Characterization of the glucose-6-phosphate isomerase gene in Phytophthora infestans reveals the presence of multiple alleles

Glucose-6-phosphate isomerase (GPI) plays a key role in both glycolysis and gluconeogenesis. Isoforms of GPI are common, and therefore, its isozyme pattern is widely used to characterize isolates of Phytophthora infestans. Despite the importance of GPI in P. infestans studies, the gene encoding this enzyme has not yet been characterized. Furthermore, it has been suggested that P. infestans contains multiple copies of the gene but this hypothesis remains to be demonstrated. We have cloned and characterized GPI in various isolates of P. infestans as well as in several species of the genus Phytophthora. The gene contains 1671bp and encodes a protein with a predicted molecular weight of 60.8kDa. Multiple different alleles were identified and Southern analysis indicated certain P. infestans isolates carry several copies of the gene. Phylogenetic analysis revealed that P. infestans GPI is most closely related to sequences from Toxoplasma gondii, Arabidopsis thaliana, and Clarkia lewisii.     

The effect of glucose-6-phosphate isomerase genotype onin vitro specific activity andin vivo flux inMytilus edulis

Four samples of the musselMytilus edulis were taken between 1984 and 1987 from Stony Brook, New York, and used to study the glucose-6-phosphate isomerase (GPI) polymorphism in this species.In vitro specific activity andin vivo flux measured in the same animals were found to be significantly correlated. A significant effect of GPI genotype on flux was observed in one of the samples; overall, significant evidence of effect of genotype on enzyme activity was also obtained. GPI activities of common genotypes tend to deviate less from the population mean than those of rare (frequency less than 5%) genotypes. This suggests the possibility that rare GPI genotypes are rare as a consequence of having biochemical properties that deviate from an optimum level and, therefore, having a lower fitness. In support of this hypothesis, we found in one of our samples that shell length is a concave function of GPI activity with an intermediate optimum activity level. The financial support provided to P.J.N.S. by the Luso-American Educational Commission (Fulbright Program), the Instituto Nacional de Investigacao Científica (Portugal), and the Faculdade de Ciências da Universidade de Lisboa during several stages of this research is gratefully acknowledged. Financial support from the Ministerio de Educatión y Ciencia (Spain) in the form of a postdoctoral Fulbright/MEC fellowship to M.S. is also gratefully acknowledged. Research was supported by National Science Foundation Grant BSR-8415060 to R.K.K. This is contribution No. 736 from the Program in Ecology and Evolution, State University of New York at Stony Brook. On leave from Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande C2, Lisboa, Portugal.     

Transformation of rapid cycling cabbage (Brassica oleracea var. capitata) with Agrobacterium rhizogenes

Summary Genetically transformed cabbage (Brassica oleracea var. capitata) roots were obtained after inoculation with two engineered Agrobacterium rhizogenes strains, each harbouring a plant selectable marker gene in their T-DNA. Axenic root clones resistant to kanamycin or hygromycin B were established, most of which did not exhibit the phenotypic characteristics of Ri-transformed roots. Shoot regeneration was induced from roots after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting plants exhibited various phenotypes: some looked normal, while others showed the transformed phenotype observed in other species. Direct evidence for genetic transformation was obtained by molecular hybridization. The trait was transmitted to the progeny. Transformed cabbage plants can be obtained within 6 months using this approach.     

Potential use of theaux2 gene fromAgrobacterium rhizogenes as a conditional negative marker in transgenic cabbage

An originalAgrobacterium tumefaciens-mediated transformation procedure, based on the actions of both wild type and disarmed bacterial strains, was developed. Theaux2 gene ofA. rhizogenes was introduced into a rapid-cycling genotype of cabbage (Brassica oleracea L.). Theaux2 gene product converts naphthalene acetamide into the auxin naphthalene acetic acid. Expression of this gene in the transgenic progeny grownin vitro led to an altered root phenotype. On a medium supplemented with napthalene acetamide (NAM), two of the three analysed progenies were characterized by the formation of callus instead of roots, whereas on a NAM-free medium all the plantlets from these progenies presented a normal phenotype. Expression of theaux2 gene was also assessed under horticultural conditions by sowing seeds in sand and watering them with a nutritive solution supplemented with NAM. Under these conditions, NAM inhibited the formation of a root system in transgenic plantlets and induced the death of the transgenic plantlets three to four weeks after germination. Thus,aux2 acts as a lethal conditional marker which could be used in negative selection of cabbage. Potential utilization of theaux2 gene to screen spontaneous androgenetic plants in order to transfer cytoplasmic male sterility in a single generation is discussed.     

Eukaryotic glucose-6-phosphate dehydrogenases: Structural screening of related proteins

Rapid assessment of structural relationships between yeast glucose-6-phosphate dehydrogenases and other eukaryotic types of this enzyme is described. Separation and size estimation of large fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis, electroblotting onto disks, and sequencer analysis provide data that permit alignment of the segments thus characterized with the related proteins, and utilize existing structural knowledge to assess new enzyme structures. Affinity labeling allows further correlations. The results establish the overall structural arrangements of the new proteins, including the location of the active-site lysine residue, even though the yeast enzyme structures are found to differ markedly from the few previously characterized glucose-6-phosphate dehydrogenases.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Response of cabbage head weight to simulated Lepidoptera defoliation

Studies were conducted to determine the effects of continuous constant amounts of artificial defoliation through the preheading and heading growth stages on head weight of cabbage. High levels of continuous artificial defoliation caused a reduction in head weight in all three years of the study, but the highest yield was always attained at some low level of preheading or heading defoliation. These results demonstrate that cabbage is tolerant to some levels of continuous defoliation before and after head formation. Results from this study are incorporated into a cost-benefit analysis to estimate an economic threshold.

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Résumé L'étude a porté sur le poids des pommes de choux (Brassica oleracea) soumis à différentes intensités de défoliations répétées avant et pendant la formation des pommes. Pendant les trois années de l'étude, huit intensités de défoliation continue ont réduit le poids des pommes, mais le poids le plus élevé a toujours été obtenu avec une faible défoliation avant et pendant la formation des pommes. Ceci montre que le chou tolère une certaine défoliation avant et pendant la formation des pommes. Les résultats de cette étude ont été utilisés dans une analyse coût-bénéfice pour estimer le seuil économique de défoliation.

     

Substrate specificity of a glucose-6-phosphate isomerase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> for monosaccharides

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.     

Mobility of three generalist predators is greater on cabbage with glossy leaf wax than on cabbage with a wax bloom

Adult coccinellids Hippodamia convergens Guerrin-Menneville, adult minute pirate bugs Orius insidiosus (Say), and larval lacewings Chrysoperla carnea (Stephens) have been reported to more effectively reduce populations of Plutella xylostella (L.) (Plutellidae: Lepidoptera) larvae on a cabbage with glossy surface waxes than on a standard cabbage variety. To examine the mechanisms, the behavior of each predator species was quantified on the two cabbage types. All three predators spent less time walking on the standard variety and more time in other activities, including grooming and scrambling (ineffective forward locomotion). In addition, walking by each predator was distributed more evenly among the parts of the leaf on the glossy cabbage than on the standard variety. In small enclosures, C. carnea and O. insidiosus found and attacked individual first instar P. xylostella more quickly on the glossy cabbage. Scanning electron micrographs showed debris, probably wax, on the tarsae of C. carnea and O. insidiosus that had walked on the standard variety, but not on those that had walked on glossy cabbage. H. convergens tarsae did not accumulate debris on either cabbage type. Predator mobility and effectiveness were apparently impeded by wax crystals, which are present only on the standard variety. This mechanism can explain the greater effectiveness of these generalist predators against P. xylostella larvae on glossy vs normal-wax cabbage.     

A study of factors affecting embryo yields from anther culture of cabbage

In cabbage (Brassica oleracea var. capitata), a thermal shock treatment of 24 h at 35 °C at the start of the culture period resulted in higher embryos per 100 anthers (30.0) compared to a treatment of 48 h. Similarly , a chilling treatment of 24 h at 4 °C resulted in a higher embryo yield (6.0) per 100 anthers compared to a treatment of 48 h. However, the embryo yields were significantly higher (p> 0.01) in thermal shock than chilling treatments in all experiments. Treatments of 6 days at either 35 °C or 4 °C gave no embryos. The most responsive cultivar was the F₁ hybrid , Hercules, in all experiments. Although anther culture was successful in the other genotypes, the open pollinated ones, the highest number of embryo yields per 100 anthers was obtained in the hybrid. High temperature treatment before culture had a beneficial effect on the embryo yields. The responsiveness of anthers to addition of increasing concentration of silver nitrate (AgN0₃) (the ethylene inhibitor) to the culture medium, showed a progressive increase in the embryo yields in all the genotypes. Since embryos were also formed in the absence of silver nitrate, probably, due to a greater genotype × medium interaction, it is noted that the presence of silver nitrate in the medium may not be essential for cabbage anther culture as reported earlier. The findings of this study may be recommended for large production of cabbage embryos in culture.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

An enzymatic fluorimetric assay for glucose-6-phosphate: Application in an in vitro Warburg-like effect

Recently, there has been a resurgence of interest in the regulatory role of cell metabolism in tumor biology and immunology. To assess changes in metabolite levels in cell populations and tissues, especially from small clinical samples, highly sensitive assays are required. Based on the reaction of glucose-6-phosphate (G6P) and the diaphorase-resazurin amplifying system, we have developed a fluorescence methodology to measure G6P concentrations in cell extracts. In this approach, G6P is oxidized by G6P dehydrogenase in the presence of NADP⁺, and the stoichiometrically generated NADPH is then amplified by the diaphorase cycling system to produce a highly fluorescent molecule—resorufin. The limit of detection (LOD) of the assay is 10 pmol. The assay has a Z′ factor of 0.81. Its usefulness is demonstrated by experiments in which the pyruvate kinase inhibitor, phenylalanine, is added to cells. After 2 h of incubation at 37 °C, G6P levels rose by 20%, thereby illustrating an in vitro Warburg-like effect on cell metabolism.     

Crystal structure of glucose-6-phosphate isomerase from Thermus thermophilus HB8 showing a snapshot of active dimeric state

Glucose-6-phosphate isomerase (GPI) is a glycolytic enzyme with ill-defined oligomeric state. In order to obtain insight into the correlation between oligomerization and the catalytic function of this enzyme, the crystal structure of GPI from the extreme thermophile Thermus thermophilus HB8 (TtGPI) has been determined at 1.95 Å resolution. The crystallographic asymmetric unit contains an apparent dimer. The core fold of protomer and the interprotomer spatial arrangement of the dimer are similar to those of already reported crystal structures of other GPIs. The active site is located on the dimer interface, and putative catalytic residues are well conserved among the GPIs. These results suggest that the observed dimeric state of TtGPI in the crystal is biologically relevant and that this enzyme uses a common catalytic mechanism for the isomerase reaction. Gel-filtration chromatography, chemical cross-linking, sedimentation equilibrium by analytical ultracentrifugation, and dynamic light-scattering experiments indicate that TtGPI exists in a dynamic equilibrium between monomeric and dimeric states in solution. Several factors potentially contributing to the thermal stability of TtGPI protomer were identified: (i) a decrease in denaturation entropy by the shorter polypeptide length and by amino acid composition, including the increased number of proline residues and a higher arginine-to-lysine ratio; (ii) a larger number of ion pairs; and (iii) a reduction in cavity volume. From these results, it is suggested that transient dimer formation is sufficient for the catalytic function and that the TtGPI protomer itself has intrinsically higher thermal stability.     

Characterization of a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans that converts monosaccharides

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co²⁺. Under these conditions, the k _cat and K _m values were 74,300 s⁻¹ and 390 mM for d-lyxose and 28,800 s⁻¹ and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield.     

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.     

Production of interspecific somatic hybrids between tuber mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) and red cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

In the present investigation, the interspecific somatic hybridization between tuber mustard and red cabbage was established in order to introduce valuable genes from red cabbage (Brassica oleracea) into Brassica juncea. Prior to fusion treatment, protoplasts of red cabbage were inactivated with 2 mM iodoacetamide to inhibit cell division. Micro-calluses were obtained at a frequency of 10.3% after approximately 5 weeks culture following protoplast fusion. Some of the fusion-derived calluses possessed red pigmented cells after being transferred to proliferation medium, and they were presumably considered to be somatic hybrid cell lines. Plantlets were regenerated from 12 cell lines, of which nine plantlets exhibited characteristics intermediate of both parents in terms of plant morphology. With the exception of common protein bands featured by two parents, there were unique banding patterns produced in the hybrids by using SDS-PAGE analysis. By chromosome countings, it was showed that they ranged approximately from 2n=30 to 42 in chromosome numbers. Their hybridity were further confirmed by RAPD analysis revealing that genes of both parents were partially incorporated into the hybrids. Positively, all these hybrids were capable of seed-setting. The pod-setting was 4.2 in somatic hybrid H₇ when backcrossed with tuber mustard.     

X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Two X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase (G6PD) were found among the progenies of isogenic strains which had been subjected to selection for high G6PD activity. Mapping of the high-activity factor in these mutants was carried out using car Zw ^B sw males of low G6PD activity. As a result, the factor mapped 0.02–0.04 unit to the left of the Zw locus. The amount of the G6PD gene was also quantitated utilizing a cloned G6PD gene as a probe, but no significant difference was found between the mutants and low-G6PD activity flies which shared the same X, second, and third chromosomes with the mutants. These findings are consistent with our notion that the mutations might be regulatory mutations, possibly resulting from the insertion of a novel class of transposable genetic elements.This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Predators mediate host plant resistance to a phytophagous pest in cabbage with glossy leaf wax

This study examined the role of generalist predators in producing higher mortality ofPlutella xylostella L. (Plutellidae) larvae on glossy vs. normal-wax cabbage,Brassica oleracea var.capitata L. To test this, survival and feeding ofP. xylostella were measured on individually caged glossy and normal-wax plants with and without each of three generalist predators,Chrysoperla carnea (Stephens) (Chrysopidae),Orius insidiosus (Say) (Anthocoridae), andHippodamia convergens Guerin-Meneville (Coccinellidae). In the greenhouse, predators always significantly reduced survival ofP. xylostella larvae on glossy plants, but never on normal-wax plants. In the field, predators significantly reducedP. xylostella survival on glossy plants, but onlyC. carnea was effective on normal-wax plants. In similar experiments with excised leaves,O. insidiosus andC. carnea were more effective predators on the glossy leaves, whileH. convergens was equally effective on both kinds of leaves. Patterns for feeding were similar, but significance levels differed from those forP. xylostella survival. The greater effectiveness of predators on glossy plants is apparently due to the reported improved mobility of these animals on glossy leaf surfaces. The data also suggest that reduced mining byP. xylostella exposes the larvae to more predation on glossy plants and contributes some to the resistance. Regardless of the mechanism, resistance toP. xylostella on glossyB. oleracea appears to depend on the action of generalist predators for its full expression. This dependence on predation must be considered in the development and deployment of glossy insect-resistantB. oleracea.     

Waxes enhance Plutella xylostella oviposition in response to sinigrin and cabbage homogenates

Oviposition by the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), on substrates treated with host stimuli (cabbage homogenate or sinigrin) and/or waxes (paraffin or a mixture of 10 single chain n-alkanes) was quantified using continuous observations and endpoint bioassays. Paraffin or an n-alkane mixture applied over cabbage homogenate or sinigrin caused an increase in oviposition compared to that on any single stimulus in choice tests. Sinigrin alone at 10^–5 M to 10^–2 M is an ovipositional stimulant; addition of alkane over sinigrin made all sinigrin concentrations (10^–6 M to 10^–2 M) significantly more stimulatory than controls. Waxes alone do not stimulate oviposition. In choice tests, insect movement between sinigrin/alkane treatment combinations was random, however, once encountered, visit duration was significantly longer on sinigrin with alkane than on sites treated with either stimulus alone. Given the ubiquity of waxes on plant surfaces and the interaction between waxes and host-specific chemical stimuli, waxes should be included when considering factors that significantly influence herbivore host acceptance.     

Glucosinolate polymorphism in wild cabbage (Brassica oleracea) influences the structure of herbivore communities

Natural plant populations often show substantial heritable variation in chemical structure of secondary metabolites. Despite a great deal of evidence from laboratory studies that these chemicals influence herbivore behaviour and life history, there exists little evidence for the structuring of natural herbivore communities according to plant chemical profiles. Brassica oleracea (Brassicaceae) produces aliphatic glucosinolates, which break down into toxins when leaf tissue is damaged. Structural diversity in these glucosinolates is heritable, and varies considerably at two ecological scales in the UK: both within and between populations. We surveyed herbivore attack on plants producing different glucosinolates, using 12 natural B. oleracea populations. In contrast to the results of previous studies in this system, which suffered low statistical power, we found significant differential responses of herbivore species to heritable glucosinolates, both within and between plant populations. We found significant correlations between herbivore infestation rates and the presence or absence of two heritable glucosinolates: sinigrin and progoitrin. There was variation between herbivore species in the direction of response, the ecological scale at which responses were identified, and the correlations for some herbivore species changed at different times of the year. We conclude that variation in plant secondary metabolites can structure the community of herbivores that attack them, and propose that herbivore-mediated differential selection deserves further investigation as a mechanism maintaining the observed diversity of glucosinolates in wild Brassica.