title: Virus-induced gene silencing of the two squalene synthase isoforms of apple tree (Malus × domestica L.) negatively impacts phytosterol biosynthesis, plastid pigmentation and leaf growth.
abstract: The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.|Laboratorio de Biotecnologıa, Sede de Investigacion Universitaria, Universidad de Antioquia, Medellin, Colombia.
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Navarro Gallón
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Carolina
authors.initials: C
authors.lastname: Elejalde-Palmett
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Daudu
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Franziska
authors.initials: F
authors.lastname: Liesecke
authors.affiliation: EA3061 Laboratoire de Biotechnologies Végétales appliquées aux plantes aromatiques et médicinales, Université Jean Monnet de Saint Etienne, Saint Etienne, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Jullien
authors.affiliation: EA3142 Groupe d'Etude des Interactions Hôte-Pathogène, Université d'Angers, Angers, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Papon
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dugé de Bernonville
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Courdavault
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Lanoue
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Oudin
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Glévarec
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Pichon
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Clastre
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: St-Pierre
authors.affiliation: Laboratorio de Biotecnologıa, Sede de Investigacion Universitaria, Universidad de Antioquia, Medellin, Colombia.
authors.forename: Lucia
authors.initials: L
authors.lastname: Atehortùa
authors.affiliation: Plant Pathology Laboratory, Iwate University, Morioka, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Yoshikawa
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Giglioli-Guivarc'h
authors.affiliation: EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France. sebastien.besseau@univ-tours.fr.
authors.forename: Sébastien
authors.identifier: http://orcid.org/0000-0002-5518-2580
authors.initials: S
authors.lastname: Besseau
mesh_terms: D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology*; D027845:Malus / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D010840:Phytosterols / Q000096:biosynthesis*; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D018087:Plastids / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D019180:Secoviridae / Q000235:genetics*; D014315:Triterpenes / Q000378:metabolism
chemical_list: D010840:Phytosterols; D020033:Protein Isoforms; D014315:Triterpenes; D013186:Farnesyl-Diphosphate Farnesyltransferase
keywords: Apple latent spherical virus (ALSV); Apple tree; Phytosterol; Squalene synthase; Triterpene; VIGS