LINC-complex mediated positioning of the vegetative nucleus is involved in calcium and ROS signaling in Arabidopsis pollen tubes

Nuclear movement and positioning play a role in developmental processes throughout life. Nuclear movement and positioning are mediated primarily by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes are comprised of the inner nuclear membrane SUN proteins and the outer nuclear membrane (ONM) KASH proteins. In Arabidopsis pollen tubes, the vegetative nucleus (VN) maintains a fixed distance from the pollen tube tip during growth, and the VN precedes the sperm cells (SCs). In pollen tubes of <i>wit12</i> and <i>wifi</i>, mutants deficient in the ONM component of a plant LINC complex, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in <i>wit12</i> and <i>wifi</i> is due to decreased sensitivity to reactive oxygen species (ROS). Here, we show that <i>wit12</i> and <i>wifi</i> are hyposensitive to exogenous H₂O₂, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca²⁺ peaks in growing pollen tubes, which are disrupted in the <i>wit12</i> mutant. In the <i>wit12</i> mutant, nuclear Ca²⁺ peaks are reduced in response to exogenous ROS, but these peaks are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca²⁺ fluctuations.

细胞核运动与定位在生物体整个生命周期的发育进程中均发挥重要作用。该过程主要由核骨架与细胞骨架连接物（linker of nucleoskeleton and cytoskeleton, LINC）复合物介导，此类复合物由内核膜SUN蛋白与外核膜（outer nuclear membrane, ONM）KASH蛋白组成。在拟南芥花粉管的生长过程中，营养核（vegetative nucleus, VN）与花粉管尖端保持固定间距，且始终领先于精子细胞（sperm cells, SCs）。在植物LINC复合物外核膜组分功能缺陷的wit12与wifi突变体的花粉管中，精子细胞会领先于营养核生长，且营养核与尖端的固定间距消失。后续此类花粉管在接收信号时通常无法正常破裂。本研究旨在探究wit12与wifi突变体中观察到的花粉管接收缺陷，是否源于对活性氧（reactive oxygen species, ROS）敏感性的降低。本研究证实，wit12与wifi突变体对外源过氧化氢（H₂O₂）表现为低敏感性，且该低敏感性与营养核与花粉管尖端的邻近程度降低相关。此外，本研究首次报道了生长中花粉管内存在核钙离子峰，且该峰在wit12突变体中被破坏。在wit12突变体中，核钙离子峰在响应外源活性氧时的幅度降低，但此类钙离子峰与花粉管破裂并无关联。本研究发现，维持营养核与花粉管尖端的邻近关系，是花粉管同时响应外源活性氧与核内钙离子波动的必要条件。