Effects of diquat on pigment-protein complexes of thylakoid membranes in soybean and maize plants

Soybean (Glycine max Merrill) and maize (Zea mays L.) plants were exposed for 5 to 48 h to the herbicide diquat under "white light" (WL) or far-red radiation (FR) (photon fluence rate of 30 μmol m^-2 s^-1). The WL enhanced diquat effect on chlorophyll content in soybean plants, while FR had the same effects on maize plants. After 5 h, diquat increased the content of polypeptides bound to light-harvesting proteins in both plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chlorophyll-protein and polypeptide composition of Mn-deficient sugar beet thylakoids

The chlorophyll-protein and polypeptide composition of manganese deficient and control sugar beet thylakoids was examined using three different detergent-electrophoresis systems. On a per chlorophyll basis, manganese deficiency reduced the amounts of CPa complex (separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis), and CP 47 and CP 43 complexes (separated by octylglucoside/SDS-polyacrylamide gel electrophoresis) without decreasing the amounts of light harvesting complexes. Lithium dodecylsulfate/Triton X-100 polyacrylamide gel electrophoresis showed that manganese deficiency decreased several thylakoid polypeptides, including a chlorophyll b containing 30 kilodalton chlorophyll-protein complex, but did not decrease the amounts of 28 and 29 kilodalton light-harvesting chlorophyll b-containing polypeptides.     

Effects of canopy shade on the lipid composition of soybean leaves

The effect of canopy shade on leaf lipid composition was examined in soybeans ( Glycine max cv. Young) grown under field conditions. Expanding leaves were tagged at 50, 58 and 65 days after planting (DAP) in plots with either a high (10 plants m⁻¹ row) or low (1 plant m⁻¹ row) plant density. At 92 DAP, light conditions ranged from a pho-tosynthetic photon flux density (PPFD) of 87% of full sun with a far-red/red (735 nm/645 nm) ratio of 0.9 at upper canopy leaves to extreme shade where the PPFD was 10% of full sun with a far-red/red ratio greater than 6. Highly shaded leaves in the high plant density treatment accumulated triacylglycerol (TG) up to 25% of total leaf lipid, a 2.4-fold increase in TG on a chlorophyll basis compared to leaves in the upper canopy. Although total polar lipid content was reduced up to 50% in shaded leaves, shade had little affect on the lipid content or composition of thylakoid membranes. Shade did not affect leaf chlorophyll content. Therefore, the changes in leaf lipid composition were not related to senescence. These findings suggest that conditions of low irradiance and/or a high FR/R ratio cause a shift in carbon metabolism toward the accumulation of TG, a storage lipid. Eighteen-carbon fatty acid desaturation was also affected in highly shaded leaves where a reduction in linolenic acid (18:3) content was accompanied by a proportional increase in oleic (18:1) and linoleic (18:2) acids.     

Iron Deficiency Induced Changes on Electron Transport Rate in Pisum Sativum Chloroplasts

Iron deficiency induced decrease in the rate of whole electron transport chain in chloroplasts of pea (Pisum sativum L.). Such reduction was mainly due to the loss of photosystem (PS) 2 activity. The same result was obtained when the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) was evaluated. The loss in PS 2 activity was primarily due to a loss of 33, 23 and 17 kDa polypeptides. In contrast, iron deficiency induced the synthesis of 28 and 29 kDa polypeptides.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

Comparison of thylakoid structure and organization in sun and shade Haberlea rhodopensis populations under desiccation and rehydration

The resurrection plant, Haberlea rhodopensis can survive nearly total desiccation only in its usual low irradiation environment. However, populations with similar capacity to recover were discovered recently in several sunny habitats. To reveal what kind of morphological, structural and thylakoid-level alterations play a role in the acclimation of this low-light adapted species to high-light environment and how do they contribute to the desiccation tolerance mechanisms, the structure of the photosynthetic apparatus, the most sensitive component of the chlorophyll-retaining resurrection plants, was analyzed by transmission electron microscopy, steady state low-temperature fluorescence and two-dimensional Blue-Native/SDS PAGE under desiccation and rehydration.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

The action of lipases on chloroplast membranes I. The release of plastocyanin from galactolipase-treated thylakoid membranes

Bean chloroplasts treated with galactolipase (lipolytic acyl hydrolase) isolated from bean leaves showed an inhibition of photosystem I activity as measured by methyl viologen-mediated oxygen uptake and NADP⁺ photoreduction. This inhibition was partially reversed by exogenous plastocyanin added to galactolipase-treated thylakoid membranes. Galactolipase released substantial amounts of endogenous plastocyanin (about 40%) from bean chloroplasts. The results are discussed with regard to the localization of plastocyanin in thylakoid membranes.Abbreviations chlf chlorophyll - DCMU 3-(2,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - MV methyl viologen - NADP⁺ nicotinamide dinucleotide phosphate - PC phosphatidylcholine - PG phosphatidylglycerol - PE phosphatidylethanolamine - PI phosphatidylinositol - SQDG sulphoquinovosyldiacylglycerol - SDS sodium dodecyl sulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethane     

The action of lipases on chloroplast membranes. III. The effect of lipid hydrolysis on chlorophyll-protein complexes in thylakoid membranes

Bean thylakoid membranes treated with various lipolytic enzymes (bean galactolipase, phospholipases A₂, C, D) showed marked changes in their acyl lipid composition. As a consequence of acyl lipids hydrolysis, destruction of some chlorophyll a-protein complexes (CP1a, CP1, CPa) or monomerization of the oligomeric of light harvesting chlorophyll a/b protein complex (LHCP) was observed. It is concluded that galactolipids and phosphatidylcholine are responsible for the stability of CP1a, CP1 and CPa, respectively. Phosphatidylglycerol and to some extent monogalactosyldiacylglycerol are essential for the stabilization of oligomeric structures of light harvesting chlorophyll a/b protein complex.Abbreviations chl chlorophyll - CP1a, CP1 chl a-protein complexes, of PSI - CPa chl a-protein complex of PSII - DGDG diagalactosyldiacylglycerol - FC free chl - GL galactolipase - LHCP^1–3 light harvesting chl a/b protein complex - MGDG monogalactosyldiacylglycerol - PAGE polyacrylamide gel electrophoresis - PC phosphatidylcholine - PG phosphatidylglycerol - PLA₂ phospholipase A₂ - PL phospholipase C - PLD phospholipase D - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - SQDG sulfoquinovosyl-diacylglycerol - TCA trichloroacetic acid - Tricine N-tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

The action of proteolytic enzymes on chloroplast thylakoid membranes

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were incubated with trypsin or pronase for several hours. The indigestible residue was analyzed by polyacrylamide gel electrophoresis. Trypsinization resulted in a complete digestion of all proteins with the exception of the pigment-protein complexes as well as a polypeptide not yet characterized. Yet, as compared with untreated material, Complex II was found to have higher electrophoretic mobility. Electron-microscopic studies illustrate that the indigestible residue still has a preserved membrane structure. Disintegration of the thylakoid membranes by sodium dodecyl sulfate followed by trypsinization also resulted in the two complexes while all the other proteins were found to be digested. However, after removal of the lipids the protein moieties of the complexes proved to be easily digestible. From these results it is concluded that pigment-protein interaction may be an important factor in maintaining a conformation rather resistant to perturbants and proteases. In contrast to trypsin, pronase completely digested the polypeptides of the thylakoid membranes including the protein moieties of the pigment-protein complexes leaving an amorphous lipid mass. The results support the assumption that the complexes are necessary to maintain the membrane structure.     

The effect of the dark interval in intermittent light on thylakoid development: photosynthetic unit formation and light harvesting protein accumulation

Etiolated bean plants were grown in intermittent light with dark intervals of shorter or longer duration, to modulate the rate of chlorophyll accumulation, relative to that of the other thylakoid components formed. We thus produced conditions under which chlorophyll becomes more or less a limiting factor. We then tested whether LHC complexes can be incorporated in the thylakoid. It was found that an equal amount of chlorophyll, formed under the same total irradiation received, may be used for the stabilization of few and large-in-size PS units containing LHC components (short dark-interval intermittent light), or for the stabilization of many and small-in-size PS units with no LHC components (long dark-interval intermittent light). The size of the PS units diminishes as the dark-interval duration is increased, with no further change after 98 minutes. The PSII/cytf ratio remains constant throughout development in intermittent light and equal to that of mature chloroplasts (PSII/cytf = 1) except in the case of very long dark-interval regimes, where about half PSII units per cytf are present. The PSII/PSI ratio was found to be correlated with the PSII unit size (the larger the size, the lower the ratio). The number of PSI units operating on the same electron transfer chain varied depending on the size of the PSII unit (the larger the PSII unit size, the more the PSI units per chain). The results suggest that it is not the chlorophyll content per se which regulates the stabilization of LHC in developing thylakoids and consequently the size of the PS units, but rather the rate by which it is accumulated, relative to that of the other thylakoid components.Abbreviations Chl Chlorophyll - CL Continuous light - CPa the reaction center complex of PSII - CPI the reaction center complex of PSI - CPIa Chlorophyll protein complex containing the CPI and the light harvesting complex of PSI - fr w fresh weight - LDC Light dark cycles - LHC-I Light-harvesting complex of PSI - LHC-II Light harvesting complex of PSII - PS photosystem - PSI photosystem I - PSII photosystem II     

Phycoerythrins of the oxyphotobacterium Prochlorococcus marinus are associated to the thylakoid membrane and are encoded by a single large gene cluster

An intrinsic divinyl-chlorophyll a/b antenna and a particular form of phycobiliprotein, phycoerythrin (PE) III, coexist in the marine oxyphotobacterium Prochlorococcus marinus CCMP 1375. The genomic region including the cpeB/A operon of P. marinus was analysed. It encompasses 10153 nucleotides that encode three structural phycobiliproteins and at least three (possibly five) different polypeptides analogous to cyanobacterial or red algal proteins involved either in the linkage of subunits or the synthesis and attachment of chromophoric groups. This gene cluster is part of the chromosome and is located within a distance of less than 110 kb from a previously characterized region containing the genes aspA-psbA-aroC. Whereas the Prochlorococcus phycobiliproteins are characterized by distinct deletions and amino acid replacements with regard to analogous proteins from other organisms, the gene arrangement resembles the organization of phycobiliprotein genes in some other cyanobacteria, in particular marine Synechococcus strains. The expression of two of the Prochlorococcus polypeptides as recombinant proteins in Escherichia coli allowed the production of individual homologous antisera to the Prochlorococcus and PE subunits. Experiments using these sera show that the Prochlorococcus PEs are specifically associated to the thylakoid membrane and that the protein level does not significantly vary as a function of light irradiance or growth phase.     

Alteration of thylakoid membrane structure in Brassica rapa ssp. oleifera during ageing in high and low light

Abstract Alterations in the composition and structure of thylakoids were studied in Brassica rapa ssp. oleifera grown under high and low irradiance (800 μmol m^?2 s^?1 and 80 μmol m^?2 s^?1). During ageing, both high and low light induced a decrease in total protein particle density and in the relative amount of 80–90 Å cytochrome b6/f and 90–100 Å ATP-synthetase. The density of PSII complexes in stacked (EFs) and unstacked (EFu) thylakoids also decreased. In high light, a shift was noted towards smaller PSII complexes in the EFs face with decreasing attached antenna complex CP29, but the relative amount of the antenna chlorophyll a-protein complexes of photosystem II (CPa) remained stable. In contrast, the proportion of peripheral LHCH on the PFs face and the density of PFs particles increased together with an increase in grana size. In low light, a shift occurred towards larger PSII complexes on the EFs face, along with a decrease in the proportion of CPa complexes and the PFs particle density (peripheral LHCH), though a marked increase was observed in the proportion of chlorophyll a/b-protein complexes in SDS-PAGE. The amount of photosystem I in green gel remained fairly stable, although the density of PFu particles (including PSI) increased in low and slightly diminished in high light. The results indicate that the organization of thylakoid components depends strongly on the light conditions and stage of development.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

The coupling factor of photophosphorylation and the electric properties of the thylakoid membrane

The rate of ATP synthesis of illuminated chloroplasts is correlated with the electric conductance of their inner membranes. In agreement with previous studies it is shown that ATP synthesis is paralleled by an increased conductance of the thylakoid membrane. This conductance together with the ability to form ATP is abolished if chloroplasts are treated with an antibody against the coupling factor CF₁. It is not influenced by the fragmented monovalent antibody. This parallels the lack of influence of the fragmented antibody on ATP synthesis in contrast to its influence on hydrolysis and exchange reactions. We conclude that there are different sites for the interaction of the coupling factor with adenine nucleotides.Extraction of the coupling factor is shown to increase the membrane conductance by more than two orders of magnitude. Reincorporation of the crude coupling factor partially restores the net conductance of the membrane (increase in resistance by a factor of 2.5), while a higher degree of restoration was observed for ATP synthesis and the proton conductivity of the membrane. We conclude that the extraction procedure opens different conductive channels in the membrane; a proton specific one, possibly associated with the binding protein for the coupling factor, plus other channels for “non-protons” which in contrast to the proton channel cannot be plugged by reincorporation of the coupling factor.     

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.     

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.     

Spectroelectrochemistry of P700 in native photosystem I particles and diethyl ether-treated thylakoid membranes from spinach and Thermosynechococcus elongatus

The redox potentials (E(composite function')) of P700 in intact and diethyl ether-treated thylakoid membranes as well as native photosystem (PS) I particles from spinach and Thermosynechococcus elongatus have been measured by a spectroelectrochemistry with an error range of +/-2-3 mV. Stepwise removal of antenna pigments by ether treatment caused distinct shifts of the E( composite function') value with increasing degree of water saturation in ether; negatively from +471 to +428 mV for spinach, but positively from +423 to +436 mV for T. elongatus. Such a contrasting behavior is discussed by invoking the mode of action of ether on the microenvironments around P700.     

Reconstitution of energy transfer and electron transfer between solubilised pigment-protein complexes from thylakoid membranes. The role of acyl lipids

Solubilisation of thylakoid membranes from young leaves of Pisum sativum in the presence of Triton X-100 resulted in an almost complete loss of quenching of light-harvesting chlorophyll-protein (LHCP) fluorescence, as measured at 77°K. There were concomitant changes in the kinetics of light-saturation curves of electron transport from 2,6-dichlorophenolindophenol/ascorbate to methyl viologen. These effects were accompenied by a physical dissociation of LHCP polypeptides from photosystem I (PSI) and photosystem II (PSII) polypeptides, as determined by polyacrylamide gel-electrophoresis. Detergent-dialysis in the presence of exogenous purified galactolipids, about 80% of which were linoleoyl molecular species, only partially reversed these effects. However, detergent-dialysis using the phospholipids, phosphatidylglycerol and phosphatidylcholine, resulted in the substantial restoration of 77°K fluorescence quenching and the restoration of both emission spectra and electron transport kinetics of both Photosystems I and II that were typical of native membranes.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DGD digalactosyldiacylglycerol - LHCP light-harvesting chlorophyll-protein - MGD monogalactosyldiacylglycerol - PCi phosphatidylcholine — Sigma grade NS - PCii -oleoyl, -palmitoyl phosphalidylcholine - PG phosphatidylglycerol - PSI photosystem I - PSII photosystem II