‘Real time’ genetic manipulation: a new tool for ecological field studies

Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene‐related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose‐dependent effects. We applied the sensitive dexamethasone (DEX)‐inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin‐based DEX application method to facilitate ectopic gene expression and RNA interference‐mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field‐grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant–herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.     

The holocentric species Luzula elegans shows interplay between centromere and large‐scale genome organization

In higher plants, the large‐scale structure of monocentric chromosomes consists of distinguishable eu‐ and heterochromatic regions, the proportions and organization of which depend on a species' genome size. To determine whether the same interplay is maintained for holocentric chromosomes, we investigated the distribution of repetitive sequences and epigenetic marks in the woodrush Luzula elegans (3.81 Gbp/1C). Sixty‐one per cent of the L. elegans genome is characterized by highly repetitive DNA, with over 30 distinct sequence families encoding an exceptionally high diversity of satellite repeats. Over 33% of the genome is composed of the Angela clade of Ty1/copia LTR retrotransposons, which are uniformly dispersed along the chromosomes, while the satellite repeats occur as bands whose distribution appears to be biased towards the chromosome termini. No satellite showed an almost chromosome‐wide distribution pattern as expected for a holocentric chromosome and no typical centromere‐associated LTR retrotransposons were found either. No distinguishable large‐scale patterns of eu‐ and heterochromatin‐typical epigenetic marks or early/late DNA replicating domains were found along mitotic chromosomes, although super‐high‐resolution light microscopy revealed distinguishable interspersed units of various chromatin types. Our data suggest a correlation between the centromere and overall genome organization in species with holocentric chromosomes.     

Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis

Light‐harvesting complex II (LHCII) contains three highly homologous chlorophyll‐a/b‐binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo‐ and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so‐called state‐transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10 sec of ‘state 2 light’ results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non‐phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super‐ and mega‐complexes that comprised both photosystem (PS)I and PSII, and the state 2‐specific PSI–LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting.     

Population genomic footprints of selection and associations with climate in natural populations of Arabidopsis halleri from the Alps

Natural genetic variation is essential for the adaptation of organisms to their local environment and to changing environmental conditions. Here, we examine genomewide patterns of nucleotide variation in natural populations of the outcrossing herb Arabidopsis halleri and associations with climatic variation among populations in the Alps. Using a pooled population sequencing (Pool‐Seq) approach, we discovered more than two million SNPs in five natural populations and identified highly differentiated genomic regions and SNPs using F_ST‐based analyses. We tested only the most strongly differentiated SNPs for associations with a nonredundant set of environmental factors using partial Mantel tests to identify topo‐climatic factors that may underlie the observed footprints of selection. Possible functions of genes showing signatures of selection were identified by Gene Ontology analysis. We found 175 genes to be highly associated with one or more of the five tested topo‐climatic factors. Of these, 23.4% had unknown functions. Genetic variation in four candidate genes was strongly associated with site water balance and solar radiation, and functional annotations were congruent with these environmental factors. Our results provide a genomewide perspective on the distribution of adaptive genetic variation in natural plant populations from a highly diverse and heterogeneous alpine environment.     

Embryos,polyps and medusae of the Early Cambrian scyphozoan Olivooides

The Early Cambrian organism Olivooides is known from both embryonic and post-embryonic stages and, consequently, it has the potential to yield vital insights into developmental evolution at the time that animal body plans were established. However, this potential can only be realized if the phylogenetic relationships of Olivooides can be constrained. The affinities of Olivooides have proved controversial because of the lack of knowledge of the internal anatomy and the limited range of developmental stages known. Here, we describe rare embryonic specimens in which internal anatomical features are preserved. We also present a fuller sequence of fossilized developmental stages of Olivooides, including associated specimens that we interpret as budding ephyrae (juvenile medusae), all of which display a clear pentaradial symmetry. Within the framework of a cnidarian interpretation, the new data serve to pinpoint the phylogenetic position of Olivooides to the scyphozoan stem group. Hypotheses about scalidophoran or echinoderm affinities of Olivooides can be rejected.     

The global nitrogen cycle in the twenty-first century

Global nitrogen fixation contributes 413 Tg of reactive nitrogen (N_r) to terrestrial and marine ecosystems annually of which anthropogenic activities are responsible for half, 210 Tg N. The majority of the transformations of anthropogenic N_r are on land (240 Tg N yr⁻¹) within soils and vegetation where reduced N_r contributes most of the input through the use of fertilizer nitrogen in agriculture. Leakages from the use of fertilizer N_r contribute to nitrate (NO₃⁻) in drainage waters from agricultural land and emissions of trace N_r compounds to the atmosphere. Emissions, mainly of ammonia (NH₃) from land together with combustion related emissions of nitrogen oxides (NO_x), contribute 100 Tg N yr⁻¹ to the atmosphere, which are transported between countries and processed within the atmosphere, generating secondary pollutants, including ozone and other photochemical oxidants and aerosols, especially ammonium nitrate (NH₄NO₃) and ammonium sulfate (NH₄)₂SO₄. Leaching and riverine transport of NO₃ contribute 40–70 Tg N yr⁻¹ to coastal waters and the open ocean, which together with the 30 Tg input to oceans from atmospheric deposition combine with marine biological nitrogen fixation (140 Tg N yr⁻¹) to double the ocean processing of N_r. Some of the marine N_r is buried in sediments, the remainder being denitrified back to the atmosphere as N₂ or N₂O. The marine processing is of a similar magnitude to that in terrestrial soils and vegetation, but has a larger fraction of natural origin. The lifetime of N_r in the atmosphere, with the exception of N₂O, is only a few weeks, while in terrestrial ecosystems, with the exception of peatlands (where it can be 10²–10³ years), the lifetime is a few decades. In the ocean, the lifetime of N_r is less well known but seems to be longer than in terrestrial ecosystems and may represent an important long-term source of N₂O that will respond very slowly to control measures on the sources of N_r from which it is produced.     

Discovering the electronic circuit diagram of life: structural relationships among transition metal binding sites in oxidoreductases

Oxidoreductases play a central role in catalysing enzymatic electron-transfer reactions across the tree of life. To first order, the equilibrium thermodynamic properties of these proteins are governed by protein folds associated with specific transition metals and ligands at the active site. A global analysis of holoenzyme structures and functions suggests that there are fewer than approximately 500 fundamental oxidoreductases, which can be further clustered into 35 unique groups. These catalysts evolved in prokaryotes early in the Earth''s history and are largely responsible for the emergence of non-equilibrium biogeochemical cycles on the planet''s surface. Although the evolutionary history of the amino acid sequences in the oxidoreductases is very difficult to reconstruct due to gene duplication and horizontal gene transfer, the evolution of the folds in the catalytic sites can potentially be used to infer the history of these enzymes. Using a novel, yet simple analysis of the secondary structures associated with the ligands in oxidoreductases, we developed a structural phylogeny of these enzymes. The results of this ‘composome’ analysis suggest an early split from a basal set of a small group of proteins dominated by loop structures into two families of oxidoreductases, one dominated by α-helices and the second by β-sheets. The structural evolutionary patterns in both clades trace redox gradients and increased hydrogen bond energy in the active sites. The overall pattern suggests that the evolution of the oxidoreductases led to decreased entropy in the transition metal folds over approximately 2.5 billion years, allowing the enzymes to use increasingly oxidized substrates with high specificity.     

Grazers: biocatalysts of terrestrial silica cycling

Silica is well known for its role as inducible defence mechanism countering herbivore attack, mainly through precipitation of opaline, biogenic silica (BSi) bodies (phytoliths) in plant epidermal tissues. Even though grazing strongly interacts with other element cycles, its impact on terrestrial silica cycling has never been thoroughly considered. Here, BSi content of ingested grass, hay and faeces of large herbivores was quantified by performing multiple chemical extraction procedures for BSi, allowing the assessment of chemical reactivity. Dissolution experiments with grass and faeces were carried out to measure direct availability of BSi for dissolution. Average BSi and readily soluble silica numbers were higher in faeces as compared with grass or hay, and differences between herbivores could be related to distinct digestive strategies. Reactivity and dissolvability of BSi increases after digestion, mainly due to degradation of organic matrices, resulting in higher silica turnover rates and mobilization potential from terrestrial to aquatic ecosystems in non-grazed versus grazed pasture systems (2 versus 20 kg Si ha⁻¹ y⁻¹). Our results suggest a crucial yet currently unexplored role of herbivores in determining silica export from land to ocean, where its availability is linked to eutrophication events and carbon sequestration through C–Si diatom interactions.     

Structure and content of pollutants in temporal surface sediments in small watersheds

ABSTRACT

Formation of gel-like surface sediments has been observed during spring and autumn in small watersheds in boreal areas with pH <5.7 and concentrations of humic substances above 3 mg L^?1. This structure efficiently accumulates dissolved, colloidal and particulate constituents. The gel consists of ferric (hydr)oxides and fulvic acid and has high viable counts of bacteria (notably Gallionella spp.) as well as fungal hyphae. The solid/solution distribution (log K_d; l kg^?1) for trace metals (Cd, Cu, Pb, V, Zn) ranges from 4 to 6.5 which indicate an efficient accumulation during periods with gel. The concentrations of adsorbable organic halogens in the gel were not different from forest soils in general.     

Two strains of Pseudomonas fluorescens bacteria differentially affect survivorship of waxworm (Galleria mellonella) larvae exposed to an arthropod fungal pathogen,Beauveria bassiana

Two strains of Pseudomonas fluorescens were found contaminating a biopesticide used in a previous study against Varroa destructor infestations in honey bee hives. In that study, the biopesticide, a formulation of a fungal pathogen of arthropods, Beauveria bassiana, failed to have any negative impact on the mite infestation despite successful results in previous studies using uncontaminated batches of the same biopesticide. The objective of the present research was to determine whether the bacteria may have interfered with the infectivity and/or virulence of B. bassiana in a simplified system; positive results in that system would then provide a rationale for further work under more complex conditions. Galleria mellonella late instar larvae treated topically with both a bacterial suspension of 6.8 to 7.0×10⁷ cfu/ml and a fungal suspension of 2.5×10⁷ or 2.5×10⁸ B. bassiana conidia/ml showed, in the case of one of the bacterial strains, significantly increased survivorship compared to larvae treated with just the B. bassiana suspension. When larvae were immersed in a bacterial suspension prior to application of B. bassiana suspension using a spray tower, a significant positive effect of the same P. fluorescens strain on larval survivorship was observed at 2.5×10⁸ conidia/ml. Neither the bacterial suspensions alone nor blank control solutions had any effect on larval survivorship. These results show that an interaction between the bacteria and the pathogen may explain some of the results from the prior field trial.