title: The Vacuolar Proton-Cation Exchanger EcNHX1 Generates pH Signals for the Expression of Secondary Metabolism in Eschscholzia californica.
abstract: Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine alkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 at the plasma membrane, resulting in a peak of lysophosphatidylcholine, and (2) a subsequent, transient efflux of vacuolar protons, resulting in a peak of cytosolic H(+). This study demonstrates that one of the Na(+)/H(+) antiporters acting at the tonoplast of E. californica cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from complementary DNA (EcNHX1-EcNHX4). RNA interference-based, simultaneous silencing of EcNHX1, EcNHX3, and EcNHX4 resulted in stable cell lines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggered overproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na(+)-dependent H(+) efflux in a Δnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene displayed Na(+)-dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of vacuolar H(+). This finding was supported by three-dimensional protein homology models that predict a plausible recognition site for lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated expression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process.
authors.affiliation: Institute of Pharmacy, Department of Pharmaceutical Biology, Laboratory of Molecular Cell Biology (S.W., W.R.), and Institute of Genetics, Department of Molecular Genetics (R.L.), Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany; andLeibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, 06120 Halle (Saale), Germany (W.B.).
authors.forename: Sophie
authors.identifier: http://orcid.org/0000-0002-0125-1974
authors.initials: S
authors.lastname: Weigl
authors.affiliation: Institute of Pharmacy, Department of Pharmaceutical Biology, Laboratory of Molecular Cell Biology (S.W., W.R.), and Institute of Genetics, Department of Molecular Genetics (R.L.), Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany; andLeibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, 06120 Halle (Saale), Germany (W.B.).
authors.forename: Wolfgang
authors.identifier: http://orcid.org/0000-0002-0825-1491
authors.initials: W
authors.lastname: Brandt
authors.affiliation: Institute of Pharmacy, Department of Pharmaceutical Biology, Laboratory of Molecular Cell Biology (S.W., W.R.), and Institute of Genetics, Department of Molecular Genetics (R.L.), Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany; andLeibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, 06120 Halle (Saale), Germany (W.B.).
authors.forename: Renate
authors.identifier: http://orcid.org/0000-0003-2687-5844
authors.initials: R
authors.lastname: Langhammer
authors.affiliation: Institute of Pharmacy, Department of Pharmaceutical Biology, Laboratory of Molecular Cell Biology (S.W., W.R.), and Institute of Genetics, Department of Molecular Genetics (R.L.), Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany; andLeibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, 06120 Halle (Saale), Germany (W.B.) werner.roos@pharmazie.uni-halle.de.
authors.forename: Werner
authors.initials: W
authors.lastname: Roos
mesh_terms: D000595:Amino Acid Sequence; D002412:Cations; D002463:Cell Membrane Permeability; D031675:Eschscholzia / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011522:Protons; D034622:RNA Interference; D064210:Secondary Metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000378:metabolism*; D040681:Structural Homology, Protein; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D002412:Cations; D008244:Lysophosphatidylcholines; D010940:Plant Proteins; D011522:Protons; D017923:Sodium-Hydrogen Exchangers