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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.
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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.
  • Molecular Plant 11/2013;
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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;
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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.
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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;
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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;
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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.
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    ABSTRACT: Even though Darwin's "On the Origin of Species" implied selection being the main driver of species formation, the role of natural selection in speciation remains poorly understood. In particular, it remains unclear how selection at a few genes can lead to genome-wide divergence and the formation of distinct species. We used a particularly attractive clear-cut case of recent plant ecological speciation to investigate the demography and genomic bases of species formation driven by adaptation to contrasting conditions. High altitude Senecio aethnensis and low altitude Senecio chrysanthemifolius live at the extremes of a mountain slope on Mt. Etna, Sicily and form a hybrid zone at intermediate altitudes, but remain morphologically distinct. Genetic differentiation of these species was analysed at the DNA polymorphism and gene expression levels by high-throughput sequencing of transcriptomes from multiple individuals. Out of ca. 18,000 genes analysed, only a small number (90) displayed differential expression between the two species. These genes showed significantly elevated species differentiation (FST and Dxy), consistent with diversifying selection acting on these genes. Genome-wide genetic differentiation of the species is surprisingly low (FST = 0.19), while ca. 200 genes showed significantly higher (FDR < 1%; mean outlier FST > 0.6) interspecific differentiation and evidence for local adaptation. Diversifying selection at only a handful of loci may be enough for the formation and maintenance of taxonomically well-defined species, despite on-going gene flow. This provides an explanation of why many closely-related species (in plants in particular) remain phenotypically and ecologically distinct despite on-going hybridisation, a question that has long puzzled naturalists and geneticists alike.
    Molecular Biology and Evolution 09/2013;
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    ABSTRACT: High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ETHYLENE OVERPRODUCER1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ETHYLENE RESISTANT1-CONSTITUTIVE TRIPLE RESPONSE1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated HIGH-AFFINITY K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.
    The Plant Cell 09/2013;
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