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An analysis of the fucoxanthin-chlorophyll proteins and the genes encoding them in the unicellular marine.. Durnford, Dion Glenn 1995

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AN ANALYSIS OF THE FUCOXANTHIN-CHLOROPHYLLPROTEINSAND THE GENES ENCODING THEM IN THEUNICELLULAR MARINERAPHIDOPHYTE, Heterosigma carterae:CHARACTERIZATION AND EVOLUTIONbyDION GLENN DURNFORDB.Sc. (Biology) Daihousie University 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENTOFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF BOTANYWe accept this thesis as conformingto the required standardA..THE UNIVERSITY OF BRITISH COLUMBIAJune 1995©Dion Glenn Durnford,1995In presentingthis thesisin partial fulfilmentof therequirements foran advanceddegree atthe Universityof BritishColumbia,I agree thatthe Libraryshall makeitfreely availablefor referenceand study.I further agreethat permissionfor extensivecopying ofthis thesisbr scholarlypurposesmay be grantedby the headof mydepartmentor by hisor her representatives.It is understoodthat copyingorpublication ofthis thesis for financialgain shall notbe allowedwithout mywrittenpermission.(Signature)____________________________Departmentofr9NThe Universityof British ColumbiaVancouver, CanadaDate__________DE-G (2/88)ABSTRA CTThe light-harvesting complexes (LHC) of the unicellularmarine chromophyte,Heterosigma carterae, were fractionated by sucrose-densitygradient centrifugation, followingdigitonin solubilization, and by non-denaturing SDS-PAGE.The sucrose gradient allowed forthe isolation of a major light-harvesting complex fraction,containing approximately53% ofthe total chlorophyll, the majority of the chlorophyllc and a single polypeptide of 19.5kDa.Up to 12 different polypeptides immunologically related toboth the fucoxanthin-Chi a/ccomplexes (FCPs) and to the chlorophyll a + b-binding proteins(CABs) were detected inthylakoids and in the lower photosystem I(PS I) enriched fractions. Using a modificationofthe non-denaturing gel system of Allen and Staehelin(1991 Anal. Biochem. 194, 2 14-222)allowed the resolution of a number of large pigment-proteincomplexes which included severalPS I and PS II fractions along with a predominant LHC fraction,an improvement overpreviously published methods.A Fcp eDNA from Heterosigina carterae has been cloned andsequenced. It encodes a210 amino acid polypeptide that has similarity to otherFCPs and to the CABs of terrestrialplants and green algae. Comparison of the FCP sequenceto the recently determined 3-dimensional structure of the pea LHC II complex indicates thatmany of the key amino acidsthought to participate in the binding of chlorophyll andin the formation of complex-stabilizingionic interactions between hydrophobic regionsof the protein are well conserved. In addition,the Fcp genes are part of a large multigene family withgreater than 20 related members inHeterosigina. Phylogenetic analyses of the LHC protein sequencesshows that the FCPs form anatural group separate from the iPCPsof the dinoflagellates. Though there are obvioussimilarities between the FCPs and the CABs, the relationships arevery distant.Analyses of polypeptides in the red algae Aglaotharnnion neglectuinand Porphyridiumcruentum, in collaboration with Greg Wolfe and Beth Gantt, werethe first to demonstrate thatpolypeptides immunologically related to the CABsand the FCPs are present within theRhodophyceae. In addition, CAB/FCP-related LHCshave not been detected in a11cyanobacterium (Nostoc) and a prochiorophyte(Prochlorothrix). This suggests the CAB/FCPrelated LHCs arose only once after the establishmentof the chioroplast and provides someevidence that suggests chioroplasts evolved from asymbiotic cyanobacterium-like organismonly once (monphyletic).The organization of the antennae in Heterosigmacarterae is equally as complex as thatin the terrestrial plants. This is indicated by thedetection of at least 12 LHC-relatedpolypeptides and the presence of a large multigenefamily encoding the FCPs. In addition,theimmunological relatedness and the sequenceconservation of the FCPs with theCABs indicatesthat the structure of the LHCs has been conservedthroughout evolution and that thesedifferentantennae complexes share a common ancestor.111TABLE OF CONTENTSAbstractiiTable of contentsivList of figuresviiiList of tablesxList of abbreviationsxiAcknowledgmentsxiiDedicationxiiiCHAPTER 1 General introduction1.1 Heterosigma carterae: characteristics and taxonomy21.2 Photosynthesis--an overview41.3 Chioroplast characteristics and thylakoid ultrastructureof the major algal taxa. . .61.3.1 Terrestrial plants/green algae61.3.2 Red algae61.3.3 Glaucophytes71.3.4 Euglenophytes71.3.5 Dinoflagellates81.3.6 Heterokonts/Haptophytes81.3.7 Cryptophytes91.4 Main photosynthetic components of the thylakoid membrane131.4.1 Photosystem II141.4.2 Cytochrome b6fcomplex and plastocyanin161.4.3 Photosystem I171.5 Light-harvesting antennal systems191.5.1 Function of antennal complexes19iv1.5.2 Intrinsic light-harvesting antennae221.5.2.1 Bacterial LHCs221.5.2.2 Eukaryotic LHCs231.5.3 Extrinisic light-harvesting antennae331.6 Concepts in chloroplast evolution351.7 Methods in molecular phylogeny381.8 Scope of this thesis41CHAPTER 2 Characterization of the light-harvestingproteins from Heterosigmacarterae2.1 Introduction422.2 Materials and Methods432.21 Heterosigma cultures432.2.2 Heterosigma thylakoid fractionation432.2.3 Denaturing SDS-PAGE and western blotting442.2.4 Non-denaturing gel system462.2.5 Spectroscopy and fluorescence measurements462.3 Results472.3.1 Fractionation of digitonin-solubilized membranesby sucrose gradientcentrifugation472.3.2 Fractionation by non-denaturing PAGE542.4 Discussion59CHAPTER 3 An immunologicalcharacterization of LHC related-polypeptides in red algae3.1 Introduction643.2 Materials and Methods3.2.1 Aglaothamnion cultures65v3.2.2 Aglaotharnnion neglectuin thylakoid fractionation663.2.3 SDS-polyacrylarnide gel electrophoresis673.3 Results683.4 Discussion78CHAPTER 4 Characterization of the Fcp cDNAsof Heterosigma carterae4.1 Introduction864.2 Materials and Methods4.2.1 Tryptic fragment sequencing874.2.2 Heterosigma DNA and RNA isolation874.2.3 cDNA library construction and screening884.2.4 Rapid Amplification of cDNAEnds (RACE)904.2.5 Southern blots924.2.6 Northern blots934.3 Results4.3.1 Identification and characterization of the Fcp1 and Fcp2 cDNAs 934.3.2 Characterization of the Fcp gene family1024.3.3 Characterization of the FCP protein sequence1074.3.4 Analysis of the FCP transit sequence1184.4 Discussion4.4.1 Fcp cDNA structure and multigene families1204.4.2 Structural aspects of sequence comparisons1234.4.3 The transit sequence and protein import126CHAPTER 5 Phylogenetic analysis of the LHCs5.1 Introduction129vi5.2 Materials and Methods5.2.1 Protein alignment1305.2.2 Phylogenetic analysis1315.2.3 Terms and concepts1335.3 Results5.3.1 Assessment of phylogenetic signal1355.3.2 Phylogeny of the tomato chlorophyll a+ b-binding protein family1435.3.3 Phylogeny of the chlorophylla + b-binding protein family1455.3.4 Phylogeny of the chlorophyll a + c-bindingproteins1535.3.5 Evolution of the LHC family of proteins1555.4 Discussion5.4.1 CAB protein evolution1595.4.2 Evolutionary relationships amongst theCab gene family1625.4.3 Evolution of the Chl a + b and Chi a + cgene families 1645.4.4 Evaluation of species relationships basedon the LHC protein trees 1655.4.5 Light-harvesting protein evolution: pathwaysand evolutionary sources 169CHAPTER 6 Summary173REFERENCES176viiLIST OF FIGURESFigure 1.1 Heterosigma carterae photograph3Figure 1.2 Thylakoid membrane componentsand electron transfer5Figure 1.3 Structure of the main chiorophyllsand carotenoids in plants andalgae 21Figure 1.4 Schematic diagram of proposedendosymbioses leading to the chioroplasts36Figure 2.1 Schematic diagram of the sucrosegradient fractionion of Heterosigma48Figure 2.2 Spectral characteristics of the sucrosegradient fractions49Figure 2.3 SDS-PAGE separation of sucrosegradient fractions50Figure 2.4 Immunological analyses of the sucrosegradient fractions53Figure 2.5 Mildly denaturing SDS-PAGEseparation of Heterosigma thylakoids54Figure 2.6 Analysis of pigment-proteincomplexes separated by SDS-PAGE55Figure 2.7 Spectral analysis of pigment-proteincomplexes 1, lOa and 1157Figure 3.1 Agloothamnion sucrose gradientfractionation68Figure 3.2 Absorption spectrum of Aglaothamnionsucrose gradient fractions69Figure 3.3 Analyses of LHC-related polypeptidesin Aglaothainnion71Figure 3.4 Immunological detection of Dl in Aglaotharnnionfractions 72Figure 3.5 Immunological analysesof thylakoids from diverse organisms73Figure 3.6 Analyses of CAB and FCP-relatedpolypeptides in Porphyridium andNostoc....75Figure 3.7 Immunological analysis of LHC-relatedpolypeptides in Prochlorothrix77Figure 4.1 Rapid amplification of cDNA ends techniqueillustrated 91Figure 4.2 FCP tryptic fragmentsequences94Figure 4.3 Schematic representation of the Fcpl cDNA96Figure 4.4 Nucleotide sequence of the Fcp 1 cDNA97Figure 4.5 Alignment of nucleic acid sequences ofthe Fcp 1 and Fcp2 cDNAs99Figure 4.6 Northern blot of Heterosigma totalRNA100Figure 4.7 Alignment of the nucleic acid sequencesfrom the 3 end of the cDNA clones103viiiFigure 4.8Figure 4.9Figure 4.10Figure 4.11Figure 4.12Figure 4.13Figure 4.14Figure 4.15Figure 4.16Figure 5.1Figure 5.2Figure 5.3Figure 5.4AFigure 5.4BFigure 5.5Figure 5.6Figure 5.7AFigure 5.7BSouthern blots of Heterosigma genomicDNA probed with the Fcpl and Fcp2cDNAs104Southern blots of Heterosigma genomic DNAprobed with the Fcpl eDNA106Topological analysis of the Fcpl sequence109Amino acid alignment of the first and third putativeMSR of the FCP 110Amino acid alignment of the Chi a + c-bindingproteins 111Amino acid alignment of the Chi a + c andChi a + b-binding proteins 112Structural model of the pea LHCII116Structural model of Heterosigma FCP117Alignment of the known chromophyte transitsequences 119Amino acid alignment of sequences used in the phylogeneticanalysis 137Random tree length distributions for the data sets usedin the analyses 141Phylogenetic analyses of the tomato CABproteins144Distance matrix analysis of the LHCs from selectedChi a + b containingtaxa146Parsimony analysis of the LHCs from selected Chia + b containing taxa 147Phylogenetic analyses of the LHCs from terrestrialplants and green algae 150Phylogenetic analyses of the Chi a + ccontaining proteins 154Distance matrix analysis of the LHC I and LHC II proteinsfrom the CAB, FCPand iPCP containing taxa156Parsimony analysis of the LHC I and LHC II proteins from theCAB, FCP andiPCP containing taxa158ixLIST OF TABLESTable 1.1Table 1.2Table 1.3Table 2.1Table 3.1Table 4.1ATable 4. lBTable 5.1Table 5.2Characterization of the major algal taxa10Summary of the tomato CAB proteins24Characteristics of chromophyte LHCs31Summary of immunologic cross-reactivity of Heterosigmathylakoid proteins.. .53Some genes commonly used to infer phylogeneticrelationships amongstphotosynthetic organisms80Codon usage of the Heterosigma Fcp cDNA101Third codon position data101Amino acid characters used in phylogeneticanalysis 131Species used in the phylogenetic analyses and their taxonomicaffiliation 134xLIST OF ABBREVIATIONSAP Allophycocyaninbp base pairsCAB chlorophyll a + b-binding proteinChi chlorophyllFCP fucoxanthin-chiorophyll proteinkb kilobasekDa kiloDaltonLHC light-harvesting complexMSR membrane spanning regionPBS phycobilisomeiPCP intrinisic peridinin-chlorophyll-proteinsPCP soluble peridinin-chlorophyll proteinPC phycocyaninPE phycoerythrinPS I photosystem IPS II photosystem IISDS-PAGE sodium dodecyl sulfate-polyacrylamide gelelectrophoresisxiA CKNOWLEDGMENTSThere are a few people I wish to acknowledgefor help on various parts of thisproject. I thank Drs. Reudi Aebersold for tryptic fragmentsequencing, Art Grossman and AnnEastman for antibodies, Tom Cavalier-Smith for helpwith the phylogenetic analysisand forallowing me access to his computer, andCarl Douglas for providing access to hislaboratory andequipment. I wish to thank Dr. Ingo Damm for helpfulcomments, friendship, andfor somepretty humorous times. Dingren Shen provided endlessamounts of advice and hispresence inthe lab (along with his command of ‘Leisure Suit Larry’)always kept things interesting. I alsowish to thank Dr. Hema Bandaranayake who,without hesitation, helped out with a numberthings directly and indirectly pertaining to this study.I also extend my gratitude toDr.Laurence Baeza for assistance during her shortstay in the lab. I wish I could have done moretoassist her. Dr. Abdul Beihadri had helpful suggestionson PCR and was a very challengingchess partner. I thank those at the front linesof research who have provided adviceand help onmethods and techniques. In particular, those pastand present members of the Douglas labwhohave consistently provided cheerful advice on demand.I also thank Dr. Beverley Green fortaking the time to edit several versions of this dissertationalong with other manuscripts. Inaddition, I thank her for helpful comments, encouragementand financial support throughout theduration of this study.I express my appreciation to Dr. Frank (Franz) Shaughnessyfor many interestingdiscussions on statistics and ecology. In addition, I thankFrank, Christel Rasmussen and BevBenedict for being my field guides to the algae and theterrestrial plants (dry algae), though theycould use a few lessons in compass navigation.I particularly thank Bev Benedict who, in herown way, provided support and encouragement.xiiTo my parentsGordon and Sarah DurnfordxiiiCHAPTER]IntroductionThe goal of this study was primarily to characterize the light harvestingantennae from theRaphidophycean alga, Heterosigma carterae. This involvedcharacterization of the fucoxanthinchlorophyll protein (FCP) complexes, cloning of thegenes encoding them, and comparingthemto the chlorophyll a + b-binding (CAB) family of proteins.The eukaryotic algae are diverse andhave been studied very little in regards to theirphotosynthetic structure and function.At the startof this study, the antennae from oniy three main groupsof chromophytes had been examined inany detail: the brown algae, the diatoms and the dinoflagellates(discussed in section 1.5.2). Thestructural relatedness of the FCPs to the CABs had notbeen demonstrated until the first FCPgene from a diatom was sequenced (Grossmanet al. 1990).Heterosigina carterae was chosen for this study for severalreasons. First, the LHCs froma member of this class of algae had not been previouslyexamined. Given the diversity amongstthe chrornophytes (see section 1.3 and Table1.1), I thought it would be useful to examineadifferent representative from oneof the other major algal taxa in order to assess the evolutionaryrelationships amongst the light-harvesting antennae.Second, like other chromophytes, thepresence of four membranes surrounding the chioroplastin Heterosigma (see section1.3)indicated that nuclear encoded, chloroplast localizedproteins probably have a differentmechanism for the targeting and translocation of theseproteins into the chioroplast. Since achloroplast targeted-nuclear encoded gene had beendetermined from only a single chromophyte(the diatom FCP), I was interested in examining thetransit sequence of the Heterosigma FCPsequence to get an idea as to possible mechanismsof chloroplast targeting. Third, the chloroplastgenome from Heterosigma carterae (publishedunder the name of Olisthodiscus luteus)had been1well characterized and a few chioroplast genes had beensequenced by Rose-Anne Cattolico’s lab.In addition, easily maintainable, axenic cultures wereavailable.1.1 Heterosigma carterae: characteristics andtaxonomyHeterosigma carterae is a unicellular alga thatis approximately 11-20imlong, 9-12 Imwide, has anywhere from 9-25 yellowish-brown chioroplastsand possesses two flagella (see Fig.1.1; Hulburt 1965; Hara and Chihara 1987). The chioroplastshave four surrounding membranes;two chioroplast envelope membranes and two membranesof the chioroplast E.R. (CER). Theouter CER membrane is not continuous with the nuclearenvelope, as occurs withsomechromophytes (Gibbs 1981). The chioroplasts are alsodistributed along the periphery of thecell.H. carterae possesses Chi a + c along with abundant amounts offucoxanthin (74% of totalcarotenoid) followed by significant levels of diatoxanthin(14%) and13-carotene (12%) (Riley andWilson 1967). H. carterae was originally grouped withthe xanthophytes due to its yellowishcolour but ultrastructure analyses and other data indicatesthat it is a member of theRaphidophyceae (Hara et al. 1985). H. carterae is widely distributedin coastal marine habitats.It is an important component in red tides and has been implicatedin fish kills, causing the loss ofmillions of dollars within the aquaculture industry of British Columbia(Taylor 1993).There is confusion in the literature regarding the taxa referredto as Olisthodiscus luteusCarter, Olisthodiscus carterae Hulburt (Hulburt 1965) and Heterosigmaakashiwo Hada.Morphological and ultrastructural studies on Heterosigma akashiwoHada and on various culturecollections identified as Olisthodiscus luteus Carter indicated thatthey were all very similar andcould be combined under the genus, Heterosigma(Hara and Chihara 1987). However, thesecollections were different from the originally described0. luteus Carter culture (Hara et al. 1985)and appear to be the same as 0. carterae, as describedby Hulburt (1965). As this appears to bethe case, it has been suggested that the genus Heterosiginabe retained and the previous speciesname (carterae) be used (Taylor 1992). A numberof studies have been published on thechloroplast genome of H. carterae (e.g. Reith and Cattolico1986) and some physiological work2on this alga has been done. However,most of this work has beenpublished under the name 0.luteus due to a misidentification of the isolates in anumber of culturecollections (Taylor andHaigh 1993).Figure 1.1Photograph of Heterosigma carterae cells. In order to give a clearerview of the numberof chioroplasts, the prepared slide wasallowed to dry partially to cause the cells to flatten.31.2 Photosynthesis—an overviewCyanobacteria, algae and terrestrial plants utilizea chlorophyll a based system for thelight reactions of photosynthesis. In these organisms thereare two membrane integralphotochemical reaction centers: photosystem I (PS I) andphotosystem II (PS II). Thesecomplexes, along with the membrane soluble plastoquinones(PQH-PQH2),the cytochrome b6fcomplex and plastocyanin (Pc) are responsible for the non-cyclicelectron transfer. This electrontransport mechanism through the two photosystems is oftenreferred to as the Z scheme and isillustrated in Figure 1.2 (Hill and Bendel 1960). Thepurpose of the process is for the productionof the reducing agent NADPH and for the generationof an electrochemical gradient throughthenet transfer of protons from the stroma into the lumen.This gradient is then utilized for thegeneration of ATP via the thylakoid membrane boundATP synthase (ATPase) (Fig. 1.2).NADPH and ATP are used for the fixation of carbon dioxide viathe carbon reduction (Calvin)cycle, for the production of carbohydrate, and formany other cellular reactions. In addition,there is also a form of cyclic electron flow around PSI and the cytochrome bf complex mediatedby the ferredoxin NADP-reductase (FNR), as indicatedby the dashed line in Figure 1.24Figure 1.2Thylakoid protein complexes involved in photosynthetic electrontransport and ATPgeneration. The complexes include photosystem II,the cytochrome b6fcomplex, photosystem I,and the ATP synthase complex. Black filled areas represent the peripheralantennae complexesof PS I and PS II. Grey filled areas represent the coreantennae of the reaction center (PS II) orthe chlorophyll binding reaction centercomplex of PS I.Photosystem IILumenCytochrome b6 f Photosystem IATPase51.3 Chloroplast characteristics and thylakoid ultrastructureof the major algal taxaMany of the plastid and cytosolic features of the main algal groupsare summarized inTable 1.1. This was included in order to emphasize thewide diversity amongst the algae and toprovide the necessary background that will allow oneto assess the relationships between them.In this section I will briefly compare and contrast someof the key traits of the different algalgroups.1.3.1 Terrestrial plants/green algaeThe terrestrial plant and green algal chioroplast structure is wellknown. There are twomembranes of the chloroplast envelope surrounding a network of thylakoidmembranes that forma unit with a single connecting lumenal space. The two chioroplastenvelope membranes are notequivalent. The outer membrane is more permeable to low molecularmass substances and theinner chloroplast envelope membrane contains 5-10 timesmore intramembrane particles(Staehelin 1986). The thylakoids form both appressed (stacked, granalamellae) andnonappressed (unstacked, stroma lamellae) regions. The grana lamellaecan consist of a few ormany stacked thylakoids, depending on the conditions andthe species. The external surfaces ofnonappressed thylakoids are directly exposed to the stroma. The thylakoidcomponents arenonrandomly distributed between the appressed and nonappressedregions (discussed insection 1.4). The green algae and terrestrial plants areunique in that starch (amylose,amylopectin—an cd-4 glucan) is stored inside the chloroplasts.1.3.2 Red algaeThylakoids of the red algae, like those of the cyanobacteria, havea parallel arrangementof unstacked thylakoids. However, in some taxa the thylakoids areconcentrically arranged, forma close association with the inner envelope of the chioroplast,and/or have one or several girdlethylakoid bands surrounding the internal parallel thylakoids(Staehelin 1986; Mörschel and Rhiel1987; Pueschel 1990). All red algae have the main antennae,phycobilisomes, attached to the6outer surfaces of the thylakoid membrane. Accessorychiorophylls, equivalent toChi b or Chi c,are not present in the red algae. At one time, Chl d wasthought to be an accessory chlorophyllinthe red algae, though it had only been observedin Gigartina (Gigartenalies) extractsand had notbeen shown to exist in vivo (Holt 1966). If Chid is not an artifact of preparation, it isin a verylow concentration and will not make a significantcontribution to the action spectrum (Gantt1990). Red algae store various forms of starch (cd,4-glucan)outside the chioroplast,in thecytoplasm.1.3.3 GlaucophytesThe glaucophytes are a group of unicellular algaethat possess inclusions referred to ascyanelles, which are considered by some to be modifiedcyanobacteria functioningas plastids.However, the cyanelles have a reduced genome comparedto free-living cyanobacteria and aredependent upon the host. In this regard, they resembleplastids. Most, but not all, contain arudimentary cell wall made up of peptidoglycansurrounding the cyanelle (for a reviewsee Kiesand Kremer, 1990). This group possesses phycobilisomes,like the red algae, and the thylakoidsin the cyanelle are unstacked and usuallyconcentrically arranged. The photosynthatereserve,starch, is stored in the cytoplasm.1.3.4 EuglenophytesEuglenophytes comprise a large group with both photosynthetic(approximately 1/3) andnonphotosynthetic representatives. The photosynthetictaxa contain Chi’s a + b and haveappressed thylakoid membrane regions in bands ofthree to many, though the thylakoids neverform grana like those in terrestrial plants(Gibbs 1970). Euglenophytes have a chioroplastenvelope with three membranes. The outermost membrane of the chioroplast envelope does notbind ribosomes (Gibbs 1978). Interestingly,the main xanthophylls in this group of algae—diadinoxanthin and diatoxanthin—are more typicalof the chromophyte algae than the ChI a +bcontaining algae and terrestrial plants. Euglenophytesstore paramylon (a131-3 linked glucan) asa reserve in crystalline granules outside the chioroplast.71.3.5 DinoflageliatesThe dinoflagellates are considered chromophyte algae becauseof the presence of Chl c;however, there are also non-photosynthetic taxa. Theprimary xanthophyll, peridinin, gives theseorganisms the reddish-brown colour associated withred tides. Like the euglenophytes, someofthe dinoflagellates have a total of three membranes surroundingthe chioroplast, the outer(third)membrane lacking bound ribosomes on its cytoplasmicsurface. The thylakoids are usuallyarranged in three appressed bands, resembling thoseof the euglenophytes . Theydiffer from thethylakoids of other chromophytes by having a reduceddiameter of the appressedregions and bythe lack of a surrounding thylakoid band (girdle thylakoid)(Staehelin 1986). Dinoflagellatesstore an a.1-4 glucan outside the chioroplast.1.3.6 Heterokonts/HaptophytesThe brown algae, diatoms, chrysophytes, xanthophytes,other heterokonts, and thehaptophytes possess a total of four membranes surroundingthe chloroplast. The outer twomembranes are collectively referred to as the chloroplastER (CER) due to the presence ofribosomes bound to the cytoplasmic side of the outermostmembrane. The outer membrane ofthe CER tends to be continuous with the nuclear envelopewhen the number of chloroplasts arelow (1 or 2). Frequently, a girdle thylakoidof three appressed membranes surroundsthe internalthylakoid membranes (Staehelin 1986), though thisdoes not occur in the haptophytes(Gibbs1970). The internal thylakoid membranes usuallyform appressed regions of threebands. Theheterokonts and haptophytes typically store af3 1-3 glucan outside the chioroplast, within thecytoplasm.81.3.7 CryptophytesThe cryptophytes are unique in that within the spacebetween the CERand the chioroplastenvelope there is a putative vestigial nucleus(the nucleomorph). This is thought tobe a remnantfrom a permanent endosymbiosis of a heterotrophic organismwith a photosynthetic eukaryote(Ludwig and Gibbs 1987). The thylakoids are usuallyin pairs and appear thicker thanotherthylakoid membranes (Gibbs 1970). Interestingly, thecryptophytes contain thephycobiliproteins, phycoerytherin orphycocyanin, within the thylakoid lumen(Spear-Bernsteinand Miller 1989). Cryptophytes store starch(an al-4 glucan) in the areabetween the CER andchloroplast envelope.9Table 1.1 Characteristics of the major algal groupsPlastid CharacteristicsTaxon Chis and maincarotene2thylakoid girdle# chipPBP1 xanth.2iamellae lameliae membr’sCyanophyta a z (3 1x napc,peapc,pecEiiiorophyta a, b, c * z J3x naGlaucophyta3a, pc, ape z, c131x 2, pepRhEta z,n, xpc, pe, apcChiorophytaa)Chlorophyceae a, b n, z, 1,vCX,133+x2b)Prasinophyceae a, b n, z, 1,ai(X, (3 3+x2Mg_2,4DDinophyta a,c2 p, d3 13x 3(f) (2)Euglenophyta a, b d1,d2, n CX, (3 3+x 3iChiorarachnida4 a, b 1-3x 4. nmtCiptopht a,c2, (cj) a2, c]3T‘pe*, pc*nmtI)dLd3,n T3II) f, d, d2(x)Chryhyta a,cj,c — f, z, a13YT(c3) (d1,d2)T— 2(4)Bacillariophytaa,c],c2,c3 f,d1,d2 1F *___anthophyta a,c],cEli, d2, v2 13T,c2 f4(z, d1,d2)Eustigmatophyta a -— v,v2 13”Tx4Ttophyta,c],c2,c3 7, d, d23’’** *_10Table 1.1 continued.mito.char.flagellarfeaturesstorageproductsTaxon type of number! featurestype! alternate namescristae type locationCyanophyta na x x na cyanobacteriablue green algaeProchiorophytana x x naGlaucophyta F 2 mastig. ciJO GlaucophyceaeRhodophyta F x x a/0 red algaeChiorophyta a/Ia)ChlorophyceaeF 2 (4) (M) (hairs) cuTgreen algaeb)PrasinophyceaeF 2 (1,4, 8) scalesmicromonadsDinophyta T 2 hairs a/0 dinoflagellatesEuglenophyta D 2(4) hairs13/0 EuglenoidsChiorarachnida T 1 hairs a/B green amoebaCryptophyta F T 2 mastig. a/B cryptomonadsRaphidophyta T 2 mastig. ?/0 chlorornonadsH golden-brown algaeChrysophyta T 2 mastig. /0H golden-brown algaei11E 2 mastig. - J3i0 * often gioupedH scales with ChrysophytesBaci11aiophyta 1 * mastig J37E * dToms —iit T T mastig. flenTPhaeophE 2 — mastig. J37D brown algaeEustigmatophyta T 1 (2) mastig. 7/0(H)T——7?:5Ha11Key to abbreviations/explanations for Table 1.1heading descriptionplastidfeatures:Chl’s/PBP -Chlorophyll’s and phycobiliproteinspresent. a, Chi a; b, Chi b;c1..3, Chi cj3’;pc, phycocyanin; pe, phycoerytherin; apc, allophycocyanin;pec,phycoerythrocyaninmain xanth. prominant xanthophylls present inorder of relative abundance: a, anteroxanthin;a, alloxanthin;ci, Crocoxanthin; C2, f3-cryptoxanthin; d1, diadinoxanthin; d2,diatoxanthin; d3,dinoxanthin; f, fucoxanthin;1, lutein; p, peridinin;vi,violaxanthin; n, neoxanthin;v2, vaucheriaxanthin; z, zeaxanthincarotene-main carotenoid present: o,13,s-carotene;13,f3,13-carotene;y,E,e-carotenethylakoids - number of appressed or looselyappressed thylakoid membranesgirdle -presence(‘v)or absence (x) of a surrounding thylakoidmembranelamellae-number of membranes surrounding the chloroplast:pep, petidoglycan wallpresent-Type of stored carbohydrate: a, cij-4 glucan;13,131,-3glucan: Location of storedreserve; I, inside chioroplast; 0, outside chioroplast;B, between chloroplastenvelope and chioroplast periplasmic membraneOther symbols used and notesooccasional occurrence/reports*located in thylakoid lumentnucleomorph (nm) presentno ribosomes bound to outer membranefGlaucophyta contain a cyanelle andnot a “true” chloroplastna not applicable? unknownx absentJ presentReferences1(Jeffery 1989)2(Bjorland and Liaaen-Jensen 1989)(Kies and Kremer 1990)(Hibberd and Norris 1984)This table is a modification of tables found in (Sleigh1989) and (Lee 1989).# chlpmembrMitochondrial Features:type of -F, flat cristae; T, tubularcristae; D, discoidal cristaecristeaFlagellar Features:number/ type -M, many; A, anisokont (flagella ofunequal length); H, heterokont (organismwith a hairy and smooth flagellum); Ha, haptonemafeatures -hairs, fibrous flagellar hairs; mastig.,mastigonemes or tripartite, tubular hairs.Storage Products:type/location121.4 Main photosynthetic components of the thylakoidmembraneThe main complexes involved in the electron transport processare PS II, thecytochrome b6fcomplex (Cyt b6/), and PS I.An ATPase is also present within the thylakoidmembrane and it produces ATP using the proton gradientgenerated by the light reactions.Thesecomplexes are not equally distributed throughout the thylakoidmembrane of the Chia + bcontaining organisms. In many cases, the complexesare preferentially localizedto eitherappressed or nonappressed thylakoid regions, referredto as lateral heterogeneity. In terrestrialplants, PS TI-LHC II complexes are primarily locatedin the appressed regions ofthe thylakoid(PS“a),though some PS II complexes are found in theunstacked sections and havea smallercross sectional absorbance (PS II). PS I is primarilyfound in the nonappressed (stroma) regionsalong with the ATP synthase complex. The Cyt bfcomplex,on the other hand, is equallydistributed in either region (Staehelin 1986). LHC IIis found mainly in the appressed regionsofthe thylakoid, though it can be found in the nonappresseddomains in a phosphorylationdependent manner. This reversible associationof LHC II with PS II (appressed region) isbelieved to mediate the distribution of excitation energybetween the two photosystems and tobeimportant in the adaptation of the organism to varyinglight conditions (Allen 1992).In the chromophyte algae, lateral heterogeneity ofthe thylakoid membrane complexes,similar to that found in the terrestrial plants, does notoccur. In the brown algae (Lichtlé et al.1992b), diatoms (Pyszniak and Gibbs 1992) and cryptomonads(Lichtld et al. 1992) the Chi a + cbinding proteins were demonstrated to be homogeneously distributedthroughout the thylakoidmembrane, in both appressed and non-appressed regions,by immunolocalization. PS Icomplexes, however, were slightly enrichedin the nonappressed regions of the chromophytethylakoids but this distribution did not resemble thealmost exclusive localization of PS Tin thenonappressed regions of the terrestrial plant and greenalgal thylakoids. Moreover, the thylakoidmembranes of the chromophytes are typically associatedin groups of three loosely appressedthylakoids; grana stacks similar to those found in terrestrial plantsare not present. Thehomogeneous localization of PS I and the antennaein chromophytes is in agreement with astudy13in diatoms that shows energy absorbedby the main LHC complex is distributedequally to PS Iand PS II (Owens 1986b). A roleof light dependent phosphorylation inthe regulation ofexcitation energy between the complexeshas not been demonstrated. It isapparent that the modeof adaptation to light in the chromophyticalgae is different from that in theterrestrial plants. Abrief discussion of the characteristics of each majorcomplex in the thylakoidmembrane is givenbelow.1.4.] Photosystem IIThe electron transfer reactions are initiatedwhen an electron from a specialchlorophyll a(P680), which is probably a dimer,is excited to the singlet state by light. Thedonation of anelectron from the excited P680 to pheophytin (Ph)results in a charge separationwithin thereaction center that is the driving forcefor the oxidation of water.This occurs via a manganesecluster (Mn) and is enhanced by the presenceof an oxygen evolving complex(OEC). A series ofoxidation state changes in the manganese cluster,referred to as the water oxidationclock,eventually result in the oxidation ofH2O(Rutherford 1989). A redox activetyrosine residue onDl mediates the transfer of electrons fromthe Mn cluster toP680+.This process results in thereduction of P680 and the liberation of 4 protons(Hj and oxygen within the lumen.P680 isthen able to undergo another round of light inducedcharge separation in the reactioncenter.From pheophytin, the electron is donatedto the first bound plastoquinone,QA(on D2), then tothe second,QB(on Dl). An associated non-heme iron moleculemay influence the stability of thequinones.The PS II complex of terrestrial plants consists ofa reaction center and a core antennaesurrounded by more peripheral antennal complexes.The reaction center is a heterodimerconsisting of the Dl and D2 polypeptides, cytochromeb559 (Cytb559)and the psbl protein. BothDl and D2 are predicted to have five transmembrane helicies witha molecular mass of 32 and34 kDa, respectively. The Dl and D2 polypeptides are relatedto the M and L subunits of thepurple bacterial reaction center and, by analogy, arethought to associate in a similar fashion(Michel and Deisenhofer 1986; Trebst 1987). Sincethe determination of the 3-D structureof the14bacterial reaction center (Deisenhofer et al. 1985),it has been accepted that thecore polypeptides(Dl + D2) bind the primary reactants involved inthe initial light-induced charge separation,asdiscussed above. Evidence that Dl and D2form the photosynthetic reactioncenter was providedthrough the isolation of a photochemically activecore complex including these polypeptidesandtwo hydrophobic polypeptides (4 and 9 kDa)of Cyt b559 (Nanba and Satoh 1987). Thesubunitsof the Cyt b559 complex probably assemble ina heterodimeric fashion and cooperativelybind theheme molecule via a conserved His residuein each (Tae and Cramer 1994). Thefunction ofCyt b559 is not known at the present time andits presence is a major difference betweenPS II andthe bacterial reaction center. In addition,there are a number of other low molecularmasspolypeptides associated with the PS II core reactioncenter (Hansson and Wydrzynski1990).Also associated with the PS II complex are the core antennaeCP43 and CP47. Theseantennae bind only Chl a and have fluorescenceemission peaks at 685 and695 nm, respectively.Though not in general agreement, it has recentlybeen determined that CP43 binds20 Chl a and 513-carotenewhile CP47 binds 2 1-22 Chl a and 413-carotenemolecules (Alfonso et a!. 1994).These complexes are responsible for the couplingof the energy transfer from the peripheralantennae to the reaction center. In addition, lumenexposed regions of CP43 and CP47may beimportant for the water splitting processas determined through inactivation and mutagenesisstudies (see Vermaas et al., 1993).In terrestrial plants and green algae there are threemajor extrinisic polypeptidesassociated with PS II on the lumenal side of the thylakoidmembrane which influence theproperties of°2evolution. They have apparent molecular massesof 33 (OEC1, psbO), 23(OEC2, psbP) and 17 kDa (OEC3, psbQ). Thesepolypeptides are not directly involvedinoxygen evolution though they probably have a structuralor regulatory function. Incyanobacteria, OEC1 is not essential for02evolution though OEC1 deletion mutants are moresensitive to photoinhibition (Mayes et a!. 1991).In contrast, Chlamydomonas mutants with lowexpression of OEC1 lack02evolution capabilities and have low PS II stability(Mayfield et al.1987). Despite these differences, which may be relatedto the organization of the complexes inthe different organisms, it is generally agreed that OEC1may help to stabilize the Mn cluster15(Ghanotakis and Yocum 1990; Vermaas et al. 1993).OEC2 and OEC3, in combination withOEC1, may be involved in concentratingspecific ions near the catalytic site forenhanced wateroxidation properties. Interestingly, homologues ofthe OEC2 and OEC3 complexesare not foundin the cyanobacteria (Stewart et al. 1985). However,OEC1 is present and isimmunologicallyrelated to the OEC 1 of terrestrial plants.1.4.2 Cytochrome b6fcomplex and plastocyaninAs theQBsite on Dl becomes reduced twice(PQ—*PQH2), the plastoquinone diffusesfrom its binding site and becomes part of the plastoquinonepool’. An oxidized plastoquinonereplaces the reduced molecule in order to continuethe electron transfer process.The transfer ofthe electrons from PS II to PS I is mediatedby the thylakoid membrane intrinsic cytochromeb6fcomplex (Cyt b6f) and by the soluble plastocyanin (orcytochrome c553) protein.The Cyt b6fcomplex oxidizes plastoquinone and subsequently reducesplastocyanin in the non-cyclic electrontransfer pathway. It is also involved in theprocess of cyclic-electron flow aroundPS I. There isa net transfer of two protons from the chloroplaststroma to the thylakoid lumen for everyelectron donated to the Cyt b6fcomplex. The mechanismof plastoquinone oxidation is not fullyunderstood but is thought to occur via a ‘Q-cycle’ where oxidationof the plastoquinone occurs ina two step process (Malkin 1992). One electronis transferred to the Rieske iron-sulfur center —>cytochromef— plastocyanin pathway. The secondelectron is passed to the two b cytochromesin succession which, after two turns of thecycle, reduces a quinone. The net resultafter twocycles is the transfer of four protons intothe lumen, the transfer of two electrons to plastocyanin,the oxidation of two plastoquinones and the reductionof a single plastoquinone (Malkin 1992).The purpose of the electron transfer process is in thegeneration of a proton gradient that is usedin the formation of ATP via a thylakoid membraneATPase (Fig. 1.2).The Cyt bfcomplex is composed of four main polypeptides inboth spinach andcyanobacteria. These include cytochromef (34 kDa),the 23 kDa polypeptide of cytochrome b6,the 20 kDa polypeptide of the Rieske FeS-protein anda 17 kDa subunit (subunit IV) (Hauska1986). Cytochromef(CytJ) has a membrane spanningregion, anchoring the subunit to the16thylakoid membrane, and a lumen exposed portion that providesa heme binding site. It is a basicdomain on Cytf that is thought to interact with a acidic domainon plastocyanin to allow electrontransfer to occur between the two components (Cramer et a!.1994). As well, electron transfer toplastocyanin is thought to be mediated by a Tyr residue nearthe heme molecule of Cytf.Plastocyanin is a small copper-binding protein (10 kDa) thatis located in the thylakoid lumenand functions as an electron carrier between the Cyt b6fcomplexand PS I, where it reducesP700+.Plastocyanin has been found in all terrestrial plantsand is present in many algae.However, in some algae, cytochrome c553 can replace plastocyanin.1.4.3 Photosystern IA second light induced charge separation occurs at PS I and starts withthe photo-oxidation of P700, which is probably a chlorophyll a dimer(Golbeck and Bryant 1991). Theelectron released in this process follows a linear array of acceptors/donorsstarting with A0,which is a Chl a molecule. The electron is then donatedto A1 (vitamin K1) and toF(a 4Fe-4Scluster). The exact order of electron transfer fromFx to FA (the second 4Fe-4S cluster) or to FB(the third 4Fe-4S cluster) (reviewed by Golbeck and Bryant1991) has not been determinedconclusively. The presence of three 4Fe-4S clusters arrangedin a triangular fashion has beenconfirmed by the 3-D structure of PS I at a resolutionof 6A (Krauss et al. 1993). The terminalelectron acceptor is the soluble iron-sulfur protein, ferredoxin(Fd, Fig. 1.2). This protein ispresent in the stroma of the chioroplast and interacts withthe solubleFerredoxinNADP+Reductase (FNR) that reduces NADP to NADPH.The primary reactants P700, A0,A1 andF are located on the core complex of PS I(CP1), which is composed of a heterodimer with polypeptidesof approximately 83 (psaA) and82 kDa (psaB) (Golbeck and Bryant 1991; Krausset al. 1993). However, CP I usually migratesat 60-70 kDa on SDS-polyacrylamide gels, probablydue to the hydrophobic nature of thecomplex (Green 1988).The two iron-sulfur centers,FA and FB, are bound to PS I subunit VII (psaC) which isclosely associated with CP1 on the stromal side of themembrane. This 9 kDa protein is highly17conserved between cyanobacteria and terrestrial plants. There aremany other smaller, non-pigmented polypeptides, in the size range of 4-22 kDa(PsaD—N in plants), associated with PSI.The functions of many of these subunits have not been elucidated.However, the stroma locatedsubunits, PsaD (22 kDa) and PsaE (10 kDa), have been shown tobe closely associated with eachother and with PsaC by cross-linking and reconstitution studies(Golbeck 1992). They arethought to be important in the ‘docking’ of ferredoxin and possiblyfor the binding and orientationof the PsaC subunit. Furthermore, PsaE has been suggestedto be important for cyclic electronflow around PS Tin cyanobacteria (Yu et al. 1993). PsaF, locatedin the thylakoid lumen, hasbeen crosslinked to plastocyanin in plants and is thought tobe a plastocyanin docking protein(see Globeck 1992). However, inactivation of the psaF genein a cyanobacterium didn’t alter theability to grow photoautotrophically and is apparentlydispensable (Chitnis et al. 1991).The total molecular mass of PS I is estimated to be 340 kDa.It is thought to bind around100 chiorophylls. Almost half of the chlorophylls havebeen located around the transmembraneregions of the cyanobacterial PsaA and PsaB polypeptidesin the 3-D structure (Krauss et al.1993). Tn terrestrial plants, the PS I core and LHC Itogether bind approximately 200 chlorophyllmolecules.Overall, there is remarkable conservation of the corereaction center complexes and otherthylakoid complexes directly involved in the electron transfer process.In fact, all of the majorcomplexes discussed above are quite highly conservedamongst the plants, eukaryotic algaeandthe cyanobacteria. This is in contrast to a considerable amountof variation in the antennacomplexes which includes differences in size, chlorophylland carotenoid content, and in theirstructural organization around the reaction centers.1.5 Light-harvesting antenna systems1.5.1 Function of antenna complexesThe light-harvesting antennae, located on the peripheryof the reaction centers, increasethe absorptive cross-section of the photosystems therebyincreasing the probability of absorbing18the available solar radiation. The antennae areprotein complexes that specificallybind pigments(chiorophylls and carotenoids) in a manner thatdetermines the position andorientation of thechromophores; this allows for efficient captureand transfer of the excitation energy.The proteinenvironment greatly influences theabsorptive properties of the non-covalentlyboundchromophores.Plants and algae typically use solar radiation inthe visible range (350-700 nm) and areable to absorb these low energy photons due tothe conjugated bond system of the chlorophyllmolecule. The delocalization of electrons throughoutthis conjugated system lowersthe energydifference between the ground and excited state allowingfor the absorption of the photonsin thevisible range. Chlorophyll a is present in all oxygenicphotosynthetic organisms and isthe onlychlorophyll type within the core complex of eitherPS I or PS II. Though Chl a isassociated withall integral membrane (intrinisic) antennae,there is significant variation in the type ofaccessorychiorophylls and/or carotenoids that are alsobound to the complex.The two main accessory chiorophyllsin oxygenic organisms are chlorophyll b,(interrestrial plants, green algae, euglenophytesand the prochlorophytes—see Table 1.1)andchlorophyll c(cl-c3)(in the chromophytic algae). However, Chlc-like pigments have beenfound in the ancient green alga, Mantoniella and insome prochlorophytes (Table 1.1). Structuresof the different chiorophylls are shown in Figure 1.3.The accessory chlorophylls increase thelight capturing ability of the organism by broadeningthe absorption profile of the antennae.Chlorophylls absorb primarily in the blue (Soret band) and red(a-band) regions with lowabsorption in the green. Chl b, with respect to Chl a,is red shifted in the Soret band and blueshifted in the a-band, illustrating how the combinationof the two chlorophylls results in abroadened absorption spectrum. Chl c absorptionis similar to that of Chi b except absorption inthe a-band is lower (relative to the Soret band)and blue shifted.Carotenoids are important for both photoprotection and for lightharvesting. In terrestrialplants the photoprotective role is of primary importance as thecarotenoids do not make asignificant contribution to the absorption spectrum.Carotenoids act as photoprotectorsbyquenching chlorophyll triplet states, that can result inthe production of highly reactive singlet19oxygen, or they can quench singlet oxygenstates directly (Rau 1988). In addition, oxygenatedcarotenoids (xanthophylls) may also take part in the xanthophyllcycle which is involved indissipation of excess light energy.In the chromophytes carotenoids are important for light-harvesting,in addition to theirphotoprotective functions. In these cases, the carotenoidsare abundant and make significantcontributions to the absorption properties of the cells.Carotenoids that play a dominantrole inlight capture typically absorb in the 480-560nm range, significantly broadening the absorptioncapabilities in a region where chlorophylls have poorabsorption. The structures of someof themain carotenoids are shown in Figure 1.3 and will bediscussed in the sections that follow.Increasing the antennal size surroundingthe reaction centre necessitatesan efficientprocess of excitation energy transfer inward towardsthe reaction center. Whena chlorophyllabsorbs a photon, an electron in the chlorophyllis knocked from a ground state orbitalto a higherenergy, excited state orbital. It is possible thatthe excitation may not be localized to theorbitalsof a single pigment but may be delocalized over severalpigments (a delocalized exciton) whichmay contribute to the migration of excitation energy(Sauer 1986). Energy transfer from excitedchlorophyll complexes may also occur by inductivetransfer (Förster transfer) whichis effectiveover longer distances. In this mechanism,an excited donor pigment relaxes tothe ground stateafter transferring the excitation energy toa neighboring acceptor, which is then excited(Sauer1986). In either energy transfer process, a separationof a positive and negative charge betweendonor and acceptor pigments (electron transfer) doesnot occur. This only occurs atthe reactioncenter chlorophyll of PS I and PS II.The antennae are closely associated with the reactioncenter complexes in the thylakoidmembrane. The following discussion has separatedthe different LHCs into two main categoriesdepending on whether they are integral membrane complexes(intrinisic) or whether they areexternally bound to the thylakoid membrane (extrinisic).I will concentrate on the LHCs from theoxygen evolving organisms, both prokaryotic and eukaryotic.I will not cover the LHCs of theanoxygenic photosynthetic bacteria.20C IdHOOHaCH,OHOHH°°OCOC HhiHO<HOJOH- —-----------------HOCOMe HMe HFigure 1.3Structure of the main chlorophylls (a,b) and carotenoids (c-j) inthe algae. (a)ChiorophyllsCl + c2. Inc1 R is —C2H5;inc2 Ris —CH=CH2.(b) Chiorophylls a+ b. Inchlorophyll b the —CH3 on ring II is replaced by —CHO. Carotenoidsare as follows: (c)13-carotene, (d) lutein, (e) siphonaxanthin, (f) vaucheriaxanthin, (g) fucoxanthin, (h)diadinoxanthin, (i) peridinin, and(j)alloxanthin.bMe,211.5.2 Membrane-intrinsic light-harvesting antennae1.5.2.1 Prokaryotic light harvesting complexesThe prochiorophytes are oxygen evolving prokaryotes that lackphycobisilomes andcontain Chi a + b. This similarity to the terrestrial plant chloroplastslead to suggestions that theprochlorophytes were the ancestors of chioroplasts containingChis a + b (Lewin, 1975). Thefirst identified prochlorophyte, Prochloron was not free-livingand was found endosymbioticallyassociated with didemnid ascidians. The Chi a + b antenna fromProchloron is 34 kDa, has aChl a/b ratio of around 2.4, and can be phosphorylated (Schusteret al. 1984; Hiller and Larkum1985). Interestingly, Prochloron has recently been shownto contain significant amounts of aChl c-like pigment, in addition to Chl’s a and b (Larkumet al. 1994). The first discovered free-living prochlorophyte, Prochlorothrix hollandica,was a fresh water dwelling species.InProchlorothrix, there are several Chi a + b antennae containing proteinsin the 32 to 38 kDarange having a Chi a/b ratio of 4 (Bullerjahn et a!. 1987;van der Staay 1992; van der Staay andStaehelin 1994). The LHC polypeptides from the above two prochiorophytesareimmunologically related to each other (Bullerjahn et al. 1990) butneither shows relatedness tothe CAB proteins of terrestrial plants, determined by a lackof cross reactivity with CAB directedantibodies (Hiller and Larkum 1985; Bullerjahn et al. 1990).Interestingly, the Prochlorothrix LHC reacts with an antibody directedagainst a Chi abinding, iron-stress induced protein from cyanobacteria(isiA) (Bullerjahn et al. 1987).Preliminary protein sequence of the LHCs from Prochlorococcus(LaRoche and Partensky,unpubl.), Prochioron (Hiller and Larkum, unpubi.)and Prochlorothrix (van der Staay and Green,unpubi.) indicates that the prochiorophyte LHC genesare related to the isiA gene product and tothe Chi a core antenna, CP43. This confirms a lack of relatednessto LHC II of the terrestrialplants and green algae.221.5.2.2 Eukaryotic light harvesting complexes (LHC)The main intrinisic eukaryotic LHCs are a family of functionallyanalogous complexesthat are evolutionarily related; discussed more thoroughlyin Chapter 5. As the different pigmentcompositions of these complexes probably reflect significantevolutionary divergence,I willconsider each major complex separately. The main intrinisicLHCs are the Chlorophyll a + bbinding proteins (CABs), the fucoxanthin-chlorophyll proteins(FCPs), and the intrinisicperidinin-chlorophyll a + c-proteins (iPCPs)of dinoflagellates. As well, the antennaeof otherchromophytes containing abundant xanthophylls other thanfucoxanthin, will be reviewed.The chlorophyll a + b-binding proteins (CABs)The chlorophyll a + b-binding proteins are found in all terrestrialplants (vascular plants,ferns, mosses). They are also found in the Chiorophyta(Green algae) and in the photosyntheticmembers of the Euglenophyta. The green amoeba, Chlorarachnion,also contains Chl a + b(Hibberd and Norris 1984), but nothing is known about the LHCs ofthis organism. The bestcharacterized plants in terms of chlorophyll proteincomplexes are the angiosperms, particularlytomato, spinach and barley. Some work has alsobeen done on the green alga Chlarnydomonas.The chlorophyll protein complexes and the differentmildly-denaturing gel systems used toisolate them have been extensively reviewed (Green1988; Thornber et al. 1991; Jansson 1994).The literature has been confused with a number of differentlabeling systems though there hasbeen a recent revision of the gene nomenclature (Janssonet al. 1992). I will adhere to this systemwhen referring to the Cab genes and will use the systemof Green et al. (1991) when referring tothe protein complex. One exception is in the designationof CP29 type I and II which I will referto as CP26 and CP29, respectively (Bassi et al. 1990).This will be done due to the lack of closerelatedness between the two proteins and to avoid confusion.A table reviewing the CABnomenclature is shown in Table 1.2.23Table 1.2 Summary of the tomato CAB proteinsgene complex size amino functionChi a/b gene # ofChr.(kDa) acids location ratio copy# introns locationLhcbl LHCII 27 2658 0 3&2type ILhcb2 LHC II 26 265 major1.3 2 1 7&12type IIPS IILhcb3 LHC II 25 265 antenna3 2 12type IIILhcb4* CP2928 287 core? ? ?(CP29-I)PS II2.6Lhcb5 CP26 26 286 antennal.9 1 5?(CP29-II)Lhcb6 CP24 24 210 minorPS II 2 17antennapsbSCP2222 276 minor3-4 1 3(22 kDa) antenna?Lhcal LHC I 22 246 LHCI-7302 3 5type ILhca2LHC I23 270 LHC 1-6801 4 10type II2.2-3.5Lhca3 LHC I 25 273LHC 1-680 1 210type IIILhca4 LHC I 21 250-251 LHC1-730 2 2 3& 6type IVArabidopsis gene;§immature polypeptide; Chr.=chromosomeTable 1.1 is a modification of Green at al. (1991) andJansson (1994).The crystal structure of the pea LHC II complexhas been determined at a 3.4 Aresolution. It contains three transmembrane ci-heliciesand the complex is stabilizedby ionicbonds between buried residues within the first and thirdtransmembrane ct-helicies. The complexcontains 12 chlorophylls (tentatively identifiedas 7 Chl a and 5 Chl b) and two lutein moleculesat the center of the complex for photoprotection (Ktihlbrandtet al. 1994). The other xanthophyllsassociated with the complex were not localized thoughthere is expected to be a neoxanthin and aviolaxanthin molecule also associated with LHCII (Peter and Thornber 1991).LHC II is the most predominant antenna in the terrestrial plantsand green algae and formstrimers that are specifically associated with PS II (Kühlbrandtand Wang 1991). In terrestrialplants LHC II consists of three polypeptides with anunequal stoichiometry. Of these, the 28 kDaLHC II type I protein (Lhcb 1) is most abundant followedby the 27 kDa polypeptide (LHC IItype II; Lhcb2). These are encodedby a multigene family consisting of anywhere from3 to 16members for Lhcbl and 1 to 4 for members for Lhcb2,depending on the plant (Greenet al. 1991;24Jansson 1994). The 25 kDa LHC II type III complex (Lhcb3)is the next most abundant LHC IIcomponent. There are reports of different subcomplexesof the main LHC II complex with theLHC II type I and type II (Lhcbl and Lhcb2) proteinsforming a more peripheral, mobile LHCIIantenna (Larsson et al. 1987; Larsson et al. 1987b;Peter and Thornber 1991; Jansson1994). Thiswould be distal to a LHC II antenna complex withprimarily LHC II type I (Larsson et al.1987;Larsson et al. 1987b) and possibly LHC II typeIII polypeptides (Peter and Thornber 1991).Thissecond subcomplex would be more closely associatedwith the core complex of PS II.Presumably the excitation of PS TI is partiallycontrolled by the reversible detachmentof thedistal LHC II subcomplex (Larsson et al. 1987;Larsson et al. 1987b; Peter and Thornber1991).Changes in light intensity, redox state of the plastoquinonepool or temperature can result in thephosphorylation of LHC II (Allen 1992). This leadsto migration of the phosphorylated LHCIIfrom PS II in the appressed regions to PS Tin thenonappressed parts of the thylakoid membrane(Anderson and Andersson 1988).Plants grown under intermittent-light conditions (limitsChl b production) preferentiallyaccumulate LHC IT type III (Lhcb3) overthe other LHC TI polypeptides indicatingthe productionof these complexes is differentially regulated (Whiteand Green 1988; Mawson et al. 1994).Because of this, it has also been suggested thatLHC TI type ITT is more proximally locatedto thereaction center and may function as alinker for the bulk LHC TI antennae containingtypes I andIT (Harrison and Melis 1992; Mawsonet al. 1994). Tn agreement with this, regreeningexperiments with intermittent-light grownbarley have shown that elevated levelsof LHC IItype III accumulate early in the continuouslight phase, relative to type I and II (Dreyfuss andThornber 1994).The minor antennal complexes associated with PSII include CP29 (Lhcb4), CP26(Lhcb5), CP24 (Lhcb6) and the 22 kDa polypeptide (psbS).They account for 6% of the totalPS TI associated Chl (Peter and Thornber1991). CP29 (aka CP29 type IT) and CP26 (aka CP29type I) are often not recognized as distinct complexessince they comigrate in some gel systems.The Chl a/b ratio of CP29 and CP26are different and estimated to be 2.6 and 1.9, respectively(Bassi et al. 1993). As these complexes(CP29 and CP26) were not as readily dissociatedfrom25PS II with detergents as compared to LHC II, they were consideredmore tightly associated withthe reaction center (Barbato et al. 1989; Camm andGreen 1989; Peter and Thornber1991). Inaddition, CP29/CP26 remain present in the thylakoidsof a barley Chi b-deficient mutant thatotherwise fails to accumulate LHC II (White andGreen 1987b), indicating there is differentialstability of some of the LHC II complexes in theabsence of Chl b. Both CP29 andCP26 arerelated to LHC II and to each other (Picherskyet al. 1991; Morishige and Thornber1992), thoughthe relationship is distant (Chapter 5).The third minor chlorophyll-protein complex (CP24,Lhcb6) has a low Chi a/b ratio(1),a molecular mass of 24 kDa (Dunahay and Staehelin1986) and is quite divergent from eitherLHC I or LHC II polypeptides (Jansson 1994). CP24is not tightly associated with the PSIIcomplex and has been proposed to connect the LHCII complex to the PS II reactioncenter(Barbato et al. 1989). The final minor complex, CP22 (with a single 22 kDa apoprotein)isassociated with the core complex of PS II andhas recently been shown to bind chlorophyll (Funket al. 1994). Sequence of the gene encoding this protein(psbS) revealed limited similarity to theCABs and showed four putative membranespanning regions (Kim et al. 1992).Evidence for an antennal complex specifically associatedwith PS I (LHC I) was firstshown in pea PS I preparations (Mullet et al. 1980).Since then it has been isolatedand morethoroughly investigated in a number of terrestrial planttaxa (Haworth et al. 1983; Lamet al.1984; Lam et al. 1984b). The presence ofa Chl a + b containing PS I associatedLHC (CP 0) hasalso been demonstrated in the green alga Chlamydomonas(Ish-Shalom and Ohad 1983). Twomain subcomplexes of LHC I, termed LHCI 680 and LHC I 730, have been isolated and found tohave Chi a/b ratios in the range of 2.2-3.5 (Lamet al. 1984; Bassi and Simpson 1987). They areso named due to their 77 K fluorescence emission at680 and 730 nm, respectively. In barley,LHC I 680 has been shown to consist of two separate polypeptidesencoded by the Lhca2(LHC I type II) and Lhca3 (LHC I type III) genes which have apparentmolecular masses of 23and 25 kDa, respectively. LHC I 730 consistsof two polypeptides, with apparent molecularmasses of 22 and 21 kDa, and N-terminal sequencinghas demonstrated that these polypeptidescorrespond to LHC I type I (Lhcal) and LHCI type IV (Lhca4), respectively (Knoetzel etal.261992). The instability of PS I and the easy detachmentof LHC I 730 in a LHC I 680 depletedbarley mutant indicates that LHC I 680 is involved inbinding LHC I 730 to the reaction centercore (Knoetzel and Simpson 1991).The CAB proteins ofgreen algaeThe pigment-protein complexes from Chi a + bcontaining algae (other thanChlamydornonas) have been studied very little. TheLHC II sequences from Chlamydomonas,Dunaliella and the terrestrial plants are allhighly conserved; these evolutionary relationshipswillbe more thoroughly considered in Chapter 5. The organizationof the inner LHC II antennaeshould be quite similar between the green algae and theterrestrial plants as the same complexeshave been identified in each. However, thereare indications of novel regulatory mechanismsinthe green alga, Dunaliella, which involve modificationsin the Chi a/b ratio in changing lightintensities (Sukenik et al. 1987). Furtherwork may unveil significant differences inthe terrestrialplant-green algal CAB organization and regulation.Nonetheless, there are some greenalgal taxathat have a pigment composition significantly differentfrom those of Chlamydomonas andDunaliella; these will be discussed below.In the green alga, Codium sp. (Siphonales), the CAB proteinscontain the unusualcarotenoid siphonaxanthin (instead of lutein)which increases the absorbance in the 500-550 nmrange. The LHC II component has a lowerChl a/b ratio than the terrestrial plant LHC11(0.7)and contains four polypeptides from 27-35.5kDa (Anderson 1985). Two of these polypeptides(34 and 35.5 kDa) have sizes significantly different fromthe terrestrial plant CABs.Furthermore, a PS I specific antenna was isolated fromthis alga. It contained siphonaxanthin,had a Chl a/b ratio of 1.7 and a polypeptide compositionresembling the LHC I polypeptides ofterrestrial plants (19-25 kDa) (Chu and Anderson 1985).The Codium CAB proteins areimmunologically related to those of terrestrial plants,despite the pigment differences betweenthem (Anderson et al. 1987).The Prasinophyte algae (Micromonadophyceae) have uniqueLHC antennae that containChl a, Chl b, and a Chl c-like pigment (Mg 2,4-divinylpheoporphryin a monomethyl ester)27(Jeffery 1989). The carotenoid prasinoxanthin was alsoassociated with the antennacomplex.The LHC complexes from the prasinophyte, Mantoniellasquamata, containat least twopolypeptides of 20.5 and 22 kDa (Fawley et al. 1986b)and are arranged into larger oligomericcomplexes of 80 kDa (possibly trimers) (Rhielet al. 1993). The presenceof a Chl c-like pigmentand the smaller size suggested a closer relationshipto the Chl a + c-bindingproteins; however,sequencing of the a gene encoding this LHC has confirmedits relatedness (though distant)to theCABs (Rhiel and Mörschel 1993). There issome evidence that a unique PSI associated antennaemay not exist in Mantoniella and that the same proteincomplex excites both photosystems(Schmitt et al. 1993). The significance of this interms of regulation and distributionof thecomplexes remains to be seen.Euglena gracilis also has Chi a + b-binding antennaethat have sizes in the 26-28 kDarange (Cunningham and Schiff 1986). Thepredominant LHC has been reportedto have amolecular ratio of 12 Chl a: 6 Chl b: 4 diadinoxanthin:1 neoxanthin (Cunningham andSchiff1986b). Interestingly, the xanthophyll diadinoxanthinis more commonly found in thechromophytes rather than in Chl a + b-containing organisms.Nevertheless, sequencing of genesencoding LHC II and LHC I proteins from Euglenahas confirmed their relatednessto the CABs(Houlné and Schantz 1988; Muchhal andSchwartzbach 1992). In spite of the sequencesimilarities, the LHCs from Euglena are uniquelytranslated into large polyprotein precursorsfrom unusually large mRNAs.The fucoxanthin-chlorophyll proteins (FCPs)Fucoxanthin is an oxygenic, allenic-xanthophyll (Fig.1.3) that absorbs in the 450-550 nmrange. Its presence in some algae extendsthe absorption range of the LHC into the green regionof the spectrum. This would be particularlyuseful as the coastal ocean waters are usually limitedin the blue wavelengths of the spectrum andare more light limited than the terrestrial plants(Larkum and Barrett 1983). However, thereis no correlation between light quality (duetoattenuation of specific wavelengths of lightat different depths) and the nature of the lightharvesting system used by the algae growingthere (Saffo 1987).28Fucoxanthin-chiorophyll proteins occurin the diatoms, chrysophytes, phaeophytes,haptophytes, and some members of the raphidophytes(including Heterosigma). The exactmolarratio of the pigments associated with the FCPare not precisely known. However,analyses ofcomplexes isolated using milder detergents suggeststhat there are 13 Chi a: 3Chl c:10 fucoxanthin: 1 violaxanthin for a brown algaFCP (Katoh et al. 1989) and approximately12.5 Chl a: 5 Chl c: 24 fucoxanthin for a diatomFCP (Friedman and Alberte 1984).It is clearthat xanthophylls are much more abundant in thesechlorophyll-proteins than in thoseof theterrestrial plants. Usually, no attempt is made todistinguish between the differentChl c forms(Cl, c2, c3), though in one study a Chl c2/cl ratio of 3.1 was observed for a PhaeodactylumFCP(Owens and Wold 1986). Both fucoxanthin andChi c transfer excitation energyto Chi a,indicating their function in the harvesting of lightenergy (Duval et al. 1983).Typically, one or two polypeptides with a molecular massof 15-2 1 kDa have beenidentified in FCP-containing fractions (see Table1.3). Earlier studies reported the isolationof aChl a + c pigment-protein complex without fucoxanthin(Barrett and Anderson 1980; Alberte eta!. 1981; Peyriere et al. 1984; Owens and Wold 1986;Hsu and Lee 1987; Boczarand Prezelin1989). As the pigment content in these complexeswas variable and thepolypeptides had thesame apparent size as the FCPs, the nature ofthese complexes is uncertain. However,theChl a + c complexes were isolated using eitherthe detergent triton X-100 or SDSand there is thepossibility that they are the resultof pigment loss from the main FCP complex (Hilleret a!.1991). In this study, a specific Chi a +C-containing complex lacking fucoxanthinwas not found.Structural relatedness of the FCPs tothe CABs has been suggested on the basis ofimmunological cross-reactions of these polypeptidesto antibodies directed towards one of thetwo LHC types (Caron et al. 1988; Passaquetet a!. 1991; Plumley et al. 1993). In thisdissertation, antisera specific for the two groups of LHCswere used to investigate the relatednessof the Heterosigma FCPs to the CABs and other FCP complexes.The FCPs, like the CABs, arealso nuclear encoded, translated on cytoplasmicribosomes, and processed into the maturepolypeptide (Fawley and Grossman 1986).Their relatedness to the CABs was firstconfirmed bythe sequencing of a cDNA encoding theFCP from Phaeodactylum (Grossman etal. 1990). This29protein possessed four hydrophobicregions, three of which are present in themature protein andmay potentially form membrane spanning regions.Since then, cDNAs encodingFCPs fromanother diatom (Odontella-gb X81054) a brownalga (Apt et al. 1994) and ahaptophyte(LaRoche et al. 1994) have been sequenced, in additionto the Heterosigma Fcp cDNA.Other chromophyte antennaeOther taxa where xanthophylls, other than fucoxanthin,play a significant role in lightabsorption include the cryptophytes, the xanthophytesand the eustigmatophytes. Thecryptophytes possess a Chi a +c2containing antenna complex whichis abundant in thexanthophyll alloxanthin. The molar ratioof these pigments in an antenna fractionfromCryptomonas rufescens has been estimatedto be 10 Chi a: 2Chl C: 3.4 alloxanthin (Lichtléet al.1987). The polypeptides of these antennae arein the 18-24 kDa size range, similar to theFCPs,and Chi a/c ratios of 1.4 (Chroomonas), 1.7 (Cryptomonasmaculata) and 4.8 (Cryptomonasrufescens) have been recorded (Ingram and Hiller 1983;Lichtlé et al. 1987; Rhiel et al. 1987). Asmall peptide sequence fragment from a cryptomonadLHC (Sidler et al. 1988) showedsimilarities to the diatom and brown algal FCP sequences.A 23 kDa LHC from the xanthophyte Pleurochiorismeiringensis contains Chla, Chl cand three abundant xanthophylls: diadinoxanthin, vaucheriaxanthin,and heteroxanthin (Wilhelmet al. 1988). These pigments have been reported toexists in the following molar ratio:1000 Chl a, 224 Chl c, 148 heteroxanthin, 264 diadinoxanthin,and 129 vaucheriaxanthin. A PS Iassociated antenna with a fluorescence emission differentfrom the main LHC has also beenisolated from this alga (Büchel and Wilhelm 1993).In the eustigmatophytes there is a lack of Chi c while the xanthophyll,violaxanthin, isabundant and plays a significant light-harvesting role.The main LHC fraction from anothereustigmatophyte, Monodus subterraneus, was enriched ina 23 kDa polypeptide. The pigmentratios of this complex were estimated to be 10Chi a: 2.8 violaxanthin: 1.3 vaucheriaxanthin ester(Arsalane et al. 1992). The 23 kDa polypeptideis immunologically related to the main FCP froma brown alga indicating a structural relatednessto this group of LHCs (Arsalane et al. 1992).30PaviovaIsochrysisChiCl + C2fucoxanthinChi C45diadinoxanthinvaucheriaxanthinheteroxanthinEustigmatophyta Nannochioropsis violaxanthinvaucheriaxanthin2.7 17.5, 18.5 52.3+5.0 15 63.0 18, 19, 19.52.7 16.4, 16.91.8-3.2 15-202.8 16, 181.5-1.7 =18nd 16.5, 17, 18, 1220.521 1318,20,24 14noChiC 26 17,25DinophytaChroornonas Chi C2alloxanthinCryptomonas1(Barrett and Anderson 1980)2(Caron et al. 1988)3(Berkaloff et a!. 1990)4(Katoh et al. 1989)5(Friedrnan and Alberte 1984)6(Gugliemelli 1984)7(Fawley and Grossman 1986)8(Owens and Wold 1986)9(Caron and Brown 1987)10(Hsu and Lee 1987)11(Brown 1988)‘2(Fawley et al. 1987)13(Hiller et al. 1988)14(LaRoche et al. 1994)15(Wilhelm et al. 1988)16(Büchel and Wilhelm 1993)17(Livne et al. 1992)18(Arsalane et al. 1992)‘9(Ingram and Hiller 1983)20(Rhiel et al. 1987)21(Boczar and Prezelin 1987)22(Knoetzel and Rensing 1990)23(Hiller et al. 1993)24(Iglesias-Prieto et al. 1993)25(Chrystal and Larkum 1987)Table 1.3 Summary of characteristics from chromophyteLHCsgroup organism accessory pigmentChl a/C polypeptides Refsratio kDaPhaeophyta(brown algae)B acilliarophyta(Diatoms)HaptophytaChiCl + C2fucoxanthinChiCl + C2fucoxanthinACrocarpia4 taxaFUCUSLaminariaDictyotaPhaeodactylumCylindrotheca5 taxa2nd5.63.34.3nd20-2321205493347891011Xanthophyta PleuroChioris4.722-23 1516MondusVisheriaesterCryptophytaGonyaulax Chi C2peridininAmphidinium3 taxa23 1820? 181.4 20,24 191.7 18, 19, 22? 201-4 17-24 2119? 221.7 19 23nd 19-20 2431Preliminary peptide sequence information froma LHC from Nannochioropsis (eustigmatophyte)(Livne et al. 1992) indicates that these polypeptidesare related to the FCPs.The intrinisic peridinin-chiorophyll proteins (iPCPs)The dinoflagellate LHCs bind Chi a, Chic2 (not c)and the xanthophyll, peridinin.Peridinin is brick red in colour andhas aC37-skeleton (instead of C40 as withother xanthophylls)and an oxidized in-chain methyl group (see Fig.1 .3i). There are two main light-harvestingcomplexes in the dinoflagellates: an intrinisicperidinin-chiorophyll a + c complex(iPCP) and awater-soluble PCP (sPCP). The latter LHC willbe discussed in the next section. Thereis someevidence for the occurrence of a Chi a +c2 complex with a high proportion of Chlc2(Chl a/cratio 0.3) and devoid of peridinin (Boczaret al. 1980; Boczar and Prezelin 1987).However,the status of this complex is uncertain due to the useof SDS in its isolation, variationsin pigmentcontent and lack of its detection in other studies (Hilleret a!. 1991; Hiller et a!. 1993; IglesiasPrieto et al. 1993). The iPCP is the main light-harvestingantenna in the dinoflagellate,Symbiodinium, with a Chl a, Chl c, peridinin ratio of1:1:2 and comprising 45, 75 and70% ofthe total cellular levels of these pigments, respectively(Iglesias-Prieto eta!. 1993). InAmphidinium, the Chl a/c ratio is a little higher(1.7) though a similar ratio of Chi a/peridininoccurs (Hiller et al. 1993). In all studies, the polypeptidesfor the iPCPs are 19-20 kDa in size.Immunological cross-reactivity of FCP polypeptideswith iPCP directed antibodies andtheprotein sequence of the iPCP of Amphidinium demonstratessimilarity to the FCPs, suggestive ofa common evolutionary origin (Hiller et a!. 1993;Hiller, unpubl. data-gb Z47563). Theindividual iPCP polypeptides from Aniphidiniuinare encoded by a putative polyprotein (Hiller,unpubl. data). This would be similar to codingarrangement of the Euglena LHC proteins(Houlnd and Schantz 1988). Significantly, photosyntheticmembers of the euglenophytes and thedinoflage!lates have a total of three membranes surroundingthe chloroplast.321.5.3 The extrinisic light-harvesting antennaeThe extrinisic light-harvesting antennae are soluble complexes thatare easily detachedfrom the membrane. This type of complex includesthe soluble peridinin-chlorophyll proteincomplexes of the dinoflagellates, as well as the phycobilisomesof both the red algae andcyanobacteria.Soluble Peridinin- Chlorophyll Complex (sPCP)Soluble peridinin-chlorophyll a complexes werethe first photosynthetic proteinscharacterized from the dinoflagellates. Early studies indicatedthat the complexes wereextrinisically associated with the thylakoid membrane,bound significant amounts of thetotalperidinin and transferred excitation energy efficientlyfrom peridinin to Chl a (Prézelinand Haxo1976; Song et al. 1976). The sPCPs also had a molarratio of Chi a to peridinin of 1:4 andwerebound to a monomeric complex of 31-35 kDa or to anapparent 15 kDa homodimer, dependingon the species (Govind et al. 1990). Recent studies onthe sPCPs have estimated that they bind 5and 15% of the total cellular Chi a and peridinin, respectively(Iglesias-Prieto et al. 1993), thoughmuch higher estimates have been reported. This suggeststhat, though the sPCPs makesignificant contributions to the absorption of light, theiPCPs are the main antennal complexes.Recently the sequence of a gene encoding a sPCP fromSymbiodinium was determined. Itencoded a 35 kDa polypeptide with a internal duplication,suggesting it arose from the fusion ofgenes encoding the 15 kDa sPCP form (Norris andMiller 1994). No similarity to any other LHCwas found. Interestingly, the resemblance of part ofthe sPCP transit sequence to the transitsequence of a thylakoid lumen localized polypeptideof terrestrial plants has lead to thesuggestion that the sPCPs may be localized within thiscompartment (Norris and Miller 1994).PhycobilisoinesThe phycobilisomes are large antennal complexes consisting of manychromophorebinding proteins which are responsible for light absorptionin the 450-655 nm range, the33wavelengths where there is poor absorptionby chlorophyll. The primary phycobiliproteinsmaking up the phycobilisome are phycoerytherin(PE)(Amax560 nm), Phycocyanin (PC)(Amax620 nm) and allophycocyanin (AP)(Amax 650nm). Each phycobiliproteinhas two differentsubunits (o and13)with a molecular mass in the 17-22kDa range that form dimers. Thec and13polypeptides of each phycobiliprotein share a degreeof sequence similarity andare evolutionarilyhomologous (Zuber 1986). Each subunit binds1-4 open chain tetrapyrrole chromophores(phycobilins) that are attached by a thioetherbond to a cysteinyl residue on the protein.Thespectral characteristics of the phycobiliprotein arepartly influenced by the proteinenvironment ofthe chromophore (Glazer 1989), asis the case with other pigment-protein complexes.Each phycobiliprotein dimer(ct,13)is arranged into cyclic trimers whichform the buildingblocks of the phycobilisome. Twophycobiliprotein cyclic trimers ({a,13}3) areassembled intohexameric protein aggregates({a,j3}6)of PE and PC, which make up the rodlike structures ofthe phycobilisomes. These are boundto a usually triangular shaped core assemblageof AP. Thephycobiliprotein aggregates are assembled andheld together with colourless linker polypeptides(Mörschel and Rhiel 1987). The exact composition ofPBS rods depends upon the organism,growth and light conditions and has been previously reviewed (Mörscheland Rhiel 1987; Glazer1989; Gantt 1990; Grossman et al. 1993). Typically,the PBSs are organized in two ways:(1) ina hemi-discoidal fashion with a central coreof AP from which the PE and PC rods radiate, or (2)in a hemi-ellipsoidal arrangement which issimilar to the above organization except itis twice asthick (Gantt 1981). Both types of pycobilisomesare found in the cyanobacteria and red algae.The cryptomonads also contain phycobilins (phycoerythrinor phycocyanin) that arelocated within the thylakoid lumen (Spear-Bernsteinand Miller 1989). This phycobiliproteindoes not form phycobilisomes but doesspecifically transfer excitation energy to PS II (Lichtlé etal. 1980).341.6 Concepts in chioroplast evolutionUnder the assumption that the endosymbiotic originof organelles is generally accepted(Gray and Doolittle 1982; Taylor 1987), there are twomain hypotheses as to the origin of thedifferent chioroplast types described in section 1.3. The idea ofan endosymbiotic origin of thechioroplast was first proposed by Mereschowsky (1905) wherehe suggested that the differentplastid types were the result of endosymbioses of different cyanobacteria-likeorganisms withnonphotosynthetic phagotrophic protists. These ideas were revisedmore recently when it wassuggested that the diversity of the plastid types were the directresult of numerous endosymbioticevents with different prokaryotes already divergentin pigment biosynthesis, antennal systemsand biochemical pathways (Sagan 1967; Raven 1970; Whatleyand Whatley 1981); this isreferred to as the polyphyletic view of chloroplast evolution.An alternative hypothesis is thatthere was only one primary endosymbiotic event betweena cyanobacterium-like organism and aphagotrophic, nonphotosynthetic eukaryotic host. Subsequentdivergence of this organism leadto the different plastid types observed in the plants and algaetoday. This view is referred to asthe monophyletic origin of plastids (Cavalier-Smith1982; Taylor 1987)I am making a distinction between what I call the primary and secondaryendosymbiosesleading to the chioroplasts (see Fig. 1.4). The termprimary endosymbiosis refers to theestablishment of a chloroplast from a prokaryotic source. This generallyis thought to haveresulted in the generation of an alga with a double membranearound the chloroplast, namely thered and green algae (Fig. 4.1, top). Debate tendsto center around whether the chloroplast fromthese two organisms share a common ancestor(monophyletic) or have separate origins(polyphyletic). With the term secondary endosymbiosis, I amreferring to events involved in theestablishment of chioroplasts directly from photosynthetic,eukaryotic sources (Fig. 4.1, bottom).This is generally thought to have occurred in the algae wherethere are more than two membranessurrounding the chloroplast (chromophytes, cryptophytes, Chiorarachnion,euglenophytes andthe dinoflagellates), as reviewed in section 1.3 (see McFaddenand Gilson, 1995).35Primary endosymbiosis0PhotosyntheticprokaryotePhagotrophiceukaryote[0®1Photosyntheticeu karyote[0®1Photosyntheticeu karyotePhagotrophiceukaryotePhotosyntheticeukaryoteFigure 1.4Schematic diagram illustrating the proposed acquisitionof chioroplasts (c) througha primary or a secondary endosymbiosis. Thenuclei of the different organisms areindicated (ni or n2). The nucleus of the photosyntheticeukaryote in a secondaryendosymbiosis (ni) is thought to have givenrise to the nucleomorph (if maintained).Secondary endosymbiosis36Traditional classification of the different algal groupswas heavily basedon their pigmentcontent. This resulted in the separation of threemain taxonomic groups: the Rhodophyta(redalgae) with Chi a and phycobilisomes,the Chlorophyta (green algae) withChl’s a and b, and theChromophyta (coloured algae) with Chi a, c andsignificant amounts of xanthophyllssuch asfucoxanthin, vaucheriaxanthin and peridinin.The accessory pigments andthe proteins bindingthem have been assigned considerable weightin the speculations as to the numberof prokaryotesinvolved in primary endosymbiotic events. Thediscovery of a chlorophyll a+ b containingprokaryote (prochlorophyte) (Lewin 1975) withthylakoid stacking similar togreen algae andterrestrial plants was interpreted as evidence fora polyphyletic chloroplast origin. Presumably,the green algae would have acquired a chloroplast froma prochlorophyte (or ‘green’ prokaryote)(Raven 1970). Moreover, the red algae were thoughtto have acquired a chloroplast fromacyanobacterium based on the presence ofphycobilisomes in both these organisms.Along thesame line of reasoning, the chromophyteswere thought to have separately acquireda chloroplastfrom a ‘yellow’ prokaryote (Raven1970) with chlorophylls a and c (Whatley andWhatley 1981)or from the anaerobic photoheterotrophic eubacteriumHeliobacterium chiorum (MargulisandObar 1985), based on apparent pigment similarities.These comparisons were the foundationupon which the arguments for a polyphyletic view ofchloroplast evolution were built.Though it is generally accepted that thered algal chloroplast evolved from acyanobacterium-like organism, the evolutionof the green algal and chromophyte chloroplastfrom Chl a + b and Chl a + c-containing prokaryotes,respectively, is more controversial. Theproposed evolution of the chromophyte plastidfrom Heliobacteriuin was not widely accepted andhas subsequently been disproved by SSU rRNA analysis(Witt and Stackebrandt 1988). Theevolution of the green algal chloroplast froma prochlorophyte is still debated and will bediscussed in Chapter 3.Gene organization and phylogenetic analysesof chloroplast encoded gene sequencesprovided some evidence for a close relationship betweenthe red and chromophyte chloroplasts.Chloroplast gene organization and gene localizationof the ATPase subunits (Kowallik1993),Rubisco large and small subunit (rbcL/S)operon (Douglas and Durnford 1989), psbA(Dl)37sequences (Winhauer et al. 1991) and other gene clusters(Reith and Munholland 1993;Douglas 1994b) have been primarily used for examiningthese evolutionary relationships. Inaddition, phylogenetic analysis of the psbA, rbcLIS , tufA (elongationfactor Tu), (Morden et al.1992), and plastid 16s rRNA (Douglas and Turner 1991b) sequencesalso supports therelationship between the red and chromophyte plastids. Thiswould indicate that thechromophyte plastid may have evolved from an associationof a red algal-like ancestor with aphagotropic eukaryotic host, leading to the chromophyte lineage.The only clear evidence suggesting that a secondary endosymbiosis occurredwas withrecent work on Cryptomonas (Douglas et al.1991) and Chiorarachnion (McFadden et a!. 1994;McFadden and Gilson 1995). Like the chromophytes, bothof these organisms have fourmembranes around the chioroplast. However, the cryptophytes andchiorarachniophytes have amembrane bound, nucleic acid containing organelle (called a nucleomorph)located in the spacebetween the outer two CER membranes and the inner two membranesof the chioroplast envelope(the periplastidal space) (Greenwood et al. 1977; Gillott and Gibbs 1980; Hibberdand Norris1984). It is generally thought that this organelle is the vestigial nucleusof the eukaryoticendosymbiont (Ludwig and Gibbs 1987). Inthe cryptomonads, the presence of phycobiliproteins(phycoerytherin or phycocyanin) and the storage of starch in the periplastidalspace (the formercytoplasm of the putative endosymbiont) lead to the suggestion thatthe endosymbiont was a redalgal-like ancestor (Gillott and Gibbs 1980).This was supported by an analysis of the nuclearSSU rRNA sequence from the nucleomorph and the nucleus of Cryptomonas .Phylogeneticanalysis showed a loose affiliation of the Cryptomonas nucleomorph sequencewith the red algaewhile the cryptomonad nuclear sequence was more distant in the tree (Douglaset al. 1991). Thenucleomorph rRNA transcripts were also localized in the nucleomorphand the periplastidal space(McFadden et al. 1994a). In a similar strategy, the nucleomorph rRNA sequencefromChiorarachnion was localized in the periplastidal space and was shownto be distinct from thenuclear rRNA sequence (McFadden et al. 1994b). The endosymbiont leadingto the chioroplastof Chiorarachnion has been hypothesized to be a green alga (Hibberd and Norris1984);however, phylogenetic studies were inconclusive (McFadden et al.1994b).381.7 Methods used in molecular phylogenyThere are only a few reports of phylogenetic analysesof the Cab genes. Demmin et al.(1989) examined the relationships of the LHC II Cabgene family within the angiosperms. Fromtheir maximum likelihood analysis of Lhcbl (LHC II typeI) and Lhcb2 (LHC II type II)sequences, they found that the angiosperm taxa grouped withintheir traditional taxonomicfamilies. Their trees suggested that the Lhcbl andLhcb2 divergence occurred prior to themonocot/dicot separation. Matsuoka (1990) suggested the Lhcbl/2divergence occurred prior tothe angiosperm and gymnosperm separation. Jansson(1994) also examined the evolutionaryrelatedness of the CAB proteins in a recent review andfound there was a close association ofCP29 (Lhcb4) and LHC I type I (Lhcal). These treesalso showed a separation between LHC I(Lhca) and LHC II (Lhcb). Within the LHC II assemblage,the green algal sequences formed aseparate branch from the Lhcbl-3 (LHC II types I-ITT) genes ofthe terrestrial plants.Unfortunately, little information was given on the alignment, charactersutilized or specificsabout the method used in the analysis. As no indicationof reliability was given, it is not possibleto judge which relationships may be significant. Recently, ananalysis of a FCP from thehaptophyte, Jsochrysis galbana, suggested it was more relatedto a tomato LHC I sequence thanto a LHC IT sequence; therefore, the FCPs were suggestedto have been derived from a LHC I-like sequence, before the appearance of the predominant LHC II antennae(LaRoche et al. 1994).One goal of this study is to examine the evolution of the Cab andFcp gene families andrelate these gene relationships back to the function of the protein complex (Chapter5). As well, Ihope to get a better idea of the Cab and Fcp gene relationships in order to determinewhen theymay have separated in relation to the functional separation of LHCI and LHC IT. Two methodswere used for the determination of phylogenetic relationships amongstthe CABs and FCPs:maximum parsimony and distance matrix. Both methods are availablein the PHYLIP computerpackage (Felsenstein 1992). Maximum parsimony is a character method basedon the principlethat the evolutionary pathway requiring the fewest“steps” is the most likely. Parsimony attempts39to generate a tree which can be explained with the smallest number of mutational events (shortestlength). The characters used in parsimony can be of a molecular or morphological nature. In thisstudy, a character is a specific amino acid (e.g. Ala, Ser, etc.) at a particular position (e.g. the49thposition of an alignment). Parsimony evaluates each character site separately on all possibleunrooted trees and records the number of changes required to explain the character distribution.The optimal tree is selected by adding the number of changes over each character site, for allpossible trees, and choosing the one with the fewest steps (see reviews by Swofford and Olsen,1991; Hillis et al., 1993 and Stewart, 1993). With large datasets, not all possible treecombinations can be tested as the number of possible trees increases exponentially with anincrease in the number of taxa. For example, with 13 taxa or more there will be over 13 billionpossible trees, making the calculations impractical (Hillis et al. 1993).To overcome this, PHYLIP uses a heuristic algorithm which starts with the first two (orthree) taxa and creates a tree. The remaining taxa are then added individually in a stepwisefashion to all possible positions on the tree. Each of the trees is evaluated at each step and theshortest tree is kept. This process continues until all the taxa have been added to the tree. Inorder to improve the chances of finding the most optimal tree, the branches undergo a series ofglobal rearrangements whereby groups (subtrees) are removed and added back to the tree in allpossible positions. After each addition, the length of the tree is again assessed and the shorterone is retained. This process continues until no further improvements in the tree topology havebeen recorded (Felsenstein 1992). The evaluation of the trees is based on the chosen optimalitycriteria. This refers to the method by which the evolutionary change of the characters is assessedor weighted (Swofford and Olsen 1990). In this study, an amino acid change is weightedaccording to the number of mutations required to explain the substitution, which is based on thegenetic code. This point is explained further in the methods section of Chapter 5.Distance methods are based on pairwise comparisons between taxa, the end result being asingle value which reflects the dissimilarity (or distance) between the two sequences. Distancesare calculated between all possible pairs of taxa and referred to as the distance matrix. With thedistance methods, characters are not considered individually between all taxa as they are in the40character based methods (parsimony). No ancestralstate is implied or required as no distinctionis made between derived or ancestral character states (Sober1988). The distance matrixcalculation can be based on three modes of amino acid substitutionin the PROTDIST program,which is included in the PHYLIP package (Felsenstein1992). In this study, distances werecalculated using the PAM (accepted point mutation)100 matrix of Dayhoff (1978) which weightsamino acid changes on the basis of their probable occurrence.This is an empirically determinedmatrix calculated through the comparison of cytochromec sequences from different species.This distance matrix is then used to infer a treeby any of several methods. This study routinelyused the Neighbor-Joining method (Saitou and Nei1987) for tree construction as described inChapter 5.1.8 Scope of this thesisThis dissertation is primarily concerned with the characterizationof the FCP complexesand the genes that encode them. I am also interested inusing this information in an analysis ofevolutionary relatedness between the different light harvesting antennae.The FCPs areseparated and immunologically analyzed for structural relatednessto both the CABs and theFCPs in Chapter 2. The immunological analysis ofthe LHCs in the red alga Aglaothamnionneglectum is presented in Chapter3. In addition, work done on the red alga, Porphyridiumcruentum, in collaboration with Beth Gantt and GregWolfe at the University of Maryland, ispresented in this Chapter. Characterization of the Heterosigma Fcpsequence and an analysis ofthe size and complexity of the nuclear encoded multigene familyis presented in Chapter 4.Finally, Chapter 5 involves an analysis of the evolutionary relationshipsamongst the CABs andFCPs which combines many of the ideas from the previous chapters.41CHAPTER 2Characterization of the light-harvesting proteins from Heterosigma carterae2.1 IntroductionAs mentioned in the general introduction, there have not been anyinvestigations into thechlorophyll-protein complexes of any raphidophycean alga. Evenamongst the otherchromophyte algae, there have been relatively few studies characterizingthe fucoxanthinchlorophyll proteins as compared to the CABsof terrestrial plants. As I am interested in thesimilarities and differences amongst the diverse antennalsystems of the algae, I decided to workon a representative of the raphidophytes, Heterosigina carterae.In the initial part of this study I isolated and characterized the different chlorophyll-proteins contributing to the antennae of this organism. This study usedtwo different methods forthe fractionation of the thylakoid membrane and subsequentcharacterization of its components,particularly the FCPs. One method was the fractionation of digitonin solubilizedthylakoids on asucrose gradient, a method that has been used on chromophyte algae with successin the past(Hiller et al. 1991). The other method involved fractionation ofsolubilized thylakoids by apartially denaturing SDS-polyacrylamide gel electrophoresis (PAGE).This method has beeninvaluable in the characterization of the terrestrial plant CABs but has,so far, had limitedapplications for the non-green algal LHCs as the complexes aremuch more unstable under theseconditions.422.2 Materials and Methods2.2.] Heterosigma culturesAn axenic culture of Heterosigina carterae was maintainedin an artificial sea watermixture as previously described (Cattolico et al. 1976). Themedia was preparedby adding saltsto distilled water at the following concentrations : 0.35M NaC1, 0.02 M MgSO4,0.021MMgC12,7.8 mM CaC12,7.5 mM KNO3,0.37 mM KH2PO4and0.37 mM NaHCO3.To this1 M Trizma base pH=7.6 (Sigma) was added to a final concentrationof 1.9 mM. Stock A(50 mM Na2EDTA and 9 mlvi FeC13)was added at 0.76 mill followedby stock B (0.29 mMZnC12,9.7 mMH3B0,0.12 mM CoC12, 0.24 mM CuC12,2.5 mMMnC12and 0.03 mM(NH4)6M07024),again at 0.76 mill. A 0.1 iglml vitamin B12 solutionwas then added at0.38 mill. The media was made up to the appropriate volume andan additional 42 mill ofdistilled water was added to account for evaporationduring autoclaving. Media was autociavedat 12 1°C for 30 minutes. Cells were grown in 1200 ml of mediain three litre fernbach flasks.These were grown with continuous agitation on an orbital shakerat 75 rpm. The light was kepton a 12:12 hour light/dark cycle to induce synchronicity (Cattolicoet al. 1976). Light levelswere maintained at 60 JiE/m2/min and the temperature was constantat 18°C throughout the lightand dark cycles. Cultures were routinely tested for contaminationwhen inoculated by adding0.5 ml of culture to 5 ml of nutrient marine media (2.0g nutrient broth and 1.25 g yeast extractper 250 ml artificial sea water). Cell counts were done usinga standard hemacytometer. Cellcounts were made after they were killed by adding 0.5% formaldehyde(50i1/lml culture)2.2.2 Heterosigma thylakoidfractionationLate log phase cells were harvested at 400 xg for 12 minutes, resuspended in cold0.33 M sorbitol, 1 mM MgC12, 50 mM HEPES pH 7.6 and proteaseinhibitors (1 mM43phenylmethyl sulfonyl fluoride, 5 mM E-amino-n-caproicacid, 1 mM benzamidine-HC1,1 mg/mI leupeptin). Protease inhibitors were routinelyused in solutions, being addedfrom stocksolutions prior to use. Cells were lysed under 4000 kPa(600 psi) nitrogen in a YedaPress (YedaResearch and Development Co. Ltd. Rehovot,Israel) to release the chloroplasts. Thechloroplasts were separated by differential centrifugation ina swinging bucket rotor at 6500x gfor 12 minutes at 4°C. Chioroplasts were washedthree times in cold 0.1 M NaC1,5 mM MgCl,20 miM Tricine pH 8.0 (including proteaseinhibitors) yielding a washed thylakoidfraction.Thylakoids to be used for non-denaturing gel electrophoresis weremade up to 10% glycerolprior to quick freezing in liquid nitrogen and storageat -80°C (Allen and Staehelin 1991).Thylakoids used for sucrose gradient fractionationwere used fresh and solubilizedwithdigitonin at a detergent to chlorophyll ratio of100:1, on ice for four hours with a constantgentlestirring. After centrifugation at 40 000 xg for 30 minutes, the supernatant was loaded onto a0.3 M-1.2 M linear sucrose gradient on top of 1.3 M and1.6 M sucrose cushions. Sucrosesolutions were made up in 10 mM Tricine pH 8.0 containing 0.05%(w/v) digitonin. Sampleswere centrifuged for 24 hours at 250 000 xg in a swinging bucket rotor at 4°C. Fractions 2 and3 (Fig. 2.1) were precipitated at 40000 xg following dialysis at 4°C in 0.1 M CaC12,10mMMgC12,10 mM Tricine pH 8.0 including protease inhibitors(as above). Due to the large amountof detergent at the top of the gradient, Fraction 1(Fig. 2.1) was pelleted at 100 000 xg followingextended periods of dialysis with many changes of thedialysis buffer. Chlorophyllconcentrations were determined in 90% acetone using theequations of Jeffrey and Humphreyshown below (Jeffrey and Humphrey 1975).Chl a = 11.47 (A664)-0.4 (A630) Chl c+ c2 = 24.36 (A630)-3.73(A664)2.2.3 Denaturing SDS-PAGE and Western BlottingFor denaturing gel electrophoresis, samples were solubilized in 2%SDS, 65 mlvi TrisHC1 pH 6.8, 50 mlvi dithiothreitol, 10% glycerol and heat denaturedfor 1 minute at 100°C.44Thylakoid and sucrose gradient fractions were loadedon the basis of chlorophyll.Gel slicesfrom non-denaturing PAGE were incubated in 2X samplebuffer (4% SDS, 132 mM Tris-HC1pH 6.8, 0.1 M dithiothreitol, 20% glycerol) at room temperaturefor 2 hours then heated to 80°Cfor 20 minutes. Polypeptides were separated on 12-16%or 7.5-15% SDS polyacrylamidegels(acrylamide: bis-acrylamide 37.5:1) containing 0.05%SDS and 1.32 M Tris-HC1 pH8.8, with a2cm stacking gel containing 5% acrylamide,0.1 M Tris-HC1 pH 6.1 and 0.1% SDS.Gels wererun for 18 hours at 4°C with the Laemmli buffer system(Laemmli 1970). Coloured molecularmass standards (Amersham) were used to estimate molecularmass.Proteins were electrotransferred to nitrocellulosein 50 mlvi sodium acetate pH 7.0overnight at 200 mA and 4°C. As a guideline,115ththe amount of chlorophyll loadedon gels tobe stained was used for the same samples destined to be used for westernblotting. Westernblotting was carried out as previously described (Whiteand Green 1987). Western blots werereblotted after stripping the nitrocellulose membrane in0.1 M glycine-HC1 pH 2.2, 20 mM Mg-acetate, 50 mM KC1 (Legocki and Verma 1981) followedby reblocking in 3% Hipure liquidgelatin (Norland Products Inc. New Brunswick, N.J.)in phosphate buffered saline (1.37 MNaCl,27 mM KC1, 81 mM Na2HPO4,15 mM KH2PO4,pH7.4). Proteins were Coomassie stainedfor2 hours using 0.1% Coomassie brilliant blue-R, 50%methanol and 7% glacial acetic acid.Gels were destained in several changes of a 20% methanol,7% glacial acetic acid. Polyclonalantibodies used in this study were anti-CP1a (c-CP1a),specific for barley CP I plus LHC I(White and Green 1987) and anti-FCP (c-FCP), specific for Phaeodactylumtricornutumfucoxanthin-chiorophyll a + c (FCP) protein complex(Fawley and Grossman 1986), which wasprovided by Dr. Art Grossman. Other antibodies includethe ct-PsaD antibody specific for a PS Iassociated subunit (also called PS I subunit #2) (Bengis and Nelson1975) and ci-D1, specific forthe PsbA polypeptide of PS II, provided by L. McIntosh.452.2.4 Non-Denaturing Gel SystemThylakoids were solubilized with a mixture of0.9% octyiglucoside, 0.9%decylmaltoside and 0.2% lithium dodecyl sulphate (in2 mM Tris-maleate pH 8.0, 10%glycerol andwith protease inhibitors) and resolved on a non-denaturinggel system according to AllenandStaehelin (Allen and Staehelin 1991) exceptthat a 7% acrylamide gel was usedwith anacrylamide to bisacrylamide ratio of 150:1. A stackinggel was not used becauseit resulted indegradation of the pigment-protein complexes.Samples were solubilized on icefor 30 minutesat an anionic detergent to chlorophyll ratio of 30:1with occasional mixing, thencentrifuged inan microfuge for 20 minutes at 4°C. Sampleswere electrophoresed at 10 mAfor 1.5 to 2.5hours at 4°C. Estimations of molecular mass were doneusing non-denatured, high molecularmass markers (Pharmacia). Gel bands were excisedand electrophoresed on a denaturinggradient gel as described above. Samples to be used forfluorescence data were excisedfrom thegel and quick frozen in liquid nitrogen prior tostorage at -80°C.2.2.5 Spectroscopy and Fluorescence measurementsAbsorption spectra were recorded on a Cary 210Spectrophotometer at room temperature.P700 content was measured from the sucrose gradientfractions directly by monitoringtherecovery of absorption at 700 nm after photo-oxidationby saturating red light with 1.7 mMascorbate and 0.075 mM methylviologen present in the reactionmixture (Marsho and Kok1972).Fluorescence emission spectra were recorded with aPerkin Elmer LS5O fluorometer withthe 77°K low temperature attachment and red sensitive photomultiplier.Excitation wavelengthwas 440 nm and the excitation and emission slit widths wereadjusted to 10 nm and 5 nm,respectively. Spectra shown are an averageof three scans. A 530 nm cut off filter helpedtoremove Rayleigh scatter in the 620 nm range.Gel slices from the non-denaturing gel system46were fitted in the cuvette with 60% glyceroland frozen in liquid nitrogen prior tomeasuring.Emission spectra were corrected for the drop in photomultipliersensitivity in the 600-800 nmrange using an averaged correction factor provided byPerkin Elmer. Excitation spectrawererecorded from similarly prepared samples at 77 K.Emissions from the excitationspectra weredetected at 680 nm in all samples. The excitation andemission slit widths were2.5 nm and10 nm, respectively. Scan rate was 300 nmlminuteand the spectra shown are an average oftwoscans.2.3 Results2.3.] Fractionation ofdigitonin-solubilized membranesby sucrose gradient centrifugationThylakoid membranes solubilized with digitoninwere resolved into three major fractionson a sucrose gradient (Fig. 2.1). The top dark brownfraction (fraction 1) was richin fucoxanthinand chlorophyll c as demonstrated by a broad shoulderfrom 488-540 nm and a prominentshoulder at 460 nm, respectively (Fig. 2.2A). Fraction1 was removed from the 21% sucroselevel and contained approximately 53% of the total chlorophyll.It also showed visible redfluorescence upon excitation with long wavelengthUV light, indicating the detachment of thelight-harvesting complex from the reaction center.A Chi a emission maximum of 675 nm, witha secondary peak at 732 nm, was recorded(Fig. 2.2B, solid line). A small peak at637.5 nm isprobably the result of the partial uncoupling of Chl cfluorescence which is preventing completetransfer of excitation energy from Chic to Chl a. Residual P700 activity was detected infraction 1, giving a Chi aIP700 ratio around 1200.47SucroseConcentration____Appearance Chi a I c Total Chi (%) Abs. Maxima0.2M-nfl1Brown 4 53 672, 440,460*,488*2Green-brown 14 25677,437,460*,:}3 light-brown1011 675,437,460*,1.6 MFigure 2.1Schematic representation of sucrose gradient fractionationof digitonin solubilizedthylakoids, with Chi a / c ratios, percentage of totalchlorophyll in each fraction andabsorption maxima data for the three major fractions.Asterisk in absorption dataindicates a shoulder.Fractions 2 (30% sucrose level) and 3 (34% sucroselevel) contained 25 and 11% of thetotal chlorophyll, respectively (Fig. 2.1). Both containedsignificant amounts of carotenoid asthere is a prominent absorption at 496 nmin both fractions (Fig. 2.2A). An absorbance shoulderat 460 nm also indicates the presence of Chlc though the Chl a/c ratios were 14 and 10 forfraction 2 and 3, respectively. This indicated significantamounts of antennae were stillassociated with the complexes. Both fractions (2and 3) were enriched in PS I with Chla / P700ratios of 340 and 420. Both fractions 2 and3 had emission maxima at 687 nmand significantshoulders at 717 nm, though fraction 2 had the greatestfluorescence in the 717 nm region48Aci)C-)Ccci0C))-Dci)>cclci)ci)0Cccl.00Cl).0ci)>Cclci)600 650 700 750 800Wavelength (nm)Wavelength (nm)Figure 2.2Spectral characteristics of the sucrose gradient fractions.(A) Room temperatureabsoption and (B) fluorescence emission spectra. Spectraare fraction 1(.—),fraction 2(---),fraction 3().A room temperature absorption spectrum (C) and fluorescenceemissionspectrum (D) of thylakoid membranes is also shown.Emission spectra were excited with437 nm light at 77°K.C600 650 700 750wavelength (nm)Wavelength (nm)8004946-.3014.3 -—28.0—19.5—18.58.0—-— 7.5Figure 2.3Polypeptides of digitonin sucrose gradient fractions separated by denaturing 12-16%gradient SDS-PAGE. (A) Stained with Coomassie blue. Thy.: Whole thylakoids; 1:Fraction 1; 2: Fraction 2; 3: Fraction 3. (B) Western blot immunoprobed withthe o-FCPantibody. Lanes labeled as in panel A. MW, molecular mass standards in kDa.Arrowheads on left of Panel B denote faint bands at 28.0, 17.0 and 17.5 kDa in wholethylakoids (Thy.) only.(Fig. 2.2B). Fluorescence in the 717 nm area is attributed to fluorescence from PS I, indicatingthat fractions 2 and 3 are enriched in PS I. The strong emission at 687 nm is probably from theantennae complexes associated with this fraction. In contrast, thylakoids had a fluorescenceemission maximum at 691 with a broad shoulder towards 740 nm (Fig. 2.2D). RoomMWAThy. 3 2 1 Thy.B1 2 3 MW21.5 —50temperature absorbance peaks at 440 and 674nm, along with prominent shouldersat 460, 492and 534 nm, were also characteristic of the thylakoids(Fig. 2.2C). Thylakoids solubilizedwithdigitonin have a blue shifted fluorescence emission maximumthat is more susceptibletochanges in the assay buffer conditions (not shown).Because of this, fluorescenceemissionmaxima have to be interpreted with caution.SDS-PAGE (Fig. 2.3A) showed that there wasa single polypeptide in Fraction1. Itcross-reacted with an antibody specific for the FCP fromthe diatom Phaeodacrylum tricornutum(Fig. 2.3B). The purity of this fraction was ideal for obtainingtryptic fragments fromthe FCP,as will be discussed in Chapter 4. Thoughone polypeptide was usually found inthis fraction,some extractions removed smaller amountsof the other three main FCP polypeptides,that areobvious in the thylakoid fraction (Fig. 2.3B).Fractions 2 and 3 showed a similar polypeptidepattern with a number of bands of 16-22 kDa, a sharp band at 37 kDa and diffusebands in the 49-55 kDa range (Fig. 2.3A).The fourpolypeptides estimated as 20.5, 19.5, 18.5and 18.0 kDa cross-reacted strongly withthe o-FCPantiserum (Fig. 2.3B). Two minor polypeptidesat about 17.5 kDa and 16.5 kDa werefaintlyimmunostained (lower arrowheads). The FCP antibodyalso detected a polypeptide with anapparent molecular mass of 28 kDa, found onlyin the thylakoid fraction (upper arrowhead,seealso Fig. 2.4A). Note that in this Figure and subsequentfigures, the apparent molecular massesdetermined by SDS-PAGE are used as labelsto identify distinguishable polypeptides,and arenot meant to imply accurate molecularmass determinations.Using an antibody specific for barleyCP 1 a (PS I core complex plus its correspondinglight-harvesting polypeptides (White andGreen 1987)), a different subset of cross-reactingpolypeptides was found (Fig. 2.4). The c-CP1a antibodydetected five major bandsatapproximately 16, 17.5, 18.5, 19 and 21.5 kDa in fractions2 (Fig. 2.4C, left) and 3, while anadditional four bands of about 18, 20, 22,28 kDa could be resolved in thylakoids(Fig. 2.4A,left). To identify which of the polypeptidesreacting with the c&CP1a antiserum werealsoimmunostained with the a-FCP antiserum, the immunoblotsshown in the left panels of Figure512.4A-C were stripped and reblotted with the o-FCP (shownin Fig. 2.4A-C, right panels). Thebands of 20.5, 19.5, 18.5, and 18.0 that were prominentin thylakoids immunostained oniy withcx-FCP (Fig. 2.3) are heavily stained in Figure 2.4A (rightpanel) and are clearly distinguishedfrom the bands labeled 21.5 and 22.0 above them andthe 16.0 band below which onlycrossreacts with the cx-CP1a antiserum. Similar results wereobtained with fraction 2 whenit wasreblotted with o-FCP (Fig. 2.4C, right panel). Note thatthe major light-harvesting polypeptidein fraction 1, which was immunostained with the cx*FCP,was not detected usingthe x-CP1a(Fig. 2.4B). Only four polypeptides at 28, 18.5, 18 and17.5 kDa appeared to react with bothantisera. Results of the immunoblotting withthe two antisera are summarized inTable 2.1.These results show that there are up to 12 polypeptidesin the FCP/CAB family in Heterosigina,a larger number than previously reported for any chromophytealga.52Western blot of sucrose gradient fractions immunoprobed withc-CPla (left panels) thenstripped, blocked and immunoprobed withc-FCP (right panels) on the same blot. (A)Whole thylakoids (B) Fraction 1 fromsucrose gradient (C) Fraction 2 from sucrosegradient. Approximate molecular masses (kDa) are used as labelsto distinguishindividual bands.Table 2.1 Summary of cross-reactivity with the o-CPlaand o-FCP antiseraMolecular Mass (kDa)antiserum 28.0 22.0 21.520.5 20.0 19.5 19.0 18.5x-CP1a± + + - + - ++*x-FCP*An apparent single band at 1-18.5 kDamay be a doubletBOP1 aCP1a +FOPMWCCP1aCP1a +FOPMW—28.0-- /20.5_z’9•5—18.517.0AMW OPlaOPlaFOP28.0—22.019.017.5//’ ‘16.0”Figure 2.4MW21.5\p20.5____190\--;-—19.518.5— --18.518017.517.516.0”‘‘17.0+ -- + - + - + + + +18.0 17.5 17.0 16.0+*+ - +C,-,3-,2.3.2 Fractionation by Non-Denaturing PAGEPigment-protein complexes were isolated from Heterosigmathylakoids solubilized in0.9% octylglucoside, 0.9% decyl maltoside and 0.2% lithium dodecyl sulphate,and separated bymeans of the non-denaturing gel system of Allen and Staehelin (Allen andStaehelin 1991) (Fig.2.5). With this system, eleven pigment-protein complexes were resolved(Fig. 2.5A). Bands 1and 10 were resolved into two Complexes (ib, lOb) with longer periodsof electrophoresis(Fig. 2.5B), but this resulted in some degradation of the central pigment-proteincomplexes. Thefirst ten pigment-protein complexes were green and lacked noticeable fucoxanthinwhileComplex 11 was a brown fraction making up approximately 40% of the totalprotein.Figure 2.5Unstained 7% polyacrylamide gel separating pigment-protein complexes of thylakoidssolubilized with 0.9% octylgiucoside, 0.9% decyl maltoside, 0.2% lithium dodecylsulfate (Allen and Staehelin 1991). (A) Electrophoresed for 1.5 hours at 10 mA at 4°C.(B) Similar gel electrophoresed for 2.5 hours. Apparent molecular masses are given inkDa.AB54AMW M 1 23 4 5 6 7 8 9 10 11 Thy--46IMW1 2 3 4[53_nw’ri5 67 8 9lOalObliicpIFigure 2.6Analysis of isolated pigment-protein complexes. (A) 7.5-15% denaturing SDS-PAGE ofpigment-protein Complexes 1-11 separated from non-denaturing PAGE.(B) Westernblot analysis of the same pigment-protein complexes. Antibody used in sequentialimmunoprobing and area of cross reactivity indicated on the right; Molecularmasses inkDa on the left. The bottom panel (CP I) is a western blot of the same gel fractions withthe cL-CP1 (PS I core complex) antiserum.3014.3BThy 1 23 4 5 6 7 8 9 lOa lOb 11 Thy322114psaD55Denaturing SDS-PAGE of Complexes 1-3 (Fig.2.6A) showed a broad stainedband ofabout 53 kDa, a 37 kDa band, and a number of sharperbands in the molecular mass range (10-21 kDa) typical of the non-pigmented subunits ofPS I (Golbeck and Bryant 1991).Animmunoblot of samples from a similar gel was sequentiallyprobed with several antibodies todetermine the composition of the various bands in Figure 2.5.Immunodetection of the PS I-D(psaD gene product, Fig. 2.6B), and CP I polypeptides (Fig.2.6, bottom panel) indicated thatpigment-protein Complexes 1-3 were PS I complexes.PS I core polypeptides werealso detectedin Complex 4 but were lacking in the other fractions.When blots were probed withthe c-FCPantibody, there appeared to be a number of antennalpolypeptides in the 17-20.5kDa rangeassociated with the PS I core complex (Fig. 2.6B). Theimmunologically detected LHCsinComplexes 1-3 had the same molecular massas those in Complex 11 and may be similarto theLHC polypeptides found in sucrose gradient fraction2 and 3. However, unlike sucrose gradientfractions 2 and 3, there did not appear to be any fucoxanthinor Chl c associated with Complexes1-3 (Fig. 2.7). This may have been the result of thedetergents used in the extraction andtheelectrophoretic forces partially denaturing the complexesbut allowing some LHCsto remainassociated with the PS I core.Complex 1 had the long wavelength absorption maximumat 677 nm typical of PS I(Fig. 2.7A) and a long wavelength chlorophyll a fluorescenceemission maximum at 717 nm(Fig. 2.7B), similar to that attributed to thePS I specific light-harvesting complexof land plants(Haworth et al. 1983; Murata and Satoh 1986). It alsohad a second fluorescence emissionmaxima at 676 nm which may indicate uncoupledchlorophyll resulting from the detergenttreatment (Fig. 2.7B). Excitation spectra of Complex1 showed a Chi a peak at 437 nm and asecond minor peak at 535 nm (Fig. 2.7C).Complexes 6- lOa appeared to be PS TI-related, as they had a numberof polypeptidesmigrating as somewhat diffuse bands in the30-50 kDa range. An antibody specific to Dl (PS IIreaction center polypeptide encoded by psbA gene) (Fig.2.6B) showed that it was found in allthese fractions but was absent from Complexes 1-5. Complexes5-7 had long wavelength560)0Cci)0(I)0)1_0DUa)>ccici)Bci)0Cci)0C’)ci)0DLL0)>ccici)400 450 500 550Wavelength (nm)600 650 700750Wavelength (nm)600800Figure 2.7Spectral characteristics of pigment-protein Complexes 1, lOa, and 11 in gel slices fromthe non-denaturing SDS-PAGE. (A) Room temperature absorption spectrum. Spectra areoffset for blarity. (B) Fluorescence emission spectra at 77 K of pigment-proteincomplexes. Spectra are Complex 1(—);Complex lOa();Complex 11( ).(C)Fluorescence excitation spectra of the same three complexes and thylakoids at 77 K.Emission was detected at 680 nm.ci)0Ccci-Q0C,)-Qa)>ccici)400 500600 700Wavelength (nm)C57maxima at 674 and Complexes 8-lOb at 671 nm (data not shown; except lOa, Fig. 2.7).Complex lOa had a fluorescence emission maximum at 686 nm (Fig. 2.7) which is typical ofPS II core complexes (Murata and Satoh 1986; Brown 1988). An excitation spectrum ofComplex lOa has a main Chl a peak at 437 nm and minor shoulder at approximately 470and502 nm, indicating minor amounts of Chl c and a carotenoid (probably fucoxanthin). Thesepeaks are probably from minor amounts of FCPs detected in this fraction (Fig. 2.6B). Variabilityin the resolution of pigment-protein Complexes 4-9 was observed between different preparationsof thylakoids, possibly the result of different cell culture densities which could have altered lightconditions.Complex lOb, detectable after longer periods of electrophoresis, was enrichedin the28 kDa polypeptide that cross reacted with the cx-FCP (Fig. 2.6B). It had absorbance peakstypically associated with Chi a, Chl c, and fucoxanthin but since it also containedsome of thelower molecular mass light-harvesting polypeptides, I cannot say whether the 28kDapolypeptide binds these pigments or not. This polypeptide may be analogous to the31 kDapigment-protein complex containing only Chi a characterized fromanother chromophyte,Ochromonas (Gibbs and Biggins 1991).Complex 11 contained the majority of the fucoxanthin and had a lowChl a/Chl c ratio asestimated from the peaks at 440 nm and 460 nm respectively(Fig. 2.7A). It appeared to besimilar to fraction 1 from the sucrose gradient, but wasnot as pure. Rather than a singlepolypeptide, it had the three major light-harvesting polypeptides at 20.5,19.5 and 18.5 kDa thatcross reacted with the diatom o-FCP antibody. Moreover, polypeptidesin the 2 1-33 kDa rangewere also weakly stained with Coomassie blue in this fraction(Fig. 2.6A). One-third the amountof Complex 11 was loaded onto the gel in Figure 2.6 in orderto prevent overloading; therefore,comparison of absolute protein amounts can not be made. Complex11 had a fluorescenceemission maximum at 681 nm at 77°K (Fig. 2.7) which is comparableto the LHC II of landplants (Murata and Satoh 1986). The excitationspectrum of Complex 11 has peaks at 437 and460 nm from Chl a and Chi c, respectively. As well,a broad area from 480 to 540 nm results58from excitation energy transfer of xanthophylls (fucoxanthin)to Chi a (Fig. 2.7C).Thisindicates that the accessory pigments arestill coupled to Chl a. Although someDl isimmunodetected in Complexes lOb and 11,it would be premature to concludethat the 28 kDapolypeptide or any of the FCPs are preferentiallyassociated with PS II, as itis impossible to ruleout comigration of individual polypeptides in thisregion of the gel.An orange free pigment zone migrated justahead of Complex 11. Absorbancespectraindicated that it contained carotenoids anda small amount of chlorophyll(data not shown). Nopolypeptides were detected following Coomassiestaining.2.4 DiscussionSucrose gradient separation followingdigitonin solubilization has beensuccessfully usedin the isolation of light-harvestingcomplexes from a number of algae(Berkaloff et al. 1990;Hiller et al. 1991; Arsalane et al.1992). This fractionation techniqueusually results in a light-harvesting antennal fraction at thetop of the gradient and a few additionaldenser pigment-protein complexes. I have foundthat the raphidophycean alga, Heterosigma,like otherchromophyte algae, has a predominant fucoxanthin-chiorophylla/c pigment-protein complexreleased by digitonin solubilization.This complex has a single polypeptide withan apparentmolecular mass of 19.5 kDa and spectral characteristicscomparable to the predominant LHCfrom other chromophytes (Hilleret al. 1991). It appears to be the more abundantof fourpredominant FCPs in the thylakoidsas determined immunologically with the ct-FCPantiserum.Since it is easily dissociated from the corecomplexes, this suggests it maybe peripherallylocated and possibly analogous to landplant LHC II. In addition, the 19.5kDa polypeptide ispreferentially removed in most digitonin extractions,suggesting it is even more distal totheother predominant FCPs. In contrast, the FCPfraction obtained by non-denaturingSDS-PAGE59(Complex 11) appeared to contain all the majorFCP polypeptides. The detergentused for thesolubilization of thylakoids in the non-denaturinggel system were obviously morepenetrating’and this resulted in a more vigorous extractionof the FCPs.The denaturing SDS-PAGE system used allowedfor the resolution of up to12 separatecross-reacting LHC related polypeptides in Heterosigina.They were in the samesize range (15-22 kDa) as those reported from otherchromophytes (Hiller et al. 1991).Most published workhas reported one to four light-harvestingpolypeptides (Hiller et al. 1991),although as many assix polypeptides from four chromophyte specieshave been reported to cross reactwith anantibody raised to Chiatnydoinonas (Chlorophyceae)LHC (Plumley et al. 1993).Differences inthe number of polypeptides detected may partlybe due to the different electrophoreticsystemsused to resolve the complexes and the natureof the antiserum. In order to rule out the possibilitythat some of the immuno-reactivebands were the result of proteolytic cleavageof largerpolypeptides, Heterosigina thylakoids wereisolated and incubated at 37°C,in the presence orabsence of protease inhibitors, withno difference in the number of LHCbands detected. Wholecells solubilized directly in 2X SDSsample buffer also showed the same patternas thylakoids(data not shown). The FCP antenna familyin Heterosigma may therefore be as complexas theCAB antenna family in land plants (Greenet al. 1991; Green et al. 1992).It is also interesting to note that these light-harvestingpolypeptides were detected usingLHC specific antibodies from a different classin the Chromophyta and antibodies fromterrestrial plants. Other studies using antibodiesspecific for land plant and chiorophyte LHCsshow cross-reactivity with various membersof the Chromophyta (Caron et al. 1988; Passaquet etal. 1991; Plumley et al. 1993); others show cross-reactivitywithin the Chromophyta (Fawley etal. 1987). These results indicate the presenceof commonly conserved antigenic determinantsassociated with all light-harvesting polypeptides, suggestiveof a common evolutionary origin.This structural similarity is confirmed by sequences of the FCPgenes from the diatom,Phaeodactylum tricornutum (Grossman etal. 1990). Protein sequences of tryptic fragmentsfrom a Cryptomonad (Sidler et al. 1988),the dinoflagellate Amphidinium (Hiller et al.1993) and60from Heterosigma (Green et al. 1992, Chapter 4) also demonstratethe apparent structuralsimilarity between the Chi a + b binding proteins and theChl a + c binding proteins.Solubilized chromophyte thylakoids have previously beenfractionated by non-denaturingPAGE, especially in sodium deoxycholate (Caronand Brown 1987; Brown 1988) or Deriphat160 gel systems (Boczar et al. 1980; Peyriereet al. 1984; Boczar and Prezelin 1989; Knoetzeland Rensing 1990). I was unable to obtain satisfactoryresults with either of thesegel systems orwith the non-denaturing gel systems used successfullywith land plants (Cammand Green 1989;Thornber et al. 1991). This indicates the labile natureof these complexes as comparedto theLHCs of terrestrial plants; a problem that is impedingcharacterizations of the number andtypesof chlorophyll-proteins in the chromophytes. However,a modification of the non-denaturinggelsystem devised by Allen and Staehelin (Allen and Staehelin1991) proved to be successful in theseparation of Heterosigma pigment-protein complexes,allowing the preservation ofa number oflarge complexes with apparent molecular massesof over 200 kDa. This system representsanimprovement over other electrophoretic separationtechniques for chromophyte algal pigment-protein complexes, being able to separate severalPS I fractions, a number of PS II fractions,anda dominant LHC fraction.An important feature of the non-denaturing electrophoreticseparation technique is theability to isolate light-harvesting antennal proteinsstill associated with the core complexes.Complexes 1-3 were PS I fractions and appearedidentical except that the slowest migratingappeared to have larger amounts of associated light-harvestingpolypeptides. Complexes 2 and 3lacked the majority of the LHC and tendedto retain high levels of the lower cross reactingpolypeptide (17 kDa) suggesting it may be closely associatedwith the PS I core complex.Previous studies on isolated PS I complexesof chromophyte algae found a 715-720nmfluorescence emission peak which is usually assumedto be due to PS I reaction center inassociation with its light-harvesting antenna (Brown1988; Berkaloff et al. 1990). As well,aPS I specific antenna with different fluorescence emissioncharacteristics has been identified inthe xanthophyte alga, Pleurochioris(BLichel and Wilhelm 1993). It appears thatthe presence of61a light harvesting complex associated with PS I is a common featureof the Chromophyta(Berkaloff et al. 1990), as is the case with green algaeand land plants where a numberof uniqueantennal proteins in the size range of 2 1-24kDa are specifically associated withPS I (Mullet etal. 1980; Haworth et al. 1983; Lam et al. 1984b).Complexes 1-3 do contain associated LHC-relatedpolypeptides though the total amountis low compared to the overwhelming occurrenceof these polypeptides in Complex11. Inaddition, these polypeptides do not appear to bindsignificant amounts of fucoxanthinor Chi c.As these polypeptides are of comparable sizesto the other LHCs in Complex 11and inthylakoids, it would seem that these polypeptidesare partially denatured andhave lost theaccessory pigments without being removedfrom their association with the corecomplex. Thismay be a result of the detergents used, the forcesexerted during electrophoresis,or acombination of both. This would not be too surprisingas the LHCs are quite susceptible todegradation. Because of the possibility of a nonspecificinteraction with the highmolecular masscomplex and an inability to distinguishbetween LHCs associated with this PS Ifraction andthose in the LHC fraction (Complex 11), I can notyet assign any specific LHC polypeptidesexclusively to PS Tin Heterosigma.The ability to resolve a number of PSI and PS II complexes is comparable tothe resultsobtained with the green alga, Chiarnydomonas, (Allenand Staehelin 1991) thoughthere aredifferences in the associations of the LHCswith these complexes. The numberof LHCsresolved also appears to differ, illustratingthe differences between the chromophytes,greenalgae and land plants. At the present time Iam unable conclude, with any certainty,the nature ofthe complex organization in the thylakoid withregard to the localization of PS I and PS II orwhether the pigment-protein complexesseparated on the non-denaturing gel system representdifferent environments within the thylakoid membrane.The recent immunocytochemical localization ofFCP and PS I complexes within thethylakoids of members of the Chromophyta (Lichtléet al. 1992; Pyszniak and Gibbs 1992;Lichtlé et al. 1992b) show that PS I andPS II are not as highly segregated within appressedand62non-appressed regions as theyare in terrestrial plants. Though therewas only a slight preferencefor PS Tin the ‘nonappressedregions of the chromophyte thylakoid,the FCPs werehomogeneously distributed throughoutall parts of the membrane.In Heterosigma, there isanassociation of a large number of antennaepolypeptides with the PSI enriched fractions fromthesucrose gradient. It is unlikely that theseare all specific PS I antennae asmost of the prominentLHC polypeptides detected in thylakoidsare present in these fractions.At the moment it is notclear which of these polypeptidesmay be preferentially associatedwith PS Ito the exclusionof aPS TI association. The consistent associationof the FCPs with theselower fractions suggeststhatthe separation of the main antennaeis not as sharply defined as isthe case with LHC I andLHC II in terrestrial plants. Thehomogeneous distributionof the FCPs in appressed andnonappressed thylakoid regions of chromophytesmay explain their prevalent associationwiththe PS I enriched fractions of the sucrosegradient. This would agree wellwith work showingthat the excitation energy capturedby the main antennae of a diatom wasequally distributed toboth photosystems (Owens 1986b). However,this remains speculative at themoment since theassociation of the FCPs with PS Imay be a result of contamination duringthe fractionationprocedure.63CHAPTER 3An immunological characterization of LHCrelated-polypeptides in red algae3.1 IntroductionThis Chapter is concerned with the immunologicalcharacterization of LHC proteins fromtwo red algae. In this Chapter I will firstdescribe the immunological analysesI did withAglaothamnion. This will be followed by the immunologicalwork done with Porphyridiumincollaboration with Beth Gantt’s group at the Universityof Maryland. Greg Wolfe, in thelab ofBeth Gantt, was the first to demonstratethat a PS I fraction from the red algaPorphyridiumcontained the core complex and an arrayof smaller polypeptides in the 11-24 kDarange, typicalof the PS I polypeptide distribution of chlorophytes andterrestrial plants (Wolfe et al. 1992;Wolfe et al. 1994b). In his work, PS I and PSII fractions from Porphyridiuin were isolated.Thespectral characteristics and the immunologicaldetection of D2, CP43 and CP47 were usedtoidentify the PS II fraction. The discoveryof a putative LHC I complex in Porphyridiumlead toa collaboration with our lab in order to examinethe immunological relatedness of thesered algalchlorophyll-proteins to other antennae. This collaborationshowed that these polypeptides wereindeed structurally related to the CABsand FCPs of the terrestrial plants and diatoms,respectively (Wolfe et al. 1994). In addition,I examined a more distantly related red alga,Aglaotharnnion neglectum, to look for similar immunologicallyrelated polypeptides todetermine whether the CAB-related antennae werea general occurrence amongst thered algaeor, alternatively, if they were unique tothe primitive red algal class representedbyPorphyridiurn.Light-harvesting polypeptides resembling the CABsof the Chi a + b-containing64organisms have not been previously discovered in thered algae. Many of the earlier attemptsatisolating pigment-protein complexes involved solubilizationof the thylakoids withSDSfollowed by polyacrylamide gel electrophoresis. Thisfrequently resulted in the release ofaconsiderable amount of free chlorophyll, thougha PS I fraction usually remained intact (Hillerand Larkum 1981; Redlinger and Gantt1983). More recently, a PS I fractionwas isolated fromthe unicellular red alga, Cyanidium caldarium,and was found to contain a highmolecular massband and four smaller polypeptides in the 13-18kDa range (Yurina et al. 1991).In a separatestudy, the thylakoid membrane compositionof Porphyridiurn was analyzed by fractionationofdetergent solubilized thylakoid membraneson a sucrose gradient. This resultedin the separationof PS I and PS II fractions (Marquardt andRied 1992). Neither of the abovetwo studiesreported the occurrence of LHC polypeptidesassociated with either PS I or PSII. The latterstudy used a partially-denaturing gelsystem to examine the thylakoid compositionwhich wouldnot be expected to resolve the LHCpolypeptides. In addition, the lack of LHCdetection ineither of these studies may have been due tothe degradation of the complexesas a result of themethods used to fractionate the thylakoids (Wolfeet al. 1992).3.2 Materials and Methods3.2.] Aglaothamnion culturesAglaotharnnion neglectum Feldmann-Mazoyeris a filamentous red alga (classRhodophyceae) originally collected off the shores of Hawaiiand belonging to the subclassFlorideophycidae—order Ceramiales (in the family Ceramiaceae).An axenic culture ofAglaothamnion was provided by Dr. KirkApt to whom I am grateful. These cultures weremaintained in the same artificial sea water medium describedin Chapter 2. No difference wasseen when Aglaothamnion was culturedin Provasoli’s enriched sea water mediaas described byMagruder (1984), so the artificial media wasused. These algae were kept on a 16 hourlight: 865hour dark cycle at 24°C and 30 jJE/m2/min, with constantbubbling of air through0.2 jimmillipore filters to maintain an axenic culture. The cultureswere kept in six litreflasks with fourlitres of media. Bubbling air at 4000 cm3/minwith an aquarium pump wassufficient to keep thecultures agitated. In order to subculture the algae andto maintain an even growthof the tissue,the algae were fragmented in a sterilized blender for 10-15seconds before inoculating freshcultures.3.2.2 Aglaothamnion neglectum thylakoidfractionationAglaothamnion was harvested bypouring the culture through four layersof cheese cloth.Thylakoids were prepared using a modificationof a method used for Porphyridiurn(Wolfe et al.1992). The material was quick frozen in liquid nitrogenand ground to a fine powderin a prefrozen mortar. This powder was resuspended in cold50 mM NaPO4pH 7.0, withthe proteaseinhibitors (1 mM phenylmethyl sulfonyl fluoride, 5mM E-amino-n-caproic acid,1 mlvibenzamidine-HC1, 1 mg/mI leupeptin). The extractwas then put through a pre-chilledFrenchpress at 1300 psi. This was repeated three timesand the effluent was kept on ice betweenruns.The extract was centrifuged at 1000 xg for 10 minutes at 4°C to remove unbroken cells andcellular debris. The supernatant was then centrifugedat 28 000 rpm in a SW28 rotorfor 45minutes at 4°C. The green pellet was resuspended in cold10 mM NaPO4pH 7.6, with proteaseinhibitors (PIs) included in the buffer. The fraction wasthen centrifuged through a sucrose stepgradient (0.5 M/ 0.8 M/ 1.6 M sucrosesteps in 10 mM NaPO4pH 7.0, plus PIs) at 27000 rpm ina SW28 rotor for three hours at 4°C. Thepellet was removed and resuspended in cold 10 mMNaPO4, 150mM NaC1, plus PIs. If necessary, the samplewas then quick frozen in liquidnitrogen and stored at -80°C for use at a later time.Thylakoids were resuspended in 4 ml ice-cold 1 MNaBr and centrifuged at 15 000 rpmin a SS34 rotor for 10 minutes. The pellet was resuspendedin 1 ml cold 50 mM HEPES pH 7.4,10 mM NaCl, 10% glycerol, and 0.75 M sucrose(+PIs). Thylakoids were solubilized in3-Dodecylmaltoside(13-DM)at a detergent:chlorophyll a ratio of 24:1for 2 hours at 4°C, in the66dark. Solubilized thylakoids were diluted1:1 with 50 mM HEPESpH 7.4(+PIs) and loadedonto a linear 15-30% (w/v) sucrose gradient(in 50 mM HEPES pH 7.4,10 mM NaC1,10% glycerol, 0.04% (wlv)13-DMand PIs). The gradient was centrifugedin a SW41 rotor at36 000 rpm for 20 hours. Pigmentedbands were dialyzed against2 x 2 liters of ice-cold 50mMHEPES pH 7.4, 10 mM NaCl plusPIs. Samples were stored at -80°Cuntil required.Chlorophyll concentrations were determinedin N-N-dimethyl formamideusing the followingequation: ChI =(A664)10.65 (a=83.9 mM1cm)(Moran 1982).3.2.3 SDS-polyacrylamide gel electrophoresisAglaothainnion thylakoid sampleswere denatured as described inChapter 2. Proteinswere separated on 14% acrylamide gels(acrylamide: bis-acrylamide37.5:1) containing 0.8 MTris-HC1 pH 8.8 and 0.1% SDS.A 2 cm stacking gel containing 5%acrylamide, 0.1 M Tris-HC1pH 6.1, and 0.1% SDS was used. Gels weretypically run at 18 mA for 17hours at 4°C.Electrophoresis buffer used was the standardSDS-Tris-glycine bufferdescribed in Chapter 2.Staining, electrotransfer to nitrocelluloseand western blotting of thepolypeptides were doneasdescribed in Chapter 2 (section 2.2.3).The o-D1 (from pea) ando-OEC3 (from spinach) were agift from Ann Eastman. The c-OEC2antiserum was donated by Dr. Ekramadoullah.Thespinach o-PsaD and cx-PsaF antisera (fromspinach) were from Bengis and Nelson(1975).67percent chlorophyll absorbancemaximaI14%417, 435,482*,672 nmII86%421,437,493,678nmFigure 3.1.Schematic diagram of linear 15-30% Sucrosegradient used to fractionated f3-dodecylmaltoside solubilized thylakoids of A. neglectum.Resolved chlorophyll-bindingcomplexes areindicated as fraction I or II. The percentageof chlorophyll and the absorptionmaxima of eachfraction are given. An(*)indicates the presence of an absorbanceshoulder.3.3 ResultsFractionation of the thylakoids of Aglaothamnionneglectuin on a 15-30% linear sucrosegradient resolved two green bands: band I andband II (fig. 3.1). The top fraction (I)was lightgreen and contained only 14% of thetotal chlorophyll present on the gradient. Thedarker greenbottom fraction (II) contained the majority ofthe chlorophyll (86%). A very small reddish-brown pellet present at the bottomof the gradient contained very little or nochlorophyll. Roomtemperature absorption spectra of the twofractions were taken (Fig. 3.2). Fraction Ihadabsorbance maxima at 417 nm, 435 nm andwith a long-wavelength absorbance maximum of672 nm. A broad shoulder at 482 nm was alsopresent. Fraction II had an absorption maxima inthe 493 nm range and a long-wavelength formof chlorophyll a at 678 nm. Soret peaks at437 nm and 421 nm were also observed. Thedistinct absorbance properties in the 480-550nmregion between the two fractions indicates differencesin carotenoid distribution.68C000U)Wavelength (nm)Figure 3.2Room temperature absorption spectrumof sucrose gradientfractions I (top spectra)and II(bottom spectra). Spectraare offset for clarity.In Figure 3.3 (panel B), thepolypeptide compositionof fraction II and thylakoidsfrom A.neglectum are shownalong with a thylakoid preparationfrom Heterosigmaand a PS I fractionfrom spinach. Analyses werelimited to fraction IIand to thylakoids ofA. neglectum because thepresence of excessive amountsof detergent prevented properresolution of the polypeptideconstituents in fractionI. Polypeptides in the size rangeof 18-22 kDa, 30-34 kDa,and 40100 kDa were especiallyabundant in the thylakoid lane(fig. 3.3 B, lane 6). FractionII had adistinct band at 66kDa and diffuse polypeptides inthe 18-22, 30-34 and40-46 kDa regions(fig.3.3 B, lane 5). The LHCsof Heterosigma carteraein the 15-22 kDa rangewere the mostabundant thylakoid proteins(fig. 3.3 B, lane 7; Chapter2). In the spinach PSI preparation therewere a number of polypeptidesin the 14-69 kDa rangebut the 2 1-27 kDa antennaewere themost prevalent (fig.3.2 B, lane 8).400 500 600 70069A western blot with the same fractions was immunostained with thex-CP1a antiserum(fig. 3.3 A). This antibody (described in Chapter 2) cross-reactedwith four polypeptides inthespinach PS I enriched fraction, the two lower polypeptidesbelonging to LHC I (25 and 22 kDa)and the two upper polypeptides (26.5 and 27kDa) to contaminating LHC II. This antiserumrecognizes epitopes from both LHC I and LHCII polypeptides (White and Green 1987).The H.carterae thylakoid fraction cross-reacts with upto six polypeptides in the size rangeof 17-23 kDa (fig. 3.3 A, lane 3). The pattern of cross-reactingpolypeptides appears alittle differentfrom similar immunoblots in Chapter 2 becausea different gel system was used toresolve them.Of particular interest was the detection of four cross-reactingpolypeptides (19.0, 18.5,18.0 and 17.5 kDa) in the A. neglectum thylakoidfraction (fig. 3.3 A, lane 2). Inaddition, theremay be a fifth immunoreactive polypeptide witha molecular mass of 19.5 kDa, as the19.0-19.5 kDa band appears to be a doublet. FractionII contains two cross-reactingpolypeptides witha size of 19.0 and 19.5 kDa. The other cross reactingpolypeptides were probablyremovedduring fractionation. It is interesting to note that thesepolypeptides are significantlysmallerthan the corresponding polypeptides in terrestrialplants and green algae (21-24 kDa). Intermsof size, they more closely resemble the FCPsfrom the chrornophytes (16-2 1 kDa). The c&CP1aantibody also cross-reacts with the core complex of PSI, as seen in the 66 kDa areaof all fourfractions (fig. 3.3 B).Using an antibody specific to the pea Dl protein, the 32 kDacore complex polypeptideof PS II (Dl) was detected in both fractionII and in the thylakoid fraction from Aglaothamnion(fig. 3.4, lanes 1 and 2). The Dl polypeptide was alsodetected in the Heterosigina thylakoidfraction (lane 3) but was absent from the spinach PSI preparation (lane 4). Though fraction IIcontains both PS I and PS II specific polypeptides, itappears to be enriched in PS I complexes.Immunological analyses have demonstrated that Dland CP47 (PS II core antenna) arestructurally conserved in Synechocystis(cyanobacterium), Prochlorothrix, Heterosigma, andAglaothamnion (data not shown). The immunologicalsimilarities of these polypeptides in thered alga, Porphyridium have also beendemonstrated (Marquardt and Ried 1992). The putative70Aco\çç\?s\—0B=9746946Figure 3.31 2 343021.514.3Composition and immunological analysis of polypeptides in Aglaothamnion thylakoidsand fraction II. A) Western blot immunostained with the o-CPla antiserum (describedin text).Samples are; 1, Aglaothamnion fraction II; 2, Aglaothamnion thylakoids;3, Heterosigmacarterae thylakoids; 4, spinach PS I fraction. B) Gel stained with coomassieblue. Samples are;5, Aglaothamnion fraction II; 6, Aglaothamnion thylakoids; 7, Heterosigma carteraethylakoids;8, spinach PS I preparation; 9, markers. Size of markers in kDa are indicated onthe right.5 6 78 971A.n.A.n. H.c. Sp.MF IIThy. Thy. PSI1 2x-D1— 3OKDaFigure 3.4Immunological detection of the Dl protein in Aglaothamnionsucrose gradient fraction II(lane 1), Aglaothamnion thylakoids (lane 2), Heterosigma thylakoids(lane 3) and in a spinachPSI fraction (lane 4). The molecular mass marker (30 kDa) is in lane5. An anti-Di antiserum(a-Dl) derived from spinach is used.ferredoxin docking and PsaC stabilizing protein, PsaD (PS I subunitII, psaD), was also presentand immunologically reactive to a spinach PsaD specific antibody in Synechocystis,Heterosigma, and Aglaothannion(fig. 3.5, psaD). However, there wereconsiderable variationsin size (14-22 kDa) and the reactions were weak in the Aglaothamnionand Heterosigmathylakoid lanes (fig. 3.5, psaD). In contrast, only Heterosigma andAglaothamnion thylakoidsshowed a cross-reaction with an antibody directed to the putative plastocyanindocking protein,PsaF (PS I subunit III), of spinach (fig. 3.5, psaF). A polypeptide immunologicallyrelated toPsaF was not detected in Synechocystis, though it may have beenremoved during isolation sinceit is extrinisically associated with the membrane.3 45721 2 34OEC1—301 2 34—30OEC21 2 34—— 21psaD—141 2 34—21psaF—14Figure 3.5Immunological analyses of thylakoids from Heterosigma (lane 1), Aglaothamnion(lane 2), Synechocystis (lane 3), and spinach (lane 4). Western blots were immunostainedwiththe anti-OEC1 (oxygen evolving complex 1), the anti-OEC2 (oxygen evolvingcomplex 2), theanti-psaD (PS I subunit II) and anti-psaF (PS I subunit III) antisera as indicated.Molecular massmarkers are indicated on the right.73Western blots were done to examine the presence or absence of polypeptides making upthe oxygen evolving complex (fig. 3.5). This was attempted immunologically usingspinachderived antibodies specific to the OEC1 (33 kDa) and OEC2 (23 kDa) polypeptides. SincetheOEC2 polypeptide is not present in cyanobacteria (Stewart et al. 1985), it was thoughtthat anexamination of the red algae for the presence or absence of this complex would provide someuseful phylogenetic information. The OEC1 polypeptide was only very weakly detectedinSynechocystis and Heterosigma and this antibody did not react significantly withanything in theAglaothamnion thylakoid fraction (fig. 3.5, OEC1). In addition, only the spinachthylakoidfraction cross-reacted with the OEC2 antibody, even though four times the normal amount ofchlorophyll was loaded on the gel (fig. 3.5, OEC2). Unfortunately, I am unable toconclude ifAglaothamnion or Heterosigma have a protein homologous to the OEC2 polypeptideas the lackof immunological cross-reaction could be due to sequence divergence andnot complete absence.Greg Wolfe et al. (1992, 1994) at the University of Maryland analyzed the pigment-protein complexes of the unicellular red alga, Forphyridiuin cruentum. They werethe first todemonstrate the presence of a putative chlorophyll-binding complex analogousto LHC I ofterrestrial plants. In a collaboration with this group, we were able to analyze immunologicallythe structural relatedness of these polypeptides to those from the CAB and FCPfamily. Thisalga is a member of the class Bangiophycidae (order Porphyridiales), whichis considered to beprimitive with respect to the Florideophycidae, of which Aglaothamnion is amember.Figure 3.6 shows the results of immunoblotting two identical gels with differentantisera; the cxjCP1a polyclonal antibody and the o-FCP antibody (described in Chapter 2).At least fivepolypeptides in the P. cruentum PS I fraction cross-reacted with the a-CPla antibody; they hadapparent molecular masses of 19.5, 20, 22, 23 and 23.5 kDa (Fig. 3.6, panelB, lane 3). Thereare two additional polypeptides cross-reacting in the thylakoid lane at 19 and20.5 kDa (Fig. 3.6,panel B, lane 4). The spinach PS I fraction showed a number of cross-reacting polypeptides(22-29 kDa) from LHC I and contaminating LHC II and CP29 (Fig. 3.6, lane 2,panel B) as thesample was overloaded. A major cross-reacting band at 65 kDa in all four lanes isthe corecomplex of PS I. It is significant that thylakoid proteins from the cyanobacterium,Nostoc, lack74Figure 3.612341234Immunological analysis of polypeptides from: 1, Nostoc thylakoids; 2,spinach PS Ifraction; 3, Porphyridium cruentum PS I fraction; 4, Porphyridium cruentumthylakoid fraction.Molecular mass markers are on the right. Antisera used included thea-FCP antiserum specificfor a diatom FCP complex (panel A) and the o-CPla antiserum(panel B).75cross-reactivity with the c-CP1a antiserum in the size range typical for a LHC(Fig. 3.6, lane 1,panel B).A second antibody, c-FCP, was used to assess relatedness of the low molecular masspolypeptides of Porphyridium to the FCPs of the chromophytic algae (Fig. 3.6,panel A). Thea-FCP antibody cross-reacted with five polypeptides from the P. cruenturn thylakoidfraction(Fig. 3.6, panel A, lane 4). Interestingly, the 19 and 19.5 kDa bands appearlessimmunologically reactive to this antiserum compared to their reaction withthe c-CP1aantiserum. The 20 and 23.5 kDa polypeptides did not appear to cross-react atall with the a-FCPantiserum. These observations indicated a degree of structural divergence betweensome of thepolypeptides. At least three polypeptides (19.5, 22, and 23 kDa) in the P. cruentumPS I fractioncross-reacted with the o-FCP antiserum (Fig. 3.6, panel A, lane 3) . Again, the 20.5kDapolypeptide in the PS I complex was only weakly detected and must have beenremoved duringpurification.These results show that the complex pattern of polypeptides immunologically related tothe LHCs in the unicellular red alga, Porphyridium, is comparable to that in the filamentous redalga, Aglaothamnion, except for differences in the apparent size of the polypeptides.This maybe due to the use of a different SDS-gel system. No polypeptides in the 18-23 kDarange weredetected in the thylakoids of the cyanobacterium, Nostoc, with the ct-FCP antiserum(Fig. 3.6,panel A, lane 1). This supports the idea that the cyanobacteria do not possessintrinisic antennaerelated to those of algae or terrestrial plants. However, a large molecular masspolypeptide(69 kDa), of unknown identity, was weakly immunostained with the x-FCP antiserum.Thylakoid and PS I fractions from the Chl a + b-containing prochlorophyte,Prochlorothrix hollandica (a gift from Georg van der Staay), were immunoblotted with thebarley o-CP1a antiserum (Fig. 3.7, lanes 1 and 2) to search for evidence of any CAB relatedLHCs. Neither of these fractions showed any immunologically reactive polypeptides in the sizerange of a typical LHC (18-34 kDa). However, the CAB polypeptides were detected in thespinach thylakoid control, as expected (fig. 3.7, lane 3). In both the thylakoid and the PS Ifractions of Prochlorothrix, the core complex of PS I was detected at 65 kDa.76Figure 3.7123M66453121Immunological analysis of Prochlorothrix hollandica PS 1(1) andthylakoid (2) fractions.A spinach control (thylakoid fraction) is also shown (3). Molecular mass standards (M)areshown on the right. The blot was probed with the o-CPla antiserum.773.4 DiscussionAnalyses with the unicellular alga, P. cruentum, demonstratedthat there are severalpolypeptides that are immunologically related to both the CABsand FCPs (Wolfe et al. 1994).Furthermore, some of these are specifically associated witha purified PS I complex (Wolfe et al.1992; Wolfe et al. 1994). This is significant as it is the firstdemonstration of such membraneintrinisic light-harvesting antennae within the Rhodophyta.Despite recent analyses of red algalthylakoid membrane polypeptide composition, CAB-relatedLHCs have not been previouslyreported. The LHCs in Porphyridiurn also cross-reactto different extents with theCAB andFCP-specific antisera. This indicates that though the differentpolypeptides share commonimmunologically reactive epitopes, there is structural variabilitybetween these antennal proteins;this may be related to different functions. These observations suggestcloser evolutionaryrelationships between the red, green and chromophyticplastids than previously thought. Recentprotein sequencing data from these polypeptides have confirmedtheir relatedness to the LHC Iproteins of terrestrial plants (Beth Gantt, unpubl. data).When I examined a second red alga, Aglaothamnionneglectum, up to five polypeptidesin the thylakoid membrane fraction were antigenicallyrelated to the Chl a + b-binding proteinsof terrestrial plants. Fewer cross-reacting polypetides were foundin the sucrose gradientfraction, probably a result of selective loss during the detergentsolubilization procedure. It hasbeen immunologically determined that fraction II fromthe sucrose gradient containspolypeptides from both PS I and PS II; therefore, it isnot possible to conclude whether they areassociated specifically with PS I or PS II. By analogywith Porphyridium, I would expect anassociation of these LHCs with PS I in A. neglectum. However,this does not exclude thepossibility that CAB/FCP-related polypeptides are associated withPS II. In both red algae, therewere fewer detectable LHCs in the detergent solubilized fractionsas compared to the thylakoidfractions. This potential for degradation may be one reason thesepolypetides were notpreviously reported.It is significant that CAB/FCP-related polypeptides were foundin representatives of both78the Porphyridiales (Porphyridiuin) and the Ceramiales (Aglaothamnion), as they representverydiverse lineages separated by large evolutionary distances (Garbary and Gabrielson1990). Thissuggests that these LHC-related polypeptides are not limited to a specific taxon; likelybeing acommon feature of the red algae. I suspect that similar polypeptides will be detectedin other redalgal orders.Plastids are thought to have evolved from endosymbiotic cyanobacteriaand/orprochlorophytes; therefore, it was significant to find a lack of CAB/FCP-relatedpolypeptides inNostoc and Prochlorothrix. This is particularly significant with theprochlorophytes as it hasbeen demonstrated that they possess Chi a + b + c-binding antennae. The lackof immunologicalrelatedness of these antennae has been demonstrated by others (Hiller andLarkum 1985;Bullerjahn et al. 1990). In addition, preliminary sequence informationfrom the prochiorophytes,Prochioron (Hiller & Larkum, unpubl.), Prochiorococcus (LaRoche& Partensky, unpubl.) andProchlorothrix (van der Staay & Green, unpubl.) showsthat the 34 kDa antennae are related tothe isiA gene product and to the inner Chl a antenna, CP43. Therewere no similarities to theCABs or the FCPs. This proves that the prochiorophyte LHCs evolvedindependently of theCABs and FCPs.The presence of CAB/FCP-related polypeptides in the red algae andtheir absence in thecyanobacteria and prochlorophytes is significant in termsof both the evolution of the light-harvesting antennal proteins and the evolution of the chioroplast.The significance with regard tothe evolution of the light-harvesting proteins as an extended genefamily will be dealt with inChapter 5. Much of the remaining discussion will deal withthe implications of theseobservations for the different hypotheses of chloroplastevolution (discussed in section 1.6),especially with regards to the red and green algae. In analyzing chloroplastevolution, bothchioroplast and nuclear characters can be used (see table 3.1). Plastidcharacters, includingplastid encoded genes and plastid localized gene products encoded inthe nucleus (such as theCABs and FCPs), were probably present in the original endosymbiontand comparisons willnormally reflect the ancestry of the chloroplast. Cytoplasmic/nuclearcharacters will generallyreflect the ancestry of the original phagotrophic host.Comparing evidence of both types is79necessary to develop a better understanding of the possible evolutionarypathways leading to thechloroplast.Many of the plastid encoded characters point toward a monophyleticscheme ofchloroplast evolution. These include the plastid 1 6s rRNA, the psbA,tufA and atpB genes (seetable 3.1) which have been reviewed by Morden et al. (1992). Recentphylogenetic analysis ofthe plastid encoded atpB gene (H-ATPase, f3-subunit) suggeststhat there is a deep divisionbetween the green and non-green algal chioroplast lineage’s,though they cluster together withinthe cyanobacterial line, indicating a single origin for the chloroplast(Kowallik 1993; Douglasand Murphy 1994).Table 3.1 Some genes commonly used to infer phylogeneticrelationships amongstphotosynthetic organismsgene protein /functionlocationpsbA Di-core PS II reaction centre proteinChioroplastatpB 13-subunit of chloroplast ATPaseChioroplastrbcL rubisco large subunitChloroplastChloroplastrbcS rubisco small subunit/nucleartufA elongation factor TuChioroplast1 6s rRNA small subunit rRNAChloroplastglyceraldehyde-3-phosphateGapAJBdehydrogenasenucleus1 8s rRNA small subunit rRNA nucleusHowever, there is evidence that is consistent with a polyphyletic interpretationofchloroplast evolution. A seven amino acid deletion in the C-terminus ofDl (psbA gene) inProchlorothrix, the green algae and in the terrestrial plants but not in theDl protein ofcyanobacteria, red algae and the chromophytes is suggestive of a polyphyleticorigin of thechloroplast (Morden and Golden 1989; Golden et al. 1993). This emphasizesa character sharedby a prokaryotic! chioroplast pair containing Chl a and b to the exclusionof another prokaryotic!80chioroplast pair containing PBSs or Chi a + c —satisfyingthe criterion for a polyphyletic origin.However, it has recently been reported that theprochiorophyte, Prochioron dideinni,lacks thisdeletion, indicating that it is not a common occurrenceamongst the prochlorophytes (Lockhartetal. 1993).It is reasonable that insertion/deletion (indel) eventswould be consideredgoodphylogenetic indicators as two independent deletionevents in an identical areaseems unlikely.However, I argue that the significance of thisparticular indel may have beenover estimated.Besides having representative sequences fromrelatively few taxa, there are twoknownvariations in the Dl indel location; one in the greenalga, Chlamydomonasreinhardtii (Ericksonet al. 1984), and the other in a euglenophyte,Euglena gracilis (Karabin et al.1984). In theseexamples, there is an eight and 16 aminoacid deletion at the C-terminal end of Dl,respectively.The fact that these algae possess additional or separatedeletions different from the otherChl a + b-containing organisms suggests that there arelower constraints on the 9-16 aminoacidsat the C-terminal end. Significantly, this region of Dlis post-translationally removedduringprocessing into the mature form.In addition, the C-terminal region is neitherrequired forprotease binding nor for recognition (Taguchiet al. 1993). Phylogenetic analysis ofthe wholepsbA gene is consistent with the monophyleticview of chloroplast evolution withProchlorothrixclustering with the cyanobacteria, separatefrom the eukaryotic algal groups (Mordenet al.1992). All things considered, itseems that the deleted region has an ambiguousevolutionaryhistory of questionable importance. Furthermore,based on the accumulation of molecularevidence from plastid/cyanobacterial characters,a direct evolutionary relationship between theprochlorophytes and the green algal chioroplasts arenow considered less likely (Turner et al.1989; Morden et al. 1992; Golden et al. 1993; Swiftand Palenik 1993).Plastid characters that do offer strong evidence fora polyphyletic origin of thechloroplast are the plastid encoded rbcL and rbcS (rubisco largeand small subunit) data whichclearly show the Chl a + b-containing eukaryotes in onelineage and the red algae andchromophytes in the other. In these studies,the cyanobacteria are closely related totheChi a + b-containing lineage, while the most similarprokaryotic ancestor to the red algal/81chromophyte line is the f3-purple bacterium,Alcaligenes eutrophus (Douglaset al. 1990).However, due to the lack of phycobilisomes,Chi a, and oxygenic photosynthesis,a 3-purplebacterium is not a logical choice fora hypothetical chioroplast ancestor.Several hypotheseshave been put forth to explain the relatednessbetween the 3-purple bacterialand chromophyterbcLIS sequences: (1) lateral gene transferof the rbcLIS operon froma purple bacterium toanalga leading to the red algal/chromophytelineage (Boczar et a!.1989b); (2) the possibilitythattwo organisms provided genes to thephagotrophic host (Assali etal. 1990); (3) since somepurple bacteria possess two rbcL genes, ithas been suggested that theoriginal eukaryoticphotoautotroph had two copies ofthe rbcLIS operon with thegreen algal and chromophytelineages each retaining a different copy;(4) finally, transfer of rbcLfrom an oL-purple bacterium,the proposed mitochondrial ancestor,to the chloroplast ancestor hasalso been suggested (Martinet al. 1992). The significanceof the rbcL data will remainan issue that will need tobe resolvedthrough the analysis of otherdata.The evidence supporting or refutingeither hypothesis of chloroplastevolution (asdiscussed in section 1.6 and above)has been plagued by conflictingand ambiguous evidence.Deciding between alternativehypotheses of chioroplast evolutioncan be done byapplying thefollowing two criteria, as emphasizedby Reith and Munholland (1993):first, the polyphyleticview of chloroplast evolutionwould be supported when characters(morphological or molecular)are found in one chloroplasttype that are shared with a putativeprokaryotic ancestor, totheexclusion of another chloroplasttype-prokaryote pair. Second,if chloroplast evolutionis to bedeemed monophyletic then thedifferent chloroplast types shouldall share a certain trait orcharacter, which is more relatedto each other than eitheris to a prokaryotic (cyanobacterial)ancestor (Reith and Munholland1993). This trait or charactercould then be interpretedashaving been derived withina single lineage, following the primaryendosymbiotic event that leadto the first chloroplast containingeukaryote.The immunological relatednessof the red algal polypeptidesto the CABs and FCPs issignificant because it providesevidence that links organismspossessing the three majorantennalsystems: the Chi a + b-containingantennae, the Chi a + c-containingantennae, and the82phycobilisomes. Previously, these characters formedthe basis of an algal taxonomicsystem thatseparated the Chl a -- b containing green algae (Chiorophyta),the Chi a + c containingchromophytic algae (Chromophyta), and thePBS containing red algae (Rhodophyta)into majordivisions. This finding also demonstrates thatthe thylakoid membrane intrinisicLHCs and thesoluble phycobilisome types of antennal systems canbe present in the same organism.The lack of detectable, immunologically relatedLHCs from the cyanobacterium,Nostoc,and the prochlorophyte, Prochlorothrix,suggests that the intrinisic light-harvestingantennaeevolved after the primary endosymbiotic eventthat gave rise to a photosyntheticeukaryote. Thepresence of CAB/FCP related LHCs inall eukaryotic algae, to the exclusion ofthe putativechloroplast ancestors (cyanobacteria and prochiorophytes),is consistent with the monophyleticview of chloroplast evolution because itseems unlikely that structurally similarproteins wouldevolve independently in the red, greenand chromophyte algae from prokaryoticprecursors.These data also support the idea that both thePBS and the CAB/FCP antennal systemsexisted inthe first photosynthetic eukaryote from whichthe green algae and red algae diverged.Divergence of the intrinisic LHCs withthe gain of Chl c (in the chromophytesandprasinophytes) and the loss of PBSs(in the chiorophytes and chromophytes)could explain thedifferences in antennal systems presently observed(Cavalier-Smith 1982).There can be alternative interpretationsor hypotheses invoked to explain the LHCdistributions between the prokaryotes and eukaryotes.One alternative is that the cyanobacteriaand prochlorophytes both contained CAB/FCP-relatedantennae but they were selectively lostover time while being retained in the immediatechloroplast ancestor (Bryant 1992). Such ascenario does not necessarily exclude a monophyleticview of chloroplast evolution.Alternatively, the different LHCs may have evolvedindependently following separatechloroplast acquisitions (polyphyletic) leading to themajor algal divisions. However, thisalternative requires the acceptance ofadditional evolutionary steps and seems less likely.Nevertheless, the independent evolutionof LHC proteins from related cyanobacterial precursorsis possible and could explain the overall low degreeof similarity between the CABs and FCPs.Some possible molecular sources from whichthe LHC antennae may have evolved are discussed83in section 5.4.6 (Chapter 5).The acceptance of a monophyletic chioroplast origin requires the assumptionthat the redand green algae are related and have diverged from a commonancestor. Such a relationship wasproposed by Cavalier-Smith (1981, 1987) who grouped the red and green algaetogether in thekindom Plantae. In order to demonstrate a evolutionary relationshipbetween the red and greenalgae it will be necessary to show similarities between nuclear encodedcharacters in addition tothe chloroplast characters.More recently, the nuclear encoded (but chloroplast localized) glyceraldehyde-3-phosphate dehydrogenases (GapAIB) from two different red algae have beensequenced andphylogenetic analyses suggests that the red and green algae form a monophyleticgroup with acyanobacterial ancestor (Zhou and Ragan 1993; Liaud et al. 1994). Thesestudies also indicatethat the green algal and red algal lineages separated very earlyin evolution. Moreover, thetransit peptide from the GapAfB precursors in red algaeresembles the chioroplast targetingtransit peptides of terrestrial plants (Zhou and Ragan 1993;Liaud et al. 1994). In vitro transportstudies with the transit peptide of the nuclear encoded y-subunitof phycoerytherin inAglaothamnion has shown that it can direct rubisco (RbcS)into pea chloroplasts (Apt et al.1993), suggesting the red and green algae have a similarchioroplast import mechanism. Thismay be more easily explained by a monophyleticorigin of the red and green algal chloroplastsdue to the perceived difficulty in acquiring import capabilities(Cavalier-Smith 1982; Cavalier-Smith et al. 1994b).A better idea of the relationships between the redand green algae can be achievedthrough the analysis of nuclear encoded charactersfrom the two groups. This can giveinformation on the nature of the eukaryotic ‘hostfor the chloroplast. The most widely utilizednuclear character to date has been the small subunitrRNA (SSU, 18s) (Bhattacharya et al. 1990;Douglas et al. 1991; Hendricks et al. 1991; Maier etal. 1991; Cavalier-Smith et al. 1994b) . Thenuclear encoded large subunit rRNA (LSU, 23s) hasbeen used infrequently (Perasso et al. 1989)due to its larger size and the smaller dataset. Most ofthese studies did not support a commonorigin of the red and green algae. A recent maximumlikelihood analysis of nuclear encoded84SSU rRNA sequences, however, showed that the red and green algae grouped together on thesame branch as would be expected for a monophyletic origin (Cavalier-Smith et al. 1994b). Thisgrouping does not occur when a distance matrix method is used, which was the case in all thestudies mentioned above (Cavalier-Smith, pers. comm.; Cavalier-Smith et al. 1994). Comparingthe studies mentioned above, the relationships between the red algae and other groups were notconsistent. This was probably due to the inclusion of different taxa, the number of charactersincluded in the analysis and the limitations of the method used. Many of these studies also didnot give an estimate of the tree node reliability, so it is hard to determine how consistent thedeeper branches of these trees are.Overall, there is accumulating evidence suggesting a monophyletic origin of thechloroplast, though the question is far from settled. If true, this would mean that the red andgreen algae share a common ancestor and that the acquisition of a chioroplast from a prokaryoticancestor occurred only once. Divergences over a long period of time would have resulted in thedifferences between the two groups today. The presence of CAB and FCP-related LHCs inthered algae provides some evidence for the monophyletic origin of the different chloroplasttypes,though other alternative evolutionary pathways can be envisaged. At the moment,there isinsufficient evidence to clearly decide whether chloroplasts with twosurrounding membraneshave arisen through one or multiple endosymbiotic events. The analysis of morecharacters froma diverse array of red, green and chromophytic algae along withcyanobacteria should help todetermine relationships amongst these groups and help to resolve conflictinginterpretations ofsome data.85CHAPTER 4Characterization of Fcp cDNAs from Heterosigma carterae4.1 IntroductionThis section examines cDNAs encoding the FCP familyof proteins in Heterosigma. Atthe beginning of this project only a single chromophytealgal FCP sequence had beencharacterized (Grossman et al. 1990). Asthe chromophytes consist of many distinctphyla, Ithought this research would beginto fill a gap in the literature and aid in ourunderstanding ofthe relationships between the different antennae andtheir diversity. As well, it wouldbe animportant piece of information for assessing the evolutionaryrelationships amongst the FCPsand the CABs of the terrestrial plants and green algae.In this chapter I will first describe thecloning and sequencing of a number of cDNAs encodingFCPs. I will then examine thecomplexity of the Fcp gene family and concentrateon the relationships of the Heterosigmasequence to other FCPs and the CABs. A cDNA librarywas constructed and screened with anucleic acid probe in order to clone cDNAs encodingthe FCPs. This will complement theprotein characterization work in Chapter 2.In addition, I was interested in the processesinvolved in the targeting and translocationof proteins into the chloroplast. As these organismspossess two additional membranes around the chloroplast,an examination of the leadersequences of nuclear encoded, chioroplast localized precursorsshould provide some clues on thenature of the transport mechanism in the chromophytes.864.2 Materials and Methods4.2.1 Tryptic fragment sequencingMaintenance of Heterosigma cultures and preparationof thylakoids havebeen previouslydescribed in Chapter 2. Sixty jig of chlorophyll (fourlanes at 15 jig/lane) from sucrosegradientfraction one (Fl, Fig. 4.2; chapter 2) was separatedon a 12-17% gradient gel (Chapter 2) andtransferred to nitrocellulose (Biorad) in 50 mM sodiumacetate pH 7.0 at 200 mA, for 1 8hours at4°C. The transferred band was stained with amidoblack (0.1% amido black, 45%H20, 45%methanol, and 10% acetic acid) and destained in 50%methanol/l0% acetic acid. The proteinband was cut out from the membrane, digested on thesupport with trypsin (Aebersold et al.1987) and separated by narrow-bore reverse phaseHPLC on a Waters peptide analyzer equippedwith a Vydac C-4 column. Individual peptides werecollected manually and sequenced usingstandard pulsed-liquid phase or solid-phase sequencingprocedures (Aebersold et al 1990).4.2.2 Heterosigma DNA and RNA isolationGenomic DNA was isolated from 4 liters of late logphase cells. Cells were harvested bycentrifugation at 1500 xg for 10 minutes. The pellet was resuspended in 2 ml extraction buffer(0.05 M Tris-HC1 pH 8.0 and 0.05 M EDTA pH 8.0) thatwas added while vortexing gently.16 ml of lysis buffer (2% sarkosyl, 10mM EDTApH 8.0, and 20 mlvi Tris-HC1 pH 7.6) wasthen added and gently mixed. Proteinase K(8 mg) was added and swirled at 37°C for 1 hour tobreak down proteins. Four ml of 5 M NaC1 was addedand mixed. This mixture was extractedwith an equal volume of Tris equilibrated phenol (pH7.6) for 30 minutes at room temperature.Following centrifugation at 9000 xg for 20 minutes, the supernatant was extracted with an87equal volume of chloroform:butanol (4:1),centrifuged as above and repeated. TheDNA wasethanol precipitated with an equal volumeof 95% ethanol and spooledout with a glass rod. TheDNA was resuspended in T.E. pH 8.0 andre-extracted twice with phenol:chloroform,as above.The supernatant was ethanol precipitatedand resuspended in a total of 11 ml T.E.pH 8.0. DNAwas purified by CsC1 density gradient ultracentrifugation,using standard protocols(Sambrooketal. 1989).Total RNA was isolated from 4liters of log phase cells of Heterosiginausing aguanidinium thiocyanate extraction method (Chromczynskiand Sacchi 1987). Cellswereharvested at 1500 xg for 10 minutes and resuspended in guanidinium solution(4 Mguanidinium thiocyanate, 25 mM sodiumcitrate pH 7, 0.5% sarcosyl, and 0.1%13-mercaptoethanol) at 1 ml per 0.1g cells, followed by hand homogenization in a 5 mlPotterElvehjem homogenizer. The followingsolutions were then added to thehomogenized extract:1/lOthvolume of 2 M sodium acetate pH 4,1 volume water saturated phenol, and115thvolumechloroform:isoamyl alcohol (49:1). Thesample was mixed following each addition.Thesample was vigorously vortexed for 10seconds, cooled on ice for 15 minutes andcentrifuged at10 000 x g for 20 minutes. An equal volumeof isopropanol was added to the supernatantinorder to precipitate the nucleic acids. Aftercentrifugation at 10 000 xg for 20 minutes thepellet was resuspended in 1.5 ml guanidinium solutionand again precipitated with an equalvolume of isopropanol. Precipitated sampleswere resuspended in T.E. and storedat -80°C. Thepoly A mRNA used in the constructionof the cDNA library was isolated fromtotal RNA usingan oligo (dT) cellulose (Pharmacia) column(Sambrook et al. 1989).4.2.3 cDNA library construction and screeningThe cDNA library was constructed from5 ig of poly A mRNA. The poiy AmRNAwas considered to be of high quality sincein vitro translation using a wheat germsystem(Promega) yielded many proteins in the 14-70kDa range. The library was constructedusing the88lambda ZAP II kit from Stratagene accordingto the manufacturers instructions. Thesynthesis ofcDNA was initiated with a poly-T primer with an XhoI adapter and after the secondstrandsynthesis, the ends were blunt ended and EcoRI linkers were ligated to the cDNA.Directionalcloning was achieved by ligating the cDNAsinto an Xho 1/EcoR I cut 2ZAP vector.Screening of the amplified library wasdone using a nucleic acid probe createdthrough apolymerase chain reaction. The amino acid sequenceinformation from the trypticfragments(Fig. 4.2) was used to create degenerateprimers for amplification of a Fcp-specificgeneproduct. The degenerate primers were basedon tryptic fragments Ti (TVEIK)and T3(YDLAGDQ) as shown in Figure 4.2. This wouldgive an amplified fragment of approximately100 bp if the alignments with the diatomFCP were correct. The primers includeda nonspecificadapter sequence with an Xba I or Pst Irestriction site in the 5’ region (indicatedin bold). Thesequences of the primers used were as follows:5’-GCTCTAGA AC(GICIT/A)T(TIGICIA)GA(AJG) AT(T/C/A) AA(A/G) CA(T/C)GG-3 for P1 and 5’-CGCTGCAG TG(AIG)TC(AIGICIT) CC(AIG/CIT) GC(AIGICIT) A(G/A)(G/A)TCA (A/G)A -3’ for P2. The 36 cyclePCR reaction profile was as follows: cycle1—4, 95°C-2 mm., 52°C-i .5 mm.,and 72°C-15 sec.;cycle 5—24, 95°C-i mm., 52°C-i .5 mm.,and 72°C-30 sec.; cycle 25—>36, 95°C-imm., 52°C-1 mm.; and 72°C-i mm.. The reaction included2.0mMMgC12, 100 pmol of each primer (P1and P2), 50 mM KC1, 10 mM Tris-HC1 pH8.3, 0.2 mM dNTPs and 0.5 mg Heterosigmacarterae genomic DNA as the target sequence. Thereaction was done under mineral oil with0.5 units of Taq polymerase (AmpliTaq, Perkin-Elmer-Cetus).The reaction mixture wasextracted with phenol-chloroform (1:1), ethanol precipitatedand run on a 4% Nusieve agarosegel (Mandel Sci.) along side Xi74-Haeifi markers (NEBL). A 118 bp fragment was isolatedusing Whatman DE-81 paper and directlylabeled withct-32P-dCTP (3000 mCi/mmolAmersham) using a random primer labeling kit (BRL),according to the manufacturersinstructions. The cDNA library was screened at highplaque densities (10 000 pfu/plate) for thefirst round of screening. Further roundsof screening were done at low densities so thatindividual plaques were well separated. Plaque lifts,denaturation, fixing and hybridization were89done as descibed in the Stratagene cDNA kitinstruction manual and accordingto Sambrook etal. (1989).Prospective positive clones went through threeto four rounds of screening until plaqueswere homogeneous. The recombinant pBluescriptphagemid (with insert) wasexcised from thek-ZAP vector using the ExAssist/SOLR system (Stratagene).Miniprep plasmid DNAwasprepared using an alkaline lysis method(Sambrook et al. 1989) and was oftensequenced directlyafter RNAse treatment, two phenol-chloroformextractions and ethanolpreciptation. Large scaleplasmid preparations were also done using analkaline lysis method followedby plasmidprecipitation with polyethylene glycol (Sambrooket al. 1989). Double stranded sequencingwith35S-dATP was done using the dideoxy chain terminatingmethod with a T7 polymerase(Pharmacia). Standard sequencing gelreceipes and running conditions were used(Sambrook etal. 1989). Sequencing gels were lifted directly offplates onto Whatman 3MM paper,withoutfixing, and dried for 45 minutes at 80°Cunder vacuum.4.2.4 Rapid Amplification of cDNA Ends(RACE)As the isolated cDNA clones were truncated nearthe N-terminus, it was necessary to usealternative methods to clone this region.The 5’ ends of the truncated cDNA clonesweredetermined using the rapid amplificationof cDNA ends (RACE) technique (asillustrated inFig. 4.1) using a modification of previouslydescribed methods (Frohman 1990; lainet al. 1992;Schuster et al. 1992). Reverse transcriptionof 1 tg of poly-A mRNA (heated at70°C, 10 mmand quenched on ice) was done using 200units superscript II reverse transcriptase(BRL),200IIMdNTPs, 20 units RNAse inhibitor (RNasin,Promega), 10 mM DTT, and 10 pmol ofagene specific primer (gspl) located near the5’ end of the truncated cDNA clone[5’AATGAAGCCGATGGTCT3’j. Thereverse transcription reaction was incubatedat 42°C for60 minutes followed by a 50°C incubationfor 15 minutes. The cDNA was treatedwithRNAse H (Pharmacia) at 42°Cfor 15 minutes to remove the RNA template.The reverse90gsp 1mRNA —Areverse transcribecDNAAadd poiy A tail withterminal transferaseaaaaaaaaAgsp2-adpaaaaaaaattttttttdt-adpAamplifyFigure 4.1 Rapid amplification of cDNAends (RACE) technique,illustrated. The gene specific primers aregspl and gsp2.The adapter portion of the primer, adp, is indicated.Mechanism is explained in the text.transcription primer and dNTPs were removedby ultra-filtration through ultrafree-MCcentrifugation filters (30,000 NMWL, Millipore) (Jamet al. 1992). Following a washing step inthe ultra-filtration unit, the samples were concentratedunder vacuum to approximately 10 pi. Apoly-A tail was then added to the extended cDNA byusing terminal deoxy transferase(Pharmacia) in a standard PCR buffer (10 mM Tris-HclpH8.3, 50 mMKCI, 25 mM MgCI2)with2 mM dATP (Schuster et al. 1992).Amplification of the cDNA was doneusing a poly T primer with a 5’ adapter (indicatedin bold) [5’ GACTCGAGTCGACATCGTTTTTTTTTTTTTTTTT3’] and a second, nested91gene specific primer (gsp2) closer to the 5’end of the truncated clone, also witha 5’ adapter[GACTCGAGTCGACATCGAGCAGGCAGAGCAGACA3’].The amplification reactionwascarried out as before (section 4.2.3) but with 2.5 mMMgCI2and 10 pmol of each primer.Thereaction was denatured at 95°C for 5 mm.and brought down to 80°C when2 units of Taqpolymerase were added and the reaction layered withoil. The reaction then continuedwith thefollowing profile: cycle 1, 55°C-5 mm., 72°C-20 mm.;cycle 2—>37, 95°C-45 sec., 55°C-imm.,72°C-2 mm.; cycle 38, 95°C-45 sec., 55°C-i mm., 72°C-lOmm. The amplified productwasisolated, as previously described, and clonedin a pBluescript II vector (Stratagene)that was cutwith EcoR V and tailed with ddTTP (Holtonand Graham 1991).4.2.5 Southern blotsFor a Southern blot, 4 jig of genomic DNA wasdigested with an appropriate restrictionenzyme using the conditions recommendedby the manufacturer. In order to ensure completedigestion, 10 units of enzyme were addedat one hour intervals for approximatelythree hours,mixing gently at each addition to avoid mechanicalshearing. DNA was run on large (20X28 cm) 0.8% agarose gels in a Tris-Borate-EDTAelectrophoresis buffer (Sambrooket al. 1989)with 100 jig/mi ethidium bromide.Gels were transferred onto Hybond-N nylonmembrane(Amersham) by capillary action in 20XSSC, as described in Sambrook et al. (1989). Nucleicacids were fixed onto the membraneby baking for 2 hours at 80°C. Membranes wereprehybridized in Church buffer (0.25 Msodium phosphate, 7% SDS, 1 mM EDTA) for 1hourat 65°C (Church and Gilbert 1984).Hybridization buffer was changed prior to addingdenaturedprobe and then incubated for 16 hours. Membraneswere typically washed at low stringency(1X SSC/0.i% SDS at 65°C) three times (15 minuteseach) or at a higher stringency wash(0.1X SSC/ 0.1%SDS at 65°C), as required.924.2.6 Northern blotsRNA was quantitated spectrophotometrically, and an appropriateamount precipitatedand resuspended in a RNA loading buffer. Samples were heatedat 70°C for 10 minutes andquenched on ice for 5 minutes prior to loading the gel.A 1.5% agarose-formaldehyde gel madewith a lx MOPS buffer (20 mM MOPS pH7, 5 mM sodium acetate,1 mlvi EDTA) and 6% (v/v)formaldehyde (2.2 M) was used. The gel was run ina 1X MOPS buffer with 2.2 Mformaldehyde (Douglas et al. 1990). RNA was transferred to Hybond-N,fixed and hybridizedas in section 4.2.5.4.3 Results4.3.1 Identification and characterizationof the Fcpl and Fcp2 cDNAsA cDNA expression library was constructed from HeterosiginamRNA. Because of this,the initial several rounds of screening were attemptedwith both the cx-FCP and o-CP 1 a antisera.However, this method was not successful and wasabandoned. An alternative was to screen thelibrary with a nucleic acid probe. A heterologous Fcpgene probe from a diatom did nothybridize to Heterosigma genomic DNA; therefore,I had to obtain protein sequence informationdirectly from a Heterosigma FCP in order to generateprimers for the amplification of ahomologous nucleic acid probe.The sequences of seven tryptic fragments were obtainedfrom the 19.5 kDa polypeptidein fraction 1 (Fl) of the sucrose gradient (Fig. 4.2,Fl). This polypeptide was easily purifiedfrom the other three dominant FCPs normally presentin the thylakoids (Fig. 4.2, Chapter 2).The 19.5 kDa polypeptide was immunologically relatedto the diatom FCP sequence (Fig. 4.2,93Fl). A total of 60 amino acid positions were obtained from the sequencingof seven trypticfragments. Only two of these tryptic fragments (Ti and T2) could be unambiguouslyalignedwith the Phaeodactylum FCP sequence and both were within the firstputative membranespanning region. The sequence information from tryptic fragments Ti andT2 was used to createdegenerate primers for the amplification of a Fcp specific probe; this wasused to screen thecDNA library.30 —21.5 —Figure 4.2Thy Fl Tryptic FragmentsT1- T V E I KT2-.XSMLAYLG?FLVTFAGT3- LPGAYDLAGDQFSSLP1’T4- S/LPGAYDLAGDQFT5- E E L/A E/A G/A GX EAT6- IINSLLGSPVEFT7- IINSLLGSPVE/DFNAGFHTryptic fragment sequences determined from the abundant protein in fraction 1 (Fl) ofthe sucrose gradient (Chapter 2). The left panel shows a western blot of a thylakoid fraction(Thy) and fraction 1 (Fl) immunostained with the ct-FCP antiserum. Molecular mass standardsin kDa are indicated on the left. Sequences from the tryptic fragments Ti -T7 areindicated onthe right. Ambiguous amino acids are indicated by a question mark.94More than 90 000 recombinant cDNA clones werescreened with the PCR amplifiedprobe resulting in the detection of 3 clones. However,all were truncated upstream of the firstputative membrane spanning region. A re-screening withone of the truncated cDNA clonesdetected an additional 30 positive clones of which 15were isolated; most were smallerthan theoriginal. The RACE technique was used to amplifythe 5’ end of the Fcp cDNA directly frompolyA+rnRNA using a reverse transcription reaction followedby PCR amplification (see Fig.4.1, methods). A number of nearly identical fragmentsdiffering in only a few nucleotides,mainly within the third codon position, were obtained fromthe amplification reaction. TheRACE product that was identical to the truncated cDNAclone in the 200 bp overlapping regionwas used to generate the full length sequence. The sequencingstrategy and the proportion of thefull length sequence determined from the RACE productand the cDNA clone are indicated inFigure 4.3.The cDNA encoding the FCP (Fcp 1) is 858 base pairs (bp) long withan open readingframe of 625 nucleotides (Fig. 4.4). This gives an immaturepolypeptide with 210 amino acidresidues which is typical for the FCPs (Grossman et al. 1990; Aptet al. 1994). The proposedtransit peptide cleavage site is at serine 36 (Fig 4.4),by analogy to the Phaeodactylumprocessing site (Bhaya and Grossman 1991). Withthe cleavage site at Ser 36, the mature proteinwould have a calculated molecular mass of18.9 kDa. This is very close to the estimated size of19.5 kDa based on the SDS polyacrylamide gel (Fig.4.2; Chapter 2). A typical polyadenylationsignal at the 3’ end of the cDNAs (AATAAA) was not present inany of the cDNA clones.In line with the proposed nomenclature for the Cab genes (Jansson etal. 1992), I use theterm Fcp 1 to refer to a specific cDNA/gene type (type 1) that includesall the nearly identicalmembers. As I have only a single complete cDNA of Fcp type 1,this term refers directly to thesequence shown in Figure 4.4.The tryptic fragment sequences were identical to the inferred proteinsequence in allpositions except for two from fragment T2. These residuesare indicated by square brackets [unFigure 4.4. The first conflict has a Tyr [Y] instead ofthe inferred Ile 82 (I). However, the latter95ORFnon-codingBstXBstElIregionBsmAStyl BssHII PvuIl Apal-100 200 300 400 500 600 700800___________RACE_ProductcDNA cloneFigure 4.3Schematic representation of the Fcp 1 cDNA and the sequencing strategy used. Shading representsthe FCP open reading frame (ORF). The sections of the Fcp sequence determined from theRACEproduct and the cDNA clone are indicated. Key restriction sites are given and the arrows indicatethe sequencing done on both strands.96•+l/1 •28/1ONucleotide sequence of the Fcp 1 cDNA from Heterosigma. The nucleotide and amino acidpositions are given above the first nucleotide of the codon(•).Tryptic fragment sequences areshown in bold below the appropriate amino acid. Ambiguous amino acids are indicated by aquestion mark (?). Amino acids not matching the derived gene sequence are indicated by squarebracketsEl.A putative processing site is indicated by an open triangle (A.aaa atg tct ctc aag ctc gcc acc ctc gct gcc gcc ctc atg ggtgcc tcc gcc ttc gtgmet ser leu lys leu ala thr leu ala ala ala leu met glyala ser ala phe val•58/20 •88/30gcc ccc aac aag atg ggc gtg gcc atg agc ttc gagaacala pro asn lys met gly val ala met ser phe glu asnzX.118/40gag atc ggc gtc cag gcc cct ctg ctt ctg gat gag gccglu ile gly val gln ala pro leu leu leu asp glu ala•178/60gac cag gag cgg ttc gac cgc ctc ggc cgc atc tcc atgasp gln glu arg phe asp arg leu gly arg ile ser metX S M.238/80ctt gcc atc ctt ggc cac ctg gtg cct ggc gct tac gacleu ala ile leu gly his leu val pro gly ala tyr aspL A [Y] L G F? L V P G A Y D.298/100ctg gct ggc gat cag ttc tcc agc ctg tct gct ctg cctleu ala gly asp gin phe ser ser leu ser ala leu proL A G DQF S S•358/120gct gct ggt gtg gcc cag acc atc ctg ggt ttt gct cagala ala gly val ala gln thr ile leu gly phe ala gln•4l8/140atc aag gag gag ctg gag gct gac gcc ggg tgg gat gacile lys glu glu leu glu ala asp ala gly trp asp aspE E L? E? G? G?.478/160gag aag aag gac tcc aag cgc gca gcc gcc cag atg ggtglu lys lys asp ser lys arg ala ala ala gin met gly•538/180atc ctt gcc ttg atg gtc cac gag atc atc aac tct ctgile leu ala leu met val his glu ile ile asn ser leuI I N S L•598/200ctg ggc tcc cct gtg gac ttc aac ttt ttt cct tct ccaleu gly ser pro val asp phe asnL G S P VE/D F Naaa att ttt gac ttt ctc ttg accggg ttg ctg tgc tgg gtt agt tcgtct ttt gtt ggt atc ttg act tgattt gaa cta tag tac cct tct ttcFigure 4.4aag tcc tcc tcc gcc ctg aaglys ser ser ser ala leu lys•l48/50ggc ttc tgg gac cct ctg ggcgly phe trp asp pro leu gly•208/70cgt acc gtc gag atc aag cacarg thr val glu ile lys hisT V El K•268/90acc acc gct ggt gtg cgt ctgthr thr ala gly val arg leuT [F]A G? L•328/llOctg ccc acc ggc ctg aag gctleu pro thr gly leu lys alaL PT.388/130ggc ttc att ggt ctg att gaggly phe ile gly leu ile glu.448/150tgc gag gcc cgc atg gac gctcys glu ala arg met asp alaX E A.508/170att gag ctg aac aac ggc cgtile glu leu asn asn gly arg•568/190cag ctg gac aac aac cct tacgin leu asp asn asn pro tyr•628/210gct ggc ttc taa aca gtg tttala gly phe OCHA G F H?ttc tga ctt ttc tgg gcc cag gct ctt tct agt gttcag ttt gta gcc tga tgc gtg ttt cat cat ata gacctt ctt ttg ctt gac tgc tca caa tct ttc ata cac.85897is consistent with the other FCP sequencesas there is an Tie and a Val at anhomologous positionin the FCPs from both Macrocystis andPhaeodactylum, respectively. The otherconflictinvolves a Phe [Fj in the sequence of T2 but a Thr 89(T) in the inferred protein.At homologouspositions in the other FCP sequences, there is either aglutamate (E), a glutamine(Q)or anarginine (R) residue. These conflicts couldbe the result of sequencing ambiguities.Alternatively, since the FCPs are encodedby several nearly identical multigenefamily members,the differences may reflect true sequence polymorphisms.A second cDNA clone from the firstround of screening was significantlydivergent fromthe first cDNA (Fcpl). An alignment ofthis second clone (Fcp2) with Fcplis shown inFigure 4.5. Because Fcp2 is not full length,the alignment starts at amino acidposition 73. Thetwo clones are 75% identical at the nucleotidelevel and 70% identical at the aminoacid level.When the chemical similarities of the aminoacids are considered, Fcp 1 and Fcp2areapproximately 82% similar. Most of the differencesbetween the two clones occurin theconnectors linking the putative membranespanning regions (MSR). Several changesin thesecond MSR were also observed, though most wereconservative substitutions.On the otherhand, few amino acid changes are observedin the first and third MSRs. Becausethis sequenceshows significant differences from the mainFcp 1 it may represent a different Fcptype;therefore, it is referred to as Fcp2 (Fcp type 2). However,such a designation requires furthercharacterization.A wide band with an average size of 0.98kb is detectedusing the Fcpl clone to probetotal RNA (Fig. 4.6); this is consistent with the sizeof the cDNA clone. The wide hybridizationsignal indicates that there is probably a collectionof related transcripts with smallsizevariations. This is consistent with the variable sizesof the 3’ non-coding region in differentFcpcDNA clones (not shown). The stronghybridization signal obtained in a short periodof time(8 hrs) indicates the Fcp genes are abundantly expressed.An analysis of the codon usage is given in table 4. 1A.In the third position of the codonthere is an overall preference for pyrimidines(C + T), representing over 67% of the codons98•217/73 .247/83K H G R I S M L A I L G H L V T T AG Vaag cac ggc cgc atc tcc atg ctt gcc atc ct ggc cac ctg gtg acc acc gct ggt gtgaag cat ggc cgc atc tct atg ctt gcc atc ctt ggc cac atc ctc acc act gccggt gctK H G R I S M L A I L G H I L T T AG A.457/153 •487/163gac gct gcc ggg tgg gat gac gag aag aag gac tcc aag cgc gca attgag ctg aac aacgat gag cag ggc tgg gat gag gcc aag aag gac tcc aag cgc gcc att gagctg aac aacD EQG W D E A K K D S K R A I E LNN•517/173 .547/183G R A AQM G I L A L N V H SQggc cgt gcc gcc cag atg ggt atc ctt gcc ttg atg gtc cac gag cagggc cgt gct gct cag atg ggt atc ctc gct ctg atg gtg cac gag accG R A A 0 M C I L A L M V H E Tgtgtttttttttccttctccaaaaatttttgactttctct>>tcctttatcctgtaacataattttaaatgaccccatagag4tFigure 4.5L D N Nctg gac aac aacatc aac aac gatI N N DComparison of the Fcpl sequence (fig.4.4) with the Fcp2partial sequence. Fcpl aminoacid and nucleotide sequences (top two lines) are displayed starting at aminoacid 73, accordingto the labeling in figure 4.4. The partial Fcp2 nucleotide andamino acid sequences are in thebottom two lines of each row. Nucleotide/amino acid positions are indicated abovetheappropriate residues. Lines above and below selected residues indicate thepotential membranespanning regions. Amino acids and nucleotides in bold indicate areasdiffering between the twosequences. The # at the end of Fcp2 indicates the start of the polyA-tail. The>> symbol at theend of Fcpl indicates the presence of additional sequence thatis not shown..277 /93 •307/103R L P G A Y D L A G DQF S S L P T C Lcgt ctg cct ggc gct tac gac ctg gct ggc gat cag ttc tcc agc ctg ccc accggc ctgcgc tgg cct ggt gcc gtg gat ctg tcc ggc aag aca tac gcc gag atc cctgct ggt atcR W P C A V C L S C K T Y A E IP A G I•337/113 .367/123K A L S A L P A A G V AQT I G F I G Laag gct ctg tct gct ctg cct gct gct ggt gtg gcc cag acc atc ggc ttcatt ggt ctgaag gcc ctt ggt gcc ctt cct ttt gct ggc gtc tgc cag att gtg gcc ttcatt ggt ctgK A L G A L P F A G V C 0 I V A F T 0•397/133I E L C F Aatt gag ctg ggt ttt gctatt gag ctt ggt ttc tccI E L C F S•427/143D A A G WQI K E E L E A D C E AR Ncag atc aag gag gag ctg gag gct gac tgc gaggcc cgc atgaag tgc cag gat gac gtg gca gcg ttc tgc gag ggc aagatgK CQD D V A A F C E GK ND D E K K C S K R A I SL N N•577/193 •607/203P Y I I N S L L G S PV D F N A G F Zcct tac atc atc aac tct ctg ctg ggc tcc cct gtg gacttc aac gct ggc ttc taa acacct tat gtg atc aac tct ctc ctc ggt gcc cct gtg gac ttcaac gcc ggc ttc taa tttP Y V I N S L L G A P V DF N A C F Z99lOpg 2Opg— 4.5— 2.4— 0.24Figure 4.6Northern blotof Heterosigma total RNA probed withthe Fcpl cDNA probe.10 and20 .tg of total RNA were loaded.Molecular mass markers (kb)are shown.100Table 4.1A Codon usage of the Heterosigma Fcp 1 cDNA210 codons MM: 22314 DaltonTTT phe FTTC phe FTTA leu LTTG leu LCTT leu LCTC leu LCTA leu LCTG leu LTCT ser STCC ser STCA ser STCG ser S4 CCTproP5 CCCproP- CCAproP18 CCGproP3 TATtyrY8 TACtyrY- TAAOCHZ- TAGAMBZ6 CAThisH2 CAChisH- CAAg1nQ- CAGg1nQ3413 ACTthrT9 ACCthrT- ACAthrT8 ACGthrT- AGTserS8 AGCserS- AGAargR10 AGGargRGTT val VGTC val VGTA val VGTG val VGCT alaAGCC ala AGCA ala AGCG ala A12 GAT asp D17 GACaspD1 GAAg1uE- GAGg1uE6131Table 4. lB Third Codon Position Datanucleotide group total (%) G+ C%Pyrimidines 67C 71T 2980Purines 33G 97A 318112- TGTcysC2 TGCcysC1 TGAOPAZ- TGGtrpW- CGTargR3 CGCargR- CGAargR7 CGGargRATT ile IATC ile IATA ile IATG met M- AATasnN6 AACasnN- AAA1ysK- AAGIysK3623 GGTg1yG10 GGCg1yG- GGAg1yG13 GGGg1yG101(Table 4. 1B). The pyrimidine bias is particularlyevident in the alanine andglycine codonusage. Cytosine accounts for 71% of the codonsin the third position when apyrimidine occurs.Likewise, purines (G + A) occur only 33%of the time in the third codon position,though thereis an extreme bias (97%) for guanine overadenine. Overall, there is a strongbias for guanineand cytosine in the third position, occurring80% of the time. Comparatively,the G + C contentof the entire Fcp 1 cDNA is 63%, though it isonly approximately 40% in the non-codingregion.The codon usage in the partial Fcp2 clonedoes not appear to be significantlydifferent from thatin the Fcp 1 clone.4.3.2 Characterization of the Fcpgene familyFifteen truncated cDNA clones of various lengthswere isolated and sequenced.Six werederived from unique genes as determinedby differences in the untranslated regionat the 3 endof the cDNA (Figure 4.7). Of these six, fivewere nearly identical in the codingregion and weretherefore considered to be the same type (Fcp1*15).Preliminary sequencing evidenceindicatesthere is at least one additional unique FcpcDNA clone(Fcpl*6).The asterisk followed by anumber indicates a different cDNA representativeof the same gene type based onsequencecomparison. However, as these cDNAsare not full length, the designationof these cDNAs asbeing nearly identical to the full length FcplcDNA clone requires further analysis.The valuesto the right of Figure 4.7 indicate the numberof identical copies isolated for each of the eDNAclones. Three copies of Fcp 1 (Fcp 1*1 in Fig. 4.7) were isolated. In addition,six clones with anuntranslated region identical toFcpl*3were isolated and two equivalent cDNA clonesofanother type were observed (Fcpl*5,Fig. 4.7). The isolation of these different cDNAs indicatesthat the major FCP protein is encodedby a multigene family as in the terrestrial plants (Green etal. 1991) and diatoms (Grossman et al. 1990).Hybridization of the Fcp2 probe to genomic DNAdetected 4-7 bands, depending on therestriction enzyme used (Fig. 4.8, lane2). When the Fcpl cDNA was used as a hybridization102No.copiesdetectedFcpl*13Fcpl*26Fcpl*32Fcpl*41Fcpl*51Fcp21SequencealignmentoftruncatedcDNAclonesstarting18bpbeforethestopcodon(bold).Codingregionisshowninuppercase,whilethenoncodingregionisinlowercase.ThenumberofidenticalcDNAclonesisolatedforeachisgiven.A(#)denotesthesiteofthepoiyA-tail.A(>>)indicatesonlyaportionofthe3’endhasbeenshown.codingregionEFNAGFZ•.GAcTTcAAcccTGGcTTcTAAacagtgttttttttttccttctccaaaaatttttgactttctcttgaccttctgacttttctgggcc>>•..GAcTTcAAcocTGGcTTcTAAatcaagtctctaattgtaacattagtgtgctgtttttggcctcgtttcattttggagctggctgaca>>•GAcTTcAAcGccGoTTTcTAAatacaatctagttggtctggattgcgatcccgcctctgggttggcgttgaggatgccatagtggact>>•GAGTTcAAcGccGGcTTcTAgcgatgttcctgttctcatgacttgcaaattttccaactggaatggactttcttgtaacctttcggg>>•..GAcTTczAcGcTGGcTTcTAAgcagtgttttttcttctctttcccaaaattgacccccctgaccttctgacttttctgggcttgggcc>>•.•GAcTTcAAcGccGGcTTcTAtttcctttatcctgtaacataattttaaatgaccccatagag#Figure4.71.35—1.1—Figure 4.8Southern blots of Heterosigma genomic DNA probed with theFcpl cDNA (1) or theFcp2 cDNA (2). Genomic DNA was digested with Sac I, Dra I or bothSac I and Dra I, asindicated. Molecular mass markers (kb) are indicated on theleft. Open arrows indicate twoDNA fragments that appear to hybridize to both Fcp cDNAprobes.12129.4—6.4—2.3—2.0—12DralSadSadD ra I104probe there were approximately 11 to 17 fragments detected (lane 1). Thesefragments ranged insize from ito 18 kb. However, not all the hybridization signals were of thesame intensity.This could be due to multiple gene copies on the fragment and/or to the closemigration of morethan one DNA segment. At this washing stringency (iX SSC, 65°C) the Fcpland Fcp2 probesappear to hybridize to fragments of the same size such as those indicatedby the open triangles inFigure 4.8. This could occur as the result of the two genes being linked onthe same fragment.Alternatively, hybridization to genes of intermediate sequence divergencecould account forfragments of weaker intensity and for some of the shared hybridizing bandsin both lanes. Inaddition, bands with strong hybridization signals may have multiplecopies of one gene type onthat fragment. Overall, there are multiple copies of both Fcp gene types (Fcpland Fcp2), theFcp 1 type having more members. This indicated the existence of a very largeFcp multigenefamily in Heterosigma.A series of genomic Southern blots were done in order to investigate the complexityofthis multigene family more thoroughly. Figure 4.9 shows a single blot that wassequentiallywashed at increasingly stringent conditions, as labeled. The probe was aSty IJBssH II fragmentfrom Fcpl (see Fig 4.3), which included sequences from the middle portionof the putativeMSR1 to the middle of MSR3. At the low stringency wash (1X SSC, 65°C)there are nineprominently hybridizing bands in the 3.6-18 kb size range and a few smallerhybridizing bands(1.4-3.3 kb) in the Sac I digest. The Dra 1(D) digest shows over 20 hybridizingbands, as doesthe Sac L’Dra I double digest. At the next level of stringency (0.iX SSC,0.18 M Na) there waslittle obvious change in the number of hybridizing bands or in the signal ratiobetween them inthe Sac I digest. In the Dra I digest one hybridizing band was lost (4.1 kb)and the signalintensity of five other bands decreased relative to the others (12, 8, 5.5, 5.4,and 3.6 kb). In thedouble digest, three hybridizing bands were removed (8, 5.8 and 2.2 kb) anda fourth had areduced signal (3.8 kb). The hybridization conditions described above wereboth below thetheoreticalTmof the probe, which was 93°C at 0.18 M Na (1X SSC) and 77°C at 0.0 18M Na(0.iX SSC).1056.44.423—2.0—1 35—1.1Figure 4.9SSID D SS/DDe0.01X SSC65°CSouthern blots of Heterosigma genomic DNA probed with a32P-labeledSty L[BssH IIFcp 1 cDNA fragment. The blot was successively washed under increasinglystringentconditions as indicated on the bottom of each panel. Molecular sizemarkers (kb) are indicatedon the left of the first panel. The restriction enzymes used were as follows:5, Sac I; S/D,Sac JIDra I; D, Dra I.94—SS/D D1XSSC 0.1XSSC65°C 65°C106The final washing condition (0.0 1XSSC, 65°C) exceeded the theoreticalTm of the probe(6 1°C at 0.00 18 M Na). After this wash,the probe was selectively removedfrom a number offragments. Three fragments with a strongsignal (6.6, 5.5, and 3.6 kb) remainedin the Sac Idigest after the 0.O1X SSC wash, alongwith one weakly hybridizingfragment (4.5 kb). In theDral lane, 8 prominent hybridizingbands remained with a sizeof 4.5, 3.8, 3.0, 2.5, 1.9, 1.8, 1.3,and 1.2 kb. Six main bands were detectablein the Sac IJDra I digest (4.5,3, 2.5, 1.9, 1.3, and1.2 kb). Though it is difficult to getan accurate estimate of gene copynumber using genomicSoutherns, there appear to be 6-8 copiesof the Fcp 1 gene. This is ingood agreement with thenumber of characterized cDNA clones.There seem to be as many as 20 or more relatedFcp genes present on the nucleargenome of Heterosigma, as seen in the numberof hybridizing fragments at the lowerstringencywashes. These may represent different Fcpgene types similar to the gene typesin the Cabfamily. However, the presence of additionalhybridizing bands due to theexistence ofpseudogenes cannot be ruled out. Thoughcare was taken to achieve completedigestion and thepattern was repeatable, some of the weaklyhybridizing bands may representintermediates of anincomplete digestion. Neither Sac I or DraI cut within the full length Fcpl cDNAclone,however, internal restriction sites in othergenes could cause an over-representationof detectablebands.4.3.3 Characterization of the FCP proteinsequenceThe FCP protein is predicted to have three membranespanning regions using the Kyteand Doolittle scale of amino acid hydrophobicity(Kyte and Doolittle 1982) witha window sizeof 19 (K-D plot, Fig. 4. bA). The hydrophobicregions of the mature protein detectedin thehydropathy plot include residues73-96, 106-141, 173-192, and the C-terminus(200-210) (aslabeled in Fig. 4.4, 4.10, 4.12). A fourth hydrophobicregion is also detected in theaminoterminus of the protein (residues 1-28),which is entirely within the predictedtransit sequence107(TS) domain. The membrane spanning regions(MSR), as labeled in Figure 4.bA, werepredicted by comparison to the K-D plot andby comparing the FCP sequencewith the CABs(Fig. 4.13). The regions that span the thylakoidmembrane in the pea LHCTI structure(Kühlbrandt et al. 1994) are conserved in theFCPs; therefore, a similar topology wasthought tobe a reasonable assumption (see Fig. 4.15 for model).A feature of the prediction isthe presenceof distinct hydrophilic domains at the start ofthe putative membrane spanning regions1 and 3(Fig. 4.10, 4.15). These areas may protrudefrom the bilayer in a continuationof the ct—helicalstructure, as appears to be the case with thepea LHC II (KUhlbrandt and Wang1991;Kühlbrandt et al. 1994). Figure 4. lOBshows the distribution of acidicand basic amino acidswithin the protein by vertical bars. Acidicamino acids (top panel (A) of Fig. 4. lOB)whichinclude glutamic acid (full bar) and asparticacid (intermediate bar). The positionsof the basicamino acids (bottom panel (B) of Fig. 4. lOB)are also shown and include arginine (fullbar),lysine (intermediate bar), and histidine (shortbar). There is an enrichment for acidic andbasicamino acids in the regions preceding, and at the startof, the predicted MSRs whichare locatedon the stromal side of the membrane. Stromaexposed areas are indicated by shadingin Figure4.10. The areas exposed to the thylakoidlumen are indicated by the hatched regions(Fig. 4.10).Few acidic and basic residues are presentin the predicted lumen exposed sectionsof the FCP(Fig. 4. lOB). There are a few occurrencesof acidic and basic residues within themembraneexposed portion of the protein, someof which are probably involved in the bindingofchlorophyll (discussed later).The first and third putative membrane spanningregions of the FCP are related as is thecase for the CABs; first recognized in atomato Cab gene by Hoffman et al. (1987).Thisinternal similarity between the two regionsis suggestive of a gene duplication. These regions(MSR1 and MSR3) are approximately 49% similarto one another (Fig. 4.11), comparable to thedegree of relatedness between MSR1 andMSR3 of the CAB proteins. With the CABs,similarity between MSR1 and MSR3 extendsinto the domains immediately preceding the startof the putative transmembrane c-helix.This is not the case with the FCPs.108A100 200BIIIII,,JIIJI 11)11AiiitABiIIiI1ii1!.iiIiiiI!iii.IqiII,t;::” BTopological analysis of theHeterosigma Fcp 1 full lengthsequence. A) Kyte-Doolittlehydropathy plot doneusing a sliding window of 19 aminoacids. Hydrophobic areas areassigned a positive value and hydrophilicones are negative The transitsequence (TS) and theputative membrane spanning regions(MSR1-3) are labeled and roughlycorrespond to the clearareas. B) an acidic-basic mapof the FCP protein. The position ofthe acidic amino acids, areindicated in the top panel (A) bya full vertical bar (E-glutamic acid)and an intermediate verticalbar (D-aspartic acid). Thebottom panel (B) shows the positionof the basic amino acids with afull bar (R-arginine), an intermediatebar (K-lysine), and a shortbar (H-histidine). In all threepanels, shading represents regionsof the protein exposed tothe stroma of the chioroplast.Hatches indicate areasthat are exposed to the thylakoidlumen.TSMSR1 MSR2 MSR3-2-1.5—1-0.500.511.52Figure 4.10100 200109MSR1 61 DQERFDRLRTVEIKKHGRISMLAILGHLVTTAGVRLP95MSR3 159 DDEKKDSKRAIELNNGRA1QMGILALMVHEQLDNNP 193Figure 4.11Alignment of the first and third putative membranespanning regions of theHeterosigma FCP. Similar amino acids are in bold.Numbers indicate the positions of thefirst and last amino acids shown, relative to the entireprotein.The alignments in figures 4.12 and 4.13 were done withMACAW (Schuler et al. 1990)and adjusted by hand to maximize similarity. TheChi a + c-binding proteins (FCPs/iPCPs)arevery well conserved between the different algal taxa.The Heterosigma FCP sequence isroughly77% similar to the other known algal FCP sequences. Theboxed regions in Figure 4.12represent similarities between the FCPs and the iPCPs.Shaded regions are amino acidsconserved only in the FCPs, excluding the Isochrysissequence. The greatest similarity betweenthe Chl a + c-binding proteins is within the putative membranespanning regions; particularlyMSR1 and MSR2 (Fig. 4.12). The N-terminal portionpreceding MSR1 is also very highlyconserved, though the analogous region in front of MSR3is not. This is contrary to therelationships between the stromal exposed regions in frontof MSR 1 and MSR3 in the CABs(see Fig. 4.13).Amino acid similarities between the CABs, FCPs andiPCPs are indicated by the boxedregions in Figure 4.13. The Heterosigma FCP sequenceis approximately 40% similar to boththe tomato LHC I and LHC II sequences, when the unambiguouslyaligned positions arecompared (see Fig. 5.1, chapter 5). For comparison, the LHCI and LHC II sequences fromtomato are approximately 57% similar using the same amino acidpositions in the calculation.The Heterosigina FCP sequence shows the greatest similarity to thegreen algal and terrestrialplant CAB sequences, almost exclusively within the predictedmembrane spanning regions ofthe mature protein, primarily within the first and thirdMSRs. However, the sequenceconservation between the CABs and the FCPs does extendinto the stromal side of the first MSR110iPcp-AcFcp-HcFcpl-MpFcp-LsFcp-OsFcpl-PtFcp-IgFcp-P1MSR3,.WZWZZZWZZZZZZZZWZZi160 170180 190 200SK1DLER [4MIIG!4F ET-GSA1t)S jKIELNNGR A IIAL 7IELD-NNPMS1’QASKPELNNGR Jt4-NKPAMS$TEASjKBNGRAfl illTF*rKLS 11 $L . ttV43-GSI PIVGEMTFS1KKLQ C1J1ELNQGR tL2LI4jVHE-VSI LPPSDEFRLWGPYWGDATFFigure 4.12Amino acid alignment ofthe Chi a+c-binding proteins fromchromophytic algae. Taxainclude: Ac, Amphidinium carterae;He, Heterosigma carterae; Mp, Macrocystispyrfera;Ls, Laminaria saccharina;Os, Odontella sinensis; Pt, Phaeodactylumtricornutum; 1g.Isochrysis galbana and P1,Paviova lutherii. Hatched boxes indicateputative membranespanning regions (MSR1-3). Boxed regions indicate sequence similaritiesbetween theFCP and iPCP sequences. Shadedregions indicate FCP similar regions, exceptIg and P1.Amino acids are numbered with referenceto the Heterosigma Fcpl sequenceas in fig. 4.4.iPep-AcFcp-HcFcpl-MpFcp-LsFcp-OsFcpl-PtFcp-IgFcp-P1ipcp-AcFcp-HcFcpl -MpFcp-LsFcp-OsFcpl-PtFcp-IgFcp-P1v/I1 10 2030 4050 60IESELGVIPTGFWDPLGL AKMKKNSLKLAT FAAALM- -GA SAFVAPNKNGVAKSSSALKM ENEZGVIDPG WEI4I4DMKSAVMAVA CAAAPGFRGP SAFNGAALSAKACSANKME2IGA {GpPGI1A442EI*DPt.GILDtIFMKL--- AIAALLAGSA AAF-APAQ-- SGKASTALNMEiLG NMKF- - - AVFAFLLASA AAF-APAQQ-SARTSVATNMGA 1tGt VADtMMTL--- ASLPSTAIAG LASAAPKVQ--PRMAANDEF -GLG GDPAGLKGDLEVYA ILGFYDPLGL LDNEEYEMSR170 8090 100 110R IKHG RI TPEL KFPG -YLSPSMG L1EDIPIL IWTTAG t1 -AYDL-AG DSSLPjtQQN- LPG-MLSNSANLFtADMPRLRVàVKHG RI r4I’QQN--NLSNSARIRYVEVKRG RIAIW1VTRNG IDYAGNSF DPNGW I PPtVYJHG R QEAGP -DIDYS-G TESIP F{GQEK- - --PLFSGDNGPAIEQIPRMVELMSR2-wzw/zw//zzz//zI120 130 140150siz] IJjY cQDQSEGSAGEAGDFGF KVLTSGI A IGEX I FAQIKE ELEAD--CEA RNSdXAGI I I VMKNVEGS---FPGF TLG----GNPDIG I AFV I VMKDVT G-EGEFPGFR NGASd IAFI E VMKDIT G--GEFVGDFR NNYQI.PYWLW I IG- f...RIQKGW AKVNPETGKA DSALREGYEPEV1JQAFV LEL V-KDVT G-EGDFPGDFDAAGWD.FGASW---- --LDFGW1-. -LDFGWP—--- -GDLGFDPLGL A200iPcp-AcFcp-HcFcpl-MpFcp-LsFcp-OsFcpl-PtFcp- IgFcp-P1YGDWANFTAS PLRYIINSLLGSP VDFNAGFYVINDLVGAS YTFN111Figure 4.13Amino acid alignment of select Chl a+b and Chl a+cbinding proteins from terrestrialplants, green algae, and chromophytes. Taxa include: Le, Lycopersiconesculentum; Cr,Chlainydomonas reinhardtii, Egi, Euglena gracilis;Ms, Mantoniella squamata; Ac,Amphidinium carterae; Hc. Heterosigma carterae;Mp, Macrocystis pyrifera and Pt,Phaeodaciylum tricornutum. Hatched boxes indicate putativemembrane spanning regions(MSR 1-3). Boxes indicate similarities between Fcps, iPcpsand Cabs. Dashes(-)represent gaps introduced to increase similarity. ProposedChi a(A)and b(, )bindingresidues of the pea LHCII are indicated. Aminoacids are numbered with reference tothe Lhcbl-Le sequence, consistent with the numberingused in figure 4.14.11210 2030 40 50Lhcal-LeLhcbl-LeLhcb-CrLhcb-EglLhcb-MsiPcp-AcFcp-HcFcpl-MpFcpl-PtMSR1-////-60 70 80LFYESI*VP IIVPLGLARELE\I CPNLGL GDVFPLLAR1RlETEII{APNI4rLPLLAKL*tREAIEt1I4ZNLGL7\1t11PtELLAGS AE I G GTG90 100GN- -WVKAQE WAAIPGGQATNGV-KFGEAV WFKAGSGT-KFGEAV WFKAGNGVPFGEGAV WYKGIP WFTAGTLCTPFPGLPGLPGLPGSEGGLDYLGNSEGGLDYLGNSADGLNYLGNP-GAVAPLAPSMGLKYEDIP-AGDQFSSLPSANLSFADMPS-GTSFESIPI I ILhcal-Le PSFLSSTKSK FAAAMPVSVG ATNSNSRFSMSADWMPGQPRLhcbl-Le GNGRITNRKA VARSA PSSSPWYGPI)RVKYLGPFSGLhcb-Cr LQVTCKATGK KTAAKARAPK SSGVEFYGPN RAKWLGPYSELhcb-Egl MLATSGRKA KAAPKS DNLSQWYGPDRARWLGPLLhcb-Ns N ACIASSFVGS VAALKATKVQAKSVSTVVKAiPcp-AcFcp-Hc MSLKL--AT FA-AALMGASFcpl-Mp MKSAVNAVAC AAAPGFRGPSFcpl-Pt MKF AVFAFLLASA- -- PSYLIDES-PSYLTNATPAYLTEV-PSYLIDIYPEFGTFESEL-- -FENEI---FESEI-- -FENEIAFVAPNKMGVAFNGAALTTSAAFAPAQ-QS333‘3‘33 APGDFG3 FPGDYC3 FPGDYC3 LPGDYC- YPG--CT AAPTGFJ QAPLGP QAPLGQQPLGAKSSSALFiSACSAMFJISARTSVATNMA3EVPA--NtrA IPE--TWLtTA IPE--TWP’TA 43SPT--TGESP IIPF GSCAfrDPIIG14DQERfrJDPLIGLL4ArDQER13DQEK110YLGQPVPW- -SQIFAQIFAQIFDDCTAVADKFYLSPAYDLMLSNDIDYMSR2120GIPSLVHAQIPSLVHAQIPSLIHAQEGSGY-PSFNGLGAISTGLKALSNGVAALS INGFAALS130? TILAIEFI -iw vfvfr -JIrSA’IILM -I LTFLSTIIM -1 jAvLAIEv\4rV -IGWIILY?P tGVAII3F ‘P PAGLAIFAF IP GIAI1kF IGCCFCCCYPGL YPGL YPGDTL----YPGVGDV---SPGFGFKVLT-- -CEARMGDFTLG-GNPGDFR---NNY160GA--FDPLGYGS--FDPLGLES--FDPLGLGP--FDPLGLGR- -FDSLGLDAAGWD- - - -FGASWD- - --LDFGWD---Lhcal-LeLhcbl-LeLhcb-CrLhcb-EglLhcb-MsiPcp-AcFcp-HcFcpl-MpFcpl-PtLhcal-LeLhcbl-LeLhcb-CrLhcb-EglLhcb-MsiPcp-AcFcp-HcFcpl-MpFcpl-Pt140 150FfQR-S NEKDSEKKKIEYRIA GGPLGEVVDPLIEYRVN GGPAGEGLDPTEYRYG GGVGDFGRELUEkYRTG LSDSPFEELTEj2SQDQ SEGSAGEAGDL1IE1LGFAQ IKEELEAD- -FILE1LAVMK NVEGS- --FPFAVMK DITG- -GEFVQQSAYPGTGPQAIjT-GKGPQAIIvT-GKGPQPL4P-KAGPQALAT-QEGPQDc4T-GSAYHE-NNPYHEIL-NKPYHEJ3-VSIL200MSR3170SKDPAKFEELAEDPFAELADDPLDjrFAELANDLAELAESG-LEELSKDEF43LERK-- -LEKKDSKAMSE14I’QASKTFSKKLQK210 220 230LLLADHLADVDDHLANVETFHLADIAJWQAHVADGDWANFTASPIILLGSPVVIDLVGASYPLiIPKGIFPNFVPGKTKFTPSAGFAASGFGFYPWHNNIGDVIPVNNNAWAFAPTVNNAPAFAPSANIFFTSPVHANVLTNALRDFNAGFTFN113(amino acid residues 44-5 1, Fig. 4.13). This primarilyincludes the WDPLGL motifof the FCPsto the (F/Y)DPLGL motif of the LHC I CABsor the (W/F)DTAGL motif of theLHC II CABs.In this case, the PL of the (F/Y)DPLGL motifis a signature sequence of the LHCI CABswhich, interestingly, also occurs in the FCPs. In theCAB sequences, there is an additionalFDPLGL motif in front of the third predictedMSR (residues 16 1-166; Fig.4.13),which is highlyconserved and is part of a local 2-fold symmetry betweenthe MSR1 and MSR3 (Kühlbrandt etal. 1994). In the FCP/iPCP sequences, thereare no obviously conserved areas infront of thethird putative MSR. However, the IsochrysisFCP sequence is an exception to thisstatement asit does have a FDPLGL motif in front of thethird MSR, more closely resemblinga CAB protein(Fig. 4.12).Figures 4.14 and 4.15 are designed todemonstrate the location of the conservedaminoacids with respect to the proposed structure ofthe LHC. A model of the pea sequenceis shownin Figure 4.14 along with the identified chlorophyllmolecules (represented by porphyrin rings)and the approximate location of the thylakoid membrane(hatched regions), thoughthe latter hasnot been accurately determined. Thisdiagram is labeled to show the amino acidsconservedamongst all LHC types (CABs, FCPs, and iPCPs).The residues conserved between all knownCAB, FCP and iPCP sequences are indicated withblack circles; most of these are within MSR1and MSR3. Significantly, many of the conserved residuesare within the area of close contactbetween the two transmembrane c-helicies(MSR1 and MSR3). The area of closecontactincludes residues Ser 69 to Ala 76 and Gly 184 to Met191 (Fig. 4.14), as defined by Kühlbrandtet al. (1994). Other conserved residuesare thought to function as Chl a ligands inpea, whichinclude the following residues: Glu 65, His68, Gly 78 (backbone carbonyl), Glu 139, Glu 180,Asn 183 and Gin 197 (His in FCPs) (Fig. 4.13 and 4.14).All ligands thought to bind Chl a inpea (six in all) are conserved in the Chl a + c-bindingproteins; presumably they would also beinvolved in Chl a binding.A putative model of the Heterosigma FCP sequenceis illustrated in Figure 4.15. Thetopology was predicted by aligning the FCPsequence with the CAB LHC II sequences and then114modeled by analogy to the pea LHC II 3-D structure. It also showsthe location of the conserved(chemically similar) amino acids when the FCPs andthe iPCPs are compared. The FCP/iPCPsequences are mainly conserved within MSR1 and MSR3. Thesimilarity within the secondputative MSR is significant but lower than when the first and thirdMSRs are compared. Thereis a lack of conserved residues in the lumen exposedportions of the protein and in the connectorbetween MSR2 and MSR3. Residues thought to bind Chl a, by analogyto the pea structure, arewell conserved (solid triangles, Fig. 4.13). The degreeof sequence conservation in themembrane spanning regions is significant enough tosuggest that the FCPs may have a similarstructural topology, though the aqueous exposed areasare not conserved and may be structurallydistinct.115Figure 4.14Amino acid comparisons based on the structural models for the pea LHCII complex.Porphyrin rings represent the approximate location of chlorophyll moleculesdetermined fromthe pea LHC II structure. The approximate location of the thylakoid membranein each Figure isindicated by the hatched regions. The pea LHC II sequence is compared toboth the CABs andthe FCPs. The key to the conserved (similar) amino acids is given in the Figure.The Figure is amodification of Figure 4 from Kühlbrandt et al. (1994).3O©®®®©NMSR1 MSR3pea LHCII model150®Lumen120 1104(D)®®®®®®®®®®CConserved in all CABs and FCP/PCPs220©Conserved in all CABsQConserved in most CABs116Heterosigma FCP model®®120®(j))D190©dOO•Conserved nail FCPsand PCPs‘-‘ ‘o QConserved in all (6/7), except 1g.QConserved in all FCPs. except 1g.Figure 4.15Model proposedfor the Heterosigma FCP protein.Porphyrin rings represent theapproximate locationof putative chlorophyll molecules byanalogy to the pea LHC II structure.The location of thechlorophylls in the FCP modelare purely speculative and are includedonlyto illustrate the conservationof the residues binding theseparticular molecules in the peastructure. The approximatelocation of the thylakoid membraneis indicated by the hatchedregions. The HeterosigmaFCP sequence is compared tothe FCPs/iPCPs. The key to theconserved residues is indicatedin the Figure.MSR1 MSR3(B) (A)50Thylakoidmembrane(C)Lumen1174.3.4 Analysis of the FCP transit sequenceThe N-terminus of the Heterosigma FCP was blocked;therefore, the precise cleavagesite of the transit peptide is not known. However,comparison to the PhaeodaciylumFCPcleavage site (Bhaya and Grossman 1991) wouldsuggest that it occurs after Met 35(Fig. 4.16),which is within a well conserved region. A comparisonof the Heterosigma N-terminal regiontothat of other chromophyte transit sequencesis shown in Figure 4.16.Most of the chromophyte transit sequences havea basic amino acid within the first fourresidues (Fig. 4.16). This is followed immediatelyby a hydrophobic region (15-17amino acidslong) emphasized by the black sectionof Figure 4.16. The CAB transit peptideslack both astrongly hydrophobic region and a positively chargedamino acid at the N-terminus.A prolineresidue followed by a positively charged aminoacid usually occurs at or near theend of thehydrophobic section (Fig 4.16). This proline mayfunction as a helix breaker should thehydrophobic region form an cr-helix. After the hydrophobicregion there is an increase in thenumber of hydrophilic residues, such as basic (Arg,Lys) and hydroxylated (Ser, Thr) aminoacids. Few, if any, acidic residues (Glu, Asp) are presentwithin the targeting sequences. Abasic N-terminus, a hydrophobic region followedby a more polar section are all characteristicsof a signal sequence (von Heijne 1990). However, thesetraits are not present in the IsochrysisFCP sequence (LaRoche et al. 1994). Though thereis a hydrophobic region (Fig 4.16), a basicamino acid in the first four residues is not present. This sequencealso has a number ofhydroxylated amino acids at the beginning ofthe transit peptide (Fig 4.16), unlike the otherchromophyte sequences.Putative processing sites for the signal sequence-like regionsare indicated in Figure 4.16(open triangles) and are based on the (-3,-1)-rules for the predictionof signal sequence cleavagesites (von Heijne 1986). There are other processingsites that can be predicted using the (-3,-i)-rules though only one is shown. The section immediatelyfollowing the hypothetical processing118site in the FCPs, and prior to thestart of the mature polypeptide, is enriched inhydroxylated andbasic amino acids (Fig 4.16). This amino acidcomposition is similar to the transit peptide ofchioroplast localized proteins of terrestrial plants.Presumably, the remaining region would thentarget the polypeptide to the chloroplast envelopeonce it crossed the ER-like membrane. Such abipartite transit sequence in the chromophytes has been suggestedby Bhaya and Grossman(1991) and expanded on by Pancic and Strotmann (1993). Inaddition, the Chroomonasphycoerythrin transit sequence has two hydrophobic domainswithin it (Fig. 4.16). The firstregion is similar to a signal sequence and the second hydrophobic region resembles the transitsequence of a thylakoid lumen localized protein (Hiller et al. 1990).VVASSSALKM FTSAKSSANKM:VATNN AFSKASTALNM AFF.PAVGGATSNVFSESSSPAHRNRRTIVM DGVQPMA.ANDE FASNDMGEVV____________GAPAGAAN•AGAAAAVTPFigure 4.16Analysis of the N-terminal transit peptide from somechromophyte nuclear encoded sequences.The sequences are from the FCPs of; Hc,Heterosigma carterae; Mp, Macrocystis pyrifera;Pt, Phaeodactylum tricornutum; Os,Odontella sinensis; and Ig, Isochrysis galbana.Othersequences are the the Odontella sinensis- y-atpase(Os-a) and the Chroomonas (cryptophyte)phycoerythrin a-subunit (C-pe). Areas enclosedin black indicate hydrophobic regions.Shadedregions emphasize hydroxylated amino acids, serineand threonine. Basic amino acids(Arg, R;Lys, K) are underlined. Transit peptide cleavagesites are indicated by solid triangles (y).Opentriangles(V)represent possible signal peptide cleavage sitesbased on the -1 ,-3 rule.119HcMpPt IvOsOs-aIgC -peGAEPFNGAALLATFAAALMGASAFVAPAVMAIACAAAPGI4_AVIASLIAGAZAFAPALAIAALLAGSAAAFAPA.AIAGLASAAPLASLAVIGSAAAYVpNNIv]NMFZ4.4 Discussion4.4.1 Fcp cDNA structure and multigene familiesThe cloned Fcp cDNA (Fcp 1) encodes one ofthe predominant polypeptidespresent inthe thylakoids of Heterosigma, as confirmedby protein sequencing. The Fcp genesare nuclearencoded by a multigene family and thenorthern blot indicates they are highlyexpressed.Southern blot analyses of genomic DNAand cDNA sequencing indicate thatthere are at least 6-8 nearly identical copies of the Fcp 1 genein Heterosigma. In addition, there appearto be over20 closely related Fcp gene sequences. Thisis comparable to the complexity observedin thetomato Cab gene family (table 1.2) wherethere are eight LHC II type I genes andgreater than13 genes encoding LHC II proteins in total(Green et al. 1991).Similarly large LHC gene families have been reportedin many other terrestrial planttaxa, such as petunia, where there are an estimated16 Lhcbl genes (Dunsmuiret al. 1983).Multi-Lhcbl gene families are also present inmoss (Long et al. 1989), green algae(Imbault etal. 1988; LaRoche et al. 1990) and in Euglena gracilis(Muchhal and Schwartzbach 1992). Inthe diatom Phaeodactylum there are at least six relatedmembers of the Fcp genefamily (Bhayaand Grossman 1993). A similar numberof Fcp cDNAs have been cloned and sequencedfromthe brown alga, Macrocystis (Apt et al. 1994). Moreover,the presence of multiple copies of themain LHC appears to be a general occurrence.The gene copy number of the primaryLHC II (Lhcb 1) is quite variable betweendifferentterrestrial plant taxa. One mechanismfor the generation of multiple gene copiesis throughunequal cross-over events which usuallyresult in the creation of tandem repeats (Langridge1991). When this occurs, subsequent unequalcross-over events can lead to increases ordecreases in the size of the gene family. Ahigh degree of similarity between the duplicatedmembers of the Fcp or Cab gene familiesmay be the result of either a recent geneduplication120event, if there is similarity in the surroundingnon-coding sequences, or throughconcertedevolution (Tanksley and Pichersky 1988; Bhaya andGrossman 1993).Large numbers of duplicated genes are usually assumedto be required for the generationof sufficient mRNA transcripts for the productionof abundant proteins (Li 1983). Asthe FCPsare the most abundant proteins in the thylakoidmembrane, this explanation seems reasonable.Alternatively, the presence of multiple gene copiesmay be less related to abundantproteinproduction but more to the lack of negative selection against multiplecopies on the genome. Inthis scenario, only a minimum number of Cabgenes may be required and selected for.I have named the more divergent Fcp sequence, Fcp2,to emphasize its distinction fromthe Fcpl sequences. The categorizing of the Fcp2sequence as being a unique gene ‘typefromthe Fcp 1 sequences requires more detailed examinationof the different FCP complexes and theirfunctions. However, as the immunological analysis inChapter 2 suggested, there is quite anintricate antennal system in Heterosigma. This wouldsuggest that many other Fcp gene ‘types’exist. If the sequence divergence between the various Cabgene types is any indication, it isunlikely that Southern blots using either the Fcp 1 or Fcp2 cDNA probes,under the previouslydescribed hybridization conditions, have detected the full extent ofthe Fcp gene family inHeterosigma. If distinct LHC I and LHC II antennae existin Heterosigma, as is likely, then thesequence divergence between them may be too large tobe easily detect by hybridization underthe conditions used in this study. The differentialcross-reactions of the FCPs with the twodifferent antisera would also suggest this (Chapter 2).Bhaya and Grossman (1993) identifiedseveral Fcp genes that were present on two different genomic clones,which may be on the samechromosome. These sequences range in amino acid similarity from86-99%. It would beinteresting to examine the functional differences between these clones,if any. The Fcp genefamily in Phaeodactylum does not appear tobe as large as that in Heterosigma, based onreported Southern hybridizations (Bhaya and Grossman1993).The Fcp genes of Heterosigma are highly expressed as indicated by thestronghybridization signal on the northern blot. The cDNA preferentiallyutilizes 28 of the possible121codons in a manner similar to the Phaeodactylurn Fcp(Grossman et al. 1990). The trendsseenin the analysis of highly expressed yeastgenes are generally consistent withthe Fcp codon usagepatterns (Bennetzen and Hall 1982). The codon usagein the Heterosigma Fcp cDNAshows avery strong bias for G + C in the third codonposition (80%, Table 4.1 B) andthis trend isevident in the other Fcp cDNAs. However, thebias is not as prominent for the diatomFcpgenes (Phaeodaciylum, 64%; Odontella, 72%)or the Isochrysis Fcp sequence(67%). TheMacrocystis Fcp cDNA shows extreme bias forG + C in the third position with approximately91% occurrence for the five Fcp genes. Thetotal G + C content for all codon positionsof theMacrocystis Fcp cDNA is, however, similar tothe that of Heterosigma. The analysisof Cabgenes in angiosperms has shown a bias forG + C occurrence in the third codon of gymnosperms(Jansson and Gustafsson 1990) and monocots (Brinkmannet al. 1987) but not dicots.A high degree of codon bias for a particular gene hasbeen correlated with the relativeconcentrations of particular tRNA molecules withinthe cell (Ikemura 1982). Codonbias is alsomore pronounced in abundantly expressed proteinsin bacteria (Sharp and Li 1986) and yeast(Bennetzen and Hall 1982). Together, the coincidenceof tRNA poois with non-random codonpreference in abundantly expressed proteins would beconsistent with an increase in translationalefficiency. For highly expressed genes, the useof rare codons may result in a decreased rate orpremature termination of translation (Robinsonet al. 1984) and be selected against.However,abundance of a particular protein did not appearto be correlated to the degree of codon bias interrestrial plants (monocots) (Campbell andGown 1990) so the universality of such a proposalremains uncertain. Alternatively, the differencesin G + C bias may be more related to theoccurrence of different G/C ratios within certain regionsof the eukaryotic genorne, calledisochores (for example, see Sharp 1991), asoccurs in mammalian nuclear DNA.The gene encoding the P-type ATPase fromHeterosigma akashiwo (which is equivalentto H. carterae, see section 1.1) has just recently beenpublished (Wada et al. 1994). It alsoshows codon usage bias, with 73% of the third codonpositions being G or C. However, there isno bias for pyrimidines in the third codonposition, as is the case with the HeterosigmaFcpl122cDNA. In addition, the degree of preferencefor the predominant codons in theATPase gene isalso not as distinct as in the Fcp] cDNA. Thesignificance of the difference in biasbetween thetwo genes from Heterosigma is not known.4.4.2 Structural aspects of sequence comparisonThe similarity of the FCPs to the CABs wasconfirmed when the sequenceof the firstFcp cDNA was determined (Grossman et al. 1990). Inaddition, there were threeputativemembrane spanning regions within the mature protein.As membrane spanning regions typicallyform ct-helicies, the prediction of three transmembranedomains in the FCPs was consistent withthe amount of measured o-helica1 structure (Hilier et al.1987). When the Heterosigma FCP iscompared to other FCPs or to the CABs, most of theconserved amino acids are within themembrane spanning, hydrophobic regions.This is particularly obvious within thefirst and thirdmembrane spanning regions where there are probablyconsiderable selective pressures againstchanges due to their importance in chlorophyll and carotenoidbinding. The four amino acidsthat form the ionic bonding pairs between MSR1 and MSR 3 in the pea LHC II structure areconserved in the Chi a + c-binding proteins (E 71—R174; R 76—E169, Fig. 4.15). In additionto binding chlorophyll, these residues are thought tobe the main protein stabilizing force withinthe lipid bilayer (Kühlbrandt et al. 1994). Moreover,within the area of close contact betweenthese two c-helical membrane spanningregions—on the sides that face one another—thereare anumber of conserved smaller residues,probably selected for close packing (GreenandKühlbrandt 1995). In addition, one of the two Metresidues (Met 73, Fig. 4.14), involved inthesterospecific binding of the internally located carotenoidsin pea, is conserved in the Chi a + cbinding proteins. However, the position analogous toMet 188 in the pea structure is notconserved in the FCPs (or in all CABs) andits role may be fulfilled by a conserved Gin residue.Overall, this indicates that the structure and topologyof the FCPs and the iPCPs are very similarto the CABs.123However, there are some noticeable differences betweenthe two main LHC types (CABsand FCPs). First, compared to the CABs, the FCPsare shorter in the connectorsjoining thetransmembrane regions, in the C-terminus andin the N-terminus, which accountsfor theirsmaller size on polyacrylamide gels. The shorter connectingregions may be an indicationthatthe FCPs helicies are more tightly packed togetherthan those of the CABs(Green andKühlbrandt 1995). Second, the two-foldsymmetry that exists between the surfaceexposedregions just before the first and third MSRs inpea appears to be lacking in the FCPs sincetheseareas are not conserved. What effect thismay have on the organization of theFCPs is notknown. Despite these differences, the overallstructural relatedness clearly indicates thatthedifferent LHC types (CAB/FCPs etc.) are evolutionarilyrelated and share a commonancestor.This also confirms the immunological work in chapter2, and elsewhere (Caron etal. 1988;Passaquet et al. 1991; Hiller et al.1993; Plumley et al. 1993; Durnford and Green1994), thatindicated the CABs and the FCPs are structurally relatedand share common antigenic epitopes.Since the 3-D structure of the pea LHC II has been determined(Kühlbrandt et al. 1994),it is possible to use this information in locating putativepigment binding residuesin other LHCs.Overall, the conservation of six of the seven putativeChi a-binding residues in the FCPs wouldsuggest that these residues also function in thebinding of chlorophyll. Presumably theseresidues would bind Chi a, but the possibilityof some of these residues ligating Chi cor nochlorophyll at all can not be dismissed. The ligandof Chi a7 in pea is not knownand the regionunder this Chl in the CABs (WFKAG; Fig. 4.14)is not conserved in the FCPs (Green andKühlbrandt 1995). As this Chl is not bound by a sidechain group, it is not possible to accuratelyassess whether it may be present in the FCPs.Three Chi b ligands in the pea LHC II structure have beententatively identified(Kühlbrandt et al, 1994). The alignment in Figure 4.13 suggeststhat two of these putative Chi bligands, within MSR2 in pea (Gin 131 and Giu 139, indicatedby open triangles), are conservedin the FCPs and iPCPs. However, a gap was insertedin the CAB sequence to maximizesimilarity to the other LHCs. In an os-helical structure,these residues would be on opposite124sides; therefore, it is unlikely that both would bind chlorophyll in the FCPs/iPCPs (Green andKühlbrandt 1995).The first of these conserved amino acids (E134, Fig. 4.12, 4.15) probably does not bindchlorophyll in the FCPs since most lack an Arg residue at position 137. In the pea LHC II, anArg in a homologous position interacts with the conserved Glu to bind Chi b5 (Kühlbrandt et al.1994). However, the presence of an Arg three positions away from the conserved Glu in thesecond MSR of the Isochrysis FCP, suggests that this residue may be a chlorophyll ligand in thiscase (see Fig. 4.12). Nonetheless, this Glu is very conserved in all FCPs and iPCPs and,if notbinding chlorophyll, probably has another important function (Green and KUhlbrandt 1995).Another amino acid with the potential to bind chlorophyll in the Heterosigma FCP isposition 123 (V123 Fig. 4.12,4.15). This could occur via a backbone carbonyl group because itwould not have a H-bonding partner in the ct-helix due to a conserved proline residueat position119 in the FCPs (Fig 4.12, 4.15). Interestingly, this residue would be on the same sideas theconserved Glu in the second MSR if an ct-helix is formed (E 134, Fig. 4.12,4.15). As well, theconserved Gin (Q125, Fig. 4.15) could provide a chlorophyll ligand, but notin addition to theformer two because they would be on different faces of the ct-helix. The FCPsprobably lack achlorophyll binding residue near the C-terminus, as occurs in the pea LHC II(position 211, Fig.4.13), because this region is not conserved and it is absent in the PhaeodactylumFCP sequence.An accurate determination of the Chl a/c ratio of the FCPs has not been done althoughestimates in the range of 1.4-5.6 have been reported (table 1.3). With an averageChi a/c ratioaround 3 and assuming at least six bound Chl a molecules (since six of theresidues thought tobind Chi a in the pea LHC II are conserved in the FCPs), there would be approximatelytwo Chic molecules per FCP polypeptide. In addition, anywhere from 5 to 12 fucoxanthinmoleculesmay be present based on calculated molar ratios of Chl a, c and fucoxanthin(Friedman andAlberte 1984; Katoh et al. 1989). Although there are a few putative chlorophyllligands in thesecond MSR of the FCPs, it is not possible to conclude if they would bindaccessorychlorophylls (Chi c) as they are suspected of doing in the CABs (Chi b).125Other domains besides the membrane spanning regions may participatein thechlorophyll or carotenoid binding. The conserveddomain in front of MSR1 in both theFCPsand CABs suggests that this region may be important for thispurpose. In the pea LHC IIstructure, this region shields Chl a4 and lutein2 from the aqueousenvironment and may beinvolved in binding one of the centrally located luteinmolecules (Kühlbrandt et al. 1994;Greenand Kühlbrandt 1995). Presumably this region hasan analogous function in the FCPs andmay,in addition, provide a site for the binding of fucoxanthin. Similarly,in pea LHC lithe positionimmediately preceding MSR3 is positioned aboveChl al and luteini and is thoughtto beimportant in their binding (Kühlbrandt et al.1994; Green and Kühlbrandt 1995). Althoughthereis no obvious sequence conservation amongst the FCPs inthis domain, it still may be importantfor both chlorophyll and carotenoid binding. It is likely that thepolar groups at each end offucoxanthin and the other carotenoids form hydrogenbonds to polar groups within the MSRconnector regions. Similar interactions with the carboxylgroup on Chl c, due to the lack of aphytol tail, could help bind or stabilize this molecule. Thoughnot well conserved in the FCPs,the other domain linking MSR1 to MSR2 and theC-terminal section may have a role in eitherchlorophyll or carotenoid binding, as suspected for thepea LHC II structure (Kühlbrandt et al.1994).4.4.3 The transit sequence and protein importThe N-terminal region of the FCP protein from Heterosigma resemblesa signal sequencethat targets proteins to the ER in eukaryotes. The targetingof nuclear encoded, plastid localizedproteins in chromophyte algae was first hypothesized tobe mediated by a eukaryotic-like signalsequence by Sarah Gibbs (1979). The presence of asignal sequence-like transit peptide inplastid localized precursors was confirmed by Grossmanet al. (1990) when the first nuclearencoded Fcp gene from a chromophyte was sequenced. The presequenceof the HeterosigmaFCP can be separated into several regions: a positivelycharged amino terminus (residues 1-4),a126hydrophobic section (residues 5-16), followed by a morepolar region preceding the cleavagesite. Though primary sequence conservation is not usually apparent,these are generalcharacteristics of all signal peptides (von Heijne1990).The presence of a signal sequence would correlatewell with the ultrastructure of thechromophyte plastid which has two additional membranessurrounding it (Gibbs 1970). Theoutermost membrane has ribosomes bound to theouter surface, like ER, and is commonlycalledthe chioroplast ER (CER). The presence of a signalsequence offers an explanation for hownuclear encoded precursors may cross these two additionalmembranes. This was followedupby Bhaya and Grossman (1991) where cotranslationaltransport and processing of the FCPprecursor was observed in an in vitro microsomal (ER)membrane system. In addition,terrestrial plant chloroplasts were not able to importthe FCP precursor, illustrating the differencein transit sequence specificity. These observationslead to the proposal that the synthesisofchioroplast precursors occurred on the CER bound ribosomesand were cotranslationallytransported through the membrane (Bhayaand Grossman 1991).The presence of a putative bipartite transit sequencein the Odontella y-ATPase subunitsuggests that after the removal of the signal sequence,the remaining peptide directs the proteinacross the two membranes of the chloroplast envelope(Pancic and Strotmann 1993). Thepresequence of this protein is much larger than thoseof the FCP sequences shown in Figure4.16. If the FCP presequence is bipartite,then the resulting transit peptide after the cleavageofthe putative signal sequence would share some characteristicswith the chloroplast transitpeptides of plants (Bhaya and Grossman 1991; Apt etal. 1994). This similarity includes a highproportion of hydroxylated and basic amino acids. However, withthe FCPs, this putativesecond leader sequence is predicted to be quite smallcompared to a typical plant transit peptide.It would be interesting to test various portionsof the last half of the Heterosigma FCPpresequence for transport competence intothe pea in vitro chloroplast system.In addition, it has been suggested that the transportof chloroplast precursors, followingtranslocation through the outer membrane, occurs viamembranous vesicles. These vesicles have127been observed between the two sets of surroundingchioroplast membranes in the chromophytesand are referred to as the periplastidal reticulum (Gibbs1979). The periplastidal reticulumwashypothesized to pinch off from the CER and fuse with the outerchioroplast envelope, apparentlydepositing protein precursors into the lumen of thechioroplast envelope. If so, the bipartitesequence may still be important in directing the precursoracross the inner chioroplast envelope.An interesting exception is with the haptophyte FCP presequence.Though ahydrophobic region is present, the N-terminal regiondoes not contain a basic aminoacid whichis a standard part of a signal sequence. Thisregion is relatively rich in hydroxylatedamino acidsas compared to the other FCPs. It has been suggestedthat an unformylated initiation Metresidue can compensate for the lack of a basic aminoacid and that there is not an absoluterequirement for a positive charge (von Heijne 1990).Otherwise, the Isochrysis FCPpresequence is unlike that of other chromophytes. The significanceof these differences, in termsof the transport mechanism, needs to be investigated.128CHAPTER 5A phylogenetic analysis of the LHCs5.1 IntroductionIn this study I was interested in investigating the evolutionof the two main LHC genefamilies: the Cabs and the Fcps. This analysisinvolves three lines of investigation.(1) I wasinterested in analyzing the relationships amongstthe different gene members of theCab family.Since most of the genes from the tomatoCab gene family have been cloned,sequenced and wellcharacterized (Green et al. 1991), it provided an opportunityto examine the evolutionaryrelationships amongst them and relate thesetrends to the proposed functionsof the differentprotein complexes. (2) I wanted to examinethe relationships between the known chromophyteFcp and iPcp gene sequences; however,as there are few known Fcp sequences,this analysis willbe limited. (3) I also wanted to examinethe evolutionary relatedness of theFCPs to the CABs.Analysis of the relationships between thedifferent Cab gene types to the Fcp genesmay provideinformation as to when the divergence of thechromophyte antennae occurred. In addition,theusefulness of the LHCs in assessing organismal phylogenywill be discussed.1295.2 Methods5.2.1 Protein AlignmentPhylogenetic analyses were carried out on the inferred protein sequencesfrom a numberof different light harvesting antenna! proteins, from a diverse rangeof organisms. An originalalignment was done using MACAW 2.02 (Schuler et al. 1990)with the 250 PAM matrix(Dayhoff et al. 1978) scale. Refinements to this alignmentwere made by hand using the GeneticData Environment (GDE) software on a SUN workstation.The process of generating anysequence alignment carries with it the assumption of positionalhomology between the residuesbeing compared. Highly divergent areas of the protein, which wereimpossible to alignunambiguously, were omitted from the analysis. This was doneto avoid the comparison of non-homologous regions of the protein which would violatethe first assumption, positionalhomology, and could significantly alter the resulting tree topology(see Schlegel, 1991).Furthermore, regions of ambiguous similarity and multiple insertion/deletionevents (indels)probably represent areas of ‘multiple hits’ and contain little, ifany, phylogenetic signal (Swoffordand Olsen 1990). Gaps were introduced in the alignmentsto maximize similarity. Those thatwere larger than one amino acid were treated as a singledeletion event in the analysis.The exact residues used in each analysis are indicated in Table5.1 by residue numberaccording to the labeling in Figure 5.1. The numberof residues varies depending onthe degreeof sequence conservation between the taxa selected for the analysis.Amino acids were analyzed,rather than nucleotides, for a three reasons: (1) thethird nucleotide of the codon tends to becomerandomized over large phylogenetic distances and isexpected to have little phy!ogenetic signal,(2) the protein sequence is somewhat less sensitiveto biases in the G + C content, and (3) itallowed the inclusion of LHC sequences that were determined onlyat the amino acid level(Swofford and Olsen 1990).130Table 5.1 Amino acid characters used inthe phylogenetic analysesdatasetTomato CABs (Fig. 5.3)Total CABs (Fig. 5.4)Green algal CABs (Fig. 5.5)FCP analysis (Fig. 5.6)Total LHC analysis (CABs/FCPs) (Fig. 5.7)*aslabeled in Figure 5.15.2.2 Phylogenetic analysischaracters*48-71; 79-111;160-192; 219-223; 225-232, 241-286as above48-71; 79-111;160-192; 219-231,241-30148-73; 79-111;140-188; 191-198; 241-27548-71: 79-111:159-194: 241-281Phylogenetic analyses were done with PHYLIP version3.5 (Felsenstein 1992) using bothparsimony (PROTPARS) and distance matrix (PROTDIST) algorithms.Both algorithms wereused to determine if putative taxon relationshipswere consistent between each treeconstructionmethod. However, consistency between methodsis not necessarily a good indicationthat thederived tree topology is the correct one (Felsenstein1992). Comparing trees generatedusing thedistance method should help in assessing which taxamay be rapidly evolving, based onthelength of the branches in the tree. Thisshould give some idea as to the likelihood ofartifactsoccurring in the parsimony tree since parsimony tendsto fail under circumstances when the rateof change between different taxa is large (Schlegel1991). In such cases, there is a tendency togroup faster changing sequences together; a case referredto as long branches attracting(Felsenstein 1978). As well, distance methods tend tobe sensitive to the number of taxa in theanalysis, which can alter tree topology (Schiegel 1991).Parsimony trees were done with the jumble option ineffect, which randomizes the orderin which each taxon is added to the analysis. This was repeatedten times for each tree since the131input order of the taxa can influence the final treetopology (Felsenstein 1992). Changes inamino acids were assigned a value conesponding tothe number of minimal mutational stepsrequired to cause such a change, based on the geneticcode. Mutations resulting in a synonymousamino acid change are not included in the calculation.It is assumed that they occur muchmorefrequently than non-synonymous amino acid changesand are phylogenetically unimportant(Felsenstein 1992). With the exception of omittingambiguously aligned regions, whichassigns aweight of zero to these areas, no external weightingwas used. If more than one mostparsimonious tree was found then a consensus tree wasshown. Consensus trees includebranchtopologies that occur most frequently; branch relationshipsappearing in more than 50%of thetrees of equal length (equally parsimonious)are definitely included (Felsenstein 1978).Distance matrix analyses utilized the Dayhoff acceptedpoint mutation (PAM) matrix(Dayhoff et al. 1978) in the calculation of distancesbetween the different taxa. This isanempirical matrix which assigns a probability for the conversionof one amino acid to another.Gaps are treated as unknown amino acids and droppedfrom the calculation (Felsenstein 1992).From the distance matrix, the tree topology wasdetermined using the neighbor-joiningmethod(Saitou and Nei 1987). The neighbor-joining methoddetermines tree topology by startingwith astar-like tree and successively clustering taxa into neighbors.Neighbors are taxa that areconnected by a single interior node and are clustered onthe basis of a calculated minimumdistance between them. The joining of neighborscontinues until only one possible unrootedtreeexists. The distance values between each taxon wererandomly inputted using the jumbleoption.An estimate of branch stability was assessed by a bootstrap analysiswith 100 replicates.Bootstrapping is a process that randomly resamplesthe character sites (eg. amino acids at aspecific position) until a dataset the same size as theoriginal is obtained; the number of taxa inthe analysis does not change. This is repeated any numberof times, as determined by the user.Depending on the size of the dataset and the numberof taxa, bootstrap trials can take aconsiderable amount of computer time. Because of this,trials are routinely limited to 100replicates. The phylogenetic analysis is then done oneach of the replicated datasets and the132results are expressed as the number of times,out of 100, a particular node on the resulting treewas supported (Felsenstein 1985). The bootstrapvalues are typically displayeddirectly on thebranches of the tree they correspond to. It isan estimate of whether the tree is likelyto change ifthe number of characters in the analysis had beenlarger. Bootstrap values shouldbe consideredas an indication of the consistency of the individualcharacters in determining aspecific treetopology and not a probability that the relationshipsdepicted are the true phylogeny.It is alsoimportant to realize that bootstrap analysis is notable to detect errors in phylogenythat are theresult of systematic errors. These are errorsthat result when the evolutionary processesviolatethe assumptions of the phylogenetic methodused (Swofford and Olsen 1990).5.2.3 Terms and conceptsA phylogenetic tree is a type of dendrogram, ie., a branchingdiagram depictinghypothesized genealogical relationships of the taxa.The taxa are displayed at terminal nodesandthe branches joining them are connected at internalnodes. The branching of thetree normallyoccurs in a bifurcating fashion. The trees in either methodare constructed based on a specificcriterion. This refers to the manner in whichthe evolutionary characters are weightedandassessed. Characters generally refer to a measurableobservation of any trait pertaining to anorganism or gene (taxon). In this study, a characterrefers to an amino acid residue locatedat aspecific position within the protein. The identity ofa character at a specific position (whetheritis Ala, Leu, or Ser, etc.) is called the characterstate. A monophyletic group refers to twoor moretaxa that share the same ancestral taxon.Alternatively, groups are considered polyphyletic whenthe taxa within them are derived from twoor more distinct ancestral genes or species. Paralogoussequences (eg. Lhcal and Lhcbl) do not share the same evolutionaryhistory as duplication anddivergence of the gene occurred prior to thedivergence of the organism (ie., a speciation event)(Schlegel, 1991). Genes of the same type(e.g. all Lhca4 sequences) are considered orthologous133because they diverged from a common ancestralsequence and should parallel the evolution of theorganism (Schlegel, 1991).The taxonomic positions of the organisms, from whichthe LHC sequences usedin thisanalysis originated, are given in Table 5.2. Thereader should also refer to Table1.1, in thegeneral introduction (Chapter 1), for further characteristicsof the algal groups in question.Table 5.2 Species and genes used in the phylogeneticanalysesOrganism Taxomonic position/geneReferenceLycopersicon esculentum Angiosperm: dicot(tomato) LhcblPichersky et al., 1985Lhcb2Pichersky et al., 1987Lhcb3 Schwartzet al., 1991aLhcb5 Picherskyst al., 1991Lhcb6a/b Schwartzand Pichersky, 1990Lhcal Hoffmanet al., 1987Lhca2 Picherskyat al., 1987Lhca3 Picherskyet al., 1989Lhca4Schwartzetal., 1991bpsbSWallbraun et al., 1994Spinacia oleracea (spinach) Angiosperm: dicotLhcb6Spangfort et al., 1991psbS Kim et al., 1992Arabidopsis thaliana Angiosperm: dicotLhcal Jensenet al., 1992Lhca2 Zhanget al., 1992Lhca3 Wang etal., 1993Lhca4 Zhangetal.,1991Lhcb4 Greenand Pichersky, 1993Lemna gibba (duckweed) Angiosperm: monocotLhcb2 Karlin-Neumannet al., 1985Gossypium hirsutum (cotton) Angiosperm: dicotLhcb2 Sagliocci etal., 1992Oryza sativa (rice) Angiosperm: monocotLhcbl Matsouka, 1990Pinus sylvestris (Scots pine) GymnospermLhcbl Jansson and Gustafsson,1990Lhcb2 Jansson andGustafsson, 1990Lhcb5 Jansson and Gustafsson,1992Lhcal Jansson and Gustafsson,1991Lhca2 Jansson and Gustafsson,1991Lhca3 Jansson and Gustafsson,1991Lhca4 Jansson andGustafsson, 1992Ginkgo biloba GymnospermLhcbl Chinn and Silverthorn,1993Polystichum munitum Pteridophyta(fern) Pichersky et al., 1990Physcomitrella patens Bryophyta(moss) Long et al., 1989134Chlainydomonas reinhardtii ChiorophytaLhcbImbault et al., 1988LhcaHwang and Herrin, 1993Chlamydomonas moewusii ChiorophytaLhcbLarouche et al., 1991Chiamydomonas stellata ChiorophytaLhcb Wolfeet al.,1993-unpubl.Lhca (2C.stellata) pir S33466,S31393Chiamydomonas eugametos ChiorophytaGagné and Guertin, 1992Dunaliella tertiolecta ChlorophytaLhcbLaRoche et al., 1990Dunaliella sauna ChiorophytaLhcbLongetal., 1989Mantoniella squamata Chlorophyta:Rheil and Mörschel, 1993PrasinophyceaeEuglena gracilis EuglenophytaMuchhal and Schwartzbach,Lhcb 1-41992Lhca (35 & 38)Houlné and Schantz, 1988Phaeodactylum tn cornutum Chromophyta: Diatom Grossmanet al., 1990Bhaya and Grossman, 1993Odontella sinensis Chromophyta: Diatom Thelen& Pancic -gb 81054Macrocystis pyrfera Chromophyta: Brown algaApt et al., 1994Heterosigina carterae Chromophyta:Durnford, D. this studyRaphidophytaIsochrysis galbana HaptophytaLaRoche et al., 1994Pavlova lutherii Haptophyta Hiller,R.unpublished*Amphidinium carterae Dinophyta: DinoflagellateHiller et al., 1993**Sequence determined at the protein level. However, recently the nucleicacid sequence of tnegene encoding the dinoflagellate iPCP protein has been determined(gb Z47562, Z47563).5.3 Results5.3.1 Assessment ofphylogentic signalThe datasets used in each analysis include the sequences shownin each Figure (Fig. 5.3-5.7) and the characters outlined in Table 5.1. The phylogenetic signalof the dataset was assessedby calculating the skewness of tree-length distributions from 10,000randomly generated treesusing the ‘random tree” option in the computer program PAUP (PhylogeneticAnalysis UsingParsimony) (Swofford 1991). The random tree option randomly selectsa tree topology and135calculates the length of that tree based on the dataset. It repeatsthis process 10 000 times andplots the tree lengths against the frequency of their occurrence.Random sequences producenearly symmetrical distributions from a parsimony analysisof all possible tree lengths whilethose that contain a phylogenetic signal have a skeweddistribution (Hillis et al. 1993). Thecharacters in the FCP sequence dataset (usedin Fig. 5.6) were manually randomizedand therandom tree distribution calculated (Fig. 5.2A) to compareit to the random tree distributionproduced with the non-randomized sequences (Fig.5.2D). As expected, the distributionwasnormal as compared to the very skewed distributionof the non-randomized dataset (compareFig. 5.2A & B). This indicates that this is auseful method of assessing the potentialphylogeneticsignal in the datasets.The tomato CAB (Fig. 5.2B) and FCP (Fig. 5.2D) datasetshad a stongly skewed randomtree distribution. The green algal dataset also showeda strongly skewed distribution,though to alesser extent (Fig. 5.2C). This indicates that thesedatasets have not diverged beyondthe point ofbeing a potentially useful phylogenetic indicator. Thelast two datasets (Fig. 5.2E and F)giveonly a moderately skewed distribution. Though thisis a conservative estimate, it indicatesthatthe dataset may be approaching a limit of changewhere convergent or back mutationshavebecome as frequent as divergent mutations (Meyeret al. 1986) and is approaching itslimit as aphylogenetic indicator. However, PAUP does not assignweights to amino acid changes,nordoes it reflect the genetic code, as PHYLIP does.Without weights, PAUP essentially calculatesthe similarity based on amino acid identity. Thisunderestimates the true relatedness of thesequences and the random tree distributions should beconsidered a lower limit estimate ofphylogenetic signal. In addition, since PAUP doesnot use a character weighting scheme,therandom tree lengths in Figure 5.2 can not be compareddirectly to the length of the trees I willshow.136Figure 5.1Amino acid sequence alignment of selected sequences used in the phylogenetic analysis.Numbers on top of the alignment indicate character positions and are not an indication of theprotein size. Regions of some proteins have been omitted to save space, and have been indicatedby the presence of a*•The sequences are arranged by gene/protein type followed by the speciesit is from. Gene types are: Lhca and b, Light harvesting complexes associated withPS I or PS II,respectively, according to the nomenclature of Jansson et al. (1992); iPCP, intrinisic peridininchlorophyll protein; FCP, fucoxanthin-chiorophyll protein. The taxa are: Ac, Amphidiniumcarterae; At, Arabidopsis thaliana; C.eugam., Chiamydoinonas eugametos; Cr, Chiamydomonasreinhardtii; Cs, Chiamydomonas stellata; Ds, Dunaliella sauna; Eg, Euglena gracilis;Gb,Ginkgo biloba; Hc, Heterosigma carterae; Ig, Isochrysis galbana; Le, Lycopersicon esculentum(tomato); Lg, Lemna gibba; Mp, Macrocystis pyrifera, Ms, Mantoniella squalnata;Os, Odentellasinensis; P1, Pavlova lutherii (partial sequence); Pm, Polystichuni muniturn; Pp, Physcomitrellapatens; Ps, Pinus sylvestris; Pt, Phaeodactyluin tricornutum; So, Spinacia oleracea. Gaps insequence are indicated by a -.137MSR111121314151IIIIIIII6171819110111100Lhcb6a-LeV--PP---KRFVVAAAAVAPKKSWIPAVXSGGNLVDPEWL00SLPGDFGFDPLGLGKDPAFLKWYREAELINGRWAMAAVLGIFVGQAWSGIP---psbS-So*KStJAPKCVEKPKLK-VEDGLFGTSG01GFTKENELFVGRVANIGFAASLLGEGITGKGIL-Lhcb5-Le*JtJ(7ppKAKAAAVSPADDELAKWYGPDRRIFLPEGLLDRSEI-PEYLNGEVPGDYGYDPFGLSKKPEDFAEYQAYELIHARWAMLGAAGFIIPEAFNKFGA--Lhcb4-At*GSGRFTAVFGFGKKX-AAPKKSAKKPV’ITDRPLWYPGAISPOWLOGSLVGDYGFDPFGLGKPAEYLQFDI*GIQRPRECELIHGRWAATLGALSVEWLTGVT---Lhcal-Le*LSSTKSKFAA)MPVYVGATNFMSRFSMSADWMPGQPRPSYL00SAPGD9’GFDSLGLGEVPANLERYKESELIHCRWAML.AVPGIIVPEALGLGN----Lhca2-Le*pPLRVSKYSPTPTARSATVCVAADPDRPLWFPGSTPPPWLDGSLPGDFGFDPLGLASDPESLRWNQQAELVHCRWANIGAAGIFIPELLTKIG---Lhcal-Le*LSSGFJREVSFRPSTSSSYNS1ICVEAK(GQWLPGLASPDYLDGSLPGDNGFDPLGLVEDPENLKWPIQAELVNGRWAMLGVAG1LPEVFTSIG---Lhca3-Le*IGSRISQSVT!RKASFVVRAASTPPVKQGANRQLWFASKQSLSYLDGSLPGDFGFDPLGL-SDPEGTGGFI-EPKWLAYGEVINGRFAMLGAAGAIAPEILGKAGLIPLhcbl-Le*GNGRIKAVAI(SAPSSSPWYGPDRVKYLGPFSGES---PSYLPGEFPGDYGWDTAGLSADPETFAXNRELEVIHCRWANLGALGCVFPELLAflNGV---Lhcb2-Le*GEGRIRTVKSAPQSIWYGEDRPKYLGPFSEQT----PSYLTGEFPGDYGWDTAGLSADPETFARNRELEVIRCRWAMLGALGCVFPEILSKNGV--Lhcb3-Le*--NANPLRDWAMGS-ARFTMSNDLWYGPDRVXYLGPFSAQT---PSYLNGEFPGDYGWDTAGLSADPEAFAXNRALEVINGRWAMLGALGCIFPEVLEKWVKV--Lhcb-GbGEGRITJ(TASKKV---VASSGSPWYGPDRVKYLGPFSGEA----PSYLTGEFPGDYGWDTAGLSADPETFA)DRELEVIHSRWANLGALGCVFPELLSRNGV--LhCb2-Lq*S00RF5RWKAVPQSIWYGADRPKFLGPFSEQT----PSYLTGEFPGDYGWDTAGLSADPETFA1RELEVIHSRWAMLGALGCIFPELLSKNGV--Lhcb-PpTVSKSAGSD’PIWYGADRPKFLGPFSGET----PSYLNGEFAGDYGWDTAGLSSD?EPPAT<ELEVIHARWANLGALGCLTPELLAKSCV--Lhcbl-Ps*GEARVEWRKAITKXL--TASASTSPWYGPDRVLYLGPFDGEP----PSYLTGEFPGDYGWDTAGLSADPENPAiCNRELEVIHCRWANLGALGCVFPELLARNCV--Lhcb-Pm*GEARVQMMAPI<SKAPSGSIWYGSDRPLYLGPFSGSP----PSYLSGEFPGDYGWDTAGLSADPETFAC1RELEVIHSRWAMLGALGCVT?ELLAEWGV--Lhcb-Cr*LQ(*A3KKTAAXAAAPKSSGVEFYGPWRAI(WLGPYSEWAT--PAYLTGEFPGDYGWDTAGLSAOPETFKRYELELIHARWAMLGALGCQTPELLAXSCT--Lhb-0sQGGRAGKSTKKGAKAVSKSSSSANQFYGPDATSGWDLQHQHPRLPTGEFPGDYGWDTAGLSADPEPPKRYRELELIHARCGLLGALGNVTPELLADE001K-Lhca-Cs*_7(AALGFRAPAQARRSWARAEQRASWPPGWPAPEYLKGELAGDYGFDPLGFGDEPRRLEWMVQAELVHCRWANLGAAGILLPEIGNKTCMQSALhca-Cr*RS7TJPRGPSGRRVAAVSNGSRVTMKAGNWLPGSDDAPAWLPDDLPGNYGFDPLSLGKEPASLKAFTESEVINGRWAGVAGSLAVELLGYGN---Lheb-EglWLATSGRXAKAAPKSDNLSQWYGPDRAKWLGPLT0EV--PSYLTGELPGDYGWDTAGLGSDPTTLARYREAEVIHARWAMLGALGVVTPELLAGNGVP--Lhea-Eg2MFASSGHKDGLWPPNAEPPALLTGEYPADRGPDPLNLAADP0VYAFMRVAEVPNGRLANLCIVGCIVPELLGKGA---C.eugam.*VRVQAAGKTVSGSKTVSGGKTVGSASAESRRVAEVQAYLATL----PGCGVESG?FKGVWDPLSLAATATVGDVRRWRESEITHGRVAMLAALGFVVGEQLEDFPA--Lheb-MsMACIASSFVGSVAALKATKVQAXSVSTVVXADIY---PEPGT--YPG--GGESPIIPFGSEICNAEREVIRGRWPffGVTGAWrl’ENGTGIP---iPcp-AcFEWERGVQDPVGFFDPLGFTADGSVENPKALAQTEIKMGRVANLATMGYITPEITGKLC---Fop-HeMSLKL--ATPA-AALMGASAPVAPN!GVAKSSSALEWSPENEIGVQAPLGFWDPLGLLDEADQERFDRLRTVEIKHGRISMLAILGHLV’rFAGVRLP0---Fcpl-Mp*MXSAVVACAAAPGFRGPSAFNGAALTTSAXCSCMSFESEIGAQAPLGFWDPLGLLADAOQERFERLRYVEVKHGRIAAIAGHLTQQN-TRLP0---Pep-OsMXLAIAALLAGSAAAFAPAQS--GKASTALNNAFESELGAQPPLGFFOPLGMLADADOERFDRLRYVEVGGRIABVAFLGQIVTENGIHLSG--Fcpl-PtMXFAVFAFLLASAAAFAPAQ-QSARTSVATNMAPENEIGAQQPLGYWDPLGLVADGDOEK?DRLRWEIKHGRICNLAVAGYLTQEAGIRLPG---Fcp-IgMWFLASLPSTAIAGLASAAPKVQ-PRMAANDEPAY-GLPGGANILGEPDPAGFLKGKDXLEVYLRFAMThGRVAMLASLGFVVQEK-FHFcp-P1APLGFYDPLGLLDNADEEYFERM-YVELMSR2psbS-SoLhcb5-Le Lhcbd-AtLhcal-Le Lhca2-LeLhca4-Le Lhca3-LeLhcbl-Le Lhcb2-Le Lhcb3-LeLhcb-GbLhcb2-LqLhcb-PpLhcbl-PsLhcb-PmLhcb-Cr Lhcb-Ds Lhca-Cs Lhea-CrLhcb-Egl Lhca-Eg2 C.eugamLheb-Ms iPcp-AcPcp-}{cFCpl-MpFcp-OsFcpl-PtFep-Ig Fcp-P1-NCGPRAVWFRTGWQDAG--WVKAQEWAAIPGGQATYL--ILNTPSWYTAGRQEYF---ILNVPKWYDAGKSEYF---QETALAWFQTG--KFGRAVWFRAG--RFGF.AVWFRAG--DFKEPVWFRAG--KFGEAVWFRAG--QFGEAVWPRAG--KFGRAVWFRAG--KFGEAVWFRAG--RFGRAVWFRAG--RFGEAVWFRAG--FGDAAIWFRAGAPGLAFPHWWRAG--WYDAPLWAVNG--FGEGAVWYRAGWFEAGDSPFNWFTAGTLCTPDD----FSFGSLLGTQLLLM-G----AEPLLLFFILFTLL-GI--—NLILAVVA-EVVLV-GP-——SISTLIWI-EVLVI—G----GTLPTILAIEFLAI-A----TDPrrLFIVELVLI-GASSSTLFVIEFILF-HGNYWADNYTLFVLEMALM-GWVESRRWVDFFDNDSQSIDWAIGALGDRGRFVDEPrrGLEGAEYYRIINGLDLEDKL--YIEFQR-NAELDSEKRL---FVENQR-SMERDSEKKZ---WAEGP.RWADIIRPGCVNTDPYVEIRRWQDIRNPGSVNQDPFAEHRRFQDWAKPGSMGRQYLhcb6a-LeWFEAGADPGAIAPIIIIII121131141151161171181191201211TGIPIYE---TLNYFGRNIPGSSYLGQPLP“C----SQLNLE---ALLLDGN----RVELVD GQPVPW----VIPPA—---SQIFSE ----SQIFSR ----SQIFSD----SQIFSD----AQIFSE ----AQIFS8 ----AQIFSE----SQIFAE ----AQIFSR----AAIFQD----RV-VAEATPWSRTAFANTGEQGRAV IFPNNRLTGTDVGIFJYSLPPNKCGFLGLEKGLGGSGDPAGGLDYLGNPSLVRAQSILAIWACQVVLM-GGGLDYLGNPNLVHAQSILAIWACQVVLM-GGGLDYLGNPNLVHAQSILAVLGFQVVLM-GGGLD(LGNPSLIHAQSILAIWACQVILN-GGGLDYLGNPNLVHAQSILATWATQVVLM-GGGLDYLGNPSLVRAQSILAIWACQVVLM-GGGLDYLGNPSLVHAQSILAIWACQVILM-GGGLDYLGNPSLVKAQSXLAIWACQVILM-GGGLDYLGNPSLVHAQNIVATSAVQVILN-GGGLNYLGNPSLIHAQNIVATLAVQVVLM-GSGPWSNGSLTFTLLM-GAVEGYRIAGG FVEGYRVGGG LVEGFRUGL AVEGYRIAGG LIEGYRVGGG AVEGYRVAGG AVRGYRIAGG AVEGYRVAGG LIEGYRVNGG LVEGYRVNGGPLGEVVDPL PLGEGLDRI-PGVGEGNDL PLGEITDPI PLGEGLDPL PLGEVTDPI PLGEVTDPI PLGEVEDPI-PAGEGLDPL PAGEGLDPLWAEHKRLYDFVRPGSQAVGWAWAPLGLGSITGVEEPLVAXENGGRA’IWFGIEVPFDLNALLAFEFVAN-AAAEGQRGDEGQRGDAGGVV----AQIFSADGLNYLGNPSLIHAQSVVLTFLSTLAIM-GAVEAYRYGGGVGDFGRELDTVDGLKLGEITMAIA-APTEYWRGQGGFNWQKGE-LDRSFDGHI--TGQAIRQFDQVQQGFWEPLLIAI-GLAESYRVSLGWATPTGTGFNNLKDECTAVADRFP-GAVAPLAPEGSGY-PSFWAVLAEVVLV-GLAEAYRTGLSDSPFEELTVGDV----YLSPSTGVXYDDtPNGLGAISKVPAAGWGQMtAYAAFSELSQDQSASTPAAEGAFGFKVLT----AYDL-AGDQFSSLPTGLRALSALPAAGVAQTIGFIGLIELGFAQIKEELE--ADCEARM----SNSANLSFADMPNGVAALSKIPPAGLAQIFAFIGFLELAVMENVEGS---FPGDFTLG--NIDYAGNSFDSFPNGWAAISGPDAIPQAGLLQIVAFVGILELAVMKDVTGEG-EFPGDFR----DIDYS-GTSFESIPNGFAALSAVPGAGIAQIXAFIGFLEIAV(DITGG--EFVGDFR---PLFSGDNGPAIEQIPQLPYWLWIVMTIG-IGRAELFRIQXGWAXVNPETGKADSALREGLEVVPAAGLAQIVAFVGFLELPV-RDVTEG-DFPGDFMSR3221231241251261271281291301C.eugamLhcb-Ms iPcp-Ac Fcp-Hc Fcpl-Mp Fcp-Os Fcpl-PtRSALGL FDPLGL FDPLGL FDPLGY FDPLGWG FNPLNF FNPLGP FDPLGL FDPLGL FDPLGL FDPLGLADDPDAFAELFDPLGLADDPEAPAELFDPLGLADDPUFAELFCPLGLAEDPEAFA.ELFDPLGLADDPEAFAELFDPLGLADDPDTFAELFDPLGLADDPIY1’FAELFDPFQMSKNEAXYKWYFDPLGFAKI3SSKSGELFDPLGL.NDPDALAELFDPLNL1IDYTYDLGNLYFDPLGLKPEDPEELRLSPGGR--FDSLGLAESGD-LEEL SSDPAELEKK---DAAGWODEKKDSKGNPFGASWDP.MSEETQASKNGALDFGWDTFDEETKLSKNNYLDPGWDTFSEDKXLQKANLIFYFEAGQ--GKTPAQLGFAFSLIGEIIT-GKGA?MPSGFFIQAYV’I’-GQGP.NVAFLGFAVQA7.AT-GKGPALLAIVGFCVQQSAYLGTGPP.MLAVMGAWFQHIYT-GP1LAFLGPXVQIINVT-GKGPMLAILGYFIQALVT-GVGPMFSNFGFFVQAIVT-GKGPKVKEIKNGRL)MPS14FGFFVQAIVT-GKGPKVKEIIGRLAMFSMPGFFVQAIV’r-GKGPKVKELKNGRL.NFSNFGFFVQAIV’r-GKGPKVKXIcNGRLMFSMFGFPVQAIVT-GKGPKVKEIGRL?MFSMFGFFVQAIv’r-GKGPKVKEIIGRLFSMPGFFVQAIVT-GKGPKVKELKNGRLAMPSMPGFFVQAIVT-GKGPLGALGL LAQLNI-ETcVPINEIEPLVVENLA.HLSDPFGN4LLTVILN1WAThLSDPLRT1’IITFLENLATHLADPWNIGPVIIDNLF}LADPGHATIFAAFFDNLLQHLSDPWHNTIIQTLYQNLLDHLADPVNNNVLTSLLENLADHLADPVNNNAWAFAENLSDHINDPVNAWAYALENLLDWLEWPVP.1NAWVYAIENLADHLADPVNNNAWAFAIENLADHIADPVANNAWAFALLNDADPVNAWAYAILADHLADPVNNNAWAYAIENLSDHLADPAVNNAWAYAVQNLDJHLANPTVNAFAFAIENLTDHLANPAENNAFAYAIDNLVDHVSNP1KVTFATNGIAAGEWLANPWGANFAGVENLTFHLAE3PSANIFFTSLANLSAHLANPIGPNIrrNL‘rE-IFQHLFFTIEKDIVEEIIJWQAHVADPVHANVLTNAGDWASYThSPLIINSLLGSPVPFNAGFVINDLVGASYTFNIVG4 pLLNVVPFFIAAINPGTGKFIPDDEEEDGGASERVPTL sss IPKGIFPN SPK s KFH TNFVPGK TNEVPGK TKFVPGA TNFVPGK TNFVPGK PTSPPGTRI’NFVPGK‘IThIFTPGK TKYTPSA TKFTPQ VSVPNFIEYH ISVPFF GFA A DIIEKDLGLPPSFPVPTLPNLSSASGFGFYFDPLALAGTLNNGVYVPDTEKLERLKLAEIKHSRLKTKGPLFGFTKSNELFVGRLAKDPDQAAILKVKEIGRLAAPPEKTAQL SKDPAKFEEL SGSPAXIKEL ----APTEEA GKDEKSMKEL AEDPEAFAEL ADDPEAFAEL ADt3P’ITFAELLhcb6a-Le psbS-So Lhcb5-Le Lhcb4-At Lhcal-Le Lhca2-Le Lhca4-Le Lhca3-Le Lhcbl-Le Lhcb2-Le Lhcb3-Le Lhcb-Gb Lhcb2-LgLhcb-Pp Lhcbl-Ps Lhcb-PmLhcb-Cr Lhcb-Ds Lhca-Cs Lhca-Cr Lhcb-Egl Lhca-Eg2QLAEIKHARL KVKEIKNGRL RTKEIa4GRL KEKELANGRL KLKEIKNGP.L KVKEIKNGP.LYPGGK-F IPPGKDV HPGGP- YPGGK-F YPGGA- YPGGL-W YPGGI--YPGGPL YPGGS- YPGGA- YPGGQ-Y YPGGS--YPGGA- YPGGS- YPGGS--YPGGS--YPOES--YPGEA--YPGGI--YPGGA--YPGGP--YPG---KVKEIGRLASFSNPGFFVKVKEIKNGRL.MFACLGFFVKQSEVTNGRLIT4FGFVSKLKEIKNGRLAMVAFLGFVAKVKELKNGRLAMVAilGFYVRAAEVKNGRLALTAVAGLTAQTKELNNGRLNIAIAGPVLKKELKHCRLSMFAWLGCIFLSAPIANGPLAAIIGMFFRAIELNNGRAAQHGILALMVAIELNNGRAAQNGILALMVRAIELNNGRAAGILGU4VRAIELWQGRAAQMGILALMVQAIV1’-GKGP QAIV’l’-GSGP QHIAYPGKGP Q}{AAT-GKGP QPLVT-KAGP QYLAT-GESP QEV---AEPG QALAT-QEGP QOGLT-GSAR HEQLD-NNPY HEELN-NKPY HEQLG-GSIP HEQLG-VSILFcp-IgYEPGDLGFDPLGLAPSDPDEFRLMQEKELSHGRLIAAAGPLAQEAVS-GTWGT’GDATPFcp-P1KRNIELNQG-AA-N-ILALMVFigure 5.2The random tree distributions of the datasets usedin the phylogenetic analyses (figures5.3-5.7). For each dataset, the lengths of 10000 randomly generated trees were calculated andplotted against their frequency of occurrence. (A) Theorder of the amino acids for eachtaxa inthe FCP dataset (used in Fig. 5.6) were randomizedmanually and the distributionsof therandomly generated tree lengths were calculated.The other graphs are the randomtreedistributions of the datasets used in the analysisof the following sequences: (B)the tomato CABproteins (Fig. 5.3), (C) the green algal sequences (Fig.5.5), (D) the FCP sequences (Fig.5.6), (E)the CAB and FCP sequences (total LHC) sequences(Fig. 5.7 A/B), and (F) the CABsequences(Fig. 5.4 A/B). The mean (&L) and the median(SI-) of each distribution is indicated on thegraph.1415007006000C soca)400L300200100450400350300Dci)250 —ci)200ci) ILI_100 —100500Tree LengthTree Length500 —700—600ci)0C 500 —ci)0- 400 —a)300 —200 —100300 —250200D 1600-ci)U-100500 —Tree Length400-1--350 T3002502004-ISO-f10050Tree Length45040035030020020015010000F1I-ujTree LengthTree Length1425.3.2 Phylogeny of the tomato Chlorophyll a + b-binding protein family:Most of the genes encoding Chi a + b-binding proteins fromtomato have been cloned andsequenced. This provides an opportunity to examine the intraspeciesrelationships between thedifferent Cab gene members of this multigene family (Table1.2). The phylogenetic trees inFigure 5.3 are based on 147 amino acid residues, representing approximately67% of the matureprotein. The amino acids used in the analysis are indicated in Table5.1. These characters makeup portions of the mature protein that are part of the membrane spanningregions (MSR 1-3) andinclude the stroma exposed areas in front of them.The distance (Fig. 5.3, A) and the parsimony (Fig. 5.3, B) methodswere fairly consistentin their predicted relationships between the different tomatoCab genes. The clustering of theLHC II sequences on a branch with Lhcb5 (CP26)is robust and consistent between analyses.This relationship is also supported by high bootstrap values. Withinthis branch, Lhcb5 andLhcb3 form the deepest nodes with Lhcb2 and Lhcblconsistently clustering together (Fig. 5.3, A&B).The Lhcb4 gene from Arabidopsis was used in the alignmentbecause the correspondinggene from tomato has not been cloned. It was expectedthat the relationship of tomato Lhcb4(CP29) would not deviate significantly from what isobserved with the Arabidopsis gene, as willbecome obvious in the next section. In both trees, the Lhcb4gene has a closer evolutionaryrelationship to the Lhcb6alb (CP24) gene than it doesto the other Lhcb sequences.The Lhca (LHC I) sequences are quite divergent and their depictedrelationships are notwell supported by bootstrap analysis. The Lhca sequencestypically have a 45% identity to Lhcbland are 50-60% identical to each other. There is moderatesupport for the closer associationofLhca2, 3 and 4 in both trees. This is to the exclusionof Lhcal, which frequently forms amonophyletic group with Lhcb4 and Lhcb6 in the distancetree (Fig. 5.3A). The clusteringofLhcb6 and Lhcb4 is considered robustbecause the branch is well supported by bootstrapreplicates (89 and 83%) and is consistent between methods.143Lhcb3BFigure 5.3Lhcb5Lhcb4 ArabidopsisLhcb6bLhcb6aLhcb2LhcblPhylogenetic analyses of theCAB proteins from tomato(Lycopersicon esculentum).(A)Distance matrixanalysis using the neighbor-joiningtree construction method.(B) Parsimonyanalysis using an identicaldataset as in (A) andas described in the resultssection. As labeled,the Lhcb4 gene wasfrom Arabidopsis thaliana.Gene names listedin the Figure correspondtothe following protein complexes:Lhcbl—3 (LHC IItypes I—>III), Lhcal—4(LHC I types I—IV), Lhcb4(CP29), Lhcb5 (CP26),Lhcb6alb (CP24),psbS (the 22 kDa protein).Further explanations andcharacterizationsof the complexes aregiven in Table 1.2.Bootstrapvalues calculated from100 replicates are givenat the appropriate nodes.ALhcalLhca3Lhca2Lhca4psbS0.10psbSLhca4Lhca3Lhca2Lhcb6bLhcb6aLhcb4ArabidOPSiSLhcalLhcb5Lhcb3144Changing the outgroup from PsbS (CP22) to Lhcb6 (CP24)did not significantly alter therelationships presented in Figure 5.3. The relationship ofthe Lhcb4/6 branch, with respect to themain Lhca branch (Lhca2-4), could be altered dependingon the alignment used and number ofcharacters selected. However, no significant change in tree topologywas observed when theamino acid changes were weighted according to their chemicalsimilarity. Little change was alsoobserved when a tree was constructed with the same distance databut using a different algorithm(eg. Fitch). Despite this, rates of divergence are relatively large inthe Lhca portion of the tree asindicated by the long branch lengths in Figure 5.3A. Bootstrapvalues are also low, suggestingthe characters used are inconsistent in their phylogeny prediction.5.3.3 Phylogeny of the Chlorophyll a + b-binding protein family:This analysis shows the relationships between the diverse CAB proteinsfrom differenttaxa. In this case, I intentionally included LHC I and LHC II sequencesto assess theirrelatedness to one another and to make inferencesabout the evolution of this gene family. Thefirst analysis used 146 amino acid positions (characters), definedin Figure 5.1. Figure 5.4B is aconsensus of four most parsimonious trees with a length of 1469.There is an apparent separation of the LHC II proteins (Lhcbl, 2,3 and 5) and the LHC Iproteins (Lhcal-4) in the distance (Fig. 5.4A) and the parsimonytrees (Fig. 5.4B). The mainexception to this is the presence of Lhcb4 and Lhcb6within the LHC I branch, which was thecase in the previous analysis (Fig. 5.3). The EuglenaLhca sequences are also outside this maindivision. However, the separation of LHC I and LHC II is not wellsupported by the bootstrapvalues in either tree. Other branch variation, indicatedby low bootstrap values, was within theestablished branches of the tree and did not alter the separation of theLhca and Lhcb lineages (ie.within the Lhcbl-2 branches).1450.10C P24/C P29LHCI-IIFigure 5.4ADistance matrix analysis of the LHC proteins from select Chlorophyll a + b containingtaxa, including both PS I associated (Lhca) and PS II associated (Lhcb) antennalcomplexes. Thetree was constructed from the distance matrix using the neighbor-joining method describedin theresults section. Sequences are labeled according to their gene names and the identificationof thecorresponding complexes are indicated in the figure legend of Figure 5.3and in table 1.2. Referto Table 5.2 for references and full species names. Bootstrap values calculated from 100replicates are given at the appropriate nodes.__________n—Spinach-psbSLTomato-psbS412LhcbEugle99Lhcb-MantoniellalLLhcb-Mantoniella2C.eugametosCP26Lhcb5-TomatoLhcb5-PineLhcb-EuglenalLhcb-D.tertidecta ILhcb-C.moewusiiLHCII- Lhcb-D.salinaGreen AlgaeLhcb-C.reinhardtii— Lhcb3-TomatoILHcII-IIILhcb-MossLhcb-FemLhcb-GinkgoLhcbl-PineLHCII-ILhcbl -TomatoLhcbl -RiceLhcb2-CottonLHCII-IILhcb2-TomatoLhcal -Tomato ILhcal-ArabidopsisLHC-I- Lhcal-PineILhca-C.reinhardtiiILhcb6b-TomatoI- Lhcb6a-Tomato1Lhcb6-SpinachILhcb4-Arabiciopsisi— Lhca2-Pine— Lhca2-TomatoI— Lhca2-ArabidopsisLhca4-TomatoLhca4-ArabidopsisLHCI-IVLhca4-PineLhca3-TomatoI— Lhca3-ArabidopsisI LHCI-IlILhca3-PineILHCILhca-Euglenal4914637—Lhcb-C.moewusiiLHCII45 Lhcb-C.reinhardtii6Lhcb-D.saliGreen AlgaeLhcb-D.tertiolecta97Lhcb2-TomatoLhcb2-Cotton622724Lhcbl-RiceLhcbl -Tomato601 35 Lhcbl -PineLH8 61Lhcbl-Ginkgo6Lhcb-FernLhcb-moss29Lhcb3-TomatoI LHCII-IIILhcb-Euglenal17100Lhcb5-PineCP26Lhcb5-Tomato100Lhcb-Mantoniellal31Lhcb-Mantoniella2C.eugametosLhcal -PineLhcal -ArabidopsisLHC -Lhcal -TomatoLhca-C.reinhardtiiLhcb4-Arabidopsis ICP29Lhcb6a-TomatoLhcb6b-Tomato CP2420 Lhcb6-Spinach99Lhca2-Tomato100Lhca2-Pine LHCI-tI70Lhca2-Arabidopsis69Lhca3-ArabidopSiS100 Lhca3-Tomato LHCI-III76Lhca3-Pne58Lhca4-Pine100Lhca4-Tomato LHCI-IVLhca4-ArabidopsisLhcb-Euglena2Lhca-EuglenalLhca-Eugena2psbS-SpinachpsbS-TomatoFigure 5.4BParsimony analysis of the LHC proteins from select Chlorophyll a +b containing taxa,including both PS I associated (Lhca) and PS II associated (Lhcb) antennalcomplexes. Analysiswas done using an identical dataset as in (A), as described in the resultssection. Refer to Table5.2 for references and full species names. Bootstrap values calculated from100 replicates aregiven at the appropriate nodes.147The distance and parsimony trees were consistent in the clustering of the sameCAB types(e.g. Lhca4-pine with Lhca4-tomato), and this was true for all known angiosperm andgymnosperm taxa. This suggests that the major LHC Cab gene lineages divergedbefore theangiosperm / gymnosperm separation. The association of the chiorophyte Lhcbsequences,however, does not follow this pattern. In both trees, they form a monophyleticbranch separatefrom the Lhcbl, Lhcb2, and Lhcb3 sequences of the terrestrial plants. The separationof theLhcbl/2 sequences from the green algal Lhcb sequences is supported(bootstrap value 75 or 62,Fig. 5.4), though the separation of Lhcb3 is not as well supported in the parsimonytree(Fig. 5.4B). This suggests that Lhcbl-3 diverged after the chlorophyte and terrestrialplantlineages had separated.The fern Lhcb sequence clusters with the Lhcbl dade in the distance treebut before theLhcbl/2 separation in the parsimony tree. The bootstrap values coincidingwith these branchesare also low. Because of this, the relationship is considered unstable andconclusions regardingthe divergence of the fern sequence, before or after the Lhcb 1/2 functionalseparation, areinconclusive (Fig. 5.4 A/B). Divergence of the moss Lhcb sequence beforethe Lhcbl/2divergence is moderately supported by the bootstrap analysis (Fig.3, A/B), though this is underthe assumption that the moss sequence represents the main LHC II antennalprotein and is not aparalogous gene. Some caution must be exercised in the analysisof the Lhcb relationshipsbetween the fern, moss, and chlorophyte CABs as a limited numberof sequences have beencharacterized; these are often assumed to be homologous to thetype 1 LHC II (Lhcbl) sequenceof the angiosperms but they could just as well be paralogoussequences.Both trees show that the Lhcb3 sequence has separated at an earlierpoint than the Lhcbland 2 gene types. Although this is a consistent relationship, it is not wellsupported (bootstrapvalue 67 or 61, Fig. 5.4AJB). This is suggestive of a divergenceof this CAB type around, or justafter, the chiorophyte/terrestrial plant separation. Lhcb5 divergedearly in the Cab geneevolution, before the separation of the chlorophytes. The preciseposition of Lhcb5, however, isnot well supported by bootstrap analysis. The close associationof Lhca types 2, 3 and 4 are148supported in both trees (Fig. 5.4). The clustering of Lhcal, Lhcb4, and Lhcb6 is consistentbetween methods but is not substantiated with the bootstrap trials.The relative relationships ofthe Euglena (Euglenophyte) and Mantoniella (Prasinophyceae) LHCs werenot well resolved onthese trees.A second analysis was done to resolve the relationships amongst thegreen algae andselected terrestrial plant sequences (Fig. 5.5, A/B). This analysis included20 taxa and a largernumber of amino acids (170), making up approximately 75%of the mature protein (Fig. 5.2A).The outgroup was the Chiamydomonas eugalnetos light responsivegene that has similarity to theCAB proteins (Gagne and Guertin 1992). This was used as the outgroupbecause it is one of themost divergent green algal sequences. Figure 5.5 A and A’ are bothunrooted trees and areidentical except that A’ is displayed in the radial format.The distance tree (Fig. 5.5, A) shows a topology that strongly supportsthe association ofthe green algal LHC II sequences with three out of four Euglena LHCII sequences. However,there is a fair degree of divergence between these sequences,as indicated by the length of thebranches. The Euglena2 sequence is more distantly related thanthe others but it is stillassociated with the LHC II branch. The Euglena sequences were isolatedby heterologoushybridization with a plant LHC II probe (Muchhal and Schwartzbach1992). Their identificationas LHC II sequences was by analogy to the terrestrialplant system. The LHC I sequences fromthe green algae and euglenoids tend to form a monophyletic assemblageseparate from the LHC IIgroup but the branches are long and not strongly supportedby the bootstrap analysis. TheEuglena-Lhca sequences were cloned by immunoscreeninga cDNA library with a LHC IIpolyclonal antibody. However, sequence comparisonsand immunoprecipitation with a barleyLHC I monoclonal antibody provided some evidencethat they are LHC I sequences (Houlné andSchantz 1988). The identification of the C. reinhardtiiLhca sequence as a PS I associatedantenna was confirmed by N-terminal sequencingof a PS I associated polypeptide (p22) (Bassi etal. 1992; Hwang and Herrin 1993). The characterizationof the Lhca gene from C. stellata hasnot been published and little informationon its function or organization is known. However, it149Figure 5.5Phylogenetic analysis of the LHC proteins fromselect green algae and terrestrial plantCAB proteins, including both PS I associated (Lhca) and PSII associated (Lhcb) antennalcomplexes. The tomato sequence is a Lhcbl and theothers are Lhcb types unless otherwiseindicated. (A) Distance matrix analysis using the neighbor-joiningtree construction method.The inset (A) is identical to (A) except it is displayed ina radial format to facilitateinterpretation. (B) Parsimony analysis using an identical dataset as in(A), as described in theresults section. Refer to Table 5.2 for references and fullspecies names. Bootstrap valuescalculated from 100 replicates are given at the appropriate nodes.150C slellala-LOcaMantoniella5Mantoniella2C.reinhardtii-LhcaTomato-LhcalArabidopsis-Lhcb4CstelIata-LhcaTomato-Lhca4Tomato-Lhca2Tomato-Lhca3Eug!enal-LhcaEuglona2-LhcaC.eugametoBTomato-Lhcblmoss-LhcbD.tertiolectaC.moewusllD.salinaC.reinhardAA’Arebidopss.1.hcb4TOmato4J,ealC. reinhard(ü-LhceC.ugareooD.salinaTomatomossC.stellataEugfena4Euglena2CieILetEogeea40.100.10Mantoniella5Mantoniefla2Etiglena3EuglenalTomato-Lhca4C.steIata-LhcaTomato-LIica2Tomato-Lhca3Tomato-LhcalC.eugametosArab Id opsis-Lhcb4Creinhardtii-Lhca151does share a degree of similarity to the N-terminal sequenceof a C. reinhardtii LHC I protein,p15.1 (Bassi et al. 1992).The Mantoniella Lhcb sequences form a branch at the base ofthe LHC II cluster in thedistance tree, as they did in Figure 5.4. However, a large divergencebetween the MantoniellaLHCs and the Lhca and Lhcb sequences are indicatedby the length of the branches separatingthem. This is clearly evident in the radial tree (Fig.5.5 A’). The position of the Mantoniellasequence is not supported in a bootstrap trial (28%)and this low value may reflect a fair amountof noise within the sequence. This noise maybe the result of non-divergent mutations (parallel,non-divergent changes) occurring at a significant rate.Parsimony analysis of this dataset gave two trees witha length of 1356. The differencesbetween the two were within the green algal LHC II branchand no significant deviations fromthe consensus tree (Fig. 5.5 B) were observed. Theoverall topology of the parsimony treeconcurs with the distance tree. The main differencebetween the two methods is in the placementof the Mantoniella LHC branch. With parsimony, the Mantoniellasequence separates before thealgal LHC II and LHC I divergence. I am skepticalabout this topology because ofthe longbranches connecting the Mantoniella and C. eugametos sequences(Fig. 5.5 A’). The presence ofthese very divergent sequences can result in treeing artifactswith a parsimony analysis(Felsenstein 1978), which is probably the case here.Though the distance tree is probably a moreaccurate reflection of the true gene phylogeny,I am unable to conclude whether the MantoniellaLHC is more closely related to either LHCI or LHC II type sequences.In both trees (Fig 5.5), the two Chiamydomonas Lhcasequences cluster with a specificterrestrial plant Lhca sequence and not witheach other (Fig. 5.4 and 5.5). The bootstrap valuesare low for these branches so the exact associationof the two sequences can not be concluded.Nevertheless, this offers preliminary evidencethat the divergence of the LHC I sequencesoccurred early in the evolutionary history;before the green algal/terrestrial plant separation.1525.3.4 Phylogeny of the chlorophyll a + c-binding proteinsThis analysis examines the relationships of the chlorophyll a +c binding proteins (FCPs,iPCPs) from several groups of non-green algae. Thedataset consists of 9 sequences (from sixtaxa) and includes 153 amino acids (90% of the matureprotein) in the analysis, indicated inTable 5.1. The protein sequences are from two diatoms(Phaeodactylurn and Odontella), a brownalga (Macrocystis), a raphidophyte (Heterosigma) and a haptophyte(Isochrysis). All of thesealgae contain fucoxanthin, Chi a and Chl c. The other proteinsequence is from thedinoflagellate, Amphidinium, which contains the predominantcarotenoid peridinin, instead offucoxanthin. The Amphidinium iPCP sequence wasdetermined directly by amino acidsequencing, while the FCP protein sequences were inferredfrom the gene sequence.Both the distance (Fig. 5.6 A) and parsimony (Fig.5.6 B) trees showed a clustering of theFCP sequences separate from the iPCP (intrinisic peridinin-chlorophyllprotein) of thedinoflagellate, Amphidinium. This is with the exception of the Isochrysissequence which wasdistant from the other sequences, even though it has a fucoxanthinbinding LHC (FCP). Thedistance analysis did not resolve the relationships amongstthe FCP sequences of the brown alga,diatoms or the raphidophyte.The parsimony analysis (Fig. 5.6 B) gave only one mostparsimonious tree with a lengthof 490. The pennate (Phaeodactylum)and centric (Odontella) diatoms form amonophyleticgroup in agreement with traditional morphologicalcharacters (Round and Crawford 1990),though the length of the branches separating them (Fig.5.6 A) indicate an early divergence. TheHeterosigma sequence is also grouped on this branch,which is separated from the brown algaldade. These relationships are supported by high bootstrapvalues and the branch is consideredrobust. The Isochrysis sequence is very divergent andis at the base of the tree. Interestingly, theHeterosigma sequence groups with the brown algae whenthe Amphidinium sequenceis removedfrom both the parsimony and distanceanalyses (not shown).153ABCFigure 5.6HeterosigmaOdontellaPhaeodactylum2Phaeodactyum 1Macrocystis2MacrocystislPhaeodactylum2Phaeodactyum 1PaviovaOdontellaPhylogenetic analysis of the FCP proteins from various Chi a + c containing organisms.(A) Distance matrix analysis using the neighbor-joining tree construction method. (B) Parsimonyanalysis using an identical dataset as in (A) and described in the results section. (C) Parsimonyanalysis using a limited number of characters (87) in order to incorporate the partial sequenceinformation from Paviova lutherii(.).Refer to Table 5.2 for references and full species names.Bootstrap values calculated from 100 replicates are given at the appropriate nodes.Isochrysis0.10Amphidinium2AmphidiniumiMacrocysfis2MacrocystisiIsochrysisOdontellaHeterosigmaAmphidinium2AmphidiniumisochrysisPhaeodactylum2PhaeodactylumlMacrocystisiMacrocystis2HeterosigmaAmphidinium2Amphidiniumi154The very distant placement of the Isochrysissequence was odd in light of the partialsequence information from Paviova, another haptophyte(Roger Hiller, unpubi.). I ran aparsimony tree with the partial Paviova sequenceincluded in the alignment (Fig. 5.6 C•).Because of its inclusion, only 87 amino acids (49%of mature protein) were used in the analysis(Fig. 5.1). There are some obvious differences in topologybetween Figure 5.6 B and5.6 C. Themain point is the close affinity of the Paviova partialsequence to the main FCP cluster,which isseparate from the Isochrysis sequence. Though theseare preliminary data, it seems unusualthattwo taxonomically related species show such a remarkabledifference. The most obviousexplanation is that the Isochrysis sequence is paralogousand not the predominant FCPin theorganism.5.3.5 Evolution of the LHCfainily ofproteinsThis analysis looks at the evolutionary relationships betweenthe CABs, FCPs and theiPCPs; the main intrinisic light-harvesting antennae.A dataset of 39 taxa and 135 amino acids(61-79% of mature polypeptides) was constructed. Theexact characters used in the analysis aregiven in Table 5.1. The taxa included werefrom the main LHC proteins of the non-green algae(FCP/iPCPs), the green algae and the terrestrial plants(CABs). In addition, representativesfromother LHC types, such as LHC I (Lhcal-4), CP24(Lhcb6), CP26 (Lhcb5) and CP29 (Lhcb4),were included.With this dataset, the interpretation can vary dependingon whether parsimony or distancemethods are used. In the distance tree thereare three major groups (Fig. 5.7 A, A):(1) thechlorophyll a + c proteins (FCP, iPCP)that group with the C. eugalnetos (CAB) sequence,(2) theLHC II (Lhcb) sequences, and (3)the LHC I (Lhca) sequences that group withLhcb6 (CP24)and Lhcb4 (CP29). The Euglena LHCI (Lhca-Euglenal/2) sequences form the earliestbranchin the tree. In this tree it appears thatChl a + b sequences from LHC I andII are more closelyrelated to each other than either is tothe Chl a + c lineage. This is clearly evidentin the radial155Figure 5.7ADistance matrix analysis of all LHC proteins from select Chia + b-containing and Chla + c-containing taxa, including both PS I associated (Lhca)and PS II associated (Lhcb) antenna!complexes. The tree was constructed from the distancematrix using the neighbor-joining methodas described in the results. Inset (A’) is a radial display of tree (A)and is otherwise identical.Refer to Table 5.2 for full species names and references. Bootstrapvalues calculated from 100replicates are given at the appropriate nodes.1569—psbS-SpinachLpsbS-Tomato— Pcp-ArnphidiniumlPcp-Amphidinium2Fcpl -MacrocystisFcp2-MacrocystisFcp-OdonteliaFcpl -Phaeodactylum— Fcp1-PhaeocactyIumFcp-Heterosigma169Lhcb-EuglenalI —Lhcb-D.tertiolectajyLhcb-D.salinajjLlhcb-C.moewusii44jLhcb-C.reinhardtiiI P—Lhcb-C.stellata61 r-Lhcb3-Tomato23U r- Lhcb-MossLhcb-Fern47-1i1- Lhcb-GinkgoLhcbl -Tomato{j-Lhcb2-Tomato74LLhcb2-LemnaLhca3-TomatoLhca2-TomatoLhca4-Tomato—Lticb6a-TomatoLhcb6b-Tomato- Lhcb4-ArabidopsisLhcal -TomatoLhca-C. reinhardtii93ELhca-Euglenalh— Lhca-Euglena20.10Fcp-IsochrysisC.eugametosLhcb-Euglena217Lhcb5-TomatoAPepFcp651Lhcb-MantoniellalLhcb-Mantoniella21520GreenAlgaeTerrestrialP Ia ntsLHCIILHCICP24!CP29Macrocystis1 AmptiidinitsoehrysisC.ougamatos MantoniellapsbS-SpinachsbS -TomatoLhca-EuglenLhca3Lhca20.10157BLhcbl -TomatoGinkgoLhcb-MossLhcb-FemLhcb2-TomatoLhcb2-LemnaLhcb3-TomatoLhcb-C.stellataLhcb-C.reinhardtiiLhcb-D.salinaLhcb-C.moewusiiLhcb-D.tertiolectaLhcb-Euglenal100Fcpl -Macrocystis47Fcp2-Macrocystis29Fcpl -Heterosigma32Fcpl-Phaeodactylum88100Fcp-Odontefla56 100Pcp-Amphidiniuml69Fcp-lsochrysis21c.eugametos100Lhcb6a-TomatoL__Lhcb6b-Tomato— Lhcb4-ArabidopsisLhca-C.reinhardtiiLhcal -TomatoLhca2-Tomatoi—Lhca3-TomatoLhca4-Tomato1Lhcb-Euglena2Parsimony analysis of all LHC proteins from select Chi a + b-containingand Chl a + ccontaining taxa including both PS I associated (Lhca) and PSII associated (Lhcb) antennalcomplexes. Analysis was done using an identical dataset as in(A), as described in the resultssection. The tomato PsbS protein is used as the outgroup. Referto Table 5.2 for references andfull species names. Bootstrap values calculated from 100 replicatesare given at the appropriatenodes.io—----psbS-Tomato— psbS-SpinachLhcb-MantoniellalLLhcb-Mantoniella2Lhcb5-TomatoLHCIIGreenAlgae161313Figure 5.7BFcpPcpFcp60LHCIC P24/CP29158tree (Fig. 5.7 A’). Because of the low bootstrap values on the main branches leading to thesebranches, however, interpretations have to be made with caution since the resolution in thisregion of the tree is poor.With parsimony, six equally parsimonious trees were found with a length of 1792.Theconsensus of these six trees is shown in Figure 5.7 B. Most of the variationswere within theinternal nodes of the main branches and were considered insignificant. This analysisproduced atree topology where the Chi a + c sequences and the LHC I (Lhca) lineage formed amonophyletic group; this relationship was not supported by bootstrap replicates.As the branchlengths are very different between the FCPs and the CABs, I am skeptical of this association andsuspect it may be the result of treeing artifacts that are common to parsimony; particularlyincases where there are unequal rates between the lineages. The low bootstrap valuesseparatingthe two lineages (16%) makes any firm conclusions regarding the relationships of the CABsandFCPs unsupported. Relationships within the major lineages are generally the same as withdistance analysis.1595.4 Discussion5.4.1 CAB protein evolutionThe trees suggest that the two main LHCII types (Lhcb 1-2) form a monophyleticdade inagreement with the analysis of Jansson (1994).The Lhcbl and Lhcb2polypetides make up themain LHC II complex and are generally90% identical in the maturepolypeptide (excluding theN-terminal region) so their close associationon the tree is not surprising. Together,Lhcbl andLhcb2 make up the main peripheralantennae of PS II. Its principal functionis the capture oflight followed by the transfer of the excitationenergy to the core reaction center of PSII. It hasalso been implicated in the mediationof thylakoid appression, the regulationof energydistribution between the photosystems andin photoprotection, all of whichhave been previouslyreviewed (Anderson and Andersson 1988; Bassiet al. 1990; Jansson 1994). LHC IIis thought toexist as a trimeric unit (Kühlbrandtand Wang 1991) composed of Lhcb 1 andLhcb2polypeptides at a ratio of approximately 2:1(Jansson 1994). Both Lhcbl and Lhcb2 havephosphorylatable threonine residues in theamino terminus of the mature polypeptidethat arethought to be responsible for thestate transition observed in thylakoids (Mullet1983). The closeevolutionary relatedness of these twocomplexes is reflected in their functionalsimilarities.However, the Lhcb2 complex is enrichedin a peripheral subpopulation of LHCII, has differentphosphorylation kinetics and appears laterin development (Larsson et al. 1987). Thesedifferences are thought to beimportant in the adaptation to different light intensities.It is likelythat the appearance of the Lhcbl I Lhcb2lineages was due to a fine tuning of the light adaptationresponse.The more distantly related Lhcb3 (LHC IItype III) sequence is a minor antennalcomponent that is about 80% identicalto the Lhcbl and Lhcb2 complexes. This proteinhas ashorter N-terminus and is often found inthe LHC II complex (Green et al. 1992b).Lhcb3 mayfunction as a linker between the bulk trimericLHC II complex and the PS II core. Thisis160suggested because of its close association with the core complex of PS II in barley Chi b-lessmutants (Harrison and Melis 1992). The more proximal location of Lhcb3 to PS II, with respectto Lhcbl/2, would accommodate the hypothesis of an earlier evolutionary history of this protein,as depicted in the tomato CAB tree (Fig. 5.3). The addition of the peripherally located Lhcbl andLhcb2 complexes could occur without much change in the preexisting inner antennaorganization.The Lhcb5 (CP26) sequence is consistently found at the base of the Lhcb dade,suggesting it has evolved prior to the peripheral LHC II complex. CP26 remains associated withthe PS II core when the main peripheral LHC II antennae are removed (Camrn and Green 1989)and is considered an inner antenna. It is likely that the peripheral antennal proteins of LHC IIarose from the divergence of a gene encoding a inner antennal, CP26-like, protein. Duplicationand divergence of the Lhcb5 gene would have to be accompanied by a change in size andchlorophyll binding capabilities. The Chl a/b ratio of LHC II is 1.3 compared to approximately3.3 for CP26 (van Amerongen et al. 1994; Table 1.2). This suggests that changes inthe amountsor relative proportion of Chl a and b bound by each polypeptide has changed in thecourse ofevolution (Green and Kühlbrandt 1995). Evolution of Lhcbl/2 at a later pointwould also suggestan increased level of regulation by phosphorylation as CP26 is not reversibly phosphorylated.An interesting finding is the close association of Lhcb6 (CP24) and Lhcb4 (CP29) in thetrees, separate from Lhcb5 (CP26). This is somewhat surprising as theCP29 and CP26 pigment-protein complexes often copurify (Green 1988) and both are associated withthe core complex ofPS II (Camm and Green 1989). Their presence in both grana (cc) andstromal(13)localized PS IIcenters (Allen and Staehelin 1992) suggests that they are part of thebasic PS II unit. Because ofthe biochemical similarities, the complexes are also known as CP29type I (CP26) and CP29type II (Cp29) (Pichersky et al. 1991). The distant relationship betweenLhcb4 and Lhcb5 hasbeen suggested through direct sequence comparison (Morishige andThornber 1992; Green andPichersky 1993) and by phylogeny construction (Jansson 1994). Lhcb6and Lhcb4 consistentlygroup together, though the long branches joining them (Fig.5.3 A) suggest they diverged from161one another long ago. The minor CP24 complex binds little chlorophyll and, like CP29, isenriched in PS II fractions (Dunahay and Staehelin 1986).The Lhcal sequence (LHC I type I) is closely related to the PS II inner core antennae,CP29 and CP24. Although consistent between methods, this relationship is only moderatelysupported by bootstrap replicates in the distance trees (Fig. 5.3/4 A). A clustering of the CP29(Lhcb4) and LHC I (Lhcal) sequences was also supported in a recent dendrogram (Jansson1994). PS I antennae, Lhca2-4, appear to be more related to one another than either is to Lhcalor Lhcb6/4. However, all the Lhca sequences are quite divergent from one another as indicatedby the long branch lengths separating them on the distance trees.The relationship amongst the different LHC I antennae are unstable and not stronglysupported by bootstrap replicates in most cases. Parsimony analysis consistently places Lhca2and Lhca3 as the closest relatives within the LHC I lineage; this is supported by bootstrapreplicates in Figure 5.3B. Distance methods, however, consistently place Lhca2 and Lhca4 as theclosest relatives, although this is not strongly supported by bootstrap trials in any of the treesshown. The latter relationship has been observed in another dendrogram that is based on adistance method (Jansson 1994). However, no indication of reliability or significance of thisbranching order was given. It is tempting to conclude a direct evolutionary relationshipbetweenLhca2 and Lhca3 as they are the polypeptides making up the LHC 1-680 complex.In tomato, theLhca2 and Lhca3 genes are linked and have been mapped to chromosome 10,suggesting a closeraffinity due to a more recent duplication event (Pichersky et al. 1989). On theother hand, thegenes encoding Lhcal and Lhca4, which make up the LHC 1-730 pigment-proteincomplex, areon different chromosomes in tomato (Pichersky et al. 1987; Schwartz et al.1991) and despite theapparent functional association, there does not seem to be any direct evolutionaryrelationshipbetween the two complexes.5.4.2 Evolutionary relationships amongst the Cab gene family162The analysis of the Cab gene family from a diverse array of organismsreinforces the ideathat two particular CAB types (eg. Lhcbl) from different organismsare more similar to oneanother than either is to different CAB types (eg. Lhca4)from the same organism. Comparisonof Lhcb 1 and Lhcb2 sequences previously revealed that the sameCAB type encodes a nearlyidentical polypeptide with similar numbers of introns (Chitnisand Thornber 1988). Thedistinction between Lhcb 1 and Lhcb2 was also madeby detecting signature amino acids (Janssonand Gustafsson 1990), which are conserved residuesspecific to a particular CAB type. Inaddition, a phylogenetic analysis of angiosperm Lhcbland Lhcb2 sequences (Demmin et al.1989; Matsuoka 1990) also revealed this trend.Sequencing of a fern (Polystichum munitum) (Picherskyet al. 1990) and moss Lhcb(Physcomitrella patens) (Long et al. 1989) provided usefulmarkers in estimates of the mainLHC II divergence. The analysis of the fern sequence wasunable to clearly resolve thebranching order. However, the moss Lhcb sequence is consistentlyat a branch before the fernand Lhcb2 separations, indicating that the functional separation ofthe Lhcbl and Lhcb2complexes occurred after the bryophyte lineage separated.This is a tentative conclusion untilmore representatives from bryophytes are sequenced, in orderto rule out the possibility ofmisleading tree topology as a result of comparing paralogousgenes. Overall, the duplication andseparation of the Lhcb2 (LHC II type II) sequencesprobably occurred after the bryophyte lineageseparated and at about the same time as the lineage leadingto the pteridophytes (ferns) diverged.This is in agreement with earlier predictions by Picherskyet al. (1990). It is apparent that thedivergence of all the major CAB types occurred early.This definitely occurred before theangiospermlgymnosperm separation, as thesame CAB types from pine consistently grouped withthe same CAB types from the angiosperms.It is not possible to draw further conclusions as tothe earliest separation of most the CABs because thereare too few sequences known from theother taxonomically distinct groups such as the Chiorophytaand the Euglenophyta.There is a distinct separation of the greenalgal LHC II sequences from the terrestrial plantLhcb 1-3 sequences suggesting that theminor Lhcb3 antenna appeared just afterthe green algal163lineage diverged. This may indicate a fundamental difference inthe regulation (Sukenik et al.1987) and organization of the peripheral LHC II antennae in greenalgae. The lack of Lhcbl andLhcb2 type antennal proteins in green algae suggests the Lhcbl/2duplication and functionaldivergence occurred in the lineage leading to the terrestrial plantsafter the green algal lineageshad separated.The identification of an LHC I sequence from thegreen alga Chiamydoinonas reinhardtiiwas confirmed by peptide sequencing of a PS I associated protein(Hwang and Herrin 1993).This analysis confirms its identity as a Lhcal type, asthis sequence tends to cluster with theangiosperm Lhcal sequences; however, the relationshipis not supported (bootstrap value 30 or54, Fig. 5.5). In this case, the Chiamydomonas sequenceis more closely related to tomato Lhcalthan to the Lhca sequence from Chiamydomonas stellata. Shouldthis relationship hold as moreLHC I sequences from green algae are determined,it would suggest that the LHC I sequenceshad diverged before the chlorophyte/land plant separation. Thisbrings up the interestingsuggestion that the different LHC I complexes evolvedprior to the establishment of the differenthomologous LHC II complexes (Hwang and Herrin1993), which will be discussed in thefollowing sections.The Euglena sequences for LHC I and LHC II are very divergent,as compared to theirgreen algal and terrestrial plant counter parts, indicatingthat this alga separated from the greenalgal lineage very early. Of the four complete LHCII proteins, three are more relatedto oneanother (Euglena sequences 1, 3 and 4) andform a branch just before the green algal! land plantLhcbl/2 sequences. The respective LHC II and LHCI proteins from the green algae and Euglenagroup together, although the branch lengthsin the LHC I lineage are very long onthe distancetrees. This suggests that LHC I and LHC II polypeptideshad functionally separated prior to theappearance of the euglenoid chloroplast.It also shows a close direct link betweenthe green algaland Euglena chloroplasts, previously hypothesized onthe basis of the presence of chlorophyllaandb.1645.4.3 Evolution of the Chia + b and Chi a + c gene families:The relative position of the Chia + c-binding proteins in relation tothe Chi a + b-bindingproteins of LHC I and LHC II is notclear from an examination of eitherthe parsimony ordistance trees. The distance trees (Fig.5.7 A, A’) suggest that LHC Isequences are more closelyrelated to the CAB LHC II sequenceswhereas the parsimony tree (Fig.5.7 B) supports the ideathat the FCPs are more related to theCAB LHC I sequences; neither treeis supported bybootstrap replicates. I am inclined tobelieve that the distance tree is amore accurate reflection ofthe true relationships because the datasethas a fair bit of noise and thebranches between sometaxa are very long. Under theseconditions, the parsimony tree maynot be reliable (Stewart1993). However, these proteins havediverged to such an extent that theresolution of suchdistant events may not be possible.Of importance is the time of divergenceof LHC I and LHC II CAB complexesand theseparation of the lineage leading to the Chi a +c-binding proteins. It would beinteresting toknow if the FCP complex had evolved fromancestral PS II associated or PSI associatedantennae. It has been suggested that the FCPsevolved from a LHC I or CP24-likeancestor andthat the LHC II complexes of higherplants evolved after the divergence ofthe chromophytes andthe chlorophytes (LaRoche et al. 1994).I agree that the major peripheral LHCII CAB sequencesevolved after the divergence of the chlorophytesand chromophytes. However, thereisinsufficient (convincing) evidence, at the moment,to suggest a closer relationship ofthe FCPs tothe Lhca-CABs. In fact, the distance treesindicate that the CAB LHC II andLHC I sequencesare more closely related to one another thaneither is to the FCPs. This wouldsuggest that theFCPs diverged from the ancestral LHC beforetheir was a separation of the LHCI and LHC IItype genes.It is not possible to make a firm conclusion regardingthe ancestry of the FCPs except tosay that they diverged from the CABs veryearly. The long branches (largedivergences) betweenthese taxa, the possibility of comparingparalogous genes, and the limitedinformation on other165members of the FCP family make any conclusionstentative. A clearer picture of the FCP/CABrelationships will develop when LHC I sequences fromthe chromophytes are characterized.5.4.4 Evaluation of species relationships basedon the LHC protein trees:The usefulness of the nuclear encoded CAB proteins asan index of phylogeneticrelationships is limited and will depend on severalfactors: the distance between the organismsstudied, the evolutionary questions asked, and theavailability of the appropriate sequenceinformation. A significant obstacle in the utilizationof CAB proteins for phylogenyis their smallsize. The relatively small number of useful charactersraises questions about the reliabilityorsignificance of the topology observed. This “uncertainty”is often reflected by low bootstrapvalues. One also has to be cautious in the constructionand interpretation of phylogenies basedona single gene/protein, which could lead to erroneoustrees (Cao et al. 1994). As mostof theconserved residues are thought to be membranespanning, there is probably a functionalconstraint on these regions for being hydrophobic.If so, the more distant relationships maymorereadily reach a point where non-divergent (homoplasmic)mutations occur at a significantrate,which can mask true evolutionary relationships(Meyer et a!. 1986). Another problem inusingthe CAB proteins for phylogeny, which isa common concern with nuclear encoded proteins,isthe possibility of comparing paralogousgenes. Since the CABs and FCPs are encodedby amultigene family, care must be takento assure that the sequences used have sharedthe sameevolutionary pathway. This is not always possibleto judge due to the fragmentary nature ofthesequence information. In most cases, thereis only one LHC polypeptide identified from aparticular organism and is assumed that it is analogousto Lhcb 1 of the terrestrial plants.However, this assumption is dubious because thereis insufficient characterization ofthe LHCsfrom anything other than select terrestrialplants and Chiamydornonas. With the Chia + ccontaining organisms, the cloned sequences typicallyencode the most abundant LHC in theorganism. However, there is little direct evidencefor a preferential association with either166photosystem. Without structural/functional informationit is difficult to judge whichsequencesmay be orthlogous or paralogous. Nonetheless,the observed gene relationships give someimportant clues as to taxon relationships in combinationwith morphological, biochemicalandother molecular sequence studies; some of thesecases are mentioned below.This study, and others (Muchhal and Schwartzbach1992; Jansson 1994), clearly showthat the light harvesting proteins fromEuglena are homologous to those of thegreen algae andland plants. The presence of three membranesaround the chloroplast and thepossession ofChls a + b suggests that the euglenoid chloroplastwas acquired secondarily; evolvingfrom asymbiotic green algae (Gibbs 1978). Phylogeneticanalyses of psbA (Dl), rbcL/S (Rubiscolargeand small subunit), tufA (Morden et al. 1992), chloroplast5S rRNA (Somerville et al. 1992), andpsaB (PS I core complex) (Assali and Loiseaux-deGoër 1992) also provides evidence fora closerelationship between the green algal and euglenoidchioroplasts.Euglena contains the xanthophylls diadinoxanthinand diatoxanthin and stores a13-1,3-glucan, paramylon, in the cytoplasm. These characteristicsmore closely resemble thechromophytes rather than the green algae. As well,an analysis of the chloroplast encodedSSUrRNA points toward a closer affinity of the Euglenachioroplast with those of the chromophytes(Douglas and Turner 199lb; Giovannoniet al. 1993). However, there may beproblemsinvolving biased base composition in thesestudies that may have caused this association(seediscussion by Lockhart et al. 1994).A couple of statements can be made concerning the branchtopology for the Mantoniellasequences (Fig. 5.4, 5.5). First, the early branchingof the Mantoniella sequences is in agreementwith the early divergence of the Prasinophyceae fromthe green algal lineage based onmorphological characteristics, such as the synthesis ofa Chl c-like pigment (Mg-2,4 D) and thepresence of scales on the cell body and flagella (Melkonian1990), and from rRNA phylogeneticanalysis (Steinkötter et al. 1994). Second,based on the tree topology the acquisitionof achloroplast (via a secondary endosymbiosis with agreen alga), or genes, by a phagotrophichostleading to the euglenophytes would haveoccurred after the separation of the prasinophytesfrom167the other green algal lineages. An alternativeinterpretation that could explainthe early branchingof Mantoniella is that the LHC evolved froma paralogous member of the LHCfamily that wasdifferent from the gene leading to the chlorophyteLHC II gene lineage.The relationships between the fucoxanthin-containingalgae are not well resolved andthere are too few complete sequences tomake it interesting. However, the fucoxanthincontaining chromophytes form a distinctgroup separate from the peridinin-containingdinoflagellate, Amphidinium. This is in agreementwith the traditional view of adistantrelationship between the dinoflagellates andthe other chromophytes. This wasbased onmorphological characters, such as differencesin the xanthophyll content, presenceof a uniquesoluble LHC complex, the presence of only threemembranes around the chioroplast,the apparentlack of histones, and persistently condensedchromosomes (Taylor, 1990). In addition,Phylogenetic analysis of nuclear rRNA consistentlyshows a deep divergence betweenthedinoflagellates and the other chromophytes (Bhattacharyaet al. 1990; Hendricks et al. 1991;Cavalier-Smith et al. 1994b). Though the iPCPs aredefinitely FCP-related, whether thisarose asthe result of a divergence from the chromophyteline or from an independent evolution ofthechloroplast, can not be resolved with this data (butsee Cavalier-Smith, 1994).The positions of the both haptophytes depicted inthe FCP I iPCP trees (Fig. 5.6) are notconsistent with standard taxonomic positionof this group. The first is the earlybranching ofIsochrysis before the dinoflagellate, and theother chromophytes. This would notbe an accuratereflection of the organismal relationshipsas a number of studies indicate that the haptophytesform a sister group to the heterokontloomycetelineage (Andersen 1991; Bhattacharyaet al. 1992;Cavalier-Smith 1994; Medlin et al. 1994; Cavalier-Smithet al. 1994b). A likely explanation forthe odd position of Isochrysis is that theFCP sequence is a paralogous gene, resultingin anerroneous tree topology. The sequence was isolatedby immuno-screening a cDNA library with aFCP specific antibody that could have detecteda product paralogous to the other sequences sincemost of the antennal proteins are immunologicallyrelated. Furthermore, there is evidencethatthere is only a single copy of this JsochrysisFcp gene (LaRoche et al. 1994), thoughin terrestrial168plants and all other known chromophytes,the main antennal protein isencoded by a multigenefamily (Green et al., 1991; Bhaya et al.,1993; Apt et al., 1994; Chapter 4).Furthercharacterization of the haptophytefamily of antennae will have to be donebefore this can beresolved.The second unusual relationship is the very closeassociation of Paviova withthe diatomswhen the tree is constructed with theavailable Pavlova FCP protein sequencedata (87characters). The Pavlova polypeptidebinds both ChI’s a, c and fucoxanthin,and is biochemicallyand immunologically quite similar to otherFCPs (Fawley et al. 1987; Hilleret al. 1988). itscurrent tree position is not expected. However,being a haptophyte, I would haveexpected it toform a deep branch at the base of the FCPlineage if it is a true reflectionof organismalphylogeny. Nevertheless, the datasetis small and the species relationships may changewhen thecomplete sequence is determined.5.4.5 Light-harvesting protein evolution:pathways and evolutionary sourcesThe FCP and CAB proteins are clearly homologousand were derived from a singleancestral gene, though the trees show therewas probably an early separation of theFCP and CABlineages. I propose that the antennalproteins associated with PS I weresome of the first proteinsthat acquired the function oflight-harvesting, probably from one of a photoprotectiverole. Itwould have been from this complex thatthe CABs and FCPs diverged atseparate times. This issuggested for a few reasons: first, the LHCI proteins in the terrestrial plantsoriginated from veryearly duplications as indicated by the largedivergence between them, as comparedto the smallerdivergence between the LHC II antennal proteins.Second, the different LHC I proteinsfrom thegreen algae seem to have a greater affinityfor specific LHC I types of the terrestrialplants, ratherthan to each other. Furthermore, the greenalgal LHC II proteins are clearlyseparated from thoseof the terrestrial plants. This suggest that theLHC I genes had diverged into the differenttypesbefore the separation of the green algae andland plants and prior to the duplication and169divergence of the LHC IT-related genes (Hwangand Herrin 1993). Third, thepresence of aCAB/FCP related LHC I complex in the redalgae (Chapter 3; Wolfe et al. 1994)along withPBSs, also suggests that the LHCI antennal proteins originated prior to themembrane intrinsicLHC II protein complex. Though none havebeen reported, it remains to be seenwhether there isan intrinisic PS II associated antennae in redalgae. As well, the sequence of the redalgal LHC Iantennae needs to be determinedto get a better idea of possible relationships.If the presence of a suitable LHC I associated antennaecomplex had been established,then the loss of PBS, due to light or nutrient stresses,may have necessitated the adaptationof anLHC I-related antennae to associate with PSII. This seems plausible as somerepresentatives ofthe PS II associated inner antennae of land plants (CP24and CP29) are evolutionarily closer tothe LHC I proteins than to LHC II proteins. The factthat some inner antennae of PS IIareLHC I-related suggests that as additional complexes wererecruited, they were addedon andbecame more peripherally located. In addition,the LHC 1-680 (Lhca2 &3) (Knoetzel et al.1992) and the LHC II complexes have similar fluorescenceemission maxima (680 nm)making afunctional transition from a PS I associationto a PS II association seem plausible. Alternatively,a separate LHC I-related complex may havebeen stressed induced for a photoprotective roleintimes of PBS degradation.Since the chioroplast is generally thought to haveevolved from a cyanobacterium, theCABIFCP related LHCs must have become the mainantennae after the establishmentof thechloroplast. In order to explain the present day pigmentationof the algae, there must have beenat least two independent losses of phycobilisomes fromthe ancestral organisms; once leading tothe green algae and at least one other timeleading to the chromophyte plastid. Moreover,therewould have to a gain in the ability to synthesizeChl b and Chi c in the green algae andchromophytes, respectively.It is probable that the phycobilisomes were replacedby a LHC I-related complex that wasinduced for photoprotection during timesof stress. The presence of an inducibleLHC I-relatedsystem could act to protect the photosystemin the event of a loss or reduction of thePBS due to170either high light or nutrient deprivation (Bryant 1992).The PBSs are efficient but metabolicallyexpensive, requiring about ten times more amino acidsper chromophore (Bryant 1992), so aninitial loss of this antenna due to a nutrient deficiency(Grossman et al. 1993) or other stressrelated events seems reasonable. In time and withsufficient modifications, this system couldeventually replace the PBS system of the cyanobacterialor red algal endosymbiont.Such a senario seems more likely since there are a numberof LHC related complexes thatare induced during either a light or nutrient stress.These include the early light-inducibleproteins (ELIP5) in the terrestrial plants (Grimm et al.1989; Adamska and Kloppstech 1994),thecarotenoid biosynthesis-related (Cbr) proteins in Dunaliella(Levy et al. 1992) and the highlight-inducible proteins (HLIPs) in cyanobacteria (Dolganovet al. 1994). These proteins havesequence similarities to the CAB proteins in the hydrophobicdomains, primarily in the first andthird transmembrane regions. Though a number ofputative chlorophyll a ligands are wellconserved in the ELIPS and HLIPS (Green and Kühlbrandt1995), it has not been conclusivelydetermined whether they bind any chlorophyll orcarotenoids.Though unrelated to the CAB proteins, there is aniron stress inducible protein (isiA) incyanobacteria that is homologous to CP43 (psbC) (Laudenbachand Straus 1988) and bindschlorophyll (Burnap et al. 1993). It has been postulatedto act as a chlorophyll reserve (Burnapetal. 1993) or as a antennal replacement in the absenceof PBS (Pakrasi et al. 1985). The similarityof the isiA gene to the LHCs of the prochlorophytes(Hiller & Larkum; LaRouche& Partensky;van der Staay & Green; unpubi.) demonstratesthe potential of stress induced proteins inthecreation of novel antennal systems.There are two potential cyanobacterial molecular sourcesfrom which the eukaryoticLHCs may have evolved after the evolutionof the chioroplast; the psbS gene andthe HLIPs.Though the psbS gene product has beenimmunologically detected in a cyanobacterium(Nilssonet al. 1990), two other groups have failedto do so (Kim et al. 1994; Vermaas, pers.comm.). Atthe moment it has only been cloned from tomato andspinach so its presence in cyanobacteriaisuncertain. Nonetheless, the psbS proteinbinds chlorophyll and is predicted tospan the171membrane four times (Kim et al. 1992; Funk et al. 1994).If present in the cyanobacteria, a C-terminal deletion could give a LHC precursor with three transmembranehelicies (Green andPichersky 1994).The HLIPs are another potential source for the evolutionof the LHCs as they are knownto occur in cyanobacteria (Dolganov et al. 1994) and they havehomologues in red algae (Reith,unpubi.; gb X62578) and the Glaucophyta (Stirewaltand Bryant, unpubl.) (see Green andKühlbrandt 1995). The HLIPs are only 72 amino acidslong, yet there is sequence similaritytothe first or third membrane spanning region ofthe ELIPs and CABs. It has been proposed thatthese proteins function as homodimers and were the evolutionarysource of the eukaryotic LHCs.One can envisage a series of gene duplications and fusions thatcould give a LHC that spannedthe membrane three times and adopted the role of light-harvesting(Dolganov et al. 1994; Greenand Kühlbrandt 1995).With the available sequence information it is not possibleto make any firm conclusionsregarding the ancestry of the FCPs. This will haveto wait until more diverse FCPfamilymembers have been identified and sequenced. Thesequence of the red algal LHC proteins willalso be an important piece of information. I wouldpredict that they will be more closely relatedto the FCPs rather than to the CABs.172CHAPTER 6SummaryThis dissertation demonstrates that the light-harvestingantennae of Heterosigmacarteraeform an intricate system comparable to thecomplexity of the LHC antennae seenin the terrestrialplants. Heterosigma possesses up to 12 differently migratingpolypeptides that crossreact todifferent extents with CAB and FCP specific antisera.There are four prominentLHCs inHeterosigma with apparent molecular massesof 18-21 kDa. The gene encodingone of theseantennae, the 19.5 kDa polypeptide, was cloned andsequenced. Based on Southernhybridizationand eDNA sequencing, there are approximately6-8 gene copies encoding thispolypeptide.Overall, there are probably over 20 relatedgenes encoding the FCPs in Heterosigma.The FCPs are structurally related to the CABsas determined by the immunologicalcross-reaction data and through direct sequence comparisons.This implies they are evolutionarilyrelated and had evolved from the same ancestral gene.Both the CABs and the FCPs have threeputative membrane spanning regions. Inthe pea LHC II two of the three membranespanningregions (MSR1 and 3) interact to bind carotenoidsand chlorophyll. The corresponding regionsinthe FCPs are very conserved. Some of thehighly conserved amino acids in the FCPsare thoughtto bind chlorophyll and/or are importantin helix-helix interactions that can help tostabilize thecomplex. These striking similarities indicatethat the FCPs and the CABs are structurallyquitesimilar.The LHCs from the diverse algal taxa that utilize fucoxanthinas an accessory chlorophyll(the FCPs) are related and form a natural monophyleticgroup. The intrinisic peridininchlorophyll protein complexes (iPCPs) fromthe dinoflagellates are definitely morecloselyrelated to the FCPs than they are to theCABs and they form a sister group to theFCP dade. Itseems that the acquisition of xanthophylls for a primaryrole in light harvesting, rather than solely173a photoprotective one, occurred early in the evolution of these LHCs. The useof differentprimary xanthophylls in the distinct antennae complexes (fucoxanthin, peridinin,vaucherioxanthin, etc.) was then the result of divergence following the separationof the mainalgal taxa. This was probably related to the light-environment the algal groupexperiences in themarine habitat.The presence of CAB/FCP-related LHCs in the red algae provides a link betweentheantennal systems of the three major groups of photosynthetic organisms.In addition, the lack ofsuch immunologically related LHCs in the cyanobacteria and prochiorophytessuggests that themembrane intrinisic LHCs originated following the endosymbiotic originof the chloroplast thatgave rise to the first true photosynthetic eukaryote. Such a scenario implies amonophyleticorigin for the chloroplast as it is unlikely that related proteins could evolve independentlyindifferent lineages.This work could continue in a number of directions. It would be interesting tofurthercharacterize the chlorophyll protein complexes in Heterosigmain addition to the sequencing ofthe genes encoding them. This would give an indication of thegene family complexity and howmuch diversity exists between the different members. This will allow thedetermination of thenature of the divergence that is responsible for the differential immunologicalcross-reactivityseen in Chapter 2. This information would be useful if antennae genecharacterization studiesfrom representatives of other major algal taxa were also beingdone. This comparison would bevery useful in determining if specific gene types areconserved between the diverse algal groupssimilar to the conservation of the Cab gene types betweenthe angiosperms and gymnosperms.Should specific gene types exist it would make an interestinggene evolution study; it wouldallow one to determine at what point certain gene duplicationshad occurred in relation to thephylogeny of the algae being compared. This would also complementthe work currently beingdone with the Cab gene family and may help to determine which specificCab gene types aremore closely related to the Fcp genes.The analyses of the gene family complexity have to be done in conjunctionwith structuraland functional studies on the pigment-protein complexes.Such functional/structural studies174include whether the complex is specificallyassociated with either PS I orPS II, more accurateestimates of the pigment content of the differentantennae complexes and theregulatory role ofthe divergent FCPs in the adaptationto different light regimes.These studies will allowfor abetter understanding of how the antennalsystems of divergent organismsare organized and givean idea of the similarities and differencesin the ways they adapt to harvestlight. 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