Auxin Immunolocalization Implicates Vesicular Neurotransmitter-Like Mode of Polar Auxin Transport in Root Apices

Immunolocalization of auxin using a new specific antibody revealed, besides the expected diffuse cytoplasmic signal, enrichments of auxin at end-poles (cross-walls), within endosomes and within nuclei of those root apex cells which accumulate abundant F-actin at their end-poles. In Brefeldin A (BFA) treated roots, a strong auxin signal was scored within BFA-induced compartments of cells having abundant actin and auxin at their end-poles, as well as within adjacent endosomes, but not in other root cells. Importantly, several types of polar auxin transport (PAT) inhibitors exert similar inhibitory effects on endocytosis, vesicle recycling, and on the enrichments of F-actin at the end-poles. These findings indicate that auxin is transported across F-actin-enriched end-poles (synapses) via neurotransmitter-like secretion. This new concept finds genetic support from the semaphore1, rum1 and rum1/lrt1 mutants of maize which are impaired in PAT, endocytosis and vesicle recycling, as well as in recruitment of F-actin and auxin to the auxin transporting end-poles. Although PIN1 localizes abundantly to the end-poles, and they also fail to support the formation of in these mutants affected in PAT, auxin and F-actin are depleted from their end-poles which also fail to support formation of the large BFA-induced compartments.Key Words: actin, auxin, maize, secretion, vesicles, neurotransmitter     

Antifungal effects and mechanism of action of viscotoxin A3

Viscotoxins are cationic proteins, isolated from different mistletoe species, that belong to the group of thionins, a group of basic cysteine-rich peptides of approximately 5 kDa. They have been shown to be cytotoxic to different types of cell, including animal, bacterial and fungal. The aim of this study was to obtain information on the cell targets and the mechanism of action of viscotoxin isoform A3 (VtA3). We describe a detailed study of viscotoxin interaction with fungal-derived model membranes, its location inside spores of Fusarium solani, as well as their induced spore death. We show that VtA3 induces the appearance of ion-channel-like activity, the generation of H2O2, and an increase in cytoplasmic free Ca2+. Moreover, we show that Ca2+ is involved in VtA3-induced spore death and increased H2O2 concentration. The data presented here strongly support the notion that the antifungal activity of VtA3 is due to membrane binding and channel formation, leading to destabilization and disruption of the plasma membrane, thereby supporting a direct role for viscotoxins in the plant defence mechanism.     

ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis

Background: High resolution melting (HRM) is an emerging new method for interrogating and characterizing DNA samples. An important aspect of this technology is data analysis. Traditional HRM curves can be difficult to interpret and the method has been criticized for lack of statistical interrogation and arbitrary interpretation of results. Methods: Here we report the basic principles and first applications of a new statistical approach to HRM analysis addressing these concerns. Our method allows automated genotyping of unknown samples coupled with formal statistical information on the likelihood, if an unknown sample is of a known genotype (by discriminant analysis or “supervised learning”). It can also determine the assortment of alleles present (by cluster analysis or “unsupervised learning”) without a priori knowledge of the genotypes present. Conclusion: The new algorithms provide highly sensitive and specific auto-calling of genotypes from HRM data in both supervised an unsupervised analysis mode. The method is based on pure statistical interrogation of the data set with a high degree of standardization. The hypothesis-free unsupervised mode offers various possibilities for de novo HRM applications such as mutation discovery.     

Metamorphosis of Hydractinia echinata (Cnidaria) is caspase-dependent

Apoptotic cell death plays an important role in many developmental pathways in multicellular animals. Here, we show that metamorphosis in the basal invertebrate Hydractinia echinata (Cnidaria) depends on the activity of caspases, the central enzymes in apoptosis. Caspases are activated during metamorphosis and this activity can be measured with caspase-3 specific fluorogenic substrates. In affinity labelling experiments 23/25 kDa bands were obtained, which represented active caspase. Specific inhibition of caspase activity with caspase-3 inhibitors abolished metamorphosis completely, reversibly and in a dose-dependent manner. This suggests that caspase activity is indispensable for metamorphosis in Hydractinia echinata.     

Synthesis and characterization of biocompatible polymer interlayers on titanium implant materials

A bifunctional copolymer series of (4-vinylbenzyl)phosphonic acid diethylester and N-acryloxysuccinimide was developed as an interlayer with the aim of immobilizing proteins on titanium surfaces. Copolymers with varying compositions were synthesized, and an alternating copolymerization of the two monomers was found. The copolymers form ultrathin films of about 2-8 nm on titanium surfaces in a simple dipping process, as estimated from the attenuation of the titanium X-ray photoelectron spectroscopy (Ti-XPS) signal. The films were characterized by infrared spectroscopy, XPS, and time-of-flight secondary ion mass spectrometry. The results indicate that the immobilization is due to phosphonate groups, and thus the phosphonate content of the copolymers is decisive for the final film thickness. These polymer films were examined for their potential protein binding capacity by using trifluoroethylamine derivatization and subsequent XPS analysis as a reactivity assay.     

Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.     

A plant's perspective of extremes: terrestrial plant responses to changing climatic variability

We review observational, experimental, and model results on how plants respond to extreme climatic conditions induced by changing climatic variability. Distinguishing between impacts of changing mean climatic conditions and changing climatic variability on terrestrial ecosystems is generally underrated in current studies. The goals of our review are thus (1) to identify plant processes that are vulnerable to changes in the variability of climatic variables rather than to changes in their mean, and (2) to depict/evaluate available study designs to quantify responses of plants to changing climatic variability. We find that phenology is largely affected by changing mean climate but also that impacts of climatic variability are much less studied, although potentially damaging. We note that plant water relations seem to be very vulnerable to extremes driven by changes in temperature and precipitation and that heatwaves and flooding have stronger impacts on physiological processes than changing mean climate. Moreover, interacting phenological and physiological processes are likely to further complicate plant responses to changing climatic variability. Phenological and physiological processes and their interactions culminate in even more sophisticated responses to changing mean climate and climatic variability at the species and community level. Generally, observational studies are well suited to study plant responses to changing mean climate, but less suitable to gain a mechanistic understanding of plant responses to climatic variability. Experiments seem best suited to simulate extreme events. In models, temporal resolution and model structure are crucial to capture plant responses to changing climatic variability. We highlight that a combination of experimental, observational, and/or modeling studies have the potential to overcome important caveats of the respective individual approaches.     

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.     

The genetic structure of the Swedish population

Patterns of genetic diversity have previously been shown to mirror geography on a global scale and within continents and individual countries. Using genome-wide SNP data on 5174 Swedes with extensive geographical coverage, we analyzed the genetic structure of the Swedish population. We observed strong differences between the far northern counties and the remaining counties. The population of Dalarna county, in north middle Sweden, which borders southern Norway, also appears to differ markedly from other counties, possibly due to this county having more individuals with remote Finnish or Norwegian ancestry than other counties. An analysis of genetic differentiation (based on pairwise F(st)) indicated that the population of Sweden's southernmost counties are genetically closer to the HapMap CEU samples of Northern European ancestry than to the populations of Sweden's northernmost counties. In a comparison of extended homozygous segments, we detected a clear divide between southern and northern Sweden with small differences between the southern counties and considerably more segments in northern Sweden. Both the increased degree of homozygosity in the north and the large genetic differences between the south and the north may have arisen due to a small population in the north and the vast geographical distances between towns and villages in the north, in contrast to the more densely settled southern parts of Sweden. Our findings have implications for future genome-wide association studies (GWAS) with respect to the matching of cases and controls and the need for within-county matching. We have shown that genetic differences within a single country may be substantial, even when viewed on a European scale. Thus, population stratification needs to be accounted for, even within a country like Sweden, which is often perceived to be relatively homogenous and a favourable resource for genetic mapping, otherwise inferences based on genetic data may lead to false conclusions.     

Changes in first flowering dates and flowering duration of 232 plant species on the island of Guernsey

Climate change has affected plant phenology; increasing temperatures are associated with advancing first flowering dates. The impact on flowering duration, however, has rarely been studied. In this study, we analysed first flowering dates and flowering durations from a 27 year dataset of weekly flower observations on 232 plant species from the island of Guernsey in the English Channel. The aim of this study was to explore variation in trends and relationships between first flowering dates, flowering duration and temperature. We specifically looked for evidence that traits, such as life forms and phylogenetic groups, explained variation in sensitivity of first flowering and flowering duration among species. Overall trends revealed significantly earlier flowering over time, by an average of 5.2 days decade^?1 since 1985. A highly significant shortening of flowering duration was observed by an average of 10 days decade^?1. Correlations between first flowering, flowering duration and year varied between different species, traits and flowering periods. Significant differences among traits were observed for first flowering and to a lesser degree for flowering duration. Overall, in comparison to first flowering, more species had significant trends in flowering duration. Temperature relationships revealed large differences in strength and direction of response. 55% of the species revealed a significant negative relationship of first flowering dates and temperature. In contrast, only 19% of flowering durations had a significant negative temperature relationship. The advance in first flowering date together with a shortening of flowering duration suggests potentially serious impacts on pollinators, which might pose a major threat to biodiversity, agriculture and horticulture. Human health, in terms of pollen allergies, however, might benefit from a shortening of specific plant pollen seasons.