In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes.
Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP-C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP-C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo.
Section of Plant Physiology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, NL-1098XH, Amsterdam, The Netherlands.|Department of Plant Molecular Biology, University of Lausanne-Sorge, Lausanne 1015, Switzerland.|Department of Plant and Microbial Biology, University of Zürich, Zürich 8008, Switzerland.
Joop E M
JEM
Vermeer
Section of Plant Physiology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, NL-1098XH, Amsterdam, The Netherlands.|Section of Plant Cell Biology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, NL-1098XH, Amsterdam, The Netherlands.
Ringo
R
van Wijk
Section of Molecular Cytology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, NL-1098XH, Amsterdam, The Netherlands.
Joachim
J
Goedhart
Department of Plant Molecular Biology, University of Lausanne-Sorge, Lausanne 1015, Switzerland.
Niko
N
Geldner
Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Joanne
J
Chory
Section of Molecular Cytology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, NL-1098XH, Amsterdam, The Netherlands.
Theodorus W J
TWJ
Gadella
Section of Plant Physiology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, NL-1098XH, Amsterdam, The Netherlands.|Section of Plant Cell Biology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, NL-1098XH, Amsterdam, The Netherlands.
Teun
T
Munnik
D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D048749:Cytokinesis; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D010703:Phorbol Esters / Q000378:metabolism; D019441:Plant Epidermis / Q000166:cytology / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000378:metabolism; D000072417:Protein Domains; D011493:Protein Kinase C / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
C011439:1,2-diacylglycerol; D004075:Diglycerides; D010703:Phorbol Esters; C119222:phorbol-12-myristate; D011493:Protein Kinase C
Arabidopsis thaliana; Biosensor; Diacylglycerol; Phospholipase C; Tobacco BY-2