Composition Of Thylakoid Membranes Research Articles

Specific Role of Glycolipids in the Regular Stacking of Membranes Reconstituted from Thylakoid Lipid Extracts

Chloroplasts contain stacks of flattened photosynthetic membranes (thylakoids) that host the photosynthetic protein complexes. Using neutron diffraction under controlled temperature and humidity, we observe that solid supported films prepared from protein-free thylakoid lipid extracts self-organize as regular bilayer stacks, the only functional supra-molecular organization for photosynthesis. We show that this lamellar order is close to the reversible phase transition from hexagonal (HII) to lamellar (Lα). This transition and the observed phase coexistence can be modulated by fine-tuning the lipid profile and the hydration of the lipid mixture. In a developmental context, like the etioplast-to-chloroplast transition, or in response to environmental variations (stress), this property might contribute to the highly dynamic flexibility of the structure of plastids. Furthermore, our analyses highlight the critical role of galactolipids - most importantly DGDG - as the contributing components to membrane stacking via hydrogen bonds between polar heads of adjacent bilayers. This role of galactolipids might be determinant in other glycolipid-rich membrane-ordered systems such as myelin sheath.Diffraction patterns obtained on the D16 instrument (ILL, Grenoble, France) will be presented together with a detailed analysis of phases and pressure-distance curves obtained for various lipid compositions ranging from purified individual thylakoid lipids to complex mixtures approaching the natural thylakoid membrane composition.

Molecular dynamics of the diatom thylakoid membrane under different light conditions

During the last years significant progress was achieved in unraveling molecular characteristics of the thylakoid membrane of different diatoms. With the present review it is intended to summarize the current knowledge about the structural and functional changes within the thylakoid membrane of diatoms acclimated to different light conditions. This aspect is addressed on the level of the organization and regulation of light-harvesting proteins, the dissipation of excessively absorbed light energy by the process of non-photochemical quenching, and the lipid composition of diatom thylakoid membranes. Finally, a working hypothesis of the domain formation of the diatom thylakoid membrane is presented to highlight the most prominent differences of heterokontic thylakoids in comparison to vascular plants and green algae during the acclimation to low and high light conditions.

Alteration in growth and thylakoid membrane lipid composition of Azolla caroliniana under phosphate deficiency

The changes in the fresh biomass accumulation, photosynthetic and anthocyanin pigments, photosystem 2 (PS 2) activity, ultrastructure of chloroplast, total lipids and fatty acid composition of thylakoid membrane were followed in the aquatic fern Azolla caroliniana grown on medium either deficient or supplied with various phosphorus concentrations. The content of photosynthetic pigments and the anthocyanin/chlorophyll ratio increased significantly with increasing PO4 3− concentration. Phosphate deficiency inhibited growth and PS 2 activity and decreased content of total lipids and phospholipids in isolated thylakoids. This was accompanied with a significant increase in the percentage of galalactolipids.

Adaptational changes in the lipids and fatty acid profile of the cell and thylakoid membrane of rice plants exposed to sunlight

Adaptational changes occurring in the lipids and fatty acids of the cell and the thylakoid membrane in response to high light treatment, was studied in 30days old rice (Oryza sativa L. cv. Jyothi) plants grown under low (150-200μmolm(-2)s(-1)) or moderate (600-800μmolm(-2)s(-1)) light conditions. Results were compared with rice plants grown in high (1200-2200μmolm(-2)s(-1)) light conditions. Exposure of rice plants and isolated chloroplast to high light, resulted in an increase in the amount of malonaldehyde, indicating oxidation of membrane lipids. Qualitative and quantitative changes in the phosphoglycolipids and quantitative changes in neutral lipids were observed in rice plants grown under the different growth conditions. A few of the phosphoglycolipids and neutral lipids were present exclusively in plants grown at low or moderate or high light, indicating requirement of different type of lipid composition of rice plants in response to their different growth irradiances. However, no significant quantitative changes were observed in the different saturated and unsaturated fatty acid groups of total lipids in low, moderate and high light grown rice plants, as a result of exposure to high light. No qualitative changes in the fatty acid composition due to difference in growth irradiance or high light treatment were seen. The changes observed in the phosphoglycolipids and neutral lipid composition of cell and thylakoid membrane of low, moderate and high light grown rice plants in response to high light, are probably the result of physiological changes in the rice plants, to sustain optimum structure and function of the cell and thylakoid membrane to maintain active physiological functions to endure high light conditions.

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.

The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane

The present study shows that thylakoid membranes of the diatom Cyclotella meneghiniana contain much higher amounts of negatively charged lipids than higher plant or green algal thylakoids. Based on these findings, we examined the influence of SQDG on the de-epoxidation reaction of the diadinoxanthin cycle and compared it with results from the second negatively charged thylakoid lipid PG. SQDG and PG exhibited a lower capacity for the solubilization of the hydrophobic xanthophyll cycle pigment diadinoxanthin than the main membrane lipid MGDG. Although complete pigment solubilization took place at higher concentrations of the negatively charged lipids, SQDG and PG strongly suppressed the de-epoxidation of diadinoxanthin in artificial membrane systems. In in vitro assays employing the isolated diadinoxanthin cycle enzyme diadinoxanthin de-epoxidase, no or only a very weak de-epoxidation reaction was observed in the presence of SQDG or PG, respectively. In binary mixtures of the inverted hexagonal phase forming lipid MGDG with the negatively charged bilayer lipids, comparable suppression took place. This is in contrast to binary mixtures of MGDG with the neutral bilayer lipids DGDG and PC, where rapid and efficient de-epoxidation was observed. In complex lipid mixtures resembling the lipid composition of the native diatom thylakoid membrane, we again found strong suppression of diadinoxanthin de-epoxidation due to the presence of SQDG or PG. We conclude that, in the native thylakoids of diatoms, a strict separation of the MGDG and SQDG domains must occur; otherwise, the rapid diadinoxanthin de-epoxidation observed in intact cells upon illumination would not be possible.

A chloroplast cyclophilin functions in the assembly and maintenance of photosystem II in Arabidopsis thaliana.

Photosynthetic light reactions rely on the proper function of large protein complexes (including photosystems I and II) that reside in the thylakoid membrane. Although their composition, structure, and function are known, the repertoire of assembly and maintenance factors is still being determined. Here we show that an immunophilin of the cyclophilin type, CYP38, plays a critical role in the assembly and maintenance of photosystem II (PSII) supercomplexes (SCs) in Arabidopsis. Mutant plants with the CYP38 gene interrupted by T-DNA insertion showed stunted growth and were hypersensitive to high light. Leaf chlorophyll fluorescence analysis and thylakoid membrane composition indicated that cyp38 mutant plants had defects in PSII SCs. Sucrose supplementation enabled the rescue of the mutant phenotype under low-light conditions, but failed to mitigate hypersensitivity to high-light stress. Protein radiolabeling assays showed that, although individual thylakoid proteins were synthesized equally in mutant and wild type, the assembly of the PSII SC was impaired in the mutant. In addition, the D1 and D2 components of the mutant PSII had a short half-life under high-light stress. The results provide evidence that CYP38 is necessary for the assembly and stabilization of PSII.

Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves

The changes induced in the photosynthetic apparatus of spinach (Spinacia oleracea L.) seedlings exposed to iron deficiency shortly after germination were characterized with two proteomic approaches coupled with chlorophyll and xanthophyll analysis and in vivo measurements of photosynthesis. During the first 10 d of iron deficiency the concentrations of chlorophyll b and violaxanthin were greatly reduced, but all xanthophylls recovered after 13-17 d of iron deficiency, when both chlorophylls were negatively affected. No new protein was formed in iron-deficient leaves, and no protein disappeared altogether. Photosystem I (PSI) proteins were largely reduced, but the stoichiometry of the antenna composition of PSI was not compromised. On the contrary, PSII proteins were less affected by the stress, but the specific antennae Lhcb4 and Lhcb6, Lhcb2 and its isoform Lhcb1.1 were all reduced, while the concentration of Lhcb3 increased. A strong reduction in thylakoid bending and an altered distribution pattern for the reduced PSI and PSII complexes were observed microscopically in iron-deficient leaves. Supercomplex organization was also affected by the stress. The trimeric organization of Lhcb and the dimerization of Lhca were reduced, while monomerization of Lhcb increased. However, the trimerization of Lhcb was partially recovered after 13-17 d of iron deficiency. In iron-deficient leaves, photosynthesis was strongly inhibited at different light intensities, and a high de-epoxidation status of the xanthophylls was observed, in association with a strong impairment of photochemical efficiency and an increase of heat dissipation as monitored by the non-photochemical quenching of fluorescence. All these negative effects of iron deficiency were attenuated but not fully reversed after again supplying iron to iron-deficient leaves for 7-13 d. These results indicate that iron deficiency has a strong impact on the proteomic structure of spinach photosystems and suggest that, in higher plants, adaptive mechanisms common in lower organisms, which allow rapid changes of the photosystem structure to cope with iron stress, are absent. It is speculated that the observed changes in the monomer-trimer equilibrium of major PSII antennae, which is possibly the result of xanthophyll fluctuations, is a first adaptative adjustment to iron deficiency, and may eventually play a role in light dissipation mechanisms.

Effect of glycinebetaine on function of thylakoid membranes in wheat flag leaves under drought stress

Two wheat cultivars, HF9703 (drought tolerant) and SN215953 (drought sensitive) were used to examine the effects of glycinebetaine (GB, 100 mM) on lipid composition and function of thylakoid membranes under drought stress. GB application mitigated negative effect of drought on Ca2+-ATPase and Hill reaction activities, chlorophyll content, gas exchange and photosynthesis. These positive effects of GB application maybe, in part, correlated with improving the lipid composition of the thylakoid membranes.

Molecular mechanisms of stress resistance of the photosynthetic apparatus

The mechanisms of action of environmental stress-inducing factors on the photosynthetic apparatus (PA) of plants are considered. The basic targets for stress produced by heat, cold, salinity, osmotic imbalance, and high irradiance are analyzed. It is suggested that stress factors have an influence on the composition of thylakoid membranes and inhibit photosynthetic processes. However, recent studies demonstrated that strong light induces the photodamage to photosystem II (PS II) due to direct action of light on the oxygen-evolving complex. Stress-induced accumulation of reactive oxygen species (ROS) leads to inhibition of the recovery of the PSII by suppressing thede novo synthesis of photosynthetic proteins. In addition, stress stimulates the synthesis of protective low-molecular weight compounds (e.g., glycine betaine) and stress proteins. The major mechanisms of acclimation and protection of the PA against damaging effects of environmental stress-inducing factors are analyzed with special reference to cyanobacterial cells and mutants with high or low stress resistance.

Cold acclimation of the Arabidopsis dgd1 mutant results in recovery from photosystem I-limited photosynthesis

We compared the thylakoid membrane composition and photosynthetic properties of non- and cold-acclimated leaves from the dgd1 mutant (lacking >90% of digalactosyl–diacylglycerol; DGDG) and wild type (WT) Arabidopsis thaliana. In contrast to warm grown plants, cold-acclimated dgd1 leaves recovered pigment-protein pools and photosynthetic function equivalent to WT. Surprisingly, this recovery was not correlated with an increase in DGDG. When returned to warm temperatures the severe dgd1 mutant phenotype reappeared. We conclude that the relative recovery of photosynthetic activity at 5 °C resulted from a temperature/lipid interaction enabling the stable assembly of PSI complexes in the thylakoid.

Glycoengineering of Cyanobacterial Thylakoid Membranes for Future Studies on the Role of Glycolipids in Photosynthesis

The lipid composition of thylakoid membranes is conserved from cyanobacteria to angiosperms. The predominating components are monogalactosyl- and digalactosyldiacylglycerol. In cyanobacteria, thylakoid membrane biosynthesis starts with the formation of monoglucosyldiacylglycerol which is C4-epimerized to the corresponding galactolipid, whereas in plastids monogalactosyldiacylglycerol is formed at the beginning. This suggests that galactolipids have specific functions in thylakoids. We wanted to investigate whether galactolipids can be replaced by glycosyldiacylglycerols with headgroups differing in their epimeric and anomeric details as well as the attachment point of the terminal hexose in diglycosyldiacylglycerols. For this purpose putative glycosyltransferase sequences were identified in databases to be used for functional expression in various host organisms. From 18 newly identified sequences, four turned out to encode glycosyltransferases catalyzing final steps in glycolipid biosynthesis: two alpha-glucosyltransferases, one beta-galactosyltransferase and one beta-glucosyltransferase. Their functional annotation was based on detailed structural characterization of the new glycolipids formed in the transformant hosts as well as on in vitro enzymatic assays. The expression of alpha-glucosyltransferases in the cyanobacterium Synechococcus resulted in the accumulation of the new alpha-galactosyldiacylglycerol which is ascribed to epimerization of the corresponding glucolipid. The expression of the beta-glucosyltransferase led to a high proportion of new beta-glucosyl-(1-->6)-beta-galactosyldiacylglycerol almost entirely replacing the native digalactosyldiacylglycerol. These results demonstrate that modifications of the glycolipid pattern in thylakoids are possible.

Reversed-phase HPLC determination of chlorophyll a' and naphthoquinones in photosystem I of red algae: existence of two menaquinone-4 molecules in photosystem I of Cyanidium caldarium.

Chlorophyll (Chl) a', the C13(2)-epimer of Chl a, is one of the two Chl molecules constituting the primary electron donor (P700) of photosystem (PS) I of a thermophilic cyanobacterium Synechococcus elongatus. To examine whether PS I of other oxygenic photosynthetic organisms in general contain one Chl a' molecule in P700, the pigment composition of thylakoid membranes and PS I preparations isolated from red algae Porphyridium purpureum and Cyanidium caldarium was examined by reversed-phase HPLC with particular attention to Chl a' and phylloquinone (PhQ), the secondary electron acceptor of PS I. The two red algae contained one Chl a' molecule at the core part of PS I. In PS I of C. caldarium, two menaquinone-4 (MQ-4) molecules were detected in place of PhQ used by higher plants and cyanobacteria. The 1:2:1 stoichiometry among Chl a', PhQ (MQ-4) and P700 in PS I of the red algae indicates that one Chl a' molecule universally exists in PS I of oxygenic photosynthetic organisms, and two MQ-4 molecules are associated with PS I of C. caldarium.

Characterization of the stimulating effect of low-dose stressors in maize and bean seedlings

The effect of some more or less harmful compounds like Cd, Pb, Ni, Ti salts and DCMU at low concentrations on the development of chloroplasts in maize and bean seedlings was investigated. Chlorophyll content, chlorophyll a/b ratio, photosynthetic activity (14CO2 fixation), chlorophyll-protein composition of thylakoid membranes, fluorescence spectra of chloroplasts, fluorescence induction parameters of leaves and electron microscopic structure of maize and bean chloroplasts as well as growth parameters were studied. Stimulation of chlorophyll synthesis and photosynthetic activity was observed at different intervals during all of the treatments, while chlorophyll a/b ratios and fluorescence properties of leaves or chloroplasts did not change considerably except in DCMU treated plants. Heavy metal treatments increased the amount of photosystem I and light-harvesting complex II, while decreased amount of photosystem I and higher amount of light-harvesting complex II was found in DCMU treated thylakoids. Electron microscopy showed only sligth differences in the morphology of chloroplast lamellar system (mostly in DCMU treated plants), while the status of the plasmalemma and tonoplast seemed to be altered as a result of certain metal treatments. Results showed the expression of a cytokinin-like effect on the development of chloroplasts. It is assumed, that these low-dose stressors generate non-specific alarm reactions in plants, which may involve changes of the hormonal balance.

Effects of slow clinorotation on lipid contents and proton permeability of thylakoid membranes of pea chloroplasts.

Photochemical characteristics and lipid composition of thylakoid membranes from 12 day-old pea leaves that were exposed to slow clino-rotation were examined and compared with a vertical control. Proton permeability of thylakoid membranes was estimated from light-induced proton uptake (delta H+) and post-illumination proton efflux in chloroplast suspensions. The delta pH magnitude was calculated from the level of light-induced quenching of 9-aminoacridine fluorescence. Proton permeability of thylakoid membranes increased during exposure to clino-rotation. When subsequently transferred to darkness, proton efflux increased almost 2-fold in clinorotated leaves. The results were compared with data on pigment and polar lipid composition of photosynthetic membranes in clino-rotated and control plants. It was concluded that both the increase of proton permeability and the decrease of polar lipid content in chloroplasts were induced by clino-rotation.

Alteration in chloroplast structure and thylakoid membrane composition due to in vivo heat treatment of rice seedlings: correlation with the functional changes

Exposure of 25 °C-grown, seven-day-old rice seedlings to mild heat stress of 40 °C for 24 h in dark did not cause any change in protein or pigment content of the thylakoids, but produced major disorganization of chloroplast ultrastructure. This heat induced disorganization of thylakoid structure/organization caused significant (∼65 percnt;) loss in PSII activity, slight loss in PSI activity, and brought about a decrease in relative quantum efficiency of PSII. The herbicide 14C atrazine binding assay revealed a decreased number of binding sites of the herbicide and altered the herbicide dissociation constant, suggesting that the heat induced disorganization of the thylakoids affects the acceptor side of PSII. Cation induced Chla fluorescence analyses at room temperature and low temperature indicated thatin vivo heat exposure of rice seedlings altered the extent of energy transfer in favor of PSI. Immunoblotting analysis of several PSII polypeptides such as D1/D2 reaction dimer and Cyt b559 showed no major changes due to mild heat exposure except for the PSII core antenna polypeptide (CP43), which could reflect the reduction in PSII activity observed in light saturation studies. Similarly, haeme staining did not indicate any change in other cytochrome related polypeptides. Our results therefore clearly suggest thatin vivo exposure of rice seedlings to elevated (40 °C) temperature caused thylakoid structural disorganization, and this disorganization of some of the thylakoid complexes resulted in a loss in thylakoid photochemical function.

Alterations in thylakoid membrane composition induced by iron starvation in sunflower plants

Previous analysis of photosynthetic electron transport of isolated thylakoid membranes in iron‐deficient sunflower plants showed a decrease in the whole chain and photosystem I (PSI) activities, whereas photosystem II (PSII) activity resulted to be unaffected (Tognini et al., 1996). On the basis of these results, the analysis of the molecular composition of thylakoid membranes was carried out. By using immuno‐blotting methods, a decrease in the protein content of PSI in iron‐deficient plants was detected, while light‐harvesting complex II (LHCII) increased and PSII core complex (CC) did not change compared to the controls. In iron‐deficient plants, the tetramethylbenzidine staining of thylakoid protein gels showed a decrease in the content of cytochrome f and cytochrome b6, while cytochrome bS59 remained unchanged in control and stressed plants. Green gels of thylakoid membranes seemed to confirm the above results; the green band corresponding to PSI holocomplex resulted less intense in chlorotic samples and the green bands of the lowest molecular weight, corresponding to LHCII, were increased in iron‐deficient thylakoid membranes in comparison to the controls. The HPLC analysis of the pigments associated with the green bands separated by Deriphat PAGE showed that the xanthophylls associated with both photosystems shifted toward the de‐epoxidated form following iron starvation, indicating the onset of a protective mechanism against photoinhibition at the levels of both photosystems.

Isolation and characterization of the thylakoid membranes from the NaCl-resistant (NaCl(r)) mutant strain of the cyanobacterium Anabaena variabilis.

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl(r) mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl(r) mutant strain and its wild-type counterpart respectively at wavelengths of 400-850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl(r) mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl(r) mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and beta-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl(r) mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl(r) mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl(r) mutant strain also showed morphological variations under NaCl stress.

UV-B Induced Alterations in Composition of Thylakoid Membrane and Amino Acids in Leaves of Rhizophora Apiculata Blume

Seedlings of Rhizophora apiculata were exposed to UV-B radiation at four doses equivalent to 10, 20, 30, and 40 % ozone depletion. The seedlings irradiated with high doses of UV-B had characteristic decline in contents of specific proteins with molecular masses of 33, 23, and 17 kDa. On the contrary, proteins of 55, 33, 25, 23, and 17 kDa were accumulated in the seedlings exposed to low doses of UV-B. The UV-B, in general, enhanced formation of saturated fatty acids and reduced unsaturated fatty acids, to a maximum extent of 88 and 26 %, respectively. The low dose of UV-B increased content of oleic acid by 9 %, and the high dose reduced it by 34 %. The high dose of UV-B enhanced the lipid peroxidation by 48 %, whereas the low dose of UV-B did not show any significant effect. The contents of amino acids such as aspartate, glutamate, asparagine, serine, glutamine, threonine, and histidine were increased in low UV-B doses by 53, 86, 142, 72, 3, 119, and 32 %, respectively; while in high doses they were reduced significantly.

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.