Biogenesis and Origin of Thylakoid Membranes

Review

Biogenesis and origin of thylakoid membranes

Ute C. Vothknecht a;*, Peter Westho b

a Botanisches Institut der Christian-Albrechts-Universitat Kiel, Am Botanischen Garten 1^9, D-24118 Kiel, Germanyb Institut fur Entwicklungs- und Molekularbiologie der Panzen, Heinrich-Heine-Universitat Dusseldorf, Universitatsstrasse 1,

D-40225 Dusseldorf, Germany

Received 25 July 2001; accepted 1 August 2001

Abstract

Thylakoids are photosynthetically active membranes found in Cyanobacteria and chloroplasts. It is likely that theyoriginated in photosynthetic bacteria, probably in close connection to the occurrence of photosystem II and oxygenicphotosynthesis. In higher plants, chloroplasts develop from undifferentiated proplastids. These contain very few internalmembranes and the whole thylakoid membrane system is built when chloroplast differentiation takes place. During cell andorganelle division a constant synthesis of new thylakoid membrane material is required. Also, rapid adaptation to changes inlight conditions and long term adaptation to a number of environmental factors are accomplished by changes in the lipid andprotein content of the thylakoids. Thus regulation of synthesis and assembly of all these elements is required to ensureoptimal function of these membranes. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Chloroplast ; Photosynthesis ; Plastid development; Organelle evolution; Thylakoid formation

1. Introduction

Evolution of oxygenic photosynthesis with its CO2xation and oxygen release enabled life on earth aswe experience it today. It is assumed that oxygenicphotosynthesis developed several billion years ago inan ancestor of todays cyanobacteria most likelyfrom an already existing anoxygenic photosynthesisapparatus [1]. The capacity to perform oxygenic pho-tosynthesis was passed on to algae and higher plantsby an endosymbiotic event that turned a cyanobac-terium into a cell organelle, the chloroplast. The pho-tosynthetic machinery of both, cyanobacteria and

chloroplasts, is located on a special internal mem-brane system, the thylakoids. Thanks to the uniquearchitecture of this membrane cyanobacteria andchloroplasts convert solar energy into chemical en-ergy with an eciency signicantly better than anyman-made photovoltaic system. Therefore, the abil-ity of the cell to build and alter this membrane sys-tem is essential for ecient oxygenic photosynthesis.Resulting from the combination of structural, bio-chemical, and genetic analysis, we have a wellfounded knowledge of the ultrastructure and compo-sition of thylakoid membranes, but despite the im-portance that the thylakoid membrane system hasfor photosynthesis and the energy metabolism ofplants and cyanobacteria, the molecular processesconnected to the origin, synthesis, maintenance andadaptation of the thylakoids remain elusive. In thisreview we will discuss recent ndings on thylakoid

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* Corresponding author. Fax: +49-431-880-4222.E-mail address: [email protected] (U.C.

Vothknecht).

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biogenesis and evolution and their impact on ourunderstanding. Since most studies concerning thebiogenesis of thylakoids have been performed onchloroplasts of higher plants and green algae, thisreview will focus on these organisms. The last sectionwill deal with internal membrane systems in bacteria,especially the thylakoids of cyanobacteria, and theevolution of the thylakoid membrane in these organ-isms.

2. Form and function in plastids

In 1848 chloroplasts were rst described by Ungersimply as pigment bound structures. Later in the19th century Schimper [2,3] characterized these struc-tures, which he called Chlorophyllkorner, in greaterdetail. With only the resolution of the light micro-scope available, he described plastids as cell compo-nents containing chlorophyll or other pigmentswhich develop from colorless precursors. He alreadyperceived the existence of various types of plastidsand made the observation that they can pass throughdierent stages during their development. Shortlyafter the invention of the electron microscope, therst electron micrographs of chloroplasts were pub-lished [4], and soon thereafter, this new techniquewas used for the rst detailed studies of dierentplastid forms and their development. In the late 50sthe basic structure of thylakoids had been described[5^7] and the means of thylakoid biogenesis werediscussed.

Thylakoids are the dominating structure insidefully mature chloroplasts. The formation and alter-ation of the thylakoid membrane structure and com-position are closely connected to the development ofthe chloroplasts from simple, undierentiated pro-plastids. These are small round shaped organelleshardly distinguishable from mitochondria (Fig. 1),that contain very few internal membranes that areoften found as vesicles or small saccular structures[7^9]. Occasionally these membranes are observedcontinuous with the inner envelope.

In the presence of light proplastids develop intomature chloroplasts. This transition has been inten-sively studied in grasses. The leaves of these mono-cotyledonous plants grow with a basal meristem andhence form a developmental gradient. Cells found at

the base of the leaf are youngest and contain mainlyproplastids while the oldest cells with fully developedchloroplasts are found close to the tip. Ultra-thinsections revealed that during the progress of chloro-plast maturation the internal membrane systembuilds up in consecutive phases. First, long lamellaare formed which are later complemented by smaller,disc-shaped structures that associate into so-calledgrana stacks (Fig. 1). At the same time the typicallens-shape form of the chloroplasts develops. Finally,mature plant chloroplasts contain a complex and in-tertwined internal membrane system which wasnamed thylakoids according to the Greek word3eVKUYASNRf (sack-like) [10]. In fully mature chlo-roplast no continuation between the inner envelopeand the thylakoids has been observed.

In the absence of light proplastids turn into etio-plasts which contain very few internal membranesbut a characteristic prolamellar body [11,12]. Theprolamellar body is a paracrystalline structure con-sisting of lipids and essentially a single protein, theNADPH-dependent protochlorophyllide oxidoreduc-tase [13,14]. Shortly after the onset of illuminationthe prolamellar body is dispersed and thylakoids be-gin to form [7,12,15]. Since the start of illuminationcan easily be controlled in experimental setups, thissystem has often been used to study chloroplast de-velopment. Prolamellar bodies are mainly consideredin connection with etioplasts but they are not re-stricted to them. Already after a short period ofdarkness secondary prolamellar bodies form insidefully matured chloroplasts [16,17]. This results in thecoexistence of prolamellar bodies and thylakoids andraises questions about the function of the prolamellarbody for the mature chloroplasts.

Proplastids can further develop into chromoplastsor leucoplasts. These are specialized forms of plastidsused for coloration or storage [18]. Chromoplasts arecarotenoid-containing plastids found in many owerpetals, fruits and roots. Coloration of these organs isoften ascribed to chromoplasts and this might evenbe their main function. Leucoplasts are characterizedby a lack of coloration and they can be distinguishedby the substance that is stored, i.e. amyloplasts, pro-teoplasts or elaioplasts. The nal stage of a plastidslife is the senescent or gerontoplast. These are plas-tids that have reached a stage of senescence that isnot reversible. All plastids, independent of their sta-

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tus, retain the ability to develop into each other (Fig.1). Interconversion of dierent plastid forms requiresdramatic changes of the ultrastructure, including thebiogenesis, reorganization and regression of internalmembranes.

3. Structure and composition of thethylakoid membrane

The additional compartment that the thylakoidnetwork creates in cyanobacterial cells and in chlo-roplasts is an important feature that distinguishesthese from bacteria performing anoxygenic photo-synthesis. In these latter organisms, the internal

membranes are invaginations still continuous withthe plasma membrane [19,20]. In mature chloroplastsand in cyanobacteria it is assumed that the thyla-koids are no longer connected to the inner envelopeor the plasma membrane, respectively, because nosuch continuum can be observed in electron micro-scopic pictures.

How is this unique structural compositionachieved? In cyanobacteria and many algae, thyla-koids consist mainly of single layers formed bylong lamellae. The structure of the thylakoid mem-brane in a fully mature chloroplast is more complex(Fig. 1). Initiated by earlier electron microscopicstudies a model for the thylakoid structure as ahuge intertwined network of stroma lamellae con-

Fig. 1. Overview of the development of chloroplasts. Chloroplast develop from undierentiated proplastids. During maturation thecomplex internal thylakoid membrane network is formed. Proplastids can also develop into other plastids forms, such as etioplasts,chromoplasts and leucoplasts. Moreover, fully dierentiated plastids retain the ability to develop into each other. Gerontoplasts are anal stage in plastid development in which a level of senescence is reached that is irreversible. The electron microscopic pictures ofthin sections show several stages in the development of a chloroplast.

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necting grana stacks was proposed which with littlealterations is still valid today [21,22]. One can distin-guish two major parts, the grana and the stromalamellae. Grana are short, disc-shaped lamellaeclosely packed to form stacks. These stacks are in-terconnected by stroma lamellae which also formprolonged extensions into the stroma. Thus, the ar-rangement of the thylakoid membrane system createsa single huge compartment inside the chloroplast, thethylakoid lumen. Additionally to creating a singleinternal space this structure builds a membrane sur-face that is much larger than a simple invagination ofthe inner envelope would generate.

To understand the complexity of the task that theformation of thylakoids presents to the cell, it isimportant to gure the components that are requiredto build up this special photosynthetic membrane.Thylakoids are lipid bilayers with a unique glycero-lipid composition dierent from other cell mem-branes. Thylakoid lipids have a high content, about70^80%, of galactosyl diglycerides and both mono-galactosyl diacylglycerol and digalactosyl diacylgly-cerol are lipids nearly exclusively found in plastidalmembranes [23]. Notably, these galactolipids containtwo highly unsaturated fatty acyl chains instead ofone as is common in membrane lipids and are bothnon-bilayer forming lipids. Additionally the thyla-koids contain phosphatidylglycerol and sulfoquino-vosyl diacylglycerol together with other minor com-ponents [23]. All these lipids are not evenlydistributed along the thylakoid membrane. Insteadthe lipid distribution diers between the leaet thatis exposed to the stroma and the inner leaet thatfaces the thylakoid lumen [23]. It is not clear howthis asymmetrical arrangement of the lipid distribu-tion is achieved. Yet it has to be assumed that it isimportant for the function of the thylakoid mem-brane.

The dominant protein complexes of the thylakoidsare photosystems I [24] and II [25] and their associ-ated light harvesting antenna, the cytochrome b6fcomplex [26] and the proton-translocating ATP syn-thase [27]. These complexes comprise not only manyperipheral and integral proteins but also a variety ofpigments and co-factors [28]. Their assembly is,therefore, a complex process and requires a largernumber of auxiliary and regulatory factors [28,29].These factors are involved in the membrane integra-

tion, modication and later degradation of the pro-teinaceous components and are also required for theaddition of the pigments and co-factors. To compli-cate matters, certain components, like the two photo-systems, are unevenly distributed in the thylakoidmembrane network. While photosystem I is mostabundant in the non-stacked stroma lamellae, photo-system II is the dominating component of the granastacks [30]. Thus thylakoid biogenesis and mainte-nance have to assure not only the arrangement of afunctional but at the same time asymmetric architec-ture of both the lipid and the protein components ofthis membrane.

4. Thylakoid membrane formation

One of the most elusive aspects of thylakoid for-mation is the exact mechanism by which the mem-brane itself is formed. In young, not yet dierenti-ated plastids a continuum can sometimes be observedbetween the inner envelope and the developing inter-nal membrane structures [7^9]. Thus the synthesis ofearly thylakoid membranes might be achieved by in-vagination of the inner envelope. Even in fully ma-ture chloroplasts the thylakoid membrane is a verydynamic system. Short-term adaptation to changinglight conditions is obtained by movement of proteins,especially the light harvesting complex, within thethylakoid membrane. Long-term adaptation on theother hand is achieved by a change in the proteinand lipid content of the thylakoids. Although in ma-ture chloroplasts a continuum between the inner en-velope and the thylakoids can no longer be observed,the membrane material required for synthesis andmaintenance of the thylakoids originates from thechloroplasts inner envelope [7,31,32] and not fromde novo synthesis on already existing thylakoids.

How these lipids are transferred from the innerenvelope to the thylakoids is controversially dis-cussed. One possibility would be the transfer byvesicles which is a common phenomenon in the cy-tosol, where vesicle trac is involved in many dier-ent cellular processes including the secretory path-way, endocytosis, neural transmission and vacuoleformation [33]. A similar vesicle transfer fromthe inner envelope to the thylakoids has been impli-cated in the synthesis of thylakoid membranes

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[7,31,32,34,35]. Vesicles inside plastids have been ob-served in early electron microscopic studies [7^9].They are common in proplastids and have alsobeen observed on the inner envelope of etioplastsin dark-grown cells of the Chlamydomonas y-1 mu-tant, shortly after illumination when chloroplast de-velopment sets in [36]. On the other hand vesicles arevery rarely detected in mature chloroplasts. They doaccumulate in the stromal space between the innerenvelope and the thylakoids after a low temperatureincubation of leaf tissue [34,35]. A similar phenom-enon is described for vesicle transport in animal cells,i.e. enodplasmic reticulum to Golgi and Golgi toplasma membrane, where low temperature blocksthe fusion of vesicles with their target membrane[37]. Further indication for vesicle transfer in chlo-roplasts comes from mutant analysis. In several plantmutants that are aected in thylakoid biogenesis, anaccumulation of vesicles can be observed. Others,like the vipp1 mutant of Arabidopsis, no longer ex-hibited low temperature vesicle accumulation [35].

The possibility of vesicle transfer inside the chlo-roplast raised the additional question whether solelymembrane lipids would be transported by thesevesicles. As in vacuole formation the vesicle trans-port in chloroplasts could be limited to the supplyof thylakoid lipids that are either synthesized at theinner envelope, i.e. galactolipids, or imported fromthe cytosol. It is also possible that non-lipid compo-nents of the thylakoid membrane might be trans-ported by means of vesicle trac [38,39]. Several ofthe non-lipid components required for the biogenesisand maintenance of thylakoids are synthesized onthe envelope, i.e. carotenoids, or in the cytosol[40,41]. Especially hydrophobic components wouldrequire a system to travel through the aqueous stro-ma.

During chloroplast maturation an extensive for-mation of thylakoid membranes occurs in concertwith the accumulation of the photosynthetic com-plexes. Several of the proteinaceous components arenuclear encoded and post-translationally importedinto the chloroplasts. It was suggested that in Chla-mydomonas the nuclear encoded light harvestingcomplex proteins are inserted into newly developingmembranes at the inner envelope immediately upontheir entrance in the organelle [42]. Later on, thedevelopment of the thylakoid system continues with

the formation of grana stacking. Again, integrationof the light harvesting complex into the thylakoidmembrane might play an important role in this struc-tural reconstruction [43]. This early speculation wassupported recently by Simidjiev and coworkers, whoshowed that delipidated light harvesting complexeswould restructure into ordered lamellae by the addi-tion of monogalactosyl diacylglycerol [44]. They con-cluded that the light harvesting complex togetherwith monogalactosyl diacylglycerol is responsiblefor lamellae organization of the thylakoid mem-brane. Therefore interaction between thylakoid pro-teins and thylakoid lipids seems important for theformation of the lipid bilayer in a membrane whosemain components are non-bilayer forming lipids.

5. Regulation of thylakoid biogenesis

How is the formation of the thylakoid lipid bilayercoordinated with the expression of proteins and thebiosynthesis of pigments and co-factors? It becameobvious quite early after the identication of DNAand genome structure that plastid development andthylakoid formation is controlled by both the ge-nome of the cell (nucleome) and the organelle (plas-tome). Plastids contain up to several hundred copiesof a circular chromosome with a size between 120and 220 kb. Encoded on the plastome is an averageof about 100^200 proteins in addition to a full set ofribosomal and transfer RNAs [45,46]. Chloroplastsare, however, estimated to house about 2000^5000dierent proteins; consequently only 5^10% of theplastidal proteins are encoded within the plastome[46,47] and the majority of proteins required for plas-tid development and function are encoded in the nu-cleus. These nuclear encoded proteins are translatedon cytoplasmic ribosomes and have to be post-trans-lationally transported to the chloroplast ([48]; Jarvisand Soll, this issue).

Many protein complexes and biosynthetic path-ways of the chloroplast contain components encodedboth in the nucleome and in the plastome and vir-tually all chloroplast functions require the concertedaction of nuclear and plastidal encoded factors (Fig.2). Complex regulatory processes are required to en-sure that gene expression of proteins encoded in thenucleome is properly coordinated with the expression

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of plastome encoded proteins. At the same time thecoordinated development of all plastids in one cellhas to be guaranteed.

Thylakoids become photochemically competentvery early in their development [49,50]. The level oftranscription, which is quite low in proplastids, in-creases drastically when the chloroplast begins tomature [51]. At the same time the translational ap-paratus inside the plastids is built up [52]. It is be-lieved that the nucleus has the control over the onsetof chloroplast dierentiation and also takes the lead-ing part in further developmental stages. To executethis control most regulatory components have beentransferred to the nucleus [53]. At the same time theplastids signal back their development stage and con-dition to the nucleus. These signals, often called theplastidal factor, inuence the expression of nuclearencoded plastid proteins [54^56]. The biogenesis andfunction of the chloroplast are therefore an integralpart of the plant cell and the development of the celland its organelle are interdependent [57,58]. This issupported by the fact that plastids cannot easily beexchanged into a dierent cell background [59^61].

This interdependence of the cell and its organelle isfurther strengthened by the fact that two dierentRNA polymerases are required to transcribe plasti-

dal genes [62,63]. This includes a phage-type RNApolymerase of nuclear origin [64] and an eubacterial,multisubunit enzyme whose core subunits are en-coded by the plastome [63] while its sigma factorsubunits are encoded by nuclear genes [65]. The nu-clear encoded RNA polymerase is primarily respon-sible for transcription of so-called housekeepinggenes of the chloroplasts, while the bacterial-typeenzyme preferentially transcribes genes encodingcomponents of the photosynthetic machinery [66].

Very little is known so far about the regulation ofplastidal import in relation to plastid development.Most studies on the regulation of plastidal importhave been done on fully mature, photosyntheticallyactive chloroplasts (Jarvis and Soll, this issue). Arecent publication indicates a direct inuence of as-sembly of the light harvesting complex on the importof the chlorophyll binding protein into the chloro-plast [42]. A similar regulation could be envisionedfor other nuclear encoded chloroplast proteins sincealso in mature chloroplasts the thylakoid composi-tion is very dynamic and undergoes constant changesin order to adapt to changing environmental condi-tions. The ability for adaptation is specially impor-tant since plants are not mobile and can thereforenot escape unfavorable conditions. Only a constant

Fig. 2. Schematic display of nucleus^chloroplast interaction. Synthesis of plastid encoded proteins is regulated by nuclear encoded fac-tors from the point of gene expression and translation until the nal incorporation into the thylakoid membrane. At the same timethe chloroplast signals the nucleus about its state of development. This signal inuences the expression of nuclear encoded genes. Thisgure is based on a scheme presented by Rochaix [102].

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communication between the organelle and the nu-cleus can ensure a coordinated supply of all the dif-ferent factors required.

6. Analysis of thylakoid biogenesis through mutants

Mutants are a powerful tool to study the involve-ment of gene products on specic processes. Manydierent mutants that display deciencies in plastiddevelopment and thylakoid formation exist in a widerange of species. Many of these mutants are ran-domly occurring natural variations, others are man-made. In early work, new mutants were produced bytreatment of plants or algae with radiation or chem-ical mutagens [43,67]. Later, the necessary genetictools became available for random insertional muta-genesis by T-DNA of Agrobacterium tumefaciens[68^70] or transposable elements. Only a limited setof these mutants can be discussed within this section.For a more detailed summary of mutants see[43,71,72].

There are many dierent types of mutations thataect both plastid development and thylakoid for-mation and the eect that a mutation has on eitheris often dicult to distinguish. Often these mutantsare blocked in a step of a biosynthetic pathway lo-cated inside the chloroplasts. The resulting loss of afunctional component of the plastid then extends itseect on the macromolecular structures. In othermutants structural components of the thylakoidmembrane are missing or defective. Mutations canaect plastids in all stages of thylakoid formation.In several cases plastids are blocked very early indevelopment. These mutants include dcl from tomato[73], dag from Antirrhinum [74], cla1-1 from Arabi-dopsis thaliana [75] and several albina mutants ofbarley [65,76]. Plastids in dcl, dag and cla1-1 seemto be arrested in the proplastid stage while plastids insome of the barley albina mutants can reach the sizeof mature chloroplasts but remain fully depleted ofinternal membrane structures except for vesicles thataccumulate in some of them.

A similar phenotype can be observed in vrpoA, Band C1 mutants that lack the bacterial-type RNApolymerase and consequently the ability to transcribethe photosynthetic genes which encode subunits ofthylakoid protein complexes [62,77]. Other mutants

can be found that are blocked in later stages of plas-tid development, anywhere from the proplastid tomature chloroplasts. Because of the close connectionbetween plastid development and thylakoid forma-tion it is often dicult to distinguish pleiotropic ef-fects of these mutations. In some cases mutants seemto suer from a secondary destruction of the internalmembrane structure rather than a defect in thylakoidsynthesis [78,79].

Defects in thylakoid formation are often caused bymutations that result in a depletion of major protein-aceous components of the thylakoid membrane, e.g.major components of the photosystems. For in-stance, the hcf136 mutant of A. thaliana cannot as-semble a functional photosystem II, and this defect isassociated with a drastically disturbed thylakoidmembrane system [80]. Mutations of the protein im-port apparatus of chloroplasts cause similar defectsin thylakoid formation [81]. Other mutations thathave a great impact on thylakoid formation are mu-tations that aect the import pathways by whichproteins are inserted into the thylakoid membrane[82^84]. Examples for such mutants can be foundin maize in the form of tha1 and tha5 which inhibitthe SecA-type import pathway and hcf106 and tha4where the vph or Tat pathway is disrupted [85,86].Not surprisingly, mutants that aect the synthesis ofimportant thylakoid lipids display alterations in thechloroplast ultrastructure. Arabidopsis dgd1 andmdg1 mutants lack the enzymes monogalactosyl di-acylglycerol synthase or digalactosyl diacylglycerolsynthase that are required for the formation of thetwo major thylakoid membrane lipids. These mu-tants show a wide range of alterations includingchanges in the chloroplast ultrastructure and proteincomposition [87^89].

Also very common is the connection between de-ciencies in thylakoid formation and disruption ofpigment biosynthesis [43,67,90]. While pleiotropic ef-fects of these mutations cannot be excluded in somecases, many investigations have supported the poten-tial inuence of chlorophyll production on chloro-plast development [43,90^93]. This connection is es-pecially interesting in light of the plastidal factorthat is discussed as a signal from the chloroplaststo the nucleus (Fig. 2). As described above, the plas-tidal factor is thought to signal the developmentalstage of the plastid to the nucleus and aect the

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expression of many dierent nuclear encoded genes[55,56]. So far the nature of this plastidal factor re-mained elusive and indication for the existence ofmore than one signaling pathways exists. A recentpaper by Chory and coworkers on the Arabidopsismutant uncoupled 5, together with earlier studies byother groups, provides evidence that one of thesefactors might have been found ([94] and referencestherein). Their ndings indicate that a subunit ofMg-chelatase, the enzyme that converts protopor-phyrin IX into Mg-protoporphyrin, has an addition-al, distinct function in the plastid^nucleus signalingpathway.

Especially interesting are mutants that aect thy-lakoid formation in otherwise fully developed chlo-roplasts. One recent example is the vipp1 mutants ofArabidopsis and Synechocystis. Mutant analysisshowed that the gene product of vipp1 is involvedin the biogenesis of thylakoids in Arabidopsis andcyanobacteria [35,95]. Interruption of the vipp1gene locus results in a complete loss of thylakoidmembranes. It seems that Vipp1 is directly involvedin the process of thylakoid biogenesis. Even more,phylogenetic analysis indicated that the presence ofthis protein might be a prerequisite to the ability ofcyanobacteria and chloroplasts to form internalmembranes. Interestingly, the Arabidopsis vipp1 mu-tant additionally lost the ability for vesicle forma-tion. A vesicle transport system might thus be impor-tant for thylakoid formation in mature chloroplasts.

7. Evolution of the thylakoid membrane system

Cyanobacteria are the only phototrophic prokary-otes that carry out oxygenic photosynthesis with twophotosystems. They very much resemble chloroplastsand it is assumed that at the time of the endosym-biotic event they had already invented oxygenic pho-tosynthesis and developed most of the photosyn-thetic features found in chloroplasts today. Likechloroplasts, most cyanobacteria contain an internalmembrane system in which the photosynthetic appa-ratus is located. Extensive stacking of grana lamellaeis not found in these organisms. Their thylakoids areorganized in layers often paralleling the contour ofthe cells. Algae are probably the organism most sim-ilar to the early endosymbiotic cells. Similar to cya-

nobacteria, most algae do not contain grana stacks.Chloroplasts of red algae contain a simple thylakoidstructure similar to cyanobacteria. In green andbrown algae regions of closely appressed thylakoidmembranes occur similar to grana stacks in chloro-plasts of higher plants [15]. Also many algae containonly a single chloroplast per cell. These structuralsimilarities t well with an evolutionary position be-tween the cyanobacterial endosymbiont and higherplants.

It is still a point of debate where photosynthesisdeveloped in the rst place. Recent results favor anorigin of photosynthesis in anoxygenic bacteria [1].Phototropic green and purple bacteria carry out an-oxygenic photosynthesis with a single photosystemstrongly resembling photosystem I. In green-sulfurbacteria the photosynthetic machinery is located inthe cytoplasmic membrane and the antenna com-plexes reside in a special non-membranous structure,the chlorosomes, closely attached to the cytoplasmicmembrane [96]. Purple bacteria on the other handoften display strong invagination of the cytoplasmicmembrane and their photosystems are concentratedin these intracytoplasmic membrane regions [97,98].It is believed that these membranes are not fullyseparated from the cytoplasmic membrane and stillform a continuum with the latter [20,21]. It is there-fore tempting to speculate that the development ofoxygenic photosynthesis is connected to two dier-ent events: the invention of the second photosystemand the biogenesis of an internal membrane systemdisconnected from the cell membrane. Support forthis speculation arose from the identication ofVipp1, a protein essential for thylakoid formationin higher plant chloroplasts and cyanobacteria[35,95]. Phylogenetic analysis showed that Vipp1can be found in organisms that carry out oxygenicphotosynthesis, i.e. plants, algae and cyanobacteria.No Vipp1 homologue has been found so far in bac-teria including those that are capable of anoxygenicphotosynthesis, such as Rhodobacter or Chlorobium.Vipp1 shares sequence homology with a subunit ofthe bacterial phage shock, pspA, and might haveoriginated from a gene duplication of the latter inan ancestor of cyanobacteria. It subsequently ob-tained an additional C-terminal domain that seemsessential for its function in thylakoid formation. InArabidopsis the vipp1 mutation also interrupts

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vesicle trac between the inner envelope and thethylakoids. No such vesicle transport has yet beenshown in any prokaryotic organism including cya-nobacteria. Further studies are needed to showwhether vesicle transport is a feature that developedonly in chloroplasts.

At least one cyanobacterium performs oxygenicphotosynthesis without having thylakoids. Gloeo-bacter violaceus was rst isolated in 1972 from alimestone rock in Switzerland [99]. Electron micro-scopic studies revealed the complete lack of internalmembranes. Not even invaginations of the plasmamembrane were observed. Nevertheless, these cellsperform oxygenic photosynthesis [100,101]. The pho-tosystems are located on the plasma membrane and,similar to purple and green-sulfur bacteria, they formtheir proton gradient along the plasma membrane.This organism might be a cyanobacterium at a stagebefore biogenesis of thylakoids was invented or hasresulted from a secondary loss of thylakoid mem-branes. Compared to cyanobacteria with thylakoidstheir photosynthetic capacity is very low. Thus, e-cient oxygenic photosynthesis may require the pres-ence of an internal membrane system.

While it is easy to envision the evolution of chlo-roplasts from a cyanobacterium, it is much moredicult to understand the evolutionary processesthat created the multiple forms of plastids. There isno indication that the structures found in proplas-tids, chromoplasts or leucoplasts have been part ofthe genetic plan that the endosymbiont transferred tothe host cell. It must be assumed that this develop-ment took place after the endosymbiotic event andwas imposed on the plastid by the host cell. It will befor future research to elucidate the evolutionary trueorigin of the thylakoid membrane and its evolutionfrom simple single membrane layers to the complexsystem present in plant chloroplasts.

Acknowledgements

The authors would like to thank Prof. Dr. J. Sollfor helpful suggestions and discussions. We wouldalso like to thank S. Westphal and C. Glockmanfor the electron microscopic pictures in Fig. 1. Finan-cial support by the Deutsche Forschungsgemein-schaft SFB TR1 is acknowledged.

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