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    ABSTRACT: The analysis of stable isotope incorporation following feeding of (13)C-labeled precursors to plant tissues provides the constraints necessary for metabolic flux analysis. This protocol describes the use of one-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy for the quantification of (13)C enrichments and isotopomer abundances in mixtures of metabolites or hydrolyzed biomass components.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1090:73-86. DOI:10.1007/978-1-62703-688-7_5
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    ABSTRACT: While steady-state (13)C metabolic flux analysis is a powerful method for deducing multiple fluxes in the central metabolic network of heterotrophic and mixotrophic plant tissues, it is also time-consuming and technically challenging. Key steps in the design and interpretation of steady-state (13)C labeling experiments are illustrated with a generic protocol based on applications to plant cell suspension cultures.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1090:53-72. DOI:10.1007/978-1-62703-688-7_4
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    ABSTRACT: One crucial feature of zygotic linkage disequilibrium (LD) analysis is its direct use of diploid genotyping data, irrespective of the type of mating system. Previous theories from an evolutionary perspective mainly focus on gametic LD, but the equivalent development for zygotic LD is not available. Here I study the evolution of zygotic LD and the covariances between gametic and zygotic LDs or between distinct zygotic LDs in a finite local population under constant immigration from a continent population. I derive the analytical theory under genetic hitchhiking effects or in a neutral process. Results indicate that zygotic LDs (diploid level) are more informative than gametic LD (haploid level) in indicating the effects of different evolutionary forces. Zygotic LDs may be greater than or comparable to gametic LD under the epistatic selection process, but smaller than gametic LD under the non epistatic selection process. The covariances between gametic and zygotic LDs are strongly affected by the mating system, linkage distance, and genetic drift effects, but weakly affected by seed and pollen flow and natural selection. The covariances between different zygotic LDs are generally robust to the effects of gene flow, selection, and linkage distance, but sensitive to the effects of genetic drift and mating system. Consistent patterns exist for the covariances between the zygotic LDs for the two-locus genotypes with one common genotype at one locus or without any common genotype at each locus. The results highlight that zygotic LDs can be applied to detecting natural population history.
    PLoS ONE 11/2013; 8(11):e80538. DOI:10.1371/journal.pone.0080538
  • Molecular Plant 11/2013; 7(4). DOI:10.1093/mp/sst167
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    ABSTRACT: Inoculations with the saprophytic fungus Trichoderma spp. are now extensively used both to promote plant growth and suppress disease development. The underlying mechanisms for both roles have yet to be fully described so that the use of Trichoderma could be optimised. We here show that Trichoderma asperelloides effects include the manipulation of host NO production. NO was rapidly formed in Arabidopsis roots in response to soil borne necrotrophic pathogen Fusarium. oxysporum and persisted for ~1 h but is only transiently produced ( ~ 10 min) when roots interact with T. asperelloides (T203). However, inoculation of F. oxysporum infected roots with T. asperelloides suppressed F. oxysporum initiated NO production. A transcriptional study of 78 NO modulated genes indicated most genes were suppressed by single and combinational challenge with F. oxysporum or T. asperelloides. Only two F. oxysporum induced genes were suppressed by T. asperelloides inoculation undertaken either 10 min prior to or post pathogen infection; a concanavlin A-like lectin protein kinase (At4g28350) and the receptor-like protein, RLP30. Thus T. asperelloides can actively suppress NO production elicited by F. oxysporum and impacts on the expression of some genes reported to be NO-responsive. Of particular interest was the reduced expression of receptor-like genes which may be required for F. oxysporum dependent necrotrophic disease development.
    Molecular Plant-Microbe Interactions 11/2013; DOI:10.1094/MPMI-06-13-0160-R
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    ABSTRACT: Chloroplasts are the organelles that define plants, and they are responsible for photosynthesis as well as numerous other functions. They are the ancestral members of a family of organelles known as plastids. Plastids are remarkably dynamic, existing in strikingly different forms that interconvert in response to developmental or environmental cues. The genetic system of this organelle and its coordination with the nucleocytosolic system, the import and routing of nucleus-encoded proteins, as well as organellar division all contribute to the biogenesis and homeostasis of plastids. They are controlled by the ubiquitin-proteasome system, which is part of a network of regulatory mechanisms that integrate plastid development into broader programmes of cellular and organismal development.
    Nature Reviews Molecular Cell Biology 11/2013; 14(12):787-802. DOI:10.1038/nrm3702
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    ABSTRACT: Metal hyperaccumulating plants are able to accumulate exceptionally high concentrations of metals, such as zinc, nickel, or cadmium, in their aerial tissues. These metals reach concentrations that would be toxic to most other plant species. This trait has evolved multiple times independently in the plant kingdom. Recent studies have provided new insight into the ecological and evolutionary significance of this trait, by showing that some metal hyperaccumulating plants can use high concentrations of accumulated metals to defend themselves against attack by pathogenic microorganisms and herbivores. Here, we review the evidence that metal hyperaccumulation acts as a defensive trait in plants, with particular emphasis on plant-pathogen interactions. We discuss the mechanisms by which defense against pathogens might have driven the evolution of metal hyperaccumulation, including the interaction of this trait with other forms of defense. In particular, we consider how physiological adaptations and fitness costs associated with metal hyperaccumulation could have resulted in trade-offs between metal hyperaccumulation and other defenses. Drawing on current understanding of the population ecology of metal hyperaccumulator plants, we consider the conditions that might have been necessary for metal hyperaccumulation to be selected as a defensive trait, and discuss the likelihood that these were fulfilled. Based on these conditions, we propose a possible scenario for the evolution of metal hyperaccumulation, in which selective pressure for resistance to pathogens or herbivores, combined with gene flow from non-metallicolous populations, increases the likelihood that the metal hyperaccumulating trait becomes established in plant populations.
    Frontiers in Plant Science 10/2013; 4:395. DOI:10.3389/fpls.2013.00395
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    ABSTRACT: Flux is one of the most informative measures of metabolic behavior. Its estimation requires integration of experimental and modeling approaches and, thus, is at the heart of metabolic systems biology. In this review, we argue that flux analysis and modeling of a range of plant systems points to the importance of the supply of metabolic inputs and demand for metabolic end-products as key drivers of metabolic behavior. This has implications for metabolic engineering, and the use of in silico models will be important to help design more effective engineering strategies. We also consider the importance of cell type-specific metabolism and the challenges of characterizing metabolism at this resolution. A combination of new measurement technologies and modeling approaches is bringing us closer to integrating metabolic behavior with whole-plant physiology and growth.
    Trends in Plant Science 10/2013; 19(4). DOI:10.1016/j.tplants.2013.09.005
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    ABSTRACT: Plants respond to pathogen attack via a rapid burst of reactive oxygen species (ROS). However, ROS are also produced by fungal metabolism and are required for the development of infection structures in Magnaporthe oryzae. To obtain a better understanding of redox regulation in M. oryzae, we measured the amount and redox potential of glutathione (EGSH ), as the major cytoplasmic anti-oxidant, the rates of ROS production, and mitochondrial activity using multi-channel four-dimensional (x,y,z,t) confocal imaging of Grx1-roGFP2 and fluorescent reporters during spore germination, appressorium formation and infection. High levels of mitochondrial activity and ROS were localized to the growing germ tube and appressorium, but EGSH was highly reduced and tightly regulated during development. Furthermore, germlings were extremely resistant to external H2 O2 exposure ex planta. EGSH remained highly reduced during successful infection of the susceptible rice cultivar CO39. By contrast, there was a dramatic reduction in the infection of resistant (IR68) rice, but the sparse hyphae that did form also maintained a similar reduced EGSH . We conclude that M. oryzae has a robust anti-oxidant defence system and maintains tight control of EGSH despite substantial oxidative challenge. Furthermore, the magnitude of the host oxidative burst alone does not stress the pathogen sufficiently to prevent infection in this pathosystem.
    New Phytologist 10/2013; 201(2). DOI:10.1111/nph.12530
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    ABSTRACT: The hybrid zone on Mount Etna (Sicily) between Senecio aethnensis and Senecio chrysanthemifolius (two morphologically and physiologically distinct species) is a classic example of an altitudinal cline. Hybridization at intermediate altitudes and gradients in phenotypic and life-history traits occur along altitudinal transects of the volcano. The cline is considered to be a good example of ecological selection with species differences arising by divergent selection opposing gene flow. However, the possibility that the cline formed from recent secondary contact following an allopatric phase is difficult to exclude. We demonstrate a recent split between S. aethnensis and S. chrysanthemifolius (as recent as ∼32,000 years ago) and sufficient gene flow (2Nm > 1) to have prevented divergence (implicating a role for diversifying selection in the maintenance of the cline). Differentially expressed genes between S. aethnensis and S. chrysanthemifolius exhibit significantly higher genetic divergence relative to "expression invariant" controls, suggesting that species differences may in part be mediated by divergent selection on differentially expressed genes involved with altitude-related adaptation. The recent split time and the absence of fixed differences between these two ecologically distinct species suggest the rapid evolution to an altitudinal cline involving selection on both sequence and expression variation.
    Evolution 10/2013; 67(10):3032-3042. DOI:10.1111/evo.12157
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