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Journal articleFeinberg H, Rambaruth ND, Jégouzo SA, et al., 2021,
Binding sites for acylated trehalose analogs of glycolipid ligands on an extended carbohydrate-recognition domain of the macrophage receptor mincle
, Journal of Biological Chemistry, Vol: 291, Pages: 21222-21233, ISSN: 1083-351XThe macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis. Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work, the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate-recognition domain, also constrains the way that the binding domains may interact on the surface of macrophages.
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Journal articleMorris WL, Alamar MC, Lopez-Cobollo RM, et al., 2019,
A member of the TERMINAL FLOWER1/CENTRORADIALIS gene family controls sprout growth in potato tubers
, Journal of Experimental Botany, Vol: 70, Pages: 835-843, ISSN: 0022-0957Potato tuber bud dormancy break followed by premature sprouting is a major commercial problem which results in quality losses and decreased tuber marketability. An approach to controlling premature tuber sprouting is to develop potato cultivars with a longer dormancy period and/or reduced rate of sprout growth. Our recent studies using a potato diploid population have identified several QTLs that are associated with tuber sprout growth. In the current study we aim to characterise a candidate gene associated with one of the largest effect QTL for rapid tuber sprout growth on potato chromosome 3. Underlying this QTL is a gene encoding a TERMINAL FLOWER 1/ CENTRORADIALIS homologue (PGSC0003DMG400014322). Here we use a transgenic approach to manipulate the expression level of the CEN family member in a potato tetraploid genotype (cv. Désirée). We demonstrate a clear effect of StCEN expression manipulation, with decreased expression levels associated with an increased rate of sprout growth, and over-expressing lines showing a lower rate of sprout growth than controls. Associated with different levels of StCEN expression were different levels of ABA and cytokinins implying a role in controlling the levels of plant growth regulators in the apical meristem.
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Journal articleNielsen CD-T, Mooij WJ, Sale D, et al., 2019,
Reversibility and reactivity in an acid catalyzed cyclocondensation to give furanochromanes - a reaction at the "oxonium-Prins' vs. "ortho-quinone methide cycloaddition' mechanistic nexus
, Chemical Science, Vol: 10, Pages: 406-412, ISSN: 2041-6520Herein we report a combined experimental and computational investigation of the acid catalyzed cyclocondensation reaction between styrenyl homoallylic alcohols and salicylaldehyde to form furanochromanes. We disclose a previously unreported isomerisation of the ‘unnatural’ trans-fused products to the diastereomeric ‘natural’ cis-fused congeners. Notwithstanding the appeal of assuming this corresponds to endo to exo isomerisation of Diels–Alder (D–A) adducts via concerted retro-cycloaddition/cycloaddition reactions of an in situ generated ortho-quinone methide with the styrenyl alkene, our combined Hammett/DFT study reveals a stepwise Prins-like process via discrete benzylic carbocation intermediates for all but the most electron deficient styrenes. As these reactions fortuitously lie at the intersection of these two mechanistic manifolds, it allows us to propose an experimentally determined indicative ρ+ value of ca. −3 as marking this nexus between a stepwise Prins-type pathway and a concerted cycloaddition reaction. This value should prove useful for categorising other reactions formally involving ‘ortho-quinomethides’, without the need for the extensive computation performed here. Logical optimisation of the reaction based upon the mechanistic insight led to the use of HFIP as an additive which enables exclusive formation of ‘natural’ cis-fused products with a ∼100-fold reaction rate increase and improved scope.
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Book chapterShao S, Yu J, Nixon PJ, 2019,
Selective replacement of the damaged D1 reaction center subunit during the repair of the oxygen-evolving photosystem II complex
, Oxygen Production and Reduction in Artificial and Natural Systems, Editors: Barber, Ruban, Nixon, Publisher: World Scientific, Pages: 319-338The multi-subunit photosystem II (PSII) pigment-protein complex found in plants, algae and cyanobacteria is nature’s biological catalyst for producing oxygen from water. PSII needs sunlight to drive water oxidation but too much light can cause irreversible damage to pigments and proteins within PSII and loss of enzyme activity. Damaged PSII complexes can, however, be repaired in a highly selective process involving the replacement of damaged components by newly synthesized copies and the recycling of undamaged protein subunits and co-factors. Although substantial progress has been made to identify the enzymes and accessory factors involved in repair, many fundamental questions remain unanswered. In this chapter we discuss recent ideas on how the damaged D1 reaction center subunit, which is the subunit most prone to damage in PSII, is specifically recognized for replacement. Detachment of CP43 allowing access by FtsH proteases to the N-terminal tail of D1 seems to underpin selective degradation.
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Journal articleReijne A-M, Bordeu I, Pruessner G, et al., 2018,
Linear stability analysis of morphodynamics during tissue regeneration in plants
, Journal of Physics D: Applied Physics, Vol: 52, Pages: 1-9, ISSN: 0022-3727One of the key characteristics of multicellular organisms is the ability to establish and maintain shapes, or morphologies, under a variety of physical and chemical perturbations. A quantitative description of the underlying morphological dynamics is a critical step to fully understand the self-organising properties of multicellular systems. Although many powerful mathematical tools have been developed to analyse stochastic dynamics, rarely these are applied to experimental developmental biology.Here, we take root tip regeneration in the plant model system Arabidopsis thaliana as an example of robust morphogenesis in living tissue, and present a novel approach to quantify and model the relaxation of the system to its unperturbed morphology. By generating and analysing time-lapse series of regenerating root tips captured with confocal microscopy, we are able to extract and model the dynamics of key morphological traits at cellular resolution. We present a linear stability analysis of its Markovian dynamics, with the stationary state representing the intact root in the space of morphological traits. This analysis suggests the intriguing co-existence of two distinct temporal scales during the process of root regeneration in Arabidopsis.We discuss the possible biological implications of our specific results, and suggest future experiments to further probe the self-organising properties of living tissue.
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Conference paperDell A, Lu D, Haslam SM, et al., 2018,
Towards a novel cancer vaccine: Characterisation of the glycome of canine melanoma cells
, Annual Meeting of the Society-for-Glycobiology (SFG), Publisher: OXFORD UNIV PRESS INC, Pages: 1038-1039, ISSN: 0959-6658 -
Journal articleBarlow N, Kusumaatmaja H, Salehi-Reyhani A, et al., 2018,
Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers
, Journal of the Royal Society Interface, Vol: 15, ISSN: 1742-5662For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.
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Journal articleBerdeni D, Cotton TEA, Daniell TJ, et al., 2018,
The effects of arbuscular mycorrhizal fungal colonisation on nutrient status, growth, productivity, and canker resistance of apple (Malus pumila)
, Frontiers in Microbiology, Vol: 9, ISSN: 1664-302XWe assess whether arbuscular mycorrhizal fungi (AMF) improve growth, nutritional status, phenology, flower and fruit production, and disease resistance in woody perennial crops using apple (Malus pumila) as a study system. In a fully factorial experiment, young trees were grown for 3 years with or without AMF (Funneliformis mosseae and Rhizophagus irregularis), and with industrial standard fertiliser applications or restricted fertiliser (10% of standard). We use two commercial scions (Dabinett and Michelin) and rootstocks (MM111 and MM106). Industrial standard fertiliser applications reduced AMF colonisation and root biomass, potentially increasing drought sensitivity. Mycorrhizal status was influenced by above ground genotypes (scion type) but not rootstocks, indicating strong interactions between above and below ground plant tissue. The AMF inoculation significantly increased resistance to Neonectria ditissima, a globally economically significant fungal pathogen of apple orchards, but did not consistently alter leaf nutrients, growth, phenology or fruit and flower production. This study significantly advances understanding of AMF benefits to woody perennial crops, especially increased disease resistance which we show is not due to improved tree nutrition or drought alleviation. Breeding programmes and standard management practises can limit the potential for these benefits.
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Journal articleCardona T, Shao S, Nixon PJ, 2018,
Enhancing photosynthesis in plants: the light reactions
, Essays in Biochemistry, Vol: 62, Pages: 85-94, ISSN: 0071-1365In this review, we highlight recent research and current ideas on how to improve the efficiency of the light reactions of photosynthesis in crops. We note that the efficiency of photosynthesis is a balance between how much energy is used for growth and the energy wasted or spent protecting the photosynthetic machinery from photodamage. There are reasons to be optimistic about enhancing photosynthetic efficiency, but many appealing ideas are still on the drawing board. It is envisioned that the crops of the future will be extensively genetically modified to tailor them to specific natural or artificial environmental conditions.
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Journal articleLandrein B, Formosa-Jordan P, Malivert A, et al., 2018,
Nitrate modulates stem cell dynamics in Arabidopsis shoot meristems through cytokinins
, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: 1382-1387, ISSN: 0027-8424The shoot apical meristem (SAM) is responsible for the generation of all the aerial parts of plants. Given its critical role, dynamical changes in SAM activity should play a central role in the adaptation of plant architecture to the environment. Using quantitative microscopy, grafting experiments, and genetic perturbations, we connect the plant environment to the SAM by describing the molecular mechanism by which cytokinins signal the level of nutrient availability to the SAM. We show that a systemic signal of cytokinin precursors mediates the adaptation of SAM size and organogenesis rate to the availability of mineral nutrients by modulating the expression of WUSCHEL, a key regulator of stem cell homeostasis. In time-lapse experiments, we further show that this mechanism allows meristems to adapt to rapid changes in nitrate concentration, and thereby modulate their rate of organ production to the availability of mineral nutrients within a few days. Our work sheds light on the role of the stem cell regulatory network by showing that it not only maintains meristem homeostasis but also allows plants to adapt to rapid changes in the environment.
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Journal articleHerraiz A, Stokes L, Turnbull C, et al., 2018,
Developing a new variety of kentia palms (Howea forsteriana): up-regulation of cytochrome b561 and chalcone synthase is associated with the red coloration of the stems
, Botany Letters, Vol: 165, Pages: 241-247, ISSN: 2381-8115The kentia palm (Howea forsteriana) is endemic to a 12 km2 volcanic island in the Tasman Sea, Lord Howe Island. It is one of the most traded houseplants in the world. The typical kentia palm presents a dark green-coloured stem. Note that the stem is made of leaf rachis and petioles, which are intermingled towards the base. However, we discovered on Lord Howe Island a new biological variety that has a red stem. Red-stemmed palm species are known and highly demanded as decorative plants. However, these red palm horticultural varieties require tropical or subtropical conditions to grow, hence commercialization is limited. Thus, a red-stemmed variety of H. forsteriana may have tremendous market potential. Nonetheless, palm trees grow generally slowly and often reach maturity at 15–20 years old or later, which may make conventional strategies unsuitable for the propagation of a new variety. This difficulty needs to be addressed before commercialization can be achieved. Here, we found that anthocyanin is responsible for the red colouration of the stem in the new variety. Using RNA sequencing and quantitative PCR, we identified two gene isoforms displaying altered expression associated with this red colouration, encoding a cytochrome b561 and a chalcone synthase. The latter protein is known to be part of the anthocyanin biosynthesis pathway, which plays a central role in pigmentation in plants. The levels of cytochrome b561 transcripts accumulated were found to be well correlated with an increased anthocyanin concentration in the red stems.
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Book chapterBaesso P, Randall RS, Sena G, 2018,
Light Sheet Fluorescence Microscopy Optimized for Long-Term Imaging of Arabidopsis Root Development.
, Pages: 145-163Light sheet fluorescence microscopy (LSFM) allows sustained and repeated optical sectioning of living specimens at high spatial and temporal resolution, with minimal photodamage. Here, we describe in detail both the hardware and the software elements of a live imaging method based on LSFM and optimized for tracking and 3D scanning of Arabidopsis root tips grown vertically in physiological conditions. The system is relatively inexpensive and with minimal footprint; hence it is well suited for laboratories of any size.
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Journal articleHoysted GA, Kowal J, Jacob A, et al., 2017,
A mycorrhizal revolution.
, Current Opinion in Plant Biology, Vol: 44, Pages: 1-6, ISSN: 1369-5266It has long been postulated that symbiotic fungi facilitated plant migrations onto land through enhancing the scavenging of mineral nutrients and exchanging these for photosynthetically fixed organic carbon. Today, land plant-fungal symbioses are both widespread and diverse. Recent discoveries show that a variety of potential fungal associates were likely available to the earliest land plants, and that these early partnerships were probably affected by changing atmospheric CO2 concentrations. Here, we evaluate current hypotheses and knowledge gaps regarding early plant-fungal partnerships in the context of newly discovered fungal mutualists of early and more recently evolved land plants and the rapidly changing views on the roles of plant-fungal symbioses in the evolution and ecology of the terrestrial biosphere.
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Journal articleBarlow NE, Bolognesi G, Haylock S, et al., 2017,
Rheological Droplet Interface Bilayers (rheo-DIBs): Probing the Unstirred Water Layer Effect on Membrane Permeability via Spinning Disk Induced Shear Stress
, Scientific Reports, Vol: 7, ISSN: 2045-2322A new rheological droplet interface bilayer (rheo-DIB) device is presented as a tool to apply shear stress on biological lipid membranes. Despite their exciting potential for affecting high-throughput membrane translocation studies, permeability assays conducted using DIBs have neglected the effect of the unstirred water layer (UWL). However as demonstrated in this study, neglecting this phenomenon can cause significant underestimates in membrane permeability measurements which in turn limits their ability to predict key processes such as drug translocation rates across lipid membranes. With the use of the rheo-DIB chip, the effective bilayer permeability can be modulated by applying shear stress to the droplet interfaces, inducing flow parallel to the DIB membranes. By analysing the relation between the effective membrane permeability and the applied stress, both the intrinsic membrane permeability and UWL thickness can be determined for the first time using this model membrane approach, thereby unlocking the potential of DIBs for undertaking diffusion assays. The results are also validated with numerical simulations.
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Journal articleZheng RB, Jégouzo SAF, Joe M, et al., 2017,
Insights into Interactions of Mycobacteria with the Host Innate Immune System from a Novel Array of Synthetic Mycobacterial Glycans.
, ACS Chemical Biology, Vol: 12, Pages: 2990-3002, ISSN: 1554-8929An array of homogeneous glycans representing all the major carbohydrate structures present in the cell wall of the human pathogen Mycobacterium tuberculosis and other mycobacteria has been probed with a panel of glycan-binding receptors expressed on cells of the mammalian innate immune system. The results provide an overview of interactions between mycobacterial glycans and receptors that mediate uptake and survival in macrophages, dendritic cells, and sinusoidal endothelial cells. A subset of the wide variety of glycan structures present on mycobacterial surfaces interact with cells of the innate immune system through the receptors tested. Endocytic receptors, including the mannose receptor, DC-SIGN, langerin, and DC-SIGNR (L-SIGN), interact predominantly with mannose-containing caps found on the mycobacterial polysaccharide lipoarabinomannan. Some of these receptors also interact with phosphatidyl-myo-inositol mannosides and mannose-containing phenolic glycolipids. Many glycans are ligands for overlapping sets of receptors, suggesting multiple, redundant routes by which mycobacteria can enter cells. Receptors with signaling capability interact with two distinct sets of mycobacterial glycans: targets for dectin-2 overlap with ligands for the mannose-binding endocytic receptors, while mincle binds exclusively to trehalose-containing structures such as trehalose dimycolate. None of the receptors surveyed bind furanose residues, which often form part of the epitopes recognized by antibodies to mycobacteria. Thus, the innate and adaptive immune systems can target different sets of mycobacterial glycans. This array, the first of its kind, represents an important new tool for probing, at a molecular level, biological roles of a broad range of mycobacterial glycans, a task that has not previously been possible.
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Journal articleOsborne OG, De-Kayne R, Bidartondo MI, et al., 2017,
Arbuscular mycorrhizal fungi promote coexistence and niche divergence of sympatric palm species on a remote oceanic island
, New Phytologist, Vol: 217, Pages: 1254-1266, ISSN: 0028-646XMicrobes can have profound effects on their hosts, driving natural selection, promoting speciation and determining species distributions. However, soil-dwelling microbes are rarely investigated as drivers of evolutionary change in plants.We used metabarcoding and experimental manipulation of soil microbiomes to investigate the impact of soil and root microbes in a well-known case of sympatric speciation, the Howea palms of Lord Howe Island (Australia). Whereas H. forsteriana can grow on both calcareous and volcanic soils, H. belmoreana is restricted to, but more successful on, volcanic soil, indicating a trade-off in adaptation to the two soil types.We suggest a novel explanation for this trade-off. Arbuscular mycorrhizal fungi (AMF) are significantly depleted in H. forsteriana on volcanic soil, relative to both H. belmoreana on volcanic soil and H. forsteriana on calcareous soil. This is mirrored by the results of survival experiments, where the sterilization of natural soil reduces Howea fitness in every soil–species combination except H. forsteriana on volcanic soil. Furthermore, AMF-associated genes exhibit evidence of divergent selection between Howea species.These results show a mechanism by which divergent adaptation can have knock-on effects on host–microbe interactions, thereby reducing interspecific competition and promoting the coexistence of plant sister species.
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Journal articleKanvil S, Pham J, Lopez-Cobollo R, et al., 2017,
Cucurbit extrafascicular phloem has strong negative impacts on aphids and is not a preferred feeding site.
, Plant, Cell and Environment, Vol: 40, Pages: 2780-2789, ISSN: 0140-7791Cucurbits have long been known to possess two types of phloem: fascicular (FP) within vascular bundles and extrafascicular phloem (EFP) surrounding vascular bundles and scattered through the cortex. Recently, their divergent composition was revealed, with FP having high sugar content consistent with conventional phloem, but EFP having much lower sugar levels and a very different proteome. However, the evolutionary advantages of possessing both FP and EFP have remained unclear. Here, we present four lines of quantitative evidence that together support the hypothesis that FP represents a typical phloem and is an attractive diet for aphids, whereas aphids avoid feeding on EFP. First, aphid stylet track endings were more abundant near the abaxial FP element of minor veins, suggesting a feeding preference for FP over EFP. Second, sugar profiles from stylet exudates were wholly consistent with FP origins, further supporting preference for FP and avoidance of EFP. Third, supplementation of EFP exudate into artificial diets confirmed an aversion to EFP in choice experiments. Finally, EFP exudate had negative effects on aphid performance. On the basis of aphids' inability to thrive on EFP, we conclude that EFP is atypical and perhaps should not be classed as a phloem system.
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Conference paperSim S, Sowley H, Kidley N, et al., 2017,
Investigation of inhibitor-protein interactions in plants & mammalians from EVV 2DIR data
, 254th National Meeting and Exposition of the American-Chemical-Society (ACS) on Chemistry's Impact on the Global Economy, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727 -
Journal articleFoyer CH, Ruban AV, Nixon PJ, 2017,
Photosynthesis solutions to enhance productivity
, Philosophical Transactions of the Royal Society of London: Biological Sciences, Vol: 372, ISSN: 0962-8436The concept that photosynthesis is a highly inefficient process in terms ofconversion of light energy into biomass is embedded in the literature. It isonly in the past decade that the processes limiting photosynthetic efficiencyhave been understood to an extent that allows a step change in our ability tomanipulate light energy assimilation into carbon gain. We can thereforeenvisage that future increases in the grain yield potential of our majorcrops may depend largely on increasing the efficiency of photosynthesis.The papers in this issue provide new insights into the nature of current limitationson photosynthesis and identify new targets that can be used for cropimprovement, together with information on the impacts of a changingenvironment on the productivity of photosynthesis on land and in ouroceans.This article is part of the themed issue ‘Enhancing photosynthesis in cropplants: targets for improvement’.
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Journal articleNoble E, Kumar S, Gorlitz F, et al., 2017,
In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime
, Plant Methods, Vol: 13, ISSN: 1746-4811BackgroundIn order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe.ResultsOur studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (~μm), single point (~1 mm2) and macroscopic (~8 × 6 mm2 of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC—a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620–710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For
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Journal articleAbas H, Linsdall SM, Mamboury M, et al., 2017,
Total Synthesis of (+)-Lophirone H and Its pentamethyl ether utilizing an oxonium-prins cyclization
, Organic Letters, Vol: 19, Pages: 2486-2489, ISSN: 1523-7052The first total synthesis of (+)-lophirone H (1) and its pentamethyl ether 29, featuring an oxonium–Prins cyclization/benzylic cation trapping reaction, is described.
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Conference paperKhanna T, Barter L, Gould I, 2017,
Development and application of the AMBER molecular mechanics force field to investigate herbicide interaction in plants
, 253rd National Meeting of the American-Chemical-Society (ACS) on Advanced Materials, Technologies, Systems, and Processes, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727 -
Journal articleBarlow NE, Smpokou E, Friddin MS, et al., 2017,
Engineering plant membranes using droplet interface bilayers
, Biomicrofluidics, Vol: 11, ISSN: 1932-1058Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.
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Journal articleBarlow NE, Bolognesi G, Flemming AJ, et al., 2016,
Multiplexed droplet Interface bilayer formation
, Lab on a Chip, Vol: 16, Pages: 4653-4657, ISSN: 1473-0197We present a simple method for the multiplexed formation ofdroplet interface bilayers (DIBs) using a mechanically operatedlinear acrylic chamber array. To demonstrate the functionality ofthe chip design, a lipid membrane permeability assay is performed.We show that multiple, symmetric DIBs can be created andseparated using this robust low-cost approach.
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Journal articleChan CL, Bolognesi G, Bhandarkar A, et al., 2016,
DROPLAY: laser writing of functional patterns within biological microdroplet displays
, Lab on a Chip, Vol: 16, Pages: 4621-4627, ISSN: 1473-0197In this study, we introduce an optofluidic method for the rapid construction of large-area cell-sized droplet assemblieswith user-defined re-writable two-dimensional patterns of functional droplets. Light responsive water-in-oil dropletscapable of releasing fluorescent dye molecules upon exposure were generated and self-assembled into arrays in amicrofluidic device. This biological architecture was exploited by the scanning laser of a confocal microscope to ‘write’ userdefined patterns of differentiated (fluorescent) droplets in a network of originally undifferentiated (non-fluorescent)droplets. As a result, long lasting images were produced on a droplet fabric with droplets acting as pixels of a biologicalmonitor, which can be erased and re-written on-demand. Regio-specific light-induced droplet differentiation within a largepopulation of droplets provides a new paradigm for the rapid construction of bio-synthetic systems with potential as tissuemimics and biological display materials.
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Journal articleBarretto S, Michoux F, Hellgardt K, et al., 2016,
Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals
, Biochemical Engineering Journal, Vol: 11, Pages: 73-81, ISSN: 1369-703XTransplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Planttissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated withsuspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotianatabacum shoots from callus tissue in RITA® temporary immersion bioreactors has been previously demonstrated.In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium duringtemporary immersion culture (4 minutes aeration every 8 hours), and the impact on biological responses andtransplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aerationrates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yieldswere correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium).A critical aeration rate of 440 ml min-1 was identified, corresponding to a shear rate of 96.7 s-1, pneumatic powerinput of 8.8 mW kg-1and initial 20-day pneumatic energy dissipation of 127 J kg-1, at which significant reductionsin biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetCyields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observationshave important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocessesfor biopharmaceutical production.
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Journal articleAhmad N, Michoux F, Lossl AG, et al., 2016,
Challenges and perspectives in commercializing plastid transformation technology
, Journal of Experimental Botany, Vol: 67, Pages: 5945-5960, ISSN: 1460-2431Plastid transformation has emerged as an alternative platform to generate transgenic plants. Attractive features of this technology include specific integration of transgenes—either individually or as operons—into the plastid genome through homologous recombination, the potential for high-level protein expression, and transgene containment because of the maternal inheritance of plastids. Several issues associated with nuclear transformation such as gene silencing, variable gene expression due to the Mendelian laws of inheritance, and epigenetic regulation have not been observed in the plastid genome. Plastid transformation has been successfully used for the production of therapeutics, vaccines, antigens, and commercial enzymes, and for engineering various agronomic traits including resistance to biotic and abiotic stresses. However, these demonstrations have usually focused on model systems such as tobacco, and the technology per se has not yet reached the market. Technical factors limiting this technology include the lack of efficient protocols for the transformation of cereals, poor transgene expression in non-green plastids, a limited number of selection markers, and the lengthy procedures required to recover fully segregated plants. This article discusses the technology of transforming the plastid genome, the positive and negative features compared with nuclear transformation, and the current challenges that need to be addressed for successful commercialization.
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Journal articlede Saint Germain A, Clavé G, Badet-Denisot MA, et al., 2016,
An histidine covalent receptor and butenolide complex mediates strigolactone perception
, Nature Chemical Biology, Vol: 12, Pages: 787-794, ISSN: 1552-4469Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.
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Journal articleLopez-Cobollo RM, Filippis I, Bennett MH, et al., 2016,
Comparative proteomics of cucurbit phloem indicates both unique and shared sets of proteins
, The Plant Journal, Vol: 88, Pages: 633-647, ISSN: 1365-313XCucurbits are well studied phloem biology models but unusually possess both fascicular phloem (FP) within vascular bundles and additional extrafascicular phloem (EFP). Although the functional differences between the two systems are not yet clear, sugar analysis and limited protein profiling previously established that FP and EFP have divergent composition. Here we report a detailed comparative proteomics study of FP and EFP in two cucurbits, pumpkin and cucumber. We re-examined the sites of exudation by video microscopy, and confirmed that in both species, the spontaneous exudate following tissue cutting derives almost exclusively from EFP. Comparative gel electrophoresis and mass spectrometry-based proteomics of exudates, sieve element contents and microdissected stem tissues established that EFP and FP profiles are highly dissimilar, and that there are also species differences. Searches against cucurbit databases enabled identification of more than 300 FP proteins from each species. Few of the detected proteins (~10%) were shared between sieve element contents of FP and EFP, and enriched Gene Ontology categories also differed. To explore quantitative differences in the proteomes, we developed multiple reaction monitoring methods for cucumber proteins that are representative markers for FP or EFP and assessed exudate composition at different times after tissue cutting. Based on failure to detect FP markers in exudate samples, we conclude that FP is blocked very rapidly and therefore contributes minimally to the exudates. Overall, the highly divergent contents of FP and EFP indicate that they are substantially independent vascular compartments. This article is protected by copyright. All rights reserved.
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Journal articleKral N, Hanna Ougolnikova A, Sena G, 2016,
Externally imposed electric field enhances plant root tip regeneration
, Regeneration, Vol: 3, Pages: 156-167, ISSN: 2052-4412In plants, shoot and root regeneration can be induced in the distinctive conditions oftissue culture (in vitro), but is also observed in intact individuals (in planta) recoveringfrom tissue damage. Roots, for example, can regenerate their fully excised meristems inplanta, even in mutants with impaired apical stem cell niches. Unfortunately, to date acomprehensive understanding of regeneration in plants is still missing.Here, we provide evidence that an imposed electric field can perturb apical rootregeneration in Arabidopsis. Crucially, we explored both spatial and temporalcompetences of the stump to respond to electrical stimulation, respectively by varyingthe position of the cut and the time interval between excision and stimulation.Our data indicate that a brief pulse of an electric field parallel to the root is sufficient toincrease by up to two-fold the probability of its regeneration, and to perturb the localdistribution of the hormone auxin, as well as cell division regulation. Remarkably, theorientation of the root towards the anode or the cathode is shown to play a role.
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