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.

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.

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 CaCl(2) 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 (CaCl(2) 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.

Analysis of gun phenotype in barley magnesium chelatase and Mg-protoporphyrin IX monomethyl ester cyclase mutants.

The ability of barley (Hordeum vulgare L.) chlorophyll biosynthetic mutants to regulate the expression of Lhc genes was analyzed by a microarray approach. The Lhc genes are located in the nucleus and encode chlorophyll a/b binding proteins of the light-harvesting complex. The chlorophyll a/b binding proteins are some of the many proteins, which are imported to the chloroplast. It has been suggested that the chloroplast can regulate expression of nuclear genes encoding chloroplast proteins, using a chlorophyll biosynthetic intermediate such as Mg-protoporphyrin IX (MP) or Mg-protoporphyrin IX monomethyl ester (MPE) as a signal molecule. These compounds are intermediates between the two enzymes magnesium-chelatase (EC 6.6.1.1) and Mg-protoporphyrin IX monomethyl ester cyclase (EC 1.14.13.81) in the chlorophyll biosynthetic pathway. Genomes uncoupled (gun) mutants are defective in the chloroplast-to-nucleus signal transduction and express Lhc even when chloroplast development is inhibited by the herbicide norflurazon. We show that barley xantha-f, -g and -h mutants, defective in the three Mg-chelatase genes, have a gun phenotype. In contrast, a xantha-l mutant, defective in a gene of Mg-protoporphyrin monomethyl ester cyclase did not. Genome uncoupling in the xantha-f, -g, -h and -l mutants was also analyzed in absence of norflurazon. All mutants showed transcription of Lhc. This was unexpected in the case of xantha-l as this mutant showed accumulation of MPE, which has been suggested to be one of the two negative regulators of Lhc transcription. We suggest that chlorophyll intermediates may only function as signal molecules at an early developmental stage of chloroplast development.

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.