Zhu Jingen, Wu Xiaorong, Yuan Shunjie, Qian Dong, Nan Qiong, An Lizhe, Xiang Yun
MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China.
PLoS One. 2014 Jul 14;9(7):e102407. doi: 10.1371/journal.pone.0102407. eCollection 2014.
The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative "linker" between Ca2+ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of Arabidopsis annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca2+ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of Ann5-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of Ann5 promoted Ca2+-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of Ann5 did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca2+-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.
花粉发育和花粉管生长的调控是一个复杂的生物学过程，对开花植物的有性生殖至关重要。膜联蛋白广泛分布于原生生物到高等真核生物中，并通过充当Ca2+信号、肌动蛋白细胞骨架和膜之间的假定“连接物”，在众多细胞事件中发挥多种作用，而这些对于花粉发育和花粉管生长是必需的。我们最近的报告表明，转基因Ann5-RNAi系中拟南芥膜联蛋白5（Ann5）功能的下调导致花粉粒严重不育。然而，关于Ann5在花粉中功能的潜在机制知之甚少。本研究表明，Ann5与磷脂膜结合，并且这种结合在体外受到Ca2+的刺激。布雷菲德菌素A（BFA）干扰内膜运输并抑制花粉萌发和花粉管生长。Ann5过表达植物的花粉萌发和花粉管生长对BFA处理均表现出增强的抗性，并且这种效应受钙调节。Ann5的过表达促进了体内花粉管中响应BFA的Ca2+依赖性细胞质流动。Latrunculin（LatB）通过与单体肌动蛋白高亲和力结合并优先靶向动态肌动蛋白丝阵列并阻止肌动蛋白聚合，从而显著抑制花粉萌发和花粉管生长。与野生型相比，Ann5的过表达对LatB响应的花粉萌发或花粉管生长没有影响，尽管Ann5以类似于某些动物膜联蛋白的方式与肌动蛋白丝相互作用。此外，与野生型Ann5完全恢复相比，Ann5在Ca2+结合位点的突变体只能部分挽救不育花粉表型。这些数据表明，Ann5通过促进钙调节的内膜运输参与花粉发育、萌发和花粉管生长。我们的结果为Ann5在花粉中的功能提供了可靠的分子机制。
