Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.     

Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4

N-terminal truncation of NPY has important physiological consequences, because the truncated peptides lose their capability to activate the Y1-receptor. The sources of N-terminally truncated NPY and related peptides are unknown and several proline specific peptidases may be involved. First, we therefore provide an overview on the peptidases, belonging to structural and functional homologues of dipeptidyl peptidase 4 (DP4) as well as aminopeptidase P (APP) and thus, represent potential candidates of NPY cleavage in vivo. Second, applying selective inhibitors against DP4, DP8/9 and DP2, respectively, the enzymatic distribution was analyzed in brain extracts from wild type and DP4 deficient F344 rat substrains and human plasma samples in activity studies as well as by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry. Third, co-transfection of Cos-1 cells with Dpp4 and Npy followed by confocal lasermicroscopy illustrated that hNPY-dsRed1-N1 was transported in large dense core vesicles towards the membrane while rDP4-GFP-C1 was transported primarily in different vesicles thereby providing no clear evidence for co-localization of NPY and DP4. Nevertheless, the review and experimental results of activity and mass spectrometry studies support the notion that at least five peptidases (DP4, DP8, DP9, XPNPEP1, XPNPEP2) are potentially involved in NPY cleavage while the serine protease DP4 (CD26) could be the principal peptidase involved in the N-terminal truncation of NPY. However, DP8 and DP9 are also capable of cleaving NPY, whereas no cleavage could be demonstrated for DP2.     

Na+ -D-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.     

Crop evolution: from genetics to genomics

The advent of the genomics age has greatly facilitated the study of crop evolution. While full-scale genome sequencing projects are underway for just a handful of crop plants, recent years have witnessed a tremendous increase in the availability of DNA sequence data for virtually all major crops. Such resources have bolstered 'traditional' genetic approaches such as QTL mapping and candidate gene-based association studies. They have also allowed us to undertake genome-wide analyses in which we simultaneously consider the importance of a large and essentially random collection of genes. These sorts of analyses promise a more or less unbiased view of the genetic basis of crop evolution and will probably result in the identification of agronomically important genes that would have otherwise been overlooked.     

Spontaneous regeneration of dry grasslands on set-aside fields

Dry grasslands are among the most threatened plant communities of Central Europe. We explore the time scale of spontaneous regeneration of dry grasslands on abandoned fields in an area of Central Europe, where also ancient grassland communities occur (Saxony-Anhalt, Germany). On three permanent plots with shallow soils we monitored during 10 years change of species composition and analysed whether spontaneous succession leads to assemblages similar to the ancient dry grassland communities in the direct surroundings. We found that dry grassland species are able to invade the permanent plots and during the 10 years of succession the number of dry grassland species increased. But even after 10 years there was a clear difference between ancient dry grassland communities and the assemblages on the permanent plots. Our findings suggest two important conclusions: First, spontaneous succession on abandoned fields is a cheap possibility for the conservation of some dry grassland species, at least on shallow soils. Second, the time scale of the regeneration process, however, is rather long. Hence, conservation of remnants of ancient grassland communities needs special attention.     

Genital damage in the orb-web spider Argiope bruennichi (Araneae: Araneidae) increases paternity success

The morphology of male genitalia often suggests functions besidessperm transfer that may have evolved under natural or sexualselection. In several species of sexually cannibalistic spiders,males damage their paired genitalia during mating, limitingthem to one copulation per pedipalp. Using a triple-mating experiment,we tested if genital damage in the orb-web spider Argiope bruennichiincreases male fitness either through facilitating his escapefrom an aggressive female or by obstructing the female's inseminationducts against future copulation attempts from other males. Wefound no survival advantage for males damaging their pedipalps;however, copulations into a previously used insemination ductwere significantly shorter when the previous male had left partsof his genitalia inside the insemination duct. Because copulationduration determines paternity in this species, our result suggeststhat male genital damage in A. bruennichi is sexually selected.By breaking off parts of their intromittent organs inside avirgin female, males can reduce sperm competition and therebyincrease their paternity success.     

Genetic variation among different populations of Aster tripolium grown on naturally and anthropogenic salt-contaminated habitats: implications for conservation strategies

The sea aster, Aster tripolium L., grows naturally in temperate regions, mainly in the salt meadows close to the coast. The species is also found in naturally and anthropogenically salt-contaminated inland habitats, such as potash mine dumps. The genetic relationships among populations from different habitats and correlations of the genotype with physiological and vegetational parameters were investigated. A. tripolium plants from five different sites close to the seashore on the North Sea island Baltrum, from five different potash mine dumps and, as an outgroup, from the seashore in Japan were probed. DNA was extracted from five plants from each of the 11 A. tripolium populations and analyzed for random amplified polymorphic DNA (RAPD). Altogether 35 polymorphic bands in 51 individuals and 45 different detectable genotypes could be identified. For evaluation of the genetic variation using RAPD bands, the neighbor-joining method, the principal coordinate analysis, and the analysis of molecular variance were applied, resulting in the classification into three genetic groups. A. tripolium plants from different ecological habitats on Baltrum were closely related while the plants growing at the deposit dumps showed a higher genetic diversity. The Japanese population was genetically very different from the German populations. Correlations between phytosociological and soil parameters and the respective genotype were not significant. The results argue for a conservation of anthropogenically salt-contaminated habitats to maintain genetic variability not only on the species level, but also within a species.     

Thyroid hormone receptor α1 is a critical regulator for the expression of ion channels during final differentiation of outer hair cells

Cochlear outer hair cells (OHCs) terminally differentiate prior to the onset of hearing. During this time period, thyroid hormone (TH) dramatically influences inner ear development. It has been shown recently that TH enhances the expression of the motor protein prestin via liganded TH receptor β (TRβ) while in contrast the expression of the potassium channel KCNQ4 is repressed by unliganded TRα1. These different mechanisms of TH regulation by TRα1 or TRβ prompted us to analyse other ion channels that are required for the final differentiation of OHCs. We analysed the onset of expression of the Ca²⁺ channel Ca_V1.3, and the K⁺ channels SK2 and BK and correlated the results with the regulation via TRα1 or TRβ. The data support the hypothesis that proteins expressed in rodents prior to or briefly after birth like Ca_V1.3 and prestin are either independent of TH (e.g. Ca_V1.3) or enhanced through TRβ (e.g. prestin). In contrast, proteins expressed in rodents later than P6 like KCNQ4 (∼P6), SK2 (∼P9) and BK (∼P11) are repressed through TRα1. We hypothesise that the precise regulation of expression of the latter genes requires a critical local TH level to overcome the TRα1 repression. Harald Winter and Claudia Braig contributed equally to this work.     

Bacterial regulatory networks include direct contact of response regulator proteins: interaction of RegA and NtrX in Rhodobacter capsulatus

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.