Data_Sheet_2_Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.]).xlsx

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.

体细胞胚胎发生（somatic embryogenesis）技术已在多数针叶树种中得以建立，但目前仅能使用幼龄材料。为拓展该技术的应用范围，亟需对细胞胚胎发生状态的关键生物学、生理学及分子特征形成更为全面的整合认知。为此，研究人员在增殖过程中多维度比较了胚性细胞团（embryonal masses, EMs）与非胚性愈伤组织（non-embryogenic calli, NECs）。
本分析选取了3个无关基因型的单一体细胞胚胎来源的等基因系（isogenic lines）的胚性细胞团与非胚性愈伤组织，分析手段包括：通过透射光显微镜进行细胞解剖结构观察、通过Illumina RNA测序开展转录组分析、通过自由凝胶电泳进行蛋白质组分析、通过液相色谱-质谱（LC-MS）检测内源植物激素（吲哚-3-乙酸、细胞分裂素与脱落酸（ABA））含量，以及通过高效液相色谱（HPLC）测定可溶性糖含量。
相较于非胚性愈伤组织，胚性细胞团表现为与细胞分化相关的转录本、蛋白质、转录因子及活性细胞分裂素显著上调，同时伴随细胞分裂相关的组织学、碳水化合物含量及遗传标志物特征。与之相反，非胚性愈伤组织则相较于胚性细胞团显著上调与刺激响应（ABA、细胞分裂素降解产物、酚类物质）、氧化应激（活性氧）及碳水化合物储存（淀粉）相关的转录本、蛋白质及代谢产物。亚网络富集分析（Sub-Network Enrichment Analyses）对胚性细胞团与非胚性愈伤组织间差异显著的转录本及蛋白质的功能与相互作用进行了揭示，进一步佐证了上述研究结果。
本研究证实了一种整合多维度转录组、蛋白质组、生物化学、组织学及解剖学分析的全新方法的实用性，该方法可用于解析与胚胎发生相关的分子事件，更具体地说，可用于解析花旗松（Douglas-fir）细胞的胚胎发生状态。