Reverse Genetics Approach to Examine Myosin XI Functions in Pollen Tube Growth

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which non-motile sperm cells are delivered to ovules thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth depends on actin filaments. Plants encode class VIII and class XI myosins as actin-based motor proteins, of which class XI myosins are required for cell expansion in vegetative tissues.

In Arabidopsis thaliana, 6 of 13 myosin XI genes are expressed in pollen: XIA, XIB, XIC, XID, XIE, and XIJ. Initially, two artificial microRNA constructs were designed to target multiple class XI myosins; however, plants expressing the artificial microRNAs had no reduction in overall fertility and only a slight reduction in root hair elongation. Therefore, to explore the functions of individual pollen myosins, homozygous T-DNA insertion mutants were isolated for all six pollen myosin genes. Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, xic xie pollen tubes grew more slowly than WT pollen tubes, which resulted in reduced fitness compared to WT and a drastic reduction in seed set. Organelle motility was significantly reduced in xic xie pollen tubes; however, vesicle accumulation and actin filament dynamics were not altered in the double mutant. Thus, it remains unclear how reduced organelle motility in xic xie pollen tubes leads to a slower growth rate.

A novel role of myosin XI in pollen germination was also revealed in this study. Pollen from mutants of XIJ, the only short-tailed myosin XI in Arabidopsis, germinated poorly in vitro. This in vitro pollen germination defect was rescued by the addition of diffusible components from female tissues. Interestingly in pollen grains, YFP-XIJ and YFP-XIA localized to the future site of germination, suggesting a role in pollen germination for multiple myosin XI isoforms. In summary, this study provided the first direct evidence that class XI myosins are involved in pollen tube growth and pollen germination.