Comparison of α-acetolactate synthase and α-acetolactate decarboxylase in Lactococcus spp. and Leuconostoc spp.

Summary Cell-free extracts of Leuconostoc and Lactococcus species were tested for their -acetolactate synthase and -acetolactate decarboxylase activities. In Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. mesenteroides and Leuconostoc lactis, the Km of -acetolactate synthase for pyruvate was close to 10 mM whereas it was 30 mM in Lactococcus lactis subsp. lactis biovar. diacetylactis. The Km of -acetolactate decarboxylase for -acetolactic acid was very low (0.3 mM) in Leuconostoc species in comparison to Lactococcus lactis subsp. lactis biovar. diacetylactis (60 mM). In the latter bacterium, -acetolactate decarboxylase showed a sigmoidal dependance upon -acetolactic acid and was activated by the three branchedchain amino acids: leucine, isoleucine and valine.     

Enzymatic synthesis and characterization of N5-(carboxymethyl)-L-ornithine and N6-(carboxymethyl)-L-lysine

Summary This report describes the enzyme-catalyzed synthesis, characterization, and chromatographic separation of N⁶-(carboxymethyl)-L-lysine and N⁵-(carboxymethyl)-L-ornithine. The two N -(carboxyalkyl)amino acids are formed via a reductive condensation between glyoxylate and the- or-amino groups of lysine and ornithine, respectively. Both reactions are catalyzed by the NADPH-dependent enzyme, N⁵-(carboxyethyl)ornithine synthase [EC 1.5.1.24], found in some strains of the lactic acid bacteriumLactococcus lactis subsp.lactis.     

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of -keto--methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 °C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.     

Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains

Lactic acid bacteria (LAB) have the enzyme potential to transform amino acids into aroma compounds that contribute greatly to cheese flavour. Generally, amino acid conversion by LAB is limited by their low production of -ketoglutarate since this -ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous -ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing -ketoglutarate from glutamate, greatly increased the conversion of amino acid to potent aroma compounds in cheese. Here we report the presence of glutamate dehydrogenase (GDH) activity required for the conversion of glutamate to -ketoglutarate in several natural LAB strains, commonly used in cheese manufacturing. Moreover, we show that the ability of LAB to produce aroma compounds from amino acids is closely related to their GDH activity. Therefore, GDH activity appears to be a major criterion for the selection of flavour-producing LAB strains, which could be used as a starter or as an adjunct to intensify flavour formation in some cheeses.     

Genotype effects on plant regeneration in callus and suspension cultures of Avena

Regenerative potential of the calli of nineteen genotypes of Avena sativa, Avena nuda, Avena byzantina and one interspecific hybrid were compared over three successive cultures. Highly significant genotype and genotype × subculture interactions were observed. Among the highest plant regenerable genotypes were Corbit (first subculture); GAF/Park and 88Ab3073 (second subculture); and GAF/Park and 87Ab5932 (third subculture). These genotypes regenerated on an average 10 to 17 plants each from a 200 mg callus mass after a 30 to 45 proliferation period. GAF/Park, a progeny of an interspecific cross, regenerated plants at a significantly higher level (11.85 plants/rep), followed by the similarly performing A. sativa (6.23 plants) and A. nuda (5.06 plants) genotypes, which were significantly higher than the A. byzantina genotypes (2.07 plants). Four genotypes were tested for their adaptability to suspension culture and plant regeneration potential by separating their cells and cell clusters into two sizes: larger and smaller than 3 mm. Larger clusters yielded plants for three genotypes GAF/Park, 88Ab3073, and Tibor. The smaller clusters only regenerated plants for GAF/Park and 88Ab3073. From one gram of callus used to initiate suspensions of GAF/Park and 88Ab3073, 119.9 and 18.8 plants, respectively, were regenerated. The plants regenerated for various genotypes from agar-solidified or suspension culture experiments had normal growth and seed set. This study confirms high and sustained regenerative capabilities of GAF/Park, a restricted genotype due to the weedy Avena fatua genetic background and identifies alternative genotypes, especially 88Ab3073 for future tissue culture and transformation studies.     

Efficient secretion of human lysozyme from the yeast, <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis, was achieved by using more stable vectors in the order of S11 replication origin-containing episomal vector < full-length K. lactis plasmid pKD1-containing vector < centromeric vector < chromosome-integrated vectors. Cells containing a PGK (phosphoglycerate kinase) promoter-driven integration vector grown in non-selective rich medium achieved the highest level of secretion, 100 g lysozyme secretion ml ^–1 culture: this level was 10-fold higher than that achieved by episomal vectors. An additional copy of the protein disulfide isomerase gene further facilitated the secretion.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Exploration and exploitation of soil by apple,kiwifruit, peach,Asian pear and grape roots

Measurements of root-length density (RLD) in a range of 31 apple, kiwifruit, peach, Asian pear and grape orchards were used to derive indices to describe the exploration and exploitation of rooting volumes. Orchards were of various ages and located on a range of soil types, geographic regions, management systems etc. Data were obtained from core samples of volume 1.66×10^-4 m³ randomly taken within a standard volume, determined by average planting grids, of 2 m radius centred on tree stems, and 1 m depth. Root systems were described using an exploitation index, E(), and an exploration index, E(0). E() is defined as the proportion of the soil volume which contains roots at RLD greater than or equal to some specified value, . E(0) is defined as the proportion of the soil volume which contains roots at any RLD greater than zero. These indices are dependent on sample size, as are all volumetric or soil-coring data.Estimates of E(0) for each orchard were obtained as the proportions of cores containing any RLD>O and assessed for dependence on species. Peach trees had a significantly higher value of E(0), equal to almost 1.0, compared to the other four species where E(0) was approximately 0.8 (p0.01) or less. There was also some variation with age. E(0) was lower for very young plants which had not fully occupied the sampled soil volume. Exploration indices for woody roots increased with rootstock age but otherwise did not explain large differences in E() between species for given values.For example at =0.05×10⁴ m.m^-3, E() was approximately 0.45 for peach and kiwifruit, and 0.05 for apple, Asian pear and grape, whereas at =0.5×10⁴ m.m^-3 the corresponding values were 0.1 and almost zero. Negative exponential curves relating E(), scaled by dividing by E(0), to were fitted for each of the 31 orchards. Exponents for these curves, k, were significantly smaller for kiwifruit and peaches than apples, grapes and Asian pears (p0.05), and smaller for apples than grapes and Asian pears (p0.05). A larger k implies a rapid fall-off in E() as increases. Although all five species contained zero and low RLD samples, only kiwifruit and peaches contained higher RLD values and consequently have higher mean RLD. This trend was consistent across all soils, regions, sampling dates, and plant ages.The analyses demonstrate that core sampling can give useful insights into macro-scale root-system distribution, such as the proportion of a soil volume explored and how it is exploited. If positions of core samples are noted during sampling using angular direction, depth and radial distance as spatial coordinates the method can be used to describe root-system structures.     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis

We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs Davis and Red River, respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The integration of Agrobacterium hormone-related genes into susceptible cv Red River can bring the activation of defense processes to a stable competence as assessed by the inhibition of mycelial growth in dual culture and gem-tube elongation of Fusarium conidia, the determination of callose contents, peroxidase induction and ion leakage in the presence of fusaric acid. This is particularly true when the transformation results in a change of phytohormone equilibria towards an higher cytokin in concentration. On the contrary, in resistant cv Davis the inhibition of both fungal growth in dual culture and conidia germination is higher when the hormone balance is modified in favour of the auxins. No significant effect was observed for ion leakage and peroxidase induction, probably because of a constitutive overproduction of cytokinins in Davis cells.     

Requirement for nonprotonated drug molecules in the direct lethal action of miconazole against Candida albicans

At 10^–5 M, miconazole (MCZ) can exert a direct physicochemical cell-damaging lethal action against logarithmic phase yeasts of Candida albicans. The imidazole moiety of MCZ has a pKa 6.5. Thus, in media of pH >6.5 most drug molecules are nonprotonated (MCZ). Conversely, at pH < 6.5 the majority are protonated and carry a positive charge (MCZH⁺). Our earlier work suggesting that MCZ is required for direct lethal action was tested further. In support of such a requirement, we established a minimal lethal concentration of MCZ (i.e. 5×10^–6 M) that was relatively independent of pH, MCZ concentration, and MCZMCZH⁺ ratio.     

Physiological responses of Lactococcus lactis ML3 to alternating conditions of growth and starvation

Lactococcus lactis species can survive periods of carbohydrate starvation for relatively long periods of time. In the first hours of starvation, however, the maximal glycolytic and arginine deiminase (ADI) pathway activities decline rapidly. The rate of decrease of the pathway activities diminishes as soon as the cells become depleted of energy-rich intermediates. Loss of glycolytic activity is associated with loss of glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase and pyruvate kinase activities. Upon addition of sugar to starved cultures these enzymatic, and thus the glycolytic, activities can be restored to 100% values. The recovery of enzymatic activities is inhibited by chloramphenicol, indicating that protein synthesis is involved. In contrast, restoration of ADI pathway activity does not require de novo synthesis of proteins. General protein degradation and synthesis have been studied in growing and starving cells using [³⁵S]methionine-labeling of proteins and two-dimensional gel analysis. The breakdown of bulk proteins in exponentially growing cells shows first-order rate kinetics (t1/2 of approximately 5 h). Following an initial breakdown of proteins with a t1/2 of 5 h during the first hour(s) of starvation, bulk proteins are degraded very slowly in starving energy-depleted cells. The breakdown of proteins during starvation appears to be (largely) nonspecific. The rate of synthesis of proteins decreases rapidly in the first hour(s) of starvation. From the onset of starvation on at least 45 proteins are no longer synthesized. During starvation relative induction of fourteen to fifteen proteins can be observed.Abbreviations ADI Arginine deiminase - ATP adenosine triphosphate - PEP phosphoenolpyruvate - membrane potential - pH pH gradient - PTS sugar phosphotransferase system - CDM chemically defined medium - TCA trichloro-acetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

Inheritance of resistance to potato viruses Y and A in progeny obtained from potato cultivars containing gene Ry:evidence for a new gene for extreme resistance to PVA

Extreme resistance in cultivated potato (Solanum tuberosum) to potato viruses Y and A (PVY and PVA) conditioned by the presence of Ry genes introduced from Solanum stoloniferum was described by Cockerham (1970). Cockerham detailed a number of genes which controlled a variety of reactions, including extreme resistance to both viruses (i.e. little or no visible reaction of plants and no viral replication following graft and manual inoculation) controlled by gene Ry _sto. In the present study, cvs Pirola and Barbara, which contain a Ry gene, were found to have extreme resistance to PVY isolates from the ordinary (PVY°), veinal necrosis (PVY^N) and potato tuber necrotic ringspot (PVY^NTN) subgroups, and PVA. The inheritance of this phenotype was examined in seedling progenies obtained by crossing Barbara and Pirola with susceptible cultivars. Segregation data for resistance to PVY and PVA in a progeny involving cv Pirola best fitted a genetical model of one gene controlling extreme resistance to both PVY and PVA, although the possibility that there are two genes, each controlling resistance to one virus but closely linked, cannot be excluded. Segregation data from progenies involving cv Barbara best fitted a genetical model in which there are two independent genes, one controlling extreme resistance to PVA and PVY and a second gene controlling extreme resistance to PVA but not to PVY. This previously unrecognised gene conferring extreme resistance to PVA only, should be given the notation Ra in keeping with nomenclature used for other resistance genes.     

Nuclear and cytoplasmic gene control of resistance to loose smut (Ustilago tritici (Pers.) Rostr.) in wheat (Triticum aestivum L.)

Summary Using disomic chromosome substitution lines based on the susceptible wheat cultivar Chinese Spring, loose smut resistance of wheat cultivars Hope and Thatcher was shown to be conferred in each case by a single dominant major gene carried on chromosome 7 A (Hope) or 7 B (Thatcher). Partial resistance was determined by genes on an additional eight Hope or seven Thatcher chromosomes, and similarities were evident between the partial resistance genotypes ofHope and Thatcher. Chinese Spring exhibited a mean infection value of approximately 50%, indicating a significant level of partial resistance, which was found to be due, in part, to genes on the homoeologous chromosome arms 1 As, 1 Es and 1 Ds, and to cytoplasmic genes. Substitution of the Chinese Spring nucleus into the cytoplasm of Aegilops squarrosa, Ae. variabilis or Ae. mutica resulted in increased susceptibility to Ustilago tritici. Several alloplasmic lines of the resistant wheat cultivars Selkirk and Chris exhibited race-specific susceptibility to U. tritici.     

Diacetyl production by different strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp.

Summary Several strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp. were compared for product formation from citrate in milk cultures. Most strains produced acetoin and butanediol. Some strains derived from buffer starter cultures produced, in addition, -acetolactate. Lactococcus lactis strain C17, which produced acetoin and butanediol but no -acetolactate in culture, was compared physiologically with L. lactis strain Ru4, which produced only -acetolactate. Activities of enzymes involved in citrate metabolism were almost identical in both strains, with the exception of -acetolactate decarboxylase, which was missing in strain Ru4. The formation of -acetolactate, acetoin and diacetyl was further analysed in cell-free extracts. -Acetolactate synthase activity saturated at a high pyruvate concentration (100 mm). This is in agreement with the observed accumulation of pyruvate externally, and probably internally, during -acetolactate, acetoin and butanediol production by L. lactis cells.Correspondence to: J. Hugenholtz