ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant. DOI: http://dx.doi.org/10.7554/eLife.26770.001

[1]  G. Mader,et al.  Rapid fractionation of wheat leaf protoplasts using membrane filtration : the determination of metabolite levels in the chloroplasts, cytosol, and mitochondria. , 1982, Plant physiology.

[2]  T. Dick,et al.  Dissecting Redox Biology Using Fluorescent Protein Sensors. , 2016, Antioxidants & redox signaling.

[3]  Takeharu Nagai,et al.  Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators , 2009, Proceedings of the National Academy of Sciences.

[4]  L. Xiong,et al.  Analyses of Ca2+ dynamics using a ubiquitin-10 promoter-driven Yellow Cameleon 3.6 indicator reveal reliable transgene expression and differences in cytoplasmic Ca2+ responses in Arabidopsis and rice (Oryza sativa) roots. , 2015, The New phytologist.

[5]  P. Geigenberger,et al.  Regulation of Respiration and Fermentation to Control the Plant Internal Oxygen Concentration1[OA] , 2008, Plant Physiology.

[6]  S. Persson,et al.  V-ATPase activity in the TGN/EE is required for exocytosis and recycling in Arabidopsis , 2015, Nature Plants.

[7]  M. Fricker.  Quantitative Redox Imaging Software. , 2016, Antioxidants & redox signaling.

[8]  M. Schwarzländer,et al.  Chloroplast-Specific in Vivo Ca2+ Imaging Using Yellow Cameleon Fluorescent Protein Sensors Reveals Organelle-Autonomous Ca2+ Signatures in the Stroma1 , 2016, Plant Physiology.

[9]  S. Adams,et al.  FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis , 2014, eLife.

[10]  Hiromi Imamura,et al.  Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA , 2012, PLoS pathogens.

[11]  P. Gardeström,et al.  Mitochondrial Contribution to Photosynthetic Metabolism (A Study with Barley (Hordeum vulgare L.) Leaf Protoplasts at Different Light Intensities and CO2 Concentrations) , 1993, Plant physiology.

[12]  D A Day,et al.  The impact of oxidative stress on Arabidopsis mitochondria. , 2002, The Plant journal : for cell and molecular biology.

[13]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[14]  M. Steup,et al.  Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L. , 1997, The Plant journal : for cell and molecular biology.

[15]  R. Ratcliffe.  IN VIVO NMR STUDIES OF THE METABOLIC RESPONSE OF PLANT TISSUES TO ANOXIA , 1997 .

[16]  H. Noji,et al.  Changes in cytosolic ATP levels and intracellular morphology during bacteria-induced hypersensitive cell death as revealed by real-time fluorescence microscopy imaging. , 2012, Plant & cell physiology.

[17]  Takeharu Nagai,et al.  Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators. , 2010, ACS chemical biology.

[18]  M. Fricker,et al.  The EF-Hand Ca 2 + Binding Protein MICU Choreographs Mitochondrial Ca 2 + Dynamics in Arabidopsis , 2015 .

[19]  J. Kudla,et al.  FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics. , 2012, The Plant journal : for cell and molecular biology.

[20]  L. Vidali,et al.  Polarized cell growth in higher plants. , 2001, Annual review of cell and developmental biology.

[21]  Klaus Palme,et al.  A quantitative ratiometric sensor for time-resolved analysis of auxin dynamics , 2013, Scientific Reports.

[22]  Dirk Inzé,et al.  GATEWAY vectors for Agrobacterium-mediated plant transformation. , 2002, Trends in plant science.

[23]  C. Pikaard,et al.  Gateway-compatible vectors for plant functional genomics and proteomics. , 2006, The Plant journal : for cell and molecular biology.

[24]  H. Heldt,et al.  On the Role of Mitochondrial Oxidative Phosphorylation in Photosynthesis Metabolism as Studied by the Effect of Oligomycin on Photosynthesis in Protoplasts and Leaves of Barley (Hordeum vulgare). , 1991, Plant physiology.

[25]  L. Sweetlove,et al.  Isolation of intact, functional mitochondria from the model plant Arabidopsis thaliana. , 2007, Methods in molecular biology.

[26]  A. Vianello,et al.  Involvement of apyrase in the regulation of the adenylate pool by adenylate kinase in plant mitochondria , 2001 .

[27]  Jim Berg,et al.  A genetically encoded fluorescent reporter of ATP/ADP ratio , 2008, Nature Methods.

[28]  K. Busch,et al.  Characterization of adenylate kinase in intact mitochondria of fertile and male sterile potato (Solanum tuberosum L.) , 1996 .

[29]  T. Pozzan,et al.  Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²⁺ handling relationship in plant cells. , 2012, The Plant journal : for cell and molecular biology.

[30]  G. Stacey,et al.  Identification of a Plant Receptor for Extracellular ATP , 2014, Science.

[31]  F. Rébeillé,et al.  Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration , 2014, Proceedings of the National Academy of Sciences.

[32]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[33]  G. Grossmann,et al.  The biosensor toolbox for plant developmental biology. , 2016, Current opinion in plant biology.

[34]  David A. Orlando,et al.  Protonophore- and pH-insensitive glucose and sucrose accumulation detected by FRET nanosensors in Arabidopsis root tips. , 2008, The Plant journal : for cell and molecular biology.

[35]  M. Fricker,et al.  Confocal imaging of glutathione redox potential in living plant cells , 2008, Journal of microscopy.

[36]  Masasuke Yoshida,et al.  Structures of the thermophilic F1-ATPase ε subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1 , 2007, Proceedings of the National Academy of Sciences.

[37]  D. D. Perrin,et al.  The stability constants of MgATP -2 ion. , 1961, Biochimica et biophysica acta.

[38]  T. Dick,et al.  Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes. , 2016, Nature chemical biology.

[39]  C. Foyer,et al.  The intracellular distribution of adenylate kinase in the leaves of spinach, wheat and barley , 1982, Planta.

[40]  M. Ravier,et al.  The Mitochondrial Ca2+ Uniporter MCU Is Essential for Glucose-Induced ATP Increases in Pancreatic β-Cells , 2012, PloS one.

[41]  Jean-Pierre Jacquot,et al.  Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer. , 2007, The Plant journal : for cell and molecular biology.

[42]  J. Xia,et al.  Lactic Acid efflux as a mechanism of hypoxic acclimation of maize root tips to anoxia. , 1992, Plant physiology.

[43]  P. Raymond,et al.  Adenine Nucleotide Ratios and Adenylate Energy Charge in Energy Metabolism , 1983 .

[44]  S. Gilroy,et al.  Imaging of the Yellow Cameleon 3.6 Indicator Reveals That Elevations in Cytosolic Ca2+ Follow Oscillating Increases in Growth in Root Hairs of Arabidopsis1[W][OA] , 2008, Plant Physiology.

[45]  Simon Gilroy,et al.  Salt stress-induced Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in plants , 2014, Proceedings of the National Academy of Sciences.

[46]  Lichuan Yang,et al.  Measurements of ATP in mammalian cells. , 2002, Methods.

[47]  M. Fricker,et al.  Pulsing of Membrane Potential in Individual Mitochondria: A Stress-Induced Mechanism to Regulate Respiratory Bioenergetics in Arabidopsis[W] , 2012, Plant Cell.

[48]  Joseph P. McKenna,et al.  Phase Analysis of Metabolic Oscillations and Membrane Potential in Pancreatic Islet β-Cells. , 2016, Biophysical journal.

[49]  U. Flügge.  Metabolite transporters in plastids. , 1998, Current opinion in plant biology.

[50]  M. Stitt,et al.  Adenine nucleotide levels in the cytosol, chloroplasts, and mitochondria of wheat leaf protoplasts. , 1982, Plant physiology.

[51]  R. Bligny,et al.  Cooperation and Competition between Adenylate Kinase, Nucleoside Diphosphokinase, Electron Transport, and ATP Synthase in Plant Mitochondria Studied by 31P-Nuclear Magnetic Resonance , 1997, Plant physiology.

[52]  Production of cysteine for bacterial and plant biotechnology: Application of cysteine feedback-insensitive isoforms of serine acetyltransferase , 2003, Amino Acids.

[53]  Alexander M. Jones,et al.  Abscisic acid dynamics in roots detected with genetically encoded FRET sensors , 2014, eLife.

[54]  I. Lorenzen,et al.  A novel fluorescent pH probe for expression in plants , 2006, Plant Methods.

[55]  D. Logan,et al.  Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. , 2000, Journal of experimental botany.

[56]  G. Yellen,et al.  Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio , 2013, Nature Communications.

[57]  Alisdair R. Fernie,et al.  Transcript and metabolite profiling of the adaptive response to mild decreases in oxygen concentration in the roots of arabidopsis plants , 2008, Annals of botany.

[58]  D. Dickinson.  Germination of Lily Pollen: Respiration and Tube Growth , 1965, Science.

[59]  P. Renault,et al.  Mapping meristem respiration of Prunus persica (L.) Batsch seedlings: potential respiration of the meristems, O2 diffusional constraints and combined effects on root growth. , 2000, Journal of experimental botany.

[60]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[61]  J. Schroeder,et al.  Live Cell Imaging with R-GECO1 Sheds Light on flg22- and Chitin-Induced Transient [Ca(2+)]cyt Patterns in Arabidopsis. , 2015, Molecular plant.

[62]  M. Stitt,et al.  Increased levels of adenine nucleotides modify the interaction between starch synthesis and respiration when adenine is supplied to discs from growing potato tubers , 2001, Planta.

[63]  A. Fernie,et al.  Adenine nucleotide transport in plants: much more than a mitochondrial issue. , 2011, Trends in plant science.

[64]  D. Day,et al.  Enzyme Distribution in Potato Mitochondria , 1979 .

[65]  A. Igamberdiev,et al.  The origin of cytosolic ATP in photosynthetic cells. , 2016, Physiologia plantarum.

[66]  K. Jiang,et al.  A fluorometer-based method for monitoring oxidation of redox-sensitive GFP (roGFP) during development and extended dark stress. , 2010, Physiologia plantarum.

[67]  A. Mustroph,et al.  Organ-specific analysis of the anaerobic primary metabolism in rice and wheat seedlings. I: Dark ethanol production is dominated by the shoots , 2006, Planta.

[68]  W. Frommer,et al.  Mitochondrial biosensors. , 2014, The international journal of biochemistry & cell biology.

[69]  A. Igamberdiev,et al.  The role of photorespiration in redox and energy balance of photosynthetic plant cells: A study with a barley mutant deficient in glycine decarboxylase. , 2001, Physiologia plantarum.

[70]  E. Russinova,et al.  Plant Chemical Genetics: From Phenotype-Based Screens to Synthetic Biology[OPEN] , 2017, Plant Physiology.

[71]  R. Tsien,et al.  Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. , 1999, The Plant journal : for cell and molecular biology.

[72]  G. Stacey,et al.  Extracellular ATP in Plants. Visualization, Localization, and Analysis of Physiological Significance in Growth and Signaling1[W] , 2006, Plant Physiology.

[73]  Y. Bazel,et al.  Methods for the determination of adenosine triphosphate and other adenine nucleotides , 2009 .

[74]  T. Dick,et al.  Multiple glutathione disulfide removal pathways mediate cytosolic redox homeostasis. , 2013, Nature chemical biology.

[75]  A. Igamberdiev,et al.  Membrane potential, adenylate levels and Mg2+ are interconnected via adenylate kinase equilibrium in plant cells. , 2003, Biochimica et biophysica acta.

[76]  J. Carde,et al.  Oxidative phosphorylation by mitochondria extracted from dry sunflower seeds. , 1991, Plant physiology.

[77]  P. Falkowski,et al.  Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms , 2015, Nature.

[78]  Zhen-Ming Pei,et al.  Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells , 2000, Nature.

[79]  Masasuke Yoshida,et al.  Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1. , 2007, Proceedings of the National Academy of Sciences of the United States of America.

[80]  P. Rich.  Chemiosmotic coupling: The cost of living , 2003, Nature.

[81]  A. Cornish-Bowden,et al.  Concentration of MgATP2- and other ions in solution. Calculation of the true concentrations of species present in mixtures of associating ions. , 1976, The Biochemical journal.

[82]  A. Kakizuka,et al.  BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells , 2016, Scientific Reports.

[83]  D. D. Perrin,et al.  The stability constant of MgATP2 , 1961 .

[84]  Alan M. Jones,et al.  Extracellular ATP signaling in plants. , 2010, Trends in cell biology.

[85]  Y. Tanami.  Studies on lysogenesis , 1956 .

[86]  R. Fluhr,et al.  Organelles Contribute Differentially to Reactive Oxygen Species-Related Events during Extended Darkness1[C][W][OA] , 2011, Plant Physiology.

[87]  Joost T. van Dongen,et al.  Phloem Metabolism and Function Have to Cope with Low Internal Oxygen1 , 2003, Plant Physiology.

[88]  A. Odermatt,et al.  Green fluorescent protein-based monitoring of endoplasmic reticulum redox poise , 2013, Front. Genet..

[89]  A. Murphy,et al.  Plant physiology and development , 2015 .

[90]  J. V. van Dongen,et al.  Oxygen sensing and signaling. , 2015, Annual review of plant biology.

[91]  Tom Beeckman,et al.  A novel sensor to map auxin response and distribution at high spatio-temporal resolution , 2012, Nature.

[92]  E. Moudrianakis,et al.  Control of complex metal ion equilibria in biochemical reaction systems. Intrinsic and apparent stability constants of metal-adenine nucleotide complexes. , 1978, The Journal of biological chemistry.

[93]  M. Stitt,et al.  Metabolic Activity Decreases as an Adaptive Response to Low Internal Oxygen in Growing Potato Tubers , 2000, Biological chemistry.

[94]  A. Hyman,et al.  ATP as a biological hydrotrope , 2017, Science.

[95]  P. Benfey,et al.  High-Resolution Expression Map of the Arabidopsis Root Reveals Alternative Splicing and lincRNA Regulation. , 2016, Developmental cell.

[96]  H. Neuhaus,et al.  Molecular Physiological Analysis of the Two Plastidic ATP/ADP Transporters from Arabidopsis12 , 2004, Plant Physiology.

[97]  G. Bertani,et al.  STUDIES ON LYSOGENESIS I , 1951, Journal of bacteriology.

[98]  D. Gibbs,et al.  Learning To Breathe: Developmental Phase Transitions in Oxygen Status. , 2017, Trends in plant science.

[99]  I. Mandl,et al.  Solubilization of insoluble matter in nature; I. The part played by salts of adenosinetriphosphate. , 1952, Biochimica et biophysica acta.

[100]  M. Fricker,et al.  Monitoring the in vivo redox state of plant mitochondria: effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge. , 2009, Biochimica et biophysica acta.

[101]  M. Fricker,et al.  The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis , 2009, Proceedings of the National Academy of Sciences.

[102]  Frank Reimann,et al.  Submembrane ATP and Ca2+ kinetics in α-cells: unexpected signaling for glucagon secretion , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[103]  R. Bligny,et al.  Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. Carbon-13 and phosphorous-31 nuclear magnetic resonance studies. , 2001, Plant physiology.

[104]  In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects , 2015, BMC Plant Biology.

[105]  G. Valentini,et al.  Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana , 2017, Plant & cell physiology.

[106]  Y. Stierhof,et al.  H+-Independent Glutamine Transport in Plant Root Tips , 2010, PloS one.

[107]  Alex Costa,et al.  Calcium Dynamics in Root Cells of Arabidopsis thaliana Visualized with Selective Plane Illumination Microscopy , 2013, PloS one.

[108]  Amy E Palmer,et al.  Measuring calcium signaling using genetically targetable fluorescent indicators , 2006, Nature Protocols.

[109]  Takeharu Nagai,et al.  Ca²⁺ regulation of mitochondrial ATP synthesis visualized at the single cell level. , 2011, ACS chemical biology.

[110]  M. Fricker,et al.  The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis[OPEN] , 2015, Plant Cell.

[111]  M. Fricker,et al.  Analysis of plant mitochondrial function using fluorescent protein sensors. , 2015, Methods in molecular biology.

[112]  W. Frommer,et al.  Rapid Metabolism of Glucose Detected with FRET Glucose Nanosensors in Epidermal Cells and Intact Roots of Arabidopsis RNA-Silencing Mutants[W][OA] , 2006, The Plant Cell Online.

[113]  P. Geigenberger,et al.  Response of plant metabolism to too little oxygen. , 2003, Current opinion in plant biology.

[114]  G. Schulz.  Structural and functional relationships in the adenylate kinase family. , 1987, Cold Spring Harbor symposia on quantitative biology.

[115]  A. Fernie,et al.  Functional characterization and organ distribution of three mitochondrial ATP-Mg/Pi carriers in Arabidopsis thaliana. , 2015, Biochimica et biophysica acta.

[116]  M. Fricker,et al.  The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide 'flashes'. , 2011, The Biochemical journal.

[117]  J. Nielsen,et al.  Engineering central metabolism – a grand challenge for plant biologists , 2017, The Plant journal : for cell and molecular biology.

[118]  P. Gardeström,et al.  Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, Studied by Rapid Fractionation of Barley (Hordeum vulgare) Protoplasts. , 1988, Plant physiology.