Influence of Ca2+ on the thylakoid lumen violaxanthin de-epoxidase activity through Ca2+ gating of H+ flux at the CFo H+ channel

Part of the chloroplast photoprotection response to excess light absorption involves formation of zeaxanthin (and antheraxanthin) via the violaxanthin deepoxidase enzyme, the activity of which requires lumen acidity near or below pH 6.0. Clearly, the violaxanthin de-epoxidase activity is strongly regulated because at equivalent energization levels (including the parameters of H⁺ accumulation and ATP formation rates), there can be either low or high violaxanthin de-epoxidase enzyme activity. This work shows that the factor or factors responsible for regulating the violaxanthin deepoxidase correlate directly with those which regulate the expression of membrane-localized or delocalized proton gradient (Δ~μ_H+) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca²⁺ binding to the lumen side of the thylakoid membrane; in particular, Ca²⁺ binding to the 8 kDA subunit III of the CF_o H⁺ channel. The activity of violaxanthin deepoxidase in pea (Pisum sativa) and spinach (Spinacea oleracea) thylakoids is shown here to be strongly correlated with conditions known from previous work to displace Ca²⁺ from the CF_o H⁺ channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intrathylakoid pH in Isolated Pea Chloroplasts as Probed by Violaxanthin Deepoxidation

Light-driven violaxanthin deepoxidation was measured in isolated pea (Pisum sativum) chloroplasts without ATP synthesis (basal conditions) and with ATP synthesis (coupled conditions). Thylakoids stored in high salt (HS) or low salt (LS) storage medium were tested. In previous experiments, HS thylakoids and LS thylakoids were related to delocalized and localized proton coupling, respectively.Light-driven deepoxidase activity was compared to the pH dependence of deepoxidase activity established in dark reactions. At an external pH of 8, light-driven deepoxidation indicated effective pH values close to pH 6 for all reaction conditions. Parallel to deepoxidation, the thylakoid lumen pH was estimated by the fluorescent dye pyranine.In LS thylakoids under coupled conditions the lumen pH did not drop below pH 6.7. At pH 6.7, no deepoxidase activity is expected based on the pH dependence of enzyme activity. The results suggest that deepoxidation activity is controlled by the pH in sequestered membrane domains, which, under localized proton coupling, can be maintained at pH 6.0 when the lumen pH is far above pH 6.0. The extent of violaxanthin conversion (availability), however, appeared to be regulated by lumenal pH. Dithiothreitol-sensitive nonphotochemical quenching of chlorophyll fluorescence was dependent on zeaxanthin and not related to lumenal pH. Thus, zeaxanthin-dependent quenching[mdash]known to be pH dependent[mdash]appeared to be triggered by the pH of localized membrane domains.     

Protein dynamics and lipid affinity of monomeric,zeaxanthin-binding LHCII in thylakoid membranes

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from ¹³C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.     

Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.     

Influence of ascorbate and the Mehler peroxidase reaction on non-photochemical quenching of chlorophyll fluorescence in maize mesophyll chloroplasts

 Non-photochemical quenching of chlorophyll fluorescence (NPQ) and quantum yield of photosystem II (PSII) were studied with intact mesophyll chloroplasts of maize (Zea mays L.) during the initial minutes of illumination using the pulse-modulated chlorophyll fluorescence technique. Non-photochemical quenching was rapidly reversible in the dark at any point during illumination, which is indicative of energy-dependent dissipation of energy (mediated via thylakoid ΔpH changes and ascorbate-dependent synthesis of zeaxanthin). In chloroplasts suspensions including 15 mM ascorbate in the medium, with addition of oxaloacetate and pyruvate, the PSII yield, rate of reduction of oxaloacetate and phosphorylation of pyruvate reached a maximum after approximately 2 min of illumination. Under these conditions, which promote phosphorylation and a decreased ΔpH across the thylakoid membrane, NPQ rose to a maximum after 2–3 min of illumination, dropped to a minimum after about 6 min, and then increased to a steady-state level. A rather similar pattern was observed when leaves were illuminated following a 30-min dark period. Providing chloroplasts with higher levels of ascorbate (60 mM), prevented the transient drop in NPQ. Anaerobic conditions or addition of potassium cyanide caused a decrease in PSII yield, providing evidence for operation of the ascorbate-dependent Mehler-peroxidase reaction. These conditions also strongly suppressed the transient drop in NPQ. Dithiothreitol, an inhibitor of violaxanthin de-epoxidase, caused a large drop in NPQ even in the presence of high levels of ascorbate. The results suggest that the decline of NPQ occurs in response to an increase in lumen pH after initiation of phosphorylation, that this decline can be suppressed by conditions where ascorbate is not limiting for violaxanthin de-epoxidase, and that the increase of NPQ after such a decline is the result of development of energy dissipation in PSII reaction centers. Received: 13 August 1999 / Accepted: 17 September 1999     

The xanthophyll cycle, its regulation and components

During the last few years much interest has been focused on the photoprotective role of zeaxanthin. In excessive light zeaxanthin is rapidly formed in the xanthophyll cycle from violaxanthin, via the intermediate antheraxanthin, a reaction reversed in the dark. The role of zeaxanthin and the xanthophyll cycle in photoprotection, is based on fluorescence quenching measurements, and in many studies a good correlation to the amount of zeaxanthin (and antheraxanthin) has been found. Other suggested roles for the xanthophylls involve, protection against oxidative stress of lipids, participation in the blue light response, modulation of the membrane fluidity and regulation of abscisic acid synthesis. The enzyme violaxanthin de-epoxidase has recently been purified from spinach and lettuce as a 43-kDa protein. It was found as 1 molecule per 20–100 electron-transport chains. The gene has been cloned and sequenced from Lactuca sativa, Nicotiana tabacum and Arabidopsis thaliana. The transit peptide was characteristic of nuclear-encoded and lumen-localized proteins. The activity of violaxanthin de-epoxidase is controlled by the lumen pH. Thus, below pH 6.6 the enzyme binds to the thylakoid membrane. In addition ascorbate becomes protonated to ascorbic acid (pK_a= 4.2) the true substrate (K_m= 0.1 m M ) for the violaxanthin de-epoxidase. We present arguments for an ascorbate transporter in the thylakoid membrane. The enzyme zeaxanthin epoxidase requires FAD as a cofactor and appears to use ferredoxin rather than NADPH as a reductant. The zeaxanthin epoxidase has not been isolated but the gene has been sequenced and a functional protein of 72.5 kDa has been expressed. The xanthophyll cycle pigments are almost evenly distributed in the thylakoid membrane and at least part of the pigments appears to be free in the lipid matrix where we conclude that the conversion by violaxanthin de-epoxidase occurs.     

Mechanical freeze-thaw damage and frost hardening in leaves and isolated thylakoids from spinach. I. Mechanical freeze-thaw damage in an artificial stroma medium

Abstract Freeze-thaw damage to thylakoids in spinach leaves has been simulated in vitro, using a complex, defined artificial stroma medium. The resulting mechanical damage was quantified by measuring the loss of the marker protein plastocyanin from the thylakoid lumen, which is released as a result of membrane rupture. Loss of plastocyanin was already apparent at 0°C and became more severe at subzero temperatures. The time course of plastocyanin loss during freezing was biphasic: after an initial rapid loss, plastocyanin release was linearly dependent on incubation time. In short-term experiments a linear dependence on freezing temperature was observed. Solute diffusion into the thylakoids, leading to influx of water and eventually membrane rupture, has been observed in vitro as well as after freezing of leaves.     

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

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

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

The effects of moderately high temperature on zeaxanthin accumulation and decay

Moderately high temperature reduces photosynthetic capacities of leaves with large effects on thylakoid reactions of photosynthesis, including xanthophyll conversion in the lipid phase of the thylakoid membrane. In previous studies, we have found that leaf temperature of 40°C increased zeaxanthin accumulation in dark-adapted, intact tobacco leaves following a brief illumination, but did not change the amount of zeaxanthin in light-adatped leaves. To investigate heat effects on zeaxanthin accumulation and decay, zeaxanthin level was monitored optically in dark-adapted, intact tobacco and Arabidopsis thaliana leaves at either 23 or 40°C under 45-min illumination. Heated leaves had more zeaxanthin following 3-min light but had less or comparable amounts of zeaxanthin by the end of 45?min of illumination. Zeaxanthin accumulated faster at light initiation and decayed faster upon darkening in leaves at 40°C than leaves at 23°C, indicating that heat increased the activities of both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). In addition, our optical measurement demonstrated in vivo that weak light enhances zeaxanthin decay relative to darkness in intact leaves of tobacco and Arabidopsis, confirming previous observations in isolated spinach chloroplasts. However, the maximum rate of decay is similar for weak light and darkness, and we used the maximum rate of decay following darkness as a measure of the rate of ZE during steady-state light. A simulation indicated that high temperature should cause a large shift in the pH dependence of the amount of zeaxanthin in leaves because of differential effects on VDE and ZE. This allows for the reduction in ΔpH caused by heat to be offset by increased VDE activity relative to ZE.     

Full-length plastocyanin precursor is translocated across isolated thylakoid membranes

In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.     

Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis

Regulation of violaxanthin de-epoxidase (VDE) involves a conformational change at low lumenal pH, followed by binding of the enzyme to the thylakoid membrane. The role of histidine residues in this process was studied by release of unbound enzyme from thylakoids upon sonication, on a pH scale from 4.7 to 7.1. The co-operativity for binding of spinach VDE (four histidines) to the membrane was found to be 3.8, with respect to protons, and had an inflexion point at pH 6.6, whereas VDE from wheat (three histidines) showed a co-operativity of 2.9 and had an inflexion point at pH 6.2. Mutant forms of VDE were constructed and probed for their binding to the outside of thylakoid membranes. With one or two histidines substituted for alanine or arginine, a lower co-operativity (1.6–2.3) was found, compared with the wild type. Based on these findings, and that the pKa value for histidine is within the range where the VDE binding takes place, we propose that protonation of the histidine residues at low pH induces the conformational change of VDE, and hence indirectly regulates binding of the enzyme to the thylakoid membrane.     

Cytochrome f: Structure,function and biosynthesis

Cytochrome f is an intrinsic membrane component of the cytochrome bf complex, transferring electrons from the Rieske FeS protein to plastocyanin in the thylakoid lumen. The protein is held in the thylakoid membrane by a single transmembrane span located near its C-terminus with a globular hydrophilic domain extending into the lumen. The globular domain of the turnip protein has recently been crystallised, offering the prospect of a detailed three-dimensional structure. Reaction with plastocyanin involves localised positive charges on cytochrome f interacting with the acidic patch on plastocyanin and electron transfer via the surface-exposed tyrosine residue (Tyr83) of plastocyanin. Apocytochrome f is encoded in the chloroplast genome and is synthesised with an N-terminal presequence which targets the protein to the thylakoid membrane. The synthesis of cytochrome f is coordinated with the synthesis of the other subunits of the cytochrome bf complex.     

Dynamic Mechanical Responses of Arabidopsis Thylakoid Membranes during PSII-Specific Illumination

Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes.     

Effect of Irradiance on the Thermal Stability of Thylakoid Membrane Isolated from Acclimated Wheat Leaves

Thermal stability of thylakoid membranes isolated from acclimated and non-acclimated wheat (Triticum aestivum L. cv. HD 2329) leaves under irradiation was studied. Damage to the photosynthetic electron transport activity was more pronounced in thylakoid membranes isolated from non-acclimated leaves as compared to thylakoid membrane isolated from acclimated wheat leaves at 35 °C. The loss of D1 protein was faster in non-acclimated thylakoid membrane as compared to acclimated thylakoid membranes at 35 °C. However, the effect of elevated temperature on the 33 kDa protein associated with oxygen evolving complex in these two types of thylakoid membranes was minimal. Trypsin digestion of the 33 kDa protein in the thylakoid membranes isolated from control and acclimated seedlings suggested that re-organisation of 33 kDa protein occurs before its release during high temperature treatment.     

Adenocarcinoma of the human exocrine pancreas: presence of secretin and caerulein receptors

We have measured the cytochrome compositions of subfractions derived from appressed and non-appressed thylakoids by centrifugation and aqueous two-phase partition. Cytochrome $\text{b}$-559 (HP) was not detectable in the fraction derived from non-appressed thylakoids. Cytochromes $\text{f}\text{,}\text{b}$-563 and $\text{b}$-559 (LP) were all evenly distributed throughout the thylakoid membrane. This distribution points to plastocyanin as a possible lateral shuttle of reducing equivalents between spatially separated photosystems.Cytochrome $\text{f}$ was accessible to externally added plastocyanin in the inside-out vesicles but not in vesicles of normal sidedness. This strongly supports a location at the inner side of the thylakoid membrane. Cytochrome $\text{b}$-563 was slowly reduced by dithionite in vesicles with both normal and inside-out orientation suggesting a location within the membrane interior.     

Comparison of violaxanthin de-epoxidation from the stroma and lumen sides of isolated thylakoid membranes from Arabidopsis: implications for the mechanism of de-epoxidation

The enzyme violaxanthin de-epoxidase (VxDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of membrane-bound violaxanthin (Vx) to zeaxanthin. De-epoxidation from the opposite, stroma side of the membrane has been investigated in the npq1 mutant from Arabidopsis thaliana (L.) Heynh. - which lacks VxDE - by adding partially purified VxDE from spinach thylakoids. The accessibility of Vx to the exogenously added enzyme (exoVxDE) and the kinetics of Vx conversion by the exoVxDE in thylakoids from npq1 plants were very similar to the characteristics of Vx conversion by the endogenous enzyme (endoVxDE) in thylakoids from wild-type plants. However, the conversion of Vx by exoVxDE was clearly retarded at lower temperatures when compared with the reaction catalyzed by endoVxDE. Since the exoVxDE - in contrast to the endoVxDE - has no access to the stacked regions of the membrane, where the xanthophylls bound to photosystem II are located, these results support the assumption of pronounced diffusion of xanthophylls within the thylakoid membrane.     

Protein transport towards the thylakoid lumen: post-translational translocation in tandem

Many proteins found in the chloroplast are synthesized in the cytoplasm as precursor molecules containing transit peptides. Proteins targeted to the stroma must pass through the two envelope membranes to reach their destination. Proteins located in the chloroplast lumen also have to be transferred across the thylakoid membrane. That is, lumen proteins must cross three biological membranes in order to reach their final location. Recent evidence shows that the routing of plastocyanin towards the lumen involves two post-translational transport processes mediated by two different regions of the transit peptide and two different processing proteases. It is postulated that the genetic information for the plastocyanin precursor, which already contained a signal peptide, was transferred from the endosymbiont to the nucleus. Then a chloroplast-specific targeting-peptide was added.     

Mechanical freeze-thaw damage and frost hardening in leaves and isolated thylakoids from spinach. II. Frost hardening reduces solute permeability and increases extensibility of thylakoid membranes

Abstract Thylakoids isolated from cold-acclimated spinach (Spinacia oleracea L.) leaves were more resistant against mechanical freeze-thaw injury measured as plastocyanin release, than thylakoids from non-acclimated leaves. They were more resistant against solute influx during freezing and they were able to re-expand to a larger volume in comparison to non-hardy controls. Likewise, plastocyanin was released from thylakoids of non-acclimated but not of frost-hardy leaves under conditions of mild in situ freezing stress for several days.