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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 A N D IMMUNOLOGICAL STUDIES ON C H L O R O P H Y L L PROTEINS by MICHAEL JOHN B.Sc,  WHITE  McMaster University,  1981  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS  FOR T H E 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  ABSTRACT  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 isolated  a+b  protein complexes  and characterized. These  included  associated a  with  photosystem  light-harvesting  antenna  I were 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  ii  were  reacted  with  the  entire  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  Affinity-purification cross-reactions.  against of  The  the  data  the  photosystem  anti-CP suggest  Ia  I  antibodies  there  are  antenna did  amino  acid  not  complex, eliminate  sequence  LHCI. these  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  chlorophyll a+b  on.  The  sequence  similarities  predicted  to  exist  among  the  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  proteins, including those of CP 29 barley  (Hordeum vulgare  L.)  belonging  to  and LHCI,  mutant  chlorina  each were f2.  of  the  three  found in the Similar  Chi a + b Chi Mess  experiments  were  performed on barley grown under intermittent light, which also completely lacks  iii  Chi  b.  belonging to the Chi a+b  Only two polypeptides  under these conditions. Taken together with published these results provide evidence for the existence  proteins  were found  studies in the literature,  of differing posttranscriptional  controls among the various chlorophyll proteins, and between these two systems.  Antibodies chlorophyll-binding barley.  The  were also used to determine the kinetics of synthesis polypeptides  chlorophyll  a+b  during antenna  the light-induced polypeptides  absent in the dark. They were first detected then increased identical  greening  of both  of the  of dark-grown  photosystems were  after 1-2 hours of illumination, and  in amount exponentially. The kinetics of accumulation are almost  for all the polypeptides  belonging  complexes, suggesting their synthesis may despite the existence  to the chlorophyll  a+b  protein  be regulated by the same mechanism  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  important role in its synthesis  increase, showing that light plays an  or degradation, even if it is not an  requirement for transcription and translation.  iv  absolute  T A B L E O F CONTENTS  Abstract  ii  List of Figures  viii  List of Abbreviations  ix  Acknowledgements  x  I. INTRODUCTION 1. Photosynthesis and electron transport 2. Chi proteins a. b. c. d.  1 1 8  Discovery of the first Chi proteins: CP I and CP II Discovery of the other Chl proteins Further detail on the Chl a proteins Further detail on the Chl a + b proteins  8 10  14 17 23  3. Factors affecting Chl protein synthesis a. b. c. d. e. f.  Experimental systems for studying Chl protein synthesis The Chl b-less barley mutant chlorina fZ Plants grown under intermittent light The etioplast to chloroplast conversion Transcriptional control of protein synthesis Posttranscriptional control of protein synthesis  4. Molecular techniques for studying Chl proteins and their synthesis 5. Properties of antibodies and antigens: Immunological specificity and cross-reactivity  23 24 26 26 28 31  33 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 b. Electrophoresis and Immunoblotting c. Quantitation of Immunoblots  III. RESULTS A. PREPARATION AND CHARACTERIZATION CHLOROPHYLL A+B PROTEIN COMPLEX v  OF  50 51 51  A  PS  53 I 53  . B.  C.  D.  E.  F.  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 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 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 T H E 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 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 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 1. Characterization of Chi a+b proteins 2. Immunological relatedness of the Chi a+b proteins 3. Modelling immunological cross-reaction  vi  129 129 133 148  4. Regulation of Chi a+b apoprotein intermittent light barley 5. Greening of etiolated barley a. Chi a+b proteins b. Chi aproteins V. R E F E R E N C E S Appendix  synthesis:  Chlorina  f2  and 157 162 162 168 172  1: List of Addresses  190  vii  List of Figures Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.  1. The four major macromolecular complexes of thylakoid membranes 3 2. Unstained gel of thylakoids solubilized in 300 mM octylglucoside 12 3. CP Ia contains CP I and the PSI antenna CP .55 4. Polypeptide composition of CP Ia and the PSI antenna CP 56 5. Visible absorption spectra of CP Ia, CP I and the PSI antenna CP. ..58 6. Anti-CP Ia cross-reacts with polypeptides of LHCII and CP 29 62 7. Dot blots reacted with anti-CP II and anti-CP Ia 64 8. Cross-reaction is due to antibodies to the PSI antenna CP 67 9. Affinity-purified anti-CP Ia also cross-reacts 70 10. Purified CP II and CP 29 used as antigens 72 11. Dot blots reacted with anti-CP II and anti-CP 29 73 12. Immunoblot reacted with anti-CP II 75 13. Polypeptide composition of LHCII vs. CP II 76 14. Immunoblot reacted with anti-CP 29 77 15. Summary figure of immunological cross-reactions 79 16. Chl Mess barley vs. anti-CP II 85 17. Chl 6-less barley vs. anti-CP 29 86 18. Chl 6-less barley vs. anti-CP Ia 88 19. Chl 6-less barley: CP la-like bands 90 20. Chl 6-less barley: CP la-like bands lack PSI antenna polypeptides 91 21. Etiolated barley: Total Chl and Chl alb ratio vs. time in light 95 22. Etiolated barley: Immunoblot reacted with anti-CP 1 96 23. Etiolated barley: Control proteins vs. time in light 97 24. Etiolated barley: Chl a+b apoproteins vs. time in light 99 25. Etiolated barley: CP 43 apoprotein vs. time in light 101 26. Etiolated barley: CP I apoprotein in normal and extreme dark 104 27. Etiolated barley processed as in Kreuz et al vs. anti-CP 1 105 28. Etiolated barley processed as in Takabe et al. vs. anti-CP 1 107 29. Intermittent light barley: immunoblots of whole cell extracts 112 30. Intermittent light barley: immunoblots of thylakoids 113 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.  A  INTRODUCTION  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 questions  plant  development.  This  thesis  does  not  attempt  to  answer  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 were  observed  and  among antibodies  characterized,  and  the  to  the  investigation  Chl a + b proteins 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 , N 0 " 2  and S O ,  2  1  2 -  [Salisbury and Ross 1978].  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 " is reduced to NO " 3  and  N0 "  is reduced to N H | ,  2  +  2  in the cytoplasm,  by nitrite reductase in plastids  [Oaks 1985].  Both nitrogen and sulfur are ultimately incorporated into amino acids, nucleotides and many other compounds.  The and  energy and reducing power for these processes is supplied by ATP  NADPH,  direct  products  of  photosynthesis.  Light  energy  harvested  chlorophyll (Chi) proteins is used to split water into oxygen (0 ), 2  electrons. In the currently accepted Z scheme of photosynthesis, (PSII)  provides  provides the  the  energy  needed  electrons with the  to  split  water,  by  protons and  photosystem II  and photosystem  I  (PSI)  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 and coupling factor. This figure is adapted from Anderson [1986] 6  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 Q g " , one 2  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 reason,  the water-splitting enzyme and because  cytochrome  is operational [Cramer et al. b559  is  readily  photooxidized  1986]. For this 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  cross  until  the  protons  membrane  from  the  this  stroma.  charge  is  The electron  transferred to the cytochrome fb  6  neutralized  pair  by  carried by  charged. It cannot the  uptake  the  of  two  plastoquinone  is  complex and the proton pair released into the  thylakoid lumen. This net deposition of protons within the thylakoid lumen creates a  transmembrane  gradient  is  proton  then  used  gradient.  to  The  synthesize  electrochemical  energy  stored  ATP from ADP and inorganic  in  this  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 membranes.  complex is the fourth major macromolecular complex in thylakoid  6  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 S 2  lumenal  surface  reduction cytochrome  of  of  the f  by  the  thylakoid membrane  Rieske-FeS  protein  plastocyanin,  membrane surface [Haehnel 1984].  a  by  blue  2  cluster are located on the  [Hauska et al.  plastoquinol copper  and  protein,  1983]. the  occur  Thus the  oxidation at  the  of  inner  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 b Wraight  1983,  Haehnel  Q cycle 1984].  [Cramer  and Whitmarsh  Following reduction of the  cytochrome f is reduced with a half-time of about 10-20 and Whitmarsh of  1977,  Crofts and  plastoquinone pool, milliseconds [Cramer  1977, Crofts and Wraight 1983]. Due to the lateral segregation  PSII and PSI into granal  Arntzen  1977,  Anderson  and stromal lamellae  and Melis  1983],  respectively  plastoquinone  [Armond and  would have to move  from the granal to the stromal lamellae in the 10-20 milliseconds. However, the measured diffusion coefficient for quinones (D = be  sufficiently  Wraight  large to  allow  plastoquinone  to  10"  9  cm /s) does not appear to 2  move  this  quickly  [Crofts and  1983, Haehnel 1984]. It has therefore been suggested that a molecule  such as plastocyanin (D =  3-4 x 10"  [Crofts and Wraight 1983,  Haehnel 1984]. Nevertheless,  9  cm /s) is also involved in this processs 2  been reduced there appears to be a 5-15  after cytochrome f has  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  diffusion coefficient of plastoquinone may be as large as 10"  suggested that 7  the  cm /s, and thus 2  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 photosynthetic  and P700 reaction centers [Senger et al. 1987]. It is found in all 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 , A 0  particles with a 1.5  1 (  X, B and A. After illumination of PSI  picosecond laser flash, P700*A" is formed in 13.7  picoseconds [Wasielewski et al. 1987]. P700, A ratio. It has been suggested that A  0  0  ±  0.8  and A , occur in a 1:1:1 molar  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 , is likely an iron sulfur cluster located on 2  CP I [Sakurai and San Pietro 1985,  Golbeck and Cornelius 1986,  Golbeck 1986]. A and B are likely closely interacting Fe S|, 4  Warden and  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] (LHCII),  the  major  Chi a + b antenna  complex  associated  and CP II  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  unless  special  conditions  are  not  covalently  precautions  described  above  are the  attached, taken  to  most keep  Chi proteins  Chi proteins them  other  intact. than  readily Thus  CP I  dissociate under  and  the  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 CP  the  SDS extraction of thylakoids is performed at 4 ° C , oligomers of  II can also be obtained [Hiller  Hayden and Hopkins  1976,  Thornber  et al.  1974,  Anderson and Levine  1979]. CP II is  also known as  1974, LHCII,  LHCP or L H C depending on the method of preparation or on arbitrary usage. The  terms LHCP  LHCI  (discussed  and L H C are now less appropriate due to the discovery of 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 and  Jay  1985].  or polypeptide composition occur between The  composition. Barley LHCII minor  25  kDa  method  of  purification  can  species  also  affect  [e.g.  Thaler  polypeptide  prepared by cation precipitation contains an additional  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 octylglucoside  and to  Green  [1980]  extracted  solubilize preferentially the  thylakoids  at  Chi proteins  4 ° C with associated  30 mM  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 complex.  The  it  under  use  of  nondenaturing  300  conditions  mM octylglucoside,  solubilizes thylakoid membranes including the Under  these  conditions  two  additional  mobility than CP I, designated  to  obtain  instead  of  a  30  green  mM, completely  Chl proteins associated  Chl proteins  with  CP I  lower  with PSI.  electrophoretic  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  electrophoretic techniques  solubilizing  thylakoids  and  the  use  of  improved  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) the  polypeptides  rather  than  the  complete  is therefore used to refer to  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 Staehelin  0.5  % of the  1986]. Its  total  Chl loaded  on preparative gels  absorption and fluorescence  weight of its polypeptides and its antigenicity  emission  [Dunahay and  spectra, the molecular  with certain monoclonal antibodies  suggest that it may be part of LHCI [David Simpson, pers. comm.]. However, its  association  antibodies  to  with  PSII  Chl a+b  particles  proteins  and  the  (discussed  later)  immunological leave the  cross-reaction  of  exact identity and  function of CP 24 unresolved at present.  The bryophytes  same  Chl  proteins  appear  to  be  and vascular plants studied to date.  present  in  all  green  Thylakoids of the  algae,  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 contain P700,  been  found in all organisms  known to  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, Nevertheless,  LHCII  shows  a  substantial  degree  Hiller and Larkum 1985].  of  immunological  across a large variety of species. Monoclonal antibodies L.)  LHCII  angiosperms, Jay  react the  with  the  corresponding  fern Nephrolepsis  relatedness  to pea (Pisum sativum  polypeptide(s)  in  thylakoids  and Chlamydomonas reinhardii  of  [Thaler and  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. Lichtenthaler  1980].  and phylloquinone  (vitamin K , ) [Interschick-Niebler and  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  weights  polyacrylamide similar  to  gel  those  electrophoresis,  of  the  green  their  apoproteins  complexes.  These  have  molecular  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,  polypeptides  Morris are  and  readily  Herrmann detected  silver-staining or immunoblotting, are  1984].  if  more  used  These  lower  sensitive  [Morris  molecular  techniques,  and Herrmann  weight  such  as  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  cross-reaction of antibodies to  homology  [Alt et al.  these proteins has  1984]  and immunological  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  predicted amino acid sequences of the  sequence  homology  CP I genes psIAl  exist between  the  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 [Michel center  and Deisenhofer bacteriochlorophyll  of the photosystem  1987].  in purple photosynthetic  The L and M polypeptides  between  them,  along  with  other  bind the  bacteria reaction  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 * is 2  added to increase the stability of these bands [Irrgang et al. 1986]. However, it was  not  conclusively  demonstrated  that  Dl  and D2  actually  bind chlorophyll.  Nanba and Satoh [1986] recently isolated a pigment-protein complex, consisting of Dl,  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 participate  suggests that CP 47  in binding either P680 or the  and CP 43 need not  pheophytin associated with the  reaction center, but that this function is served by D l and D2 instead.  PSII  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 those of the  20-30 kDa range, lower than  Chl a apoproteins. In addition to ^-carotene,  xanthophylls lutein, neoxanthin  LHCII contains  and violaxanthin. [Rawyler et al.  et al. 1983]. The carotenoid composition of the other Chl a+b been investigated. that the  1980,  proteins  indicated by cross-reaction  Landis,  proteins has not  A major part of my work is directed towards  Chl a+b  the  demonstrating  also comprise an immunologically related group, as of antibodies  to these polypeptides.  While this work  was being published, similar results were obtained by Evans and Anderson [1986] and  by  Sylvia  discussed  Darr  [Darr  in detail in the  chapter of the Results,  et  al.  1986].  last section  and has been  This  of this  immunological  relatedness  Introduction and in the  is  second  published by White and Green [1987a,  1987b, 1987c].  The and  are  polypeptides synthesized  of the Chl a + b proteins are encoded in nuclear genes  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 family.  polypeptides  For example,  of LHCII  are  encoded  by  a  large  nuclear multigene  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  termini of the  peptides  are less  than  mature cab polypeptides  50  % homologous  show considerable  and the  divergence  amino  [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  clearly discernible between the particular amino acids  high  sequence conservation  which are  Type I and Type II amino acid sequences, in  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 they  show substantial homology within a sequence, i.e.  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  (Lycopersicon esculentum L.) and petunia [Hoffmann et al. 1987,  in  tomato  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 granal  additional functions of LHCII polypeptides are their role in mediating  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 ) or higher concentrations of monovalent cations 2+  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  granal stacking [Steinback et al.  from LHCII  and also eliminates  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  electrophoresis  polypeptides  as determined by SDS polyacrylamide gel  and freeze fracture of thylakoid membranes [Armond et al.  1977]. The intermittent light plastids have full photochemical activities  1976,  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 an  Chi 6-less barley mutant chlorina f2 has been and continues to be  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 )  to  2 +  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  sensitive immunological techniques indeed  present  amounts.  in this  Bassi  et al.  mutant.  to show  mutant,  although  [1986]  attributed  However,  that most  Ryrie  some LHCII are present  stacking  in  [1983]  used  polypeptides  were  in greatly reduced  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  absorbed  by  Chi 6),  decreases  increases  the amount of light  light  the  at  amount  645  nm (light  of light  H, a  wavelength  absorbed by PSII and  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  SATTKK  an amino terminal hexapeptide  in which one or both of the  from LHCII  threonine residues  of sequence  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  supplementary  reverse 705  mechanism  is  stimulated  by  addition  of  cations  nm light, except that in the latter case a phosphatase  activated in place of a kinase.  or is  23 Evidence distinct  from  that  LHCII  CP 29 has  is  not  recently  involved in state transitions  been  presented  by  and thus  Dunahay  and  is  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  OY-YG which shows elevated  et al.  [1987] studied  the  maize  double  mutant  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  cation-regulated  LHCII  antenna  which  thylakoid stacking and the  is  phosphorylated  concomitant  and  involved in  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 include  mutants  that  have  been  and various  used  growth  to  study  conditions  Chi protein or light  synthesis  regimes.  By  in vivo 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.  system lacking only a single  For example,  Chi 6-less mutants  provide a  factor (Chi 6), while dark-grown (etiolated) plants  24 lack light and therefore cause of differences  Chls a and 6.  between etiolated  The absence of light is  the  and normal plants, but these  primary 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  Chl-binding  f2  is  polypeptide  not but  a  mutation  appears  to be  in  a  structural  blocked at  gene  coding  for  a  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  molecules to 50 Chl a molecules for PSII and from 185 Chl a+b 150  Chl a  molecules  for  PSI  [Ghirardi  et al.  1986].  In  Chl  a+b  molecules to  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  These  apoprotein on  assessments  were  the  based  basis of its on  SDS  molecular weight  polyacrylamide  gel  [Machold 1981]. 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]  to  that  used  short-term  7-day-old  etiolated  labeling  in  vivo  with  35 [  S]-methionine  show  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, L H C n 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  Therefore these sorts of questions which  both  chapters  Chi 6-less  mutants  C and E of the  absence  of  Chi a/6-binding  polypeptides.  are best answered by comparative studies in and  Results  intermittent  section  light  report results  plants  are  from  used,  experiments  and 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  plants contain a special type of plastid known as an etioplast  plant. These  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  germinating seeds or seedlings exposed  conversion  can  be  studied  in  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.  those of control plants,  However, it was  by comparing the found that  growth of the leaf in a statistically  the  growth  of these plants  measuring process  slowed  to the  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  Etiolated control  of  control of protein synthesis  plants  plant  gene  have  been  widely  transcription.  used  Perhaps  in the  studying most  the  phytochrome  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  flanking these genes [J. Simpson et al. 1985,  with  the  5' -DNA  sequence  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' [J. Simpson et al. 1985,  region of cab genes  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),  discovered.  and  was,  in  The size of  the  fact,  the  way  in  which  these  genes  transcript pools for most of these  genes reached a maximum size after  were  first  photoregulated  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  Dl  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 attributed to differences  between species, differences  could be  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,  carboxylase  which  code  for  the  large  subunit  of  ribulose  bisphosphate  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  reported  by  increase  of  Rodermel and  transcription of Bogorad  7-day-old  [1985] and the  maize  grown  light-induced  at  30 °C  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  NADPH factor  the  dark.  These  : protochlorophyllide reductase [Vierling and  polypeptides 1986]  in  Alberte  of the water  1983,  include  [Apel  Selstam  splitting complex  and cytochromes f and b  6  1981], and  the  genes  the  subunits  Sandelius  [Ryrie et al.  coding  of coupling  1984],  1984,  for  the  three  Liveanu et al.  [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  by  in  these  studies  as  the  methods  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 [ nevertheless  S]-methionine and chased in the light. This kind of result is  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  sufficient  protein  if  a  immunological techniques  lag  time  for  for the detection  synthesis  is  allowed,  of these Chi a/fe-binding  sensitive  polypeptides  are among the best controls available for studies on dark-grown plants.  33  A  major concern in studies  dark-grown  plants  should  be  the  where  CP I apoprotein is not detected in  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 differences  and Kreuz et al. 1986]. This presented the opportunity to discuss in  our  experimental  results  and  the  possible  causes  for  these  differences.  4. M o l e c u l a r techniques  In  for studying C h l proteins a n d their synthesis  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  properties) to visualize or quantitate the macromolecule.  on  its  chemical  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.  greening the Chi protein complexes  During the early stages of  are even more labile, and tend to dissociate  during electrophoresis [Tanaka and Tsuji 1983, Tanaka and Tsuji 1985]. Thirdly, systems lacking Chi b, such intermittent  light,  as  do not have  Chi 6-less mutants detectable green  and plants  grown under  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 molecular staining.  weight This  can  using be  to identify SDS  done  these polypeptides  polyacrylamide by  purifying  gel  on the  electrophoresis  Chi proteins  from  basis of their followed mature  by  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 identification.  This  is not  for proper  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  sequence-specific  plant  cell  proteins.  These  advantages  exist  because  the  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 would otherwise above,  not be  immunoblotting  plants, in which its identification and quantitation  possible. methods  In  addition to  are  currently  all of the  the  most  advantages  sensitive  listed  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 [ Brown 1984, is  perhaps  S]-methionine-labeled proteins [Nielsen and  Heukeshoven and Dernick 1985]. In my experiments, silver-staining 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  molecular  a polypeptide  weight. It also  reaction  (as described  prevents  in the  Consequently, the combined  relative  to using  misidentification  appropriate  techniques  a single  property  such as  due to immunological  chapters  of the Results  cross  section).  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  the antibody Western  combining  site. In a linearized, denatured  blot, the basis of the immunological  acid  sequence  which  both  epitopes  and antibody  chapter used of  amino  acids  specificity  SDS-protein  combining  sites  the probability of specified  are discussed  work  presented  is termed  on a gel or  is the particular  constitutes the epitope. The size, shape  of the Results. The theoretical  to estimate  site on the antibody  amino  and properties of  in detail  in this  in the last  chapter  can be  that similar or identical epitopes, i.e. sequences  length,  will  arise  among  two or more  different  proteins due to chance alone.  Similar or identical epitopes may or  more  convergent  also be due to a common  proteins, or to functional or structural evolution of these  proteins. Antibodies  factors which  origin of two  have  to proteins which  caused the 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 Lazarides show  immunological  [Shaw  phosphatase relatedness phosphatase performed  in a variety of studies  [reviewed by  1982]. Both monoclonal and polyclonal antibodies have been  close  proteins  been used  et  al.  relatedness  1984].  phosphodiesterase  proteins [Culp  and the phosphodiesterase to  test  if  certain  vertebrate  Cross-reacting polyclonal  and 5' -nucleotide of these two  among  removal  or  were  antibodies  used  and Butler 1985].  used to  neurofilament to  alkaline  to demonstrate  the  Since the alkaline  are both glycoproteins, experiments were  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  [Borroto exists  genes  and Dure among  the  which 1987].  existed  at  the  A complicated  various  seed  beginning  pattern  storage  of  angiosperm  of immunological relatedness  proteins.  Immunoblotting  endosperm storage proteins with polyclonal antibodies raised against fraction  showed  reaction  with  a  14  evolution  of  maize  a glutelin-2  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  this  and what  their  properties  were.  During  number of other research groups have investigated  the  course  of  this PSI-associated  work  a  Chl a+b  40  protein, which is now known as the PSI antenna, LHCI.  The antibodies effort.  purification to  The  them  of  was  observation  the  various  Chi proteins  and  the  production of  a major undertaking involving considerable time and 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  available. This  the  mathematics  mathematics  as  of biological sequence  developed by Arratia  comparison became  et al. [1986] was made  available to us in manuscript form two years in advance of publication, courtesy of  Dr.  Michael  Mathematics immunological chance biological  Waterman,  department.  through  Joe  Our theoretical  cross-reactions  cross-reactions  are  significance  should  will  occur  infrequent, not  be  Watkins, results  formerly  predict  due  to  chance  the  results  automatically  of  the  U.B.C.  the  probability  that  alone.  Although  such  demonstrate  that  clearly 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 result was grown  investigated  under  polypeptides  CP I apoprotein were detected in dark-grown barley, and this in a series of experiments.  intermittent  present  were  light  revealed  different  from  that those  the  Finally, set  present  in  studies on barley of  Chl a/6-binding  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 A N D METHODS  1. Plant material and growth conditions  All experiments were done using barley (Hordeum vulgare L . cv. Bonanza). Barley was  chosen  developmental  primarily because it is the most commonly used plant for  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  grown in a  thylakoid preparation, normal and  greenhouse in potting  seeds were soaked  overnight  (16  chlorina f2  soil. For developmental  barley  experiments,  h) in aerated distilled water  were barley  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  experiments followed  by  covering  the  door  and  its  perimeter.  Intermittent  light  were performed in the same way but using cycles of 2 min light 118  min of darkness. In both experiments  using white fluorescent  seedlings were greened  light (Sylvania F14T12) which produced a quantum flux  of approximately 75 microEinsteins m ~ s ~ 2  at soil level (before germination) and  1  approximately 90-120 microEinsteins m " s " 2  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 42  to  16-day-old  barley  seedlings  were  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 Na P«0 2  7  (pH 7.4),  Tris-maleate  (pH  was  followed  by two  washes each  of  10 mM  0.3 M sucrose (pH 7.4) and finally 2 mM EDTA, 10 mM  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 gels  with  Coomassie  polypeptides,  blue  temperature. It is necessary to stain these  or silver  or to use them  et  [Wray  al.  1984] in order  for immunoblotting  if one  to see the  is interested  in a  particular polypeptide(s).  For  Coomassie blue staining,  w/v Coomassie Brilliant Blue R acid  for 20-30  1.5 mm  (Sigma), 50 %  min (or longer for thicker  several changes of 20 %  thick  methanol,  7 %  in  40 %  methanol,  the equilibration by  in 50 %  the method  determined  acetic  methanol  of Wray  et  v/v acetic  1-2 days at  Unstained gels to be silver-stained silver-stained  gels to be silver-stained  by the  were  soaked  acid to reduce or remove blue bands prior to  al.  and silver-staining. Silver staining was done [1984].  by reference to the mobilities  carbonic anhydrase  Both  10 %  were  overnight and then  procedure. Coomassie blue-stained  and 7 %  %  acetic acid over the next  equilibrated  standard  in 0.2  destained in  were  methanol  soaked  then  time they were stored or photographed. %  were  v/v methanol  gels). Gels  which  in 50  gels  Apparent  of Sigma  molecular  standards  weights  were  (No. SDS-6 with  added or No. SDS-7).  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  polyacrylamide running gels for two 3 mm  mm  to make 10  thick preparative gels, or four 1.5  thick analytical gels: 40.0 ml acrylamide : bisacrylamide (30 : 0.8 % w/v), 9.8, and 0.6 ml 20 % w/v SDS. This gel solution  78.4 ml 2 M  Tris-HCl p H  was  in an Erlenmeyer  deaerated  flask  connected  to a  vacuum  line. Following  45 deaeration,  1.0  ml  10  %  w/v  N N N ' N ' -tetramethylethylenediamine polymerization,  and  the  gels  ammonium (TEMED)  poured  persulfate  were  immediately.  and  added  Gels  of  50  to  lower  fil  initiate 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 bisacrylamide (30 6.1, and 0.15  : 0.8  ml acrylamide :  %), 21.7 ml distilled water, 3.0 ml 1 M Tris-HCl pH  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. P r e p a r a t i o n  o f C h l o r o p h y l l P r o t e i n 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  stacking gels. Chi proteins were excised, mashed in 65 ethylene under  glycol,  5  mM  nondenaturing  separate  dithiothreitol conditions, using  nongreen co-migrating  electrophoresis  was  heat-denatured  pH a  and  higher  mM  gels  with  Tris-HCl, 10  electrophoresed polyacrylamide  a  2  cm  %  (v/v)  second  time  concentration  to  contaminants. Purity of complexes from the second  verified  in the  6.8  %  on  a  totally  presence of 2  %  denaturing  SDS.  Samples  gel,  using  were purified  samples  by  further  electrophoresis if necessary.  CP 10  %  II was  polyacrylamide  (25-30 kDa it  polypeptides %  CP CP  polyacrylamide  migration  and  into  in the  dependent on  from  to  II,  of CP  29  on  gels. For  Ia 10  %  is  gels.  Since  then 29  the  II* (50-60 kDa  polypeptides  29. When CP  which  in  the  range) on  CP  H  region  II* is re-electrophoresed, some of well  was  separated  prepared  by  from  co-migrating  re-electrophoresis on  mobility  of  Chi  concentration [Chua et  al.  1975], the relative rate of  proteins  is  strongly  is slower during the second electrophoresis. This separates it  contaminants  was  co-migrating  II* region. CP  also from intact CP  bottom  re-electrophoresis of CP  avoid  polyacrylamide  non-green  CP  by  range), especially CP  dissociates  14  prepared  which  by  re-electrophoresis of both  polyacrylamide  each purification, a SDS  during  the  first electrophoresis,  II.  purified  the presence of 2 %  co-migrated  gels, and  CP  I by  CP  for 30  polyacrylamide, Coomassie blue-stained gels.  min  and  and  re-electrophoresis on  sample from the second gel was  at 65 °C  Ia top  CP 12  Ia %  heat-denatured in  checked for purity on  10  %  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  Chlorophyll  Protein  centrifugation and cation precipitation [Ryrie et al. 1980].  5.  Spectra  and Chlorophyll  a/b ratios  of Purified  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. P r e p a r a t i o n  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  injection. Clotted blood was  followed  by  bleeding  about  10  cooled to 4°C. The serum was  days  after  each  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 was  near  the  bottom  of the  gel.  density) or the front of dissociated Chi  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 H P O « , 2  15 mM K H P O „ 2  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  heat-denatured  as at  a  cryoprotectant,  65 °C  for  30  and  the  min (or frozen  supernatant  was  and heat-denatured  immediately later). To  remove SDS for protein assay, 50 p\ of sample was precipitated with 950 u\ acetone and spun at water  and  protein  15,000g for 60 determined  using  s.  Pellets were resuspended  the  BioRad  protein  assay.  in 1.00 ml 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  probed  % gradient  with  gels  anti-coupling  [Chua  factor  or  1980]  were used  antisera  against  to  prepare blots  the  Chi a+b  to be  proteins.  Samples to be screened with other antibodies were run on 10 % gels [Kirchanski and  Park  1976].  oxygen-evolving  Polyclonal  complex  antibodies  and the  to  the  33  kDa  herbicide-binding polypeptide  polypeptide  of  the  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 sometimes necessary  pg. It  was  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. P R E P A R A T I O N A N D CHARACTERIZATION OF A PS I C H L O R O P H Y L L A + B PROTEIN C O M P L E X  1. Isolation  and characterization of the  two  C P Ia  bands  and  studies,  it  the  PSI  antenna complex  To necessary  obtain  antibodies  for  use  in  to develop or optimize methods  developmental  was  first  for purifying the Chl proteins to be  used as antigens. Since these purifications are very labour intensive, the amount of  time  crude  involved would be considerably  preparations  of  all  Chl  reduced by finding methods  proteins  could  be  obtained  from  in which 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 PSI  complexes  min.  In the  preferentially solubilized the Chl proteins associated with PSII. The could then be pelleted by ultracentrifugation at course of determining the best methods  100,000g for 30  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  53  higher  concentration  completely  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 LHCII or CP  rounds of electrophoresis did not contain any  polypeptides in the  29 regions of the gel (square brackets in Fig. 4b).  Several other polypeptides with molecular masses in the 9-19 were associated with CP  Ia complexes but were completely  kDa  range  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 et al.  1975, Acker et al.  The  absorption  maxima at 436 All CP and  have been iron sulfur proteins associated with PSI  and  nm  spectrum 671.5  la bands had  675  1982, Lagoutte et al.  nm  of  PSI-associated  absorption spectra similar to CP  Chi  which  oxidizes Chi  b  protein nm  shoulder in both the CP  had  (Fig. 5).  I, with maxima at  but with a Chi b contribution in the 470-480 nm  PSI-associated Chi protein disappeared 2  new  1984].  a Chi b shoulder around 472  and  vertical arrow). This 470-480 nm  NH OH  the  [Nelson  437  region (Fig. 5,  Ia complexes and the  when methanol extracts were treated with  [Ogawa  and  Shibata  1965]. Both  complexes  therefore contained Chi b.  Using the method of MacKinney [1941], the PSI antenna CP alb  ratio of 2.5  ±  1.5  based on  had  a Chi  six measurements made on separate samples.  This variability could be due to the small amounts of purified material obtainable  58  Fig. PSI that red the  5. Visible absorption spectra of gel slices of CP Ia bottom, CP I and the antenna CP. The absorption spectrum of CP Ia top is indistinguishable from of CP Ia bottom. Numbers indicate the positions (in nm) of the blue and absorption maxima. A Chl b shoulder (large vertical arrow) is visible in both 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 C P 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. C P Ia complexes can be obtained directly from intact chloroplasts  Intact MgCl  2  to  chloroplasts  maintain  were  prepared in  granal stacking.  When  grinding media these  containing  chloroplasts  were  5 mM  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  66 45  '36 .29 • 25  ant.{  a b  -20  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  electrophoresis  the  are  right  ideal,  amount of  and if  the  protein  is  development  used, time  if  the  of the  conditions of 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 blots  indicated  immunoenzymatic detection  that  antibodies  could  of purified  detect  16  pg  Chl proteins Chl,  on dot  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 composition of LHCII,  is close to that of the  approximately 4 Chl a,  3 Chl b and  consensus  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  thylakoid  eliminated  polypeptides  the possibility  that  the cross-reactions observed  were caused by reaction with  unelicited  with  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  polypeptides  CP  in the molecular  Ia preparation  did not contain  weight range of the LHCII and C P 29 apoproteins  (25-29 kDa), it was surprising to find that polypeptides detected  at these  positions were  when total thylakoid protein was blotted and reacted with  (Fig. 6b). The anti-CP by  any detectable  the method  Ia also cross-reacted with  of Burke  et  al.  polypeptides  [1978] and with  CP  this  antibody  of LHCII  29 purified  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  with  or L H C n ,  CP  CP  29  29 and LHCII.  approximately  The  cross-reacted  The amounts  complex with  which  antigenic  of Chi loaded  showed  contamination  determinants  in lanes  6c, d  in purified and e were  equivalent.  reaction of anti-CP Ia with C P II and C P 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 apoprotein  no  on blots of total  thylakoid protein  was  very  antibody weak,  with  CP I  as the CP I  66 apoprotein  was  reaction was  not  as  antigenic  as  the  Chi a + 6-binding  polypeptides.  This  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  a+6-binding polypeptides  cross  is therefore  reactions the  most  to  relatedness  of  the  Chi  probable interpretation of these  results.  3. Specificity of cross-reactions observed with anti-CP Ia  Since quantitative  the  argument presented  considerations,  in the  experiments  were  previous  paragraph  performed to  is  based on  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  -66  -45 -36  ant.{  •  -29  I  -25 -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  anti-CP Ia by this procedure removed the  Ia.  Repeated  anti-CP I activity  extraction  of the  (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  Antibodies synthesized  obtained  with  the  alkaline  phosphatase  conjugate.  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. reacting  with  the  The purified PSII  antiserum showed no depletion of the antibodies  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 C P II and C P 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  70  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  used to check for purity  not resolved when relatively large amounts were  (as  in this figure). Western blots of total thylakoid  protein incubated with preimmune serum showed no reaction (Fig. 10).  5. Antibodies to C P II and C P 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 might  be  due  to  anti-Chl  activity.  that some of the Even  if samples  cross-reaction observed  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  -66 -45 -36 -29 -25  29[ II [  -20 a  b  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  (Fig.  Unlike  13).  in  LHCII  CP II  purified by  which  is  the  method  purified  by  of  Ryrie  et al.  electrophoresis  of  [1980]  thylakoids  solubilized in detergent (see Materials and Methods for details), LHCII is prepared from  Triton-solubilized thylakoids  precipitation  [Burke et al.  1978,  by  sucrose-gradient  Ryrie  et al.  centrifugation  and  1980]. Barley LHCII  cation  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  purified  LHCn  (purified  (Fig.  CP 29)  apoprotein (Fig. 16f) 16e),  including the  contained the  and with all of the polypeptides in minor 25  kDa polypeptide.  same amount of protein as lane  LHCII), showing the relative strengths  Lane  16f  16e (purified  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). amounts of protein were used it cross-reacted with the CP n  When larger  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 -45 —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 and  [1980]  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. resolved  they  Both were  appeared to be legitimate always  present  in  CP 29  polypeptides,  approximately a  1:1  since  when  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— ^  20- •  ]29 •^  S 29 n i  a n t  Ia 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  that  these  polypeptides  approaches were  generally  therefore  below),  consumed  purified  and  one of the major limitations being most  of  submitted  the  sample.  separately  for  These  two  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.  extent, cleaving perhaps  10  All these chemical cleavages % of the  worked to  a limited  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  method was generated  improve  yield  experimentally  abandoned. Proteases  suitable sized fragments  consumed  such as  still  Staphylococcus  more  sample),  this  aureus V8 protease  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 barley.  A  comparison  of  purified by the method of Ryrie et al. [1980] from 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 B A R L E Y M U T A N T L A C K I N G C H L O R O P H Y L L 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  Previous  Chi b  studies  in  (see  regulating  the  Introduction)  accumulation  have  of  reported that  the this  Chi a + b mutant  proteins. 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  Triton-solubilized  referred  thylakoids  to by  here  as  ' LHCII'  sucrose-gradient  was  centrifugation  prepared and  from 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, raised  against  CP II reacted with both the  Machold 1981]. The antibodies  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  although there  the was  27  kDa range  a stainable  (CP H) polypeptide  reacted  anti-CP  II,  at  to  wild-type  barley  band corresponding to the  major LHCII with  compared  (Fig.  13),  lower part of the  band. When transferred to nitrocellulose and  least  two  polypeptides  were  detected,  one  of  approximately 26-27 kDa and one at about 25 kDa. (Fig. 16b and c). If smaller amounts LHCII  of thylakoid protein from the polypeptide  was  detected  (Fig.  mutant were used, 16d).  This  was  only the surprising,  25 kDa as  this  polypeptide was the least abundant of the three L H C n 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 C P 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  -45 -36  29 c  -29 —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  —45 -36 — 29  -25  4a  -20  b  c  d e  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, CP  29  apoprotein.  These  15 and 17d and e), verifying that this was the  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 (Fig.  the  CP  I  polypeptides  were  detected  only  at  higher  concentrations  18e).  The  cross-reaction of anti-CP Ia with LHCII and CP 29 polypeptides  seen in the blots  of normal and mutant thylakoids in Fig. 18b and e.  cross-reaction provided confirmation that the major LHCII polypeptides CP ft)  is  This  (those of  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  polypeptide found in LHCII but not CP II (e.g. Fig. 13) were still present.  kDa  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. C P 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 SDS.  Preparation  of  therefore done on 0.1  green  Chl protein  % (w/v)  deoxycholate  bands  from  in the presence of  Chl 6-less  barley  was  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.  THE  SYNTHESIS  OF  CHLOROPHYLL-BINDING  POLYPEPTIDES  DURING GREENING OF E T I O L A T E D 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. important  question for each  Chi protein is  whether  or not  its  An  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  belonging to  not  CP 29  used  antibodies  and the  to  study  PSI antenna  the  (LHCI).  synthesis It is  of polypeptides  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 barley  until it reached a value of 3 at  leaves  (Fig.  throughout the  21).  Leaf  height  12 h identical to that of mature  and weight did not  change  significantly  12 h following exposure to light, and SDS-extractable protein per  g leaf increased about 40 %. All quantitative immunoblotting measurements therefore  determined  using  equal  amounts  normalized by multiplying by the  of protein per  gel  were  lane,  and then  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 samples alpha,  abundances collected beta  of several  before  well-studied  and during the  and gamma  subunits  thylakoid proteins 12  were measured in  h illumination. This  of coupling factor,  the  33  included the  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, et  al.  1984,  Liveanu  et  al.  1986,  Selstam  Vierling and Alberte 1983, Ryrie and  Sandelius  remained roughly constant or increased slightly during the  1984].  Amounts  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  95  Chlorophyll a/b and Total Chlorophyll vs. Time 250  12  10  a  o  ^  _  A  Chi a/b  200  /Total Chi  s  g> H50  o _©  100 Q-  6^  CD , Z3,  u h50  •A *  4  6  Time [hours]  8  10  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-  Legend A alpho + beta x jjamma •  33 kDa £rotsin  B Dl  3  6  9  12  Time in Light [hours]  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  Antibodies to the Chl a+b antenna  CP  (LHCI)  were  proteins CP II (LHCII), CP 29 and the PSI  used  accumulation of these polypeptides.  to  determine  (Fig.  24).  None  of  these  the  kinetics  Of these three Chl a+b  synthesis of CP II and its polypeptides previously.  proteins  polypeptides  The apoprotein of CP II  in response were  showed  light-induced  proteins, only the  to light has been studied  detected its  of  typical  in  dark-grown barley  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 C P 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 Chlamydomonas Unfortunately, anti-keratin  antibodies (courtesy  this  activities, in the  bands  denatured  successfully [Morris  and  the  corresponding  of Dr. Nam-Hai  antibody  polypeptide in  to  and  was it  found  reacted  60-70 kDa range. protein  extracts.  PSII  core  protein  Chua) were therefore to  contain  most This  anti-coupling  strongly  with  antibody reacted  Surprisingly,  used  this  5  of  instead.  factor  and  an  unidentified  with  up to nine  antibody  has  been  used to identify the CP 47 gene from chloroplast DNA in spinach 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  accumulation anti-5  extracts  of CP 47  antibody)  showed  under  these  during  greening of etiolated  that  they  conditions.  Studies  on  the  kinetics  spinach (using  the  of  same  were similar to those of CP 43 [Reinhold  Herrmann pers. comm.].  5. Synthesis of the C P 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.  amounts in the  The  CP I  dark and showed  apoprotein  was  at most a two-fold  present  in  increase  substantial  after  12 h of  illumination (Figs. 22 and 26). These results are similar to those of Nechushtai and  Nelson  [1985] with  detected little  wheat,  but different  from  most  other  studies which  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  chamber covered with additional layers of dark night  as  described  in  Materials  and  grown  in  a  dark  material and watered  Methods.  Experiments  were  growth once at 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  etiolated seedlings (Fig. 26,  ±  4 % of the  12  h level  was  detected in the  ' 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  etiolated  liquid nitrogen evaporated. barley using this  method  Since CP I apoprotein was  (Fig. 27),  still seen in  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 age or growth conditions, or the methods  in the species used, its  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  c o E  0.6-  b  0.4-  D  c o o o  A-  Extreme Dark  0.2  Legend a GROWTH CHAMBER X METAL CAN  —|— 10  12  Time in Light [hours]  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  <  -66 -45 -36 -29 -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  proportion of CP I detected in the decreases sequentially  (Fig. 28b),  supernatant  (Fig. 28b)  at  0 h. The  15,800g supernatant is largest at 0 h and  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 for  the  if differences  discrepancy  in the sensitivity  between our results  of immunodetection might account  and those of Takabe  et al.  [1986],  another Western blot was done with the samples prepared by their methods. In this  experiment  development these  time  conditions  the  amount  of the CP I  of  sample  loaded  immunoblot in the apoprotein  was  not  on  gels was  enzyme detected  substrate in  the  halved  and  the  reduced. Under 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  «-  Pellets  0  12 2 4 4 8  - * «-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 C h l  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. light  regimes  such  [1980b] and most other intermittent light studies. Other 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,  et al. 1976,  Armond  1977, Mullet et al. 1980b]. These plants have both PSI and P S n  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  mutant which also lacks Chl fe.  equivalent  to  the  chlorina f2  barley  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  thylakoids  f2  thylakoids should be present  from  plants  grown  immunoblotting experiments  show  under  in roughly equivalent intermittent  light.  amounts in  The  following  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  Barley seedlings were above  for 6 days.  extracts  polypeptides  grown under intermittent white  Seedlings  were  harvested  and used  light as described  to prepare whole cell  by freezing in liquid nitrogen and extraction with 2 % SDS, 50 mM  dithiothreitol,  65  greening  etiolated  of  mM Tris-HCl barley.  pH 7.4 In  another  as  described  experiment,  for  the  seedlings  experiments were  used  on 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  Ill 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 clearly  indicated  that  this  band was  with blots of intermittent  light  thylakoids  the  CP 29  apoprotein  (Fig.  30a-d). The unidentified polypeptide of lower molecular weight also reacted  112  N IL N IL N IL  I  |! -66 r  u  ^ -45 -36  II^I-I antfc:  • -25 •  1  -20  Ia antibodies against  n  29  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  6645362925-  ••••  .13? r }ant.  20anti-29  anti-la  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. M A T H E M A T I C A L MODELING O F 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  polypeptides  due  to  chance  polypeptides  have  evolved  proteins) alone.  from  a  might  This  independently  differs  from  common ancestral  arise  cases  in  in  different  which  two  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 occur,  as  biological is  the  significance  to  current practice.  immunological cross-reactions Instead,  it  is  necessary  to  whenever determine  they 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  polypeptides  in  and the  White  1987].  presence  of  However,  reducing  it  is  agent  possible  and  SDS  to  heat-denature  (as  described  in  Materials and Methods) and essentially linearize them for gel electrophoresis and immunoblotting. probability  that  For situations two  such  as  these it  or more polypeptides  is  will  identical) subsequence of amino acids sufficiently  possible  share  an  to  determine  identical  (or  the  almost  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  for the combining site [Kabat 1978]. Antibody binding affinities  with antigen  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 anti-dextran (TM5),  a  antibody  dextran  about 2.5  exhibited  containing  nm long [Stryer  1981]. However,  maximum binding affinity five  glucoses  [Kabat  another  with isomaltopentaose  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  antibody combining site would correspond to approximately 2.5 nm/0.35  longest 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 would be about 1.9 nm or 5.4  glucoses it  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  dimensions of about 3 by 2 nm [Amit et al. 1986]. In deriving the  maximum equations  which follow, the length of the antibody combining site (in amino acids) has been i  left as  a variable. However, for sample calculations  combining sites recognize  sequences about  5  to  6  we amino  assume that antibody 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.)  proteins or nucleic acids, then p <  If the sequences  being compared are  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  simple  of the  result  was  longest match  [Arratia and Waterman 1985], although this  by no means intuitively  obvious  but required detailed  complicated proof. The general case in which mismatches  can intervene  and  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 calculations  results  performed  nonprotein-coding 1985].  of  These  Arratia on  sequences  extensive  a  et  al.  large  taken  have  number  from  calculations  the  required  been of  empirically vertebrate  GenBank 170  confirmed  protein-coding  database  min to  using  be  [Smith  and  et  performed by  al. 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]  arbitrarily-selected disjoint nucleic acid sequences of length 2 , 7  2  8  also ...2  1 2  hold  for  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 natural extension  of the  suggested by Dr. Michael Waterman,  and is a  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 another  antibodies  polypeptide  polyclonal cross-react  or  raised due  monoclonal  with  one  or  against  a  chance  alone?  2)  antibodies  raised  against  to  more  given  polypeptides  polypeptide  in  What  a  is  will  cross-react  the  probability that  a  given  pool  of  polypeptide  with  will  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  comparison of the sequences will reveal whether or not they subsequence  sufficiently  done  by visual inspection  either  homology  known,  a  direct  share a common  long to allow antibody binding. This comparison can be  matrix [Pustell  or by using a computer program to plot a  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 th and u and v are the relative frequencies of each i i i 1  1  amino acid in M  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  distribution function exp(-exp(-x)) (Arratia et al. [1986]).  variable  having  cumulative  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(p (l-p)mn)} k U  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  method of calculation does not  are  used  change.  antibodies had been used P{CR} = 2.2  x  m = u = 5.5,  but otherwise  the  For this example, if monoclonal -5 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  polypeptides is considered next.  against  a pool of  S independent  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 (P{no CR}) i=0 F  1  1  S P{at least one CR}=l-(P{no CR}) This  approach  is  ideally  suited  (3) 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  Anderson  1979,  suggested  that  complexes  Andersson et some  of  be  FeS  may  polypeptides of 8, Lagoutte  et  al.  [Siefermann-Harms and  the  16 and 1984].  al. lower  proteins  1982,  Ninneman  Noben  et  molecular  weight  associated  with  18 kDa [Nelson et al.  This  might  al.  account  for  1983].  Waldron It  polypeptides PSI,  1975, the  1979,  9,  in  has in  particular  and  been  CP Ia  Acker et al. 16.5  and  the 1982,  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  129  grana [Andersson and Anderson  130 1980,  Anderson et al.  1983,  Peters et al.  1983]  found in stromal vesicles  [Henry et al.  antenna therefore  PSI with a wider absorption cross-section [Anderson  et  al.  1983]  endows  both  by  increasing  1983,  while little or no LHCII is  antenna  Peters  size  and  et al.  1983]. The PSI  allowing  more  efficient  absorption around 470 and 650 nm.  The PSI antenna CP described in the Results contains four polypeptides between  21  and  correspond to the  24  kDa  when  polypeptides  run  in the  on  silver-stained  peripheral  gradient  gels.  These  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 Results  demonstrate  isolated  as  completion  a of  that  single, this  proteins, CP II and CP 29. The data presented in the all four PSI antenna polypeptides in barley can be  intact  Chi protein  work,  Kuang SDS  obtained  from  pea  using  contained  the  four  polypeptides  et al.  on  polyacrylamide  [1984] reported a  gels.  PSI  polyacrylamide gel  electrophoresis,  of  results  LHCI.  Similar  were  Following  antenna CP, which  also  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 similar  1984, to  Sarvari  et al.  one  described  the  1984,  Kuang et al.  in  the  Results  1984]. PSI antenna CPs have  been  isolated  by  re-electrophoresis of pea and bean CP Ia [Argyroudi-Akoyunoglou 1984], bean PSI particles Anderson Other  [Sarvari et al.  1984]  or  spinach  PSI  particles  [Lam et  1984]. They were reported to contain only one or two  workers  also  report  only  one  or  two  complexes  [Remy and Ambard-Bretteville 1984,  Thornber  1983]. Since some of these complexes  gels, it is possible  polypeptides Brandt et al.  in  al.  1984,  polypeptides. PSI-associated  1983,  Skrdla and  were not analyzed on gradient  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 has  been  no further report on the  [Brandt et al.  former, which was  presented  1983]. There 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  polypeptides  molecular  of  19-24  weight  kDa  range  reported  for  than pea  those  in  [Haworth  barley, et  al.  with  three  1983],  while  polypeptides of 23, 24, 25 and 25.8 kDa have been reported for spinach [Evans and all  Anderson 1986]. In contrast there are four barley PSI antenna polypeptides in the  resolve  21 or 22-24 kDa range,  than the  polypeptides  can  so that they  PSI antenna polypeptides only  be  resolved  on  are even more difficult to  in dicots. I have  certain gradient  gels  found that these 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 such  as  in diffusion),  and a sensitive method of detection  is employed,  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 precipitation  29  is  clearly  method  of  distinct Burke  from LHCII. et  al.  [1978]  LHCII does  prepared by not  contain  the  the  cation 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 [1978]. None of three anti-Chl a + b protein antisera detected CP in LHCII prepared Burke et CaCl  al.  29 polypeptides  [1980] or by the method of  [1978] even if very high divalent cation concentrations (200  and  2  by the method of Ryrie et al.  al.  10  mM  mM  MgCl ) were used in the cation precipitation step. Since 2  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  LHCII migration between granal and  stromal  include thylakoid stacking,  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. I m m u n o l o g i c a l 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  10), and in particular did not contain any  immunoblotting (e.g. Figs. 6  contaminating  and  polypeptides from other  Chi a + b protein complexes. Anti-CP Ia reacted strongly with both purified LHCII (Fig.  6d),  thylakoids activity  purified  CP  (Fig. 6b).  was  29  This  removed  from  (Fig. 6c)  and  cross-reaction was the  anti-CP  the  corresponding  still  Ia, and  observed  polypeptides  if the  antibodies to CP  anti-CP  in I  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 the the  against the PSI antenna  PSII  antenna  anti-CP  (LHCI) polypeptides. These cross-reactions with  polypeptides were  Ia  (Fig. 9),  not removed by the affinity  thereby  providing  further  purification of  evidence  that  these  showed  some  cross-reactions were not due to contamination of the injected antigen.  The  polyclonal  immunological  antisera  cross-reaction  Although  these  respective  antigens, they  protein  were  used  used  of 21-24 on  with  CP  II  each  cross-reactions were  routinely  polypeptides  to  and  other's weaker  were  readily  for  immunoblotting.  kDa  were  gels or dot blots  (e.g. Figs.  29  antigen than  detected  not observed  CP  with  both  (Figs. the  12,  13, and 16).  reactions  the amounts  Cross-reactions unless larger  with  their  of thylakoid  with  thylakoid  amounts of protein  7, 11, 13). The  objection could be  raised that the 21-24 kDa polypeptides which reacted with anti-CP 29 might be proteolytic  fragments  thylakoid  isolation  of CP did  29. However, the use of protease inhibitors during  not  eliminate  these  cross-reactions only occurred at the position  cross-reactions.  of the PSI antenna  also inconsistent with proteolysis. Digestion of CP V8  protease  proteolytic  or trypsin,  fragments  with  followed a  wide  by  of molecular  unlikely that the reaction in the 21-24 kDa  range  fact  with  anti-CP  weights.  that  polypeptides is  29 with Staphylococcus  immunoblotting  range  The  aureus  29, reveals  It is therefore  is due to proteolytic cleavage  of CP 29.  It has been recently reported that antibodies to spinach CP II and CP also  show  small amounts  and  Staehelin 1987],  of cross-reaction  as reported here  with  each  29  other's antigen [Dunahay  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  reaction of these antibodies to CP II with  risk  of contamination. Thus  anti-CP  29  is  the  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 with  not only cross-react with other LHCII the  polypeptides  of  LHCI.  Most  of  polypeptides, but also cross-react these  monoclonal  antibodies  categorized into one of six classes based on their binding specificities. these classes  recognized various combinations  remaining two classes recognized both LHCII  of LHCII  polypeptides  were  Four of while  the  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  antigens,  proteolytic  causing  the  fragments resulting  contaminated antibodies  to  the  LHCI  react  with  polypeptides LHCII.  used  as  They further  reported that three monoclonal antibodies to LHCI did not cross-react with LHCII. Williams and Ellis homology  between  [1986] therefore LHCI  and  conclude that there  LHCII  polypeptides,  but  is do  no major sequence 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 discussed  above.  is  clearly demonstrated  by  the  experiments  Some of their monoclonal antibodies  of Darr  et al.  reacted only with  LHCII  137 polypeptides, as they recognize sites found only on LHCII. A smaller number of these monoclonals recognize recognize  polypeptides  belonging  noncross-reacting monoclonals may of  sites shared between LHCII  to  to  LHCI  both  these  and LHCI, and thus  complexes.  described by Williams  The  three  and Ellis  [1986]  simply be specific for sites unique to LHCI polypeptides. This interpretation 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 Finally,  between species will not account for the differences  the  recent  sequencing  of two  LHCI  cDNAs  in their results.  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  polypeptide(s)  less  abundant than L H C n  from those of LHCII  but it is  difficult to  resolve  its  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 desired results than  the  are obtained, so that weaker reactions with polypeptides other  antigen  polypeptides  may  publishable.  These  ' nonspecific',  a and  appropriate  here.  of  may go  not  reactions  not  be  unnoticed,  vague  polypeptides  numbers  stopped when the researcher's  are  term. others,  are  or  may  Weak remain  frequently  Clearly  the  although  Such reactions  proteins  detected.  are  screened  antibodies  perhaps  with  unexplained  referred to  to be  with  reactions  the  expected  unidentified  and  thus  not  in scientific jargon as are  reacting  term  ' less  on occasion  polyclonal antisera,  due  with  some  specific' when  is  large  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  mathematical  to  a  given  cross-reaction  protein  but  not  model one of the  with  another  antiserum.  simplifying assumptions  In  was  the 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  antisera. The genetics determining the immune response  recognized  by  different  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 shows  two  et al.  1987].  segments,  The amino acid  comprising  sequence predicted from  approximately  50  % of  the  this  cDNA  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  produce  three  sequences clearly shows how Williams and Ellis [1986] could  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 to  results given in the last chapter of the results section can be used  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.  among  the  Although  there  various polypeptides  will  be  within a  a small proportion of unique sites 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  1987, Stayton et al. 1987] to LHCII proteins  can  be  demonstrated  to  be  sequences [Hoffmann et al.  sequences, the relatedness biologically  significant  by  among Chl a + b an  alternative  method. Arratia et al. [1986] give the expectation of the length of the longest expected match between two sequences as  \og(qmn) +  All  kloglogiqmn) +  klogiq/p) - log(k!) +  7log(e) -  1/2  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  mathematical amino  acid  acids,  since  it  is  not  predictions. The longest sequences  of  currently possible  to  model these in  observed matching sequence  the- tomato LHCI  gene  (coding  for  between  the  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 with  primary sequence  denatured  polypeptides,  homology indicated by immunological cross-reaction 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.  evolution  imposed  divergent by  evolution.  structural  or  It  could  functional  also  be  constraints.  due  might be binding of Chl b or certain carotenoids,  harvesting  and transfer  photosystems. account  for  the  internal Chl o  convergent  Examples  constraints  of light to  to  of  or the  antennae  such  efficient  of the  two  It is unlikely that a structural requirement for Chl c binding could this  sequence  homology,  as  the  Chl a  quantities of Chl a but are not related to the Chl a+b  proteins  bind  substantial  proteins.  The origin of the Chl 0 + 6 proteins from a common ancestral gene seems to  be  the  most  probable  unequivocally. First,  explanation,  all the Chl a+b  although  this  cannot  be  demonstrated  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  hypothesis.  gene  transfer  event  Alternatively, the  is  required to  Chl a+b  explain  the  proteins may always  divergent  evolution  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  necessary Chl  become  Chl a + b proteins.  to obtain Chl 0 + 6 proteins,  a + b proteins,  even  though  they  A eukaryotic gene organization is since  the prokaryote Prochloron  may  be  different  from  not  contains  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  polypeptides  the  protein. The light-harvesting  purple  photosynthetic  molecular masses than the  bacteria 21,  24  polypeptides  [Okamura et al. 1974,  polypeptides  of the  green  bacteriochlorophyll antenna Rhodospeudomonas  spp.  have  much  in  lower  and 28 kDa L, M and H reaction center Youvan et al. 1984]. Similarly, the antenna  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 alb ratio, the  capacity  to  proteins differ in some properties such as the Chl 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 function of a set  ' fine-tuning'  and specialization of  of similar proteins derived from a common ancestor,  simply to the possibility that they  have always been different  or due  and that these  particular properties are not subject to convergent evolution.  The location of the Chl a+b tightly separate  linked genes per cluster chromosomes  [Polans  protein genes within the nucleus as several  [Pichersky et al.  et al.  1985,  1985], but with clusters on  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  'fine-tuning'  species.  This  Mendelian  inheritance  therefore  constitutes  a  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. and tomato,  the  However, it should be kept in mind that pea  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  preserved and expressed,  chance.  This  beneficial  mutation  may  ultimately  and thus incorporated into the photosynthetic  be  apparatus  of the species.  In contrast the Chi a proteins are much more evolutionarily conserved and not  subject  mutations  to  the  in the  same mechanisms Chi a+b  proteins.  chloroplast DNA within a single  which propagate However,  there  beneficial are  or  deleterious  300-1000  copies of  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  immunological intuitive  mean  cross-reaction  guesses  cross-reacts  not  with  may a  be  second  that  all  are invalid, correct. protein  presently since  For not  there  example,  published are if  drawn from  on  some cases in which a  a  conclusions  monoclonal pool  of  intuitive guess that this is biologically significant agrees with the  antibody  proteins,  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  accurate  and of the monoclonal antibodies.  mathematical  polyclonal  antisera  procedures  are  predictions  are  used  routine  in  to  is  most  screen  Western  The ability to make reasonably important  a large  blotting  in  experiments  where  number of polypeptides.  where  antibodies  are  often  Such 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. equations  to  assumptions  modelling were  antibody  made.  These  1985,  Arratia  et al.  binding sites  on  assumptions  may  1986]. To apply these  proteins, increase  a  few or  simplifying  decrease  the  predicted probability of immunological cross-reaction. The degree to which these assumptions  apply  mathematical  has  not  adjustments  yet  been  experimentally  could perhaps be  becomes available. All of these assumptions  made  when  determined, the  and  some  empirical evidence  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 polypeptides.  difference,  should  provide  insight  into  the  nature  of  biological  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  frequently  than  binding sites  expected  due  to  in  these  chance  regions  may  arrangement  of  be the  shared more 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  localized at certain regions other  evidence  indicates  [Benjamin et al. 1984,  arise.  Some  in proteins  that  the  [e.g.  entire  Jemmerson  researchers  claim that  Atassi and Atassi  exposed  protein  1987]. However,  1986], whereas  surface  studies  such  Atassi and Atassi [1986] have used folded proteins as antigens.  epitopes are  is  antigenic  as  those of  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  Note that this effect is opposite  the probability of cross-reaction.  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 linearized  polypeptides,  such  as  the model is intended to apply only to  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  Glycosylations  amino may  acid,  not  be  moiety  may be sufficiently  apply  to  antibodies  to  which  will  change  the  dealt  with  in this  fashion  large to be an epitope. glycoproteins  when  they  value  of p  since  Thus the are  only  slightly.  the carbohydrate model does not  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].  nonglycosylated  These  proteins.  The  antibodies  could  model  applies  also  nevertheless to  be  antibodies  used  raised  with against  nonglycosylated proteins. Shared antibody sites are infrequent and the probability that such a site would also be glycosylated antibody binding, is exceedingly  by chance alone,  thus preventing  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,  antibody  for  antibody  would  population,  as  the  and  cross-reacting  these  cross-reacting  represent  at  would  site.  best  a  In  Thus to have  antibody  must  likely  small  of  will be specific  a  the  antisera  percentage  a reasonable  possess  reduce  polyclonal  the majority of antibodies  were raised against. cross-reacting  even  affinity the  the  of  cross-reacting total  antibody  for the antigen  chance of being detected,  relatively  site. Consequent^ even a conservative  high  affinity  substitution  the  in the  for  they the 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  predicting the probability of cross-reactions polyclonal antibodies.  towards  due to chance alone as detected by  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  conservative  amino  acid.  replacements  by  It  would  therefore  increasing the  be  value  more  of p,  accurate the  to  model  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  estimate.  A  nonessential,  of  interpretation  and  decrease  antibody  this  purpose  good  nonconserved  would be antibodies  for  polypeptide from  to random synthetic  the  accuracy  would  be  one  of  the  raised  resulting against  some obscure organism. Best polypeptides  a  of all  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  nonglycosylated  would  proteins  be is  possible  to  hindered to  see  if  any  the  binding  significant  of  antibodies  to  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  consecutive  nonglycosylated  polypeptides  which  share  exactly  5  or  6  identical  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  psbB, was  amino acids). The spinach CP 47 nucleotide incorrectly translated  in Morris  and Herrmann  sequence, known as [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  identical amino acids followed by one nonconservative  (five  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  polyclonal  has been isolated  antisera to these proteins  significant,  these  antisera  might  still  polyclonal antisera to these proteins  is  as well. Although the  presently provide  will  too  useful  small to insights.  likely increase  be  number of statistically  The number of  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  terminal hexapeptide  SATTKK  which is phosphorylated in LHCII  the amino  [Mullet 1983].  Not only does it contain the threonine pair phosphorylated in LHCII, but it also contains  a  consecutive  corresponding  to  the  pair two  of strongly lysines.  basic  The  amino acids,  existence  of  i.e.  two  arginines  phosphorylated  and  156 nonphosphorylated forms of CP I and CP 47 has been suggested as a reason for the  multiplicity of slightly  polyacylamide there  is  unlikely  as  gels yet  that  under  different totally  denaturing  no experimental  the  in charge  relative  to the  weight bands and  evidence to  small difference  groups would be significant  molecular  observed  on SDS  reducing conditions.  support this idea  However,  and it seems  or mass imparted by  phosphate  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 protein  the  kinase  bottom activities  to the were  top of the  leaf.  detected  etioplasts  in  The highest specific and  the  early  thylakoid stages of  light-induced development,  and were not due solely to phosphorylation of LHCII.  As  LHCII  in  Chlamydomonas,  stages, while  is  phosphorylated  a number of unidentified  polypeptides  predominantly  at  the  later  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. cross-reaction,  this  would be  If such an incubation eliminated immunological  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  sequence is conserved not only between psIAl  DPTTR  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 shows  that  immunoblotting this  mutant  of  Chl 6-less  contains  most  thylakoids of  the  isolated  from  Chl a/6-binding  chlorina  f2  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  prepared by cation precipitation. The two major polypeptides separated as two distinct green complexes binds Chi [Green and Camm  and  LHCII  of CP II can be  in spinach, suggesting that each one  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  reported by Ryrie [1983] using anti-LHCII and by others  was  based on Coomassie  blue staining [Anderson and Levine 1974, Thornber and Highkin 1974, Henriques and  Park  1978, The  1975,  Miller et al.  Burke et al. 1979, anti-LHCH  1976,  Machold et al.  1977,  Apel and Kloppstech  Simpson 1979, Machold 1981, Bellemare et al. 1982].  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 [Ryrie  1983]. It detected three of these polypeptides 1983].  (anti-CP II)  Thus the in this  Ryrie's antibodies  polypeptides  detected by  in the chlorina f2 mutant  antibodies  to  barley  CP II*  thesis, cannot be directly compared to those detected by  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  translated  in  vitro, taken  and  up  by  Kloppstech intact  1983].  mutant  or  These  mRNAs  can  be  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 thesis is  version of this idea that would explain the results presented in this that  the  stability  of  the  various  polypeptides  is  dependent  amount of Chl b which they bind. Thus CP 29 (Chl alb = antenna (Chl alb =  on the  3) and the PSI  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  interpretation formation  not  bind  would be  Chi a that  of labile complexes  the  in  the  absence  binding of Chi c  which dissociate  of  Chi  b.  An  alternative  occurs but results  upon the  addition of  in the  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  number  antenna  of PSI  sizes  inferred from  and PSII reaction centers  the  light  harvesting  per given  capacity and  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  intermittent  light  barley,  results  from  the  Chi fe-less barley mutant with the  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  polypeptides were not detected in either system. over in the  absence  in thylakoids.  Some  LHCII  These likely are rapidly turned  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, shown that LHCII [Apel  and LHCI  and Kloppstech 1978,  mRNAs  are  present  in  mRNAs  are present  Bellemare et al.  barley  grown  under  as  other studies have  in the  chlorina f2 mutant  1982], and at least the intermittent  light  LHCII  [Cuming  and  Bennett 1981, Viro and Kloppstech 1982]. (I do not know of any studies which have looked for the LHCI have  ever  looked for  transcripts in intermittent light plants.)  CP 29  mRNA.  Fortunately, the  CP 29  No studies  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 There  is  a lag of several  hours prior to substantial  synthesis of Chl b lags behind the  12 hours (Fig. 21).  Chl synthesis,  and the  synthesis of Chl a. The early stages of  163 SUMMARY TABLES FOR STUDIES ON T H E CHL 6-LESS BARLEY MUTANT CHLORINA F2 AND BARLEY GROWN UNDER INTERMITTENT LIGHT Complex:  CP 29 LHCII LHCI  Polypeptides present in chlorina f2  +  some +  Polypeptides present in intermittent light  +  some  Complex:  Requirements for synthesis and accumulation: Chlorophyll 6 Continuous light (more than 2 min every 2 hours)  CP 29 LHCII LHCI  no some no  no some 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 minor Chi a+b  show  that the polypeptides of the two  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 roughly constant throughout the first  apoproteins is  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 1979]. have  Many since  studies on the been  performed.  regulation of LHCII Cab  gene  to continuous light [Apel  synthesis in etiolated  transcripts  are  easily  plants  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  synthesis for all Chl a+b  a  common mechanism  of light-regulated  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 this  a lag in the synthesis of the two major LHCII polypeptides. In contrast to expectation  the  Chl a+b  apoproteins  maintain  throughout the light-induced greening (Fig. 21),  a  constant  despite the  stoichiometry  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  attributed to the barley,  control  presence  intermittent  light  among  the  three  Chl 6-less  or absence of specific barley  and etiolated  light-induced greening all contain carotenoids  was  grown  under  a  prosthetic  barley  in  cannot  be  groups. Chl 6-less  the  early  stages of  and Chl a but lack Chl 6. The  factor most likely responsible for these differences 6-less mutant  systems  day-night  is the light regime? The Chl  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  Cuming and Bennett  1981,  light  [Apel  Bellemare et al. 1982,  and Kloppstech  1978,  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 cannot  be  conversion  explained with  posttranslational kinds  of  in  the level.  terms  resulting These  light-regulated  light-regulated  of  the  Chi  light-induced  acting  differences  of  certain  a  protochlorophyllide  to Chi  stabilizing  at  could however  (post)translational  degradation  as  controls.  Chi .a/6-binding  be  factor  explained  One  might  dark-induction,  or  be even  specific in  proteases light-induced  which  polypeptides,  differ  synthesis  or  by  possibility  different polypeptides may be degraded under different light regimes. mechanism  differences  in  their  the other  is in  the which  A possible light-  degradation.  or  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 against  to  protein  CP 43  6 of  Chlamydomonas is  from higher plants.  not  CP 43  especially  exhibits  accumulation similar to that of the Chl a+b  sensitive  when  used  a pattern of light-induced  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  apoproteins cytoplasm.  and  are  CP  encoded  I by  Furthermore, as  are  chloroplast  nuclear plastid  genes genes  encoded  whereas  and  must  coding  for  be  the  Chl  imported  thylakoid  a+b  from  proteins,  the both  CP 43 and CP I are constitutively transcribed [Herrmann et al. 1985, Klein and Mullet et al. explanations  1986, which  Kreuz et al. would  1986]. However, there are still a number of  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.  light-induced factors  may  In  the  case  of  the  Chl a  be required for translation, or the  proteins,  light  polypeptides  or  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 considered  in as  the  dark  internal  under these  controls.  Thus  conditions, this  and  hypothesis  they would  therefore  can be  require that  the  170 posttranscriptional synthesis of CP I be extremely  sensitive  to  light, more so  mRNA  and  than that of the other Chl proteins.  Alternatively, dark-grown  plants  transcription  or  the  presence  suggests  translation  of  that of  both  light  the  CP I  is  CP  not  I  an  absolute  apoprotein.  apoprotein  requirement  Kreuz  et  al.  in for  [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 continuous  CP I in etiolated  light and then  plants  which have  transferred back into darkness  been  exposed  to  [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. PSI activity has been detected even earlier in  other systems, often hours before the detection of PSII activity [Bradbeer 1981, Herrmann  et  al.  1985].  However,  the  possibility  of  translational  and  posttranslational controls are not mutually exclusive, and the presence of CP I in  171  dark-grown plants cannot be used to distinguish a light effect on the translation from effects (only) at a posttranslational level.  rate of  V. REFERENCES 1.  Acker, S., Lagoutte, B., Picaud, A. and J . 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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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