Spatial relationship of photosystem I, photosystem II, and the light- harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent- light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment- protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

Hydrogenase of purple bacteria: properties and regulation of synthesis

Physico-chemical properties of homogeneous preparations of soluble and membrane-bound hydrogenases from the purple sulfur bacterium Thiocapsa roseopersicina BBS and membrane-bound hydrogenase of Rhodopseudomonas capsulata, strain B10 have been studied. Compared to the enzymes from other sources, the hydrogenase of Thiocapsa roseopersicina is more stable to O₂ and products of its reduction (O ₂ ^- , H₂O₂), temperature and a number of other factors of the medium. A natural electron donor for T. roseopersicina hydrogenase is a low-potential cytochrome C₃, while the natural electron acceptors for hydrogenases of R. capsulata, T. roseopersicina, Ectothiorhodospira shaposhnikovii and Anabaena cylindrica are cytochromes of groups c and b.In different phototrophs, synthesis of hydrogenase can be inhibited by the presence of high concentrations of O₂. In some microorganisms (e.g. Rhodopseudomonas capsulata, strain B10) the repressing effect on hydrogenase formation is also exhibited by organic compounds. H₂ may not necessarily be present for hydrogenase synthesis by purple bacteria, but its presence may considerable increase the level of the enzyme.Abbreviations SDS sodium dodecylsulfate - Hipip high-potential iron — sulfur protein - R Rhodopseudomonas - T Thiocapsa - Rh Rhodospirillum - C Chromatium This paper is dedicated to Professor Dr. H.G. Schlegel in honour of his sixtieth birthday and in recognition of his great contribution in the field of physiology and biochemistry of microorganisms capable of using H₂. Professor H.G. Schlegel had a profound and most fuitful influence on the progress in the research of the laboratory headed by the author     

Biogenesis,assembly and turnover of photosystem II units

Assembly of photosystem II, a multiprotein complex embedded in the thylakoid membrane, requires stoichiometric production of over 20 protein subunits. Since part of the protein subunits are encoded in the chloroplast genome and part in the nucleus, a signalling network operates between the two genetic compartments in order to prevent wasteful production of proteins. Coordinated synthesis of proteins also takes place among the chloroplast-encoded subunits, thus establishing a hierarchy in the protein components that allows a stepwise building of the complex. In addition to this dependence on assembly partners, other factors such as the developmental stage of the plastid and various photosynthesis-related parameters exert a strict control on the accumulation, membrane targeting and assembly of the PSII subunits. Here, we briefly review recent results on this field obtained with three major approaches: biogenesis of photosystem II during the development of chloroplasts from etioplasts, use of photosystem II-specific mutants and photosystem II turnover during its repair cycle.     

Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition,have unstable photosystem I complexes,and altered redox homeostasis in the chloroplast stroma

The PSI-D subunit of photosystem I is a hydrophilic subunit of about 18 kDa, which is exposed to the stroma and has an important function in the docking of ferredoxin to photosystem I. We have used an antisense approach to obtain Arabidopsis thaliana plants with only 5-60% of PSI-D. No plants were recovered completely lacking PSI-D, suggesting that PSI-D is essential for a functional PSI in plants. Plants with reduced amounts of PSI-D showed a similar decrease in all other subunits of PSI including the light harvesting complex, suggesting that in the absence of PSI-D, PSI cannot be properly assembled and becomes degraded. Plants with reduced amounts of PSI-D became light-stressed even in low light although they exhibited high non-photochemical quenching (NPQ). The high NPQ was generated by upregulating the level of violaxanthin de-epoxidase and PsbS, which are both essential components of NPQ. Interestingly, the lack of PSI-D affected the redox state of thioredoxin. During the normal light cycle thioredoxin became increasingly oxidized, which was observed as decreasing malate dehydrogenase activity over a 4-h light period. This result shows that photosynthesis was close to normal the first 15 min, but after 2-4 h photoinhibition dominated as the stroma progressively became less reduced. The change in the thiol disulfide redox state might be fatal for the PSI-D-less plants, because reduction of thioredoxin is one of the main switches for the initiation of CO2 assimilation and photoprotection upon light exposure.     

Relationship between photoinhibition of photosynthesis, D1 protein turnover and chloroplast structure: effects of protein synthesis inhibitors

Irradiation of Spinach oleracea intact leaf tissue and of mesophyll protoplasts of Valerianella locusta at 20° C with strong light resulted in severe (40–80%) inhibition of photosynthesis, measured as photosystem II electron transport activity in isolated thylakoids or as fluorescence parameter F_V/F_M on intact leaf disks. No net degradation of the D1 protein of photosystem II was seen under these conditions. However, in the presence of streptomycin, an inhibitor of chloroplast protein synthesis, net D1 degradation (up to about 80%) did occur with a half-time of 4–6h, and photoinhibition was enhanced. Thylakoid ultrastructure remained stable during photoinhibition, even when substantial degradation of D1 took place in the presence of streptomycin. When leaf disks were irradiated at 2°C, streptomycin did not influence the degree of photoinhibition, and net Dl degradation did not occur. These results suggest that in excess (photoinhibitory) light at 20°C, turnover (coordinated degradation and synthesis) of D1 diminished the degree of photoinhibition. The observed photoinhibition is thought to be due to the accumulation of inactive photosystem II reaction centres still containing D1. In the presence of streptomycin, the Dl protein was degraded (probably in the previously inactivated centres), but restoration of active centres via D1 synthesis was blocked, leading to more severe photoinhibition. Low temperature (2°C), by restricting both degradation and resynthesis of D1, favoured the accumulation of inactive centres. Streptomycin and chloramphenicol (another inhibitor of chloroplast protein synthesis) were tested for side-effects on photosynthesis. Strong inhibitory effects of chloramphenicol, but much less severe effects of streptomycin were observed.     

Structural and functional properties of the cyanobacterial photosystem I complex

Photosystem I (PSI) complexes have been isolated from two cyanobacterial strains, Synechococcus sp. PCC 7002 and 6301. These complexes contain six to seven low molecular mass subunits in addition to the two high molecular mass subunits previously shown to bind the primary reaction center components. Chemical cross-linking of ferredoxin to the complex identified a 17.5-kDa subunit as the ferredoxin-binding protein in the Synechococcus sp. PCC 6301-PSI complex. The amino acid sequence of this subunit, deduced from the DNA sequence of the gene, confirmed its identity as the psaD gene product. A 17-kDa subunit cross-links to the electron donor, cytochrome c-553, in a manner analogous to the cross-linking of plastocyanin to the higher plant PSI complex. Using antibodies raised against the spinach psaC gene product (a 9-kDa subunit which binds Fe-S centers A and B), we identified an analogous protein in the cyanobacterial PSI complex.     

A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells

We have previously shown that when cultured fibroblasts are briefly incubated at 2 degrees C with a fluorescent (NBD) analogue of ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]-D-e rythro- sphingosine, fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occurs. During further incubation at 37 degrees C, the Golgi apparatus and later the plasma membrane become intensely fluorescent. Concomitantly, the fluorescent ceramide is metabolized to fluorescent analogues of sphingomyelin and glucosylceramide [Lipsky, N. G., & Pagano, R. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2608-2612]. In the present study we synthesized fluorescent N-acylsphingosine analogues using various long-chain bases (D-erythro-sphingosine, L-erythro-sphingosine, D-threo-sphingosine, L-threo-sphingosine, D-erythro-dihydrosphingosine, L-threo-dihydrosphingosine, phytosphingosine, and 3-ketosphingosine) and fluorescent fatty acids (epsilon-NBD-aminohexanoic acid; D- or L-alpha-OH-epsilon-NBD-aminohexanoic acid; D- or L-alpha-NBD-aminohexanoic acid). Using previously described resonance energy transfer assays, we examined the rates of spontaneous transfer of these compounds between liposomes and their ability to undergo transbilayer movement. The fluorescent N-acylsphingosine analogues had half-times for spontaneous transfer of 0.3-4.0 min at 25 degrees C, and all were capable of transbilayer movement in lipid vesicles. The metabolism and intracellular distribution of analogues in cultured fibroblasts were also studied. While most of the fluorescent N-acylsphingosines were significantly metabolized to the corresponding sphingomyelin analogues, metabolism to glucosylceramide was strongly dependent on the long-chain base and the stereochemistry of the fluorescent fatty acid moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

The low-energy forms of photosystem I light-harvesting complexes: spectroscopic properties and pigment-pigment interaction characteristics

In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line-narrowing experiments. It was found that the red-most absorption bands of Lhca3, Lhca4, and Lhca1-4 peak, respectively, at 704, 708, and 709 nm and are responsible for 725-, 733-, and 732-nm fluorescence emission bands. These bands are more red shifted compared to "normal" chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400-450 cm(-1)), which is the result of both large homogeneous and inhomogeneous broadening. The observed optical reorganization energy is untypical for Chl a and resembles more that of BChl a antenna systems. The large broadening and the changes in optical reorganization energy are explained by a mixing of an Lhca excitonic state with a charge transfer state. Such a charge transfer state can be stabilized by the polar residues around Chl 1025. It is shown that the optical reorganization energy is changing through the inhomogeneous distribution of the red-most absorption band, with the pigments contributing to the red part of the distribution showing higher values. A second red emission form in Lhca4 was detected at 705 nm and originates from a broad absorption band peaking at 690 nm. This fluorescence emission is present also in the Lhca4-N-47H mutant, which lacks the 733-nm emission band.     

The chloroplast and cytoplasmic ribosomes of euglena: I. Stability of chloroplast ribosomes prepared by an improved procedure

A new isolation procedure has resulted in an improved yield of stable 68S chloroplast ribosomes from Euglena gracilis var. bacillaris. Chloroplasts are isolated by suspending the cells in buffer I (sorbitol, 250 mm; sucrose, 250 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm) and passing through a French press at less than 1500 pounds per square inch. The crude chloroplasts are purified by three washings with buffer II (sorbitol, 150 mm; sucrose, 150 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm). Stable 68S chloroplast ribosomes are obtained when the isolated chloroplasts are resuspended in ribosome buffer (tris-HCI, pH 7.6, 10 mm; magnesium acetate, 12 mm; KCI, 60 mm) containing spermidine, 0.5 mm; mercaptoethanol, 14 mm; sucrose, 8% (w/w), passed through a French press at 4000 pounds per square inch and extracted with either 0.1% (w/v) sodium deoxycholate or 1.0% (v/v) Triton X-100. At 0 to 4 C in ribosome buffer, the purified 68S chloroplast monosome forms a 53S particle while the 35S particle, an expected product of monosome dissociation, cannot be detected. Spermidine and mercaptoethanol prevent the formation of 53S particles from 68S monosomes. The purified 53S particles derived from 68S monosomes contain 23S RNA as well as a significant amount of 16S RNA, suggesting that this particle may not be a true ribosomal subunit.     

Heat stress-induced effects of photosystem I: an overview of structural and functional responses

Temperature is one of the main factors controlling the formation, development, and functional performance of the photosynthetic apparatus in all photoautotrophs (green plants, algae, and cyanobacteria) on Earth. The projected climate change scenarios predict increases in air temperature across Earth’s biomes ranging from moderate (3–4?°C) to extreme (6–8?°C) by the year 2100 (IPCC in Climate change 2007: The physical science basis: summery for policymakers, IPCC WG1 Fourth Assessment Report 2007; Climate change 2014: Mitigation of Climate Change, IPCC WG3 Fifth Assessment Report 2014). In some areas, especially of the Northern hemisphere, even more extreme warm seasonal temperatures may occur, which possibly will cause significant negative effects on the development, growth, and yield of important agricultural crops. It is well documented that high temperatures can cause direct damages of the photosynthetic apparatus and photosystem II (PSII) is generally considered to be the primary target of heat-induced inactivation of photosynthesis. However, since photosystem I (PSI) is considered to determine the global amount of enthalpy in living systems (Nelson in Biochim Biophys Acta 1807:856–863, 2011; Photosynth Res 116:145–151, 2013), the effects of elevated temperatures on PSI might be of vital importance for regulating the photosynthetic response of all photoautotrophs in the changing environment. In this review, we summarize the experimental data that demonstrate the critical impact of heat-induced alterations on the structure, composition, and functional performance of PSI and their significant implications on photosynthesis under future climate change scenarios.     

The 8 kDa polypeptide in photosystem I is a probable candidate of an iron-sulfur center protein coded by the chloroplast gene frxA

The N-terminal sequence of the 8 kDa polypeptide isolated from spinach photosystem I (PS I) particles was determined by a gas-phase sequencer. The sequence showed the characteristic distribution of cysteine residues in the bacterial-type ferredoxins and was highly homologous to that deduced from the chloroplast gene frxA of liverwort, Marchantia polymorpha. It is strongly suggested that the 8 kDa polypeptide has to be an apoprotein of one of the iron-sulfur center proteins in PS I particles.     

Structure, function and regulation of plant photosystem I

Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the 'red' chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.     

Structure, function and regulation of plant photosystem I

Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the ‘red’ chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.     

Regulation of synthesis and turnover of an interferon-inducible mRNA.

Regulation of synthesis and turnover of an interferon (IFN)-inducible mRNA, mRNA 561, in HeLa monolayer cells was studied. Cytoplasmic levels of this mRNA were estimated by hybridization analyses with a cDNA clone that we have isolated as a probe. IFN-alpha A induced a high level of this mRNA in a transient fashion, whereas no induction was observed in response to IFN-gamma. Surprisingly little mRNA 561 was induced in cells treated simultaneously with IFN-alpha A and an inhibitor of protein synthesis, suggesting that in addition to IFN-alpha A, an interferon-inducible protein was needed for induction of this mRNA. Apparently this putative protein could be induced by IFN-gamma as well. Thus, although little mRNA 561 was synthesized in cells treated either with IFN-gamma alone or with IFN-alpha A and cycloheximide, a large quantity of this mRNA was induced in cells which had been pretreated with IFN-gamma and then treated with IFN-alpha A and cycloheximide. Once mRNA 561 was induced by IFN-alpha A, it turned over rapidly. This rapid turnover could be blocked by actinomycin D or cycloheximide indicating that another IFN-inducible protein may mediate this process.