Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.     

Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore

Herbivory induces both direct and indirect defenses in plants; however, some combinations of these defenses may not be compatible. The jasmonate signal cascade activated both direct (nicotine accumulations) and indirect (mono- and sesquiterpene emissions) whole-plant defense responses in the native tobacco Nicotiana attenuata Torr. Ex Wats. Nicotine accumulations were proportional to the amount of leaf wounding and the resulting increases in jasmonic acid (JA) concentrations. However, when larvae of the nicotine-tolerant herbivore, Manduca sexta, fed on plants or their oral secretions were applied to leaf punctures, the normal wound response was dramatically altered, as evidenced by large (4- to 10-fold) increases in the release of (i) volatile terpenoids and (ii) ethylene, (iii) increased (4- to 30-fold) accumulations of endogenous JA pools, but (iv) decreased or unchanged nicotine accumulations. The ethylene release, which was insensitive to inhibitors of induced JA accumulation, was sufficient to account for the attenuated nicotine response. Applications of ethylene and ethephon suppressed the induced nicotine response and pre-treatment of plants with a competitive inhibitor of ethylene receptors, 1-methylcyclopropene, restored the full nicotine response. This ethylene burst, however, did not inhibit the release of volatile terpenoids. Because parasitoids of Manduca larvae are sensitive to the dietary intake of nicotine by their hosts, this ethylene-mediated switching from direct to a putative indirect defense may represent an adaptive tailoring of a plant's defense response. Received: 13 June 1999 / Accepted: 21 August 1999     

Mechanisms of breathing instability in patients with obstructive sleep apnea.

The response to chemical stimuli (chemical responsiveness) and the increases in respiratory drive required for arousal (arousal threshold) and for opening the airway without arousal (effective recruitment threshold) are important determinants of ventilatory instability and, hence, severity of obstructive apnea. We measured these variables in 21 obstructive apnea patients (apnea-hypopnea index 91 +/- 24 h(-1)) while on continuous-positive-airway pressure. During sleep, pressure was intermittently reduced (dial down) to induce severe hypopneas. Dial downs were done on room air and following approximately 30 s of breathing hypercapneic and/or hypoxic mixtures, which induced a range of ventilatory stimulation before dial down. Ventilation just before dial down and flow during dial down were measured. Chemical responsiveness, estimated as the percent increase in ventilation during the 5(th) breath following administration of 6% CO(2) combined with approximately 4% desaturation, was large (187 +/- 117%). Arousal threshold, estimated as the percent increase in ventilation associated with a 50% probability of arousal, ranged from 40% to >268% and was <120% in 12/21 patients, indicating that in many patients arousal occurs with modest changes in chemical drive. Effective recruitment threshold, estimated as percent increase in pre-dial-down ventilation associated with a significant increase in dial-down flow, ranged from zero to >174% and was <110% in 12/21 patients, indicating that in many patients reflex dilatation occurs with modest increases in drive. The two thresholds were not correlated. In most OSA patients, airway patency may be maintained with only modest increases in chemical drive, but instability results because of a low arousal threshold and a brisk increase in drive following brief reduction in alveolar ventilation.     

An improved method for direct gene transfer and subsequent regeneration ofArabidopsis thaliana Landsbergerecta and two marker lines

A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 μg plasmid DNA and 28 μg carrier DNA per 6 x 10⁵ protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10⁻⁴, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.     

Molecular phylogeny of the springhare, Pedetes capensis, based on mitochondrial DNA sequences

The phylogenetic position of the Pedetidae, represented by a single species Pedetes capensis, is controversial, reflecting in part the retention of both Hystricomorphous and Sciurognathous characteristics in this rodent. In an attempt to clarify the species evolutionary relationships, mtDNA gene sequences from 10 rodent species (representing seven families) were analyzed using phenetic, parsimony, and maximum-likelihood methods of phylogenetic inference; the rabbit, Oryctolagus cuniculus (Order Lagomorpha), and cow, Bos taurus (Order Artiodactyla), were used as outgroups. Investigation of 714 base pairs of the protein-coding cytochrome b gene indicate strong base bias at the third codon position with significant rate heterogeneity evident between the three structural domains of this gene. Similar analyses conducted on 816 base pairs of the 12S rRNA gene revealed a transversion bias in the loop sections of all taxa. The cytochrome b gene sequences proved useful in resolving associations between closely related species but failed to produce consistent tree topologies at the family level. In contrast, phylogenetic analysis of the 12S rRNA gene resulted in strong support for the clustering of Pedetidae/Heteromyidae/Geomyidae and Muridae in one clade to the exclusion of the Hystricidae/Thryonomyidae and Sciuridae, a finding which is concordant with studies of rodent fetal membranes as well as reproductive and other anatomical features.      

Electrophoretic Karyotype of the Obligate Biotrophic Parasite Plasmodiophora brassicae Wor.

Classical genetic analysis is not possible with the protist Plasmodiophora brassicae due to the intracellular life of this obligate biotrophic parasite . An electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis to facilitate gene mapping of P. brassicae. Using two different separation conditions 16 chromosomal bands of P. brassicae were distinguished ranging in approximate size from 2.2 Mb to 680 kb. According to this determination of chromosome number and size, the total genome size of P. brassicae was estimated to be 20.3 Mb. The chromosomal bands were further designated by their hybridization pattern with repetitive elements of P. brassicae . The repetitive element H4 (1800 bp) hybridized with 14 chromosomal bands, but the sequence of H4 showed no homology to known centromere or telomere structures and revealed no repetitive motifs.     

Quaternary ammonium compounds in soil: implications for antibiotic resistance development

Quaternary ammonium compounds (QACs) are surface-active, antimicrobial, high production volume (HPV) chemicals with a broad application in agriculture. This review provides a comprehensive overview of (1) predicted and measured concentrations of QACs in soils including their analysis, (2) sequestration mechanisms in soils based on their physicochemical properties and chemical structure, and (3) implications of concentrations and fate of QACs in soils for the proliferation of antibiotic resistance in the environment. Predicted environmental concentrations (PEC) for QACs that are applied to soils with manure are in the order of 3.5 mg kg^?1. Based on literature data, the median PEC of QAC in sewage sludge amended soils is 25 µg kg^?1. The positively charged QACs are mainly sorbed to clay minerals. We propose that QACs might be sequestered in the interlayer regions of layered silicates in clay-rich soils, reducing their acute toxicity, while increasing their persistence. The release of sequestered QACs from soil can still potentially maintain concentration levels that are sufficient to develop antibiotic resistance in the environment.