Chlorophyll a/b-binding proteins: an extended family

A large proportion of the chlorophyll in a plant is engaged in harvesting light energy and transferring it to the photochemical reaction centres. These 'antenna' chlorophylls are non-covalently bound to specific proteins to form chlorophyll-protein complexes. The chlorophyll a/b-binding (CAB) polypeptides are encoded by an extended family of nuclear genes. It has recently been discovered that other proteins not known to bind chlorophyll, the early light-inducible proteins (ELIPs), are also related and could be considered part of this family. We suggest that the latter proteins may be involved in pigment biosynthesis or in assembly of the thylakoid membrane.     

A Cyanobacterial Chlorophyll Synthase-HliD Complex Associates with the Ycf39 Protein and the YidC/Alb3 Insertase

Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChlG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChlG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAG-ChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigment-protein complexes with minimal risk of accumulating phototoxic unbound chlorophylls.     

低温胁迫期间水稻光合膜色素与蛋白水平的变化

对４℃和１１℃两种低温胁迫过程中水稻类囊体膜色素与蛋白组成的变化进行了比较研究。结果表明：４℃低温不仅使类囊体膜中的光合色素（叶绿素、类胡萝卜素）含量降低,而且还引起膜蛋白组成的深刻变化,表现在大部分原有膜蛋白组分的含量在低温下明显降低,同时在低温处理的第３天诱导出一条３２．５ＫＤ的新蛋白带。与４℃处理相比,１１℃低温处理只引起了光合色素含量的降低,而对类囊体膜蛋白组成的影响不大,另外发现,两种低     

Replacement of Histidines of Light Harvesting Chlorophyll a/b Binding Protein II Disrupts Chlorophyll-Protein Complex Assembly

Eukaryotic light harvesting proteins (LHCPs) bind pigments and assemble into complexes (LHCs) that channel light energy into photosynthetic reaction centers. The structures of several prokaryotic LHCPs are known and histidines are important for the binding of the associated pigments. It has been difficult to predict how the eukaryotic LHCPs associate with pigments as the structure of the major LHCP of photosystem II is not yet known. While each LHCPII binds approximately 13 chlorophylls the protein contains only three histidines, one in each putative transmembrane helix. Experiments that use isolated pea (Pisum sativum L.) chloroplasts and mutant LHCPII synthesized in vitro show that the substitution of either an alanine or an arginine for each histidine residue inhibits some aspect of LHCII assembly. The histidine of the first membrane helix, but not the second or third, may be involved in the transport across the chloroplast envelope. No histidine alone is essential for the insertion of LHCP into thylakoid membranes, yet arginine substitutions are more inhibitory than those of alanine. The histidine replacements have their most pronounced effect on the assembly of LHCP into LHCII.     

Immunological identification and quantification of light harvesting chlorophyll a/b protein of Chlorella protothecoides

The alteration of photosynthetic membrane proteins in relation to the disappearance of pigments during the heterotrophic growth of Chlorella protothecoides was investigated. Chlorophylls and certain polypeptides associated with the LHC II disappeared after 50 hr of heterotrophic growth but the 24 kDa apoprotein constituting LHC II was not affected. Immunological analysis indicated that the chlorophylls and the light harvesting complex proteins of the thylakoid membranes are not tightly coupled and the latter is retained in its native form irrespective of the presence or absence of the former. The circumstantial evidence that the other photosynthetic membrane polypeptides are degraded along with the pigments due to increased proteolytic activity in the rapidly dividing heterotrophic cells indicate that chlorophyll synthesis is not a pre-requisite for the synthesis of the LHC II apoprotein.     

Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana.

The glycerolipid digalactosyl diacylglycerol (DGDG) is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus. Here we employ a genetic approach based on the dgd1 mutant of Arabidopsis thaliana to investigate the function of DGDG in thylakoid membranes. The primary defect in the genetically well-characterized dgd1 mutant resulted in a 90% reduction of the DGDG content. The mutant showed a decreased photosystem II (PSII) to photosystem I ratio. In vivo room- and low-temperature (77 K) chlorophyll fluorescence measurements with thylakoid preparations are in agreement with a drastically altered excitation energy allocation to the reaction centers. Quantification of pigment-binding apoproteins and pigments supports an altered stoichiometry of individual pigment-protein complexes in the mutant. Most strikingly, an increase in the amount of peripheral light-harvesting complexes of PSII relative to the inner antenna complexes and the PSII reaction center/core complexes was observed. Regardless of the severe alterations in thylakoid organization, photosynthetic oxygen evolution was virtually not compromised in dgd1 mutant leaves.     

Spectroscopic and molecular characterization of the oligomeric antenna of the diatom Phaeodactylum tricornutum

The photosynthetic antenna system of diatoms contains fucoxanthin chlorophyll a/c binding proteins (FCPs), which are membrane intrinsic proteins showing high homology to the light harvesting complexes (LHC) of higher plants. In the present study, we used a mild solubilization of P. tricornutum thylakoid membranes in combination with sucrose density gradient centrifugation or gelfiltration and obtained an oligomeric FCP complex (FCPo). The spectroscopic characteristics and pigment stoichiometries of the FCPo complex were comparable to FCP complexes that were isolated after solubilization with higher detergent per chlorophyll ratios. The excitation energy transfer between the FCP-bound pigments was more efficient in the oligomeric FCPo complexes, indicating that these complexes may represent the native form of the diatom antenna system in the thylakoid membrane. Determination of the molecular masses of the two different FCP fractions by gelfiltration revealed that the FCP complexes consisted of trimers, whereas the FCPo complexes were either composed of six monomers or two tightly associated trimers. In contrast to vascular plants, stable functional monomers could not be isolated in P. tricornutum. Both types of FCP complexes showed two protein bands in SDS-gels with apparent molecular masses of 18 and 19 kDa, respectively. Sequence analysis by MS/MS revealed that the 19 kDa protein corresponded to the fcpC and fcpD genes, whereas the 18 kDa band contained the protein of the fcpE gene. The presence of an oligomeric antenna in diatoms is in line with the oligomeric organization of antenna complexes in different photoautotrophic groups.     

Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+. II. Cell-cycle variations in the synthesis and assembly of pigment

Synthesis of the chlorophyll and the major carotenoid pigments and their assembly into thylakoid membrane have been studied throughout the 12-h light/12-h dark vegetative cell cycle of synchronous Chlamydomonas reinhardtii 137+ (wild-type). Pulse exposure of cells to radioactive acetate under conditions in which labeling accurately reflects lipogenesis, followed by cellular fractionation to purify thylakoid membrane, allowed direct analysis of the pigment synthesis and assembly attendant to thylakoid biogenesis. All pigments are synthesized and assembled into thylakoids continuously, but differentially, with respect to cell-cycle time. Highest synthesis and assembly rates are confined to the photoperiod (mid-to-late G1) and support chlorophyll and carotenoid accretion before M-phase. The lower levels at which these processes take place during the dark period (S, M, and early-to- mid G1) have been ascribed to pigment turnover. Within this general periodic pattern, pigment synthesis and assembly occur in a "multi- step" manner, i.e., by a temporally-ordered, stepwise integration of the various pigments into the thylakoid membrane matrix. The cell-cycle kinetics of pigment assembly at the subcellular level mirror the kinetics of pigment synthesis at the cellular level, indicating that pigment synthesis not only provides chlorophyll and carotenoid for thylakoid biogenesis but may also serve as a critical rate-determinant to pigment assembly.     

Structure, function and regulation of plant photosystem I

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.     

Novel mechanisms for the targeting of proteins into and across chloroplast membranes

The assembly of the photosynthetic apparatus requires the translocation of numerous proteins from the cytosol, initially into the stroma and thereafter into or across the thylakoid membrane. Recent studies have shown that proteins are transported into this membrane by a variety of mechanisms, some of which are derived from a cyanobacterial-type ancestor, whereas others have evolved in response to the more complex transport pathway used by cytosolically synthesized chloroplast proteins. It is now apparent that some of the targeting pathways are used exclusively by hydrophobic thylakoid membrane proteins; here we review recent progress in our understanding of the biogenesis of this important class of protein.     

Light stress photodynamics of chlorophyll-binding proteins in Arabidopsis thaliana thylakoid membranes revealed by high-resolution mass spectrometric studies

In higher plants the light energy is captured by the photosynthetic pigments that are bound to photosystem I and II and their light-harvesting complex (LHC) subunits. In this study, we examined the photodynamic changes within chlorophyll-protein complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to low light and subsequently exposed to light stress. Chlorophyll-protein complexes were isolated using sucrose density gradient centrifugation and blue-native polyacrylamid gel electrophoresis (BN-PAGE). Proteome analysis was performed using SDS-PAGE, HPLC and high resolution mass spectrometry. We identified several rarely expressed and stress-induced chlorophyll-binding proteins, showed changes in localization of early light-induced protein family and LHC protein family members between different photosynthetic complexes and assembled/disassembled subcomplexes under light stress conditions and discuss their role in a variety of light stress-related processes.     

Biotic stress induced demolition of thylakoid structure and loss in photoelectron transport of chloroplasts in papaya leaves.

Papaya mosaic virus (PMV) causes severe mosaic symptoms in the papaya (Carica papaya L.) leaves. The PMV-induced alterations in photosystem II (PS II) structure and photochemical functions were probed. An increase in chlorophyll a (Chl a) fluorescence polarization suggests pathogen-induced transformation of thylakoid membrane to a gel phase. This transformation in physical state of thylakoid membrane may result in alteration in topology of pigments on pigment-binding proteins as reflected in pathogen-induced loss in the efficiency of energy transfer from carotenoids to chlorophylls. The fast Chl a fluorescence induction kinetics of healthy and PMV-infected plants by F(O)-F(J)-F(I)-F(P) transients revealed pathogen-induced perturbation on PS II acceptor side electron transfer equilibrium between Q(A) and Q(B) and in the pool size of electron transport acceptors. Pathogen-induced loss in photosynthetic pigments, changes in thylakoid structure and decrease in the ratio of F(V)/F(M) (photochemical potential of PS II) further correlate with the loss in photoelectron transport of PS II as probed by 2,6-dichlorophenol indophenol (DCPIP)-Hill reaction. Restoration of the loss by 1,5-diphenyl carbazide (DPC), an exogenous electron donor, that donates electron directly to reaction centre II bypassing the oxygen evolving system (OES), leads towards the conclusion that OES is one of the major targets of biotic stress. Further, the data suggest that chlorophyll fluorescence could be used as a non-invasive handy tool to assess the loss in photosynthetic efficiency and symptom severity in infected green tissues vis-a-vis the healthy ones.     

The chloroplast signal recognition particle (CpSRP) pathway as a tool to minimize chlorophyll antenna size and maximize photosynthetic productivity

The concept of the Truncated Light-harvesting chlorophyll Antenna (TLA) size, as a tool by which to maximize sunlight utilization and photosynthetic productivity in microalgal mass cultures or high-density plant canopies, is discussed. TLA technology is known to improve sunlight-to-product energy conversion efficiencies and is hereby exemplified by photosynthetic productivity estimates of wild type and a TLA strain under simulated mass culture conditions. Recent advances in the generation of TLA-type mutants by targeting genes of the chloroplast signal-recognition particle (CpSRP) pathway, affecting the thylakoid membrane assembly of light-harvesting proteins, are also summarized. Two distinct CpSRP assembly pathways are recognized, one entailing post-translational, the other a co-translational mechanism. Differences between the post-translational and co-translational integration mechanisms are outlined, as these pertain to the CpSRP-mediated assembly of thylakoid membrane protein complexes in higher plants and green microalgae. The applicability of the CpSRP pathway genes in efforts to generate TLA-type strains with enhanced solar energy conversion efficiency in photosynthesis is evaluated.     

Structure, function and regulation of plant photosystem I

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.     

Consecutive binding of chlorophylls a and b during the assembly in vitro of light-harvesting chlorophyll-a/b protein (LHCIIb)

The apoprotein of the major light-harvesting chlorophyll a/b complex (LHCIIb) is post-translationally imported into the chloroplast, where membrane insertion, protein folding, and pigment binding take place. The sequence and molecular mechanism of the latter steps is largely unknown. The complex spontaneously self-organises in vitro to form structurally authentic LHCIIb upon reconstituting the unfolded recombinant protein with the pigments chlorophyll a, b, and carotenoids in detergent micelles. Former measurements of LHCIIb assembly had revealed two apparent kinetic phases, a faster one (tau1) in the range of 10 s to 1 min, and a slower one (tau2) in the range of several min. To unravel the sequence of events we analysed the binding of chlorophylls into the complex by using time-resolved fluorescence measurements of resonance energy transfer from chlorophylls to an acceptor dye attached to the apoprotein. Chlorophyll a, offered in the absence of chlorophyll b, bound with the faster kinetics (tau1) exclusively whereas chlorophyll b, in the absence of chlorophyll a, bound predominantly with the slower kinetics (tau2). In double-jump experiments, LHCIIb assembly could be dissected into a faster chlorophyll a and a subsequent, predominantly slower chlorophyll b-binding step. The assignment of the faster and the slower kinetic phase to predominantly chlorophyll a and exclusively chlorophyll b binding, respectively, was verified by analysing the assembly kinetics with a circular dichroism signal in the visible domain presumably reflecting the establishment of pigment-pigment interactions. We propose that slow chlorophyll binding is confined to the exclusively chlorophyll b binding sites whereas faster binding occurs to the chlorophyll a binding sites. The latter sites can bind both chlorophylls a and b but in a reversible fashion as long as the complex is not stabilised by proper occupation of the chlorophyll b sites. The resulting two-step model of LHCIIb assembly is able to reconcile the highly specific binding sites containing either chlorophyll a or b, as seen in the recent crystal structures of LHCIIb, with the observation of promiscuous binding sites able to bind both chlorophyll a and b in numerous reconstitution analyses of LHCIIb assembly.     

Native architecture and acclimation of photosynthetic membranes in a fast-growing cyanobacterium

Efficient solar energy conversion is ensured by the organization, physical association, and physiological coordination of various protein complexes in photosynthetic membranes. Here, we visualize the native architecture and interactions of photosynthetic complexes within the thylakoid membranes from a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) using high-resolution atomic force microscopy. In the Syn2973 thylakoid membranes, both photosystem I (PSI)-enriched domains and crystalline photosystem II (PSII) dimer arrays were observed, providing favorable membrane environments for photosynthetic electron transport. The high light (HL)-adapted thylakoid membranes accommodated a large amount of PSI complexes, without the incorporation of iron-stress-induced protein A (IsiA) assemblies and formation of IsiA–PSI supercomplexes. In the iron deficiency (Fe⁻)-treated thylakoid membranes, in contrast, IsiA proteins densely associated with PSI, forming the IsiA–PSI supercomplexes with varying assembly structures. Moreover, type-I NADH dehydrogenase-like complexes (NDH-1) were upregulated under the HL and Fe⁻ conditions and established close association with PSI complexes to facilitate cyclic electron transport. Our study provides insight into the structural heterogeneity and plasticity of the photosynthetic apparatus in the context of their native membranes in Syn2973 under environmental stress. Advanced understanding of the photosynthetic membrane organization and adaptation will provide a framework for uncovering the molecular mechanisms of efficient light harvesting and energy conversion.     

The yeast split-ubiquitin system to study chloroplast membrane protein interactions

Each photosynthetic complex within the thylakoid membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein–protein interactions of thylakoid membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the thylakoid membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF₀III, suggesting an important role of Alb3 in the assembly of photosynthetic thylakoid membrane complexes.     

Early steps in the assembly of light-harvesting chlorophyll a/b complex: time-resolved fluorescence measurements

The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.     

Orientation of pigments and pigment-protein complexes in the diatom Cylindrotheca fusiformis. A linear-dichroism study

The orientation of pigments and pigment-protein complexes of the marine diatom Cylindrotheca fusiformis was studied by linear dichroism at 77 K. The technique of polyacrylamide gel squeezing was used to orient the diatom intact cells, their isolated thylakoid membranes and the three pigment-protein complexes: chlorophyll ac-fucoxanthin, chlorophyll ac and PS I complexes. The data indicate that specific orientation of various pigments exists at all structure levels. Tentative assignments of various features of the linear-dichroism spectra to the major photosynthetic pigments are presented. The orientation of the three pigment-protein complexes with respect to the thylakoid membrane plane and the major axis of the cell is also discussed.