these homologous sequences code for LHCII, and it is further possible that some of them may not be expressed at all. They are therefore referred to as cab genes (cab is short for chlorophyll a/6-binding polypeptide) rather than strictly LHCII genes. These cab genes are present in tightly-linked genetic assemblages, and clusters of cab genes have been mapped onto two or more chromosomes in the same organism [Pichersky et al. 1985, Polans et al. 1985, Vallejos et al. 1986]. Two distinct types of cab genes have been characterized. Each of the two types of cab proteins is highly conserved across-species, but they differ by about 19 15 % in their amino acid sequence of the mature polypeptide within the same organism. This suggests that the Type I and Type II cab polypeptide lineages diverged from each other before the split of monocots and dicots [Pichersky et al. 1986]. The transit peptides are less than 50 % homologous and the amino termini of the mature cab polypeptides show considerable divergence [Pichersky et al. 1986, Hoffmann et al. 1987]. Both Type I and Type II cab genes encode precursor polypeptides of 264-267 amino acids, which consist of a transit peptide of 34-36 amino acids and a mature polypeptide of approximately 229-231 amino acids [Dunsmuir 1985, Karlin-Neumann et al. 1985, Pichersky et al. 1985, Pichersky et al. 1986]. Unlike the Type I genes, the Type II genes contain a short intron of about 84 nucleotides [Karlin-Neumann et al. 1985, Pichersky et al. 1986, Stayton et al. 1986]. There are segments of relatively high sequence conservation which are clearly discernible between the Type I and Type II amino acid sequences, in particular amino acids 70-120 and 190-240 (numbering from the start of the precursor sequence) [Pichersky et al. 1986]. Not only are these regions conserved between sequences, but they show substantial homology within a sequence, i.e. they are partial internal repeats or duplications. These same regions show a substantial degree of sequence homology with the predicted amino acid sequences of the newly discovered cDNAs coding for LHCI polypeptides in tomato (Lycopersicon esculentum L.) and petunia [Hoffmann et al. 1987, Stayton et al. 1987], although the homology among the petunia sequences is slightly less than in tomato. 20 A light-harvesting antenna function has been demonstrated for both LHCII and LHCI [Thornber and Highkin 1979, Ortiz et al. 1984]. Such a function has not been demonstrated for CP 29, but is likely given that this is true for all the other Chl fc-containing proteins. CP 29 is associated with PSII particles [Goodman Dunahay et al. 1984], and may be an internal antenna associated with PSII [Dunahay and Staehelin 1987, Greene et al. 1987]. Two additional functions of LHCII polypeptides are their role in mediating granal membrane stacking and their associated role in the distribution of excitation energy between photosystems I and II. Washing thylakoids in low salt media causes granal unstacking. Restacking will result if low concentrations of divalent cations (e.g. 5 mM M g 2 +) or higher concentrations of monovalent cations are added to the unstacked thylakoids. These cation binding sites reside on LHCII [Davis and Gross 1975, Davis and Gross 1976]. Mild trypsin treatment of thylakoids removes an amino terminal peptide from LHCII and also eliminates granal stacking [Steinback et al. 1979]. LHCII can be cation precipitated from normal thylakoids solubilized with Triton X-100. [Burke et al. 1976, Ryrie et al. 1980]. Other thylakoid proteins are not precipitated by this procedure. Plants grown under intermittent light (2 min of light every 2 hours) lack Chl b and lack LHCII polypeptides as determined by SDS polyacrylamide gel electrophoresis and freeze fracture of thylakoid membranes [Armond et al. 1976, 1977]. The intermittent light plastids have full photochemical activities but are nearly agranal. Continuous illumination of the intermittent light plants induces the synthesis of LHCII, granal stacking, an increase in photosynthetic unit size and 21 a corresponding decrease in whole chain electron transport [Armond et al. 1976, 1977]. The Chi 6-less barley mutant chlorina f2 has been and continues to be an important tool in studies on the function of LHCII in granal stacking and light-harvesting. It contains stacked thylakoids in vivo but requires much higher cation concentrations (e.g. 25 mM M g 2 + ) to maintain stacking or restack thylakoids in vitro compared to normal thylakoids [Burke et al. 1979, Ryrie 1983, Bassi et al. 1985]. This mutant is greatly depleted in LHCII polypeptides and requires almost twice the amount of light required to saturate the photochemistry in normal barley [Thornber and Highkin 1979]. Since it was originally assumed to be completely deficient in LHCII polypeptides, it was not understood how any thylakoid stacking could occur in this mutant. However, Ryrie [1983] used sensitive immunological techniques to show that some LHCII polypeptides were indeed present in this mutant, although most are present in greatly reduced amounts. Bassi et al. [1986] attributed stacking in this mutant to other polypeptides in PSII particles, but did not eliminate the possibility of a role for the residual LHCII polypeptides in the thylakoid stacking. The addition of cations to chloroplasts or the addition of supplementary light at 705 nm (light I) increase the rate of electron transport through PSII while decreasing that through PSI (State I). Conversely, the removal of cations or the addition of supplementary light at 645 nm (light H, a wavelength absorbed by Chi 6), decreases the amount of light absorbed by PSII and increases the amount of light absorbed by PSI [Barber 1982, Brecht 1986]. 22 These observations can be explained by the mobile antenna hypothesis [Kyle et al. 1983, Kyle et al. 1984]. The removal of cations increases the net negative charge on LHCII and the thylakoid surface. This loosens the interaction of LHCII units, resulting in (partial) unstacking, and loosens the interaction of LHCII units with the PSII core [Butler 1978, Barber 1982]. In normal thylakoids most of the PSII is located in the granal regions and PSI is concentrated in the stromal lamellae [Armond and Arntzen 1977, Anderson and Melis 1983]. Consequently the electrostatic repulsion results in the migration of some LHCII from the granal lamellae to the stromal lamellae [Kyle et al. 1984]. This decreases the absorption cross-section (light-harvesting capacity) of PSII and increases that of PSI. These state transitions occur in normal barley but were not detected in intermittent light barley or the chlorina f2 Chl 6-less barley mutant [Haworth et al. 1983]. Light at 645 nm is absorbed largely by LHCII, which is mostly associated with PSII, resulting in increased electron transport through PSII. This reduces the plastoquinone pool, activating a kinase which phosphorylates LHCII [Bennett 1977, Bennett 1979, Allen and Holmes 1986]. Mullet [1983] has shown that trypsin removes an amino terminal hexapeptide from LHCII of sequence SATTKK in which one or both of the threonine residues are phosphorylated. (Most ca6 genes contain at least one threonine near the amino terminal of the mature polypeptide.) The phosphorylation increases the net negative surface charge, analogous to the removal of cations and eliciting the same result [Bnecht 1986]. The reverse mechanism is stimulated by addition of cations or supplementary 705 nm light, except that in the latter case a phosphatase is activated in place of a kinase. 23 Evidence that CP 29 is not involved in state transitions and thus is distinct from LHCII has recently been presented by Dunahay and Staehelin [1987] with spinach and by Greene et al. [1987] with maize. CP II* (LHCII) migrates from grana to stroma following its phosphorylation in state I-state II transitions, whereas CP 29 remains primarily within the grana regions [Dunahay and Staehelin 1987]. Greene et al. [1987] studied the maize double mutant OY-YG which shows elevated Chi alb ratios, loss of mobile CP II* (LHCII) and of CP Ia, greatly reduced granal stacking and loss of M g 2 + mediated control of excitation energy distribution when grown at high light intensities. Although this mutant is depleted in LHCII and LHCI at these intensities it contains levels of CP 29 comparable to those in normal maize. This suggests that CP 29 is not part of the mobile LHCII antenna which is phosphorylated and involved in cation-regulated thylakoid stacking and the concomitant distribution of excitation energy between photosystems [Greene et al. 1987]. 3. Factors affecting Chi protein synthesis a. Experimental systems for studying Chi protein synthesis Systems that have been used to study Chi protein synthesis in vivo include mutants and various growth conditions or light regimes. By choosing systems which lack one or more factors required for Chi protein synthesis, such as light or Chi, it is possible to deduce or at least postulate the effects of these factors on Chi protein synthesis. For example, Chi 6-less mutants provide a system lacking only a single factor (Chi 6), while dark-grown (etiolated) plants 24 lack light and therefore Chls a and 6. The absence of light is the primary cause of differences between etiolated and normal plants, but these differences may or may not result from the absence of one or more of the Chls. Clearly, the researcher is limited to those systems which are available. Thus while it would be extremely useful to have a Chl a-less mutant to complement work on a Chl Mess mutant, viable Chl a-less mutants have not been found, presumably because such a mutation is lethal. b. The Chl b-less barley mutant chlorina f2 As already mentioned, the chlorina f2 Chl 6-less mutant was important in determining the role of LHCII in light-harvesting and thylakoid stacking. Unlike similar mutants which contain reduced levels of Chl 6, chlorina f2 is unusual in that it completely lacks Chl 6 under all growth conditions [D. Simpson et al. 1985]. Chlorina f2 is not a mutation in a structural gene coding for a Chl-binding polypeptide but appears to be blocked at a late stage of Chl 6 synthesis, possibly in the conversion of Chl c to Chl 6 [D. Simpson et al. 1985]. The photosynthetic unit size in chlorina f2 is reduced from 250 Chl a+b molecules to 50 Chl a molecules for PSII and from 185 Chl a+b molecules to 150 Chl a molecules for PSI [Ghirardi et al. 1986]. In addition to lacking substantial quantities of LHCII polypeptides, it has been reported to be deficient in all the PSI antenna (LHCI) polypeptides [Mullet et al. 1980b]. However, chlorina f2 thylakoids have been reported to contain a polypeptide identified as the CP 29 apoprotein on the basis of its molecular weight [Machold 1981]. These assessments were based on SDS polyacrylamide gel electrophoresis of 25 heat-denatured thylakoids, followed by Coomassie blue staining. This technique unfortunately contains a number of limitations which will be discussed in the last section of this Introduction. Despite the reported depletion of many Chl a/6-binding polypeptides in this mutant, it contains relatively normal levels of mRNAs for the LHCII and LHCI polypeptides [Apel and Kloppstech 1978, Bellemare et al. 1982]. These mRNAs can be translated in vitro and the resulting radioactively labeled precursor polypeptides taken up by isolated normal or mutant chloroplasts and incorporated into thylakoids. It was therefore postulated that the LHCII and LHCI polypeptides are rapidly turned over in the absence of Chl 6, and therefore do not accumulate in the Chl 6-less thylakoids [Bellemare et al. 1982]. Such a turnover of LHCII has been observed in the absence of light in normal plants. Bennett [1981] used short-term labeling in vivo with 35 [ S]-methionine to show that 7-day-old etiolated pea seedlings exposed to continuous light for 24 h continued to incorporate radioactivity into LHCII for up to 48 h after transfer of the plants from light to darkness. Although synthesis continued, there was an 84 % decrease of LHCII in the dark accompanied by a loss of 38 % of the Chl c and 74 % of the Chl 6. However, when the young plants were exposed to three days of light, LHCn was stabilized and turnover no longer occurred upon transfer back into darkness [Bennett 1981]. 26 c. Plants grown under intermittent light A second useful in vivo system for studying the absence of Chi b are plants grown under intermittent light. These plants contain Chi a and Chi a proteins but lack Chi b and the polypeptides of both LHCII and the PSI antenna (LHCI) polypeptides as judged by Coomassie blue staining [Armond et al. 1976, 1977, Mullet et al. 1980b]. Despite the absence of LHCII, the intermittent light plants nevertheless contain relatively normal levels of LHCII mRNA [Cuming and Bennett 1981, Viro and Kloppstech 1982]. Thus although intermittent light is sufficient for transcription, continuous light is required for the accumulation of LHCII in thj'lakoid membranes. Since plants grown under intermittent light lack both continuous light and Chi b, it is not clear which of these two factors is directly responsible for the reported absence of Chi a/6-binding polypeptides. Therefore these sorts of questions are best answered by comparative studies in which both Chi 6-less mutants and intermittent light plants are used, and chapters C and E of the Results section report results from experiments on these two systems respectively. cf. The etioplast to chloroplast conversion Perhaps the most frequently used system for studying the effects of light on plant genes and protein synthesis is the dark-grown etiolated plant. These plants contain a special type of plastid known as an etioplast which contains carotenoids but lacks Chi and many of the thylakoid proteins found in mature chloroplasts. Unlike chloroplasts, which contain granal and stromal lamellae, 27 etioplasts contain one or more paracrystalline prolamellar bodies [Liitz 1986, Kesselmeier and Laudenbach 1986]. The prolamellar body is a branched tubular lattice constructed of four- or six-armed units [Kesselmeier and Laudenbach 1986, Liitz 1986, Selstam and Sandelius 1984]. Upon illumination the prolamellar body is gradually transformed into the usual thylakoid arrangement seen in mature chloroplasts. Both etioplasts and chloroplasts originate from proplastids. Proplastids are small, colourless or pale green, undifferentiated plastids with little internal structure [Kirk and Tilney-Bassett 1978]. The type of plastid which results during plant development is dependent on whether or not light is present. Since etioplasts can be transformed into chloroplasts, the etioplast can be thought of as an additional stage (or optional stage from the researcher's point of view) in the proplastid to chloroplast transformation. Chloroplasts can also revert to etioplasts if green plants are returned to the dark for extended periods. The direct proplastid to chloroplast conversion can be studied in germinating seeds or seedlings exposed to light and in developing leaves. The leaves of grasses are especially useful for this purpose. These leaves grow from a basal meristem by cell division so that the oldest cells are at the top of a leaf while the youngest cells are at the base [Boffey et al. 1979]. Boffey et al. [1980] measured cell age ascending the wheat (Triticum aestivum L.) leaf by placing a small ink spot just above the basal meristem each day and measuring the distance the ink spots moved over days 4 to 7. I tried this same method with barley seedlings. However, by comparing the growth of these plants to those of control plants, it was found that the measuring process slowed the growth of the leaf in a statistically significant fashion. This is not surprising 28 since in addition to handling, a small puncture must be made in the coleoptile with a pen tip in order to apply the ink spot. It was therefore not possible to repeat the results of Boffey et al. [1980] and assign definite cell ages along the barley leaf gradient. This is one reason why I chose to study Chl protein synthesis during the etioplast to chloroplast conversion instead. A second reason is that even the youngest portion of a grass leaf gradient is exposed to light and contains small amounts of Chl. Therefore determining the effects of these factors on Chl protein synthesis is more complicated than in systems such as mutants, intermittent light plants or etiolated plants where one or more of these factors is completely lacking. Finally, very little work has been done with leaf gradients relative to the abundance of information available on the etioplast to chloroplast conversion. However, it is important to remember that what is true for one type of plastid interconversion is not necessarily true for another. e. Transcriptional control of protein synthesis Etiolated plants have been widely used in studying the phytochrome control of plant gene transcription. Perhaps the most intensively studied phytochrome responses are those of the nuclear multigene families of cab genes and genes coding for the small subunit of ribulose bisphosphate carboxylase. The phytochrome control of transcription is associated with the 5' -DNA sequence flanking these genes [J. Simpson et al. 1985, Nagy et al. 1986]. At least two cis-acting elements involved in light-induced transcription have been identified 5' 29 to the rbcS genes. One is a conserved sequence between the transcription start site and the 5' boundary of the TATA box. The second is an enhancer-like element of 240-280 base pairs located upstream of the first region. It has a significant influence on the level of light-induced transcription and on organ-specific expression. A similar enhancer-like element occurs in the 5' region of cab genes [J. Simpson et al. 1985, Nagy et al. 1986]. Cab genes respond to red light in the low and very low fluence ranges whereas probes for small subunit mRNAs detect only a low fluence response [Kaufman et al. 1985]. Both of these responses are reversible by far-red light in pea [Kaufman et al. 1985]. In contrast to these nuclear genes, all chloroplast genes studied to date which code for thylakoid proteins appear to be constitutively transcribed [Herrmann et al. 1985]. However, light can regulate the level of transcription, at least under certain conditions and for certain genes. Rodermel and Bogorad [1985] identified six regions of the plastid genome, accounting for 19 % of the unique plastid DNA, which showed increased transcription in response to light. This included the two CP I genes psIAl and psIA2 (more generally known as psaA and psaB), and was, in fact, the way in which these genes were first discovered. The size of the transcript pools for most of these photoregulated genes reached a maximum size after 10, 20 or 44 h of illumination and then declined to approximately the preillumination levels [Rodermel and Bogorad 1985]. The PSI transcripts psaA and psaB peak around 10 h while the PSII transcripts psbC and psbD, which code for CP 43 and D2 respectively, reach a maximum size after about 20 h of illumination [Rodermel et al. 1987]. The psbA transcript which codes for D l continues to increase for at least 68 h of continuous 30 illumination. These light-induced increases appear to be mediated by phytochrome [Rodermel et al. 1987]. Mullet et al. [1987] have recently shown that transcripts of the psaA and psaB genes are also present in etiolated barley as are transcripts of the PSII genes psbA, psbC and psbD. This is despite the fact that at least the PSII genes are not translated in the absence of light. The psbA (Dl) transcript levels remain relatively high in illuminated barley but the other transcript levels show a dramatic decrease following illumination [Mullet et al. 1987]. These results are in direct contrast with those of Rodermel and Bogorad [1985] and Rodermel et al. [1987] mentioned above. These differences could be attributed to differences between species, differences in experimental protocol, or differences in the length of time plants were grown in the dark and the growth temperature. Mullet et al. [1987] report that the level of transcription of plastid genes in etiolated plants can be dependent on their age. This includes factors such as growth temperature which determine the physiological age of the plants. RbcL transcripts, which code for the large subunit of ribulose bisphosphate carboxylase and do not require light for translation, show a steady decline in both dark-grown and illuminated plants with age. Thus a similar pattern of transcription for the other plastid genes would explain the discrepancy between the light-induced increase of transcription of 7-day-old maize grown at 30 °C reported by Rodermel and Bogorad [1985] and the light-induced decrease in 4.5-day-old barley grown at 23°C reported by Mullet et al. [1987]. However, the psaA-psaB, psbA and psbC transcript levels remain at a high level in 4.5 to 9-day-old dark-grown barley plants, opposite to the situation with the rbcL transcripts [Mullet et al. 1987].. Thus it is not clear whether light increases or decreases the transcription of these plastid genes, but all workers agree that the transcripts are present in etiolated plants. f. Posttranscriptional control of protein synthesis A number of nuclear and plastid genes coding for thylakoid proteins are also translated in the dark. These include the genes coding for NADPH : protochlorophyllide reductase [Apel 1981], the subunits of coupling factor [Vierling and Alberte 1983, Selstam and Sandelius 1984], the three polypeptides of the water splitting complex [Ryrie et al. 1984, Liveanu et al. 1986] and cytochromes f and b 6 [Takabe et al. 1986]. There is controversy as to whether or not small amounts of CP I apoprotein are translated in etiolated plants. It has been detected in dark-grown Spirodela oligorrhiza [de Heij et al. 1984], bean, spinach, oat [Nechushtai and Nelson 1985] barley, wheat and rye [Shlyk et al. 1986], but was not detected in dark-grown pea, wheat and barley in other studies [Vierling and Alberte 1983, Takabe et al. 1986, Kreuz et al. 1986]. This suggests that these differences are not so much due to the species used in these studies as the methods by which these experiments were performed. Klein and Mullet [1986] did not detect synthesis of CP I apoprotein in pulse-chase experiments where 5-day-old etiolated barley seedlings were labeled 35 in the dark with [ S]-methionine and chased in the light. This kind of result is nevertheless compatible with immunoblotting experiments which detect CP I apoprotein in etiolated tissue, since the former monitors synthesis over a period of hours while the latter measures total accumulation since the plants began 32 growing. Indeed, Kreuz et al. [1986] have isolated polysomes containing CP I mRNA from 6-day-old etiolated barley seedlings, even though they did not detect the apoprotein on immunoblots. This implies either that these polysomes are in a state of arrested translation [e.g. Mullet et al. 1986], that the levels of translation are too low to detect, or that the translated polypeptide is turned over. The question of whether or not CP I apoprotein is present in dark-grown barley is investigated in my experiments on the greening of etiolated plants. A major concern in studies where it is detected should be ensuring that the growth conditions are completely dark, as even a small light leak in the growth chamber might promote synthesis. The presence of Chi a+b apoproteins indicates light leakage and these proteins can therefore serve as internal controls in dark-grown plants, in particular because LHCII is absent from dark-grown plants [reviewed in Tobin and Silverthorne 1985, Anderson 1986, Ellis 1986]. The transcription of cab genes is a sensitive phytochrome response [Kaufman et al. 1985, Tobin and Silverthorne 1985] and it is generally believed that LHCII also requires continuous light to accumulate in thylakoids as already discussed. Even a small light leak in a growth chamber will be a source of continuous light. As I will show in experiments on barley grown under intermittent light, even 2 min of light every 2 h is sufficient to promote synthesis of some of the Chi a/6-binding polypeptides. Since even brief exposure to light can promote synthesis, although only if a sufficient lag time for protein synthesis is allowed, sensitive immunological techniques for the detection of these Chi a/fe-binding polypeptides are among the best controls available for studies on dark-grown plants. 33 A major concern in studies where CP I apoprotein is not detected in dark-grown plants should be the sensitivity of the detection techniques. As discussed earlier, the particular species used in a study, how it was grown, and the experimental protocol adopted might influence the outcome of a study as well. By using the same methods and the same species (barley) used in studies where CP I apoprotein was not detected in dark-grown plants, possible sources of differences between these studies and those studies which detect CP I apoprotein, such as my studies, can be eliminated. Some of my studies on the detection of CP I apoprotein in dark-grown barley were presented at the same conferences at which other workers reported the detection of CP I transcripts in dark-grown barley, but did not detect synthesis of the protein [now published in Klein and Mullet 1986 and Kreuz et al. 1986]. This presented the opportunity to discuss differences in our experimental results and the possible causes for these differences. 4. Molecular techniques for studying C h l proteins and their synthesis In order to study the effects of light on the synthesis of a particular kind of compound or macromolecule, it is necessary to be able to detect it. Most molecular techniques require that this molecule first be separated or isolated from the biological material on the basis of its physical or chemical properties. For example, gel electrophoresis can be used to separate macromolecules from each other on the basis of molecular weight (a physical property). In this case, it is then necessary to employ a staining procedure (dependent on its chemical properties) to visualize or quantitate the macromolecule. 34 Chi proteins possess several properties which can be used for identification and subsequent quantitation. When thylakoids are solubilized in nonionic detergents such as octylglucoside, these proteins retain their bound Chi, allowing them to be separated on gels containing small amounts of SDS. The Chi proteins can then be identified on the basis of both their apparent molecular weight and green colour. An intact Chi protein can be further characterized by measuring its Chi alb ratio and determining its absorption spectrum. This is the way in which many of the Chi proteins were first identified. There are three reasons why this approach is not well suited to developmental studies. Firstly, etiolated plants lack Chi. Thus if an etiolated plant contained the polypeptide component of a Chi protein, it could not be identified on the basis of its Chi binding properties. Secondly, Chi proteins are labile and some of the Chi dissociates during electrophoresis. During the early stages of greening the Chi protein complexes are even more labile, and tend to dissociate during electrophoresis [Tanaka and Tsuji 1983, Tanaka and Tsuji 1985]. Thirdly, systems lacking Chi b, such as Chi 6-less mutants and plants grown under intermittent light, do not have detectable green Chi a + b proteins, but might contain the corresponding polypeptides. It is therefore necessary to use properties other than Chi binding in a developmental study, especially if one is to determine whether or not these polypeptides are present in etiolated plants. One could attempt to identify these polypeptides on the basis of their molecular weight using SDS polyacrylamide gel electrophoresis followed by staining. This can be done by purifying Chi proteins from mature tissue, 35 heat-denaturing them to dissociate the Chl, and determining their polypeptide composition by standard denaturing SDS gel electrophoresis methods. Samples of heat-denatured thylakoids can be electrophoresed in adjacent gel lanes and the polypeptides of the purified Chl proteins aligned with the corresponding polypeptides in thylakoids. Unfortunately, this method of identifying polypeptides also has problems. The correct alignment of polypeptides is critical for proper identification. This is not a problem for very abundant Chl proteins such as CP I or CP II, where the stained band is a dominant feature of the thylakoid electrophoretic profile. However, it is more difficult to identify the less abundant Chl proteins, and correct identification is particularly difficult in developmental systems, in which only extremely small amounts of these polypeptides may be present. This procedure is also somewhat unreliable, as polypeptides of similar molecular weight co-migrate with the. Chl-binding polypeptides. Again, this is not a problem for abundant Chl proteins in mature tissue, but is a major impediment to developmental studies. Chl-binding polypeptides which are completely absent might appear to be present due to co-migrating polypeptides of similar molecular weight. It is for these and other reasons that the use of antibodies to identify or quantitate polypeptides has become increasingly popular in recent years. One advantage of immunoblotting techniques is that Chl does not have to be attached to a polypeptide in order to identify it. Antibodies can also be used to detect antigen in unresolved mixtures such as SDS-solubilized membranes or even SDS extracts of whole plant cell proteins. These advantages exist because the sequence-specific nature of the antigenicity is used to identify the antigen instead 36 of more general properties, such as Chl-binding and molecular weight, which are shared by other molecules. It is necessary to purify the antigen thoroughly in order to make the antibody, so there is no saving of time involved. However, this antigen can be purified from mature tissue, and the resulting antibody used to detect the antigen in systems such as etiolated plants, in which its identification and quantitation would otherwise not be possible. In addition to all of the advantages listed above, immunoblotting methods are currently the most sensitive techniques available. They are frequently used to detect antigen in the 0.1 to 1 ng range. This is much more sensitive than either Coomassie blue or silver staining. The newly developed silver-staining techniques for polypeptides in gels are reported to be 50-100 times more sensitive than Coomassie blue, comparable to 35 the sensitivity of autoradiography of [ S]-methionine-labeled proteins [Nielsen and Brown 1984, Heukeshoven and Dernick 1985]. In my experiments, silver-staining is perhaps 10-50 times as sensitive as Coomassie blue staining, unless long development times are used which produce a very high background. The chemical bases of silver staining and Coomassie blue staining have only recently begun to be understood [e.g. Tal et al. 1985]. The combined techniques of electrophoresis and immunoblotting are especially powerful because they allow the antigen to be detected on the basis of both its antigenicity and molecular weight. The simultaneous identification of polypeptides on the basis of both these properties greatly reduces the chance of 37 error in identifying a polypeptide relative to using a single property such as molecular weight. It also prevents misidentification due to immunological cross reaction (as described in the appropriate chapters of the Results section). Consequently, the combined techniques of electrophoresis and immunoblotting are the method of choice for detection of small quantities of Chl-binding polypeptides in most developmental systems. 5 . Properties of antibodies and antigens: Immunological specificity and cross-reactivity The basic principle underlying the use of antibodies in immunoblotting is the recognition of a specific site on the antigen. This site is referred to as an antibody binding site or epitope. The corresponding site on the antibody is termed the antibody combining site. In a linearized, denatured SDS-protein on a gel or Western blot, the basis of the immunological specificity is the particular amino acid sequence which constitutes the epitope. The size, shape and properties of both epitopes and antibody combining sites are discussed in detail in the last chapter of the Results. The theoretical work presented in this chapter can be used to estimate the probability that similar or identical epitopes, i.e. sequences of amino acids of specified length, will arise among two or more different proteins due to chance alone. Similar or identical epitopes may also be due to a common origin of two or more proteins, or to functional or structural factors which have caused the convergent evolution of these proteins. Antibodies to proteins which share such 38 epitopes will not only react with their antigen but will also show immunological cross-reaction with other proteins containing the epitopes. These proteins are then said to be immunologically related. Immunological cross-reactions among related proteins have been observed in a number of systems. Keratins comprise a complex multigene family, and widely cross-reacting antibodies have been used in a variety of studies [reviewed by Lazarides 1982]. Both monoclonal and polyclonal antibodies have been used to show close immunological relatedness among certain vertebrate neurofilament proteins [Shaw et al. 1984]. Cross-reacting polyclonal antibodies to alkaline phosphatase and 5' -nucleotide phosphodiesterase were used to demonstrate the relatedness of these two proteins [Culp and Butler 1985]. Since the alkaline phosphatase and the phosphodiesterase are both glycoproteins, experiments were performed to test if removal or modification of the carbohydrate moieties eliminated the immunological cross-reaction. It was found that antibodies to the native proteins cross-reacted only when the carbohydrate moiety was present, but that antibodies to denatured 5' -nucleotide phosphodiesterase cross-reacted with denatured alkaline phosphatase following removal of the carbohydrate [Culp and Butler 1985]. These kinds of experiments are necessary in order to establish if immunological cross-reaction between glycoproteins is due to amino acid sequence homology, but they are clearly not required in systems which lack glycoproteins, such as chloroplasts [Keegstra and Kline 1982]. Many plant proteins coded for by nuclear genes are not just present in single copies but appear to be coded for by multigene families [Apel et al. 1986, 39 Vallejos et al. 1986]. It might be expected that the polypeptides encoded by a nuclear multigene family would be immunologically related. The multigene families which code for the seed storage proteins, found in the seed protein bodies of flowering plants, provide one such example [Apel et al. 1986]. One subgroup of these proteins, the globulin seed storage proteins, is believed to be derived from two ancestral genes which existed at the beginning of angiosperm evolution [Borroto and Dure 1987]. A complicated pattern of immunological relatedness exists among the various seed storage proteins. Immunoblotting of maize endosperm storage proteins with polyclonal antibodies raised against a glutelin-2 fraction showed reaction with a 14 kDa zein polypeptide but not other zein polypeptides [Ludevid et al. 1985]. To demonstrate that the same antibodies were reacting with these two polypeptides, antibodies were eluted from a nitrocellulose blot at low pH and then reused [Ludevid et al. 1985]. It has also been shown that purified anti-zein IgG cross-reacted with hordeins, but that anti-hordein IgG did not cross-react with zeins, and that both cross-reacted with a few proteins of the gliadin family [Dierks-Ventling and Cozens 1982]. The original intention of this thesis was to study Chl protein synthesis using whatever systems were available. It was decided that antibodies to specific Chl proteins would be an extremely useful tool in this endeavor for the reasons outlined above. The discovery of a Chl a+b protein associated with CP I (PSI) during antigen preparation necessitated the isolation and characterization of this complex in order to complete our understanding of what Chl a+b proteins were present and what their properties were. During the course of this work a number of other research groups have investigated this PSI-associated Chl a+b 40 protein, which is now known as the PSI antenna, LHCI. The purification of the various Chi proteins and the production of antibodies to them was a major undertaking involving considerable time and effort. The observation of immunological cross-reactions among the resulting antisera was at first an unanticipated result. It necessitated careful study of the nature of these cross-reactions and the determination of the specificities of each antiserum individually. Since it could be shown that these immunological cross-reactions were not due to contamination of the injected antigens, they were of considerable interest and became a major focus of my immunological studies. Evaluating the significance and possible causes for these cross-reactions gave rise to the theoretical work presented in the last chapter of the results. This could not be done until the mathematics of biological sequence comparison became available. This mathematics as developed by Arratia et al. [1986] was made available to us in manuscript form two years in advance of publication, courtesy of Dr. Michael Waterman, through Joe Watkins, formerly of the U.B.C. Mathematics department. Our theoretical results predict the probability that immunological cross-reactions will occur due to chance alone. Although such chance cross-reactions are infrequent, the results clearly demonstrate that biological significance should not be automatically attributed to immunological cross-reactions whenever they are observed. It is predicted that all the major Chi a+b proteins share some primary sequence homology and that they may have arisen from a common ancestral gene. Immunological studies on the Chi 6-less barley mutant chlorina f2 gave rise to the interesting discovery that most of the Chl a/6-binding polypeptides were present in these Chl 6-less thylakoid membranes. This demonstrated that Chl 6 was not an absolute requirement for transcription or translation of those polypeptides which were present. Immunological studies on Chl protein synthesis during greening of etiolated barley indicated that all the Chl a+b apoproteins showed almost identical kinetics of light-induced accumulation. Surprisingly, small amounts of the CP I apoprotein were detected in dark-grown barley, and this result was investigated in a series of experiments. Finally, studies on barley grown under intermittent light revealed that the set of Chl a/6-binding polypeptides present were different from those present in Chl 6-less barley, indicating that these two systems are not physiologically equivalent at all. These data, taken together with results in the literature, indicate that the synthesis or accumulation of almost every Chl protein studied is regulated differently from the other Chl proteins, at least under the conditions in certain in vivo systems. The factors regulating the synthesis or accumulation of Chl proteins include Chl a and Chl 6 likely acting at the posttranscriptional level, and light acting at several levels, depending on which Chl protein is involved. II. MATERIALS AND METHODS 1. Plant material and growth conditions All experiments were done using barley (Hordeum vulgare L. cv. Bonanza). Barley was chosen primarily because it is the most commonly used plant for developmental studies and is easy to grow. In addition, a number of barley mutants are available including the well-studied Chi Mess mutant, chlorina f2. For routine thylakoid preparation, normal and chlorina f2 barley were grown in a greenhouse in potting soil. For developmental experiments, barley seeds were soaked overnight (16 h) in aerated distilled water unless indicated otherwise, and grown in wooden flats of potting soil for six days in the dark at 20°C. This was done in a controlled environment chamber with additional layers of dark material covering the door and its perimeter. Intermittent light experiments were performed in the same way but using cycles of 2 min light followed by 118 min of darkness. In both experiments seedlings were greened using white fluorescent light (Sylvania F14T12) which produced a quantum flux of approximately 75 microEinsteins m~ 2 s~ 1 at soil level (before germination) and approximately 90-120 microEinsteins m" 2 s" 1 at the top of a 12 cm leaf. 2. Preparation and Detergent Solubilization of Thylakoid Membranes Thylakoids were prepared using a method modified from Camm and Green [1980]. Thirty g of leaves from 7 to 16-day-old barley seedlings were 42 43 homogenized for 4 x 2 s in a chilled blender in 150 ml of 350 mM sorbitol, 50 mM tricine pH 7.8 and the homogenate filtered through two layers of Miracloth. Chloroplasts were sedimented at 4°C and 4,000g for one min, and washed once with this same medium. This was followed by two washes each of 10 mM N a 2 P « 0 7 (pH 7.4), 0.3 M sucrose (pH 7.4) and finally 2 mM EDTA, 10 mM Tris-maleate (pH 8.0). The thylakoids were recovered after each wash by centrifugation at 12,000g for 5 min at 4°C. Chl was determined according to the method of Arnon [1949]. Thylakoids required for immediate use were solubilized with 300 mM octyl-/3-D-glucopyranoside (Sigma) at a detergent to Chl ratio (w/w) of 30. Remaining thylakoids were stored at -70°C in 65 mM Tris-HCl (pH 6.8), 10 % (v/v) ethylene glycol, 5 mM dithiothreitol or 0.1 % (v/v) /J-mercaptoethanol. 3. Polyacrylamide Gel Electrophoresis The terms nondenaturing gel and denaturing gel apply to gels loaded with native or heat-denatured samples respectively. A nondenaturing gel is prechilled, loaded with thylakoids or native Chl proteins, and run at 4°C under low light as described in Camm and Green [1980]. Since the Chl proteins migrate as green bands they can be seen without staining, and such a gel is often referred to as a "green gel". Partial denaturation of some Chl proteins usually occurs due primarily to the small amounts of SDS in the gel and running buffer. However, this SDS is necessary in order to separate the Chl proteins from each other and to prevent smearing or streaking. Heat-denatured samples may also be run on the same gel provided that they have been cooled before loading. A denaturing gel contains only samples which have been heat-denatured (as in section 7) and 44 can be run in the cold or at room temperature. It is necessary to stain these gels with Coomassie blue or silver [Wray et al. 1984] in order to see the polypeptides, or to use them for immunoblotting if one is interested in a particular polypeptide(s). For Coomassie blue staining, 1.5 mm thick gels were soaked in 0.2 % w/v Coomassie Brilliant Blue R (Sigma), 50 % v/v methanol and 7 % v/v acetic acid for 20-30 min (or longer for thicker gels). Gels were then destained in several changes of 20 % methanol, 7 % acetic acid over the next 1-2 days at which time they were stored or photographed. Unstained gels to be silver-stained were equilibrated in 50 % methanol overnight and then silver-stained by the standard procedure. Coomassie blue-stained gels to be silver-stained were soaked in 40 % methanol, 10 % acetic acid to reduce or remove blue bands prior to the equilibration in 50 % methanol and silver-staining. Silver staining was done by the method of Wray et al. [1984]. Apparent molecular weights were determined by reference to the mobilities of Sigma standards (No. SDS-6 with carbonic anhydrase added or No. SDS-7). Both nondenaturing and denaturing gels were made in the same way. Gels containing a single polyacrylamide concentration were made using the method of Kirchanski and Park [1976]. The following ingredients were used to make 10 % polyacrylamide running gels for two 3 mm thick preparative gels, or four 1.5 mm thick analytical gels: 40.0 ml acrylamide : bisacrylamide (30 : 0.8 % w/v), 78.4 ml 2 M Tris-HCl pH 9.8, and 0.6 ml 20 % w/v SDS. This gel solution was deaerated in an Erlenmeyer flask connected to a vacuum line. Following 45 deaeration, 1.0 ml 10 % w/v ammonium persulfate and 50 fil NNN' N ' -tetramethylethylenediamine (TEMED) were added to initiate polymerization, and the gels poured immediately. Gels of lower or higher polyacrylamide concentration were made by substituting the appropriate volumes of water or 3 M Tris-HCl pH 9.8 for some of the 2 M Tris-HCl pH 9.8. All running gels were overlaid with water-saturated isobutanol until polymerization occurred. The polymerized running gels were overlaid with 5 % polyacrylamide stacking gels. The solution for the stacking gels contained 5.0 ml acrylamide : bisacrylamide (30 : 0.8 %), 21.7 ml distilled water, 3.0 ml 1 M Tris-HCl pH 6.1, and 0.15 ml 20 % SDS. Following deaeration, polymerization was initiated by adding 0.1 ml 10 % ammonium persulfate and 50 jil TEMED. Slot formers were inserted immediately after pouring. Electrophoresis buffer for these gels was made using 382 ml 1 M glycine, 50 ml 1 M Tris and 10 ml of 20 % SDS made up to 2 1 with distilled water. Gradient gels were made of various polyacrylamide concentrations and electrophoresed as described in detail by Chua [1980]. The exact polyacrylamide concentrations used for a particular experiment are indicated in the appropriate text or figure legend of the Results section. 4. Preparation of Chlorophyll Protein Complexes Polyacrylamide gel electrophoresis for isolation of Chi proteins was performed at 4°C under low light and low current as described in the previous section. The initial separation of Chi proteins from octylglucoside-solubilized 46 thylakoids was performed on 3 mm thick, prechilled 7.5 % gels with 2 cm stacking gels. Chi proteins were excised, mashed in 65 mM Tris-HCl, 10 % (v/v) ethylene glycol, 5 mM dithiothreitol pH 6.8 and electrophoresed a second time under nondenaturing conditions, using a higher polyacrylamide concentration to separate nongreen co-migrating contaminants. Purity of complexes from the second electrophoresis was verified on a totally denaturing gel, using samples heat-denatured in the presence of 2 % SDS. Samples were purified by further electrophoresis if necessary. CP II was prepared by re-electrophoresis of CP II* (50-60 kDa range) on 10 % polyacrylamide to avoid co-migrating polypeptides in the CP H region (25-30 kDa range), especially CP 29. When CP II* is re-electrophoresed, some of it dissociates into CP II, which is then well separated from co-migrating polypeptides in the CP II* region. CP 29 was prepared by re-electrophoresis on 14 % polyacrylamide gels. Since the mobility of Chi proteins is strongly dependent on polyacrylamide concentration [Chua et al. 1975], the relative rate of migration of CP 29 is slower during the second electrophoresis. This separates it from non-green contaminants which co-migrated during the first electrophoresis, and also from intact CP II. CP Ia was purified by re-electrophoresis of both CP Ia top and CP Ia bottom on 10 % polyacrylamide gels, and CP I by re-electrophoresis on 12 % gels. For each purification, a sample from the second gel was heat-denatured in the presence of 2 % SDS at 65 °C for 30 min and checked for purity on 10 % polyacrylamide, Coomassie blue-stained gels. 47 For electroelution, slices containing Chl proteins were excised from gels on ice, chopped into small cubes and placed into the collecting cup of an ISCO model 1750 electrophoretic sample concentrator. Chl proteins were electroeluted at 4°C in 4 mM Tris-acetate, 0.2 mM EDTA pH 8.6 at 1.5 Watts for 2 h, or longer for larger Chl proteins and higher polyacrylamide concentrations. LHCII was prepared from Triton-solubilized thylakoids by sucrose centrifugation and cation precipitation [Ryrie et al. 1980]. 5. Spectra and Chlorophyll a/b ratios of Purified Chlorophyll Protein Complexes Spectra of Chl protein complexes were determined directly from gel slices and from electroeluted Chl proteins using a Cary 210 spectrophotometer. Chl alb ratios were determined from methanol extracts of gel slices and of electroeluted Chl proteins using the methods of MacKinney [1941] or Ogawa and Shibata [1965]. 6. Preparation of antisera Chl proteins were electroeluted or the gel strips mashed and injected as a slurry. Rabbits were given intramuscular injections of 15 to 20 ng Chl (estimated to be 75 to 100 ug protein) in Freund's complete adjuvant and then in Freund's incomplete adjuvant 10 days after. Successive injections were given monthly in 48 Freund's incomplete adjuvant followed by bleeding about 10 days after each injection. Clotted blood was cooled to 4°C. The serum was then decanted and spun at 4,000g for 5 min to remove erythrocytes. Western blots of denatured thylakoids were used to screen antisera for activity. None of the preimmune sera reacted with any thylakoid proteins when tested as 50-fold dilutions. 7. Western Blotting and Immunoenzymatic Detection of Chlorophyll Proteins All samples used for Western blotting were heat-denatured in 2 % SDS, 50 mM dithiothreitol, 65 mM Tris-HCl, pH 7.4 at 65 °C for 30 min before electrophoresis. Gels were electrophoresed until the tracking dye (bromphenol blue with ethylene glycol added to increase density) or the front of dissociated Chi was near the bottom of the gel. On gels of relatively high polyacrylamide concentration (e.g. 15 %) the Chi front ran well behind the tracking dye, so the tracking dye was often electrophoresed off the bottom of these gels although the Chi was not. Transfer of proteins from analytical gels onto nitrocellulose was done overnight at 8 V and 220 mA in 50 mM Na acetate pH 7.0 at 4°C using an apparatus of the Bio-Rad Transblot type. The Western blot was cut into strips corresponding to gel lanes or left intact, and incubated in blocker (3 % (v/v) Norland Hipure fish skin gelatin [Saravis 1984] in phosphate-buffered saline (1.37 M NaCl, 27 mM KCI, 81 mM N a 2 H P O « , 15 mM K H 2 P O „ pH 7.4) for 1 h. Blots were incubated in antiserum diluted 100 to 500 x in the same solution for 1 h, then washed 3 x in phosphate-buffered saline pH 7.4, each wash taking 10 49 min. This was followed by 1 h of incubation with alkaline phosphatase conjugated to goat anti-rabbit antibody (Kirkegaard and Perry Laboratories Inc.) in blocker and another 3 washes in phosphate-buffered saline. Blots were then washed for 10 min in 50 mM Tris-HCl pH 8.0 followed by addition of substrate (0.1 % w/v Naphthol AS MX phosphoric acid, disodium salt, 0.2 % w/v Fast Red TR salt (both from Sigma) dissolved in the same buffer). Blots were developed until the optimum colour intensity was obtained (15 min to 1 h or more) and dried between paper towels. Blotting with peroxidase conjugated to goat anti-rabbit antibody (Kirkegaard and Perry Laboratories Inc.) was done using the method of Polvino et al. [1984]. Silver staining of polyacrylamide gels after blotting did not show any remaining protein. Bands on immunoblots were aligned using stained lanes of normal and mutant thylakoids and purified Chl proteins run on the same gel. In' addition, approximate molecular weights were determined from Sigma SDS-7 molecular weight standards. 8. Affinity purification of anti-CP Ia Blank nitrocellulose strips were incubated in a solution of highly purified CP Ia for 30 min. Strips were transferred to blocker for 30 min, then put into anti-CP Ia diluted in blocker for 30 min. Strips were washed 10 min in lx phosphate-buffered saline pH 7.4, 0.05 % Tween 20 and then washed 10 min in blocker. Both washes were accompanied by frequent vortexing. Anti-CP Ia was desorbed from the nitrocellulose in 10 mM glycine-HCl pH 2.3 for 30 min, and 50 neutralized to pH 7.4 using 0.2 N NaOH. 9. Developmental Experiments a. Preparation of Whole Cell Protein Extracts Dark controls (0 h samples) were processed immediately upon opening the growth chamber door as described for the other samples below. For subsequent data points, etiolated seedlings were greened under white fluorescent light (Sylvania F14T12) for varying lengths of time, rapidly cut and placed into liquid nitrogen, and ground in a mortar and pestle. SDS buffer at room temperature (2 % SDS, 50 mM dithiothreitol, 65 mM Tris-HCl pH 7.4) was added to the powdered leaves before thawing occurred. After additional grinding and a 5 min solubilization, the mixture was centrifuged at 12,100g for 5 min. Ethylene glycol (5 or 10 % v/v) was added to increase sample density for future electrophoresis and to serve as a cryoprotectant, and the supernatant was immediately heat-denatured at 65 °C for 30 min (or frozen and heat-denatured later). To remove SDS for protein assay, 50 p\ of sample was precipitated with 950 u\ acetone and spun at 15,000g for 60 s. Pellets were resuspended in 1.00 ml water and protein determined using the BioRad protein assay. Chl was determined by the method of Arnon [1949] using the supematants directly, or by grinding leaves in liquid nitrogen and extracting directly with 80 % acetone. Standard deviations were at most 10 % for the protein assays and about 1 % for the Chl assays of samples from leaves greened 3 h or longer. 51 b. Electrophoresis and Immunoblotting Ten or twenty ug of the total cell protein extracts were loaded per gel lane. 10-20 % gradient gels [Chua 1980] were used to prepare blots to be probed with anti-coupling factor or antisera against the Chi a+b proteins. Samples to be screened with other antibodies were run on 10 % gels [Kirchanski and Park 1976]. Polyclonal antibodies to the 33 kDa polypeptide of the oxygen-evolving complex and the herbicide-binding polypeptide D l of PSII were gifts from Drs. David Allred and Lee Mcintosh respectively. Polyclonal antibodies to polypeptide 6 of Chlamydomonas PSII, which corresponds to CP 43 in higher plants, was provided by Dr. Nam-Hai Chua. Antiserum to coupling factor CF1 subunits was made by Elaine Moase, formerly of our lab. c. Quantitation of Immunoblots Blots were photographed with RA 71 OP graphic film using a copy stand, the 5x7 inch negatives scanned using a Helena Quick Scan R&D densitometer, and the areas under the scans measured using an Apple II graphics tablet. Plots of the area under the densitometer scans as a function of the amount of antigen were constructed from two-fold dilution series of the leaf extracts. They showed that the combination of electrophoresis and blotting procedures, antibody reactions, and the procedure for quantitating blots resulted in a linear response when the amount of total cell protein per gel lane was between 0 and 20 pg. It was sometimes necessary to correct for the width of polypeptide bands on the blots, as the larger amount of antigen at later times often increased the band width. 52 (Increases in the height or intensity of the band were measured by the densitometer.) The net standard deviations for protein assay and all subsequent steps including quantitation of the blots were approximately 20 %. III. RESULTS A. PREPARATION AND CHARACTERIZATION OF A PS I CHLOROPHYLL A + B PROTEIN COMPLEX 1. Isolation and characterization of the two CP Ia bands and the PSI antenna complex To obtain antibodies for use in developmental studies, it was first necessary to develop or optimize methods for purifying the Chl proteins to be used as antigens. Since these purifications are very labour intensive, the amount of time involved would be considerably reduced by finding methods in which crude preparations of all Chl proteins could be obtained from the first electrophoresis. While purifying CP I, I also developed a protocol for purifying its Chl a + b antenna complex, the existence of which had been postulated by Mullet et al. [1980a, 1980b]. Many of the results in this chapter, along with parts of the following chapter on immunological cross-reaction, were published by White and Green [1987a]. Camm and Green [1980] found that treatment of thylakoids with 30 mM octylglucoside preferentially solubilized the Chl proteins associated with PSII. The PSI complexes could then be pelleted by ultracentrifugation at 100,000g for 30 min. In the course of determining the best methods for isolating Chl proteins from barley, I found that using the same amount of octylglucoside (i.e. the same detergent to Chl ratio w/w) at a ten-fold higher concentration completely 53 54 solubilized barley thylakoids. Under these conditions the 300 mM octylglucoside solubilizes intact PSI complexes in addition to the PSII complexes solubilized by 30 mM octylglucoside. When these solubilized thylakoids were electrophoresed on polyacrylamide gels containing 0.05 % SDS at 4°C, two CP Ia bands (designated CP Ia top and CP Ia bottom) were obtained in addition to the Chi proteins I, II*, 47, 43, 29 and II mentioned in the Introduction (Fig. 2). If either CP Ia band was excised and re-electrophoresed on a second nondenaturing gel, three green bands were obtained (in descending order of apparent molecular weight): i) a CP Ia band, ii) CP I, and iii) a new Chi protein (Fig. 3b). This new Chi protein had an apparent molecular mass of 43 kDa on 10 % acrylamide gels and 35 kDa on 7.5 % gels. Its position on polyacrylamide gels clearly distinguished it from the Chi a+b proteins CP II and CP 29 (Fig. 3a, c). Some of the new Chi protein remained associated with CP I as CP Ia throughout at least two cycles of electrophoresis, indicating a tight binding. Under optimum conditions almost all the CP I existed in CP Ia complexes and there was little free CP I. These results suggest that the new Chi protein was associated with CP I in vivo. When this Chi protein was completely heat-denatured in the presence of 2 % SDS and re-electrophoresed, it dissociated into four polypeptides of 21-24 kDa (arrows in Fig. 4a) and free Chi. The same four polypeptides were present in CP Ia complexes run through two cycles of electrophoresis (Fig. 4b). On gels consisting of a single polyacrylamide concentration they appeared as two bands of about 22 and 24 kDa. The position of the lowest of the four bands on gradient PSI ant. CP CP29 CPIa C P A Fig. 3. Unstained 10 % polyacrylamide SDS. gel showing the dissociation of CP Ia bottom into CP I and a new PSI antenna CP (arrow). The green complexes were excised from a 7.5 % polyacrylamide gel such as that shown in Fig. 2, and then re-electrophoresed on the gel shown in this figure, a) CP 29. b) CP Ia bottom, c) CP II. CP Ia top produces an identical electrophoretic pattern to CP Ia bottom. Note the different positions of the three Chi a+b protein complexes. 56 Fig. 4. Silver-stained 10 to 15 % polyacrylamide gradient gel. a) Denatured PSI antenna CP. Four polypeptides are visible (arrows), b) CP Ia bottom run through two cycles of electrophoresis. Some CP I has dissociated into its 64 and 66 kDa apoproteins (apo CP I), and the PSI antenna has dissociated into its constituent polypeptides in the 21-24 kDa range. Note that not all of the CP Ia complex has dissociated. There are no polypeptides in the LHCII and CP 29 regions of the gel (square brackets). The masses of the molecular weight standards (in kDa) are indicated by the numbers on the left. 57 gels was somewhat variable with respect to the other polypeptides. It could be recognized by its characteristic yellow-orange colour when silver-stained, which distinguished it from the brown colour of the other polypeptides. Note that CP Ia purified by two rounds of electrophoresis did not contain any polypeptides in the LHCII or CP 29 regions of the gel (square brackets in Fig. 4b). Several other polypeptides with molecular masses in the 9-19 kDa range were associated with CP Ia complexes but were completely released by the end of the second electrophoresis. They therefore were more loosely bound constituents of the CP Ia complex or contaminants present during the first electrophoresis. Some of these may have been iron sulfur proteins associated with PSI [Nelson et al. 1975, Acker et al. 1982, Lagoutte et al. 1984]. The absorption spectrum of the new PSI-associated Chi protein had maxima at 436 and 671.5 nm and a Chi b shoulder around 472 nm (Fig. 5). All CP la bands had absorption spectra similar to CP I, with maxima at 437 and 675 nm but with a Chi b contribution in the 470-480 nm region (Fig. 5, vertical arrow). This 470-480 nm shoulder in both the CP Ia complexes and the PSI-associated Chi protein disappeared when methanol extracts were treated with NH 2OH which oxidizes Chi b [Ogawa and Shibata 1965]. Both complexes therefore contained Chi b. Using the method of MacKinney [1941], the PSI antenna CP had a Chi alb ratio of 2.5 ± 1.5 based on six measurements made on separate samples. This variability could be due to the small amounts of purified material obtainable 58 Fig. 5. Visible absorption spectra of gel slices of CP Ia bottom, CP I and the PSI antenna CP. The absorption spectrum of CP Ia top is indistinguishable from that of CP Ia bottom. Numbers indicate the positions (in nm) of the blue and red absorption maxima. A Chl b shoulder (large vertical arrow) is visible in both the CP Ia and PSI antenna CP spectra. 59 or to a preferential loss of Chl a during gel electrophoresis as occurs with CP II and its oligomers [Green and Camm 1982, Remy et al. 1977]. CP Ia complexes which remained undissociated throughout two cycles of electrophoresis had a Chl alb ratio of 4.9 ± 0.6 % based on three samples. Since the PSI antenna CP was tightly associated with CP I (i.e. PSI) in the CP Ia complexes, contained Chls a and b, and four polypeptides of 21-24 kDa, it most closely resembles the LHCI isolated by Haworth et al. [1983] from pea. 2. Effect of detergents on the dissociation of CP Ia complexes Various detergents were tested to see if any would cause a complete dissociation of CP Ia into CP I and PSI antenna CP without removing the Chl from Chl proteins. CP Ia bands were cut out of 7.5 % acrylamide gels and strips of equal length mashed in the appropriate detergent at 4°C. The resulting slurry was run on nondenaturing gels. Neither 0.3-1.0 % SDS nor 15-90 mM octylglucoside had any detectable effect on CP Ia, CP I or the PSI antenna CP. High octylglucoside concentrations (150 mM), 1 % deoxycholate and all Triton X-100 concentrations (0.025-2.5 %) were detrimental to all three Chl proteins, causing at least a partial dissociation into free Chl and apoproteins. None of these detergents separated CP Ia completely into CP I and its antenna without damaging the Chl proteins. This is further evidence of the tight binding of the PSI antenna CP to CP I, i.e. the binding of some of the PSI antenna CP to CP I appears to be stronger than the binding of Chl to these proteins. 60 3. CP Ia complexes can be obtained directly from intact chloroplasts Intact chloroplasts were prepared in grinding media containing 5 mM MgCl 2 to maintain granal stacking. When these chloroplasts were solubilized directly with 300 mM octylglucoside, the same electrophoretic Chl protein pattern was obtained as with thylakoids prepared and solubilized by the usual method (see Fig. 2). These results differ from those of Markwell [1980] where extraction of intact chloroplasts with 1 % SDS produced a different electrophoretic pattern from similarly extracted thylakoids. Thylakoids washed in 10 mM Tris-maleate buffer pH 8.0 and thylakoids washed in this buffer containing 0.75 mM EDTA produced a pattern of Chl proteins identical to those prepared by washing in 10 mM Tris-maleate, 2.0 mM EDTA, pH 8.0. Thus CP Ia complexes were obtained whether or not divalent cations were present prior to detergent extraction. Chloroplasts or thylakoids solubilized with 300 mM octylglucoside in distilled water were the same as those extracted with octylglucoside in buffer. It is therefore unlikely that the CP Ia complexes are an artefact generated by the preparative procedure. B. IMMUNOLOGICAL CROSS-REACTION AMONG CHLOROPHYLL A + B-BINDING POLYPEPTIDES 1. Preparation of antibodies to CP Ia CP Ia was used for raising antibodies to the PSI antenna polypeptides, because it was relatively easy to prepare the amounts of purified complex needed, and more importantly because this complex migrates far away from the other Chi 6-containing complexes. The purity of the antigen is crucial for the experiments which follow. Fig. 2 shows that CP Ia was well separated from both CP 29 and both forms of LHCII (CP II and CP II*) by the first electrophoresis on 7.5 % polyacrylamide. Fig. 3 shows that it was still very well separated when re-electrophoresed on 10 % polyacrylamide. When CP Ia purified this way was denatured, electrophoresed on gradient gels and silver-stained, it did not show any contaminating polypeptides in the LHCII and CP 29 regions (marked with square brackets in Fig. 4b). This technique is 50-100 times more sensitive than Coomassie blue staining [Nielsen and Brown 1984, Heukeshoven and Dernick 1985]. Any minor contaminants which were antigenic should have been detected when purified CP Ia was electrophoresed, blotted, and reacted with the antibody raised against it. Such an experiment is shown in Fig. 6c. There were very intense bands at the positions of the CP I apoprotein and the PSI antenna polypeptides, but absolutely nothing was visible at the positions of the CP 29 and LHCII polypeptides. The PSI antenna polypeptides in barley have very similar molecular 62 M ant.{ a b 66 45 '36 .29 • 25 - 2 0 Fig. 6. Immunoblot (lanes b-e) of a 15 % polyacrylamide gel reacted with anti-CP Ia. All samples have been heat-denatured in 2 % SDS, 50 mM dithiothreitol, 65 mM Tris-HCl, pH 7.6 at 65°C for 30 min. a) Thylakoids, stained, b-e): Gel lanes Western blotted and reacted with anti-CP Ia. b) Thylakoids containing 0.22 yg Chl. c) Purified CP Ia. d) Purified LHCII. e) Purified CP 29. Lanes c, d and e each contain about 1 yg protein, f) Blot of thylakoids (containing 0.5 yg Chl), Western blotted and reacted with preimmune serum. The masses of the molecular weight standards (in kDa) are indicated by the numbers on the right. 63 weights and thus are difficult to resolve on gels or blots. Since the completion of this work, PSI antenna complexes have been isolated from dicot species such as pea and spinach [e.g. Haworth et al. 1983, Evans and Anderson 1986]. The PSI antenna polypeptides in these species are spread over a substantially larger range of molecular weights, and thus are much easier to resolve by electrophoresis. The PSI antenna complexes isolated from other species are discussed in detail in the Discussion. However, the PSI antenna polypeptides in barley form a band which is sufficientl}' broad to see if the upper or lower polypeptides are missing. Fortuitous resolution of some of these polypeptides occurs on occasion. This may occur if exactly the right amount of protein is used, if the conditions of electrophoresis are ideal, and if the development time of the silver-stain or immunoblots is optimal. If too little protein is used, development times must be increased to allow visualization and this results in an increased background. If too much protein is used, the electrophoretic bands widen and merge together. Quantitative immunoenzymatic detection of purified Chl proteins on dot blots indicated that antibodies could detect 16 pg Chl, corresponding to approximately 80 pg protein (Fig. 7). This assumed a protein to Chl weight ratio of approximately 5:1, i.e. about six, 900 Da chlorophyll molecules per 27,000 Da polypeptide (27,000/(6x900) = 5). This estimate is close to that of the consensus composition of LHCII, approximately 4 Chl a, 3 Chl b and 1-2 xanthophyll molecules per polypeptide, although some controversy about the exact pigment to protein ratio remains [Thornber 1986]. Thus when 0.5 ug of antigen protein was loaded in a gel slot, electrophoresed and immunoblotted, one could have detected 0.1 % (500 pg) or less of a contaminant using these antibodies. The preimmune 64 Ag Ab Fig. 7. Dot blots of native Chi a + b proteins probed with antibodies to CP II (from barley II*) and CP Ia (also from barley). Each strip contains six spots of a five-fold serial dilution. Amounts of antigen in the 1 pi spots going from left to right are approximately 400 pg, 80 pg, 16 pg, 320 fg and 64 fg Chi. All four Chi a + b proteins show at least some reaction with both antisera. Ag: antigen on dot blot, Ab: antibody dot blot was incubated in. 65 serum in lane 5f gave no reaction at all with total thylakoids containing 0.5 Mg of Chi. This eliminated the possibility that the cross-reactions observed with thylakoid polypeptides were caused by reaction with unelicited components of the rabbit serum, or with the second antibody-enzyme conjugate, substrate or dye. 2. Antibodies to CP Ia cross-react with LHCII and CP 29 polypeptides Since the purified CP Ia preparation did not contain any detectable polypeptides in the molecular weight range of the LHCII and CP 29 apoproteins (25-29 kDa), it was surprising to find that polypeptides at these positions were detected when total thylakoid protein was blotted and reacted with this antibody (Fig. 6b). The anti-CP Ia also cross-reacted with polypeptides of LHCII purified by the method of Burke et al. [1978] and with CP 29 purified by three successive electrophoretic runs (Fig. 6d and e). This showed that these other Chi a + fe-binding polypeptides were responsible for the reaction observed with polypeptides of whole thylakoids. It also showed that the anti-CP Ia, even though it was raised against a highly purified complex which showed no contamination with CP 29 or L H C n , cross-reacted with antigenic determinants in purified CP 29 and LHCII. The amounts of Chi loaded in lanes 6c, d and e were approximately equivalent. The reaction of anti-CP Ia with CP II and CP 29 polypeptides was quite strong, such that about 50 % of the antibody recognized these polypeptides under some conditions (Fig. 6b). In contrast, the reaction of this antibody with CP I apoprotein on blots of total thylakoid protein was very weak, as the CP I 66 apoprotein was not as antigenic as the Chi a + 6-binding polypeptides. This reaction was clearly visible when longer development times are used (Figs. 8a and 9d), but was always weaker than the cross-reactions with the PSII antenna polypeptides. However, anti-CP Ia readily detected the CP I apoprotein on blots of purified CP Ia (Figs. 6c, 8d and 9c), because some of the PSI antenna CP had dissociated during electrophoretic purification of the native complex, resulting in a higher ratio of CP I to antenna polypeptides. Thus anti-CP Ia reacted more strongly with CP II and CP 29 polypeptides than with those of CP I (e.g. Figs. 6b and 8a), a major component of the injected antigen. It was therefore very unlikely that the subpopulation of anti-CP Ia antibodies directed against CP I could account for the intense reactions with CP n and CP 29 polypeptides. Attributing the immunological cross reactions to relatedness of the Chi a+6-binding polypeptides is therefore the most probable interpretation of these results. 3. Specificity of cross-reactions observed with anti-CP Ia Since the argument presented in the previous paragraph is based on quantitative considerations, experiments were performed to demonstrate directly that the reaction of anti-CP Ia with the Chi a+6-binding polypeptides was not due to the subpopulation of antibodies directed against CP I. First of all, antisera to CP I raised in two different rabbits did not react with any of the Chi o+6-binding polypeptides (e.g. Fig. 8b). Furthermore, when the anti-CP I activity was removed from anti-CP Ia, this antiserum still reacted with the Chi c+6-binding polypeptides. This was done by blotting a mixture of native and 67 ant.{ • I -66 - 4 5 -36 - 2 9 - 2 5 -20 a b c d e f Fig. 8. Experiment showing that cross-reaction of anti-CP Ia with the PSII antenna polypeptides (arrows) is not due to antibodies specific for CP I (I) but due to antibodies to the PSI antenna polypeptides (ant), a-c): Denatured thylakoids containing 0.16 ug Chl. d-f): Denatured CP Ia. Western blots were cut into strips corresponding to gel lanes and reacted with anti-CP Ia (a and d), anti-CP I (b and e) and anti-CP Ia from which the anti-CP I activity had been removed by repeated extraction with CP I (c and f). All samples were electrophoresed on 10 % polyacrylamide gels prior to blotting. The masses of the molecular weight standards (in kDa) are indicated by the numbers on the right. 68 heat-denatured CP I onto nitrocellulose strips which were blocked with 3 % fish skin gelatin and then incubated with anti-CP Ia. Repeated extraction of the anti-CP Ia by this procedure removed the anti-CP I activity (compare Fig. 8 lanes c and f to lanes a and d), but did not remove the cross-reactions with the PSII antenna polypeptides (Fig. 8c). Some nonspecific removal of all antibody activities accompanied the specific removal of anti-CP I activity, presumably due to protein binding to glass, plastic and unblocked sites on the nitrocellulose (since even an excellent blocking agent such as fish skin gelatin will not block 100 % of the sites). These experiments showed that the cross-reactions with the LHCII and CP 29 polypeptides were due to antibodies against the PSI antenna polypeptides and not due to antibodies against CP I. All the samples in Figs. 6, 8 and 9 were totally denatured by heating in the presence of 2 % SDS, so the reactions were not due to antibodies against Chi. Similarly, anti-SDS activity was not responsible since all the thylakoid proteins would have been expected to bind SDS and react if this were the case. In addition, there was no reaction at the free pigment front, where anti-Chl or anti-galactolipid activity would have been observed. None of the antisera reacted with dot blots of monogalactosyldiacylglycerol or digalactosyldiacylglycerol. Experiments performed using peroxidase conjugated to goat anti-rabbit antibodies gave the same results obtained with the alkaline phosphatase conjugate. Antibodies synthesized against the PS II proteins 5 and 6 of Chlamydomonas (which recognize the corresponding CP 47 and CP 43 of higher plants) did not react with any of the Chi a + 6-binding polypeptides. They did react with proteins in the 35-50 kDa range as expected. 69 The objection could still be raised that the anti-CP Ia antiserum contained antibody to trace contaminants that just happened to be much more antigenic than the CP Ia polypeptides. If so, it should be possible to select preferentially antibodies to CP Ia proteins by affinity purification of the antiserum using highly purified CP Ia. Due to the much higher amounts of CP Ia protein present, any antibodies to trace contaminants should be greatly depleted in the resulting purified antiserum. To prepare affinity-purified antiserum, nitrocellulose strips were soaked in highly purified CP Ia, blocked, and soaked in CP Ia antiserum for 30 min. After thorough washing, the antibodies were desorbed from the nitrocellulose strips. Fig. 9c shows a sample of the purified CP Ia used in affinity purification of the antiserum, blotted against the original antiserum. The original antiserum and the affinity purified antiserum, reacted with blots of total thylakoids, are compared in lanes 9d and e. The purified antiserum showed no depletion of the antibodies reacting with the PSII antenna polypeptides (arrows). It was therefore the antibodies to the PSI antenna polypeptides which recognized antigenic determinants on LHCII and CP 29 polypeptides. 4. Preparation of Antibodies to CP II and CP 29 Antibodies were raised against CPU which is a form of LHCII prepared by gel electrophoresis [see methods]. It contained two polypeptides which migrated as a single band of 26-27 kDa in the gel system used here. CP 29, the minor Chl a+b protein complex of PSII, was purified the same way. The apoproteins 7 0 Fig. 9. Experiment showing that anti-CP Ia purified by pre-adsorption onto CP Ia still reacts with LHCII and CP 29 polypeptides, a) molecular weight standards, Coomassie blue stained. Their masses (in kDa) are given at left, b) Denatured thylakoids, Coomassie-blue stained, c) Denatured CP Ia used for affinity purification of anti-CP Ia, blotted and reacted with the original anti-CP Ia. d) Denatured thylakoids containing 0.20 ug Chl, blotted and reacted with anti-CP Ia. e) Denatured thylakoids containing 0.20 yg Chl, blotted and reacted with affinity purified anti-CP Ia. The relative strength of reaction with the PSII antenna polypeptides (arrows at right) is unaltered. All samples were electrophoresed on 10 % polyacrylamide gels prior to blotting. of the purified CP II and CP 29 used as antigens were pure as judged by silver-staining and immunoblotting (Fig. 10), the two most sensitive methods of protein detection. There were no detectable CP II polypeptides in the purified CP 29, and no CP 29 polypeptides in the purified CP II (Fig. 10). The two polypeptides of CP 29 were not resolved when relatively large amounts were used to check for purity (as in this figure). Western blots of total thylakoid protein incubated with preimmune serum showed no reaction (Fig. 10). 5. Antibodies to CP II and CP 29 also cross-react The reaction of anti-CP II and anti-CP 29 with native Chi a + b proteins was tested in a series of dot blots shown in Figs. 7 and 11. Anti-CP II and anti-CP 29 reacted most strongly with their respective antigens, but also showed some cross-reaction with other Chi a+b proteins. Both these antibodies reacted more strongly with each other's antigen than with the PSI antenna CP (Figs. 7 and 11). The anti-CP II in Fig. 11 was raised using CP II from Lemna gibba, and was a gift from Dr. Elaine Tobin. Although dot blots are useful for screening large numbers of samples at one time, it is unlikely but possible that some of the cross-reaction observed might be due to anti-Chl activity. Even if samples are heat-denatured, the pheophytins resulting from the heating of Chls are still present on the dot blot, opposite to the situation with Western blots. Therefore experiments with Western blots were also performed. 72 29[ II [ a b -66 -45 -36 -29 -25 -20 e Fig. 10. Controls showing purity of the antigens used to make anti-CP II and anti-CP 29. All samples were heat-denatured in 2 % SDS, 50 mM dithiothreitol, 65 mM Tris-HCl, pH 7.4 at 65°C for 30 min, and electrophoresed on a 10-20 % polyacrylamide gradient gel. a) Blot of wild-type thylakoids reacted with preimmune serum from the rabbit used to make anti-CP H. b) Blot of pure CP II reacted with anti-CP II. c) Pure CP II, silver-stained, d) Blot of pure CP 29 reacted with anti-CP 29. e) Pure CP 29, silver-stained, f) Blot of wild-type thylakoids reacted with preimmune serum from the rabbit used to make anti-CP 29. Masses of the molecular weight standards (in kDa) are given at right. 73 Fig. 11. Dot blots of native PSI antenna CP, CP II and CP 29 probed with antibodies produced to CP II (from Lemna gibba, courtesy of Elaine Tobin) and CP 29 (from barley). Each strip contains six spots of a five-fold serial dilution. Amounts of antigen in the 1 ul spots going from left to right are approximately 2 ng, 400 pg, 80 pg, 16 pg, 320 fg and 64 fg Chl. Anti-CP II and anti-CP 29 react with their respective antigens at concentrations about 5-25 times lower than those of the other Chl a + b proteins. Ag: antigen on dot blot, Ab: antibody dot blot was incubated in. 74 When small amounts of denatured thylakoid protein were electrophoresed, Western blotted and reacted with anti-CP II, the antiserum appeared to react only with the CP II apoprotein (Fig. 12d). However, when larger amounts of thylakoids were used, anti-CP H also detected polypeptides immediately above and below the CP II apoprotein (Fig. 12c-d). These cross-reacting polypeptides had the same molecular weights as the CP 29 apoprotein (28-29 kDa) and a 25 kDa polypeptide found in LHCII purified by the method of Ryrie et al. [1980] (Fig. 13). Unlike CP II which is purified by electrophoresis of thylakoids solubilized in detergent (see Materials and Methods for details), LHCII is prepared from Triton-solubilized thylakoids by sucrose-gradient centrifugation and cation precipitation [Burke et al. 1978, Ryrie et al. 1980]. Barley LHCII not only contains the polypeptides found in CP II, but also contains this minor 25 kDa polypeptide as discussed in detail in the following chapter. In addition to reacting with the 29 and 25 kDa polypeptides in thylakoids, anti-CP II also reacted with the purified CP 29 apoprotein (Fig. 16f) and with all of the polypeptides in purified LHCn (Fig. 16e), including the minor 25 kDa polypeptide. Lane 16f (purified CP 29) contained the same amount of protein as lane 16e (purified LHCII), showing the relative strengths of the reactions with the polypeptides of the two complexes. Anti-CP 29 detected only the CP 29 apoprotein on immunoblots of gel lanes containing small amounts of denatured thylakoids (Fig. 14f). When larger amounts of protein were used it cross-reacted with the CP n polypeptides and with polypeptides running at the position of the PSI antenna (Fig. 14d and e). These results were not affected by the inclusion of protease inhibitors (0.1 mM 75 "66 - 4 5 —42 Fig. 12. Immunoblot (lanes b-d and f) of a 7.5-15 % gradient gel reacted with anti-CP II. All samples were heat-denatured as in Fig. 10. a) Thylakoids, stained with Coomassie blue, b-d) Thylakoids containing b) 0.80 c) 0.27 and d) 0.09 ag Chl. e) Purified CP II (from II*), silver-stained, f) Purified CP II (from II*), immunoblotted with anti-CP II. g) Blot of thylakoids reacted with preimmune serum. The masses of the molecular weight standards (in kDa) are indicated by the numbers on the right. 76 a b e d e f Fig. 13. Polypeptide composition of LHCII and CP II. Samples were heat-denatured and electrophoresed on 15 % polyacrylamide gels. Lanes a-c and f are Coomassie blue-stained; lanes d and e are Western blots, a) Chi 6-less barley thylakoids. b) Wild-type barley thylakoids. c) LHCII. d) LHCII, blotted and reacted with anti-CP II. e) CP- II, blotted and reacted with anti-CP II. f) CP H, stained. Masses of the molecular weight standards (in kDa) are given at left. 77 Fig. 14. Immunoblot (lanes d-h) of a 10-15 % gradient gel reacted with anti-CP 29. All samples were heat-denatured as in Fig. 10. a-c) Silver-stained gel lanes showing a) Purified CP II (from II*). b) Purified CP 29. c) Thylakoids (wild-type barley) containing 11.5 ug Chi, stained with Coomassie blue, d-f) Thylakoids containing d) 0.90 e) 0.30 and f) 0.10 ug Chi. g) Purified CP 29. The faint high molecular weight band is an oligomer as it is absent prior to denaturation. h) Purified CP II. i) Blot of thylakoids reacted with preimmune serum. The masses of the molecular weight standards (in kDa) are on the right. 78 phenylmethylsulfonyl fluoride, 6 mM p-aminobenzamidine and 40 mM e-amino-n-caproic acid in all solutions) during thylakoid preparation. In addition, anti-CP 29 reacted with LHCII purified by the method of Ryrie et al [1980] and with CP II purified by gel electrophoresis (Fig. 14h). Approximately equal amounts of Chl were loaded in lanes 13g and 13h. It should be noted that the reaction of this antiserum with CP II polypeptides in thylakoids is deceptively strong because they make up such a large proportion of total thylakoid protein. A comparison of the three anti-Chl a + b protein antisera specificities at low levels of thylakoid proteins is shown in Fig. 15. To demonstrate that the immunological cross-reaction was due to sequence homology in the primary sequence, attempts were made to sequence CP 29. The first problem to arise was the resolution of CP 29 on some occasions into two polypeptides by extremely prolonged electrophoresis on totally denaturing 10 % polyacrylamide gels or certain gradient gels (e.g. Fig. 12a). These polypeptides were almost identical in molecular weight and frequently would be resolved on one side of a gel but not the other, or insufficiently resolved for purification, thus requiring a fourth cycle of electrophoresis. (A minimum of two cycles of electrophoresis on a large number of gels was first required to purify the green complex, followed by a third cycle of denaturing electrophoresis as the first attempt to resolve these polypeptides.) Neither polypeptide co-migrated with LHCII polypeptides. Both appeared to be legitimate CP 29 polypeptides, since when resolved they were always present in approximately a 1:1 stoichiometry. A contaminant would be expected to vary in amount between one purification and the next. However, it was not clear whether or not this was two forms of one 79 a b e d 4 5 — 36' 29— ^ , ]29 20- • • ^ a n t S 29 n Ia i 1 antibodies against Fig. 15. Cross-reaction of Chl a+b protein antibodies. Denatured thylakoids were electrophoresed on a 7.5-15 % polyacrylamide gradient gel, and then stained or transferred to nitrocellulose, a) Thylakoids containing 3.0 ug Chl, stained (S) with Coomassie blue, b-d) Immunoblots of thylakoids (0.22 ug Chl) reacted with anti-CP 29 (b), anti-CP II (c) and anti-CP Ia (d). 80 polypeptide, two homologous polypeptides or two unrelated polypeptides. Chemical cleavage and limited proteolysis did not work well or at best gave ambiguous answers to this question (discussed below), one of the major limitations being that these approaches generally consumed most of the sample. These two polypeptides were therefore purified and submitted separately for sequencing, although this doubled the required purification time. Substantial quantities of CP 29 were purified on two separate occasions and submitted for sequencing on the gas phase sequenator at the University of Victoria. Edman degradation was not successful since in each case the CP 29 polypeptides were blocked at the amino terminal. This occurred despite the fact that only the purest chemicals available were used, including charcoal-filtered and recrystallized SDS [Hunkapiller et al. 1983] and the highest quality acrylamide and bisacrylamide. The blockage may be a posttranslational modification of the mature amino terminus, as blocked amino termini have been reported from most attempts to sequence LHCII polypeptides [Dunsmuir 1985, Ken Hoober pers. comm.]. A variety of chemical cleavages was therefore employed in an attempt to generate and purify a peptide with a free amino terminal and thus obtain at least a partial sequence. This would not eliminate the problem of blockage if it were a consequence of the methods of purification, but would generate free amino termini if the cause of blockage were posttranslational modification of the mature amino terminus in plants. All these chemical cleavages worked to a limited extent, cleaving perhaps 10 % of the total protein. The formic acid used in 81 limited acid hydrolysis (cleavage of Asp-Pro bonds) and in CNBr cleavage (cleavage predominantly at methionine) was difficult to remove from the sample and incomplete removal caused distortion of the electrophoretic patterns. Following the use of hydroxylamine-HCl (cleavage at Asn-Gly), the pH of the sample must be adjusted with NaOH, introducing salt which must removed by dialysis. This may result in the loss of peptides through the dialysis membrane. The major problems for all these methods was that they were designed for use with relatively large amounts of protein. Since the yield was not nearly sufficient (and attempts to improve yield experimentally consumed still more sample), this method was abandoned. Proteases such as Staphylococcus aureus V8 protease generated suitable sized fragments when used at optimal concentration for the right length of time. However, the protease itself contained two bands at 29 kDa and 6 or 7 peptides of lower molecular weight. Most commercial proteases are not very pure and thus there is a risk that the contaminants introduced into the sample with the protease may be mistaken for, or copurified with, a fragment of CP 29. The various attempts were ultimately abandoned due to time constraints. Instead, the CP 29 antibodies are now being used by Gopal Subramaniam of our lab to screen cDNA libraries made from barley and tomato, in order to obtain a nucleic acid sequence for CP 29. At this time several clones have been isolated from the tomato library. These clones were selected using anti-CP 29 .which had been treated with LHCII purified by the method of Ryrie et al. [1980] from barley. A comparison of nucleic acid sequences (or predicted amino acid sequences) of CP II and CP 29 will provide more information on the relatedness 82 of their polypeptides than further immunoblotting experiments would. 83 C. STUDIES ON A BARLEY MUTANT LACKING CHLOROPHYLL B 1. Chlorina f2 is depleted in the major LHCII polypeptides The chlorina f2 mutant provides a unique opportunity to examine the role played by Chi b in regulating the accumulation of the Chi a + b proteins. Previous studies (see Introduction) have reported that this mutant lacks PSI antenna (LHCI) and LHCII polypeptides on the basis of Coomassie blue staining. However, Ryrie [1983] used more sensitive immunological techniques to detect some of these L H C n polypeptides using an antibody to spinach LHCII. Following this example, antibodies to the three Chi a+b proteins were used to look for the corresponding polypeptides in Chi 6-less barley. This work is also described in White and Green [1987c]. The complex referred to here as ' LHCII' was prepared from Triton-solubilized thylakoids by sucrose-gradient centrifugation and cation precipitation [Burke et al. 1978, Ryrie et al. 1980]. It contained the 26-27 kDa band of CP II (Fig. 13e and f) and a minor polypeptide of 25 kDa (Fig. 13c and d). In most plants, the 26-27 kDa band can be resolved into two closely-spaced polypeptides [Green and Camm 1982, Machold 1981], but in barley the two polypeptides run as a single band unless a gel system containing a high urea concentration is used [Machold et al. 1977, Machold 1981]. The antibodies raised against CP II reacted with both the major 26-27 kDa band and the minor 25 kDa polypeptide of LHCII, even though the latter was not found in CP n. (Fig. 13d). When combined with evidence that the two major polypeptides 84 in the 26-27 kDa band are immunologically related [Chua and Blomberg 1979], this suggests that all three LHCII polypeptides are closely related. The 25 kDa polypeptide seen on immunoblots appeared to co-migrate with a Coomassie blue-stainable polypeptide in both normal and Chl 6-less thylakoids (Fig. 13a and b). Thylakoids of the chlorina f2 mutant were conspicuously deficient in LHCII polypeptides in the 27 kDa range compared to wild-type barley (Fig. 13), although there was a stainable band corresponding to the lower part of the major LHCII (CP H) polypeptide band. When transferred to nitrocellulose and reacted with anti-CP II, at least two polypeptides were detected, one of approximately 26-27 kDa and one at about 25 kDa. (Fig. 16b and c). If smaller amounts of thylakoid protein from the mutant were used, only the 25 kDa LHCII polypeptide was detected (Fig. 16d). This was surprising, as this polypeptide was the least abundant of the three LHCn polypeptides in wild-type barley. Thus the two major LHCII polypeptides, which ran as one band, were depleted to a much greater extent in the chlorina f2 mutant than the 25 kDa polypeptide (compare Fig. 16b-d and g). 2. Chlorina f2 contains the CP 29 polypeptides When Chl 6-less thylakoids were electrophoresed, blotted and reacted with anti-CP 29, a strong reaction was observed at the position of the two CP 29 polypeptides (Fig. 17b-d). If larger amounts of protein were loaded, a weaker reaction with the 25 kDa LHCII polypeptide (arrow) was also visible (Fig. 17b). 85 29 c [ - 4 5 - 3 6 - 2 9 —25 —20 b c d e f g h Fig. 16. Detection of LHCII polypeptides in Chl 6-less barley. Samples were heat-denatured and electrophoresed on 15 % polyacrylamide gels, a) Coomassie blue-stained Chl 6-less thylakoids containing 10 Mg Chl. b-g) Western blot reacted with anti-CP II: b-d) Chl 6-less thylakoids containing b) 0.36, c) 0.12 and d) 0.04 ug Chl. e) Purified LHCII (about 1 ug protein), prepared by the method of Burke, Ditto and Arntzen [19]. f) Purified CP 29 (about 1 ug protein), g) Wild-type thylakoids containing 0.16 ug Chl. h) Western blot of normal thylakoids reacted with preimmune serum. Masses of the molecular weight standards (in kDa) are given at right. 86 4 a b c d e —45 - 3 6 — 29 - 2 5 - 2 0 f Fig. 17. Detection of CP 29 polypeptides in Chl 6-less barley. Samples were heat-denatured and electrophoresed on 15 % polyacrylamide gels, a) Coomassie blue-stained Chl 6-less thylakoids containing 10 ug Chl. b-e) Western blot reacted with anti-CP 29: b-c) Chl 6-less thylakoids containing b) 0.25 and c) 0.08 ng Chl. d) Purified CP 29 (about 1 ug protein), e) Wild-type thylakoids containing 0.16 ug Chl. f) Western blot of normal thylakoids reacted with preimmune serum. Masses of the molecular weight standards (in kDa) are given at right. The arrow marks the position of the 25 kDa LHCII polypeptide. 87 Purified CP 29 and normal thylakoids showed strong immunoenzymatic staining in the 29 kDa region (Figs. 14, 15 and 17d and e), verifying that this was the CP 29 apoprotein. These results confirm the earlier suggestion of CP 29 polypeptides in chlorina f2 thylakoids based on Coomassie blue staining [Machold 1981], and show that the Coomassie stained band was not just an unrelated co-migrating polypeptide. They further suggest that the CP 29 apoprotein and the 25 kDa polypeptide are immunologically related (see below). 3. Chlorina f2 contains the PSI antenna (LHCI) polypeptides Gel lanes of normal and chlorina f2 barley thylakoids were blotted onto nitrocellulose and reacted with anti-CP Ia in an experiment identical to those performed with anti-CP H and anti-CP 29. PSI antenna polypeptides were clearly present in Chi 6-less barley (Fig. 18d and e). The antennal polypeptides were visible when thylakoids equivalent to only 0.075 ug Chi are loaded (Fig. 18d), while the CP I polypeptides were detected only at higher concentrations (Fig. 18e). The cross-reaction of anti-CP Ia with LHCII and CP 29 polypeptides is seen in the blots of normal and mutant thylakoids in Fig. 18b and e. This cross-reaction provided confirmation that the major LHCII polypeptides (those of CP ft) were the ones depleted in these Chi 6-less thylakoids. It also provided additional evidence that the apoprotein of CP 29 and the minor 25 kDa polypeptide found in LHCII but not CP II (e.g. Fig. 13) were still present. 88 a b e d e f Fig. 18. Detection of PSI antenna polypeptides in Chi 6-less barley. Samples were heat-denatured and electrophoresed on 15 % polyacrylamide gels, a) Western blot of normal thylakoids reacted with preimmune serum, b-e) Western blot reacted with anti-CP Ia. b) Wild-type thylakoids containing 0.22 jug Chi. c) Purified CP Ia. d) Chi 6-less thylakoids containing 0.075 Mg Chi. e) Chi 6-less thylakoids containing 0.225 Mg Chi. f) Coomassie-blue stained Chi 6-less thylakoids. Masses of the molecular weight standards (in kDa) are given at right. 89 4. CP la-like complexes in Chl b-less barley Gels of Chl 6-less barley produced a number of CP la-like bands running above a double CP I band. However, these bands were more labile than the CP la-like bands in wild-type barley and rapidly dissociated in the presence of SDS. Preparation of green Chl protein bands from Chl 6-less barley was therefore done on 0.1 % (w/v) deoxycholate gels [Waldron and Anderson 1979]. Under these conditions a number of CP la-like bands, two CP I bands, CP 47, CP 43 and a very faint band in the position of CP II* were visible on unstained gels (Fig. 19a). When these CP la-like bands were excised and re-run on nondenaturing gels they produced three green bands (in descending order of apparent molecular weight: i) a CP la-like band ii) CP I and iii) a faint green band with a slightly higher apparent molecular weight than the PSI antenna CP in wild-type barley (Fig. 19b) The faint green complex appeared to be only loosely associated with CP I in the CP la-like bands and present in small amounts. Both the CP la-like band and the faint green band lack polypeptides in the 21 to 25 kDa range (Fig. 20c and d). Thus the PSI antenna CP was not present in the CP la-like complexes of Chl 6-less barley. The faint green band (Fig. 19b) appeared to contain CP 47 and a number of other colourless polypeptides (Fig. 20d), but not the PSI antenna polypeptides. It was therefore possible that this association was artefactual. It was also possible that the CP la-like bands in chlorina f2 are or contain oligomers of CP I, and that the PSI antenna polypeptides were at best loosely attached and dissociated upon detergent solubilization of thylakoids, or 90 Fig. 19. Unstained 8 % polyacrylamide deoxycholate gels, a) Native thylakoids from Chi 6-less barley solubilized in 300 mM octylglucoside. Many CP la-like bands (unlabeled thin arrows) are seen in addition to a double CP I band, CP 47, CP 43 and a faint band thought to be CP II* (unlabeled thick arrow), b) Dissociation of Chi 6-less CP la-like bands into CP I and a faint green CP band (unlabeled thick arrow). 91 Ia ap wt b-less Fig. 20. Silver-stained 10 to 15 % polyacrylamide gradient gel. a) Denatured PSI antenna CP from normal barley, b) CP Ia bottom from normal barley, purified by two cycles of electrophoresis. Some CP I has dissociated into its 64 and 66 kDa apoproteins (apo CP I), c) CP la-like band from Chl Mess barley, d) Polypeptide composition of a Chl protein derived from a CP la-like band in Chl 6-less barley. At least six polypeptides are present (arrows), but they are not those found in the PSI antenna of normal barley (lane a). during the first electrophoresis. 93 D. T H E SYNTHESIS OF CHLOROPHYLL-BINDING POLYPEPTIDES DURING GREENING OF ETIOLATED B A R L E Y 1. Rationale and Experimental Objectives The experiments described in this chapter describe the accumulation of Chl-binding polypeptides during greening of etiolated plants under continuous white light. These dark-grown plants contain carotenoids but lack Chls c and b. An important question for each Chi protein is whether or not its apoprotein is present in the absence of both Chls and light. The use of antibodies is essential to address this question properly since it is only in this way that the presence of small amounts of certain polypeptides can be detected or ruled out. This is one major difference between these experiments and many of the studies reported in the literature. In addition, published studies have concentrated on CP I and CP H but have not used antibodies to study the synthesis of polypeptides belonging to CP 29 and the PSI antenna (LHCI). It is also of interest to compare the kinetics of accumulation for the various Chi proteins, in particular to determine if the Chi a+b proteins share similar kinetics, and whether the Chi o proteins behave as a single group in their pattern of light-induced accumulation. Much of the following work is also described in White and Green [1987d, 1987e]. 94 2. Controls Illumination of etiolated barley seedlings produced a typical sigmoidal pattern of Chl accumulation (Fig. 21). The Chl a/b ratio was initially high and decreased until it reached a value of 3 at 12 h identical to that of mature barley leaves (Fig. 21). Leaf height and weight did not change significantly throughout the 12 h following exposure to light, and SDS-extractable protein per g leaf increased about 40 %. All quantitative immunoblotting measurements were therefore determined using equal amounts of protein per gel lane, and then normalized by multiplying by the total protein per g of leaf fresh weight. A typical immunoblot is shown in Fig. 22. To compare our system to those used in other developmental studies, the relative abundances of several well-studied thylakoid proteins were measured in samples collected before and during the 12 h illumination. This included the alpha, beta and gamma subunits of coupling factor, the 33 kDa polypeptide involved in water splitting, and the herbicide-binding protein D l . The alpha, beta and gamma subunits of coupling factor and the 33 kDa protein of the oxygen-evolving complex were present in dark-grown barley (Fig. 23), as reported for a number of plants [de Heij et al. 1984, Vierling and Alberte 1983, Ryrie et al. 1984, Liveanu et al. 1986, Selstam and Sandelius 1984]. Amounts remained roughly constant or increased slightly during the 12 h of greening in agreement with published data for barley [Vierling and Alberte 1983, Ryrie et al. 1984] and spinach [Liveanu et al. 1986]. The standard deviations for quantitative immunoblotting, as shown in Fig. 23 and the following figures, were about 9 5 Chlorophyll a/b and Total Chlorophyll vs. Time 12 10 a s o ^ 6^ _ A Chi a/b •A * /Total Chi 4 6 8 Time [hours] 10 H50 250 200 g> o _© 100 Q-CD , Z3, u h50 12 Fig. 21. Accumulation of total Chi and decrease in the Chi a/b ratio following illumination of 6-day-old etiolated barley. 96 Time in light (h) RS. 0 1 2 3 6 9 12 ••-66 -45 -36 -29 -25 -20 Fig. 22. A typical Western blot of whole cell proteins reacted with anti-CP I. Protein extracts were prepared from 6-day-old etiolated barley exposed to light for various lengths of time, heat-denatured, electrophoresed on a 10-20 % gradient gel, transferred onto nitrocellulose and reacted with anti-CP I (as described in Materials and Methods). All gel lanes were loaded with equal amounts of protein. The nitrocellulose strip at left was reacted with preimmune serum (P.S.) from the rabbits used to obtain the antiserum to CP I. The masses of the molecular weight standards (in kDa) are indicated by the numbers at right. 97 Accumulation of Thylakoid Membrane Proteins vs. Time 1.6 1.4-3 6 9 Time in Light [hours] 12 Legend A alpho + beta x jjamma • 33 kDa £rotsin B Dl Fig. 23. Control experiments showing the effects of light on the amounts of various plastid membrane proteins. The proteins shown are the 33 kDa polypeptide of the oxygen evolving complex (33 kDa), the alpha and beta subunits of coupling factor (CFl), the gamma subunit of C F l (CF1 gamma) and the herbicide-binding protein of PSII (Dl). 98 20 %. D l , the 32 kDa herbicide-binding polypeptide of PSII, appeared to be absent in the dark (Fig. 23) but showed the expected light-induced accumulation [Klein and Mullet 1986, Liveanu et al. 1986, Fromm et al. 1985, Bedbrook et al. 1978]. 3. Synthesis of polypeptides of the three Chl a+b proteins Antibodies to the Chl a+b proteins CP II (LHCII), CP 29 and the PSI antenna CP (LHCI) were used to determine the kinetics of light-induced accumulation of these polypeptides. Of these three Chl a+b proteins, only the synthesis of CP II and its polypeptides in response to light has been studied previously. None of these polypeptides were detected in dark-grown barley (Fig. 24). The apoprotein of CP II showed its typical sigmoidal increase in response to light [reviewed in Tobin and Silverthorne 1985, Anderson 1986, Ellis 1986]. The apoproteins of both CP 29 and the PSI antenna CP showed almost identical patterns of accumulation to the CP H apoprotein (Fig. 24), suggesting that their synthesis was coordinately regulated by light. Experiments conducted using 5-day-old barley gave similar results to those shown in Fig. 24, which were obtained from 6-day-old barley. 4. Synthesis of the CP 43 apoprotein CP 43 (also known as CPa-2), the Chl o antennal core protein of PSII, was detected with anti-6 antibody (courtesy of Dr. Nam-Hai Chua) raised against Accumulation of Chlorophyll a+b Apoproteins vs. Time 1.2-1 I Legend A PSI_antenna CP •< CP 29„ • CPII Time in light [hours] Fig. 24. Polypeptides belonging to the three Chi a+b proteins, CP II, CP 29 and the PSI antenna, accumulate with almost identical kinetics following the exposure of etiolated barley to light. 100 the corresponding PSII core protein 6 of Chlamydomonas [Chua and Blomberg 1979]. Anti-6 is monospecific for the CP 43 apoprotein in spinach, and does not react with the apoprotein of CP 47 [Chua and Blomberg 1979]. No CP 43 apoprotein was detected in protein extracts of dark-grown barley (Fig. 25), but it increased rapidly over the 12 h illumination period, displaying a pattern of light-induced increase similar to those of the Chl a+b antenna polypeptides. Experiments to determine the kinetics of accumulation of CP 47 following illumination of dark-grown plants were unsuccessful. Technical difficulties and time limitations were encountered in the purification of CP 47. Included among these was the production of multiple polypeptide bands in the 40-51 kDa range upon denaturation of CP 47, and the co-migration of contaminating polypeptides with these bands. Previous studies with Coomassie blue staining failed to detect all of these bands and have consequently presented an oversimplified view. Polyclonal antibodies to the corresponding PSII core protein 5 of Chlamydomonas (courtesy of Dr. Nam-Hai Chua) were therefore used instead. Unfortunately, this antibody was found to contain anti-coupling factor and anti-keratin activities, and it reacted most strongly with an unidentified polypeptide in the 60-70 kDa range. This antibody reacted with up to nine bands in denatured protein extracts. Surprisingly, this antibody has been successfully used to identify the CP 47 gene from chloroplast DNA in spinach [Morris and Herrmann 1984]. Monoclonal antibodies to spinach CP 47 were obtained from Dr. Terry Bricker. Shipment of these antibodies appeared to reduce their activity, but reactions were obtained with dilutions of spinach PSII particles 101 Accumulation of CP43 Apoprotein vs. Time 1.2 T : 1 Time in Light [hours] Fig. 25 Accumulation of the CP 43 apoprotein during the light-induced greening of etiolated barley. The accumulation of the CP II apoprotein is included for purposes of comparison. 102 on ELISA plates. However, no reaction was obtained with native or denatured barley protein extracts under these conditions. Studies on the kinetics of accumulation of CP 47 during greening of etiolated spinach (using the same anti-5 antibody) showed that they were similar to those of CP 43 [Reinhold Herrmann pers. comm.]. 5 . Synthesis of the CP I apoprotein To study the synthesis of a Chi a reaction center protein, antibodies to CP I, which contains two polypeptides of 64-66 kDa, were used in a series of immunoblotting experiments. The CP I apoprotein was present in substantial amounts in the dark and showed at most a two-fold increase after 12 h of illumination (Figs. 22 and 26). These results are similar to those of Nechushtai and Nelson [1985] with wheat, but different from most other studies which detected little or no CP I apoprotein in the dark [Vierling and Alberte 1983, Takabe et al. 1986, Klein and Mullet 1986, Kreuz et al. 1986]. In the preceding experiments, plants were grown in a dark growth chamber covered with additional layers of dark material and watered once at night as described in Materials and Methods. Experiments were therefore performed to test if the CP I apoprotein detected in dark-grown plants might be due to very small amounts of light entering the growth chamber, possibly during the single night-time watering. Flats of seedlings were placed in a large metal can covered with five layers of black cloth, placed in the same dark growth chamber, and not watered. The soil remained moist despite the lack of watering, 103 presumably due to higher levels of humidity maintained within the metal can. The amount of CP I apoprotein detected was decreased, but even under these conditions approximately 19 ± 4 % of the 12 h level was detected in the etiolated seedlings (Fig. 26, ' Extreme dark'). To test the possibility that exposure to light during soaking of the seeds might account for the small amount of CP I apoprotein in dark-grown plants, the soaking was omitted and plants were simply grown for six days as described above. The immunoblot from this experiment was almost identical to that shown in Fig. 27, but lacked the low molecular weight band at 0-2 h. Another set of plants grown in the same way was processed by the method of Kreuz et al. [1986], who did not detect CP I polypeptides in dark-grown barley. This method was very similar to the one used m the preceding experiments (see Materials and Methods), differing mainly in that the SDS-buffer was heated to 95 °C before it was added to the sample. In both methods the buffer was added immediately after the liquid nitrogen evaporated. Since CP I apoprotein was still seen in etiolated barley using this method (Fig. 27), presoaking of the seeds was not responsible for the CP I apoprotein detected in etiolated barley. 6. Effect of Sample Preparation It is not known whether the conflicting reports on the presence of CP I apoprotein in dark-grown plants are due to differences in the species used, its age or growth conditions, or the methods used to conduct these studies. Some studies of CP I synthesis following illumination of etiolated plants have examined 104 Accumulation of CPI apoprotein 1.2-CN H §5 0.8- Normal Dark ~ (1 A-0.6-c o E D b 0.4-c o o o 0.2 Extreme Dark —|— 10 12 Time in Light [hours] Legend a GROWTH CHAMBER X METAL CAN Fig. 26. Accumulation of CP I apoprotein following illumination of etiolated barley. ' Normal dark': Barley grown in a dark growth chamber and watered once at night. ' Extreme dark': Barley grown as described above, and additionally placed in a large metal can with five layers of black cloth covering the opening. Watering was not necessary due to the increased humidity. :o5 Time in light (h) 0 1 2 3 6 91224 < - 6 6 - 4 5 -36 - 2 9 -25 Fig. 27. Immunoblot of heat-denatured whole cell proteins probed with anti-CP I. Etiolated barley was grown in extreme dark as described in Fig. 26 but without presoaking the seeds, and exposed to continuous light for the times (in hours) indicated at the top of the figure. Samples were prepared as described by Kreuz et al. [1986]. All gel slots contained 5 ug of protein. The masses of the molecular weight standards (in kDa) are indicated by the numbers at right. 106 pelleted membrane fractions instead of whole cell proteins. Takabe et al. [1986] did not detect the CP I apoprotein in membrane fractions pelleted at 15,800g from dark-grown pea, wheat or barley. Barley plants were therefore grown under conditions of extreme darkness (without presoaking the seeds) as described above, processed by this method, and immunoblotted using anti-CP I. CP I apoprotein was found both in the pellet and the supernatant (Fig. 28b) at 0 h. The proportion of CP I detected in the 15,800g supernatant is largest at 0 h and decreases sequentially (Fig. 28b), suggesting that the density of the membranes containing the CP I (apoprotein) increases following exposure to light. When these experiments were repeated with the omission of SDS from the homogenization buffer, the 5 min 12,100g spin (which would pellet mature thylakoids) did not pellet the CP I apoprotein found in dark-grown tissue. The CP I apoprotein in this supernatant was be pelleted by spinning at 40,000g for 15 min. This suggests the CP I apoprotein is on small membrane fragments or that, unlike chloroplasts, etioplasts are not pelleted at 12,100g. To see if differences in the sensitivity of immunodetection might account for the discrepancy between our results and those of Takabe et al. [1986], another Western blot was done with the samples prepared by their methods. In this experiment the amount of sample loaded on gels was halved and the development time of the immunoblot in the enzyme substrate reduced. Under these conditions CP I apoprotein was not detected in the 0 h pellet or supernatant (Fig. 28a). The resulting immunoblot then looked like those obtained by other workers [Takabe et al. 1986, Kreuz et al. 1986]. These data suggested 107 Time in light (h) O 12 2 4 4 8 0 12 2 4 4 8 «- Pellets -* «-Supernatants-* Fig. 28. Immunoblots of samples prepared by the method of Takabe et al. [1986] from etiolated barley grown in extreme dark as in Fig. 27. Both pellets and supernatants from the 15,800g centrifugation were heat-denatured, electrophoresed, blotted and probed with anti-CP I as in Fig. 27. a) 5 ug protein per gel lane; anti-CP I diluted 300x; blot developed 10 min in substrate, b) 10 ug protein per gel lane; anti-CP I diluted lOOx; blot developed 30 min in substrate. Widths of the gel slots were twice those used in Fig. 27. The masses of the molecular weight standards (in kDa) are indicated by the numbers at right. 108 that the detection of CP I apoprotein in etiolated tissue did not depend on the species used, but did depend on such factors as the tissue fractionation methods and centrifugation speeds, the growth conditions, and especially on the sensitivity of immunoenzymatic detection. 109 E . CHLOROPHYLL-BINDING POLYPEPTIDES IN B A R L E Y GROWN UNDER INTERMITTENT LIGHT 1. Plants grown under intermittent light have many similarities to Chl b-less mutants The term 'intermittent light' is used to refer to brief periods of white light which interrupt much longer periods of dark at regular intervals. All the following intermittent light experiments were performed using a light regime consisting of 2 min of white light every 2 h, i.e. alternating periods of 2 min white light and 118 min dark, as used by Armond et al. [1976], Armond et al. [1977], Mullet et al. [1980b] and most other intermittent light studies. Other light regimes such as alternating 2 min white light with 98 min dark [Argyroudi-Akoyunoglou and Akoyunoglou 1980] have also been used with similar results. Plants grown under intermittent light contain relatively normal amounts of Chl a but lack Chl fe [Argyroudi-Akoyunoglou and Akoyunoglou 1970, Armond et al. 1976, 1977, Mullet et al. 1980b]. These plants have both PSI and PSn activity and the Chl a proteins [Armond et al. 1976, 1977, Mullet et al. 1980b]. However, the LHCI and LHCH polypeptides cannot be detected in intermittent light thylakoids by Coomassie blue staining [Armond et al. 1976, 1977, Mullet et al. 1980b]. For these two reasons it is generally believed that barley grown under intermittent light is physiologically equivalent to the chlorina f2 barley mutant which also lacks Chl fe. 110 It was previously believed that this Chl 6-less barley mutant lacked the Chl o+6-binding polypeptides. However, as shown in a previous chapter of this thesis, these polypeptides can be detected in this mutant by using sensitive immunoblotting techniques. Thus if barley grown under intermittent light and the chlorina f2 mutant are truly physiologically equivalent, those polypeptides present in chlorina f2 thylakoids should be present in roughly equivalent amounts in thylakoids from plants grown under intermittent light. The following immunoblotting experiments show that chlorina f2 barley and intermittent light barley do not contain the same set of Chl o+6-binding polypeptides. 2. Intermittent light barley lacks LHCI and most LHCII polypeptides Barley seedlings were grown under intermittent white light as described above for 6 days. Seedlings were harvested and used to prepare whole cell extracts by freezing in liquid nitrogen and extraction with 2 % SDS, 50 mM dithiothreitol, 65 mM Tris-HCl pH 7.4 as described for the experiments on greening of etiolated barley. In another experiment, seedlings were used to prepare thylakoids as described in Materials and Methods. The whole cell extracts or thylakoids were electrophoresed, Western blotted and reacted with anti-CP 29, anti-CP II and anti-CP Ia. Gel lanes of normal and intermittent light samples were loaded on an equal chlorophyll basis, which means that lanes of intermittent light samples contained more total protein. Both whole cell extracts and thylakoids from intermittent light barley showed no reaction at the position of the LHCI polypeptides when reacted with I l l anti-CP Ia (Figs. 29 and 30e,f). This was not due to a problem with the anti-CP Ia since the LHCI polypeptides were detected in adjacent lanes of normal and chlorina f2 thylakoids on the same blot. Large amounts of some LHCII polypeptides were also missing from intermittent light plants as can be seen in blots reacted with anti-CP II (Fig. 29). 3. Intermittent light barley contains the C P 29 polypeptides Immunoblots of whole cell extracts reacted with each of the three anti-Chl a+b protein antisera revealed the presence of a band at the position of the CP 29 polypeptides (Fig. 29). This band reacted most intensely with anti-CP 29. The anti-CP 29 also reacted with an unidentified polypeptide of lower molecular weight. Although this polypeptide occurred at the position of the LHCI polypeptides, it could not belong to LHCI since anti-CP Ia showed no reaction at this position (Figs. 29 and 30e,f). To confirm that the band detected by all three antisera was the CP 29 apoprotein, LHCII purified by the method of Ryrie et al. [1980] was used to remove anti-LHCII activity from anti-CP 29. This was done by incubating sheets of nitrocellulose in a phosphate-buffered saline solution containing LHCII. These sheets were then put into Petri dishes of anti-CP 29 antiserum which had been diluted 25 times, and many repeated extractions were performed. Reaction of this purified anti-CP 29 at a final concentration of 1/200 with blots of intermittent light thylakoids clearly indicated that this band was the CP 29 apoprotein (Fig. 30a-d). The unidentified polypeptide of lower molecular weight also reacted 112 N IL N IL N IL I | ! r - 6 6 u ^ - 4 5 - 3 6 I I ^ I - I • - 2 5 a n t f c : • 1 - 2 0 n 29 Ia antibodies against Fig. 29. Western blots of normal thylakoids and whole cell protein extracts from intermittent light barley reacted with anti-CP II, anti-CP 29, and anti-CP Ia. Samples were electrophoresed on a 10 % polyacrylamide gel with a running gel at pH 8.8. This produces sharper bands but requires a longer electrophoresis time and slightly alters the pattern of electrophoretic migration. N: Normal thylakoids containing 0.12 Mg Chl, included for comparison and as controls. IL: Intermittent light samples containing 0.12 Mg Chl. Polypeptides of CP 29 (29) LHCII (II) and the PSI antenna (ant.) are indicated by the arrows at left. Masses of the molecular weight standards (in kDa) are indicated at right. 113 66-4 5 -36-29-25-• • • • .13? r }ant. 20-ant i -29 a n t i - l a Fig. 30. Western blots of intermittent light (a,b,e,f), chlorina f2 (c,g) and normal thylakoids (d,h) reacted with anti-CP 29 (a-d) and anti-CP I (e-h), electrophoresed on a standard 15 % polyacrylamide gel. a,b,e,f) Intermittent light thylakoids containing 0.05 ug Chi (a,e) or 0.10 ug Chi (b,f). c,g) Chi 6-less (chlorina f2) thylakoids containing 0.14 ug Chi. d,h) Normal thylakoids containing 0.14 ug Chi. These blots were developed for a longer time than those in Fig. 24 to demonstrate the absence of PSI antenna polypeptides. Polypeptides of CP 29 (29) LHCII (II) and the PSI antenna (ant.) are indicated by the arrows at right. The high molecular weight bands in lanes b and c are likely oligomers of CP 29, as such oligomers are frequently formed upon heat denaturation of Chi proteins. Masses of the molecular weight standards (in kDa) are indicated at left. with this antiserum (Fig. 30a,b). 115 F. MATHEMATICAL MODELING OF IMMUNOLOGICAL CROSS-REACTION 1. A null hypothesis for immunological cross-reaction: Some cross-reaction is due to chance alone A necessary condition for antibody binding to polypeptides is the recognition of a sequence of amino acids, either as a consecutive sequence in a linearized polypeptide or as a composite epitope in a folded polypeptide. The length of this sequence in a linearized polypeptide is approximately 5-6 amino acids (discussed in the next section). In most cases polypeptides are made up of the twenty most common amino acids in various arrangements. It is therefore possible that the same sequence of 5-6 amino acids (or arrangement of amino acids in the case of folded proteins) might independently arise in different polypeptides due to chance alone. This differs from cases in which two polypeptides have evolved from a common ancestral gene or cases where a convergent evolution has occurred due to functional or structural constraints. All of the above cases may result in sequence homology which, if sufficiently extensive, may result in common antibody binding sites. Therefore it is wrong to attribute biological significance to immunological cross-reactions whenever they occur, as is the current practice. Instead, it is necessary to determine the probability that this cross-reaction might occur due to chance alone, before other explanations can be considered. The situation involving folded polypeptides appears to be quite complicated mathematically and sufficient empirical evidence to allow modelling is currently 116 lacking [Sibbald and White 1987]. However, it is possible to heat-denature polypeptides in the presence of reducing agent and SDS (as described in Materials and Methods) and essentially linearize them for gel electrophoresis and immunoblotting. For situations such as these it is possible to determine the probability that two or more polypeptides will share an identical (or almost identical) subsequence of amino acids sufficiently long to allow antibody binding, 1. e. a subsequence 5-6 amino acids or more in length. Using these methods it is possible to model the probability of chance cross-reaction for both soluble and membrane polypeptides. 2. Dimensions of the antibody combining site Antibody molecules specifically recognize areas on protein surfaces of 3-4 2 nm [Novotny and Haber 1986]. The antibody combining site is the region of the antibody which binds the antigen. It contains the variable and hypervariable regions of the heavy and light immunoglobulin chains. It should not be confused with the antibody binding site referred to in the following sections of this chapter, which is a site on the antigen. Antibody combining sites have roughly the same surface area as the corresponding binding sites on their antigen [Amit et al. 1986]. Detailed studies on antibodies to a large number of polysaccharides have been used to elucidate the size and shape of antibody combining sites. This was done by identifying the structure which competed most effectively with antigen for the combining site [Kabat 1978]. Antibody binding affinities were measured 117 for each member of a polysaccharide series of cUffering chain lengths. For one well-studied antibody the full binding affinity was obtained with isomaltohexaose (IM6), a polysaccharide containing six glucose residues. It was inferred that the combining site was a groove into which any six a-1,6 linked glucoses in the interior of a linear chain could fit [Rabat 1978]. Thus the binding site of this antidextran antibody was about 2.5 nm long [Stryer 1981]. However, another anti-dextran antibody exhibited maximum binding affinity with isomaltopentaose (TM5), a dextran containing five glucoses [Kabat 1978]. The axial distance between adjacent amino acids in an extended polypeptide conformation such as |3-pleated sheet is approximately 0.35 nm [Stryer 1981]. Thus the longest antibody combining site would correspond to approximately 2.5 nm/0.35 nm per amino acid=7 amino acids. It is also possible to estimate a lower limit for the length of an antibody combining site. For example, the antibody which exhibited maximum binding affinity with the five-glucose dextran, TM5, would have a combining site at most five glucoses in length. If the site had a length equivalent to 4.5 glucoses it would be about 1.9 nm or 5.4 amino acids in length. These upper and lower limits of about 2.5 nm and 1.9 nm respectively agree with recent x-ray crystal data for a Fab fragment from a monoclonal antibody to lysozyme. The interface between this antigen and antibody extended over an area with maximum dimensions of about 3 by 2 nm [Amit et al. 1986]. In deriving the equations which follow, the length of the antibody combining site (in amino acids) has been i left as a variable. However, for sample calculations we assume that antibody combining sites recognize sequences about 5 to 6 amino acids in length on 118 average. 3. Comparing two polypeptides for matching amino acids is analogous to flipping a row of coins If two sequences of elements, such as strings of amino acids, are aligned and compared sequentially element by element, each of these comparisons will result in either a match (identical amino acids) or a mismatch (different amino acids) at any given point between these two sequences. The outcome of this series of comparisons can be expressed as a string of matches and mismatches. Mathematically this is analogous to flipping a row of (biased) coins where the outcome of each toss is either a head of value 1 occurring with probability p, or a tail of value 0 and probability q = 1-p [Arratia et al. 1986]. The length of the longest expected match between two such sequences is therefore analogous to the longest head run in a sequence of flipped coins. (It does not matter whether this is a spatial or temporal sequence.) If the sequences being compared are proteins or nucleic acids, then p < 0.5, analogous to the flipping of a biased coin. In this way the problem of comparing two sequences of elements is reduced to a single sequence of outcomes. This simplifies the approach to the problem so that mathematical solutions can be obtained. The length of the longest run of heads in the first n tosses of a coin was first considered by Erdos and Renyi [1970]. A number of refinements and reviews deriving from this original work have since been made [see Arratia and Waterman 1985]. DNA sequences and polypeptides which are highly homologous 119 may be compared simply by alignment with the homologous sequence followed by scoring matches and mismatches, without allowing for shifts between sequences. However, sequences that are this homologous would not likely arise by chance alone. Furthermore, for any such sequence that might arise due to chance alone there would be many more sequences containing much less homology for which the proper alignment between sequences would not be clear. For example, consider the comparison of the following two amino acid sequences. 1. DGALVIEAWSPSWAIQK 2. TTKKGALVISGALVPEA One possible alignment between these two sequences of amino acids is given below. This alignment results in five consecutive matches (denoted by asterisks). - DGALVIEAWSPSWAIQK * * * * * TTKKGALVISGALVPEA However, an alternative alignment producing six matches with an intervening mismatch is also possible. DGALVIEAWSPSWAIQK * * * * ** TTKKGALVISGALVPEA Since any alignment of two sequences might produce matching subsequences sufficiently long to allow antibody binding, all such alignments must be considered. This can be done by considering all possible alignments between these two sequences. This procedure is routinely used in sequence comparison programs which search for homology between sequences. For a discussion of one such 120 program see Pustell and Kafatos [1982]. Motivated by comparison of DNA sequences, Arratia and Waterman [1985] determined the length of the longest head run M r when all possible shifts of one sequence relative to the other are allowed. Allowing shifts roughly doubles the length of the longest match [Arratia and Waterman 1985], although this simple result was by no means intuitively obvious but required detailed and complicated proof. The general case in which mismatches can intervene in the longest matching subsequence is dealt with by Arratia et al. [1986]. Their results also give the expectation and variance of the longest matching subsequence between two DNA sequences or two polypeptides of known length. The results of Arratia et al. have been empirically confirmed using calculations performed on a large number of vertebrate protein-coding and nonprotein-coding sequences taken from the GenBank database [Smith et al. 1985]. These extensive calculations required 170 min to be performed by a Fortran program executed on a Cray-1 supercomputer. A linear relationship exists between the length of the longest matching subsequence between two nucleic acid sequences and the logarithm of the product of sequence length. The longest matching subsequences are not distributed according to a normal distribution but rather by an extreme value distribution [Smith et al. 1985]. The mathematical predictions of Arratia et al. [1986] also hold for arbitrarily-selected disjoint nucleic acid sequences of length 2 7 , 2 8 ...21 2 selected from the complete lambda phage genome. This process was repeated 6 times to 121 generate a total of 36 nonoverlapping fragments containing 48,378 of the 48,502 nucleotides in lambda. These data were used to show that the lambda nucleotides were independently and identically distributed (i.i.d.) [Arratia et al. 1986]. The extent to which the assumption of i.i.d. sequences applies at a protein level has not yet been established. 4. Determining the probability of shared antibody binding sites due to chance alone The following theoretical work was done as a collaboration with Peter Sibbald [Sibbald and White 1987]. The use of the extreme value random variable approach to this problem was suggested by Dr. Michael Waterman, and is a natural extension of the solution to the longest matching subsequence problem presented in Arratia, Gordon and Waterman [1986]. To determine the probability of immunological cross-reactions for the most commonly encountered experimental situations we have derived equations to answer the following two questions: 1) What is the probability that polyclonal or monoclonal antibodies raised against a given polypeptide will cross-react with another polypeptide due to chance alone? 2) What is the probability that polyclonal or monoclonal antibodies raised against a given polypeptide will cross-react with one or more polypeptides in a pool of polypeptides? The equations are intended to provide order of magnitude estimates. Since no other models are available, these estimates are required for determining the probability of chance cross-reaction, and for providing a theoretical framework to allow 122 interpretation of experimental results. Throughout the following discussion it is assumed that one or more of the polypeptide sequences is not known. If the sequences are known, a direct comparison of the sequences will reveal whether or not they share a common subsequence sufficiently long to allow antibody binding. This comparison can be done either by visual inspection or by using a computer program to plot a homology matrix [Pustell and Kafatos 1982]. If sequences are not known but information regarding the amino acid composition of the polypeptides is available, this can be used to obtain a slightly more accurate estimate of the probability of cross-reaction. This is done by modifying the value of p as defined below. Finally, the length of the polypeptides being considered can be determined by gel electrophoresis or immunoblotting. It should be remembered that the equations are intended to apply only to linearized proteins. Polypeptides are modelled as linear, independently and identically distributed (i.i.d.) [Arratia et al. 1986] strings of amino acids. Linear in this context means unfolded such as is the case following reduction of disulfide bonds and treatment with SDS. This means that only continuous antibody binding sites exist on the polypeptide - not composite ones (ones produced by protein folding). I.i.d. sequences result if each element (amino acid) in a frequency distribution (pool of amino acids) is selected independently of other elements. The assumptions implicit in this model and the cases of glycoproteins and folded proteins are covered in the discussion. 123 Since only continuous sites are considered, we assume that if cross-reaction is to occur, the polypeptides must share an amino acid sequence sufficiently long to allow antibody binding. Arratia et al. [1986], Smith et al. [1985] and Waterman [1986] have estimated the longest expected match between two strings M and N, made up of m and n (respectively) i.i.d. elements from a finite alphabet. Starting from Theorem 2b of Arratia et al. [1986], we derive a simple expression for the probability that the longest expected match between two polypeptides M and N is greater than the length of an antibody binding site. This is equivalent to the probability that an antibody to M will cross-react with N. Theorem 2b states that the probability, P, that the longest match M , k between two sequences of length m and n is greater than some value u is: P{M (m,n) > u} = P{|W/log( 1/p) + log((l-p)mn)/log(l/p)| > u} k where i = imax p = Z u v where imax = the number of different amino acids i= l 1 1 th and u and v are the relative frequencies of each i amino acid in M i i and N respectively. Below we assume imax = 20. p is the probability that two randomly chosen amino acids, one from M and the other from N, will be identical. log is natural log W is a standard extreme value random variable having cumulative distribution function exp(-exp(-x)) (Arratia et al. [1986]). 124 | | means the greatest integer part. e.g. |2.7| = 2.0 To facilitate algebraic manipulation of this expression it is desirable to eliminate the greatest integer function. This can simply be done by rounding up u on the right hand side of the equation to the next integer value if it is a non-integer. To do this, u on the right hand side is replaced by u so that if u = 5 u = 5 but if u = 5.1 then u = 6. Then P{M (m,n) > u}=P{W/log( 1/p) + log((l-p)mn)/log( 1/p) > u} k Rearrange P{M (m,n) > u} = P{W> u(log(l/p))-log(( l-p)mn)} k P{M (m,n)>u} = P{W>-log(pU(l-p)mn)} k Substitute, P{W>x}= l-exp(-exp(-x)) u P{M (m,n)>u}= l-exp-(p (l-p)mn) k So the probability that two polypeptides m and n amino acids long share a subsequence which is u or more amino acids in length is u approximately l-exp-(p (l-p)mn). If a cross-reaction is to occur, according to this model, u must be set at the length (in amino acids) of an antibody binding site. An antibody binding site (epitope) is about 5.5 amino acids, i.e. u = 5.5 and u = 6. Therefore the probability of a cross-reaction (P{CR}) 125 for two polypeptides of lengths m and n due to chance alone is: 6 P{CR}=l-exp-(p (l-p)mn) (1) Example _1_: Polyclonal antibodies to a linear 29 kDa polypeptide M, are tested against a linear 27 kDa polypeptide N. The antibodies cross-react. We can formulate a null hypothesis that this is probable just due to chance alone. Solution: The average amino acid is about 110 Da so m = 29,000/110 = 263.6 and n = 27,000/110 = 245.5. (The decimal places are retained only for the duration of the calculation, although rounding to the nearest integer would not significantly affect the result.) Nothing is known about the amino acid composition so assume p = 0.05. The probability that antibodies to M will 6 cross-react with N is P{CR}= l-exp-((.05) (l-.05)(263.6)(245.5)) = 0.0010. The probability that this cross-reaction is not due to chance is 0.9990. Therefore we reject the null hypothesis that the cross reaction is likely due to chance alone and are in a position to consider other explanations. If monoclonal rather than polyclonal antibodies are used m = u = 5.5, but otherwise the method of calculation does not change. For this example, if monoclonal -5 antibodies had been used P{CR} = 2.2 x 10 . A useful prediction of this model is that when polyclonal antibodies are used and the product mn < 6 10 , then it is at least 98 % certain that the cross-reaction is not due to chance, given the assumptions involved. However, this is only true if the two polypeptides are considered in isolation and have not been selected from a pool of polypeptides. 126 When antibodies to M are reacted against many different polypeptides, there is an increased chance of a cross-reaction. The case where antibodies to M are reacted against a pool of S independent polypeptides is considered next. Two approaches are possible - one for which it is assumed that all S polypeptides are the same length and one for which differing lengths are taken into account. Approach _1_: All S polypeptides are n amino acids in length. S P{at least one CR}=l-(P{no CR}) 6 S P{at least one CR}= l-(l-exp-(p (l-p)mn)) (2) Example 2\ For Example 1 given above, the probability that polyclonal antibodies to a 29 kDa polypeptide would react (due to chance alone) with any of ten independent polypeptides, each of 27 kDa, is 10 P{at least one CR}= l-(0.9990) =0.010. Approach 2: In protein extracts from whole cells or organelles, n is not a constant but rather, polypeptides of many lengths are found with varying abundance. For example, this information is available for E. coli [Savageau 1986], and can be obtained from two-dimensional gels for other systems. To take into account this distribution, the number of polypeptides F of each I length n are counted and a set of (n, F) pairs is constructed. Note that i i i LF =S. Using equation (1), (n, P{no CR}) pairs are calculated for all i I i values of n. The weighted average P{no CR} is given by: 127 l = nmax P{no CR}=1/S I F(P{no CR}) i=0 1 1 S P{at least one CR}=l-(P{no CR}) (3) This approach is ideally suited to a microcomputer which can easily generate and manipulate the pairs involved. The probabilities of cross-reactions for various values of m and n using both monoclonal and polyclonal antibodies are depicted in Fig. 31. As expected, P{CR} increases both with increasing polypeptide length and with increased pool size. Monoclonals are much less likely to cross-react than are polyclonals. 128 Fig. 31. The log (base 10) P{CR} increases as a function of polypeptide length (n). For the two polyclonal cases illustrated above, m = n and pool size (S) is 1 for the lower curve and 100 for the upper curve. A pool size of 100 polypeptides increases the P{CR} about two orders of magnitude over the case where a single polypeptide is being reacted. For the two monoclonal curves, n is as labelled and m is effectively u, independent of the actual size of m. This is because monoclonal antibodies only recognize a single epitope on an antigenic polypeptide and consequently the actual length of m, provided that m > 5, is unimportant. Above , P{CR} is shown for pool sizes of 1000 polypeptides and 1 polypeptide. Note that 1) monoclonals are much less likely to cross-react than are polyclonals, 2) increases in pool size increase the P{CR}. This figure is taken from Sibbald and White [1987] with the permission of both authors. IV. DISCUSSION 1. Characterization of Chi a+b proteins The CP Ia complexes in octylglucoside-solubilized thylakoids (Fig. 2) are not just oligomers of CP I since they contain the PSI antenna complex. Both also contain nonpigmented polypeptides in addition to the four which constitute the antenna. However, CP Ia top may be an oligomer of CP Ia bottom since their spectra are indistinguishable. Alternatively, they may possess i) different stoichiometrics of CP I, antenna and nonpigmented polypeptides or ii) different carotenoids, lipids or other bound molecules or iii) various combinations of the above. More than one band in the CP Ia region of gels has been observed by several other workers [Siefermann-Harms and Ninneman 1979, Waldron and Anderson 1979, Andersson et al. 1982, Noben et al. 1983]. It has been suggested that some of the lower molecular weight polypeptides in CP Ia complexes may be FeS proteins associated with PSI, in particular the polypeptides of 8, 16 and 18 kDa [Nelson et al. 1975, Acker et al. 1982, Lagoutte et al. 1984]. This might account for the 9, 16.5 and 19 kDa polypeptides which dissociated from the CP Ia complexes during re-electrophoresis. On green gels in which the Chi proteins are not dissociated, almost all the CP I exists in CP Ia complexes and there is very little free CP I. This suggests that CP I is associated with its antenna CP in vivo. PSI is located in the stroma and PSII almost entirely in the grana [Andersson and Anderson 129 130 1980, Anderson et al. 1983, Peters et al. 1983] while little or no LHCII is found in stromal vesicles [Henry et al. 1983, Peters et al. 1983]. The PSI antenna therefore endows PSI with a wider absorption cross-section [Anderson et al. 1983] both by increasing antenna size and allowing more efficient absorption around 470 and 650 nm. The PSI antenna CP described in the Results contains four polypeptides between 21 and 24 kDa when run on silver-stained gradient gels. These correspond to the polypeptides in the peripheral PSI antenna hypothesized by Mullet et al. [1980a, 1980b] and isolated on sucrose gradients by Haworth et al. [1983] or Lam et al. [1984]. The PSI antenna CP is associated with CP I, contains Chi a and small amounts of Chi b, and is clearly distinct from the PSII antenna Chi- a+b proteins, CP II and CP 29. The data presented in the Results demonstrate that all four PSI antenna polypeptides in barley can be isolated as a single, intact Chi protein on polyacrylamide gels. Following completion of this work, Kuang et al. [1984] reported a PSI antenna CP, obtained from pea using SDS polyacrylamide gel electrophoresis, which also contained the four polypeptides of LHCI. Similar results were obtained by Dunahay and Staehelin [1985] using a CP Ia complex, designated CP I*, from spinach. During the last 2-3 years, PSI antenna complexes have been isolated from several plant species, and contain anywhere from one to four polypeptides [Haworth et al. 1983, Lam et al. 1984, Ortiz et al. 1984, Argyroudi-Akoyunoglou 1984, Lam et al. 1984b, Metz et al. 1984, Remy and Ambard-Bretteville 1984, 131 Anderson 1984, Sarvari et al. 1984, Kuang et al. 1984]. PSI antenna CPs similar to the one described in the Results have been isolated by re-electrophoresis of pea and bean CP Ia [Argyroudi-Akoyunoglou 1984], bean PSI particles [Sarvari et al. 1984] or spinach PSI particles [Lam et al. 1984, Anderson 1984]. They were reported to contain only one or two polypeptides. Other workers also report only one or two polypeptides in PSI-associated complexes [Remy and Ambard-Bretteville 1984, Brandt et al. 1983, Skrdla and Thornber 1983]. Since some of these complexes were not analyzed on gradient gels, it is possible that other unresolved polypeptides were present. It is also possible that some differences in polypeptide composition may be accounted for by different methods of fractionation or purification [Anderson 1984]. In particular two of these PSI-associated Chl proteins contained a single polypeptide of unusual molecular weight, specifically 29 kDa in tobacco [Skrdla and Thornber 1983] and 32 kDa in the green alga Chlamydobotrys stellata [Brandt et al. 1983]. There has been no further report on the former, which was presented only in an abstract, whereas the green alga may actually contain a PSI antenna with a different composition from higher plants. CP I and an analogous PSI antenna called CP 0 exist together as CP Ia complexes in Chlamydomonas [Ish-Shalom et al. 1983] and occur as single large particles in freeze-fractured thylakoids [Olive et al. 1983]. However, CP 0 has two polypeptides in common with CP II [Ish-Shalom et al. 1983], unlike the PSI antenna CP (LHCI) of higher plants. A complex with some similarities to the CP Ia of higher plants is also found in the prokaryote Prochloron. It contains mostly Chl a, some Chl b, a principal polypeptide of 60-70 kDa analogous to 132 that of CP I, and polypeptides of 22, 20, 27 and 16 kDa [Hiller and Larkum 1985]. Most of the PSI antenna complexes isolated to date have been from dicot species such as spinach and pea. The PSI antenna polypeptides in these species have a wider molecular weight range than those in barley, with three polypeptides of 19-24 kDa reported for pea [Haworth et al. 1983], while polypeptides of 23, 24, 25 and 25.8 kDa have been reported for spinach [Evans and Anderson 1986]. In contrast there are four barley PSI antenna polypeptides all in the 21 or 22-24 kDa range, so that they are even more difficult to resolve than the PSI antenna polypeptides in dicots. I have found that these polypeptides can only be resolved on certain gradient gels if just the right amount of sample is loaded, the gel is run for a long time Oout not too long so that it results in diffusion), and a sensitive method of detection is employed, such as silver-staining or immunoblotting. If too much material is loaded the polypeptides will run together, but if not enough material is loaded the less abundant upper polypeptides will not be detected. Those instances in the Results where these bands are resolved are usually the result of more than one experiment. CP 29 is clearly distinct from LHCII. LHCII prepared by the cation precipitation method of Burke et al. [1978] does not contain the CP 29 polypeptides as judged by Coomassie blue staining [Machold 1981]. Similar results have been reported by Suss [1983] in which LHCII was prepared by column chromatography followed by cation aggregation. The method of Ryrie et al. [1980] 133 used in this work for preparing LHCII is modified from that of Burke et al. [1978]. None of three anti-Chl a + b protein antisera detected CP 29 polypeptides in LHCII prepared by the method of Ryrie et al. [1980] or by the method of Burke et al. [1978] even if very high divalent cation concentrations (200 mM CaCl 2 and 10 mM MgCl2) were used in the cation precipitation step. Since immunoblotting can detect very small amounts of antigen, these data suggest that CP 29 is not cation-precipitable and thus is likely not directly responsible for the cation-induced responses attributed to LHCII. These include thylakoid stacking, LHCII migration between granal and stromal lamellae, and the distribution of excitation energy between photosystems I and H. These data further support the similar conclusions of Dunahay and Staehelin [1987] and Greene et al. [1987] discussed in the Introduction. 2. Immunological relatedness of the C h i a + b proteins Polyclonal antisera to each of the three Chi a + b proteins all showed at least some immunological cross-reaction. All three antigens were pure as judged by Coomassie blue staining, silver-staining and immunoblotting (e.g. Figs. 6 and 10), and in particular did not contain any contaminating polypeptides from other Chi a + b protein complexes. Anti-CP Ia reacted strongly with both purified LHCII (Fig. 6d), purified CP 29 (Fig. 6c) and the corresponding polypeptides in thylakoids (Fig. 6b). This cross-reaction was still observed if the anti-CP I activity was removed from the anti-CP Ia, and antibodies to CP I did not recognize the polypeptides belonging to the Chi a + b proteins (Fig. 8). Thus the cross-reactions with LHCE and CP 29 polypeptides were due to antibodies 134 directed against the PSI antenna (LHCI) polypeptides. These cross-reactions with the PSII antenna polypeptides were not removed by the affinity purification of the anti-CP Ia (Fig. 9), thereby providing further evidence that these cross-reactions were not due to contamination of the injected antigen. The polyclonal antisera to CP II and CP 29 both showed some immunological cross-reaction with each other's antigen (Figs. 12, 13, and 16). Although these cross-reactions were weaker than the reactions with their respective antigens, they were readily detected with the amounts of thylakoid protein routinely used for immunoblotting. Cross-reactions with thylakoid polypeptides of 21-24 kDa were not observed unless larger amounts of protein were used on gels or dot blots (e.g. Figs. 7, 11, 13). The objection could be raised that the 21-24 kDa polypeptides which reacted with anti-CP 29 might be proteolytic fragments of CP 29. However, the use of protease inhibitors during thylakoid isolation did not eliminate these cross-reactions. The fact that cross-reactions only occurred at the position of the PSI antenna polypeptides is also inconsistent with proteolysis. Digestion of CP 29 with Staphylococcus aureus V8 protease or trypsin, followed by immunoblotting with anti-CP 29, reveals proteolytic fragments with a wide range of molecular weights. It is therefore unlikely that the reaction in the 21-24 kDa range is due to proteolytic cleavage of CP 29. It has been recently reported that antibodies to spinach CP II and CP 29 also show small amounts of cross-reaction with each other's antigen [Dunahay and Staehelin 1987], as reported here for antibodies to the same proteins in 135 barley. My antibodies to CP II as well as those of Dunahay and Staehelin were raised to the oligomeric form CP II*. This complex migrates much more slowly than the CP 29 complex. It is therefore isolated from a different region of the gel than CP 29, thus greatly reducing the risk of contamination. Thus the reaction of these antibodies to CP II with anti-CP 29 is almost certainly a legitimate cross-reaction. The reaction of anti-CP 29 with CP II polypeptides is also likely to be legitimate, given the apparent purity of the antigen. Polyclonal antibodies raised against individual PSI antenna (LHCI) polypeptides in pea and spinach cross-react with other LHCI polypeptides [Evans and Anderson 1986, Williams and Ellis 1986]. Polyclonal antibodies to these polypeptides also cross-react with LHCII polypeptides [Evans and Anderson 1986, Williams and Ellis 1986]. This has been demonstrated for antisera to three of four denatured spinach LHCI polypeptides, by affinity purifying the cross-reacting antibodies using LHCII and demonstrating that the affinity-purified antibodies still react with both LHCI and LHCII polypeptides [Evans and Anderson 1986]. Darr et al. [1986] have recently shown that monoclonal antibodies to pea LHCII not only cross-react with other LHCII polypeptides, but also cross-react with the polypeptides of LHCI. Most of these monoclonal antibodies were categorized into one of six classes based on their binding specificities. Four of these classes recognized various combinations of LHCII polypeptides while the remaining two classes recognized both LHCII and LHCI polypeptides. Thus not only do antibodies to LHCI polypeptides react with polypeptides in LHCII, but antibodies to LHCII also react with the LHCI polypeptides. A monoclonal 136 antibody to LHCI which cross-reacts with CP 29 has recently been discovered [David Simpson, pers. comm.]. Williams and Ellis [1986] attributed the cross-reaction they observed with pea to contamination of their PSI antenna antigens with proteolytic fragments of LHCII. It was postulated that the anti-LHCII reacted with these proteolytic fragments of identical molecular weight to the LHCI polypeptides, thus appearing to cross-react with the LHCI polypeptides instead. Conversely, it was postulated that these proteolytic fragments contaminated the LHCI polypeptides used as antigens, causing the resulting antibodies to react with LHCII. They further reported that three monoclonal antibodies to LHCI did not cross-react with LHCII. Williams and Ellis [1986] therefore conclude that there is no major sequence homology between LHCI and LHCII polypeptides, but do not rule out the possibility of small patches of homology. Although these experiments reiterate the well-known fact that the purity of an injected antigen is critical, they have no bearing as to the purity of the same antigens prepared by different methods or in different species in the studies discussed above. Furthermore, this sort of contamination cannot be used to explain the cross-reactivity observed with monoclonal or affinity-purified polyclonal antibodies. Instead, the discrepancy among these various studies can be simply explained by the existence of both shared and unique antigenic sites among the LHCI and LHCII polypeptides. The existence of both shared and unique sites on these polypeptides is clearly demonstrated by the experiments of Darr et al. discussed above. Some of their monoclonal antibodies reacted only with LHCII 137 polypeptides, as they recognize sites found only on LHCII. A smaller number of these monoclonals recognize sites shared between LHCII and LHCI, and thus recognize polypeptides belonging to both these complexes. The three noncross-reacting monoclonals to LHCI described by Williams and Ellis [1986] may simply be specific for sites unique to LHCI polypeptides. This interpretation of experimental results is further supported by the fact that both Darr et al. [1986] and Williams and Ellis [1986] did all their experiments on pea, so that differences between species will not account for the differences in their results. Finally, the recent sequencing of two LHCI cDNAs clearly demonstrates the existence of regions of homology shared between LHCI and LHCII polypeptides, and is discussed in detail later in this section. There are many possible reasons which would explain why immunological cross-reactions have not been reported with antibodies to Chi a+b proteins in earlier studies. First, LHCII is by far the most abundant Chi a+b protein and researchers were not even aware of the existence of the other Chi a+b proteins before 1980 (and some researchers still appear to be unaware). Western blotting techniques are also relatively new. The LHCII polypeptides are so abundant that they are likely to produce an intense staining reaction on immunoblots long before weaker cross-reactions with less abundant polypeptides are apparent. Not only is CP 29 less abundant than LHCn but it is difficult to resolve its polypeptide(s) from those of LHCII on gels of denatured barley thylakoids. In species such as spinach this is even more difficult or impossible as the LHCII and CP 29 polypeptides are similar in molecular weight [Camm and Green 1980, Dunahay and Staehelin 1987]. In such cases cross-reaction cannot be 138 demonstrated with thylakoids but must be demonstrated using the purified complexes. The development of immunoblots is often stopped when the researcher's desired results are obtained, so that weaker reactions with polypeptides other than the antigen may not be detected. Weak reactions with unidentified polypeptides may go unnoticed, or may remain unexplained and thus not publishable. These reactions are frequently referred to in scientific jargon as ' nonspecific', a vague term. Clearly the antibodies are reacting with some polypeptides and not others, although perhaps the term ' less specific' is appropriate here. Such reactions are to be expected on occasion when large numbers of proteins are screened with polyclonal antisera, due to biological reasons or due to chance alone as predicted by the immunological cross-reaction model in the last chapter of the Results. My experiments have shown that the ability to observe cross-reactivity with antibodies to Chl proteins is a function of the amount of protein loaded on gels (Figs. 12, 13, 16-18) or dot blots (Figs. 7 and 11), at least for certain antibodies. Similar results for anti-CPU* and anti-CP 29 have been reported by Dunahay and Staehelin [1987]. Thus the proper antigen for a given antibody may be the only protein immunostained if smaller amounts of thylakoids are used than those required to detect cross-reaction. This is not surprising as the majority of antibodies in the antiserum will have the highest affinity for the antigen against which they were raised. Thus, although some may bind to the small amounts of other polypeptides present, these cross-reactions may be lowered 139 below the limit of detection when smaller amounts of thylakoid protein are used. Finally, the statistical nature of producing antibodies to certain sites on polypeptides may partially explain why cross-reaction is sometimes detected with one antiserum to a given protein but not with another antiserum. In the mathematical cross-reaction model one of the simplifying assumptions was that antibodies were made to all sites on a polypeptide. Whether or not this is true is still an area of controversy among immunologists. (This will be discussed in more detail later with reference to the mathematical modelling.) In any case it is conceivable that some antisera to Chi 0+6 proteins will contain fewer antibodies to the small proportion of shared sites, and thus produce little or no detectable cross-reaction when used. The method of antigen preparation may affect which epitopes are recognized and to what extent they are recognized by different antisera. The genetics determining the immune response may also differ among the animals in which the antisera were raised. Any or all of the above factors may account for discrepancies in the literature. A tomato cDNA coding for an LHCI polypeptide has just been sequenced [Hoffmann et al. 1987]. The amino acid sequence predicted from this cDNA shows two segments, comprising approximately 50 % of the protein, where homology to the PSII cab proteins is 50 % and 65 % respectively. Within these homologous regions are several stretches of five or more identical amino acids, i.e. regions long enough to allow antibody binding. (The reader is referred to the last chapter of the Results section for discussion and calculations regarding the length of epitopes and antibody combining sites). Similar results have just been 140 obtained for a different LHCI cDNA from petunia [Stayton et al. 1987]. In this case there is slightly less homology between the LHCI and LHCII polypeptides, but there are still short runs of matching amino acids which are long enough to result in shared antibody binding sites. It could therefore be predicted from these sequences that polyclonal antisera raised against LHCI or LHCII polypeptides consist mostly of antibodies to the unique sites, but would also contain some antibodies which cross-react at the shared sites. Specifically, comparison of these LHCI and LHCII sequences clearly shows how Williams and Ellis [1986] could produce three monoclonal antibodies to LHCI which did not cross-react with LHCII, while Darr et al. [1987] obtained four classes of monoclonal antibodies to unique sites on LHCII and two classes against sites shared among LHCII and LHCI polypeptides. The results given in the last chapter of the results section can be used to determine whether or not the immunological cross-reaction displayed by antibodies to Chi proteins is biologically significant, i.e. it is possible to estimate the probability that it is not due to chance arrangement of amino acids. As shown in Example 1 in the last chapter of the Results section, there is only a 0.1 % chance that cross-reaction will occur with a polyclonal antiserum to one of these polypeptides and one other specified polypeptide of similar molecular weight. However, there are at least 2 polypeptides in CP 29, at least 3 in LHCII and at least 4 in LHCI for a total of at least 9 polypeptides. These are seldom if ever resolved on a single polyacrylamide gel, and certainly not all resolved on gels of single polyacrylamide concentration. Given that there are about 16 genes coding for LHCII alone in petunia [Dunsmuir et al. 1983], there are probably 141 many more than 9 Chl a+6-binding polypeptides in barley. These LHCII polypeptides differ by 5-15 % in predicted amino acid sequence and thus are not independent as required for the model. For purposes of estimating the probability of a shared antibody binding site between these polypeptides and those in other Chl proteins, polypeptides which are 85-95 % homologous probably behave like a single independent polypeptide which is slightly longer. In general the polypeptides belonging to a single Chl a+b protein are likely quite homologous, and in the absence of other information the polyclonal antiserum to such an apoprotein may be treated as a polyclonal antiserum to a single polypeptide. Although there will be a small proportion of unique sites among the various polypeptides within a single Chl a + b protein, in reality antibodies may not be made to all the sites on all the polypeptides, so these factors likely compensate for each other to a reasonable approximation. If this is so then it is not even necessary to model the polypeptides within a Chl protein as a single polypeptide of slightly longer length, but as a single polypeptide of identical length. This correction can nevertheless be applied if one wants to obtain a conservative estimate of the probability that a shared antibody-binding site would not arise due to chance alone. This kind of minor alteration is probably not significant when one considers the approximate and statistical nature of the mathematical prediction. Therefore it is possible to estimate an upper limit for the probability that a polyclonal antiserum to a Chl a+b protein will cross-react due to chance. As shown in Example 2 in the last chapter of the Results section, the chance that 142 a polyclonal antiserum to a 29 kDa polypeptide cross-reacts with one or more of 10 independent 27 kDa polypeptides due to chance alone is approximately 1 %. It therefore seems reasonable to attribute the immunological relatedness among the Chl a + b proteins to a biological cause, such as divergent evolution from a common ancestral gene, or convergent evolution due to functional or structural constraints as discussed below. This conclusion is reinforced by the fact that a number of cross-reactions have been detected among the various Chl proteins by several research groups, as detailed earlier. These include the immunological cross-reactions among LHCI polypeptides, among LHCII polypeptides, and between LHCI and LHCII polypeptides. Also included are the reactions of anti-CP Ia with LHCII and CP 29 polypeptides, and the cross-reactions displayed by antibodies to CP II and CP 29. Finally, this conclusion is firmly established by the newly discovered (and as yet unpublished) LHCI sequences discussed below. By comparing the recently determined LHCI sequences [Hoffmann et al. 1987, Stayton et al. 1987] to LHCII sequences, the relatedness among Chl a + b proteins can be demonstrated to be biologically significant by an alternative method. Arratia et al. [1986] give the expectation of the length of the longest expected match between two sequences as \og(qmn) + kloglogiqmn) + klogiq/p) - log(k!) + 7log(e) - 1/2 All variables are as defined in the last chapter of the Results section except that k = the number of mismatches permitted log denotes log base 1/p. 143 (Remember q = l-p.) Consider the case where no mismatches are allowed, i.e. k = 0. The equation then simplifies to log(gmn) + 7log(e) - 1/2 Thus between the precursor polypeptides of tomato LHCI (246 amino acids) and LHCII (265 amino acids) we would expect a string of 3.4 consecutive identical amino acids in common to these two sequences due to chance arrangement of amino acids alone. Any alignment of these two sequences may be used to obtain this match but no insertions or deletions are allowed within the longest run of matching amino acids, since it is not currently possible to model these in mathematical predictions. The longest observed matching sequence between the amino acid sequences of the- tomato LHCI gene (coding for the precursor polypeptide of cafc-6A) and tomato Type I and Type II cab genes (coding for the precursor polypeptides of cab-ZC and cab-4 respectively) is 13 consecutive identical amino acids in length. In this particular instance no mismatches, insertions or deletions are associated with the longest matching subsequence. This sequence is much longer than the 3.4 consecutive identical amino acids expected due to chance alone and is highly statistically significant well beyond the 99.9 % level. The primary sequence homology indicated by immunological cross-reaction with denatured polypeptides, and by direct comparison of DNA sequences, suggests that the genes coding for the Chi a+6-binding polypeptides form a large nuclear multigene family. The PSI antenna polypeptides and likely CP 29 as well are included in this family in addition to LHCII. Such sequence homology at the 144 primary sequence level (as opposed to shared antibody binding sites created by folding of proteins) could be due to the origin of these sequences from a common ancestral gene, i.e. divergent evolution. It could also be due to convergent evolution imposed by structural or functional constraints. Examples of such constraints might be binding of Chl b or certain carotenoids, or the efficient harvesting and transfer of light to the internal Chl o antennae of the two photosystems. It is unlikely that a structural requirement for Chl c binding could account for this sequence homology, as the Chl a proteins bind substantial quantities of Chl a but are not related to the Chl a+b proteins. The origin of the Chl 0 + 6 proteins from a common ancestral gene seems to be the most probable explanation, although this cannot be demonstrated unequivocally. First, all the Chl a+b apoproteins are of nuclear origin whereas the Chl o apoproteins are chloroplast encoded. The convergent evolution hypothesis does not explain why all the Chl a/6-binding polypeptides are nuclear encoded. If these polypeptides were of chloroplast origin in the distant past, then the convergent evolution hypothesis is consistent only if multiple chloroplast to nucleus transfers of every single Chl a + b protein gene have occurred. In contrast only one such gene transfer event is required to explain the divergent evolution hypothesis. Alternatively, the Chl a+b proteins may always have been encoded by nuclear genes. In this case the convergent evolution hypothesis should explain why only nuclear genes are observed to undergo this sort of convergent evolution in order to become Chl a + b proteins. A eukaryotic gene organization is not necessary to obtain Chl 0 + 6 proteins, since the prokaryote Prochloron contains Chl a + b proteins, even though they may be different from those of higher 145 plants. The only remaining possibility consistent with convergent evolution is that chloroplast genes would have been transferred to the nucleus every time they underwent convergent evolution to become Chi a + b protein genes. Secondly, all the Chi a/6-binding polypeptides have molecular masses in the 20-30 kDa range, whereas the apoproteins of the Chi a proteins CP I, CP 47 and CP 43 are approximately 64, 51 and 44 kDa respectively. There is no known reason why a certain length of polypeptide should be associated with Chi a or Chi b binding. It is not known how the Chi is bound in the Chi proteins of higher plants, except that this binding is noncovalent. However, it is not inconceivable that there may be an optimal size for an efficient light-harvesting antenna protein. The light-harvesting bacteriochlorophyll antenna polypeptides in the purple photosynthetic bacteria Rhodospeudomonas spp. have much lower molecular masses than the 21, 24 and 28 kDa L, M and H reaction center polypeptides [Okamura et al. 1974, Youvan et al. 1984]. Similarly, the antenna polypeptides of the green photosynthetic bacterium Chloroflexus aurantiacus are also much smaller than the reaction center polypeptides [Wechsler et al. 1987]. The antennal polypeptides of both green and purple photosynthetic bacteria are in the 6-12 kDa range [Youvan et al. 1984, Wechsler et al. 1987], suggesting that the molecular masses of the higher plant Chi a/fc-binding polypeptides cannot be explained in terms of their light-harvesting function alone. The phycobiliproteins of cyanobacteria and red algae generally consist of a polypeptides in the 12-20 kDa range and (3 polypeptides in the 15-22 kDa range, the molecular masses differing with the organism [Gantt 1981]. 146 Although the Chl a+b proteins differ in some properties such as the Chl alb ratio, the capacity to be phosphorylated, or cation precipitability, this is equally well explained by both the convergent and divergent evolution hypotheses. These differences could be due to evolutionary ' fine-tuning' and specialization of function of a set of similar proteins derived from a common ancestor, or due simply to the possibility that they have always been different and that these particular properties are not subject to convergent evolution. The location of the Chl a+b protein genes within the nucleus as several tightly linked genes per cluster [Pichersky et al. 1985], but with clusters on separate chromosomes [Polans et al. 1985, Vallejos et al. 1986], has many implications for the evolution of these genes in this regard. The clusters behave as discrete linkage units showing classical Mendelian inheritance. Thus they are subject to all the advantages of meiotic gene mixing conferred by the pollination of outcrossing species. This Mendelian inheritance therefore constitutes a 'fine-tuning' mechanism by which a very large number of Chl a+b protein gene clusters, differing to various degrees in different members of a plant population, can be grouped in numerous arrangements in different individual plants. Each of these plants is subject to a potentially unique set of environmental selection pressures, and thus can be thought of as a vehicle for propagating the divergent evolution of these gene clusters. However, it should be kept in mind that pea and tomato, the two species in which these gene clusters have been mapped [Polans et al. 1985, Vallejos et al. 1986], are both dicot species, whereas barley is a more evolutionarily distant monocot. It is therefore possible that the Chl a + b protein genes may be differently organized within the barley genome. At 147 the same time it should be remembered that the divergence of type I and type II cab sequences likely preceded the split of monocot and dicot lineages [Pichersky et al. 1987]. The Chi a + b genes within a cluster would be expected to behave as a single linkage group in most but not all meiotic events. Thus they experience a slower rate of divergence among themselves relative to genes located in other clusters [Pichersky et al. 1985, Vallejos et al. 1986, Pichersky et al. 1987]. This allows genes to be duplicated so that the additional copies may gradually undergo mutation, perhaps as a temporarily unexpressed gene or as an additionally expressed gene, without being detrimental or lethal to the organism. It can be speculated that a series of such detrimental mutations, of many different sorts in the various individuals of a species, may on occasion give rise to a beneficial mutation simply due to chance. This beneficial mutation may ultimately be preserved and expressed, and thus incorporated into the photosynthetic apparatus of the species. In contrast the Chi a proteins are much more evolutionarily conserved and not subject to the same mechanisms which propagate beneficial or deleterious mutations in the Chi a+b proteins. However, there are 300-1000 copies of chloroplast DNA within a single chloroplast, and a number of chloroplasts per cell [Sellden and Leech 1981]. Thus it is possible for mutations to coexist with normal chloroplast DNA sequences within a single plastid, and to be passed on to progeny by means of cytoplasmic inheritance. Intermolecular recombination events might then give rise to second copy of the gene within a given circle of 148 plastid DNA. Such a mechanism could be invoked to account for the pairwise sequence homology between the two CP I genes, between CP 47 and CP 43, and between D l and D2. 3. Modelling immunological cross-reaction The mathematical modelling of immunological cross-reaction due to chance alone contains several simplifying assumptions, which may increase or decrease the predicted probability of this cross-reaction relative to the true probability for antibodies to biological polypeptides. Before examining these assumptions, it is extremely important to realize that such a mathematical model is an absolutely essential calculation which must be made before a cause can be postulated to explain the cross-reaction. These calculations are not optional. If it is not known whether a cross-reaction is expected due to chance arrangement of amino acids or not, it is not correct to attribute biological significance to it, as is the current practice. To do so is analogous to attempting to evaluate statistical information in the absence of a null hypothesis. This does not mean that all presently published conclusions on immunological cross-reaction are invalid, since there are some cases in which intuitive guesses may be correct. For example, if a monoclonal antibody cross-reacts with a second protein not drawn from a pool of proteins, the intuitive guess that this is biologically significant agrees with the mathematics. However, even in this case the researcher is making a mathematical evaluation or approximation as to whether this event is expected, given the properties of 149 the antigen and of the monoclonal antibodies. The ability to make reasonably accurate mathematical predictions is most important in experiments where polyclonal antisera are used to screen a large number of polypeptides. Such procedures are routine in Western blotting where antibodies are often used against a large number of linearized polypeptides which have been separated by totally denaturing SDS polyacrylamide gel electrophoresis. As discussed in the last chapter of the Results, the equations of Arratia et al. and the assumptions of independent sequences consisting of independently and identically distributed (i.i.d.) elements have been successfully shown to apply to DNA sequences [Smith et al. 1985, Arratia et al. 1986]. To apply these equations to modelling antibody binding sites on proteins, a few simplifying assumptions were made. These assumptions may increase or decrease the predicted probability of immunological cross-reaction. The degree to which these assumptions apply has not yet been experimentally determined, and some mathematical adjustments could perhaps be made when the empirical evidence becomes available. All of these assumptions are of a biological rather than a mathematical nature. Thus these equations provide a valid null hypothesis which estimates the probability of cross-reaction due to chance arrangement of amino acids alone. What is unclear is the extent to which the probability or frequency of the cross-reactions observed with biological polypeptides differs from the probability or frequency expected for independent sequences containing i.i.d. amino acids. The evaluation of the magnitude of this difference, if indeed there is any significant difference, should provide insight into the nature of biological polypeptides. 150 If polypeptides are not independent and i.i.d., the actual probability of cross-reaction observed with biological polypeptides will likely be greater than that predicted by the model. For example, certain amino acids might be more frequent in hydrophobic regions or membrane spanning regions in the case of membrane proteins. Thus antibody binding sites in these regions may be shared more frequently than expected due to chance arrangement of the amino acids. However, even in cases such as these it is known that any amino acid can occur within hydrophobic regions, that most membrane proteins have soluble regions, and that most soluble proteins have hydrophobic regions. Thus the extent to which biological systems differ from the model is limited in this sense. One assumption made in order to predict the probability of cross-reactions observed with polyclonal antisera is that antibodies are made to all sites on a polypeptide. This assumption appears to be generally valid, but some controversy remains and exceptions will arise. Some researchers claim that epitopes are localized at certain regions in proteins [e.g. Atassi and Atassi 1986], whereas other evidence indicates that the entire exposed protein surface is antigenic [Benjamin et al. 1984, Jemmerson 1987]. However, studies such as those of Atassi and Atassi [1986] have used folded proteins as antigens. Thus antibodies would not be elicited against the buried regions of the folded antigen but would be elicited only to those sites present at the protein surface. These problems could be avoided by using a linearized SDS-protein as an antigen. If antibodies are not made to all sites on a polypeptide, the probability of cross-reaction will be decreased, i.e. the model will overestimate the probability of cross-reaction. Note that this effect is opposite to some of the assumptions discussed earlier, 151 which would underestimate the probability of cross-reaction if invalid. This consideration does not apply to monoclonal antibodies. It should be remembered that the model is intended to apply only to linearized polypeptides, such as totally denatured and linearized proteins on Western blots. At present there is not sufficient information to determine whether or not it could also be applied to folded proteins. The examination of 23 x-ray crystal structures of proteins by Rose et al. [1985] determined that an average of 72 % of a protein surface is rendered inaccessible to the solvent upon folding. Since solvent accessibility would appear to be a minimum requirement for antibody binding or for eliciting antibodies to a site, many sites are lost upon protein folding. At the same time new composite sites are created in addition to sites along the primary amino acid sequence. Based on such considerations it can therefore be speculated that the probability of cross-reaction between linear and folded polypeptides does not differ by more than an order of magnitude [Sibbald and White 1987]. Modified amino acids can be readily incorporated into the model. For example, a phosphorylated or hydroxylated amino acid is simply treated as one more kind of amino acid, which will change the value of p only slightly. Glycosylations may not be dealt with in this fashion since the carbohydrate moiety may be sufficiently large to be an epitope. Thus the model does not apply to antibodies to glycoproteins when they are reacted with other glycoproteins. In such cases the carbohydrate groups must be removed in order to determine if cross-reaction is due to sites shared on the polypeptides [Culp 152 and Butler 1985]. These antibodies could nevertheless be used with nonglycosylated proteins. The model also applies to antibodies raised against nonglycosylated proteins. Shared antibody sites are infrequent and the probability that such a site would also be glycosylated by chance alone, thus preventing antibody binding, is exceedingly small. These problems need not be considered in systems which lack glycoproteins, such as chloroplasts [Keegstra and Kline 1982]. Using the results of Arratia et al. [1986] it is possible to allow for mismatches in the antibody binding site. However, there are several reasons why it is probably not necessary to do so. First of all, antibodies are extremely specific and even one mismatching amino acid in the epitope will often eliminate immunological reactivity. Thus only extremely conservative replacements are likely to permit binding, and even these would likely reduce the affinity of the antibody for the cross-reacting site. In polyclonal antisera the cross-reacting antibody would represent at best a small percentage of the total antibody population, as the majority of antibodies will be specific for the antigen they were raised against. Thus to have a reasonable chance of being detected, the cross-reacting antibody must possess a relatively high affinity for the cross-reacting site. Consequent^ even a conservative substitution in the epitope which reduced the binding affinity would likely prevent immunological detection of the cross-reaction, even if some binding occurred. It therefore seems unnecessary to model mismatches which allow antibody binding for a first attempt towards predicting the probability of cross-reactions due to chance alone as detected by polyclonal antibodies. 153 In the case of monoclonal antibodies it might be reasonable to allow for a mismatch if immunoblots are incubated for a long time in antibody or if long blot development times are used. In this case the number of mismatches still should not exceed more than one or two conservative mismatches within a single epitope, since it is probable that only those polypeptides with a low number of mismatches relative to the antigen would show sufficiently strong antibody binding to be detected. These kinds of conservative mismatches are vastly different from those modelled by Arratia et al. in which any amino acid is allowed to replace the matching amino acid. It would therefore be more accurate to model conservative replacements by increasing the value of p, the probability of a match, by a very small amount. However, this sort of refinement of the model seems unjustified at present, since the model is only intended to give order of magnitude results and is presently untested. There are a number of methods by which the model could be tested and the true probability of cross-reaction due to chance alone determined. One method would be to raise antibodies against non-glycoprotein polypeptides and screen a number of different subcellular fractions from organisms that were as distantly related as possible. Antibodies to composite proteins consisting of two or more closely related polypeptides (such as my antibodies to chlorophyll proteins) are not ideal for this purpose, as the more complex nature of the antigen would increase the difficulty of interpretation and decrease the accuracy of the resulting estimate. A good antibody for this purpose would be one raised against a nonessential, nonconserved polypeptide from some obscure organism. Best of all would be antibodies to random synthetic polypeptides of known sequence. The 154 results for subcellular fractions containing glycoproteins should initially be tabulated separately from subcellular fractions, such as chloroplasts, which do not. In this way it would be possible to see if the binding of antibodies to nonglycosylated proteins is hindered to any significant degree by carbohydrate moieties on the potentially cross-reacting polypeptides. A shared epitope of 5 to 6 amino acids between two polypeptides seems to be a necessary condition for immunological cross-reaction to occur and is likely a sufficient condition as well. The postulate that a single shared epitope of 5 to 6 identical amino acids is sufficient to cause immunological cross-reaction detectable by polyclonal antisera could also be tested. One method would be to find two nonglycosylated polypeptides which share exactly 5 or 6 identical consecutive amino acids but share no other common antibody binding sites. By making a set of polyclonal antibodies to each in different rabbits and reacting them with the other polypeptide, it would be possible to determine how many of these polyclonal antisera cross-react with a polypeptide that shares only one antibody-binding site (with no mismatches) with their antigen. It might seem that this is a very demanding set of requirements which would be difficult to satisfy. However, exactly this situation exists between the CP I polypeptides and that of CP 47 (508 amino acids). The spinach CP 47 nucleotide sequence, known as psbB, was incorrectly translated in Morris and Herrmann [1984], with codons specifying threonine mistakenly translated as serine. Thus the sequence DPTTRR (rather than DPSSRR) occurs at positions 500 to 505, just three amino acids to the left of the carboxyl terminal. The sequence DPTTRR is also found at amino acids 15 to 20 near the amino terminus of maize psIA2 (735 amino acids) and 155 the sequence DPTTRY (five identical amino acids followed by one nonconservative mismatch) is found at amino acids 416 to 421 of maize psIAl (751 amino acids) [Fish et al. 1985]. Polyclonal antibodies have already been made to these same psIAl and psIA2 polypeptides [Fish et al. 1985]. Antibodies to the spinach CP 47 sequence described by Herrmann et al. are currently being made in our lab, and a barley cDNA coding for CP 47 has been isolated as well. Although the number of polyclonal antisera to these proteins is presently too small to be statistically significant, these antisera might still provide useful insights. The number of polyclonal antisera to these proteins will likely increase over the next couple years, as both proteins are part of a photosystem core complex and are being intensively investigated at present. The function of the DPTTRR sequence is not known. Since it is present in both CP I and CP 47, Fish et al. [1985] suggested that it may be related to reaction center function. However, the strongly hydrophilic nature of this sequence and its double positive charge, combined with its location near amino or carboxyl termini suggest that this sequence is located in a peripheral or soluble portion of these proteins. For this and the following reasons I speculate that this sequence may be involved in phosphorylation instead. This sequence resembles the amino terminal hexapeptide SATTKK which is phosphorylated in LHCII [Mullet 1983]. Not only does it contain the threonine pair phosphorylated in LHCII, but it also contains a consecutive pair of strongly basic amino acids, i.e. two arginines corresponding to the two lysines. The existence of phosphorylated and 156 nonphosphorylated forms of CP I and CP 47 has been suggested as a reason for the multiplicity of slightly different molecular weight bands observed on SDS polyacylamide gels under totally denaturing and reducing conditions. However, there is as yet no experimental evidence to support this idea and it seems unlikely that the small difference in charge or mass imparted by phosphate groups would be significant relative to the many SDS molecules which would bind to a polypeptide over 500 amino acids in length. The apoprotein of CP 47 and a number of polypeptides, including ones with the same molecular weight as the CP I apoprotein, are phosphorylated in the early stages of light-induced development in Chlamydomonas reinhardii, while LHCII is phosphorylated predominantly at later stages [Owens and Ohad 1983]. Baker et al. [1983] looked at phosphorylation of thylakoid proteins in etiolated wheat leaves and in normally greened wheat leaves ascending a gradient of cell ages from the bottom to the top of the leaf. The highest specific thylakoid protein kinase activities were detected in etioplasts and the early stages of light-induced development, and were not due solely to phosphorylation of LHCII. As in Chlamydomonas, LHCII is phosphorylated predominantly at the later stages, while a number of unidentified polypeptides of higher molecular weight were phosphorylated in thylakoids from 7-day-old etiolated leaves and from early developmental stages in the basal 1 cm of 7-day-old light-grown leaves [Baker et al. 1983]. In both of the above studies no attempts were made to identify the phosphorylated polypeptides in the CP I apoprotein region of the gel. If the DPTTRR/Y sequences are phosphorylated, although this is by no 157 means certain, it would be necessary to remove the phosphate groups before performing the cross-reaction experiments described above. This could be done by prior incubation with phosphatase. If such an incubation eliminated immunological cross-reaction, this would be evidence that phosphorylation does occur at the DPTTRR sequence. Alternatively, the above sequences are or will be available in expression vectors and it seems less likely that these particular sequences would be phosphorylated in E. coli. Both the DPTTRR and DPTTRY sequences are also found in the recently sequenced psIAl and psIA2 of pea [Lehmbeck et al. 1986]. Thus the DPTTR sequence is conserved not only between psIAl and psIA2, but also between a monocot and a dicot species. Since psIAl and psIA2 code for 45 % homologous polypeptides, the chance that this sequence would be conserved due to chance 5 alone is approximately (0.45) = 0.018. The probability that CP I (psIA2) and CP 47 (psbB) share the DPTTRR sequence due to chance alone is the probability that two proteins of this length share an antibody binding site, approximately 6 l-exp-((.05) (l-.05)(735)(508)) = 0.0055. 4. Regulation of Chl a+b apoprotein synthesis: Chlorina f2 and intermittent light barley The immunoblotting of Chl 6-less thylakoids isolated from chlorina f2 shows that this mutant contains most of the Chl a/6-binding polypeptides, although some of these may be present in reduced amounts. Relatively normal levels of the CP 29 apoprotein are present, confirming the results of Machold 158 [1981] which were based on Coomassie blue staining. The 25 kDa polypeptide of LHCII is also present in relatively normal amounts, unlike the 26 and 27 kDa polypeptides found in both CP II prepared by gel electrophoresis and LHCII prepared by cation precipitation. The two major polypeptides of CP II can be separated as two distinct green complexes in spinach, suggesting that each one binds Chi [Green and Camm 1982]. However, the minor 25 kDa polypeptide of barley LHCII is not present in CP II. Thus it is cation precipitable but does not necessarily bind Chi. If it does not bind Chi, there is no apparent reason why it should be lacking in a Chi 6-less mutant. The absence of one or more LHCII polypeptide(s) from chlorina f2 was reported by Ryrie [1983] using anti-LHCII and by others based on Coomassie blue staining [Anderson and Levine 1974, Thornber and Highkin 1974, Henriques and Park 1975, Miller et al. 1976, Machold et al. 1977, Apel and Kloppstech 1978, Burke et al. 1979, Simpson 1979, Machold 1981, Bellemare et al. 1982]. The anti-LHCH used by Ryrie was raised against a spinach LHCII preparation which contained three incompletely resolved polypeptides and an additional fourth polypeptide of higher molecular weight [Ryrie et al. 1980, Andersson et al. 1982, Ryrie 1983]. It detected three of these polypeptides in the chlorina f2 mutant [Ryrie 1983]. Thus the polypeptides detected by antibodies to barley CP II* (anti-CP II) in this thesis, cannot be directly compared to those detected by Ryrie's antibodies to spinach LHCII. These results nevertheless agree with his finding that some of the LHCII polypeptides are present in chlorina f2, and that at least one of the major LHCII polypeptides may be completely lacking. 159 The PSI antenna polypeptides are readily detected with immunoblotting techniques. They were not detected in prior work using Coomassie blue staining [Mullet et al. 1980b, and Bellemare et al. 1982] Messenger RNAs coding for both LHCII and LHCI polypeptides are present in relatively normal amounts in chlorina f2 [Apel and Kloppstech 1978, Bellemare et al. 1982, Viro and Kloppstech 1983]. These mRNAs can be translated in vitro, taken up by intact mutant or normal chloroplasts and incorporated into thylakoids. This led Bellemare et al. [1982] to suggest that these polypeptides were rapidly turned over in the absence of Chl 6 and thus did not accumulate in chlorina f2 thylakoids. (They thought these polypeptides were absent since they had not been detected by Coomassie blue staining). A modified version of this idea that would explain the results presented in this thesis is that the stability of the various polypeptides is dependent on the amount of Chl b which they bind. Thus CP 29 (Chl alb = 3) and the PSI antenna (Chl alb = 3-4) are present, as is the 25 kDa polypeptide of LHCII, which has not been demonstrated to bind Chl. In contrast the major 26 and 27 kDa polypeptides of LHCTI/CP H (Chl alb = 1.0-1.5) are almost completely absent, with only a small amount of a single polypeptide detectable in chlorina f2. Thus the polypeptides most depleted in these Chl 6-less thylakoids are those of LHCII/CP H which bind most of the Chl fe in higher plants. Green Chl a+b protein complexes are not detected in chlorina f2 by the standard or even modified mildly denaturing SDS polyacrylamide gel electrophoresis procedures which readily detect these complexes in normal barley 160 [Waldron and Anderson 1979, Results]. These data could also be interpreted as indicating that those polypeptides of the Chi a + b proteins present in Chi t-less thylakoids do not bind Chi a in the absence of Chi b. An alternative interpretation would be that the binding of Chi c occurs but results in the formation of labile complexes which dissociate upon the addition of detergent. Unstable green bands from this mutant have been detected in the LHCII region of sucrose gradients [Edith Camm, pers. comm.] and a green CP H/LHCII band has recently been isolated in two independent studies by the use of ultrasensitive electrophoretic procedures [Bassi et al. 1985, Duranton and Brown 1987]. In the study of Bassi et al. 1985 this was accomplished by growing the Chi 6-less mutant under continuous light, but such a green band was not recovered when the mutant was grown under a regular day/night cycle as done for the plants used in this thesis. In both studies the mutant LHCII/CP II band contains only Chi a and those LHCII polypeptides present in the chlorina f2 mutant, and is reduced in amount relative to normal barley. The detection of the PSI antenna polypeptides in chlorina f2 is interesting in light of the results of Ghirardi et al. [1986], who showed that the size of the functional PSI antenna in chlorina f2 was only decreased from 185 Chi a + b molecules to 150 Chi a molecules per P700 relative to normal barley, while the antenna size of PSII was reduced from 250 Chi a+b molecules to 50 Chi a molecules. In the mobile LHCH hypothesis most of the LHCII is associated with PSII but some is associated with PSI as well. The relative absence from chlorina f2 of the two major 26 and 27 kDa LHCII polypeptides, which constitute CP II, could therefore account for both the large decrease in PSII antenna size and the 161 small decrease in PSI antenna size found in this mutant. This situation is rather analogous to that of the OY-YG maize mutant [Greene et al. 1987] which has a high Chi a/b ratio and is greatly depleted in the mobile LHCII population at high light intensities, but does not lack CP 29. The detection of small amounts of green Chi fe-less LHCII in chlorina f2 [Bassi et al. 1985, Duranton and Brown 1987] could also be consistent with the reduced antenna sizes measured by Ghirardi et al. [1986] in this mutant. These are functional antenna sizes inferred from the light harvesting capacity and number of PSI and PSII reaction centers per given amount of Chi in the mutant. Thus if any unstable Chi a-containing LHCII, LHCI or CP 29 antennae in this mutant were nonfunctional or even slightly less efficient in function, this would be measured as a decrease in the size of functional PSI and PSII antennae. The PSI and PSII antenna sizes measured by Ghirardi et al. [1986] would therefore be consistent not only with a functional LHCI complex containing only Chi a, but also with a reduced, impaired or nonfunctional LHCII complex. By comparing the results from the Chi fe-less barley mutant with the intermittent light barley, which lacks Chi b and has not been exposed to continuous light, it is possible to arrive at the following conclusions. The CP 29 apoprotein was present under both sets of conditions, suggesting that it requires neither continuous light nor Chi b to accumulate in thylakoids. Some LHCII polypeptides were not detected in either system. These likely are rapidly turned over in the absence of Chi b. Therefore it is not possible to tell from the intermittent light plants whether or not they have an additional requirement for 162 continuous light. Finally, the PSI antenna (LHCI) polypeptides were present in Chl fe-less barley, although perhaps in reduced amounts, but were missing from the intermittent light barley. Thus they do not have an absolute requirement for Chl b but do require continuous light to accumulate in thylakoids. These results are likely due to control at a posttranscriptional level, as other studies have shown that LHCII and LHCI mRNAs are present in the chlorina f2 mutant [Apel and Kloppstech 1978, Bellemare et al. 1982], and at least the LHCII mRNAs are present in barley grown under intermittent light [Cuming and Bennett 1981, Viro and Kloppstech 1982]. (I do not know of any studies which have looked for the LHCI transcripts in intermittent light plants.) No studies have ever looked for CP 29 mRNA. Fortunately, the CP 29 apoprotein is present in both chlorina f2 and intermittent light barley, so it seems reasonable to postulate that it is transcribed and translated in the absence of Chl b and/or continuous light. These conclusions and the data on the chlorina f2 mutant and intermittent light barley are summarized in the tables on the following page. 5 . Greening of etiolated barley a. Chl a+b proteins Etiolated plants contain carotenoids but completely lack both Chls a and b. Following the illumination of etiolated barley the Chl alb ratio is very high and then declines to a value of 3, that of mature tissue, by 12 hours (Fig. 21). There is a lag of several hours prior to substantial Chl synthesis, and the synthesis of Chl b lags behind the synthesis of Chl a. The early stages of 163 SUMMARY TABLES FOR STUDIES ON THE CHL 6-LESS BARLEY MUTANT CHLORINA F2 AND BARLEY GROWN UNDER INTERMITTENT LIGHT Complex: Polypeptides present in chlorina f2 Polypeptides present in intermittent light CP 29 LHCII LHCI + some + + some Complex: Requirements for synthesis and accumulation: Chlorophyll 6 Continuous light (more than 2 min every 2 hours) CP 29 no no LHCII some some LHCI no yes 164 greening therefore resemble Chi 6-less mutants or plants grown under intermittent light in this respect. A major difference between the early stages of light-induced greening and these other Chi 6-less systems is that the etiolated plants have only recently been exposed to light. Thus transcripts and polypeptides may have only had a few hours in which to accumulate, unlike the intermittent light-grown barley or the Chi 6-less barley mutant. Clearly, there are many other differences between the etiolated plants, intermittent light plants, and plants exposed to a day-night cycle. However, these differences are results of their respective light regimes and thus not due to independent causes. In contrast, the absence of Chi 6 in the chlorina f2 mutant results from a genetic cause not associated with its light regime. The data presented in Fig. 24 show that the polypeptides of the two minor Chi a+b proteins, CP 29 and LHCI, accumulate with the same kinetics as CP II (LHCII) polypeptides following the illumination of etiolated barley. The data are normalized to the 12 hour amounts in order to illustrate this point, and do not suggest that all these polypeptides accumulate to the same extent. However, they do indicate that the stoichiometry of the Chi a+b apoproteins is roughly constant throughout the first 12 hours of greening. Since the Chi alb ratio at 12 hours is the same as that of mature tissue, and since the relative intensities of the various polypeptides on Western blots was also similar to that of mature tissue, it is likely that a stoichiometry similar to that of mature tissue is maintained throughout the greening process. None of the Chi a + b apoproteins were detected by immunoblotting of SDS 165 extracts from 5-day-old or 6-day-old dark-grown barley. Apel and Kloppstech [1978] did not detect LHCII transcripts or LHCII protein in etiolated barley. A 15 second pulse of red light induced transcription but the protein could not be detected in thylakoids unless the plants were exposed to continuous light [Apel 1979]. Many studies on the regulation of LHCII synthesis in etiolated plants have since been performed. Cab gene transcripts are easily detectable in dark-grown pea seedlings but are at the limit of detection in dark-grown barley leaves [Ellis 1986]. As discussed in the Introduction many studies have shown that cab gene transcription is a phytochrome response. Given the absence of the CP 29 and PSI antenna (LHCI) polypeptides from dark-grown plants it can be stated that these polypeptides require light in order to accumulate. The similar light-induced appearance and accumulation of LHC H, CP 29 and PSI antenna polypeptides would be consistent with a common mechanism of light-regulated synthesis for all Chl a+b proteins. It is possible that the transcription of the nuclear genes encoding the CP 29 and PSI antenna polypeptides requires light and it is also possible that this transcription may be due to phytochrome. This question can only be resolved by studies done at a transcriptional level, which will likely be one of the major topics investigated during my postdoctoral studies with Dr. William F. Thompson. Since the high Chl alb ratios during the early stages of greening resemble the situation in chlorina f2 or barley greened under intermittent light, it might be expected that the pattern of Chl a/6-binding polypeptide accumulation would resemble that present in one of these two systems. In particular the Chl 6-less mutant grown under continuous light lacks or is greatly depleted in the two 166 major LHCII polypeptides. Therefore it might be expected that the lag in Chl b synthesis during light-induced greening of etiolated barley might be accompanied by a lag in the synthesis of the two major LHCII polypeptides. In contrast to this expectation the Chl a+b apoproteins maintain a constant stoichiometry throughout the light-induced greening (Fig. 21), despite the constantly changing Chl alb ratios (Fig. 24). As already mentioned, this stoichiometry is that found in mature thylakoids. This implies that Chl b is not the major factor influencing the accumulation of these polypeptides during greening. The differences between the kinetics of accumulation of the Chl a + b proteins during the greening of etiolated barley, and those predicted solely from consideration of the Chl alb ratio, indicate that factors other than Chl are involved in the regulation of Chl a+b protein accumulation. Thus the differences in (post)translational control among the three Chl 6-less systems cannot be attributed to the presence or absence of specific prosthetic groups. Chl 6-less barley, intermittent light barley and etiolated barley in the early stages of light-induced greening all contain carotenoids and Chl a but lack Chl 6. The factor most likely responsible for these differences is the light regime? The Chl 6-less mutant was grown under a day-night cycle and therefore experienced prolonged periods of continuous light. The intermittent light plants were exposed to 2 minutes of light every 2 hours for 6 days, and the early stages of greening in etiolated plants experienced 6 days of darkness and were harvested after 1, 2, 3 or 6 hours of continuous light. (By arbitrary definition I consider the 9 and 12 hour stages as the later stages of greening since the Chl alb ratios have decreased considerably by this time.) Each light regime produces a 167 unique set of controls. These controls are posttranscriptional and thus cannot be explained by phytochrome acting at a transcriptional level. Transcripts for all three Chi a+b proteins must be present during the early stages of light-induced greening in etiolated barley considering the rapid accumulation of these proteins at this time. Other studies have shown that LHCII mRNAs are present in both Chi Mess barley and barley grown under intermittent light [Apel and Kloppstech 1978, Cuming and Bennett 1981, Bellemare et al. 1982, Viro and Kloppstech 1982]. CP 29 transcripts must also be present in both Chi 6-less barley and barley grown under intermittent light at some point, given that the protein is present in both systems. The same argument applies to the PSI antenna polypeptides present in Chi 6-less barley, and Bellemare et al. [1982] showed that mRNAs encoding these polypeptides were present and could be translated, even though the polypeptides themselves were not detected by Coomassie blue staining. Since all three systems contain Chi c and lack Chi 6, these differences cannot be explained in terms of the light-induced protochlorophyllide to Chi conversion with the resulting Chi acting as a stabilizing factor at the posttranslational level. These differences could however be explained by other kinds of light-regulated (post)translational controls. One possibility is the light-regulated degradation of certain Chi .a/6-binding polypeptides, in which different polypeptides may be degraded under different light regimes. A possible mechanism might be specific proteases which differ in their light- or dark-induction, or even in light-induced synthesis or degradation. Other 168 explanations are possible as well but only future research will provide a definite answer. b. Chl aproteins CP 43, an internal Chl a protein antenna associated with the PSII core, was not detected in dark-grown barley. It is however possible that very small amounts below the limit of detection were present, or that the anti-6 antibody raised to protein 6 of Chlamydomonas is not especially sensitive when used against CP 43 from higher plants. CP 43 exhibits a pattern of light-induced accumulation similar to that of the Chl a+b proteins. This is in contrast to CP I which was detected in small amounts in dark-grown barley and is the PSI reaction center protein which binds P700. Both CP 43 and the Chl a + b proteins serve a light harvesting function. CP 43 and CP I are chloroplast encoded whereas the Chl a + b apoproteins are encoded by nuclear genes and must be imported from the cytoplasm. Furthermore, as plastid genes coding for thylakoid proteins, both CP 43 and CP I are constitutively transcribed [Herrmann et al. 1985, Klein and Mullet et al. 1986, Kreuz et al. 1986]. However, there are still a number of explanations which would account for the similar kinetics of light-induced accumulation between CP 43 and the Chl a + b proteins. One such explanation is that light is required for Chl a synthesis and that all these proteins require Chl a in order to accumulate. In the case of the Chl a proteins, light or light-induced factors may be required for translation, or the polypeptides may 169 simply turn over in the absence of Chi a. It is also possible that certain Chi a proteins must be present before certain Chi a + b proteins can accumulate. However, this last possibility would not explain the existence of some mutants which lack PSII but nevertheless contain LHCII [Bennoun et al. 1981, Metz and Miles 1984]. The detection of small amounts of CP I apoprotein in dark-grown barley disagrees with some reports in the literature. The most likely explanation which accounts for this discrepancy would be a difference in the sensitivity of the immunological techniques used for detection. Results identical to those reported in these studies [Kreuz et al. 1986, Takabe et al. 1986] could be obtained using shorter blot development times, but longer blot development times revealed the presence of CP I apoprotein in plants which had never • been exposed to light (e.g. Fig. 28.). Since it is possible to get these different results on the same samples from the same plants, it is not necessary to postulate that differences between species or differences in the methods of sample preparation account for the discrepancies in the literature. The detection of CP I apoprotein in dark-grown plants therefore has two possible explanations. The first is that the synthesis of CP I apoprotein is very sensitive to light, and that an extremely small light leak was present during growth despite the many extra precautions taken. Although it is not possible to demonstrate the complete absence of light, none of the other Chi proteins were synthesized in the dark under these conditions, and they therefore can be considered as internal controls. Thus this hypothesis would require that the 170 posttranscriptional synthesis of CP I be extremely sensitive to light, more so than that of the other Chl proteins. Alternatively, the presence of both CP I mRNA and apoprotein in dark-grown plants suggests that light is not an absolute requirement for transcription or translation of the CP I apoprotein. Kreuz et al. [1986] demonstrated that the concentration of CP I mRNA present in polysomes isolated from dark-grown barley was the same as in polysomes from dark-grown barley which had been illuminated for 6 hours. These data are also consistent with the continued synthesis of CP I in etiolated plants which have been exposed to continuous light and then transferred back into darkness [Vierling and Alberte 1983, Akoyunoglou and Akoyunoglou 1985]. By analogy to the role of Chl b in LHCII accumulation, Chl a produced by intermittent or continuous light might act as a stabilizing factor which reduces the rate at which CP I polypeptides are degraded (assuming they are turned over). This would explain why transcription and translation can occur in darkness without the accumulation of large amounts of CP I apoprotein. It would also explain how CP I accumulates so rapidly following illumination, well before any of the other Chl proteins. Tanaka and Tsuji [1985] have shown that the green CP I complex and its associated P700 activity can be detected 45-60 min after illumination of etiolated barley. 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Biochem. 225. Williams, R.S. and R.J. Ellis. 1986. Immunological studies on the light-harvesting polypeptides of photosystems I and n. FEBS Lett. 203: 295-300. 226. Wray, W.T., Boulikas, T., Wray, V.P. and R. Hancock. 1981. Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118: 197-203. 227. Yamada, Y., Itoh, N. and K. Satoh. 1985. A versatile chromatographic procedure for purifying PSII reaction center complex from digitonin extracts of spinach thylakoids. Plant Cell Physiol. 26: 1263-1271. 228. Youvan, D . C , Alberti, M., Begusch, H., Bylina, E.J. and J.E. Hearst. 1984. Reaction center and light-harvesting genes from Rhodospeudomonas  capsulata. Proc. Natl. Acad. Sci. USA 81: 189-192. APPENDIX 1: LIST OF ADDRESSES Allred, Dr. David R. Dept. of Molecular, Cellular and Developmental Biology, University of Colorado, Campus Box 347, Boulder, Colorado 80309 USA Apel, Dr. Klaus Botanisches Institut der Universitat, Olshausenstrasse 40, D-2300 Kiel, Federal Republic of Germany Bricker, Dr. Terry M. Dept. of Chemistry, University of Southern Missisipi, Hattiesburg, Missisipi 39406 USA Briggs, Dr. Winslow R. Director, Plant Biology Dept., Carnegie Institute of Washington, 290 Panama Street, Stanford, California 94305 USA Chua, Dr. Nam-Hai 1230 York Avenue, Box 301, Rockefeller University, New York, New York 10021 USA Mcintosh, Dr. Lee MSU/DOE Plant Research Lab, Michigan State University, East Lansing, Michigan 48824 USA Staehelin, Dr. L. Andrew Box 347, University of Colorado, Boulder, Colorado 80309 USA 190 191 Thompson, Dr. William F. Dept. of Botany, North Carolina State University Raleigh, North Carolina 27695 USA Waterman, Dr. Michael S. Dept. of Mathematics, University of Southern California, Los Angeles, California 90089-1113 USA 


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