Time-dependent decay and anisotropy of fluorescence from diphenylhexatriene embedded in the chloroplast thylakoid membrane

The hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene has been incorporated into the membranes of isolated thylakoids, separated granal and stromal lamellae and aqueous dispersions of extracted thylakoid galactolipids. Time-resolved fluorescence decays have been recorded on a nanosecond scale using single-photon counting in order to assess the motional properties of the probe. All the experimental systems used showed biphasic decay kinetics and the anisotropies of the decays have been interpreted in terms of a model for wobbling diffusion confined to a cone. The analysis has given information about dynamic and structural restraints of the lipid acyl chains. In the intact thylakoid membrane the degree of order of the fatty acid acyl chains is higher and their rate of motion slower than for isolated lipids. Even so, the dynamic and structural parameters indicate that the thylakoids can be considered as a relatively fluid membrane system when compared with many other biological membranes, a property which is probably required to facilitate efficient long-range diffusion of lipophilic mobile electron-transport components. It is suggested that the optimization of thylakoid fluidity is linked to regulation of the membrane protein/lipid ratio which is also likely to be responsible for the higher fluidity of stromal membranes relative to those of the grana.     

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C-terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35–HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35–HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes.     

Altered rates of protein transport in Arabidopsis mutants deficient in chloroplast membrane unsaturation

Protein transfer across membranes is mediated by protein machinery embedded in the membrane. The complement of different lipid classes within a membrane is known to influence the efficiency of some protein translocation processes, but very little is known about whether the fatty acid composition of the membrane bilayer also affects protein transport. We investigated this issue using three mutants of Arabidopsis, fad6, fad5, and fad3 fad7 fad8, that have reduced levels of fatty acid unsaturation in their thylakoid membranes. Interestingly, the effect of reduced unsaturation was different for three distinct pathways of protein transport. In thylakoids from all three mutants, transport of the OE17 protein on the DeltapH/Tat pathway was reduced by up to 50% relative to wild-type controls, when assays were run at 10, 20 or 30 degrees C. By contrast, transport of the OE33 protein on the Sec pathway was substantially increased in all the mutants at the three temperatures. Transport of the CF(O)II protein (ATPg) on the 'spontaneous' pathway was largely unaffected by reduced unsaturation of the thylakoid membranes. Experiments with intact chloroplasts from wild-type Arabidopsis and the three mutants confirmed these changes in thylakoid transport reactions and also demonstrated an increased rate of protein import across the chloroplast envelope in each of the mutants. This increased capacity of chloroplast protein import may partially compensate for the reduced capacity of thylakoid transport by the DeltapH/Tat pathway. The fad5, fad6 and fad3 fad7 fad8 mutants used in this study grow normally at 15-20 degrees C, but exhibit reduced photosynthesis and growth, relative to wild-type controls, at low temperatures (4 degrees C). The results reported here indicate that protein transport and chloroplast biogenesis may well contribute to these low-temperature phenotypes.     

Membrane dynamics as seen by fourier transform infrared spectroscopy in a cyanobacterium, Synechocystis PCC 6803. The effects of lipid unsaturation and the protein-to-lipid ratio

The roles of lipid unsaturation and lipid-protein interactions in maintaining the physiologically required membrane dynamics were investigated in a cyanobacterium strain, Synechocystis PCC 6803. The specific effects of lipid unsaturation on the membrane structure were addressed by the use of desaturase-deficient (desA(-)/desD(-)) mutant cells (which contain only oleic acid as unsaturated fatty acid species) of Synechocystis PCC 6803. The dynamic properties of the membranes were determined from the temperature dependence of the symmetric CH(2) stretching vibration frequency, which is indicative of the lipid fatty acyl chain disorder. It was found that a similar membrane dynamics is maintained at any growth temperature, in both the wild-type and the mutant cell membranes, with the exception of mutant cells grown at the lower physiological temperature limit. It seems that in the physiological temperature range the desaturase system of the cells can modulate the level of lipid desaturation sufficiently to maintain similar membrane dynamics. Below the range of normal growth temperatures, however, the extent of lipid disorder was always higher in the thylakoid than in the cytoplasmic membranes prepared from the same cells. This difference was attributed to the considerable difference in protein-to-lipid ratio in the two kinds of membranes, as determined from the ratio of the intensities of the protein amide I band and the lipid ester C&z.dbnd6;O vibration. The contributions to the membrane dynamics of an ab ovo present 'structural' lipid disorder due to the protein-lipid interactions and of a thermally induced 'dynamic' lipid disorder could be distinguished.     

PEROXIREDOXIN Q stimulates the activity of the chloroplast 16:1Δ3trans FATTY ACID DESATURASE4

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1^Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid‐associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild‐type levels of 16:1t. The FAD4–PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co‐production of PRXQ with FAD4 was required to produce Δ3‐desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site‐directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.     

Specific role of phosphatidylglycerol and functional overlaps with other thylakoid lipids in Arabidopsis chloroplast biogenesis

Key message

With phosphate deficiency, the role of phosphatidylglycerol is compensated by increased glycolipid content in thylakoid membrane biogenesis but not photosynthetic electron transport in Arabidopsis chloroplasts.

Abstract

In plants and cyanobacteria, anionic phosphatidylglycerol (PG) is the only major phospholipid in thylakoid membranes, where neutral galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are predominant. In addition to provide a lipid bilayer matrix, PG plays a specific role in photosynthetic electron transport. Non-phosphorous sulfoquinovosyldiacylglycerol (SQDG) is another anionic lipid in thylakoids; it substitutes for PG under phosphate (Pi) deficiency to maintain proper balance of anionic charge in thylakoid membranes. Although the crucial role of PG in photosynthesis has been deeply analyzed in cyanobacteria, its physiological function in seed plants other than photosynthesis remains unclear. To reveal specific roles of PG and functional overlaps with other thylakoid lipids, we characterized a PG-deficient Arabidopsis mutant (pgp1-2) under Pi-controlled conditions. Under Pi-sufficient conditions, the proportion of PG and other thylakoid lipids was decreased in pgp1-2, which led to severe disruption of thylakoid membrane biogenesis. Under Pi-deficient conditions, the proportion of all glycolipids in the mutant was greatly increased, with that of PG further decreased. In Pi-deficient pgp1-2, thylakoid membranes remarkably developed, which was accompanied by a change in nucleoid morphology and restored expression of nuclear- and plastid-encoded photosynthesis genes. Increase in glycolipid content with Pi deficiency may compensate for the loss of PG in terms of thylakoid membrane biogenesis. Although Pi deficiency increased chlorophyll and photosynthesis protein content in pgp1-2, it critically decreased photochemical activity in PSII. Further deprivation of PG in photosynthesis complexes may abolish the PSII activity in Pi-deficient pgp1-2, which suggests that glycolipids cannot replace PG in photosynthesis.

     

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 small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid import

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5' of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram- bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis.     

Cryoprotectin protects thylakoids during a freeze-thaw cycle by a mechanism involving stable membrane binding

Chloroplast thylakoid membranes of higher plants are damaged by freezing both in vivo and in vitro. The resulting inactivation of photosynthetic electron transport has been related to transient membrane rupture, leading to the loss of soluble electron transport proteins and osmotically active solutes from the thylakoid lumen. We have recently purified and sequenced a protein from cold acclimated cabbage, that protects thylakoids from this freeze-thaw damage. The protein belongs to the WAX9 family of nonspecific lipid transfer proteins, but has no detectable lipid transfer activity. Conversely, other transport-active lipid transfer proteins show no cryoprotective activity. We show here that cryoprotectin binds to thylakoid membranes. Both cryoprotective activity and membrane binding were inhibited in the presence of specific sugars, most effectively by Glc-6-S. The binding of cryoprotectin to thylakoids reduced the fluidity of the membrane lipids close to the membrane/solution interface, but not in the hydrophobic core region. Using immobilized liposomes we could show that cryoprotectin was able to bind to pure lipid membranes.     

The fluidity of chloroplast thylakoid membranes and their constituent lipids: A comparative study by ESR

Comparative measurements were made of the fluidity of chloroplast thylakoids, total membrane lipids and polar lipids utilizing the order parameter and motion of spin labels.No significant differences were found in the fluidity of membranes or total membrane lipids from a wild type and a mutant barley (Hordeum vulgare chlorina f₂ mutant) which lacks chlorophyll $\text{b}$ and a 25 000 dalton thylakoid polypeptide. Redistribution of intrinsic, exoplasmic face (EF) membrane particles by unstacking thylakoid membranes in low salt medium also had no effect on membrane fluidity. However, heating of isolated thylakoids decreased membrane fluidity.The fluidity of vesicles composed of membrane lipids is much greater than that of the corresponding membranes. Fluidity of the membranes, however, increased during greening indicating that the rigidity of the membranes, compared with that of total membrane lipids, is not caused by chlorophyll or its associated peptides. It is concluded that the restriction of motion in the acyl chains in the thylakoids is not caused by chlorophyll or the major intrinsic polypeptide but by some other protein components.     

The topology of phosphatidylglycerol populations is essential for sustaining photosynthetic electron flow activities in thylakoid membranes

The transmembrane distribution of phosphatidylglycerol (PG) was determined in rightside-out (RO) and inside-out vesicles (IO) obtained by fragmentation of spinach thylakoids in a Yeda press, followed by partition in an aqueous dextran-polyethyleneglycol two-phase system. Using the phospholipase A(2) from porcine pancreas to digest selectively PG molecules in the outer monolayer (exposed to the incubation medium) of the membrane, we found the molar outside/inside distribution to be 70/30+/-5 in RO and 40/60+/-3 in IO. The transmembrane distribution of PG in IO was the opposite of that in intact thylakoids (molar ratio 58/42+/-3). The phospholipid population which sustained most of the uncoupled photosystem II electron flow activity was localized in the inner monolayer (exposed to the thylakoid lumen) of both thylakoid and RO membranes. In contrast, the activity in IO membranes was highly dependent on the PG population located in the outer monolayer. This finding brings the first direct demonstration of the dependence of the photosynthetic electron flow activity on the integrity of the inner topological pool of PG in the thylakoid membrane.     

Three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography

We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20° to 25° as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slit-like architecture, but their size varies tremendously from approximately 15 × 30 nm to approximately 15 × 435 nm, which is approximately 5 times larger than seen in chemically fixed thylakoids. The variable slit length results in less periodicity in grana/stroma thylakoid organization than proposed in the original helical model. The stroma thylakoids also exhibit considerable architectural variability, which is dependent, in part, on the number and the orientation of adjacent grana stacks to which they are connected. Whereas some stroma thylakoids form solid, sheet-like bridges between adjacent grana, others exhibit a branching geometry with small, more tubular sheet domains also connecting adjacent, parallel stroma thylakoids. We postulate that the tremendous variability in size of the junctional slits may reflect a novel, active role of junctional slits in the regulation of photosynthetic function. In particular, by controlling the size of junctional slits, plants could regulate the flow of ions and membrane molecules between grana and stroma thylakoid membrane domains.     

Effect of Cold-Hardening on Thylakoid Membrane Lipids and Proteins of Spring Wheat and Winter Wheat

By comparison of thylakoid membrane lipids and their fatty acid composition, the supermolecular structure of light harvesting chlorophyll a/b-protein complex of Photosystem Ⅱ (LHC Ⅱ ) and the spectroscopic characteristics of thylakoids in winter wheat (Yanda 1817) with those in spring wheat (8901) before and after cold-hardening, it was found that after cold-hardening: (1)The trans-3-hexadeeenoic acid content of phosphatidyl alycerol (PG) in both cultivars decreased significantly, the ratio of monogalactosyl diglyceride (MGDG)/digalactosyl diglyceride (DGDG) in the thylakoid of Yanda 1817 decreased, but had no distinct change in 8901. (2)The lipid/chlorophyll ratio in thylakoids of Yanda 1817 increased significantly, but had no distinct change in 8901. (3) The LHC Ⅱ oligomer content decreased in thylakoids of both cultivars. (4) The A683/A652 ratio of the 4th derivative absorption spectra increased in both cultivars. (5)The F685/F738 ratio of low temperature (77K) fluorescence spectra of thylakoids in 8901 increased but was not affected in Yanda 1817. It was concluded that one of the major strategies of wheat to adapt low temperature was the increase of thylakoid membrane fluidity, and that the decrease of MGDG content may play an important role in stabilizing the bilayer structure of the thylakoid membrane at low temperature.     

Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites

Plant chloroplasts contain an intricate photosynthetic membrane system, the thylakoids, and are surrounded by two envelope membranes at which thylakoid lipids are assembled. The glycoglycerolipids mono- and digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol as well as phosphatidylglycerol, are present in thylakoid membranes, giving them a unique composition. Fatty acids are synthesized in the chloroplast and are either directly assembled into thylakoid lipids at the envelope membranes or exported to the ER (endoplasmic reticulum) for extraplastidic lipid assembly. A fraction of lipid precursors is reimported into the chloroplast for the synthesis of thylakoid lipids. Thus polar lipid assembly in plants requires tight co-ordination between the chloroplast and the ER and necessitates inter-organelle lipid trafficking. In the present paper, we discuss the current knowledge of the export of fatty acids from the chloroplast and the import of chloroplast lipid precursors assembled at the ER. Direct membrane contact sites between the ER and the chloroplast outer envelopes are discussed as possible conduits for lipid transfer.     

Effect of Temperature on Growth of Bacillus sp. T-4, a Thermophilic Acidophilic Bacterium

The lipid distribution and function in the thylakoid membranes from a thermophilic cyanobacterium, Mastigocladus laminosus, were investigated. The thylakoid membranes were treated with digitonin and separated on a DEAE-cellulose column into fractions enriched in photosystem I or II complex. Lipid analyses showed a specific distribution of anionic lipids among the fractions. A mild delipidation of the membranes with cholate indicates that monogalactosyl diacylglycerol (MGDG) and sulfoquinovosyl diacylglycerol (SQDG) are released rapidly, while the major parts of digalactosyl diacylglycerol (DGDG) and phosphatidylglycerol (PG) are tightly associated with membranes, suggesting a different distribution between the two groups of lipids. Measurements of fluorescence of delipidated and reconstituted thylakoids showed the contribution of lipids to energy transfer. MGDG enhanced all the original fluorescence of thylakoids, while acidic PG and SQDG stimulated fluorescence of photosystem I and antena chlorophyll-protein complexes. DGDG was less effective under the conditions tested.     

Low Temperature Development Induces a Specific Decrease in trans-Delta-Hexadecenoic Acid Content which Influences LHCII Organization

Lipid and fatty acid analyses were performed on whole leaf extracts and isolated thylakoids from winter rye (Secale cereale L. cv Puma) grown at 5°C cold-hardened rye (RH) and 20°C nonhardened rye (RNH). Although no significant change in total lipid content was observed, growth at low, cold-hardening temperature resulted in a specific 67% (thylakoids) to 74% (whole leaves) decrease in the trans-Δ³-hexadecenoic acid (trans-16:1) level associated with phosphatidyldiacylglycerol (PG). Electron spin resonance and differential scanning calorimetry (DSC) indicated no significant difference in the fluidity of RH and RNH thylakoids. Separation of chlorophyll-protein complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the ratio of oligomeric light harvesting complex:monomeric light harvesting complex (LHCII₁:LHCII₃) was 2-fold higher in RNH than RH thylakoids. The ratio of CP1a:CP1 was also 1.5-fold higher in RNH than RH thylakoids. Analyses of winter rye grown at 20, 15, 10, and 5°C indicated that both, the trans-16:1 acid levels in PG and the LHCII₁:LHCII₃ decreased concomitantly with a decrease in growth temperature. Above 40°C, differential scanning calorimetry of RNH thylakoids indicated the presence of five major endotherms (47, 60, 67, 73, and 86°C). Although the general features of the temperature transitions observed above 40°C in RH thylakoids were similar to those observed for RNH thylakoids, the transitions at 60 and 73°C were resolved as inflections only and RH thylakoids exhibited transitions at 45 and 84°C which were 2°C lower than those observed in RNH thylakoids. Since polypeptide and lipid compositions of RH and RNH thylakoids were very similar, we suggest that these differences reflect alterations in thylakoid membrane organization. Specifically, it is suggested that low developmental temperature modulates LHCII organization such that oligomeric LHCII predominates in RNH thylakoids whereas a monomeric or an intermediate form of LHCII predominates in RH thylakoids. Furthermore, we conclude that low developmental temperature modulates LHCII organization by specifically altering the fatty composition of thylakoid PG.     

Dimerization constant and single-channel conductance of Gramicidin in thylakoid membranes

Summary The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5×10¹⁴ cm²/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the upconcentration of gramicidin in the small fractional area of protein free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nm (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mm NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.We would like to thank Ms. H. Kenneweg for photographs. financial support by the DFG (SFB 171/B3) is gratefully acknowledged.This paper is dedicated to the Late Prof. Peter Läger.     

Temperature dependence of the lipid packing in thylakoid membranes studied by time- and spectrally resolved fluorescence of Merocyanine 540

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above -30 degrees C and that a phase transition occurs only above 45 degrees C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases--upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 degrees C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 degrees C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.     

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Increases in lutein/carotenoid ratios lead to decreased thylakoid fluidity and hamper grana folding due to carotenoid-dependent changes in both photosynthetic complexes and lipid matrix organization.     

Localization and integration of thylakoid protein translocase subunit cpTatC

Thylakoid membranes have a unique complement of proteins, most of which are nuclear encoded synthesized in the cytosol, imported into the stroma and translocated into thylakoid membranes by specific thylakoid translocases. Known thylakoid translocases contain core multi-spanning, membrane-integrated subunits that are also nuclear-encoded and imported into chloroplasts before being integrated into thylakoid membranes. Thylakoid translocases play a central role in determining the composition of thylakoids, yet the manner by which the core translocase subunits are integrated into the membrane is not known. We used biochemical and genetic approaches to investigate the integration of the core subunit of the chloroplast Tat translocase, cpTatC, into thylakoid membranes. In vitro import assays show that cpTatC correctly localizes to thylakoids if imported into intact chloroplasts, but that it does not integrate into isolated thylakoids. In vitro transit peptide processing and chimeric precursor import experiments suggest that cpTatC possesses a stroma-targeting transit peptide. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate, suggesting that it might integrate through one of the known thylakoid translocation pathways. However, chemical inhibitors to the cpSecA-cpSecY and cpTat pathways did not impede cpTatC localization to thylakoids when used in import assays. Analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration or assembly into the cpTat receptor complex. These data suggest the existence of another translocase, possibly one dedicated to the integration of chloroplast translocases.