title: Comparative analysis of MAMP-induced calcium influx in Arabidopsis seedlings and protoplasts.
abstract: Rapid transient elevation of cytoplasmic calcium (Ca(2+)) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca(2+) levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca(2+) sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca(2+) biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca(2+) levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca(2+) signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca(2+)signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca(2+) signaling pathways in plant cells.
authors.affiliation: Max Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Maintz
authors.affiliation: RWTH Aachen University, Institute for Biology I, Unit of Plant Molecular Cell Biology, 52056 Aachen, Germany.
authors.forename: Meltem
authors.initials: M
authors.lastname: Cavdar
authors.affiliation: RWTH Aachen University, Institute for Biology I, Unit of Plant Molecular Cell Biology, 52056 Aachen, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Tamborski
authors.affiliation: Max Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Kwaaitaal
authors.affiliation: Max Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Carl-von-Linné-Weg 10, 50829 Cologne, Germany Present address: Wageningen University, Laboratory of Molecular Biology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Rik
authors.initials: R
authors.lastname: Huisman
authors.affiliation: Max Planck Institute for Plant Breeding Research, Chemical Biology Laboratory, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Meesters
authors.affiliation: Max Planck Institute for Plant Breeding Research, Chemical Biology Laboratory, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
authors.forename: Erich
authors.initials: E
authors.lastname: Kombrink
authors.affiliation: Max Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Carl-von-Linné-Weg 10, 50829 Cologne, Germany RWTH Aachen University, Institute for Biology I, Unit of Plant Molecular Cell Biology, 52056 Aachen, Germany panstruga@bio1.rwth-aachen.de.
authors.forename: Ralph
authors.initials: R
authors.lastname: Panstruga
mesh_terms: D017360:Arabidopsis / Q000254:growth & development / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D017136:Ion Transport; D018515:Plant Leaves / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism*
chemical_list: D002118:Calcium
keywords: Aequorin; Arabidopsis thaliana; Calcium signature • Elicitor; Lanthanum chloride; Mesophyll protoplasts; Staurosporine; Transfection; flg22