Major Light-harvesting Complex Research Articles

Three-Dimensional Structure of Spinach Major Light-Harvesting Complex

Three-Dimensional Structure of Spinach Major Light-Harvesting Complex

Light Absorption by the Chlorophyll a–b Complexes of Photosystem II in a Leaf with Special Reference to LHCII¶

To investigate the light-harvesting properties of the Photosystem II chlorophyll (chl) a-b complexes (major light-harvesting complex of Photosystem II [LHCII], CP24, CP26, CP29) in a mature leaf under natural "daylight" illumination, the absorption spectra of the isolated complexes were converted into the photon absorption spectrum (1-T) within a leaf, using the approach of Rivadossi et al. ([1999] Photosynth. Res. 60, 209-215). In the Qy region, significant enhancement of light harvesting by the chl b electronic transitions, with respect to the absorption spectra (optical density [OD]), as well as a large and generalized increase (between two- and four-fold) associated with the vibrational bands of both chl a and b, was observed, which acquires an important light-harvesting role (approximately 30-40% of total). In the Soret region, a small increase in light harvesting by chl b was indicated. To gain more detailed information on these aspects the light harvesting of LHCII in a leaf was investigated. This required describing the pigment absorption (chl a and b, carotenoids) in the LHCII OD spectrum in terms of spectral subbands, which were subsequently used to estimate the relative light harvesting of each pigment type in LHCII of a leaf. When the entire visible spectral interval between 400 and 730 nm is considered, the chl a light harvesting is essentially unchanged with respect to the absorption spectrum (OD) of isolated LHCII, whereas the chl b contribution is 20% higher and the carotenoids are 33% lower. The relative enhancement of the chl b absorption is principally associated with the Qy electronic transition region, the light-harvesting contribution of which becomes prominent in the leaf.

A genome?s-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a valuable model system for defining the structure and function of polypeptides of the photosynthetic apparatus and the dynamic aspects of photosynthesis. Recently, a genome-wide analysis of cDNAs and a draft genome sequence that covers approximately 90% of the genome were made available, providing a clear picture of the composition of specific gene families, the relationships among the gene family members, and the location of each member on the genome. We used the available sequence information to analyze the extensive family of light-harvesting genes in C. reinhardtii. There are nine genes encoding polypeptides of the major light-harvesting complex of photosystem II, two genes encoding the minor light-harvesting polypeptides of photosystem II, and nine genes encoding polypeptides predicted to comprise the photosystem I light-harvesting complex. Furthermore, there are five genes encoding early light-induced proteins and two genes encoding LI818 polypeptides. A candidate for the PsbS gene has also been found in the raw genome sequence data (Niyogi, personal communication), although no genes encoding homologues of the Sep, or Hli polypeptides have been identified. In this manuscript, we identify and classify the family of light-harvesting polypeptides encoded on the C. reinhardtii genome. This is an important first step in designing specific genetic, biochemical, and physiological studies aimed at characterizing the composition, function, and regulation of the light-harvesting complexes.

Absence of the Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II - effects on photosynthesis, grana stacking and fitness.

We have constructed Arabidopsis thaliana plants that are virtually devoid of the major light-harvesting complex, LHC II. This was accomplished by introducing the Lhcb2.1 coding region in the antisense orientation into the genome by Agrobacterium-mediated transformation. Lhcb1 and Lhcb2 were absent, while Lhcb3, a protein present in LHC II associated with photosystem (PS) II, was retained. Plants had a pale green appearance and showed reduced chlorophyll content and an elevated chlorophyll a/b ratio. The content of PS II reaction centres was unchanged on a leaf area basis, but there was evidence for increases in the relative levels of other light harvesting proteins, notably CP26, associated with PS II, and Lhca4, associated with PS I. Electron microscopy showed the presence of grana. Photosynthetic rates at saturating irradiance were the same in wild-type and antisense plants, but there was a 10-15% reduction in quantum yield that reflected the decrease in light absorption by the leaf. The antisense plants were not able to perform state transitions, and their capacity for non-photochemical quenching was reduced. There was no difference in growth between wild-type and antisense plants under controlled climate conditions, but the antisense plants performed worse compared to the wild type in the field, with decreases in seed production of up to 70%.

Zeaxanthin accumulation in the absence of a functional xanthophyll cycle protects Chlamydomonas reinhardtii from photooxidative stress.

Xanthophylls participate in light harvesting and are essential in protecting the chloroplast from photooxidative damage. To investigate the roles of xanthophylls in photoprotection, we isolated and characterized extragenic suppressors of the npq1 lor1 double mutant of Chlamydomonas reinhardtii, which lacks zeaxanthin and lutein and undergoes irreversible photooxidative bleaching and cell death at moderate to high light intensities. Here, we describe three suppressor strains that carry point mutations in the coding sequence of the zeaxanthin epoxidase gene, resulting in the constitutive accumulation of zeaxanthin in a range of concentrations. The presence of zeaxanthin in these strains was sufficient to prevent photooxidative damage in the npq1 lor1 background. The size of the light-harvesting antenna in the suppressors decreased in high light in a manner that was proportional to the relative content of zeaxanthin, with the strain having the most zeaxanthin showing a severe reduction in levels of the major light-harvesting complex II proteins in high light. We show that the effect of constitutive zeaxanthin on light harvesting is not the main cause of increased photoprotection, because in the absence of zeaxanthin, a strain with a smaller light-harvesting antenna showed only minor protection against photobleaching in high light. Furthermore, the zeaxanthin-accumulating suppressors were able to tolerate higher levels of exogenous reactive oxygen than their parental strain under conditions that did not affect light harvesting. Our results are consistent with an antioxidant role of zeaxanthin in the quenching of singlet oxygen and/or free radicals in the thylakoid membrane in vivo.

Determination of Relative Chlorophyll Binding Affinities in the Major Light-harvesting Chlorophyll a/b Complex

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.

A molecular understanding of complementary chromatic adaptation.

Photosynthetic activity and the composition of the photosynthetic apparatus are strongly regulated by environmental conditions. Some visually dramatic changes in pigmentation of cyanobacterial cells that occur during changing nutrient and light conditions reflect marked alterations in components of the major light-harvesting complex in these organisms, the phycobilisome. As noted well over 100 years ago, the pigment composition of some cyanobacteria is very sensitive to ambient wavelengths of light; this sensitivity reflects molecular changes in polypeptide constituents of the phycobilisome. The levels of different pigmented polypeptides or phycobiliproteins that become associated with the phycobilisome are adjusted to optimize absorption of excitation energy present in the environment. This process, called complementary chromatic adaptation, is controlled by a bilin-binding photoreceptor related to phytochrome of vascular plants; however, many other regulatory elements also play a role in chromatic adaptation. My perspectives and biases on the history and significance of this process are presented in this essay.

The major light-harvesting pigment protein of Acaryochloris marina

The major light-harvesting protein complex containing chlorophyll (Chl) d was isolated from Acaryochloris marina thylakoid membranes. Isolation was achieved by detergent solubilisation followed by separation on 6–40% sucrose gradients using ultracentrifugation. The best Chl d yield (70%) used 0.3% dodecyl maltoside, 0.15% octyl glucoside, 0.05% zwittergent 3-14 with the detergent:total Chl d ratio around 10:1 (w/w). Characterisation of the light-harvesting pigment protein complex (lhc) involved non-denaturing electrophoresis, SDS–PAGE, absorbance and fluorescence spectroscopy. The main polypeptide in the lhc was shown to be ca. 34 kDa and to contain Chl d and Chl a, indicating that the Acaryochloris lhc is similar to that of prochlorophytes. The Chl a level varied with the culture conditions, which is consistent with previous findings.

Functional architecture of the major light-harvesting complex from higher plants

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.

The Major Antenna Complex of Photosystem II Has a Xanthophyll Binding Site Not Involved in Light Harvesting

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.

Acclimation of Arabidopsis thaliana to the light environment: the existence of separate low light and high light responses

The capacity for photosynthetic acclimation in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was assessed during growth over a broad range of irradiance. Discontinuities in the response to growth irradiance were revealed for the light- and CO2-saturated rate of photosynthesis (Pmax) and the ratio of chlorophyll a to chlorophyll b (Chl a/b). Three separate phases in the response of Pmax and Chl a/b to growth light were evident, with increases at low and high irradiance ranges and a plateau at intermediate irradiance. By measuring all chlorophyll-containing components of the thylakoid membrane that contribute to Chl a/b we reveal that distinct strategies for growth at low and high irradiance underlie the discontinuous response. These strategies include, in addition to changes in the major light-harvesting complexes of photosystem II (LHCII), large shifts in the amounts of both reaction centres as well as significant changes in the levels of minor LHCII and LHCI components.

Differential expression of antenna and core genes in Prochlorococcus PCC 9511 (Oxyphotobacteria) grown under a modulated light-dark cycle.

The continuous changes in incident solar light occurring during the day oblige oxyphototrophs, such as the marine prokaryote Prochlorococcus, to modulate the synthesis and degradation rates of their photosynthetic components finely. How this natural phenomenon influences the diel expression of photosynthetic genes has never been studied in this ecologically important oxyphotobacterium. Here, the high light-adapted strain Prochlorococcus sp. PCC 9511 was grown in large-volume continuous culture under a modulated 12 h-12 h light-dark cycle mimicking the conditions found in the upper layer of equatorial oceans. The pcbA gene encoding the major light-harvesting complex showed strong diel variations in transcript levels with two maxima, one before the onset of illumination and the other near the end of the photoperiod. In contrast, the mRNA level of psbA (encoding the reaction centre II subunit D1), the monocistronic transcript of psbD (encoding D2) and the dicistronic transcript of psbDC were all tightly correlated with light irradiance, with a minimum at night and a maximum at noon. The occurrence of a second peak during the dark period for the monocistronic transcript of psbC (encoding one of the PS II core Chl a antenna proteins) suggested the involvement of post-transcriptional regulation. Differential expression of the external antenna and core genes may constitute a mechanism of regulation of the antenna size to cope with the excess photon fluxes that Prochlorococcus cells experience in the upper layer of oceans around midday. The 5' ends of all transcripts were mapped, and a conserved motif, 5'-TTGATGA-3', was identified within the putative psbA and pcbA promoters.

Carotenoid-to-Chlorophyll Energy Transfer in Recombinant Major Light-Harvesting Complex (LHCII) of Higher Plants. I. Femtosecond Transient Absorption Measurements

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 ∼50 and 75 fs for the two luteins, and ∼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.

The major light-harvesting protein complex in Acaryochloris marina

The major light-harvesting protein complex with chlorophyll d has been isolated from Acaryochloris marina. The separation was achieved by solubilising the thylakoid membranes with a mixture of detergents: dodecyl maltoside, octyl glucoside and zwittergent 3-14; the best solubilisation rate was ca.70% with the detergent:chlorophyll ratio of 10:1. Once solubilised, the chlorophyll-binding complexes were separated using linear sucrose density gradients (6-40%) and ultracentrifuged at 150 000 x g for 16 h. The major light-harvesting complexes from the gradients were then characterised using non-denaturing and SDS-PAGE electrophoresis; the pigment content was estimated by HPLC and spectral analysis used both absorbance and fluorescence spectroscopy. The data indicate that the main polypeptide of the light-harvesting complex is ca. 35kDa, and the main pigment is chlorophyll d; however, carotenoids and a small amount of chlorophyll a are present. The level of chlorophyll a appears to be variable depending on the culture conditions.

Changes in chlorophyll spectral characteristics in rye seedlings grown under heavy metal stress

Heavy metals produce many severe toxic effects while taken up by plants in higher concentrations. Kupper et al. (1996, 1998) demonstrated that the substitution of the central Mg ion in chlorophyll by heavy metals in vivo is one of the most important damages observed in the photosynthetic apparatus of stressed plants. Here we present the results of the effects of Cd, Cu, Hg, Ni, Pb and Zn on spectral characteristics of chlorophyll in rye seedlings grown under heavy metals stress. Heavy metals were applied to the nutrient medium at 500 mM concentration after 4 days of growth at light intensity of 150 mmol m-2s-1, 16/8 h day/night regime. After next 7 days of growth at the above conditions second leaves were subjected to subsequent analyses. Absorption, fluorescence induction and emission spectra were taken from the leaves, isolated chloroplasts and chlorophyll extracted from metal-treated seedlings. We also performed the measurements of chlorophyll a fluorescence induction kinetics by the modulated fluorescence technique. Chlorophyll spectral changes, disturbances in energy transfer in photosynthetic antennae, as well as changes in structural properties of the major light-harvesting complex (LHCII) were observed in heavy-metal treated plants. Kupper H, Kupper F, Spiller M, 1996. J.Exp.Bot. 47: 259-266. Kupper H, Kupper F, Spiller M, 1998. Photosynth. Res. 58: 123-133.

Functional studies on the small Cab-like proteins of Synechocystis sp. PCC 6803

In contrast to higher plants, cyanobacteria appear to lack chlorophyll b as well as multi-helix Cab proteins. The major peripheral light-harvesting complex in cyanobacteria is the phycobilisome, which is associated with the thylakoid membrane. But recently a small protein of 8 kDa was identified in the cyanobacterium Synechococcus sp. PCC 7942. This protein is predicted to have a single membrane-spanning helix, which shows significant sequence similarity to the first and third membrane-spanning region of proteins belonging to the cab family (Dolganov et al., Proc. Natl. Sci. USA 92, 636-640, 1995). By homologous searching, five genes have been identified in cyanobacterium Synechocystis PCC 6803, which are named as small cab like proteins (Scps). In view of their primary structure, these Scps may be involved in pigment binding (Funk and Vermaas, Biochemistry, 38, 9397-9404, 1999). In vitro reconstitution methods involved in overexpression of these proteins in E. coli and reconstituting the proteins with pigments extracted from cyanobacteria are adopted to test if these proteins bind pigment. In vivo overexpression of these proteins with His tag in cyanobacteria and then isolation of these proteins are also under way. Thus, we are allowed to investigate if the Scps are really involved in pigment binding and indeed a so far not detected family of additional antenna proteins in cyanobacteria. As different Scp mutants are available, we will also be able to find the conditions under which these proteins bind pigment.

Understanding the Energy Transfer Function of LHCII, the Major Light-Harvesting Complex of Green Plants

Since the crystal structure of the major light-harvesting complex II (LHCII) of green plants was obtained by Kühlbrandt, Wang and Fujiyoshi (Nature 1994, 367, 614−621), this chlorophyll-containing trimeric membrane protein has been the subject of intensive investigation. The complex contains between 36 and 42 chlorophyll molecules per trimer (Chl a and Chl b) and 10 to 12 xanthophyll molecules (lutein, neoxanthin and violaxanthin). The protein displays a rich spectrum of interactions, both between pigments and between the pigments and the protein, and these interactions have been studied with a multitude of different techniques. In this article we present an overview of the most important experimental results that have become available over the past decade and relate these to the structural knowledge. Emphasis will be put on the pigment identities, their spectroscopic features, and the interactions between the pigments, which determine both steady-state (polarized) properties and singlet and triplet energy transfer dynamics. Remaining questions will be pinpointed and hopefully they can help direct research in the near future.

Folding, assembly, and stability of the major light-harvesting complex of higher plants, LHCII, in the presence of native lipids.

The influence of thylakoid lipids on the association kinetics and thermal stability of the major light-harvesting complex of photosytem II (LHCII) has been studied in vitro. The apoprotein, light-harvesting chlorophyll a/b-binding protein (Lhcb1), can be refolded and complexed with pigments in detergent solution even in the absence of lipids. Two thylakoid lipids, phosphatidyl glycerol and digalactosyl diacylglycerol, are known to interact specifically with LHCII in vivo. Here we show that both of these lipids, as well as monogalactosyl diacylglycerol, stabilize reconstituted LHCII toward thermal denaturation. Two slow kinetic phases are connected with the establishment of energy transfer between chlorophyll b and chlorophyll a and, thus, are thought to reflect the formation of the pigment-protein complex with tightly coupled chlorophylls. The lipids studied here all have the same effect on the rate of complex assembly in vitro and slow these two kinetic phases by the same degree. Both kinetic phases also slow when reactant concentrations are decreased, suggesting that the corresponding reaction step(s) involve(s) pigment binding.

Generation of fluorescence quenchers from the triplet states of chlorophylls in the major light-harvesting complex II from green plants.

Laser flash-induced changes of the fluorescence yield were studied in aggregates of light-harvesting complex II (LHCII) on a time scale ranging from microseconds to seconds. Carotenoid (Car) and chlorophyll (Chl) triplet states, decaying with lifetimes of several microseconds and hundreds of microseconds, respectively, are responsible for initial light-induced fluorescence quenching via singlet-triplet annihilation. In addition, at times ranging from milliseconds to seconds, a slow decay of the light-induced fluorescence quenching can be observed, indicating the presence of additional quenchers generated by the laser. The generation of the quenchers is found to be sensitive to the presence of oxygen. It is proposed that long-lived fluorescence quenchers can be generated from Chl triplets that are not transferred to Car molecules. The quenchers could be Chl cations or other radicals that are produced directly from Chl triplets or via Chl triplet-sensitized singlet oxygen. Decay of the quenchers takes place on a millisecond to second time scale. The decay is slowed by a few orders of magnitude at 77 K indicating that structural changes or migration-limited processes are involved in the recovery. Fluorescence quenching is not observed for trimers, which is explained by a reduction of the quenching domain size compared to that of aggregates. This type of fluorescence quenching can operate under very high light intensities when Chl triplets start to accumulate in the light-harvesting antenna.

Contribution of LHC II complex to the electric properties of thylakoid membranes: an electric light scattering study of Chl b-less barley mutant

Electric light scattering measurements demonstrate a strong decline in the permanent electric dipole moment and electric polarizability of both thylakoid membranes and photosystem II-enriched particles of the Chlorina f2 mutant which has severely reduced levels of light-harvesting chlorophyll a/ b-binding proteins compared to the wild type barley chloroplasts. The shift in the electric polarizability relaxation to higher frequencies in thylakoids and photosystem II particles from Chlorina f2 reflects higher mobility of the interfacial charges of the mutant than that of the wild type membranes. The experimental data strongly suggest that the major light-harvesting complex of photosystem II directly contribute to the electric properties of thylakoid membranes.