title: Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.
abstract: Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.
authors.affiliation: Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen and Danish Basic Research Foundation Centre of excellence: Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Kisten Sisse Krag
authors.initials: KS
authors.lastname: Gjetting
authors.forename: Cecilie Karkov
authors.initials: CK
authors.lastname: Ytting
authors.forename: Alexander
authors.initials: A
authors.lastname: Schulz
authors.forename: Anja Thoe
authors.initials: AT
authors.lastname: Fuglsang
mesh_terms: D000269:Adhesives / Q000494:pharmacology; D000468:Alkalies; D017360:Arabidopsis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D002138:Calibration; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics*; D006706:Homeostasis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007210:Indoleacetic Acids / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D058503:Mesophyll Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009928:Organ Specificity / Q000187:drug effects; D010477:Perfusion; D018517:Plant Roots / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D036226:Seedlings / Q000166:cytology / Q000187:drug effects
chemical_list: D000269:Adhesives; D000468:Alkalies; D007210:Indoleacetic Acids