Arbuscular Mycorrhizal Fungal Networks Vary throughout the Growing Season and between Successional Stages

To date, few analyses of mutualistic networks have investigated successional or seasonal dynamics. Combining interaction data from multiple time points likely creates an inaccurate picture of the structure of networks (because these networks are aggregated across time), which may negatively influence their application in ecosystem assessments and conservation. Using a replicated bipartite mutualistic network of arbuscular mycorrhizal (AM) fungal-plant associations, detected using large sample numbers of plants and AM fungi identified through molecular techniques, we test whether the properties of the network are temporally dynamic either between different successional stages or within the growing season. These questions have never been directly tested in the AM fungal-plant mutualism or the vast majority of other mutualisms. We demonstrate the following results: First, our examination of two different successional stages (young and old forest) demonstrated that succession increases the proportion of specialists within the community and decreases the number of interactions. Second, AM fungal-plant mutualism structure changed throughout the growing season as the number of links between partners increased. Third, we observed shifts in associations between AM fungal and plant species throughout the growing season, potentially reflecting changes in biotic and abiotic conditions. Thus, this analysis opens up two entirely new areas of research: 1) identifying what influences changes in plant-AM fungal associations in these networks, and 2) what aspects of temporal variation and succession are of general importance in structuring bipartite networks and plant-AM fungal communities.     

Long-term ecological study (palaeoecology) to chronicle habitat degradation and inform conservation ecology: an exemplar from the Brecon Beacons, South Wales

For ecosystems perceived as degraded, but for which the causal factors or timescales for the degradation are disputed or not known, long-term (palaeo-)ecological records may aid understanding and lead to more meaningful conservation approaches. To help ‘bridge the gap’ between (very) long-term ecology and contemporary ecology for practical application, there have been calls for working relationships to be established between palaeoecologists and conservation ecologists. One environment in which this has been attempted is blanket mire. Many blanket mires in Europe are degraded and contain few sphagna. In South Wales, almost all exhibit symptoms of degradation, with dominance by purple moor grass (Molinia caerulea) widespread. We used palaeoecological techniques on three peat profiles in the Brecon Beacons to investigate vegetation history of high-altitude blanket mire to help assess the relative contribution of various factors in mire degradation and to inform strategies for mire conservation and restoration management. We found that declines in sphagna preceded the rise to dominance of monocotyledons. Macrofossil records showed that although Molinia was already present on the Beacons before the start of the industrial revolution, its major rise to dominance in one profile was within the 20th Century, coincident with evidence for local fire. In another profile, it was out-competed by Eriophorum vaginatum after the start of the industrial revolution; there is circumstantial evidence to suggest that a reduction in burning contributed to the rise in E. vaginatum. Conservation management to reduce the current local dominance of both Eriophorum and Molinia is supported by the palaeoecological data, but severe erosion and hagging of peat will constrain practical methods for achieving this on the Beacons until the peat is stabilised. We suggest that palaeoecological techniques have wider applicability in conservation.     

Multigene engineering: dawn of an exciting new era in biotechnology

Development of a rice variety enriched in provitamin A, the accumulation of polyhydroxybutyrate polyester in Arabidopsis nuclear transgenic plants (with enzymes targeted to chloroplasts in both), and the expression of bacterial operons via the chloroplast genome are recent landmark achievements in multigene engineering. Hyper-expression of transgenes has resulted in the formation of insecticidal protein crystals or inclusion bodies of pharmaceutical proteins in transgenic chloroplasts, achieving the highest level of transgene expression ever reported in transgenic plants. These achievements illustrate the potential of multigene engineering to realize benefits of the post-genomic revolution.     

Field production and functional evaluation of chloroplast-derived interferon-alpha2b

Type I interferons (IFNs) inhibit viral replication and cell growth and enhance the immune response, and therefore have many clinical applications. IFN-α2b ranks third in world market use for a biopharmaceutical, behind only insulin and erythropoietin. The average annual cost of IFN-α2b for the treatment of hepatitis C infection is $26 000, and is therefore unavailable to the majority of patients in developing countries. Therefore, we expressed IFN-α2b in tobacco chloroplasts, and transgenic lines were grown in the field after obtaining United States Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS) approval. Stable, site-specific integration of transgenes into chloroplast genomes and homoplasmy through several generations were confirmed. IFN-α2b levels reached up to 20% of total soluble protein, or 3 mg per gram of leaf (fresh weight). Transgenic IFN-α2b had similar in vitro biological activity to commercially produced PEG-Intron™ when tested for its ability to protect cells against cytopathic viral replication in the vesicular stomatitis virus cytopathic effect (VSV CPE) assay and to inhibit early-stage human immunodeficiency virus (HIV) infection. The antitumour and immunomodulating properties of IFN-α2b were also seen in vivo . Chloroplast-derived IFN-α2b increased the expression of major histocompatibility complex class I (MHC I) on splenocytes and the total number of natural killer (NK) cells. Finally, IFN-α2b purified from chloroplast transgenic lines (cpIFN-α2b) protected mice from a highly metastatic tumour line. This demonstration of high levels of expression of IFN-α2b, transgene containment and biological activity akin to that of commercial preparations of IFN-α2b facilitated the first field production of a plant-derived human blood protein, a critical step towards human clinical trials and commercialization.     

Mind the sustainability gap

Despite increasing efforts to reach sustainability, key global biophysical indicators such as climate change and biodiversity loss continue to deteriorate rather than improve. Ongoing failure to move towards sustainability calls into question the focus of current research and policy. We recommend two strategies for progress. First, sustainability must be conceptualized as a hierarchy of considerations, with the biophysical limits of the Earth setting the ultimate boundaries within which social and economic goals must be achieved. Second, transdisciplinary research programs must confront key normative questions facing modern consumer societies. The humanities should have a key role in such programs. Assisted by these strategies, ambitious targets that realistically reflect the biophysical limits of the life-support system of the Earth must be set and relentlessly worked towards.     

Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis

Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 +/- 0.5 g (means +/- SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system.     

Complete chloroplast genome sequences of Hordeum vulgare , Sorghum bicolor and Agrostis stolonifera , and comparative analyses with other grass genomes

Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5′ end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19-37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16–21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C–U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complete chloroplast genome sequences of <Emphasis Type="Italic">Hordeum vulgare</Emphasis>, <Emphasis Type="Italic">Sorghum bicolor</Emphasis> and <Emphasis Type="Italic">Agrostis stolonifera</Emphasis>, and comparative analyses with other grass genomes

Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5' end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19-37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16-21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C-U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae.     

Impaired macrophage function underscores susceptibility to Salmonella in mice lacking Irgm1 (LRG-47)

IRG proteins, or immunity-related GTPases (also known as p47 GTPases), are a group of IFN-regulated proteins that are highly expressed in response to infection. The proteins localize to intracellular membranes including vacuoles that contain pathogens in infected macrophages and other host cells. Current data indicate that the IRG protein Irgm1 (LRG-47) is critical for resistance to intracellular bacteria. This function is thought to be a consequence of regulating the survival of vacuolar bacteria in host cells. In the current work, the role of Irgm1 in controlling resistance to Salmonella typhimurium was explored to further define the mechanism through which the protein regulates host resistance. Irgm1-deficient mice displayed increased susceptibility to this bacterium that was reflected in increased bacterial loads in spleen and liver and decreased maturation of S. typhimurium granulomas. The mice also displayed an inability to concentrate macrophages at sites of bacterial deposition. In vitro, the ability of Irgm1-deficient macrophages to suppress intracellular growth of S. typhimurium was impaired. Furthermore, adhesion and motility of Irgm1-deficient macrophages after activation with IFN-gamma was markedly decreased. Altered adhesion/motility of those cells was accompanied by changes in cell morphology, density of adhesion-associated proteins, and actin staining. Together, these data suggest that in addition to regulating the maturation of pathogen-containing vacuoles, Irgm1 plays a key role in regulating the adhesion and motility of activated macrophages.