Exposure of Norway spruce (Picea abies) somatic embryos and those of many other conifers to post-maturation desiccation treatment significantly improves their germination. An integration analysis was conducted to understand the underlying processes induced during the desiccation phase at the molecular level. Carbohydrate, protein and phytohormone assays associated with histological and proteomic studies were performed for the evaluation of markers and actors in this phase. Multivariate comparison of mature somatic embryos with mature desiccated somatic embryos and/or zygotic embryos provided new insights into the processes involved during the desiccation step of somatic embryogenesis. Desiccated embryos were characterized by reduced levels of starch and soluble carbohydrates but elevated levels of raffinose family oligosaccharides. Desiccation treatment decreased the content of abscisic acid and its derivatives but increased total auxins and cytokinins. The content of phytohormones in dry zygotic embryos was lower than in somatic embryos, but their profile was mostly analogous, apart from differences in cytokinin profiles. The biological processes "Acquisition of desiccation tolerance", "Response to stimulus", "Response to stress" and "Stored energy" were activated in both the desiccated somatic embryos and zygotic embryos when compared to the proteome of mature somatic embryos before desiccation. Based on the specific biochemical changes of important constituents (abscisic acid, raffinose, stachyose, LEA proteins and cruciferins) induced by the desiccation treatment and observed similarities between somatic and zygotic P. abies embryos, we concluded that the somatic embryos approximated to a state of desiccation tolerance. This physiological change could be responsible for the reorientation of Norway spruce somatic embryos toward a stage suitable for germination.

Department of Experimental Plant Biology Faculty of Science Charles University Prague Czechia

INRAE ONF BioForA Orléans France

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

Plateforme Proteome University of Bordeaux Bordeaux France

Zobrazit více v PubMed

Amara I., Zaidi I., Masmoudi K., Ludevid M., Pagès M., Goday A., et al. (2014). Insights into late embryogenesis abundant (LEA) proteins in plants: from structure to the functions. DOI

Angelovici R., Galili G., Fernie A. R., Fait A. (2010). Seed desiccation: a bridge between maturation and germination. PubMed DOI

Attree S. M., Fowke L. C. (1993). Embryogeny of gymnosperms: advances in synthetic seed technology of conifers. DOI

Attree S. M., Moore D., Sawhney V. K., Fowke L. C. (1991). Enhanced maturation and desiccation tolerance of white spruce [

Attree S. M., Pomeroy M. K., Fowke L. C. (1995). Development of white spruce ( DOI

Balbuena T. S., Jo L., Pieruzzi F. P., Dias L. L. C., Silveira V., Santa-Catarina C., et al. (2011). Differential proteome analysis of mature and germinated embryos of PubMed DOI

Balbuena T. S., Silveira V., Junqueira M., Dias L. L. C., Santa-Catarina C., Shevchenko A., et al. (2009). Changes in the 2-DE protein profile during zygotic embryogenesis in the Brazilian Pine ( PubMed DOI

Bomal C., Le V. Q., Tremblay F. M. (2002). Induction of tolerance to fast desiccation in black spruce ( PubMed DOI

Brunoni F., Collani S., Casanova-Sáez R., Šimura J., Karady M., Schmid M., et al. (2020). Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis. PubMed DOI

Brunoni F., Ljung K., Bellini C. (2019). Control of root meristem establishment in conifers. PubMed DOI

Brzobohatý B., Moore I., Kristoffersen P., Bako L., Campos N., Schell J., et al. (1993). Release of active cytokinin by a β-glucosidase localized to the maize root meristem. PubMed DOI

Businge E., Bygdell J., Wingsle G., Moritz T., Egertsdotter U. (2013). The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos. PubMed DOI

Carpenter C. V., Koester M. K., Gupta P. K. (2000).

Carrier D. J., Kendall E. J., Bock C. A., Cunningham J. E., Dunstan D. I. (1999). Water content, lipid deposition, and (+)-abscisic acid content in developing white spruce seeds. DOI

Chatelain E., Hundertmark M., Leprince O., Le Gall S., Satour P., Deligny-Penninck S., et al. (2012). Temporal profiling of the heat-stable proteome during late maturation of PubMed DOI

Chiwocha S., von Aderkas P. (2002). Endogenous levels of free and conjugated forms of auxin, cytokinins and abscisic acid during seed development in Douglas fir. DOI

Crowe J. H., Hoekstra F. A., Crowe L. M. (1992). Anhydrobiosis. PubMed DOI

Djilianov D. L., Dobrev P. I., Moyankova D. P., Vankova R., Georgieva D. T., Gajdosova S., et al. (2013). Dynamics of endogenous phytohormones during desiccation and recovery of the resurrection plant species DOI

Downie B., Bewley J. D. (2000). Soluble sugar content of white spruce ( DOI

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

Eliášová K., Vondráková Z., Gemperlová L., Nedìěla V., Runštuk J., Fischerová L., et al. (2018). The response of PubMed DOI PMC

Eliášová K., Vondráková Z., Malbeck J., Trávníčková A., Pešek B., Vágner M., et al. (2017). Histological and biochemical response of Norway spruce somatic embryos to UV-B irradiation. DOI

Fait A., Angelovici R., Less H., Ohad I., Urbanczyk-Wochniak E., Fernie A. R., et al. (2006). Arabidopsis seed development and germination Is associated with temporally distinct metabolic switches. PubMed DOI PMC

Fehér A., Pasternak T. P., Dudits D. (2003). Transition of somatic plant cells to an embryogenic state. DOI

Filonova L. H., Bozhkov P. V., Brukhin V. B., Daniel G., Zhivotovsky B., von Arnold S. (2000). Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce. PubMed

Find J. I. (1997). Changes in endogenous ABA levels in developing somatic embryos of Norway spruce ( DOI

Finnie C., Bak-Jensen K. S., Laugesen S., Roepstorff P., Svensson B. (2006). Differential appearance of isoforms and cultivar variation in protein temporal profiles revealed in the maturing barley grain proteome. DOI

Flinn B. S., Roberts D. R., Newton C. H., Cyr D. R., Webster F. B., Taylor I. E. P. (1993). Storage protein gene expression in zygotic and somatic embryos of interior spruce. DOI

Gösslová M., Svobodová H., Lipavská H., Albrechtová J., Vreugdenhil D. (2001). Comparing carbohydrate status during Norway spruce seed development and somatic embryo formation.

Grigová M., Kubeš M., Drážná N., Řezanka T., Lipavská H. (2007). Storage lipid dynamics in somatic embryos of Norway spruce ( PubMed DOI

Gupta P., Durzan D. (1986). Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos of DOI

Gutmann M., Charpentier J. P., Doumas P., Jay-Allemand C. (1996). Histological investigation of walnut cotyledon fragments for a better understanding of in vitro adventitious root initiation. PubMed DOI

Hakman I. (1993). Embryology in Norway spruce ( DOI

Hakman I., Stabel P., Engström P., Eriksson T. (1990). Storage protein accumulation during zygotic and somatic embryo development in DOI

Hazubska-Przybyl T., Wawrzyniak M., Obarska A., Bojarczuk K. (2015). Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce. DOI

Hoekstra F. A., Golovina E. A., Tetteroo F. A. A., Wolkers W. F. (2001). Induction of desiccation tolerance in plant somatic embryos: how exclusive is the protective role of sugars? PubMed DOI

Huang H., Møller I. M., Song S.-Q. (2012). Proteomics of desiccation tolerance during development and germination of maize embryos. PubMed DOI

Hudec L., Konrádová H., Hašková A., Lipavská H. (2016). Norway spruce embryogenesis: changes in carbohydrate profile, structural development and response to polyethylene glycol. PubMed DOI PMC

Iraqi D., Tremblay F. M. (2001). Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development. PubMed DOI

Jing D. L., Zhang J. W., Xia Y., Kong L. S., OuYang F. Q., Zhang S. G., et al. (2017). Proteomic analysis of stress-related proteins and metabolic pathways in PubMed DOI PMC

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

Joy R. W., IV, Yeung E. C., Kong L., Thorpe T. A. (1991). Development of white spruce somatic embryos: I. Storage product deposition. DOI

Käll L., Canterbury J., Weston J., Noble W. S., MacCoss M. J. (2007). Semi-supervised learning for peptide identification from shotgun proteomics datasets. PubMed DOI

Keller F., Pharr D. M. (1996). “Metabolism of carbohydrates in sinks and sources: galactosyl-sucrose oligosaccharides,” in DOI

Kermode A. R., Dumbroff E. B., Bewley J. D. (1989). The role of maturation drying in the transition from seed development to germination. 7. Effects of partial and complete desiccation on abscisic acid levels and sensitivity in DOI

Keunen E., Peshev D., Vangronsveld J., van den Ende W., Cuypers A. (2013). Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept. PubMed DOI

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

Klimaszewska K., Morency F., Jones-Overton C., Cooke J. (2004). Accumulation pattern and identification of seed storage proteins in zygotic embryos of DOI

Kong L., Yeung E. C. (1992). Development of white spruce somatic embryos: II. Continual shoot meristem development during germination. DOI

Kong L., Attree S. M., Evans D. E., Binarova P., Yeung E. C., Fowke L. C. (1999). “Somatic embryogenesis in white spruce: studies of embryo development and cell biology,” in DOI

Konradova H., Grigova M., Lipavska H. (2003). Cold-induced accumulation of raffinose family oligosaccharides in somatic embryos of Norway spruce ( DOI

Koster K. L. (1991). Glass formation and desiccation tolerance in seeds. PubMed DOI PMC

Koster K. L., Bryant G. (2005). “Dehydration in model membranes and protoplasts: contrasting effects at low, intermediate and high hydrations,” in DOI

Koster K. L., Leopold A. C. (1988). Sugars and desiccation tolerance in seeds. PubMed DOI PMC

Kubes M., Drazna N., Konradova H., Lipavska H. (2014). Robust carbohydrate dynamics based on sucrose resynthesis in developing Norway spruce somatic embryos at variable sugar supply. DOI

Lara-Chavez A., Egertsdotter U., Flinn B. S. (2012). Comparison of gene expression markers during zygotic and somatic embryogenesis in pine. DOI

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

Leal I., Misra S., Attree S. M., Fowke L. C. (1995). Effect of abscisic acid, osmoticum and desiccation on 11s storage protein gene expression in somatic embryos of white spruce. DOI

Lelu M. A., Label P. (1994). Changes in the levels of abscisic acid and its glucose ester conjugate during maturation of hybrid larch ( DOI

Lelu M. A., Klimaszewska K., Pflaum G., Bastien C. (1995). Effect of maturation duration on desiccation tolerance in hybrid larch ( DOI

Lelu-Walter M. A., Bernier-Cardou M., Klimaszewska K. (2008). Clonal plant production from self- and cross-pollinated seed families of DOI

Leprince O., Buitink J. (2010). Desiccation tolerance: from genomics to the field. DOI

Leprince O., Buitink J. (2015). Introduction to desiccation biology: from old borders to new frontiers. PubMed DOI

Leprince O., Pellizzaro A., Berriri S., Buitink J. (2017). Late seed maturation: drying without dying. PubMed DOI

Leprince O., Vanderwerf A., Deltour R., Lambers H. (1992). Respiratory pathways in germinating maize radicles correlated with desiccation tolerance and soluble sugars. DOI

Letham D. S., Palni L. M. S. (1983). The biosynthesis and metabolism of cytokinins. DOI

Li Q., Wang B. C., Xu Y., Zhu Y. X. (2007). Systematic studies of 12S seed storage protein accumulation and degradation patterns during Arabidopsis seed maturation and early seedling germination stages. PubMed DOI

Liao Y., Juan I. P. (2015). Improving the germination of somatic embryos of DOI

Lin T.-P., Huang N.-H. (1994). The relationship between carbohydrate composition of some tree seeds and their longevity. DOI

Lipavská H., Konrádová H. (2004). Invited review: somatic embryogenesis in conifers: The role of carbohydrate metabolism. DOI

Lipavska H., Svobodova H., Albrechtova J., Kumstyrova L., Vagner M., Vondrakova Z. (2000). Carbohydrate status during somatic embryo maturation in Norway spruce. DOI

Lippert D., Yuen M., Bohlmann J. (2009). Spruce proteome DB: a resource for conifer proteomics research. DOI

Liu Y., Müller K., El-Kassaby Y. A., Kermode A. R. (2015). Changes in hormone flux and signaling in white spruce ( PubMed DOI PMC

Marques A., Nijveen H., Somi C., Ligterink W., Hilhorst H. (2019). Induction of desiccation tolerance in desiccation sensitive PubMed DOI PMC

Maruyama T. E., Hosoi Y. (2012). Post-maturation treatment improves and synchronizes somatic embryo germination of three species of Japanese pines. DOI

Morel A., Trontin J.-F., Corbineau F., Lomenech A.-M., Beaufour M., Reymond I., et al. (2014). Cotyledonary somatic embryos of PubMed DOI

Nagmani R., Diner A., Garton S., Zipf A. (1995). “Anatomical comparison of somatic and zygotic embryogeny in conifers,” in

Ntuli T. M., Finch-Savage W. E., Berjak P., Pammenter N. W. (2011). Increased drying rate lowers the critical water content for survival in embryonic axes of English oak ( PubMed DOI

Oliver M. J., O’Mahony P., Wood A. J. (1998). “To dryness and beyond” – Preparation for the dried state and rehydration in vegetative desiccation-tolerant plants. DOI

Pammenter N. W., Berjak P. (1999). A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. DOI

Pérez H., Hill L. M., Walters C. (2020). “A protective role for accumulated dry matter reserves in seeds during desiccation: implications for conservation,” in

Personat J.-M., Tejedor-Cano J., Prieto-Dapena P., Almoguera C., Jordano J. (2014). Co-overexpression of two heat shock factors results in enhanced seed longevity and in synergistic effects on seedling tolerance to severe dehydration and oxidative stress. PubMed DOI PMC

Peterbauer T., Richter A. (2001). Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds. DOI

Pokorná E., Hluska T., Galuszka P., Hallmark H. T., Dobrev P. I., Záveská Drábková L., et al. (2021). Cytokinin N-glucosides: occurrence, metabolism and biological activities in plants. PubMed DOI PMC

Pond S. E., von Aderkas P., Bonga J. M. (2002). Improving tolerance of somatic embryos of DOI

Pullman G. S., Bucalo K. (2014). Pine somatic embryogenesis: analyses of seed tissue and medium to improve protocol development. DOI

Pullman G. S., Buchanan M. (2008). Identification and quantitative analysis of stage-specific carbohydrates in loblolly pine ( PubMed DOI

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

Righetti K., Vu J. L., Pelletier S., Vu B. L., Glaab E., Lalanne D., et al. (2015). Inference of longevity-related genes from a robust coexpression network of seed maturation identifies regulators linking seed storability to biotic defense-related pathways. PubMed DOI PMC

Roberts D. R. (1991). Abscisic acid and mannitol promote early development, maturation and storage protein accumulation in somatic embryos of interior spruce. DOI

Roberts D. R., Sutton B. C. S., Flinn B. S. (1990b). Synchronous and high-frequency germination of interior spruce somatic embryos following partial drying at high relative humidity. DOI

Roberts D. R., Flinn B. S., Webb D. T., Webster F. B., Sutton B. C. S. (1990a). Abscisic acid and indole-3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce. DOI

Roberts D. R., Lazaroff W. R., Webster F. B. (1991). Interaction between maturation and high relative humidity treatments and their effects on germination of sitka spruce somatic embryos. DOI

Sass J. E. (1958).

Sauter J. J., van Cleve B. (1991). Biochemical and ultrastructural results during starch-sugar-conversion in ray parenchyma cells of DOI

Sghaier B., Kriaa W., Bahloul M., Novo J. V. J., Drira N. (2009). Effect of ABA, arginine and sucrose on protein content of date palm somatic embryos. DOI

Shi J., Zhen Y., Zheng R.-H. (2010). Proteome profiling of early seed development in PubMed DOI PMC

Silveira V., Balbuena T. S., Santa-Catarina C., Floh E. I. S., Guerra M. P., Handro W. (2004). Biochemical changes during seed development in DOI

Silveira V., Santa-Catarina C., Balbuena T. S., Moraes F. M. S., Ricart C. A. O., Sousa M. V., et al. (2008). Endogenous abscisic acid and protein contents during seed development of DOI

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

Stasolla C., Belmonte M. F., Van Zyl L., Craig D. L., Liu W., Yeung E. C., et al. (2004a). The effect of reduced glutathione on morphology and gene expression of white spruce ( PubMed DOI

Stasolla C., Bozhkov P. V., Chu T. M., Van Zyl L., Egertsdotter U., Suarez M. F., et al. (2004b). Variation in transcript abundance during somatic embryogenesis in gymnosperms. PubMed DOI

Stasolla C., van Zyl L., Egertsdotter U., Craig D., Liu W. B., Sederoff R. R. (2003). The effects of polyethylene glycol on gene expression of developing white spruce somatic embryos. PubMed DOI PMC

RStudio (2021).

Tereso S., Zoglauer K., Milhinhos A., Miguel C., Oliveira M. M. (2007). Zygotic and somatic embryo morphogenesis in PubMed 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

Teyssier C., Maury S., Beaufour M., Grondin C., Delaunay A., Le Metté C., et al. (2014). In search of markers for somatic embryo maturation in hybrid larch ( PubMed DOI

Vágner M., Vondráková Z., Strnadová Z., Eder J., Macháčková I. (1998). Endogenous levels of plant growth hormones during early stages of somatic embryogenesis of

Vales T., Feng X., Ge L., Xu N., Cairney J., Pullman G. S., et al. (2007). Improved somatic embryo maturation in loblolly pine by monitoring ABA-responsive gene expression. PubMed DOI

Välimäki S., Paavilainen L., Tikkinen M., Salonen F., Varis S., Aronen T. (2020). Production of Norway spruce embryos in a temporary immersion system (TIS). 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.

von Aderkas P., Lelu M. A., Label P. (2001). Plant growth regulator levels during maturation of larch somatic embryos.

von Aderkas P., Rohr R., Sundberg B., Gutmann M., Dumont-BéBoux N., Lelu M. A. (2002). Abscisic acid and its influence on development of the embryonal root cap, storage product and secondary metabolite accumulation in hybrid larch somatic embryos.

von Arnold S., Hakman I. (1988). Regulation of somatic embryo development in DOI

von Arnold S., Clapham D., Abrahamsson M. (2019). Embryology in conifers. DOI

von Arnold S., Clapham D., Egertsdotter U., Mo L. H. (1996). Somatic embryogenesis in conifers - A case study of induction and development of somatic embryos in

Vondrakova Z., Dobrev P. I., Pesek B., Fischerova L., Vagner M., Motyka V. (2018). Profiles of endogenous phytohormones over the course of Norway spruce somatic embryogenesis. PubMed DOI PMC

Vondráková Z., Eliášová K., Vágner M., Martincová O., Cvikrová M. (2015). Exogenous putrescine affects endogenous polyamine levels and the development of DOI

Vondráková Z., Trávníčková A., Malbeck J., Cvikrová M. (2013). “The effect of different desiccation treatments on polyamine metabolism of spruce somatic embryos,” in

Wang W.-Q., Ye J.-Q., Rogowska-Wrzesinska A., Wojdyla K. I., Jensen O. N., Møller I. M., et al. (2014). Proteomic comparison between maturation drying and prematurely imposed drying of PubMed DOI

Xiao L., Koster K. L. (2001). Desiccation tolerance of protoplasts isolated from pea embryos. PubMed DOI

Yathisha N. S., Barbara P., Gügi B., Yogendra K., Jogaiah S., Azeddine D., et al. (2020). Vegetative desiccation tolerance in PubMed DOI PMC

Záveská-Drábková L., Honys D., Motyka V. (2021). Evolutionary diversification of cytokinin-specific glucosyltransferases in angiosperms and enigma of missing PubMed DOI PMC

Zhou X., Zheng R., Liu G., Xu Y., Zhou Y., Laux T., et al. (2017). Desiccation treatment and endogenous IAA levels are key factors influencing high frequency somatic embryogenesis in PubMed DOI PMC

The humidity level matters during the desiccation of Norway spruce somatic embryos