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Celiac disease (CD) develops in genetically susceptible individuals as a result of ingesting gluten-forming proteins found in cereals, such as wheat (Triticum aestivum L.), rye (Secale cereale L.) and barley (Hordeum sativum L.). There are claims that breeding practices have changed wheat protein chemistry over the years and this has resulted in modern wheat being more antigenic in terms of CD as opposed to historical wheat. The aim of this study was to detect and quantify celiac-disease-initiating peptides of α-gliadin proteins in historical and modern spring wheat cultivars. The results indicate that immunogenic epitopes are detected in both historical and modern spring wheat cultivars irrespective of release year. Quantitation indicated that the amount of immunogenic epitopes glia-α9 (PFPQPQLPY) and glia-α20 (FRPQQPYPQ), and total α-gliadin varied randomly across the cultivars that were analyzed, suggesting there is no association between cultivar release year and amounts of immunogenic epitopes and α-gliadin.

Thirty hard red spring (HRS) wheat cultivars released between 1910 and 2013 were analyzed to determine how they cluster in terms of parentage and HPLC analysis of gliadins and unreduced proteins. Markers for reduced height genes were assessed to determine associations between semidwarfism and protein composition, as well as quality characteristics. In terms of parentage, the clusters formed were grouped according to release year and semidwarfism. Farinograph development time and stability showed significant (P ≤ 0.05) differences between clusters, indicating improvement of mixing characteristics over time. Flour ash content was significantly different (P ≤ 0.05) between clusters, indicating improved milling quality. The clusters based on the gliadin reverse-phase HPLC peak binary data did not group cultivars according to release year and the semidwarf characteristic. However, cultivars were grouped according to year in the absorbance area based dendrogram. The clusters based on absorbance area of size-exclusion HPLC showed significant (P ≤ 0.05) differences for release year, the semidwarf characteristic, and farinograph stability. Overall, the results indicate that the introduction of reduced height genes accompanied the improvements of dough mixing and breadmaking quality characteristics, without altering the composition of gliadin proteins over the last 100 years of HRS wheat breeding.

Patel, J. S., Meinhardt, S. W., Sierotzki, H., Stammler, G., Gudmestad, N. C., and Adhikari, T. B. 2011. A two-step molecular detection method for Pyrenophora tritici-repentis isolates insensitive to QoI fungicides. Plant Dis. 95:1558-1564. Tan spot, caused by Pyrenophora tritici-repentis, is an important disease of wheat worldwide. To manage tan spot, quinone outside inhibitor (QoI) fungicides such as azoxystrobin and pyraclostrobin have been applied in many countries. QoI fungicides target the cytochrome b (cyt b) site in complex III of mitochondria and, thus, pose a serious risk for resistance development. The resistance mechanism to QoI fungicides is mainly due to point mutations in the cyt b gene. The objective of this study was to develop a molecular detection method for the four currently known mutations responsible for shifts in sensitivity toward QoI fungicides in P. tritici-repentis. Twelve specific primers were designed based on sequences from the National Center for Biotechnology Information accessions AAXI01000704 and DQ919068 and used to generate a fragment of the cyt b gene which possesses four known singlenucleotide polymorphisms (SNPs). These mutant clones served as positive controls because QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis have not yet been reported in the United States. The partial cyt b gene clones were sequenced to identify the SNPs at sites G143A and F129L. Genomic DNA of the mutated partial cyt b gene clones and the European QoI-insensitive and -reducedsensitive isolates of P. tritici-repentis possessing G143A (GCT) and F129L (TTA, TTG, and CTC) mutations were amplified by polymerase chain reaction (PCR) using two specific primer pairs and were further digested with three specific restriction enzymes (BsaJI, Fnu4HI, and MnlI). The results of the digested PCR product from genomic DNA of known QoI-insensitive and -reduced-sensitive isolates of P. triticirepentis had DNA bands consistent with the mutation GCT at G143A and the mutations TTA, TTG, and CTC at F129L. The amplified region at the F129 site also had 99% sequence similarity with P. teres , the net blotch pathogen of barley. To validate mutations, we further tested two isolates of P. teres known to have reduced sensitivity to QoI fungicides possessing the mutations TTA and CTC at F129L. After PCR amplification and restriction digestion, DNA bands identical to those observed for the partial cyt b mutant clones were detected. These results suggest that this newly developed two-step molecular detection method is rapid, robust, and specific to monitor QoI-insensitive and -reducedsensitive isolates of P. tritici-repentis.

Summary The nuclear‐encoded species cytoplasm specific (scs) genes control nuclear–cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of in vivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9 million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70 Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288 Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano‐mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear–cytoplasmic compatibility in wheat.

This study characterized a fragment of the cytochrome b gene from Ascochyta rabiei isolates collected in North Dakota, USA, that varied in sensitivity to quinone-outside inhibitor (QoI) fungicides. The sequenced genomic DNA fragment contained a group I intron immediately after codon 131. The size of the cytochrome b gene was estimated to be over 4·6 kb. Multiple alignment analysis of cDNA and protein sequences revealed a mutation that changed the codon for amino acid 143 from GGT to GCT, introducing an amino acid substitution from glycine to alanine (G143A), which is frequently associated with QoI resistance. Based on this mutation, a diagnostic PCR assay was developed using an approach called mismatch amplification mutation assay. This method was successfully validated by testing a total of 70 A. rabiei isolates, of which 38 isolates were found to be QoI-resistant. This fast and accurate PCR assay provides a very useful and simple screening method for QoI resistance in A. rabiei isolates.

Tan spot, caused by Pyrenophora tritici-repentis , is an important disease of wheat ( Triticum aestivum L.) in the United States (U.S). Wheat growers sometimes rely on fungicide applications to manage foliar diseases. Pyraclostrobin is one of the recommended quinone outside inhibitor (QoI) fungicides and is commonly used to manage tan spot on wheat. Although P . tritici-repentis isolates has been reported as insensitive to QoI fungicides in Europe, little is known regarding the sensitivity of the P. tritici-repentis population to fungicides in the U.S. Twenty-eight isolates collected prior to 1997 and 136 isolates collected from pyraclostrobin treated fields in North Dakota were collected in 2007 and 2009 to determine if P. tritici-repentis isolates can use alternative respiration in the presence or absence of salicylhydroxamic acid (SHAM) in vitro and to determine the effective fungicide concentration that inhibited conidia germination by 50% (EC 50 ). Six of 10 baseline isolates of P. tritici-repentis assayed had significantly higher ( P ≤ 0.05) EC 50 values when SHAM was not added to agar medium amended with pyraclostrobin, confirming that the fungus can utilize alternative respiration to overcome QoI toxicity in vitro . EC 50 values of the baseline isolates ranged from 0.0012 to 0.0024 μg/ml (mean EC 50 value of 0.0017 μg/ml and standard deviation is ±0.00039 μg/ml) while EC 50 values of the 136 fungicide exposed isolates ranged from 0.0013 μg/ml to 0.0027 μg/ml (mean EC 50 value of 0.0017 μg/ml and standard deviation is ±0.00030). These results indicate that the field exposed isolates have not shifted to reduced sensitivity to pyraclostrobin and that the baseline sensitivity values can be useful for a fungicide resistance monitoring program for P. tritici-repentis in the U.S.

Plant disease resistance is often conferred by genes with nucleotide binding site (NBS) and leucine-rich repeat (LRR) or serine/threonine protein kinase (S/TPK) domains. Much less is known about mechanisms of susceptibility, particularly to necrotrophic fungal pathogens. The pathogens that cause the diseases tan spot and Stagonospora nodorum blotch on wheat produce effectors (host-selective toxins) that induce susceptibility in wheat lines harboring corresponding toxin sensitivity genes. The effector ToxA is produced by both pathogens, and sensitivity to ToxA is governed by the Tsn1 gene on wheat chromosome arm 5BL. Here, we report the cloning of Tsn1 , which was found to have disease resistance gene-like features, including S/TPK and NBS-LRR domains. Mutagenesis revealed that all three domains are required for ToxA sensitivity, and hence disease susceptibility. Tsn1 is unique to ToxA-sensitive genotypes, and insensitive genotypes are null. Sequencing and phylogenetic analysis indicated that Tsn1 arose in the B-genome diploid progenitor of polyploid wheat through a gene-fusion event that gave rise to its unique structure. Although Tsn1 is necessary to mediate ToxA recognition, yeast two-hybrid experiments suggested that the Tsn1 protein does not interact directly with ToxA. Tsn1 transcription is tightly regulated by the circadian clock and light, providing further evidence that Tsn1 -ToxA interactions are associated with photosynthesis pathways. This work suggests that these necrotrophic pathogens may thrive by subverting the resistance mechanisms acquired by plants to combat other pathogens.

The toxin sensitivity gene Tsn1 interacts with Ptr ToxA (ToxA), a host-selective toxin produced by the necrotrophic fungus Pyrenophora tritici-repentis. The molecular mechanisms associated with cell death in sensitive wheat cultivars following ToxA application are not well understood. To address this question, we used the Affymetrix GeneChip Wheat Genome Array to compare gene expression in a sensitive wheat cultivar possessing the Tsn1 gene with the insensitive wheat cv. Nec103, which lacks the Tsn1 gene. This analysis was performed at early timepoints after infiltration with ToxA (e.g., 0.5 to 12 h postinfiltration [hpi]); at this time, ToxA is known to internalize into mesophyll cells without visible cell death symptoms. Gene expression also was monitored at later timepoints (24 to 48 hpi), when ToxA causes extensive damage in cellular compartments and visible cell death. At both early and late timepoints, numerous defense-related genes were induced (2- to 197-fold increases) and included genes involved in the phenylpropanoid pathway, lignification, and the production of reactive oxygen species (ROS). Furthermore, a subset of host genes functioning in signal transduction, metabolism, and as transcription factors was induced as a consequence of the Tsn1–ToxA interaction. Nine genes known to be involved in the host defense response and signaling pathways were selected for analysis by quantitative real-time polymerase chain reaction, and the expression profiles of these genes confirmed the results obtained in microarray experiments. Histochemical analyses of a sensitive wheat cultivar showed that H2O2 was present in leaves undergoing cell death, indicating that ROS signaling is a major event involved in ToxA-mediated cell death. The results suggest that recognition of ToxA via Tsn1 triggers transcriptional reprogramming events similar to those reported for avirulence–resistance gene interactions, and that host-derived genes play an important role in the modulation of susceptibility to P. tritici-repentis.

*Summary We recently showed that the wheat pathogen Stagonospora nodorum produces proteinaceous host-selective toxins (HSTs). These toxins include SnTox1 as well as SnToxA, a HST first identified from Pyrenophora tritici-repentis that was implicated in a very recent horizontal gene transfer event from S. nodorum to P. triticirepentis. Compelling evidence implicating SnToxA and SnTox1 in disease development has been obtained. Here, we report the partial purification and characterization of a third HST designated SnTox2, as well as the genetic characterization of the corresponding host-sensitivity gene. SnTox2 was protease sensitive and is estimated between 7 and 10 kDa in size. Sensitivity to SnTox2 was conferred by a single dominant gene designated Snn2, which mapped to the short arm of wheat chromosome 2D. Genetic analysis of reaction to conidial inoculations in a segregating wheat population indicated that both the Snn2-SnTox2 and the Tsn1SnToxA interactions were involved in disease development, and together they accounted for the majority of the phenotypic variation. Therefore, S. nodorum produces multiple toxins that rely on specific interactions with host gene products to cause disease. The identification of multiple HST‐host gene interactions important for disease development and the availability of the S. nodorum whole genome sequence indicate the potential for this pathosystem to serve as a toxin-based, inverse gene-for-gene model.

New diseases of humans, animals and plants emerge regularly. Enhanced virulence on a new host can be facilitated by the acquisition of novel virulence factors. Interspecific gene transfer is known to be a source of such virulence factors in bacterial pathogens (often manifested as pathogenicity islands in the recipient organism) and it has been speculated that interspecific transfer of virulence factors may occur in fungal pathogens. Until now, no direct support has been available for this hypothesis. Here we present evidence that a gene encoding a critical virulence factor was transferred from one species of fungal pathogen to another. This gene transfer probably occurred just before 1941, creating a pathogen population with significantly enhanced virulence and leading to the emergence of a new damaging disease of wheat.

The wheat Tsn1 gene confers sensitivity to the host-selective toxin Ptr ToxA produced by the tan spot fungus (Pyrenophora tritici-repentis). The long-term goal of this research is to isolate Tsn1 using a positional cloning approach. Here, we evaluated 54 ESTs (expressed sequence tags) physically mapped to deletion bin 5BL 0.75-0.76, which is a gene-rich region containing Tsn1. Twenty-three EST loci were mapped as either PCR-based single-stranded conformational polymorphism or RFLP markers in a low-resolution wheat population. The genetic map corresponding to the 5BL 0.75-0.76 deletion bin spans 18.5 cM and contains 37 markers for a density of 2 markers/cM. The EST-based genetic map will be useful for tagging other genes, establishing colinearity with rice, and anchoring sequence ready BAC contigs of the 5BL 0.75-0.76 deletion bin. High-resolution mapping showed that EST-derived markers together with previously developed AFLP-derived markers delineated Tsn1 to a 0.8 cM interval. Flanking markers were used to screen the Langdon durum BAC library and contigs of 205 and 228 kb flanking Tsn1 were assembled, sequenced, and anchored to the genetic map. Recombination frequency averaged 760 kb/cM across the 228 kb contig, but no recombination was observed across the 205 kb contig resulting in an expected recombination frequency of more than 10 Mb/cM. Therefore, chromosome walking within the Tsn1 region may be difficult. However, the sequenced BACs allowed the identification of one microsatellite in each contig for which markers were developed and shown to be highly suitable for marker-assisted selection of Tsn1.

A toxin, designated SnTox1, was partially purified from culture filtrates of isolate Sn2000 of Stagonospora nodorum, the causal agent of wheat leaf and glume blotch. The toxin showed selective action on several different wheat genotypes, indicating that it is a host-selective toxin (HST). The toxic activity was reduced when incubated at 50°C and activity was eliminated when treated with proteinase K, suggesting that the HST is a protein. The synthetic hexaploid wheat W-7984 and hard red spring wheat Opata 85, the parents of the International Triticeae Mapping Initiative (ITMI) mapping population, were found to be sensitive and insensitive, respectively, to SnTox1. The ITMI mapping population was evaluated for toxin reaction and used to map the gene conditioning sensitivity. This gene, designated Snn1, mapped to the distal end of the short arm of chromosome 1B. The wheat cv. Chinese Spring (CS) and all CS nullisomic-tetrasomic lines were sensitive to the toxin, with the exception of N1BT1D. Insensitivity also was observed when the 1B chromosome of CS was substituted with the 1B chromosome of an insensitive accession of Triticum dicoccoides. In addition, a series of 1BS chromosome deletion lines were used to physically localize the sensitivity gene. Physical mapping indicated that Snn1 lies within a major gene-rich region on 1BS. This is the first report identifying a putative proteinaceous HST from S. nodorum and the chromosomal location of a host gene conferring sensitivity.

A fundamental problem of plant science is to understand the biochemical basis of plant/pathogen interactions. The foliar disease tan spot of wheat (Triticum aestivum), caused byPyrenophora tritici-repentis, involves Ptr ToxA, a proteinaceous host-selective toxin that causes host cell death. The fungal gene ToxA encodes a 17.2-kD pre-pro-protein that is processed to produce the mature 13.2-kD toxin. Amino acids 140 to 142 of the pre-pro-protein form an arginyl-glycyl-aspartic (RGD) sequence, a motif involved in the binding of some animal proteins and pathogens to transmembrane receptor proteins called integrins. Integrin-like proteins have been identified in plants recently, but their role in plant biology is unclear. Our model for Ptr ToxA action predicts that toxin interacts with a putative host receptor through the RGD motif. Mutant clones of a ToxA cDNA, created by polymerase chain reaction such that the RGD in the pro-toxin was changed to arginyl-alanyl-aspartic or to arginyl-glycyl-glutamic, were expressed in Escherichia coli. Extracts containing mutated forms of toxin failed to cause host cell death, but extracts from E. coliexpressing both a wild-type pro-protein cDNA and a control mutation away from RGD were active in cell death development. In competition experiments, 2 mm RGD tripeptide reduced the level of electrolyte leakage from wheat leaves by 63% when co-infiltrated with purified Ptr ToxA (15 μg mL−1) obtained from the fungus, but the control peptide arginyl-glycyl-glutamyl-serine provided no protection. These experiments indicate that the RGD motif of Ptr ToxA is involved with toxin action, possibly by interacting with a putative integrin-like receptor in the host.

Mutation and chromosomal rearrangements are the two main forces of increasing genetic diversity for natural selection to act upon, and ultimately drive the evolutionary process. Although genome evolution is a function of both forces, simultaneously, the ratio of each can be varied among different genomes and genomic regions. It is believed that in plant mitochondrial genome, rearrangements play a more important role than point mutations, but relatively few studies have directly addressed this phenomenon. To address this issue, we isolated and sequenced the ATP6-1 and ATP6-2 genes from 46 different euplasmic and alloplasmic wheat lines. Four different ATP6-1 orthologs were detected, two of them reported for the first time. Expression analysis revealed that all four orthologs are transcriptionally active. Results also indicated that both point mutation and genomic rearrangement are involved in the evolution of ATP6. However, rearrangement is the predominant force that triggers drastic variation. Data also indicated that speciation of domesticated wheat cultivars were simultaneous with the duplication of this gene. These results directly support the notion that rearrangement plays a significant role in driving plant mitochondrial genome evolution.

Background Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line. Results The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line. Conclusions Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

The fungus Pyrenophora tritici-repentis produces a toxin (Ptr ToxA) that causes rapid cell necrosis in sensitive wheat genotypes. A single recessive gene (tsn1) on chromosome 5BL in common wheat confers insensitivity to this toxin. Our objectives were to analyze the allelic relationships of genotypes that have shown insensitivity to a P. tritici-repentis necrosis-inducing toxin, map the gene for insensitivity to the necrosis-inducing factor produced by P. tritici-repentis in a durum wheat population, and determine the reaction to P. tritici-repentis of aneuploid genotypes that do not contain the gene. Greenhouse-grown plants of seven populations from crosses of insensitive genotypes; an F2 population of durum wheat; and ‘Chinese Spring’ aneuploid, substitution, and deletion lines were infiltrated with Ptr ToxA. All crosses involving insensitive genotypes failed to produce sensitive progeny, indicating that the same gene is present in these genotypes. The gene for insensitivity in the durum population was mapped to the same region on 5BL as in common wheat using restriction fragment length polymorphism markers. ‘Chinese Spring’, its homoeologous group 5 nullisomic-tetrasomic stocks, and 5BL deletion lines were insensitive to the toxin. Substitution of a 5B chromosome from sensitive genotypes into ‘Chinese Spring’ resulted in sensitivity. Therefore, insensitivity is not conferred by a gene product per se, but rather conferred by absence of a gene for sensitivity.

(1998). Standardization of toxin nomenclature in the Pyrenophora tritici-repentis/wheat interaction. Canadian Journal of Plant Pathology: Vol. 20, No. 4, pp. 421-424.

Cultivar-specific toxic metabolites of Pyrenophora tritici-repentis are involved in the appearance of necrotic and chlorotic foliar lesions characteristic of tan spot. A P. tritici-repentis necrosis-inducing toxin, Ptr necrosis toxin, was purified from isolate 86-124, sequenced by gas-phase amino acid microsequencing, and characterized by circular dichroism (CD) spectroscopy and isoelectric focusing. The purified protein had a similar amino acid composition and molecular weight as previously reported. Analysis of the CD spectrum from 178 to 250 nm indicated a protein consisting of 13% alpha-helix, 36% antiparallel beta-sheet, 25% turns, and 25% other structures. The Ptr necrosis toxin from isolate 86-124 has an isoelectric point near pH 10. Using overlapping proteolytic fragments obtained from the toxin, a sequence of 101 continuous amino acids was obtained, but the amino terminus was blocked and 9 to 16 amino acids could not be sequenced. Secondary structure prediction based on the amino acid sequence indicated a beta-sheet protein with little alpha-helix, which is in agreement with the structure determined by CD spectroscopy. Sequence analysis indicated the presence of a possible membrane adhesion site and several possible phosphorylation sites that may be involved in phytotoxicity.

Ptr ToxA, the host-selective toxin produced by Pyrenophora tritici-repentis, is genetically associated with the development of tan spot disease of wheat. The toxin was shown previously to cause a programmed cell death in the host that requires de novo mRNA and protein synthesis. In the present study, inhibitors of plant signaling mechanisms protected wheat leaves from toxin action, as determined by electrolyte leakage bioassays, when applied to leaves with toxin. Okadaic acid, calyculin A and phenylarsine oxide, all inhibitors of protein phosphatase activity, reduced toxin-induced electrolyte leakage by more than 90%. Inorganic calcium channel blockers (LaCl3 and CoCl2 reduced toxin-induced electrolyte leakage by 78–95%, depending on inhibitor and time of measurement. By comparison, about 50% protection was achieved by the application of the protein kinase inhibitors staurosporine and K-252A. Nonetheless, the reduction in toxin-induced electrolyte leakage by protein kinase inhibitors was reproduced in multiple trials and was statistically significant. The data indicate that host signaling mechanisms, including calcium fluxes and a protein phosphorylation cascade, are required for the Ptr ToxA-induced cell death in wheat. Our current model holds that the signaling events occur between toxin perception by the cell and the toxin-directed gene expression in the host associated with cell death. As an alternative, the toxin-induced mRNA synthesis required for cell death may be for protein phosphatase and/or protein kinase genes. Additional work is required to resolve these possibilities.

Arabinoxylans (AX), xylanase, and xylanase inhibitors have an important role in many cereal food processing applications. The effects of genotype, growing location, and their interaction (G × L) on AX, apparent xylanase activity, and apparent xylanase inhibition activity of Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) were investigated for six hard red and six hard white spring wheat genotypes grown at three locations. Difference in total AX level among genotypes was not determined to a significant level by genotype. Instead, variability in total AX content was largely dependent on G × L. However, total AX content was significantly different between the two wheat classes. For bran xylanase activity, 25% of the variability could be attributed to G × L interaction. Moreover, there was significant difference between the bran xylanase activities in the two wheat classes. Bran TAXI activity and XIP activity were significantly different among genotypes. Genotype con...

This paper reports the first isolation from cultures of two endoxylanases secreted by Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch]. When F. graminearum is grown on wheat bran hydrated with a modified synthetic medium, high xylanase activity can be extracted. The two endoxylanases were identified by LC-MS/MS as the products of genes FGSG_6445 (Genbank gene id 2788192 ) (xylanase 1) and FGSG_3624 (GenBank accession no. AJ863566 ) (xylanase 2) with 61 and 51% sequence coverage, respectively. Both enzymes showed a pH optimum at pH 6, with xylanase 1 exhibiting a wider active pH range (5.5-9) than xlylanase 2 (5.5-7.5). Their temperature dependences were similar,60% between 35 and 60 °C, with optimal temperatures of 45 °C for xylanase 1 and 50 °C for xylanase 2. Kinetic studies found that both enzymes had a lower K(m) for linear beachwood xylan than arabinoxylan. For xylanase 2, the V(max) increased with arabinoxylan, but decreased for xylanase 1.

Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K+ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a Ki of 1 μM. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a Ki of 0.006 μM, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 μM and 24 μM. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 μM. NADH dehydrogenase gave Km values of 1 μM for NADH and 4 μM for reduced hypoxanthine adenine dinucleotide.

Crude membrane preparation fromFibrobacter succinogenes S85 were investigated and found to contain NADH dehydrogenase (NADH:decylubiquinone oxidoreductase) and NADH-linked fumarate reductase activities. Under aerobic conditions the maximum NADH dehydrogenase activity (252 nmoles/min/mg protein) was ten times greater than that of NADH-fumarate reductase (23 nmoles/min/mg protein). NADH-fumarate reductase was strongly inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), rotenone, HgCl2, ando-phenanthroline. Inhibition of the NADH dehydrogenase by the first three compounds, particularly rotenone, accounted for most of the effects on NADH-fumarate reductase. The α-band of ab-type cytochrome was resolved into two cytochromes, a cytochromeb 560 (oxidized by addition of HOQNO) and a cytochromeb 563 (oxidized by subsequent addition of fumarate).

We investigated the use of electron spin resonance spectroscopy (ESR) and the spin labelled fatty acid, 16 doxylstearic acid (16-DSA) in the study of starch and protein modification during extrusion processing of corn starch (CS), corn meal (CM), and their blends with sunflower protein isolate (SPI). Spectra due to binding of the spin probe to starch and protein were identified. Changes in spectra after extrusion of all materials indicated that: (1) ~90% of the starch was modified (gelatinized); (2) The globular protein structure was disrupted. These changes demonstrate that spin probes and ESR spectroscopy may be used to study starch and protein modification during extrusion and other food processes.

Gene cloning is the first step of targeted gene replacement for functional studies, discovery of gene alleles, and gene expression among other applications. In this chapter, we will describe a cloning technique suitable for fungal species where the genomic information and sequences available are limited. This strategy involves obtaining protein sequences of the gene of interest from various organisms to identify at least two conserved regions. Degenerate primers are designed from these two conserved regions and the resulting PCR products are sequenced. The sequence of the PCR products can be analyzed using suitable databases to determine their similarity to the gene/protein of interest. In cases where the entire gene cannot be cloned directly using these primers, this initial nucleotide sequence can be used as a template for further primer design and genome walking in both directions for either the cloning of a longer fragment or even the cloning of the complete gene. Here, we describe the partial cloning of a reducing polyketide synthase gene from the fungal plant pathogen Ascochyta rabiei using this strategy.

Many bacteria contain proton-translocating membrane-bound NADH-quinone oxidoreductases (NDH-1), which demonstrate significant genetic, spectral, and kinetic similarity with their mitochondrial counterparts. This review is devoted to the comparative aspects of the iron-sulfur cluster composition of NDH-1 from the most well-studied bacterial systems to date.: Paracoccus denitrificans, Rhodobacter sphaeroides, Escherichia coli, and Thermus thermophilus. These bacterial systems provide useful models for the study of coupling Site I and contain all the essential parts of the electron-transfer and proton-translocating machinery of their eukaryotic counterparts.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.

The host-selective toxin Ptr ToxA is produced by races 1 and 2 of Pyrenophora tritici-repentis, causal agent of tan spot of wheat. Ptr ToxA has been causally associated with pathogenicity by the race 2 phenotype in this system. However, the role of toxin in disease caused by race 1, the most prevalent form of the fungus in the central and northern Great Plains of North America, has not been rigorously investigated. Three independent wheat lines harboring mutations for insensitivity to Ptr ToxA were derived from ethylmethane sulfonate treatment of the hard red spring wheat cv. Kulm, possessing the single dominant gene for toxin sensitivity. Each of the three mutants was insensitive to Ptr ToxA in bioassays based on necrosis development and electrolyte leakage. Each mutant was crossed to each of the other mutants and to the wild-type Kulm. Segregation data indicate that each mutant line harbors a single recessive mutation for toxin insensitivity that maps to or near the same locus, possibly the toxin-sensitivity gene. Each toxin-insensitive mutant line was susceptible to two isolates of race 1 of P. tritici-repentis. F2 and F3 generations derived from crosses between Kulm and each mutant segregated for toxin reaction. However, segregation for fungal reaction was not evident, and all F3 families were tan spot susceptible regardless of toxin reaction. Host insensitivity to Ptr ToxA is not necessarily equivalent to resistance to race 1. Ptr ToxA should not be used alone as a proxy for fungal inoculations by breeding programs aimed at developing tan spot-resistant wheat.

Stagonospora nodorum leaf blotch is an economically important foliar disease in the major wheat-growing areas of the world. In related work, we identified a host-selective toxin (HST) produced by the S. nodorum isolate Sn2000 and determined the chromosomal location of the host gene (Snn1) conditioning sensitivity to the toxin using the International Triticeae Mapping Initiative mapping population and cytogenetic stocks. In this study, we used the same plant materials to identify quantitative trait loci (QTL) associated with resistance to fungal inoculations of Sn2000 and investigate the role of the toxin in causing disease. Disease reactions were scored at 5, 7, and 10 days postinoculation to evaluate changes in the degree of effectiveness of individual QTL. A major QTL was identified on the short arm of chromosome 1B, which coincided with the snn1 toxin-insensitivity gene. This locus explained 58% of the phenotypic variation for the 5-day reading but decreased to 27% for the 10-day reading, indicating that the toxin is most effective in the early stages of the interaction. In addition, relatively minor QTL were identified on chromosomes 3AS, 3DL, 4AL, 4BL, 5DL, 6AL, and 7BL, but not all minor QTL were significant for all readings and their effects varied. Multiple regression models explained from 68% of the phenotypic variation for the 5-day reading to 36% for the 10-day reading. The Chinese Spring nullisomic 1B tetrasomic 1D line and the Chinese Spring-Triticum dicoccoides disomic 1B chromosome substitution line, which were insensitive to SnTox1, were more resistant to the fungus than the rest of the nullisomictetrasomic and disomic chromosome substitution lines. Our results indicate that the toxin produced by isolate Sn2000 is a major virulence factor.

Culture filtrate from Pyrenophora tritici-repentis race 1 isolate 78-62 contained a genotype-specific toxin which elicited extensive chlorosis on sensitive wheat genotypes. This toxin was partially purified using gel filtration, ion exchange, and reversed-phase chromatography. The chlorosis toxin was found to be a polar, nonionic, low-molecular-weight molecule. Wheat genotypes infiltrated with crude culture filtrate and partially purified chlorosis toxin exhibited the same chlorotic symptoms seen with conidial inoculations of isolate 78-62. All tested wheat genotypes that exhibited extensive chlorosis to the toxin also exhibited extensive chlorosis to conidial inoculations, and all wheat genotypes insensitive to the toxin did not exhibit extensive chlorosis to conidial inoculations. The recombinant inbred population derived from the cross W-7984 × Opata 85 segregated for chlorosis induction from infiltration with partially purified chlorosis toxin from isolate 78-62. The locus identified by the marker XGli1, associated with resistance to conidial inoculations from race 1 isolates Pti2 and 78-62 and race 3 isolate D308, also was associated with insensitivity to infiltration of crude culture filtrate and partially purified chlorosis toxin. The marker XGli1, located on the short arm of chromosome 1A, is linked to the insensitivity locus within 5.7 cM. We propose that this chlorosis toxin be designated Ptr ToxC.

The pathogen Stagonospora nodorum produces multiple hostselective toxins (HSTs) that induce cell death and necrosis in sensitive wheat (Triticum sp.) genotypes. One such HST is SnTox1, which interacts with the host gene Snn1 on wheat chromosome arm 1BS to cause necrosis leading to disease susceptibility. Toward the positional cloning of Snn1, we developed saturated and high-resolution maps of the Snn1 locus and evaluated colinearity of the region with rice (Oryza sativa L.). An F2 population of 120 individuals derived from ‘Chinese Spring’ (CS) and the CS–T. dicoccoides chromosome 1B disomic substitution line was used to map 54 markers consisting of restriction fragment length polymorphisms (RFLPs), simple sequence repeats, and bin mapped expressed sequence tags (ESTs). Colinearity between wheat 1BS and rice was determined by aligning EST and RFLP probe sequences to the rice genome. Overall, colinearity was poorly conserved due to numerous complex chromosomal rearrangements, and of 48 wheat EST-RFLP sequences mapped, 30 had signifi cant similarity to sequences on nine different rice chromosomes. However, 12 of the wheat sequences had similarity to sequences on rice chromosome 5 and were in a colinear arrangement with only a few exceptions, including an inversion of the markers fl anking Snn1. High-resolution mapping of the Snn1 locus in 8510 gametes delineated the gene to a 0.46-cM interval. Two EST-derived markers that cosegregated with Snn1 were found to share homology to nucleotide binding site–leucine rich repeat–like genes and are considered potential candidates for Snn1.

Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8. Three of these clusters (designated as [N-1H]T, [N-2H]T, and [N-3]T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively. These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I). They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex. Two additional very low potential iron-sulfur clusters (one binuclear, [N-1L]T, and one tetranuclear, [N-2L]T) were observed in membrane particles. These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively. No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster [N-2]B was found in this bacterial system. In membrane particles isolated from T. thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials [Em9(Q/QH2) = 40, -100, -160, -300 mV] and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide. Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase. Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T. thermophilus HB-8.

We recently showed that the wheat pathogen Stagonospora nodorum produces proteinaceous host-selective toxins (HSTs). These toxins include SnTox1 as well as SnToxA, a HST first identified from Pyrenophora tritici-repentis that was implicated in a very recent horizontal gene transfer event from S. nodorum to P. tritici-repentis. Compelling evidence implicating SnToxA and SnTox1 in disease development has been obtained. Here, we report the partial purification and characterization of a third HST designated SnTox2, as well as the genetic characterization of the corresponding host-sensitivity gene. SnTox2 was protease sensitive and is estimated between 7 and 10 kDa in size. Sensitivity to SnTox2 was conferred by a single dominant gene designated Snn2, which mapped to the short arm of wheat chromosome 2D. Genetic analysis of reaction to conidial inoculations in a segregating wheat population indicated that both the Snn2-SnTox2 and the Tsn1-SnToxA interactions were involved in disease development, and together they accounted for the majority of the phenotypic variation. Therefore, S. nodorum produces multiple toxins that rely on specific interactions with host gene products to cause disease. The identification of multiple HST-host gene interactions important for disease development and the availability of the S. nodorum whole genome sequence indicate the potential for this pathosystem to serve as a toxin-based, inverse gene-for-gene model.

The wheat tan spot fungus ( Pyrenophora tritici-repentis ) produces a well-characterized host-selective toxin (HST) known as Ptr ToxA, which induces necrosis in genotypes that harbor the Tsn1 gene on chromosome 5B. In previous work, we showed that the Stagonospora nodorum isolate Sn2000 produces at least 2 HSTs (SnTox1 and SnToxA). Sensitivity to SnTox1 is governed by the Snn1 gene on chromosome 1B in wheat. SnToxA is encoded by a gene with a high degree of similarity to the Ptr ToxA gene. Here, we evaluate toxin sensitivity and resistance to S. nodorum blotch (SNB) caused by Sn2000 in a recombinant inbred population that does not segregate for Snn1. Sensitivity to the Sn2000 toxin preparation cosegregated with sensitivity to Ptr ToxA at the Tsn1 locus. Tsn1-disrupted mutants were insensitive to both Ptr ToxA and SnToxA, suggesting that the 2 toxins are functionally similar, because they recognize the same locus in the host to induce necrosis. The locus harboring the tsn1 allele underlies a major quantitative trait locus (QTL) for resistance to SNB caused by Sn2000, and explains 62% of the phenotypic variation, indicating that the toxin is an important virulence factor for this fungus. The Tsn1 locus and several minor QTLs together explained 77% of the phenotypic variation. Therefore, the Tsn1–ToxA interaction in the wheat–S. nodorum pathosystem parallels that of the wheat–tan spot system, and the wheat Tsn1 gene serves as a major determinant for susceptibility to both SNB and tan spot.

A characteristic feature of infections with the nematode parasite of mice Heligmosomoides polygyrus, is a marked IgG1 hypergammaglobulinaemia. A possible source for this immunoglobulin has recently been demonstrated, through evidence that H. polygyrus adult worm homogenate (AWH) can induce the in vitro production of non-specific IgG1 from mouse lymphocytes. To determine the interactions between this immunoglobulin and the parasite, the ability of IgG1 to bind to AWH of H. polygyrus was investigated. Protein (Western) blotting indicated that mouse monoclonal antibodies are able to bind non-specifically to selected parasite antigens. Furthermore, by binding H. polygyrus adult worm homogenate to cyanogen bromide (CNBr)-activated Sepharose CL-4B, an affinity column was prepared which could be used to efficiently purify mouse IgG1 monoclonal antibodies. These antibodies were eluted from the affinity column and still retained their original specificity. These results indicate that H. polygyrus not only induces the production of non-specific IgG1 by the host, it can also bind this immunoglobulin to its own specific proteins. Thus, it is possible that IgG1 produced during a primary infection with H. polygyrus may not entirely benefit the host.

The technique of distance measurement, utilizing spin relaxation enhancement by an external probe, has been extended to the study of intrinsic semiquinone radicals through the use of holmium-EDTA complexes and continuous wave electron paramagnetic resonance spectroscopy. This technique has been used to determine the distance of the semiquinone anion, Qi (also designated as Qn.- or Qc.-), from the surface of the ubiquinone cytochrome c oxidoreductase, consisting of only three subunits, in membrane particles from Rhodobacter capsulates. The location of the semiquinone anion is 6-10 A from the N side protein, establishing that there are two separate quinone reaction sites, i.e., 'Qi' and 'Qo', within this complex on opposite sides of the membrane. The results are discussed in relation to reported ENDOR, EPR, and optical studies of the mitochondrial counterpart.

We have examined the spatial organization of the redox active centers in the Site II segment of the bovine heart respiratory chain by using reconstituted proteoliposomes of ubiquinol-cytochrome c oxidoreductase (Complex III or cytochrome bc1 complex) and EPR techniques. 1) Mutual spin-spin interactions between intrinsic redox active centers were detected. The spin relaxation of the Rieske iron-sulfur cluster was enhanced by the paramagnetic cytochrome c1 and b566 hemes but not by cytochrome b562. 2) Relative distances of the individual redox active centers to the P-side and N-side surfaces of the reconstituted Complex III proteoliposome were measured by our paramagnetic probe method (Blum, H., Bowyer, J. R., Cusanovich, M. A., Waring, A. J., and Ohnishi, T. (1983) Biochim. Biophys. Acta 748, 418-428). The cytochrome b562 heme was shown to be close to the middle of the phospholipid bilayer, while the Rieske iron-sulfur cluster and cytochrome b566 heme were assigned to be near the P-side surface level of the membrane. This probe method is a low resolution technique from the structural viewpoint; however, it can provide direct and reliable assignment of the topographical locations of redox active centers within the membrane. This is the first direct demonstration of the transmembranous location of the two cytochrome b hemes, although electron transfer between these two hemes crosses only half of the membrane thickness. Our data support the assignment of transmembranous distribution of the redox active centers based on electrochromic measurements (Robertson, D.E., and Dutton, P.L. (1988) Biochim, Biophys. Acta 935, 273-291). The implication of these results on the mechanism of Site II energy coupling is discussed.

We have examined the thermodynamic and EPR properties of one of the ubiquinol oxidase systems (the cytochrome d complex) of Escherichia coli, and have assigned the EPR-detectable signals to the optically identified cytochromes. The axial high spin g = 6.0 signal has been assigned to cytochrome d based on the physicochemical properties of this signal and those of the optically defined cytochrome d. A rhombic low spin species at gx,y,z = 1.85, 2.3, 2.5 exhibited similar properties but was present at only one-fifth the concentration of the axial high spin species. Both species have an Em7 of 260 mV and follow a -60 mV/pH unit dependence from pH 6 to 10. The rhombic high spin signal with gy,z = 5.5 and 6.3 has been assigned to cytochrome b-595. This component has an Em7 of 136 mV and follows a -30 mV/pH unit dependence from pH 6 to 10. Lastly, the low spin gz = 3.3 signal which titrates with an Em7 of 195 mV and follows a -40 mV/pH unit dependence from pH 6 to 10 has been assigned to cytochrome b-558. Spin quantitation of the high-spin signals indicates that cytochrome d and b-595 are present in approximately equal amounts. These observations are discussed in terms of the stoichiometry of the prosthetic groups and its implications on the mechanism of electron transport.

Spectrophotometric, kinetic, thermodynamic and stoichiometric properties of the low-potential b-type cytochrome of chromatophores from Rhodopseudomonas sphaeroides are reported. Cytochrome b-566 has a double α-band with maxima at 559 and 566 nm. Resolution of the spectrum by full-spectral redox potentiometry showed no indication that the two peaks represent more than one component. The component titrated with Em,7 ≈ −80 ± 10 mV. By appropriate choice of wavelength pairs and by subtraction of the contribution due to other components, the kinetics of cytochrome b-566 absorbance changes following flash excitation have been resolved from those of other components. Time-resolved flash spectra corrected for the contributions of other components are consistent with the behavior of both peaks of the α-band as a single kinetic species. The kinetics of cytochrome b-566 in the presence of antimycin show that the reduction of this cytochrome occurred only if cytochrome b-561 was reduced before the flash, either chemically, by poising the ambient redox potential (Eh) below the Em of cytochrome b-561 (Em,7 ≈ 50 mV), or photochemically at higher redox potentials by a previous flash. The rate of reduction of cytochrome b-566 varied with Eh. At low Eh (approx. 0 mV) reduction on the first flash showed t 1 2 ≈ 1.25 ms ; at high Eh (approx. 180 mV) reduction on the second flash showed t 1 2 ≈ 10 ms . In the absence of antimycin at Eh ≈ 0 mV, cytochrome b-566 was observed to become rapidly reduced ( t 1 2 ≈ 500 μ s ) and then reoxidized ( t 1 2 ≈ 2 ms ) after a single flash. At higher redox potentials (Eh > 80 mV) no kinetic changes which could be unambiguously attributed to cytochrome b-566 were observed following a single flash. The results are interpreted in terms of a Q-cycle mechanism in which the reductant for cytochrome b-566 is the semiquinone formed on oxidation of ubiquinol from the quinone pool. The oxidation of the ubiquinol occurs by a concerted reaction in which one electron is accepted by the Rieske-type FeS center and the other by cytochrome b-566. We suggest that the kinetic characteristics may indicate a pathway for reduction of the b-type cytochromes in which cytochrome b-566 is the immediate electron acceptor and donates to cytochrome b-561 in a serial pathway. The experimental results in the presence of antimycin are compared with data from a computer simulation of the thermodynamic behavior of the chain, and the computer model is shown to provide an excellent fit.

The ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans exhibits a thermodynamically stable ubisemiquinone radical detectable by EPR spectroscopy. The radical is centered at g = 2.004, is sensitive to antimycin, and has a midpoint potential at pH 8.5 of +42 mV. These properties are very similar to those of the stable ubisemiquinone (Qi) previously characterized in the cytochrome bc1 complexes of mitochondria. The micro-environment of the Rieske iron-sulfur cluster in the Paracoccus cytochrome bc1 complex changes in parallel with the redox state of the ubiquinone pool. This change is manifested as shifts in the gx, gy, and gz values of the iron-sulfur cluster EPR signal from 1.80, 1.89, and 2.02 to 1.76, 1.90, and 2.03, respectively, as ubiquinone is reduced to ubiquinol. The spectral shift is accompanied by a broadening of the signal and follows a two electron reduction curve, with a midpoint potential at pH 8.5 of +30 mV. A hydroxy analogue of ubiquinone, UHDBT, which inhibits respiration in the cytochrome bc1 complex, shifts the gx, gy, and gz values of the iron-sulfur cluster EPR signal to 1.78, 1.89, and 2.03, respectively, and raises the midpoint potential of the iron-sulfur cluster at pH 7.5 from +265 to +320 mV. These changes in the micro-environment of the Paracoccus Rieske iron-sulfur cluster are like those elicited in mitochondria. These results indicate that the cytochrome bc1 complex of P. denitrificans has a binding site for ubisemiquinone and that this site confers properties on the bound ubisemiquinone similar to those in mitochondria. In addition, the line shape of the Rieske iron-sulfur cluster changes in response to the oxidation-reduction status of ubiquinone, and the midpoint of the iron-sulfur cluster increases in the presence of a hydroxyquinone analogue of ubiquinone. The latter results are also similar to those observed in the mitochondrial cytochrome bc1 complex. However, unlike the mitochondrial complexes, which contain eight to 11 polypeptides and are thought to contain distinct quinone binding proteins, the Paracoccus cytochrome bc1 complex contains only three polypeptide subunits, cytochromes b, c1, and iron-sulfur protein. The ubisemiquinone binding site and the site at which ubiquinone and/or ubiquinol bind to affect the Rieske iron-sulfur cluster in Paracoccus thus exist in the absence of any distinct quinone binding proteins and must be composed of domains contributed by the cytochromes and/or iron-sulfur protein.

The physicochemical properties of the iron-sulfur clusters present in the NADH:ubiquinone oxidoreductase of Paracoccus denitrificans have been examined in the cytoplasmic membrane particles by redox potentiometry and EPR spectroscopy. Analogous to the iron-sulfur clusters present in the mitochondrial NADH: ubiquinone oxidoreductase, we have found two binuclear and three tetranuclear EPR detectable iron-sulfur clusters, namely, N-1a, N-1b, N-2, N-3, and N-4. In the bacterial system, the two binuclear clusters differ in line shape and in Em values; the cluster with more rhombic symmetry (gx,y,z = 1.918, 1.937, 2.029) has the Em7.0 value of -150 while the almost axial one (gx,y,z = 1.929, 1.941, 2.019) has Em7.0 of -270 mV. The Em of the former cluster is pH dependent (-60 mV/pH) as in the case of mammalian N-1a while the latter is pH independent as is the mammalian cluster N-1b. The pH-dependent P. denitrificans [2Fe-2S] cluster, which we have labeled N-1a, has an Em7.0 as high as that of N-2, in contrast to the mammalian N-1a. Thus N-1a is reducible with a physiological reductant, NADH in this bacterial system. The Em of the cluster N-2 is also pH dependent (Em7.0 = -130 mV) with a pK value near 7.7. The Em values of all other clusters exhibit no pH dependence as in the case of their mammalian counterparts. We have found that the cluster N-1a is the most labile component among the five iron-sulfur clusters and may give rise to variable relative spin concentrations and extremely low Em values due to the facile modifications of the microenvironment of the cluster. The P. denitrificans NADH:ubiquinone oxidoreductase provides a unique and useful site I model system where redox composition is similar to the mitochondrial enzyme but with fewer numbers of polypeptides (Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311).

The energy coupled NADH-ubiquinone (Q) oxidoreductase segment of the respiratory chain of Escherichia coli GR19N has been studied by EPR spectroscopy. Previously Matsushita et al. [(1987) Biochemistry 26, 7732-7737] have demonstrated the presence of two distinct NADH-Q oxidoreductases in E. coli membrane particles and designated them NADH dh I and NADH dh II. Although both enzymes oxidize NADH, only NADH dh I is coupled to the formation of the H+ electrochemical gradient. In addition to NADH, NADH dh I oxidizes nicotinamide hypoxanthine dinucleotide (deamino-NADH), while NADH dh II does not. In membrane particles we have detected EPR signals arising from four low-potential iron-sulfur clusters, one binuclear, one tetranuclear, and two fast spin relaxing g perpendicular = 1.94 type clusters (whose cluster structure has not yet been assigned). The binuclear cluster, temporarily designated [N-1]E, shows an EPR spectrum with gx,y,z = 1.92, 1.935, 2.03 and the Em7.4 value of -220 mV (n = 1). The tetranuclear cluster, [N-2]E, elicits a spectrum with gx,y,z = 1.90, 1.91, 2.05 and an Em7.4 of -240 mV (n = 1). These two clusters have been shown to be part of the NADH dh I complex by stability and inhibitor studies. When stored at 4 degrees C, both clusters are extremely labile as is the deamino-NADH-Q oxidoreductase activity. Addition of deamino-NADH in the presence of piericidin A results in nearly full reduction of [N-2]E within 17 s. In membrane particles pretreated with piericidin A, the cluster [N-1]E is only partly reducible by deamino-NADH and shows an altered line shape.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to a single complementation group (G12) have been determined to have lesions in the iron-sulfur protein (Rieske protein) of ubiquinol: cytochrome c reductase. Mutants capable of expressing the protein were chosen for further studies. The genes from 13 independent isolates were cloned and their mutations sequenced. Twelve mutations were ascertained to cause single amino acid substitutions in the carboxyl-terminal regions of the protein between residues 127 and 173. This region is proposed to be part of the catalytic domain with the ligands responsible for co-ordinating the two irons of the 2Fe-2S cluster. Based on the catalytic properties of the ubiquinol: cytochrome c reductase complex and the electron paramagnetic resonance (e.p.r.) signals of the iron-sulfur protein, the mutants describe two different phenotypes. A subset of mutants have no detectable iron-sulfur cluster and are completely deficient in ubiquinol: cytochrome c reductase activity. These strains identify mutations in residues considered to be essential for binding of the iron or for maintaining a proper tertiary structure of the catalytic domain. A second group of mutants have reduced levels of enzymatic activity and exhibit e.p.r. spectra characteristic of the Rieske iron-sulfur cluster. The mutations in the latter strains have been ascribed to residues that influence the redox properties of the cluster by distorting the iron-binding pocket. A secondary and tertiary structure model is presented of the carboxyl-terminal 65 residues constituting the catalytic domain of the iron-sulfur protein. It is postulated that the two irons of the cluster are co-ordinated by three cysteine and a single histidine residue located in a loop structure. The catalytic domain also contains two short a-helices and three β-strands that form a partial β-barrel. Most of the hydrophilic amino acids are present in turns that map to one pole of the domain. When viewed in the context of the model, mutations that abolish the iron-sulfur cluster are mostly in residues defining the boundaries of the α-helices and β-strands. The notable exception is a cysteine residue that has been assigned to the loop with the iron ligands. This cysteine residue is proposed to co-ordinate one iron of the cluster. Mutations that reduce ubiquinol: cytochrome c reductase activity and alter the redox potential of the cluster occur in residues located in the loop that contains the ligands of the cluster.

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c(2) oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c(1) and c(2), the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c(1) and c(2), and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c(2) oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c(1) and c(2) is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.

Cytochrome b-561 of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides is reduced after flash illumination in the presence of myxothiazol in an antimycin-sensitive reaction. Flash-induced reduction was observed over the redox range in which cytochrome b-561 and the Q-pool are both oxidized before the flash. The extent of reduction increased with increasing pH, and was maximal at pH greater than 10.0 where the extent approached that observed in the presence of antimycin following a group of flashes. Reduction of cytochrome b-561 in the presence of myxothiazol showed a lag of approximately 1 ms after the flash, followed by reduction with t 1/2 approximately 6 ms; by analogy with the similar kinetics of the quinol oxidase site, we suggest that the rate is determined by collision with the QH2 produced in the pool on flash excitation.