Conifer somatic embryogenesis (SE) is a process driven by exogenously supplied plant growth regulators (PGRs). Exogenous PGRs and endogenous phytohormones trigger particular ontogenetic events. Complex mechanisms involving a number of endogenous phytohormones control the differentiation of cells and tissues, as well as the establishment of structures and organs. Most of the mechanisms and hormonal functions in the SE of conifers have not yet been described. With the aim to better understand these mechanisms, we provided detailed analysis of the spectrum of endogenous phytohormones over the course of SE in Norway spruce (Picea abies). Concentrations of endogenous phytohormones including auxins, cytokinins (CKs), abscisic acid (ABA), jasmonates, and salicylic acid (SA) in somatic P. abies embryos were analyzed by HPLC-ESI-MS/MS. The results revealed that the concentrations of particular phytohormone classes varied substantially between proliferation, maturation, desiccation, and germination. Endogenous ABA showed a maximum concentration at the maturation stage, which reflected the presence of exogenous ABA in the medium and demonstrated its efficient perception by the embryos as a prerequisite for their further development. Auxins also had concentration maxima at the maturation stage, suggesting a role in embryo polarization. Endogenous jasmonates were detected in conifer somatic embryos for the first time, and reached maxima at germination. According to our knowledge, we have presented evidence for the involvement of the non-indole auxin phenylacetic acid, cis-zeatin- and dihydrozeatin-type CKs and SA in SE for the first time. The presented results represent the currently most comprehensive overview of plant hormone levels in embryos throughout the whole process of conifer SE. The differences in concentrations of various classes of phytohormones over the proliferation, maturation, desiccation, and germination in somatic P. abies embryos clearly indicate correlations between endogenous phytohormone profiles and particular developmental stages of the SE of conifers.

Directional intercellular transport of the phytohormone auxin mediated by PIN FORMED (PIN) efflux carriers plays essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. Multilevel regulations of PIN activity under internal and external cues are complicated; however, the underlying molecular mechanism remains elusive. Here we demonstrate that 3'-Phosphoinositide-Dependent Protein Kinase1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub integrating the upstream lipid signalling and the downstream substrate activity through phosphorylation.Genetic analysis uncovers that loss-of-function Arabidopsis mutant pdk1.1 pdk1.2 exhibits a plethora of abnormalities in organogenesis and growth, due to the defective PIN-dependent auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 Protein Kinase to facilitate its activity towards PIN proteins. Our studies establish a lipiddependent phosphorylation cascade connecting membrane composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes. two auxin maxima sites for the fused cotyledon were observed (Supplementary Fig. 7a, e), while none for the lateral root primordia was found in pdk1.1 pdk1.2 (Supplementary Fig. 7b, f).There was a dramatic depletion of the DR5rev::GFP reporter signal at the concave side of the apical hook of pdk1.1 pdk1.2 etiolated seedlings under dark (Fig. 2f, g). Similarly, ACC treatment induced increase of DR5rev::GFP in the apical hook accompanied with the exaggerated curvature (Supplementary Fig. 7c) or roots under dark (Supplementary Fig. 7d) was undetectable (Supplementary Fig. 7g) or dramatically decreased (Supplementary Fig. 7h) in pdk1.1 pdk1.2. Collectively, PDK1 deficiency abolished the plants' ability of maintaining a functional local auxin concentration during various organogenesis processes, as well as that of redirecting the auxin flow in tropic responses.Based on the scenario that PDK1 acts as a master regulator of AGC kinases in different organisms 21 , and the crucial roles of AGC kinases in regulating auxin transport 30,43 , we speculate that PDK1 might function in auxin pathway through regulating specific AGC kinases.Of the 39 AGC members, PID/WAGs 6-8,44 , D6PKs 9 and PAX 11 have been reported to regulate either the polarity (by PID/WAGs) or the activity (by all of these) of PIN proteins respectively through phosphorylation, and hence modulate auxin fluxes. Therefore, we firstly measured the basipetal auxin transport in intact etiolated hypocotyls with [ 3 H]-IAA. Results showed that there was indeed a decrease in IAA transport in pdk1.2 and pdk1.1 pdk1.2, but not in pdk1.1, compared to that of Col-0 (Fig. 2h).These observations suggest that PDK1s are required for polar auxin transport, and the subsequent asymmetric auxin distribution, in which PIN auxin exporters play a critical role 3,5 . Thus, we examined the polarity of PIN proteins firs...

The somatic embryogenesis of conifers is a process susceptible to exogenous phytohormonal treatments. We report the effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the auxin inhibitor p-chlorophenoxyisobutyric acid (PCIB) on the endogenous level of the auxin indole-3-acetic acid (IAA) and on the anatomical composition of early somatic embryos of Abies alba (European silver fir). The embryogenic suspensor mass (ESM) of Abies alba proliferated on a medium supplemented by 2,4-D as well as on an auxin-free medium. The endogenous level of IAA was significantly higher in the ESM cultivated on a medium supplemented by 2,4-D. The decrease in the endogenous level of IAA in the first week of maturation is one of the most important stimuli responsible for the subsequent development of embryos. However, suppression of IAA synthesis by an auxin inhibitor did not stimulate the development of embryos. The maturation of somatic embryos from the globular to the cotyledonary stage occurs when the concentration of endogenous auxin in the ESM (including the embryos) increases. Early somatic embryos proliferating on a medium supplemented by auxin had an increased probability of maturing successfully. Exogenous auxin treatment during maturation did not compensate for the auxin deficiency during proliferation.

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.

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