H2O2调控水曲柳体胚发生中细胞程序性死亡的机理

Plant programmed cell death (PCD), especially PCD in plant somatic embryogenesis was paid great attention worldwide. In earlier research, we found that addition of high concentration of sucrose to medium increased somatic embryo (SE) induction of Fraxinus mandshurica. The browning of cotyledon explants during SE induction typically anticipates the entry of cells into embryogenesis. The programmed cell death in cells of browning cotyledon explants was assessmented. The occurrence of H2O2 burst anticipates the entry of cells into PCD. Thus we speculate that reactive oxygen (ROS) released was involved in the induction of PCD during SE induction. However, ROS regulatory mechanism in induced programmed cell death by exogenous carbohydrates during somatic embryogenesis of F. mandshurica has been questioned. In this research, we will investigat the involvement of the H2O2 and NO in the signal transduction of PCD triggered by these exogenous carbohydrates during SE induction of F. mandshurica. We will investigat the roles of the H2O2 and NO in PCD during SE induction of F. mandshurica and further explore the Intracellular biosynthetic pathway of NO in explant. On the basis of above research results, we could elucidate the involvement of H2O2 regulatory mechanism of PCD during somatic embryogenesis of F. mandshurica, determine involvement of the key signaling molecules and regulatory mechanism during SE induction of F. mandshuric. The results of this study will supply experimental evidence to further elucidate PCD regulatory mechanism during plant cell totipotency express.

植物细胞程序性死亡（PCD），尤其是植物体胚发生中的PCD现象受到国内外研究者热切关注。前期研究中发现培养基中较高蔗糖浓度有利于水曲柳体胚的诱导，外植体褐变现象可作为水曲柳体胚产生的早期标志性事件，褐变外植体发生PCD之前细胞内H2O2含量显著增加。因此推测活性氧（ROS）参与了水曲柳体胚发生中外植体PCD的激活。但对ROS调控外植体PCD的关键信号分子和机理尚不清楚。本研究将分析外源糖类诱导水曲柳体胚发生中外植体细胞PCD中涉及到的细胞内H2O2以及NO的信号转导，分析H2O2和NO在水曲柳外植体细胞PCD和体胚发生中的作用，并进一步探索水曲柳体胚发生中外植体细胞内NO的合成途径。在上述研究结果的基础上，分析总结H2O2调控水曲柳体胚发生中外植体PCD的机理，阐述水曲柳体胚发生中PCD的关键信号分子及其调控作用。研究结果将为深入了解植物细胞全能性表达中PCD的调控机制提供科学依据

本项目以我国东北地区重要的珍贵阔叶树种水曲柳为研究对象，通过组织培养手段重点研究了以水曲柳成熟合子胚为外植体的体细胞胚诱导，并对水曲柳体细胞胚胎发生的生物化学进行了分析，在此基础上分析了外源过氧化氢和外源一氧化氮对水曲柳体细胞胚胎发生以及外植体细胞程序性死亡、外植体细胞内活性氧代谢和一氧化氮合成的调控作用，并对水曲柳体细胞胚胎发生中一氧化氮信号的合成途径进行了初步探讨。研究发现：（1）H2O2和NO在水曲柳体细胞胚胎发生中起着重要的作用。适当增加外植体细胞内H2O2含量有利于体细胞胚诱导，增加外植体细胞内NO含量有利于体细胞胚胎发生和发育。在应用中，可利用低浓度H2O2和外源NO供体处理外植体以获得更高的体细胞胚诱导率和更多高质量体细胞胚；（2）活性氧爆发可认为是PCD的前兆。细胞内H2O2含量增加可诱导PCD的发生。在胚性细胞向体细胞胚分化发生的阶段、球胚大量出现的阶段和体细胞胚趋向形态成熟阶段均发生了外植体细胞死亡量的增加；（3）H2O2和NO均参与了水曲柳体细胞胚胎发生过程中的外植体细胞死亡过程，且与外植体细胞的PCD关系密切，可以作为水曲柳体细胞胚胎发生中外植体细胞PCD的指示性信号，NO信号位于H2O2上游；外植体细胞内NO/H2O2的平衡影响体细胞胚胎发生结果；（4）水曲柳体细胞胚胎发生中内源NO由一氧化氮合酶和硝酸还原酶两种途径共同合成。二者起到的主导作用时间节点和对体细胞胚胎发生的功能不相同：在体细胞胚诱导培养早期阶段，NOS合成途径起主导作用；在大量非胚性向胚性细胞转化阶段，NR合成途径起主导作用。研究结果阐述了H2O2调控水曲柳体胚发生中外植体PCD的机理，可为阐述水曲柳体细胞胚胎发生调控机理提供理论和实验依据。