title: Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.
abstract: Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.
authors.affiliation: INRA, UR 0588 Unité Amélioration, Génétique et Physiologie Forestières, 2163 Avenue de la Pomme de Pin, CS 4001, Ardon, 45075, Orléans Cedex 2, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Morel
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Trontin
authors.forename: Françoise
authors.initials: F
authors.lastname: Corbineau
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Lomenech
authors.forename: Martine
authors.initials: M
authors.lastname: Beaufour
authors.forename: Isabelle
authors.initials: I
authors.lastname: Reymond
authors.forename: Claire
authors.initials: C
authors.lastname: Le Metté
authors.forename: Kevin
authors.initials: K
authors.lastname: Ader
authors.forename: Luc
authors.initials: L
authors.lastname: Harvengt
authors.forename: Martine
authors.initials: M
authors.lastname: Cadene
authors.forename: Philippe
authors.initials: P
authors.lastname: Label
authors.forename: Caroline
authors.initials: C
authors.lastname: Teyssier
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Lelu-Walter
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis*; D016000:Cluster Analysis; D018548:Cotyledon / Q000196:embryology* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D028223:Pinus / Q000196:embryology* / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods; D012639:Seeds / Q000145:classification / Q000196:embryology* / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D013997:Time Factors; D014867:Water / Q000378:metabolism
chemical_list: D015415:Biomarkers; D002241:Carbohydrates; D010940:Plant Proteins; D020543:Proteome; D014867:Water; D005632:Fructose; D013395:Sucrose; D008320:Maltose; D005947:Glucose