Functional Analyses of the Plant Photosystem I-Light-Harvesting Complex II Supercomplex Reveal That Light-Harvesting Complex II Loosely Bound to Photosystem II Is a Very Efficient Antenna for Photosystem I in State II

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.     

The Effects of Illumination on the Xanthophyll Composition of the Photosystem II Light-Harvesting Complexes of Spinach Thylakoid Membranes

The xanthophyll composition of the light-harvesting chlorophyll a/b proteins of photosystem II (LHCII) has been determined for spinach (Spinacia oleracea L.) leaves after dark adaptation and following illumination under conditions optimized for conversion of violaxanthin into zeaxanthin. Each of the four LHCII components was found to have a unique xanthophyll composition. The major carotenoid was lutein, comprising 60% of carotenoid in the bulk LHCIIb and 35 to 50% in the minor LHCII components LHCIIa, LHCIIc, and LHCIId. The percent of carotenoid found in the xanthophyll cycle pigments was approximately 10 to 15% in LHCIIb and 30 to 40% in LHCIIa, LHCIIc, and LHCIId. The xanthophyll cycle was active for the pigments bound to all of the LHCII components. The extent of deepoxidation for complexes prepared from light-treated leaves was 27, 65, 69, and 43% for LHCIIa, -b, -c, and -d, respectively. These levels of conversion of violaxanthin to zeaxanthin were found in LHCII prepared by three different isolation procedures. It was estimated that approximately 50% of the zeaxanthin associated with photosystem II is in LHCIIb and 30% is associated with the minor LHCII components.     

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)–tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2⁻ mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.     

Organization of the Light-Harvesting Complex of Photosystem I and Its Assembly during Plastid Development

Photosystem I (PSI) holocomplexes were fractionated to study the organization of the light-harvesting complex I (LHC I) pigment-proteins in barley (Hordeum vulgare) plastids. LHC Ia and LHC Ib can be isolated as oligomeric, presumably trimeric, pigment-protein complexes. The LHC Ia oligomeric complex contains both the 24- and the 21.5-kD apoproteins encoded by the Lhca3 and Lhca2 genes and is slightly larger than the oligomeric LHC Ib complex containing the Lhca1 and Lhca4 gene products of 21 and 20 kD. The synthesis and assembly of LHC I during light-driven development of intermittent light-grown plants occurs rapidly upon exposure to continuous illumination. Complete PSI complexes are detected by nondenaturing Deriphat (disodium N-dodecyl-[beta]-iminodipropionate-160)-PAGE after 2 h of illumination, and their appearance correlates with that of the 730- to 740-nm emission characteristic of assembled LHC I. However, the majority of the newly synthesized LHC I apoproteins are present as monomeric complexes in the thylakoids during the early hours of greening. We propose that during development of the protochloroplast the LHC I apoproteins are first assembled into monomeric pigmented complexes that then aggregate into trimers before becoming attached to the pre-existing core complex to form a complete PSI holocomplex.     

Steady-State Phosphorylation of Light-Harvesting Complex II Proteins Preserves Photosystem I under Fluctuating White Light

According to the “state transitions” theory, the light-harvesting complex II (LHCII) phosphorylation in plant chloroplasts is essential to adjust the relative absorption cross section of photosystem II (PSII) and PSI upon changes in light quality. The role of LHCII phosphorylation upon changes in light intensity is less thoroughly investigated, particularly when changes in light intensity are too fast to allow the phosphorylation/dephosphorylation processes to occur. Here, we demonstrate that the Arabidopsis (Arabidopsis thaliana) stn7 (for state transition7) mutant, devoid of the STN7 kinase and LHCII phosphorylation, shows a growth penalty only under fluctuating white light due to a low amount of PSI. Under constant growth light conditions, stn7 acquires chloroplast redox homeostasis by increasing the relative amount of PSI centers. Thus, in plant chloroplasts, the steady-state LHCII phosphorylation plays a major role in preserving PSI upon rapid fluctuations in white light intensity. Such protection of PSI results from LHCII phosphorylation-dependent equal distribution of excitation energy to both PSII and PSI from the shared LHCII antenna and occurs in cooperation with nonphotochemical quenching and the proton gradient regulation5-dependent control of electron flow, which are likewise strictly regulated by white light intensity. LHCII phosphorylation is concluded to function both as a stabilizer (in time scales of seconds to minutes) and a dynamic regulator (in time scales from tens of minutes to hours and days) of redox homeostasis in chloroplasts, subject to modifications by both environmental and metabolic cues. Exceeding the capacity of LHCII phosphorylation/dephosphorylation to balance the distribution of excitation energy between PSII and PSI results in readjustment of photosystem stoichiometry.Plant acclimation to different quantities and qualities of light has been extensively investigated. The light quality experiments have usually concerned the red/blue and far-red light acclimation strategies, which have been closely related to the state transitions and the phosphorylation of the light-harvesting complex II (LHCII) proteins, Lhcb1 and Lhcb2, by the state transition7 (STN7) kinase (Allen, 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006; Rochaix, 2007). Such studies on acclimation to different qualities of light have uncovered key mechanisms required for the maintenance of photosynthetic efficiency in dense populations and canopies (Dietzel et al., 2008). However, the role of LHCII phosphorylation under fluctuations in the quantity of white light has been scarcely investigated. Light conditions in natural environments may be very complex with respect to the quantity of white light, which constantly fluctuates both in short- and long-term durations (Smith, 1982; Külheim et al., 2002). Thus, the acclimation strategies to natural environments must concomitantly meet the challenges of both high- and low-light acclimation. Changing cloudiness, for example, would initiate both the high-light and low-light acclimation signals in the time scale of minutes and hours, whereas the movements of leaves in the wind or the rapid movement of clouds would initiate even more frequent light acclimation signals. The kinetics of reversible LHCII phosphorylation is far too slow to cope with rapid environmental changes.The phosphorylation level of LHCII proteins in the thylakoid membrane is regulated by both the STN7 kinase and the counteracting PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010). No definite results are available about regulation of the PPH1/TAP38 phosphatase, but the STN7 kinase is strongly under redox regulation (Lemeille et al., 2009) and controls the phosphorylation level of LHCII proteins under varying white light intensities as well as according to chloroplast metabolic cues, as described already decades ago (Fernyhough et al., 1983; Rintamäki et al., 2000; Hou et al., 2003). So far, research on the role of the STN7 kinase and LHCII phosphorylation in the light acclimation of higher plants has heavily focused on reversible LHCII phosphorylation and concomitant state transitions. The state 1-to-state 2 transition, by definition, means the phosphorylation of LHCII proteins, their detachment from PSII in grana membranes, and migration to the stroma membranes to serve in the collection of excitation energy to PSI (Fork and Satoh, 1986; Williams and Allen, 1987; Wollman, 2001; Rochaix, 2007; Kargul and Barber, 2008; Murata, 2009; Lemeille et al., 2010; Minagawa, 2011). Concomitantly, the absorption cross section of PSII decreases and that of PSI increases (Canaani and Malkin, 1984; Malkin et al., 1986; Ruban and Johnson, 2009). Indeed, state transitions have been well documented when different qualities (blue/red and far red) of light, preferentially exciting either PSII or PSI, have been applied.Different from state transitions, the white light intensity-dependent reversible LHCII phosphorylation does not result in differential excitation of the two photosystems (Tikkanen et al., 2010). Instead, both photosystems remain nearly equally excited independently whether the LHCII proteins are heavily phosphorylated or strongly dephosphorylated. Moreover, it is worth noting that the different qualities of light generally used to induce reversible LHCII phosphorylation and state transitions (blue/red and far-red lights) have usually been of very low intensity (for review, see Haldrup et al., 2001), and apparently, minimal protonation of the lumen takes place under such illumination conditions. Yet another difference between induction of LHCII protein phosphorylation by different qualities of light or different quantities of white light concerns the concomitant induction of PSII core protein phosphorylation. In the former case, the level of PSII core protein phosphorylation follows the phosphorylation pattern of LHCII proteins, whereas under different quantities of white light, the phosphorylation behavior of PSII core and LHCII proteins is the opposite (Tikkanen et al., 2008b).To gain a more comprehensive understanding of the physiological role of white light-induced changes in LHCII protein phosphorylation, we have integrated Arabidopsis (Arabidopsis thaliana) LHCII phosphorylation with other light-dependent regulatory modifications of light harvesting and electron transfer in the thylakoid membrane, which include the nonphotochemical quenching of excitation energy (for review, see Niyogi, 1999; Horton and Ruban, 2005; Barros and Kühlbrandt, 2009; de Bianchi et al., 2010; Jahns and Holzwarth, 2012; Ruban et al., 2012) and the photosynthetic control of electron transfer by the cytochrome b₆f (Cytb6f) complex (Rumberg and Siggel, 1969; Witt, 1979; Tikhonov et al., 1981; Bendall, 1982; Nishio and Whitmarsh, 1993; Joliot and Johnson, 2011; Suorsa et al., 2012; for review, see Foyer et al., 1990, 2012), both strongly dependent on lumenal protonation.It is demonstrated that the steady-state LHCII phosphorylation is particularly important under rapidly fluctuating light (FL) conditions. This ensures equal energy distribution to both photosystems, prevents the accumulation of electrons in the intersystem electron transfer chain (ETC), eliminates perturbations in chloroplast redox balance, and maintains PSI functionality upon rapid fluctuations in white light intensity.     

Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex

The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf^*-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [³⁵S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf^*-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts.     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

Processing of the Precursors for the Light-Harvesting Chlorophyll-Binding Proteins of Photosystem II and Photosystem I during Import and in an Organelle-Free Assay

We have investigated whether the precursors for the light-harvesting chlorophyll a/b binding proteins (LHCP) of photosystems II and I (PSII and PSI) are cleavable substrates in an organelle-free reaction, and have compared the products with those obtained during in vitro import into chloroplasts. Representatives from the tomato (Lycopersicon esculentum) LHCP family were analyzed. The precursor for LHCP type I of PSII (pLHCPII-1), encoded by the tomato gene Cab3C, was cleaved at only one site in the organelle-free assay, but two sites were recognized during import, analogous to our earlier results with a wheat precursor for LHCPII-1. The relative abundance of the two peptides produced was investigated during import of pLHCPII-1 into chloroplasts isolated from plants greened for 2 or 24 hours. In contrast to pLHCPII-1, the precursors for LHCP type II and III of PSI were cleaved in both assays, giving rise to a single peptide. The precursor for LHCP type I of PSI, encoded by gene Cab6A, yielded two peptides of 23.5 and 21.5 kilodaltons during import, whereas in the organelle-free assay only the 23.5 kilodalton peptide was found. N-terminal sequence analysis of this radiolabeled peptide has tentatively identified the site cleaved in the organelle-free assay between met40 and ser41 of the precursor.     

Thylakoid FtsH Protease Contributes to Photosystem II and Cytochrome b6f Remodeling in Chlamydomonas reinhardtii under Stress Conditions

FtsH is the major thylakoid membrane protease found in organisms performing oxygenic photosynthesis. Here, we show that FtsH from Chlamydomonas reinhardtii forms heterooligomers comprising two subunits, FtsH1 and FtsH2. We characterized this protease using FtsH mutants that we identified through a genetic suppressor approach that restored phototrophic growth of mutants originally defective for cytochrome b₆f accumulation. We thus extended the spectrum of FtsH substrates in the thylakoid membranes beyond photosystem II, showing the susceptibility of cytochrome b₆f complexes (and proteins involved in the c_i heme binding pathway to cytochrome b₆) to FtsH. We then show how FtsH is involved in the response of C. reinhardtii to macronutrient stress. Upon phosphorus starvation, photosynthesis inactivation results from an FtsH-sensitive photoinhibition process. In contrast, we identified an FtsH-dependent loss of photosystem II and cytochrome b₆f complexes in darkness upon sulfur deprivation. The D1 fragmentation pattern observed in the latter condition was similar to that observed in photoinhibitory conditions, which points to a similar degradation pathway in these two widely different environmental conditions. Our experiments thus provide extensive evidence that FtsH plays a major role in the quality control of thylakoid membrane proteins and in the response of C. reinhardtii to light and macronutrient stress.     

The Photosystem II Light-Harvesting Protein Lhcb3 Affects the Macrostructure of Photosystem II and the Rate of State Transitions in Arabidopsis

The main trimeric light-harvesting complex of higher plants (LHCII) consists of three different Lhcb proteins (Lhcb1-3). We show that Arabidopsis thaliana T-DNA knockout plants lacking Lhcb3 (koLhcb3) compensate for the lack of Lhcb3 by producing increased amounts of Lhcb1 and Lhcb2. As in wild-type plants, LHCII-photosystem II (PSII) supercomplexes were present in Lhcb3 knockout plants (koLhcb3), and preservation of the LHCII trimers (M trimers) indicates that the Lhcb3 in M trimers has been replaced by Lhcb1 and/or Lhcb2. However, the rotational position of the M LHCII trimer was altered, suggesting that the Lhcb3 subunit affects the macrostructural arrangement of the LHCII antenna. The absence of Lhcb3 did not result in any significant alteration in PSII efficiency or qE type of nonphotochemical quenching, but the rate of transition from State 1 to State 2 was increased in koLhcb3, although the final extent of state transition was unchanged. The level of phosphorylation of LHCII was increased in the koLhcb3 plants compared with wild-type plants in both State 1 and State 2. The relative increase in phosphorylation upon transition from State 1 to State 2 was also significantly higher in koLhcb3. It is suggested that the main function of Lhcb3 is to modulate the rate of state transitions.     

Conformational Dynamics of Light-Harvesting Complex II in a Native Membrane Environment

Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states.     

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn₄CaO₅ cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn₄CaO₅ cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn₄CaO₅ cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S₁ state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.     

Isolation of Lamellar Aggregates of the Light-Harvesting Chlorophyll a/b Protein Complex of Photosystem II with Long-Range Chiral Order and Structural Flexibility

Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupaet al.(1987,Plant Physiol.84, 19–24), was optimized for obtaining purified lamellar aggregates with long-range chiral order and structural flexibility (the capability of undergoing light-induced reversible structural changes). By varying the concentration of the detergent Triton X-100 for the solubilization of thylakoid membranes, we obtained four types of LHCII aggregates: (i) With low detergent concentration, ≤0.6% (v/v), the aggregates contained lipids in high amount. These preparations with Chl a/b ratios of about 1.4 contained minor antenna complexes with a fingerprint of an additional CD band at (+) 505 nm; they formed disordered lamellae and exhibited no or weak psi-type CD bands (psi, polymerization- or salt-induced), which did not possess the ability to undergo light-induced changes (ΔCD). (ii) At the optimal concentration, around 0.7 ± 0.1% (v/v), the detergent removed some lipids and most of the minor complexes, and the Chl a/b ratio dropped to 1.0–1.1. LHCII formed loosely stacked two-dimensional lamellae which exhibited psi-type CD bands and large light-induced reversible structural changes (ΔCD). (iii) At detergent concentration above the optimum, around 0.8–1% (v/v), the lipid content of LHCII decreased and minor complexes could not be detected. LHCII formed disordered aggregates and showed neither psi-type CD nor ΔCD. (iv) High concentrations (≥1.1% (v/v)) Triton X-100 led to very pure but largely delipidated samples assembled into tightly stacked three-dimensional lamellar structures with intense psi-type CD but no ΔCD.     

Effects of 5-Aminolevulinic Acid on the Accumulation of Chlorophyll b and Apoproteins of the Light-Harvesting Chlorophyll a/b-Protein Complex of Photosystem II

The effects were examined of 5-aminolevulinic acid (ALA) onthe accumulation of Chl and apoproteins of light-harvestingChl a/b-protein complex of photosystem II (LHCII) in cucumbercotyledons under intermittent light. A supply of ALA preferentiallyincreased the accumulation of Chl a during intermittent illumination.However, when cotyledons were pretreated with a brief exposureto light or benzyladenine (BA), the stimulatory effect of ALAon the increase in the level of Chl b was greater than thatin the level of Chl a, resulting in decreased ratios of Chla/b. Time-course experiments with preilluminated cotyledonsrevealed that LHCII apoproteins accumulated rapidly within thefirst 30 min of intermittent illumination with a decline duringsubsequent incubation in darkness. A supply of ALA did not affectthe accumulation of LHCII apoproteins during the intermittentlight period, but it efficiently inhibited the decline in theirlevels during the subsequent darkness. After exposure to a singlepulse of light of BA-treated cotyledons, the prompt increasein levels of LHCII apoproteins was not accompanied by the formationof Ch b, which began to accumulate later. The pattern of changesin levels of LHCII apoproteins was quite similar to that inlevels of Chl a. These results suggest that LHCII apoproteinsare first stabilized by binding with Chl a and that an increasedsupply of Chl a and the accumulation of LHCII apoproteins areprerequisites for the formation of Chl b. ¹Present address: Department of Chemistry, Faculty of Scienceand Technology, Meijo University, Aichi, 468 Japan.     

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H₂O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H₂O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-Q_B-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1,-dimethylurea - DCPIP dichlorophenol-indophenol (2,6-dichloro-4((4-hydroxyphenyl)imino)-2,5-cyclohexadien-1-one) - DPC diphenyl carbazide (2,2-diphenylcarbonic dihydrazide) - FeCN ferricyanide (hexacyanoferrate(III)) - _app apparent quantum yield of photosynthetic oxygen evolution - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - PPBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PQ pool plastoquinone - Q_B secondary quinone acceptor of PS II - RT room temperature - WC whole chain electron transfer     

Thylakoid membrane protein phosphorylation leads to a decrease in connectivity between Photosystem II reaction centers

The room-temperature fluorescence induction transients from stroma-free chloroplast membranes (in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea) have been analyzed to determine the effects of membrane protein phosphorylation on the connectivity between Photosystem (PS) II centers. Chloroplast membranes which have been incubated in the light with ATP exhibit: (1) a decrease in the variable fluorescence as a function of the initial fluorescence, (2) a shift from a sigmoidal to an exponential fluorescence induction curve, and (3) a reduced amount of the fast () component of the induction transient. These phenomenona are completely reversible by dark incubation of the samples (leading to protein dephosphorylation). We conclude that connectivity between PS II centers is reduced as a function of thylakoid membrane protein phosphorylation. This may in turn be the mechanism which increases the amount of absorbed excitation energy available to PS I.