Roberto Bassi

Abstract: Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching (CTQ) have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light harvesting complex II (LHCII) in ADQ, and exclusively monomeric Lhcb proteins in CTQ. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin (Zea), a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than LHCII during aggregation, and that this is enhanced by binding to Zea, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll (Chl) 612-613, in close contact with lutein bound at site L1, are important facilitators of ADQ.

Abstract: The outer antenna system of Chlamydomonas reinhardtii Photosystem I is composed of nine gene products, but due to difficulty in purification their individual properties are not known. In this work, the functional properties of the nine Lhca antennas of Chlamydomonas, have been investigated upon expression of the apoproteins in bacteria and refolding in vitro of the pigment-protein complexes. It is shown that all Lhca complexes have a red-shifted fluorescence emission as compared to the antenna complexes of Photosystem II, similar to Lhca from higher plants, but less red-shifted. Three complexes, namely Lhca2, Lhca4 and Lhca9, exhibit emission maxima above 707 nm and all carry an asparagine as ligand for Chl 603. The comparison of the protein sequences and the biochemical/spectroscopic properties of the refolded Chlamydomonas complexes with those of the well-characterized Arabidopsis thaliana Lhcas shows that all the Chlamydomonas complexes have a chromophore organization similar to that of A. thaliana antennas, particularly to Lhca2, despite low sequence identity. All the major biochemical and spectroscopic properties of the Lhca complexes have been conserved through the evolution, including those involved in "red forms" absorption. It has been proposed that in Chlamydomonas PSI antenna size and polypeptide composition can be modulated in vivo depending on growth conditions, at variance as compared to higher plants. Thus, the different properties of the individual Lhca complexes can be functional to adapt the architecture of the PSI-LHCI supercomplex to different environmental conditions.

Abstract: Thylakoid membranes in higher plant chloroplasts are composed by two distinct domains: stacked grana and stroma lamellae. We developed a procedure for biochemical isolation of grana membranes using mild detergent to maintain membrane structure. Pigment and polypeptide analyses of membrane preparation showed the preparations were indeed enriched in grana membranes. The method was shown to be effective in four different plant species, although with small changes in detergent concentration. Electron microscopy analyses also showed that the preparation consisted of large membrane patches with roughly round shape and diameter comparable with grana membranes in vivo. Furthermore, protein complexes distribution was shown to be maintained with respect to freeze fracture studies, demonstrating that the protocol was successful in isolating membranes close to their in vivo state.

Abstract: Elucidating the molecular details of qE (energy quenching) induction in higher plants has proven to be a major challenge. Identification of qE mutants has provided initial information on functional elements involved in the qE mechanism; furthermore, investigations on isolated pigment-protein complexes and analysis in vivo and in vitro by sophisticated spectroscopic methods have been used for the elucidation of mechanisms involved. The aim of the present review is to summarize the current knowledge of the phenotype of npq (non-photochemical quenching)-knockout mutants, the role of gene products involved in the qE process and compare the molecular models proposed for this process.

Abstract: Light is the source of energy for photosynthetic organisms; when in excess, however, it also drives the formation of reactive oxygen species and, consequently, photoinhibition. Plants and algae have evolved mechanisms to regulate light harvesting efficiency in response to variable light intensity so as to avoid oxidative damage. Nonphotochemical quenching (NPQ) consists of the rapid dissipation of excess excitation energy as heat. Although widespread among oxygenic photosynthetic organisms, NPQ shows important differences in its machinery. In land plants, such as Arabidopsis thaliana, NPQ depends on the presence of PSBS, whereas in the green alga Chlamydomonas reinhardtii it requires a different protein called LHCSR. In this work, we show that both proteins are present in the moss Physcomitrella patens. By generating KO mutants lacking PSBS and/or LHCSR, we also demonstrate that both gene products are active in NPQ. Plants lacking both proteins are more susceptible to high light stress than WT, implying that they are active in photoprotection. These results suggest that NPQ is a fundamental mechanism for survival in excess light and that upon land colonization, photosynthetic organisms evolved a unique mechanism for excess energy dissipation before losing the ancestral one found in algae.

Abstract: When light absorbed by plants exceeds the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly de-epoxidized to zeaxanthin in the so-called xanthophyll cycle. Zeaxanthin plays a key role in the protection of photosynthetic organisms against excess light, by promoting rapidly reversible (qE) and long-term (qI) quenching of excited chlorophylls, and preventing lipid oxidation. The photoprotective role of zeaxanthin, either free or bound to light-harvesting complexes (Lhcs), has been investigated by using mutants lacking Chl b (ch1) and/or specific xanthophyll species (npq, lut2). The ch1 mutation causes (1) the absence of Lhcb proteins; (2) strong reduction of the feedback de-excitation (qE); and (3) accumulation of xanthophylls as free pigments into thylakoids. Ch1 mutants showed extreme sensitivity to photo-oxidative stress in high light, due to higher singlet oxygen ((1)O(2)) release. The double mutant ch1npq1 was more sensitive to photo-oxidation than ch1, showing that zeaxanthin does protect lipids even when free in the membrane. Nevertheless, lack of zeaxanthin had a much stronger impact on the level of lipid peroxidation in Lhcs-containing plants (WT vs npq1) with respect to Lhc-less plants (ch1 vs ch1npq1), implying that its protective effect is enhanced by interaction with antenna proteins. It is proposed that the antioxidant capacity of zeaxanthin is empowered in the presence of PSII-LHCs-Zea complexes, while its effect on enhancement of qE only provides a minor contribution. Comparison of the sensitivity of WT vs npq1 plants to exogenous (1)O(2) suggests that besides the scavenging of (1)O(2), at least one additional mechanism is involved in chloroplast photoprotection.

Abstract: Lhcb6 (CP24) is a monomeric antenna protein of photosystem II, which has been shown to play special roles in photoprotective mechanisms, such as the Non-Photochemical Quenching and reorganization of grana membranes in excess light conditions. In this work we analyzed Lhcb6 in vivo and in vitro: we show this protein, upon activation of the xanthophyll cycle, accumulates zeaxanthin into inner binding sites faster and to a larger extent than any other pigment-protein complex. By comparative analysis of Lhcb6 complexes violaxanthin or zeaxanthin binding, we demonstrate that zeaxanthin not only down-regulates chlorophyll singlet excited states, but also increases the efficiency of chlorophyll triplet quenching, with consequent reduction of singlet oxygen production and significant enhancement of photo-stability. On these bases we propose that Lhcb6, the most recent addition to the Lhcb protein family which evolved concomitantly to the adaptation of photosynthesis to land environment, has a crucial role in zeaxanthin-dependent photoprotection.

Abstract: Plants are able to deal with variable environmental conditions: when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by Violaxanthin De-Epoxidase (VDE), using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the VDE catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analysed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate binding sites and point to two residues, D177 and Y198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.

Abstract: The role of individual photosynthetic antenna complexes of Photosystem II (PSII) both in membrane organization and excitation energy transfer have been investigated. Thylakoid membranes from wild-type Arabidopsis thaliana, and three mutants lacking light-harvesting complexes CP24, CP26, or CP29, respectively, were studied by picosecond-fluorescence spectroscopy. By using different excitation/detection wavelength combinations it was possible for the first time, to our knowledge, to separate PSI and PSII fluorescence kinetics. The sub-100 ps component, previously ascribed entirely to PSI, turns out to be due partly to PSII. Moreover, the migration time of excitations from antenna to PSII reaction center (RC) was determined for the first time, to our knowledge, for thylakoid membranes. It is four times longer than for PSII-only membranes, due to additional antenna complexes, which are less well connected to the RC. The results in the absence of CP26 are very similar to those of wild-type, demonstrating that the PSII organization is not disturbed. However, the kinetics in the absence of CP29 and, especially, of CP24 show that a large fraction of the light-harvesting complexes becomes badly connected to the RCs. Interestingly, the excited-state lifetimes of the disconnected light-harvesting complexes seem to be substantially quenched.

Abstract: We investigated the pigment composition and the transcriptome of albina (alb-e ( 16 ) and alb-f ( 17 )) and xantha (xan-s ( 46 ) and xan-b ( 12 )) barley mutants to provide an overall transcriptional picture of genes whose expression is interconnected with chloroplast activities and to search for candidate genes associated with the mutations. Beside those encoding plastid-localized proteins, more than 3,000 genes involved in non-chloroplast localized metabolism were up-/down-regulated in the mutants revealing the network of chloroplast-dependent metabolic pathways. The alb-e ( 16 ) mutant was characterized by overaccumulation of protoporphyrin IX upon ALA (5-amino levulinic acid) feeding and down-regulation of the gene encoding one subunit of Mg-chelatase, suggesting a block of the chlorophyll biosynthetic pathway before Mg-protoporphyrin IX biosynthesis, while alb-f ( 17 ) overaccumulated Mg-protoporphyrin IX and repressed PorA expression, without alterations in Mg-chelatase mRNA level. The alb-f ( 17 )mutant also showed overexpression of several genes involved in phytochrome and in phytochrome-dependent pathways. The results indicate that the down-regulation of Lhcb genes in alb-e ( 16 ) cannot be mediated by the accumulation of Mg-protoporphyrin IX. After ALA treatment, xan-s ( 46 ) showed overaccumulation of Mg-protoporphyrin IX, while the relative porphyrin composition of xan-b ( 12 ) was similar to wild type. The transcripts encoding the components of several mitochondrial metabolic pathways were up-regulated in albina/xantha leaves to compensate for the absence of active chloroplasts. The mRNAs encoding gun3, gun4, and gun5 barley homologous genes showed significant expression variations and were used to search for co-expressed genes across all samples. These analyses provide additional evidences on a chloroplast-dependent covariation of large sets of nuclear genes.

Abstract: Energy-dependent quenching of excitons in photosystem II of plants, or qE, has been positively correlated with the transient production of carotenoid radical cation species. Zeaxanthin was shown to be the donor species in the CP29 antenna complex. We report transient absorbance analyses of CP24 and CP26 complexes that bind lutein and zeaxanthin in the L1 and L2 domains, respectively. For CP24 complexes, the transient absorbance difference profiles give a reconstructed transient absorbance spectrum with a single peak centered at approximately 980 nm, consistent with zeaxanthin radical cation formation. In contrast, CP26 gives constants for the decay components probed at 940 and 980 nm of 144 and 194 ps, a transient absorbance spectrum that has a main peak at 980 nm, and a substantial shoulder at 940 nm. This suggests the presence of two charge transfer quenching sites in CP26 involving zeaxanthin radical cation and lutein radical cation species. We also show that lutein radical cation formation in CP26 is dependent on binding of zeaxanthin to the L2 domain, implying that zeaxanthin acts as an allosteric effector of charge transfer quenching involving lutein in the L1 domain.

Abstract: PsbS plays a major role in activating the photoprotection mechanism known as "non-photochemical quenching," which dissipates chlorophyll excited states exceeding the capacity for photosynthetic electron transport. PsbS activity is known to be triggered by low lumenal pH. However, the molecular mechanism by which this subunit regulates light harvesting efficiency is still unknown. Here we show that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins (CP29 and CP24) and the trimeric LHCII-M. Dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light, strongly suggesting that protein subunits catalyzing the reaction of heat dissipation are buried into the complex and thus not available for interaction with PsbS. Consistently, we showed that knock-out mutants on two subunits participating to the B4C complex were strongly affected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpreted these results to mean that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions.

Abstract: Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid DeltapH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and alpha-carotene than the wild type. szl1 contains a point mutation in the lycopene beta-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene beta-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.

Abstract: Lhcb5 is an antenna protein that is highly conserved in plants and green algae. It is part of the inner layer of photosystem II antenna system retained in high light acclimated plants. To study the structure-function relation and the role of individual pigments in this complex, we (i) "knocked out" each of the chromophores bound to multiple (nine total) chlorophyll sites and (ii) exchanged the xanthophylls bound to the three xanthophyll sites. The occupancy and associated energy of the pigment binding sites were determined. The role of the individual pigments in protein folding, stability, energy transfer, and dissipation was studied in vitro. The results indicate that lutein has a primary role in the folding and stability of the complex, whereas violaxanthin and zeaxanthin have a negative effect on folding yield and stability, respectively. The data showed a distinct function for the L1 and L2 carotenoid binding sites, the former preferentially involved in gathering the excitation energy to chlorophyll a (Chl a), whereas the latter modulates the concentration of chlorophyll singlet excited states dependent on the xanthophylls bound to it, likely via an interaction with Chl-603. Our results also underscored the role of zeaxanthin and lutein in quenching the excitation energy, whereas violaxanthin was shown to be very effective in energy transfer. The characteristics of the isolated proteins were consistent with the observed role of Lhcb5 in vivo in catalyzing fluorescence quenching upon zeaxanthin binding.

Abstract: State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b6f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light-induced reduction of this bond may occur through a transthylakoid thiol-reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b6f assembly and heme biogenesis.

Abstract: BACKGROUND: Plant performance is affected by the level of expression of PsbS, a key photoprotective protein involved in the process of feedback de-excitation (FDE), or the qE component of non-photochemical quenching, NPQ. RESULTS: In studies presented here, under constant laboratory conditions the metabolite profiles of leaves of wild-type Arabidopsis thaliana and plants lacking or overexpressing PsbS were very similar, but under natural conditions their differences in levels of PsbS expression were associated with major changes in metabolite profiles. Some carbohydrates and amino acids differed ten-fold in abundance between PsbS-lacking mutants and over-expressers, with wild-type plants having intermediate amounts, showing that a metabolic shift had occurred. The transcriptomes of the genotypes also varied under field conditions, and the genes induced in plants lacking PsbS were similar to those reportedly induced in plants exposed to ozone stress or treated with methyl jasmonate (MeJA). Genes involved in the biosynthesis of JA were up-regulated, and enzymes involved in this pathway accumulated. JA levels in the undamaged leaves of field-grown plants did not differ between wild-type and PsbS-lacking mutants, but they were higher in the mutants when they were exposed to herbivory. CONCLUSION: These findings suggest that lack of FDE results in increased photooxidative stress in the chloroplasts of Arabidopsis plants grown in the field, which elicits a response at the transcriptome level, causing a redirection of metabolism from growth towards defence that resembles a MeJA/JA response.

Abstract: Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin beta-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.

Abstract: Photosynthetic light-harvesting complexes absorb energy and guide photoexcitations to reaction centers with speed and efficacy that produce near-perfect efficiency. Light harvesting complex II (LHCII) is the most abundant light-harvesting complex and is responsible for absorbing the majority of light energy in plants. We apply two-dimensional electronic spectroscopy to examine energy flow in LHCII. This technique allows for direct mapping of excitation energy pathways as a function of absorption and emission wavelength. The experimental and theoretical results reveal that excitation energy transfers through the complex on three time scales: previously unobserved sub-100 fs relaxation through spatially overlapping states, several hundred femtosecond transfer between nearby chlorophylls, and picosecond energy transfer steps between layers of pigments. All energy is observed to collect into the energetically lowest and most delocalized states, which serve as exit sites. We examine the angular distribution of optimal energy transfer produced by this delocalized electronic structure and discuss how it facilitates the exit step in which the energy moves from LHCII to other complexes toward the reaction center.

Abstract: The near-unity efficiency of energy transfer in photosynthesis makes photosynthetic light-harvesting complexes a promising avenue for developing new renewable energy technologies. Knowledge of the energy landscape of these complexes is essential in understanding their function, but its experimental determination has proven elusive. Here, the observation of quantum coherence using two-dimensional electronic spectroscopy is employed to directly measure the 14 lowest electronic energy levels in light-harvesting complex II (LHCII), the most abundant antenna complex in plants containing approximately 50% of the world's chlorophyll. We observe that the electronically excited states are relatively evenly distributed, highlighting an important design principle of photosynthetic complexes that explains the observed ultrafast intracomplex energy transfer in LHCII.

Abstract: In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O(2) to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O(2). As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins.

Abstract: Previous work on intact thylakoid membranes showed that transient formation of a zeaxanthin radical cation was correlated with regulation of photosynthetic light-harvesting via energy-dependent quenching. A molecular mechanism for such quenching was proposed to involve charge transfer within a chlorophyll-zeaxanthin heterodimer. Using near infrared (880-1100 nm) transient absorption spectroscopy, we demonstrate that carotenoid (mainly zeaxanthin) radical cation generation occurs solely in isolated minor light-harvesting complexes that bind zeaxanthin, consistent with the engagement of charge transfer quenching therein. We estimated that less than 0.5% of the isolated minor complexes undergo charge transfer quenching in vitro, whereas the fraction of minor complexes estimated to be engaged in charge transfer quenching in isolated thylakoids was more than 80 times higher. We conclude that minor complexes which bind zeaxanthin are sites of charge transfer quenching in vivo and that they can assume Non-quenching and Quenching conformations, the equilibrium LHC(N) <==> LHC(Q) of which is modulated by the transthylakoid pH gradient, the PsbS protein, and protein-protein interactions.

Abstract: In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called non-photochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575-578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished.

Abstract: Energy-dependent quenching of excess absorbed light energy (qE) is a vital mechanism for regulating photosynthetic light harvesting in higher plants. All of the physiological characteristics of qE have been positively correlated with charge transfer between coupled chlorophyll and zeaxanthin molecules in the light-harvesting antenna of photosystem II (PSII). We found evidence for charge-transfer quenching in all three of the individual minor antenna complexes of PSII (CP29, CP26, and CP24), and we conclude that charge-transfer quenching in CP29 involves a delocalized state of an excitonically coupled chlorophyll dimer. We propose that reversible conformational changes in CP29 can "tune" the electronic coupling between the chlorophylls in this dimer, thereby modulating the energy of the chlorophyll-zeaxanthin charge-transfer state and switching on and off the charge-transfer quenching during qE.

Abstract: To avoid photodamage, photosynthetic organisms have developed mechanisms to evade or dissipate excess energy. Lumen overacidification caused by light-induced electron transport triggers quenching of excited chlorophylls and dissipation of excess energy into heat. In higher plants participation of the PsbS protein as the sensor of low lumenal pH was clearly demonstrated. Although light-dependent energy quenching is a property of all photosynthetic organisms, large differences in amplitude and kinetics can be observed thus raising the question whether a single common mechanism is in action. We performed a detailed study of PsbS expression/accumulation in Chlamydomonas reinhardtii and investigated its accumulation in other algae and plants. We showed that PsbS cannot be detected in Chlamydomonas under a wide range of growth conditions. Overexpression of the endogenous psbs gene showed that the corresponding protein could not be addressed to the thylakoid membranes. Survey of different unicellular green algae showed no accumulation of anti-PsbS reactive proteins differently from multicellular species. Nevertheless, some unicellular species exhibit high energy quenching activity, suggesting that a PsbS-independent mechanism is activated. By correlating growth habitat and PsbS accumulation in different species, we suggest that during the evolution the light environment has been a determinant factor for the conservation/loss of the PsbS function.

Abstract: We performed transient absorption (TA) measurements on CP29 minor light-harvesting complexes that were reconstituted in vitro with either violaxanthin (Vio) or zeaxanthin (Zea) and demonstrate that the Zea-bound CP29 complexes exhibit charge-transfer (CT) quenching that has been correlated with the energy-dependent quenching (qE) in higher plants. Simulations of the difference TA kinetics reveal two-phase kinetics for intracomplex energy transfer to the CT quenching site in CP29 complexes, with a fast <500 fs component and a approximately 6 ps component. Specific chlorophyll sites within CP29 are identified as likely locations for CT quenching. We also construct a kinetic model for CT quenching during qE in an intact system that incorporates CP29 as a CT trap and show that the model is consistent with previous in vivo measurements on spinach thylakoid membranes. Finally, we compare simulations of CT quenching in thylakoids with those of the individual CP29 complexes and propose that CP29 rather than LHCII is a site of CT quenching.

Abstract: Time-resolved fluorescence measurements were performed on isolated core and intact Photosystem I (PS I) particles and stroma membranes from Arabidopsis thaliana to characterize the type of energy-trapping kinetics in higher plant PS I. Target analysis confirms the previously proposed "charge recombination" model. No bottleneck in the energy flow from the bulk antenna compartments to the reaction center has been found. For both particles a trap-limited kinetics is realized, with an apparent charge separation lifetime of approximately 6 ps. No red chlorophylls (Chls) are found in the PS I-core complex from A. thaliana. Rather, the observed red-shifted fluorescence (700-710 nm range) originates from the reaction center. In contrast, two red Chl compartments, located in the peripheral light-harvesting complexes, are resolved in the intact PS I particles (decay lifetimes 33 and 95 ps, respectively). These two red states have been attributed to the two red states found in Lhca 3 and Lhca 4, respectively. The influence of the red Chls on the slowing of the overall trapping kinetics in the intact PS I complex is estimated to be approximately four times larger than the effect of the bulk antenna enlargement.

Abstract: The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.

Abstract: BACKGROUND: In eukaryotes the photosynthetic antenna system is composed of subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. The moss Physcomitrella patens is a member of a lineage that diverged from seed plants early after land colonization and therefore by studying this organism, we may gain insight into adaptations to the aerial environment. PRINCIPAL FINDINGS: In this study, we characterized the antenna protein multigene family in Physcomitrella patens, by sequence analysis as well as biochemical and functional investigations. Sequence identification and analysis showed that some antenna polypeptides, such as Lhcb3 and Lhcb6, are present only in land organisms, suggesting they play a role in adaptation to the sub-aerial environment. Our functional analysis which showed that photo-protective mechanisms in Physcomitrella patens are very similar to those in seed plants fits with this hypothesis. In particular, Physcomitrella patens also activates Non Photochemical Quenching upon illumination, consistent with the detection of an ortholog of the PsbS protein. As a further adaptation to terrestrial conditions, the content of Photosystem I low energy absorbing chlorophylls also increased, as demonstrated by differences in Lhca3 and Lhca4 polypeptide sequences, in vitro reconstitution experiments and low temperature fluorescence spectra. CONCLUSIONS: This study highlights the role of Lhc family members in environmental adaptation and allowed proteins associated with mechanisms of stress resistance to be identified within this large family.

Abstract: We investigated the function of chlorophyll a/b binding antenna proteins Chlorophyll Protein 26 (CP26) and CP24 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout lines that completely lacked one or both of these proteins. All three mutant lines had a decreased efficiency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII and formation of PSII reaction center depleted domains in grana partitions. Photosynthesis was affected in plants lacking CP24 but not in plants lacking CP26: the former mutant had decreased electron transport rates, a lower DeltapH gradient across the grana membranes, reduced capacity for nonphotochemical quenching, and limited growth. Furthermore, the PSII particles of these plants were organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, overall electron transport, nonphotochemical quenching, and growth of the double mutant were restored to wild type. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that limitation in electron transport was due to restricted electron transport between Q(A) and Q(B), which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion.

Abstract: The location of the neoxanthin binding site in CP26 and CP29 was investigated by site-directed mutagenesis. The crystallographic structure of LHCII shows that the binding of neoxanthin to the N1 site is stabilised by an H bond with a tyrosine in the lumenal loop. This residue is conserved in CP26 and CP29. Mutation of this tyrosine into phenylalanine induced specific loss of neoxanthin without affecting violaxanthin binding. In contrast to previous proposals, it is thus concluded that also in these minor antenna complexes neoxanthin is accommodated in the N1 site. The characteristics of this binding site in the different antenna complexes are discussed.

Abstract: The ch1 mutant of Arabidopsis (Arabidopsis thaliana) lacks chlorophyll (Chl) b. Leaves of this mutant are devoid of photosystem II (PSII) Chl-protein antenna complexes and have a very low capacity of nonphotochemical quenching (NPQ) of Chl fluorescence. Lhcb5 was the only PSII antenna protein that accumulated to a significant level in ch1 mutant leaves, but the apoprotein did not assemble in vivo with Chls to form a functional antenna. The abundance of Lhca proteins was also reduced to approximately 20% of the wild-type level. ch1 was crossed with various xanthophyll mutants to analyze the antioxidant activity of carotenoids unbound to PSII antenna. Suppression of zeaxanthin by crossing ch1 with npq1 resulted in oxidative stress in high light, while removing other xanthophylls or the PSII protein PsbS had no such effect. The tocopherol-deficient ch1 vte1 double mutant was as sensitive to high light as ch1 npq1, and the triple mutant ch1 npq1 vte1 exhibited an extreme sensitivity to photooxidative stress, indicating that zeaxanthin and tocopherols have cumulative effects. Conversely, constitutive accumulation of zeaxanthin in the ch1 npq2 double mutant led to an increased phototolerance relative to ch1. Comparison of ch1 npq2 with another zeaxanthin-accumulating mutant (ch1 lut2) that lacks lutein suggests that protection of polyunsaturated lipids by zeaxanthin is enhanced when lutein is also present. During photooxidative stress, alpha-tocopherol noticeably decreased in ch1 npq1 and increased in ch1 npq2 relative to ch1, suggesting protection of vitamin E by high zeaxanthin levels. Our results indicate that the antioxidant activity of zeaxanthin, distinct from NPQ, can occur in the absence of PSII light-harvesting complexes. The capacity of zeaxanthin to protect thylakoid membrane lipids is comparable to that of vitamin E but noticeably higher than that of all other xanthophylls of Arabidopsis leaves.

Abstract: In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line-narrowing experiments. It was found that the red-most absorption bands of Lhca3, Lhca4, and Lhca1-4 peak, respectively, at 704, 708, and 709 nm and are responsible for 725-, 733-, and 732-nm fluorescence emission bands. These bands are more red shifted compared to "normal" chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400-450 cm(-1)), which is the result of both large homogeneous and inhomogeneous broadening. The observed optical reorganization energy is untypical for Chl a and resembles more that of BChl a antenna systems. The large broadening and the changes in optical reorganization energy are explained by a mixing of an Lhca excitonic state with a charge transfer state. Such a charge transfer state can be stabilized by the polar residues around Chl 1025. It is shown that the optical reorganization energy is changing through the inhomogeneous distribution of the red-most absorption band, with the pigments contributing to the red part of the distribution showing higher values. A second red emission form in Lhca4 was detected at 705 nm and originates from a broad absorption band peaking at 690 nm. This fluorescence emission is present also in the Lhca4-N-47H mutant, which lacks the 733-nm emission band.

Abstract: The lutein-5,6-epoxide (Lx) cycle operates in some plants between lutein (L) and its monoepoxide, Lx. Whereas recent studies have established the photoprotective roles of the analogous violaxanthin cycle, physiological functions of the Lx cycle are still unknown. In this article, we investigated the operation of the Lx cycle in light-harvesting antenna complexes (Lhcs) of Inga sapindoides Willd, a tropical tree legume accumulating substantial Lx in shade leaves, to identify the xanthophyll-binding sites involved in short- and long-term responses of the Lx cycle and to analyze the effects on light-harvesting efficiency. In shade leaves, Lx was converted into L upon light exposure, which then replaced Lx in the peripheral V1 site in trimeric Lhcs and the internal L2 site in both monomeric and trimeric Lhcs, leading to xanthophyll composition resembling sun-type Lhcs. Similar to the violaxanthin cycle, the Lx cycle was operating in both photosystems, yet the light-induced Lx --> L conversion was not reversible overnight. Interestingly, the experiments using recombinant Lhcb5 reconstituted with different Lx and/or L levels showed that reconstitution with Lx results in a significantly higher fluorescence yield due to higher energy transfer efficiencies among chlorophyll (Chl) a molecules, as well as from xanthophylls to Chl a. Furthermore, the spectroscopic analyses of photosystem I-LHCI from I. sapindoides revealed prominent red-most Chl forms, having the lowest energy level thus far reported for higher plants, along with reduced energy transfer efficiency from antenna pigments to Chl a. These results are discussed in the context of photoacclimation and shade adaptation.

Abstract: In this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I.

Abstract: The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.

Abstract: In this work we modeled the circular dichroism (CD) spectrum of LHCII, the main light harvesting antenna of photosystem II of higher plants. Excitonic calculations are performed for a monomeric subunit, taken from the crystal structure of trimeric LHCII from spinach [Liu, Z. F., Yan, H. C., Wang, K. B., Kuang, T. Y., Zhang, J. P., Gui, L. L., An, X. M., and Chang, W. R. (2004) Nature 428, 287-292]. All of the major features of the CD spectrum above 450 nm are satisfactorily reproduced, and possible orientations of the Chl and carotenoid transition dipole moments are identified. The obtained modeling parameters are used to simulate the CD spectra of two complexes with altered pigment composition: a mutant lacking Chls a 611-612 and a complex lacking the carotenoid neoxanthin. By removing the relevant pigment(s) from the structure, we are able to reproduce their spectra, which implies that the alteration does not disturb the overall structure. The CD spectrum of trimeric LHCII shows a reversed relative intensity of the two negative bands around 470 and 490 nm as compared to monomeric LHCII. The simulations reproduce this reversal, indicating that it is mainly due to interactions between chromophores in different monomeric subunits, and the trimerization does not induce observable changes in the monomeric structure. Our simulated spectrum resembles one of two different trimeric CD spectra reported in literature. We argue that the differences in the experimental trimeric CD spectra are caused by changes in the strength of the monomer-monomer interactions due to the differences in detergents used for the purification of the complexes.

Abstract: In this work, the spectroscopic characteristics of carotenoids associated with the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra, and laser-induced triplet-minus-singlet (T-S) spectra were determined for native LHCI from the wild type (WT) and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while beta-carotene was found to be associated only with the native LHCI preparation and recombinant Lhca3. In the native complexes, the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue-shifted to 487 and 501 nm. The "red" lutein absorbing at 510 nm was found to be associated with Lhca3 and Lhca4 which also show a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T-S maxima: one at 507 nm (corresponding to the 490 nm singlet absorption) and the second at 525 nm (with absorption at 510 nm). The "blue"-absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011, and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca, the 510 nm band is present in both the monomeric and dimeric complexes. We suggest that the large red shift observed for this xanthophyll is due to interaction with the neighbor Chl 1015. In the native T-S spectrum, the contribution of carotenoids associated with Lhca2 is visible while the one of Lhca1 is not. This suggests that in the Lhca2-Lhca3 heterodimeric complex energy equilibration is not complete at least on a fast time scale.

Abstract: The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.

Abstract: We analyze the effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size at transcriptional and post-transcriptional levels. This was approached by studying transcription and accumulation of light-harvesting complexes in wild type versus the barley mutant viridis zb63, which is depleted in photosystem I and where plastoquinone is constitutively reduced. We show that the mRNA level of genes encoding antenna proteins is almost unaffected in the mutant; this stability of messenger level is not a peculiarity of antenna-encoding genes, but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by two-dimensional PAGE shows that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that the plastoquinone redox state plays an important role in the long term regulation of chloroplast protein expression. However, its modulation is active at the post-transcriptional rather than transcriptional level.

Abstract: 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.

Abstract: Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the 'red' chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.

Abstract: Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.

Abstract: This historical review was compiled and edited by Giorgio Forti, whereas the other authors of the different sections are listed alphabetically after his name, below the title of the paper; they are also listed in the individual sections. This review deals with the research on photosynthesis performed in several Italian laboratories during the last 50 years; it includes research done, in collaboration, at several international laboratories, particularly USA, UK, Switzerland, Hungary, Germany, France, Finland, Denmark, and Austria. Wherever pertinent, references are provided, especially to other historical papers in Govindjee et al. [Govindjee, Beatty JT, Gest H, Allen JF (eds) (2005) Discoveries in Photosynthesis. Springer, Dordrecht]. This paper covers the physical and chemical events starting with the absorption of a quantum of light by a pigment molecule to the conversion of the radiation energy into the stable chemical forms of the reducing power and of ATP. It describes the work done on the structure, function and regulation of the photosynthetic apparatus in higher plants, unicellular algae and in photosynthetic bacteria. Phenomena such as photoinhibition and the protection from it are also included. Research in biophysics of photosynthesis in Padova (Italy) is discussed by G.M. Giacometti and G. Giacometti (2006).

Abstract: Photosystem II of higher plants is a multisubunit transmembrane complex composed of a core moiety and an extensive peripheral antenna system. The number of antenna polypeptides per core complex is modulated following environmental conditions in order to optimize photosynthetic performance. In this study, we used a barley (Hordeum vulgare) mutant, viridis zb63, which lacks photosystem I, to mimic extreme and chronic overexcitation of photosystem II. The mutation was shown to reduce the photosystem II antenna to a minimal size of about 100 chlorophylls per photosystem II reaction centre, which was not further reducible. The minimal photosystem II unit was analysed by biochemical methods and by electron microscopy, and found to consist of a dimeric photosystem II reaction centre core surrounded by monomeric Lhcb4 (chlorophyll protein 29), Lhcb5 (chlorophyll protein 26) and trimeric light-harvesting complex II antenna proteins. This minimal photosystem II unit forms arrays in vivo, possibly to increase the efficiency of energy distribution and provide photoprotection. In wild-type plants, an additional antenna protein, chlorophyll protein 24 (Lhcb6), which is not expressed in viridis zb63, is proposed to associate to this minimal unit and stabilize larger antenna systems when needed. The analysis of the mutant also revealed the presence of two distinct signalling pathways activated by excess light absorbed by photosystem II: one, dependent on the redox state of the electron transport chain, is involved in the regulation of antenna size, and the second, more directly linked to the level of photoinhibitory stress perceived by the cell, participates in regulating carotenoid biosynthesis.

Abstract: The first dedicated step in plant xanthophyll biosynthesis is carotenoid hydroxylation. In Arabidopsis thaliana, this reaction is performed by both heme (LUT1 and LUT5) and non-heme (CHY1 and CHY2) hydroxylases. No mutant completely abolishing alpha- or beta-carotene hydroxylation has been described to date. We constructed double and triple mutant combinations in CHY1, CHY2, LUT1, LUT5 and LUT2 (lycopene epsilon-cyclase). In chy1chy2lut2, 80% of leaf carotenoids is represented by beta-carotene. In chy1chy2lut5, beta-carotene hydroxylation is completely abolished, while hydroxylation of the beta-ring of alpha-carotene is still observed. The data are consistent with a role of LUT5 in beta-ring hydroxylation, and with the existence of an additional hydroxylase, acting on the beta-ring of alpha-, but not beta-carotene.

Abstract: BACKGROUND: Lutein is the most abundant xanthophyll in the photosynthetic apparatus of higher plants. It binds to site L1 of all Lhc proteins, whose occupancy is indispensable for protein folding and quenching chlorophyll triplets. Thus, the lack of a visible phenotype in mutants lacking lutein has been surprising. RESULTS: We have re-assessed the lut2.1 phenotypes through biochemical and spectroscopic methods. Lhc proteins from the lut2.1 mutant compensate the lack of lutein by binding violaxanthin in sites L1 and L2. This substitution reduces the capacity for regulatory mechanisms such as NPQ, reduces antenna size, induces the compensatory synthesis of Antheraxanthin + Zeaxanthin, and prevents the trimerization of LHCII complexes. In vitro reconstitution shows that the lack of lutein per se is sufficient to prevent trimerization. lut2.1 showed a reduced capacity for state I-state II transitions, a selective degradation of Lhcb1 and 2, and a higher level of photodamage in high light and/or low temperature, suggesting that violaxanthin cannot fully restore chlorophyll triplet quenching. In vitro photobleaching experiments and time-resolved spectroscopy of carotenoid triplet formation confirmed this hypothesis. The npq1lut2.1 double mutant, lacking both zeaxanthin and lutein, is highly susceptible to light stress. CONCLUSION: Lutein has the specific property of quenching harmful 3Chl* by binding at site L1 of the major LHCII complex and of other Lhc proteins of plants, thus preventing ROS formation. Substitution of lutein by violaxanthin decreases the efficiency of 3Chl* quenching and causes higher ROS yield. The phenotype of lut2.1 mutant in low light is weak only because rescuing mechanisms of photoprotection, namely zeaxanthin synthesis, compensate for the ROS production. We conclude that zeaxanthin is effective in photoprotection of plants lacking lutein due to the multiple effects of zeaxanthin in photoprotection, including ROS scavenging and direct quenching of Chl fluorescence by binding to the L2 allosteric site of Lhc proteins.

Abstract: Unlike plants, Chlamydomonas reinhardtii shows a restricted ability to develop nonphotochemical quenching upon illumination. Most of this limited quenching is due to state transitions instead of DeltapH-driven high-energy state quenching, qE. The latter could only be observed when the ability of the cells to perform photosynthesis was impaired, either by lowering temperature to approximately 0 degrees C or in mutants lacking RubisCO activity. Two main features were identified that account for the low level of qE in Chlamydomonas. On one hand, the electrochemical proton gradient generated upon illumination is apparently not sufficient to promote fluorescence quenching. On the other hand, the capacity to transduce the presence of a DeltapH into a quenching response is also intrinsically decreased in this alga, when compared to plants. The possible mechanism leading to these differences is discussed.

Abstract: Previously, we have shown that barley (Hordeum vulgare) plants carrying a mutation preventing chloroplast development are completely frost susceptible as well as impaired in the expression of several cold-regulated genes. Here we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes. First, by comparing control wild type against cold-hardened wild-type plants 2,735 probe sets with statistically significant changes (P = 0.05; > or = 2-fold change) were identified. Expression of these wild-type cold-regulated genes was then analyzed in control and cold-hardened mutants. Only about 11% of the genes cold regulated in wild type were regulated to a similar extent in all genotypes (chloroplast-independent cold-regulated genes); this class includes many genes known to be under C-repeat binding factor control. C-repeat binding factor genes were also equally induced in mutants and wild-type plants. About 67% of wild-type cold-regulated genes were not regulated by cold in any mutant (chloroplast-dependent cold-regulated genes). We found that the lack of cold regulation in the mutants is due to the presence of signaling pathway(s) normally cold activated in wild type but constitutively active in the mutants, as well as to the disruption of low-temperature signaling pathway(s) due to the absence of active chloroplasts. We also found that photooxidative stress signaling pathway is constitutively active in the mutants. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold.

Abstract: The selectively red excited emission spectrum, at room temperature, of the in vitro reconstituted Lhca4, has a pronounced non-equilibrium distribution, leading to enhanced emission from the directly excited low-energy pigments. Two different emitting forms (or states), with maximal emission at 713 and 735nm (F713 and F735) and unusual spectral properties, have been identified. Both high-energy states are populated when selective excitation is into the F735 state and the fluorescence anisotropy spectrum attains the value of 0.3 in the wavelength region where both emission states are present. This indicates that the two states are on the same Lhca4 complex and have transition dipoles with similar orientation.

Abstract: The red-most fluorescence emission of photosystem I (733 nm at 4 K) is associated with the Lhca4 subunit of the antenna complex. It has been proposed that this unique spectral feature originates from the low energy absorption band of an excitonic interaction involving chlorophyll A5 and a second chlorophyll a molecule, probably B5 (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Because of the short distances between chromophores in Lhc proteins, the possibility that other pigments are involved in the red-shifted spectral forms could not be ruled out. In this study, we have analyzed the pigment-pigment interactions between nearest neighboring chromophores in Lhca4. This was done by deleting individual chlorophyll binding sites by mutagenesis, and analyzing the changes in the spectroscopic properties of recombinant proteins refolded in vitro. The red-shifted (733 nm) fluorescence peak, the major target of this analysis, was lost upon mutations affecting sites A4, A5, and B5 and was modified by mutating site B6. In agreement with the shorter distance between chlorophylls A5 and B5 (7.9 A) versus A4 and A5 (12.2 A) in Lhca4 (Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635), we conclude that the low energy spectral form originates from an interaction involving pigments in sites A5 and B5. Mutation at site B6, although inducing a 15-nm blue-shift of the emission peak, maintains the red-shifted emission. This implies that chromophores responsible for the interaction are conserved and suggests a modification in the pigment organization. Besides the A5-B5 pair, evidence for additional pigment-pigment interactions between chlorophylls in sites B3-A3 and B6-A6 was obtained. However, these features do not affect the red-most spectral form responsible for the 733-nm fluorescence emission band.

Abstract: The regulation of light harvesting in higher plant photosynthesis, defined as stress-dependent modulation of the ratio of energy transfer to the reaction centers versus heat dissipation, was studied by means of carotenoid biosynthesis mutants and recombinant light harvesting complexes (LHCs) with modified chromophore binding. The npq2 mutant of Arabidopsis thaliana, blocked in the biosynthesis of violaxanthin and thus accumulating zeaxanthin, was shown to have a lower fluorescence yield of chlorophyll in vivo and, correspondingly, a higher level of energy dissipation, with respect to the wild-type strain and npq1 mutant, the latter of which is incapable of zeaxanthin accumulation. Experiments on purified thylakoid membranes from all three mutants showed that the major source of the difference between the npq2 and wild-type preparations was a change in pigment to protein interactions, which can explain the lower chlorophyll fluorescence yield in the npq2 samples. Analysis of the xanthophyll binding LHC proteins showed that the Lhcb5 photosystem II subunit (also called CP26) undergoes a change in its pI upon binding of zeaxanthin. The same effect was observed in wild-type CP26 upon treatment that leads to the accumulation of zeaxanthin in the membrane and was interpreted as the consequence of a conformational change. This hypothesis was confirmed by the analysis of two recombinant proteins obtained by overexpression of the Lhcb5 apoprotein in Escherichia coli and reconstitution in vitro with either violaxanthin or zeaxanthin. The V and Z containing pigment-protein complexes obtained by this procedure showed different pIs and high and low fluorescence yields, respectively. These results confirm that LHC proteins exist in multiple conformations, an idea suggested by previous spectroscopic measurements (Moya et al., 2001), and imply that the switch between the different LHC protein conformations is activated by the binding of zeaxanthin to the allosteric site L2. The results suggest that the quenching process induced by the accumulation of zeaxanthin contributes to qI, a component of NPQ whose origin was previously poorly understood.

Abstract: In higher plants many different genes encode Lhcb proteins that belong to a highly conserved protein family. Evolutionary conservation of this genetic redundancy suggests that individual gene products play different roles in light harvesting and photoprotection depending on environmental conditions. We have tested the hypothesis that expression/accumulation of individual light harvesting complex (Lhc) proteins depends on plant growth conditions. Zea mays plants were grown in different temperature (13 degrees C vs. 24 degrees C) and light (high vs. low) conditions. The thylakoid membranes were isolated and fractionated by sucrose gradient and the protein content of the different bands was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences were found in the accumulation of both the major light harvesting complex of photosystem II (LHCII) complexes and the minor antenna chlorophyll proteins CP29, CP26 and CP24. In particular, temperature seems to play a major role in driving the expression/accumulation of the different proteins: the LHCII/minor antenna ratio increases with decreasing temperature. The pigment composition and the spectroscopic properties of LHCII complexes isolated from low temperature grown plants are significantly different from those of LHCII purified from high temperature grown plants. Two-dimensional maps show that different LHCII proteins are accumulated at different levels depending on growth conditions. Moreover the low temperature/high light grown plants show an increased value of nonphotochemical quenching. These results suggest a specific role of different LHCII complexes in the organization of the potosystem II and photoprotection.

Abstract: In this study, triplet quenching, the major photoprotection mechanism in antenna proteins, has been studied in the light-harvesting complex of photosystem I (LHC-I). The ability of carotenoids bound to LHC-I subunit Lhca4, which is characterized by the presence of the red-most absorption components at wavelength >700 nm, to protect the system through quenching of the chlorophyll triplet states, has been probed, by analyzing the induction of carotenoid triplet formation. We have investigated this process at low temperature, when the funneling of the excitation toward the low-lying excited states of the Chls is stronger, by means of optically detected magnetic resonance (ODMR), which is well-suited for investigation of triplet states in photosynthetic systems. The high selectivity and sensitivity of the technique has made it possible to point out the presence of specific interactions between carotenoids forming the triplet states and specific chlorophylls characterized by red-shifted absorption, by detection of the microwave-induced Triplet minus Singlet (T-S) spectra. The effect of the red forms on the efficiency of triplet quenching was specifically probed by using the Asn47His mutant, in which the red forms have been selectively abolished (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Lack of the red forms yields into a reduced efficiency of the triplet quenching in LHC-I thus suggesting that the "red Chls" play a role in enhancing triplet quenching in LHC-I and, possibly, in the whole photosystem I.

Abstract: The kinetics of response to strong light have been examined in deeply shaded leaves of the tropical tree legume (Inga sp.) which have extraordinarily high levels of the alpha-xanthophyll lutein-epoxide that are co-located in pigment-protein complexes of the photosynthetic apparatus with the beta-xanthophyll violaxanthin. As in other species, rapidly reversible photoprotection (measured as non-photochemical chlorophyll fluorescence quenching) is initiated within the time frame of sun-flecks (minutes), before detectable conversion of violaxanthin to antheraxanthin or zeaxanthin. Photoprotection is stabilized within hours of exposure to strong light by simultaneously engaging the reversible violaxanthin cycle and a slowly reversible conversion of lutein-epoxide to lutein. It is proposed that this lutein 'locks in' a primary mechanism of photoprotection during photoacclimation in this species, converting efficient light-harvesting antennae of the shade plant into potential excitation dissipating centres. It is hypothesized that lutein occupies sites L2 and V1 in light-harvesting chlorophyll protein complexes of photosystem II, facilitating enhanced photoprotection through the superior singlet and/or triplet chlorophyll quenching capacity of lutein.

Abstract: Light-harvesting complex I (LHCI), which serves as a peripheral antenna for photosystem I (PSI) in green plants, consists mainly of four polypeptides, Lhca1-4. We report room temperature emission properties of individual reconstituted monomeric Lhca proteins (Lhca1, -2, -3, and -4) and dimeric Lhca1/4, performed by steady-state and time-resolved fluorescence techniques. The emission quantum yields of the samples are approximately 0.12, 0.085, 0.081, 0.041, and 0.063 for Lhca1, -2, -3, -4, and the -1/4 dimer, respectively, which is considerably lower than the value of 0.22 found for light-harvesting complex II (LHCII), the main peripheral antenna complex of photosystem II in green plants. The decay components of LHCI proteins can be divided in two categories: Lhca1 and Lhca3 have decay times of 1.1-1.6 ns and 3.3-3.6 ns, and Lhca2 and Lhca4 have decay times of 0.7-0.9 ns and 3.1-3.2 ns. These categories seem to correlate with the pigment composition of the samples. All decay times are faster than that observed previously for LHCII. When the absolute emission yields and the lifetimes of the Lhca samples are combined, the overall emission properties of the individual Lhca proteins are expressed in terms of their emitting dipole moment strength. In the samples without extreme red states, that is, Lhca1 and Lhca2, the emitting dipole moment has a value close to unity (relative to monomeric chlorophyll in acetone), which is similar to that for LHCII, whereas, in the samples with the red-most state (F-730), that is, Lhca3, -4, and the -1/4 dimer, the emitting dipole moment has a value less than unity (0.6-0.8), which can be explained by mixing the red-most (exciton) state with a dark charge-transfer state, as suggested in previous PSI red pigment studies. In addition, we find a lifetime component of approximately 50-150 ps in all red-pigment-containing samples, which cannot be due to "slow" energy transfer, but is instead assigned to an unrelaxed state of the pigment-protein, which, on this time-scale, is converted into the final emitting state.

Abstract: We report on the association of the antenna system to the reaction center in Photosystem I. Biochemical analysis of mutants depleted in antenna polypeptides showed that the binding of the antenna moiety is strongly cooperative. The minimal building block for the antenna system was shown to be a dimer. Specific protein-protein interactions play an important role in antenna association, and the gap pigments, bound at the interface between core and antenna, are proposed to mediate these interactions Gap pigments have been characterized by comparing the spectra of the Photosystem I to those of the isolated antenna and core components. CD spectroscopy showed that they are involved in pigment-pigment interactions, supporting their relevance in energy transfer from antenna to the reaction center. Moreover, gap pigments contribute to the red-shifted emission forms of Photosystem I antenna. When compared with Photosystem II, the association of peripheral antenna complexes in PSI appears to be more stable, but far less flexible and functional implications are discussed.

Abstract: Formate and phosphate affect substantially the rate of tyrosine D (TyrD) oxidation and the stability of the radical TyrD* in Photosystem II [Hienerwadel R, Boussac A, Breton J and Berthomieu C (1996) Biochemistry 35: 15447-15460]. This observation prompted us to analyze the influence of formate and phosphate on the environment of TyrD using FTIR spectroscopy. The nu (CO) IR mode of TyrD* at 1503 cm-1 remains unchanged whatever the buffer used at pH 6 and whether formate is present or not in the sample. Similarly, the main IR mode of reduced TyrD remains at approximately 1250 cm-1 in all tested conditions. We thus conclude that formate does not modify the hydrogen-bonded interactions of TyrD and TyrD* with neighbouring D2His189 and D2Gln164. In the TyrD-state, an IR mode of formate significantly different from that observed in solution, is detected using 13C-formate, showing that formate forms a strong electrostatic interaction within PS II. The presence of formate affects also IR bands that may be assigned to an arginine side chain. Upon TyrD* formation, formate does not protonate but its binding interaction weakens. A proton uptake by Mes or phosphate buffer is detected, which is not observed when BisTris is used as a buffer. In these latter conditions, IR bands characteristic of the protonation of a carboxylate group of the protein are detected instead. The present IR data and the recent structural model of the TyrD environment proposed by Ferreira KN, Iverson TM, Maghlaoui K, Barber J and Iwata S [(2004) Science 303: 1831-1838], suggest that the proton released upon TyrD* formation is shared within a hydrogen bonding network including D2Arg294, and CP47Glu364 and that perturbation of this network by formate - possibly binding near D2Arg294 - substantially affects the properties of TyrD.

Abstract: EET in reconstituted Lhca4, a peripheral light-harvesting complex from Photosystem I of Arabidopsis thaliana, containing 10 chlorophylls and 2 carotenoids, was studied at room temperature by femtosecond transient absorption spectroscopy. Two spectral forms of Lut were observed in the sites L1 and L2, characterized by significantly different interactions with nearby chlorophyll a molecules. A favorable interpretation of these differences is that the efficiency of EET to Chls is about two times lower from the "blue" Lut in the site L1 than from the "red" Lut in the site L2 due to fast IC in the former case. A major part of the energy absorbed by the "red" Lut, approximately 60%-70%, is transferred to Chls on a sub-100-fs timescale from the state S(2) but, in addition, minor EET from the hot S(1) state within 400-500 fs is also observed. EET from the S(1) state to chlorophylls occurs also within 2-3 ps and is ascribed to Vio and/or "blue" Lut. EET from Chl b to Chl a is biphasic and characterized by time constants of approximately 300 fs and 3.0 ps. These rates are ascribed to EET from Chl b spectral forms absorbing at approximately 644 nm and approximately 650 nm, respectively. About 25% of the excited Chls a decays very fast-within approximately 15 ps. This decay is proposed to be related to the presence of the interacting Chls A5 and B5 located next to the carotenoid in the site L2 and may imply some photoprotective role for Lhca4 in the photosystem I super-complex.

Abstract: The electric-field induced absorption changes (Stark effect) of reconstituted light-harvesting complex II (LHCII) in different oligomerisation states-monomers and trimers-with different xanthophyll content have been probed at 77 K. The Stark spectra of the reconstituted control samples, containing the xanthophylls lutein and neoxanthin, are very similar to previously reported spectra of native LHCII. Reconstituted LHCII, containing lutein but no neoxanthin, shows a similar electrooptical response in the Chl a region, but the Stark signal of Chl b around 650 nm amounts to at most approximately 25% of that of the control samples. We conclude that neoxanthin strongly modifies the electronic states of the nearby Chl b molecules causing a large electrooptical response at 650 nm stemming from one or more Chls b in the control samples. Ambiguities about the assignment of several bands in the Soret region [Biochim. Biophys. Acta 1605 (2003) 83] are resolved and the striking difference in electric field response between the two lutein molecules is confirmed. The Stark effect in the carotenoid spectral region in both control and neoxanthin-deficient samples is almost identical, showing that the neoxanthin Stark signal is small and much less intense than the lutein Stark signal.

Abstract: In green plants, the xanthophyll carotenoid zeaxanthin is synthesized transiently under conditions of excess light energy and participates in photoprotection. In the Arabidopsis lut2 npq2 double mutant, all xanthophylls were replaced constitutively by zeaxanthin, the only xanthophyll whose synthesis was not impaired. The relative proportions of the different chlorophyll antenna proteins were strongly affected with respect to the wild-type strain. The major antenna, LHCII, did not form trimers, and its abundance was strongly reduced as was CP26, albeit to a lesser extent. In contrast, CP29, CP24, LHCI proteins, and the PSI and PSII core complexes did not undergo major changes. PSII-LHCII supercomplexes were not detectable while the PSI-LHCI supercomplex remained unaffected. The effect of zeaxanthin accumulation on the stability of the different Lhc proteins was uneven: the LHCII proteins from lut2 npq2 had a lower melting temperature as compared with the wild-type complex while LHCI showed increased resistance to heat denaturation. Consistent with the loss of LHCII, light-state 1 to state 2 transitions were suppressed, the photochemical efficiency in limiting light was reduced and photosynthesis was saturated at higher light intensities in lut2 npq2 leaves, resulting in a photosynthetic phenotype resembling that of high light-acclimated leaves. Zeaxanthin functioned in vivo as a light-harvesting accessory pigment in lut2 npq2 chlorophyll antennae. As a whole, the in vivo data are consistent with the results obtained by using recombinant Lhc proteins reconstituted in vitro with purified zeaxanthin. While PSII photoinhibition was similar in wild type and lut2 npq2 exposed to high light at low temperature, the double mutant was much more resistant to photooxidative stress and lipid peroxidation than the wild type. The latter observation is consistent with an antioxidant and lipid protective role of zeaxanthin in vivo.

Abstract: The major antenna complex of higher-plant photosynthesis, LHCII, is composed by the products of three genes, namely, Lhcb1-2-3. In this paper, the biochemical and spectroscopic properties of each of the three gene products were investigated. The three complexes were obtained by overexpression of the apoproteins in bacteria and refolding in vitro with purified pigments, thus allowing detection of differences in the structure/function of the pigment-binding gene products. The analyses showed that Lhcb1 and Lhcb2 complexes have similar pigment binding properties, although not identical, while Lhcb3 is clearly different with respect to both pigment binding and spectral properties and cannot produce homotrimers in vitro. Heterotrimers containing Lhcb3 together with Lhcb1 and/or -2 proteins were obtained upon assembly with Lhcb proteins purified from thylakoids. The major functional characteristics of Lhcb3 with respect to Lhcb1 and -2 consisted in (i) a red-shift of one specific chlorophyll a chromophore, strongly affecting the red-most region of the absorption spectrum and (ii) a different specificity for xanthophylls binding to sites L2 and N1. These properties make Lhcb3 a relative sink for excitation energy in isolated heterotrimers with Lhcb1 + Lhcb2, and potentially, a preferential site of regulation of the antenna function in excess light conditions.

Abstract: A transglutaminase (TGase; EC 2.3.2.13) activity, which shared many properties with the TGase activity of the Helianthus tuberosus chloroplast, was observed in the Zea mays L. chloroplast and in its fractions. This activity was found to be prevalent in thylakoids; bis-(glutamyl) spermidine and bis-(glutamyl) putrescine were the main polyamine conjugates formed. Light stimulated the endogenous thylakoid activity. Putrescine, spermidine and spermine were conjugated to the isolated light-harvesting complex of photosystem II (LHCII) with different degrees of efficiency, spermine being the polyamine most efficiently conjugated. A TGase with a light-sensitive activity was identified in the photosystem II-enriched fraction. Its partial purification on a sucrose gradient allowed the separation of a 39-kDa band, which was immunorecognised by two anti-TGase antibodies (Ab-3 and rat prostatic gland-TGase). Both a colorimetric and a radiometric assay for TGase activity, the former carried out in the presence of biotinylated cadaverine and the latter in the presence of polyamines labelled with radioactive isotopes and resulting in the isolation of glutamyl-polyamines, further confirmed that the thylakoid enzyme is indeed a calcium-dependent transglutaminase (Thyl-TGase). At variance with guinea pig liver and erythrocyte TGases, which are insensitive to light, the activity of the thylakoid transglutaminase is affected by light. Moreover, this enzyme, when tested with purified LHCII as substrate, catalysed the production of mono- and bis-glutamyl-polyamines in equal amounts, whereas the 'animal' enzymes produced mainly mono-derivatives. Herein, it is discussed whether this light sensitivity is due to the enzyme or the substrate.

Abstract: The biochemical, biophysical, and physiological properties of the PsbS protein were studied in relation to mutations of two symmetry-related, lumen-exposed glutamate residues, Glu-122 and Glu-226. These two glutamates are targets for protonation during lumen acidification in excess light. Mutation of PsbS did not affect xanthophyll cycle pigment conversion or pool size. Plants containing PsbS mutations of both glutamates did not have any rapidly inducible nonphotochemical quenching (qE) and had similar chlorophyll fluorescence lifetime components as npq4-1, a psbS deletion mutant. The double mutant also lacked a characteristic leaf absorbance change at 535 nm (DeltaA535), and PsbS from these plants did not bind dicyclohexylcarbodiimide (DCCD), a known inhibitor of qE. Mutation of only one of the glutamates had intermediate effects on qE, chlorophyll fluorescence lifetime component amplitudes, DCCD binding, and DeltaA535. Little if any differences were observed comparing the two single mutants, suggesting that the glutamates are chemically and functionally equivalent. Based on these results a bifacial model for the functional interaction of PsbS with photosystem II is proposed. Furthermore, based on the extent of qE inhibition in the mutants, photochemical and nonphotochemical quenching processes of photosystem II were associated with distinct chlorophyll fluorescence life-time distribution components.

Abstract: Illumination of dark-adapted barley plants with low light transiently induced a large nonphotochemical quenching of chlorophyll fluorescence. This reaction was identified as a form of high-energy-state quenching. Its appearance was not accompanied by zeaxanthin synthesis but was associated with a reversible inactivation of a fraction of photosystem II (PSII) centers. Both the fluorescence quenching and PSII inactivation relaxed in parallel with the activation of the Calvin cycle. We interpret the induction of this phenomenon as due to the generation of a quenched state in the PSII core complex. This reaction is probably caused by the transient overacidification of the thylakoid lumen, whereas its dissipation results from the relaxation of both the pH gradient across the thylakoid membrane and redox pressure upon activation of carbon fixation. At saturating light intensities, inactivation of PSII was still observed at the onset of illumination, although its recovery did not result in dissipation of high-energy quenching, which presents typical characteristics of an antenna-associated quenching at steady state. Reaction-center quenching seems therefore to be a common transient feature during illumination, being replaced by other phenomena (photochemical or antenna quenching and photoinhibition), depending on the balance between light and carbon fixation fluxes.

Abstract: Photosystem I of higher plants is characterized by red-shifted spectral forms deriving from chlorophyll chromophores. Each of the four Lhca1 to -4 subunits exhibits a specific fluorescence emission spectrum, peaking at 688, 701, 725, and 733 nm, respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in Lhca3 and Lhca4, whereas the basis for the fluorescence emission at 701 nm in Lhca2 is not yet clear. We report a detailed characterization of the Lhca2 subunit using molecular biology, biochemistry, and spectroscopy and show that the 701-nm emission form originates from a broad absorption band at 690 nm. Spectroscopy on recombinant mutant proteins assesses that this band represents the low energy form of an excitonic interaction involving two chlorophyll a molecules bound to sites A5 and B5, the same protein domains previously identified for Lhca3 and Lhca4. The resulting emission is, however, substantially shifted to higher energies. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (Ben Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635). We suggest that, within the Lhca subfamily, spectroscopic properties of chromophores are modulated by the strength of the excitonic coupling between the chromophores A5 and B5, thus yielding fluorescence emission spanning a large wavelength interval. It is concluded that the interchromophore distance rather than the transition energy of the individual chromophores or the orientation of transition vectors represents the critical factor in determining the excitonic coupling in Lhca pigment-protein complexes.

Abstract: In this study, two gene products (Lhca2 and Lhca3), encoding higher plants (Arabidopsis thaliana) Photosystem I antenna complexes, were overexpressed in bacteria and reconstituted in vitro with purified chloroplast pigments. The chlorophyll-xanthophyll proteins thus obtained were characterized by biochemical and spectroscopic methods. Both complexes were shown to bind 10 chlorophyll (a and b) molecules per polypeptide, Lhca2 having higher chlorophyll b content as compared to Lhca3. The two proteins differed for the number of carotenoid binding sites: two and three for Lhca2 and Lhca3, respectively. beta-carotene was specifically bound to Lhca3 in addition to the xanthophylls violaxanthin and lutein, indicating a peculiar structure of carotenoid binding sites in this protein since it is the only one so far identified with the ability of binding beta-carotene. Analysis of the spectroscopic properties of the two pigment proteins showed the presence of low energy absorption forms (red forms) in both complexes, albeit with different energies and amplitudes. The fluorescence emission maximum at 77 K of Lhca2 was found at 701 nm, while in Lhca3 the major emission was at 725 nm. Reconstitution of Lhca3 without Chl b reveals that Chl b is not involved in originating the low energy absorption forms of this complex. The present data are discussed in comparison to the properties of the recombinant Lhca1 and Lhca4 complexes and of the native LHCI preparation, previously analyzed, thus showing a comprehensive description of the gene products composing the Photosystem I light harvesting system of A. thaliana.

Abstract: The energy transfer processes between carotenoids and Chls have been studied by femtosecond transient absorption in the CP29-WT complex, which contains only two carotenoids per polypeptide located in the L1 and L2 sites, and in the CP29-E166V mutant in which only the L1 site is occupied. The comparison of these two samples allowed us to discriminate between the energy transfer pathways from the two carotenoid binding sites and thus to obtain detailed information on the Chl organization in CP29 and to assign the acceptor chlorophylls. For both samples, the main transfer occurs from the S(2) state of the carotenoid. In the case of the L1 site the energy acceptor is the Chl a 680 nm (A2), whereas the Chl a 675 nm (A4-A5) and the Chl b 652 nm (B6) are the acceptors from the xanthophyll in the L2 site. These transfers occur with lifetimes of 80-130 fs. Two additional transfers are observed with 700-fs and 8- to 20-ps lifetimes. Both these transfers originate from the carotenoid S(1) states. The faster lifetime is due to energy transfer from a vibrationally unrelaxed S(1) state, whereas the 8- to 20-ps component is due to a transfer from the S(1,0) state of violaxanthin and/or neoxanthin located in site L2. A comparison between the carotenoid to Chl energy transfer pathways in CP29 and LHCII is presented and differences in the structural organization in the two complexes are discussed.

Abstract: The binding sites for xanthophylls in the CP29 antenna protein of higher plant Photosystem II have been investigated using recombinant proteins refolded in vitro. Despite the presence of three xanthophyll species CP29 binds two carotenoids per polypeptide. The localization of neoxanthin was studied producing a chimeric protein constructed by swapping the C-helix domain from CP29 to LHCII. The resulting holoprotein did not bind neoxanthin, confirming that the N1 site is not present in CP29. Neoxanthin in CP29 was, instead, bound to the L2 site, which is thus shown to have a wider specificity with respect to the homologous site L2 in LHCII. Lutein was found in the L1 site of CP29. For each site the selectivity for individual xanthophyll species was studied as well as its role in protein stabilization, energy transfer, and photoprotection. Putative xanthophyll binding sequences, identified by primary structure analysis as a stretch of hydrophobic residues including an acidic term, were analyzed by site-directed mutagenesis or, in one case, by deleting the entire sequence. The mutant proteins were unaffected in their xanthophyll composition, thus suggesting that the target motifs had little influence in determining xanthophyll binding, whereas hydrophobic sequences in the membrane-spanning helices are important.

Abstract: The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.

Abstract: The energy transfer processes between Chls b and Chls a have been studied in the minor antenna complex CP29 by femtosecond transient absorption spectroscopy. Two samples were analyzed: the native CP29, purified from higher plants, and the recombinant one, reconstituted in vitro with the full pigment complement. The measurements indicate that the transfer kinetics in the two samples are virtually identical, confirming that the reconstituted CP29 has the same spectroscopic properties as the native one. In particular, three lifetimes (150 fs, 1.2 ps, and 5-6 ps) were identified for Chl b-652 nm to Chl a energy transfer and at least one for Chl b-640 nm (600-800 fs). Considering that the complexes bind two Chls b per polypeptide, the observation of more than two lifetimes for the Chl b to Chl a energy transfer, in both samples, clearly indicates the presence of the so-called mixed Chl binding sites--sites which are not selective for Chl a or Chl b, but can accommodate either species. The kinetic components and spectra are assigned to specific Chl binding sites in the complex, which provides further information on the structural organization.

Abstract: The lutein-epoxide cycle (Lx cycle) is an auxiliary xanthophyll cycle known to operate only in some higher-plant species. It occurs in parallel with the common violaxanthin cycle (V cycle) and involves the same epoxidation and de-epoxidation reactions as in the V cycle. In this study, the occurrence of the Lx cycle was investigated in the two major families of mistletoe, the Loranthaceae and the Viscaceae. In an attempt to find the limiting factor(s) for the occurrence of the Lx cycle, pigment profiles of mistletoes with and without the Lx cycle were compared. The availability of lutein as a substrate for the zeaxanthin epoxidase appeared not to be critical. This was supported by the absence of the Lx cycle in the transgenic Arabidopsis plant lutOE, in which synthesis of lutein was increased at the expense of V by overexpression of epsilon-cyclase, a key enzyme for lutein synthesis. Furthermore, analysis of pigment distribution within the mistletoe thylakoids excluded the possibility of different localizations for the Lx- and V-cycle pigments. From these findings, together with previous reports on the substrate specificity of the two enzymes in the V cycle, we propose that mutation to zeaxanthin epoxidase could have resulted in altered regulation and/or substrate specificity of the enzyme that gave rise to the parallel operation of two xanthophyll cycles in some plants. The distribution pattern of Lx in the mistletoe phylogeny inferred from 18S rRNA gene sequences also suggested that the occurrence of the Lx cycle is determined genetically. Possible molecular evolutionary processes that may have led to the operation of the Lx cycle in some mistletoes are discussed.

Abstract: Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at approximately 700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.

Abstract: The light harvesting complex Lhca1, one of the four gene products comprising the photosystem I antenna system, has been analyzed by site-directed mutagenesis with the aim of determining the chromophore(s) responsible for its long wavelength chlorophyll spectral form, a specific characteristic of the LHCI antenna complex. A family of mutant proteins, each carrying a mutation at a single chlorophyll-binding residue, was obtained and characterized by biochemical and spectroscopic methods. A map of the chromophores bound to each of the 10 chlorophyll-binding sites was drawn, and the energy levels of the Q(y) transition were determined in most cases. When compared with Lhcb proteins previously analyzed, Lhca1 is characterized by stronger interactions between individual chromophores as detected by both biochemical and spectroscopic methods; most mutations, although targeted to a single residue, lead to the loss of more than one chromophore and of conservative CD signals typical of chlorophyll-chlorophyll interactions. The lower energy absorption form (686 nm at 100K, 688 nm at room temperature), which is responsible for the red-shifted emission components at 690 and 701 nm, typical of Lhca1, is associated with a chlorophyll a/chlorophyll a excitonic interaction originating from a pigment cluster localized in the protein domain situated between helix C and the helix A/helix B cross. This cluster includes chlorophylls bound to sites A5-B5-B6 and a xanthophyll bound to site L2.

Abstract: The room temperature absorption bandshape for the Q transition region of chlorophyll a is calculated using the vibrational frequency modes and Franck-Condon (FC) factors obtained by line-narrowing spectroscopies of chlorophyll a in a glassy (Rebane and Avarmaa, Chem. Phys. 1982; 68:191-200) and in a native environment (Gillie et al., J. Phys. Chem. 1989; 93:1620-1627) at low temperatures. The calculated bandshapes are compared with the absorption spectra of chlorophyll a measured in two different solvents and with that obtained in vivo by a mutational analysis of a chlorophyll-protein complex. It is demonstrated that the measured distributions of FC factors can account for the absorption bandshape of chlorophyll a in a hexacoordinated state, whereas, when pentacoordinated, reduced FC coupling for vibrational frequencies in the range 540-850 cm(-1) occurs. The FC factor distribution for pentacoordinated chlorophyll also describes the native chlorophyll a spectrum but, in this case, either a low-frequency mode (nu < 200 cm(-1)) must be added or else the 262-cm(-1) mode must increase in coupling by about one order of magnitude to describe the skewness of the main absorption bandshape.

Abstract: The biochemical properties of PsbS protein, a nuclear-encoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyll-binding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca(2+)-binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [(14)C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH.

Abstract: Three plant xanthophylls are components of the xanthophyll cycle in which, upon exposure of leaves to high light, the enzyme violaxanthin de-epoxidase (VDE) transforms violaxanthin into zeaxanthin via the intermediate antheraxanthin. Previous work () showed that xanthophylls are bound to Lhc proteins and that substitution of violaxanthin with zeaxanthin induces conformational changes and fluorescence quenching by thermal dissipation. We have analyzed the efficiency of different Lhc proteins to exchange violaxanthin with zeaxanthin both in vivo and in vitro. Light stress of Zea mays leaves activates VDE, and the newly formed zeaxanthin is found primarily in CP26 and CP24, whereas other Lhc proteins show a lower exchange capacity. The de-epoxidation system has been reconstituted in vitro by using recombinant Lhc proteins, recombinant VDE, and monogalactosyl diacylglycerol (MGDG) to determine the intrinsic capacity for violaxanthin-to-zeaxanthin exchange of individual Lhc gene products. Again, CP26 was the most efficient in xanthophyll exchange. Biochemical and spectroscopic analysis of individual Lhc proteins after de-epoxidation in vitro showed that xanthophyll exchange occurs at the L2-binding site. Xanthophyll exchange depends on low pH, implying that access to the binding site is controlled by a conformational change via lumenal pH. These findings suggest that the xanthophyll cycle participates in a signal transduction system acting in the modulation of light harvesting versus thermal dissipation in the antenna system of higher plants.

Abstract: The carotenoid species lutein, violaxanthin, and zeaxanthin are crucial in the xanthophyll-dependent nonphotochemical quenching occurring in photosynthetic systems of higher plants, since they are involved in dissipation of excess energy and thus protect the photosynthetic machinery from irreversible inhibition. Nonetheless, important properties of the xanthophyll cycle carotenoids, such as the energy of their S(1) electronic states, are difficult to study and were only recently determined in organic solvents [Polívka, T. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 4914. Frank, H. A. (2000) Biochemistry 39, 2831]. In the present study, we have determined the S(1) energies of three carotenoid species, violaxanthin, lutein, and zeaxanthin, in their LHCII (peripheral light-harvesting complex of photosystem II) protein environment by constructing recombinant Lhcb1 (Lhc = light-harvesting complex) proteins containing single carotenoid species. Within experimental error the S(1) energy is the same for all three carotenoids in the monomeric LHCII, 13,900 +/- 300 cm(-1) (720 +/- 15 nm), thus well below the Q(y)() transitions of chlorophylls. In addition, we have found that, although the S(1) lifetimes of violaxanthin, lutein, and zeaxanthin differ substantially in solution, when incorporated into the LHCII protein, their S(1) states have in fact the same lifetime of about 11 ps. Despite the similar spectroscopic properties of the carotenoids bound to the LHCII, we observed a maximal fluorescence quenching when zeaxanthin was present in the LHCII complex. On the basis of these observations, we suggest that, rather than different photochemical properties of individual carotenoid species, changes in the protein conformation induced by binding of carotenoids with distinct molecular structures are involved in the quenching phenomena associated with Lhc proteins.

Abstract: The chlorophyll a/b-xanthophyll-protein CP26 complex belongs to the Lhc protein family. It binds nine chlorophylls and two xanthophylls per 26.6 kDa polypeptide. Determination of the characteristics of each binding site is needed for the understanding of functional organization of individual proteins belonging to the photosystem II supramolecular complex. The biochemical and spectroscopic features of native CP26 are presented here together with identification of pigment binding and energy transitions in different sites. The analysis has been performed via a new approach using recombinant CP26 complexes in which the chromophore content has been experimentally modified. Data were interpreted on the basis of homology with CP29 and LHCII complexes, for which detailed knowledge is available from mutation analysis. We propose that one additional Chl b is present in CP26 as compared to CP29 and that it is located in site B2. We also found that in CP26 three chlorophyll binding sites are selective for Chl a, one of them being essential for the folding of the pigment-protein complex. Two xanthophyll binding sites were identified, one of which (L1) is essential for protein folding and specifically binds lutein. The second site (L2) has lower selectivity and can bind any of the xanthophyll species present in thylakoids.

Abstract: The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while beta-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.

Abstract: We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Abstract: The steady state absorption and fluorescence spectroscopic properties of the xanthophylls, violaxanthin, zeaxanthin, and lutein, and the efficiencies of singlet energy transfer from the individual xanthophylls to chlorophyll have been investigated in recombinant CP26 protein overexpressed in Escherichia coli and then refolded in vitro with purified pigments. Also, the effect of the different xanthophylls on the extents of static and dynamic quenching of chlorophyll fluorescence has been investigated. Absorption, fluorescence, and fluorescence excitation demonstrate that the efficiency of light harvesting from the xanthophylls to chlorophyll a is relatively high and insensitive to the particular xanthophyll that is present. A small effect of the different xanthophylls is observed on the extent of quenching of Chl fluorescence. The data provide the precise wavelengths of the absorption and fluorescence features of the bound pigments in the highly congested spectral profiles from these light-harvesting complexes. This information is important in assessing the mechanisms by which higher plants dissipate excess energy in light-harvesting proteins.

Abstract: The energy transfer kinetics from carotenoids to chlorophylls and among chlorophylls has been measured by femtosecond transient absorption kinetics in a monomeric unit of the major light-harvesting complex (LHCII) from higher plants. The samples were reconstituted complexes with different carotenoid contents. The kinetics was measured both in the carotenoid absorption region and in the chlorophyll Q(y) region using two different excitation wavelengths suitable for selective excitation of the carotenoids. Analysis of the data shows that the overwhelming part of the energy transfer from the carotenoids occurs directly from the initially excited S(2) state of the carotenoids. Only a small part (<20%) may possibly take an S(1) pathway. All the S(2) energy transfer from carotenoids to chlorophylls occurs with time constants <100 fs. We have been able to differentiate among the three carotenoids, two luteins and neoxanthin, which have transfer times of approximately 50 and 75 fs for the two luteins, and approximately 90 fs for neoxanthin. About 50% of the energy absorbed by carotenoids is initially transferred directly to chlorophyll b (Chl b), while the rest is transferred to Chl a. Neoxanthin almost exclusively transfers to Chl b. Due to various complex effects discussed in the paper, such as a specific coupling of Chl b and Chl a excited states, the percentage of direct Chl b transfer thus is somewhat lower than estimated by us previously for LHCII from Arabidopsis thaliana. (Connelly, J. P., M. G. Müller, R. Bassi, R. Croce, and A. R. Holzwarth. 1997. Biochemistry. 36:281). We can distinguish three different Chls b receiving energy directly from carotenoids. We propose as a new mechanism that the carotenoid-to-Chl b transfer occurs to a large part via the B(x) state of Chl b and to the Q(x) state, while the transfer to Chl a occurs only via the Q(x) state. We find no compelling evidence in favor of a substantial S(1) transfer path of the carotenoids, although some transfer via the S(1) state of neoxanthin can not be entirely excluded. The S(1) lifetimes of the two luteins were determined to be 15 ps and 3.9 ps. A detailed quantitative analysis and kinetic model of the processes described here will be presented in a separate paper.

Abstract: We have characterized a xanthophyll binding site, called V1, in the major light harvesting complex of photosystem II, distinct from the three tightly binding sites previously described as L1, L2, and N1. Xanthophyll binding to the V1 site can be preserved upon solubilization of the chloroplast membranes with the mild detergent dodecyl-alpha-d-maltoside, while an IEF purification step completely removes the ligand. Surprisingly, spectroscopic analysis showed that when bound in this site, xanthophylls are unable to transfer absorbed light energy to chlorophyll a. Pigments bound to sites L1, L2, and N1, in contrast, readily transfer energy to chlorophyll a. This result suggests that this binding site is not directly involved in light harvesting function. When violaxanthin, which in normal conditions is the main carotenoid in this site, is depleted by the de-epoxidation in strong light, the site binds other xanthophyll species, including newly synthesized zeaxanthin, which does not induce detectable changes in the properties of the complex. It is proposed that this xanthophyll binding site represents a reservoir of readily available violaxanthin for the operation of the xanthophyll cycle in excess light conditions.

Abstract: Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.

Abstract: Resonance Raman spectra of the native Lhcb4 antenna protein are compared with those of a recombinant protein prepared by in vitro refolding of its polypeptide, over-expressed in Escherichia coli, with added pigments [Giuffra et al. (1996) Eur. J. Biochem. 238, 112-120]. The results indicate that the native pigment conformation is reproduced almost perfectly in the reconstituted protein, with only small differences which are attributed to a slight shift in the Soret absorption peak of two or more chlorophylls. This procedure therefore represents a model system for the investigation of site-directed mutant LHC proteins, which are otherwise very difficult to obtain.

Abstract: We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.

Abstract: Light-harvesting complexes (Lhc) catalyse sunlight harvesting for photosynthesis as well as other essential functions, including photoprotection by quenching of harmful chlorophyll triplet states and prevention of photoinhibition by dissipation of excitation energy in excess. In addition, folding of Lhc proteins depends on the availability of both xanthophylls and carotenoids, thus preventing the potential formation of harmful chlorophyll-protein complexes lacking photoprotectors. We have used the mutation analysis in order to study the association of the different functions to three protein domains, each composed of a xanthophyll molecule and of neighbour chlorophylls a and b, within the major antenna complex of photosystem II, i.e. LHCII. We have found that the xanthophyll to chlorophyll energy transfer is a shared property of the whole pigment-protein complex, and occurs with similar efficiency in each of the three structural domains. Photoprotection by quenching of chlorophyll triplets is catalysed mainly by lutein bound to site L1, and occurs via energy transfer from chlorophylls A1 and B1. This domain is essential for pigment-induced protein folding. The domains L2 and N1 weakly influence either the protein stability or the photoprotection; however, replacement of xanthophyll species bound to these structural domains modulates the fluorescence quantum yield of LHCII, and suggests that non-radiative dissipation of excess energy can be regulated through allosteric modification of the protein structure by exchanging xanthophylls in these sites.

Abstract: We have identified a Ca(2+)-binding site of the 29-kDa chlorophyll a/b-binding protein CP29, a light harvesting protein of photosystem II most likely involved in photoregulation. (45)Ca(2+) binding studies and dot blot analyses of CP29 demonstrate that CP29 is a Ca(2+)-binding protein. The primary sequence of CP29 does not exhibit an obvious Ca(2+)-binding site therefore we have used Yb(3+) replacement to analyze this site. Near-infrared Yb(3+) vibronic side band fluorescence spectroscopy (Roselli, C., Boussac, A., and Mattioli, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12897-12901) of Yb(3+)-reconstituted CP29 indicated a single population of Yb(3+)-binding sites rich in carboxylic acids, characteristic of Ca(2+)-binding sites. A structural model of CP29 presents two purported extra-membranar loops which are relatively rich in carboxylic acids, one on the stromae side and one on the lumenal side. The loop on the lumenal side is adjacent to glutamic acid 166 in helix C of CP29, which is known to be the binding site for dicyclohexylcarbodiimide (Pesaresi, P., Sandonà, D., Giuffra, E. , and Bassi, R. (1997) FEBS Lett. 402, 151-156). Dicyclohexylcarbodiimide binding prevented Ca(2+) binding, therefore we propose that the Ca(2+) in CP29 is bound in the domain including the lumenal loop between helices B and C.

Abstract: A preparation consisting of isolated dimeric peripheral antenna complexes from green plant photosystem I (light-harvesting complex I or LHCI) has been characterized by means of (polarized) steady-state absorption and fluorescence spectroscopy at low temperatures. We show that this preparation can be described reasonably well by a mixture of two types of dimers. In the first dimer about 10% of all Q(y)() absorption of the chlorophylls arises from two chlorophylls with absorption and emission maxima at about 711 and 733 nm, respectively, whereas in the second about 10% of the absorption arises from two chlorophylls with absorption and emission maxima at about 693 and 702 nm, respectively. The remaining chlorophylls show spectroscopic properties comparable to those of the related peripheral antenna complexes of photosystem II. We attribute the first dimer to a heterodimer of the Lhca1 and Lhca4 proteins and the second to a hetero- or homodimer of the Lhca2 and/or Lhca3 proteins. We suggest that the chlorophylls responsible for the 733 nm emission (F-730) and 702 nm emission (F-702) are excitonically coupled dimers and that F-730 originates from one of the strongest coupled pair of chlorophylls observed in nature.

Abstract: The energy transfer rates between chlorophylls in the light harvesting complex CP29 of higher plants at room temperature were calculated ab initio according to the Förster mechanism (Förster T. 1948, Ann. Physik. 2:55-67). Recently, the transition moment orientation of CP29 chlorophylls was determined by differential linear dichroism and absorption spectroscopy of wild-type versus mutant proteins in which single chromophores were missing (Simonetto R., Crimi M., Sandonà D., Croce R., Cinque G., Breton J., and Bassi R. 1999. Biochemistry. 38:12974-12983). In this way the Q(y) transition energy and chlorophyll a/b affinity of each binding site was obtained and their characteristics supported by reconstruction of steady-state linear dichroism and absorption spectra at room temperature. In this study, the spectral form of individual chlorophyll a and b ligands within the protein environment was experimentally determined, and their extinction coefficients were also used to evaluate the absolute overlap integral between donors and acceptors employing the Stepanov relation for both the emission spectrum and the Stokes shift. This information was used to calculate the time-dependent excitation redistribution among CP29 chlorophylls on solving numerically the Pauli master equation of the complex: transient absorption measurements in the (sub)picosecond time scale were simulated and compared to pump-and-probe experimental data in the Q(y) region on the native CP29 at room temperature upon selective excitation of chlorophylls b at 640 or 650 nm. The kinetic model indicates a bidirectional excitation transfer over all CP29 chlorophylls a species, which is particularly rapid between the pure sites A1-A2 and A4-A5. Chlorophylls b in mixed sites act mostly as energy donors for chlorophylls a, whereas site B5 shows high and bidirectional coupling independent of the pigment hosted.

Abstract: Photoprotection of the chloroplast is an important component of abiotic stress resistance in plants. Carotenoids have a central role in photoprotection. We review here the recent evidence, derived mainly from in vitro reconstitution of recombinant Lhc proteins with different carotenoids and from carotenoid biosynthesis mutants, for the existence of different mechanisms of photoprotection and regulation based on xanthophyll binding to Lhc proteins into multiple sites and the exchange of chromophores between different Lhc proteins during exposure of plants to high light stress and the operation of the xanthophyll cycle. The use of recombinant Lhc proteins has revealed up to four binding sites in members of Lhc families with distinct selectivity for xanthophyll species which are here hypothesised to have different functions. Site L1 is selective for lutein and is here proposed to be essential for catalysing the protection from singlet oxygen by quenching chlorophyll triplets. Site L2 and N1 are here proposed to act as allosteric sites involved in the regulation of chlorophyll singlet excited states by exchanging ligand during the operation of the xanthophyll cycle. Site V1 of the major antenna complex LHC II is here hypothesised to be a deposit for readily available substrate for violaxanthin de-epoxidase rather than a light harvesting pigment. Moreover, xanthophylls bound to Lhc proteins can be released into the lipid bilayer where they contribute to the scavenging of reactive oxygen species produced in excess light.

Abstract: The spectral forms of the two chlorophyll species in higher plant Photosystem II antenna proteins have been experimentally determined within their protein environment. Recombinant CP29 and LHC II antenna proteins missing individual chromophores were obtained by over-expression in bacteria without any changing of the primary protein sequence and in vitro reconstitution. Difference absorption spectroscopy with respect to the corresponding proteins binding the complete pigment complement yielded the spectral shape and extinction of single chlorophyll a and b. A functional relation of their absorption was given by Gaussian subband decomposition covering the entire Q(x) and Q(y) optical region together with the absolute value of the molar extinction coefficient. With respect to analogous determinations reported in the literature for organic solvents, this information is valuable for further understanding the in-protein chlorophyll excited states and excited state dynamics: in particular, for the calculation of Förster transfer rates by means of chlorophyll-chlorophyll overlap integral employing the Stepanov relation for emission and single chromophore transition energies according to the results of mutational analysis of chlorophyll binding sites [Bassi et al. (1999) Proc Natl Acad Sci USA 96: 10056-10061; Remelli et al. (1999) J Biol Chem 274: 33510-33521].

Abstract: The absorption spectra of two light harvesting complexes from higher plants, CP29 and LHC II, have been analysed in the Soret region in order to obtain a description in terms of the absorption spectra of the individual pigments. This information is of great practical use when applying spectroscopic techniques to the study of energy transfer in photosynthesis such as time-resolved spectroscopy thus allowing determination of the relative absorption cross-section for the different chromophores in the system as a function of wavelength. In this study, recombinant Lhc proteins carrying point mutations in pigment-binding residues have been used in order to obtain the spectral shape of individual chromophores by differential spectroscopy with respect to the WT protein. Combinations of spectra thus obtained were then used to fit the absorption spectra of WT and mutant pigment-proteins according to the constraints posed by stoichiometry of pigments as derived by biochemical analysis. This procedure allowed identification of each pigment in term of its wavelength position, spectral shape and extinction coefficient. The data obtained by this procedure have been successfully applied to the description of other higher plant Lhc proteins thus supporting the view that the Lhc superfamily members share specific pigment-protein interactions as suggested by sequence homology.

Abstract: 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.

Abstract: The major light-harvesting complex of photosystem II in higher plants is known as LHCII. It is composed of a number of chlorophyll-binding proteins sharing epitopes with each other. The number of apoproteins resolved by fully denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis varies in different species. In order to know if this heterogeneity is caused by the expression of a number of homologous genes or if it is the product of post-translational modifications, we have resolved the six major apoproteins of Zea mays LHCII. Each protein is purified to homogeneity, subjected to direct protein sequencing and the sequences compared with those deduced from lhcb genes in maize and other organisms. All of the six proteins are distinct gene products, since they show differences in their primary structure. Three apoproteins are identified as products of type I lhcb genes and one each as type II and type III gene products. A sixth protein does not fit the requirements for any of the lhcb genes so far cloned and is therefore probably the product of an lhcb gene type not yet described. Our results clearly show that the major source of LHCII protein heterogeneity is the expression of many lhcb genes. Fractionation of maize LHCII by non-denaturing flat-bed isoelectric focusing resolves at least five major isoforms showing distinct differences in their polypeptide composition and also differing in their spectroscopic properties, thus suggesting that individual Lhcb gene products have distinct pigment-binding properties.

Abstract: We report the expression of the barley (Hordeum vulgare L.) COR (cold-regulated) gene cor14b (formerly pt59) and the accumulation of its chloroplast-localized protein product. A polyclonal antibody raised against the cor14b-encoded protein detected two chloroplast COR proteins: COR14a and COR14b. N-terminal sequencing of COR14a and expression of cor14b in Arabidopsis plants showed that COR14a is not encoded by the cor14b sequence, but it shared homology with the wheat (Triticum aestivum L.) WCS19 COR protein. The expression of cor14b was strongly impaired in the barley albino mutant an, suggesting the involvement of a plastidial factor in the control of gene expression. Low-level accumulation of COR14b was induced by cold treatment in etiolated plants, although cor14b expression and protein accumulation were enhanced after a short light pulse. Light quality was a determining factor in regulating gene expression: red or blue but not far-red or green light pulses were able to promote COR14b accumulation in etiolated plants, suggesting that phytochrome and blue light photoreceptors may be involved in the control of cor14b gene expression. Maximum accumulation of COR14b was reached only when plants were grown and/or hardened under the standard photoperiod. The effect of light on the COR14b stability was demonstrated by using transgenic Arabidopsis. These plants constitutively expressed cor14b mRNAs regardless of temperature and light conditions; nevertheless, green plants accumulated about twice as much COR14b protein as etiolated plants.

Abstract: Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It utilizes light for photochemical energy conversion, and is heavily involved in the regulation of the energy flow. We investigated the structural organization of photosystem II and its associated light-harvesting antenna by electron microscopy, multivariate statistical analysis, and classification procedures on partially solubilized photosystem II membranes from spinach. Observation by electron microscopy shortly after a mild disruption of freshly prepared membranes with the detergent n-dodecyl-alpha,D-maltoside revealed the presence of several large supramolecular complexes. In addition to the previously reported supercomplexes [Boekema, E. J., van Roon, H., and Dekker, J. P. (1998) FEBS Lett. 424, 95-99], we observed complexes with the major trimeric chlorophyll a/b protein (LHCII) in a third, L-type of binding position (C2S2M0-2L1-2), and two different types of megacomplexes, both identified as dimeric associations of supercomplexes with LHCII in two types of binding sites (C4S4M2-4). We conclude that the association of photosystem II and its associated light-harvesting antenna is intrinsically heterogeneous, and that the minor CP26 and CP24 proteins play a crucial role in the supramolecular organization of the complete photosystem. We suggest that different types of organization form the structural basis for photosystem II to specifically react to changing light and stress conditions, by providing different routes of excitation energy transfer.

Abstract: The localisation of the xanthophyll neoxanthin within the structure of the major light harvesting complex (LHCII) of higher plants has been investigated by site-directed mutagenesis and spectroscopic methods. Mutation analysis performed on pigment binding sites in different helix domains leads to selective loss of neoxanthin for mutations on helix C thus localising this pigment between the helix C and helix A/B domains. Recombinant proteins binding two lutein molecules per polypeptide but lacking neoxanthin have been used in order to determine the contribution of neoxanthin to the absorption and linear dichroism spectra. The data were used to derive the orientation of the neoxanthin transition moment, lying in the polyene chain, which was thus determined to form an angle of 57 +/- 1.5 degrees with respect to the normal to the membrane plane where the protein is inserted. On the basis of these results we propose a model for the localisation of the carotenoid site in the LHCII structure which is still unresolved.

Abstract: The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a-Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.

Abstract: The Q(y) transition dipole moment vectors of all eight chlorophylls in the higher-plant antenna protein CP29 were calculated by an original method on the basis of linear dichroism and absorption spectroscopy. The contribution of individual chromophores was determined from difference spectra between wild type and mutant proteins in which a single chlorophyll has been removed by mutating pigment-binding residues. Recombinant proteins were constructed by overexpressing the apoprotein in bacteria and refolding of the pigment-protein complex in vitro [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The spectroscopic data are interpreted on the basis of a protein structural model obtained via the homology with the major antenna complex LHCII [Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Nature 367, 614-621]. The results allow us to determine the orientation of six chromophores within the protein structure. The orientations of the two remaining chromophores are inferred by considering the symmetry properties of CP29 and fitting steady state absorption and linear dichroism spectra by independent chlorophyll spectral forms. As a consequence, four "mixed" sites with different chlorophyll a and b binding affinities are identified in CP29. Geometrical data and the Förster mechanism for energy transfer suggest that excitation energy equilibrates rapidly among chlorophyll "pure" sites while energy preferentially flows outward from chlorophyll "mixed" sites. The orientation of the dipole moments of two chlorophyll molecules symmetrically located at the center of the protein and parallel to the carotenoid transition vectors suggests a role in energy transfer from xanthophyll to chlorophyll.

Abstract: The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q(y) transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.

Abstract: Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in (1)O(2)(*) scavenging.

Abstract: The light-harvesting chlorophyll a/b protein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophyll a, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or beta-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyll b and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophyll a to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/b ratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions. The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophyll a molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b absorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a.

Abstract: The minor chlorophyll a/b-binding (CAB) proteins of the liverwort Marchantia polymorpha L. were investigated in order to compare the antenna organization and the light-acclimation potential in lower and higher plants. Homologues to the minor CAB proteins CP24, CP26 and CP29 were identified by the following criteria: enrichment in photosystem II preparations, immunological cross-reactivities, spectroscopic properties and protein-fragment amino acid sequences. The high violaxanthin content of the minor CAB proteins in M. polymorpha indicates that their role in protection from high light is comparable in lower and higher plants. Considerably more-alkaline isoelectric points are found for the minor CAB proteins of M. polymorpha than for their higher-plant counterparts. This might be due to a higher content of basic amino acids. While the N-terminal sequence of angiosperm CP29 contains a threonine that becomes phosphorylated during cold stress, this amino acid is substituted by valine in M. polymorpha. Therefore, the regulatory properties of this protein could differ in lower and higher plants.

Abstract: The CP29 subunit of Photosystem II is reversibly phosphorylated in Zea mays upon exposure to high light in the cold (Bergantino et al., J Biol Chem 270 (1995) 8474-8481). This phenomenon was previously proposed to be restricted to C4 plants. We present the complete sequence of the CP29 protein, deduced from a maize Lhcb4 cDNA clone, and its comparison with the previously known Lhcb4 sequences of two C3 plants: Hordeum vulgare and Arabidopsis thaliana. Despite the relatively low degree of homology in their amino-terminal region, i.e. the part of the molecule which is phosphorylated in maize, the three polypeptides conserve consensus sequences for the site of phosphorylation. We proved by immunoblotting and 33P-labelling that the same post-translational modification occurs in barley. Being thus common to C3 and C4 plant species, the phosphorylation of this minor antenna complex of Photosystem II appears now as a widespread phenomenon, possibly part of the phosphorylation cascade which signals the redox status of the plastoquinone to the nuclear transcription apparatus. Arabidopsis plants do not show phosphorylation of CP29 in the same conditions, but other low-molecular-weight phosphoproteins, whose role need to be elucidated, become evident.

Abstract: CP26 is the most recently described antenna protein in higher plants which has been reported to be involved in xanthophyll-dependent regulation of the light-harvesting function but is largely unknown due to the difficulties of purification. In this study we have overexpressed in Escherichia coli the Lhcb5 gene product and reconstituted CP26 in vitro by refolding the recombinant protein in the presence of chlorophyll a, chlorophyll b and xanthophylls. The resulting pigment-protein complex is stable enough to be isolated by partially denaturing gel electrophoresis. Reconstitution and isolation conditions for CP26 are similar to those used for other chlorophyll a/b complexes like the major light-harvesting complex of photosystem II (LHCII) and CP29; however, CP26 differs with regard to its lower specificity in carotenoid binding. Most significantly, rather stable recombinant CP26 can be reconstituted containing violaxanthin as the only carotenoid. This enhanced plasticity with respect to carotenoid binding is consistent with CP26 being the major binding protein of violaxanthin involved in the xanthophyll cycle. The availability of recombinant CP26 opens the way to a better characterisation of this pigment-protein complex with regard to its biochemistry and its physiological functions.

Abstract: A new photosystem II preparation was isolated from Marchantia polymorpha thylakoids upon solubilization with dodecyl beta-D-maltoside and glycerol gradient ultracentrifugation. Its protein composition was analyzed, and all tested polypeptides from the core complex, the oxygen-evolving enhancer and the light-harvesting complex (LHC) could be detected. The only component severely depleted compared with the grana membrane preparation was the psbS gene product. This complex was subjected to chemical cross-linking using the cleavable homobifunctional cross-linker dithiobis(sulfosuccinimidylpropionate). The overall pattern of cross-linking-products was analyzed by diagonal electrophoresis, where the cross-linking agent was cleaved by reduction of the disulfide bond between the first and second dimensions of the gel, followed by immunoblotting. Many cross-linking products were characterized and these data used in order to identify protein masses revealed by electron microscopy [Boekema, E. J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175-179]. It is concluded that the core proteins CP43 and CP47 are located at opposite sides of the D1-D2-cytochrome b559 complex. Minor CAB proteins were found to interact with core complex subunits (CP29, CP26) and LHCII (CP26), supporting the view that these proteins could interface the major LHCII with the reaction center.

Abstract: Mutation analysis of higher plants light harvesting proteins has been prevented for a long time by the lack of a suitable expression system providing chromophores essential for the folding of these membrane-intrinsic pigment-protein complexes. Early work on in vitro reconstitution of the major light harvesting complex of photosystem II (LHCII) indicated an alternative way to mutation analysis of these proteins. A new procedure for in vitro refolding of the four light harvesting complexes of photosystem II, namely CP24, CP29, CP26 and LHCII yields recombinant pigment-proteins indistinguishable from the native proteins isolated from leaves. This method allows both the performing of single point mutations on protein sequence and the exchange of the chromophores bound to the protein scaffold. We review here recent results obtained by this method on the pigment-binding properties, on the chlorophyll-binding residues, on the identification of proton-binding sites and on the role of xanthophylls in the regulation of light harvesting function.

Abstract: The relationship between the binding of dicyclohexylcarbodiimide (DCCD) to isolated light-harvesting proteins of photosystem II and the inhibition of chlorophyll fluorescence quenching by DCCD have been investigated. For a range of different complexes an approximately linear relationship was obtained between the efficiency of DCCD binding and the DCCD-dependent reversal of fluorescence quenching. The most efficient labeling was found for the minor light-harvesting complexes, CP29 and CP26. In the case of the former, five different preparations were compared including two reconstituted complexes in which a putative DCCD-binding site had been mutagenized. Again, an approximately linear relationship between DCCD binding and the extent of reversal of fluorescence quenching was found. However, the binding of DCCD was found to occur at least an order of magnitude faster than the change in fluorescence. The results are discussed in terms of the multiplicity of DCCD-binding sites and the influence of protein structure on both the binding of DCCD and the fluorescence quenching mechanism.

Abstract: Energy-dependent quenching of chlorophyll fluorescence (qE) reflects the action of a powerful mechanism of protection from photoinhibition in which the low pH in the chloroplast lumen induces dissipation of excess excitation energy. Dicyclohexylcarbodiimide (DCCD), a protein-modifying agent, is a powerful inhibitor of qE and has been shown to react with acidic residues, in a hydrophobic environment, involved in proton translocation. The CP29 subunit of photosystem II has been proposed to be the site of qE quenching and shown to bind DCCD. We have hypothesised, on the basis of the CP29 protein sequence and of the structure of light-harvesting complex II protein, that glutamic acid 166 is the DCCD binding site. In this study, we have produced recombinant proteins either with wild-type sequence or carrying a mutation on the 166 position. We show that the mutant protein does not bind DCCD. This identifies E166 as the site whose protonation may lead to a conformational change triggering qE.

Abstract: Singlet energy transfer between the carotenoids (Cars) and chlorophylls (Chls) in the light-harvesting complex II (LHC II) from higher plants has been studied using ultrafast transient absorption spectroscopy by exciting the Cars directly in the 475-515 nm wavelength range. LHC II trimers from Arabidopsis thaliana with well-defined Car compositions have been used. From HPLC, the wild type (WT) monomer contains two luteins (Ls), one neoxanthin (N), and a trace of violaxanthin (V) per 12 Chls. The ABA-3 mutant contains 1.4 Ls and 0.6 zeaxanthin (Z) per monomer. Though exploitation of the difference in Car constitution and exciting the WT at 475 and 490 nm, and the ABA-3 mutant at 490 and 515 nm, the different Car contributions to energy transfer have been probed. Evidence for energy transfer mainly from the Car to Chl b is observed in the WT. In the mutant, additional transfer from Car to Chl a correlates with the presence of Z. The results imply predominant energy transfer from the central Ls to Chl b which requires a modification of the currently accepted arrangement of Chl pigments in LHC II.

Abstract: The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.

Abstract: The effects of low temperature on the relative contributions of the reaction center and the antenna activities to photosystem II (PSII) electron transport were estimated by chlorophyll fluorescence. The inhibition of PSII photochemistry resulted from photo-damage to the reaction center and/or a reduced probability of excitation energy trapping by the reaction center. Although chill treatment did not modify the proportion of the dimeric to monomeric PSII, it destabilized its main light-harvesting complex. Full protection of the reaction center was achieved only in the presence of the phosphorylated PSII subunit, CP29. In a nonphosphorylating genotype the chill treatment led to photoinhibitory damage. The phosphorylation of CP29 modified neither its binding to the PSII core nor its pigment content. Phosphorylated CP29 was isolated by flat-bed isoelectric focusing. Its spectral characteristics indicated a depletion of the chlorophyll spectral forms with the highest excitation transfer efficiency to the reaction center. It is suggested that phosphorylated CP29 performs its regulatory function by an yet undescribed mechanism based on a shift of the equilibrium for the excitation energy toward the antenna.

Abstract: The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.

Abstract: Photosystem I with its full antenna complement (PSI-LHCI) has been prepared by mild detergent solubilization with octyl beta-D-glucopyranoside from maize thylakoids. A preliminary polypeptide analysis is presented. At room temperature, the steady-state fluorescence derives from an almost perfectly thermalized state, as demonstrated by a Stepanov analysis, in which about 90% of the excited states are associated with the red chlorophyll spectral forms absorbing above 700 nm. Equilibration is temperature-sensitive and is lost at T < 200 K. A careful analysis of fluorescence between 75 and 280 K clearly demonstrates the presence of at least three red chlorophyll spectral forms with emission maxima at 720, 730, and 742 nm, the absorption origin bands of which have been calculated at 714, 725, and 738 nm. On the basis of a minor deviation from thermal equilibration around 695 nm, it is suggested that at least 3-4 antenna chlorophylls, with an average absorption near 695 nm, are strongly coupled to P700. Thermodynamic analysis of absorption and fluorescence spectra indicates that the equilibrium, absorption-weighted excited state population of the P700 dimer is around 0.013 assuming that the low-energy exciton state possesses all the oscillator strength. The average free energy for excitation transfer from antenna to P700 is thus calculated to be -0.26 kT at room temperature. This indicates that P700 is almost isoenergetic with its antenna at room temperature when the red forms are taken fully into account. From the calculated excited state population of P700, we estimate that the primary charge separation rate in PSI is 1-2 ps-1.

Abstract: Light energy absorbed by the chloroplast membranes of higher plants is dissipated by nonradiative de-excitation in order to protect against photodamage. In photosystem II, which is the photosynthetic component most sensitive to photoinhibition, three pigment binding subunits, called CP24, CP26, and CP29, have been proposed to act in the regulation of the chlorophyll excited states concentration. In heavy stress conditions, CP29 becomes phosphorylated in its stroma-exposed portion, and this process is reversed by returning to normal conditions. In this study, we have used the pigments bound to the intramembrane hydrophobic portion of the protein as intrinsic probes to detect conformational changes induced by phosphorylation. We isolated the phosphorylated and unphosphorylated forms of the protein and showed that, although they have the same pigment complement, spectral differences can be consistently detected by absorption, linear dichroism, and circular dichroism spectroscopy. Alkaline phosphatase treatment of the phosphoprotein restores both the electrophoretic mobility and the spectral properties of the unphosphorylated CP29. The results of this study show that phosphorylation of CP29 can modulate the spectral properties of this photosystem II subunit and provide a possible mechanism for the regulation of excitation energy supply to the reaction center.

Abstract: Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformational changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474-8481). In this study, we have identified the phosphorylation site on the N-terminal, stroma-exposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transduction pathway is discussed.

Abstract: The resistance of maize plants to cold stress has been associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling treatment in the light. The cold-induced protein has been isolated, characterized by amino acid sequencing, and pulse labeled with radioactive precursors, showing that it is the product of post-translational modification by phosphorylation of the minor chlorophyll a/b protein CP29 rather than the product of a cold-regulated gene or an unprocessed CP29 precursor. We show here that the CP29 kinase activity displays unique characteristics differing from previously described thylakoid kinases and is regulated by the redox state of a quinonic site. Finally, we show that maize plants unable to perform phosphorylation have enhanced sensitivity to cold-induced photoinhibition.

Abstract: We have studied the occurrence and organization of photosystem II (PSII) in bundle sheath thylakoids and stroma lamellae from maize. As shown by non-denaturing lauryl beta-D- iminopropionidate (Deriphat)/PAGE, PSII exists in a dimeric form in grana membranes. In bundle sheath and stroma lamellae, however, only a monomeric form was found. Based on immunotitration data, we estimated the stoichiometry of the individual components of the PSII core complex and antenna systems. In stroma lamellae, all PSII antenna complexes had a stoichiometry similar to that in grana membranes, with the exception of light-harvesting complex II (LHCII) that was somewhat over-represented, while the minor antenna complexes CP26 and CP29 were under-represented. In bundle sheath, the amount of LHCII was approximately eight times higher than expected with respect to D1. The 33-kDa protein of the oxygen-evolving enhancer polypeptides was not detectable nor was the ferredoxin-NADP+ reductase, thus strongly suggesting that no significant linear electron transport occurs in bundle sheath thylakoids. Fluorescence induction data suggest that most of the PSII reaction centers in bundle sheath and stroma lamellae sustain electron transport towards a secondary acceptor pool. Stromal PSII centers are only weakly inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), whereas, unexpectedly, dichlorobenzoquinone and methyl viologen had a pronounced inhibitory effect of the QA- reoxidation. An additional specificity of these centers is the slow rate (50-ms range) of the QA to QB electron transfer. The amplitude of variable fluorescence found in stroma lamellae can only account for a small fraction (1-2%) of the variable fluorescence of whole thylakoids. This suggests that stromal PSII cannot be solely responsible for the slow beta-phase of the induction kinetics.

Abstract: The three-dimensional structure of photosystem II (PSII) has been determined by conventional transmission electron microscopy and computerized three-dimensional reconstruction. Both the complete system and that lacking the oxygen-evolving complex have been analyzed. The PSII complex has a four-lobed structure with twofold symmetry. An estimate of the molecular mass and the results of Deriphat/PAGE analysis suggest that a reaction centre is present in each half of the structure resolved by electron microscopy. Stepwise removal of components of the complex showed that the removal of CP47 (a 47-kDa chlorophyll-protein complex) induces monomerization of PSII, which indicates the importance of this subunit for the dimeric structure.

Abstract: Room temperature and 10 K absorption and linear dichroism spectra of the chlorophyll-protein complexes comprising the outer antenna of PSII (LHCII, CP29, CP26, CP24) have been analyzed in terms of a linear combination of asymmetric Gaussian bands. The results demonstrate the following: (a) The absorption and linear dichroism spectra of each sample can be described by nearly the same set of Gaussian bands at room temperature and 10 K. (b) The relative distributions of the transition moments of the major red-absorbing spectral forms seem to be similar in all four outer antenna chlorophyll-protein complexes at room temperature, with the 684-nm band being oriented closest to the particle plane at room temperature and the 677- and 669-nm bands being tilted at progressively greater angles out of the particle plane. The shorter wavelength transitions seem to be oriented close to the magic angle, but interpretation is complicated in this spectral region due to the low linear dichroism values and by overlap with vibrational bands. (c) The 684-nm band, detected in room temperature absorption and linear dichroism spectra of all complexes, vanishes at 10 K.

Abstract: Photosystem II membrane fractions from dark-adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl beta-D-maltoside. Chlorophyll-binding proteins from photosystem II were isolated either by ultracentrifugation on a sucrose gradient, or by flat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light-harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll-protein complexes 43, 47) and in the photosystem II reaction centers chlorophyll-protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll-protein complexes in this region of the photosynthetic membrane. Photosystem II core fractions prepared either by ultra-centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem II core in its components showed that it was the chlorophyll-protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll-protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll-protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids. Concerning the main light-harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration, while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light-harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26-kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing.

Abstract: The distribution of the photosynthetic pigments of the chlorophyll-binding proteins or photosystem-II membranes, isolated from dark-adapted maize leaves was determined. Most (80%) of a xanthophyll, violaxanthin, was found in the three minor chlorophyll-a/b proteins CP24, CP26 and CP29 whose function is unknown. Violaxanthin is the precursor of zeaxanthin, which is involved in dissipating excess excitation energy into heat [Demmig-Adams, B. (1991) Biochim. Biophys. Acta 1020, 1-24] under conditions of high transmembrane pH gradient [Gilmore, A. M. & Yamamoto, H. Y. (1992) Proc. Natl Acad. Sci. USA 89, 1899-1903]. We propose that a role for the minor photosystem-II chlorophyll-a/b proteins is the regulation of energy transfer to the reaction centre. It was also confirmed that the photosystem II reaction centre (D1-D2-cytochrome b559) contains beta-carotene as the only carotenoid. However, the two other chlorophyll-a-binding proteins of photosystem II, CP47 and CP43, bind not only beta-carotene, but also the xanthophyll lutein, previously thought to be restricted to chlorophyll-a/b proteins.

Abstract: The chlorophyll-protein complexes that form the antenna system of photosystem II have been purified and analyzed in terms of the commonly observed chlorophyll spectral forms. With the exception of chlorophyll b, which is known to be associated with the complexes comprising the outer antenna (LHCII, CP24, CP26, CP29), the spectral forms occur with similar absorption maxima and are present in rather similar amounts in each of the antenna complexes. On the basis of the published chlorophyll stoichiometries for the complexes in photosystem II antenna, the distribution of the spectral forms in a "reconstituted" antenna has been determined. These data were used to calculate the equilibrium population of excited states within the various chlorophyll-protein complexes within photosystem II. This was compared with the light absorption capacity of each of the complexes in the "reconstituted" antenna. The ratio of these two parameters (excited-state equilibrium distribution/absorption capacity) was determined to be 1.21 for the inner (core) antenna and 0.88 for LHCII. The standard free energy change for exciton transfer from the outer to the inner antenna was calculated to be -0.17 kcal mol-1. It is concluded that the photosystem II antenna is arranged as a very shallow funnel.

Abstract: The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM], derived from mouse liver, showed the presence of currents corresponding to low conductances (less than 50 pS), as well as of four distinct conductances of 99 pS, 152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).

Abstract: In this study we have isolated the chlorophyll a/b-binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and characterized them by N-terminal sequencing, fluorescence, and absorption spectroscopy and by immunochemical means. The results indicate that in this organism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypeptides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chlorophyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emission typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypeptides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these characteristics confirm this hypothesis.

Abstract: In this study, we report on the composition of a photosystem-II antenna preparation which contains three chlorophyll-a/b proteins (CP), CP29, CP24 and light-harvesting complex (LHC) II obtained from Zea mays grana membranes as previously described [Dainese, P. & Bassi, R. (1991) J. Biol. Chem. 266, 8136-8142]. We demonstrate that the three chlorophyll proteins are present in the preparation with a 3:3:9 molar ratio and that they form a supramolecular antenna complex which represents one third of the photosystem-II antenna system. Phosphorylation experiments show that this complex is involved in the mechanism of regulation of excitation-energy distribution between photosystems: phosphorylation of the membranes induces dissociation of the LHCII moiety from the CP29-CP24 moiety and changes in the aggregation state of LHCII components of the CP29-CP24-LHCII complex. The LHCII subpopulations of the complex are shown to be distinct from the total LHCII population by isoelectrofocusing analysis. On the basis of these data and in the light of the stoichiometry of photosystem-II chlorophyll-binding proteins, we propose a model for the organization of photosystem-II antenna system.

Abstract: Photosystem (PS) II membranes, obtained by the method of Berthold et al. (Berthold, D. A., Babcock, G. T., and Yocum, C. F. (1981) FEBS Lett. 134, 231-234), have been fractionated by a sucrose gradient ultracentrifugation method which allows the quantitative separation of the three major chlorophyll binding complexes in these membranes: the chlorophyll (chl) a binding PSII reaction center core, the major light-harvesting complex II, and the minor chl a/b proteins called CP26, CP29, and CP24. Each fraction has been analyzed for its subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results show that 12 mol of light-harvesting complex II and 1.5 mol of each of the minor chl a/b proteins are present per mol of the PSII reaction center complex in PSII membranes. These data suggest a dimeric organization of PSII, in agreement with a recent crystallographic study (Bassi, R., Ghiretti Magaldi, A., Tognon, G., Giacometti, G. M., and Miller, K. (1989) Eur. J. Cell Biol. 50, 84-93) and imply that such a dimeric complex is served by antenna chl a/b proteins whose minimal aggregation state includes three polypeptides. This was confirmed by covalent cross-linking of purified antenna complexes.

Abstract: The cytochrome b6/f complex operates in photosynthetic electron transfer either in linear electron flow from photosystem II to photosystem I or in cyclic flow around photosystem I. Using membrane fractionation and immunocytochemistry, we show a change in lateral distribution of cytochrome b6/f complexes along the thylakoid membranes during state transitions. This change is seen in maize as well as in the green algae Chlamydomonas reinhardtii. When either of the two organisms were adapted to state II in vivo, the proportion of cytochrome b6/f complexes found in the photosystem I-enriched stroma lamellae regions was significantly larger than after adaptation to state I. A similar observation was made upon state I to state II transitions done in vitro by illuminating, in the presence of ATP, broken maize chloroplasts prepared from dark-adapted leaves. This reorganization of the electron-transfer chain is concurrent with the change in light-energy distribution between the two photosystems, which requires lateral displacement of light-harvesting complex II. That the changes in lateral distribution of both cytochrome b6/f and light-harvesting II complexes seen upon state transition in vitro similarly required addition of exogenous ATP, suggests that the change in cytochrome b6/f organization also depends on kinase activity. The increased concentration of cytochrome b6/f complexes in the vicinity of photosystem I in state II is discussed in terms of an increase in cyclic electron flow, thus favoring ATP production. Because transition to state II can be triggered in vivo by ATP depletion, we conclude that state transitions should be regarded not only as a light-adaptation mechanism but also as a rerouting of photosynthetic electron flow, enabling photosynthetic organisms to adapt to changes in the cell demand for ATP.

Abstract: By detergent treatment of isolated photosynthetic membranes from maize chloroplasts, we have prepared two-dimensional crystals of the photosystem II complex. Two distinct crystal forms are produced by this treatment. Analysis of Fourier transforms of the crystals shows that each crystal type is formed from two inverted layers. Within the rectangular 17.8 x 26.7 nm unit cell of each layer is a tetrameric structure enclosing a two-fold symmetry axis, a result implying that the basic structural unit of photosystem II is dimeric. Tris-washing, which removes proteins associated with the oxygen-evolving apparatus from the inner surface of the photosynthetic membrane, causes a distinct change in the structure of these tetramers and reveals a dimeric core complex which may be directly associated with the photosystem II machinery.

Abstract: The polypeptides of the barley light-harvesting protein of photosystem I (LHCI) share certain epitopes. At least two of these common epitopes are present in chlorophyll a/b-protein 1 (Chla/b-P1 = CP29), as shown by cross-reacting monoclonal antibodies (14). These antibodies were employed for immunological identification of polypeptides translated in vitro in an mRNA-dependent cell-free rabbit reticulocyte lysate. The monoclonal antibody CMpLHCI:2 precipitated only one polypeptide of molecular weight 28 kD from in vitro translates primed with polyA+ RNA. No 28 kD precipitation band was found, if this antibody was mixed with a PSI-200 preparation before it was added to the translate. The translational capacity of the LHCI transcripts isolated from 12 hours greened barley was much higher than those isolated from 6 hours greened barley. Transcripts for LHCI polypeptides were also found among the polyA+ RNA of the mutant viridis-k23, which is devoid of LHCI polypeptides in its thylakoid membranes. The monoclonal antibody CMpCh1a/b-P1:1 precipitated a polypeptide of molecular weight 31 kD from in vitro translates primed with polyA+ RNA. Thus, the cross-reactivity the two antibodies show with the mature proteins is not found when the antibodies are reacted with the precursor proteins.

Abstract: Photosystem I (PSI) preparations with a chlorophyll a/b ratio of 6.0 were isolated from barley thylakoids using two different methods. The high-molecular-mass complex (CP1a) which is resolved by non-denaturing gel electrophoresis had the same properties as a PSI preparation (PSI-200) isolated by Triton X-100 solubilisation of thylakoids followed by sucrose gradient ultracentrifugation. This material had a chlorophyll:P700 ratio of 208:1 and was composed of three different chlorophyll-protein complexes which could be separated from each other by solubilising the PSI preparation in dodecyl maltoside followed by sucrose gradient ultracentrifugation. Approximately half of the chlorophyll, including all the chlorophyll b, was located in two antenna complexes designated LHCI-680 and LHCI-730, which were identified by their characteristic low-temperature fluorescence emission spectra. The rest of the chlorophyll a was associated with the PSI reaction centre, P700 Chla-P1, which fluoresced at 720 nm. Each chlorophyll-protein complex had a unique polypeptide composition and characteristic circular dichroic and absorption spectra. The use of dodecyl maltoside instead of dodecyl sulphate resulted in a less denatured form of LHCI-680, which fluoresced at 690 nm at 77 K. One of the sucrose gradient fractions contained a complex consisting of only LHCI-730 and P700 Chla-P1 which fluoresced at 731 nm, indicating that LHCI-730 is structurally associated with P700 Chla-P1 and quenches its fluorescence. Approximately three-quarters of the light-harvesting antenna chlorophyll was in LHCI-730, but only about one-quarter of the normal complement of LHCI-730 was required to quench the reaction centre. By reducing the amount of Triton relative to the chlorophyll concentration, a PSI preparation (chlorophyll a/b ratio of 3.5) with a chlorophyll:P700 ratio of 300:1 was isolated. It contained no photosystem II, but a significant amount of LHCII which was functionally connected to the PSI reaction centre. Reconstitution studies demonstrated that excitation energy transfer from LHCII to PSI requires the presence of LHCI-680, and we propose that, in PSI, the following linear excitation energy transfer sequence occurs: LHCII----LHCI-680----LHCI-730----P700 Chla-P1.

Abstract: The chlorophyll-protein complexes of purified maize photosystem II membranes were separated by a new mild gel electrophoresis system under conditions which maintained all of the major chlorophyll a/b-protein complex (LHCII) in the oligomeric form. This enabled the resolution of three chlorophyll a/b-proteins in the 26-31-kDa region which are normally obscured by monomeric LHCII. All chlorophyll a/b-proteins had unique polypeptide compositions and characteristic spectral properties. One of them (CP26) has not previously been described, and another (CP24) appeared to be identical to the connecting antenna of photosystem I (LHCI-680). Both CP24 and CP29 from maize had at least one epitope in common with the light-harvesting antennae of photosystem I, as shown by cross-reactivity with a monoclonal antibody raised against LHCI from barley thylakoids. A complex designated Chla.P2, which was capable of electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol, was isolated by nondenaturing gel electrophoresis. It lacked CP43, which therefore can be excluded as an essential component of the photosystem II reaction center core. Fractionation of octyl glucoside-solubilized photosystem II membranes in the presence and absence of Mg2+ enabled the isolation of the Chla . P2 complex and revealed the existence of a light-harvesting complex consisting of CP29, CP26, and CP24. This complex and the major light-harvesting system (LHCII) are postulated to transfer excitation energy independently to the photosystem II reaction center via CP43.

Abstract: Thylakoids from enzymatically separated bundle sheath and mesophyll tissue chloroplasts were examined for their chlorophyll-proteins by tube sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Differences were found in distribution of chlorophyll among peaks. The chlorophyll-protein a peak (CPa), considered to be the photosystem II (PSII) reaction centre by many authors, was seen to be absent in bundle sheath thylakoid samples. The slab SDS-PAGE revealed the absence of the polypeptides present in PSII preparations of chloroplast subfractions having only PSII activity. This finding confirms Anderson's hypothesis of the structure of grana and stroma thylakoids.