Journal Article

Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii Mirb.

Costa, Guy, Teyssier, Caroline, Gautier, Florian, Le Metté, Claire, Label, Philippe, Eliášová, Kateřina, Leplé, Jean-Charles, Vondráková, Zuzana, Lelu-Walter, Marie-Anne, Lomenech, Anne-Marie, Trontin, Jean-François

2018

Overview

Background To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2 ry ) and tertiary (3 ry ) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2 ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1 ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g − 1 f.w.). Moreover, we observed similar differences in yield between 2 ry and 3 ry lines of SD4 (2400 vs 3921 g − 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2 ry vs 1 ry and 2 ry vs 3 ry lines at histo-cytological (using LC-MS/MS) and proteomic levels. Results Repetitive somatic embryogenesis dramatically improved the proliferating lines’ cellular organization (genotype SD4’s most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2 ry and (even more) 3 ry lines. Among 2300–2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2 ry vs 1 ry and 3 ry vs 2 ry lines, respectively, with special emphasis on “Proteolysis” and “Catabolic process” Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2 ry relative to 1 ry lines, but up-regulated in 3 ry relative to 2 ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same “Plant development” and “Defense response” biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3 ry lines. Conclusions This first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines’ embryogenic status.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Abiotic stress / Abscisic acid / Agriculture / Biological activity / Biomedical and Life Sciences / Biosynthesis / Catabolism / Chitin / Conifers / Cycle protein / Cytology / Douglas fir / Embryogenic potential / Embryonic development / Embryonic growth stage / Embryos / Flavonoids / Gene expression / gene expression regulation / gene ontology / Gene Regulatory Networks / Genetic aspects / Genetic engineering / Genomes / genotype / Genotypes / Growth / Growth regulators / Jasmonic acid / Life Sciences / Lignin / liquid chromatography / Mass Spectrometry / Metabolism / Metabolites / Oxidation / Oxidation-reduction potential / Phenylalanine / Plant embryology / Plant growth / Plant growth regulators / Plant Proteins - metabolism / Plant Proteins - physiology / Plant Sciences / Plant Somatic Embryogenesis Techniques - methods / Proliferation / Proteins / Proteolysis / Proteomic / Proteomics / Pseudotsuga - embryology / Pseudotsuga - growth & development / Pseudotsuga - metabolism / Pseudotsuga menziesii / Reproductive biology / Research Article / Salicylic acid / Secondary metabolites / Seeds - growth & development / Seeds - metabolism / Somatic embryogenesis / somatic embryos / tandem mass spectrometry / Tree Biology / Vegetal Biology