Engineering sugarcane cultivars with bovine pancreatic trypsin inhibitor (aprotinin) gene for protection against top borer (Scirpophaga excerptalis Walker)

The inhibitory activity of bovine pancreatic trypsin inhibitor (aprotinin), a natural polypeptide and a proteinase inhibitor, was demonstrated on gut proteinases of three lepidopteran borers of sugarcane using commercially available aprotinin. A synthetic gene coding for aprotinin, designed and codon optimized for better expression in plant system (Shantaram 1999), was transferred to two sugarcane cultivars namely CoC 92061 and Co 86032 through particle bombardment. Aprotinin gene expression was driven by maize ubiquitin promoter and the plant selection marker used was hygromycin resistance. The integration, expression and functionality of the transgene was confirmed by Southern, Western and insect bioassay, respectively. Southern analysis showed two to four integration sites of the transgene in the transformed plants. Independent transgenic events showed varied levels of transgene expression resulting in different levels (0.16–0.50%) of aprotinin. In in vivo bioassay studies, larvae of top borer Scirpophaga excerptalis Walker (Lepidoptera: Pyralidae) fed on transgenics showed significant reduction in larval weight which indicated impairment of their development. Results of this study show the possibility of deploying aprotinin gene for the development of transgenic sugarcane cultivars resistant to top borer.     

Biostructural analysis of the metal-sensor domain of CnrX from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34

In Cupriavidus metallidurans CH34, the proteins CnrX, CnrY, and CnrH regulate the expression of the cnrCBA operon that codes for a cation-efflux pump involved in cobalt and nickel resistance. The periplasmic part of CnrX can be defined as the metal sensor in the signal transduction complex composed of the membrane-bound anti-sigma factor CnrY and the extra-cytoplasmic function sigma factor CnrH. A soluble form of CnrX was overproduced and purified. This protein behaves as a dimer in solution as judged from gel filtration, sedimentation velocity experiments, and NMR. Native crystals diffracting to 2.3 A using synchrotron radiation were obtained using the hanging-drop vapor-diffusion method. They belong to the primitive monoclinic space group P2(1), with unit cell parameters a = 31.87, b = 74.80, c = 93.67 A, beta = 90.107 degrees. NMR data and secondary structure prediction suggest that this protein is essentially formed by helices.     

Expression of N-methyl-D-aspartate (R)(GluN2B) - subunits in the brain structures of rats selected for low and high anxiety

In this paper, we studied differences in the density of N-methyl-D-aspartate (NMDA) receptor GluN2B subunits in the brains of low (LR) and high (HR) anxiety rats subjected to extinction trials and re-learning of a conditioned fear response, modeling a natural course of anxiety disorders. Classifications of animals as LR or HR was determined by fear-induced freezing responses in the contextual fear test. Increased basal concentrations of GluN2B subunits were observed in the amygdala of HR rats as compared to the unconditioned control group by Western blot analysis. Re-exposure of HR animals to the fear-conditioned context resulted in elevated concentrations of GluN2B subunits in the amygdala, hippocampus and the prefrontal cortex compared to LR rats as well as in the hippocampus and prefrontal cortex vs. the control group. In addition, it was shown that re-test of a conditioned fear increased the number of cells expressing GluN2B subunits in the basolateral amygdala, dentate gyrus of the hippocampus and secondary motor cortex (M2) in the HR group relative to the LR group. Together, these data suggest that animals that are more anxious have altered patterns of GluN2B subunit expression in the frontal cortex and limbic structures, which control emotional behaviour.     

Phylogeographic patterns in Drosophila montana

The Drosophila virilis species group offers valuable opportunities for studying the roles of chromosomal re-arrangements and mating signals in speciation. The 13 species are divided into two subgroups, the montana and virilis 'phylads'. There is greater differentiation among species within the montana phylad in both karyotype and acoustic signals than exists among members of the virilis phylad. Drosophila montana is a divergent species which is included in the montana phylad. Here, we analyse the phylogeography of D. montana to provide a framework for understanding divergence of acoustic signals among populations. We analysed mitochondrial sequences corresponding to the cytochrome oxidase I and cytochrome oxidase II genes, as well as 16 microsatellite loci, from 108 lines of D. montana covering most of the species' range. The species shows a clear genetic differentiation between North American and Scandinavian populations. Microsatellite allele frequencies and mitochondrial DNA haplotypes gave significant FST values between populations from Canada, USA and Finland. A Bayesian analysis of population structure based on the microsatellite frequencies showed four genetically distinct groups, corresponding to these three populations plus a small sample from Japan. A network based on mitochondrial haplotypes showed two Finnish clades of very different shape and variability, and another clade with all sequences from North America and Japan. All D. montana populations showed evidence of demographic expansion but the patterns inferred by coalescent analysis differed between populations. The divergence times between Scandinavian and North American clades were estimated to range from 450,000 to 900,000 years with populations in Canada and the USA possibly representing descendants of different refugial populations. Long-term separation of D. montana populations could have provided the opportunity for differentiation observed in male signal traits, especially carrier frequency of the song, but relaxation of sexual selection during population expansion may have been necessary.     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.     

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO₂ via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.     

Quantitative structure analysis of genetic diversity among spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from different geographical regions

Genetic diversity in spring bread wheat (T.␣aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as␣compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B␣genome (0.71) as compared to the A (0.68) and D␣genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H _e = 0.68), genetic diversity among European spring bread wheats was H _e = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.     

How I learned to love carrots: the role of the cytoskeleton in shaping plant cells

The carrot cell suspension was originally used because it provided a model system for studying directional cell expansion - a key process in plant morphogenesis. Early immunofluorescence studies of plant microtubules, using these cells, provided hints that the cortical array of microtubules was dynamic and this was later confirmed by microinjection studies on plant epidermal cells. A nonfixation approach for detecting F-actin was then developed on these cells and showed that, unlike animal cells, actin filaments remained associated with the nucleus throughout division and could have a role in aligning the plane of cell division. Currently, we are using detergent-extracted carrot cytoskeletons for isolating microtubule-associated proteins (MAPs). I discuss how MAPs may be involved in the oriented deposition of cellulose in the cell wall.