Thylakoid Protein Complexes Research Articles

Isolation of high-chlorophyll-fluorescence mutants of Arabidopsis thaliana and their characterisation by spectroscopy, immunoblotting and northern hybridisation.

Thirty-four recessive photosynthetic mutants of the high-chlorophyll-fluorescence (hcf) phenotype have been isolated by screening 7700 M2 progenies of ethyl methane sulfonate-treated seeds of Arabidopsis thaliana. Most of the mutants isolated were found to be seedling-lethal, but could be grown on sucrose-supplemented media. Chlorophyll (Chl) fluorescence induction, absorption changes in the reaction-centre chlorophyll of PS I (P700) at 830 nm and Chl a/Chl b ratios were recorded in order to probe the photosynthetic functions and to define the mutational lesion. These studies were complemented by immunoblot and Northern analyses which finally led to the classification of the mutants into six different groups. Four classes of mutants were affected in PS I, PS II (two different classes) or the intersystem electron-transport chain, respectively. A fifth mutant class was of pleiotropic nature and the sixth class comprised a Chl b-deficient mutant. Several of the mutants showed severe deficiencies in the levels of subunits of PS I, PS II or the cytochrome b6/f complex. Thus the mutational lesions could be located precisely. Only one mutant was defective in the transcript patterns of some plastid-encoded photosynthesis genes. Hence most of the mutants isolated appear to be affected in translational and post-translational regulatory processes of thylakoid membrane biogenesis or in structural genes encoding constituent subunits of the thylakoid protein complexes.

Complex formation in plant thylakoid membranes. Competition studies on membrane protein interactions using synthetic peptide fragments

Thylakoid membranes of pea were used to study competition between extra-membrane fragments and their parental membrane-bound proteins. Phosphorylated and unphosphorylated fragments of light harvesting complex II (LHC II) from higher plants were used to compete with LHC II for interactions with itself and with other thylakoid protein complexes. Effects of these peptide fragments of LHC II and of control peptides were followed by 80 K chlorophyll fluorescence spectroscopy of isolated thylakoids. The phosphorylated LHC II fragment competes with membrane-bound phosphoproteins in the phosphatase reaction. The same fragment accelerates the process of dark-to-light adaptation and decreases the rate of the light-to-dark adaptation when these are followed by fluorescence spectroscopy. In contrast, the non-phosphorylated LHC II peptide does not affect the rate of adaptation but produces results consistent with inhibition of formation of a quenching complex. In this quenching complex we propose that LHC II remains inaccessible to the LHC II kinase, explaining an observed decrease in LHC II phosphorylation in the later stages of the time-course of phosphorylation. The most conspicuous protein which is steadily phosphorylated during the time-course of phosphorylation is the 9 kDa (psbH) protein. The participation of the phosphorylated form of psbH in the quenching complex, where it is inaccessible to the phosphatase, may explain its anomalously slow dephosphorylation. The significance of the proposed complex of LHC II with phospho-psbH is discussed.

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b 6/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.

Uniconazole‐induced thermotolerance in wheat seedlings is mediated by transpirational cooling

Seed treatment of thermotolerant and sensitive cultivars of wheat (Triticum aestivum L. cv. Frederick and Glenlea, respectively) with uniconazole reduced shoot and increased root fresh and dry weights. When subjected to 48°C for 6 h, treated seedlings had lower leaf‐temperatures and overall higher rates of evapotranspiration. Percent survival 11 days after high temperature incubation in untreated seedlings was 53% in Frederick and 30% in Glenlea, whereas in uniconazole‐treated seedlings it was 94% and 80%, respectively. Transpiration resistance markedly increased in the control cultivars after 4 h of high‐temperature incubation, but remained relatively unchanged in the treated cultivars. The increased root to shoot ratio exhibited in treated plants may have alleviated guard cell stress, even under high evaporative demand. Chlorophyll fluorescence measurements on leaves of heat‐stressed seedlings indicated that increased photosynthetic metabolism in treated seedlings was correlated with lower leaf temperatures. High‐temperature stress resulted in a dramatic decline in pigments and proteins in thylakoid extracts of control seedlings but not in extracts from treated seedlings. Integrity of thylakoid pigment protein complexes, as illustrated by reduced relative amounts of free pigment, was maintained after exposure to high temperatures in treated seedlings. Furthermore, the treated Glenlea seedlings displayed a reduction in the monomeric form of Sight‐harvesting chlorophyll protein II (LHCP II) compared to control and Frederick seedlings. The heat‐tolerant Frederick cultivar showed a greater protective effect from uniconazole treatment than the sensitive Glenlea cultivar. Uniconazole treatment did not affect heat‐shock protein (HSP) synthesis in mesocotyl tissue.

Predictions of Mn and Fe use efficiencies of phototrophic growth as a function of light availability for growth and of C assimilation pathway

SUMMARYIron is involved in many photosynthetic, respiratory and nitrogen assimilation reactions of plants as Fe bound tightly to polypeptides catalysing redox reactions. Manganese is involved as tightly bound Mn in photoreaction II of photosynthesis and in certain superoxide dismutases, while loosely bound Mn2+ is the unique activator of some enzymes, and is an alternative to Mg2+ in activating many enzymes. This paper uses data on the quantitative role of Fe and Mn in catalysts to predict the efficiency with which Fe and Mn are used in C assimilation [mol C assimilated (mol catalytic metal in enzyme)−1 s−1] and the metal cost of C assimilation [mol catalytic metal in enzyme (mol C assimilated)−1 s−1] in photolithotrophic growth in relation to genetic and environmental variables. The genetic variables were the relative content of thylakoid proteins in major taxa (cyanobacteria and red algae, chlorophytes and chromophytes) and smaller‐scale taxonomic differences (various subtypes of C4 metabolism, and C3 metabolism, in terrestrial vascular plants). The environmental variables were the range of photon flux densities in which photolithotrophic growth of O2 evolvers can occur, and the inorganic C supply conditions controlling the repression/de‐repression of the inorganic C concentrating mechanism in cyanobacteria and microalgae.The results of the computations yield the following conclusions. The largest predicted difference in Fe and Mn costs of photolithotrophic growth is related to changes in the photon flux density for growth. The predicted Fe cost increased 50‐fold, and the Mn cost increased 80‐fold, at the lowest extreme of photon flux density compared to the highest found naturally. The increase is partly countered by the larger ratio of light‐harvesting pigments to thylakoid protein complexes assumed for the cells grown at low photon flux densities, although the extent of the increase in photosynthetic unit size is limited by considerations of efficiency of excitation energy transfer. However, the major influences are the higher pigment content in biomass enabling a larger fraction of incident light to the absorbed, and the sub‐maximal specific reaction rates of redox catalysts (whose content is constrained via excitation energy transfer considerations) at very low photon flux densities. A smaller difference, four‐fold or less, in Fe and Mn costs of photolithotrophic growth, is predicted by comparing major taxa (cyanobacteria plus red algae; chlorophytes plus chromophytes) with contrasting ratios of thylakoid redox catalysts. Differences in Fe and Mn costs of growth of less than two‐fold are predicted when the Fe‐ and Mn‐efficient organisms with CO2− concentrating mechanisms (C4 land plants; algae with active inorganic C influx) and high requirements for ATP relative to NADPH, are compared with organisms relying on CO2 diffusion from air or air‐equilibrated solutions and C3 biochemistry.These predictions of variations in Fe and Mn costs of photolithotrophy have implications for the ecology of phototrophs.

Lipid-protein interactions in stacked and destacked thylakoid membranes and the influence of phosphorylation and illumination. Spin label ESR studies

The effects of membrane destacking, protein phosphorylation, and continuous illumination have been studied in pea thylakoid membranes using ESR spectroscopy of an incorporated spin-labelled phosphatidylglycerol. This spin-labelled analogue of an endogenous thylakoid lipid has previously been shown to exhibit a selectivity of interaction with thylakoid proteins. Neither destacking, phosphorylation nor illumination was found to change the ESR spectra appreciably, suggesting that for phosphatidylglycerol at least, neither the number of protein-associated membrane lipids nor their pattern of selectivity was altered. The redistribution of the thylakoid protein complexes in the membrane, under these various conditions, therefore takes place with conservation of the properties of the lipid/protein interface.

Endopeptidases in the Stroma and Thylakoids of Pea Chloroplasts

Three endopeptidases (EP2, EP3, and EP4) were identified after fractionation of pea (Pisum sativum, var Feltham First) chloroplast stromal extracts. All three were identified by their ability to cleave in vitro-synthesized preplastocyanin to lower molecular weight forms. EP2 is inhibited by phenylmethylsulfonylfluoride, and both EP2 and EP3 are inhibited by the heavy metal chelators 1,10-phenanthroline and EDTA. A further endopeptidase, EP5, was identified in Triton X-100 extracts of thylakoid membranes. Experiments involving contrifugation through a sucrose pad indicate that EP5 either has a high molecular weight or is associated with a thylakoid protein complex. EP5 is effectively inhibited by phenylmethylsulfonylfluoride, iodoacetate, and 1,10-phenanthroline, but not by EGTA. The implications of these results for the analysis of chloroplast protein maturation are discussed, and an improved protocol for the purification of the stromal processing peptidase is described which ensures the removal of EP2, the most active of the stromal peptidases analysed in this study.

Deletion Mutagenesis of the Cytochrome b559 Protein Inactivates the Reaction Center of Photosystem II.

In green plant-like photosynthesis, oxygen evolution is catalyzed by a thylakoid membrane-bound protein complex, photosystem II. Cytochrome b559, a protein component of the reaction center of this complex, is absent in a genetically engineered mutant of the cyanobacterium, Synechocystis 6803 [Pakrasi, H.B., Williams, J.G.K., and Arntzen, C.J. (1988). EMBO J. 7, 325-332]. In this mutant, the genes psbE and psbF, encoding cytochrome b559, were deleted by targeted mutagenesis. Two other protein components, D1 and D2 of the photosystem II reaction center, are also absent in this mutant. However, two chlorophyll-binding proteins, CP47 and CP43, as well as a manganese-stabilizing extrinsic protein component of photosystem II are stably assembled in the thylakoids of this mutant. Thus, this deletion mutation destabilizes the reaction center of photosystem II only. The mutant also lacks a fluorescence maximum peak at 695 nm (at 77 K) even though the CP47 protein, considered to be the origin of this fluorescence peak, is present in this mutant. We propose that the fluorescence at 695 nm originates from an interaction between the reaction center of photosystem II and CP47. The deletion mutant shows the absence of variable fluorescence at room temperature, indicating that its photosystem II complex is photochemically inactive. Also, photoreduction of QA, the primary acceptor quinone in photosystem II, could not be detected in the mutant. We conclude that cytochrome b559 plays at least an essential structural role in the reaction center of photosystem II.

Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator.

The maize photosynthetic mutant hcf106 has a distinctive and unusual thylakoid membrane organization, and fails to accumulate three of the five thylakoid membrane protein complexes. This mutant arose in a Robertson's Mutator background, and shows somatic instability typical of a transposon-induced allele. In addition, hcf106 is suppressed when Mu1 elements are inactive and modified in their terminal inverted repeats. Thus plants homozygous for the mutant allele adopt a mutant phenotype only when Mu1 elements are active and unmodified. DNA from the mutant allele has been cloned by 'transposon-tagging' using the transposon Mu1, and the identity of the clone confirmed by observing somatic excision of the transposon in a revertant sector. A 1.2 kb transcript homologous to the cloned DNA is found in wild-type and suppressed seedlings, but is not found in mutant seedlings, suggesting that suppression is mediated at the level of transcript accumulation.

Galactolipids of Thylakoid Pigment Protein Complexes Separated Electrophoretically from Thylakoids of Dunaliella salina Labeled with Radioactive Fatty Acids

Experiments have been conducted to determine whether mono- and digalactosyldiacylglycerols (MGDG and DGDG) labeled by short term exposure of the green alga Dunaliella salina to exogenous radioactive lauric, myristic, or palmitic acids are preferentially recovered in one or more thylakoid protein complexes separated by electrophoresis on nondenaturing gels. A selective retention of certain lipid classes and molecular species by the major electrophoretically separable fractions, oligomeric form (LHCP-1) and monomeric form (LHCP-3) of hight-harvesting chlorophyll a/b protein complex, and major photosystem I protein complex (CPla), and nonprotein-bound lipids, was indicated. After 5 min of labeling with (3)H-palmitate the specific radioactivities of MGDG and DGDG molecular species recovered from LHCP-3 and CPla were slightly higher and lower, respectively, than specific radioactivities of equivalent molecular species from LHCP-1 and nonprotein-bound lipid. However, the differences were smaller than those which have been observed between whole thylakoids and the more highly labeled chloroplast envelopes from briefly labeled cells.

Transport of proteins into chloroplasts. Organization, orientation, and lateral distribution of the plastocyanin processing peptidase in the thylakoid network.

Plastocyanin is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen of the chloroplast. Maturation of preplastocyanin involves successive cleavages by a stromal peptidase and a distinct thylakoidal peptidase. In this report we have analyzed the precise location and orientation of the thylakoidal peptidase with respect to the thylakoid membrane. Experiments involving differential centrifugation of thylakoid extracts and sonication of isolated vesicles indicate that the peptidase is tightly bound to the thylakoid membrane but not intimately associated with any of the major thylakoid protein complexes. Analysis of the lateral distribution of the peptidase has shown that the enzyme is exclusively located in the non-appressed lamellae of the thylakoid network. The active site of the peptidase is on the lumenal face of the thylakoid membrane.

Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.

86] Isolation and characterization of genes for cytochrome [formula omitted] complex

This chapter describes procedures used for the isolation and preliminary characterization of genes encoding the cytochrome b 6 /f complex from the filamentous cyanobacterium Nostoc PCC 7906. The proton-translocating thylakoid membrane cytochrome b 6 /f complex catalyzes electron transfer between the two photosystems (PS) and is required for cyclic electron flow around photosystem I (PSI). In cyanobacteria as in plant chloroplasts, the complex consists of four essential polypeptides: cytochromes f and b 6 , the Rieske Fe-S protein, and a subunit IV. In the cyanobacterium Anabaena these have apparent molecular weights of 31,000, 22,500, 22,000 and 16,000, respectively. Cloned genes can now be introduced by transformation or conjugation into several strains of cyanobacteria enabling, among other applications, a molecular genetic analysis of the structure and function of thylakoid membrane protein complexes. An additional requirement for this approach is the availability of cloned genes for these proteins and the determination of their nucleotide and derived amino acid sequences. Heterologous hybridization (the use of a gene from one source as a probe for isolating a gene from another source) was the approach first used to isolate cyanobacterial genes and perhaps remains the most widely used one. Its success ultimately depends on the level of nucleotide sequence similarity between the probe and the target DNA.

Development of chloroplast membranes during light induced greening ofChlorella fusca g-2

The development of thylakoid protein complexes during light induced greening of a mutant ofChlorella fusca was studied. Separation of chlorophyll-protein complexes and thylakoid polypeptides by LDS-polyacrylamide gel electrophoresis show that cells grown in the dark contain proteins belonging to coupling factor and cytochrome f/b6 complex. Parts of both reaction centers are present also. The antennae complexes are specifically lost in yellow cells. The changes in polypeptide pattern at different stages of development in the light are related to ultrastructural changes. The beginning of membrane appression can be correlated with the appearance of the light-harvesting complex II. While the average diameter of EF-particles increases throughout the greening process, their densitiesapart from the rearrangement due to membrane stacking-remain fairly constant. The kinetics of EFu-particle enlargement are different from those of EFs-particles.

Spatial organization of the cytochrome b6- f complex within chloroplast thylakoid membranes

The spatial distribution of the chloroplast thylakoid protein complex comprised of cytochromes f and b-563, and the Rieske iron-sulfur protein (Cyt b 6- f) has been controversial because of conflicting results obtained by different techniques. We have combined the following biochemical and immunochemical techniques to approach this question: (1) French press disruption of thylakoids, followed by repeated two-phase aqueous polymer partitioning to separate inside-out grana from right-side-out stroma membrane fragments; (2) electrophoretic analysis followed by the 3,3′,5,5′-tetramethylbenzidine stain for cytochrome hemes; (3) electroblot analysis with anti-Cyt b 6- f antibodies; (4) agglutination of membrane fragments with anti-Cyt b 6- f antibodies; and (5) post-embedment thin-section immunolabeling of chemically fixed or ultrarapidly frozen chloroplasts with anti-Cyt b 6- f antibodies. Our results indicate that the complex is present in both of the isolated membrane fragment populations in similar amounts, with the bulk of the immunoreactive sites exposed to the thylakoidal lumen. Direct immunolabeling of thin-sectioned chloroplasts resulted in localization of the complex throughout the thylakoids, without specialized compartmentation. These results provide both the temporal and spatial resolution necessary for accurate localization of the complex. We concur with models proposing distribution of Cyt b 6- f throughout all thylakoid membranes.

Effect of different light qualities on the ultrastructure, thylakoid membrane composition and assimilation metabolism of Chlorella fusca

Chlorella fusca (Shihira et Krauss) strain C‐1.1.10 was grown under three different light qualities (red, white or blue light) in homocontinuous cultures. Under electron microscopy, blue light cultures showed enlarged cells, thinner cell walls and lower starch content than red light cells. Under blue light, the degree of stacking of the thylakoid membranes was significantly lower than under white or red light conditions. Changing the light from blue to red the ratio of exposed to appressed membranes was doubled. Compared to red light cells, blue light cells exhibited higher photosynthetic rates per chlorophyll molecule and contained less chlorophyll per dry weight. Blue light stimulated the content of soluble protein as well as that of soluble carbohydrates. The dry weight productivity per unit time was enhanced under blue light conditions. The thylakoid protein complexes which are generally assumed to be localized in the exposed membranes were found in higher concentrations under blue light than under red light. In blue light, both the Photosystem II/Photosystem I ratio and the ratio of light‐harvesting chlorophyll protein to P‐700 chlorophyll a‐protein were lower than in red light. Blue light cells contained twice the concentration of cytochrome f, which correlates well with their higher photosynthetic capacity. When altering the light quality, the degree of change in the reaction center complexes was much lower than expected given the corresponding degree of change in the ratio of exposed to appressed membranes. These results are discussed in light of the question as to whether the variation in the stoichiometry of the laterally distributed complexes can be explained by changes in the degree of stacking alone.

Nuclear, Virescent Mutants of Zea mays L. with High Levels of Chlorophyll (a/b) Light-Harvesting Complex during Thylakoid Assembly

We have found nuclear, recessive mutants in Zea mays L. where assembly of the major chlorophyll (a/b) light-harvesting complex (LHC) was not delayed relative to most other thylakoid protein complexes during thylakoid biogenesis. This contrasts with the normal development of maize chloroplasts (NR Baker, R Leech 1977 Plant Physiol 60: 640-644). All four mutants examined were allelic and virescent, and displayed visibly higher yields of leaf Chl fluorescence during greening. Fully greened mutants had normal leaf Chl fluorescence yield and normal levels of LHC, and grew to maturity under field conditions. Therefore, delayed LHC assembly is not an obligate feature of thylakoid differentiation.Assigning the molecular basis for the mutation should provide information concerning reguation of LHC assembly. Several possibilities are discussed. The pleiotropic mutant phenotype is not attributable to defects in thylakoid glycerolipid synthesis. Thylakoids isolated from greening mutant leaf sections had elevated glycerolipid/Chl ratios. In addition, both the molar distribution and acyl composition of four major glycerolipids were normal for developing mutant thylakoids.