花粉管发育包括花粉萌发和花粉管生长，是开花植物将精细胞运送到胚囊完成双受精的关键过程。先前的研究表明，分泌囊泡的靶向运输及融合对于花粉管发育至关重要。在囊泡的运输和融合过程中，Ca²⁺信号是一个重要的调节因子。此外，SNAREs蛋白参与调节囊泡与质膜融合的最后阶段。但花粉管发育过程中Ca²⁺信号和SNAREs蛋白调控囊泡融合的分子机制目前仍不清楚。

为了探究花粉萌发过程中Ca²⁺信号与囊泡融合之间的调控机制，本研究利用GCaMP6作为 [Ca²⁺]_cyt指示剂对花粉的萌发过程进行观察，结果表明 [Ca²⁺]_cyt在预期萌发位点呈现周期性振荡。Ca²⁺通道抑制剂GdCl₃、Ca²⁺螯合剂EGTA和Ca²⁺激动剂A23187的处理以及Ca²⁺通道CNGC18点突变体cngc18-22均导致 [Ca²⁺]_cyt振荡异常，并影响花粉萌发，这些结果表明[Ca²⁺]_cyt的周期性振荡在花粉萌发过程中具有重要作用。进一步的研究发现，AtFH5标记的分泌囊泡在经历旋转样运动后停留并聚集在预期萌发位点的时间早于 [Ca²⁺]_cyt振荡。通过微丝解聚药物Latrunculin B (LatB) 处理或AtFH5突变破坏分泌囊泡在预期萌发位点的积累，均显著抑制 [Ca²⁺]_cyt振荡。相关性分析显示，AtFH5标记的分泌囊泡聚集在预期萌发位点的荧光信号强度与 [Ca²⁺]_cyt增加的峰值正相关。药理学实验和遗传学的研究结果表明，[Ca²⁺]_cyt振荡促进AtFH5标记的分泌囊泡向预期萌发位点胞吐，[Ca²⁺]_cyt增加的峰值与AtFH5标记的分泌囊泡的胞吐速率正相关。此外，[Ca²⁺]_cyt增加之后花粉粒预期萌发位点阶梯式凸起引起花粉粒形变，并在萌发位点处发生细胞壁增厚。这些结果揭示了[Ca²⁺]_cyt振荡与AtFH5标记的分泌囊泡相互依赖，即AtFH5标记的分泌囊泡的积累和聚集引起 [Ca²⁺]_cyt升高，[Ca²⁺]_cyt升高又促进AtFH5标记的分泌囊泡的胞吐，最终导致花粉粒建立极性并萌发出花粉管。

由于花粉粒体积大，难以细致观察囊泡动态，本研究选用花粉管这一模型进行囊泡融合分子机制的探究。本研究通过生物信息学分析发现SNAREs家族蛋白VAMP726在花粉中高表达，因此对VAMP726在花粉管中的功能进行了详细探究。结果显示，缺失VAMP726导致花粉管生长速率显著下降，花粉管显著变宽，这花粉管生长缺陷的表型能被mTurquoise2-VAMP726回补，过表达VAMP726同样显著抑制花粉管生长。vamp726-1花粉管生长受损的表型与fh5-3相似，fh5-3 vamp726-1双突变体的表型与单突变体一致，暗示VAMP726和AtFH5可能协同参与花粉管生长过程。采用酵母双杂交和萤火素酶互补实验进行蛋白相互作用分析，发现VAMP726与AtFH5相互作用。细胞生物学和遗传学实验结果也显示，VAMP726与AtFH5标记的分泌囊泡高度共定位；fh5-3突变体花粉管中VAMP726的质膜定位含量下降，进一步使用LatB处理导致质膜上定位的VAMP726含量降低；vamp726-1突变体花粉管中质膜上定位的AtFH5含量同样降低。这些结果表明，VAMP726与AtFH5在花粉管中的定位存在相互依赖性。细胞生物学实验表明，VAMP726缺失引起花粉管顶端微丝动态性降低并导致微丝积累，但体外生化实验表明缺失跨膜结构的截断蛋白VAMP726△TM不直接结合微丝。这一结果暗示VAMP726可能通过调控AtFH5的定位和功能进而影响微丝动态变化。以上结果表明VAMP726和AtFH5协同调控囊泡运输与微丝动态从而参与花粉管生长过程。

综上所述，本研究发现拟南芥花粉管发育过程中AtFH5标记的分泌囊泡依赖的 [Ca²⁺]_cyt振荡促进分泌囊泡与预期萌发位点质膜逐步融合，VAMP726与AtFH5直接互作进而调节花粉管生长过程中的囊泡融合和微丝动态。本研究揭示了花粉管发育过程中分泌囊泡与质膜融合的分子机制，为深入理解微丝骨架、[Ca²⁺]_cyt信号和囊泡融合参与花粉管发育过程提供了新的实验依据。

Pollen tube development includes pollen germination and pollen tube growth, which are key processes for transporting sperm cells to the embryo sac to complete the double fertilization of flowering plants. Previous studies have shown that the targeted trafficking and fusion of secretory vesicles are essential for the establishment and maintenance of cell polarity. Calcium is an important regulator during vesicle trafficking and fusion. In addition, SNARE proteins are involved in regulating the final stages of vesicle fusion with the plasma membrane. However, the underly mechanism between calcium signaling and SNAREs involved in vesicle fusion during pollen tube development is still unclear.

To explore the underlying mechanism between calcium signaling and vesicle fusion during pollen germination, we used GCaMP6 as a [Ca²⁺]_cyt indicator to observe the [Ca²⁺]_cyt dynamic during pollen germination, and the results showed that [Ca²⁺]_cyt oscillates periodically at the prospective germination site. Treatment with Ca²⁺ channel inhibitor GdCl3, Ca²⁺ chelator EGTA, Ca²⁺ agonist A23187 and Ca²⁺ ion channel mutant cngc18-22 of all caused abnormal [Ca²⁺]_cyt oscillation and affected pollen germination. These results suggested that the periodic oscillations of [Ca²⁺]_cyt played an important role in the process of pollen germination. In addition, we found that [Ca²⁺]_cyt oscillations are associated with rotation-like movements of AtFH5-mCherry labeled secretory vesicles labeled. Correlation analysis showed that the signal intensity of AtFH5-mCherry labeled secretory vesicle accumulation at the prospective germination site relative to the whole cell was positively correlated with the peak of [Ca²⁺]_cyt increase. Cell biological experiments showed that AtFH5-mCherry labeled secretory vesicles stopped and aggregated at the prospective germination site before [Ca²⁺]_cyt oscillation, and the aggregation of AtFH5-mCherry labeled secretory vesicles at the prospective germination site caused [Ca²⁺]_cyt increased. Disruption accumulation of secretory vesicles at the prospective germination site by treating with the microfilaments depolymerization drug Latrunculin B (LatB) or AtFH5 mutation significantly disturbs [Ca²⁺]_cyt oscillations. Pharmacological and genetics experiments suggested that [Ca²⁺]_cyt oscillations promoted the exocytosis of AtFH5-mCherry labeled secretory vesicles. Furthermore, it was found that the pollen grain deformation was caused by stepwise bulge after the increase of [Ca²⁺]_cyt and the cell wall thickness of the deformed pollen grains at the germination sites was thicker than that at the non-germination sites. These results revealed that the oscillations of [Ca²⁺]_cyt and AtFH5-mCherry labeled secretory vesicles coordinated. The accumulation and aggregation of AtFH5-mCherry labeled secretory vesicles caused [Ca²⁺]_cyt increase, in turn, increased [Ca²⁺]_cyt promoted the exocytosis of AtFH5-mCherry labeled secretory vesicles, and finally led to the stepwise bulge of pollen grains.

Due to the large size of pollen grains, it is harder to observe the dynamics of vesicles in detail. In our study, pollen tube was used as a model to explore the molecular mechanism of vesicle fusion. Through bioinformatics analysis, we revealed that SNAREs family protein VAMP726 was highly expressed in pollen, so the function of VAMP726 in pollen tubes was explored in detail. Our results showed that loss of VAMP726 resulted in a significant decrease in pollen tube growth rate and width, and mTurquiose2-VAMP726 was able to rescue this phenotype. Overexpression of VAMP726 similarly significantly inhibited pollen tube development. The impaired pollen tube development phenotype of vamp726-1 was similar to that of the AtFH5 mutant fh5-3, and the phenotype of fh5-3 vamp726-1 double mutant was similar to vamp726-1 and fh5-3, suggesting that VAMP726 and AtFH5 may be synergistically involved in the pollen tube development. Protein interaction analysis revealed that VAMP726 interacted with AtFH5 in yeast two-hybrid and luciferase complementation experiments. The results of cell biology and genetic experiments showed that VAMP726 and AtFH5-mCherry labeled secretory vesicles were highly co-localized in the cytoplasm and plasma membrane of pollen tubes. Plasma membrane localization of VAMP726 decreased in fh5-3 pollen tubes, and LatB treatment also resulted in decreased plasma membrane localization of VAMP726 in fh5-3 pollen tubes. The signal of AtFH5-mCherry at the plasma membrane was similarly reduced in the vamp726-1 mutant pollen tube. These results all suggested that VAMP726 and AtFH5 coordinate their localization in pollen tubes. Further cell biology experiments showed that VAMP726 mutation caused decreased apical microfilament dynamics of the pollen tube and resulted in microfilament accumulation. However, biochemical experiments in vitro showed that the truncated protein without transmembrane domain (VAMP726△TM) did not directly bind to microfilaments, suggesting that VAMP726 may affect the dynamic changes of microfilaments by regulating the localization and function of AtFH5. In conclusion, these results indicated that VAMP726 interacted with AtFH5 to regulate vesicle transport and microfilament dynamics to regulate pollen tube growth.

In summary, our study shows that AtFH5-mCherry labeled secretory vesicle dependent [Ca²⁺]_cyt oscillation promotes the stepwise fusion of secretory vesicles with the plasma membrane at the prospective germination site, and VAMP726 directly interacts with AtFH5 to regulate vesicle fusion and microfilament dynamics during pollen tube growth. Our study reveals the molecular mechanisms underlying the fusion of secretory vesicles with the plasma membrane during pollen tube development, and provides a new experimental basis for further understanding the involvement of microfilament skeleton, [Ca²⁺]_cyt signaling, and vesicle fusion in pollen tube development.