Publications by year
In Press
Yvon-Durocher G, Buckling A, Smirnoff N (In Press). Adaptation of phytoplankton to a decade of experimental warming linked to increased photosynthesis. Nature Ecology and Evolution
Flori S, Dickenson J, Gaikwad T, Cole I, Smirnoff N, Helliwell K, Brownlee C, Wheeler G (In Press). Diatoms exhibit dynamic chloroplast calcium signals in response to high light and oxidative stress.
Abstract:
Diatoms exhibit dynamic chloroplast calcium signals in response to high light and oxidative stress
AbstractDiatoms are a group of silicified algae that play a major role in marine and freshwater ecosystems. Diatom chloroplasts were acquired by secondary endosymbiosis and exhibit important structural and functional differences from the primary plastids of land plants and green algae. Many functions of primary plastids, including photoacclimation and inorganic carbon acquisition, are regulated by calcium-dependent signalling processes. Calcium signalling has also been implicated in the photoprotective responses of diatoms, although the nature of calcium elevations in diatom chloroplasts and their wider role in cell signalling remains unknown. Using genetically encoded calcium indicators, we find that the diatomPhaeodactylum tricornutumexhibits dynamic chloroplast calcium elevations that are distinct from those found in land plants. Chloroplast calcium ([Ca2+]chl) acts independently from the cytosol and is not elevated by stimuli that induce large cytosolic calcium ([Ca2+]cyt) elevations. In contrast, high light and exogenous hydrogen peroxide (H2O2) induce large, sustained calcium elevations in the chloroplast stroma that are not replicated in the cytosol. Measurements using the fluorescent H2O2sensor roGFP2-Orp1 indicate that [Ca2+]chlelevations induced by these stimuli correspond to the accumulation of H2O2in the chloroplast. [Ca2+]chlelevations were also induced by the addition of methyl viologen, which acts to generate superoxide within the chloroplast, and by treatments that disrupt non-photochemical quenching (NPQ). The findings indicate that diatoms generate specific calcium elevations in the chloroplast in response to high light and oxidative stress that likely modulate the activity of calcium-sensitive components in photoprotection and other regulatory pathways.
Abstract.
Arnaud D, Deeks MJ, Smirnoff N (In Press). Differences between apoplastic and cytosolic reactive oxygen species production in <i>Arabidopsis</i> during pattern-triggered immunity.
Abstract:
Differences between apoplastic and cytosolic reactive oxygen species production in Arabidopsis during pattern-triggered immunity
AbstractDespite an ever-increasing interest in reactive oxygen species (ROS) signalling during plant-microbe interactions, very little information exists, mainly for technical reasons, on the molecular mechanisms regulating intracellular hydrogen peroxide (H2O2) signalling during PAMP-triggered immunity. Here, we used a sensitive fluorimetry method and the H2O2 sensor roGFP2-Orp1, which revealed unsuspected features on the regulation of cytoplasmic H2O2 and thiol redox dynamics upon pathogen-associated molecular patterns (PAMPs) perception by Arabidopsis thaliana. Extended PAMP-induced cytosolic roGFP2-Orp1 oxidation was distinct from the transient oxidative burst in the apoplast measured by luminol oxidation. Pharmacological and genetic analyses indicate that the prolonged PAMP-induced H2O2 increase in the cytoplasm was largely independent on NADPH oxidases and apoplastic peroxidases. By contrast, the NADPH oxidase mutant rbohF was hyper-sensitive to roGFP2-Orp1 oxidation by H2O2 and PAMP indicating a lower antioxidant capacity. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure and ROS production measured by a fluorescein-based probe in guard cells resulting in defects in stomatal defences against bacteria. However, stomatal closure was not correlated with an increase in roGFP2-Orp1 oxidation in guard cells. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. Altogether, our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity.Significance statementPlants mount defence responses to pathogens by detecting pathogen-associated molecular patterns (PAMPs). One response is a rapid and transient burst of reactive oxygen species (ROS, e.g. superoxide and hydrogen peroxide) in the cell wall (apoplast) produced by NADPH oxidases and cell wall peroxidases. Using a genetically-encoded hydrogen peroxide sensor roGFP2-Orp1, we found that, in contrast to the transient apoplastic ROS burst, there is also prolonged hydrogen peroxide production in the cytosol upon PAMP perception which is independent of NADPH oxidase and cell wall peroxidases. Our results suggest that apoplastic ROS rather than intracellular hydrogen peroxide is a signal triggering stomatal closure during PAMP-triggered immunity. Additionally, we re-address the relative contribution of the NADPH oxidases D and F in stomatal immunity.
Abstract.
Schaum C-E, Buckling A, Smirnoff N, Studholme DJ, Yvon-Durocher G (In Press). Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom.
Abstract:
Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom
Diatoms 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 found that evolutionary rescue under severe warming was slow but adaptation to more realistic scenarios, where temperature increases were moderate or where they fluctuated between benign and severe conditions, was rapid. Adaption to warming was linked to major phenotypic changes in metabolism and elemental composition. Whole genome re-sequencing identified significant genetic divergence both among populations adapted to the different warming regimes and between the evolved and ancestral lineages. Consistent with the phenotypic changes, the most rapidly evolving genes were associated with transcriptional regulation, cellular response to oxidative stress and redox homeostasis. These results demonstrate that evolution of thermal tolerance in marine diatoms can be rapid, particularly in fluctuating environments, and is underpinned by major genomic and phenotypic divergence.
Abstract.
Fenech M, Amorim-Silva V, del Valle AE, Arnaud D, Castillo AG, Smirnoff N, Botella MA (In Press). Organization and control of the ascorbate biosynthesis pathway in plants.
Abstract:
Organization and control of the ascorbate biosynthesis pathway in plants
ABSTRACTThe enzymatic steps involved inl-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions inNicotiana benthamianaand Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, andl-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP andl-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complementedvtc2null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes inN. benthamianasupported the roleof VTC2as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 andl-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.
Abstract.
Bunbury F, Helliwell KE, Mehrshahi P, Davey MP, Salmon D, Holzer A, Smirnoff N, Smith AG (In Press). Physiological and molecular responses of a newly evolved auxotroph of Chlamydomonas to B<sub>12</sub> deprivation.
Abstract:
Physiological and molecular responses of a newly evolved auxotroph of Chlamydomonas to B12 deprivation
AbstractThe corrinoid B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions and we previously demonstrated that experimental evolution of the non-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupted C1 metabolism, caused an accumulation of starch and triacylglycerides and a decrease in photosynthetic pigments, proteins and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species (ROS), which preceded rapid cell death. Surprisingly, survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use-efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing the environment to evolve mechanisms to better tolerate B12 limitation.
Abstract.
Singleton C, Howard TP, Smirnoff N (In Press). Synthetic metabolons for metabolic engineering.
Journal of Experimental BotanyAbstract:
Synthetic metabolons for metabolic engineering
It has been proposed that enzymes can associate into complexes (metabolons) that increase the efficiency of metabolic pathways by channelling substrates between enzymes. Metabolons may increase flux by increasing the local concentration of intermediates, decreasing the concentration of enzymes needed to maintain a given flux, directing the products of a pathway to a specific subcellular location or minimizing the escape of reactive intermediates. Metabolons can be formed by relatively loose non-covalent protein–protein interaction, anchorage to membranes, and (in bacteria) by encapsulation of enzymes in protein-coated microcompartments. Evidence that non-coated metabolons are effective at channelling substrates is scarce and difficult to obtain. In plants there is strong evidence that small proportions of glycolytic enzymes are associated with the outside of mitochondria and are effective in substrate channelling. More recently, synthetic metabolons, in which enzymes are scaffolded to synthetic proteins or nucleic acids, have been expressed in microorganisms and these provide evidence that scaffolded enzymes are more effective than free enzymes for metabolic engineering. This provides experimental evidence that metabolons may have a general advantage and opens the way to improving the outcome of metabolic engineering in plants by including synthetic metabolons in the toolbox.
Abstract.
Barton S, Jenkins J, Buckling A, Schaum C-E, Smirnoff N, Yvon-Durocher G (In Press). Universal metabolic constraints on the thermal tolerance of marine phytoplankton.
Abstract:
Universal metabolic constraints on the thermal tolerance of marine phytoplankton
AbstractMarine phytoplankton are responsible for over 45% of annual global net primary production. Ocean warming is expected to drive massive reorganisation of phytoplankton communities, resulting in pole-ward range shifts and sharp declines in species diversity, particularly in the tropics. The impacts of warming on phytoplankton species depend critically on their physiological sensitivity to temperature change, characterised by thermal tolerance curves. Local extinctions arise when temperatures exceed species’ thermal tolerance limits. The mechanisms that determine the characteristics of thermal tolerance curves (e.g. optimal and maximal temperatures) and their variability among the broad physiological diversity of marine phytoplankton are however poorly understood. Here we show that differences in the temperature responses of photosynthesis and respiration establish physiological trade-offs that constrain the thermal tolerance of 18 species of marine phytoplankton, spanning cyanobacteria as well as the red and green super-families. Across all species we found that rates of respiration were more sensitive to increasing temperature and typically had higher optimal temperatures than photosynthesis. Consequently, the fraction of photosynthetic energy available for allocation to growth (carbon-use efficiency) declined exponentially with rising temperatures with a sensitivity that was invariant among the 18 species. Furthermore, the optimal temperature of growth was generally lower than that of photosynthesis and as a result, supra-optimal declines in growth rate were associated with temperature ranges where the carbon-use efficiency exhibited accelerated declines. These highly conserved patterns demonstrate that the limits of thermal tolerance in marine phytoplankton are underpinned by common metabolic constraints linked to the differential temperature responses of photosynthesis and respiration.Significance StatementThe impacts of warming on marine phytoplankton depend on their sensitivity to rising temperatures, yet there is currently limited understanding of the mechanisms that limit thermal tolerance among the diversity of marine phytoplankton. Using a comparative study on the dominant, ecologically important lineages of marine phytoplankton – Bacillariophyceae, Dinophyceae, Cyanophyceae, Prasinophyceae, Prymnesiophyceae – we show that rates of respiration are consistently more sensitive to increasing temperature than photosynthesis. Consequently, the fraction of photosynthetic energy available for growth declines with rising temperatures with a sensitivity that is invariant among species. Our results suggest that declines in phytoplankton performance at high temperatures are driven by universal metabolic constrains linked to rising respiratory costs eventually exceeding the supply of reduced carbon from photosynthesis.
Abstract.
2023
Arnaud D, Deeks MJ, Smirnoff N (2023). <scp>RBOHF</scp> activates stomatal immunity by modulating both reactive oxygen species and apoplastic <scp>pH</scp> dynamics in Arabidopsis.
The Plant JournalAbstract:
RBOHF activates stomatal immunity by modulating both reactive oxygen species and apoplastic pH dynamics in Arabidopsis
SUMMARYStomatal defences are important for plants to prevent pathogen entry and further colonisation of leaves. Apoplastic reactive oxygen species (ROS) generated by NADPH oxidases and apoplastic peroxidases play an important role in activating stomatal closure upon perception of bacteria. However, downstream events, particularly the factors influencing cytosolic hydrogen peroxide (H2O2) signatures in guard cells are poorly understood. We used the H2O2 sensor roGFP2‐Orp1 and a ROS‐specific fluorescein probe to study intracellular oxidative events during stomatal immune response using Arabidopsis mutants involved in the apoplastic ROS burst. Surprisingly, the NADPH oxidase mutant rbohF showed over‐oxidation of roGFP2‐Orp1 by a pathogen‐associated molecular pattern (PAMP) in guard cells. However, stomatal closure was not tightly correlated with high roGFP2‐Orp1 oxidation. In contrast, RBOHF was necessary for PAMP‐mediated ROS production measured by a fluorescein‐based probe in guard cells. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP‐triggered stomatal closure resulting in defects in stomatal defences against bacteria. Interestingly, RBOHF also participated in PAMP‐induced apoplastic alkalinisation. The rbohF mutants were also partly impaired in H2O2‐mediated stomatal closure at 100 μm while higher H2O2 concentration up to 1 mm did not promote stomatal closure in wild‐type plants. Our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity.
Abstract.
Barton S, Padfield D, Masterson A, Buckling A, Smirnoff N, Yvon‐Durocher G (2023). Comparative experimental evolution reveals species‐specific idiosyncrasies in marine phytoplankton adaptation to warming.
Global Change Biology,
29(18), 5261-5275.
Abstract:
Comparative experimental evolution reveals species‐specific idiosyncrasies in marine phytoplankton adaptation to warming
AbstractA number of experimental studies have demonstrated that phytoplankton can display rapid thermal adaptation in response to warmed environments. While these studies provide insight into the evolutionary responses of single species, they tend to employ different experimental techniques. Consequently, our ability to compare the potential for thermal adaptation across different, ecologically relevant, species remains limited. Here, we address this limitation by conducting simultaneous long‐term warming experiments with the same experimental design on clonal isolates of three phylogenetically diverse species of marine phytoplankton; the cyanobacterium Synechococcus sp. the prasinophyte Ostreococcus tauri and the diatom Phaeodoactylum tricornutum. Over the same experimental time period, we observed differing levels of thermal adaptation in response to stressful supra‐optimal temperatures. Synechococcus sp. displayed the greatest improvement in fitness (i.e. growth rate) and thermal tolerance (i.e. temperature limits of growth). Ostreococcus tauri was able to improve fitness and thermal tolerance, but to a lesser extent. Finally, Phaeodoactylum tricornutum showed no signs of adaptation. These findings could help us understand how the structure of phytoplankton communities may change in response to warming, and possible biogeochemical implications, as some species show relatively more rapid adaptive shifts in their thermal tolerance.
Abstract.
2022
Schaum C-E, Buckling A, Smirnoff N, Yvon-Durocher G (2022). Evolution of thermal tolerance and phenotypic plasticity under rapid and slow temperature fluctuations.
Proc Biol Sci,
289(1980).
Abstract:
Evolution of thermal tolerance and phenotypic plasticity under rapid and slow temperature fluctuations.
Global warming is associated with an increase in sea surface temperature and its variability. The consequences of evolving in variable, fluctuating environments are explored by a large body of theory: when populations evolve in fluctuating environments the frequency of fluctuations determines the shapes of tolerance curves (indicative of habitats that organisms can inhabit) and trait reaction norms (the phenotypes that organisms display across these environments). Despite this well-established theoretical backbone, predicting how trait and tolerance curves will evolve in organisms at the foundation of marine ecosystems remains a challenge. Here, we used a globally distributed phytoplankton, Thalassiosira pseudonana, and show that fluctuations in temperature on scales of 3-4 generations rapidly selected for populations with enhanced trait plasticity and elevated thermal tolerance. Fluctuations spanning 30-40 generations selected for the formation of two stable, genetically and physiologically distinct populations, one evolving high trait plasticity and enhanced thermal tolerance, and the other, akin to samples evolved under constant warming, with lower trait plasticity and a smaller increase in thermal tolerance.
Abstract.
Author URL.
Arnaud D, Deeks MJ, Smirnoff N (2022). Organelle-targeted biosensors reveal distinct oxidative events during pattern-triggered immune responses.
Plant Physiology,
191(4), 2551-2569.
Abstract:
Organelle-targeted biosensors reveal distinct oxidative events during pattern-triggered immune responses
Abstract
. Reactive oxygen species are produced in response to pathogens and pathogen-associated molecular patterns, as exemplified by the rapid extracellular oxidative burst dependent on the NADPH oxidase isoform RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). We used the H2O2 biosensor roGFP2-Orp1 and the glutathione redox state biosensor GRX1-roGFP2 targeted to various organelles to reveal unsuspected oxidative events during the pattern-triggered immune response to flagellin (flg22) and after inoculation with Pseudomonas syringae. roGFP2-Orp1 was oxidized in a biphasic manner 1 and 6 h after treatment, with a more intense and faster response in the cytosol compared to chloroplasts, mitochondria, and peroxisomes. Peroxisomal and cytosolic GRX1-roGFP2 were also oxidized in a biphasic manner. Interestingly, our results suggested that bacterial effectors partially suppress the second phase of roGFP2-Orp1 oxidation in the cytosol. Pharmacological and genetic analyses indicated that the pathogen-associated molecular pattern-induced cytosolic oxidation required the BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) and BOTRYTIS-INDUCED KINASE 1 (BIK1) signaling components involved in the immune response but was largely independent of NADPH oxidases RBOHD and RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) and apoplastic peroxidases peroxidase 33 (PRX33) and peroxidase 34 (PRX34). The initial apoplastic oxidative burst measured with luminol was followed by a second oxidation burst, both of which preceded the two waves of cytosolic oxidation. In contrast to the cytosolic oxidation, these bursts were RBOHD-dependent. Our results reveal complex oxidative sources and dynamics during the pattern-triggered immune response, including that cytosolic oxidation is largely independent of the preceding extracellular oxidation events.
Abstract.
Lamanchai K, Smirnoff N, Salmon DL, Ngernmuen A, Roytrakul S, Leetanasaksakul K, Kittisenachai S, Jantasuriyarat C (2022). OsVTC1-1 Gene Silencing Promotes a Defense Response in Rice and Enhances Resistance to Magnaporthe oryzae.
PLANTS-BASEL,
11(17).
Author URL.
Lamanchai K, Salmon DL, Smirnoff N, Sutthinon P, Roytrakul S, Leetanasaksakul K, Kittisenachai S, Jantasuriyarat C (2022). OsVTC1-1 RNAi Mutant with Reduction of Ascorbic Acid Synthesis Alters Cell Wall Sugar Composition and Cell Wall-Associated Proteins.
AGRONOMY-BASEL,
12(6).
Author URL.
Smirnoff N (2022). SHINING a LIGHT ON ASCORBATE METABOLISM.
Author URL.
2021
Hancock RD, Smirnoff N, Lunn JE (2021). <i>Journal of Experimental Botany</i> 70th anniversary: plant metabolism in a changing world. Journal of Experimental Botany, 72(17), 5939-5941.
Littlejohn GR, Breen S, Smirnoff N, Grant M (2021). Chloroplast immunity illuminated.
New Phytol,
229(6), 3088-3107.
Abstract:
Chloroplast immunity illuminated.
The chloroplast has recently emerged as pivotal to co-ordinating plant defence responses and as a target of plant pathogens. Beyond its central position in oxygenic photosynthesis and primary metabolism - key targets in the complex virulence strategies of diverse pathogens - the chloroplast integrates, decodes and responds to environmental signals. The capacity of chloroplasts to synthesize phytohormones and a diverse range of secondary metabolites, combined with retrograde and reactive oxygen signalling, provides exquisite flexibility to both perceive and respond to biotic stresses. These processes also represent a plethora of opportunities for pathogens to evolve strategies to directly or indirectly target 'chloroplast immunity'. This review covers the contribution of the chloroplast to pathogen associated molecular pattern and effector triggered immunity as well as systemic acquired immunity. We address phytohormone modulation of immunity and surmise how chloroplast-derived reactive oxygen species underpin chloroplast immunity through indirect evidence inferred from genetic modification of core chloroplast components and direct pathogen targeting of the chloroplast. We assess the impact of transcriptional reprogramming of nuclear-encoded chloroplast genes during disease and defence and look at future research challenges.
Abstract.
Author URL.
Bömer M, Pérez-Salamó I, Florance HV, Salmon D, Dudenhoffer J-H, Finch P, Cinar A, Smirnoff N, Harvey A, Devoto A, et al (2021). Jasmonates induce Arabidopsis bioactivities selectively inhibiting the growth of breast cancer cells through CDC6 and mTOR.
New Phytol,
229(4), 2120-2134.
Abstract:
Jasmonates induce Arabidopsis bioactivities selectively inhibiting the growth of breast cancer cells through CDC6 and mTOR.
Phytochemicals are used often in vitro and in vivo in cancer research. The plant hormones jasmonates (JAs) control the synthesis of specialized metabolites through complex regulatory networks. JAs possess selective cytotoxicity in mixed populations of cancer and normal cells. Here, direct incubation of leaf explants from the non-medicinal plant Arabidopsis thaliana with human breast cancer cells, selectively suppresses cancer cell growth. High-throughput LC-MS identified Arabidopsis metabolites. Protein and transcript levels of cell cycle regulators were examined in breast cancer cells. A synergistic effect by methyljasmonate (MeJA) and by compounds upregulated in the metabolome of MeJA-treated Arabidopsis leaves, on the breast cancer cell cycle, is associated with Cell Division Cycle 6 (CDC6), Cyclin-dependent kinase 2 (CDK2), Cyclins D1 and D3, indicating that key cell cycle components mediate cell viability reduction. Bioactives such as indoles, quinolines and cis-(+)-12-oxophytodienoic acid, in synergy, could act as anticancer compounds. Our work suggests a universal role for MeJA-treatment of Arabidopsis in altering the DNA replication regulator CDC6, supporting conservation, across kingdoms, of cell cycle regulation, through the crosstalk between the mechanistic target of rapamycin, mTOR and JAs. This study has important implications for the identification of metabolites with anti-cancer bioactivities in plants with no known medicinal pedigree and it will have applications in developing disease treatments.
Abstract.
Author URL.
Feroz H, Ferlez B, Oh H, Mohammadiarani H, Ren T, Baker CS, Gajewski JP, Lugar DJ, Gaudana SB, Butler P, et al (2021). Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin.
Biochimica et Biophysica Acta - Biomembranes,
1863(8).
Abstract:
Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin
We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl− transport rate of 442 (±17.7) Cl−/protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl−/protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB12PEO8) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl−/photon and 0.28 (±0.04) Cl−/photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl−/protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl−/photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%.
Abstract.
Xiong H, Hua L, Reyna-Llorens I, Shi Y, Chen K-M, Smirnoff N, Kromdijk J, Hibberd JM (2021). Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase.
Proc Natl Acad Sci U S A,
118(25).
Abstract:
Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase.
When exposed to high light, plants produce reactive oxygen species (ROS). In Arabidopsis thaliana, local stress such as excess heat or light initiates a systemic ROS wave in phloem and xylem cells dependent on NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins. In the case of excess light, although the initial local accumulation of ROS preferentially takes place in bundle-sheath strands, little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2',7'-dichlorodihydrofluorescein diacetate, we found that, after exposure to high light, ROS were produced more rapidly in bundle-sheath strands than mesophyll cells. This response was not affected either by CO2 supply or photorespiration. Consistent with these findings, deep sequencing of messenger RNA (mRNA) isolated from mesophyll or bundle-sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H2O2-producing enzyme NADPH oxidase were more abundant in bundle-sheath strands than mesophyll cells. ROS production in bundle-sheath strands was decreased in mutant alleles of the bundle-sheath strand preferential isoform of OsRBOHA and increased when it was overexpressed. Despite the plethora of pathways able to generate ROS in response to excess light, NADPH oxidase-mediated accumulation of ROS in the rice bundle-sheath strand was detected in etiolated leaves lacking chlorophyll. We conclude that photosynthesis is not necessary for the local ROS response to high light but is in part mediated by NADPH oxidase activity.
Abstract.
Author URL.
Geigenberger P, Smirnoff N, Van Breusegem F, Dietz K-J, Noctor G (2021). Plant redox biology—on the move. Plant Physiology, 186(1), 1-3.
Helliwell KE, Kleiner FH, Hardstaff H, Chrachri A, Gaikwad T, Salmon D, Smirnoff N, Wheeler GL, Brownlee C (2021). Spatiotemporal patterns of intracellular Ca<sup>2+</sup> signalling govern hypo‐osmotic stress resilience in marine diatoms.
New Phytologist,
230(1), 155-170.
Abstract:
Spatiotemporal patterns of intracellular Ca2+ signalling govern hypo‐osmotic stress resilience in marine diatoms
Summary
Diatoms are globally important phytoplankton that dominate coastal and polar‐ice assemblages. These environments exhibit substantial changes in salinity over dynamic spatiotemporal regimes. Rapid sensory systems are vital to mitigate the harmful consequences of osmotic stress. Population‐based analyses have suggested that Ca2+ signalling is involved in diatom osmotic sensing. However, mechanistic insight of the role of osmotic Ca2+ signalling is limited.
Here, we show that Phaeodactylum Ca2+ elevations are essential for surviving hypo‐osmotic shock. Moreover, employing novel single‐cell imaging techniques we have characterised real‐time Ca2+ signalling responses in single diatom cells to environmental osmotic perturbations.
We observe that intracellular spatiotemporal patterns of osmotic‐induced Ca2+ elevations encode vital information regarding the nature of the osmotic stimulus. Localised Ca2+ signals evoked by mild or gradual hypo‐osmotic shocks are propagated globally from the apical cell tips, enabling fine‐tuned cell volume regulation across the whole cell.
Finally, we demonstrate that diatoms adopt Ca2+‐independent and dependent mechanisms for osmoregulation. We find that efflux of organic osmolytes occurs in a Ca2+‐independent manner, but this response is insufficient to mitigate cell damage during hypo‐osmotic shock. By comparison, Ca2+‐dependent signalling is necessary to prevent cell bursting via precise coordination of K+ transport, and therefore is likely to underpin survival in dynamic osmotic environments.
Abstract.
Fenech M, Amorim-Silva V, Esteban del Valle A, Arnaud D, Ruiz-Lopez N, Castillo AG, Smirnoff N, Botella MA (2021). The role of GDP-<scp>l</scp>-galactose phosphorylase in the control of ascorbate biosynthesis.
Plant Physiology,
185(4), 1574-1594.
Abstract:
The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis
AbstractThe enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff–Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.
Abstract.
2020
Alegre ML, Steelheart C, Baldet P, Rothan C, Just D, Okabe Y, Ezura H, Smirnoff N, Gergoff Grozeff GE, Bartoli CG, et al (2020). Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.
Planta,
251(2).
Abstract:
Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.
Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-L-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-L-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production.
Abstract.
Author URL.
Barton S, Jenkins J, Buckling A, Schaum C-E, Smirnoff N, Raven JA, Yvon‐Durocher G (2020). Evolutionary temperature compensation of carbon fixation in marine phytoplankton.
Ecology Letters,
23(4), 722-733.
Abstract:
Evolutionary temperature compensation of carbon fixation in marine phytoplankton
AbstractThe efficiency of carbon sequestration by the biological pump could decline in the coming decades because respiration tends to increase more with temperature than photosynthesis. Despite these differences in the short‐term temperature sensitivities of photosynthesis and respiration, it remains unknown whether the long‐term impacts of global warming on metabolic rates of phytoplankton can be modulated by evolutionary adaptation. We found that respiration was consistently more temperature dependent than photosynthesis across 18 diverse marine phytoplankton, resulting in universal declines in the rate of carbon fixation with short‐term increases in temperature. Long‐term experimental evolution under high temperature reversed the short‐term stimulation of metabolic rates, resulting in increased rates of carbon fixation. Our findings suggest that thermal adaptation may therefore have an ameliorating impact on the efficiency of phytoplankton as primary mediators of the biological carbon pump.
Abstract.
Dewhirst RA, Smirnoff N, Belcher CM (2020). Pine species that support crown fire regimes have lower leaf-level terpene contents than those native to surface fire regimes.
Fire,
3(2), 1-19.
Abstract:
Pine species that support crown fire regimes have lower leaf-level terpene contents than those native to surface fire regimes
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.
Abstract.
Bunbury F, Helliwell KE, Mehrshahi P, Davey MP, Salmon DL, Holzer A, Smirnoff N, Smith AG (2020). Responses of a Newly Evolved Auxotroph of Chlamydomonas to B12 Deprivation.
Plant Physiol,
183(1), 167-178.
Abstract:
Responses of a Newly Evolved Auxotroph of Chlamydomonas to B12 Deprivation.
The corrinoid B12 is synthesized only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions, and we previously demonstrated that experimental evolution of the non-B12-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupts C1 metabolism, causes an accumulation of starch and triacylglycerides, and leads to a decrease in photosynthetic pigments, proteins, and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species, which preceded rapid cell death. Survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to coping with B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing a suitable environment for evolving mechanisms to tolerate B12 limitation better.
Abstract.
Author URL.
Haque T, Eaves DJ, Lin Z, Zampronio CG, Cooper HJ, Bosch M, Smirnoff N, Franklin-Tong VE (2020). Self-Incompatibility Triggers Irreversible Oxidative Modification of Proteins in Incompatible Pollen.
Plant Physiol,
183(3), 1391-1404.
Abstract:
Self-Incompatibility Triggers Irreversible Oxidative Modification of Proteins in Incompatible Pollen.
Self-incompatibility (SI) is used by many angiosperms to prevent self-fertilization and inbreeding. In common poppy (Papaver rhoeas), interaction of cognate pollen and pistil S-determinants triggers programmed cell death (PCD) of incompatible pollen. We previously identified that reactive oxygen species (ROS) signal to SI-PCD. ROS-induced oxidative posttranslational modifications (oxPTMs) can regulate protein structure and function. Here, we have identified and mapped oxPTMs triggered by SI in incompatible pollen. Notably, SI-induced pollen had numerous irreversible oxidative modifications, while untreated pollen had virtually none. Our data provide a valuable analysis of the protein targets of ROS in the context of SI-induction and comprise a benchmark because currently there are few reports of irreversible oxPTMs in plants. Strikingly, cytoskeletal proteins and enzymes involved in energy metabolism are a prominent target of ROS. Oxidative modifications to a phosphomimic form of a pyrophosphatase result in a reduction of its activity. Therefore, our results demonstrate irreversible oxidation of pollen proteins during SI and provide evidence that this modification can affect protein function. We suggest that this reduction in cellular activity could lead to PCD.
Abstract.
Author URL.
Mullineaux PM, Exposito-Rodriguez M, Laissue PP, Smirnoff N, Park E (2020). Spatial chloroplast-to-nucleus signalling involving plastid-nuclear complexes and stromules.
Philos Trans R Soc Lond B Biol Sci,
375(1801).
Abstract:
Spatial chloroplast-to-nucleus signalling involving plastid-nuclear complexes and stromules.
Communication between chloroplasts and the nucleus in response to various environmental cues may be mediated by various small molecules. Signalling specificity could be enhanced if the physical contact between these organelles facilitates direct transfer and prevents interference from other subcellular sources of the same molecules. Plant cells have plastid-nuclear complexes, which provide close physical contact between these organelles. Plastid-nuclear complexes have been proposed to facilitate transfer of photosynthesis-derived H2O2 to the nucleus in high light. Stromules (stroma filled tubular plastid extensions) may provide an additional conduit for transfer of a wider range of signalling molecules, including proteins. However, plastid-nuclear complexes and stromules have been hitherto treated as distinct phenomena. We suggest that plastid-nuclear complexes and stromules work in a coordinated manner so that, according to environmental conditions or developmental state, the two modes of connection contribute to varying extents. We hypothesize that this association is dynamic and that there may be a link between plastid-nuclear complexes and the development of stromules. Furthermore, the changes in contact could alter signalling specificity by allowing an extended or different range of signalling molecules to be delivered to the nucleus. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.
Abstract.
Author URL.
Valente F (2020). Sub-cellular responses of wheat epidermal cells to Zymoseptoria tritici.
Abstract:
Sub-cellular responses of wheat epidermal cells to Zymoseptoria tritici
Zymoseptoria tritici (formerly Mycosphaerella graminicola), an Ascomycete fungus, is the main causal agent of Septoria tritici blotch, one of the main devastating wheat (Triticum aestivum) foliar diseases worldwide. The infection cycle of the hemi-biotrophic Z. tritici is divided into a symptom-less biotrophic phase followed by a necrotrophic phase characterised by pycnidia development. Little is known about molecular and cellular strategies of wheat defence during the first intimate contact with Z. tritici. Furthermore, information about immune responses in the wheat epidermal cells in pre-invasion resistance is lacking. In order to address the questions outlined above, we designed a series of novel assays on this plant patho-system to analyse the role of wheat focal immunity in response to Z. tritici hyphae. We tested the hypothesis that these wheat sub-cellular responses differed between Z. tritici virulent and avirulent strains during the fungal biotrophic symptom-less phase. High-resolution microscopy approaches combined with automated object detection recognition for organelle movement suggested a response from subsidiary cells flanking guard cells in the Z. tritici pre-invasion defence with regards to early changes of cell wall architecture and organelle re-distribution at the site of fungal interaction. These responses showed only subtle differences between compatible and incompatible strain-cultivar combinations. Our preliminary evidence suggests that the presence or absence of focal responses in subsidiary cells does not depend upon the AvrStb6 / Stb6 gene-for-gene relationship. Thus, novel applications in biology combined with live-cell imaging provided us with a promising tool to analyse the spatiotemporal dynamics of Z. tritici hyphae on infected wheat tissues. To the extent of our knowledge, this is the first report that analyses wheat subsidiary cells in response to fungal pathogens at the sub-cellular scale. This may constitute the object of future studies of wheat immunity and drive to the discovery of key factors linked to the strategies that lead to fungal invasion and circumvention of the wheat immune system.
Abstract.
2019
Drenichev MS, Bennett M, Novikov RA, Mansfield J, Smirnoff N, Grant M, Mikhailov SN (2019). A role for 3′-O-β-D-ribofuranosyladenosine in altering plant immunity.
Phytochemistry,
157, 128-134.
Abstract:
A role for 3′-O-β-D-ribofuranosyladenosine in altering plant immunity
Our understanding of how, and the extent to which, phytopathogens reconfigure host metabolic pathways to enhance virulence is remarkably limited. Here we investigate the dynamics of the natural disaccharide nucleoside, 3′-O-β-D-ribofuranosyladenosine, in leaves of Arabidopsis thaliana infected with virulent Pseudomonas syringae pv. tomato strain DC3000. 3′-O-β-D-ribofuranosyladenosine is a plant derived molecule that rapidly accumulates following delivery of P. syringae type III effectors to represent a major component of the infected leaf metabolome. We report the first synthesis of 3′-O-β-D-ribofuranosyladenosine using a method involving the condensation of a small excess of 1-O-acetyl-2,3,5-three-O-benzoyl-β-ribofuranose activated with tin tetrachloride with 2′,5′-di-O-tert-butyldimethylsilyladenosine in 1,2-dichloroethane with further removal of silyl and benzoyl protecting groups. Interestingly, application of synthetic 3′-O-β-D-ribofuranosyladenosine did not affect either bacterial multiplication or infection dynamics suggesting a major reconfiguration of metabolism during pathogenesis and a heavy metabolic burden on the infected plant.
Abstract.
Breen S, Brown H, Zabala MDT, Arnaud D, Hussain RF, Kulasekaran S, Smirnoff N, Schwarzlaender M, Littlejohn G, Grant MR, et al (2019). Chloroplasts, a major hub of immune signaling.
Author URL.
Smirnoff N (2019). Engineering of Metabolic Pathways Using Synthetic Enzyme Complexes.
Plant Physiol,
179(3), 918-928.
Abstract:
Engineering of Metabolic Pathways Using Synthetic Enzyme Complexes.
Prospects are reviewed for the use of synthetic enzyme complexes as a metabolic engineering tool.
Abstract.
Author URL.
Smirnoff N, Arnaud D (2019). Hydrogen peroxide metabolism and functions in plants.
New Phytol,
221(3), 1197-1214.
Abstract:
Hydrogen peroxide metabolism and functions in plants.
Contents Summary 1197 I. Introduction 1198 II. Measurement and imaging of H2 O2 1198 III. H2 O2 and O2·- toxicity 1199 IV. Production of H2 O2 : enzymes and subcellular locations 1200 V. H2 O2 transport 1205 VI. Control of H2 O2 concentration: how and where? 1205 VII. Metabolic functions of H2 O2 1207 VIII. H2 O2 signalling 1207 IX. Where next? 1209 Acknowledgements 1209 References 1209 SUMMARY: Hydrogen peroxide (H2 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 oxidizes specific cysteine residues of target proteins to the sulfenic acid form and, similar 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 the detection of specific proteins modified by H2 O2 , will allow a deeper understanding of its signalling roles.
Abstract.
Author URL.
Gaikwad T, Breen S, Kulasekaran S, Arnaud D, Hussain RF, Smirnoff N, Breeze E, Alfano JR, Littlejohn G, Grant MR, et al (2019). The role of chloroplasts in disease and defence.
Author URL.
Barton S (2019). Understanding the responses of marine phytoplankton to experimental warming.
Abstract:
Understanding the responses of marine phytoplankton to experimental warming
Understanding how marine phytoplankton will fare in response to the expected increases in ocean temperature over the next century is crucial for improving their inclusion in models of ocean biogeochemistry. Marine phytoplankton plays an essential role for the global carbon cycle, accounting for approximately 50% of global primary production, and provides the base of all aquatic food webs. There is currently poor understanding of what sets the limits of thermal tolerance and how quickly different species of phytoplankton can adapt to changes in environmental temperature. Furthermore, models that have previously factored for the response of phytoplankton to warming have tended to generalise their inclusion by applying the Eppley coefficient to make predictions about future ocean productivity; this is an across-species characterisation of the thermal sensitivity of phytoplankton growth rates, which assumes a monotonic, exponential, increase in maximal growth rates with temperature. To enhance our understanding of the responses of marine phytoplankton to warming we first investigated the limits of thermal tolerance, as well as the thermal performance of both photosynthesis and respiration rates, for an array of phytoplankton taxa, representing key functional groups, including: cyanobacteria, diatoms, coccolithophores, dinoflagellates and chlorophytes. We identify, qualitatively, that the limits of thermal tolerance are likely to be underpinned by the thermal performance of metabolism, whereby across all taxa respiration was more temperature dependent, and generally had a higher optimal temperature, than photosynthesis. Next, using the understanding of thermal tolerance at the species level we estimated an across-species temperature dependence of maximal growth rates that was lower than the within-species average, supporting the “partial compensation” mechanism of thermal adaptation and highlighting that the canonical Eppley coefficient is likely to under or overestimate the temperature dependence in ocean regions where particular species, or phylogenetic groups, may dominate. With this finding we were also able to associate greater thermal tolerance with covariance of other ecologically important physiological and morphological traits, highlighting that the likely restructuring of phytoplankton communities in response to warming will have strong implications for ecosystem function and biogeochemical cycles. Lastly, we investigated the pace and magnitude of thermal adaptation to a stressful supra-optimal temperature across three very different but ecologically important phytoplankton species. We found that across the three taxa there was clear variance in the rate and magnitude of thermal adaptation, with the least complex and smallest of the three taxa showing the fastest rates of thermal adaptation and the greatest improvement in thermal tolerance. Underpinning thermal adaptation across the taxa were clear metabolic adjustments, likely to be associated with overcoming the constraints of carbon allocation to growth due to the differing thermal sensitivities of photosynthesis and respiration. We conclude that each of the main findings from this research can help improve the inclusion of marine phytoplankton in models of ocean biogeochemistry and as part of wider Earth systems models, thereby aiding predictions of the likely reorganisation of phytoplankton communities and the impact of warming on the critical ecosystem services and biogeochemical cycles that phytoplankton mediate.
Abstract.
2018
Walker CE, Heath S, Salmon DL, Smirnoff N, Langer G, Taylor AR, Brownlee C, Wheeler GL (2018). An extracellular polysaccharide-rich organic layer contributes to organization of the coccosphere in coccolithophores.
Frontiers in Marine Science,
5(AUG).
Abstract:
An extracellular polysaccharide-rich organic layer contributes to organization of the coccosphere in coccolithophores
Coccolithophores are globally abundant marine microalgae characterized by their ability to form calcite platelets (coccoliths). The coccoliths are produced internally in a Golgi-derived vesicle. Mature coccoliths are extruded from the cell to form a protective covering on the cell surface, known as the coccosphere. Current evidence indicates that calcite precipitation in the coccolith vesicle (CV) is modulated by coccolith-associated polysaccharides (CAPs). Whilst previous research into CAPs has focussed on their roles in calcite precipitation within the CV, little is known of their extracellular roles. Using fluorescent lectins, we visualize the extracellular polysaccharide-rich organic layer associated with external coccoliths and demonstrate that it differs between species in structure and composition. Biochemical analysis of polysaccharide extracted from coccoliths indicated substantial differences between species in monosaccharide composition and uronic acid content. In Coccolithus braarudii our studies indicate that polysaccharide-rich material is extruded with the coccoliths, where it plays a role in the adhesion of the coccoliths to the cell surface and contributes to the overall organization of the coccosphere. Together, these results highlight the important extracellular roles of CAPs and their contribution to the dynamic nature of the coccosphere.
Abstract.
Smirnoff N (2018). Ascorbic acid metabolism and functions: a comparison of plants and mammals. Free Radical Biology and Medicine
Ishikawa T, Maruta T, Yoshimura K, Smirnoff N (2018). Biosynthesis and regulation of ascorbic acid in plants. In (Ed)
Antioxidants and Antioxidant Enzymes in Higher Plants, 163-179.
Abstract:
Biosynthesis and regulation of ascorbic acid in plants
Abstract.
Schaum E, Buckling A, Studholme D, Smirnoff N, Yvon-Durocher G (2018). Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom. Nature Communications
Schaum C-E, Buckling A, Smirnoff N, Studholme DJ, Yvon-Durocher G (2018). Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom (vol 9, 1719, 2018).
NATURE COMMUNICATIONS,
9 Author URL.
Feroz H, Ferlez B, Lefoulon C, Ren T, Baker CS, Gajewski JP, Lugar DJ, Gaudana SB, Butler PJ, Hühn J, et al (2018). Light-Driven Chloride Transport Kinetics of Halorhodopsin.
Biophysical Journal,
115(2), 353-360.
Abstract:
Light-Driven Chloride Transport Kinetics of Halorhodopsin
Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl− transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl−/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e. 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.
Abstract.
Helliwell KE, Pandhal J, Cooper MB, Longworth J, Kudahl UJ, Russo DA, Tomsett EV, Bunbury F, Salmon DL, Smirnoff N, et al (2018). Quantitative proteomics of a B12 -dependent alga grown in coculture with bacteria reveals metabolic tradeoffs required for mutualism.
New Phytol,
217(2), 599-612.
Abstract:
Quantitative proteomics of a B12 -dependent alga grown in coculture with bacteria reveals metabolic tradeoffs required for mutualism.
The unicellular green alga Lobomonas rostrata requires an external supply of vitamin B12 (cobalamin) for growth, which it can obtain in stable laboratory cultures from the soil bacterium Mesorhizobium loti in exchange for photosynthate. We investigated changes in protein expression in the alga that allow it to engage in this mutualism. We used quantitative isobaric tagging (iTRAQ) proteomics to determine the L. rostrata proteome grown axenically with B12 supplementation or in coculture with M. loti. Data are available via ProteomeXchange (PXD005046). Using the related Chlamydomonas reinhardtii as a reference genome, 588 algal proteins could be identified. Enzymes of amino acid biosynthesis were higher in coculture than in axenic culture, and this was reflected in increased amounts of total cellular protein and several free amino acids. A number of heat shock proteins were also elevated. Conversely, photosynthetic proteins and those of chloroplast protein synthesis were significantly lower in L. rostrata cells in coculture. These observations were confirmed by measurement of electron transfer rates in cells grown under the two conditions. The results indicate that, despite the stability of the mutualism, L. rostrata experiences stress in coculture with M. loti, and must adjust its metabolism accordingly.
Abstract.
Author URL.
Mullineaux PM, Exposito-Rodriguez M, Laissue PP, Smirnoff N (2018). ROS-dependent signalling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes.
Free Radic Biol Med,
122, 52-64.
Abstract:
ROS-dependent signalling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes.
Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signalling molecules to drive cellular responses to changes in their environment. In this respect, there is considerable commonality between all eukaryotes imposed by the constraints of ROS chemistry, similar metabolism in many subcellular compartments, the requirement for a high degree of signal specificity and the deployment of thiol peroxidases as transducers of oxidising equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signalling arising from oxygenic photosynthesis in chloroplasts and photoautotropism, which often induce an imbalance between absorption of light energy and the capacity to use it productively. A key means of responding to this imbalance is through communication of chloroplasts with the nucleus to adjust cellular metabolism. Two ROS, singlet oxygen (1O2) and hydrogen peroxide (H2O2), initiate distinct signalling pathways when photosynthesis is perturbed. 1O2, because of its potent reactivity means that it initiates but does not transduce signalling. In contrast, the lower reactivity of H2O2 means that it can also be a mobile messenger in a spatially-defined signalling pathway. How plants translate a H2O2 message to bring about changes in gene expression is unknown and therefore, we draw on information from other eukaryotes to propose a working hypothesis. The role of these ROS generated in other subcellular compartments of plant cells in response to HL is critically considered alongside other eukaryotes. Finally, the responses of animal cells to oxidative stress upon high irradiance exposure is considered for new comparisons between plant and animal cells.
Abstract.
Author URL.
2017
Sambles CM, Salmon DL, Florance H, Howard TP, Smirnoff N, Nielsen LR, McKinney LV, Kjær ED, Buggs RJA, Studholme DJ, et al (2017). Ash leaf metabolomes reveal differences between trees tolerant and susceptible to ash dieback disease.
Sci Data,
4Abstract:
Ash leaf metabolomes reveal differences between trees tolerant and susceptible to ash dieback disease.
European common ash, Fraxinus excelsior, is currently threatened by Ash dieback (ADB) caused by the fungus, Hymenoscyphus fraxineus. To detect and identify metabolites that may be products of pathways important in contributing to resistance against H. fraxineus, we performed untargeted metabolomic profiling on leaves from five high-susceptibility and five low-susceptibility F. excelsior individuals identified during Danish field trials. We describe in this study, two datasets. The first is untargeted LC-MS metabolomics raw data from ash leaves with high-susceptibility and low-susceptibility to ADB in positive and negative mode. These data allow the application of peak picking, alignment, gap-filling and retention-time correlation analyses to be performed in alternative ways. The second, a processed dataset containing abundances of aligned features across all samples enables further mining of the data. Here we illustrate the utility of this dataset which has previously been used to identify putative iridoid glycosides, well known anti-herbivory terpenoid derivatives, and show differential abundance in tolerant and susceptible ash samples.
Abstract.
Author URL.
Exposito-Rodriguez M, Laissue PP, Yvon-Durocher G, Smirnoff N, Mullineaux PM (2017). Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism.
Nature Communications,
8(1).
Abstract:
Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism
AbstractChloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression.
Abstract.
2016
Lim B, Smirnoff N, Cobbett CS, Goltz JF (2016). Ascorbate-Deficient vtc2 Mutants in Arabidopsis Do Not Exhibit Decreased Growth. Frontiers in Plant Science, 7, 1025-1025.
de Torres Zabala M, Zhai B, Jayaraman S, Eleftheriadou G, Winsbury R, Yang R, Truman W, Tang S, Smirnoff N, Grant M, et al (2016). Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection.
New Phytologist,
209(3), 1120-1134.
Abstract:
Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection
Pathogens target phytohormone signalling pathways to promote disease. Plants deploy salicylic acid (SA)-mediated defences against biotrophs. Pathogens antagonize SA immunity by activating jasmonate signalling, for example Pseudomonas syringae pv. tomato DC3000 produces coronatine (COR), a jasmonic acid (JA) mimic. This study found unexpected dynamics between SA, JA and COR and co-operation between JAZ jasmonate repressor proteins during DC3000 infection. We used a systems-based approach involving targeted hormone profiling, high-temporal-resolution micro-array analysis, reverse genetics and mRNA-seq. Unexpectedly, foliar JA did not accumulate until late in the infection process and was higher in leaves challenged with COR-deficient P. syringae or in the more resistant JA receptor mutant coi1. JAZ regulation was complex and COR alone was insufficient to sustainably induce JAZs. JAZs contribute to early basal and subsequent secondary plant defence responses. We showed that JAZ5 and JAZ10 specifically co-operate to restrict COR cytotoxicity and pathogen growth through a complex transcriptional reprogramming that does not involve the basic helix-loop-helix transcription factors MYC2 and related MYC3 and MYC4 previously shown to restrict pathogen growth. mRNA-seq predicts compromised SA signalling in a jaz5/10 mutant and rapid suppression of JA-related components on bacterial infection.
Abstract.
de Torres Zabala M, Zhai B, Jayaraman S, Eleftheriadou G, Winsbury R, Yang R, Truman W, Tang S, Smirnoff N, Grant M, et al (2016). Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection.
New Phytol,
209(3), 1120-1134.
Abstract:
Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection.
Pathogens target phytohormone signalling pathways to promote disease. Plants deploy salicylic acid (SA)-mediated defences against biotrophs. Pathogens antagonize SA immunity by activating jasmonate signalling, for example Pseudomonas syringae pv. tomato DC3000 produces coronatine (COR), a jasmonic acid (JA) mimic. This study found unexpected dynamics between SA, JA and COR and co-operation between JAZ jasmonate repressor proteins during DC3000 infection. We used a systems-based approach involving targeted hormone profiling, high-temporal-resolution micro-array analysis, reverse genetics and mRNA-seq. Unexpectedly, foliar JA did not accumulate until late in the infection process and was higher in leaves challenged with COR-deficient P. syringae or in the more resistant JA receptor mutant coi1. JAZ regulation was complex and COR alone was insufficient to sustainably induce JAZs. JAZs contribute to early basal and subsequent secondary plant defence responses. We showed that JAZ5 and JAZ10 specifically co-operate to restrict COR cytotoxicity and pathogen growth through a complex transcriptional reprogramming that does not involve the basic helix-loop-helix transcription factors MYC2 and related MYC3 and MYC4 previously shown to restrict pathogen growth. mRNA-seq predicts compromised SA signalling in a jaz5/10 mutant and rapid suppression of JA-related components on bacterial infection.
Abstract.
Author URL.
Bechtold U, Penfold CA, Jenkins DJ, Legaie R, Moore JD, Lawson T, Matthews JSA, Vialet-Chabrand SRM, Baxter L, Subramaniam S, et al (2016). Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.
Plant Cell,
28(2), 345-366.
Abstract:
Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.
In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.
Abstract.
Author URL.
2015
Goodey NA, Florance HV, Smirnoff N, Hodgson DJ (2015). Aphids Pick Their Poison: Selective Sequestration of Plant Chemicals Affects Host Plant Use in a Specialist Herbivore.
J Chem Ecol,
41(10), 956-964.
Abstract:
Aphids Pick Their Poison: Selective Sequestration of Plant Chemicals Affects Host Plant Use in a Specialist Herbivore.
In some plant-insect interactions, specialist herbivores exploit the chemical defenses of their food plant to their own advantage. Brassica plants produce glucosinolates that are broken down into defensive toxins when tissue is damaged, but the specialist aphid, Brevicoryne brassicae, uses these chemicals against its own natural enemies by becoming a "walking mustard-oil bomb". Analysis of glucosinolate concentrations in plant tissue and associated aphid colonies reveals that not only do aphids sequester glucosinolates, but they do so selectively. Aphids specifically accumulate sinigrin to high concentrations while preferentially excreting a structurally similar glucosinolate, progoitrin. Surveys of aphid infestation in wild populations of Brassica oleracea show that this pattern of sequestration and excretion maps onto host plant use. The probability of aphid infestation decreases with increasing concentrations of progoitrin in plants. Brassica brassicae, therefore, appear to select among food plants according to plant secondary metabolite profiles, and selectively store only some compounds that are used against their own enemies. The results demonstrate chemical and behavioral mechanisms that help to explain evidence of geographic patterns and evolutionary dynamics in Brassica-aphid interactions.
Abstract.
Author URL.
Sultana N, Florance HV, Johns A, Smirnoff N (2015). Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana.
Plant Cell and Environment,
38(2), 375-384.
Abstract:
Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana
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.
Abstract.
de Torres M, Littlejohn G, Jayaraman S, Studholme D, Bailey TC, Lawson T, Delfino L, Licht D, Truman W, Bölter B, et al (2015). Chloroplasts play a central role in plant defence
and are targeted by pathogen effectors. Nature Plants, 1, n/a-n/a.
Wheeler GL, Smirnoff N, Ishikawa T (2015). EVOLUTION OF ALTERNATIVE PATHWAYS FOR VITAMIN C SYNTHESIS FOLLOWING PLASTID ACQUISITION.
EUROPEAN JOURNAL OF PHYCOLOGY,
50, 52-52.
Author URL.
Wheeler G, Ishikawa T, Pornsaksit V, Smirnoff N (2015). Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes.
Elife,
4Abstract:
Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes.
Ascorbic acid (vitamin C) is an enzyme co-factor in eukaryotes that also plays a critical role in protecting photosynthetic eukaryotes against damaging reactive oxygen species derived from the chloroplast. Many animal lineages, including primates, have become ascorbate auxotrophs due to the loss of the terminal enzyme in their biosynthetic pathway, L-gulonolactone oxidase (GULO). The alternative pathways found in land plants and Euglena use a different terminal enzyme, L-galactonolactone dehydrogenase (GLDH). The evolutionary processes leading to these differing pathways and their contribution to the cellular roles of ascorbate remain unclear. Here we present molecular and biochemical evidence demonstrating that GULO was functionally replaced with GLDH in photosynthetic eukaryote lineages following plastid acquisition. GULO has therefore been lost repeatedly throughout eukaryote evolution. The formation of the alternative biosynthetic pathways in photosynthetic eukaryotes uncoupled ascorbate synthesis from hydrogen peroxide production and likely contributed to the rise of ascorbate as a major photoprotective antioxidant.
Abstract.
Author URL.
Littlejohn GR, Mansfield JC, Parker D, Lind R, Perfect S, Seymour M, Smirnoff N, Love J, Moger J (2015). In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.
Plant Physiol,
168(1), 18-28.
Abstract:
In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.
The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.
Abstract.
Author URL.
Wang C-C, Chandrappa D, Smirnoff N, Moger J (2015). Monitoring Lipid Accumulation in the Green Microalga Botryococcus braunii with Frequency-Modulated Stimulated Raman Scattering.
Author URL.
Proctor MCF, Smirnoff N (2015). Photoprotection in bryophytes: Rate and extent of dark relaxation of non-photochemical quenching of chlorophyll fluorescence.
Journal of Bryology,
37(3), 171-177.
Abstract:
Photoprotection in bryophytes: Rate and extent of dark relaxation of non-photochemical quenching of chlorophyll fluorescence
Vascular plants are typically endohydric and are killed by drying beyond 30% relative water content. Bryophytes are ectohydric and are typically desiccation tolerant (DT). Mosses in open sun-exposed habitats show major electron flow to oxygen and high levels of non-photochemical quenching (NPQ) in chlorophyll fluorescence measurements. This has been regarded as a main source of photoprotection for these plants. The aim of the work described in this paper was to explore the rate and extent of relaxation of this quenching, and to seek evidence of its nature and consequences. Sequences of measurements were made during illumination at various intensities and a subsequent dark period. Lightresponse curves were constructed using dithiothreitol (DTT) as an inhibitor of violaxanthin de-epoxidase to provide additional evidence of the proportion of NPQ mediated by the xanthophyll cycle. The relaxation curves were fitted by exponential decay curves. A double-exponential fit to curves for the sun-adapted species gave a fast phase with a halflife of ca 6–16 seconds, and a slow phase with a halflife of ca 100–300 seconds. Shade species were best fitted by single-exponential curves. A persistent offset remained of ca 5–23% of the pre-darkening NPQ. Light-response curves for several species showed NPQ reduced in the presence of DTT to similar proportions of the control. Around 70–95% of NPQ in the bryophytes investigated relaxed with a halflife of ca 2–5 minutes. The fast phase of the double-exponential fit is consistent with likely rates of decay of the trans-thylakoid pH gradient and re-epoxidation of zeaxanthin. This leads to the same conclusion as the effect of DTT in depressing NPQ. The contrast in physiology between bryophytes and vascular plants reflects the different selection pressures facing leaf cells of poikilohydric plants and the mesophyll cells of vascular plants, and their divergent evolutionary histories since the mid-Palaeozoic.
Abstract.
MacGregor DR, Kendall SL, Florance H, Fedi F, Moore K, Paszkiewicz K, Smirnoff N, Penfield S (2015). Seed production temperature regulation of primary dormancy occurs through control of seed coat phenylpropanoid metabolism.
New Phytol,
205(2), 642-652.
Abstract:
Seed production temperature regulation of primary dormancy occurs through control of seed coat phenylpropanoid metabolism.
Environmental changes during seed production are important drivers of lot-to-lot variation in seed behaviour and enable wild species to time their life history with seasonal cues. Temperature during seed set is the dominant environmental signal determining the depth of primary dormancy, although the mechanisms though which temperature changes impart changes in dormancy state are still only partly understood. We used molecular, genetic and biochemical techniques to examine the mechanism through which temperature variation affects Arabidopsis thaliana seed dormancy. Here we show that, in Arabidopsis, low temperatures during seed maturation result in an increase in phenylpropanoid gene expression in seeds and that this correlates with higher concentrations of seed coat procyanidins. Lower maturation temperatures cause differences in coat permeability to tetrazolium, and mutants with increased seed coat permeability and/or low procyanidin concentrations are less able to enter strongly dormant states after exposure to low temperatures during seed maturation. Our data show that maternal temperature signalling regulates seed coat properties, and this is an important pathway through which the environmental signals control primary dormancy depth.
Abstract.
Author URL.
Smirnoff N (2015). The evolution of vitamin C biosynthetic pathways in plants and algae.
Author URL.
Lewis LA, Polanski K, de Torres-Zabala M, Jayaraman S, Bowden L, Moore J, Penfold CA, Jenkins DJ, Hill C, Baxter L, et al (2015). Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.
Plant Cell,
27(11), 3038-3064.
Abstract:
Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.
Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae.
Abstract.
Author URL.
2014
Littlejohn GR, Mansfield JC, Christmas JT, Witterick E, Fricker MD, Grant MR, Smirnoff N, Everson RM, Moger J, Love J, et al (2014). An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.
Front Plant Sci,
5Abstract:
An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.
Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the "negative space" within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.
Abstract.
Author URL.
Cresswell JE, Robert FXL, Florance H, Smirnoff N (2014). Clearance of ingested neonicotinoid pesticide (imidacloprid) in honey bees (Apis mellifera) and bumblebees (Bombus terrestris).
Pest Management Science,
70(2), 332-337.
Abstract:
Clearance of ingested neonicotinoid pesticide (imidacloprid) in honey bees (Apis mellifera) and bumblebees (Bombus terrestris)
BACKGROUND: Bees in agricultural landscapes are exposed to dietary pesticides such as imidacloprid when they feed from treated mass-flowering crops. Concern about the consequent impact on bees makes it important to understand their resilience. In the laboratory, the authors therefore fed adult worker bees on dosed syrup (125 μg L-1 of imidacloprid, or 98 μg kg-1) either continuously or as a pulsed exposure and measured their behaviour (feeding and locomotory activity) and whole-body residues. RESULTS: on dosed syrup, honey bees maintained much lower bodily levels of imidacloprid than bumblebees (
Abstract.
Cresswell JE, Robert F-XL, Florance H, Smirnoff N (2014). Clearance of ingested neonicotinoid pesticide (imidacloprid) in honey bees (Apis mellifera) and bumblebees (Bombus terrestris).
Pest Manag Sci,
70(2), 332-337.
Abstract:
Clearance of ingested neonicotinoid pesticide (imidacloprid) in honey bees (Apis mellifera) and bumblebees (Bombus terrestris).
BACKGROUND: Bees in agricultural landscapes are exposed to dietary pesticides such as imidacloprid when they feed from treated mass-flowering crops. Concern about the consequent impact on bees makes it important to understand their resilience. In the laboratory, the authors therefore fed adult worker bees on dosed syrup (125 μg L(-1) of imidacloprid, or 98 μg kg(-1)) either continuously or as a pulsed exposure and measured their behaviour (feeding and locomotory activity) and whole-body residues. RESULTS: on dosed syrup, honey bees maintained much lower bodily levels of imidacloprid than bumblebees (
Abstract.
Author URL.
Chen M, MacGregor DR, Dave A, Florance H, Moore K, Paszkiewicz K, Smirnoff N, Graham IA, Penfield S (2014). Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year.
Proc Natl Acad Sci U S A,
111(52), 18787-18792.
Abstract:
Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year.
Seasonal behavior is important for fitness in temperate environments but it is unclear how progeny gain their initial seasonal entrainment. Plants use temperature signals to measure time of year, and changes to life histories are therefore an important consequence of climate change. Here we show that in Arabidopsis the current and prior temperature experience of the mother plant is used to control germination of progeny seeds, via the activation of the florigen Flowering Locus T (FT) in fruit tissues. We demonstrate that maternal past and current temperature experience are transduced to the FT locus in silique phloem. In turn, FT controls seed dormancy through inhibition of proanthocyanidin synthesis in fruits, resulting in altered seed coat tannin content. Our data reveal that maternal temperature history is integrated through FT in the fruit to generate a metabolic signal that entrains the behavior of progeny seeds according to time of year.
Abstract.
Author URL.
Madadkar Haghjou M, Colville L, Smirnoff N (2014). The induction of menadione stress tolerance in the marine microalga, Dunaliella viridis, through cold pretreatment and modulation of the ascorbate and glutathione pools.
Plant Physiol Biochem,
84, 96-104.
Abstract:
The induction of menadione stress tolerance in the marine microalga, Dunaliella viridis, through cold pretreatment and modulation of the ascorbate and glutathione pools.
The effect of cold pretreatment on menadione tolerance was investigated in the cells of the marine microalga, Dunaliella viridis. In addition, the involvement of ascorbate and glutathione in the response to menadione stress was tested by treating cell suspensions with l-galactono-1,4-lactone, an ascorbate precursor, and buthionine sulfoximine, an inhibitor of glutathione synthesis. Menadione was highly toxic to non cold-pretreated cells, and caused a large decrease in cell number. Cold pretreatment alleviated menadione toxicity and cold pretreated cells accumulated lower levels of reactive oxygen species, and had enhanced antioxidant capacity due to increased levels of β-carotene, reduced ascorbate and total glutathione compared to non cold-pretreated cells. Cold pretreatment also altered the response to l-galactono-1,4-lactone and buthionine sulfoximine treatments. Combined l-galactono-1,4-lactone and menadione treatment was lethal in non-cold pretreated cells, but in cold-pretreated cells it had a positive effect on cell numbers compared to menadione alone. Overall, exposure of Dunaliella cells to cold stress enhanced tolerance to subsequent oxidative stress induced by menadione.
Abstract.
Author URL.
2013
Leonelli S, Smirnoff, Nicholas, Moore, Jonathan, Cook, Charis, Bastow, Ruth (2013). Making Open Data Work for Plant Science. Journal of Experimental Botany, 64(14), 4109-4117.
Howard TP, Middelhaufe S, Moore K, Edner C, Kolak DM, Taylor GN, Parker DA, Lee R, Smirnoff N, Aves SJ, et al (2013). Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli.
Proc Natl Acad Sci USA,
110, 7636-7641.
Abstract:
Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli
Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e. aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.
Abstract.
Author URL.
Exposito-Rodriguez M, Laissue PP, Littlejohn GR, Smirnoff N, Mullineaux PM (2013). The use of HyPer to examine spatial and temporal changes in H2O2 in high light-exposed plants.
Methods Enzymol,
527, 185-201.
Abstract:
The use of HyPer to examine spatial and temporal changes in H2O2 in high light-exposed plants.
Exposure of photosynthetic cells of leaf tissues of Arabidopsis thaliana (Arabidopsis) to high light intensities (HL) may provoke a rapid rise in hydrogen peroxide (H2O2) levels in chloroplasts and subcellular compartments, such as peroxisomes, associated with photosynthetic metabolism. It has been hypothesized that when H2O2 is contained at or near its site of production then it plays an important role in signaling to induce acclimation to HL. However, should this discrete containment fail and H2O2 levels exceed the capacity of antioxidant systems to scavenge them, then oxidative stress ensues which triggers cell death. To test this hypothesis, the spatiotemporal accumulation of H2O2 needs to be quantified in different subcellular compartments. In this chapter, preliminary experiments are presented on the use of Arabidopsis seedlings transformed with a nuclear-encoded cytosol-located yellow fluorescent protein-based sensor for H2O2, called HyPer. HyPer allows ratiometric determination of its fluorescence at two excitation wavelengths, which frees quantification of H2O2 from the variable levels of HyPer in vivo. HyPer fluorescence was shown to have the potential to provide the necessary spatial, temporal, and quantitative resolution to study HL responses of seedlings using confocal microscopy. Chlorophyll fluorescence imaging was used to quantify photoinhibition of photosynthesis induced by HL treatment of seedlings on the microscope staging. However, several technical issues remain, the most challenging of which is the silencing of HyPer expression beyond the seedling stage. This limited our pilot studies to cotyledon epidermal cells, which while not photosynthetic, nevertheless responded to HL with 45% increase in cytosolic H2O2.
Abstract.
Author URL.
2012
Perera V, de Torres Zabala M, Florance H, Smirnoff N, Grant M, Yang ZR (2012). Aligning extracted LC-MS peak lists via density maximization.
Metabolomics,
8, 175-185.
Abstract:
Aligning extracted LC-MS peak lists via density maximization
Rapid improvements in mass spectrometry sensitivity and mass accuracy combined with improved liquid chromatography separation technologies allow acquisition of high throughput metabolomics data, providing an excellent opportunity to understand biological processes. While spectral deconvolution software can identify discrete masses and their associated isotopes and adducts, the utility of metabolomic approaches for many statistical analyses such as identifying differentially abundant ions depends heavily on data quality and robustness, especially, the accuracy of aligning features across multiple biological replicates. We have developed a novel algorithm for feature alignment using density maximization. Instead of a greedy iterative, hence local, merging strategy, which has been widely used in the literature and in commercial applications, we apply a global merging strategy to improve alignment quality. Using both simulated and real data, we demonstrate that our new algorithm provides high map (e. g. chromatogram) coverage, which is critically important for non-targeted comparative metabolite profiling of highly replicated biological datasets. © 2011 Springer Science+Business Media, LLC.
Abstract.
Cresswell JE, Page CJ, Uygun MB, Holmbergh M, Li Y, Wheeler JG, Laycock I, Pook CJ, de Ibarra NH, Smirnoff N, et al (2012). Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid).
Zoology,
115(6), 365-371.
Abstract:
Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid)
Currently, there is concern about declining bee populations and the sustainability of pollination services. One potential threat to bees is the unintended impact of systemic insecticides, which are ingested by bees in the nectar and pollen from flowers of treated crops. To establish whether imidacloprid, a systemic neonicotinoid and insect neurotoxin, harms individual bees when ingested at environmentally realistic levels, we exposed adult worker bumble bees, Bombus terrestris L. (Hymenoptera: Apidae), and honey bees, Apis mellifera L. (Hymenoptera: Apidae), to dietary imidacloprid in feeder syrup at dosages between 0.08 and 125μgl-1. Honey bees showed no response to dietary imidacloprid on any variable that we measured (feeding, locomotion and longevity). In contrast, bumble bees progressively developed over time a dose-dependent reduction in feeding rate with declines of 10-30% in the environmentally relevant range of up to 10μgl-1, but neither their locomotory activity nor longevity varied with diet. To explain their differential sensitivity, we speculate that honey bees are better pre-adapted than bumble bees to feed on nectars containing synthetic alkaloids, such as imidacloprid, by virtue of their ancestral adaptation to tropical nectars in which natural alkaloids are prevalent. We emphasise that our study does not suggest that honey bee colonies are invulnerable to dietary imidacloprid under field conditions, but our findings do raise new concern about the impact of agricultural neonicotinoids on wild bumble bee populations. © 2012 Elsevier GmbH.
Abstract.
Smirnoff N (2012). THE ROLE OF ASCORBATE IN THE ACCLIMATION OF LEAVES TO HIGH LIGHT.
PHARMACEUTICAL BIOLOGY,
50(5), 634-634.
Author URL.
Page M, Sultana N, Paszkiewicz K, Florance H, Smirnoff N (2012). The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis.
Plant, Cell & Environment,
35, 388-404.
Abstract:
The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis
Ascorbate and anthocyanins act as photoprotectants during exposure to high light (HL). They accumulate in Arabidopsis leaves in response to HL on a similar time-scale, suggesting a potential relationship between them. Flavonoids and related metabolites were identified and profiled by LC-MS/MS. The ascorbate deficient mutants vtc1, vtc2 and vtc3 accumulated less anthocyanin than wild-type during HL acclimation. In contrast, kaempferol glycoside accumulation was less affected by light and not decreased by ascorbate deficiency, while sinapoyl malate levels decreased during HL acclimation. Comparison of six Arabidopsis ecotypes showed a positive correlation between ascorbate and anthocyanin accumulation in HL. mRNA-Seq analysis showed that all flavonoid biosynthesis transcripts were increased by HL acclimation in wild-type. RT-PCR analysis showed that vtc1 and vtc2 were impaired in HL induction of transcripts of anthocyanin biosynthesis enzymes, and the transcription factors PAP1, GL3 and EGL3 that activate the pathway. Abscisic acid and jasmonic acid, hormones that could affect anthocyanin accumulation, were unaffected in vtc mutants. It is concluded that HL induction of anthocyanin synthesis involves a redox-sensitive process upstream of the known transcription factors. Because anthocyanins accumulate in preference to kaempferol glycosides and sinapoyl malate in HL, they might have specific properties that make them useful in high light acclimation.
Abstract.
2011
Perera V, De Torres Zabala M, Florance H, Smirnoff N, Grant M, Yang ZR (2011). Aligning extracted LC-MS peak lists via density maximization. Metabolomics, 1-11.
Smirnoff N (2011). Chapter 4 Vitamin C the Metabolism and Functions of Ascorbic Acid in Plants. In (Ed) Biosynthesis of Vitamins in Plants Part B, 107-177.
Proctor MCF, Smirnoff N (2011). Ecophysiology of photosynthesis in bryophytes: major roles for oxygen photoreduction and non-photochemical quenching?.
Physiol Plant,
141(2), 130-140.
Abstract:
Ecophysiology of photosynthesis in bryophytes: major roles for oxygen photoreduction and non-photochemical quenching?
CO(2) fixation in mosses saturates at moderate irradiances. Relative electron transport rate (RETR) inferred from chlorophyll fluorescence saturates at similar irradiance in shade species (e.g. Plagiomnium undulatum, Trichocolea tomentella), but many species of unshaded habitats (e.g. Andreaea rothii, Schistidium apocarpum, Sphagnum spp. and Frullania dilatata) show non-saturating RETR at high irradiance, with high non-photochemical quenching (NPQ). In P. undulatum and S. apocarpum, experiments in different gas mixtures showed O(2) and CO(2) as interchangeable electron sinks. Nitrogen + saturating CO(2) gave high RETR and depressed NPQ. In S. apocarpum, glycolaldehyde (inhibiting photosynthesis and photorespiration) depressed RETR in air more at low than at high irradiance; in CO(2) -free air RETR was maintained at all irradiances. Non-saturating electron flow was not suppressed in ambient CO(2) with 1% O(2). The results indicate high capacity for oxygen photoreduction when CO(2) assimilation is limited. Non-saturating light-dependent H(2) O(2) production, insensitive to glycolaldehyde, suggests that electron transport is supported by oxygen photoreduction, perhaps via the Mehler-peroxidase reaction. Consistent with this, mosses were highly tolerant to paraquat, which generates superoxide at photosystem I (PSI). Protection against excess excitation energy in mosses involves high capacity for photosynthetic electron transport to oxygen and high NPQ, activated at high irradiance, alongside high reactive oxygen species (ROS) tolerance.
Abstract.
Author URL.
Gao Y, Badejo AA, Shibata H, Sawa Y, Maruta T, Shigeoka S, Page M, Smirnoff N, Ishikawa T (2011). Expression analysis of the VTC2 and VTC5 genes encoding GDP-L-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana.
Biosci Biotechnol Biochem,
75(9), 1783-1788.
Abstract:
Expression analysis of the VTC2 and VTC5 genes encoding GDP-L-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana.
Arabidopsis thaliana contains two GDP-L-galactose phosphorylase genes, VTC2 and VTC5, which are critical for ascorbate (AsA) biosynthesis. We investigated the expression levels of both VTC2 and VTC5 genes in wild-type A. thaliana and the AsA deficient mutants during early seedling growth. Ascorbate accumulated to an equal extent in all genotypes up to 5 d post-germination (DPG). The transcript level of VTC2 was dominant, and increased in parallel with AsA accumulation in the wild type. On the other hand, the expression of VTC5 compensated for the reduced VTC2 transcription levels in the AsA deficient mutant vtc2-1 in young seedlings. A luciferase activity assay indicated that the VTC5 promoter was more active in young (2 DPG) cotyledons and that the VTC2 and VTC5 promoters drove a day-to-night variation in expression. The present work provides clues to the precise roles of VTC2 and VTC5 in AsA biosynthesis in A. thaliana at the young seedling stage.
Abstract.
Author URL.
Gao Y, Nishikawa H, Badejo AA, Shibata H, Sawa Y, Nakagawa T, Maruta T, Shigeoka S, Smirnoff N, Ishikawa T, et al (2011). Expression of aspartyl protease and C3HC4-type RING zinc finger genes are responsive to ascorbic acid in Arabidopsis thaliana.
J Exp Bot,
62(10), 3647-3657.
Abstract:
Expression of aspartyl protease and C3HC4-type RING zinc finger genes are responsive to ascorbic acid in Arabidopsis thaliana.
Ascorbate (AsA) is a redox buffer and enzyme cofactor with various proposed functions in stress responses and growth. The aim was to identify genes whose transcript levels respond to changes in leaf AsA. The AsA-deficient Arabidopsis mutant vtc2-1 was incubated with the AsA precursor L-galactono-1,4-lactone (L-GalL) to increase leaf AsA concentration. Differentially expressed genes screened by DNA microarray were further characterized for AsA responsiveness in wild-type plants. The analysis of 14 candidates by real-time PCR identified an aspartyl protease gene (ASP, At1g66180) and a C3HC4-type RING zinc finger gene (AtATL15, At1g22500) whose transcripts were rapidly responsive to increases in AsA pool size caused by L-GalL and AsA supplementation and light. Transgenic Arabidopsis plants expressing an AtATL15 promoter::luciferase reporter confirmed that the promoter is L-GalL, AsA, and light responsive. The expression patterns of ASP and AtATL15 suggest they have roles in growth regulation. The promoter of AtATL15 is responsive to AsA status and will provide a tool to investigate the functions of AsA in plants further.
Abstract.
Author URL.
Salmon D, Smirnoff N, Wheeler G, Brownlee C (2011). METABOLITE PROFILING INDICATES INTRASPECIFIC VARIABILITY IN THE ACCUMULATION OF COMPATIBLE SOLUTES IN THE HAPTOPHYTE ALGA EMILIANIA HUXLEYI.
EUROPEAN JOURNAL OF PHYCOLOGY,
46, 154-155.
Author URL.
Wilkins KA, Bancroft J, Bosch M, Ings J, Smirnoff N, Franklin-Tong VE (2011). Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver.
Plant Physiol,
156(1), 404-416.
Abstract:
Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver.
Pollen-pistil interactions are critical early events regulating pollination and fertilization. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants. Although data implicate the involvement of reactive oxygen species (ROS) and nitric oxide (NO) in pollen-pistil interactions and the regulation of pollen tube growth, there has been a lack of studies investigating ROS and NO signaling in pollen tubes in response to defined, physiologically relevant stimuli. We have used live-cell imaging to visualize ROS and NO in growing Papaver rhoeas pollen tubes using chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and demonstrate that SI induces relatively rapid and transient increases in ROS and NO, with each showing a distinctive "signature" within incompatible pollen tubes. Investigating how these signals integrate with the SI responses, we show that Ca(2+) increases are upstream of ROS and NO. As ROS/NO scavengers alleviated both the formation of SI-induced actin punctate foci and also the activation of a DEVDase/caspase-3-like activity, this demonstrates that ROS and NO act upstream of these key SI markers and suggests that they signal to these SI events. These data represent, to our knowledge, the first steps in understanding ROS/NO signaling triggered by this receptor-ligand interaction in pollen tubes.
Abstract.
Author URL.
Smirnoff N (2011). Vitamin C: the metabolism and functions of ascorbic acid in plants.
,
59, 107-177.
Abstract:
Vitamin C: the metabolism and functions of ascorbic acid in plants
It is widely accepted that the predominant ascorbate biosynthesis pathway in green plants is via GDP-mannose and l-galactose. d-galacturonic, d-glucuronic acid and GDP-l-gulose could be minor ascorbate precursors, but there is no definitive evidence. Arabidopsis thaliana mutants lacking ascorbate cannot grow, but it is not known which function is critical: control of reactive oxygen or the proposed roles in modulating cell expansion and division. Ascorbate is transported in the phloem, and glucose conjugates occur in the phloem of the Cucurbitaceae. Ascorbate or dehydroascorbate transporters have not been identified at the molecular level. Pathways from ascorbate to oxalate in the apoplast and tartrate in grape berries have been identified. Ascorbate-deficient (vtc) mutants tend to be smaller, more sensitive to abiotic stresses and more resistant to biotrophic pathogens. The use of mutants and overexpression shows the importance of ascorbate peroxidase, monodehydroascorbate reductase and dehydroascorbate reductase in reactive oxygen defence and signalling. Ascorbate accumulation in Arabidopsis leaves is increased by high light along with expression and activity of l-galactose phosphorylase (VTC2), reflecting multiple roles in photosynthesis. These roles are modulation of hydrogen peroxide and singlet oxygen, enzyme cofactor in the xanthophyll cycle and, speculatively, a photosystem II electron donor during photoinhibition. © 2011 Elsevier Ltd.
Abstract.
2010
Littlejohn GR, Gouveia JD, Edner C, Smirnoff N, Love J (2010). Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll.
New Phytol,
186(4), 1018-1025.
Abstract:
Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll.
*Air spaces in the leaf mesophyll generate deleterious optical effects that compromise confocal microscopy. Leaves were mounted in the nontoxic, nonfluorescent perfluorocarbon, perfluorodecalin (PFD), and optical enhancement and physiological effect were assessed using confocal microscopy and chlorophyll fluorescence. Mounting leaves of Arabidopsis thaliana in PFD significantly improved the optical qualities of the leaf, thereby enabling high-resolution laser scanning confocal imaging over twofold deeper into the mesophyll, compared with using water. Incubation in PFD had less physiological impact on the mounted specimen than water. We conclude that the application of PFD as a mounting medium substantially increases confocal image resolution of living mesophyll and vascular bundle cells, with minimal physiological impact.
Abstract.
Author URL.
Smirnoff N (2010). Tocochromanols: rancid lipids, seed longevity, and beyond.
Proc Natl Acad Sci U S A,
107(42), 17857-17858.
Author URL.
2009
Spelman K, Iiams-Hauser K, Cech NB, Taylor EW, Smirnoff N, Wenner CA (2009). Role for PPARgamma in IL-2 inhibition in T cells by Echinacea-derived undeca-2E-ene-8,10-diynoic acid isobutylamide.
Int Immunopharmacol,
9(11), 1260-1264.
Abstract:
Role for PPARgamma in IL-2 inhibition in T cells by Echinacea-derived undeca-2E-ene-8,10-diynoic acid isobutylamide.
Certain fatty acid amides from Echinacea spp. have demonstrated moderate to high cannabinoid activity. As a result, CB2 activation is currently hypothesized to be the basis of activity for immunomodulation by Echinacea spp. PPARgamma, an orphan nuclear receptor and lipid sensor, is known to inhibit IL-2 production and be activated by fatty acid derivatives such as the endocannabinoids. In these investigations, we demonstrate that undeca-2E-ene-8,10-diynoic acid, an Echinacea angustifolia-derived alkylamide lacking affinity for the CB2 receptor, inhibits IL-2 secretion in Jurkat T cells through PPARgamma activity at low micromolar concentrations (330 ng/mL). The IL-2 inhibition is reversed by the addition of the selective PPARgamma antagonist T0070907. Additionally, we show that that undeca-2-ene-8,10-diynoic acid stimulates 3T3-L1 differentiation, a process dependent on PPARgamma activity. These experiments demonstrate that PPARgamma is involved in T cell IL-2 inhibition by undeca-2-ene-8,10-diynoic acid and suggest that cytokine modulation by the alkylamides is due to polyvalent activity.
Abstract.
Author URL.
Haghjou MM, Shariati M, Smirnoff N (2009). The effect of acute high light and low temperature stresses on the ascorbate-glutathione cycle and superoxide dismutase activity in two Dunaliella salina strains.
Physiol Plant,
135(3), 272-280.
Abstract:
The effect of acute high light and low temperature stresses on the ascorbate-glutathione cycle and superoxide dismutase activity in two Dunaliella salina strains.
Dunaliella species accumulate carotenoids and their role in protection against photooxidative stress has been investigated extensively. By contrast, the role of other antioxidants in this alga, has received less attention. Therefore, the components of the ascorbate-glutathione cycle, along with superoxide dismutase (E.C. 1.15.1.1) and peroxidase (E.C. 1.11.1.11) activity were compared in two strains of Dunaliella salina. Strain IR-1 had two-fold higher chlorophyll and beta-carotene concentration than Gh-U. IR-1 had around four-fold higher superoxide dismutase, ascorbate peroxidase and pyrogallol peroxidase activities than Gh-U on a protein basis. Ascorbate and glutathione concentrations and redox state did not differ between strains and there was little difference in the activity of ascorbate-glutathione cycle enzymes (monodehydroascorbate reductase [E.C. 1.6.5.4], dehydroascorbate reductase [E.C. 1.8.5.1] and glutathione reductase [E.C. 1.8.1.7]). The response of these antioxidants to high light and low temperature was assessed by transferring cells from normal growth conditions (28 degrees C, photon flux density of 100 micromol m(-2) s(-1))to 28 degrees C/1200 micromol m(-2) s(-1); 13 degrees C/100 micromol m(-2) s(-1); 13 degrees C/1200 micromol m(-2) s(-1) and 28 degrees C/100 micromol m(-2) s(-1) for 24 h. Low temperature and combined high light-low temperature decreased chlorophyll and beta-carotene in both strains indicating that these treatments cause photooxidative stress. High light, low temperature and combined high light-low temperature treatments increased the total ascorbate pool by 10-50% and the total glutathione pool by 20-100% with no consistent effect on their redox state. Activities of ascorbate-glutathione cycle enzymes were not greatly affected but all the treatments increased superoxide dismutase activity. It is concluded that D. salina can partially adjust to photooxidative conditions by increasing superoxide dismutase activity, ascorbate and glutathione.
Abstract.
Author URL.
Galvez-Valdivieso G, Fryer MJ, Lawson T, Slattery K, Truman W, Smirnoff N, Asami T, Davies WJ, Jones AM, Baker NR, et al (2009). The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells.
Plant Cell,
21(7), 2143-2162.
Abstract:
The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells.
Previously, it has been shown that Arabidopsis thaliana leaves exposed to high light accumulate hydrogen peroxide (H2O2) in bundle sheath cell (BSC) chloroplasts as part of a retrograde signaling network that induces ASCORBATE PEROXIDASE2 (APX2). Abscisic acid (ABA) signaling has been postulated to be involved in this network. To investigate the proposed role of ABA, a combination of physiological, pharmacological, bioinformatic, and molecular genetic approaches was used. ABA biosynthesis is initiated in vascular parenchyma and activates a signaling network in neighboring BSCs. This signaling network includes the Galpha subunit of the heterotrimeric G protein complex, the OPEN STOMATA1 protein kinase, and extracellular H2O2, which together coordinate with a redox-retrograde signal from BSC chloroplasts to activate APX2 expression. High light-responsive genes expressed in other leaf tissues are subject to a coordination of chloroplast retrograde signaling and transcellular signaling activated by ABA synthesized in vascular cells. ABA is necessary for the successful adjustment of the leaf to repeated episodes of high light. This process involves maintenance of photochemical quenching, which is required for dissipation of excess excitation energy.
Abstract.
Author URL.
2008
Colville L, Smirnoff N (2008). Antioxidant status, peroxidase activity, and PR protein transcript levels in ascorbate-deficient Arabidopsis thaliana vtc mutants.
J Exp Bot,
59(14), 3857-3868.
Abstract:
Antioxidant status, peroxidase activity, and PR protein transcript levels in ascorbate-deficient Arabidopsis thaliana vtc mutants.
Ascorbate is the most abundant small molecule antioxidant in plants and is proposed to function, along with other members of an antioxidant network, in controlling reactive oxygen species. A biochemical and molecular characterization of four ascorbate-deficient (vtc) Arabidopsis thaliana mutants has been carried out to determine if ascorbate deficiency is compensated by changes in the other major antioxidants. Seedlings grown in vitro were used to minimize stress and longer term developmental differences. Comparison was made with the low glutathione cad2 mutant and vtc2-1 treated with D,L-buthionine-[S,R]-sulphoximine to cause combined ascorbate and glutathione deficiency. The pool sizes and oxidation state of ascorbate and glutathione were not altered by deficiency of the other. alpha-Tocopherol and activities of monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and catalase were little affected. Ascorbate peroxidase activity was higher in vtc1, vtc2-1, and vtc2-2. Ionically bound cell wall peroxidase activity was increased in vtc1, vtc2-1, and vtc4. Supplementation with ascorbate increased cell wall peroxidase activity. 2,6-Dichlorobenzonitrile, an inhibitor of cellulose synthesis, increased cell wall peroxidase activity in the wild type and vtc1. The transcript level of an endochitinase, PR1, and PR2, but not GST6, was increased in vtc1, vtc2-1, and vtc-2-2. Endochitinase transcript levels increased after ascorbate, paraquat, salicylic acid, and UV-C treatment, PR1 after salicylic acid treatment, and PR2 after paraquat and UV-C treatment. Camalexin was higher in vtc1 and the vtc2 alleles. Induction of PR genes, cell wall peroxidase activity, and camalexin in vtc1, vtc2-1, and vtc2-2 suggests that the mutants are affected in pathogen response signalling pathways.
Abstract.
Author URL.
Smirnoff N, Grant M (2008). Plant biology: do DELLAs do defence?.
Curr Biol,
18(14), R617-R619.
Abstract:
Plant biology: do DELLAs do defence?
DELLA proteins repress plant growth and developmental processes. Recent data suggest that DELLAs improve survival by imposing growth restraint during plant stress, enabling limited resources to be diverted to pathogen defence.
Abstract.
Author URL.
Dowdle J, Ishikawa T, Gatzek S, Rolinski S, Smirnoff N (2008). Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability (vol 52, pg 673, 2007).
PLANT JOURNAL,
53(3), 595-595.
Author URL.
2007
Macnair MR, Dowdle J, Filatov V, Ford-Lloyd B (2007). A quantitative trait loci analysis of zinc hyperaccumulation in Arabidopsis halleri. New Phytologist, 174(3), 580-590.
Smirnoff N (2007). Antioxidants and Reactive Oxygen Species in Plants.
Antioxidants and Reactive Oxygen Species in Plants, 1-302.
Abstract:
Antioxidants and Reactive Oxygen Species in Plants
Reactive oxygen species (ROS) are produced during the interaction of metabolism with oxygen. As ROS have the potential to cause oxidative damage by reacting with biomolecules, research on ROS has concentrated on the oxidative damage that results from exposure to environmental stresses and on the role of ROS in defence against pathogens. However, more recently, it has become apparent that ROS also have important roles as signalling molecules. A complex network of enzymatic and small molecule antioxidants controls the concentration of ROS and repairs oxidative damage, and research is revealing the complex and subtle interplay between ROS and antioxidants in controlling plant growth, development and response to the environment. This book covers these new developments, generally focussing on molecular and biochemical details and providing a point of entry to the detailed literature. It is directed at researchers and professionals in plant molecular biology, biochemistry and cell biology, in both the academic and industrial sectors. © 2005 by Blackwell Publishing Ltd.
Abstract.
vanderHoning, H. Nishioka, M. Uehara, Y. (2007). Armadillo repeat-containing kinesins and a NIMA-related kinase are required for epidermal-cell morphogenesis in Arabidopsis. Plant Journal, 10.1111/j.1365-313X.
Smirnoff N (2007). Ascorbate, Tocopherol and Carotenoids: Metabolism, Pathway Engineering and Functions. , 53-86.
Ishikawa T, Dowdle J, Smirnoff N (2007). Erratum: Progress in manipulating ascorbic acid biosynthesis and accumulation in plants (Physiologia Plantarum (2006) 126, (343-355)). Physiologia Plantarum, 129(4).
Talbot NJ, Egan MJ, Jones MA, Smirnoff N (2007). Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease. Proceedings of the National Academy of Sciences, 104(28), 11772-11777.
Raymond, M.J. Yang, Z. Smirnoff, N. (2007). NADPH oxidase-dependent reactive oxygen species formation required for root-hair growth depends on ROP GTPase. Journal of Experimental Botany, 58, 1261-1270.
Smirnoff N (2007). Preface. Antioxidants and Reactive Oxygen Species in Plants
Ishikawa T, Dowdle J, Smirnoff N (2007). Progress in manipulating ascorbic acid biosynthesis and accumulation in plants (vol 126, pg 343, 2006).
PHYSIOLOGIA PLANTARUM,
129(4), 831-831.
Author URL.
Jones MA, Smirnoff N (2007). Reactive Oxygen Species in Plant Development and Pathogen Defence. , 197-214.
Potocky M, Zarsky V, Jones M, Smirnoff N, Bezvoda R (2007). Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth.
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY,
146(4), S269-S270.
Author URL.
Jones, M.A. Bezvoda, R. Smirnoff, N. (2007). Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytologist, 174, 742-751.
Smirnoff N, Dowdle J, Ishikawa T (2007). The role of VTC2 in vitamin C biosynthesis in Arabidopsis thaliana.
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY,
146(4), S250-S250.
Author URL.
Ishikawa, T. Gatzek, S. Rolinski, S. (2007). Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant Journal, 52, 673-689.
Smirnoff N, Dowdle J, Gatzek S, Ishikawa T (2007). Two genes in Arabidopsis thaliana encoding GDP-l-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.
Ishikawa T, Tanimoto M, Dowdle J, Smirnoff N (2007). VTC2 encodes GDP-L-galactose phosphorylase, an enzyme involved in plant ascorbate biosynthesis.
Author URL.
2006
Smirnoff N, Jones MA, Raymond MJ (2006). Analysis of the root-hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root hair development in Arabidopsis . The Plant Journal, 45(1), 83-100.
Macnair MR, Dowdle J, Ford-Lloyd B, Smirnoff N (2006). Comparison of gene expression in segregating families identifies genes and genomic regions involved in a novel adaptation, zinc hyperaccumulation. Molecular Ecology, 15(10), 3045-3059.
Hancock JT, Smirnoff N, Foyer CH (2006). Journal of Experimental Botany: Preface. Journal of Experimental Botany, 57(8).
Smirnoff, N. (2006). Nuclear dynamics during the simultaneous and sustained tip growth of multiple root hairs arising from a single root epidermal cell. Journal of Experimental Botany, 57, 4269-4275.
Hancock JT, Smirnoff N, Foyer CH (2006). Oxygen metabolism coming up ROSes: a holistic view of the redox metabolism of plant cells - Preface.
JOURNAL OF EXPERIMENTAL BOTANY,
57(8), IV-V.
Author URL.
Dowdle, J. Smirnoff, N. (2006). Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiologia Plantarum, 125, 343-355.
Smirnoff N, Conklin PL, Gatzek S, Ishikawa T (2006). The Arabidopsis thaliana VTC4 gene encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme. Journal of Biological Chemistry, 281(23), 15662-15670.
2005
Baroja-Mazo A, Del Valle P, Rúa J, De Cima S, Busto F, De Arriaga D, Smirnoff N (2005). Characterisation and biosynthesis of D-erythroascorbic acid in Phycomyces blakesleeanus.
Fungal Genetics and Biology,
42(5), 390-402.
Abstract:
Characterisation and biosynthesis of D-erythroascorbic acid in Phycomyces blakesleeanus
D-Erythroascorbate and D-erythroascorbate glucoside have been identified in the Zygomycete fungus Phycomyces blakesleeanus. Ascomycete and Basidiomycete fungi also synthesise D-erythroascorbate instead of L-ascorbate, suggesting that d-erythroascorbate synthesis evolved in the common ancestor of the fungi. Both compounds accumulate in P. blakesleeanus at higher levels than observed in other fungal species. D-Erythroascorbate glucoside reduced dichlorophenolindophenol as effectively as L-ascorbate, but was more stable to autoxidation. D-Erythroascorbate glucoside predominated in spores and stationary phase mycelium. Free D-erythroascorbate accumulated during the exponential phase of mycelial growth and decreased to very low levels in the stationary phase. This suggests an association between growth and free D-erythroascorbate. P. blakesleeanus converted exogenous D-arabinose to D-erythroascorbate and its glucoside. A monomeric NAD-dependent D-arabinose dehydrogenase of 41 kDa was purified to near homogeneity. The enzyme oxidised D-arabinose, L-galactose, and L-fucose. Correspondingly, mycelium converted exogenous L-galactose and L-fucose to L-ascorbate and 6-deoxyascorbate, respectively. The antioxidant role of D-erythroascorbate and its glucoside is discussed. © 2005 Elsevier Inc. All rights reserved.
Abstract.
Baroja-Mazo A, del Valle P, Rúa J, de Cima S, Busto F, de Arriaga D, Smirnoff N (2005). Characterisation and biosynthesis of D-erythroascorbic acid in Phycomyces blakesleeanus.
Fungal Genet Biol,
42(5), 390-402.
Abstract:
Characterisation and biosynthesis of D-erythroascorbic acid in Phycomyces blakesleeanus.
D-Erythroascorbate and D-erythroascorbate glucoside have been identified in the Zygomycete fungus Phycomyces blakesleeanus. Ascomycete and Basidiomycete fungi also synthesise D-erythroascorbate instead of l-ascorbate, suggesting that D-erythroascorbate synthesis evolved in the common ancestor of the fungi. Both compounds accumulate in P. blakesleeanus at higher levels than observed in other fungal species. D-Erythroascorbate glucoside reduced dichlorophenolindophenol as effectively as L-ascorbate, but was more stable to autoxidation. D-Erythroascorbate glucoside predominated in spores and stationary phase mycelium. Free D-erythroascorbate accumulated during the exponential phase of mycelial growth and decreased to very low levels in the stationary phase. This suggests an association between growth and free D-erythroascorbate. P. blakesleeanus converted exogenous D-arabinose to D-erythroascorbate and its glucoside. A monomeric NAD-dependent D-arabinose dehydrogenase of 41 kDa was purified to near homogeneity. The enzyme oxidised D-arabinose, L-galactose, and L-fucose. Correspondingly, mycelium converted exogenous L-galactose and L-fucose to L-ascorbate and 6-deoxyascorbate, respectively. The antioxidant role of D-erythroascorbate and its glucoside is discussed.
Abstract.
Author URL.
Burrell M, Smirnoff N (2005). Making sense of the metabolome special issue - Preface.
JOURNAL OF EXPERIMENTAL BOTANY,
56(410), IV-IV.
Author URL.
Burrell M, Smirnoff N (2005). Making sense of the metabolome: Preface. Journal of Experimental Botany, 56(410).
2003
Smirnoff N (2003). Vitamin C booster.
Nat Biotechnol,
21(2), 134-136.
Author URL.
2002
Smirnoff N, Gatzek S, Wheeler GL (2002). Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. The Plant Journal, 30(5), 541-553.
Smirnoff N (2002). Journal of Experimental Botany: Preface. Journal of Experimental Botany, 53(372).
Smirnoff N (2002). Plants in cold climates and waterlogged soils. Annals of Botany, 90(4).
Raymond MJ, Smirnoff N (2002). Proline metabolism and transport in maize seedlings at low water potential.
Ann Bot,
89 Spec No(7), 813-823.
Abstract:
Proline metabolism and transport in maize seedlings at low water potential.
The growing zone of maize seedling primary roots accumulates proline at low water potential. Endosperm removal and excision of root tips rapidly decreased the proline pool and greatly reduced proline accumulation in root tips at low water potential. Proline accumulation was not restored by exogenous amino acids. Labelling root lips with [14C]glutamate and [14C]proline showed that the rate of proline utilization (oxidation and protein synthesis) exceeded the rate of biosynthesis by five-fold at high and low water potentials. This explains the reduction in the proline pool following root and endosperm excision and the inability to accumulate proline at low water potential. The endosperm is therefore the source of the proline that accumulates in the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [14C]Proline was transported from the scutellum to other parts of the seedling and reached the highest concentration in the root tip. Less [14C]proline was transported at low water potential but because of the lower rate of protein synthesis and oxidation, more accumulated as proline in the root tip. Despite the low biosynthesis capacity of the roots, the extent of proline accumulation in relation to water potential is precisely controlled by transport and utilization rate.
Abstract.
Author URL.
Murakeözy EP, Smirnoff N, Nagy Z, Tuba Z (2002). Seasonal accumulation pattern of pinitol and other carbohydrates in Limonium gmelini subsp. hungarica.
Journal of Plant Physiology,
159(5), 485-490.
Abstract:
Seasonal accumulation pattern of pinitol and other carbohydrates in Limonium gmelini subsp. hungarica
Concentrations of the main soluble carbohydrates were measured in leaf and root samples of Limonium gmelini subsp. hungarica (Plumbaginaceae), a species native to the Hungarian inland salty areas. Samples were collected between March and October, from plants growing in their natural habitat. D-pinitol was identified as a significant component of the carbohydrate pool. The seasonal changes in the leaf pinitol concentration were dependent on different stress factors such as decrease of soil water content, low temperature, and leaf sodium concentration. A five-fold increase in the shoot D-pinitol values were observed between mid-April (15 μmol g-1 dw-1) and August (78 μmol g-1 dw-1) concentrations. In the roots, pinitol concentration was relatively low and increased only moderately in the summer. No significant diel changes in the concentration of pinitol were observed except during the time of intensive leaf growth in the spring. The possible functions of pinitol in L. gmelini are discussed.
Abstract.
Smirnoff N (2002). Untitled - Preface.
JOURNAL OF EXPERIMENTAL BOTANY,
53(372).
Author URL.
2001
Smirnoff N, Conklin PL, Loewus FA (2001). Biosynthesis of ascorbic acid in plants: a renaissance.
ANNUAL REVIEW OF PLANT PHYSIOLOGY AND PLANT MOLECULAR BIOLOGY,
52, 437-+.
Author URL.
Takács Z, Tuba Z, Smirnoff N (2001). Exaggeration of desiccation stress by heavy metal pollution in Tortula ruralis: a pilot study.
Abstract:
Exaggeration of desiccation stress by heavy metal pollution in Tortula ruralis: a pilot study
Abstract.
Smirnoff N (2001). L-ascorbic acid biosynthesis.
Vitam Horm,
61, 241-266.
Abstract:
L-ascorbic acid biosynthesis.
Biosynthesis of L-ascorbate (vitamin C) occurs by different pathways in plants and mammals. Yeast contain D-erythroascorbate, a C5 analog of ascorbate. UDP-D-glucuronic acid is the precursor in mammals. Loss of UDP forms glucuronic acid/glucuronolactone. Reduction of these at C-1 then forms L-gulonic acid/L-gulono-1,4-lactone. The lactone is oxidized by a microsomal L-gulono-1,4-lactone oxidase to ascorbate. Only the L-gulono-1,4-lactone oxidase has been purified and cloned, and very little is known about the properties of the other enzymes. Plants form ascorbate from GDP-D-mannose via GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone. The final oxidation of L-galactono-1,4-lactone to ascorbate is catalyzed by a mitochondrial L-galactono-1,4-lactone dehydrogenase located on the inner membrane and using cytochrome c as electron acceptor. GDP-mannose pyrophosphorylase and L-galactono-1,4-lactone dehydrogenase have been cloned. Yeast synthesizes D-erythroascorbate from D-arabinose and D-arabinono-1,4-lactone in a pathway analogous to that in plants. The plant, mammalian, and yeast aldonolactone oxidase/dehydrogenases that catalyze the last step in each pathway have significant sequence homology. L-Gulono-1,4-lactone oxidase is mutated and not expressed in animals, such as primates, that have lost ascorbate biosynthesis capacity. Assessment of the literature reveals that little is known about many of the enzymes involved in ascorbate biosynthesis or about the factors controlling flux through the pathways. There is also a possibility that minor alternative pathways exist in plants and mammals.
Abstract.
Author URL.
Bachmann, K. Smirnoff, N. Macnair, M.R. (2001). The role of drought tolerance in serpentine tolerance in the Mimulus guttatus Fischer ex DC. complex. South African Journal of Science, 97, 581-586.
2000
Smirnoff N (2000). Ascorbate biosynthesis and function in photoprotection.
Abstract:
Ascorbate biosynthesis and function in photoprotection.
Abstract.
Author URL.
Smirnoff N, Wheeler GL (2000). Ascorbic acid in plants: Biosynthesis and function.
Critical Reviews in Plant Sciences,
19(4), 267-290.
Abstract:
Ascorbic acid in plants: Biosynthesis and function
Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtc 1 mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifacetted molecule.
Abstract.
Smirnoff N, Wheeler GL (2000). Ascorbic acid in plants: biosynthesis and function.
Crit Rev Biochem Mol Biol,
35(4), 291-314.
Abstract:
Ascorbic acid in plants: biosynthesis and function.
Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtc 1 mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifaceted molecule.
Abstract.
Author URL.
Smirnoff N (2000). Ascorbic acid: metabolism and functions of a multi-facetted molecule.
Curr Opin Plant Biol,
3(3), 229-235.
Abstract:
Ascorbic acid: metabolism and functions of a multi-facetted molecule.
Ascorbic acid (vitamin C) is the most abundant antioxidant in plants. Its biosynthetic pathway via GDP-D-mannose and L-galactose, which was proposed only recently, is now supported by molecular genetic evidence from Arabidopsis thaliana and transgenic potato plants. Except for the last step (which is located on the inner mitochondrial membrane) the pathway is cytosolic, sharing GDP-sugar intermediates with cell-wall polysaccharide and glycoprotein synthesis. Ascorbate peroxidase is emerging as a key enzyme in the fine control of H(2)O(2) concentration; its expression being controlled by redox signals and H(2)O(2). Convincing evidence of the involvement of ascorbate in cell division and growth is also accumulating. Its role as a cofactor in the synthesis of cell wall hydroxyproline-rich glycoproteins is one mechanism for this function.
Abstract.
Author URL.
Davey MW, Van Montagu M, Inzé D, Sanmartin M, Kanellis A, Smirnoff N, Benzie IFF, Strain JJ, Favell D, Fletcher J, et al (2000). Plant L-ascorbic acid: Chemistry, function, metabolism, bioavailability and effects of processing.
Journal of the Science of Food and Agriculture,
80(7), 825-860.
Abstract:
Plant L-ascorbic acid: Chemistry, function, metabolism, bioavailability and effects of processing
Humans are unable to synthesise L-ascorbic acid (L-AA, ascorbate, vitamin C), and are thus entirely dependent upon dietary sources to meet needs. In both plant and animal metabolism, the biological functions of L-ascorbic acid are centred around the antioxidant properties of this molecule. Considerable evidence has been accruing in the last two decades of the importance of L-AA in protecting not only the plant from oxidative stress, but also mammals from various chronic diseases that have their origins in oxidative stress. Evidence suggests that the plasma levels of L-AA in large sections of the population are sub-optimal for the health protective effects of this vitamin. Until quite recently, little focus has been given to improving the L-AA content of plant foods, either in terms of the amounts present in commercial crop varieties, or in minimising losses prior to ingestion. Further, while L-AA biosynthesis in animals was elucidated in the 1960s, it is only very recently that a distinct biosynthetic route for plants has been proposed. The characterisation of this new pathway will undoubtedly provide the necessary focus and impetus to enable fundamental questions on plant L-AA metabolism to be resolved. This review focuses on the role of L-AA in metabolism and the latest studies regarding its biosynthesis, tissue compartmentalisation, turnover and catabolism. These inter-relationships are considered in relation to the potential to improve the L-AA content of crops. Methodology for the reliable analysis of L-AA in plant foods is briefly reviewed. The concentrations found in common food sources and the effects of processing, or storage prior to consumption are discussed. Finally the factors that determine the bioavailability of L-AA and how it may be improved are considered, as well as the most important future research needs. (C) 2000 Society of Chemical Industry.
Abstract.
Proctor MC, Smirnoff N (2000). Rapid recovery of photosystems on rewetting desiccation-tolerant mosses: chlorophyll fluorescence and inhibitor experiments.
J Exp Bot,
51(351), 1695-1704.
Abstract:
Rapid recovery of photosystems on rewetting desiccation-tolerant mosses: chlorophyll fluorescence and inhibitor experiments.
In the mosses Racomitrium lanuginosum, Anomodon viticulosus and Rhytidiadelphus loreus, after a few days air dry, F:(v)/F:(m) reached, within the first minute of remoistening in the dark, two-thirds or more of the value attained after 40 min. A fast initial phase of recovery was completed within 10-20 min after which further change was slow. Initial recovery of Phi(PSII) in the light was somewhat slower, but was generally substantially complete within a similar time. Remoistening with 0.3 mM cycloheximide (CHX) or 3 mM dithiothreitol (DTT) made little difference to this short-term (40 min) recovery of either F:(v)/F:(m) or Phi(PSII); 3 mM chloramphenicol (CMP) had little effect on recovery of F:(v)/F:(m), but resulted in substantial (though not total) depression of Phi(PSII) and (14)CO(2) uptake. Effects of the protein-synthesis inhibitors and DTT were much more clearly apparent in longer-term experiments (>20 h) but only in the light. In the dark, the three inhibitors had at most only slight effects over periods of 60-100 h. In the light, CMP-treated samples of all three species showed a progressive decline of dark-adapted F:(v)/F:(m), falling to zero within 1-5 d (possibly due to blocking of the turnover of the D1 protein of PSII) and accelerated by DTT. CHX-treated samples showed a similar but slower decline. In the shade-adapted and relatively desiccation-sensitive Rhytidiadelphus loreus, slow recovery of F:(v)/F:(m) continued in the dark even in the presence of CMP and CHX for much of the 142 h of the experiment. The results indicate that in desiccation-tolerant bryophytes recovery of photosynthesis after periods of a few days air dry requires only limited chloroplast protein synthesis and is substantially independent of protein synthesis in the cytoplasm.
Abstract.
Author URL.
Spickett CM, Smirnoff N, Pitt AR (2000). The biosynthesis of erythroascorbate in Saccharomyces cerevisiae and its role as an antioxidant.
Free Radic Biol Med,
28(2), 183-192.
Abstract:
The biosynthesis of erythroascorbate in Saccharomyces cerevisiae and its role as an antioxidant.
This study investigated the ability of the yeast Saccharomyces cerevisiae to synthesize ascorbate and its 5-carbon analogue erythroascorbate from a variety of precursors, and their importance as antioxidants in this organism. Studies of ascorbate and analogues in micro-organisms have been reported previously, but their function as antioxidants have been largely ignored. Ascorbate and erythroascorbate concentrations in yeast extracts were measured spectrophotometrically, and their levels and identity were checked using liquid chromatography-electrospray mass spectrometry. The yeast was readily able to synthesize ascorbate from L-galactono-1,4-lactone or erythroascorbate from D-arabinose and D-arabino-1,4-lactone, whereas L-gulono-1,4-lactone was a much poorer substrate for ascorbate biosynthesis. In untreated cells, the concentration of ascorbate-like compounds was below the level of detection of the methods of analysis used in this study (approximately 0.1 mM). Intracellular ascorbate and erythroascorbate were oxidized at high concentrations of tert-butylhydroperoxide, but not hydrogen peroxide. Their synthesis was not increased in response to low levels of stress, however, and preloading with erythroascorbate did not protect glutathione levels during oxidative stress. This study provides new information on the metabolism of ascorbate and erythroascorbate in S. cerevisiae, and suggests that erythroascorbate is of limited importance as an antioxidant in S. cerevisiae.
Abstract.
Author URL.
Pallanca JE, Smirnoff N (2000). The control of ascorbic acid synthesis and turnover in pea seedlings.
J Exp Bot,
51(345), 669-674.
Abstract:
The control of ascorbic acid synthesis and turnover in pea seedlings.
The rate of ascorbate synthesis and turnover in pea seedling embryonic axes was investigated in relation to its pool size. Ascorbate accumulated in embryonic axes of germinating pea seeds which has been supplied with ascorbate. Incorporation of [U-14C]glucose into ascorbate after a 2 h labelling period was reduced by ascorbate loading for 3 h and 20 h, providing evidence that ascorbate biosynthesis is inhibited by endogenous ascorbate. Ascorbate turnover was estimated by following the metabolism of [1-14C]ascorbate over 2 h after ascorbate loading and by the rate of decrease of the ascorbate pool size after ascorbate loading. Ascorbate turnover rate, determined by [1-14C]ascorbate metabolism, increased as a linear function of pool size. The absolute turnover rate was higher in ascorbate-loaded embryonic axes but was always about 13% of the pool per hour. The initial rate of ascorbate turnover, estimated from the net decrease in pool size after ascorbate loading, also showed a similar turnover rate to that estimated from [1-14C]ascorbate metabolism. Ascorbate loading had no effect on ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase activity. Ascorbate oxidase activity decreased after ascorbate loading.
Abstract.
Author URL.
1999
Pallanca JE, Smirnoff N (1999). Ascorbic acid metabolism in pea seedlings. A comparison of D-glucosone, L-sorbosone, and L-galactono-1,4-lactone as ascorbate precursors.
Plant Physiology,
120(2), 453-461.
Abstract:
Ascorbic acid metabolism in pea seedlings. A comparison of D-glucosone, L-sorbosone, and L-galactono-1,4-lactone as ascorbate precursors
Ascorbic acid (AsA) accumulates in pea (Pisum sativum L.) seedlings during germination, with the most rapid phase of accumulation coinciding with radicle emergence. Monodehydroascorbate reductase and dehydroascorbic acid reductase were active in the embryonic axes before AsA accumulation started, whereas AsA oxidase and AsA peroxidase activities increased in parallel with AsA. Excised embryonic axes were used to investigate the osone pathway of AsA biosynthesis, in which D-glucosone and L-sorbosone are the proposed intermediates. [U-14C]Glucosone was incorporated into AsA and inhibited the incorporation of [U-14C]glucose (Glc) into AsA. A higher D-glucosone concentration (5 mM) inhibited AsA accumulation, L-Sorbosone did not affect AsA pool size but caused a small inhibition in the incorporation of [U-14C]Glc into AsA. Oxidase and dehydrogenase activities capable of converting Glc or Glc-6-phosphate to glucosone were not detected in embryonic axis extracts. The osones are therefore unlikely to be physiological intermediates of AsA biosynthesis, L-Galactono-1,4-lactone, recently proposed as the AsA precursor (G.L. Wheeler, M.A. Jones, N. Smirnoff [1998] Nature 393: 365-369), was readily converted to AsA by pea embryonic axes. Although L-galactono-1,4-lactone did not inhibit [14C]Glc incorporation into AsA, this does not mean that it is not a precursor, because competition between endogenous and exogenous pools was minimized by its very small pool size and rapid metabolism.
Abstract.
Pallanca JE, Smirnoff N (1999). Ascorbic acid metabolism in pea seedlings. A comparison of D-glucosone, L-sorbosone, and L-galactono-1,4-lactone as ascorbate precursors.
Plant Physiol,
120(2), 453-462.
Abstract:
Ascorbic acid metabolism in pea seedlings. A comparison of D-glucosone, L-sorbosone, and L-galactono-1,4-lactone as ascorbate precursors.
L-Ascorbic acid (AsA) accumulates in pea (Pisum sativum L.) seedlings during germination, with the most rapid phase of accumulation coinciding with radicle emergence. Monodehydroascorbate reductase and dehydroascorbic acid reductase were active in the embryonic axes before AsA accumulation started, whereas AsA oxidase and AsA peroxidase activities increased in parallel with AsA. Excised embryonic axes were used to investigate the osone pathway of AsA biosynthesis, in which D-glucosone and L-sorbosone are the proposed intermediates. [U-14C]Glucosone was incorporated into AsA and inhibited the incorporation of [U-14C]glucose (Glc) into AsA. A higher D-glucosone concentration (5 mM) inhibited AsA accumulation. L-Sorbosone did not affect AsA pool size but caused a small inhibition in the incorporation of [U-14C]Glc into AsA. Oxidase and dehydrogenase activities capable of converting Glc or Glc-6-phosphate to glucosone were not detected in embryonic axis extracts. The osones are therefore unlikely to be physiological intermediates of AsA biosynthesis. L-Galactono-1,4-lactone, recently proposed as the AsA precursor (G.L. Wheeler, M.A. Jones, N. Smirnoff [1998] Nature 393: 365-369), was readily converted to AsA by pea embryonic axes. Although L-galactono-1,4-lactone did not inhibit [14C]Glc incorporation into AsA, this does not mean that it is not a precursor, because competition between endogenous and exogenous pools was minimized by its very small pool size and rapid metabolism.
Abstract.
Author URL.
Smirnoff N, Bryant JA (1999). DREB takes the stress out of growing up.
Nat Biotechnol,
17(3), 229-230.
Author URL.
Conklin PL, Norris SR, Wheeler GL, Williams EH, Smirnoff N, Last RL (1999). Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis.
Abstract:
Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis.
Abstract.
Author URL.
Dixon KP, Xu JR, Smirnoff N, Talbot NJ (1999). Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea.
Plant Cell,
11(10), 2045-2058.
Abstract:
Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea.
The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle. To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control of cellular turgor, we analyzed the response of M. grisea to hyperosmotic stress. During acute and chronic hyperosmotic stress adaptation, M. grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol. A mitogen-activated protein kinase-encoding gene OSM1 was isolated from M. grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast. A null mutation of OSM1 was generated in M. grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions. M. grisea deltaosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium. Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Deltaosm1 null mutation, and the mutants were fully pathogenic. This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection. Consistent with this, exposure of M. grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol.
Abstract.
Author URL.
Macnair MR, Smirnoff N (1999). Use of zincon to study uptake and accumulation of zinc by zinc tolerant and hyperaccumulating plants.
Communications in Soil Science and Plant Analysis,
30(7-8), 1127-1136.
Abstract:
Use of zincon to study uptake and accumulation of zinc by zinc tolerant and hyperaccumulating plants
A quick and cheap method to digest plant material using 2% sulphosalicylic acid and to analyze the extract for zinc (Zn) using the colorimetric agent zincon is described. It is shown that the recovery of Zn is excellent, within 90% of that determined by nitric acid digestion and atomic absorption spectrophotometry (AAS). The sensitivity is similar to the AAS. Its usefulness for investigating Zn uptake by the Zn hyperaccumulator Cardaminopsis halleri is investigated. Almost 50% of the variation between samples is due to between-plant variance, with between-leaf variation making up a further 37%. Some of the between-leaf variance is due to age: young leaves contain less Zn than old leaves. In roots, root tips contain about twice as much Zn as root bases.
Abstract.
1998
Marschall M, Proctor MCF, Smirnoff N (1998). Carbohydrate composition and invertase activity of the leafy liverwort Porella platyphylla.
New Phytologist,
138(2), 343-353.
Abstract:
Carbohydrate composition and invertase activity of the leafy liverwort Porella platyphylla
The major soluble carbohydrates in the desiccation-tolerant leafy liverwort Porella platyphylla (L.) Lindb. are sucrose and a homologous series of fructans including the trisaccharide 1-kestose. Exogenous glucose and fructose (10 mol m-3) did not affect the composition of the soluble carbohydrate pool. Sucrose caused an increase in the fructan pool. Sucrose also inhibited photosynthetic oxygen evolution and respiration. The fructan pool was maintained in preference to sucrose during dark starvation. Low temperature and low water potential increased the fructan pool whereas desiccation increased the proportion of high molecular weight fructan. Acid invertase activity was detected in a taxonomically diverse range of liverworts but was very low or undetectable in a range of mosses. The invertase activity from P. platyphylla was partially purified ammonium sulphate precipitation. The reaction products of the partially purified enzyme were equimolar glucose and fructose. Kestose and higher DP fructans were not detected suggesting that, at least under the assay conditions used, the enzyme does not have sucrose:sucrose fructosyl transferase activity. The pH optimum was 4.5-5 and the K(m) for sucrose was 1.7 mol m-3. Pyridoxal hydrochloride (5 mol m-3) caused 50% inhibition. The coexistence of sucrose and invertase suggests that either the invertase is inactive in vivo or is in a different subcellular compartment from sucrose. The pH response shows that it would have very low activity at cytosolic pH. A large acidic vacuole was detected in P. platyphylla leaf cells neutral-red staining in which either invertase or sucrose could be sequestered. Rehydrating desiccated P. platyphylla for 10 min resulted in a 60% loss of extractable invertase activity. By contrast, extractable malate dehydrogenase activity increased during rehydration. Rehydrating desiccated leaves caused an increase in glucose and fructose suggesting that the sucrose pool was susceptible to invertase at this time. It is suggested that the partial inactivation of invertase during rehydration minimizes sucrose hydrolysis while membrane structure and subcellular compartmentation are re-established.
Abstract.
Barnwell P, Blanchard AN, Bryant JA, Smirnoff N, Weir AF (1998). Isolation of DNA from the Highly Mucilagenous Succulent Plant Sedum telephium.
Plant Molecular Biology Reporter,
16(2).
Abstract:
Isolation of DNA from the Highly Mucilagenous Succulent Plant Sedum telephium
The C3-CAM intermediate plant Sedum telephium is very rich in mucilages (gelling polysaccharides) which present a major obstacle to the isolation of DNA. We have developed an extraction procedure that involves a step-wise increase in the concentration of CTAB (cetyl-trimethylammonium bromide) finally yielding a nucleic acid precipitate that is free from polysaccharides. DNA may be further purified by differential precipitation with LiCl and/or RNase digestion. The DNA has excellent spectral qualities, is readily accessible to restriction endonucleases and is usable as a PCR template.
Abstract.
Smirnoff N (1998). Plant resistance to environmental stress.
Curr Opin Biotechnol,
9(2), 214-219.
Abstract:
Plant resistance to environmental stress.
A common effect of many environmental stresses is to cause oxidative damage; consequently, the antioxidant system is being intensively investigated. The use of transgenic plants to probe the role of the antioxidant system continues to be an important approach. The uncharted area of signal transduction in relation to oxidative stress is beginning to attract attention. Studies of drought response at the cellular level have focused on the role of compatible solutes (osmolytes) in acclimation to water stress. Information on signal transduction processes during drought is beginning to appear. As with the antioxidant system, there is increasing use of metabolic engineering in transgenic plants to introduce exotic compatible solutes. It is concluded that these potentially have a use in understanding, or even improving, drought resistance; however, there is a need for the assessment of stress tolerance of transgenics to be carried out at a more sophisticated level and for a critical analysis of the relevance for crop yield of the genes currently being manipulated.
Abstract.
Author URL.
Wheeler GL, Jones MA, Smirnoff N (1998). The biosynthetic pathway of vitamin C in higher plants.
Nature,
393(6683), 365-369.
Abstract:
The biosynthetic pathway of vitamin C in higher plants.
Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.
Abstract.
Author URL.
1997
Csintalan Z, Takacs Z, Tuba Z, Proctor MCF, Smirnoff N, Grace J (1997). Desiccation tolerant grassland cryptogams under elevated CO<inf>2</inf>: Preliminary findings.
Abstracta Botanica,
21(2), 309-315.
Abstract:
Desiccation tolerant grassland cryptogams under elevated CO2: Preliminary findings
The desiccation-tolerant (DT) moss Tortula ruralis and the DT lichen Cladonia convoluta, collected from a dry sandy grassland in Hungary, were grown for 4 months at present (350 μmol mol-1) and elevated (700 μmol mol-1) CO2 concentrations in open-top chambers. Subsequent measurements showed no change in total chlorophyll, soluble sugar, starch or protein-N content in either species; short-term responses of gas exchange and chlorophyll fluorescence to drying out and remoistening were also substantially unaltered. The response of net CO2 uptake to ambient CO2 concentration at saturating irradiance and optimum water content showed either no change (C. convoluta) or (in Tortula ruralis) no change in the initial slope of the A/Ca curve and a small but significant enhancement of uptake at higher CO2 concentrations (i.e. upward acclimation) in material grown at elevated CO2. These results are contrary to most published findings in vascular plants and in the endohydric moss Polytrichum formosum. They suggest that DT bryophytes and lichens may be favoured relative to vascular plants by rising atmospheric CO2 concentration.
Abstract.
De Jong JC, McCormack BJ, Smirnoff N, Talbot NJ (1997). Glycerol generates turgor in rice blast [5]. Nature, 389(6648), 244-245.
Conklin PL, Pallanca JE, Last RL, Smirnoff N (1997). L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1.
Plant Physiol,
115(3), 1277-1285.
Abstract:
L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1.
The biosynthesis of L-ascorbic acid (vitamin C) is not well understood in plants. The ozone-sensitive Arabidopsis thaliana mutant vitamin c-1 (vtc1; formerly known as soz1) is deficient in ascorbic acid, accumulating approximately 30% of wild-type levels. This deficiency could result from elevated catabolism or decreased biosynthesis. No differences that could account for the deficiency were found in the activities of enzymes that catalyze the oxidation or reduction of ascorbic acid. The absolute rate of ascorbic acid turnover is actually less in vtc1 than in wild type; however, the turnover rate relative to the pool of ascorbic acid is not significantly different. The results from [U-14C]Glc labeling experiments suggest that the deficiency is the result of a biosynthetic defect: less L-[14C]ascorbic acid as a percentage of total soluble 14C accumulates in vtc1 than in wild type. The feeding of two putative biosynthetic intermediates, D-glucosone and L-sorbosone, had no positive effect on ascorbic acid levels in either genotype. The vtc1 defect does not appear to be the result of a deficiency in L-galactono-1,4-lactone dehydrogenase, an enzyme able to convert L-galactono-1,4-lactone to ascorbic acid.
Abstract.
Author URL.
Tuba Z, Smirnoff N, Csintalan Z, Szente K, Nagy Z (1997). Respiration during slow desiccation of the poikilochlorophyllous desiccation tolerant plant Xerophyta scabrida at present-day CO<inf>2</inf> concentration.
Plant Physiology and Biochemistry,
35(5), 381-386.
Abstract:
Respiration during slow desiccation of the poikilochlorophyllous desiccation tolerant plant Xerophyta scabrida at present-day CO2 concentration
The time-course of changes in photosynthesis and respiration during long-term desiccation of the poikilohydric desiccation tolerant (PDT) monocot Xerophyta scabrida are reported. Complete leaf desiccation required 16 days. A large reduction occurred in the leaf area/leaf weight ratio that resulted in an extended period of desiccation by reducing the rate of water loss. Net CO2 assimilation declined sharply in the desiccating leaves and ceased altogether after day 3. The reduction in net CO2 assimilation was associated with a decrease in chlorophyll a+b content, a reduction in photochemistry and stomatal closure. The chlorophyll/carotenoid ratio [(a+b)/(x+c)] reflected that the disorganization of the photosynthetic pigment composition and the photosynthetic system began after net CO2 assimilation stopped. The leaves lost 86% of their chlorophyll a+b content and 70% of their carotenoids after 16 days. The reduction in the variable chlorophyll fluorescence decrease ratio Rfd690 values during desiccation indicated a decrease in the activity of the chlorophylls and the thylakoids and the eventual cessation of photochemical activity. In contrast to photosynthesis, respiration was much less affected during the desiccation period and was detectable until near the end of the desiccation period. The respiration rate was linearly related to tissue water content on a fresh weight basis. X. scabrida is able to maintain respiration for a longer period than homoiochloropyllous desiccation tolerant (HDT) plants which maintain their pigment content and chloroplast integrity during desiccation. This response in X. scabrida is suggested to maintain energy supply allowing controlled breakdown of photosynthetic pigments and disorganization of the chloroplasts into desiccoplasts. The respiration which occurs during desiccation is termed desiccation respiration.
Abstract.
1996
Smirnoff N, Pallanca JE (1996). Ascorbate metabolism in relation to oxidative stress.
Author URL.
Smirnoff N (1996). Regulation of crassulacean acid metabolism by water status in the C-3/CAM intermediate Sedum telephium.
Author URL.
Smirnoff N (1996). The function and metabolism of ascorbic acid in plants.
Annals of Botany,
78(6), 661-669.
Abstract:
The function and metabolism of ascorbic acid in plants
Ascorbate is a major metabolite in plants. It is an antioxidant and, in association with other components of the antioxidant system, protects plants against oxidative damage resulting from aerobic metabolism, photosynthesis and a range of pollutants. Recent approaches, using mutants and transgenic plants, are providing evidence for a key role for the ascorbate glutathione cycle in protecting plants against oxidative stress. Ascorbate is also a cofactor for some hydroxylase enzymes (e.g. prolyl hydroxylase) and violaxanthin de-epoxidase. The latter enzyme links ascorbate to the photoprotective xanthophyll cycle. A role in regulating photosynthetic electron transport has been proposed. The biosynthetic pathway of ascorbate in plants has not been identified and evidence for the proposed pathways is reviewed. Ascorbate occurs in the cell wall where it is a first line of defence against ozone. Cell wall ascorbate and cell wall-localized ascorbate oxidase (AO) have been implicated in control of growth. High AO activity is associated with rapidly expanding cells and a model which links wall ascorbate and ascorbate oxidase to cell wall extensibility is presented Ascorbate has also been implicated in regulation of cell division by influencing progression from G1 to S phase of the cell cycle. There is a need to increase our understanding of this enigmatic molecule since it could be involved in a wide range of important functions from antioxidant defence and photosynthesis to growth regulation.
Abstract.
1995
SMIRNOFF N (1995). ANTIOXIDANT SYSTEMS AND PLANT RESPONSE TO THE ENVIRONMENT.
Author URL.
SMIRNOFF N (1995). METABOLIC FLEXIBILITY IN RELATION TO THE ENVIRONMENT.
Author URL.
Webster J, Davey RA, Smirnoff N, Fricke W, Hinde P, Tomos D, Turner JCR (1995). Mannitol and hexoses are components of Buller's drop.
Mycological Research,
99(7), 833-838.
Abstract:
Mannitol and hexoses are components of Buller's drop
The mechanism of ballistospore self-propulsion by basidiomycetes involves the hygroscopic adsorption of water vapour by a drop of liquid (Buller's drop) on the hilar appendix of the spore until it makes contact with another expanding drop, the adaxial drop on the face of the spore above it. Aliquots of liquid from Buller's drop have been collected from Itersonilia perplexans using micropipettes. Analysis of the droplets by microfluorescence assays indicated the presence of mannitol and hexoses. Using the same technique, washings from basidiospore deposits of I. perplexans, containing the evaporated remnants of Buller's drops and the adaxial drops carried away by the spores on discharge, were further shown to contain mannitol and hexoses. Glc analyses of washings from spore deposits of a range of basidiomycetes also indicated the presence of mannitol and hexoses. Calculations based on the estimated concentrations of mannitol and hexoses in Buller's drop from Itersonilia show that the concentration of solutes is sufficient to bring about the condensation of water vapour from a saturated atmosphere and thus to cause the growth of Buller's drop at rates previously measured. When brought close to, but not in contact with, the surface of an agar plate, crystals of pure mannitol rapidly deliquesced. Mannitol and hexoses contribute significantly to the hygroscopic nature of Buller's drop in basidiomycetes. © 1995, British Mycological Society. All rights reserved.
Abstract.
POPP M, SMIRNOFF N (1995). POLYOL ACCUMULATION AND METABOLISM DURING WATER DEFICIT.
Author URL.
SAMARAS Y, BRESSAN RA, CSONKA LN, GARCIARIOS MG, DURZO MP, RHODES D (1995). PROLINE ACCUMULATION DURING DROUGHT AND SALINITY.
Author URL.
OVERY S, SMIRNOFF N (1995). PROLINE DEHYDROGENASE AND PROLINE ACCUMULATION IN PISUM-SATIVUM AT LOW WATER POTENTIAL.
PLANT PHYSIOLOGY,
108(2), 66-66.
Author URL.
NIMMO HG, CARTER PJ, FEWSON CA, NELSON JPS, NIMMO GA, WILKINS MB (1995). REGULATION OF MALATE SYNTHESIS IN CAM PLANTS AND GUARD CELLS - EFFECTS OF LIGHT AND TEMPERATURE ON THE PHOSPHORYLATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE.
Author URL.
1994
ORTHEN B, POPP M, SMIRNOFF N (1994). HYDROXYL RADICAL SCAVENGING PROPERTIES OF CYCLITOLS.
Author URL.
CONTI S, SMIRNOFF N (1994). RAPID TRIGGERING OF MALATE ACCUMULATION IN THE C-3/CAM INTERMEDIATE PLANT SEDUM-TELEPHIUM - RELATIONSHIP WITH WATER STATUS AND PHOSPHOENOLPYRUVATE CARBOXYLASE.
JOURNAL OF EXPERIMENTAL BOTANY,
45(280), 1613-1621.
Author URL.
Conti S, Smirnoff N (1994). Rapid triggering of malate accumulation in the C<inf>3</inf>/CAM intermediate plant Sedum telephium: Relationship with water status and phosphoenolpyruvate carboxylase.
Journal of Experimental Botany,
45(11), 1613-1621.
Abstract:
Rapid triggering of malate accumulation in the C3/CAM intermediate plant Sedum telephium: Relationship with water status and phosphoenolpyruvate carboxylase
Sedum telephium is a C3/CAM intermediate plant in which expression of CAM is caused by water deficit. The timing of the C3-CAM switch and its relationship with water status and phosphoenolpyruvate (PEP) carboxylase activity have been investigated. Water deficit was provided by application of polyethylene glycol (PEG) solutions so that roots were exposed to water potentials from 0 to - 2.0 MPa below that of the nutrient solution. The response of the plants was measured during the first dark period after PEG addition and 7 d later. Malic acid accumulation was triggered during the first dark period at root water potentials of -0.3 MPa or less. This corresponded with very small decreases in leaf water potential and relative water content. The capacity of PEP carboxylase was not altered at any water potential during the first dark period. After 7 d the capacity of PEP carboxylase progressively increased as water potential declined to -0.4 MPa. At this, and more negative, water potentials it was 5-fold higher than in well-watered leaves. Malic acid fluctuations increased with decreasing PEG water potential below a threshold of -0.1 MPa. Malic acid levels at the end of the light period were progressively lower as water potential decreased. NAD- and NADP-malic enzyme activity were not affected by low water potential.Leaves detached from well-watered plants in the middle of the light period and kept hydrated did not accumulate malic acid during the following dark period. Allowing the leaves to lose 10% of their water content induced malic acid accumulation during the same time. Conversely, leaves detached from long-term droughted plants (which had malate fluctuations and a PEP carboxylase capacity 5-fold higher than well-watered plants) accumulated malate during the night if maintained at the same low hydration state (82%RWC), whereas malic acid accumulation was promptly reduced if they were rehydrated. Malic acid accumulation could therefore be rapidly altered by changing the hydration state of the leaves. The short-term rehydration treatments did not alter PEP carboxylase capacity. However, alteration of leaf hydration affected the apparent Km (PEP) of PEP carboxylase extracted 1 h before the end of the dark period. The Km was increased by rehydration and decreased by dehydration. Sensitivity to feedback inhibition by malate was not affected by hydration state and was high for PEP carboxylase from well-watered leaves and lower for PEP carboxylase from long-term droughted leaves.Taken together, the responses of intact plants and detached leaves show that malic acid accumulation can be triggered very rapidly by small water deficits in the leaves. The extent of night-time malic acid accumulation is independent of PEP carboxylase capacity. However, a change in the hydration state of the leaves can rapidly alter the affinity of PEP carboxylase for PEP. The regulation of malic acid accumulation in relation to the drought-induced triggering of CAM is discussed. © 1994 Oxford University Press.
Abstract.
1993
Spickett CM, Smirnoff N, Ratcliffe RG (1993). An in Vivo Nuclear Magnetic Resonance Investigation of Ion Transport in Maize (Zea mays) and Spartina anglica Roots during Exposure to High Salt Concentrations.
Plant Physiol,
102(2), 629-638.
Abstract:
An in Vivo Nuclear Magnetic Resonance Investigation of Ion Transport in Maize (Zea mays) and Spartina anglica Roots during Exposure to High Salt Concentrations.
The response of maize (Zea mays L.) and Spartina anglica root tips to exposure to sodium chloride concentrations in the range 0 to 500 mM was investigated using 23Na and 31P nuclear magnetic resonance spectroscopy (NMR). Changes in the chemical shift of the pH-dependent 31P-NMR signals from the cytoplasmic and vacuolar orthophosphate pools were correlated with the uptake of sodium, and after allowing for a number of complicating factors we concluded that these chemical shift changes indicated the occurrence of a small cytoplasmic alkalinization (0.1-0.2 pH units) and a larger vacuolar alkalinization (0.6 pH units) in maize root tips exposed to salt concentrations greater than 200 mM. The data were interpreted in terms of the ion transport processes that may be important during salt stress, and we concluded that the vacuolar alkalinization provided evidence for the operation of a tonoplast Na+/H+-antiport with an activity that exceeded the activity of the tonoplast H+ pumps. The intracellular pH values stabilized during prolonged treatment with high salt concentrations, and this observation was linked to the recent demonstration (Y. Nakamura, K. Kasamo, N. Shimosato, M. Sakata, E. Ohta [1992] Plant Cell Physiol 33: 139-149) of the salt-induced activation of the tonoplast H+- ATPase. Sodium vanadate, an inhibitor of the plasmalemma H+- ATPase, stimulated the net uptake of sodium by maize root tips, and this was interpreted in terms of a reduction in active sodium efflux from the tissue. S. anglica root tips accumulated sodium more slowly than did maize, with no change in cytoplasmic pH and a relatively small change (0.3 pH units) in vacuolar pH, and it appears that salt tolerance in Spartina is based in part on its ability to prevent the net influx of sodium chloride.
Abstract.
Author URL.
Conti S, Smirnoff N (1993). Perception of soil water deficit and cam induction in sedu telephium:The role of roots and growth regulators. Giornale Botanico Italiano, 127(5), 943-944.
SMIRNOFF N (1993). TANSLEY REVIEW. 52. THE ROLE OF ACTIVE OXYGEN IN THE RESPONSE OF PLANTS TO WATER-DEFICIT AND DESICCATION.
NEW PHYTOLOGIST,
125(1), 27-58.
Author URL.
1992
Spickett CM, Smirnoff N, Ratcliffe RG (1992). Metabolic Response of Maize Roots to Hyperosmotic Shock : an in VivoP Nuclear Magnetic Resonance Study.
Plant Physiol,
99(3), 856-863.
Abstract:
Metabolic Response of Maize Roots to Hyperosmotic Shock : an in VivoP Nuclear Magnetic Resonance Study.
(31)P nuclear magnetic resonance spectroscopy was used to study the response of maize (Zea mays L.) root tips to hyperosmotic shock. The aim was to identify changes in metabolism that might be relevant to the perception of low soil water potential and the subsequent adaptation of the tissue to these conditions. Osmotic shock was found to result in two different types of response: changes in metabolite levels and changes in intracellular pH. The most notable metabolic changes, which were produced by all the osmotica tested, were increases in phosphocholine and vacuolar phosphate, with a transient increase in cytoplasmic phosphate. It was observed that treatment with ionic and nonionic osmotica produced different effects on the concentrations of bioenergetically important metabolites. It is postulated that these changes are the result of hydrolysis of phosphatidylcholine and other membrane phospholipids, due to differential activation of specific membrane-associated phospholipases by changes in the surface tension of the plasmalemma. These events may be important in the detection of osmotic shock and subsequent acclimatization. A cytoplasmic alkalinization was also observed during hyperosmotic treatment, and this response, which is consistent with the activation of the plasmalemma H(+)-ATPase, together with the other metabolic changes, may suggest the existence of a complex and integrated mechanism of osmoregulation.
Abstract.
Author URL.
Stewart GR, Joly CA, Smirnoff N (1992). Partitioning of inorganic nitrogen assimilation between the roots and shoots of cerrado and forest trees of contrasting plant communities of South East Brasil.
Oecologia,
91(4), 511-517.
Abstract:
Partitioning of inorganic nitrogen assimilation between the roots and shoots of cerrado and forest trees of contrasting plant communities of South East Brasil
Woody plants growing in cerrado and forest communities of south-east Brasil were found to have low levels of nitrate reductase activity in their leaves suggesting that nitrate ions are not an important nitrogen source in these communities. Only in the leaves of species growing in areas of disturbance, such as gaps and forest margins, were high levels of nitrate reductase present. When pot-grown plants were supplied with nitrate, leaves and roots of almost all species responded by inducing increased levels of nitrate reductase. Pioneer or colonizing species exhibited highest levels of nitrate reductase and high shoot: root nitrate reductase activities. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase were present in leaves and roots of the species examined.15N-labelled nitrate and ammonium were used to compare the assimilatory characteristics of two species:Enterolobium contortisiliquum, with a high capacity to reduce nitrate, and Calophyllum brasiliense, of low capacity. The rate of nitrate assimilation in the former was five times that of the latter. Both species had similar rates of ammonium assimilation. Results for eight species of contrasting habitats showed that leaf nitrogen content increased in parallel with xylem sap nitrogen concentrations, suggesting that the ability of the root system to acquire, assimilate or export nitrate determines shoot nitrogen status. These results emphasise the importance of nitrogen transport and metabolism in roots as determinants of whole plant nitrogen status. © 1992 Springer-Verlag.
Abstract.
Smirnoff N (1992). The carbohydrates of bryophytes in relation to desiccation tolerance.
Abstract:
The carbohydrates of bryophytes in relation to desiccation tolerance
Abstract.
1990
SMIRNOFF N, RATCLIFFE RG (1990). EARLY RESPONSES OF PEA ROOTS TO WATER DEFICIT STUDIED WITH P-31-NMR.
Author URL.
GROENHOF AC, SMIRNOFF N, BRYANT JA (1990). REGULATION OF THE C3 TO CAM SWITCH IN SEDUM-TELEPHIUM BY WATER-STRESS.
Author URL.
GROENHOF AC, SMIRNOFF N, BRYANT JA (1990). THE APPEARANCE OF a NEW MOLECULAR-SPECIES OF PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC) AND THE RAPID INDUCTION OF CAM IN SEDUM-TELEPHIUM L.
PLANT CELL AND ENVIRONMENT,
13(5), 437-445.
Author URL.
SLEE NJD, BRYANT JA, SMIRNOFF N, SMITH BG (1990). THE EFFECT OF WATER DEFICIT AND ABA ON PROTEIN-SYNTHESIS IN SUNFLOWERS (HELIANTHUS-ANNUUS).
Author URL.
1989
Smirnoff N, Cumbes QJ (1989). Hydroxyl radical scavenging activity of compatible solutes.
Phytochemistry,
28(4), 1057-1060.
Abstract:
Hydroxyl radical scavenging activity of compatible solutes
Compatible solutes were assessed for their hydroxyl radical scavenging activity by their ability to compete in two different hydroxyl radical generating and detecting systems. Hydroxyl radicals were generated by ascorbate-hydrogen peroxide or by xanthine oxidase-hypoxanthine-hydrogen peroxide. They were detected by hydroxylation of salicylate or by denaturation of malate dehydrogenase. of the compatible solutes tested, sorbitol, mannitol, myo-inositol and proline were effective hydroxyl radical scavengers. Glycinebetaine was ineffective. The role of compatible solutes as hydroxyl radical scavengers in vivo is discussed. © 1989.
Abstract.
HUGHES SG, BRYANT JA, SMIRNOFF N (1989). MOLECULAR-BIOLOGY - APPLICATION TO STUDIES OF STRESS TOLERANCE.
Author URL.
1988
SMIRNOFF N, COLOMBE SV (1988). DROUGHT INFLUENCES THE ACTIVITY OF ENZYMES OF THE CHLOROPLAST HYDROGEN-PEROXIDE SCAVENGING SYSTEM.
JOURNAL OF EXPERIMENTAL BOTANY,
39(205), 1097-1108.
Author URL.
Smirnoff N, Colombé SV (1988). Drought influences the activity of enzymes of the chloroplast hydrogen peroxide scavenging system.
Journal of Experimental Botany,
39(8), 1097-1108.
Abstract:
Drought influences the activity of enzymes of the chloroplast hydrogen peroxide scavenging system
Smirnoff, N. and Colombé, S. V. 1988. Drought influences the activity of enzymes of the chloroplast hydrogen peroxide scavenging system.-J. exp. Bot. 39: 1097-1108.The effect of drought on the activity of ascorbate peroxidase (AP), glutathione reductase (GR) and monodehydroascorbate reductase (MDAR) in leaves of barley (Hordeum vulgare) and tef {Eragrostis tef) was studied. These enzymes are components of the chloroplast hydrogen peroxide scavenging system. Severe leaf water deficit (
Abstract.
GROENHOF AC, SMIRNOFF N, BRYANT JA (1988). ENZYMIC ACTIVITIES ASSOCIATED WITH THE ABILITY OF AERIAL AND SUBMERGED FORMS OF LITTORELLA-UNIFLORA (L) ASCHERS TO PERFORM CAM.
JOURNAL OF EXPERIMENTAL BOTANY,
39(200), 353-361.
Author URL.
1987
SMIRNOFF N, STEWART GR (1987). GLUTAMINE SYNTHETASE AND AMMONIUM ASSIMILATION IN ROOTS OF ZINC‐TOLERANT AND NON‐TOLERANT CLONES OF DESCHAMPSIA CESPITOSA (L.) BEAUV. AND ANTHOXANTHUM ODORATUM L.
New Phytologist,
107(4), 659-670.
Abstract:
GLUTAMINE SYNTHETASE AND AMMONIUM ASSIMILATION IN ROOTS OF ZINC‐TOLERANT AND NON‐TOLERANT CLONES OF DESCHAMPSIA CESPITOSA (L.) BEAUV. AND ANTHOXANTHUM ODORATUM L.
The influence of zinc on glutamine synthetase (GS) activity and ammonium assimilation in roots of zinc‐tolerant and non‐tolerant clones of Deschampsia cespitosa (L.) Beauv. and Anthoxanthum odoratum L. was studied. One isoform of GS was resolved by ion exchange chromatography in D. cespitosa roots. However, two isoforms were found in A. odoratum roots; one of these was attributed to fungal contamination and was not studied further. There was no difference in the sensitivity of partially purified GS from zinc‐tolerant and non‐tolerant clones to inhibition by zinc. Inhibition of GS by Zn was reversible and the kinetics of inhibition were studied. Methionine sulphoximine (an inhibitor of GS) induced ammonium accumulation in the roots of control and zinc‐grown roots of Zn‐tolerant D. cespitosa. Zinc inhibited incorporation of [15N]ammonium into free amino acids (determined by combined gas chromatography–mass spectrometry) in both clones of D. cespitosa, but higher concentrations were required to achieve inhibition in the tolerant clone. The labelling pattern was unaffected by zinc in both clones. The results suggest that zinc has no effect on the pathway of ammonium assimilation in zinc‐grown roots and that assimilation is via the glutamine synthetase–glutamate synthase pathway. There was no evidence to support the hypothesis that glutamate dehydrogenase functions preferentially in ammonium assimilation under conditions of environmental stress. Copyright © 1987, Wiley Blackwell. All rights reserved
Abstract.
SMIRNOFF N, STEWART GR (1987). NITROGEN ASSIMILATION AND ZINC TOXICITY TO ZINC‐TOLERANT AND NON‐TOLERANT CLONES OF DESCHAMPSIA CESPITOSA (L.) BEAUV.
New Phytologist,
107(4), 671-680.
Abstract:
NITROGEN ASSIMILATION AND ZINC TOXICITY TO ZINC‐TOLERANT AND NON‐TOLERANT CLONES OF DESCHAMPSIA CESPITOSA (L.) BEAUV.
The effect of nitrate and ammonium nutrition on the toxicity of zinc to zinc‐tolerant and non‐tolerant clones of Deschampsia cespitosa (L.) Beauv, was investigated. Zinc and nitrogen source interacted in their effect on root growth and zinc tended to be less toxic if ammonium was the nitrogen source. Root growth of the non‐tolerant clone was inhibited by ammonium. Zinc caused chlorosis in nitrate‐grown tolerant plants but not in ammonium‐grown plants. Zinc caused a decrease in the concentrations of reduced nitrogen compounds in leaves and decreased nitrate translocation to them. The zinc concentration in roots was slightly decreased in ammonium‐grown plants. In vivo nitrate reductase activity was stimulated by high zinc concentrations in the tolerant clone. Asparagine and proline accumulated in response to zinc treatment in the non‐tolerant clone. Ammonium nutrition also induced asparagine accumulation and such accumulation was much greater in the non‐tolerant clone. The small decrease in zinc toxicity when ammonium is the nitrogen source could be only partly accounted for by lower zinc uptake. It is suggested that asparagine which accumulates in ammonium‐grown plants, forms an intracellular complex with zinc and thereby decreases its toxicity. Copyright © 1987, Wiley Blackwell. All rights reserved
Abstract.
SHAH N, SMIRNOFF N, STEWART GR (1987). PHOTOSYNTHESIS AND STOMATAL CHARACTERISTICS OF STRIGA-HERMONTHICA IN RELATION TO ITS PARASITIC HABIT.
PHYSIOLOGIA PLANTARUM,
69(4), 699-703.
Author URL.
1986
INGROUILLE MJ, SMIRNOFF N (1986). THLASPI CAERULESCENS J. & C. PRESL. (T. ALPESTRE L.) IN BRITAIN.
New Phytologist,
102(1), 219-233.
Abstract:
THLASPI CAERULESCENS J. & C. PRESL. (T. ALPESTRE L.) IN BRITAIN
Thlaspi caerulescens J. & C. Presl. (T. alpestre L.) has a restricted and disjunct distribution in Britain. Both on natural and mine sites it is confined to soils contaminated by lead and zinc. Morphological variation occurs both regionally and locally but there is no support from a taxometric cluster analysis for the taxonomic recognition of variants. In experiment the index of zinc tolerance of plants raised from seed was positively correlated with the concentration of zinc in the soils from which the seed parents originated. The distribution of T. caerulescens is discussed in relation to morphological and physiological variation between populations. Copyright © 1986, Wiley Blackwell. All rights reserved
Abstract.
1985
SMIRNOFF N, STEWART GR (1985). NITRATE ASSIMILATION AND TRANSLOCATION BY HIGHER-PLANTS - COMPARATIVE PHYSIOLOGY AND ECOLOGICAL CONSEQUENCES.
PHYSIOLOGIA PLANTARUM,
64(2), 133-140.
Author URL.
SMIRNOFF N, WINSLOW MD, STEWART GR (1985). NITRATE REDUCTASE-ACTIVITY IN LEAVES OF BARLEY (HORDEUM-VULGARE) AND DURUM-WHEAT (TRITICUM-DURUM) DURING FIELD AND RAPIDLY APPLIED WATER DEFICITS.
JOURNAL OF EXPERIMENTAL BOTANY,
36(169), 1200-1208.
Author URL.
Smirnoff N, Winslow MD, Stewart GR (1985). Nitrate reductase acivity in leaves of barley (Hordeum vulgare) and durum wheat (Triticum durum) during field and rapidly applied water deficits.
Journal of Experimental Botany,
36(8), 1200-1208.
Abstract:
Nitrate reductase acivity in leaves of barley (Hordeum vulgare) and durum wheat (Triticum durum) during field and rapidly applied water deficits
Smirnoff, N. Winslow, M. D. and Stewart, G. R. 1985. Nitrate reductase activity in leaves of barley (Hordeum vulgare) and durum wheat (Triticum durum) during field and rapidly applied water deficits.-J. exp. Bot 36: 1200-1208.The effect of field and rapidly applied water deficits on nitrate reductase activity in the leaves of two barley varieties and one durum wheat variety was investigated. In field experiments plants were subjected to irrigation at different rates in three Mediterranean environments by means of a line source sprinkler irrigation system. The environments differed in rainfall and nitrogen fertility. Plant water potentials decreased from -1.5 MPa to between -2.5 and -3.0 MPa as the irrigation rate decreased. Nitrate reductase activity in the leaves of these plants during heading was either unaffected or sometimes increased where the least water was supplied. Nitrate reductase activity was highest in the plants growing with an ample nitrogen supply irrespective of water regime. In contrast, seedlings subject to rapidly applied water stress over 6 d lost 30-85% of their nitrate reductase activity when leaf water potential fell from between -0.33 and -0.75 MPa to between -O.93 and -2.04 MPa. The decrease was less in the young leaves than in the old leaves. Polyethylene glycol induced osmotic stress resulted in a drop in leaf water potential from -0.20 MPa to between -1.05 and -1.20 MPa along with a loss of 40-85% of leaf nitrate reductase activity after 48 h.It is suggested that maintenance of nitrate reductase activity in field grown barley and durum wheat plants reflects an acclimation to water deficit Maintenance of nitrate assimilation during water stress may allow continued synthesis of nitrogenous compatible solutes using the excess photochemical energy available during stomatal closure. © 1985 Oxford University Press.
Abstract.
Smirnoff N, Stewart GR (1985). Stress metabolites and their role in coastal plants.
Vegetatio,
62(1-3), 273-278.
Abstract:
Stress metabolites and their role in coastal plants
The stress metabolites proline, glycine betaine and sorbitol were accumulated in the leaves of some angiosperms from sand dunes and shingle. Chloride, where it was measured, was not accumulated to high concentrations in leaves suggesting that these soils are not saline. Sand dunes and shingle soils have low water-holding capacity, so it is possible that solute accumulation was a response to drought which could be of adaptive significance. In sand dunes low water availability could be associated with increased leaf temperatures because of reduced transpiration rates and high soil temperatures. The role of stress metabolites in heat tolerance was considered. Proline, betaine, sorbitol and mannitol increased the heat stability of glutamine synthetase (GS) and glutamate: oxaloacetate aminotransferase from Ammophila arenaria. For GS the effect increased with solute concentration. The polyols were more effective at high temperatures. The heat stability of GS from the moss Tortula ruraliformis and the brown alga Fucus vesiculosus was increased by mannitol. The effect of the solutes was independent of plant species and type of enzyme. It is suggested that the accumulation of solutes may have ecological importance in protecting sand-dune plants from heat damage during periods of drought. © 1985 Dr W. Junk Publishers.
Abstract.
1984
Smirnoff N, Todd P, Stewart GR (1984). The occurrence of nitrate reduction in the leaves of woody plants.
Annals of Botany,
54(3), 363-374.
Abstract:
The occurrence of nitrate reduction in the leaves of woody plants
Nitrate reductase activities greater than 0·2 μmol h-1 g-1 f. wt, measured by an in vivo assay, occurred in 41 per cent of a large sample (555 species) of woody plants. If several taxonomic groups (Gymnosperms, Ericaceae and Proteaceae) with consistently low activities were discounted activities greater than 0·2 μmol h-1 g-1 f. wt occurred in 73 per cent of the species. This compares with 93 per cent in herbaceous species, suggesting that leaf nitrate reduction is of common occurrence in woody plants. In a small sample of species leaf nitrate reductase activity correlated with nitrate concentration in the xylem sap. Low activities occurred consistently in the Gymnosperms, Ericaceae and Proteaceae. Feeding cut shoots of representatives of these groups with nitrate caused induction of leaf nitrate reductase activity in the Gymnosperms and Proteaceae, but only limited induction in the Ericaceae. The Ericaceae, with the exception of two species, had low activities and low nitrate reductase inducibility. Root assimilation may predominate in the Gymnosperms and Proteaceae. It is suggested that nitrate reduction generally occurs in the leaves of trees from a variety of plant communities and that this may be related to the lower energy cost of leaf, as opposed to root, nitrate assimilation. © 1984 Annals of Botany Company.
Abstract.
1983
Smirnoff N, Stewart GR, Todd P (1983). THE OCCURRENCE OF NITRATE REDUCTION IN THE LEAVES OF WOODY PLANTS.
Author URL.
Smirnoff N, Crawford RMM (1983). Variation in the structure and response to flooding of root aerenchyma in some wetland plants.
Annals of Botany,
51(2), 237-249.
Abstract:
Variation in the structure and response to flooding of root aerenchyma in some wetland plants
The structure and response to flooding of root cortical aerenchyma (air space tissue) in a variety of wetland (flood-tolerant) species was investigated and compared with some flood-intolerant species. In some species aerenchyma consisted of enlarged schizogenous intercellular spaces and in others aerenchyma formation involved lysigeny. Two types of lysigenous aerenchyma were distinguished. In the first the diaphragms between lacunae were arranged radially and consisted of both collapsed and intact cells. In the second type, which was confined to the Cyperaceae, the radial diaphragms contained intact cells, and stretched between them were tangentially-arranged diaphragms of collapsed cells.Flooding in sand culture generally increased root porosity (air space content) although there were exceptions. The flood-intolerant species Senecio jacobaea produced aerenchyma but did not survive long-term flooding. Among the flood-tolerant species, Filipendula ulmaria did not produce extensive aerenchyma even when flooded. Eriophorum angustifolium and E. vaginatum produced extensive aerenchyma under drained conditions which was not increased by flooding. In Nardus stricta root porosity was increased by low nutrient levels as well as by flooding. © 1983 Annals of Botany Company.
Abstract.
1978
BROWN DH, SMIRNOFF N (1978). OBSERVATIONS ON EFFECT OF OZONE ON CLADONIA-RANGIFORMIS.
LICHENOLOGIST,
10, 91-94.
Author URL.
