Engineering of metabolic pathways using synthetic enzyme complexes

 This is the author accepted manuscript. The final version is available from American Society of Plant Biologists via the DOI in this recordThe prospects for the use of synthetic enzyme complexes as a metabolic engineering tool are reviewed.Biotechnology and Biological Sciences Research Council (BBSRC

Hydrogen peroxide metabolism and functions in plants

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this recordH2 O2 is produced, via superoxide and superoxide dismutase, by electron transport in chloroplasts and mitochondria, plasma membrane NADPH oxidases, peroxisomal oxidases, type III peroxidases and other apoplastic oxidases. Intracellular transport is facilitated by aquaporins and H2 O2 is removed by catalase, peroxiredoxin, glutathione peroxidase-like enzymes and ascorbate peroxidase, all of which have cell compartment-specific isoforms. Apoplastic H2 O2 influences cell expansion, development and defence by its involvement in type III peroxidase-mediated polymer cross-linking, lignification and, possibly, cell expansion via H2 O2 -derived hydroxyl radicals. Excess H2 O2 triggers chloroplast and peroxisome autophagy and programmed cell death. The role of H2 O2 in signalling, for example during acclimation to stress and pathogen defence, has received much attention but the signal transduction mechanisms are poorly-defined. H2 O2 oxidises specific cysteine residues of target proteins to the sulfenic acid form and, similarly to other organisms, this modification could initiate thiol-based redox relays and modify target enzymes, receptor kinases and transcription factors. Quantification of the sources and sinks of H2 O2 is being improved by the spatial and temporal resolution of genetically-encoded H2 O2 sensors such as HyPer and roGFP2-Orp1. These H2 O2 sensors combined with detection of specific proteins modified by H2 O2 will allow deeper understanding of its signalling roles. This article is protected by copyright. All rights reserved.The Biotechnology and Biological Sciences Research Council provided funding for research on hydrogen peroxide signalling (BB/I020004/1 and BB/N001311/1)

Pine species that support crown fire regimes have lower leaf-level terpene contents than those native to surface fire regimes

This is the final version. Available from MDPI via the DOI in this record.Fire is increasingly being recognised as an important evolutionary driver in fire-prone environments. Biochemical traits such as terpene (volatile isoprenoid) concentration are assumed to influence plant flammability but have often been overlooked as fire adaptations. We have measured the leaf-level flammability and terpene content of a selection of Pinus species native to environments with differing fire regimes (crown fire, surface fire and no fire). We demonstrate that this biochemical trait is associated with leaf-level flammability which likely links to fire-proneness and we suggest that this contributes to post-fire seedling survival. We find that surface-fire species have the highest terpene abundance and are intrinsically the most flammable, compared to crown-fire species. We suggest that the biochemical traits of surface fire species may have been under selective pressure to modify the fire environment at the leaf and litter scale to moderate fire spread and intensity. We indicate that litter flammability is driven not only by packing ratios and bulk density, but also by terpene content.European Research Counci

Ascorbic acid metabolism and functions: a comparison of plants and mammals

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Ascorbic acid is synthesised by eukaryotes, the known exceptions being primates and some other animal groups which have lost functional gulonolactone oxidase. Prokaryotes do not synthesise ascorbate and do not need an ascorbate supply, so the functions that are essential for mammals and plants are not required or are substituted by other compounds. The ability of ascorbate to donate electrons enables it to act as a free radical scavenger and to reduce higher oxidation states of iron to Fe2+. These reactions are the basis of its biological activity along with the relative stability of the resulting resonance stabilised monodehydroascorbate radical. The importance of these properties is emphasised by the evolution of at least three biosynthetic pathways and production of an ascorbate analogue, erythroascorbate, by fungi. The iron reducing activity of ascorbate maintains the reactive centre Fe2+ of 2-oxoglutarate-dependent dioxygenases (2-ODDs) thus preventing inactivation. These enzymes have diverse functions and, recently, the possibility that ascorbate status in mammals could influence 2-ODDs involved in histone and DNA demethylation thereby influencing stem cell differentiation and cancer has been uncovered. Ascorbate is involved in iron uptake and transport in plants and animals. While the above biochemical functions are shared between mammals and plants, ascorbate peroxidase (APX) is an enzyme family limited to plants and photosynthetic protists. It provides these organisms with increased capacity to remove H2O2 produced by photosynthetic electron transport and photorespiration. The Fe reducing activity of ascorbate enables hydroxyl radical production (pro-oxidant effect) and the reactivity of dehydroascorbate (DHA) and reaction of its degradation products with proteins (dehydroascorbylation and glycation) is potentially damaging. Ascorbate status influences gene expression in plants and mammals but at present there is little evidence that it acts as a specific signalling molecule. It most likely acts indirectly by influencing the redox state of thiols and 2-ODD activity. However, the possibility that dehydroascorbylation is a regulatory post-translational protein modification could be explored.NS was funded by UK Biotechnology and Biological Sciences Research Council grant numbers BB/I020004/1 and BB/N001311/

Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana

© 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens.Hazara University (Mansehra, NWFP, Pakistan)Higher Education Commission (Pakistan)BBSRCExeter University Science Strategy Fun

Monitoring lipid accumulation in the green microalga Botryococcus braunii with frequency-modulated stimulated Raman scattering

© 2015 SPIE.The potential of microalgae as a source of renewable energy has received considerable interest because they can produce lipids (fatty acids and isoprenoids) that can be readily converted into biofuels. However, significant research in this area is required to increase yields to make this a viable renewable source of energy. An analytical tool that could provide quantitative in situ spectroscopic analysis of lipids synthesis in individual microalgae would significantly enhance our capability to understand the synthesis process at the cellular level and lead to the development of strategies for increasing yield. Stimulated Raman scattering (SRS) microscopy has great potential in this area however, the pump-probe signal from two-color two-photon absorption of pigments (chlorophyll and carotenoids) overwhelm the SRS signal and prevent its application. Clearly, the development of a background suppression technique is of significant value for this important research area. To overcome the limitation of SRS in pigmented specimens, we establish a frequency-modulated stimulated Raman scattering (FM-SRS) microscopy that eliminates the non-Raman background by rapidly toggling on-and-off the targeted Raman resonance. Moreover, we perform the background-free imaging and analysis of intracellular lipid droplets and extracellular hydrocarbons in a green microalga with FM-SRS microscopy. We believe that FM-SRS microscopy demonstrates the potential for many applications in pigmented cells and provides the opportunity for improved selective visualization of the chemical composition of algae and plants.We thank Delong Zhang at Purdue University for fruitful discussion. This research was supported by grants (BB/K013602/1) from the Biotechnology and Biological Sciences Research Council (BBSRC) and Syngenta

Ascorbate-Deficient vtc2 Mutants in Arabidopsis Do Not Exhibit Decreased Growth

This is the final version of the article. Available from Frontiers Media via the DOI in this record.In higher plants the L-galactose pathway represents the major route for ascorbate biosynthesis. The first committed step of this pathway is catalyzed by the enzyme GDP-L-galactose phosphorylase and is encoded by two paralogs in Arabidopsis – VITAMIN C2 (VTC2) and VTC5. The first mutant of this enzyme, vtc2-1, isolated via an EMS mutagenesis screen, has approximately 20–30% of wildtype ascorbate levels and has been reported to have decreased growth under standard laboratory conditions. Here, we show that a T-DNA insertion into the VTC2 causes a similar reduction in ascorbate levels, but does not greatly affect plant growth. Subsequent segregation analysis revealed the growth defects of vtc2-1 mutants segregate independently of the vtc2-1 mutation. These observations suggest that it is the presence of an independent cryptic mutation that affects growth of vtc2-1 mutants, and not the 70–80% decrease in ascorbate levels that has been assumed in past studies.This research is supported by a grant from the Australian Research Council (BL, CSC, JFG) and a Biotechnology and Biological Sciences Research Council grant (NS; BB/G021678/1)

Use of Geotechnical Design Summary Report as a Management Tool for Resolving Disputes on Underground Construction Projects

The Los Angeles Metro Rail Subway project is initially planned for 30 km (18 mi) of twin, 6-m (20-ft) diameter bored tunnels under city streets and a total of 16 stations. This paper describes briefly the history of the project, the geologic setting, and the challenges encountered during design or anticipated during construction. It also introduces, as part of contract documentation, an interpretive geotechnical baseline report which establishes the basis for identification and recognition of site condition baselines . In so doing, this report (known as a GDSR ) has proven to be an effective tool for ameliorating contractual problems and facilitating conflict resolution

Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom

This is the final version of the article. Available from Springer Nature via the DOI in this recordThe publisher correction to this article is in ORE at: http://hdl.handle.net/10871/34487Diatoms contribute roughly 20% of global primary production, but the factors determining their ability to adapt to global warming are unknown. Here we quantify the capacity for adaptation to warming in the marine diatom Thalassiosira pseudonana. We find that evolutionary rescue under severe (32 °C) warming is slow, but adaptation to more realistic scenarios where temperature increases are moderate (26 °C) or fluctuate between benign and severe conditions is rapid and linked to phenotypic changes in metabolic traits and elemental composition. Whole-genome re-sequencing identifies genetic divergence among populations selected in the different warming regimes and between the evolved and ancestral lineages. Consistent with the phenotypic changes, the most rapidly evolving genes are associated with transcriptional regulation, cellular responses to oxidative stress and redox homeostasis. These results demonstrate that the evolution of thermal tolerance in marine diatoms can be rapid, particularly in fluctuating environments, and is underpinned by major genomic and phenotypic change.This study was funded by a Leverhulme Trust research grant (RPG-2013-335). Whole genome re-sequencing was carried out at Exeter Sequencing Service and Computational core facilities at the University of Exeter, where Dr. Karen Moore, Dr. Audrey Farbos, Paul O’Neill, and Dr. Konrad Paszkiewicz lead the handling of the samples. Exeter Squencing Services are supported by Medical Research Council Clinical Infrastructure award (MR/M008924/1), Wellcome Trust Institutional Strategic Support Fund (WT097835MF), Wellcome Trust Multi User Equipment Award (WT101650MA), and BBSRC LOLA award (BB/K003240/1)

Publisher Correction: Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom

The article for which this is the publisher correction is in ORE at: http://hdl.handle.net/10871/32652The PDF version of this Article was updated shortly after publication following an error which resulted in the Φ symbol being omitted from the left hand side of equation 8. The HTML version was correct from the time of publication