Accumulation of Zeaxanthin in Abscisic Acid-Deficient Mutants of Arabidopsis Does Not Affect Chlorophyll Fluorescence Quenching or Sensitivity to Photoinhibition in Vivo

Abscisic acid (ABA)-deficient mutants of Arabidopsis do not synthesize the epoxy-xanthophylls antheraxanthin, violaxanthin, or neoxanthin. However, thylakoid membranes from these mutants contain 3-fold more zeaxanthin than wild-type plants. This increase in zeaxanthin occurs as a stoichiometric replacement of the missing violaxanthin and neoxanthin within the pigment-protein complexes of both photosystem I and photosystem II (PSII). The retention of zeaxanthin in the dark by ABA-deficient mutants sensitizes the leaves to the development of nonphotochemical quenching (NPQ) during the first 2 to 4 min following a dark-light transition. However, the increase in pool size does not result in any increase in steady-state NPQ. When we exposed wild-type and ABA-deficient mutants leaves to twice growth irradiance, the mutants developed lower maximal NPQ but suffered similar photoinhibition to wildtype, measured both as a decline in the ratio of variable to maximal fluorescence and as a loss of functional PSII centers from oxygen flash yield measurements. These results suggest that only a few of the zeaxanthin molecules present within the light-harvesting antenna of PSII may be involved in NPQ and neither the accumulation of a large pool of zeaxanthin within the antenna of PSII nor an increase in conversion of violaxanthin to zeaxanthin will necessarily enhance photoprotective energy dissipation.     

Occurrence of the carotenoid lactucaxanthin in higher plant LHC II

The pigment composition of the light-harvesting complexes of Photosystem II (LHC II) has been determined for lettuce (Lactuca sativa). In common with other members of the composite, the photosynthetic tissues of this species may contain large amounts of the carotenoid lactucaxanthin (, -carotene-3,3'-diol) in addition to their normal compliment of carotenoids. The occurrence and distribution of lactucaxanthin in LHC II has been examined using isoelectric focusing of BBY particles followed by reversed-phase HPLC analysis of the pigments. The major carotenoids detected in LHC IIb, LHC IIa (CP29) and LHC IIc (CP26) purified from dark-adapted lettuce were lutein, violaxanthin, neoxanthin and lactucaxanthin. Lactucaxanthin has been shown to be a major component of PS II, accounting for 26% of total xanthophyll in both LHC IIb (23% total xanthophyll) and in the minor complexes (12–16%). In this study, LHC IIb was clearly resolved into four bands and their carotenoid composition determined. These four bands proved to be very similar in their pigment content and composition, although the relative amounts of neoxanthin and lutein in particular were found to increase from bands 1 to 4 (i.e. with increasing electrophoretic mobility). The operation of the xanthophyll cycle has also been examined in the LHC of L. sativa following light treatment. The conversion efficiency for violaxanthinzeaxanthin was nearly identical for each light-harvesting complex examined at 58–61%. Nearly half of the zeaxanthin formed in PS II was associated with LHC IIb, although the molar ratio of zeaxanthin:chlorophyll a was highest in the minor LHC.Abbreviations HPLC high performance liquid chromatography - IEF isoelectric focusing - LHCII light-harvesting complex associated with Photosystem II - PS II Photosystem II - qE pH-dependent nonphotochemical quenching of chlorophyll fluorescence     

The coleoptile chloroplast: Distinct distribution of xanthophyll cycle pigments,and enrichment in Photosystem II

Recent studies have shown that coleoptile chloroplasts operate the xanthophyll cycle, and that their zeaxanthin concentration co-varies with their sensitivity to blue light. The present study characterized the distribution of photosynthetic pigments in thylakoid pigment–protein complexes from dark-adapted and light-treated coleoptile and mesophyll chloroplasts, the low temperature fluorescence emission spectra, and the rates of PS I and PS II electron transport in both types of chloroplasts from 5-day-old corn seedlings. Pigments were extracted from isolated PS I holocomplex, LHC IIb trimeric and LHC II monomeric complexes and analyzed by HPLC. Chlorophyll distribution in coleoptile thylakoids showed 31% of the total collected Chl in PS I and 65% in the light harvesting complexes of PS II. In mesophyll thylakoids, the values were 44% and 54%, respectively. Mesophyll and coleoptile PS I holocomplexes differed in their Chl t a/Chl t b ratios (8.1 and 6.1, respectively) and -carotene content. In contrast, mesophyll and coleoptile LHC IIb trimers and LHC II monomers had similar Chl t a/Chl t b ratios and -carotene content. The three analyzed pigment–protein complexes from dark-adapted coleoptile chloroplasts contained zeaxanthin, whereas there was no detectable zeaxanthin in the complexes from dark-adapted mesophyll chloroplasts. In both chloroplast types, zeaxanthin and antheraxanthin increased markedly in the three pigment–protein complexes upon illumination, while violaxanthin decreased. In mesophyll thylakoids, zeaxanthin distribution as a percentage of the xanthophyll cycle pool was: LHC II monomers > LHC IIb trimers > PS I holocomplex, and in coleoptile thylakoids, it was: LHC IIb trimers > LHC II monomers = PS I holocomplex. Low temperature (77 K) fluorescence emission spectra showed that the 686 nm emission of coleoptile chloroplasts was approximately 50% larger than that of mesophyll chloroplasts when normalized at 734 nm. The pigment and fluorescence analysis data suggest that there is relatively more PS II per PS I and more LHC I per CC I in coleoptile chloroplasts than in mesophyll chloroplasts. Measurements of t in vitro uncoupled photosynthetic electron transport showed approximately 60% higher rates of electron flow through PS II in coleoptile chloroplasts than in mesophyll chloroplasts. Electron transport rates through PS I were similar in both chloroplast types. Thus, when compared to mesophyll chloroplasts, coleoptile chloroplasts have a distinct PS I pigment composition, a distinct chlorophyll distribution between PS I and PS II, a distinct zeaxanthin percentage distribution among thylakoid pigment–protein complexes, a higher PS II-related fluorescence emission, and higher PS II electron transport capacity. These characteristics may be associated with a sensory transducing role of coleoptile chloroplasts.     

Carotenoid distribution and deepoxidation in thylakoid pigment-protein complexes from cotton leaves and bundle-sheath cells of maize

In response to excess light, the xanthophyll violaxanthin (V) is deepoxidized to zeaxanthin (Z) via antheraxanthin (A) and the degree of this deepoxidation is strongly correlated with dissipation of excess energy and photoprotection in PS II. However, little is known about the site of V deepoxidation and the localization of Z within the thylakoid membranes. To gain insight into this problem, thylakoids were isolated from cotton leaves and bundle-sheath strands of maize, the pigment protein-complexes separated on Deriphat gels, electroeluted, and the pigments analyzed by HPLC. In cotton thylakoids, 30% of the xanthophyll cycle pigments were associated with the PS I holocomplex, including the PS I light-harvesting complexes and PS I core complex proteins (CC I), and about 50% with the PS II light-harvesting complexes (LHC II). The Chl was evenly distributed between PS I and PS II. Less than 2% of the neoxanthin, about 18% of the lutein, and as much as 76% of the -carotene of the thylakoids were associated with PS I. Exposure of pre-darkened cotton leaves to a high photon flux density for 20 min prior to thylakoid isolation caused about one-half of the V to be converted to Z. The distribution of Z among the pigment-protein complexes was found to be similar to that of V. The distribution of the other carotenoids was unaffected by the light treatment. Similarly, in field-grown maize leaves and in the bundle-sheath strands isolated from them, about 40% of the V present at dawn had been converted to Z at solar noon. Light treatment of isolated bundle-sheath strands which initially contained little Z caused a similar degree of conversion of V to Z. As in cotton thylakoids, about 30% the V+A+Z pool in bundle-sheath thylakoids from maize was associated with the PS I holocomplex and the CC I bands and 46% with the LHC II bands, regardless of the extent of deepoxidation. These results demonstrate that Z is present in PS I as well as in PS II and that deepoxidation evidently takes place within the pigment-protein complexes of both photosystems.Abbreviations A antheraxanthin - CC I, CC II Core or reaction center complex of PS I, PS II - CP Chl protein - EPS epoxidation state - F_m Chl fluorescence at closed PS II reaction centers - IEF isoelectric focussing gels - LHC I, LHC II light-harvesting complex of PS I, PS II - OE oxygen evolving polypeptide - PFD photon flux density - PS I^* PS I holocomplex - V violaxanthin - Z zeaxanthin - antibody against C.I.W.-D.P.B. Publication No. 1127.     

Seasonal changes of violaxanthin cycle pigment de-epoxidation in wintergreen and evergreen plants

We studied carotenoids composition and the activities of the xanthophylls pigments in evergreen conifers (Abies sibirica, Juniperus communis, Picea obovata) and dwarf-shrub (Vaccinium vitis-idaea), and in wintergreen herbaceous plants (Ajuga reptans, Pyrola rotundifolia) growing near Syktyvkar (61°67(/) N 50°77(/) E). The carotenoid pool consisted mainly of following xanthophylls: lutein (70%), neoxanthin (7-10%) and a xanthophylls cycle component - violaxanthin (3-15%). Zeaxanthin and antheraxanthin were found in conifer samples collected in December-March while in other species - during all year. A direct connection between xanthophyll pigment de-epoxidation level and light energy thermal dissipation was shown only for boreal conifer species. It is proposed that zeaxanthin plays a central role in the dissipation of excess excitation energy (nonphotochemical quenching) in the antenna of photosystem II (PSII). We conclude that the increase in the extent of de-epoxidation is beneficial for the retention of PSII activity for conifers in early spring and for herbs in summer.     

Light-harvesting pigment-proteins of photosystem I in maize. Subunit composition and biogenesis

Three different pigment-binding proteins of the light-harvesting complex (LHC I) of maize photosystem I (PS I) have been isolated. Absorption and fluorescence excitation spectral analyses showed that each pigment-protein can transfer absorbed energy from its carotenoid and/or chlorophyll b components to chlorophyll alpha. Their apoproteins with apparent sizes of 24 (LHC Ia), 21 (LHC Ib), and 17 (LHC Ic) kDa have been purified to homogeneity. Differences in their pigment and amino acid compositions and in their reactions with antibodies demonstrate that the two smaller pigment-proteins are not proteolytically derived from the largest one. LHC Ib's apoprotein is particularly enriched in cysteine residues. None of the three apoproteins cross-reacted with antibodies raised against the major light-harvesting chlorophyll a/b-protein of photosystem II (LHC IIb) or against the PS I core complex (CC I) subunits. Studies of the biogenesis of PS I during greening of etiolated plants showed that all of the CC I subunits accumulated to a detectable level prior to the appearance of the 17-kDa subunit of LHC I, the accumulation of which preceded those of the 24- and 21-kDa subunits of LHC I. In addition, subunit VI of CC I is shown to be differentially expressed in mesophyll and bundle sheath cells; a slightly larger form of it accumulates in mesophyll than in bundle sheath thylakoids during plastid development.     

Different roles of alpha- and beta-branch xanthophylls in photosystem assembly and photoprotection

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively alpha-branch (chy1chy2lut5) or beta-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of beta-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.     

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.     

Molecular configuration of xanthophyll cycle carotenoids in photosystem II antenna complexes

The molecular configuration of the xanthophyll cycle carotenoids, violaxanthin and zeaxanthin, was studied in various isolated photosystem II antenna components in comparison to intact photosystem II membranes using resonance Raman combined with low-temperature absorption spectroscopy. The molecular configurations of zeaxanthin and violaxanthin in thylakoids and isolated photosystem II membranes were found to be the same within an isolated oligomeric LHCII antenna, confirming our recent conclusion that these molecules are not freely located in photosynthetic membranes (Ruban, A. V., Pascal, A. A., Robert, B., and Horton, P. (2001) J. Biol. Chem. 276, 24862-24870). In contrast, xanthophyll cycle carotenoids bound to LHCII trimers had largely lost their in vivo configuration, suggesting their partial dissociation from the binding locus. Violaxanthin and zeaxanthin associated with the minor antenna complexes, CP26 and CP29, were also found to be in a relaxed configuration, similar to that of free pigment. The origin of the characteristic C-H vibrational bands of violaxanthin and zeaxanthin in vivo is discussed by comparison with those of neoxanthin and lutein in oligomeric and trimeric LHCII respectively.     

Resonance Raman spectroscopy of carotenoids in Photosystem I particles

Low-temperature resonance Raman (RR) spectroscopy was used for the first time to study the spectral properties, binding sites and composition of major carotenoids in spinach Photosystem I (PSI) particles. Excitation was provided by an argon ion laser at 457.9, 476.5, 488, 496.5, 502 and 514.5 nm. Raman spectra contained the four known groups of bands characteristic for carotenoids (called from nu(1) to nu4). Upon 514.5, 496.5 and 476.5 nm excitations, the nu(1)-nu(3) frequencies coincided with those established for lutein. Spectrum upon 502-nm excitation could be assigned to originate from violaxanthin, at 488 nm to 9-cis neoxanthin, and at 457.9 nm to beta-carotene and 9-cis neoxanthin. The overall configuration and composition of these bound carotenoid molecules in Photosystem I particles were compared with the composition of pigment extracts from the same PSI particles dissolved in pyridine, as well as to configuration in the main chlorophyll a/b light-harvesting protein complex of photosystem II. The absorption transitions for lutein, violaxanthin and 9-cis neoxanthin in spinach photosystem I particles are characterized, and the binding sites of lutein and neoxanthin are discussed. Resonance Raman data suggest that beta-carotene molecules are also present in all-trans and, probably, in 9-cis configurations.     

Photoprotective energy dissipation involves the reorganization of photosystem II light-harvesting complexes in the grana membranes of spinach chloroplasts

Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.     

Photosynthetic pigment composition and photosystem II photochemistry of wheat ears.

The characteristics of pigment composition and photosystem II (PSII) photochemistry in the flag leaf and ear parts of wheat (Triticum aestivum L.) grown in the field was compared. At the early stage of flowering, awns and the flag leaf showed the highest values in the maximal efficiency of PSII photochemistry (Fv/Fm), actual PSII efficiency (phi(PSII)), photochemical quenching (qP), and the efficiency of excitation capture by open PSII centres (Fv/F'm), followed by glumes, lemmas, and paleae, respectively except that no differences in F'v/F'm were observed among glumes, leamms, and paleae. With progressing grain filling, there was a change in the photosynthetic pigment stoichiometry. In the ear parts, neoxanthin and antheraxanthin decreased equally with chlorophyll levels. Lutein and zeaxanthin decreased less than chlorophyll levels while beta-carotene and violaxanthin decreased faster than chlorophyll levels. No big differences in pigment composition were observed among different ear parts. For the flag leaf, neoxanthin and beta-carotene decreased concomitantly with chlorophyll, whereas lutein and xanthophyll cycle pigment were less affected, leading to increases in lutein/chlorophyll and xanthophyll cycle pigment/chlorophyll ratios. Fv/Fm, phi(PSII), qP, and F'v/F'm decreased gradually in the flag leaf and ear parts but to different extents. The largest changes were observed in awns, followed by the lemmas of floret 2, the lemmas of floret 1, glumes, and the flag leaf, respectively. The results suggest that during grain filling, a down-regulation of PSII associated with an increase of the de-epoxidation state of the xanthophyll cycle carotenoids occurred in the flag leaf but not in the ear parts.     

Xanthophyll Cycle Pigment Localization and Dynamics during Exposure to Low Temperatures and Light Stress in Vinca major

The distribution of xanthophyll cycle pigments (violaxanthin plus antheraxanthin plus zeaxanthin [VAZ]) among photosynthetic pigment-protein complexes was examined in Vinca major before, during, and subsequent to a photoinhibitory treatment at low temperature. Four pigment-protein complexes were isolated: the core of photosystem (PS) II, the major light-harvesting complex (LHC) protein of PSII (LHCII), the minor light-harvesting proteins (CPs) of PSII (CP29, CP26, and CP24), and PSI with its LHC proteins (PSI-LHCI). In isolated thylakoids 80% of VAZ was bound to protein independently of the de-epoxidation state and was found in all complexes. Plants grown outside in natural sunlight had higher levels of VAZ (expressed per chlorophyll), compared with plants grown in low light in the laboratory, and the additional VAZ was mainly bound to the major LHCII complex, apparently in an acid-labile site. The extent of de-epoxidation of VAZ in high light and the rate of reconversion of Z plus A to V following 2.5 h of recovery were greatest in the free-pigment fraction and varied among the pigment-protein complexes. Photoinhibition caused increases in VAZ, particularly in low-light-acclimated leaves. The data suggest that the photoinhibitory treatment caused an enrichment in VAZ bound to the minor CPs caused by de novo synthesis of the pigments and/or a redistribution of VAZ from the major LHCII complex.     

Structural analysis and comparison of light-harvesting complexes I and II

Photosynthesis is a fundamental biological process involving the conversion of solar energy into chemical energy. The initial photochemical and photophysical events of photosynthesis are mediated by photosystem II (PSII) and photosystem I (PSI). Both PSII and PSI are multi-subunit supramolecular machineries composed of a core complex and a peripheral antenna system. The antenna system serves to capture light energy and transfer it to the core efficiently. Both PSII and PSI in the green lineage (plants and green algae) and PSI in red algae have an antenna system comprising a series of chlorophyll- and carotenoid-binding membrane proteins belonging to the light-harvesting complex (LHC) superfamily, including LHCII and LHCI. However, the antenna size and subunit composition vary considerably in the two photosystems from diverse organisms. On the basis of the plant and algal LHCII and LHCI structures that have been solved by X-ray crystallography and single-particle cryo-electron microscopy we review the detailed structural features and characteristic pigment properties of these LHCs in PSII and PSI. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.     

Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes

Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation.     

Reconstitution of light-harvesting complexes and photosystem II cores into galactolipid and phospholipid liposomes

Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous to that described for LHC-PC liposomes. Reconstitution of PSII cores into DG vesicles also produced proteoliposomes with randomly dispersed particles (approximately 7.5 +/- 1.6 nm). In contrast, PSII-PC mixtures formed convoluted networks of tubular membranes that exhibited very few fracture faces. Most of the protein particles (approximately 7.0 +/- 1.5 nm) were seen trapped between, rather than embedded in, the membranes. The interaction between the zwitterionic head group of the phosphatidyl choline and the negatively charged PSII core may be responsible for the unusual membrane structures observed.     

Association of chlorophyll a/c(2) complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c(2) proteins were found. The most common complexes have Chl a/c(2) complexes at both sides of the PSI core monomer and have dimensions of about 17x24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c(2) light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c(2) proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c(2) proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Evidence of the supercomplex organization of photosystem II and light-harvesting complexes in <Emphasis Type="Italic">Nannochloropsis granulata</Emphasis>

Diverse light-harvesting complexes (LHCs) have been found in photosynthetic microalgae that originated from secondary endosymbiosis involving primary red algae. However, the associations between LHCs and photosystem I (PSI) and photosystem II (PSII) in these microalgae are not fully understood. Eustigmatophyta is a red algal lineage that appears to have a unique organization in its photosynthetic machinery, consisting of only chlorophyll a and carotenoids that are atypical compared with other closely related groups. In this study, the supramolecular organization of pigment–protein complexes in the eustigmatophyte alga, Nannochloropsis granulata was investigated using Clear Native (CN) PAGE coupled with two-dimensional (2D) SDS-PAGE. Our results showed two slowly migrating green bands that corresponded to PSII supercomplexes, which consisted of reaction centers and LHCs. These green bands were also characterized as PSII complexes by their low temperature fluorescence emission spectra. The protein subunits of the PSII–LHC resolved by 2D CN/SDS-PAGE were analyzed by mass spectrometry, and four different LHC proteins were identified. Phylogenetic analysis of the identified LHC protein sequences revealed that they belonged to four different Lhc groups; (1) stress-related Lhcx proteins, (2) fucoxanthin chlorophyll a/c-binding Lhcf proteins, (3) red-shifted Chromera light-harvesting proteins (Red-CLH), and (4) Lhcr proteins, which are commonly found in organisms possessing red algal plastids. This is the first report showing evidence of a pigment–protein supercomplex consisting of PSII and LHCs, and to identify PSII-associated LHC proteins in Nannochloropsis.     

Absence of lutein, violaxanthin and neoxanthin affects the functional chlorophyll antenna size of photosystem-II but not that of photosystem-I in the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii double mutant npq2 lor1 lacks the beta, epsilon-carotenoids lutein and loroxanthin as well as all beta,beta-epoxycarotenoids derived from zeaxanthin (e.g. violaxanthin and neoxanthin). Thus, the only carotenoids present in the thylakoid membranes of the npq2 lor1 cells are beta-carotene and zeaxanthin. The effect of these mutations on the photochemical apparatus assembly and function was investigated. In cells of the mutant strain, the content of photosystem-II (PSII) and photosystem-I (PSI) was similar to that of the wild type, but npq2 lor1 had a significantly smaller PSII light-harvesting Chl antenna size. In contrast, the Chl antenna size of PSI was not truncated in the mutant. SDS-PAGE and Western blot analysis qualitatively revealed the presence of all LHCII and LHCI apoproteins in the thylakoid membrane of the mutant. The results showed that some of the LHCII and most of the LHCI were assembled and functionally connected with PSII and PSI, respectively. Photon conversion efficiency measurements, based on the initial slope of the light-saturation curve of photosynthesis and on the yield of Chl a fluorescence in vivo, showed similar efficiencies. However, a significantly greater light intensity was required for the saturation of photosynthesis in the mutant than in the wild type. It is concluded that zeaxanthin can successfully replace lutein and violaxanthin in most of the functional light-harvesting antenna of the npq2 lor1 mutant.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II