Laboratory of Plant Cell Biology (BIOCEL), Department of Botany, Institute of Biosciences, University of Sao Paulo, 05422-970 Sao Paulo, Brazil.
Tree Physiol. 2014 Jan;34(1):94-104. doi: 10.1093/treephys/tpt102. Epub 2013 Dec 9.
Somatic embryogenesis is an important biotechnological tool in the large-scale propagation of elite genotypes and ex situ conservation of conifer species. Protocols for the induction and proliferation of embryogenic cultures (ECs) of Brazilian pine (Araucaria angustifolia (Bert.) O. Ktze) are well established, although the proper formation of mature somatic embryos (SEs) is still problematic. Thus, the identification of molecular markers for the screening of ECs able to respond to maturation conditions (abscisic acid and osmotic agents) is highly desirable. To develop molecular markers for the early detection of ECs able to develop well-formed SEs under maturation conditions, we analyzed the proteins found during the proliferation phase of A. angustifolia cell lines with different embryogenic capabilities, with one cell line being responsive to maturation conditions (R cell line), and one cell line that presented blocked development of SEs (B cell line). In addition, based on the peptides identified, polyamine levels (free and conjugate), ethylene production and reactive oxygen species (ROS) emission were analyzed using both EC lines (R and B cell lines). A marked difference in the biochemistry of ECs between these two cell lines was observed. Eleven proteins that were differentially expressed in the cell lines were identified by the combination of two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry. Among these, S-adenosylmethionine synthase, the enzyme associated with polyamines and ethylene biosynthesis, was observed exclusively in the R cell line, while a protein linked to the oxidative stress subunit F of NADH dehydrogenase was observed exclusively in the B cell lines. Additionally, B cell lines showed higher levels of diamine putrescine and lower levels of ethylene. Higher values of ethylene and ROS were observed for the cell line that showed normal development of SEs. Altogether, our results open new perspectives in the optimization of culture conditions for A. angustifolia somatic embryogenesis, as well as establishing biochemical markers for the early selection of ECs during maturation trials.
体胚发生是大规模繁殖优良基因型和异地保护松科植物的重要生物技术工具。巴西松（Araucaria angustifolia (Bert.) O. Ktze）的胚性培养物（ECs）的诱导和增殖方案已经建立，尽管成熟体胚（SEs）的适当形成仍然存在问题。因此，鉴定能够响应成熟条件（脱落酸和渗透剂）的 ECs 的分子标记物是非常理想的。为了开发用于筛选能够在成熟条件下形成良好 SEs 的 ECs 的分子标记物，我们分析了具有不同胚性能力的 A. angustifolia 细胞系在增殖阶段发现的蛋白质，其中一个细胞系对成熟条件有反应（R 细胞系），另一个细胞系的 SEs 发育受阻（B 细胞系）。此外，基于鉴定出的肽，使用这两个 EC 系（R 和 B 细胞系）分析了多胺水平（游离和结合）、乙烯产生和活性氧（ROS）的排放。观察到这两个细胞系之间的 ECs 生化性质存在明显差异。通过二维电泳（2-DE）和 MALDI-TOF/TOF 质谱联用技术组合，鉴定出这两个细胞系中 11 种差异表达的蛋白质。其中，S-腺苷甲硫氨酸合成酶是与多胺和乙烯生物合成相关的酶，仅在 R 细胞系中观察到，而与 NADH 脱氢酶氧化应激亚基 F 相关的蛋白质仅在 B 细胞系中观察到。此外，B 细胞系表现出更高水平的二胺腐胺和更低水平的乙烯。表现出 SEs 正常发育的细胞系的乙烯和 ROS 值更高。总的来说，我们的研究结果为优化 A. angustifolia 体细胞胚胎发生的培养条件提供了新的视角，并为成熟试验中 ECs 的早期选择建立了生化标记物。
