Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases.

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs-indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase-suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that-in line with the Dynamic Exchange Model-the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery.

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. II. Structure and Functions.

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

Accumulation of geranylgeranylated chlorophylls in the pigment-protein complexes of Arabidopsis thaliana acclimated to green light: effects on the organization of light-harvesting complex II and photosystem II functions.

Light quality significantly influences plant metabolism, growth and development. Recently, we have demonstrated that leaves of barley and other plant species grown under monochromatic green light (500-590 nm) accumulated a large pool of chlorophyll a (Chl a) intermediates with incomplete hydrogenation of their phytyl chains. In this work, we studied accumulation of these geranylgeranylated Chls a and b in pigment-protein complexes (PPCs) of Arabidopsis plants acclimated to green light and their structural-functional consequences on the photosynthetic apparatus. We found that geranylgeranylated Chls are present in all major PPCs, although their presence was more pronounced in light-harvesting complex II (LHCII) and less prominent in supercomplexes of photosystem II (PSII). Accumulation of geranylgeranylated Chls hampered the formation of PSII and PSI super- and megacomplexes in the thylakoid membranes as well as their assembly into chiral macrodomains; it also lowered the temperature stability of the PPCs, especially that of LHCII trimers, which led to their monomerization and an anomaly in the photoprotective mechanism of non-photochemical quenching. Role of geranylgeranylated Chls in adverse effects on photosynthetic apparatus of plants acclimated to green light is discussed.

Modulation of non-bilayer lipid phases and the structure and functions of thylakoid membranes: effects on the water-soluble enzyme violaxanthin de-epoxidase.

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content-yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31P-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase.

Chemiluminescent detection of oligoclonal immunoglobulins after isoelectric focusing and affinity-mediated immunoblotting.

BACKGROUND AND AIMS: Detection of oligoclonal IgG (o-IgG) in the cerebrospinal fluid (CSF) not found in serum is the principal laboratory test to support a diagnosis of multiple sclerosis. The aim of this study was to compare chemiluminescent and chromogenic detection of oligoclonal immunoglobulins in the cerebrospinal fluid and serum after their separation by means of isoelectric focusing followed by immunoblotting. METHODS: A set of experiments was designed to detect oligoclonal immunoglobulins by means of alkaline phosphatase BCIP/NBT substrate and chemiluminescent peroxidase substrate. RESULTS: Based on visual evaluation of signals, chemiluminescent detection requires about a 4 times lower amount of applied protein than very sensitive BCIP/NBT chromogenic detection. Very good correlation between methods has been shown for oligoclonal IgG. Antigen-specific oligoclonal IgG could be demonstrated by both methods although the pattern was clearer using chemiluminescence. In one patient, oligoclonal IgD bands barely visible by BCIP/NBT were convincingly demonstrated by chemiluminescence. CONCLUSION: Chemiluminescent detection is a feasible option for oligoclonal immunoglobulin detection and could be used in cases when the sensitivity needs to be improved. Further studies and method optimisation are warranted.

Enhanced thermal stability of the thylakoid membranes from spruce. A comparison with selected angiosperms.

Recently, we have found that thermal stability of photosystem II (PSII) photochemistry in spruce needles is higher than in other plants (barley, beech) cultivated under the same temperatures. In this work, temperature dependences of various characteristics of PSII organization were studied in order to obtain complex information on the thermal stability of PSII function and organization in spruce. Temperature dependency of circular dichroism spectra revealed by about 6 °C higher thermal stability of macrodomain organization in spruce thylakoid membranes in comparison with Arabidopsis and barley ones; however, thermal disintegration of light-harvesting complex of PSII did not significantly differ among the species studied. These results thus indicate that thermal stability of PSII macro-organization in spruce thylakoid membranes is enhanced to a similar extent as thermal stability of PSII photochemistry. Clear-native polyacrylamide gel electrophoresis of preheated thylakoids demonstrated that among the separated pigment-protein complexes, only PSII supercomplexes (SCs) revealed considerably higher thermal stability in spruce thylakoids as compared to Arabidopsis and barley ones. Hence we suggest that higher thermal stability of PSII macro-organization of spruce is influenced by the maintenance of PSII SCs in the thylakoid membrane. In addition, we discuss possible effects of different PSII organizations and lipid compositions on the thermal stability of spruce thylakoid membranes.

Characterization of acclimation of Hordeum vulgare to high irradiation based on different responses of photosynthetic activity and pigment composition.

The ability of spring barley (Hordeum vulgare cv. Akcent) to adjust the composition and function of the photosynthetic apparatus to growth irradiances of 25-1200 mumol m(-2) s(-1) was studied by gas exchange and chlorophyll a fluorescence measurements and high-performance liquid chromatography. The increased growth irradiance stimulated light- and CO(2)-saturated rates of CO(2) assimilation expressed on a leaf area basis up to 730 mumol m(-2) s(-1) (HL730), whereas at an irradiance of 1200 mumol m(-2) s(-1) (EHL1200) both rates decreased significantly. Further, the acclimation to EHL1200 was associated with an extremely high chlorophyll a/b ratio (3.97), a more than doubled xanthophyll cycle pool (VAZ) and a six-fold higher de-epoxidation state of the xanthophyll cycle pigments as compared to barley grown under 25 mumol m(-2) s(-1) (LL25). EHL1200 plants also exhibited a long-term inhibition of Photosystem II (PS II) photochemical efficiency (F (v)/F (m)). Photosynthetic capacity, chlorophyll a/b and VAZ revealed a linear trend of dependence on PS II excitation pressure in a certain range of growth irradiances (100-730 mumol m(-2) s(-1)). The deviation from linearity of these relationships for EHL1200 barley is discussed. In addition, the role of increased VAZ and/or accumulation of zeaxanthin and antheraxanthin in acclimation of barley to high irradiance is studied with respect to regulation of non-radiative dissipation and/or photochemical efficiency within PS II.