Vetrici Mariana A, Yevtushenko Dmytro P, Misra Santosh
Department of Biological Sciences University of Lethbridge Lethbridge AB Canada.
Centre for Forest Biology Department of Biochemistry & Microbiology University of Victoria Victoria BC Canada.
Plant Direct. 2021 Jul 29;5(7):e00333. doi: 10.1002/pld3.333. eCollection 2021 Jul.
Douglas-fir () is one of the world's premier lumber species and somatic embryogenesis (SE) is the most promising method for rapid propagation of superior tree genotypes. The development and optimization of SE protocols in conifers is hindered by a lack of knowledge of the molecular basis of embryogenesis and limited sequence data. In Arabidopsis, the () gene is a master regulator of embryogenesis that induces SE when expressed ectopically. We isolated the homologue from Douglas-fir, designated as . expression in somatic embryos and developing seeds demonstrated a unique, alternating pattern of expression with the highest levels during early stages of embryogenesis. PmLEC1 protein accumulation during seed development correlated with its transcriptional levels during early embryogenesis; however, substantial protein levels persisted until 2 weeks on germination medium. Treatment of mature, stratified seeds with 2,4-epibrassinolide, sorbitol, mannitol, or NaCl upregulated expression, which may provide strategies to induce SE from mature tissues. Sequence analysis of the gene revealed a 5' UTR intron containing binding sites for transcription factors (TFs), such as ABI3, LEC2, FUS3, and AGL15, which are critical regulators of embryogenesis in angiosperms. Regulatory elements for these and other seed-specific TFs and biotic and abiotic signals were identified within the locus. Most importantly, functional analysis of showed that it rescued the Arabidopsis null mutant and, in the T2 generation, led to the development of embryo-like structures, indicating a key role of in the regulation of embryogenesis.
花旗松（Pseudotsuga menziesii）是世界上主要的木材树种之一，体细胞胚胎发生（SE）是快速繁殖优良树木基因型最有前景的方法。针叶树中SE方案的开发和优化受到胚胎发生分子基础知识的缺乏和有限序列数据的阻碍。在拟南芥中，LEC1（LEAFY COTYLEDON1）基因是胚胎发生的主要调节因子，异位表达时可诱导体细胞胚胎发生。我们从花旗松中分离出LEC1同源基因，命名为PmLEC1。PmLEC1在体细胞胚胎和发育中的种子中的表达呈现出独特的交替模式，在胚胎发生早期表达水平最高。种子发育过程中PmLEC1蛋白的积累与其在胚胎发生早期的转录水平相关；然而，在萌发培养基上培养2周后，仍存在大量蛋白质。用2,4-表油菜素内酯、山梨醇、甘露醇或氯化钠处理成熟、分层的种子会上调PmLEC1的表达，这可能为从成熟组织诱导体细胞胚胎发生提供策略。对PmLEC1基因的序列分析揭示了一个5'UTR内含子，其中含有转录因子（TFs）如ABI3、LEC2、FUS3和AGL15的结合位点，这些转录因子是被子植物胚胎发生的关键调节因子。在PmLEC1基因座内鉴定出了这些和其他种子特异性转录因子以及生物和非生物信号的调控元件。最重要的是，PmLEC1的功能分析表明它拯救了拟南芥LEC1缺失突变体，并且在T2代中导致了胚状结构的发育，表明PmLEC1在胚胎发生调控中起关键作用。
