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

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

Quantitative correlation of peripheral and intrinsic core polypeptides of photosystem II with photosynthetic electron-transport activity ofAcetabularia mediterranea in red and blue light

The high photosynthetic activity (O₂ production and CO₂ consumption) ofAcetabularia mediterranea Lamour. (=A. acetabulum (L.) Silva) characteristic of cells cultured in white light decreases slowly when cells are kept in continuous red light, and is less than 20% of the original activity after three weeks. Subsequent blue irradiation restores the original activity completely within 3–5 d. The polypeptide composition of the thylakoids from cells grown in either red or blue light and after transfer from red to blue light was analyzed mainly with regards to photosystem II (PSII). The P700-containing reaction-centre complex of photosystem I, CPI, showed only minor quantitative alterations as a consequence of the growth-light quality, which correlated well with the activity of photosystem I under these conditions. In PSII, no drastic changes occurred in the quantity of the reaction-centre components D1 (herbicide-binding polypeptide) and D2, as determined by immunoblots. Likewise, the proteins associated with the water-splitting apparatus did not change detectably in thylakoids from red- or blue-light-treated cells (the 16-kDa component could not be found inAcetabularia thylakoids). The level of the major light-harvesting complex was completely unaffected by the light quality. In contrast, the quantities of the chlorophyll a-protein complexes of the core antenna, CP43 and CP47 (and probably CP29), changed, with kinetics similar to those of total photosynthetic activity. We postulate that the function of the PSII antenna became increasingly impaired in the absence of blue light (i.e. in red light), while blue light had a restoring effect. The peripheral antenna, comprising the light-harvesting complexes, is probably functionally connected with the reaction-centre chlorophylls via the core antenna chlorophyll-protein complexes (CP43, CP47 and probably CP29). A deficiency of these complexes would lead to uncoupling of antenna and reaction centre in the majority of PSII complexes after long periods of red-light treatment.     

Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii

Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.     

LPA2 is required for efficient assembly of photosystem II in Arabidopsis thaliana

To elucidate the molecular mechanism of photosystem II (PSII) assembly, we characterized the low psii accumulation2 (lpa2) mutant of Arabidopsis thaliana, which is defective in the accumulation of PSII supercomplexes. The levels and processing patterns of the RNAs encoding the PSII subunits are unaltered in the mutant. In vivo protein-labeling experiments showed that the synthesis of CP43 (for chlorophyll a binding protein) was greatly reduced, but CP47, D1, and D2 were synthesized at normal rates in the lpa2-1 mutant. The newly synthesized CP43 was rapidly degraded in lpa2-1, and the turnover rates of D1 and D2 were higher in lpa2-1 than in wild-type plants. The newly synthesized PSII proteins were assembled into PSII complexes, but the assembly of PSII was less efficient in the mutant than in wild-type plants. LPA2 encodes an intrinsic thylakoid membrane protein, which is not an integral subunit of PSII. Yeast two-hybrid assays indicated that LPA2 interacts with the PSII core protein CP43 but not with the PSII reaction center proteins D1 and D2. Moreover, direct interactions of LPA2 with Albino3 (Alb3), which is involved in thylakoid membrane biogenesis and cell division, were also detected. Thus, the results suggest that LPA2, which appears to form a complex with Alb3, is involved in assisting CP43 assembly within PSII.     

Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in Synechocystis PCC 6803

Accumulation of monomer and dimer photosystem (PS) II reaction center core complexes has been analyzed by two-dimensional Blue-native/SDS-PAGE in Synechocystis PCC 6803 wild type and in mutant strains lacking genes psbA, psbB, psbC, psbDIC/DII, or the psbEFLJ operon. In vivo pulse-chase radiolabeling experiments revealed that mutant cells assembled PSII precomplexes only. In DeltapsbC and DeltapsbB, assembly of reaction center cores lacking CP43 and reaction center complexes was detected, respectively. In DeltapsbA, protein subunits CP43, CP47, D2, and cytochrome b559 were synthesized, but proteins did not assemble. Similarly, in DeltapsbD/C lacking D2, and CP43, the de novo synthesized proteins D1, CP47, and cytochrome b559 did not form any mutual complexes, indicating that assembly of the reaction center complex is a prerequisite for assembly with core subunits CP47 and CP43. Finally, although CP43 and CP47 accumulated in DeltapsbEFLJ, D2 was neither expressed nor accumulated. We, furthermore, show that the amount of D2 is high in the strain lacking D1, whereas the amount of D1 is low in the strain lacking D2. We conclude that expression of the psbEFLJ operon is a prerequisite for D2 accumulation that is the key regulatory step for D1 accumulation and consecutive assembly of the PSII reaction center complex.     

The STN8 kinase‐PBCP phosphatase system is responsible for high‐light‐induced reversible phosphorylation of the PSII inner antenna subunit CP29 in rice

Reversible phosphorylation of thylakoid light‐harvesting proteins is a mechanism to compensate for unbalanced excitation of photosystem I (PSI) versus photosystem II (PSII) under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high‐light (HL) response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in HL, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of AtPBCP, antagonist of AtSTN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P‐CP29. Based on these data, we propose that the activities of the OsSTN8 kinase and the antagonistic OsPBCP phosphatase, in addition to being involved in the repair of photo‐damaged PSII, are also responsible for the HL‐dependent reversible phosphorylation of the inner antenna CP29.     

The structure, function and dynamics of photosystem two

One of the greatest challenges in modern photosynthesis research is to elucidate fully the structural and functional properties of photosystem two (PSII). This water-plasto-quinone oxidoreductase is located in a membrane complex composed of more than 25 subunits. The primary and secondary structures of all known subunits which constitute the central core of PSII are reviewed. How these subunits interact with each other to produce the tertiary and quaternary structure of PSII in vivo is not fully understood. However, electron microscopy is helping to fill this gap in our knowledge both by single particle analysis and electron crystallography. These studies suggest that active PSII is dimeric, although the functional significance of this oligomeric state is not yet understood. Moreover, the elucidation of the structure of photosystem one (PSI) by X-ray crystallography has revealed features which are likely to be relevant to PSII structure. It seems highly likely that the D1 protein with CP43 and D2 protein with CP47 (summing 11 transmembrane helices in each case) will have structural similarities to the organisation of PsaA and PsaB. It is likely that the turnover of the D1 protein is aided by the relatively easy removal of CP43 from this arrangement of the PSII core.     

Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants

In higher plants, the photosystem (PS) II core and its several light harvesting antenna (LHCII) proteins undergo reversible phosphorylation cycles according to the light intensity. High light intensity induces strong phosphorylation of the PSII core proteins and suppresses the phosphorylation level of the LHCII proteins. Decrease in light intensity, in turn, suppresses the phosphorylation of PSII core, but strongly induces the phosphorylation of LHCII. Reversible and differential phosphorylation of the PSII-LHCII proteins is dependent on the interplay between the STN7 and STN8 kinases, and the respective phosphatases. The STN7 kinase phosphorylates the LHCII proteins and to a lesser extent also the PSII core proteins D1, D2 and CP43. The STN8 kinase, on the contrary, is rather specific for the PSII core proteins. Mechanistically, the PSII-LHCII protein phosphorylation is required for optimal mobility of the PSII-LHCII protein complexes along the thylakoid membrane. Physiologically, the phosphorylation of LHCII is a prerequisite for sufficient excitation of PSI, enabling the excitation and redox balance between PSII and PSI under low irradiance, when excitation energy transfer from the LHCII antenna to the two photosystems is efficient and thermal dissipation of excitation energy (NPQ) is minimised. The importance of PSII core protein phosphorylation is manifested under highlight when the photodamage of PSII is rapid and phosphorylation is required to facilitate the migration of damaged PSII from grana stacks to stroma lamellae for repair. The importance of thylakoid protein phosphorylation is highlighted under fluctuating intensity of light where the STN7 kinase dependent balancing of electron transfer is a prerequisite for optimal growth and development of the plant. This article is part of a Special Issue entitled: Photosystem II.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana

To gain insight into the processes involved in photosystem II (PSII) biogenesis and maintenance, we characterized the low psii accumulation1 (lpa1) mutant of Arabidopsis thaliana, which generally accumulates lower than wild-type levels of the PSII complex. In vivo protein labeling experiments showed that synthesis of the D1 and D2 proteins was greatly reduced in the lpa1 mutant, while other plastid-encoded proteins were translated at rates similar to the wild type. In addition, turnover rates of the PSII core proteins CP47, CP43, D1, and D2 were higher in lpa1 than in wild-type plants. The newly synthesized PSII proteins were assembled into functional protein complexes, but the assembly was less efficient in the mutant. LPA1 encodes a chloroplast protein that contains two tetratricopeptide repeat domains and is an intrinsic membrane protein but not an integral subunit of PSII. Yeast two-hybrid studies revealed that LPA1 interacts with D1 but not with D2, cytochrome b6, or Alb3. Thus, LPA1 appears to be an integral membrane chaperone that is required for efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1.     

Mass spectrometric resolution of reversible protein phosphorylation in photosynthetic membranes of Arabidopsis thaliana

The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.     

Evidence that the PsbK polypeptide is associated with the photosystem II core antenna complex CP43

PsbK is encoded by the chloroplast psbK gene and is one of the small polypeptides of photosystem II (PSII). This polypeptide is required for accumulation of the PSII complex. In the present study, we generated an antibody against recombinant mature PsbK of Chlamydomonas and used it in Western blots to localize PsbK in the PSII core complex. PsbK was found in the thylakoid membranes, and purification of the PSII core complex from detergent-solubilized thylakoid membranes showed that PsbK is tightly associated with the PSII core complex. We used potassium thiocyanate to separate PSII into subcore complexes, including the D1/D2/cytochrome b559 reaction center complex, CP47, and CP43, and we found that PsbK co-purifies with one of the core antenna complexes, CP43, during ion exchange chromatography. Subsequent gel filtration chromatography of the purified CP43 confirmed that PsbK is tightly associated with CP43. Steady-state levels of PsbK were also determined in Chlamydomonas mutants expressing various levels of PSII. Quantitative Western blotting revealed that the levels of PsbK in these mutants are approximately equal to those of CP43, suggesting that PsbK is stable only when associated with CP43 in the chloroplast. Together, our results indicate that PsbK is an integral part of the PSII complex and may participate in the assembly and stability of the PSII complex.     

Role of the PsbI protein in photosystem II assembly and repair in the cyanobacterium Synechocystis sp. PCC 6803

The involvement of the PsbI protein in the assembly and repair of the photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. Analysis of PSII complexes in the wild-type strain showed that the PsbI protein was present in dimeric and monomeric core complexes, core complexes lacking CP43, and in reaction center complexes containing D1, D2, and cytochrome b-559. In addition, immunoprecipitation experiments and the use of a histidine-tagged derivative of PsbI have revealed the presence in the thylakoid membrane of assembly complexes containing PsbI and either the precursor or mature forms of D1. Analysis of PSII assembly in the psbI deletion mutant and in strains lacking PsbI together with other PSII subunits showed that PsbI was not required for formation of PSII reaction center complexes or core complexes, although levels of unassembled D1 were reduced in its absence. However, loss of PsbI led to a dramatic destabilization of CP43 binding within monomeric and dimeric PSII core complexes. Despite the close structural relationship between D1 and PsbI in the PSII complex, PsbI turned over much slower than D1, whereas high light-induced turnover of D1 was accelerated in the absence of PsbI. Overall, our results suggest that PsbI is an early assembly partner for D1 and that it plays a functional role in stabilizing the binding of CP43 in the PSII holoenzyme.     

Charge separation and energy transfer in the photosystem II core complex studied by femtosecond midinfrared spectroscopy

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.     

Dimeric and monomeric organization of photosystem II. Distribution of five distinct complexes in the different domains of the thylakoid membrane

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.     

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.     

PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.     

Phosphorylation of PSII proteins in maize thylakoids in the presence of Pb ions

Lead is potentially toxic to all organisms including plants. Many physiological studies suggest that plants have developed various mechanisms to contend with heavy metals, however the molecular mechanisms remain unclear. We studied maize plants in which lead was introduced into detached leaves through the transpiration stream. The photochemical efficiency of PSII, measured as an Fv/Fm ratio, in the maize leaves treated with Pb was only 10% lower than in control leaves. The PSII activity was not affected by Pb ions in mesophyll thylakoids, whereas in bundle sheath it was reduced. Protein phosphorylation in mesophyll and bundle sheath thylakoids was analyzed using mass spectrometry and protein blotting before and after lead treatment. Both methods clearly demonstrated increase in phosphorylation of the PSII proteins upon treatment with Pb²⁺, however, the extent of D1, D2 and CP43 phosphorylation in the mesophyll chloroplasts was clearly higher than in bundle sheath cells. We found that in the presence of Pb ions there was no detectable dephosphorylation of the strongly phosphorylated D1 and PsbH proteins of PSII complex in darkness or under far red light. These results suggest that Pb²⁺ stimulates phosphorylation of PSII core proteins, which can affect stability of the PSII complexes and the rate of D1 protein degradation. Increased phosphorylation of the PSII core proteins induced by Pb ions may be a crucial protection mechanism stabilizing optimal composition of the PSII complexes under metal stress conditions. Our results show that acclimation to Pb ions was achieved in both types of maize chloroplasts in the same way. However, these processes are obviously more complex because of different metabolic status in mesophyll and bundle sheath chloroplasts.     

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

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

Isolation and Characterization of Photosystem Ⅱ of Porphyra yezoensis Ueda

The thylakoid membranes were isolated and purified from gametophyte of Porphyra yezoensis Ueda (P. yezoensis) by sucrose density gradient ultracentrifugation. After P. yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem Ⅱ (PSⅡ) particles were isolated and purified. The activity of PSⅡ