【作者】 孔令安；

【导师】 汪矛；

【作者基本信息】 中国农业大学 ， 植物学， 2005， 博士

【摘要】 花粉管是种子植物受精过程中雄性生殖单位的载体,具有典型的极性顶端生长模式。近年来,花粉管极性生长机理的研究引起了人们极大的兴趣。根据现有的研究表明,蛋白磷酸酶参与了ABA、病原侵染、胁迫及发育信号转导途径,但是蛋白磷酸酶在花粉管极性生长中的作用机理还不清楚。本研究以裸子植物青杆（Picea wilsonii Mast.）花粉为材料,采用细胞学手段,应用电子显微镜、激光扫描共聚焦显微镜和显微红外光谱（FTIR）等技术,以对花粉萌发和花粉管生长起关键作用的Ca²⁺作为切入点,研究PP1和（或）PP2A（以下以PP1/PP2A表示）和Ca²⁺之间的关系,以及受Ca²⁺调节的下游生理过程,探讨蛋白质可逆磷酸化在花粉萌发及花粉管极性生长中的作用机理,同时也为探讨蛋白质可逆磷酸化在其他植物细胞中作用机理的研究提供重要的理论参考。 细胞化学定位证明,青杆花粉中含有丰富的Ca²⁺,液泡是最主要的Ca²⁺库;花粉萌发过程中伴随胞内Ca²⁺释放,而蛋白磷酸酶专一性抑制剂OA和CalA抑制这一释放过程。同时,纳摩尔浓度的OA和CalA明显抑制青杆花粉萌发以及花粉管的生长,并导致部分青杆花粉管畸形生长。 荧光染料Fluo-3标记发现,标准培养基培养的花粉管呈现典型的从顶端到基部的Ca²⁺梯度,而30 nmol/L OA或30 nmol/L CalA处理的花粉管（约75%）Ca²⁺梯度消失或梯度不明显,说明PP1/PP2A参与花粉管中Ca²⁺梯度的维持。同时研究发现,外源Ca²⁺使正常生长的Fluo-3标记的花粉管30秒内荧光强度明显增加,而OA或CalA处理的花粉管荧光强度增加缓慢,说明并同时首次证实青杆花粉管生长中顶端Ca²⁺内流受PP1/PP2A的调节。 电子显微镜下观察,正常生长的花粉管顶端以及亚顶端壁旁体（paramural body）与质膜的融合现象发生频繁,而OA或CalA处理的花粉管中融合事件稀少。生理测定证明,在花粉培养的同一时间,标准培养基中酸性磷酸酶活性高于含有抑制剂的培养基,说明PP1/PP2A调控花粉管生长过程中胞外分泌活性。FM4-64标记发现,OA或CalA处理的花粉管顶端小泡胞吞活性及胞吞方式发生了改变。本研究提出了花粉管生长中可能存在的胞吐/胞吞异位的特殊胞吞方式。 苯胺兰染色荧光显微镜下观察:标准培养基中培养的花粉管,胼胝质分布于顶端和亚顶端,含量较低;而OA或CalA处理的花粉管,胼胝质在顶端大量积累。用单克隆抗体JIM5、JIM7标记,激光扫描共聚焦显微镜下观察发现,标准培养基中培养的花粉管,酸性果胶质分布于整个花粉管壁,顶端含量较低;而OA或CalA处理的花粉管,酸性果胶质主要分布于顶端,并且其含量高于正常生长的花粉管。正常生长的花粉管,酯化果胶质分布于顶端细胞壁,而OA或Calm处理的花粉管,酯化果胶质分布于基部细胞壁。FTIR显微分析技术进一步证明了两种果胶质在花粉管顶端壁上相对含量的变化。 以上研究结果表明,青杆花粉萌发及花粉管生长受PP1/PP2A的严格调控。PP1/PP2A参与花粉培养过程中花粉细胞内Ca²⁺的释放可能是其调控花粉萌发机理之一;PP1/PP2A对青杆花粉管生长的调控是通过调节青杆花粉管胞外Ca²⁺的内流、细胞内Ca²⁺动态变化、然后调节胞吞/胞吐作用以及细胞壁的构建;Ca²⁺信号是这一生理事件的中心环节。 综合上述,本文总结了PP1/PP2A在青杆花粉管生长中可能的作用机理,并绘制出了模式图。更多还原

【Abstract】 Pollen tube, a carrier of sperm cell during the process of sexual reproduction in seed plants, exhibits a typical polarized tip growth, and so its growth attracts much attention from scientists. A great number of reports have demonstrated that protein phosphatases (PPases) are involved in transduction pathway for ABA, pathogen infection, stresses and developmental signals. While the roles of PPases in pollen tube growth remain unclear due to the limited evidence. Therefore, by means of cytobiological methods and FTIR analysis, we conducted the investigation with a view to revealing the mechanism underlying which PP1/PP2A regulate the pollen tube development.The results showed that pollens contain abundant Ca~2+, and vacuole was the most important Ca~2+ resource in the Picea wilsonii pollens. When pollens were cultured in standard medium, the cytoplasmic Ca~2+ level decreased. But the decrease was efficiently blocked by treatment with 30 nM OA or 30 nM CalA. At nanomolar concentrations, OA and CalA clearly inhibited pollen germination and tube growth and led to abnormal morphology of pollen tubes.Normally growing pollen tubes of P. wilsonii displayed a typical tip-focused cytosolic free Ca~2+ gradient, whereas almost half (~45%) of the 30 nM OA- or 30 nM CalA-treated pollen tubes emitted faint fluorescence with a relatively leveled cytosolic Ca2+ gradient from the tip to the base of the tube. In some (-35%) of the 30 nM OA- or 30 nM CalA-treated tubes, the Ca2+ gradient had completely dissipated. One of important findings in the present study was that exogenous Ca2+ rapidly enhanced fluorescence intensity in the apex of control pollen tubes, but much more slowly in inhibitor-treated pollen tubes, indicating that the PP1 and/or PP2A was involved extracellular calcium uptake.TEM observations indicated that the fusion of paramural bodies with plasma membranes occurred frequently in the tip and sub-tip regions of control pollen tubes, but the fusion rarely occurred in inhibitor-treated pollen tubes. FM4-64 labeling revealed that the endocytosis in tip region of pollen tube changed with the OA or CalA treatments, and a hypothesis is proposed to explain endocytotic mode in the growth of Picea wilsonii pollen tube.Callose was observed to be greatly accumulated in the tip regions of inhibitor-treated pollen tubes. The immunolabeling of pollen tubes revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present in the tip region and on the flanks of the sub-tip in normal pollen tubes. In inhibitor-treated pollen tubes, these epitopes existed only in the extreme tip region and at higher concentration than in control pollen tube. The esterified pectin recognized by JIM7 was located preferentially at the extreme tip region in normal pollen tubes, but at basal sites in inhibitor-treated tubes. FTIR analysis further confirmed the changes in both pectin distributions and their relative contents.These data strongly indicate that the PPland/or PP2A is essential to Ca~2+ dynamics, exocytotic activity, and the biosynthesis and deposition of cell wall components, and that Ca~2+ dynamics presumably play a central role in the PP1 and/or 2A-modulated series of events in pollen tube development.更多还原