Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

BioForA INRA ONF Orléans France

BIOGECO INRA University of Bordeaux Cestas France

Centre de Génomique Fonctionnelle Plateforme Protéome University of Bordeaux Bordeaux France

FCBA Pôle Biotechnologie et Sylviculture Avancée Cestas France

Institute of Experimental Botany of the Czech Academy of Sciences Prague Czechia

PEIRENE Sylva LIM Université de Limoges Limoges France

UCA INRA UMR PIAF Clermont Ferrand France

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Adrian A., Rahnenfuhrer J. (2016).

Amsterdam A., Pitzer F., Baumeister W. (1993). Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control. PubMed PMC

Bastien J. C., Sanchez L., Michaud D. (2013). “Douglas-fir (

Benjamini Y., Hochberg Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing.

Bonga J. M., Klimaszewska K. K., von Aderkas P. (2010). Recalcitrance in clonal propagation, in particular of conifers. DOI

Bonhomme M., Peuch M., Ameglio T., Rageau R., Guilliot A., Decourteix M., et al. (2010). Carbohydrate uptake from xylem vessels and its distribution among stem tissues and buds in walnut ( PubMed DOI

Brand U., Grunewald M., Hobe M., Simon R. (2002). Regulation of CLV3 expression by two homeobox genes in PubMed DOI PMC

Bravo S., Bertín A., Turner A., Sepúlveda F., Jopia P., Parra M. J., et al. (2017). Differences in DNA methylation, DNA structure and embryogenesis-related gene expression between embryogenic and non embryogenic lines of DOI

Businge E., Brackmann K., Moritz T., Egertsdotter U. (2012). Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce ( PubMed DOI

Carlson M. (2017).

Chang S., Puryear J., Cairney J. (1993). A simple and efficient method for isolating RNA from pine trees.

Chin C. F., Tan H. S. (2018). The use of proteomic tools to address challenges faced in clonal propagation of tropical crops through somatic embryogenesis. PubMed DOI PMC

Correia S., Vinhas R., Manadas B., Lourenço A. S., Veríssimo P., Canhoto J. M. (2012). Comparative proteomic analysis of auxin-induced embryogenic and nonembryogenic tissues of the solanaceous tree PubMed DOI

Crouzet J., Trombik T., Fraysse A. S., Boutry M. (2006). Organization and function of the plant pleiotropic drug resistance ABC transporter family. PubMed DOI

Cutler A. J., Krochko J. E. (1999). Formation and breakdown of ABA. PubMed DOI

Dawson R. J. P., Locher K. P. (2006). Structure of a bacterial multidrug ABC transporter. PubMed DOI

Dobrev P. I., Kamínek M. (2002). Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. PubMed DOI

Dronne S., Label P., Lelu M.-A. (1997). Desiccation decreases abscisic acid content in hybrid larch (

Durzan D., Gupta P. (1987). Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures. DOI

Edgar R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy, and high throughput. PubMed DOI PMC

Edreva A. (2005). Pathogenesis-related proteins: research progress in the last 15 years.

Elhiti M., Stasolla C., Wang A. (2013). Molecular regulation of plant somatic embryogenesis. DOI

Etienne H., Sotta B., Montoro P., Miginiac E., Carron M. P. (1993). Relations between exogenous growth-regulators and endogenous indole-3-acetic-acid and abscisic acid in the expression of somatic embryogenesis in DOI

Fehér A. (2015). Somatic embryogenesis - Stress-induced remodeling of plant cell fate. PubMed DOI

Friml J., Vieten A., Sauer M., Weijers D., Schwarz H., Hamann T., et al. (2003). Efflux-dependent auxin gradients establish the apical-basal axis of PubMed DOI

Fujita M., Fujita Y., Maruyama K., Seki M., Hiratsu K., Ohme-Takagi M., et al. (2004). A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. PubMed DOI

Gälweiler L., Guan C. H., Muller A., Wisman E., Mendgen K., Yephremov A., et al. (1998). Regulation of polar auxin transport by AtPIN1 in PubMed DOI

Gautier F., Eliášová K., Leplé J. C., Vondráková Z., Lomenech A. M., Le Metté C., et al. (2018). Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of PubMed DOI PMC

Goncalves L. S. A., Rodrigues R., Diz M. S. S., Robaina R. R., do Amaral A. T., Carvalho A. O., et al. (2013). Peroxidase is involved in Pepper yellow mosaic virus resistance in PubMed DOI

Götz S., García-Gómez J. M., Terol J., Williams T. D., Nagaraj S. H., Nueda M. J., et al. (2008). High-throughput functional annotation and data mining with the Blast2GO suite. PubMed DOI PMC

Gournas C., Papageorgiou I., Diallinas G. (2008). The nucleobase-ascorbate transporter (NAT) family: genomics, evolution, structure-function relationships and physiological role. PubMed DOI

Haecker A., Gross-Hardt R., Geiges B., Sarkar A., Breuninger H., Herrmann M., et al. (2004). Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in PubMed DOI

Heim M. A., Jakoby M., Werber M., Martin C., Weisshaar B., Bailey P. C. (2003). The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. PubMed DOI

Helleboid S., Hendriks T., Bauw G., Inze D., Vasseur J., Hilbert J. L. (2000). Three major somatic embryogenesis related proteins in PubMed DOI

Heringer A. S., Santa-Catarina C., Silveira V. (2018). Insights from proteomic studies into plant somatic embryogenesis. PubMed DOI

Jourdain I., Lelu M. A., Label P. (1997). Hormonal changes during growth of somatic embryogenic masses in hybrid larch [

Kaminek M., Brezinova A., Gaudinova A., Motyka V., Vankova R., Zazimalova E. (2000). Purine cytokinins: a proposal of abbreviations. DOI

Klein M., Perfus-Barbeoch L., Frelet A., Gaedeke N., Reinhardt D., Mueller-Roeber B., et al. (2003). The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use. PubMed DOI

Klimaszewska K., Hargreaves C., Lelu-Walter M.-A., Trontin J.-F. (2016). “Advances in conifer somatic embryogenesis since year 2000,” in PubMed

Klimaszewska K., Overton C., Stewart D., Rutledge R. (2011). Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process. PubMed DOI

Klip A., Tsakiridis T., Marette A., Ortiz P. A. (1994). Regulation of expression of glucose transporters by glucose: a review of studies PubMed

Klubicová K., Uvácková L., Danchenko M., Nemecek P., Skultéty L., Salaj J., et al. (2017). Insights into the early stage of PubMed DOI

Kumaravel M., Uma S., Backiyarani S., Saraswathi M. S., Vaganan M. M., Muthusamy M., et al. (2017). Differential proteome analysis during early somatic embryogenesis in PubMed DOI

Kurdyukov S., Faust A., Trenkamp S., Bar S., Franke R., Efremova N., et al. (2006). Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain alpha-,omega-dicarboxylic fatty acids and formation of extracellular matrix. PubMed DOI

Laux T., Mayer K. F. X., Berger J., Jurgens G. (1996). The WUSCHEL gene is required for shoot and floral meristem integrity in PubMed

Lê S., Josse J., Husson F. (2008). FactoMineR: an R package for multivariate analysis.

Lelu-Walter M.-A., Gautier F., Eliášová K., Sanchez L., Teyssier C., Lomenech A.-M., et al. (2018). High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in DOI

Lelu-Walter M.-A., Thompson D., Harvengt L., Sanchez L., Toribio M., Pâques L. E. (2013). Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. DOI

Léran S., Varala K., Boyer J. C., Chiurazzi M., Crawford N., Daniel-Vedele F., et al. (2014). A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. PubMed DOI

Li H., Durbin R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. PubMed DOI PMC

Li Q., Zhang S., Wang J. (2015). Transcriptomic and proteomic analyses of embryogenic tissues in PubMed DOI

Li Y. D., Zhu Y. X., Yao J., Zhang S. L., Wang L., Guo C. L., et al. (2017). Genome-wide identification and expression analyses of the homeobox transcription factor family during ovule development in seedless and seeded grapes. PubMed DOI PMC

Liao Y. K., Liao C. K., Ho Y. L. (2008). Maturation of somatic embryos in two embryogenic cultures of DOI

Lipavská H., Konrádová H. (2004). Somatic embryogenesis in conifers: the role of carbohydrate metabolism.

Lippert D., Jun Z., Ralph S., Ellis D. E., Gilbert M., Olafson R., et al. (2005). Proteome analysis of early somatic embryogenesis in PubMed DOI

Lombard V., Ramulu H. G., Drula E., Coutinho P. M., Henrissat B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. PubMed DOI PMC

Long T. A., Tsukagoshi H., Busch W., Lahner B., Salt D. E., Benfey P. N. (2010). The bHLH transcription factor POPEYE regulates response to iron deficiency in PubMed DOI PMC

Love M. I., Huber W., Anders S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. PubMed DOI PMC

Lyngved R., Renaut J., Hausman J.-F., Iversen T.-H., Hvoslef-Eide A. K. (2008). Embryo-specific Proteins in DOI

Maadon S. N., Rohani E. R., Ismail I., Baharum S. N., Normah M. N. (2016). Somatic embryogenesis and metabolic differences between embryogenic and non-embryogenic structures in mangosteen. DOI

Mahdavi-Darvari F., Noor N., Ismanizan I. (2015). Epigenetic regulation and gene markers as signals of early somatic embryogenesis. DOI

Mahmud I., Shrestha B., Boroujerdi A., Chowdhury K. (2015). NMR-based metabolomics profile comparisons to distinguish between embryogenic and non-embryogenic callus tissue of sugarcane at the biochemical level. DOI

Marsoni M., Bracale M., Espen L., Prinsi B., Negri A. S., Vannini C. (2008). Proteomic analysis of somatic embryogenesis in PubMed DOI

Martin A. B., Cuadrado Y., Guerra H., Gallego P., Hita O., Martin L., et al. (2000). Differences in the contents of total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from PubMed DOI

Martinoia E., Klein M., Geisler M., Bovet L., Forestier C., Kolukisaoglu U., et al. (2002). Multifunctionality of plant ABC transporters - more than just detoxifiers. PubMed DOI

Mayer K. F. X., Schoof H., Haecker A., Lenhard M., Jurgens G., Laux T. (1998). Role of WUSCHEL in regulating stem cell fate in the PubMed DOI

Mi H., Muruganujan A., Thomas P. (2013). PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. PubMed DOI PMC

Miguel C., Rupps A., Raschke J., Rodrigues A., Trontin J. (2016). “Impact of molecular studies on somatic embryogenesis development for implementation in conifer multi-varietal forestry,” in

Morel A., Teyssier C., Trontin J.-F., Eliášová K., Pešek B., Beaufour M., et al. (2014). Early molecular events involved in PubMed DOI

Nakano R. T., Matsushima R., Ueda H., Tamura K., Shimada T., Li L., et al. (2009). GNOM-LIKE1/ERMO1 and SEC24a/ERMO2 are required for maintenance of endoplasmic reticulum morphology in PubMed DOI PMC

Navarro B. V., Elbl P., De Souza A. P., Jardim V., de Oliveira L. F., Macedo A. F., et al. (2017). Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, PubMed DOI PMC

Neale D. B., McGuire P. E., Wheeler N. C., Stevens K. A., Crepeau M. W., Cardeno C., et al. (2017). The douglas-fir genome sequence reveals specialization of the photosynthetic apparatus in pinaceae. PubMed DOI PMC

Nørgaard J. V., Krogstrup P. (1991). Cytokinin induced somatic embryogenesis from immature embryos of PubMed DOI

Ohbayashi I., Sugiyama M. (2018). Plant nucleolar stress response, a new face in the NAC-dependent cellular stress responses. PubMed DOI PMC

Palovaara J., Hallberg H., Stasolla C., Hakman I. (2010). Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8/9 in developing seeds and somatic embryos of the gymnosperm PubMed DOI

Pascual M. B., Cánovas F. M., Ávila C. (2015). The NAC transcription factor family in maritime pine ( PubMed DOI PMC

Pighin J. A., Zheng H. Q., Balakshin L. J., Goodman I. P., Western T. L., Jetter R., et al. (2004). Plant cuticular lipid export requires an ABC transporter. PubMed DOI

Pollard M., Beisson F., Li Y. H., Ohlrogge J. B. (2008). Building lipid barriers: biosynthesis of cutin and suberin. PubMed DOI

Pullman G., Johnson S., Bucalo K. (2009). Douglas fir embryogenic tissue initiation. DOI

Quesnelle P. E., Emery R. J. N. (2007). cis-Cytokinins that predominate in DOI

R Development Core Team (2011).

Reeves C., Hargreaves C., Trontin J.-F., Leu-Walter M.-A. (2018). Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir. DOI

Rosenberg-Zand S. R., Jenkins D. J. A., Diamandis E. P. (2000). Steroid hormone activity of flavonoids and related compounds. PubMed DOI

Rutledge R. G., Stewart D., Caron S., Overton C., Boyle B., MacKay J., et al. (2013). Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce ( PubMed DOI PMC

Rutledge R. G., Stewart D., Overton C., Klimaszewska K. (2017). Gene expression analysis of primordial shoot explants collected from mature white spruce ( PubMed DOI PMC

Sampedro J., Cosgrove D. J. (2005). The expansin superfamily. PubMed DOI PMC

Schmutz J., Cannon S. B., Schlueter J., Ma J. X., Mitros T., Nelson W., et al. (2010). Genome sequence of the palaeopolyploid soybean. PubMed DOI

Sharifi G., Ebrahimzadeh H., Ghareyazie B., Gharechahi J., Vatankhah E. (2012). Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron ( PubMed DOI PMC

Shih M. D., Hsieh T. Y., Jian W. T., Wu M. T., Yang S. J., Hoekstra F. A., et al. (2012). Functional studies of soybean ( PubMed DOI

Silva Rde C., Carmo L. S. T., Luis Z. G., Silva L. P., Scherwinski-Pereira J. E., Mehta A. (2014). Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm ( PubMed DOI

Stasolla C., Yeung E. (2003). Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. DOI

Steinmann T., Geldner N., Grebe M., Mangold S., Jackson C. L., Paris S., et al. (1999). Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. PubMed DOI

Su Y. H., Su Y. X., Liu Y. G., Zhang X. S. (2013). Abscisic acid is required for somatic embryo initiation through mediating spatial auxin response in DOI

Teyssier C., Grondin C., Bonhomme L., Lomenech A.-M., Vallance M., Morabito D., et al. (2011). Increased gelling agent concentration promotes somatic embryo maturation in hybrid larch ( PubMed DOI

Treutter D. (2006). Significance of flavonoids in plant resistance: a review. DOI

Trontin J. F., Aronen T., Hargreaves C., Montalbán I., Moncaleán P., Reeves C., et al. (2016a). “International effort to induce somatic embryogenesis in adult pine trees,” in

Trontin J.-F., Klimaszewska K., Morel A., Hargreaves C. L., Lelu-Walter M.-A. (2016b). “Molecular aspects of conifer zygotic and somatic embryo development: a review of genome-wide approaches and recent insights,” in PubMed DOI

Vaňková R. (1999). “Cytokinin glycoconjugates - distribution, metabolism and function,” in

Varhanikova M., Uvackova L., Skultety L., Pretova A., Obert B., Hajduch M. (2014). Comparative quantitative proteomic analysis of embryogenic and non-embryogenic calli in maize suggests the role of oxylipins in plant totipotency. PubMed DOI

Vestman D., Larsson E., Uddenberg D., Cairney J., Clapham D., Sundberg E., et al. (2011). Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression. DOI

Vizcaíno J. A., Csordas A., del-Toro N., Dianes J. A., Griss J., Lavidas I., et al. (2016). 2016 update of the PRIDE database and related tools. PubMed DOI PMC

von Aderkas P., Klimaszewska K., Bonga J. M. (1990). Diploid and haploid embryogenesis in DOI

Vondráková Z., Dobrev P., Pešek B., Fischerová L., Vágner M., Motyka V. (2018). Profiles of endogenous phytohormones over the course of Norway spruce somatic embryogenesis. PubMed DOI PMC

Vondráková Z., Eliášová K., Fischerová L., Vágner M. (2011). The role of auxins in somatic embryogenesis of DOI

Vondráková Z., Eliášová K., Vágner M. (2014). The anti-actin drugs latrunculin and cytochalasin affect the maturation of spruce somatic embryos in different ways. PubMed DOI

Vondráková Z., Krajňáková J., Fischerová L., Vágner M., Eliášová K. (2016). “Physiology and role of plant growth regulators in somatic embryogenesis,” in

Weber H., Borisjuk L., Heim U., Sauer N., Wobus U. (1997). A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds. PubMed DOI PMC

Wickham H. (2009).

Wickham H. (2017).

Williams L. E., Lemoine R., Sauer N. (2000). Sugar transporters in higher plants – a diversity of roles and complex regulation. PubMed DOI

Willigen C. V., Verdoucq L., Boursiac Y., Maurel C. (2004). “Aquaporins in Plants,” in

Withers S. G. (2001). Mechanisms of glycosyl transferases and hydrolases. DOI

Yu T., Li G., Dong S., Liu P., Zhang J., Zhao B. (2016). Proteomic analysis of maize grain development using iTRAQ reveals temporal programs of diverse metabolic processes. PubMed DOI PMC

Zhang J., Ma H., Chen S., Ji M., Perl A., Kovacs L., et al. (2009). Stress response proteins’ differential expression in embryogenic and non-embryogenic callus of DOI

Zhao J., Wang B., Wang X., Zhang Y., Dong M., Zhang J. (2015). iTRAQ-based comparative proteomic analysis of embryogenic and non-embryogenic tissues of Prince Rupprecht’s larch ( DOI