Stroma-exposed Thylakoid Research Articles

Role of serine/threonine protein kinase STN7 in the formation of two distinct photosystem I supercomplexes in Physcomitrium patens.

Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments.

Lateral heterogeneity of the proton potential along the thylakoid membranes of chloroplasts

This work is devoted to critical analysis and reexamination of the problem of lateral heterogeneity of the trans-thylakoid pH difference (ΔpH=pHout−pHin) in thylakoid membranes of chloroplasts. Correct measurements of ΔpH may be complicated by nonuniform partitioning of the protons pumped into the lumen of granal (stacked) and stroma-exposed thylakoids. We have compared results of ΔpH estimations in isolated bean chloroplasts by two different methods. One of the methods is based on the use of pH-sensitive spin-probes. Another approach relies on the analysis of pH-dependent post-illumination reduction of P700+ - oxidized reaction center of photosystem I (PSI). Both methods lead to virtually the same values of ΔpH, as measured in the state of photosynthetic control when the ATP synthase complexes are inactive (ΔpH~2.3–2.5 at pHout~8). Under the photophosphorylation conditions (metabolic state 3), ΔpH decreases due to the proton drain from the lumen to stroma via active ATP synthases (ΔpH~1–2 at pHout~8). In this state, ΔpH values derived from kinetic data are smaller than ΔpH measured with the pH-probing amines. Such a discrepancy can be explained by the coexistence of thylakoids with different pHin established in granal and stromal thylakoids. The kinetic method is equivalent to the use of a “local pH-meter”, which is sensitive to less significant decrease in pHin inside the stroma-exposed thylakoids. Otherwise, pH-indicating probes give information on pHin values averaged out at both granal and stromal thylakoids. Experimental results have been analyzed within the framework of our mathematical model developed for simulation of electron and proton transport processes in laterally heterogeneous thylakoids. The model provides a reasonable description of experimental data, supporting the notion that the long-range diffusion of protons within the lumen and obstructed diffusion of mobile electron carriers (PQH2 and Pc) influence the lateral profiles of pH along the thylakoid membranes. The model predicts significant alkalization of the inter-thylakoid gap and the establishment of nonuniform lateral profiles of ΔpH under the photophosphorylation conditions. These results are discussed in the context of the problem of energy coupling in laterally heterogeneous lamellar system of chloroplasts.

Changes in antenna sizes of photosystems during state transitions in granal and stroma-exposed thylakoid membrane of intact chloroplasts in Arabidopsis mesophyll protoplasts.

In chloroplasts of plants and algae, state transition is an important regulatory mechanism to maintain the excitation balance between PSI and PSII in the thylakoid membrane. Light-harvesting complex II (LHCII) plays a key role as the regulated energy distributor between PSI and PSII. It is widely accepted that LHCII, which is bound to PSII localized mainly in the granal thylakoid, migrates to bind with PSI localized mainly in the stroma-exposed thylakoid under preferential excitation of PSII. The phenomena have been extensively characterized by many methods. However, the exchange of LHCII between PSII and PSI has not been directly observed in vivo at physiological temperatures. Herein we applied fluorescence spectromicroscopy to Arabidopsis mesophyll protoplasts in order to observe in vivo changes in fluorescence spectra of granal and stromal thylakoid regions during the state transition. The microscopic fluorescence spectra obtained from a few sections with different depths were decomposed into PSI and PSII spectra and self-absorption effects were removed. We were able to determine amplitude changes of PSI and PSII in fluorescence spectra solely due to state transition. Subdomain analysis of granal and stromal thylakoid regions clarified variant behaviors in the different regions.

The membrane proteome of stroma thylakoids from Arabidopsis thaliana studied by successive in-solution and in-gel digestion.

From individual localization and large-scale proteomic studies, we know that stroma-exposed thylakoid membranes harbor part of the machinery performing the light-dependent photosynthetic reactions. The minor components of the stroma thylakoid proteome, regulating and maintaining the photosynthetic machinery, are in the process of being unraveled. In this study, we developed in-solution and in-gel proteolytic digestion methods, and used them to identify minor membrane proteins, e.g. transporters, in stroma thylakoids prepared from Arabidopsis thaliana (L.) Heynh Columbia-0 leaves. In-solution digestion with chymotrypsin yielded the largest number of peptides, but in combination with methanol extraction resulted in identification of the largest number of membrane proteins. Although less efficient in extracting peptides, in-gel digestion with trypsin and chymotrypsin led to identification of additional proteins. We identified a total of 58 proteins including 44 membrane proteins. Almost half are known thylakoid proteins with roles in photosynthetic light reactions, proteolysis and import. The other half, including many transporters, are not known as chloroplast proteins, because they have been either curated (manually assigned) to other cellular compartments or not curated at all at the plastid protein databases. Transporters include ATP-binding cassette (ABC) proteins, transporters for K(+) and other cations. Other proteins either have a role in processes probably linked to photosynthesis, namely translation, metabolism, stress and signaling or are contaminants. Our results indicate that all these proteins are present in stroma thylakoids; however, individual studies are required to validate their location and putative roles. This study also provides strategies complementary to traditional methods for identification of membrane proteins from other cellular compartments.

HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis

Under high-irradiance conditions, plants must efficiently protect photosystem II (PSII) from damage. In this study, we demonstrate that the chloroplast protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1) is expressed in response to high light and functions in protecting PSII against photodamage. Arabidopsis thaliana hhl1 mutants show hypersensitivity to high light, drastically decreased PSII photosynthetic activity, higher nonphotochemical quenching activity, a faster xanthophyll cycle, and increased accumulation of reactive oxygen species following high-light exposure. Moreover, HHL1 deficiency accelerated the degradation of PSII core subunits under high light, decreasing the accumulation of PSII core subunits and PSII-light-harvesting complex II supercomplex. HHL1 primarily localizes in the stroma-exposed thylakoid membranes and associates with the PSII core monomer complex through direct interaction with PSII core proteins CP43 and CP47. Interestingly, HHL1 also directly interacts, in vivo and in vitro, with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), which functions in the repair and reassembly of PSII. Furthermore, the hhl1 lqy1 double mutants show increased photosensitivity compared with single mutants. Taken together, these results suggest that HHL1 forms a complex with LQY1 and participates in photodamage repair of PSII under high light.

Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Modulation of the light-harvesting chlorophyll antenna size in Chlamydomonas reinhardtii by TLA1 gene over-expression and RNA interference

Truncated light-harvesting antenna 1 (TLA1) is a nuclear gene proposed to regulate the chlorophyll (Chl) antenna size in Chlamydomonas reinhardtii. The Chl antenna size of the photosystems and the chloroplast ultrastructure were manipulated upon TLA1 gene over-expression and RNAi downregulation. The TLA1 over-expressing lines possessed a larger chlorophyll antenna size for both photosystems and contained greater levels of Chl b per cell relative to the wild type. Conversely, TLA1 RNAi transformants had a smaller Chl antenna size for both photosystems and lower levels of Chl b per cell. Western blot analyses of the TLA1 over-expressing and RNAi transformants showed that modulation of TLA1 gene expression was paralleled by modulation in the expression of light-harvesting protein, reaction centre D1 and D2, and VIPP1 genes. Transmission electron microscopy showed that modulation of TLA1 gene expression impacts the organization of thylakoid membranes in the chloroplast. Over-expressing lines showed well-defined grana, whereas RNAi transformants possessed loosely held together and more stroma-exposed thylakoids. Cell fractionation suggested localization of the TLA1 protein in the inner chloroplast envelope and potentially in association with nascent thylakoid membranes, indicating a role in Chl antenna assembly and thylakoid membrane biogenesis. The results provide a mechanistic understanding of the Chl antenna size regulation by the TLA1 gene.

A Small Zinc Finger Thylakoid Protein Plays a Role in Maintenance of Photosystem II in Arabidopsis thaliana

This work identifies LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), a Zn finger protein that shows disulfide isomerase activity, interacts with the photosystem II (PSII) core complex, and may act in repair of photodamaged PSII complexes. Two mutants of an unannotated small Zn finger containing a thylakoid membrane protein of Arabidopsis thaliana (At1g75690; LQY1) were found to have a lower quantum yield of PSII photochemistry and reduced PSII electron transport rate following high-light treatment. The mutants dissipate more excess excitation energy via nonphotochemical pathways than wild type, and they also display elevated accumulation of reactive oxygen species under high light. After high-light treatment, the mutants have less PSII-light-harvesting complex II supercomplex than wild-type plants. Analysis of thylakoid membrane protein complexes showed that wild-type LQY1 protein comigrates with the PSII core monomer and the CP43-less PSII monomer (a marker for ongoing PSII repair and reassembly). PSII repair and reassembly involve the breakage and formation of disulfide bonds among PSII proteins. Interestingly, the recombinant LQY1 protein demonstrates a protein disulfide isomerase activity. LQY1 is more abundant in stroma-exposed thylakoids, where key steps of PSII repair and reassembly take place. The absence of the LQY1 protein accelerates turnover and synthesis of PSII reaction center protein D1. These results suggest that the LQY1 protein may be involved in maintaining PSII activity under high light by regulating repair and reassembly of PSII complexes.

Phosphorylation of Photosynthetic Antenna Protein CP29 and Photosystem II Structure Changes in Monocotyledonous Plants under Environmental Stresses

Kinetic studies of protein dephosphorylation in thylakoid membranes showed that the minor light-harvesting antenna protein CP29 could be phosphorylated in barley (C3) and maize (C4) seedlings, but not in spinach under water [Liu, W. J., et al. (2009) Biochim. Biophys. Acta 1787, 1238-1245], salt, or cold stress [Pursiheimo, S., et al. (2003) Plant Cell Environ. 26, 1995-2003], suggesting that phosphorylation of CP29 is a general phenomenon in monocots, but not in dicots under environmental stresses. Abscisic acid (ABA), reactive oxygen species (ROS), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), NO, and the scavenger of H(2)O(2) had weak effects on CP29 phosphorylation. However, three protein kinase inhibitors, U0126, W7, and K252a (for mitogen-activated protein kinase, Ca(2+)-dependent protein kinase, and Ser/Thr protein kinases, respectively), decrease the level of CP29 phosphorylation in barley apparently under environmental stresses. Therefore, these three protein kinases are involved in CP29 phosphorylation. We also found that most CP29 phosphorylation was accompanied by its lateral migration from granum membranes to stroma-exposed thylakoid regions, and the instability of PSII supercomplexes and LHCII trimers under environmental stresses.

Defects in a proteolytic step of light-harvesting complex II in anArabidopsis stay-green mutant,ore10, during dark-induced leaf senescence

During dark-induced leaf senescence (DIS), the non-functional stay-green mutantore10 showed delayed chlorophyll (Chl) degradation and increased stability in its light-harvesting complex II (LHCII). These phenomena were closely related to the formation of aggregates that mainly consisted of terminal-truncated LHCII (Oh et al., 2003). Theore10 mutant apparently lacks the protease needed to degrade the truncated LHCII. In wild-type (WT) plants, protease was found in the thylakoid fraction, but not the soluble fraction. A similar experiment using dansylated LHCII revealed that the protease degraded both WT andore10 LHCII, indicating that its stability inore10 perhaps did not result from a defect in the LHCII polypeptides themselves. Although protease activity was not present in non-senesced WT leaves, it was induced during DIS. It also was possible to diminish the high level of protease present in the thylakoids through high-salt washing, suggesting that this enzyme is extrinsically bound to the outer surface of the stroma-exposed thylakoid regions.

Changing concepts about the distribution of Photosystems I and II between grana-appressed and stroma-exposed thylakoid membranes.

Thylakoid membranes of higher plants and some green algae, which house the light-harvesting and energy transducing functions of the chloroplast, are structurally unique. The concept of the photosynthetic unit of the 1930s (Robert Emerson, William Arnold and Hans Gaffron), needing one reaction center per hundreds of antenna molecules, was modified by the discovery of the Enhancement effect in oxygen evolution in two different wavelengths of light (Robert Emerson and his coworkers) in the late 1950s, followed by the 1960 Z scheme of Robin Hill and Fay Bendall. It was realized that two light reactions and two pigment systems were needed for oxygenic photosynthesis. Changing ideas about the distribution of Photosystem II (PS II) and PS I between the green-appressed and stroma-exposed thylakoid membrane domains, which led to the concept of lateral heterogeneity, are discussed.

Dephosphorylation of photosystem II reaction center proteins in plant photosynthetic membranes as an immediate response to abrupt elevation of temperature.

Kinetic studies of protein dephosphorylation in photosynthetic thylakoid membranes revealed specifically accelerated dephosphorylation of photosystem II (PSII) core proteins at elevated temperatures. Raising the temperature from 22 degrees C to 42 degrees C resulted in a more than 10-fold increase in the dephosphorylation rates of the PSII reaction center proteins D1 and D2 and of the chlorophyll a binding protein CP43 in isolated spinach (Spinacia oleracea) thylakoids. In contrast the dephosphorylation rates of the light harvesting protein complex and the 9-kD protein of the PSII (PsbH) were accelerated only 2- to 3-fold. The use of a phospho-threonine antibody to measure in vivo phosphorylation levels in spinach leaves revealed a more than 20-fold acceleration in D1, D2, and CP43 dephosphorylation induced by abrupt elevation of temperature, but no increase in light harvesting protein complex dephosphorylation. This rapid dephosphorylation is catalyzed by a PSII-specific, intrinsic membrane protein phosphatase. Phosphatase assays, using intact thylakoids, solubilized membranes, and the isolated enzyme, revealed that the temperature-induced lateral migration of PSII to the stroma-exposed thylakoids only partially contributed to the rapid increase in the dephosphorylation rate. Significant activation of the phosphatase coincided with the temperature-induced release of TLP40 from the membrane into thylakoid lumen. TLP40 is a peptidyl-prolyl cis-trans isomerase, which acts as a regulatory subunit of the membrane phosphatase. Thus dissociation of TLP40 caused by an abrupt elevation in temperature and activation of the membrane protein phosphatase are suggested to trigger accelerated repair of photodamaged PSII and to operate as possible early signals initiating other heat shock responses in chloroplasts.

Role of phosphorylation in the repair cycle and oligomeric structure of photosystem II

The role of PSII protein phosphorylation in the oligomeric structure of the complex and in the repair of photodamaged PSII centers was studied with intact thylakoids and thylakoid membrane subfractions isolated from differentially light-treated pumpkin (Cucurbita pepo L.) leaves. A combination of sucrose gradient fractionation of thylakoid protein complexes and immunodetection with phosphothreonine and protein-specific antibodies was used. We report in this study that the extent of phosphorylation of PSII core proteins is equivalent in dimers and monomers, and directly depends on light intensity. Phosphorylated PSII monomers migrate to the stroma-exposed thylakoids, probably following damage of the D1 protein and the dissociation of the light-harvesting complex of PSII. Once in the stroma lamellae, monomers are gradually dephosphorylated to allow the reparation of the complex. First, CP43 is dephosphorylated and as a consequence of this modification it detaches from the PSII core. In addition to D1, D2 is also thereafter dephosphorylated. Phosphorylation of PSII core polypeptides probably ensures the integrity of the monomers until repair can proceed. Dephosphorylation, on the other hand, might serve the need for opening the complex and coordinating D1 proteolysis and the attachment of ribosomes.

Induction of acclimative proteolysis of the light-harvesting chlorophyll a/b protein of photosystem II in response to elevated light intensities

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

Light-induced reversible local fusions of thylakoid membranes in the presence of dibucaine or tetracaine

The dynamics of structural changes in pea chloroplasts in the presence of 25–50 μM dibucaine or tetracaine has been examined using electron microscopy. The light-induced uptake of anesthetic cations by thylakoids is attended by the appearance of local fusions of stroma-exposed thylakoid membranes. The first membrane protrusions and interthylakoid contacts are observed after 4 s illumination and they become numerous by 10 s. As a result, a network of anastomoses is formed which is maintained during at least 10 min. These effects are reversible in the dark and can be reproduced several times. The formation of membrane fusions is inhibited by the addition of protonophore. It is supposed that the energy-dependent uptake of protonated anesthetics by thylakoids leads to an increase in positive surface charge and thus a lateral pressure on the inner side of the thylakoid membrane. The appearance of membrane protrusions (crinkles) having the positive curvature of their inner surface may be considered as a way of compensating for lateral pressure. Presumably, anastomoses result from the fusion of crinkles to adjacent thylakoids.

Kinetic Resolution of the Incorporation of the D1 Protein into Photosystem II and Localization of Assembly Intermediates in Thylakoid Membranes of Spinach Chloroplasts

The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation. Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane. The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems. Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes.

Photoinhibition and D1 Protein Degradation in Peas Acclimated to Different Growth Irradiances.

The relationship between the susceptibility of photosystem II (PSII) to photoinhibition in vivo and the rate of degradation of the D1 protein of the PSII reaction center heterodimer was investigated in leaves from pea plants (Pisum sativum L. cv Greenfeast) grown under widely contrasting irradiances. There was an inverse linear relationship between the extent of photoinhibition and chlorophyll (Chl) a/b ratios, with low-light leaves being more susceptible to high light. In the presence of the chloroplast-encoded protein synthesis inhibitor lincomycin, the differential sensitivity of the various light-acclimated pea leaves to photoinhibition was largely removed, demonstrating the importance of D1 protein turnover as the most crucial mechanism to protect against photoinhibition. In the differently light-acclimated pea leaves, the rate of D1 protein degradation (measured from [35S]methionine pulse-chase experiments) increased with increasing incident light intensities only if the light was not high enough to cause photoinhibition in vivo. Under moderate illumination, the rate constant for D1 protein degradation corresponded to the rate constant for photoinhibition in the presence of lincomycin, demonstrating a balance between photodamage to D1 protein and subsequent recovery, via D1 protein degradation, de novo synthesis of precursor D1 protein, and reassembly of functional PSII. In marked contrast, in light sufficiently high to cause photoinhibition in vivo, the rate of D1 protein degradation no longer increased concomitantly with increasing photoinhibition, suggesting that the rate of D1 protein degradation is playing a regulatory role. The extent of thylakoid stacking, indicated by the Chl a/b ratios of the differently light-acclimated pea leaves, was linearly related to the half-life of the D1 protein in strong light. We conclude that photoinhibition in vivo occurs under conditions in which the rate of D1 protein degradation can no longer be enhanced to rapidly remove irreversibly damaged D1 protein. We suggest that low-light pea leaves, with more stacked membranes and less stroma-exposed thylakoids, are more susceptible to photoinhibition in vivo mainly due to their slower rate of D1 protein degradation under sustained high light and their slower repair cycle of the photodamaged PSII centers.

The domain organization of the chloroplast thylakoid membrane. Localization of photosystem I and of the cytochrome b6- f complex

The relative distribution of chlorophyll (Chl) a and Chl b, of Photosystem I (PS I), and of the cytochrome b 6- f (Cyt b 6- f) complex in different subchloroplast membrane vesicles is presented. Spinach chloroplasts ( Chl a Chl b = 2.7 , Chl PS I = 435:1 , Chl Cyt f = 970:1 ) were fractionated by Yeda press treatment and by sonication. Counter-current distribution of the resulting thylakoid vesicles in dextran-polyethylene glycol aqueous two-phase systems yielded a laterally extended range of chloroplast thylakoid membranes without loss of chloroplast material from the initial sample. In this analysis, PS I-enriched membrane vesicles moved in the leading edge of the counter-current distribution diagram, whereas PS II-enriched vesicles remained close to the trailing edge of the counter-current distribution diagram. Vesicles of mixed composition and vesicles depleted in both PS I and PS II were isolated from intermediate positions. The Chl PS I ratio in the various fractions approximated a hyperbolic function of the Chl a Chl b ratio with limiting values of Chl PS I = 190:1 attained at a Chl a Chl b = 6 and a Chl a Chl b = 1.67 attained when the Chl PS I ratio approaches infinity ([PS I] = 0). Such hyperbolic distribution is consistent with the exclusive localization of PS I in stroma-exposed thylakoids and of PS II in the membrane of the grana partition region. A depletion of the Cyt b 6- f complex from the membrane domain of PS I was detected ( Chl a Chl b = 5.1 , Chl PS I = 210:1 , sol Chl Cyt f = 1510:1 ). Moreover, a chloroplast thylakoid membrane was identified, distinct both from that of PS II and from that of PS I which was enriched in the Cyt b 6- f complex ( Chl a Chl b = 2.67 , Chl PS I = 460:1 , Chl Cyt f = 590:1 ). Our results suggested that such a Cyt b 6- f domain is localized in the region between the appressed and the non-appressed membranes, possibly in the membrane of the fret region.

Different degrees of phosphorylation and lateral mobility of two polypeptides belonging to the light-harvesting complex of Photosystem II

Several studies have shown that a subpopulation of the light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC-II) migrates from the appressed to the stroma-exposed thylakoids upon its phosphorylation. In this study we have analyzed the 27 and 25 kDa apopolypeptides of LHC-II, resolved by two-dimensional electrophoresis, with respect to their relative abundance and phosphorylation in thylakoids and subfractions derived from appressed or stroma-exposed thylakoid regions. The results show that the two polypeptides are heterogeneous with respect to both phosphate incorporation and degree of lateral migration. In intact thylakoids, the specific phosphorylation of the 25 kDa polypeptide exceeded that of the 27 kDa polypeptide by a factor of 3. Following phosphorylation, the 25 kDa polypeptide of the stroma lamellae showed as much as 4–5-times higher specific phosphorylation compared to the 27 kDa polypeptide. Moreover, there was a time-dependent increase in the amount of the 25 kDa polypeptide relative to the 27 kDa polypeptide in the stroma-exposed thylakoids. These results demonstrate a different polypeptide composition of the LHC-II tightly bound to Photosystem II and the free pool of LHC-II able to migrate laterally upon phosphorylation. The mobile pool of LHC-II is estimated to have two 27 kDa polypeptides for every 25 kDa polypeptide, while the ratio in the immobile pool is 4:1.

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C 3 plants. Agranal bundle sheath chloroplasts ( Chl a Chl b > 5.0 ) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl a b light-harvesting complex (LHC), and most of the cyt b 559. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl a b LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P 700 are closely coordinated. Thus, it is likely that both cyt f and P 700 are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.