Purification and characterization of a hydroxamic acid glucoside β-glucosidase from wheat (Triticum aestivum L.) seedlings

A beta-glucosidase (EC 3.2.1.21) with a high affinity for cyclic hydroxamic acid beta-D-glucosides was purified from 48-h-old wheat (Triticum aestivum L.) seedlings. The activity occurred transiently at a high level during the non-autotrophic stage of growth, and the nature of the transient occurrence was correlated with that of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). The glucosidase had maximum activity at an acidic pH (pH 5.5) and the purified enzyme showed a high affinity for DIMBOA-Glc, Vmax and Km being 4100 nkat/mg protein and 0.27 mM, respectively. It also hydrolyzed p-nitrophenol beta-glycosides, as well as flavone and isoflavone glucosides, but to a lesser extent. The results indicated that the primary natural substrate for the glucosidase is DIMBOA-Glc and that the enzyme is involved in defense against pathogens and herbivores in non-autotrophic wheat. The glucosidase was found to be present as oligomeric forms with a molecular mass of 260-300 kDa comprising 60- and 58-kDa monomers. The N-terminal 12-amino-acid sequences of the two monomers were identical (Gly-Thr-Pro-(Ser?)-Lys-Pro-Ala-Glu-Pro-Ile-Gly-Pro), and showed no similarity to those of other plant glucosidases. Polyacrylamide gel electrophoresis under nondenaturing condition indicated the existence of at least eight isozymes. Three cultivars of Triticum aestivum had the same zone of glucosidase activity on zymograms, but the activity zones of the Triticum species, T. aestivum L., T. spelta L. and T. turgidum L., had different mobilities.     

Typing of Yersinia pestis isolates from the state of Ceará, Brazil

AIMS: To investigate whether modifications in Yersinia pestis isolates from three plague foci from the state of Ceará, Brazil, had occurred over the years as a consequence of genetic adaptation to the environment. METHODS AND RESULTS: The isolates were studied with respect to susceptibility to antimicrobial drugs, plasmid and protein profiling, pigmentation on Congo red-agar plates, and the presence of some pathogenicity genes using PCR. Most of the expected virulence markers were detected in the cultures examined. There was no evidence of any alteration that could be associated with their origin (patients, rodents and fleas) or period of isolation (1971-1997). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic or genotypic changes were not detected in the cultures examined. However, the results obtained will serve as a reference to follow the evolution of Y. pestis in these foci.     

Biological behaviour in mice of Trypanosoma cruzi isolates from Amazonas and Paraná, Brazil

The biological behaviour of 23 Trypanosoma cruzi isolates in Swiss mice was compared. Nineteen isolates were obtained from patients in the acute phase of Chagas disease (13), sylvatic reservoir hosts (Didelphis marsupialis) (3), and triatomine bugs (Rhodnius robustus) (3) from four regions of the State of Amazonas (AM). Four isolates were obtained from chronic chagasic patients in the State of Paraná (PR): three autochthones, and one allochthone from the State of Minas Gerais. Only one isolate was unable to infect the mice. The AM and PR isolates showed the largest number of significant differences from each other. The former had lower mean values in the pre-patent (5.4 days) and patent (4.6 days) periods (PP), with the parasitaemia (Pmax) reaching a peak of 9.9×10(4) blood trypomastigotes (BT)/mL of blood by the 7th day following inoculation. The AM isolates also had higher positivity to fresh-blood examination (FBE) (84.1%) compared to haemoculture (HC) (58.7%) and polymerase chain reaction (PCR) (33.3%), in addition to higher mortality (2.9%). The PR isolates had higher values for PP (18.5 days) and Pmax (99.9×10(4)BT/mL) as well as higher positivity to FBE (87.2%), HC (100%), and PCR (83.3%). The correlations between the biological behaviour of the T. cruzi isolates and the clinical and epidemiological characteristics of Chagas disease are discussed.     

Stress treatments influence efficiency of microspore embryogenesis and green plant regeneration in hexaploid triticale (× Triticosecale Wittmack L.)

Doubled haploid (DH) production is a key technology in plant breeding and research. One emerging method of choice for DH production is microspore culture, which requires reprogramming of the microspores from their normal gametophytic development to a sporophytic development resulting in embryo formation. This commonly requires the application of stress such as cold, heat, or starvation. Here, we report the effect of different stress treatments on embryo formation and the proportion of green plants in triticale microspore culture. We observed different responses to the applied stress treatments among three studied genotypes. In general, a 3-wk cold stress treatment performed best with regard to the two criteria. For one genotype, the application of a 24- or 48-h heat stress gave similar or slightly better results and consequently may be an alternative for genotypes that are recalcitrant to the cold stress treatment.     

Toxoplasma gondii infections in cats from Paraná, Brazil: seroprevalence, tissue distribution, and biologic and genetic characterization of isolates

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete environmentally resistant oocysts. The prevalence of T. gondii was determined in 58 domestic cats from 51 homes from Santa Isabel do Ivai, Parana State, Brazil where a water-associated outbreak of acute toxoplasmosis had occurred in humans. Antibodies to T. gondii were found with the modified agglutination test in 49 of 58 (84.4%) cats at a serum dilution of 1:20. Tissues (brain, heart, and skeletal muscle) of 54 of these cats were bioassayed in T. gondii-free, laboratory-reared cats; T. gondii oocysts were excreted by 33 cats that were fed feline tissues. Brains from these 54 cats were bioassayed in mice; T. gondii was isolated from 7. Skeletal muscles and hearts of 15 cats were also bioassayed in mice; T. gondii was isolated from skeletal muscles of 9 and hearts of 13. The results indicate that T. gondii localizes in muscle tissue more than the brains of cats. In total there were 37 T. gondii isolates from 54 cats. Most isolates of T. gondii were virulent for mice. Genotyping of the 37 isolates of T. gondii, using the SAG2 locus, revealed that 15 isolates were type I and 22 were type III. The absence of type II genotype in cats in this study is consistent with the previous studies on T. gondii isolates from Brazil and is noteworthy because most T. gondii isolates from the United States are type II. These findings support the view that Brazilian and North American T. gondii isolates are genetically distinct. This is the first report of genotyping of T. gondii isolates from the domestic cat.     

Genetic characterization of Trypanosoma cruzi natural clones from the state of Paraíba, Brazil

Eighteen Trypanosoma cruzi stocks from the state of Paraíba, Brazil, isolated from man, wild mammals, and triatomine bugs were studied by multilocus enzyme electrophoresis and random primed amplified polymorphic DNA. Despite the low number of stocks, a notable genetic, genotypic, and phylogenetic diversity was recorded. The presence of the two main phylogenetic subdivisions, T. cruzi I and II, was recorded. The strong linkage disequilibrium observed in the population under survey suggests that T. cruzi undergoes predominant clonal evolution in this area too, although this result should be confirmed by a broader sample. The pattern of clonal variation does not suggests a recent origin by founder effect with a limited number of different genotypes.     

Virulence and molecular characterization of Toxoplasma gondii isolated from goats in Ceará, Brazil

One hundred and sixty-nine fragments of heart muscle collected from goats in the State of Ceará, Brazil, were analyzed by mouse bioassay with the aim of isolating Toxoplasma gondii. Two T. gondii isolates, named G1 and G2, were obtained and were characterized by PCR-RFLP. In addition, their virulence was evaluated by experimental inoculation into BALB/c mice. The G1 isolate presented high virulence leading to 100% mortality of mice after inoculations with 10¹, 10², and 10³ tachyzoites. The G2 isolate presented low virulence and none of the doses tested lead to mortality of mice. The PCR-RFLP analysis showed that the two isolates are recombinants of the types I/III. However, they differ in the haplotype combination for the genotypes analyzed.     

Experimental and modelling studies of drought‐adaptive root architectural traits in wheat (Triticum aestivum L.)

Abstract

This paper presents an interdisciplinary approach to crop improvement that links physiology with plant breeding and simulation modelling to enhance the selection of high‐yielding, drought‐tolerant varieties. In a series of field experiments in Queensland, Australia, we found that the yield of CIMMYT wheat line SeriM82 ranged from 6% to 28% greater than the current cultivar Hartog. Physiological studies on the adaptive traits revealed that SeriM82 had a narrower root architecture and extracted more soil moisture, particularly deep in the profile. Results of a simulation analysis of these adaptive root traits with the cropping system model APSIM for a range of rain‐fed environments in southern Queensland indicated a mean relative yield benefit of 14.5% in water‐deficit seasons. Furthermore, each additional millimetre of water extracted during grain filling generated an extra 55 kg ha^?1 of grain yield. Further root studies of a large number of wheat genotypes revealed that wheat root architecture is closely linked to the angle of seminal roots at the seedling stage – a trait which is suitable for large‐scale and cost‐effective screening programmes. Overall, our results suggest that an interdisciplinary approach to crop improvement is likely to enhance the rate of yield improvement in rain‐fed crops.     

Structural analysis of wheat wax (Triticum aestivum, c.v. ‘Naturastar’ L.): from the molecular level to three dimensional crystals

In order to elucidate the self assembly process of plant epicuticular waxes, and the molecular arrangement within the crystals, re-crystallisation of wax platelets was studied on biological and non-biological surfaces. Wax platelets were extracted from the leaf blades of wheat (Triticum aestivum L., c.v. ‘Naturastar’, Poaceae). Waxes were analysed by gas chromatography (GC) and mass spectrometry (MS). Octacosan-1-ol was found to be the most abundant chemical component of the wax mixture (66 m%) and also the determining compound for the shape of the wax platelets. The electron diffraction pattern showed that both the wax mixture and pure octacosan-1-ol are crystalline. The re-crystallisation of the natural wax mixture and the pure octacosan-1-ol were studied by scanning tunnelling microscopy (STM), atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Crystallisation of wheat waxes and pure octacosano-1-ol on the non polar highly ordered pyrolytic graphite (HOPG) led to the formation of platelet structures similar to those found on the plant surface. In contrast, irregular wax morphologies and flat lying plates were formed on glass, silicon, salt crystals (NaCl) and mica surfaces. Movement of wheat wax through isolated Convallaria majalis cuticles led to typical wax platelets of wheat, arranged in the complex patterns typical for C. majalis. STM of pure octacosan-1-ol monolayers on HOPG showed that the arrangement of the molecules strictly followed the hexagonal structure of the substrate crystal. Re-crystallisation of wheat waxes on non-polar crystalline HOPG substrate showed that technical surfaces could be used to generate microstructured, biomimetic surfaces. AFM and SEM studies proved that a template effect of the substrate determined the orientation of the re-grown crystals. These effects of the structure and polarity of the substrate on the morphology of the epicuticular waxes are relevant for understanding interactions between biological as well as technical surfaces and waxes.     

Antioxidant activity and ROS tolerance in triticale (×Triticosecale Wittm.) anthers affect the efficiency of microspore embryogenesis

To verify the hypothesis that cell redox status regulates the process of microspore embryogenesis (ME), reactive oxygen species (ROS) generation and the activity of enzymatic and non-enzymatic antioxidants were analyzed in eight doubled haploid lines of triticale with significant differences in embryogenic potential. The analyses were performed in anthers excised from freshly cut tillers (control) and from low temperature (LT) pre-treated tillers (3 weeks at 4 °C) in which ME has been initiated. Significant associations between ME effectiveness and the variables studied were found. In control cultures, high superoxide dismutase (SOD) activity appeared crucial for microspore viability. On the other hand, positive though non-linear correlation between ME effectiveness and H₂O₂ generation, and negative correlation with catalase (CAT) activity suggest that some threshold level of H₂O₂ is important for successful ME initiation. LT tillers pre-treatment significantly increased H₂O₂ accumulation, which had a negative effect on ME effectiveness. However, even high level of H₂O₂ did not endanger cell viability as long as the cells exhibited high activity of ROS-decomposing enzymes (SOD, CAT and POX). The ability to sustain antioxidative enzyme activity under cold stress in the dark was another important requirement for high effectiveness of ME, allowing for the generation of the signal initiating microspore reprogramming and simultaneously protecting the cells from the toxic effects of ROS production. The role of antioxidative enzymes cannot be replaced even by high activity of non-enzymatic antioxidants. In conclusion, genetically controlled but environmentally modified cell tolerance to oxidative stress seems to play an important role in triticale ME.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Monosomic analysis of heading date and spikelet number in the common wheat (Triticum aestivum L.) multispikelet line ‘Noa’

Summary Genetic analysis of heading date and spikelet number was carried out in the common wheat (Triticum aestivum L.) multispikelet line Noa, by using the monosomic series of the regular line Mara. Noa's high number of spikelets was found to be controlled by a recessive major gene on chromosome 2D; a slight reduction in spikelet number was induced by another recessive gene on Noa's 7A chromosome. Noa's late heading date was found to be controlled by two recessive genes, located on chromosome 2D (a major effect) and 6B (a minor effect). The nature of the genes located on Noa's 2D chromosome and the relationship between spikelet number and heading date are discussed.     

Clinical data and molecular analysis of Mycobacterium tuberculosis isolates from drug-resistant tuberculosis patients in Goiás, Brazil

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.     

Investigation of morphogenesis of inflorescence and determination of the nature of inheritance of “supernumerary spikelets” trait of bread wheat (Triticum aestivum L.) mutant line

Using C-banding and FISH methods, the karyotype of MC1611 induced mutant of bread wheat, which develop additional spikelets at a rachis node (trait “supernumerary spikelets”) was characterized. It was determined that the mutant phenotype is not associated with aneuploidy and major chromosomal rearrangements. The results of genetic analysis showed that supernumerary spikelets of the line are caused by a mutation of the single Bh-D.1 gene, influenced by the genetic background. The mutation causes abnormalities of inflorescence morphogenesis associated with the development of ectopic spikelet meristems in place of floral meristems in the basal part of the spikelets, causing the appearance of additional spikes at a rachis node. The mutant phenotype suggests that the Bh-D gene determines the fate of the lateral meristems in ear, which develops as floral meristem and gives rise to floral organs in wild-type inflorescences. In the bh-D.1 mutant, the floral meristem identity is impaired. The characterized mutant can be used in further studies on molecular genetic basis of development of wheat inflorescence.     

Molecular characterization of the celiac disease epitope domains in α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.)

Nineteen novel full-ORF α-gliadin genes and 32 pseudogenes containing at least one stop codon were cloned and sequenced from three Aegilops tauschii accessions (T15, T43 and T26) and two bread wheat cultivars (Gaocheng 8901 and Zhongyou 9507). Analysis of three typical α-gliadin genes (Gli-At4, Gli-G1 and Gli-Z4) revealed some InDels and a considerable number of SNPs among them. Most of the pseudogenes were resulted from C to T change, leading to the generation of TAG or TAA in-frame stop codon. The putative proteins of both Gli-At3 and Gli-Z7 genes contained an extra cysteine residue in the unique domain II. Analysis of toxic epitodes among 19 deduced α-gliadins demonstrated that 14 of these contained 1–5 T cell stimulatory toxic epitopes while the other 5 did not contain any toxic epitopes. The glutamine residues in two specific ployglutamine domains ranged from 7 to 27, indicating a high variation in length. According to the numbers of 4 T cell stimulatory toxic epitopes and glutamine residues in the two ployglutamine domains among the 19 α-gliadin genes, 2 were assigned to chromosome 6A, 5 to chromosome 6B and 12 to chromosome 6D. These results were consistent with those from wheat cv. Chinese Spring nulli-tetrasomic and phylogenetic analysis. Secondary structure prediction showed that all α-gliadins had high content of β-strands and most of the α-helixes and β-strands were present in two unique domains. Phylogenetic analysis demonstrated that α-gliadin genes had a high homology with γ-gliadin, B-hordein, and LMW-GS genes and they diverged at approximate 39 MYA. Finally, the five α-gliadin genes were successfully expressed in E. coli, and their expression amount reached to the maximum after 4 h induced by IPTG, indicating that the α-gliadin genes can express in a high level under the control of T₇ promoter.     

Differential mRNA stability to endogenous ribonucleases of the coding region and 3′ untranslated regions of wheat (Triticum aestivum L.) manganese superoxide dismutase genes

The sequences of the 3′ untranslated region (UTR) of the manganese superoxide dismutase (MnSOD) genes in wheat (Triticum aestivum) were found to be quite variable with different predicted thermostabilities. The degradation rates of the 3′ UTR variants and the coding region were measured following exposure to endogenous nucleases. The degradation rates of the 3′ UTR variants for 15 min were not significantly different, meaning the degradation rates of the 3′ UTR variants were not directly related to the thermostabilities. However, the degradation rate of the coding region was significantly faster than those of the 3’ UTR variants. Further investigation revealed the coding region seemed to have specific sites for degradation, indicating a possibility of increasing MnSOD expression by the degradation site alteration.     

Molecular differentiation of species of the genus Zungaro (Siluriformes, Pimelodidae) from the Amazon and Paraná-Paraguay River basins in Brazil

Fish species of the Zungaro genus (Siluriformes, Pimelodidae) are amongst the largest migratory fish in Latin America and have considerable economic importance for commercial fishing in Brazil. However, natural populations of this large catfish are experiencing a severe decline. There are significant taxonomical inconsistencies for this fish. Two geographically separated species of the fish were initially described, one endemic in the Amazon and another in the Paraná-Paraguay River basins. A taxonomic review had recently proposed that there is only one Zungaro species in Brazil, based on morphological data. We made a molecular study of Zungaro populations in an attempt to solve taxonomical inconsistencies and to analyze genetic diversity in natural populations of this genus. We analyzed two regions of the mitochondrial DNA (the control region and the ATPase 6 gene region) of individuals sampled from the Paraná-Paraguay River and Amazon River basins. Analyses based on p-distances and maximum likelihood phylogenetic models showed a genetic difference between populations corresponding to different species. Genetic differentiation between Zungaro populations was at the same level as that observed between other Siluriformes species, using the same DNA sequences. We conclude that Zungaro species of the Paraná-Paraguay River basin do not belong to the same species found in the Amazon basin. This finding has a significant implication for conservation of this fish, given that populations are disappearing at a high rate in the Paraná-Paraguay River basin, mainly due to impoundments.     

Genetic control of heading date and spikelet number in common wheat (T. aestivum L.) line ‘Noa’

Summary The phenology and build-up of spikelet number under 10 h day-length were studied in five wheat lines: the multispikelet line Noa, the regular line Mara, the F₁ hybrid between them and monosomics 2D of Mara and of this hybrid (lacking the 2D chromosome of Mara). Noa had a longer spike development phase, a higher initial number of spikelet primordia and a slower rate of spikelet production than Mara. The F₁ hybrid was similar to Noa in its high initial number of spikelets and to Mara in its high rate of spikelet production. This hybrid had a shorter spikelet phase than both parents. Deletion of one dose of the Mara 2D chromosome from either Mara or the F₁ hybrid caused a reduction in the rate of spikelet production and an increase in the duration of the spikelet phase. These effects were due to the reduced dosage of the 2D chromosome. However, in the F₁ hybrid this deletion also caused an increase in the spike development phase — an indication that Noa carries on its 2D chromosome a recessive gene for late heading date which acts on the spike development phase. This gene of Noa is independent of the day-length sensitive gene ppd, and is different from Noas dominant gene for large initial number of spikelets.     

Brumptomyia angelae, a new species of Phlebotominae (Diptera, Psychodidae) of the Atlantic forest of the state of Paraná, Brazil

The male of Brumptomyia angelae, sp. nov., a new species of Phlebotominae (Diptera, Psychodidae) of the Atlantic forest of the state of Paraná, Brazil, is described and illustrated. This new taxon is closely related to Brumptomyia ortizi Martins, Silva & Falc?o 1971, Brumptomyia nitzulescui (Costa Lima, 1932), and Brumptomyia troglodytes (Lutz, 1922). The male genitalia of these three latter species have also been drawn.     

Dental development and ontogenetic diet shifts of Roeboides paranensis pignalberi (osteichthyes, Characinae) in pools of the upper Rio Paraná floodplain (state of Paraná, Brazil)

Species of the characid genus Roeboides are known for their habit of tearing off and ingesting scales from other fishes. Specimens of Roeboides paranensis were taken monthly from March 1992 through February 1993 in five pools of the upper Rio Paraná floodplain and in one site in the Rio Paraná itself, with the aim of relating the dental development to ontogenetic diet shifts. Between 15-22 mm SL, fish had teeth with hypertrophied bases (mammiform) that moved to the outside of the mouth in both maxillas. During ontogeny, the diet shifted, with the smallest individuals eating more microcrustaceans and the larger ones eating more scales. Lepidophagy is non-obligatory, because the ingestion of insects and other invertebrates occurred at all sizes. Spatial variation in diet was large, however diet similarity was great for Roeboides from three floodplain pools that had similar proportions of the main diet categories.