The occurrence of a thylakoid-localized small zinc finger protein in land plants

Previous studies showed that LOW QUANTUM YIELD OF PHOTOSYSTEM II 1 (LQY1), a small thylakoid zinc finger protein was involved in maintenance and repair of Photosystem II (PSII). Here the author provide additional evidence for the role of LQY1 in PSII maintenance and repair and further commentary on the occurrence of LQY1 protein among land plants. After exposure to high light, Arabidopsis thaliana mutants lacking functional LQY1 gene (At1g75690) are more photoinhibited than wild-type control plants; display higher total non-photochemical quenching and photoinhibitory quenching. These results are consistent with the initial observation that lqy1 mutants have lower PSII efficiency than wild-type plants after high-light treatment. The low-PSII-efficiency phenotype can be suppressed upon complementation of lqy1 mutants with the LQY1 gene from wild-type plants. This further demonstrates that LQY1 is important in maintaining the activity of photosystem II in Arabidopsis. LQY1 homologs are present in land plants but are absent from sequenced genomes of green algae and cyanobacteria, which may reflect plant adaptation to excess light stress during the transition to land.     

The role of ΔpH-dependent dissipation of excitation energy in protecting photosystem II against light-induced damage in Arabidopsis thaliana

The Arabidopsis thaliana subunit PsbS of photosystem II (PSII) is essential for the non-photochemical quenching of chlorophyll fluorescence and thus for ΔpH-dependent energy dissipation (qE). As a result of the excision of an En-transposon, a frameshift mutation in the psbS gene was obtained, which results in the complete absence of the PsbS protein and of qE. Two-dimensional gel analyses of thylakoid membranes indicated that the depletion of PsbS has no effect on PSII composition, excluding a structural role for PsbS in the organization of the PSII antenna. The susceptibility of mutant plants to photoinactivation of PSII was significantly increased during exposure to high light for up to 8 h. Divergence of mutant plants from wild-type levels of photoinactivation were most pronounced during the first 2 h of illumination, while after longer exposure times the rate of PSII inactivation were similar in both genotypes. The increased PSII inactivation in the mutant was not accompanied by an increased rate of D1 protein degradation, and recovery of PSII activity in the mutant under low light was similar or even faster in comparison to wild-type plants. However, growth under high light intensities resulted in decreased growth rates of psbs mutant plants. We conclude that energy dissipation in PSII related to qE is not primarily required for the protection of PSII against light-induced destruction, but may rather be involved in reducing the electron pressure on the photosynthetic electron transport chain at saturating light intensities.     

Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.     

Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.     

Posttranslational events leading to the assembly of photosystem II protein complex: a study using photosynthesis mutants from Chlamydomonas reinhardtii

We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry. In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized.     

Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins

Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSII∆OEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSII∆OEE were modified, but when only OEC33 or PSII∆OEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, maximal efficiency of PSII photochemistry (F _v/F _m ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40°C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.     

Photosystem II Function and Dynamics in Three Widely Used Arabidopsis thaliana Accessions

Columbia-0 (Col-0), Wassilewskija-4 (Ws-4), and Landsberg erecta-0 (Ler-0) are used as background lines for many public Arabidopsis mutant collections, and for investigation in laboratory conditions of plant processes, including photosynthesis and response to high-intensity light (HL). The photosystem II (PSII) complex is sensitive to HL and requires repair to sustain its function. PSII repair is a multistep process controlled by numerous factors, including protein phosphorylation and thylakoid membrane stacking. Here we have characterized the function and dynamics of PSII complex under growth-light and HL conditions. Ws-4 displayed 30% more thylakoid lipids per chlorophyll and 40% less chlorophyll per carotenoid than Col-0 and Ler-0. There were no large differences in thylakoid stacking, photoprotection and relative levels of photosynthetic complexes among the three accessions. An increased efficiency of PSII closure was found in Ws-4 following illumination with saturation flashes or continuous light. Phosphorylation of the PSII D1/D2 proteins was reduced by 50% in Ws-4 as compared to Col-0 and Ler-0. An increase in abundance of the responsible STN8 kinase in response to HL treatment was found in all three accessions, but Ws-4 displayed 50% lower levels than Col-0 and Ler-0. Despite this, the HL treatment caused in Ws-4 the lagest extent of PSII inactivation, disassembly, D1 protein degradation, and the largest decrease in the size of stacked thylakoids. The dilution of chlorophyll-protein complexes with additional lipids and carotenoids in Ws-4 may represent a mechanism to facilitate lateral protein traffic in the membrane, thus compensating for the lack of a full complement of STN8 kinase. Nevertheless, additional PSII damage occurs in Ws-4, which exceeds the D1 protein synthesis capacity, thus leading to enhanced photoinhibition. Our findings are valuable for selection of appropriate background line for PSII characterization in Arabidopsis mutants, and also provide the first insights into natural variation of PSII protein phosphorylation.     

Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

Membrane lipid unsaturation modulates processing of the photosystem II reaction-center protein D1 at low temperatures.

The role of membrane lipid unsaturation in the restoration of photosystem II (PSII) function and in the synthesis of the D1 protein at different temperatures after photoinhibition was studied in wild-type cells and a mutant of Synechocystis sp. PCC 6803 with genetically inactivated desaturase genes. We show that posttranslational carboxyl-terminal processing of the precursor form of the D1 protein is an extremely sensitive reaction in the PSII repair cycle and is readily affected by low temperatures. Furthermore, the threshold temperature at which perturbations in D1-protein processing start to emerge is specifically dependent on the extent of thylakoid membrane lipid unsaturation, as indicated by comparison of wild-type cells with the mutant defective in desaturation of 18:1 fatty acids of thylakoid membranes. When the temperature was decreased from 33 degrees C (growth temperature) to 18 degrees C, the inability of the fatty acid mutant to recover from photoinhibition was accompanied by a failure to process the newly synthesized D1 protein, which accumulated in considerable amounts as an unprocessed precursor D1 protein. Precursor D1 integrated into PSII monomer and dimer complexes even at low temperatures, but no activation of oxygen evolution occurred in these complexes in mutant cells defective in fatty acid unsaturation.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

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

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

Knockdown of the PsbP protein does not prevent assembly of the dimeric PSII core complex but impairs accumulation of photosystem II supercomplexes in tobacco

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically found in land plants and green algae. Using PsbP-RNAi tobacco, we have investigated effects of PsbP knockdown on protein supercomplex organization within the thylakoid membranes and photosynthetic properties of PSII. In PsbP-RNAi leaves, PSII dimers binding the extrinsic PsbO protein could be formed, while the light-harvesting complex II (LHCII)-PSII supercomplexes were severely decreased. Furthermore, LHCII and major PSII subunits were significantly dephosphorylated. Electron microscopic analysis showed that thylakoid grana stacking in PsbP-RNAi chloroplast was largely disordered and appeared similar to the stromally-exposed or marginal regions of wild-type thylakoids. Knockdown of PsbP modified both the donor and acceptor sides of PSII; In addition to the lower water-splitting activity, the primary quinone Q_A in PSII was significantly reduced even when the photosystem I reaction center (P700) was noticeably oxidized, and thermoluminescence studies suggested the stabilization of the charged pair, S₂/Q_A⁻. These data indicate that assembly and/or maintenance of the functional MnCa cluster is perturbed in absence of PsbP, which impairs accumulation of final active forms of PSII supercomplexes.     

Photoinhibition of photosynthesis without net loss of photosystem II components inPopulus deltoides

The photoinhibition of photosynthesis was investigated on intact attached leaves and isolated thylakoid membranes of Populus deltoides.Our studies demonstrate that in intact leaves photoinhibition takes place under high irradiance which is more pronounced at higher temperatures. No net loss of Dl and other proteins associated with photosystem II (PSII) were observed even after 64 % photoinhibition suggesting that the degradation of polypeptides associated with PSII is not the only key step responsible for photoinhibition as observed by other workers. Electron transport studies in isolated thylakoid membranes suggested water oxidation complex as one of the damaged site during high light exposure. The possible mechanisms of photoinhibition without net loss of D1 protein are discussed.     

水稻永绿色基因突变和超表达对其叶片类囊体蛋白降解的影响

应用蛋白质免疫杂交技术分析了永绿色基因(Stay-green Rice,SGR)突变和超表达对水稻(Oryza sativa)叶片类囊体蛋白质降解的影响.结果表明,在正常生长条件下,SGR超表达降低了光系统Ⅱ(PSⅡ)、光系统Ⅰ(PS Ⅰ)和电子传递链等的蛋白质含量.暗诱导衰老处理时,SGR突变延缓了PSⅠ和PSⅡ的蛋...     

Functional analysis of the PsbP-like protein (sll1418) in Synechocystis sp. PCC 6803

A recent proteomic analysis of the thylakoid lumen of Arabidopsis thaliana revealed the presence of several PsbP-like proteins, and a homologue to this gene family was detected in the genome of the cyanobacterium Synechocystis sp. PCC 6803 (Schubert M, Petersson UA, Haas BJ, Funk C, Schröder WP, Kieselbach T (2002) J Biol Chem 277, 8354–8365). Using a peptide-directed antibody against this cyanobacterial PsbP-like protein (sll1418) we could show that it was localized in the thylakoid membrane and associated with Photosystem II. While salt washes did not remove the PsbP-like protein from the thylakoid membrane, it was partially lost during the detergent-based isolation of PSII membrane fractions. In total cell extracts this protein is present in the same amount as the extrinsic PsbO protein. We did not see any significant functional difference between the wild-type and a PsbP-like insertion mutant.     

Photoinhibition and recovery in a herbicide-resistant mutant from<Emphasis Type="Italic"> Glycine max</Emphasis> (L.) Merr. cell cultures deficient in fatty acid unsaturation

Photoinhibition and recovery were studied in two photosynthetic cell suspensions from soybean (Glycine max L. Merr): the wild type (WT) and the herbicide-resistant D1 mutant STR7. This mutant also showed an increase in saturated fatty acids from thylakoid lipids. STR7 was more sensitive to photoinhibition under culture conditions. In vivo photoinhibition experiments in the presence of chloramphenicol, in vitro studies in isolated thylakoid membranes, and immunoblot analysis indicated that the process of light-induced degradation of the D1 protein was not involved in the response of STR7 to light. At growth temperature (24°C), the recovery rate of photoinhibited photosystem II (PSII) was slower in STR7 relative to WT. Photoinhibition and recovery were differentially affected by temperature in both cell lines. The rates of photoinhibition were faster in STR7 at any temperature below 27°C. The rates of PSII recovery from STR7 were more severely affected than those of WT at temperatures lower than 24°C. The photoinhibition and recovery rates of WT at 17°C mimicked those of STR7 at 24°C. In organelle translation studies indicated that synthesis and elongation of D1 were substantially similar in both cell lines. However, sucrose gradient fractionation of chloroplast membranes demonstrated that D1 and also other PSII proteins such as D2, OEE33, and LCHII had a reduced capability to incorporate into PSII to yield a mature assembled complex in STR7. This effect may become the rate-limiting step during the recovery of photoinhibited PSII and may explain the increased sensitivity to high light found in STR7. Our data may hint at a possible role of fatty acids from membrane lipids in the assembly and dynamics of PSII.Abbreviations DCBQ 2,6-Dichloro p-benzoquinone - DM Dodecyl--d-maltoside - DTT Dithiothreitol - HL High light - LHCII Light-harvesting complex II - LL Low light - OEE Oxygen-evolving extrinsic proteins - PAGE Polyacrylamide gel electrophoresis - PG Phosphatidylglycerol - PSII Photosystem II - Q_A and Q_B Secondary quinone electron acceptors from PSII - RC Reaction center - SDS Sodium dodecyl sulfate - WT Wild type