High-light-induced superoxide anion radical formation in cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach as detected by EPR spectroscopy

The generation of superoxide anion radical (O₂ ^·−) in the cytochrome b ₆ f complex (Cyt b ₆ f) of spinach under high-light illumination was studied using electron paramagnetic resonance spectroscopy. The generation of O₂ ^·− was lost in the absence of molecular oxygen. It was also suppressed in the presence of NaN₃ and could be scavenged by extraneous antioxidants such as ascorbate, β-carotene, and glutathione. The results also indicate that O₂ ^·−, which is produced under high-light illumination of the Cyt b ₆ f from spinach, might be generated from a reaction involing ¹O₂, and the Rieske Fe-S protein could serve as the electron donor in the O₂ ^·− production. The mechanism of photoprotection of the Cyt b ₆ f complex by antioxidants is discussed.     

The peroxidase activity of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach chloroplasts

The cytochrome b ₆ f (Cyt b ₆ f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H₂O₂) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (K _m) values for H₂O₂ and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k _obs) was about 26 M⁻¹ s⁻¹ and the turnover number (K _cat) was about 60 min⁻¹ (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H₂O₂]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

Adaptive intraspecific divergence: An example using the animal cytochrome <Emphasis Type=Italic>b</Emphasis> gene

The topologies of phylogenetic trees characterized by a high level of intraspecific divergence between the phylogenetic DNA groups (clades) are often explained in terms of the theory of Pleistocene refugia. To elucidate the issue of the adaptive role of intraspecific divergence, the changes in the physicochemical properties of amino acids in the course of cladogenesis (MM01 model of the TreeSAAP 3.2 package) were analyzed in this work using as an example the nucleotide sequences of the cytochrome b gene in some species of northern animals (lemmings, redbacked voles, chipmunks, flying squirrels, ermines). It was shown that the process of intraspecific divergence was rarely accompanied by radical amino acid substitutions in cytochrome b caused by adaptation (directional selection). In connection with this, the hypothesis is discussed according to which the adaptive variants formed in the species at the peak of cold were lost with climatic warming due to the drift or selection against individuals adapted to cold.     

<Emphasis Type=Italic>Boletus kermesinus</Emphasis>, a new species of <Emphasis Type=Italic>Boletus</Emphasis> section <Emphasis Type=Italic>Luridi</Emphasis> from central Honshu,Japan

Boletus kermesinus, a new species of Boletus section Luridi, is fully described and illustrated based on the materials collected in subalpine coniferous forests of central Honshu, Japan. It has distinctive features of dark-red basidiomata having distinct viscidity in the pileus surface, usually unchanging flesh, discolorous red pores, and an entirely reticulate stipe becoming coarsely lacerate-rimose with age.     

DNA markers linked to the <Emphasis Type=Italic>R</Emphasis><Subscript><Emphasis Type=Italic>2</Emphasis></Subscript> rust resistance gene in sunflower (<Emphasis Type=Italic>Helianthus annuus</Emphasis> L.) facilitate anticipatory breeding for this disease variant

Pre-emptive breeding for host disease resistance is an effective strategy for combating and managing devastating incursions of plant pathogens. Comprehensive, long-term studies have revealed that virulence to the R ₂ sunflower (Helianthus annuus L.) rust resistance gene in the line MC29 does not exist in the Australian rust (Puccinia helianthi) population. We report in this study the identification of molecular markers linked to this gene. The three simple sequence repeat (SSR) markers ORS795, ORS882, and ORS938 were linked in coupling to the gene, while the SSR marker ORS333 was linked in repulsion. Reliable selection for homozygous-resistant individuals was efficient when the three markers, ORS795, ORS882, and ORS333, were used in combination. Phenotyping for this resistance gene is not possible in Australia without introducing a quarantinable race of the pathogen. Therefore, the availability of reliable and heritable DNA-based markers will enable the efficient deployment of this gene, permitting a more effective strategy for generating sustainable commercial cultivars containing this rust resistance gene.     

Catalysis of Methaemoglobin Reduction by Erythrocyte Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> and Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> Reductase

THE reduction of methaemoglobin is one of the few oxidation-reduction reactions known to occur in the mature red blood cell. Approximately 3% of the circulating haemoglobin of an adult man is oxidized each day to methaemoglobin¹ and so a methaemoglobin reduction system is needed in the cell to maintain a low concentration of methaemoglobin (normally less than 1% of the total haemoglobin²).     

Fine mapping and analyses of <Emphasis Type=Italic>R</Emphasis><Subscript><Emphasis Type=Italic>SC8</Emphasis></Subscript> resistance candidate genes to soybean mosaic virus in soybean

Soybean mosaic virus (SMV) in soybean [Glycine max (L.) Merr.] is a destructive foliar disease in soybean-producing countries worldwide. In this study, F₂, F_2:3, and F_7:11 recombinant inbred lines populations derived from Kefeng No.1 × Nannong 1138-2 were used to study inheritance and linkage mapping of the SMV strain SC8 resistance gene in Kefeng No.1. Results indicated that a single dominant gene (designated R _SC8 ) controls resistance, which is located on chromosome 2 (MLG D1b). A mixed segregating population was developed by selfing two heterozygous plants (RHL153-1 and RHL153-2) at four markers adjacent to the locus and used in fine mapping R _SC8 . In addition, two genomic-simple sequence repeats (SSR) markers BARCSOYSSR_02_0610 and BARCSOYSSR_02_0616 were identified that flank the two sides of R _SC8 . Sequence analysis of the soybean genome indicated that the interval between the two genomic-SSR markers is 200 kb. QRT-PCR analysis of the candidate genes determined that five genes (Glyma02g13310, 13320, 13400, 13460, and 13470) are likely involved in soybean SMV resistance. These results will have utility in cloning, transferring, and pyramiding of the R _SC8 through marker-assisted selection in soybean breeding programs.