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Photosystem II in Different Parts of the Thylakoid Membrane: A Functional Comparison between Different Domains †
Abstract
The electron transport properties of photosystem II (PSII) from five different domains of the thylakoid membrane were analyzed by flash-induced fluorescence kinetics. These domains are the entire grana, the grana core, the margins from the grana, the stroma lamellae, and the Y100 fraction (which represent more purified stroma lamellae). The two first fractions originate from appressed grana membranes and have PSII with a high proportion of O(2)-evolving centers (80-90%) and efficient electron transport on the acceptor side. About 30% of the granal PSII centers were found in the margin fraction. Two-thirds of those PSII centers evolve O(2), but the electron transfer on the acceptor side is slowed. PSII from the stroma lamellae was less active. The fraction containing the entire stroma has only 43% O(2)-evolving PSII centers and slow electron transfer on the acceptor side. In contrast, PSII centers of the Y100 fraction show no O(2) evolution and were unable to reduce Q(B). Flash-induced fluorescence decay measurements in the presence of DCMU give information about the integrity of the donor side of PSII. We were able to distinguish between PSII centers with a functional Mn cluster and without any Mn cluster, and PSII centers which undergo photoactivation and have a partially assembled Mn cluster. From this analysis, we propose the existence of a PSII activity gradient in the thylakoid membrane. The gradient is directed from the stroma lamellae, where the Mn cluster is absent or inactive, via the margins where photoactivation accelerates, to the grana core domain where PSII is fully photoactivated. The photoactivation process correlates to the PSII diffusion along the membrane and is initiated in the stroma lamellae while the final steps take place in the appressed regions of the grana core. The margin domain is seemingly very important in this process.
... All measurements except the control point (0 h of S-dep) were done under anaerobic conditions. Analysis of fluorescence decay kinetics was done using three exponential decay components (Mamedov et al., 2000;Volgusheva et al., 2016). For measurements in the presence of electron or membrane proton gradient inhibitors, the culture was split between two aliquots and the fluorescence kinetics were measured with and without inhibitor, after the cells were incubated in the dark for the time shown in Table 1. ...
... It should be noted that the millisecond phase provides information on the rate of the PQ molecule binding to the Q B site in the PSII center, and so effectively reflects the redox state of the PQ pool. The slow phases (hundreds of milliseconds to seconds) reports recombination from Q A − to the donor side of PSII (Crofts and Wraight, 1983;Crofts et al., 1993;Renger et al., 1995;Vass et al., 1999;Mamedov et al., 2000;Volgusheva et al., 2016). We have previously reported using flash-induced fluorescence decay kinetics measurements how to assess changes in the photosynthetic electron transfer during S-dep (Volgusheva et al., 2013(Volgusheva et al., , 2016. ...
... DCMU is a well-known inhibitor of the PSII activity which binds to the Q B site and effectively blocks forward electron transfer from Q A − (Bishop, 1958;Draber et al., 1991). Instead only recombination to the donor side of PSII is reflected in the fluorescence decay (Vass et al., 1999;Mamedov et al., 2000;Roose et al., 2010;Volgusheva et al., 2016). After addition of DCMU, the dominating fast and middle decay phase completely disappeared in the control sample, and only slow recombination between the Q A − and the S 2 state was observed (Fig. 4A, black triangles). ...
The redox state of the PQ-pool in sulfur deprived, H2 producing Chlamydomonasreinhardtii cells was studied using single flash-induced variable fluorescence decay kinetics. During H2 production, the fluorescence decay kinetics exhibited unusual post-illumination rise of variable fluorescence giving a wave-like appearance. The wave showed the transient fluorescence minimum at ~ 60 msec after the flash followed by a rise reaching the transient fluorescence maximum at ~1 sec after the flash, before decaying back to the initial fluorescence level. Similar wave-like fluorescence decay kinetics were reported earlier in anaerobically incubated cyanobacteria [Deak et al., 2014 Biochimica Biophysica Acta 1837, 1522-1532] but not in green alga. From several different electron and proton transfer inhibitors used, polymyxin B, inhibitor of type II NAD(P)H dehydrogenase (NDA2) had the effect of eliminating the fluorescence wave feature, indicating involvement of NDA2 in this phenomenon. This was further confirmed by the absence of the fluorescence wave in Δnda2 mutant lacking NDA2. Additionally, Δnda2 mutant have also shown delayed and diminished H2 production (only 23% compared to the wild type). Our results show that fluorescence wave phenomenon in C.reinhardtii is observed under the highly reduced conditions and is induced by the NDA2 mediated electron flow from the reduced stromal components to the PQ-pool. Therefore, the fluorescence wave phenomenon is a sensitive probe for the complex network of redox reactions at the PQ-pool level in the thylakoid membrane. It could be used in further characterization and improvement of the electron transfer pathways leading to the H2 production in C.reinhardtii.
... Monitoring the changes of the flash-induced fluorescence yield or so-called variable fluorescence is a powerful tool to study the electron transfer reactions in PSII [19,20]. Fluorescence yield reports on the charge separation and redox state of Q A in PSII when change in the fluorescence yield from the lowest ( ) electron transfer and the middle phase in few msec reflects electron transfer to Q B which first has to bind to the Q B -site) [19,20]. ...
... Monitoring the changes of the flash-induced fluorescence yield or so-called variable fluorescence is a powerful tool to study the electron transfer reactions in PSII [19,20]. Fluorescence yield reports on the charge separation and redox state of Q A in PSII when change in the fluorescence yield from the lowest ( ) electron transfer and the middle phase in few msec reflects electron transfer to Q B which first has to bind to the Q B -site) [19,20]. These two phases dominate the variable fluorescence decay in intact PSII. ...
... These two phases dominate the variable fluorescence decay in intact PSII. The residual slow phase in a few seconds originating from the recombination between Q A − and the donor side of PSII can also be detected [19,20]. The slow recombination phase can be studied in more detail if measured in the presence of DCMU, an inhibitor which binds to the Q B -site and blocks the forward electron transfer from Q A − [19,20]. ...
Redox properties of the acceptor side of Photosystem II were studied during H2 gas production in cells of Chlamydomonas reinhardtii. Flash-induced variable fluorescence changes and thermoluminescence measurements were performed in wild type and Stm6 mutant cells during different stages of sulfur (S)-deprivation. Analysis of the fluorescence decay kinetics indicated that the forward electron transfer on the acceptor side of Photosystem II was dramatically slowed down during the O2 evolution and O2 consumption stages and was completely blocked in the anaerobic stage of S-deprivation, thus, indicating a complete reduction of the PQ-pool. During the H2 formation stage, the forward electron transfer kinetics in the μsec and msec time scale re-appeared indicating partially restored electron flow from QA⁻ to QB and the PQ-pool. Thermoluminescese measurements fully confirmed the fluorescence kinetic analysis. Activation of hydrogenase in the H2 formation stage is responsible for re-oxidation of the PQ pool and reactivation of the electron flow which was found to be faster and more efficient on the Stm6 mutant due to the higher amount of functionally preserved Photosystem II.
... Flash-induced increase and subsequent relaxation of the chlorophyll fluorescence yield (variable fluorescence decay kinetics) were measured as described in [37] with a FL3300 DCMU. The kinetics were analysed in terms of several exponential components (fast, middle and slow phases) as described in [38][39][40]. ...
... However, a small fraction of inactive PSII centres seemed to be present in the mutant. [38][39][40]. Deletion of the PsbY subunit altered the electron flow from Q A -( Figure 4A, red graph, Table 2). The fast phase became shorter and faster (417 sec, 26%), but the middle phase of the exponential decay in PsbY was found to be 37 msec. ...
... When variable fluorescence decay was measured in the presence of DCMU, an inhibitor blocking the electron transfer between Q A and Q B , fluorescence decay was dominated by recombination kinetics between Q A and the S 2 state in PSII [38][39][40]. The fluorescence decay kinetics in PsbY in the presence of DCMU were slightly slower if compared to kinetics in WT and the control mutant PsbYcom ( Figure 4B, Table 2). ...
Photosystem II is a protein complex embedded in the thylakoid membrane of photosynthetic organisms and performs the light driven water oxidation into electrons and molecular oxygen that initiate the photosynthetic process. This important complex is composed of more than two dozen of intrinsic and peripheral subunits, of those half are low molecular mass proteins. PsbY is one of those low molecular mass proteins; this 4.7–4.9 kDa intrinsic protein seems not to bind any cofactors. Based on structural data from cyanobacterial and red algal Photosystem II PsbY is located closely or in direct contact with cytochrome b559. Cytochrome b559 consists of two protein subunits (PsbE and PsbF) ligating a heme-group in-between them. While the exact function of this component in Photosystem II has not yet been clarified, a crucial role for assembly and photo-protection in prokaryotic complexes has been suggested. One unique feature of Cytb559 is its redox-heterogeneity, forming high, medium and low potential, however, neither origin nor mechanism are known. To reveal the function of PsbY within Photosystem II of Arabidopsis we have analysed PsbY knock-out plants and compared them to wild type and to complemented mutant lines. We show that in the absence of PsbY protein Cytb559 is only present in its oxidized, low potential form and plants depleted of PsbY were found to be more susceptible to photoinhibition.
... Flash-Induced FluorescenceD ecay Measurements. The functional capacity of the PSII centers during the differentphases of Sdeprivation was analyzedbyflash-induced fluorescence decay kinetic measurements in WT andStm6mutantcells.Thismethodreports on the electron transport properties of PSIIa nd can be usedt o monitor both the acceptor-and the donor-side reactions (29,30). Immediatelyafter the flash,the fluorescence rises from the F 0 level to the F max level (Fig. 3).Thisreflects the charge separationinPSII andthe stabilization of the electron on the first quinone acceptor, Q A .Thisresultsinthe formation of ahigh-fluorescence state. ...
... Immediatelyafter the flash,the fluorescence rises from the F 0 level to the F max level (Fig. 3).Thisreflects the charge separationinPSII andthe stabilization of the electron on the first quinone acceptor, Q A .Thisresultsinthe formation of ahigh-fluorescence state. The consequent decay of variable fluorescenced emonstrates howt he electron leaves Q A − in the particular sample (29,30). In the controlsample fromthe WT cells,morethan80% of the fluorescence decayed in the microsecond to millisecond timerange (Fig. 3A,black circles).Thisfastdecay of the variable fluorescence is normal andre fl ects fast and efficientf orwarde lectron transfer from Q A − to Q B (resulting in Q B − )i nt he majority of the PSII centers. ...
... Insteadoft he fast decayindicativeo fforward electron transfer,t he decayk ineticsw ered ominated by slow reactionsoccurring in thehundredsofmillisecondstosecondstime range. Such slow decayk ineticsw eret ypical for recombination reactionsb etween Q A − andt he S 2 stateo ft he water-oxidizing complexi nP SII (29)(30)(31).I ndeed, very similark ineticsw ereo bserved when the flash-induced fluorescence decaywas measured in thes ames ampleb ut in thep resenceo fD CMU, whichb locks forwardelectrontransferbetween Q A − andQ B (Fig.3A,openred circles).The fluorescence decaykineticsinthe presenceofDCMU arew ellu nderstooda nd reflectr ecombination betweenQ A − and theS 2 state (29)(30)(31). Thus,f romt he almost identicalk ineticsw e conclude that thef orward electron transfer from Q A − wasc ompletelyblocked in theanaerobic phase(III) during Sdeprivation of theWTcells. ...
Photobiological H2 production is an attractive option for renewable solar fuels. Sulfur-deprived cells of Chlamydomonas reinhardtii have been shown to produce hydrogen with the highest efficiency among photobiological systems. We have investigated the photosynthetic reactions during sulfur deprivation and H2 production in the wild-type and state transition mutant 6 (Stm6) mutant of Chlamydomonas reinhardtii. The incubation period (130 h) was dissected into different phases, and changes in the amount and functional status of photosystem II (PSII) were investigated in vivo by electron paramagnetic resonance spectroscopy and variable fluorescence measurements. In the wild type it was found that the amount of PSII is decreased to 25% of the original level; the electron transport from PSII was completely blocked during the anaerobic phase preceding H2 formation. This block was released during the H2 production phase, indicating that the hydrogenase withdraws electrons from the plastoquinone pool. This partly removes the block in PSII electron transport, thereby permitting electron flow from water oxidation to hydrogenase. In the Stm6 mutant, which has higher respiration and H2 evolution than the wild type, PSII was analogously but much less affected. The addition of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea revealed that ∼80% of the H2 production was inhibited in both strains. We conclude that (i) at least in the earlier stages, most of the electrons delivered to the hydrogenase originate from water oxidation by PSII, (ii) a faster onset of anaerobiosis preserves PSII from irreversible photoinhibition, and (iii) mutants with enhanced respiratory activity should be considered for better photobiological H2 production.
... The first three domains were analysed in this study as separated fractions, as well as the entire Grana and Y100 fractions. The exact origin of the Y100 fraction within the stroma-exposed region of the thylakoid membrane is unknown (Mamedov et al. 2000). the inhibition of the electron transport reactions. ...
... These reactions correlate with the lateral movement of the new PSII centres from the stroma lamellae to the grana core region, i.e. opposite to the flow of photoinhibited PSII centres. The movement is accompanied by changes in the electron transport properties of the PSII centres (Rova et al. 1998, Magnuson et al. 1999, Mamedov et al. 2000 and finally completed by photoactivation of PSII, the light-driven process of the assembly of the Mn-cluster, finally producing PSII, fully capable of water oxidation (Ananyev et al. 2001, Ono 2001). We will focus on these reactions below, starting with the last process-photoactivation of the Mn-cluster, in order to better understand, which reaction is taking place where, during this lateral movement of PSII complexes in the thylakoid membrane. ...
... In agreement with this, photoactivation experiments on an Y D -less Table 1. Comparison of the Q B binding on the acceptor side of PSII during photoactivation of the dark-grown C. reinhardtii cells (left part of the Table, Rova et al. 1998) and in the different parts of the spinach thylakoid membrane (right part of the Table, Mamedov et al. 2000). The data were obtained from kinetic analysis of the flash-induced variable fluorescence decay. ...
Due to its unique ability to split water, Photosystem II (PSII) is easily accessible to oxidative damage. Photoinhibited PSII centres diffuse laterally from the grana core region of the thylakoid membrane to the stroma lamellae in order to allow replacement of damaged proteins and cofactors. The ‘new born’ PSII centres in this region are characterized by the absence of the water splitting capacity and very poor ability to bind the secondary quinone acceptor, QB. After the repair process PSII has to regain the water splitting capacity. This requires a set of well-defined electron transfer reactions leading to assembly of the Mn-cluster. In order to minimize the danger of photoinhibition during these earlier stages of photoactivation of PSII, auxiliary donors to the primary donor P680+, such as redox active tyrosine on D2 protein, YD, and cytochrome b559 become involved in the electron transport reactions by providing necessary electrons. Cytochrome b559 may also serve as an electron acceptor to QA– if elevated light intensities occur during the photoactivation process. These reactions lead to activation of QB binding, and finally to the assembly of the Mn-cluster. All these electron transport events occur simultaneously with the lateral movement of PSII centres back to the appressed regions of the grana core, where the pool of the most active PSII is situated.
... The thylakoid membrane system in chloroplasts has a more complex structure than the undifferentiated thylakoid membrane of cyanobacteria. SDS-PAGE of the polypeptide distribution(Wollenberger et al. 1994) and biophysical studies (Wollenberger et al. 1995; Mamedov et al. 2000) of the light-harvesting antenna attachment and the localization and specific O 2 evolution activity of PSII-WOC complexes found within different regions of the thylakoid membrane system in chloroplasts indicates that a gradient exists in the number of subunits and complexity of functional activity. PSII size and functional complexity increases in going from the individual stroma lamellae, to the periphery of the appressed multi-membrane grana lamaellae (so-called margins) to the centers of the appressed grana lamaellae (core grana), as summarized inTable 1. ...
... et al. 1994) The Mn cluster is absent or inactive in the PSII found in the stroma membrane; a majority fraction of the peripheral grana PSII-WOC are active but have slow Q A Q B electron transfer kinetics; while greater than 80-90% of the core grana PSII-WOC are fully active in O 2 evolution and have normal acceptor side kinetics. (Mamedov et al. 2000) The authors hypothesize that the " photoactivation " process (here meaning both subunit assembly and inorganic core uptake) is initiated in the stroma lamellae, diffuses into the grana periphery, and is completed within the appressed regions of the inner grana core. In summary, studies of PSII-WOC biogenesis in both cyanobacteria and chloroplasts support an " assembly line " model for the manufacturing of functional PSII core complexes. ...
... This is followed by transport to stroma lamaellae where acquisition of inefficient primary electron transport (slow Q A Q B ) occurs. Some CP43 and CP47 is present,(Mamedov et al. 2000) although gels reveal that CP43 is present at lower levels than the reaction center subunits.(Wollenberger et al. 1994) Photoactivation of O 2 evolution occurs in some centers (≤ 40%), but the activity is unstable in the dark, suggesting that stable binding of the extrinsic 33 kDa protein has not been established in those centers that do photoactivate. ...
The biogenesis of the Photosystem II (PS II) water oxidizing complex is reviewed with emphasis on the sequence and location
of assembly of the subunits within the internal membrane systems of prokaryotes and chloroplasts. The uptake and distribution
of manganese in cyanobacterial cells are discussed. The role of individual subunits in isolated PS II in light-induced assembly
of the inorganic core of the water splitting complex is discussed (photoactivation process). The roles of the inorganic cofactors
(Mn2+, Ca2+, Cl and bicarbonate) in water splitting are revealed by examining the effects of their reconstitution with non-native cofactors.
Novel instrumentation for the measurement of O2 concentration and the kinetics and mechanism of photoactivation are described.
... The flash-induced fluorescence in presence of DCMU was measured with a PAM-100 fluorimeter (Walz, Effeltrich, Germany) according to Ref. [38]. Dark-adapted PSII samples at 20 Ag Chl ml À1 were incubated for 5 min in a Cd 2+ (0-10 mM)-and DCMU (20 AM)-containing buffer. ...
... A minor part of the flash-induced fluorescence did not decay during the 60-s measuring time (~10% of F v is included in the slow phase in Table 1). The decay phases and their relative amplitudes are similar to what is commonly found in PSII enriched membrane preparations [38,43,44]. By the addition of 0.1-10 mM CdCl 2 , we observed several changes. ...
... DCMU binds to the Q B site and blocks forward electron transfer from Q A . Instead, Q A À is forced to recombine with the positively charge donor side components (after single flash, the dominating component is the S 2 state in the OEC) and the recombination kinetics reports on the integrity of the electron transfer chain on the donor side of PS II [38,[44][45][46][47]. In the absence of Cd 2+ , the flash-induced fluorescence in the presence of DCMU displayed a multi-exponential decay ( Table 1) with two decaying phases (t O c280 ms and 2.1 s) and one nondecaying phase (t O N10 s). ...
Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+). In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites. The number of sites for Cd(2+) binding and the possible nature of these sites are discussed.
... The decay was fitted with three exponential components. The fast component arises from the recombination of Q A with partially active Mn centers, and the middle component is due to S 2 Q A charge recombination (Mamedov et al. 2000(Mamedov et al. , 2002. The very slow phase (t 1/2 C 10 s) represents oxygen evolving centers that were in the S 0 state before the flash was applied. ...
... The very slow phase (t 1/2 C 10 s) represents oxygen evolving centers that were in the S 0 state before the flash was applied. In the presence of DCMU, both amplitudes and half-times in the control samples were very similar to that previously published (Mamedov et al. 2000(Mamedov et al. , 2002. The fluorescence relaxation was slowed down in thylakoids of Fe-deficient plants, and the amplitude of the fast and middle phase increased (Table 2). ...
... Indeed, the amplitude of the middle phase of this decay, which represents the recombination of Q A with S 2 , increased at the expense of the slow phase ( Table 2). The fast phase, with a 200-300 ms half-time, previously proposed to originate from Q A recombination with partially active Mn centers (Mamedov et al. 2000(Mamedov et al. , 2002 also increased. ...
The effect of iron deficiency on photosynthetic electron transport in Photosystem II (PS II) was studied in leaves and thylakoid membranes of lettuce (Lactuca sativa, Romaine variety) plants. PS II electron transport was characterized by oxygen evolution and chlorophyll fluorescence parameters. Iron deficiency in the culture medium was shown to affect water oxidation and the advancement of the S-states. A decrease of maximal quantum yield of PS II and an increase of fluorescence intensity at step J and I of OJIP kinetics were also observed. Thermoluminescence measurements revealed that charge recombination between the quinone acceptor of PS II, Q(B), and the S(2) state of the Mn-cluster was strongly perturbed. Also the dark decay of Chl fluorescence after a single turnover white flash was greatly retarded indicating a slower rate of Q(A)(-) reoxidation.
... One can distinguish between so-called PSIIa (large Chl antenna) in the grana and PSIIh (small Chl antenna) in the stroma lamellae [6] and between PSIa (larger antenna) in the margins and PSIh (smaller antenna) in the stroma lamellae and end membranes [7,8]. In addition, PSII is functionally very different in the different parts of the thylakoid membrane, probably reflecting an activity gradient due to the repair of PSII after photoinhibition [9]. ...
... To fully understand the function of the dynamic thylakoid membrane, it is necessary to have a detailed knowledge both about the function of the two photosystems and their relative abundance in the different domains of the thylakoid. We have earlier presented a functional analysis of PSII in the different thylakoid domains [9]. The dominating part of PSII in the grana region has a functional acceptor side and an active oxygen-evolving complex. ...
... In contrast, large part of PSII in the stromal region is inactive on either or both the acceptor and donor sides. Several other pools of PSII with more inhomogeneous function are also found both in the stroma lamellae and the margins of the grana [9]. ...
Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700(+) and Y(D)( .-), respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIbeta) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIalpha) 300, PSI (PSIbeta) in stroma lamellae 214, PSII in grana core (PSIIalpha) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.
... Flash-induced variable fluorescence was measured with a PAM fluorimeter (Walz, Effeltrich, Germany) according to Mamedov et al. [30]. Samples containing 10 μg Chl/mL were dark adapted for 5 min, and DCMU was added to a final concentration of 10 μM. ...
... A similar EPR experiment was performed in the presence of DCMU (Fig. 3B, dark trace), which blocks electron transfer from Q A − to Q B . This leads to recombination of the Q A − species with the CaMn 4 cluster, now in the S 2 state, after a flashinduced charge separation [30,33,34]. The CaMn 4 cluster is thereby re-reduced to the S 1 state, and no advancement in the S-cycle beyond the S 2 state is possible. ...
... The application of a single flash to an S 1 synchronised sample of PSII-enriched membranes containing DCMU induces the S 2 /Q A − state, which is highly fluorescent [40]. The decay of this variable fluorescence can therefore be used to follow the recombination reaction back to the S 1 /Q A state [30,33,34]. The process S 1 Y flash S 2 Y dark decay S 1 is thus observed. ...
The long-lived, light-induced radical Y(D) of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn(4) cluster, situated approximately 30 A away. In this study we report a transient step-change increase in Y(D) EPR intensity upon the application of a single laser flash to S(1) state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y(D) by the OEC in the S(2) state, as a consequence of the single laser flash turnover. The post-flash decay reflected S(2)-->S(1) back-turnover, as confirmed by their correlations with independent measurements of S(2) multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y(D) as a probe.
... Finally, CP43 (PsbC), PsbK, and PsbZ bind to the RC (Rokka et al., 2005;Boehm et al., 2011). This complex is photoactivated by stepwise assembling the Mn 4 O 5 Ca cluster, which is further stabilized by binding the three luminal subunits (Mamedov et al., 2000;Mamedov et al., 2008). Ultimately, two PSII monomers form a PSII dimer and bind additional LHCs (Shevela et al., 2016). ...
... In vascular plants, which display strong lateral heterogeneity of stacked grana thylakoids and unstacked stroma lamellae, no evidence exists for distinct thylakoid domains similar to "thylakoid centers" or "translation zones." Instead, the first steps of both de novo biogenesis and PSII repair take place in the stroma lamellae, with PSII then moving toward the grana core during the later steps of assembly and photoactivation (Mamedov et al., 2000;Mamedov et al., 2008). ...
The pathway of photosystem II (PSII) assembly is well understood, and multiple auxiliary proteins supporting it have been identified, but little is known about rate-limiting steps controlling PSII biogenesis. In the cyanobacterium Synechocystis PCC6803 and the green alga Chlamydomonas reinhardtii, indications exist that the biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, we modified the 5´-untranslated region of psbD via chloroplast transformation in tobacco (Nicotiana tabacum). A drastic reduction in psbD mRNA abundance resulted in a strong decrease in PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in cyanobacteria or in C. reinhardtii.
... Fluorescence decay kinetics report on the electron transfer from the primary quinone electron acceptor in PSII (Q A − ) after a single flash in PSII. [39][40][41] Fig. 1C shows the fluorescence decay traces in the control WT (black) and the C3 mutant cells (red). The WT cells show a fast fluorescence decay of more than 80% of the total variable fluorescence. ...
... This is normal, and the fast decay reflects efficient forward electron transfer from Q A − to Q B in PSII centers with an occupied Q B -site with Q B bound to its pocket in PSII (t 1/2 = 232 μs, 58% of the total amplitude) or an empty Q B -site (which first requires Q B binding, t 1/2 = 3.5 ms, 25%). [39][40][41] In the C3 mutant cells, the fluorescence decay was very different: the μsec phase was smaller (38%) and 2 times slower (t 1/2 = 462 μsec) indicating that less Q B is present in its site. The rest of the decay is dominating by a decay phase (t 1/2 = 119 ms, 44%) representing a very slow Q B binding. ...
The green alga Chlamydomonas reinhardtii can photoproduce H2 gas for only a few minutes under anaerobic conditions due to the inhibition of hydrogenase by O2 produced by Photosystem II (PSII). A few days of sustained H2 production can only be achieved when O2 and H2 production are temporally separated under two-stage processes such as sulfur deprivation. Under sulfur deprivation, H2 production is initiated after the over-reduction of plastoquinone pool and decreased PSII activity in the thylakoid membrane. As a result, activated hydrogenase consumes the excess of electrons produced by PSII [Volgusheva et al. (2013) PNAS USA 110, 7223]. Here we report that similar conditions can be achieved by simply altering the ratio between photosystem I (PSI) and PSII. In the C3 mutant of C. reinhardtii we found a lower PSI/PSII ratio than in the wild type, 0.33 vs 0.85, respectively. This imbalance of photosystems resulted in the over-reduced state of plastoquinone pool and activation of hydrogenase in the C3 mutant that allowed the photoproduction of H2 continuously for 42 days. This is an unprecedented duration of H2 production in green algae under standard growth conditions without any nutrient limitation. Photosynthetic electron flow from PSII to hydrogenase was closely regulated during this long-term H2 production. The amount of PSII was decreased and amount of PSI was increased reaching PSI/PSII ratio of more than 5 as shown by EPR and fluorescence spectroscopy. This fine-tuning of photosystems allows to sustain the long-term production of H2 in C. reinhardtii by direct photosynthetic pathway.
... The variable Chl fluorescence yield (F v ) decay is well known to reflect the efficiency of primary charge separation (P680*Q A → P680 + Q A − ), while its decay reflects electron transfer by reoxidation of Q A − (Murchie and Lawson 2013). The latter includes both forward electron transfer to Q B and charge recombination (Vass et al. 1999, Mamedov et al. 2000, Roose et al. 2010. Three decay phases were observed in the Ctrl chloroplasts: a fast decay phase with the half-time of 420 μs (48%), a middle phase of 4.4 ms (27%) and a slow phase of 1.27 s (25%, Fig. 4D, white trace). ...
... In order to probe possible involvement of the electron-acceptor side in the inefficient turnover of PSII found in 2 h etiochloroplasts, we measured the flash-induced fluorescence decay kinetics also in plastid preparations (see white circle traces in Fig. 4; for non-normalized variable fluorescence decay traces, see Fig. S3). An inspection of the obtained traces (white circles) showed that the decay kinetics in the 4-and 8-h etiochloroplasts are virtually the same as in the Ctrl chloroplasts with two dominating phases, fast and middle, indicating efficient electron transfer from Q A − to Q B -and Q B -binding, respectively ( Fig. 4B-D) (Vass et al. 1999, Mamedov et al. 2000, Roose et al. 2010. ...
High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water‐oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodelling. Employing membrane‐inlet mass spectrometry (MIMS), O2‐polarography under flashing light conditions we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these “PSII birth defects” in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de‐etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O2‐polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, QB‐inhibitor binding, and thermoluminescence studies indicate that the decline of the high light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability QA⁻ → QB during de‐etiolation. This rate depends in turn on the downstream clearing of electrons upon build‐up of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer‐range energy transfer.
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... This also holds for studies of structure and function of PSII. PSII is a large multiprotein-pigment complex which in its active form in higher plants is mostly found in the stacked granal membranes of chloroplasts [21][22][23]. It initiates the photosynthetic electron flow by using light energy to extract electrons from water and to reduce the pool of the plastoquinone molecules [24,25]. ...
... Oxygen evolution and variable fluorescence were measured as in [34]. The number of active PSII centers was determined by measuring DCIP reduction in the absence and presence exogenous electron donor DPC as described in [21]. The PSI/PSII ratio was determined by EPR measurements at room temperature as in [42]. ...
Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe²(+) and Phe- Q(A)- Fe²(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S₀- and S₂-states of CaMn₄O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn₄O(x)-cluster in the S₀-, S₁-, S₂-, and the S₃-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b₅₅₉ were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.
... Analysis of the higher organisation of PSII complexes by 2D-BN/SDS-PAGE, on the other hand, revealed in DrbcL thylakoids mostly PSII monomer complexes, which have been characterized by slow electron transfer on the acceptor side of PSII and being located in stroma lamellae [32]. Although we cannot completely exclude the possibility that the PSII centers in DrbcL mutant resemble PSIIs of stroma lamellae, not even traces of PSII centers lacking the bound Mn cluster [32] could be identified in DrbcL thylakoids; indeed, the flash-induced fluorescence relaxation in the presence of DCMU did not show any fast phase typical to PSIIs in the stroma lamellae fraction. ...
... Analysis of the higher organisation of PSII complexes by 2D-BN/SDS-PAGE, on the other hand, revealed in DrbcL thylakoids mostly PSII monomer complexes, which have been characterized by slow electron transfer on the acceptor side of PSII and being located in stroma lamellae [32]. Although we cannot completely exclude the possibility that the PSII centers in DrbcL mutant resemble PSIIs of stroma lamellae, not even traces of PSII centers lacking the bound Mn cluster [32] could be identified in DrbcL thylakoids; indeed, the flash-induced fluorescence relaxation in the presence of DCMU did not show any fast phase typical to PSIIs in the stroma lamellae fraction. ...
Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The Delta rbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q(A) and Q(B), whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of Delta rbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F0 'dark rise' in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O2 in Delta rbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the Delta rbcL mutant under growth conditions. This protective capacity was rapidly exceeded in Delta rbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.
... chlorophyll fluorescence transients, it is necessary to appropriately evaluate each of the kinetic components. Our analysis revealed the phases: (i) a fast phase related to Q -A reoxidation by a secondary quinone acceptor Q B , (ii) a middle phase associated with reoxidation by plastoquinone (PQ) binding to the Q B site after the flash, and (iii) a slow phase attributed to Q -A reoxidation via charge recombination with the S2 state of the oxygen evolving complex in PSII reaction centers that cannot transfer the electron to the PQ pool 30,78,79 . Interestingly, an additional kinetic phase (several tens of millisecond) was observed, which is characteristic of C. reinhardtii but absent or reduced in higher plants and cyanobacteria; the nature of this phase remains unclear 22 . ...
This study introduces an evaluation methodology tailored for bioreactors, with the aim of assessing the stress experienced by algae due to harmful contaminants released from antifouling (AF) paints. We present an online monitoring system equipped with an ultra-sensitive sensor that conducts non-invasive measurements of algal culture's optical density and physiological stage through chlorophyll fluorescence signals. By coupling the ultra-sensitive sensor with flash-induced chlorophyll fluorescence, we examined the dynamic fluorescence changes in the green microalga Chlamydomonas reinhardtii when exposed to biocides. Over a 24-h observation period, increasing concentrations of biocides led to a decrease in photosynthetic activity. Notably, a substantial reduction in the maximum quantum yield of primary photochemistry (FV/FM) was observed within the first hour of exposure. Subsequently, we detected a partial recovery in FV/FM; however, this recovery remained 50% lower than that of the controls. Integrating the advanced submersible sensor with fluorescence decay kinetics offered a comprehensive perspective on the dynamic alterations in algal cells under the exposure to biocides released from antifouling coatings. The analysis of fluorescence relaxation kinetics revealed a significant shortening of the fast and middle phases, along with an increase in the duration of the slow phase, for the coating with the highest levels of biocides. Combining automated culturing and measuring methods, this approach has demonstrated its effectiveness as an ultrasensitive and non-invasive tool for monitoring the physiology of photosynthetic cultures. This is particularly valuable in the context of studying microalgae and their early responses to various environmental conditions, as well as the potential to develop an AF system with minimal harm to the environment.
... Interestingly, partial photoactivation significantly increased the decay yield during the fast phase from 34% in Mn depleted cells to 43% in partially photoactivated cells, and from 43% to 40% in dark incubated cells. The fast phase in the presence of DCMU could attribute to redox-active, partially assembled Mn clusters(Mamedov et al. 2000), which could be explained by low quantum efficiency of photoactivation in D1-E189Rmutant. Photoactivation of the D1-E189K mutant with 200 flashes resulted in a decreased time constant for the slow phase (τ = 100 s, amplitude 31%) compared to HA extracted cells (τ >100 s, amplitude 41%) (fluorescence relaxation yield p = 0.001, time constant p = 0.0002) and increased half time of the middle phase from 144 ms and 18.5% to 457 ms and 27.6% of total yield respectively. ...
Photosystem II (PSII) of plants, algae, and cyanobacteria utilize solar energy to catalyze one of the most important and most thermodynamically demanding reactions in nature: the oxidation of water into protons and molecular oxygen. Oxygen produced by PSII is toxic byproduct, however it is essential for respiration, the ozone layer and the extracted electrons drive the fixation of atmospheric CO2 to create biomass. The mechanism of water splitting driven by the light-induced charge separation is relatively well studied and high-resolution crystal structures are available to reveal the molecular aspects of PSII complex, however considerably less is known about how the inorganic Mn4O5Ca cluster is assembled de novo.
... Asterisks indicate statistically significant differences between the two light regimes (t-test, P < 0.05); mean AE SE; n = 3 biological replicates; ns, not significant. In the flash-induced variable fluorescence decay, a saturating flash fully reduces Q A , the primary quinone electron acceptor in PSII, resulting in maximal fluorescence yield (Figure 5,left panels), and the following fluorescence decay kinetics depend on the Q A − re-oxidation (Mamedov et al., 2000;Vass et al., 1999). Three exponential decay components of different speeds were obtained (t 1 , t 2 and t 3 in Table S1). ...
C4 photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO2 concentration at the site of CO2 fixation. C4 plants benefit from high irradiance but their efficiency decreases under shade causing a loss of productivity in crop canopies. We investigated shade acclimation responses of a model NADP‐ME monocot Setaria viridis focussing on cell‐specific electron transport capacity. Plants grown under low light (LL) maintained CO2 assimilation rates similar to high light plants but had an increased chlorophyll and light‐harvesting‐protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen‐evolving activity and the PSII/PSI ratio enhanced in LL BS cells indicating a higher capacity for linear electron flow. Abundances of PSI, ATP synthase, Cytochrome b6f and the chloroplastic NAD(P) dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were all increased in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts was associated with the more oxidised plastoquinone pool in LL plants and the formation of PSII ‐ light‐harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results suggest that the supramolecular composition of Photosystem II is BS cells is adjusted according to the redox state of the plastoquinone pool. This discovery contributes to understanding the tuning of PSII activity in C4 plants and will support strategies for crop improvement including the engineering of C4 photosynthesis into C3 plants.
... A middle phase in a few milliseconds is typical for PSII complexes, where QA − reoxidation is limited by the diffusion of PQ molecules to an empty QB-site. A slow phase in a few seconds to tens of seconds reflects the charge recombination from the S2QA − state of water oxidation to the S1QA state (Cao and Govindjee 1990) and can also detect the donor side of PSII (Vass et al. 1999, Mamedov et al. 2000. In the present study, it was clear that the fast phase was in the majority (> 60%) in all treatments, indicating that QA − reoxidation was induced by electron transfer from QA − to QB/QB − . ...
Effects of potassium permanganate (KMnO4) on PSII of Mycrocystis aeruginosa were investigated by measuring the chlorophyll fluorescence in vivo. KMnO4 exposure reduced the rate of oxygen evolution and cell growth. High concentration of KMnO4 (10 mg L-1) decreased the fast phase and increased the slow phase of QA- reoxidation kinetics. Electron transport after QA was blocked, resulting in a considerable amount of QA- reoxidation being performed via S2(QAQB)- charge recombination. KMnO4 decreased the density of the active photosynthetic reaction centers and the maximum quantum yield for primary photochemistry and inhibited electron transport, which resulted in a decline of the performance of PSII activity and caused an increase in dissipated energy flux per reaction center and antenna size. Our results suggest that both the donor side and the acceptor on the phase of QA- to QB to PQ of PSII in M. aeruginosa were targets of KMnO4 toxicity.
... To investigate this, we performed flash-induced variable fluorescence decay and thermoluminescence measurements in isolated PSII membranes from wild type and psb33 plants. Analysis of the flash-induced fluorescence decay kinetics demonstrated faster electron transfer from Q A to Q B in psb33 mutant ( Figure 7A, left panel) (Mamedov et al., 2000;Roose et al., 2010;Vass et al., 1999). Also, thermoluminescence measurements, which are a useful complement to the flash-induced fluorescence decay measurements (Ducruet and Vass, 2009;Vass and Govindjee, 1996), also showed the presence of only a slightly up-shifted B-band (which represents the Q B - S 2 state recombination) at 38 °C. ...
Plants can quickly and dynamically respond to spectral and intensity variations of the incident light. These responses include activation of developmental processes, morphological changes, and photosynthetic acclimation that ensure optimal energy conversion and minimal photoinhibition. Plant adaptation and acclimation to environmental changes have been extensively studied, but many details surrounding these processes remain elusive. The Photosystem II (PSII) associated protein PSB33 plays a fundamental role in sustaining PSII as well as in the regulation of the light antenna in fluctuating lights. We investigated how PSB33 knock-out plants perform under different light qualities. psb33 plants displayed 88% lower fresh weight compared to wild type plants when cultivated in the border of UVA-blue light. The sensitivity towards UVA light was associated with a lower abundance of PSII proteins, which reduces psb33 plants´ capacity for photosynthesis. The UVA phenotype was further found to be linked to altered phytohormone status and changed thylakoid ultrastructure. Our results collectively show that PSB33 is involved in a UVA light-mediated mechanism to maintain a functional PSII pool in the chloroplast.
... The effect of PsbTn deletion was further characterized by measuring flash-induced variable fluorescence, which reflects the redox state of the first quinone acceptor, Q A , in PSII (Supplemental Fig. S6). The kinetics of the variable fluorescence decay thus provide information on electron transfer from Q A 2 (Mamedov et al., 2000;von Sydow et al., 2016). However, no significant differences between the wild type and the mutants could be detected (Supplemental Fig. S6A). ...
Photosystem II (PSII) is a supramolecular complex containing over 30 protein subunits and a large set of cofactors including various pigments and quinones as well as Mn, Ca, Cl, and Fe ions. Eukaryotic PSII complexes contain many subunits not found in their bacterial counterparts, including the proteins PsbP, PsbQ, PsbS, and PsbW, as well as the highly homologous, low molecular mass subunits PsbTn1 and PsbTn2 whose function is currently unknown. To determine the function of PsbTn1 and PsbTn2, we generated single and double psbTn1 and psbTn2 knock-out mutants in Arabidopsis thaliana. Cross-linking and reciprocal co-immunoprecipitation experiments revealed that PsbTn is a lumenal PSII protein situated next to the cytochrome b559 subunit PsbE. The removal of the PsbTn proteins decreased the oxygen evolution rate and PSII core phosphorylation level but increased the susceptibility of PSII to photoinhibition and the production of reactive oxygen species. The assembly and stability of PSII were unaffected, indicating that the deficiencies of the psbTn1 psbTn2 double mutants are due to structural changes. Double mutants exhibited a higher rate of non-photochemical quenching of excited states than the wild type and single mutants, as well as slower state transition kinetics and a lower quantum yield of PSII when grown in the field. Based on these results, we propose that the main function of the PsbTn proteins is to enable PSII to acclimate to light shifts or intense illumination.
... Chen et al., (2014), also indicated that the decoupling of OEC is accompanied by a reduction of reaction centers of PSII able to reduce Q B . This fraction of reaction centers, also the most active fraction in water oxidation by OEC, is denominated Q B reducing centers (Mamedov et al., 2000). The OJIP test also indicated that Q B reducing centers decreased only in IR50 under ST respect to the control condition ( Figure 5-1 B). ...
Rice (Oryza sativa L.) is one of the most important cereal crops in the global food
system feeding nearly half the world’s population and its cultivation, processing and
trade represents one of the biggest economic activities on Earth. During the months of
culture the temperatures are optimal (28-30 °C) for rice but high frequencies of
suboptimal temperatures are observed. Although some early studies demonstrated that
rice growth rate and metabolism are noticeably inhibited in the range of 15-20 °C, the
physiological bases for rice growth delay by suboptimal temperatures remain fairly
unexplored. Hence, the aim of this Thesis was to study the response of rice seedlings
from contrasting cultivars to suboptimal temperatures of growth at different organization
levels and elucidate some mechanisms related with tolerance.
In order to achieve our goal we designed a screening method for detecting
variability in sensitivity among rice cultivars at the seedling stage subjected to
suboptimal temperatures usually found in the field (13/21 °C d/n, Entre Ríos,
Argentina). This method was based on the reduction of the growth of the third leaf and
it had some advantages, for instance, it was specifically designed for testing
suboptimal temperatures, it was a quick method easy to set up in a growth chamber
where multiple cultivars can fit at once, and it is quantitative. It is noteworthy that the
measurements done in growth chambers were highly correlated with the ones in the
field, pointing out that the proposed system was able to predict what happened in the
field and so it works as a model system of the stress.
The main characteristic of the suboptimal temperatures stress was the reduction of
the seedling growth at the shoot as well as at the leaf level. The growth analysis of the
fourth leaf together with the transcriptome, proteome and redox system analyses
accorded that processes related with cell division were inhibited and that damage
occurred in the division zone so enzymatic antioxidant systems were triggered in this
zone as a response. The diminution of growth was associated with a detriment of the
Photosystem II evidenced in fast-transient chlorophyll analyses that impacted also in
the photosynthetic rate and stomata conductance. Besides, carbon assimilation,
storage and usage were also compromise. Different comparative analysis between
tolerant and sensitive cultivars allowed us to distinguish some responses to the stress
that could be associated with its variability in sensitivity. Hence, we associated the
tolerance against suboptimal temperatures stress with the capacity of maintaining a
healthy photosynthetic apparatus, the protection of the meristematic zone that leads
cell division rate, and the balance between carbon storage and usage. For first time we
here described the major effects of the suboptimal temperatures stress in rice
seedlings and detected possible mechanism of tolerance that should be further studied.
This work will help to design future strategies for stacking tolerant mechanisms that will
help in the obtaining of high yield cultivars that perform better under this stress. The
creation of new marker assisted breeding programs could be a good way to address
this issue and, hence, genome-wide association studies based on the described
mechanism could help for finding new markers.
... PSII activity of M and BS thylakoids of Z. mays, D. sanguinalis, and E. crus-galli was monitored spectrophotometrically as dichlorophenol-indophenol (DCPIP) photoreduction as described in Mamedov et al. (2000). Photoreduction was measured as the decrease in absorbance at 590 nm in a medium containing 330 mM sorbitol, 40 mM Hepes-KOH (pH 7.6), 1 mM KH 2 PO 4 , 5 mM NaCl, 5 mM MgCl 2 , 5 mM NH 4 Cl, and 0.1 mM DCPIP. ...
Main conclusion
Three species chosen as representatives of NADP-ME C4 subtype exhibit different sensitivity toward photoinhibition, and great photochemical differences were found to exist between the species. These characteristics might be due to the imbalance in the excitation energy between the photosystems present in M and BS cells, and also due to that between species caused by the penetration of light inside the leaves. Such regulation in the distribution of light intensity between M and BS cells shows that co-operation between both the metabolic systems determines effective photosynthesis and reduces the harmful effects of high light on the degradation of PSII through the production of reactive oxygen species (ROS).
Abstract
We have investigated several physiological parameters of NADP-ME-type C4 species (e.g., Zea mays, Echinochloa crus-galli, and Digitariasanguinalis) grown under moderate light intensity (200 µmol photons m⁻² s⁻¹) and, subsequently, exposed to excess light intensity (HL, 1600 µmol photons m⁻² s⁻¹). Our main interest was to understand why these species, grown under identical conditions, differ in their responses toward high light, and what is the physiological significance of these differences. Among the investigated species, Echinochloa crus-galli is best adapted to HL treatment. High resistance of the photosynthetic apparatus of E. crus-galli to HL was accompanied by an elevated level of phosphorylation of PSII proteins, and higher values of photochemical quenching, ATP/ADP ratio, activity of PSI and PSII complexes, as well as integrity of the thylakoid membranes. It was also shown that the non-radiative dissipation of energy in the studied plants was not dependent on carotenoid contents and, thus, other photoprotective mechanisms might have been engaged under HL stress conditions. The activity of the enzymes superoxide dismutase and ascorbate peroxidase as well as the content of malondialdehyde and H2O2 suggests that antioxidant defense is not responsible for the differences observed in the tolerance of NADP-ME species toward HL stress. We concluded that the chloroplasts of the examined NADP-ME species showed different sensitivity to short-term high light irradiance, suggesting a role of other factors excluding light factors, thus influencing the response of thylakoid proteins. We also observed that HL affects the mesophyll chloroplasts first hand and, subsequently, the bundle sheath chloroplasts.
... These results are presented in Fig. 8B and also in Table 1. When blocking reduction of Q B by Q A − using DCMU, the fast decay phase in the 150-350 ms time scale originates from centres without a functional Mn cluster [23,49] and these are considered as inactive RCs for O 2 evolution or non-O 2 -evolving PSII centres. These inactive RCs are still able to accumulate the P680 + state but the charge recombination occurs between Q A − and the oxidized secondary electron donor, Y + Z [24]. ...
Lead is a toxic, non-essential, heavy metal which affects photosynthesis by interacting with the essential metals integrated in photosynthetic apparatus or in metalloenzymes necessary for photosynthesis. However, studies of its effect on algal photophysiology, including the functional organization of PSII, are scarce. In this study, we used various non-invasive chlorophyll fluorescence measurements to investigate the changes in structural and functional aspects of the photosynthetic apparatus in two freshwater green algae, Chlorella and Scenedesmus, brought about under lead toxicity. PSII performance was inhibited by lead, as indicated by the decrease in maximum quantum yield and performance index. Flash fluorescence induction curves revealed that there was a significant alteration in antenna heterogeneity in which the decrease of PSII energetic connectivity (Joliot's p, Strasser's p2G, Paillotin's Ω, Lavergne's J) and the increase of antenna size under lead was remarkable. QA reoxidation and OJIP-derived parameters showed an increase in QB non-reducing reaction centres in Chlorella, but not in Scenedesmus, when grown in the presence of lead. Analysis of QA-reoxidation in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) providing data about O2-evolving/non-O2 evolving RCs, also showed the increase of inactive RCs under lead treatment in Chlorella but not in Scenedesmus. In addition, from the heterogeneity measurement results, differences between QB reducing vs PSIIα and QB non-reducing vs PSIIβ could clearly be distinguished.
... In some experiments, the PSII activity of M and BS thylakoids of maize and A. thaliana thylakoids was monitored spectrophotometrically as dichlorophenol-indophenol (DCPIP) photoreduction described in Mamedov et al. (2000). Agranal chloroplasts present in the BS cells of maize have PSII with oxygen evolving complex (OEC) and LHCII. ...
Main conclusion:
Light quality has various effects on photochemistry and protein phosphorylation in Zea mays and Arabidopsis thaliana thylakoids due to different degrees of light penetration across leaves and redox status in chloroplasts. The effect of the spectral quality of light (red, R and far red, FR) on the function of thylakoid proteins in Zea mays and Arabidopsis thaliana was investigated. It was concluded that red light stimulates PSII activity in A. thaliana thylakoids and in maize bundle sheath (BS) thylakoids, but not in mesophyll (M) thylakoids. The light quality did not change PSI activity in M thylakoids of maize. FR used after a white light period increased PSI activity significantly in maize BS and only slightly in A. thaliana thylakoids. As shown by blue native (BN)-PAGE followed by SDS-PAGE, proteins were differently phosphorylated in the thylakoids, indicating their different functions. FR light increased dephosphorylation of LHCII proteins in A. thaliana thylakoids, whereas in maize, dephosphorylation did not occur at all. The rate of phosphorylation was higher in maize BS than in M thylakoids. D1 protein phosphorylation increased in maize and decreased in A. thaliana upon irradiation with both R and growth light (white light, W). Light variations did not change the level of proteins in thylakoids. Our data strongly suggest that response to light quality is a species-dependent phenomenon. We concluded that the maize chloroplasts were differently stimulated, probably due to different degrees of light penetration across the leaf and thereby the redox status in the chloroplasts. These acclimation changes induced by light quality are important in the regulation of chloroplast membrane flexibility and thus its function.
... The same approach was used to test an additional fraction that was purified in our samples, that is, the BBY margins and end membranes (MAR fraction; Fig. 2). This fraction is defined as the portion of the appressed thylakoids in contact with the stroma and, according to previous studies, represents an essential compartment for photosynthetic activity (70). When performing differentially abundance analysis between margin fraction samples and BBY samples we identified a number of 296 differentially abundant proteins (q Ͻ 0.05), among which 238 are more abundant in margins (LogFC Ͼ 0). ...
Photosynthesis has shaped atmospheric and ocean chemistries and probably changed the climate as well, as oxygen is released from water as part of the photosynthetic process. In photosynthetic eukaryotes, this process occurs in the chloroplast, an organelle containing the most abundant biological membrane, the thylakoids. The thylakoids of plants and some green algae are structurally inhomogeneous, consisting of two main domains: the grana, which are piles of membranes gathered by stacking forces, and the stroma-lamellae, which are unstacked thylakoids connecting the grana. The major photosynthetic complexes are unevenly distributed, within these compartments, due to steric and electrostatic constraints. Although proteomic analysis of thylakoids has been instrumental to define its protein components, no extensive proteomic study of sub-thylakoid localization of proteins in the BBY (grana) and the stroma-lamellae fractions has been achieved so far. To fill this gap, we performed a complete survey of the protein composition of these thylakoid sub-compartments using thylakoid membrane fractionations. We employed semi-quantitative proteomics coupled with a data analysis pipeline and manual annotation to differentiate genuine BBY and stroma-lamellae proteins from possible contaminants. About 300 thylakoid (or potentially thylakoid) proteins were shown to be enriched in either the BBY or the stroma-lamellae fractions. Overall present findings corroborate previous observations obtained for photosynthetic proteins that used non-proteomic approaches. The originality of the present proteomic relies in the identification of photosynthetic proteins whose differential distribution in the thylakoid sub-compartments might explain already observed phenomenon such as LHCII docking. Besides, from the present localization results we can suggest new molecular actors for photosynthesis-linked activities. For instance, most PsbP-like subunits being differently localized in stroma-lamellae, these proteins could be linked to the PSI-NDH complex in the context of cyclic electron flow around PSI. In addition we could identify about a hundred new likely minor thylakoid (or chloroplast) proteins, some of them being potential regulators of the chloroplast physiology.
... The composition of EFu particles is less clear but the results indicate it to be PS II in its monomeric form. Monomeric PS II complexes have been isolated from the stroma region (Santini et al. 1994; Bassi et al. 1995; Dekker et al. 2002) and showed different structural and functional properties from the granal counterparts (Melis 1991; Jansson et al. 1997; Mamedov et al. 2000). These results are in good agreement with a 'PS II-repair cycle' which involves a monomerization and transfer of the damaged PS II complexes from grana to stroma region, partial disassembly , replacement of newly synthetized D1 protein and finally return of the rebuilt complex to grana membranes (Barbato et al. 1992; Barber and Andersson 1992; Aro et al. 1993; Kruse 2001; Baena-Gonzales and Aro 2002). ...
Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment-protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II.
The effect of the toxicant 2,3',4,4',6‐pentachlorobiphenyl (PCB‐119) on the growth, chlorophyll content, and PSII activity of C. sorokiniana cells was investigated. A strong negative effect of the toxicant was observed at PCB concentration of 0.05 μg mL–1, when culture growth ceased, chlorophyll strongly bleached, and cell death occurred. The use of original highly sensitive fluorimeter to measure three types of high‐resolution chlorophyll fluorescence kinetics allowed us to detect an initial dramatic decrease in the activity of primary photosynthetic reactions, followed by their almost complete recovery at the end of the incubation period when most cells were dead. The study of the distribution of individual cells in culture in terms of Fv/Fm parameter, which reflects the quantum yield of PSII photochemistry, revealed the existence of 2–3% of cells retaining high Fv/Fm (> 0.7) in the presence of the toxicant. The treated cultures were able to resume growth after prolonged incubation in fresh medium. The high sensitivity fluorescence methods used made it possible to identify stress‐resistant cells which maintain high photosynthetic activity in the presence of lethal doses of toxic substances; these cells provide recovery of the population after stress.
Elysia chlorotica is a kleptoplastic sea slug that preys on Vaucheria litorea, stealing its plastids which continue to photosynthesize for months inside the animal cells. We investigated the native properties of V. litorea plastids to understand how they withstand the rigors of photosynthesis in isolation. Transcription of specific genes in laboratory-isolated V. litorea plastids was monitored up to seven days. The involvement of plastid-encoded FtsH, a key plastid maintenance protease, in recovery from photoinhibition in V. litorea was estimated in cycloheximide-treated cells. In vitro comparison of V. litorea and spinach thylakoids was applied to investigate ROS formation in V. litorea. In comparison to other tested genes, the transcripts of ftsH and translation elongation factor EF-Tu (tufA) decreased slowly during incubation of isolated V. litorea plastids. Higher level of FtsH was also evident in cycloheximide-treated cells during recovery from photoinhibition. Charge recombination in PSII of V. litorea was found to be fine-tuned to produce only small quantities of singlet oxygen ( 1O2), and the plastids also contain ROS-protective compounds. Our results support the view that the genetic characteristics of the plastids themselves are crucial in creating a photosynthetic sea slug. The plastid’s autonomous repair machinery is likely enhanced by low 1O2 production and by elevated expression of FtsH in the plastids.
Elysia chlorotica is a kleptoplastic sea slug that preys on Vaucheria litorea , stealing its plastids which then continue to photosynthesize for months inside the animal cells. We investigated the native properties of V. litorea plastids to understand how they withstand the rigors of photosynthesis in isolation. Transcription of specific genes in laboratory-isolated V. litorea plastids was monitored up to seven days. The involvement of plastid-encoded FtsH, a key plastid maintenance protease, in recovery from photoinhibition in V. litorea was estimated in cycloheximide-treated cells. In vitro comparison of V. litorea and spinach thylakoids was applied to investigate ROS formation in V. litorea . Isolating V. litorea plastids triggered upregulation of ftsH and translation elongation factor EF-Tu ( tufA ). Upregulation of FtsH was also evident in cycloheximide-treated cells during recovery from photoinhibition. Charge recombination in PSII of V. litorea was found to be fine-tuned to produce only small quantities of singlet oxygen ( ¹ O 2 ). Our results support the view that the genetic characteristics of the plastids themselves are crucial in creating a photosynthetic sea slug. The plastid’s autonomous repair machinery is likely enhanced by low ¹ O 2 production and by upregulation of FtsH in the plastids.
Highlight
Isolated Vaucheria litorea plastids exhibit upregulation of tufA and ftsH , key plastid maintenance genes, and produce only little singlet oxygen. These factors likely contribute to plastid longevity in kleptoplastic slugs.
The pathway of photosystem II assembly is well understood and multiple auxiliary proteins supporting it have been identified. By contrast, little is known about rate-limiting steps controlling PSII biogenesis. In the green alga Chlamydomonas reinhardtii , biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, the 5’-untranslated region of psbD was modified via chloroplast transformation in tobacco. A drastic reduction in psbD mRNA abundance resulted in a strong decrease of PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD . Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants, and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in Chlamydomonas .
One sentence summary
PSII biogenesis in tobacco is neither limited by transcript accumulation nor translation of psbD and psbC .
Charge separation is a key component of the reactions cascade of photosynthesis, by which solar energy is converted to chemical energy. From this photochemical reaction, two radicals of opposite charge are formed, a highly reducing anion and a highly oxidising cation. We have previously proposed that the cation after far-red light excitation is located on a component different from PD1, which is the location of the primary electron hole after visible light excitation. Here, we attempt to provide further insight into the location of the primary charge separation upon far-red light excitation of PS II, using the EPR signal of the spin polarized 3P680 as a probe. We demonstrate that, under far-red light illumination, the spin polarized 3P680 is not formed, despite the primary charge separation still occurring at these conditions. We propose that this is because under far-red light excitation, the primary electron hole is localized on ChlD1, rather than on PD1. The fact that identical samples have demonstrated charge separation upon both far-red and visible light excitation supports our hypothesis that two pathways for primary charge separation exist in parallel in PS II reaction centres. These pathways are excited and activated dependent of the wavelength applied.
Tyrosine D (TyrD) is an auxiliary redox active tyrosine residue in photosystem II (PsiI). The mechanism of TyrD oxidation was investigated by EPR spectroscopy, flash-induced fluorescence decay and thermoluminescence measurements in PsiI enriched membranes from spinach. PsiI membranes were chemically treated with 3mM ascorbate and 1mM diaminodurene and subsequent washing, leading to the complete reduction of TyrD. TyrD oxidation kinetics and competing recombination reactions were measured after a single saturating flash in the absence and presence of DCMU (inhibitor of the QB-site) in the pH range of 4.7-8.5. Two kinetic phases of TyrD oxidation were observed by the time resolved EPR spectroscopy - the fast phase (msec-sec time range) and the pH dependent slow phase (tens of seconds time range). In the presence of DCMU, TyrD oxidation kinetics was monophasic in the entire pH range, i.e. only the fast kinetics was observed. The results obtained from the fluorescence and thermoluminescence analysis show that when forward electron transport is blocked in the presence of DCMU, the S2QA(-) recombination outcompetes the slow phase of TyrD oxidation by the S2 state. Modelling of the whole complex of these electron transfer events associated with TyrD oxidation fitted very well with our experimental data. Based on these data, structural information and theoretical considerations we confirm our assignment of the fast and slow oxidation kinetics to two populations of PsiI centers with different water positions (proximal and distal) in the TyrD vicinity.
The effect of exposing Photosystem II (PSII) enriched membranes to far-red laser flashes has been investigated by flash-induced variable Chla fluorescence and EPR spectroscopy. An enhancement of variable fluorescence of PSII together with a slowing down of the variable fluorescence decay kinetics was observed after application of laser flashes at a defined wavelength between 650–800 nm. In addition, the EPR signals from the S0−, S1− and S2-states of the water oxidizing complex were detected after application of far-red laser flashes.
Influence of the modification of the cyanobacterial light-harvesting complex
[i.e. phycobilisomes (PBS)] on the surface electric properties and the
functions of photosynthetic membranes was investigated. We used four PBS
mutant strains of Synechocystis sp. PCC6803 as follows: PAL (PBS-less),
CK (phycocyanin-less), BE (PSII-PBS-less) and PSI-less/apcE− (PSI-less with
detached PBS). Modifications of the PBS content lead to changes in the cell
morphology and surface electric properties of the thylakoid membranes as
well as in their functions, such as photosynthetic oxygen-evolving activity,
P700 kinetics and energy transfer between the pigment–protein complexes.
Data reveal that the complete elimination of PBS in the PAL mutant causes
a slight decrease in the electric dipole moments of the thylakoid membranes,
whereas significant perturbations of the surface charges were registered in
the membranes without assembled PBS–PSII macrocomplex (BE mutant) or
PSI complex (PSI-less mutant). These observations correlate with the detected
alterations in the membrane structural organization. Using a polarographic
oxygen rate electrode, we showed that the ratio of the fast to the slow
oxygen-evolving PSII centers depends on the partial or complete elimination
of light-harvesting complexes, as the slow operating PSII centers dominate in
the PBS-less mutant and in the mutant with detached PBS.
The action of the environmental toxic Pb(2+) on photosynthetic electron transport was studied in thylakoid membranes isolated from spinach leaves. Fluorescence and thermoluminescence techniques were performed in order to determine the mode of Pb(2+) action in photosystem II (PSII). The invariance of fluorescence characteristics of chlorophyll a (Chl a) and magnesium tetraphenylporphyrin (MgTPP), a molecule structurally analogous to Chl a, in the presence of Pb(2+) confirms that Pb cation does not interact directly with chlorophyll molecules in PSII. The results show that Pb interacts with the water oxidation complex thus perturbing charge recombination between the quinone acceptors of PSII and the S2 state of the Mn4Ca cluster. Electron transfer between the quinone acceptors QA and QB is also greatly retarded in the presence of Pb(2+). This is proposed to be owing to a transmembrane modification of the acceptor side of the photosystem.
This review analyzes various alternative pathways of chloroplast electron transport mediated by photoreactions of photosystem
I (PSI) and unrelated to activity of photosystem II (PSII). The mechanisms and functional significance of the alternative
pathways are considered. These pathways are complexly organized and comprise ferredoxin-dependent electron recycling around
PSI, as well as electron donation to noncyclic chain in the region between PSII and PSI from reduced substances localized
in the chloroplast stroma. For each of the alternative pathways, the origin of corresponding enzymes and their compartmentalization
in the complex membrane system of the chloroplast are discussed. It is shown that operation of alternative electron transport
pathways contributes to energy transduction and cell defense function, facilitates the absorption of inorganic carbon, and
is significant for chloroplast respiration. Multiple mechanisms for regulation of alternative pathways have been revealed.
It is concluded that PSI-related alternative electron transport pathways constitute an integral part of entire system of photosynthetic
electron transport, this system being principally responsible for energy supply of phototrophic cells and whole plants.
Influence of the modification of the cyanobacterial light-harvesting complex [i.e. phycobilisomes (PBS)] on the surface electric properties and the functions of photosynthetic membranes was investigated. We used four PBS mutant strains of Synechocystis sp. PCC6803 as follows: PAL (PBS-less), CK (phycocyanin-less), BE (PSII-PBS-less) and PSI-less/apcE(-) (PSI-less with detached PBS). Modifications of the PBS content lead to changes in the cell morphology and surface electric properties of the thylakoid membranes as well as in their functions, such as photosynthetic oxygen-evolving activity, P700 kinetics and energy transfer between the pigment-protein complexes. Data reveal that the complete elimination of PBS in the PAL mutant causes a slight decrease in the electric dipole moments of the thylakoid membranes, whereas significant perturbations of the surface charges were registered in the membranes without assembled PBS-PSII macrocomplex (BE mutant) or PSI complex (PSI-less mutant). These observations correlate with the detected alterations in the membrane structural organization. Using a polarographic oxygen rate electrode, we showed that the ratio of the fast to the slow oxygen-evolving PSII centers depends on the partial or complete elimination of light-harvesting complexes, as the slow operating PSII centers dominate in the PBS-less mutant and in the mutant with detached PBS.
The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800 nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn(4) cluster. Far-red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic resonance spectroscopy. Signals from the S(2)-, S(3)-, and S(0)-states could be detected after one, two, and three far-red flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm. In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and energy levels of the charge separated states in PSII are discussed in light of the presented results.
Genome sequence of Arabidopsis thaliana (Arabidopsis) revealed two psbO genes (At5g66570 and At3g50820) which encode two distinct PsbO isoforms: PsbO1 and PsbO2, respectively. To get insights into the function of the PsbO1 and PsbO2 isoforms in Arabidopsis we have performed systematic and comprehensive investigations of the whole photosynthetic electron transfer chain in the T-DNA insertion mutant lines, psbo1 and psbo2. The absence of the PsbO1 isoform and presence of only the PsbO2 isoform in the psbo1 mutant results in (i) malfunction of both the donor and acceptor sides of Photosystem (PS) II and (ii) high sensitivity of PSII centers to photodamage, thus implying the importance of the PsbO1 isoform for proper structure and function of PSII. The presence of only the PsbO2 isoform in the PSII centers has consequences not only to the function of PSII but also to the PSI/PSII ratio in thylakoids. These results in modification of the whole electron transfer chain with higher rate of cyclic electron transfer around PSI, faster induction of NPQ and a larger size of the PQ-pool compared to WT, being in line with apparently increased chlororespiration in the psbo1 mutant plants. The presence of only the PsbO1 isoform in the psbo2 mutant did not induce any significant differences in the performance of PSII under standard growth conditions as compared to WT. Nevertheless, under high light illumination, it seems that the presence of also the PsbO2 isoform becomes favourable for efficient repair of the PSII complex.
The present contribution describes a new experimental setup that permits time-resolved monitoring of the rise kinetics of the relative fluorescence yield, Phi(rel)(t), and simultaneously of the decay of delayed light emission, L(t), induced by strong actinic laser flashes. The results obtained by excitation of dark-adapted samples with a train of eight flashes reveal (a) in suspensions of spinach thylakoids, Phi(rel)(t) exhibits a typical period four oscillation that is characteristic for a dependence on the redox states S(i)() of the water oxidizing complex (WOC), (b) the relative extent of the unresolved "instantaneous" rise to the level (100 ns) at 100 ns and the maximum values of Phi(rel)(t) attained at about 45 s after each actinic flash, (45 s) synchronously oscillate and exhibit the largest values at flash nos. 1 and 5 and minima after flash nos. 2 and 3, (c) opposite effects are observed for the normalized extent of the rise kinetics in the 100 ns to 5 s time domain of relative fluorescence yield, Phi(rel)(5 s) - Phi(rel)(100 ns), i.e., both parameters attain minimum and maximum values after the first/fifth and second/third flash, respectively, and (d) analogous features for the "fast" and "slow" ns-kinetics of the fluorescence rise were observed in suspensions of Chlamydomas reinhardtii cells. A slight phase shift by one flash is ascribed to physiological differences. The applicability of this noninvasive technique to study reactions of photosystem II, especially the reduction kinetics of P680(*)(+) and their dependence on the redox state S(i)() of the WOC, is discussed.
In photosynthesis, light-harvesting chlorophyll molecules are shunted between photosystems by phosphorylation of the protein to which they are bound. An anchor for the phosphorylated chlorophyll-protein complex has now been identified in the reaction centre of chloroplast photosystem I. This finding supports the idea that molecular recognition, not membrane surface charge, governs the architecture of the chloroplast thylakoid membrane. We describe a model for the chloroplast thylakoid membrane that is consistent with recent structural data that specify the relative dimensions of intrinsic protein complexes and their dispositions within the membrane. Control of molecular recognition accommodates membrane stacking, lateral heterogeneity and regulation of light-harvesting function by means of protein phosphorylation during state transitions--adaptations that compensate for selective excitation of photosystem I or photosystem II. High-resolution structural description of membrane protein-protein interactions is now required to understand thylakoid structure and regulation of photosynthesis.
Assembly of the inorganic core (Mn(4)O(x)Ca(1)Cl(y)) of the water oxidizing enzyme of oxygenic photosynthesis generates O(2) evolution capacity via the photodriven binding and photooxidation of the free inorganic cofactors within the cofactor-depleted enzyme (apo-WOC-PSII) by a process called photoactivation. Using in vitro photoactivation of spinach PSII membranes, we identify a new lower affinity site for bicarbonate interaction in the WOC. Bicarbonate addition causes a 300% stimulation of the rate and a 50% increase in yield of photoassembled PSII centers when using Mn(2+) and Ca(2+) concentrations that are 10-50-fold larger range than previously examined. Maintenance of a fixed Mn(2+)/Ca(2+) ratio (1:500) produces the fastest rates and highest yields of photoactivation, which has implications for intracellular cofactor homeostasis. A two-step (biexponential) model is shown to accurately fit the assembly kinetics over a 200-fold range of Mn(2+) concentrations. The first step, the binding and photooxidation of Mn(2+) to Mn(3+), is specifically stimulated via formation of a ternary complex between Mn(2+), bicarbonate, and apo-WOC-PSII, having a proposed stoichiometry of [Mn(2+)(HCO(3)(-))]. This low-affinity bicarbonate complex is thermodynamically easier to oxidize than the aqua precursor, [Mn(2+)(OH(2))]. The photooxidized intermediate, [Mn(3+)(HCO(3)(-))], is longer lived and increases the photoactivation yield by suppressing irreversible photodamage to the cofactor-free apo-WOC-PSII (photoinhibition). Bicarbonate does not affect the second (rate-limiting) dark step of photoactivation, attributed to a protein conformational change. Together with the previously characterized high-affinity site, these results reveal that bicarbonate is a multifunctional "native" cofactor important for photoactivation and photoprotection of the WOC-PSII complex.
The relative proportion of stroma lamellae and grana end membranes was determined from electron micrographs of 58 chloroplasts from 21 different plant species. The percentage of grana end membranes varied between 1 and 21% of the total thylakoid membrane indicating a large variation in the size of grana stacks. By contrast the stroma lamellae account for 20.3 +/- 2.5 (sd)% of the total thylakoid membrane. A plot of percentage stroma lamellae against percentage of grana end membranes fits a straight line with a slope of zero showing that the proportion of stroma lamellae is independent of the size of the grana stacks. That stroma lamellae account for about 20% of the thylakoid membrane is in agreement with fragmentation and separation analysis (Gadjieva et al. Biochim. Biophys. Acta 144: 92-100, 1999). Chloroplasts from spinach, grown under high or low light, were fragmented by sonication and separated by countercurrent distribution into two vesicle populations originating from grana and stroma lamellae plus end membranes, respectively. The separation diagrams were very similar lending independent support for the notion that the proportion of stroma lamellae is constant. The results are discussed in relation to the composition and function of the chloroplast in plants grown under different environmental conditions, and in relation to a recent quantitative model for the thylakoid (Albertsson, Trends Plant Sci. 6: 349-354, 2001).
Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and QA− to QB(QB−) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin−) and the EPR signal from the QA− Fe2+ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S2 state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S2 multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S2 multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b
559 was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed.
The Ala344 residue of the D1 protein has been identified as a crucial residue of the catalytic cluster of the water-oxidizing complex, however, its function has not been fully clarified. Here we have used thermoluminescence and flash-induced chlorophyll fluorescence measurements to characterize the effect of the D1-Ala344stop mutation on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutant cannot grow photoautotrophically it shows flash-induced thermoluminescence and chlorophyll fluorescence signals reflecting the stabilization of negative and positive charges on the Q(A) and Q(B) quinone electron acceptors, and stable Photosystem II donors, respectively. Decay of flash induced chlorophyll fluorescence yield is multiphasic in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with 6 ms, 350 ms, and 26 s time constants. When cells are illuminated with repetitive flashes, fired at 1 ms intervals, the 6 ms phase is gradually decreased with the concomitant increase of the 350 ms phase. After 45 min dark adaptation of mutant cells the 6 ms and 350 ms phases were significantly decreased and a very slow decaying component was formed. Flash induced oscillation of the thermoluminescence B band, which reflects the redox cycling of the water-oxidizing complex in the wild-type cells, was completely abolished in the D1-Ala344stop mutant. The results demonstrate that low efficiency photooxidation of Mn occurs in about 60% of the PSII centers. The photooxidizable Mn is unstable in the dark, and formation of higher S states is inhibited. In addition, the Q(A) to Q(B) electron transfer step is slowed down as an indirect consequence of the donor side modification. Our data indicate that the stabilization of a Mn ion by the alpha-carboxylate chain of the D1-Ala344 residue might represent one of the final steps in the assembly of functional catalytic sites for water oxidation.
The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane,
the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers,
PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by
blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes
and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma
lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is
totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in
each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all
of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did
not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.
It is well known that two photosystems, I and II, are needed to transfer electrons from H2O to NADP+ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS IIα and PS IIβ units, (b) RC complex containing electron acceptor Q1 or Q2; and (c) electron acceptor complex: QA (having two different redox potentials QL and QH) and QB (QB-type; Q'B type; and non-QB type); additional components such as iron (Q-400), U (Em,7=−450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.
The D1 reaction center protein in photosystem II (PSII) has a high turnover rate due to light-induced inactivation of the redox components. We have studied the reactivation kinetics of the redox components of PSII after strong illumination and compared these kinetics with the turnover of the D1 protein and translation kinetics of the plastid-encoded PSII core proteins in Chlamydomonas reinhardtii cells. Repair of PSII was to a large extent dependent on protein translation. During the first hours of repair, D1 translation was highly accelerated as compared to the other PSII core proteins. By addition of protein synthesis inhibitors during the recovery process, it was found that the time from protein synthesis to full reassembly and reactivation of the individual PSII complexes was about 55 +/- 10 min. Inactivation and reactivation of the redox components in PSII were followed by electron spin resonance and electron transport measurements. Combining the data shows that reactivation of the individual components proceeded together or shortly after one another. Thus, no accumulation of any partially active reactivation intermediate occurred. We conclude that the rate-limiting step of the repair cycle of PSII lies in the degradation and synthesis of the PSII reaction center proteins. Once stable synthesis of the PSII core proteins is achieved, reactivation of the redox components occurs very quickly.
The process of photoactivation has been studied in dark grown cells of Chlamydomonas reinhardtii. A mutant, FUD 39, lacking the Cl−-concentrating 23-kDa psbP protein of photosystem II was found to have a decreased capability to perform photoactivation.
The yield of the process never reached wild type level, and contrary to the wild type, it was highly dependent on the intensity
of the activating light, with a very narrow optimum around 1 μE m−2 s−1. The different behavior in the mutant can be explained by a requirement for a longer dark period, between the two photoacts,
during the photoactivation. This is proposed to reflect the decreased Cl− affinity in the mutant. Photoactivation in the mutant was also found to be very sensitive to competing photoinhibitory processes.
The inhibition was located to the donor side of photosystem II and affected the photoactivation capability before electron
transfer from Tyrz was inhibited. We propose an extended model for photoactivation in which an intermediate that is sensitive to photoinhibition
is formed if Cl− is not functionally bound to the manganese cluster.
Photoinhibition of photosynthesis was studied in isolated photosystem II membranes by using chlorophyll fluorescence and electron paramagnetic resonance (EPR) spectroscopy combined with protein analysis. Under anaerobic conditions four sequentially intermediate steps in the photoinhibitory process were identified and characterized. These intermediates show high dark chlorophyll fluorescence (Foi) with typical decay kinetics (fast, semistable, stable, and nondecaying). The fast-decaying state has no bound QB but possesses a single reduced QA species with a 30-s decay half-time in the dark (QB, second quinone acceptor; QA, first quinone acceptor). In the semistable state, Q-A is stabilized for 2-3 min, most likely by protonation, and gives rise to the Q-A Fe2+ EPR signal in the dark. In the stable state, QA has become double reduced and is stabilized for 0.5-2 hr by protonation and a protein conformational change. The final, nondecaying state is likely to represent centers where QA H2 has left its binding site. The first three photoinhibitory states are reversible in the dark through reestablishment of QA to QB electron transfer. Significantly, illumination at 4 K of anaerobically photoinhibited centers trapped in all but the fast state gives rise to a spinpolarized triplet EPR signal from chlorophyll P680 (primary electron donor). When oxygen is introduced during anaerobic illumination, the light-inducible chlorophyll triplet is lost concomitant with induction of D1 protein degradation. The results are integrated into a model for the photoinhibitory process involving initial loss of bound QB followed by stable reduction and subsequent loss of QA facilitating chlorophyll P680 triplet formation. This in turn mediates light-induced formation of highly reactive and damaging singlet oxygen.
The stoichiometric amounts and the photoactivity kinetics of photosystem I (PSI) and of the alpha and beta components of photosystem II (PSII(alpha) and PSII(beta)) were compared in spinach chloroplast membrane (thylakoid) fractions derived from appressed and nonappressed regions. Stroma-exposed thylakoid fractions from the nonappressed regions were isolated by differential centrifugation following a mechanical press treatment of the chloroplasts. Thylakoid vesicles derived mainly from the appressed membranes of grana were isolated by the aqueous polymer two-phase partition method. Stroma-exposed thylakoids were found to have a chlorophyll a/chlorophyll b ratio of 6.0 and a PSII(beta)/PSI reaction center ratio of 0.3. Kinetic analysis of system II photoactivity revealed the absence of PSII(alpha) from stroma-exposed thylakoids. The photoactivity of system I in stroma-exposed thylakoids showed a single kinetic component identical to that of unfractionated chloroplasts, suggesting that PSI does not receive excitation energy from the PSII-chlorophyll ab light-harvesting complex. Thus, stroma-exposed thylakoids are significantly enriched in both PSI and PSII(beta). Inside-out vesicles from the appressed membranes of grana-partition regions had a chlorophyll a/chlorophyll b ratio of 2.0 and a PSII/PSI reaction center ratio of 10.0. The photoactivity of system II showed the membranes of the grana-partition regions to be significantly enriched in PSII(alpha). We conclude that PSII(alpha) is exclusively located in the membranes of the grana partitions while PSII(beta) and PSI are located in stroma-exposed thylakoids. The low PSI reaction center (P700) content of vesicles derived from grana partitions and the kinetic homogeneity of the PSI complex suggest total exclusion of P700 as a functional component in the membrane of the grana-partition region.
Photosystem-two (PSII) in the chloroplasts of higher plants and green algae is not homogeneous. A review of PSII heterogeneity is presented and a model is proposed which is consistent with much of the data presented in the literature. It is proposed that the non-quinone electron acceptor of PSII is preferentially associated with the sub-population of PSII known as PSIIß.
A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (−196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 μmol O2 per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.
The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.
Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.
Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14-18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.
Thylakoids from spinach were fragmented by sonication and the viscles so obtained were separated into different populations by aqueous two-phase partitioning using the dextran-poly(ethylene glycol) system. The different vesicle populations were analyzed with respect to the concentrations of P700 and cytochrome. The P700 content varied between 0.93 (PS-II-enriched grana vesicles named BS) and 4.85 (stroma membrane vesicles named Y100) mmol per mol chlorophyll. The cytochrome f content varied between 1.92 (vesicles originating from the grana periphery, named 120S vesicles) and 3.12 (PS-II-enriched grana vesicles named BS) mmol/mol chlorophyll. A plot of the P700 content against the cytochrome f content of the different vesicle populations was compared with hypothetical models of membrane vesicles. The results show that the thylakoid membrane consists of at least three different domains with respect to cytochrome f. These are suggested to be: (1) the stroma lamellae; (2) the core of the partition region of the grana; and (3) a peripheral annulus of the grana discs including the margins (and perhaps also the end membranes).
A membrane fraction composed of right-side-out vesicles and deriving from the periphery of the granal stacked region of spinach thylakoid membranes has been isolated by means of sonication and separation in an aqueous two-phase system, and its biochemical and photochemical properties examined. This vesicle population, referred to as the chloroplast granal margins, is enriched in PSI as compared to the central core of the grana, has a Chl ratio of between 3.0 and 3.3, and on a per chlorophyll basis contains the least amount of cytochrome f as compared to both the central core of the grana and the stroma lamellae domains. Notably, however, the margins represent the region of the thylakoid most enriched in the 64 kDa kinase. P700 and room temperature fluorescence induction kinetics show that the granal margins contain PS I with a larger antenna size than the PS I from the stroma lamellae and that the PS II of the margins is, with respect to the antenna size, more like the PS II from the stroma lamellae (i.e., PS IIβ). The margin vesicles also contain the ATPase complex as do the other stroma exposed areas of the thylakoid.
The decay kinetics for the S2 and S3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S2- and S3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S3 decays via S2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S0 to S1 220–225 K, for S1 to S2 135–140 K, for S2 to S3 230 K and for the S3-to-S0 transition 235 K.
The chloroplast grana margins of spinach thylakoids were isolated by sonication and aqueous-two-phase partitioning and their electron transport properties examined. Photosystem II and I electron transport activities were measured and compared to the appressed and non-appressed grana core and stroma lamellae, respectively, as well as to whole thylakoids. The results show that the PS II complexes in the grana margins are of the PS IIβ subtype with respect to antenna size, but are QB reducing with respect to the acceptor side properties, while the PS I centers in the grana margins have slightly larger antennae as compared to the PS I centers in the stroma lamellae and are more like the PS Iα centers located in the grana domain. The ability to reduce ferredoxin and NADP+ was also tested and it was found that the grana margin membrane fraction was unable to reduce ferredoxin, even in the presence of added artificial electron donors. The stroma lamellae and whole thylakoid fractions both reduced ferredoxin at high rates. However, the grana margins could catalyze the reduction of NADP+ when supplied with the necessary components (ferredoxin, ferredoxin:NADP+ oxidoreductase, and an electron source). The results suggest that the PS I populations located in the margins of the grana domain are functionally different from the PS I centers located in the stroma lamellae. The data support a model whereby the PS I centers in the grana are primarily involved in non-cyclic electron transport, while the PS I centers located in the stroma lamellae are capable of participating in both cyclic and non-cyclic electron transport.
Electron paramagnetic resonance (EPR) spectroscopy has been applied in an investigation on the mechanism for photoinhibition of the electron transport in Photosystem II. The experiments were performed in vitro in thylakoid membranes and preparations of Photosystem-II-enriched membranes. Photoinhibition resulted in inhibition of the oxygen evolution and EPR measurements of the S2 state multiline EPR signal show that its induction by illumination at 198 K was decreased with the same kinetics as the oxygen evolution. Further EPR measurements show that the reduction of QA was inhibited with the same kinetics as the oxygen evolution. The amount of photoreducible pheophytin was estimated from photoaccumulation experiments under reducing conditions and the results show that the primary charge separation reaction was inhibited much slower than the oxygen evolution or the reduction of QA. These results indicate that photoinhibition inhibits the electron transfer between pheophytin and QA probably by impairment of the function of QA. In the inhibited centers the primary charge separation reaction is still operational. It is suggested that the event leading to photoinhibition of the electron transport is the double reduction of QA which then leaves its site. Photoinhibition also results in rapid oxidation of cytochrome b-559 and a change of cytochrome b-559 from its high potential form to its low potential form. The reaction is quantitative and proceeds with the same kinetics as the inhibition of oxygen evolution. The potential shift of cytochrome b-559 suggests that photoinhibition induces early conformational changes in Photosystem II.
Reoxidation of the fluorescence quencher “Q” in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylureaReoxidation of the fluorescence quencher Q in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea shows the following properties:It is sensitive to very low concentrations of hydroxylamine (a few μM).It corresponds to a back reaction between Q− and the primary oxidant Z+ formed in the light. A part of this back reaction gives rise to luminescence emission.Within the range we studied the kinetic of reoxidation is second order with regards to Q−.RésuméLa réoxydation du quencher de fluorescence Q en présence de 3-(3,4-dichlorophényl)-1,1-diméthylurée présente les propriétés suivantes:Elle est sensible à de très faibles concentrations d'hydroxylamine (quelques μM).Elle résulte d'une recombinaison de charges entre Q− et l'oxydant primaire Z+ formé à la lumière. Cette recombinaison s'effectue, au moins en partie, avec émission de luminescence.Dans le domaine étudié, la cinétique de réoxydation est d'ordre 2 par rapport à la concentration de Q−.
The rapid response of the photosynthetic system to changes in light intensity (response within less than 30 min) is condidered. Variations in light intensity result in concentration changes in photosynthetic intermediates or protein states. These changes in turn affect Photosystem II (PS II) by a ‘backpressure effect’, resulting in the accumulation of PS II products (reduced plastoquinone, lumen protons). The product backpressure produces PS II states especially susceptible to photoinactivation and photodamage. By activation of special adaptation mechanisms, the efficiency of the photosynthetic system is optimized and photodamage is minimized. The following aspects are discussed: 1.long-term vs. short-term adaptation;2.analysis of short-term adaptation by measurement of chlorophyll a fluorescence and photosynthetic oxygen evolution;3.kinetics of the response of the photosynthetic system to changes in light intensity (induction curves, assignment of phases, time constants);4.the ‘product backpressure’ on PS II (accumulation of reduced QA, lumen pH effect on PS II donor side reactions, thylakoid voltage effect on PS II photochemistry);5.the molecular mechanisms of short-term adaptation (pH-dependent energy quenching, reversible inactivation of the manganese complex, light-harvesting complex (LHC) phosphorylation);6.induction of photoinactivation and photodamage;7.relation between product backpressure, adaptation and photodamage.
Thylakoid vesicles containing photosystem II from either the appressed or non-appressed thylakoid region were subjected to photoinhibitory illumination. Photosystem II from the non-appressed region was found to be less sensitive to photoinhibition compared to photosystem II from the oppressed region under both aerobic and anaerobic conditions.
When the photosystem II quinone acceptor complex has been singly reduced to the state QAQ−B, there is a 22 s half-time back-reaction of Q−B with an oxidized photosystem II donor (S2), directly measured here for the first time. From the back-reaction kinetics with and without inhibitors, kinetic and equilibrium parameters have been estimated. We suggest that the state QAQ−B of the complex is formed by a second-order reaction of vacant reaction centers in the state Q−A with plastoquinone from the pool, and discuss the physico-chemical parameters involved.
Redox titrations of QA, the first quinone electron acceptor, have been performed on Photosystem II (PS II) membranes which were either active or inactive in terms of oxygen evolution. The redox state of QA was monitored by measuring the chlorophyll fluorescence yield. When titrations were done at room temperature in the absence of mediators, an Em value of approx. −80 mV was obtained for active centres and approx. +65 mV for inactive centres. These values confirm earlier reports (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98) in which measurements were made under comparable conditions. In addition, we found that these Em values were independent of pH from pH 5.5 to pH 7.5, the range of pH over which the O2-evolving enzyme is stable. To understand better the scattered values for the Em of QA which exist in the literature and to assess the validity of the present values, experiments were performed under a range of different titration conditions. Two main experimental factors were found to have a strong influence on the measured Em of QA. First, the presence of redox mediators at low ambient potentials led to an irreversible shift from the low-potential (active) form to the high-potential (inactive) form. This is attributed to the reduction of the Mn cluster which is thought to remain out of equilibrium when titrations are done without mediators. Secondly, upon freezing of samples poised at low potential a change in the redox state of QA occurred, as measured by EPR and fluorescence at low temperature. Freezing and thawing of active PS II at potentials where QA is chemically reduced results in an irreversible change in the Em of QA from the low-potential to the high-potential form. This is accompanied by inhibition of oxygen evolution. It is suggested that this effect might also be related to the reduction of the Mn cluster which is, in this case, induced by freeze-thawing in the presence of chemically reduced QA−. Based on these observations, it is suggested that most titrations of QA in active PS II that have been reported previously suffer from one or both of these unexpected technical difficulties. Thus, the Em values obtained at room temperature and without mediators are probably those which should be taken into account in understanding the energetics of PS II.
A study of electron paramagnetic resonance (EPR) signals from components on the electron donor side of photosystem II has been performed. By measurement of EPR signal IIslow (D+) it is shown that, after three flashes, D+ decays slowly in the dark at room temperature in the fraction of the centers that was in the S0 state (t1/2 of 20 min in thylakoid membranes and 50 min in photosystem II enriched membranes). This reaction is accompanied by a conversion of S0 to S1. The concentration of S1 was estimated from the amplitude of the S2-state multiline EPR signal that could be generated by illumination at 200 K. These observations indicate that D+ accepts an electron from S0 in a dark reaction in which D and S1 are formed. In addition, the reactions by which D donates an electron to S2 or S3 have been directly measured by monitoring both signal IIslow and the multiline signal. The redox interactions between the D/D+ couple and the S states are explained in terms of a model in which D/D+ has a midpoint potential between those of S0/S1, and S1/S2. In addition, this model provides explanations for a number of previously unrelated phenomena, and the proposal is put forward that the reaction between D+ and Mn2+ is involved in the so-called photoactivation process.
The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that QA of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of QA than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.
Photosystem II in green plant chloroplasts displays heterogeneity both in the composition of its light-harvesting antenna and in the ability to reduce the plastoquinone pool. These two features are discussed in terms of chloroplast development and in view of a proposed photosystem II repair cycle.
An improved, non-detergent, method for preparative isolation of PS II membrane vesicles from spinach chloroplasts is presented. Thylakoids (chlorophyll (Chl) a/b ratio 2.8, Chl/P700 435) were fractionated by Yeda press treatment and aqueous two-phase partition to yield inside-out vesicles (1) (chl a/b 2.2, chl/P700 700). These vesicles were subjected a sonication — phase partitioning procedure; steps of sonication of inside-out vesicles, while still present in a dextran-polyethylene glycol two-phase system were alternated by phase partition. These steps selectively removed P700-containing membrane fragments from the inside-out vesicles and yielded a membrane fraction with improved PS II purity (Chl a/b ratio 1.9, Chl/P700 1500) and retained oxygen evolving capacity (295 mol O2 mg Chl-1 h-1).
Ca2+ depletion of Photosystem II from spinach results in reversible retardation of electron transfer on both donor and acceptor sides. On the donor side, a decrease of the electron transfer rate from TyrZ results in an enhanced charge recombination between the oxidized primary donor, P680+, and the reduced acceptor quinone, QA−, which in turn leads to a decrease in the amplitude of the fluorescence yield. In addition, slow electron transfer from the manganese cluster in the dark-stable S2 state results in the appearance of a transient EPR signal from TyrZox which decays with half-times of 600 ms and 5 s. On the acceptor side, the disappearance of the 400 μs decay transient in the fluorescence yield indicates that the electron transfer from QA− to QB has been severely inhibited. These results suggests that removal of a Ca2+ ion from the donor side in PS II, which results in the inhibition of oxygen evolution and in the appearance of an EPR signal in the S′3 state leads to structural changes which are transmitted to the acceptor side. The strikingly similar behavior after depletion of Ca2+ of the TyrZox EPR signal and the split radical signal from the S′3 state suggests that both signals involves the same oxidized amino acid residue, TyrZox. The absence of large effects on the EPR properties of the non-heme iron suggests that the structural changes on the acceptor side are subtle in nature. Chemical modification of histidine results in inhibition of QA− to QB electron transfer and to changes in the magnetic properties of the oxidized non-heme iron but only to minor perturbations of the donor-side. This suggests that histidine, susceptible to chemical modification, is located mainly on the acceptor side of PS II.
The spectra of the absorbance changes due to the turnover of the so-called S-states of the oxygen-evolving apparatus were determined. The changes were induced by a series of saturating flashes in dark-adapted Photosystem II preparations, isolated from spinach chloroplasts. The electron acceptor was 2,5-dichloro-p-benzoquinone. The fraction of System II centers involved in each S-state transition on each flash was calculated from the oscillation pattern of the 1 ms absorbance transient which accompanies oxygen release. The difference spectrum associated with each S-state transition was then calculated from the observed flash-induced difference spectra. The spectra were found to contain a contribution by electron transfer at the acceptor side, which oscillated during the flash series approximately with a periodicity of two and was apparently modulated to some extent by the redox state of the donor side. At the donor side, the S0 → S1, S1 → S2 and S2 → S3 transitions were all three accompanied by the same absorbance difference spectrum, attributed previously to an oxidation of Mn(III) to Mn(IV) (Dekker, J.P., Van Gorkom, H.J., Brok, M. and Ouwehand, L. (1984) Biochim. Biophys. Acta 764, 301–309). It is concluded that each of these S-state transitions involves the oxidation of an Mn(III) to Mn(IV). The spectrum and amplitude of the millisecond transient were in agreement with its assignment to the reduction of the oxidized secondary donor Z+ and the three Mn(IV) ions.
An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg2+. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS IIα. (5) An Em = − 10 mV has been measured at pH 7.8 for the primary electron acceptor Qα of PS IIα. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll . In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.
In vivo photoactivation of Photosystem II was studied in the FUD39 mutant strain of the green alga Chlamydomonas reinhardtii which lacks the 23 kDa protein subunit involved in water oxidation. Dark grown cells, devoid of oxygen evolution, were illuminated at 0.8 μE m−2 s−1 light intensity which promotes optimal activation of oxygen evolution, or at 17 μE m−2 s−1, where photoactivation compete with deleterious photodamage. The involvement of the two redox active cofactors tyrosineD and cytochrome b559 during the photoactivation process, was investigated by EPR spectroscopy. TyrosineD on the D2 reaction center protein functions as auxiliary electron donor to the primary donor P680 + during the first minutes of photoactivation at 0.8 μE m−2 s−1 (compare with Rova et al., Biochemistry, 37 (1998) 11039–11045.). Here we show that also cytochrome b559 was rapidly oxidized during the first 10 min of photoactivation with a similar rate to tyrosineD. This implies that both cytochrome b559 and tyrosineD may function as auxiliary electron donors to P680 + and/or the oxidized tyrosine⋅Z on the D1 protein, to avoid photoinhibition before successful photoactivation was accomplished. As the catalytic water-oxidation successively became activated, TyrosineD remained oxidized while cytochrome b559 became rereduced to the equilibrium level that was observed prior to photoactivation. At 17 μE m−2 s−1 light intensity, where photoinhibition competes significantly with photoactivation, tyrosineD was very rapidly completely oxidized, after which the amount of oxidized tyrosineD decreased due to photoinhibition. In contrast, cytochrome b559 became reduced during the first 2 min of photoactivation at 17 μE m−2 s−1. After this, it was reoxidized, returning to the equilibrium level within 10 min. Thus, during in vivo photoactivation in high-light cytochrome b559 serves two functions. Initially, it probably oxidizes the reduced primary acceptor pheophytin, thereby relieving the acceptor side of reductive pressure, and later on it serves as auxiliary electron donor, preventing donor-side photoinhibition.
(1) Thylakoids from spinach chloroplasts were phosphorylated, fragmented by sonication, and then fractionated by aqueous two-phase partitioning to yield membrane fragments, deriving from different structural domains of the membrane: grana, grana margins, grana core and stroma lamellae. The photochemical activities of PS Iα and PS IIα, located in the grana, and PS Iβ and PS IIβ, located in the stroma lamellae, were compared for phosphorylated and control thylakoids. The antenna size (reflected by the Km value) and maximum activity (Vmax) of PS IIα declined by 19 and 23%, respectively, while for PS IIβ the antenna size and Vmax decreased by 4 and 12%, respectively. No significant changes in antenna size were detected for either grana PS Iα or stroma lamellae PS Iβ. Counter-current distribution was used for the quantitative separation of grana and stroma lamellae vesicles. Upon phosphorylation, the stroma lamellae fraction increased from 30% to 35% of the total, based on total absorbance at 680 nm. This increase can be explained by partial unstacking of the grana periphery and appressed membranes near the fret regions. Portions of the previously stacked membranes can therefore break and separate with the stroma exposed membrane. In addition, since the grana margins contain PS Iα (with 40% larger antennae than PS Iβ), which is functionally connected to LHC II, it is to be expected that some of these PS Iα units will also enter the stroma lamellae fraction and thus help contribute to a lower chlorophyll a/b ratio and a small increase in the average PS I antenna size of the stroma lamellae fraction from phosphorylated thylakoids. It is concluded that the incidence of partial destacking of the grana, which occurs due to the phosphorylation of LHC II and PS II polypeptides, may promote the exposure of the granal PS Iα centers to the aqueous stroma and increase cyclic electron flow around Photosystem I and thereby ATP production over NADPH production. (2) Subthylakoid vesicles, representing the different structural domains, were also examined for their properties following an incubation in presence of light and ATP. Phosphorylation of membrane proteins including LHC II and PS II associated polypeptides was observed in membrane fractions deriving from the grana lamellae and, to a lesser extent, the grana core. Three unidentified polypeptides of 15, 20 and 22 kDa were the most abundantly labeled polypeptides in the stroma lamellae fraction. No membrane proteins became phospho-labeled in the grana margin fraction.
The effect of photoactivation (the assembly of the Mn cluster involved in oxygen evolution) in Photosystem II (PS II), on the redox midpoint potential of the primary quinone electron acceptor, QA, has been investigated. Measurements of the redox state of QA were performed using chlorophyll fluorescence. Cells of Scenedesmus obliquus were grown in the dark to obtain PS II lacking the oxygen-evolving complex. Growth in the light leads to photoactivation. The midpoint potential of QA was shifted, upon photoactivation, from + 110 mV to −80 mV. In cells of a low-fluorescence mutant, LF1, that is unable to assemble the oxygen-evolving complex but that has an otherwise normal PS II, the higher potential form of QA was found. NH2OH treatment of spinach PS II, which releases the Mn and thus inactivates the oxygen-evolving complex, causes an upshift of the redox potential of QA (Krieger and Weis (1992) Photosynthetica 27, 89–98). Oxygen evolution can be reconstituted by incubation in the light in the presence of MnCl2 and CaCl2. Such photoactivation caused the midpoint potential of QA to be shifted back from around +55 mV to lower potentials (−80 mV), typical for active PS II. The above results indicate that the state of the donor side of PS II has a direct influence on the properties of the acceptor side. It is suggested that the change from the high- to the low-potential form of QA may represent a mechanism for protection of PS II during the assembly of the O2-evolving enzyme.
The activity of Photosystem (PS) IIβ was monitored for the first time in the absence of added herbicides in isolated chloroplast samples and in leaves of spinach in vivo. The new approach was implemented following identification of the initial chloroplast fluorescence rise from F0 to Fp1 (Forbush, B. and Kok, B. (1968) Biochim. Biophys. Acta 162, 243–253) as the variable fluorescence yield controlled by PS IIβ. Evidence is presented indicating the absence of the intermediate plastoquinone pool from the thylakoid membrane in which PS IIβ is localized. A two-step developmental process for PS II assembly and grana formation is proposed. According to this working hypothesis, PS IIβ is the precursor form of PS IIα, structurally and functionally complete except for the absence of the peripheral chlorophyll light-harvesting antenna and the complement of the plastoquinone pool. It is postulated that addition of these two hydrophobic components converts PS IIβ into PS IIα and initiates the incorporation of the thylakoid membrane into grana.
A rapid procedure to fractionate the thylakoid membrane into two well-separated vesicle populations, one originating from the grana and the other from the stroma-membrane region, has been developed. This was achieved by sonication of thylakoids present in an aqueous two-phase system followed by partitioning either by countercurrent distribution or by a batch procedure in three steps. The membrane populations were analysed according to their composition and photochemical activities. The grana membranes comprise, on chlorophyll basis, about 60% of the thylakoid material and are enriched in PS II, but also contain some PS I, while the stroma membranes comprise about 40% and are enriched in PS I, but also contain some PS II. Cytochrome f was slightly enriched in the grana-derived vesicle fraction. The properties of both PS I and PS II differ between the two populations. The PS I of the grana fraction (PS Iα) reached half-saturation at about half the light intensity of the PS I in the stroma-membrane fraction (PS Iβ). The rate of P-700 photooxidation under low light illumination was higher for PS Iα than for PS Iβ (30% larger rate constant), showing that PS Iα has a larger antenna. The PS II of the grana fraction (PS IIα) reached half-saturation at half the light intensity compared to the PS II of the stroma-membrane fraction (PS IIβ). The results show that the grana-derived membranes contain PS Iα and PS IIα which have larger functional antenna sizes than the corresponding PS Iβ and PS IIβ of the stroma membranes. The results suggest that the photosystems of the grana are designed to allow effective electron transport both at low and high light intensities, while the stroma-membrane photosystems mainly work at high light intensities as a supplement to the grana systems.
Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone QB- with the S2 state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S2QA- recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50 degreesC in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a TyrD+-QA- recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42 degreesC. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52 degreesC) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45 degreesC (C-band) slightly increased. It is proposed that the 42 to 52 degreesC band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII QB-nonreducing centers.
To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.
Eleven site-directed mutations were constructed at aspartate 170 of the D1 polypeptide of the photosystem II (PSII) reaction center of the cyanobacterium Synechocystis sp. PCC 6803. The light-saturated rates of O2 evolution (VO2) measured in whole cells range from close to that of wild-type for Asp170Glu to zero for Asp170Ser and Ala. Those mutant strains that are best able to evolve O2 are also those that show the lowest Km in PSII core complexes for the oxidation of Mn2+ by oxidized Tyr161, the normal oxidant of the Mn cluster responsible for O2 evolution. To a first approximation, the lower the pKa of the residue at position 170, the higher the VO2 and the lower the Km. D1-Asp170 appears to participate in the early steps associated with the assembly of the Mn cluster. It is also the first reported example of an amino acid residue critical to the function and assembly of the oxygen-evolving complex.
A model of the photosynthetic membrane from higher plants is presented. The different photosystems, PSI alpha, PSI beta, PSII alpha and PSII beta, are located in separate domains. The photosystems with the largest antenna systems, the alpha systems, are in the grana and the other in the stroma lamellae. In each grana disc PSI alpha is located in a flat annulus surrounding a circular PSII alpha domain. In this the PSII alpha units with the largest antennae are found in the center. The model is consistent with results from recent membrane fractionation experiments.
Upon illumination, a dark-adapted photosynthetic sample shows time-dependent changes in chlorophyll (Chl) a fluorescence yield, known as the Kautsky phenomenon or the OIDPS transient. Based on the differential effects of electron acceptors such as 2,5-dimethyl-p-benzoquinone (DMQ) and 2,6-dichloro-p-benzoquinone (DCBQ) on Chl a fluorescence transients of spinach thylakoids, we suggest that the OID phase reflects the reduction of the electron acceptor QA to QA- in the inactive PS II (see Graan, T. and Ort, D. (1986) Diochim. Biophys. Acta 852, 320-330). In spinach thylakoids, heat-induced increase of the Chl a fluorescence yield is also differentially sensitive to the addition of DMQ and DCBQ suggesting that this increase is mainly on the 'I' level, and thus heating is suggested to convert active PS II to inactive PS II centers. The kinetics of decay of QA-, calculated from variable Chl a fluorescence, was analyzed into three exponential components (365-395 microseconds; 6-7 ms; and 1.4-1.7 s). In heated samples, the decay rate of variable Chl a fluorescence is slower than the normal back-reaction rate; there is a preponderance of the slow component that may be due, partly, to the active centers undergoing slow back reaction between QA- and the S2 state of the oxygen-evolving complex.
In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R.J., Barry, B.A., Babcock, G.T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA- occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA- occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.
The possibility of reconstituting a functionally competent endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles (ISO-vesicles), and PS II core complexes was analyzed by measuring (i) the characteristic period four oscillation of the oxygen yield due to excitation of dark-adapted samples with a train of short flashes and (ii) laser flash-induced transients of the relative quantum yield of chlorophyll fluorescence. The data obtained revealed that (a) an endogenous pool capacity comparable to that of intact thylakoids can be restored in PS II membrane fragments and ISO-vesicles by a sonication treatment using native plastoquinone-9, (b) a more pronounced oxygen oscillation pattern arises in PS II core complexes after application of the same reconstitution procedure, (c) the extent of the endogenous pool restoration at a ratio of 15 quinone molecules per PS II in the reconstitution assay strongly depends on the nature of the quinone molecule [maximum effects can be only achieved with PQ-9, while at the same concentration ubiquinone-45 (UQ-9) is almost inefficient], and (d) a sonication step is required for stable insertion of PQ-9 into PS II preparations. Measurements of the reconstruction degree as a function of the structure of different quinones with selected properties lead to the conclusion that specific binding domains exist in PS II in addition to the QB site. These domains exhibit a surprisingly high specificity for the type of quinone that can be bound. On the basis of a comparison of the results obtained, the structure of the quinone head group seems to be more important than the large hydrophobic side chain and/or the general lipophilicity of the compound.
Even though light is the source of energy for photosynthesis, it can also be harmful to plants. Light-induced damage is targetted mainly to Photosystem II and leads to inactivation of electron transport and subsequent oxidative damage of the reaction centre, in particular to the D1 protein. Inactivation and protein damage can be induced by two different mechanisms, either from the acceptor side or from donor side of P680. The damaged D1 protein is triggered for degradation and digested by at least one serine-type proteinase that is tightly associated with the Photosystem II complex itself. The damaged Photosystem II complex dissociates from the light-harvesting antenna and migrates from appressed to non-appressed thylakoid regions where a new D1 protein is co-translationally inserted into the partially disassembled Photosystem II complex. D1 protein phosphorylation probably allows for coordinated biodegradation and biosynthesis of the D1 protein. After religation of cofactors and assembly of subunits, the repaired Photosystem II complex can again be found in the appressed membrane regions. Various protective mechanisms and an efficient repair cycle of Photosystem II allow plants to survive light stress.
The inhibition of DPC-mediated DCIP photoreduction by exogenous MnCl2 in Tris-treated photosystem II (PSII) membrane fragments has been used to probe for amino acids on the PSII reaction center proteins, including D1His337, that provide ligands for binding manganese [Preston, C., & Seibert, M. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. I, pp 925-928, Kluwer Academic Publishers, Dordrecht, The Netherlands; Preston, C., & Seibert, M. (1991) Biochemistry 30, 9615-9624 and 9625-9633]. At a concentration of 200 microM, DPC is photooxidized at both a high-affinity and a low-affinity site in PSII at approximately the same initial rate. Addition of 10 microM MnCl2 noncompetitively inhibits DPC photooxidation at the high-affinity site, with a Ki of 1.5 microM, causing a decrease of about 50% in the overall DCIP photoreduction rate. The high-affinity site for Mn binding was deconvoluted into four independent components. In earlier work, the inhibition was attributed to the tight association of either Mn2+ or Mn3+ with the PSII membrane. We report here that inhibition of DPC photooxidation may involve two different types of high-affinity, Mn-binding components: (a) one that is specific for Mn, and (b) others that bind Mn, but may also bind additional divalent cations, such as Zn and Co, that are not photooxidized by PSII. These conclusions are based on the observations that (a) DPC photooxidation can be inhibited by Zn2+ and Co2+; (b) Zn2+ and Co2+ interact with Mn2+ in a nonmutually exclusive manner, suggesting that they may share some binding components with Mn2+; (c) high-affinity Mn2+ (but not Zn2+ or Co2+) inhibition of DPC photooxidation is accompanied by nondecaying fluorescence emission, following a single saturating flash, indicating efficient electron donation by Mn2+ to YZ+; (d) Mn2+ photooxidation in the presence of DPC is not inhibited by Zn2+ or Co2+; and (e) kinetic modeling of the interaction between high-affinity Mn2+ and DPC in PSII indicates inhibition of steady-state Mn2+ photooxidation by DPC, but allows for a single photooxidation of Mn2+. We conclude that Mn inhibition of DPC photooxidation can be used to identify Mn-binding sites of physiological importance, and suggest that the Mn-specific component of the high-affinity, Mn-binding site involves the ligand to the first Mn bound during photoactivation (i.e., Asp170 on D1, as found by other investigators).
The reaction center protein D1 in photosystem II shows a high turnover during illumination. The degradation of the D1 protein is preceded by photoinhibition of the electron transport in photosystem II. There are two distinct mechanisms for this: acceptor-side- and donor-side-induced photoinhibition. Here, donor-side-induced photoinhibition was studied in photosystem II membranes after Cl- depletion or washing with tris(hydroxymethyl)aminomethane (Tris) which destroys water oxidation, reversibly or irreversibly, respectively. Photoinhibition after these treatments leads to fast degradation of the D1 protein, and the mechanism behind this was investigated. Illumination of Cl- depleted photosystem II membranes resulted in a rapid and simultaneous inhibition of Cl(-)-reconstitutable oxygen evolution, loss of 2 Mn ions per photosystem II center, increase in the electron transfer between the electron donor diphenylcarbazide and electron acceptor 2,6-dichlorophenolindophenol, and an increase in the EPR signal IIfast from tyrosine-Zox. The destruction of the Mn cluster leads to the loss of oxygen evolution and to an increased accessibility for diphenylcarbazide to donate electrons to Tyr-Zox. The increase in the EPR signal from Tyr-Zox can be explained by slower reduction kinetics of Tyr-Zox due to the Mn release. On a longer photoinhibition time scale, a decrease in the amplitude of Tyr-Zox and inhibition of the electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol occurred simultaneously in both Cl(-)-depleted and Tris-washed photosystem II membranes. These slower photoinhibition reactions were then studied in detail in Tris-washed photosystem II membranes. Compared to photoinhibition of Tyr-Zox, the EPR signal from tyrosine-Dox decreased much slower. Tyr-Dox was photoinhibited in parallel with the EPRsignals from reduced QA, reduced pheophytin, and an oxidized chlorophyll radical (chlorophyllz). This shows that the acceptor side components and the primary charge separation reaction (P680+ pheophytin-) were operational although Tyr-Z was inactivated. The amount of the D1 protein also declined in parallel with Tyr-Dox, which shows that the D1 protein is not damaged until long after the Mn complex and Tyr-Z have become inactivated. Instead, it is likely that the strongly oxidizing P680+ is responsible for the damage to the D1 protein.
The proton transfer reactions induced by the oxidation and reduction of the secondary donor, tyrosine YZ, have been studied in photosystem II after inactivation (Mn-depletion) of the oxygen-evolving complex. The rate of the recombination reaction of YZox with the reduced primary acceptor QA- appears modulated by a protonatable group with pK approximately 6 in the presence of YZox. The finding of monophasic recombination kinetics requires that the proton equilibration of this group is faster than the recombination rate. The same group modulates the extent of proton release, from 0 below pH 5 to 1 per center above pH 7. The kinetics of proton appearance and disappearance in the bulk medium are markedly dependent on the material used. In PSII core particles, the release is observed in the 100 micros range and the uptake accompanies the recombination reaction. In PSII membranes, both of these reactions are markedly delayed, so that the uptake considerably lags behind the completion of the recombination reaction. An electrochromic shift of a chlorophyll is present during the whole lifetime of YZox, suggesting a charged character of this species. A fast decreasing phase of this signal was observed in particles in the same time range as proton release. These results are discussed in the framework of a model where the proton originating from the formation of the neutral oxidized tyrosine radical (YZ.) remains locally trapped. In turn, this proton shifts the pK of a nearby group from a value >/=9 to a value of 6.
Photoactivation of photosystem II has been studied in the FUD 39 mutant of Chlamydomonas reinhardtii that lacks the 23 kDa extrinsic subunit of photosystem II. We have taken advantage of the slow photoactivation rate of FUD 39, earlier demonstrated in Rova, E. M., et al. [(1996) J. Biol. Chem. 271, 28918-28924], to study events in photosystem II during intermediate stages of the process. By measuring the EPR multiline signal, the decay of the variable fluorescence after single flashes, and electron transfer from water to the QB site, we found a good correlation between the building of a tetrameric Mn cluster, longer recombination times between QA- and the donor side of photosystem II, and the achievement of water splitting ability. An increased rate of electron transfer from QA- to the QB site on the acceptor side of photosystem II, mainly due to enhanced efficiency of binding of QB to its site, was found to precede the building of the Mn cluster. We also showed that TyrD was oxidized simultaneously with this increase in electron-transfer rate. Thus, it appears that photoactivation is sequential, with an increased rate of electron transfer on the acceptor side occurring together with the oxidation of TyrD in the first step, followed by the assembly of the Mn cluster. We suggest that a conformational change of photosystem II is induced early in the photoactivation process facilitating electron transfer from the primary donor to the acceptor side. As a consequence, TyrD, an auxiliary electron donor to P680+/TyrZ*, is oxidized. That this occurs before the Mn cluster is fully functional serves to protect photosystem II against donor side induced photodamage.
Flash-induced chlorophyll fluorescence kinetics from photosystem II in thylakoids from the dark-grown wild type and two site-directed mutants of the D1 protein His190 residue (D1-H190) in Chlamydomonas reinhardtii have been characterized. Induction of the chlorophyll fluorescence on the first flash, reflecting electron transport from YZ to P680(+), exhibited a strong pH dependence with a pK of 7.6 in the dark-grown wild type which lacks the Mn cluster. The chlorophyll fluorescence decay, measured in the presence of DCMU, which reflects recombination between QA- and YZox, was also pH-dependent with a similar pK of 7.5. These results indicate participation by the same base, which is suggested to be D1-H190, in oxidation and reduction of YZ in forward electron transfer and recombination pathways, respectively. This hypothesis was tested in the D1-H190 mutants. Induction of chlorophyll fluorescence in these H190 mutants has been observed to be inefficient due to slow electron transfer from YZ to P680(+) [Roffey, R. A., et al. (1994) Biochim. Biophys. Acta 1185, 257-270]. We show that this reaction is pH-dependent, with a pK of 8. 1, and at pH >/=9, the fluorescence induction is efficient in the H190 mutants, suggesting direct titration of YZ. The efficient oxidation of YZ ( approximately 70% at pH 9.0) at high pH was confirmed by kinetic EPR measurements. In contrast to the wild type, the H190 mutants show little or no observable fluorescence decay. Our data suggest that H190 is an essential component in the electron transfer reactions in photosystem II and acts as a proton acceptor upon YZ oxidation. In the H190 mutants, this reaction is inefficient and YZ oxidation only occurs at elevated pHs when YZ itself probably is deprotonated. We also propose that H190 is able to return a proton to YZox during electron recombination from QA- in a reaction which does not take place in the D1-H190 mutants.
Thylakoids isolated from tobacco were fragmented by sonication and the vesicles so obtained were separated by partitioning in aqueous polymer two-phase systems. By this procedure, grana vesicles were separated from stroma exposed membrane vesicles. The latter vesicles could be further fractionated by countercurrent distribution, with dextran-polyethylene glycol phase systems, and divided into two main populations, tentatively named 'stroma lamellae' and 'end membrane'. Both these vesicle preparations have high chlorophyll a/b ratio, high photosystem (PS) I and low PS II content, suggesting their origin from stroma exposed regions of the thylakoid. The two vesicle populations have been compared with respect to biochemical composition and photosynthetic activity. The 'end membrane' has a higher chlorophyll a/b ratio (5.7 vs. 4.7), higher P700 content (4.7 vs. 3.3 mmol/mol of chlorophyll). The 'end membrane' has the lowest PS II content, the ratio PS I/PS II being more than 10, as shown by EPR measurements. The PS II in both fractions is of the beta-type. The decay of fluorescence is different for the two populations, the 'stroma lamellae' showing a very slow decay even in the presence of K3Fe(CN)6 as an acceptor. The two vesicle populations have very different surface properties: the end membranes prefer the upper phase much more than the stroma lamellae, a fact which was utilized for their separation. Arguments are presented which support the suggestion that the two vesicle populations originate from the grana end membranes and the stroma lamellae, respectively.
- H H Robinsson
- A R Crofts
Robinsson, H. H., and Crofts, A. R. (1983) FEBS Lett. 153,
221-226.
- G H Krause
- E Weis
Krause, G. H., and Weis, E. (1991) Annu. ReV. Plant Physiol.
Plant Mol. Biol. 42, 313-349.