title: Alternative splicing-mediated targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to mitochondria affects organelle morphology.
abstract: Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Enrico
authors.initials: E
authors.lastname: Teardo
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Luca
authors.initials: L
authors.lastname: Carraretto
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Sara
authors.initials: S
authors.lastname: De Bortoli
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Alex
authors.initials: A
authors.lastname: Costa
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Smrutisanjita
authors.initials: S
authors.lastname: Behera
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Richard
authors.initials: R
authors.lastname: Wagner
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Fiorella
authors.initials: F
authors.lastname: Lo Schiavo
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.).
authors.forename: Elide
authors.identifier: http://orcid.org/0000-0002-7994-4666
authors.initials: E
authors.lastname: Formentin
authors.affiliation: Department of Biology, University of Padova, 35121 Padua, Italy (E.T., L.C., S.D.B., F.L.S., E.F., I.S.);Department of Biosciences, University of Milan, 20133 Milan, Italy (A.C., S.B.); andBiophysics, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (R.W.) ildi@civ.bio.unipd.it.
authors.forename: Ildiko
authors.initials: I
authors.lastname: Szabo
mesh_terms: D017398:Alternative Splicing / Q000235:genetics* / Q000502:physiology; D017360:Arabidopsis / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000235:genetics / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D016922:Cellular Senescence / Q000235:genetics / Q000502:physiology; D002736:Chloroplasts / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D008928:Mitochondria / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000502:physiology / Q000648:ultrastructure; D017470:Receptors, Glutamate / Q000235:genetics* / Q000502:physiology
chemical_list: D029681:Arabidopsis Proteins; D017470:Receptors, Glutamate; C000601589:glutamate receptor 3.5, Arabidopsis; D002118:Calcium