Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.])

Abstract

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.

Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee

“…To date, many authors have distinguished EC from NEC based on morphological and histological studies [51,52,53]. Biochemical comparison of these tissues showed that carbohydrates, phytohormones and free amino acids differentially accumulated in EC and NEC [32,54,55]. When comparing metabolomic and hormonal profiles of EC and NEC, we were able to identify metabolic markers of the embryogenic state that can be directly used to pilot the optimization of the composition of the culture medium.…”

“…Metabolite profiles in our study showed that NEC accumulated starch reserves, while EC highly accumulated maltotriose, a product of starch hydrolysis. Gautier et al [32] also observed large structures of starch reserves in NEC of Douglas-fir ( P. menziesii ) and hypothesized that NEC main fate was survival while EC was a transient state before fate transition. We highlight here for the first time that maltotriose is the preferred way to store sugars in EC for a faster usage during fate transition.…”

“…Consequently, exogenous hormones play an essential role to maintain the stem cell niche [31]. Only sucrose content showed a sharp decrease followed by an increase in glucose and fructose meaning that cells are getting their energy from the exogenous sucrose as recently showed by Gautier et al [32] in Douglas-fir ( P. menziesii) .…”

“…In the same species, using liquid chromatography-mass spectrometry (LC-MS), Vondrakova et al [31] drew endogenous phytohormone profiles in nine sampled stages covering proliferation, maturation, desiccation and germination. Using the same technique, Gautier et al [32] compared phytohormone profiles between embryogenic calli and non-embryogenic calli in Douglas-fir ( Pseudotsuga menziesii) . All these authors assumed that clear correlations may exist among the different metabolome or phytohormone profiles and some specific SE stages.…”

Douglas‐fir LEAFY COTYLEDON1 (PmLEC1) is an active transcription factor during zygotic and somatic embryogenesis

“…Forests composed of superior genotypes will yield genetic gains much faster than those that could occur by natural selection (Pullman et al., 2003 ). However, the molecular regulatory mechanisms that induce SE in conifers remain unknown (Gautier et al., 2019 ; Pullman et al., 2015 ). Many conifer genotypes are recalcitrant to SE induction (Pullman et al., 2005 ).…”