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Summary: In natural ecosystems, plants compete for space, nutrients and light. The optically dense canopies limit the penetration of photosynthetically active radiation and light often becomes a growth‐limiting factor for the understory. The reduced availability of photons in the lower leaf layers is also a major constraint for yield potential in canopies of crop monocultures. Traditionally, crop breeding has selected traits related to plant architecture and nutrient assimilation rather than light use efficiency. Leaf optical density is primarily determined by tissue morphology and by the foliar concentration of photosynthetic pigments (chlorophylls and carotenoids). Most pigment molecules are bound to light‐harvesting antenna proteins in the chloroplast thylakoid membranes, where they serve photon capture and excitation energy transfer toward reaction centers of photosystems. Engineering the abundance and composition of antenna proteins has been suggested as a strategy to improve light distribution within canopies and reduce the gap between theoretical and field productivity. Since the assembly of the photosynthetic antennas relies on several coordinated biological processes, many genetic targets are available for modulating cellular chlorophyll levels. In this review, we outline the rationale behind the advantages of developing pale green phenotypes and describe possible approaches toward engineering light‐harvesting systems. [ABSTRACT FROM AUTHOR]

The necessary reduction of greenhouse gas (GHG) emissions may lead in the future to an increase in solar irradiance (solar brightening). Anthropogenic aerosols (and their precursors) that cause solar dimming are in fact often co‐emitted with GHGs. While the reduction of GHG emissions is expected to slow down the ongoing increase in the greenhouse effect, an increased surface irradiance due to reduced atmospheric aerosol load might occur in the most populated areas of the earth. Increased irradiance may lead to air warming, favour the occurrence of heatwaves and increase the evaporative demand of the atmosphere. This is why effective and sustainable solar radiation management strategies to reflect more light back to space should be designed, tested and implemented together with GHG emission mitigation. Here we propose that new plants (crops, orchards and forests) with low‐chlorophyll (Chl) content may provide a realistic, sustainable and relatively simple solution to increase surface reflectance of large geographical areas via changes in surface albedo. This may finally offset all or part of the expected local solar brightening. While high‐Chl content provides substantial competitive advantages to plants growing in their natural environment, new plants with low‐Chl content may be successfully used in agriculture and silviculture and be as productive as the green wildtypes (or even more). The most appropriate strategies to obtain highly productive and highly reflective plants are discussed in this paper and their mitigation potential is examined together with the challenges associated with their introduction in agriculture. [ABSTRACT FROM AUTHOR]

Summary: High‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth. [ABSTRACT FROM AUTHOR]

Summary: High‐throughput RNA sequencing (RNA‐seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages – protonema, gametophore and sporophyte – allowed us to define both general transcriptional patterns and stage‐specific transcripts. As an example of variation of physico‐chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter‐laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories. [ABSTRACT FROM AUTHOR]

Reversible phosphorylation of thylakoid light-harvesting proteins is a mechanism to compensate for unbalanced excitation of photosystem I ( PSI) versus photosystem II ( PSII) under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high-light ( HL) response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in HL, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of At PBCP, antagonist of At STN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P- CP29. Based on these data, we propose that the activities of the Os STN8 kinase and the antagonistic Os PBCP phosphatase, in addition to being involved in the repair of photo-damaged PSII, are also responsible for the HL-dependent reversible phosphorylation of the inner antenna CP29. [ABSTRACT FROM AUTHOR]

Plants evolved photoprotective mechanisms in order to counteract the damaging effects of excess light in oxygenic environments. Among them, chloroplast avoidance and non-photochemical quenching concur in reducing the concentration of chlorophyll excited states in the photosynthetic apparatus to avoid photooxidation. We evaluated their relative importance in regulating excitation pressure on photosystem II. To this aim, genotypes were constructed carrying mutations impairing the chloroplast avoidance response ( phot2) as well as mutations affecting the biosynthesis of the photoprotective xanthophyll zeaxanthin ( npq1) or the activation of non-photochemical quenching ( npq4), followed by evaluation of their photosensitivity in vivo. Suppression of avoidance response resulted in oxidative stress under excess light at low temperature, while removing either zeaxanthin or PsbS had a milder effect. The double mutants phot2 npq1 and phot2 npq4 showed the highest sensitivity to photooxidative stress, indicating that xanthophyll cycle and q E have additive effects over the avoidance response. The interactions between non-photochemical quenching and avoidance responses were studied by analyzing the kinetics of fluorescence decay and recovery at different light intensities. phot2 fluorescence decay lacked a component, here named as q M. This kinetic component linearly correlated with the leaf transmittance changes due to chloroplast relocation induced by white light and was absent when red light was used as actinic source. On these basis we conclude that a decrease in leaf optical density affects the apparent non-photochemical quenching ( NPQ) rise kinetic. Thus, excess light-induced fluorescence decrease is in part due to avoidance of photon absorption rather than to a genuine quenching process. [ABSTRACT FROM AUTHOR]

BACKGROUND In 2008-2009, a survey in the Emilia Romagna region of Italy collected information on the farm use of plant protection products ( PPPs) and evaluated whether the provisions of the Directive for the Sustainable Use of Pesticides are applicable. It was concluded that the provisions can be implemented, even if some gaps need to be filled and also the behaviour of farmers needs to be improved. Moreover, it was observed that all stages in the use of PPPs on farms could generate risks for the operator and/or the environment. One of the recommendations is to promote training for operators and to adopt good agronomic practices in order to improve sustainable use of PPPs. RESULTS The findings were used, in the following years, to develop a Guideline for Sustainable Use of PPPs to help the user in identifying the flaws in current practices at farm level as well as their corresponding corrective actions. The Guidelines are accompanied by free online software to be used as a diagnostic tool as well as to provide recommendations for improvements. CONCLUSION The approach adopted, taking into account the variability in farm structure, cropping pattern, risk attitude and economic availability, is not an instrument to identify the most suitable protection strategy for a given crop in a given period, but to help professional users to improve their practices in managing PPPs on farms and to make the most appropriate choices leading to reduced environmental and human risk, without compromising the profitability of agricultural production and food standards. This work has, as an underlying principle, a holistic approach to link the different elements of the three pillars of sustainability (environment, economy and society) and to enhance knowledge, which represents one of the main aspects of the Directive. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

as1, for antenna size mutant 1, was obtained by insertion mutagenesis of the unicellular green alga Chlamydomonas reinhardtii. This strain has a low chlorophyll content, 8% with respect to the wild type, and displays a general reduction in thylakoid polypeptides. The mutant was found to carry an insertion into a homologous gene, prokaryotic arsenite transporter ( ARSA), whose yeast and mammal counterparts were found to be involved in the targeting of tail-anchored ( TA) proteins to cytosol-exposed membranes, essential for several cellular functions. Here we present the characterization in a photosynthetic organism of an insertion mutant in an ARSA-homolog gene. The ARSA1 protein was found to be localized in the cytosol, and yet its absence in as1 leads to a small chloroplast and a strongly decreased chlorophyll content per cell. ARSA1 appears to be required for optimal biogenesis of photosynthetic complexes because of its involvement in the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon ( TOC) complex, which, in turn, catalyzes the import of nucleus-encoded precursor polypeptides into the chloroplast. Remarkably, the effect of the mutation appears to be restricted to biogenesis of chlorophyll-binding polypeptides and is not compensated by the other ARSA homolog encoded by the C. reinhardtii genome, implying a non-redundant function. [ABSTRACT FROM AUTHOR]

ABSTRACT Functions of α- and β-branch carotenoids in whole-plant acclimation to photo-oxidative stress were studied in Arabidopsis thaliana wild-type (wt) and carotenoid mutants, lutein deficient ( lut2, lut5), non- photochemical quenching1 ( npq1) and suppressor of zeaxanthin- less1 ( szl1) npq1 double mutant. Photo-oxidative stress was applied by exposing plants to sunflecks. The sunflecks caused reduction of chlorophyll content in all plants, but more severely in those having high α- to β-branch carotenoid composition ( α/ β-ratio) ( lut5, szl1npq1). While this did not alter carotenoid composition in wt or lut2, which accumulates only β-branch carotenoids, increased xanthophyll levels were found in the mutants with high α/ β-ratios ( lut5, szl1npq1) or without xanthophyll-cycle operation ( npq1, szl1npq1). The PsbS protein content increased in all sunfleck plants but lut2. These changes were accompanied by no change ( npq1, szl1npq1) or enhanced capacity (wt, lut5) of NPQ. Leaf mass per area increased in lut2, but decreased in wt and lut5 that showed increased NPQ. The sunflecks decelerated primary root growth in wt and npq1 having normal α/ β-ratios, but suppressed lateral root formation in lut5 and szl1npq1 having high α/ β-ratios. The results highlight the importance of proper regulation of the α- and β-branch carotenoid pathways for whole-plant acclimation, not only leaf photoprotection, under photo-oxidative stress. [ABSTRACT FROM AUTHOR]

Although light is the source of energy for photosynthetic organisms, it causes oxidative stress when in excess. Plants and algae prevent reactive oxygen species ( ROS) formation by activation of nonphotochemical quenching ( NPQ), which dissipates excess excitation energy as heat. Although NPQ is found in both algae and plants, these organisms rely on two different proteins for its activation, Light harvesting complex stress-related (LHCSR) and Photosystem II subunit S (PSBS). In the moss Physcomitrella patens, both proteins are present and active., Several P. patens lines depleted in or over-expressing PSBS and/or LHCSR at various levels were generated by exploiting the ability of Physcomitrella to undergo homologous recombination., The analysis of the transgenic lines showed that either protein is sufficient, alone, for NPQ activation independently of the other, supporting the idea that they rely on different activation mechanisms. Modulation of PSBS and/or LHCSR contents was found to be correlated with NPQ amplitude, indicating that plants and algae can directly modulate their ability to dissipate energy simply by altering the accumulation level of one or both of these proteins., The availability of a large range of P. patens genotypes differing in PSBS and LHCSR content allowed comparison of their activation mechanisms and discussion of implications for the evolution of photoprotection during land colonization. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms respond to strong illumination by activating several photoprotection mechanisms. One of them, non-photochemical quenching (NPQ), consists in the thermal dissipation of energy absorbed in excess. In vascular plants NPQ relies on the activity of PSBS, whereas in the green algae Chlamydomonas reinhardtii it requires a different protein, LHCSR. The moss Physcomitrella patens is the only known organism in which both proteins are present and active in triggering NPQ, making this organism particularly interesting for the characterization of this protection mechanism. We analysed the acclimation of Physcomitrella to high light and low temperature, finding that these conditions induce an increase in NPQ correlated to overexpression of both PSBS and LHCSR. Mutants depleted of PSBS and/or LHCSR showed that modulation of their accumulation indeed determines NPQ amplitude. All mutants with impaired NPQ also showed enhanced photosensitivity when exposed to high light or low temperature, indicating that in this moss the fast-responding NPQ mechanism is also involved in long-term acclimation. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

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. [Copyright &y& Elsevier]

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 FROM AUTHOR]

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 FROM AUTHOR]

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 α- or β-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 β-carotene. In chy1chy2lut5, β-carotene hydroxylation is completely abolished, while hydroxylation of the β-ring of α-carotene is still observed. The data are consistent with a role of LUT5 in β-ring hydroxylation, and with the existence of an additional hydroxylase, acting on the β-ring of α-, but not β-carotene. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Plant thylakoids have a highly conserved xanthophyll composition, consisting of β-carotene, lutein, neoxanthin and a pool of violaxanthin that can be converted to antheraxanthin and zeaxanthin in excess light conditions. Recent work has shown that xanthophylls undergo dynamic changes, not only in their composition but also in their distribution among Lhc proteins. Xanthophylls are released from specific binding site in the major trimeric LHCII complex of photosystem II and are subsequently bound to different sites into monomeric Lhcb proteins and dimeric Lhca proteins. In this work we review available evidence from in vivo and in vitro studies on the structural determinants that control xanthophyll exchange in Lhc proteins. We conclude that the xanthophyll exchange rate is determined by the structure of individual Lhc gene products and it is specifically controlled by the lumenal pH independently from the activation state of the violaxanthin de-epoxidase enzyme. The xanthophyll exchange induces important modifications in the organization of the antenna system of Photosystem II and, possibly of Photosystem I. Major changes consist into a modulation of the light harvesting efficiency and an increase of the protection from lipid peroxidation. The xanthophyll cycle thus appears to be a signal transduction system for co-ordinated regulation of the photoprotection mechanisms under persistent stress from excess light. [ABSTRACT FROM AUTHOR]

Examines the effects of the recombinant photosystem II antenna complex on the zeaxanthin and phosphorylation. Formation of the chlorophyll emission; Decrease of the fluorescence quantum yields; Enhancement of the nonradiative deactivation.

Studies the occurrence and organization of photosystem II in bundle sheath thylakoids and stroma lamellae from maize. Fluorescence induction; Photosystem II aggregation state; Electron transfer; Induction kinetics.

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 heal [Demmig-Adams, B. (1991) Biochim. Biophys. Acta 1020, 1–241 under conditions of high transmembrane pH gradient [Giimore, 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 β-carotene as the only carotenoid. However, the two other chlorophyll-a-binding proteins of photosystem II, CP47 and CP43, bind not only β-carotene, but also the xanthophyll lutein, previously thought to be restricted to chlorophyll-a/b proteins. [ABSTRACT FROM AUTHOR]

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-LH CII complex. The LHCII subpopulations of the complex arc 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 FROM AUTHOR]

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 threequarters 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... [ABSTRACT FROM AUTHOR]

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 m 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. [ABSTRACT FROM AUTHOR]

A new photosystem II preparation was isolated from Marchantia polymorpha thylakoids upon solubilization with dodecyl β-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]. lt 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 FROM AUTHOR]

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 FROM AUTHOR]

The light-harvesting proteins (LHC) constitute a multigene family including, in higher plants, at least 12 members whose location, within the photosynthetic membrane, relative abundance and putative function appear to be very different. The major light-harvesting complex of photosystem II (LHCII) is the most abundant membrane protein in the biosphere and fulfil a constitutive light-harvesting function for photo- system H while the early light-induced proteins (ELIPs) are expressed in low amounts under stress conditions. Primary sequence analysis suggests that all these proteins share a common structure which was resolved at 3.7 Å resolution by electron crystallography in the case of the major LHCII complex: Three transmembrane helices connected by hydrophilic loops coordinate seven chlorophyll a and five chlorophyll b molecules by histidine, glutamine, asparagine lateral chains as well as by charge compensated ionic pairs of glutamic acid and arginine residues; moreover, at least two xantophyll molecules are located at the centre of the structure in close contact with seven porphyrins, tentatively identified as chlorophyll a. The antenna system is also involved in the regulation of excitation energy transfer to reaction centre II. This function has been attributed to three members of the protein family, namely CP29, CP26 and CP24 (also called minor chlorophyll proteins) which have been recently characterised and shown to bind most of the xantophyll cycle carotenoids, thus suggesting that the non-photochemical quenching mechanism is acting in these proteins. Further support to this assignment comes from the recent identification of protonation sites in CP29 and CP26 by covalent dicyclohexhylcarbodiimide binding suggesting that these respond to low lumenal pH. In addition, CP29 is reversibly phosphorylated under light and cold stress conditions, undergoing confoiniational change, supporting the hypothesis that these subunits, present in low amounts in photosystem II, have a major regulatory role in the light-harvesting function and are thus important in environmental stress resistance. [ABSTRACT FROM AUTHOR]

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., Rumler, U. & Rudiger, 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 FROM AUTHOR]

Photosystem II membrane fractions from dark-adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl β-D-maltoside. Chlorophyll-binding proteins from photosystem Il were isolated either by ultracentrifugation on a sucrose gradient, or by fiat 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 Il 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 FROM AUTHOR]

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 FROM AUTHOR]