title: Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.
abstract: Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.|These authors contributed equally to this work.
authors: Ambroise
authors: A
authors: Testard
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.|These authors contributed equally to this work.
authors: Daniel
authors: D
authors: Da Silva
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.
authors: Mélanie
authors: M
authors: Ormancey
authors: Institut de Pharmacologie et de Biologie Structurale IPBS CNRS, Fédération de Recherche 3450 Agrobiosciences Interactions et Biodiversités, Plateforme Protéomique Génopole Toulouse Midi Pyrénées, Toulouse, France.
authors: Carole
authors: C
authors: Pichereaux
authors: Institut Fédératif de Recherche 3450, Plateforme Imagerie-Microscopie, Pôle de Biotechnologie Végétale, 31326, Castanet-Tolosan, France.
authors: Cécile
authors: C
authors: Pouzet
authors: Institut Fédératif de Recherche 3450, Plateforme Imagerie-Microscopie, Pôle de Biotechnologie Végétale, 31326, Castanet-Tolosan, France.
authors: Alain
authors: A
authors: Jauneau
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.
authors: Sabine
authors: S
authors: Grat
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.
authors: Eugénie
authors: E
authors: Robe
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.
authors: Christian
authors: C
authors: Brière
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.
authors: Valérie
authors: V
authors: Cotelle
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France.
authors: Christian
authors: C
authors: Mazars
authors: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, BP42617, 31326, Castanet-Tolosan, France thuleau@lrsv.ups-tlse.fr.
authors: Patrice
authors: P
authors: Thuleau
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology*; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D017343:Genes, Plant; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D009569:Nitric Oxide / Q000494:pharmacology*; D015538:Nitrosation; D009696:Nucleic Acids / Q000378:metabolism; D059828:Plant Cells / Q000187:drug effects / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014026:Nicotiana / Q000166:cytology* / Q000201:enzymology / Q000235:genetics
chemical_list: D009696:Nucleic Acids; D010940:Plant Proteins; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D013110:Sphingosine; C005682:safingol; D002118:Calcium
keywords: 
  S-nitrosylation; Dihydrosphingosine; GAPC; GAPDH; LCBs; Nitric oxide; Sphinganine; Sphingolipids; Tobacco BY-2 cells