title: Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.
abstract: Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes.
authors.affiliation: UMR5546, Toulouse III Paul Sabatier University-CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France.
authors.forename: Anna
authors.initials: A
authors.lastname: Plasencia
authors.affiliation: UMR5546, Toulouse III Paul Sabatier University-CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France.
authors.forename: Marçal
authors.initials: M
authors.lastname: Soler
authors.affiliation: UMR5546, Toulouse III Paul Sabatier University-CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France.
authors.forename: Annabelle
authors.initials: A
authors.lastname: Dupas
authors.affiliation: UMR5546, Toulouse III Paul Sabatier University-CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Ladouce
authors.affiliation: UMR5546, Toulouse III Paul Sabatier University-CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France.
authors.forename: Guilherme
authors.initials: G
authors.lastname: Silva-Martins
authors.affiliation: FRAIB, CNRS, Cell Imaging Plateform, Castanet Tolosan, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Martinez
authors.affiliation: INRA/AgroParisTech, UMR1318, Saclay Plant Science, Jean-Pierre Bourgin Institute (IJPB), Versailles, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Lapierre
authors.affiliation: Rhizogenesis, UMR DIADE, IRD, Montpellier, France.
authors.forename: Claudine
authors.initials: C
authors.lastname: Franche
authors.affiliation: UMR5546, Toulouse III Paul Sabatier University-CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Truchet
authors.affiliation: UMR5546, Toulouse III Paul Sabatier University-CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Grima-Pettenati
mesh_terms: D018533:Biomass; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005052:Eucalyptus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D018745:Genome, Plant; D008031:Lignin / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000254:growth & development / Q000378:metabolism; D046509:Tissue Culture Techniques; D014934:Wood / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D052584:Xylem / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D008031:Lignin
keywords: Agrobacterium rhizogenes; Eucalyptus; hairy roots; lignin; secondary cell wall; xylem