Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Regulatory factors for the assembly of thylakoid membrane protein complexes

Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.     

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.     

Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: their specificity and evolution

Sulfoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG) are lipids with negative charges, distributed among membranes of chloroplasts of plants and their postulated progenitors, cyanobacteria, and also widely among membranes of anoxygenic photosynthetic bacteria. Thus, these acidic lipids are of great interest in terms of their roles in the function and evolution of the photosynthetic membranes. The physiological significance of these lipids in photosynthesis has been examined through characterization of mutants defective in their abilities to synthesize SQDG or PG, and through characterization of isolated thylakoid membranes or photosynthetic particles, the acidic lipid contents of which were manipulated in vitro, for example, on treatment with phospholipase to degrade PG. Responsibility of SQDG or PG has been clarified so far in terms of the structural and/or functional integrity of photosystems I and/or II in cyanobacterial, green algal, and higher plant species. Also implied were distinct levels of the responsibility in the different photosynthetic organisms. Extreme cases involved the indispensability of SQDG for photosynthesis and growth in two prokaryotic, photosynthetic organisms and the contribution of PG to construction of the photosystem-I trimer exclusively in cyanobacteria. Here, roles of these acidic lipids are discussed with a focus on their specificity and the evolution of photosynthetic membranes.Norihiro Sato is the recipient of the Botanical Society Award for Young Scientist, 2003.     

Damage and protection of the photosynthetic apparatus from UV-B radiation. I. Effect of ascorbate

In this work, the effect of the exogenously added ascorbate (Asc) against the UV-B inhibition of the photosystem II (PSII) functions in isolated pea thylakoid membranes was studied. The results reveal that Asc decreases the UV-B induced damage of the donor and the acceptor side of PSII during short treatment up to 60 min. The exogenous Asc exhibits a different UV-protective effect on PSII centers in grana and stroma lamellae, as the effect is more pronounced on the PSIIβ centers in comparison to PSIIα centers. Data also suggest that one of the possible protective roles of the Asc in photosynthetic membranes is the modification of the oxygen-evolving complex by influence on the initial S₀–S₁ state distribution in the dark.     

Mechanism and regulation of the violaxanthin cycle: The role of antenna proteins and membrane lipids

The violaxanthin cycle describes the reversible conversion of violaxanthin to zeaxanthin via the intermediate antheraxanthin. This light-dependent xanthophyll conversion is essential for the adaptation of plants and algae to different light conditions and allows a reversible switch of photosynthetic light-harvesting complexes between a light-harvesting state under low light and a dissipative state under high light. The photoprotective functions of zeaxanthin have been intensively studied during the last decade, but much less attention has been directed to the mechanism and regulation of xanthophyll conversion. In this review, an overview is given on recent progress in the understanding of the role of (i) xanthophyll binding by antenna proteins and of (ii) the lipid properties of the thylakoid membrane in the regulation of xanthophyll conversion. The consequences of these findings for the mechanism and regulation of xanthophyll conversion in the thylakoid membrane will be discussed.     

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1‐2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient‐sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi‐starved mgd1‐2 leaves, biogenesis of thylakoid‐like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress‐induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light‐harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1‐2 mutant. Moreover, the reduced expression of nuclear‐ and plastid‐encoded photosynthetic genes observed in the mgd1‐2 mutant under Pi‐sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid‐ and nuclear‐encoded photosynthetic genes, independently of photosynthesis.     

Structural changes of the thylakoid membrane network induced by high light stress in plant chloroplasts

Land plants live in a challenging environment dominated by unpredictable changes. A particular problem is fluctuation in sunlight intensity that can cause irreversible damage of components of the photosynthetic apparatus in thylakoid membranes under high light conditions. Although a battery of photoprotective mechanisms minimize damage, photoinhibition of the photosystem II (PSII) complex occurs. Plants have evolved a multi-step PSII repair cycle that allows efficient recovery from photooxidative PSII damage. An important feature of the repair cycle is its subcompartmentalization to stacked grana thylakoids and unstacked thylakoid regions. Thus, understanding the crosstalk between stacked and unstacked thylakoid membranes is essential to understand the PSII repair cycle. This review summarizes recent progress in our understanding of high-light-induced structural changes of the thylakoid membrane system and correlates these changes to the efficiency of the PSII repair cycle. The role of reversible protein phosphorylation for structural alterations is discussed. It turns out that dynamic changes in thylakoid membrane architecture triggered by high light exposure are central for efficient repair of PSII.     

Ammonium tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 and the role of the psbA multigene family

Ammonium is one of the major nutrients for plants, and a ubiquitous intermediate in plant metabolism, but it is also known to be toxic to many organisms, in particular to plants and oxygenic photosynthetic microorganisms. Although previous studies revealed a link between ammonium toxicity and photodamage in cyanobacteria under in vivo conditions, ammonium‐induced photodamage of photosystem II (PSII) has not yet been investigated with isolated thylakoid membranes. We show here that ammonium directly accelerated photodamage of PSII in Synechocystis sp. strain PCC6803, rather than affecting the repair of photodamaged PSII. Using isolated thylakoid membranes, it could be demonstrated that ammonium‐induced photodamage of PSII primarily occurred at the oxygen evolution complex, which has a known binding site for ammonium. Wild‐type Synechocystis PCC6803 cells can tolerate relatively high concentrations of ammonium because of efficient PSII repair. Ammonium tolerance requires all three psbA genes since mutants of any of the three single psbA genes are more sensitive to ammonium than wild‐type cells. Even the poorly expressed psbA1 gene, whose expression was studied in some detail, plays a detectable role in ammonium tolerance.     

The case for chloroplast thylakoid carbonic anhydrase

Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C₃ and C₄ plants possess the ability to reversibly hydrate CO₂. That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution.     

An improved purification method and a further characterization of the 33-kilodalton protein of spinach chloroplasts

The 33-kDa protein was purified in a high yield from thylakoid membranes of spinach chloroplasts. The extinction coefficient and A^1%_1cm value at 276 nm of the protein were 22000 M^?1·cm^?1 and 6.8, respectively. The 33-kDa protein and a polypeptide appearing at 32 kDa in the SDS-polyacrylamide gel electrophoresis of thylakoid membranes were compared by peptide mapping after limited proteolysis. This indicates that the 32-kDa band is entirely due to the 33-kDa protein. The molar ratio of chlorophyll to the 33-kDa protein in the chloroplasts was estimated to be 300. This suggests that one photosynthetic unit possesses one or two molecules of the 33-kDa protein.     

Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis

The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged -323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.     

Thylakoid Membrane Protein Kinase Activity as a Signal Transduction Pathway in Chloroplasts

In plants external stimuli are perceived through a cascade of signals and signal transduction pathways. Protein phosphorylation and de-phosphorylation is one of the most important transduction paths for the perception of signals in plants. The highest concentrations of plant phospho-proteins are located in chloroplasts. This facilitates the protection of thylakoid membranes from stress-induced damage and augments adaptive strategies in plants. In this review, the protein kinases associated with phosphorylation of thylakoid membrane protein, and the adaptive changes in thylakoid membrane architecture and developmental cues are given. The presence of membrane bound kinases in thylakoid membranes have evolutionary implications for the signal transduction pathways and the photosynthetic gene expression for thylakoid membrane protein dynamics. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of the Specific Roles of Anions in Electron Transport and Energy Transfer Reactions in Photosynthesis

Ionic environment is important in regulating photosynthetic reactions. The roles of cations, Mn²⁺, Mg²⁺, Ca²⁺, Na⁺, and K⁺ as cofactors in electron transport, energy transfer, phosphorylation, and carbon assimilation are better known than the roles of anions, except for chloride and bicarbonate. Only a limited information exists on the roles and effects of nitri formate, sulphate, and phosphate. In this review, we evaluate and highlight the roles of some specific anions on electron transport as well as on excitation energy transfer processes in photosynthesis. Anions exert significant effects on thyla membrane conformation and membrane fluidity, possibly by redistributing the thylakoid membrane surface charges. The anion/cation induced phase transitions in the hydrophilic domains of the thylakoid membranes are probably responsible for the various structural and co-related functional changes under stress. Anions are also important in regulation of energy distribution between the two photosystems. Anions do not only divert more energy from photosystem (PS) 2 to PS1, but can also reverse the effect of cations on energy distribution in a valence-dependent manner. Anions affect also the structure of the photosynthetic apparatus and excitation energy distribution between the two photosystems.     

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH_in established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pH_out = 7.8, we found that pH_in ≈ 5.4-5.7 in the state of photosynthetic control, and pH_in ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H⁺/ATP is n ≥ 4-4.7.     

Temperature modulation and the presence of C20 fatty acids in mono‐ and digalactosyldiacylglycerol of Euglena gracilis and Lepocinclis acus: A modern interpretation of euglenid galactolipids using positive‐ion electrospray ionization/mass spectrometry

Mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively) are important galactolipids that comprise photosynthetic membranes in almost all photosynthetic organisms. Intact forms of MGDG and DGDG of Euglena gracilis and Lepocinclis acus, two example euglenids with secondary plastids of green algal origin, were elucidated with fatty acid regiochemistry via positive‐ion electrospray ionization/mass spectrometry at two growth temperatures. At 20°C, E. gracilis and L. acus produced predominantly 18:3/16:4 (sn‐1/sn‐2) MGDG, whereas at 30°C this was supplanted by 18:2/16:2 MGDG. At both temperatures were also observed a variety of other MGDG and DGDG forms, including C₂₀ fatty acid‐containing forms not expected in a green algal‐derived plastid. In addition to providing structural details of MGDG and DGDG not available in past studies, these results suggest a previously unknown relationship between these two organisms and the red algae. This study also illustrates that temperature modulation of galactolipids occurs via modification of unsaturation of both the sn‐1 and sn‐2 fatty acids; this is fundamentally different from previously published studies from our laboratory on other algal classes.     

LIGHT-HARVESTING COMPLEX AF'OPROTEINS IN CYTOPLASMIC VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Cells of Chlamydomonas reinhardtii Dangeard strain cw15arg7A contain electron-opaque material, often in the form of large granules, within cytoplasmic vacuoles. Immunoelectron microscopy with antibodies to polypeptide 11, a component of the major light-harvesting chlorophyll (Chl) a/b-protein complex (LHCII,) of thylakoid membranes, revealed the presence of LHCII Polypeptides within the chloroplast and in vacuolar material in cells grown in the light. Vacuolar material was also heavily immunodecorated in dark-grown cells that did not synthesize Chl. Accumulation of LHCII polypeptides was further studied in greening and light-grown cells of a pale green mutant, deficient in LHCII, that was derived from cu15arg7A by insertional mutagenesis. Light-grown cells of this mutant strain contained relatively few thylakoid membranes and synthesized LHCII polypeptides at a low rate. However, cytoplasmic vacuoles were immunoreactive. Appearance of mature-sized LHCII polypeptides in vacuoles suggested that these proteins were partially translocated across the envelope but not retained by the chloroplast without assembly of LHCII.     

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023 Å^− 1, corresponding to spatially and statistically averaged repeat distance values of about 275–330 Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D₂O in dim laboratory light or transpired with D₂O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.     

Formation of cross-linking between photosystem 2 proteins during irradiation of thylakoid membranes at high temperature

Irradiation of thylakoid membranes at 40 °C resulted in complete inhibition of photosystem (PS) 2 activity measured as 2,6-dichlorophenol indophenol (DCIP) photoreduction either in the absence or presence of 1,5-diphenylcarbazide (DPC). Concomitant with the inactivation of PS2 activity, several thylakoid proteins were lost and high molecular mass cross-linking products appeared that cross-reacted with antibodies against proteins of PS2 but not with antibodies against proteins of other three complexes PS1, ATP synthase, and cytochrome b₆f. Irradiation of thylakoid membranes suspended in buffer of basic pH or high concentration of Tris at 25 °C resulted in the formation of cross-linking products similar to those in thylakoid membranes irradiated at 40 °C. Presence of radical scavengers and DPC during the high temperature treatment prevented the formation of cross-linking products. These results suggest the involvement of oxygen evolving co mplex (OEC) in the formation of cross-linking between PS2 proteins in thylakoid membrane irradiated at high temperature. This revised version was published online in September 2006 with corrections to the Cover Date.