The PsbP-like protein (sll1418) of Synechocystis sp. PCC 6803 stabilises the donor side of Photosystem II

The PsbP-like protein of the cyanobacterium Synechocystis sp. PCC 6803 is a peripheral component of Photosystem II, located at the lumenal side of the thylakoid membrane. Removal of this protein leads to decreased competitive potential of a PsbP-like deletion mutant when grown in a mixture with wild-type cells. Flash-induced oxygen evolution traces of the mutant show a higher probability of misses, correlated with increased amplitudes of the S-states decay in the dark. Thermoluminescence emission traces demonstrate a changed charge recombination pattern in the mutant, the S(3)Q(B)(-) couple becoming the major species instead of the S(2)Q(B)(-). Our data suggest a possible role of the PsbP-like protein in stabilisation of the charge separation in Photosystem II of cyanobacteria through interaction with the Mn cluster.     

Cytochrome f: Structure,function and biosynthesis

Cytochrome f is an intrinsic membrane component of the cytochrome bf complex, transferring electrons from the Rieske FeS protein to plastocyanin in the thylakoid lumen. The protein is held in the thylakoid membrane by a single transmembrane span located near its C-terminus with a globular hydrophilic domain extending into the lumen. The globular domain of the turnip protein has recently been crystallised, offering the prospect of a detailed three-dimensional structure. Reaction with plastocyanin involves localised positive charges on cytochrome f interacting with the acidic patch on plastocyanin and electron transfer via the surface-exposed tyrosine residue (Tyr83) of plastocyanin. Apocytochrome f is encoded in the chloroplast genome and is synthesised with an N-terminal presequence which targets the protein to the thylakoid membrane. The synthesis of cytochrome f is coordinated with the synthesis of the other subunits of the cytochrome bf complex.     

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.     

Chloroplasts ofArabidopsis thaliana homozygous for the ch-1 locus lack chlorophyllb,lack stable LHCPII and have stacked thylakoids

We are interested in the mechanism of insertion of proteins into the chloroplast thylakoid membrane and the role that accessory pigments may play in this process. For this reason we have begun a molecular analysis of mutant plants deficient in pigments that associate with thylakoid membrane proteins. We have characterized plants that are homozygous for the previously isolated, recessive mutation chlorina-1 (ch-1) or Arabidopsis thaliana. Despite the lack of chlorophyll b and light-harvesting proteins of photosystem II (LHCPII) near normal levels of LHCPII mRNA are found in the mutant, in contrast to LHCPII mRNA levels in carotenoid-deficient mutants. The LHCPII mRNA of chlorina-1 plants can be translated in vitro so it is likely that LHCPII is not stable in ch-1 plants. Moreover, the thylakoid membranes of ch-1 plants remain appressed even though LHCPII levels are drastically reduced.     

Effect of Irradiance on the Thermal Stability of Thylakoid Membrane Isolated from Acclimated Wheat Leaves

Thermal stability of thylakoid membranes isolated from acclimated and non-acclimated wheat (Triticum aestivum L. cv. HD 2329) leaves under irradiation was studied. Damage to the photosynthetic electron transport activity was more pronounced in thylakoid membranes isolated from non-acclimated leaves as compared to thylakoid membrane isolated from acclimated wheat leaves at 35 °C. The loss of D1 protein was faster in non-acclimated thylakoid membrane as compared to acclimated thylakoid membranes at 35 °C. However, the effect of elevated temperature on the 33 kDa protein associated with oxygen evolving complex in these two types of thylakoid membranes was minimal. Trypsin digestion of the 33 kDa protein in the thylakoid membranes isolated from control and acclimated seedlings suggested that re-organisation of 33 kDa protein occurs before its release during high temperature treatment.     

An Arabidopsis thaliana protein homologous to cyanobacterial high-light-inducible proteins

An Arabidopsis thaliana cDNA clone encoding a novel 110 amino acid thylakoid protein has been sequenced. The in vitro synthesized protein is taken up by intact chloroplasts, inserted into the thylakoid membrane and the transit peptide is cleaved off during this process. The mature protein is predicted to contain 69 amino acids, to form one membrane-spanning -helix and to have its N-terminus at the stromal side of the thylakoid membrane. The protein showed similarity to the LHC, ELIP and PsbS proteins of higher plants, but more pronounced to the high-light-inducible proteins (HLIPs) of cyanobacteria and red algae, to which no homologue previously has been detected in higher plants. As for HLIP and ELIP, high light increases the mRNA levels of the corresponding gene. Sequence comparisons indicate that the protein may bind chlorophyll and form dimers in the thylakoid membrane. The level of expression of the protein seems to be far lower than that of normal PSI and PSII subunits.     

Studies on the expression of NDH-H,a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher-plant chloroplasts

The plastid genomes of higher plants contain eleven reading frames (ndhA-K) that are homologous to genes encoding subunits of the mitochondrial NADH-ubiquinone-oxidoreductase (complex I). The carboxyterminal end of the NDH-H subunit from rice (Oryza sativa L.) was expressed as a fusion protein in Escherichia coli and antibodies against the fusion protein were generated in rabbits. The antibody was used to study the expression of NDH-H, and the following results were obtained: (i) NDH-H is expressed in mono- and dicotyledonous plants, (ii) NDH-H is localized on the stroma lamellae of the thylakoid membrane and (iii) NDH-H is expressed in etioplasts. Together with the finding that two other ndh genes (ndhI and ndhK) are expressed in plastids, these results point to the existence of an NAD(P)H-plastoquinone-oxidoreductase on the thylakoid membrane. The possible function of the enzyme in plastids is discussed and it is suggested that it works in balancing the ATP/ADP and the NADPH/NADP ratios during changing external (i.e. light) or internal (i.e. ATP and NADPH demands of biosynthetic pathways of the plastid) conditions.Abbreviations PVDF polyvinylidene difluoride - SDS sodium dodecyl sulfate We thank Professor M. Sugiura for the rice plastid DNA clone bank, Oliver Buchholz for Sorghum plastid membranes, Pioneer Hi-Bred Inc. for maize and Sorghum seeds and the Deutsche Forschungsgemeinschaft for financial support (SFB189).     

叶绿体类囊体腔蛋白功能研究进展

【目的】类囊体是叶绿体光合作用中光反应进行的重要场所。类囊体腔是由类囊体膜包围形成的一个狭小空间。在类囊体腔中存在多种不同的蛋白家族,包括高叶绿素荧光(high chlorophyll fluorescence, HCF)蛋白、亲免蛋白、放氧复合物(oxygen-evolving complex, OEC)蛋白、PsbP类蛋白等,它们对植物的光合作用、核酸代谢以及氧化还原反应等都起着重要作用。【评论】文章分类综述了参与光合作用调控的类囊体腔蛋白在光系统组装、植物生长发育调节和高光逆境响应等生理活动中发挥的重要作用。【展望】文章可为未来研究类囊体腔蛋白的生理功能提供理论参考。     

Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus

Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635–2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion. Received: 28 July 1997 / Accepted: 26 November 1997     

Physcomitrella patens as a model for the study of chloroplast protein transport: conserved machineries between vascular and non-vascular plants

A single general import pathway in vascular plants mediates the transport of precursor proteins across the two membranes of the chloroplast envelope, and at least four pathways are responsible for thylakoid protein targeting. While the transport systems in the thylakoid are related to bacterial secretion systems, the envelope machinery is thought to have arisen with the endosymbiotic event and to be derived, at least in part, from proteins present in the original endosymbiont. Recently the moss Physcomitrella patens has gained worldwide attention for its ability to undergo homologous recombination in the nuclear genome at rates unseen in any other land plants. Because of this, we were interested to know whether it would be a useful model system for studying chloroplast protein transport. We searched the large database of P. patens expressed sequence tags for chloroplast transport components and found many putative homologues. We obtained full-length sequences for homologues of three Toc components from moss. To our knowledge, this is the first sequence information for these proteins from non-vascular plants. In addition to identifying components of the transport machinery from moss, we isolated plastids and tested their activity in protein import assays. Our data indicate that moss and pea (Pisum sativum) plastid transport systems are functionally similar. These findings identify P. patens as a potentially useful tool for combining genetic and biochemical approaches for the study of chloroplast protein targeting. Abbreviations: EST, expressed sequence tag; LHCP, light-harvesting chlorophyll-binding protein; NIBB, National Institute for Basic Biology; OE17, 17 kDa subunit of the oxygen-evolving complex; PC, plastocyanin; PEP, Physcomitrella EST Programme; SPP, stromal processing peptidase; SRP, signal recognition particle; Tat, twin-arginine translocation; Tic, translocon at the inner membrane of the chloroplast envelope; Toc, translocon at the outer membrane of the chloroplast envelope; TPP, thylakoid processing peptidase; TPR, tetratricopeptide repeatSupplementary material to this paper is available in electronic form at .This revised version was opublished online in July 2005 with corrected page numbers.     

Effect of carbon dioxide on pigment and membrane content in Synechococcus lividus

The effect of carbon dioxide on pigment and membrane content in Synechococcus lividus was studied by depriving cells of CO₂ and examining cell populations biochemically and by electron microscopy. After 120 h of CO₂ deprivation, S. lividus lost all detectable chlorophyll a and C-phycocyanin. Such bleached cultures were mustard yellow, the result of approximately 1.8 times more carotenoid per cell than green control cultures.Although cells from beached cultures appeared morphologically identical to control green cells when examined by light microscopy, electron microscopic examination revealed them to be devoid of detectable thylakoid membrane. Thylakoid membrane could not be recovered by physical isolation or revealed by freeze etching of bleached S. lividus. In addition, inclusion bodies characteristically found in S. lividus were also absent.Reintroduction of CO₂ into bleached cultures resulted in a rapid resynthesis of both chlorophyll a and C-phycocyanin. Electron microscopic examination of these regreening cultures revealed that thylakoid membrane was also rapidly resynthesized. Growth of regreened cultures did not occur until there was the synthesis of a full complement of chlorophyll a, C-phycocyanin, and thylakoid membrane.A time course study of the cytological events occurring during bleaching and regreening is presented.     

Immunogold localization of acyl carrier protein in plants and Escherichia coli: Evidence for membrane association in plants

Immunogold labelling was used to study the distribution of acyl carrier protein (ACP) in Escherichia coli and a variety of plant tissues. In E. coli, ACP is distributed throughout the cytoplasm, confirming the observation of S. Jackowski et al. (1985, J. Bacteriol., 162, 5–8_. In the mesocarp of Avocado (Persea americana) and maturing seeds of oil-seed rape (Brassica napus cv. Jet Neuf), over 95% of the ACP is localised to plastids. The protein is almost exclusively located in the chloroplasts of leaf material from oil-seed rape. Approximately 80% of the gold particles associated with the ACP were further localized to the thylakoid membrane of the chloroplast. Since acetyl-CoA carboxylase has been reported to be localized to the thylakoid membrane (C.G. Kannangara and C.J. Jensen, 1975, Eur. J. Biochem., 54, 25–30), these results are consistent with the view that the two sequential enzymes in fatty-acid synthesis are in close spacial proximity.Abbreviations ACC acetyl CoA carboxylase - ACP acyl carrier protein - FAS fatty-acid synthetase     

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25°C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.     

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem Q_B protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants. Xiaoping Jia and Jincheng Yuan have equal contribution.     

Impact of UV-B Radiation on Thylakoid Membrane and Fatty Acid Profile of <Emphasis Type="Italic">Spirulina platensis</Emphasis>

Ultraviolet-B (UV-B) radiation to thylakoid membrane and fatty acid profile has been investigated in cyanobacterium, Spirulina platensis. The thylakoid membrane was isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells followed by differential density gradient centrifugation and morphological variations were examined. UV radiation distorted the membrane on the outer side with reduced chlorophyll a (chl a) content compared to its untreated counterpart. Liquid chromatography-mass spectrometry (LC-MS) was used for characterization of chl a of the thylakoid membrane. UV-B exposure resulted in alterations in the pigment-protein complexes 47 kDa and 43 kDa. Furthermore, 94 kDa and 20 kDa protein appeared in UV-B-exposed thylakoid membrane of S. platensis. The composition of fatty acid in response to UV-B radiation was detected by gas chromatography–mass spectrometry having 23.5% saturated fatty acid (SFA), 76.4% monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA). In contrast to its UV-B-untreated counterpart, SFA was 46.6%, and MUFA and PUFA were 53.3%. Our findings suggest that UV-B radiation not only affects membrane morphology and its protein profile but also reduces saturated fatty acid and increases unsaturated fatty acids in S. platensis.     

Molecular analysis of the Chlamydomonas nuclear gene encoding PsbW and demonstration that PsbW is a subunit of photosystem II,but not photosystem I

PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Oligonucleotide-directed mutagenesis of psbB,the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis sp. PCC 6803

A mutant strain of the cyanobacterium Synechocystis sp. PCC (Pasteur Culture Collection) 6803 has been developed in which psbB, the gene coding for the chlorophyl a-binding protein CP47 in Photosystem II (PSII), has been deleted. This deletion mutant can be used for the reintroduction of modified psbB into the cyanobacterium. To study the role of a large hydrophilic region in CP47, presumably located on the lumenal side of the thylakoid membrane between the fifth and sixth membrane-spanning regions, specific deletions have been introduced in psbB coding for regions within this domain. One psbB mutation leads to deletion of Gly-351 to Thr-365 in CP47, another psbB mutation was targeted towards deletion of Arg-384 to Val-392 in this protein. The deletion from Gly-351 to Thr-365 results in a loss of PSII activity and of photoautotrophic growth of the mutant, but the deletion between Arg-384 and Val-392 retains PSII activity and the ability to grow photoautotrophically. The mutant strain with the deletion from Gly-351 to Thr-365 does not assemble a stable PSII reaction center complex in its thylakoid membranes, and exhibits diminished levels of CP47 and of the reaction center proteins D1 and D2. In contrast to the Arg-384 to Val-392 portion of this domain, the region between Gly-351 and Thr-365 appears essential for the normal structure and function of photosystem II.     

FREEZE-FRACTURED CHLOROPLAST MEMBRANES OF GONYAULAX POLYEDRA (PYRROPHYTA)1

The chloroplast membranes of Gonyaulax polyedra Stein were studied in replicas of rapidly frozen and fractured cells. The thylakoid EF_s face lacked the large 15–16 nm particles characteristic of plants with the light-harvesting chlorophyll a/b protein, presumably because the principal light-harvesting protein of Gonyaulax is the small water-soluble peridinin-chlorophyll-protein and the chlorophyll a/b protein is absent. As in other plants, the EF_s thylakoid fracture face carried more particles (4 ×) than EF_uface. The PF faces of the thylakoid showed twice as many particles as did the EF_s faces. No circadian differences in the number or size of thylakoid membrane particles could be detected. Three membranes comprise the chloroplast envelope in Gonyaulax. They could be clearly differentiated in freeze-fractured cells. The middle envelope membrane carried many fewer particles on both the EF and PF faces than did the other two envelope membranes. The PF faces of both the outer and inner envelope membranes showed more particles than the EF faces, as do many other membranes which have been examined.     

Multiple fates of non‐mature lumenal proteins in thylakoids

Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N–terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid‐transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling‐lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1‐null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid‐transfer signal removal on protein targeting and accumulation by examining the localization of non‐mature lumenal proteins in the Arabidopsis plsp1‐null mutant and performing a protein import assay using pea chloroplasts. In plsp1‐null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non‐mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid‐transfer signal removal did not disrupt cpSEC‐ and cpTAT‐dependent translocation, but prevented release of proteins from the membrane. Interestingly, non‐mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440‐kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1‐null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.