Photoinhibition as a function of the ambient redox potential in Tris-washed PS II membrane fragments

The mode of photoinhibition as a function of the ambient redox potential (E_ambient) in suspensions of Tris-washed PS II membrane fragments has been analyzed by monitoring flash-induced absorption changes at 830 nm. It was found: (a) the detectable initial amplitude, ΔA^total ₈₃₀, as a measure of the capacity to form the `stable' radical pair, P680^+ċ Q^−ċ _A, drastically decreases during a 10 min photoinhibition at E_ambient values below +350 mV; (b) conversely, the normalized extent of the 18 μs relaxation kinetics, ΔA^{18 μ s} ₈₃₀ as a measure of the electron transfer from Y_Z to P680^+ċ becomes highly susceptible to light stress when E_ambient exceeds values of about +350 mV; (c) effects of the ambient redox potentials are highly pronounced during light exposure under anaerobic conditions, while much smaller differences arise under aerobic conditions; (d) the extent of damage does not correlate with the total concentration of K₃[Fe(CN)₆] and K₄[Fe(CN)₆] in the suspension during photoinhibition but rather depends on the E_m-values; (e) qualitatively similar features are observed when the redox buffer system K₃[Fe(CN)₆]/Na₂S₂O₄ is replaced by K₂[IrCl₆]/Na₂S₂O₄; (f) the characteristic E_ambient-dependence of photoinhibition is observed only under anaerobic conditions. The results are discussed with respect to different redox components that might be involved, including brief comments on a possible role of Cyt b559. This revised version was published online in June 2006 with corrections to the Cover Date.     

EPR characterization of photosystem II from different domains of the thylakoid membrane

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.     

Hebeloma species associated with Cistus

The genus Hebeloma has a number of species highly specific to Cistus and others that occur with several host genera. This paper discusses the species of Hebeloma that appear to be ectomycorrhizal with Cistus, judging from their occurrence when Cistus is the only available host. The previously unknown species H. plesiocistum spec. nov. is described. We also provide a key to the known Hebeloma associates of Cistus. Molecular analyses based on ITS sequence data further illustrate the distinctness of the newly described species and difficulties in the species delimitation with view to H. erumpens. Specific associations with Cistus may have evolved more than once within the genus Hebeloma.     

Nonsense-mediated mRNA decay in barley mutants allows the cloning of mutated genes by a microarray approach.

We have previously described a microarray approach to identify and clone genes from mutants of higher organisms. In the method cDNA of two mutants with similar phenotype are competitively hybridized to DNA clones arrayed on a glass slide. Clones corresponding to an mRNA that is not expressed in one of the strains due to a mutation will be specifically highlighted in the hybridization, which provides a possibility to identify and eventually clone the mutated gene. The approach is dependent on mutations that affect the amount of mRNA. Nonsense mutations, which prematurely terminate translation, can be such mutations as a surveillance system known as nonsense-mediated decay (NMD) has been developed by organisms to reduce the abundance of mRNA with nonsense codons. In the present study, we have analysed the barley (Hordeum vulgare L.) magnesium chelatase mutants xantha-f26, xantha-f27 and xantha-f40 in order to investigate the presence of NMD in barley, as well as the importance of the position of the stop codon for NMD. Both nonsense-mutants xantha-f27 and xantha-f40, but not the missense mutant xantha-f26, showed NMD. This was not expected for xantha-f27 as its mutation is in the last exon of the gene. We conclude the NMD expands the number of mutants that can be used for gene cloning by our described microarray approach.     

Interconversion of low- and high-potential forms of cytochrome b(559) in Tris-washed photosystem II membranes under aerobic and anaerobic conditions.

In this study, the reversible conversion between the high- (HP) and low-potential (LP) forms of Cytb(559) has been analyzed in Tris-washed photosystem II (PSII) enriched membranes. These samples are deprived of the Mn cluster of the water-oxidizing complex (WOC) and the extrinsic regulatory proteins. The results obtained by application of optical and EPR spectroscopy reveal that (i) under aerobic conditions, the vast majority of Cytb(559) exhibits a low midpoint potential, (ii) after removal of O(2) in the dark, a fraction of Cytb(559) is converted to the high-potential form which reaches level of about 25% of the total Cytb(559), (iii) a similar dark transformation of LP --> HP Cytb(559) occurs under reducing conditions (8 mM hydroquinone), (iv) under anaerobic conditions and in the presence of 8 mM hydroquinone, about 60% of the Cytb(559) attains the HP form, (v) the interconversion is reversible with the re-establishment of aerobic conditions, and (vi) aerobic and oxidizing conditions (2 mM ferricyanide or 0.5 mM potassium iridate) induce a decrease of the amount of the HP form, also showing that the conversion is reversible. This reversible interconversion between LP and HP Cytb(559) is not observed in PSII membrane fragments with an intact WOC. On the basis of these findings, the possibility is discussed that the O(2)-dependent conversion of Cytb(559) in PSII complexes lacking a functionally competent WOC is related to a protective role of Cytb(559) in photoinhibition and/or that it is involved in the regulation of the assembly of a competent water-oxidizing complex in PSII.     

Fractionation of the thylakoid membranes from tobacco. A tentative isolation of 'end membrane' and purified 'stroma lamellae' membranes

Thylakoids isolated from tobacco were fragmented by sonication and the vesicles so obtained were separated by partitioning in aqueous polymer two-phase systems. By this procedure, grana vesicles were separated from stroma exposed membrane vesicles. The latter vesicles could be further fractionated by countercurrent distribution, with dextran-polyethylene glycol phase systems, and divided into two main populations, tentatively named 'stroma lamellae' and 'end membrane'. Both these vesicle preparations have high chlorophyll a/b ratio, high photosystem (PS) I and low PS II content, suggesting their origin from stroma exposed regions of the thylakoid. The two vesicle populations have been compared with respect to biochemical composition and photosynthetic activity. The 'end membrane' has a higher chlorophyll a/b ratio (5.7 vs. 4.7), higher P700 content (4.7 vs. 3.3 mmol/mol of chlorophyll). The 'end membrane' has the lowest PS II content, the ratio PS I/PS II being more than 10, as shown by EPR measurements. The PS II in both fractions is of the beta-type. The decay of fluorescence is different for the two populations, the 'stroma lamellae' showing a very slow decay even in the presence of K3Fe(CN)6 as an acceptor. The two vesicle populations have very different surface properties: the end membranes prefer the upper phase much more than the stroma lamellae, a fact which was utilized for their separation. Arguments are presented which support the suggestion that the two vesicle populations originate from the grana end membranes and the stroma lamellae, respectively.     

Spatial structure of Thr-Pro-Ala-Glu-Asp-Phe-Met-Arg-Phe-NH2 molecule

The spatial structure of cardioactive Thr-Pro-Ala-Glu-Asp-Phe-Met-Arg-Phe-NH₂ molecule has been investigated using a theoretical conformational analysis. The low-energy conformations of the molecule were found, the values of the backbone and side T-T chain dihedral angles of amino acid residues constituting the peptide were determined, and the energies of intra- and interresidual interactions were estimated. It is revealed that the spatial structure of this molecule can exist in 11 stable backbone forms.     

Oxygen-induced changes in the redox state of the cytochrome b559 in photosystem II depend on the integrity of the Mn cluster

The effect of oxygen and anaerobiosis on the redox properties of Cyt b ₅₅₉ was investigated in PSII preparations from spinach with different degree of disintegration of the donor side. Comparative studies were performed on intact PSII membranes and PSII membranes that were deprived of the 18-kDa peripheral subunit (0.25 NaCl washed), the 18- and 24-kDa peripheral subunits (1 M NaCl washed), the 18-, 24- and 33-kDa peripheral subunits (1.2 M CaCl₂ washed), Cl depleted and after complete depletion of the Mn cluster (Tris washed). In active PSII centers, about 75% of Cyt b ₅₅₉ was found in the high-potential form and the rest in the intermediate potential form. With decomposition of the donor side, the intermediate potential form started to dominate, reaching more than 90% after Tris treatment. The oxygen-dependent conversion of the intermediate potential form of Cyt b ₅₅₉ into the low-potential and high-potential forms was only observed after treatments that directly affect the Mn cluster. In PSII membranes, deprived of all three extrinsic subunits (CaCl₂ treatment), 21% of the intermediate potential form was converted into the low-potential form and 14% into the high-potential form by the removal of oxygen. In Tris-washed PSII membranes, completely lacking the Mn cluster, this conversion amounted to 60 and 33%, respectively. In intact PSII membranes, the oxygen-dependent conversion did not occur. The possible physiological role of this oxygen-dependent behavior of the Cyt b ₅₅₉ redox forms during the assembly/photoactivation cycle of PSII is discussed.