The 1.49 A resolution crystal structure of PsbQ from photosystem II of Spinacia oleracea reveals a PPII structure in the N-terminal region

We report the high-resolution structure of the spinach PsbQ protein, one of the main extrinsic proteins of higher plant photosystem II (PSII). The crystal structure shows that there are two well-defined regions in PsbQ, the C-terminal region (residues 46-149) folded as a four helix up-down bundle and the N-terminal region (residues 1-45) that is loosely packed. This structure provides, for the first time, insights into the crucial N-terminal region. First, two parallel beta-strands cross spatially, joining the beginning and the end of the N-terminal region of PsbQ. Secondly, the residues Pro9-Pro10-Pro11-Pro12 form a left-handed helix (or a polyproline type II (PPII) structure), which is stabilized by hydrogen bonds between the Pro peptide carbonyl groups and solvent water molecules. Thirdly, residues 14-33 are not visible in the electron density map, suggesting that this loop might be very flexible and presumably extended when PsbQ is free in solution. On the basis of the essential role of the N-terminal region of PsbQ in binding to PSII, we propose that both the PPII structure and the missing loop are key secondary structure elements in the recognition of specific protein-protein interactions between PsbQ and other oxygen-evolving complex extrinsic and/or intrinsic proteins of PSII. In addition, the PsbQ crystal coordinates two zinc ions, one of them is proposed to have a physiological role in higher plants, on the basis of the full conservation of the ligand protein residues in the sequence subfamily.     

Cross-reconstitution of the extrinsic proteins and Photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea

Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.     

Isolation of sperm cells from mature pollen grains of Spinacia oleracea L.

Summary When mature pollen grains of Spinacia oleracea were squashed in a 25% sucrose solution and subsequently centrifuged on a percoll layer, sperm cells were isolated in high numbers. All steps were carried out at 4° C. Isolated sperm cells could be kept alive for several hours.     

The donor side of Photosystem II as the copper-inhibitory binding site

We have measured, under Cu (II) toxicity conditions, the oxygen-evolving capacity of spinach PS II particles in the Hill reactions H₂OSiMo (in the presence and absence of DCMU) and H₂OPPBQ, as well as the fluorescence induction curve of Tris-washed spinach PS II particles. Cu (II) inhibits both Hill reactions and, in the first case, the DCMU-insensitive H₂O SiMo activity. In addition, the variable fluorescence is lowered by Cu (II). We have interpreted our results in terms of a donor side inhibition close to the reaction center. The same polarographic and fluorescence measurements carried out at different pHs indicate that Cu (II) could bind to amino acid residues that can be protonated and deprotonated. In order to reverse the Cu (II) inhibition by a posterior EDTA treatment, in experiments of preincubation of PS II particles with Cu (II) in light we have demonstrated that light is essential for the damage due to Cu (II) and that this furthermore is irreversible.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenilcarbazide - F_o initial non-variable fluorescence - F_I intermediate fluorescence yield - F_m maximum fluorescence yield - F_v variable fluorescence yield - Mes 2,-(N-morpholino)ethanosulfonic acid - OEC oxygen-evolving complex - P680 Primary electron donor chlorophyll - Pheo pheophytin - PPBQ phenyl-p-benzo-quinone - PS II Photosystem II - SiMo Silicomolybdate - Q_B secondary quinone acceptor - Q_A primary quinone aceptor - Tris N-tris(hydroxymethyl)amino ethane - Tyr_z electron carrier functioning between P680 and the Mn cluster This article is dedicated to Prof. Dr. Harmut Lichtenthaler on the occasion of his 60th birthday.     

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.     

Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

Solution NMR and molecular dynamics reveal a persistent alpha helix within the dynamic region of PsbQ from photosystem II of higher plants

The extrinsic proteins of photosystem II of higher plants and green algae PsbO, PsbP, PsbQ, and PsbR are essential for stable oxygen production in the oxygen evolving center. In the available X‐ray crystallographic structure of higher plant PsbQ residues S14‐Y33 are missing. Building on the backbone NMR assignment of PsbQ, which includes this “missing link”, we report the extended resonance assignment including side chain atoms. Based on nuclear Overhauser effect spectra a high resolution solution structure of PsbQ with a backbone RMSD of 0.81 Å was obtained from torsion angle dynamics. Within the N‐terminal residues 1–45 the solution structure deviates significantly from the X‐ray crystallographic one, while the four‐helix bundle core found previously is confirmed. A short α‐helix is observed in the solution structure at the location where a β‐strand had been proposed in the earlier crystallographic study. NMR relaxation data and unrestrained molecular dynamics simulations corroborate that the N‐terminal region behaves as a flexible tail with a persistent short local helical secondary structure, while no indications of forming a β‐strand are found. Proteins 2015; 83:1677–1686. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Characterization of photosystem II in salt-stressed cyanobacterial Spirulina platensis cells

PSII activity was inhibited after Spirulina platensis cells were incubated with different salt concentrations (0-0.8 M NaCl) for 12 h. Flash-induced fluorescence kinetics showed that in the absence of DCMU, the half time of the fast and slow components decreased while that of the middle component increased considerably with increasing salt concentration. In the presence of DCMU, fluorescence relaxation was dominated by a 0.6s component in control cells. After salt stress, this was partially replaced by a faster new component with half time of 20-50 ms. Thermoluminescence measurements revealed that S₂Q_A⁻ and S₂Q_B⁻ recombinations were shifted to higher temperatures in parallel and the intensities of the thermoluminescence emissions were significantly reduced in salt-stressed cells. The period-four oscillation of the thermoluminescence B band was highly damped. There were no significant changes in contents of CP47, CP43, cytochrome c550, and D1 proteins. However, content of the PsbO protein in thylakoid fraction decreased but increased significantly in soluble fraction. The results suggest that salt stress leads to a modification of the Q_B niche at the acceptor side and an increase in the stability of the S₂ state at the donor side, which is associated with a dissociation of the PsbO protein.     

The effects of light-induced reduction of the photosystem II reaction center

Accumulation of reduced pheophytin a (Pheo-D1) in photosystem II reaction center (PSII RC) under illumination at low redox potential is accompanied by changes in absorbance and circular dichroism spectra. The temperature dependences of these spectral changes have the potential to distinguish between changes caused by the excitonic interaction and temperature-dependent processes. We observed a conformational change in the PSII RC protein part and changes in the spatial positions of the PSII RC pigments of the active D1 branch upon reduction of Pheo-D1 only in the case of high temperature (298 K) dynamics. The resulting absorption difference spectra of PSII RC models equilibrated at temperatures of 77 K and 298 K were highly consistent with our previous experiments in which light-induced bleaching of the PSII RC absorbance spectrum was observable only at 298 K. These results support our previous hypothesis that Pheo-D1 does not interact excitonically with the other chlorins of the PSII RC, since the reduced form of Pheo-D1 causes absorption spectra bleaching only due to temperature-dependent processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

The role of the gas phase in the greening and growth of illuminated cell suspension cultures of spinach (Spinacia oleracea,L.)

Summary The gas phase developed above spinach suspension cultures critically affected their growth and greening. Ethylene accumulation inhibited greening; this effect of ethylene was antagonised when the culture gas phase was enriched with carbon dioxide. Greening was enhanced by reducing the partial pressure of oxygen below the air level; this effect was observed when oxygen supply did not restrict growth. One of the authors (C.C.D.) was supported by an S.R.C. studentship grant during this work.     

Role of 5-aminolevulinic acid (ALA) on active oxygen-scavenging system in NaCl-treated spinach (Spinacia oleracea)

ALA is a key precursor in the biosynthesis of porphyrins such as chlorophyll and heme, and was found to induce temporary elevations in the photosynthesis rate, APX, and CAT; furthermore, treatment with ALA at a low concentration might be correlated to the increase of NaCl tolerance of spinach plants. The photosynthetic rate and the levels of active oxygen-scavenging system in the 3rd leaf of spinach (Spinacia oleracea) plants grown by foliar treatment with 0, 0.18, 0.60 and 1.80 mmol/L 5-aminolevulinic acid under 50 and 100 mmol/L NaCl were analyzed. Plants treated with 0.60 and 1.80 mmol/L ALA showed significant increases in the photosynthetic rate at 50 and 100 mmol/L NaCl, while that of 0.18 mmol/L ALA did not show any changes at 50 mmol/L NaCl and a gradual decrease at 100 mmol/L NaCl. In contrast, the rate with 0 mmol/L ALA showed reduction at both concentrations of NaCl. The increase of hydrogen peroxide content by treatment with 0.60 and 1.80 mmol/L ALA were more controlled than that of 0 mmol/L ALA under both NaCl conditions. These ALA-treated spinach leaves also exhibited a lower oxidized/reduced ascorbate acid ratio and a higher reduced/oxidized glutathione ratio than the 0 mmol/L-treated spinach leaves when grown at both NaCl conditions. With regard to the antioxidant enzyme activities in the leaves, ascorbate peroxidase, catalase, and glutathione reductase activities were enhanced remarkably, most notably at day 3, by treatment with 0.60 and 1.80 mmol/L ALA under both NaCl conditions in comparison to that of 0 and 0.18 mmol/L ALA. These data indicate that the protection against oxidative damage by higher levels of antioxidants and enzyme activities, and by a more active ascorbate-glutathione cycle related to the increase of the photosynthesis rate, could be involved in the increased salt tolerance observed in spinach by treatment with 0.60 to 1.80 mmol/L ALA with NaCl.     

Association of chlorophyll a/c2 complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c₂ proteins were found. The most common complexes have Chl a/c₂ complexes at both sides of the PSI core monomer and have dimensions of about 17 × 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c₂ light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c₂ proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c₂ proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

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.     

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.     

Alterations in photochemical efficiency of photosystem II in wheat plant on hot summer day

In this study the effect of increasing temperature on photochemical efficiency of PS II in wheat plants has been studied on a hot summer day (9:00 AM (Control)–7:00 PM) by measuring Chl a fluorescence. Increasing temperature for a short period of time (2–4 h), in nature affects the efficiency of PS II complex reversibly and does not cause permanent damage to any of the components of photosystem II. A scheme has been provided to demonstrate the sequence and severity of events which get affected maximum by temperature stress.     

Lethal and sublethal effects of a granulovirus on the tomato moth Lacanobia oleracea

A Scottish isolate of Lacanobia oleracea granulovirus (LoGV) was tested against larvae from a laboratory colony of L. oleracea (Lepidoptera: Noctuidae) using the peroral bioassay technique. The median lethal doses ranged from 10(1.38) occlusion bodies in first instar larvae to 10(7.45) in fifth instars. Sublethal effects on development and reproduction were observed among the survivors of virus challenge. Larvae dosed as first and second instars had faster developmental rates, and those dosed as fourth and sixth instars had reduced larval and pupal weights, compared to their respective controls. There was a 15% reduction in the number of eggs laid by adults that developed from infected larvae, but no reduction in egg viability. The F(1) generation did not show significantly greater mortality than controls, suggesting a lack of transovarial transmission. The significance of these results in terms of pest management of the tomato moth is discussed.     

Molecular evidence that the genes for dioecism and monoecism in Spinacia oleracea L. are located at different loci in a chromosomal region

Spinach (Spinacia oleracea L.) is widely known to be dioecious. However, monoecious plants can also occur in this species. Sex expression in dioecious spinach plants is controlled by a single gene pair termed X and Y. Our previous study showed that a single, incompletely dominant gene, which controls the monoecious condition in spinach line 03–336, should be allelic or linked to X/Y. Here, we developed 19 AFLP markers closely linked to the monoecious gene. The AFLP markers were mapped to a 38.2-cM chromosomal region that included the monoecious gene, which is bracketed between flanking markers with a distance of 7.1 cM. The four AFLP markers developed in our studies were converted into sequence-characterized amplified region (SCAR) markers, which are linked to both the monoecious gene and Y and are common to both populations segregating for the genes. Linkage analysis using the SCAR markers suggested that the monoecious gene (M) and Y are located in different intervals, between different marker pairs. Analysis of populations segregating for both M and Y also directly demonstrates linkage of the genes at a distance of ∼12 cM. The data presented in this study may be useful for breeding dioecious and highly male monoecious lines utilized as the pollen parents for hybrid seed production, as well as for studies of the evolutionary history of sexual systems in this species, and can provide a molecular basis for positional cloning of the sex-determining genes.     

Shikonin isovalerate: An inhibitor of photosystem II in Chlorogloeopsis fritschi

Shikonin isovalerate, extracted from the roots of the desert plant Arnebia decumbens, was tested for its effect on photosynthetic electron transport system of Chlorogloeopsis fritschii. The ferricyanide-Hill reaction with water and DPC as electron donors was inhibited completely with 10^-5 M shikonin isovalerate. The photoreduction of DCPIP through photosystem II was only slightly inhibited. Photosystem I from durohydroquinone to methyl viologen was not affected using 10^-6 M shikonin isovalerate. The same concentration caused 49% inhibition of cyclic photophosphorylation. These results suggest that shikonin isovalerate inhibits photosynthetic electron flow at the plastoquinone pool.Abbreviations DCMU 3-(3,4-dichlorophenyl)-N,N-dimethyl urea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-P-benzoquinone - DCPIP 2–6-dichlorophenolindophenol - DPC Diphenylcarbazide - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine