双受精作用作为开花植物特有的一种受精方式，对于植物的有性生殖以及增加子代适应性方面具有重要意义。与动物精子不同，开花植物的精细胞不能自由移动，需要借助花粉管的极性生长才能运输至胚囊，从而完成双受精。在花粉中，两个精细胞与营养核连接组成雄性生殖单位，以整体的方式运输。虽然前人利用固定染色和基因枪瞬时转化微管结合蛋白的方法观察到雄性生殖单位周围的微管骨架结构，并通过药理学实验初步揭示了微管骨架系统可能参与雄性生殖单位的迁移，但是其具体作用方式还有待深入研究。此外，花粉中也存在大量的微丝骨架，目前对于两种细胞骨架的互作关系及其在雄性生殖单位迁移中的作用还未见报道。

本研究通过大量筛选微管蛋白和微管结合蛋白的亚细胞定位，首次获得了稳定的花粉微管活体荧光标记材料。利用该材料以及微丝和雄性生殖单位的双色或三色荧光标记材料，对花粉萌发以及花粉管生长过程中微管、微丝和雄性生殖单位的时空动态关系进行了系统观察：在花粉萌发过程中，微管组装起始于营养核和精细胞周围，形成笼状结构将雄性生殖单位稳定在花粉中央区域，而微丝在细胞质膜起始组装，沿花粉周缘旋转式运动；当微丝在花粉萌发位点形成刷状环结构时，微管及其包裹的营养核向萌发位点相反方向位移。在快速生长的花粉管中，营养核前缘与微管保持长时间接触，引领精细胞稳定地向花粉管顶端迁移，而只与微丝发生间断的共定位。药理学实验结果表明，微丝解聚后恢复的过程是沿着微管进行的，并且微管对于微丝的旋转式运动起阻碍作用。这些结果表明微管和微丝在花粉中存在紧密的互作关系，二者可能协同参与雄性生殖单位的迁移。

为了进一步解析微管和微丝骨架系统在雄性生殖单位迁移中的功能，分别使用特异性抑制剂对花粉进行处理。与对照组相比，解聚微管后，花粉粒中雄性生殖单位位移幅度明显增大，甚至能够移动到花粉边缘位置。快速生长的花粉管中营养核不再维持在距花粉管顶端一定距离的位置，表现出更加快速的不规则往复运动。这些表型在TUB1、TUB4和TUB9功能缺失的三突变体中得到了验证，并且抑制驱动蛋白活性后花粉管中营养核迁移的表型与微管解聚后的表型相似。以上结果表明，微管和驱动蛋白对于雄性生殖单位/营养核的精细定位和稳定的定向运输是必不可少的。微管解聚后恢复的实验显示，营养核只有在接触到重新聚合的微管后，才能从剧烈的往复运动变为相对稳定的向前运动，并迅速移动到距花粉管顶端相对稳定的位置，表明微管骨架为营养核稳定的定向迁移提供轨道。与解聚微管后观察到的结果不同的是，解聚微丝后，花粉粒中营养核位移幅度明显减小，花粉管的生长和雄性生殖单位的运动很快停止。抑制肌球蛋白活性后，花粉粒中营养核位移幅度明显减小，花粉管中营养核正负向运动速度和幅度均显著减小，但并不改变其前缘沿微管向花粉管顶端运动的模式，表明微丝骨架系统为雄性生殖单位的运动提供主要驱动力。

基于上述结果，本论文提出了微丝-肌球蛋白系统为雄性生殖单位/营养核迁移提供主要驱动力、微管-驱动蛋白系统为雄性生殖单位/营养核运输提供主要轨道并稳定其定向迁移的工作模型，为深入理解花粉雄性生殖单位迁移的分子机制提供了新的实验证据，对于认知植物细胞中的细胞器运动和植物有性生殖过程具有重要的科学意义。

Double fertilization is a unique and feature-defining process of flowering plants. Unlike animals, whose sperm cells travel through self-propelled flagellum, flowering plants rely on the polarized growth of pollen tubes to deliver sperm cells to the embryo sac for double fertilization. Typically, the two sperm cells are physically connected, through which with the pollen vegetative nucleus (VN) via cytoplasmic projection, they form the male germ unit (MGU) and co-migrate in pollen tube. Although previous studies showed that the microtubule bundles around MGU were labeled by indirect immunofluorescence assays and transient transformation of GFP-tagged AtEB1c and inhibition of microtubule polymerization caused severe impairment of MGU migration. These results implied that microtubule network played a role in MGU migration. Despite of crucial roles for plant reproduction, the mechanisms underlying the migration of MGU have not been well understood so far. Pollen tubes also have extensive actin filaments. However, the molecular mechanisms of MGU migration mediated by microtubule and actin filaments have not been studied so far.

In this study, we developed a fluorescently tagged marker line for visualizing microtubules in pollen and investigated the dynamic interplays among microtubules, actin filaments and MGUs. This study reported that shortly after hydration, the two cytoskeleton systems initiated their assembly at different sites: microtubules rapidly assembled around MGU with ring- and glasses-shaped series structures, whereas the strongest fluorescence of actin filaments appeared near the plasma membrane. Before germination, actin filaments rotated mainly on the edge of pollen grains. In contrast, microtubules formed a cage-like meshwork, located in the relatively central region and did not rotate with the actin filaments. When actin filaments developed the collar-like construction at the future germination site, the cage-like network of microtubules and VN contracted behind the actin structure, opposite to the future site of pollen tube outgrowth. After the emergence of pollen tubes, actin filaments entered the tube first, then microtubules followed. In fast growing pollen tubes, the leading edge of VN was always contacted with microtubules followed by SCs, whereas the front region of VN was only occasionally co-localized with actin filaments. These results suggested that microtubules and actin filaments might play distinct roles in MGU migration in pollen.

To dissect the function of microtubules in MGU migration, we used inhibitor of microtubules polymerization to fully depolymerize microtubules in pollen. The VN was able to travel to the edge region of pollen grain during pollen germination, resulting in a significantly increased maximal movement distance compared to that of control. During pollen tube elongation, inhibition of microtubule polymerization caused an increased velocity and magnified amplitude of VN bidirectional movement and an unfixed distance of VN to the tube tip. These results were verified by the loss-of-function mutations of tub1 tub4 tub9. Pharmacological data further revealed that inhibition of kinesin activities resulted in impaired VN movement similar with oryzalin treatment. These data suggested that microtubules were essential for maintaining the fine-positioning and the steady movement of VN. Consistently, the wash-out experiment showed that during microtubule recovery, the VN moved back and forth in the pollen tube before it got in touch with the microtubules. As long as the tip of the VN came into a contact with microtubules, it started a steady forward movement and returned to the front position of pollen tube. These data supported important roles of microtubules cytoskeleton served as the track for the VN directional transport in pollen tubes.

To explore the role of actomyosin in MGU migration, inhibitors of actin polymerization and myosin activities were applied in pollen, respectively. The backward movement of the VN during pollen germination was markedly inhibited in the presence of either inhibitor. After LatB treatment, the growth of pollen tube was halted soon, and VN stopped moving. Inhibition of myosin activities resulted in decreased velocity and amplitude of VN bidirectional movement without moving pattern change.

Based on these data, we propose that the successful delivery of MGUs requires an elaborate cooperation between the actin cytoskeleton and microtubule system: actomyosin provides the major driving force for MGU migration, and microtubule and kinesins fine-tune the directional movement of MGU in pollen. These data not only provide important evidence for the regulatory mechanism of MGU migration, but also promote our understanding of the sexual reproduction process in flowering plants.