Ca2+和CLPs在替码板栗枝条芽体PCD信号转导途径中的级联反应研究

The apical-bud-senescence chestnut is characterized by the apical mixed bud of fruit bearing shoot, which usually senescence and abscission after flowering and the basal latent bud will develop to new fruit bearing shoot in next year. This has the advantages of facilitating intensive cultivation and increasing production. We had confirmed that PCD did occur in mixed bud of apical-bud-senescence chestnut, and what's more the type of PCD is vacuolar cell death rather than necrosis. Hormone were the control signals in mixed buds undergoing PCD of apical-bud-senescence chestnut, but the next signal transducer still remained unknown by now. In this study, ultracytochemical localization of Ca2+ and Ca2+-ATPase and CLPs activity were tested in the progress of mixed buds undergoing PCD in apical-bud-senescence chestnut, and then the interactive of Ca2+ , Ca2+-ATPase and CLPs in the buds of apical-bud-senescence chestnut Under Ca2+ Stress in order to further investigate the relationships between the Ca2+ and CLPs in mixed buds undergoing PCD of apical-bud-senescence chestnut.The aim of this experiment will be useful for study the whole signal transduction of buds in plants, and lay the foundation for artificial regulation of mixed bud senescence so as to remodel the development pattern of fruit branches of chestnut.

替码板栗具有枝条前段在果实成熟后自然衰亡的独特性状，其树体具有自然更新控冠、修剪省工的天然优势。前期研究表明，替码板栗枝条自然衰亡是由其上部芽体发生一种由激素参与的液泡途径细胞死亡类型PCD所导致，但对激素在芽体内部启动的下游Ca2+信号转导途径及级联反应尚不清楚。已知Ca2+、Ca2+-ATPase和CLPs在植物PCD信号转导途径中发挥重要作用。本项目以替码板栗处于PCD过程中的芽体为材料，通过电镜法检测胞内Ca2+、Ca2+-ATPase时空变化，解析二者在芽体PCD信号转导中的互作效应；特异底物比色法检测胞内Ca2+信号转导后相关CLPs（尤其VPEs）活性变化，解析二者在芽体PCD信号转导中的级联；Ca2+胁迫剂处理替码板栗芽体，检验胁迫条件下芽体PCD中Ca2+的级联应答效应。对进一步揭示植物芽体完整PCD信号转导途径具有重要意义，并对人为调控替码性状具有重要理论及应用价值。

替码板栗具有枝条前段在果实成熟后自然衰亡的独特性状，其树体具有自然更新控冠、修剪省工的天然优势。前期研究表明，替码板栗枝条自然衰亡是由其上部芽体发生一种由激素参与的液泡途径细胞死亡类型PCD所导致，但对激素在芽体内部启动的下游Ca2+信号转导途径及级联反应尚不清楚。本项目以替码板栗处于PCD过程中的芽体为材料，通过电镜法检测胞内Ca2+、Ca2+-ATPase时空变化，解析二者在芽体PCD信号转导中的互作效应；特异底物比色法检测胞内Ca2+信号转导后相关CLPs活性变化，解析二者在芽体PCD信号转导中的级联；Ca2+抑制剂处理替码板栗芽体，检验胁迫条件下芽体PCD中Ca2+的级联应答效应。结果表明：替码板栗芽体正常时，胞内Ca2+浓度较低；随着时间推移，胞外细胞间隙和细胞壁上的Ca2+进入胞内，并部分高浓度集聚于质膜、液泡膜和内质网膜上以及附近，同时质膜和液泡膜上Ca2+-ATPase活性增强，并且液泡内Ca2+含量增加，胞内Caspase—1、5、8、10活性升高；之后胞内质膜和液泡膜部分破裂，其上Ca2+-ATPase活性降低，Caspase-3、Caspase-6活性升高，导致细胞器开始降解；最后Ca2+沉淀在细胞器降解碎片上，膜上Ca2+-ATPase活性消失，胞内CLPs活性亦消失，PCD过程结束。该研究结果对进一步揭示植物芽体完整PCD信号转导途径具有重要意义，并对人为调控替码性状具有重要理论及应用价值。