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 sustain tip growth and fertility. However, investigating pollen tube growth is challenging in non-model plants due to the lack of transgenic tools. Here, we developed 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.

Acknowledgements

The authors gratefully acknowledge financial support from the MEXT Scholarship with Embassy Recommendation via the China Scholarship Council to X.J. This work was also supported by grants from the Japan Society for the Promotion of Science (21K15119 to A.G.M., 22H05172, 22H05178 to S.O., and 21K18235, 22H04980, 22K21352 to T.H.) and the Japan Science and Technology Agency (JPMJCR20E5 to T.H.).

Author contributions

X.J., A.G.M., S.O., and T.H. designed the project. X.J. and A.G.M. performed most of the cytology experiments. X.J. conducted the bioinformatics analysis. X.J., S.O., and T.H. analyzed the data. X.J. and T.H. drafted the manuscript.

Data availability

The sequences of the genes used in this study were listed in Table S2 and can be accessed in the T. fournieri cDNA database (http://dandelion.liveholonics.com/torenia/).

Conflict of interests

The authors declare that they have no conflicts of interest to report.

Supplementary information

Supplementary data is available at Horticulture Research online.

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