Structural and functional characterization of a winter malting barley.

The development of winter malting barley (Hordeum vulgare L.) varieties is emerging as a worldwide priority due to the numerous advantages of these varieties over spring types. However, the complexity of both malting quality and winter hardiness phenotypes makes simultaneous improvement a challenge. To obtain an understanding of the relationship between loci controlling winter hardiness and malt quality and to assess the potential for breeding winter malting barley varieties, we structurally and functionally characterized the six-row accession "88Ab536", a cold-tolerant line with superior malting quality characteristics that derives from the cross of NE76129/Morex//Morex. We used 4,596 SNPs to construct the haplotype structure of 88Ab536 on which malting quality and winter hardiness loci reported in the literature were aligned. The genomic regions determining malting quality and winter hardiness traits have been defined in this founder germplasm, which will assist breeders in targeting regions for marker-assisted selection. The Barley1 GeneChip array was used to functionally characterize 88Ab536 during malting. Its gene expression profile was similar to that of the archetypical malting variety Morex, which is consistent with their similar malting quality characteristics. The characterization of 88Ab536 has increased our understanding of the genetic relationships of malting quality and winter hardiness, and will provide a genetic foundation for further development of more cold-tolerant varieties that have malt quality characteristics that meet or exceed current benchmarks.

Effect of reducing and oxidizing agents and pH on malt endoproteolytic activities and brewing mashes.

The activities of the four endoproteinase classes of malted barley are known to vary with pH, and it seemed likely that the cysteine enzyme activities could be altered by redox agents. This study determined how altering the pH and adding redox agents to mashes influenced the worts that were produced during the brewing process. The reducing agents cysteine.HCl, dithiothreitol, and beta-mercaptoethanol increased the proteolysis that occurred in malt extracts and mashes. This increased proteolysis was negated by the addition of the oxidizing agents diamide or hydrogen peroxide. The addition of reducing agents to mashes increased the soluble protein, free amino nitrogen (FAN), and extract values of their resultant worts, and this effect was abolished by the concomitant addition of oxidizing agents. Raising the pH values of the mashes strongly reduced their proteolytic activities, soluble protein, FAN, and extract values, but not their beta-glucan levels. These results show that several of the major aspects of malting and brewing quality can be adjusted by varying the pH and redox qualitites of mashes, which could be helpful to brewers. These results also strengthen the previous proposal made by Buchanan et al. that the redox status of plants may play a significant part in controlling their physiology.

Characterization of the Ustilago maydis sid2 gene, encoding a multidomain peptide synthetase in the ferrichrome biosynthetic gene cluster.

Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation of L-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase. sid1 encodes L-ornithine N(5)-oxygenase, which catalyzes hydroxylation of L-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. In this report we characterize sid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream of sid1 and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulated sid2 transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption of urbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis in Ustilago. Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1 of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation of sid1, altered the differential regulation of sid2 by iron.

sid1, a gene initiating siderophore biosynthesis in Ustilago maydis: molecular characterization, regulation by iron, and role in phytopathogenicity.

Iron uptake in Ustilago maydis is mediated by production of extracellular hydroxamate siderophores. L-Or-nithine N5-oxygenase catalyzes hydroxylation of L-ornithine, which is the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. We have characterized sid1, a gene coding for this enzyme, by complementation in trans, gene disruption, and DNA sequence analysis. A comparison of genomic DNA and cDNA sequences has shown that the gene is interrupted by three introns. The putative amino acid sequence revealed similarity with Escherichia coli lysine N6-hydroxylase, which catalyzes the hydroxylation of lysine, the first step in biosynthesis of aerobactin. Two transcription initiation points have been determined, both by PCR amplification of the 5' end of the mRNA and by primer extension. A 2.3-kb transcript which accumulates in cells grown under low iron conditions was detected by Northern hybridization. A less abundant 2.7-kb transcript was observed in cells grown in iron-containing medium. By contrast, constitutive accumulation of the 2.3-kb transcript was observed in a mutant carrying a disruption of urbs1, a gene involved in regulation of siderophore biosynthesis. Analysis of the pathogenicity of mutants carrying a null allele of sid1 suggests that the biosynthetic pathway of siderophores does not play an essential role in the infection of maize by U. maydis.

Genome organization of Magnaporthe grisea: genetic map, electrophoretic karyotype, and occurrence of repeated DNAs.

A genetic map of Magnaporthe grisea (anamorph=Pyricularia oryzae and P. grisea), the causal agent of rice blast disease, was generated from segregation data utilizing 97 RFLP markers, two isoenzyme loci and the mating type locus among progeny of a cross between parental strains Guy 11 and 2539. Of the seven chromosomes of M. Grisea, three were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis, while the remaining four migrated as two doublet bands. By utilizing differences between CHEF mobilities of unresolved chromosomes from the parental strains, Southern analysis with selected markers allowed the chromosomal assignment of all linkage groups. A small translocation involving 1 marker was found in the parental strains used to produce the segregating population from which the map was constructed. Nine classes of repetitive DNA elements were found in the genome of a fungal isolate pathogenic to rice. These occurred only a few times or not at all in the genomes of isolates showing reduced virulence on rice. One repetitive DNA was shown to have structural similarity to the Alu sequences found in primates, a sequence similarity to the copia-like elements of Drosophila, and peptide similarity to transposable elements found in Drosophila, other fungi, and higher plants.

Characterization of siderophores from Ustilago maydis.

Two siderophores, ferrichrome and ferrichrome A, were found in cultures of Ustilago maydis (DC) Corda. Both siderophores were found intracellularly and extracellularly. Their authenticity was confirmed by thin layer chromatography, HPLC, UV-visible spectrometry, paper electrophoresis, amino acid analysis, NMR and fast atom bombardment mass spectroscopy. Regulation of siderophore production by iron was examined. Repression of biosynthesis of extracellular siderophores occurred at 10(-5) M iron. Ferrichrome was found intracellularly at all iron concentrations employed; in general, ferrichrome A was not found to be cell-associated.

Analysis of ferrichrome biosynthesis in the phytopathogenic fungus Ustilago maydis: cloning of an ornithine-N5-oxygenase gene.

By using a non-enterobactin-producing enb-7 mutant of Salmonella typhimurium LT2 as a biological indicator, a novel screening method was developed for identifying mutants of Ustilago maydis defective in the biosynthesis of the siderophores ferrichrome and ferrichrome A. Two classes of siderophore mutations, both recessive, were isolated after mutagenesis of haploid cells of the corn smut fungus. Class I mutants no longer produced ferrichrome while retaining the ability to produce ferrichrome A; class II mutants were defective in the production of both ferrichrome and ferrichrome A. Genetic and biochemical data suggest that class II mutants are defective in the ability to hydroxylate L-ornithine to delta-N-hydroxyornithine, the first step in the biosynthesis of these siderophores. A genomic library of wild-type U. maydis DNA was constructed in the cosmid transformation vector pCU3, which contains a dominant selectable marker for hygromycin B resistance. Two cosmids, pSid1 and pSid2, were identified in this library by their ability to complement class II siderophore auxotrophs. The production of both siderophores was concomitantly restored in the majority of the resultant transformants. Transforming DNA could be recovered from the fungal, cosmid-containing transformants by in vitro packaging with lambda bacteriophage extracts. Alternatively, the clones could be identified by a sib selection procedure. Cotransformation was found to occur at a high frequency in the fungus and was used to determine that a 2.5-kilobase HindIII-NruI fragment in pSid1 was responsible for complementing the class II siderophore biosynthetic mutation.

Modification of disease resistance of tobacco callus tissues by cytokinins.

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a "hypersensitive" reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.