Interaction of N,N,N',N'-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the Q(B) niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (muM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S(2)Q(B)(-) and S(3)Q(B)(-) (B-band), S(2)Q(A)(-) (Q-band), and Y(D)(+)Q(A)(-) (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y(n)) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q(B) confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q(B) niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q(B)-site and to reduce the higher S-states of the Mn cluster.     

Seasonal responses of photosynthetic electron transport in Scots pine (Pinus sylvestris L.) studied by thermoluminescence

The potential of photosynthesis to recover from winter stress was studied by following the thermoluminescence (TL) and chlorophyll fluorescence changes of winter pine needles during the exposure to room temperature (20 degrees C) and an irradiance of 100 micromol m(-2) s(-1). TL measurements of photosystem II (PSII) revealed that the S(2)Q(B)(-) charge recombinations (the B-band) were shifted to lower temperatures in winter pine needles, while the S(2)Q(A)(-) recombinations (the Q-band) remained close to 0 degrees C. This was accompanied by a drastically reduced (65%) PSII photochemical efficiency measured as F(v)/ F(m,) and a 20-fold faster rate of the fluorescence transient from F(o) to F(m) as compared to summer pine. A strong positive correlation between the increase in the photochemical efficiency of PSII and the increase in the relative contribution of the B-band was found during the time course of the recovery process. The seasonal dynamics of TL in Scots pine needles studied under field conditions revealed that between November and April, the contribution of the Q- and B-bands to the overall TL emission was very low (less than 5%). During spring, the relative contribution of the Q- and B-bands, corresponding to charge recombination events between the acceptor and donor sides of PSII, rapidly increased, reaching maximal values in late July. A sharp decline of the B-band was observed in late summer, followed by a gradual decrease, reaching minimal values in November. Possible mechanisms of the seasonally induced changes in the redox properties of S(2)/S(3)Q(B)(-) recombinations are discussed. It is proposed that the lowered redox potential of Q(B) in winter needles increases the population of Q(A)(-), thus enhancing the probability for non-radiative P680(+)Q(A)(-) recombination. This is suggested to enhance the radiationless dissipation of excess light within the PSII reaction center during cold acclimation and during cold winter periods.     

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.     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Photosystem II associated carbonic anhydrase activity in higher plants is situated in core complex

The thylakoid membrane containing photosystem II (PSII membranes) from pea and wheat leaves catalyzed the reaction of CO2 hydration with low rate, which increased after their incubation either with Triton X-100, up to Triton/chlorophyll ratio 1:1, or 1 M CaCl2. The presence of the inhibitor of CAs, p-aminomethylbenzensulfonamide (mafenide), at the start line in the course of electrophoresis of PSII membranes solubilized by n-dodecyl-beta-maltoside (DM) decreased the amount of PSII core complex in the gel. The elution of PSII core complex from the column with immobilized mafenide occurred only either by mafenide or another inhibitor of CAs, ethoxyzolamide. The above results led to a conclusion that membrane-bound CA activity associated with PSII is situated in the core complex.     

Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.     

Phosphatidylglycerol requirement for the function of electron acceptor plastoquinone Q(B) in the photosystem II reaction center

Phosphatidylglycerol (PG), a ubiquitous constituent of thylakoid membranes of chloroplasts and cyanobacteria, is demonstrated to be essential for the functionality of plastoquinone electron acceptor Q(B) in the photosystem II reaction center of oxygenic photosynthesis. Growth of the pgsA mutant cells of Synechocystis sp. PCC6803 that are defective in phosphatidylglycerolphosphate synthase and are incapable of synthesizing PG, in a medium without PG, resulted in a 90% decrease in PG content and a 50% loss of photosynthetic oxygen-evolving activity as reported [Hagio, M., Gombos, Z., Várkonyi, Z., Masamoto, K., Sato, N., Tsuzuki, M., and Wada, H. (2000) Plant Physiol. 124, 795-804]. We have studied each step of the electron transport in photosystem II of the pgsA mutant to clarify the functional site of PG. Accumulation of Q(A)(-) was indicated by the fast rise of chlorophyll fluorescence yield under continuous and flash illumination. Oxidation of Q(A)(-) by Q(B) plastoquinone was shown to become slow, and Q(A)(-) reoxidation required a few seconds when measured by double flash fluorescence measurements. Thermoluminescence measurements further indicated the accumulation of the S(2)Q(A)(-) state but not of the S(2)Q(B)(-) state following the PG deprivation. These results suggest that the function of Q(B) plastoquinone was inactivated by the PG deprivation. We assume that PG is an indispensable component of the photosystem II reaction center complex to maintain the structural integrity of the Q(B)-binding site. These findings provide the first clear identification of a specific functional site of PG in the photosynthetic reaction center.     

Protecting effect of phosphorylation on oxidative damage of D1 protein by down-regulating the production of superoxide anion in photosystem II membranes under high light

The physiological significance of photosystem II (PSII) core protein phosphorylation has been suggested to facilitate the migration of oxidative damaged D1 and D2 proteins, but meanwhile the phosphorylation seems to be associated with the suppression of reactive oxygen species (ROS) production, and it also relates to the degradation of PSII reaction center proteins. To more clearly elucidate the possible protecting effect of the phosphorylation on oxidative damage of D1 protein, the degradation of oxidized D1 protein and the production of superoxide anion in the non-phosphorylated and phosphorylated PSII membranes were comparatively detected using the Western blotting and electron spin resonance spin-trapping technique, respectively. Obviously, all of three ROS components, including superoxide anion, hydrogen peroxide and hydroxyl radical are responsible for the degradation of oxidized D1 protein, and the protection of the D1 protein degradation by phosphorylation is accompanied by the inhibition of superoxide anion production. Furthermore, the inhibiting effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a competitor to Q(B), on superoxide anion production and its protecting effect on D1 protein degradation are even more obvious than those of phosphorylation. Both DCMU effects are independent of whether PSII membranes are phosphorylated or not, which reasonably implies that the herbicide DCMU and D1 protein phosphorylation probably share the same target site in D1 protein of PSII. So, altogether it can be concluded that the phosphorylation of D1 protein reduces the oxidative damage of D1 protein by decreasing the production of superoxide anion in PSII membranes under high light.     

Interactions between thylakoid electron transfer complexes. II. Modification studies with glutaraldehyde

Photosystem I (PSI) and photosystem II (PSII) complexes have been isolated from stacked spinach thylakoid membranes that had been treated with varying amounts of glutaraldehyde. The concentrations of cytochrome f, Q, and P700 have been determined by spectrophotometric methods. It was found that at low concentrations of glutaraldehyde, the amount of cytochrome f associated with either PSII or PSI increased significantly while the amounts of Q and P700 stayed relatively constant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analyses indicated the presence of cytochrome f and other components of the cytochrome b6-f complex in the PSII and PSI preparations after glutaraldehyde treatment, but no intermolecular cross-linked polypeptides could be detected. Solubilization of the cytochrome b6-f complex was also inhibited after thylakoid membranes were treated with low concentrations of glutaraldehyde. These results are discussed in relation to current models for the organization of the membrane complexes, and relate to the location of the cytochrome b6-f complex in appressed and nonappressed membrane regions of thylakoids.     

Gross morphological changes in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp. PCC 6803

Cells of Synechocystis sp. PCC 6803 lacking photosystem I (PSI-less) and containing only photosystem II (PSII) or lacking both photosystems I and II (PSI/PSII-less) were compared to wild type (WT) cells to investigate the role of the photosystems in the architecture, structure, and number of thylakoid membranes. All cells were grown at 0.5μmol photons m(-2)s(-1). The lumen of the thylakoid membranes of the WT cells grown at this low light intensity were inflated compared to cells grown at higher light intensity. Tubular as well as sheet-like thylakoid membranes were found in the PSI-less strain at all stages of development with organized regular arrays of phycobilisomes on the surface of the thylakoid membranes. Tubular structures were also found in the PSI/PSII-less strain, but these were smaller in diameter to those found in the PSI-less strain with what appeared to be a different internal structure and were less common. There were fewer and smaller thylakoid membrane sheets in the double mutant and the phycobilisomes were found on the surface in more disordered arrays. These differences in thylakoid membrane structure most likely reflect the altered composition of photosynthetic particles and distribution of other integral membrane proteins and their interaction with the lipid bilayer. These results suggest an important role for the presence of PSII in the formation of the highly ordered tubular structures.     

A mutant small heat shock protein with increased thylakoid association provides an elevated resistance against UV-B damage in synechocystis 6803

Besides acting as molecular chaperones, the amphitropic small heat shock proteins (sHsps) are suggested to play an additional role in membrane quality control. We investigated sHsp membrane function in the model cyanobacterium Synechocystis sp. PPC 6803 using mutants of the single sHsp from this organism, Hsp17. We examined mutants in the N-terminal arm, L9P and Q16R, for altered interaction with thylakoid and lipid membranes and examined the effects of these mutations on thylakoid functions. These mutants are unusual in that they retain their oligomeric state and chaperone activity in vitro but fail to confer thermotolerance in vivo. We found that both mutant proteins had dramatically altered membrane/lipid interaction properties. Whereas L9P showed strongly reduced binding to thylakoid and model membranes, Q16R was almost exclusively membrane-associated, properties that may be the cause of reduced heat tolerance of cells carrying these mutations. Among the lipid classes tested, Q16R displayed the highest interaction with negatively charged SQDG. In Q16R cells a specific alteration of the thylakoid-embedded Photosystem II (PSII) complex was observed. Namely, the binding of plastoquinone and quinone analogue acceptors to the Q(B) site was modified. In addition, the presence of Q16R dramatically reduced UV-B damage of PSII activity because of enhanced PSII repair. We suggest these effects occur at least partly because of increased interaction of Q16R with SQDG in the PSII complex. Our findings further support the model that membrane association is a functional property of sHsps and suggest sHsps as a possible biotechnological tool to enhance UV protection of photosynthetic organisms.     

EVIDENCE FOR A LACK OF PHOTOSYSTEM SEGREGATION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

The distribution of photosystems I and II (PSI and PSII) in cells of Chlamydomonas reinhardtii Dangeard was studied by immunogold electron microscopy using cultures grown autotrophically at moderate irradiance and harvested in the middle of the light period. Sections of Lowicryl-embedded cells were labeled with monospecific heterologous antisera raised against the reaction center proteins of PSI (CP1-e) or the core antenna proteins of PSII (CP40 and CP47). All three antisera labeled both the appressed and the nonappressed thylakoid membranes at essentially similar densities. Labeling with both PSI and PSII antisera was slightly more concentrated over the outer nonappressed membranes of the thylakoid bands (1.7- to 2.4-fold with anti-CP1- e and 1.5- to 1.8-fold with anti-CP47 and anti-CP40). However, since appressed membranes comprised 73% of the total thylakoid membranes, 50%–62% of the PSI and 58%–65% of the PSII labeling were localized on appressed membranes. We conclude that photosystem distribution in C. reinhardtii is similar to that reported for other algae and different from the lateral heterogeneity observed in higher plants.     

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.     

Localisation and processing of the precursor form of photosystem II protein D1 inSynechocystis 6803

Pure plasma membrane and thylakoid membrane fractions from Synechocystis 6803 were isolated to study the localisation and processing of the precursor form of the D1 protein (pD1) of photosystem II (PSII). PSII core proteins (D1, D2 and cytb559) were localised both to plasma and thylakoid membrane fractions, the majority in thylakoids. pD1 was found only in the thylakoid membrane where active PSII is known to function. Membrane fatty acid unsaturation was shown to be critical in processing of pD1 into mature D1 protein. This was concluded from pulse-labelling experiments at low temperature using wild type and a mutant Synechocystis 6803 with a low level of membrane fatty acid unsaturation. Further, pD1 was identified as two distinct bands, an indication of two cleavage sites in the precursor peptide or, alternatively, two different conformations of pD1. Our results provide evidence for thylakoid membranes being a primary synthesis site for D1 protein during its light-activated turnover. The existence of the PSII core proteins in the plasma membrane, on the other hand, may be related to the biosynthesis of new PSII complexes in these membranes.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of external electric field on the electron transport system of spinach chloroplasts

Since the thylakoid membranes of an active chloroplast are constantly exposed to the electric fields generated by the electron transport system inside the membranes, we have studied the effects of pretreating chloroplasts of spinach ( Spinacia oleracea L.) leaves with an external AC (alternating current) electric field on their electron transport system. It was found that a few minutes electric field pretreatment (333 V cm^-1 across chloroplast samples), especially at low frequency, irreversibly inhibited the activity of photosystem II (PSII), but under certain conditions, stimulated that of photosystem I (PSI). From the measurements of fluorescence from PSII, we ascribe the inhibition to a lesion close to its reaction center P680, leading to increased dissipation of excitation energy to heat. The effect on PSI was investigated by the reduction of its reaction center, P700 by various artificial donors. We suggest that the stimulative effect can be attributed to a positive shift of the surface charge density of thylakoid membranes that brings about an increase in the accessibility of exogenous electronegative donors.     

Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga,Chlamydomonas reinhardtii having his-tagged CP47

Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni(2+)-affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) and Cl(-) when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q(A)(-) to Q(B) was significantly inhibited, and the electron transfer from Q(A)(-) to ferricyanide was largely stimulated in the presence of Ca(2+). These results indicate that the acceptor side, Q(B) site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl(2). The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl(2)-treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.     

Protective Action of Spermine and Spermidine against Photoinhibition of Photosystem I in Isolated Thylakoid Membranes

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O₂ uptake rates, P700 photooxidation and the accumulation of superoxide anions (O₂ ⁻) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O₂ ⁻ generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O₂ ⁻ generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.     

Active and resting states of the O2-evolving complex of photosystem II

During dark adaptation, a change in the O2-evolving complex (OEC) of spinach photosystem II (PSII) occurs that affects both the structure of the Mn site and the chemical properties of the OEC, as determined from low-temperature electron paramagnetic resonance (EPR) spectroscopy and O2 measurements. The S2-state multiline EPR signal, arising from a Mn-containing species in the OEC, exhibits different properties in long-term (4 h at 0 degrees C) and short-term (6 min at 0 degree C) dark-adapted PSII membranes or thylakoids. The optimal temperature for producing this EPR signal in long-term dark-adapted samples is 200 K compared to 170 K for short-term dark-adapted samples. However, in short-term dark-adapted samples, illumination at 170 K produces an EPR signal with a different hyperfine structure and a wider field range than does illumination at 160 K or below. In contrast, the line shape of the S2-state EPR signal produced in long-term dark-adapted samples is independent of the illumination temperature. The EPR-detected change in the Mn site of the OEC that occurs during dark adaptation is correlated with a change in O2 consumption activity of PSII or thylakoid membranes. PSII membranes and thylakoid membranes slowly consume O2 following illumination, but only when a functional OEC and excess reductant are present. We assign this slow consumption of O2 to a catalytic reduction of O2 by the OEC in the dark. The rate of O2 consumption decreases during dark adaptation; long-term dark-adapted PSII or thylakoid membranes do not consume O2 despite the presence of excess reductant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Movement of newly imported light-harvesting chlorophyll-binding protein from unstacked to stacked thylakoid membranes is not affected by light treatment or absence of amino-terminal threonines

In higher plants and algae, the transduction of captured light energy is highly regulated as excess excitation of photosystem II (PSII) reaction centers can be redirected to photosystem I (PSI) reaction centers. Models that attempt to explain this phenomenon involve light-harvesting chlorophyll-protein complexes (LHCII) that capture light energy and migrate between PSII and PSI. This report shows that in pea chloroplasts, the major protein component of LHCII, light-harvesting chlorophyll-binding protein (LHCP), can indeed migrate within the thylakoid membrane. We show, however, that although newly imported LHCP inserts into both stacked and unstacked thylakoid membranes, it then moves only from the unstacked, PSI-rich membranes to the stacked, PSII-rich membranes. The observed migration is not affected by light treatment that induces a redistribution of captured light energy (state I-state II transition) that previously was thought to induce LHCP to migrate in the opposite direction, from stacked to unstacked membranes. A mutation that removes the site of LHCP phosphorylation, the proposed trigger of state transitions, also has no effect on the integration and movement of LHCP, but does render LHCP more susceptible to proteolytic degradation. These results are not consistent with current models that deal with the short-term change in the distribution of light energy.