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.

• Klíčová slova
• embryonal mass, histo-cytology, network, non-embryogenic callus, phytohormone, proteomic, soluble carbohydrate, transcriptomic,
• Publikační typ
• časopisecké články MeSH

Ultraviolet-B (UV-B) radiation is a key environmental signal which initiates diverse responses that affect the metabolism, development, and viability of plants. In keeping with our previous studies, we concentrated primarily on how UV-B radiation affects Norway spruce [Picea abies (L.) Karst.] somatic embryo maturation and how phenolics and polyamines (PAs) are linked to the defense response invoked by UV-B irradiation. We treated clusters of Norway spruce embryogenic culture (EC) with UV-B during the five stages of embryo maturation (early, cylindrical, precotyledonary, cotyledonary, and mature embryos). For the first time, we take an advantage of the unique environmental scanning electron microscope AQUASEM II to characterize somatic embryos in their native state. The severity of the irradiation effect on embryonal cell viability was shown to be dependent on the intensity of radiation as well as the stage of embryo development, and might be related to the formation of protoderm. The response of early embryos was characterized by an increase in malondialdehyde (MDA), a marked decrease in PA contents and a decline in phenolics. The reduced ability to activate the defense system seems to be responsible not only for the severe cell damage and decrease in viability but also for the inhibition of embryo development. The significant reduction in spermidine (Spd), which has been reported to be crucial for the somatic embryo development of several coniferous species, may be causally linked to the limited development of embryos. The pronounced decrease in cell wall-bound ferulic acid might correspond to failure of somatic embryos to reach more advanced stages of development. Embryos at later stages of development showed stress defense responses that were more efficient against UV-B exposure.

• Klíčová slova
• Picea abies (L.) Karst., ferulic acid, polyamines, somatic embryogenesis, viability,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry 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 2ry and 3ry 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 2ry vs 1ry and 2ry vs 3ry 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 2ry and (even more) 3ry lines. Among 2300-2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on "Proteolysis" and "Catabolic process" Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry 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 3ry 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.

• Klíčová slova
• Cytology, Douglas-fir, Embryogenic potential, Plant growth regulators, Proliferation, Proteomic,
• MeSH
• genové regulační sítě MeSH
• hmotnostní spektrometrie MeSH
• proteomika MeSH
• Pseudotsuga embryologie   růst a vývoj   metabolismus   MeSH
• rostlinné proteiny metabolismus   fyziologie   MeSH
• semena rostlinná růst a vývoj   metabolismus   MeSH
• somatická embryogeneze rostlin metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• rostlinné proteiny MeSH