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  • Journal article
    Michoux F, Ahmad N, Wei Z-Y, Belgio E, Ruban A, Nixon PJet al., 2016,

    Testing the role of the N-terminal tail of D1 in the maintenance of photosystem II in tobacco chloroplasts

    , Frontiers in Plant Science, Vol: 7, ISSN: 1664-462X

    A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction centre subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25 % of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress.
  • Journal article
    Dunning LT, Hipperson H, Baker WJ, Butlin RK, Devaux C, Hutton I, Igea J, Papadopulos AST, Quan X, Smadja CM, Turnbull CGN, Savolainen Vet al., 2016,

    Ecological speciation in sympatric palms: 1. Gene expression, selection and pleiotropy

    , Journal of Evolutionary Biology, Vol: 29, Pages: 1472-1487, ISSN: 1420-9101

    Ecological speciation requires divergent selection, reproductive isolation, and a genetic mechanism to link the two. We examined the role of gene expression and coding sequence evolution in this process using two species of Howea palms that have diverged sympatrically on Lord Howe Island, Australia. These palms are associated with distinct soil types and have displaced flowering times, representing an ideal candidate for ecological speciation. We generated large amounts of RNA-Seq data from multiple individuals and tissue types collected on the island from each of the two species. We found that differentially expressed loci as well as those with divergent coding sequences between Howea species were associated with known ecological and phenotypic differences, including response to salinity, drought, pH and flowering time. We identified loci with potential dual function in flowering time and soil adaptation, which effect on flowering time was validated by knocking orthologous genes in a model plant species. These results indicate that pleiotropy could have favoured the evolution of barrier traits in this system, despite ongoing gene flow.
  • Journal article
    Larrieu A, Champion A, Legrand J, Lavenus J, Mast D, Brunoud G, Oh J, Guyomarc H S, Pizot M, Farmer EE, Turnbull C, Vernoux T, Bennett MJ, Laplaze Let al., 2016,

    Corrigendum: A fluorescent hormone biosensor reveals the dynamics of jasmonate signalling in plants

    , Nature Communications, Vol: 7, ISSN: 2041-1723
  • Conference paper
    Ces O, Elani Y, Karamdad K, Friddin MS, Barter LMC, Bolognesi G, Law RV, Chan CL, Brooks NJ, Seddon JMet al., 2016,

    Novel microfluidic technologies for the bottom-up construction of artificial cells

    This talk will outline novel microfluidic strategies for biomembrane engineering that are capable of fabricating vesicles [1], droplet interface bilayer networks [2], multisomes [3] and artificial tissues [4] where parameters such as membrane asymmetry, membrane curvature, compartment connectivity and individual compartment contents can be controlled. Various bulk methods, such as extrusion, gentle hydration and electroformation, have been synonymous with the formation of lipid vesicles over recent years. However these strategies suffer from significant shortcomings associated with these processes including limited control of vesicle structural parameters such as size, lamellarity, membrane composition and internal contents. To address this technological bottleneck we have developed novel microfluidic platforms to form lipid vesicles in high-Throughput with full control over the composition of both the inner and outer leaflet of the membrane thereby enabling the manufacture of symmetric and asymmetric vesicles. This is achieved by manufacturing microfluidic channels with a step junction, produced by double-layer photolithography, which facilitates the transfer of a W/O emulsion across an oil-water phase boundary and the self-Assembly of a phospholipid bilayer. These platforms are being used to explore the role of asymmetry in biological systems [1] and study the engineering rules that regulate membrane mediated protein-protein interactions [5]. In addition, these technologies are enabling the construction of biological machines capable of acting as micro-reactors [6], environmental sensors and smart delivery vehicles [5] as well as complex multi-compartment artificial cells where the contents and connectivity of each compartment can be controlled. These compartments are separated by biological functional membranes that can facilitate transport between the compartments themselves and between the compartments and external environment. This approach has led to the deve
  • Book chapter
    Barretto S, Michoux F, Nixon PJ, 2016,

    Temporary Immersion Bioreactors for the Contained Production of Recombinant Proteins in Transplastomic Plants.

    , Recombinant Proteins from Plants: Methods and Protocols, Publisher: Springer, Pages: 149-160, ISBN: 978-1-4939-3288-7

    Despite the largely maternal inheritance of plastid genomes, the risk of transgene dissemination from transplastomic plants can limit the scope for field cultivation. There is a need for a cost-effective, scalable process to grow large quantities of transplastomic plant biomass for biosynthesis of biopharmaceuticals and other high-value heterologous proteins. Temporary immersion culture is a means of achieving this under fully contained conditions. This method describes the organogenesis of transplastomic Nicotiana tabacum callus in RITA(®) temporary immersion bioreactors to produce rootless leafy biomass, and subsequent total soluble protein extraction, SDS-PAGE, and Western immunoblot analysis of heterologous protein expression. This method can be used for propagation of plastid or nuclear transformants, though is especially suitable for transplastomic biomass, as organogenesis leads to greater expression and accumulation of transplastomic proteins due to increases in chloroplast number and size.
  • Journal article
    Antoniadi I, Plackova L, Simonovik B, Dolezal K, Turnbull C, Ljung K, Novak Oet al., 2015,

    Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex

    , PLANT CELL, Vol: 27, Pages: 1955-1967, ISSN: 1040-4651
  • Journal article
    Kanvil S, Collins CM, Powell G, Turnbull CGNet al., 2015,

    Cryptic Virulence and Avirulence Alleles Revealed by Controlled Sexual Recombination in Pea Aphids

    , GENETICS, Vol: 199, Pages: 581-593, ISSN: 0016-6731
  • Journal article
    Kanvil S, Powell G, Turnbull C, 2014,

    Pea aphid biotype performance on diverse Medicago host genotypes indicates highly specific virulence and resistance functions

    , Bulletin of Entomological Research, Vol: 104, Pages: 689-701, ISSN: 1475-2670

    Aphid–plant interactions depend on genotypes of both organisms, which determine the two-way molecular exchange that leads to compatible or incompatible outcomes. The underlying genes are mostly unknown, making it difficult to predict likelihood of aphid success or host resistance, and hampering crop genetic improvement. Here we screened eight pea aphid clonal genotypes collected from diverse legume hosts, on a species-wide panel of Medicago truncatula (Mt) genotypes. Aphid virulence was measured by survival, fecundity and growth rate, together with scores for chlorosis and necrosis as host response indicators. Outcomes were highly dependent on the specific aphid–host genotype combinations. Only one Mt line was fully resistant against all clones. Aphid-induced host chlorosis and necrosis varied greatly, but correlated with resistance only in a few combinations. Bi-clustering analysis indicated that all aphid clones could be distinguished by their performance profiles across the host genotypes tested, with each clone being genetically differentiated and potentially representing a distinct biotype. Clones originating from Medicago sativa ranged from highly virulent to almost completely avirulent on both Medicago species, indicating that some were well adapted, whereas others were most likely migrants. Comparisons of closely related pairs of Australian Mt genotypes differing in aphid resistance revealed no enhanced resistance to European pea aphid clones. Based on the extensive variation in pea aphid adaptation even on unfamiliar hosts, most likely reflecting multiple biotype-specific gene-for-gene interactions, we conclude that robust defences require an arsenal of appropriate resistance genes.
  • Journal article
    Young NF, Ferguson BJ, Antoniadi I, Bennett MH, Beveridge CA, Turnbull CGNet al., 2014,

    Conditional Auxin Response and Differential Cytokinin Profiles in Shoot Branching Mutants.

    , Plant physiology, Vol: 165, Pages: 1723-1736, ISSN: 0032-0889

    Strigolactone (SL), auxin, and cytokinin (CK) are hormones that interact to regulate shoot branching. For example, several ramosus (rms) branching mutants in pea (Pisum sativum) have SL defects, perturbed xylem CK levels, and diminished responses to auxin in shoot decapitation assays. In contrast with the last of these characteristics, we discovered that buds on isolated nodes (explants) of rms plants instead respond normally to auxin. We hypothesized that the presence or absence of attached roots would result in transcriptional and hormonal differences in buds and subtending stem tissues, and might underlie the differential auxin response. However, decapitated plants and explants both showed similar up-regulation of CK biosynthesis genes, increased CK levels, and down-regulation of auxin transport genes. Moreover, auxin application counteracted these trends, regardless of the effectiveness of auxin at inhibiting bud growth. Multivariate analysis revealed that stem transcript and CK changes were largely associated with decapitation and/or root removal and auxin response, whereas bud transcript profiles related more to SL defects. CK clustering profiles were indicative of additional zeatin-type CKs in decapitated stems being supplied by roots and thus promoting bud growth in SL-deficient genotypes even in the presence of added auxin. This difference in CK content may explain why rms buds on explants respond better to auxin than those on decapitated plants. We further conclude that rapid changes in CK status in stems are auxin dependent but largely SL independent, suggesting a model in which auxin and CK are dominant regulators of decapitation-induced branching, whereas SLs are more important in intact plants.
  • Journal article
    O'Donnelly K, Zhao G, Patel P, Butt MS, Mak LH, Kretschmer S, Woscholski R, Barter LMCet al., 2014,

    Isolation and kinetic characterisation of hydrophobically distinct populations of form I Rubisco

    , Planet Methods, Vol: 10, ISSN: 1746-4811

    BackgroundRubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. It is thought to be a major cause of photosynthetic inefficiency, suffering from both a slow catalytic rate and lack of specificity due to a competing reaction with oxygen. Revealing and understanding the engineering rules that dictate Rubisco’s activity could have a significant impact on photosynthetic efficiency and crop yield.ResultsThis paper describes the purification and characterisation of a number of hydrophobically distinct populations of Rubisco from both Spinacia oleracea and Brassica oleracea extracts. The populations were obtained using a novel and rapid purification protocol that employs hydrophobic interaction chromatography (HIC) as a form I Rubisco enrichment procedure, resulting in distinct Rubisco populations of expected enzymatic activities, high purities and integrity.ConclusionsWe demonstrate here that HIC can be employed to isolate form I Rubisco with purities and activities comparable to those obtained via ion exchange chromatography (IEC). Interestingly, and in contrast to other published purification methods, HIC resulted in the isolation of a number of hydrophobically distinct Rubisco populations. Our findings reveal a so far unaccounted diversity in the hydrophobic properties within form 1 Rubisco. By employing HIC to isolate and characterise Spinacia oleracea and Brassica oleracea, we show that the presence of these distinct Rubisco populations is not species specific, and we report for the first time the kinetic properties of Rubisco from Brassica oleracea extracts. These observations may aid future studies concerning Rubisco’s structural and functional properties.
  • Journal article
    Miller D, Booth PJ, Seddon JM, Templer RH, Law RV, Woscholski R, Ces O, Barter LMCet al., 2013,

    Protocell design through modular compartmentalization

    , JOURNAL OF THE ROYAL SOCIETY INTERFACE, Vol: 10, ISSN: 1742-5689
  • Journal article
    Feinberg H, Jegouzo SAF, Rowntree TJW, Guan Y, Brash MA, Taylor ME, Weis WI, Drickamer Ket al., 2013,

    Mechanism for Recognition of an Unusual Mycobacterial Glycolipid by the Macrophage Receptor Mincle

    , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 28457-28465
  • Journal article
    Michoux F, Ahmad N, Hennig A, Nixon PJ, Warzecha Het al., 2013,

    Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes

    , PLANTA, Vol: 237, Pages: 903-908, ISSN: 0032-0935
  • Journal article
    Windram O, Madhou P, McHattie S, Hill C, Hickman R, Cooke E, Jenkins DJ, Penfold CA, Baxter L, Breeze E, Kiddle SJ, Rhodes J, Atwell S, Kliebenstein DJ, Kim Y-S, Stegle O, Borgwardt K, Zhang C, Tabrett A, Legaie R, Moore J, Finkenstadt B, Wild DL, Mead A, Rand D, Beynon J, Ott S, Buchanan-Wollaston V, Denby KJet al., 2012,

    Arabidopsis defense against Botrytis cinerea: Chronology and regulation deciphered by high-resolution temporal transcriptomic analysis

    , Plant Cell, Vol: 24, Pages: 3530-3557, ISSN: 1040-4651

    Transcriptional reprogramming forms a major part of a plant’s response to pathogen infection. Many individual components and pathways operating during plant defense have been identified, but our knowledge of how these different components interact is still rudimentary. We generated a high-resolution time series of gene expression profiles from a single Arabidopsis thaliana leaf during infection by the necrotrophic fungal pathogen Botrytis cinerea. Approximately one-third of the Arabidopsis genome is differentially expressed during the first 48 h after infection, with the majority of changes in gene expression occurring before significant lesion development. We used computational tools to obtain a detailed chronology of the defense response against B. cinerea, highlighting the times at which signaling and metabolic processes change, and identify transcription factor families operating at different times after infection. Motif enrichment and network inference predicted regulatory interactions, and testing of one such prediction identified a role for TGA3 in defense against necrotrophic pathogens. These data provide an unprecedented level of detail about transcriptional changes during a defense response and are suited to systems biology analyses to generate predictive models of the gene regulatory networks mediating the Arabidopsis response to B. cinerea.
  • Journal article
    Ahmad N, Michoux F, Nixon PJ, 2012,

    Investigating the production of foreign membrane proteins in tobacco chloroplasts: expression of an algal plastid terminal oxidase

    , PLoS One, Vol: 7, Pages: 1-10, ISSN: 1932-6203

    Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.
  • Journal article
    Komenda J, Sobotka R, Nixon PJ, 2012,

    Assembling and maintaining the Photosystem II complex in chloroplasts and cyanobacteria

    , CURRENT OPINION IN PLANT BIOLOGY, Vol: 15, Pages: 245-251, ISSN: 1369-5266
  • Journal article
    Wormit A, Butt SM, Chairam I, McKenna JF, Nunes-Nesi A, Kjaer L, O'Donnelly K, Fernie AR, Woscholski R, Barter MCL, Hamann Tet al., 2012,

    Osmosensitive Changes of Carbohydrate Metabolism in Response to Cellulose Biosynthesis Inhibition

    , PLANT PHYSIOLOGY, Vol: 159, Pages: 105-117, ISSN: 0032-0889
  • Journal article
    Charalambous K, Booth PJ, Woscholski R, Seddon JM, Templer RH, Law RV, Barter LMC, Ces Oet al., 2012,

    Engineering <i>de Novo</i> Membrane-Mediated Protein-Protein Communication Networks

    , JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 134, Pages: 5746-5749, ISSN: 0002-7863
  • Journal article
    Ahmad N, Michoux F, McCarthy J, Nixon PJet al., 2012,

    Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts

    , PLANTA, Vol: 235, Pages: 863-871, ISSN: 0032-0935
  • Conference paper
    Michoux F, Nixon PJ, 2011,

    Towards the sustainable and continuous <i>in</i>-<i>vitro</i> production of active pharmaceutical ingredients from medicinal plants

    , 59th International Congress and Annual Meeting of the Society-for-Medicinal-Plant-and-Natural-Product-Research, Publisher: GEORG THIEME VERLAG KG, Pages: 1281-1281, ISSN: 0032-0943
  • Journal article
    Michoux F, Ahmad N, McCarthy J, Nixon PJet al., 2011,

    Contained and high-level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor

    , PLANT BIOTECHNOLOGY JOURNAL, Vol: 9, Pages: 575-584, ISSN: 1467-7644
  • Journal article
    Fournier F, Gardner EM, Guo R, Donaldson PM, Barter LMC, Palmer DJ, Barnett CJ, Willison KR, Gould IR, Klug DRet al., 2008,

    Optical fingerprinting of peptides using two-dimensional infrared spectroscopy: Proof of principle

    , ANALYTICAL BIOCHEMISTRY, Vol: 374, Pages: 358-365, ISSN: 0003-2697
  • Journal article
    Donaldson PM, Guo R, Fournier F, Gardner EM, Barter LMC, Barnett CJ, Gould IR, Klug DR, Palmer DJ, Willison KRet al., 2007,

    Direct identification and decongestion of Fermi resonances by control of pulse time ordering in two-dimensional IR spectroscopy (vol 127, art no. 114513, 2007)

    , JOURNAL OF CHEMICAL PHYSICS, Vol: 127, ISSN: 0021-9606
  • Journal article
    Barter LMC, Klug DR, Woscholski R, 2007,

    Does history repeat itself? The emergence of a new discipline (vol 1, pg 737, 2006)

    , ACS CHEMICAL BIOLOGY, Vol: 2, Pages: 271-271, ISSN: 1554-8929
  • Journal article
    Donaldson PM, Guo R, Fournier F, Gardner EM, Barter LMC, Palmer DJ, Barnett CJ, Willison KR, Gould IR, Klug DRet al., 2007,

    Direct identification and decongestion of Fermi Resonances by control of pulse time-ordering in 2D-IR Spectroscopy

    , Journal Of Chemical Physics
  • Journal article
    Barter LMC, Klug DR, Woscholski R, 2006,

    Does history repeat itself? The emergence of a new discipline

    , ACS CHEMICAL BIOLOGY, Vol: 1, Pages: 737-740, ISSN: 1554-8929
  • Journal article
    Barter LMC, Klug DR, 2005,

    A unified picture of energy and electron transfer in primary photosynthesis

    , CHEMICAL PHYSICS, Vol: 319, Pages: 308-315, ISSN: 0301-0104
  • Book chapter
    Barter LMC, van Grondelle R, Klug DR, 2005,

    Energy trapping and equilibration: a balance of regulation and efficiency

    , Photosystem II: the light-driven water: plastoquinone oxidoreductase, Editors: Wydrzynski, Satoh, Wydrzynski, Satoh, Publisher: Springer, Pages: 491-514, ISBN: 9781402042492
  • Journal article
    Barter LMC, Durrant JR, Klug DR, 2003,

    A quantitative structure-function relationship for the Photosystern II reaction center: Supermolecular behavior in natural photosynthesis

    , PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 100, Pages: 946-951, ISSN: 0027-8424
  • Journal article
    Barter LMC, Schilstra MJ, Barber J, Durrant JR, Klug DRet al., 2001,

    Are the trapping dynamics in Photosystem II sensitive to Q<sub>A</sub> redox potential?

    , JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEMISTRY, Vol: 142, Pages: 127-132, ISSN: 1010-6030

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