Plant species associations in alpine-subnival vegetation patches in the Central Caucasus

Abstract. Patches of herbaceous vegetation found at the boundary of the alpine and subnival belts in the Central Caucasus (3000 m) were analysed for species composition, interspecific associations, as well as for relations between age, size and diversity of the patches. 34 plant species were recorded. Positive and negative associations were found among the most frequent species. A model is presented that describes the cumulation of species in patches during succession. In the final and stable successional stage, the maximum number of species within a patch was nine. Species composition varies in patches of different age, typical alpine species being more frequent in older ones. The results are interpreted as evidence for niche differentiation and selection of species composition through the 'sieve’ of interspecific relations, taking place during succession in the studied patches.     

ZFN‐mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium‐mediated floral dip transformation

Previously, we showed that ZFN‐mediated induction of double‐strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium‐mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild‐type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T‐DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10^?3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10^?3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so‐called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T‐DNA copies. Two plant lines did not contain extra T‐DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.     

Do biotic interactions shape both sides of the humped‐back model of species richness in plant communities?

A humped-back relationship between species richness and community biomass has frequently been observed in plant communities, at both local and regional scales, although often improperly called a productivity-diversity relationship. Explanations for this relationship have emphasized the role of competitive exclusion, probably because at the time when the relationship was first examined, competition was considered to be the significant biotic filter structuring plant communities. However, over the last 15 years there has been a renewed interest in facilitation and this research has shown a clear link between the role of facilitation in structuring communities and both community biomass and the severity of the environment. Although facilitation may enlarge the realized niche of species and increase community richness in stressful environments, there has only been one previous attempt to revisit the humped-back model of species richness and to include facilitative processes. However, to date, no model has explored whether biotic interactions can potentially shape both sides of the humped-back model for species richness commonly detected in plant communities. Here, we propose a revision of Grime's original model that incorporates a new understanding of the role of facilitative interactions in plant communities. In this revised model, facilitation promotes diversity at medium to high environmental severity levels, by expanding the realized niche of stress-intolerant competitive species into harsh physical conditions. However, when environmental conditions become extremely severe the positive effects of the benefactors wane (as supported by recent research on facilitative interactions in extremely severe environments) and diversity is reduced. Conversely, with decreasing stress along the biomass gradient, facilitation decreases because stress-intolerant species become able to exist away from the canopy of the stress-tolerant species (as proposed by facilitation theory). At the same time competition increases for stress-tolerant species, reducing diversity in the most benign conditions (as proposed by models of competition theory). In this way our inclusion of facilitation into the classic model of plant species diversity and community biomass generates a more powerful and richer predictive framework for understanding the role of plant interactions in changing diversity. We then use our revised model to explain both the observed discrepancies between natural patterns of species richness and community biomass and the results of experimental studies of the impact of biodiversity on the productivity of herbaceous communities. It is clear that explicit consideration of concurrent changes in stress-tolerant and competitive species enhances our capacity to explain and interpret patterns in plant community diversity with respect to environmental severity.     

Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked “hyperpolarization,” i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.     

Facilitation in subnival vegetation patches

Abstract. We examined spatial relationships among species in the subnival zone of the central Caucasus. The species composition of 300 vegetation patches was analysed. 144 of them contained only one species, whereas the other 156 contained 2.36 ± 1.31 species, with species numbers distributed as follows: 59 patches with two species, 41 with three, 39 with four and 17 with five species. In the multi-species patches, the 22 most frequent species were examined and 46 statistically significant species associations, 36 positive and 10 negative, were found. Ten of these 22 species were typical subnival plants very rarely occurring at lower altitudes. The other 12 species are ‘invaders’ as they have broader altitudinal ranges of occurrence and are common in alpine and even in subalpine belts. Contrary to the typical subnival species the invaders were found exclusively in the patches with more than one species. Invader species were significantly more associated with subnival species while they were significantly less associated with other species than expected by chance. Our interpretation is that typical subnival species nurse plants from lower altitudes and facilitate their invasion to more adverse subnival environments.     

Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene

Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the -glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related, endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.     

DNA hybridization with hardjobovis-specific recombinant probes as a method for type discrimination of Leptospira interrogans serovar hardjo

Restriction endonuclease analysis of DNA of Leptospira interrogans, serovar hardjo, showed two distinct types within this serovar. These two types, hardjoprajitno and hardjobovis, cannot be differentiated by monoclonal antibodies. Application of 32P- or biotin-labelled total DNA probes in dot-blot or in situ hybridization assays showed a high sensitivity of the assays but also considerable cross-hybridization. Therefore, a genomic library of hardjobovis was constructed and a number of hardjobovis-specific recombinant clones were isolated. Finally, four clones were selected on the basis of a strong hybridization signal and a high specificity for hardjobovis as compared to hardjoprajitno. In a dot-blot assay as well as in in situ hybridization experiments all four clones gave strong signals, and no cross-hybridization with hardjoprajitno was observed in either type of assay. Our results indicate that specific recombinant DNA probes might provide tools for routine diagnosis and classification in cases of hardjo infections.     

Reorganization of microtubule nucleation during muscle differentiation

Skeletal muscle differentiation involves a complete reorganization of the microtubule network. Nearly 20 years ago, Tassin et al. [1985: J Cell Biol 100:35-46] suggested a mechanism for this reorganization by showing a redistribution of the microtubule organizing center from the centrosome to the nuclear membrane. Little progress has been made since. It is still not clear whether centrosomal proteins are redistributed together, whether microtubules are nucleated at the nuclear membrane or transported there post-nucleation, and whether gamma-tubulin (gammatub) remains necessary for nucleation in myotubes. To investigate these questions, we have examined the redistribution of the centrosomal proteins pericentrin (PC), gammatub, and ninein in the C2 muscle cell line. Immunofluorescence of differentiated myotubes shows PC along the nuclear membrane whereas gammatub is only detected there after pre-fixation detergent extraction. After expression of a GFP-tagged gammatub, we observe a weak fluorescence along the nuclear membrane, confirming the presence of gammatub at a low concentration relative to PC. Microinjection of anti-gammatub antibodies into myotubes blocks microtubule growth from both nuclear membranes and centrosomal sites. The centrosomal microtubule-anchoring protein, ninein, is found at the nuclear membrane as well and its distribution appears independent of microtubule integrity. We conclude that centrosomal proteins are redistributed independently during muscle differentiation, to sites that nucleate microtubules both along the nuclear membranes and through the cytoplasm.