Investigating Vesicle-Mediated Regulation of Pollen Tube Growth through BFA Inhibition and AS-ODN Targeting of TfRABA4D in Torenia fournieri

Abstract

In flowering plants, pollen tube growth is essential for delivering immotile sperm cells during double fertilization, directly influencing seed yield. This process relies on vesicle-mediated trafficking to drive tip growth and maintain fertility. However, investigating pollen tube growth is challenging in non-model plants due to the lack of transgenic tools. Here, we established a method to transiently inhibit vesicle activity in pollen tubes of the wishbone flower (Torenia fournieri), a classic plant for sexual reproduction studies, using brefeldin A (BFA) and antisense oligodeoxynucleotides (AS-ODNs) targeting key genes. BFA broadly disrupted vesicle gradient homeostasis in T. fournieri pollen tubes, leading to widespread changes in cell wall deposition, ROS distribution, and pollen tube morphology. To assess the role of specific genes, we designed AS-ODNs against TfANX, the sole ANXUR homolog in T. fournieri, which successfully penetrated cell membranes and suppressed TfANX expression. This inhibition impaired pollen tube tip growth, causing pollen tube leakage at the shank region and, in some cases, multiple leakages. Similarly, AS-ODN targeting TfRABA4D, a pollen-specific vesicle regulator, induced a bulging phenotype and disrupted pectin deposition and reduced ROS distribution, mirroring BFA effects. These findings elucidate vesicle-mediated regulation in pollen tube tip growth and introduce an accessible method for genetic manipulation in reproductive research of non-model plants.