Hydroxyproline O-Arabinosylation in Pollen Fertility

Abstract

Sexual reproduction in flowering plants relies on the successful delivery and fusion of the male and female gametes. Ovules contain the female gametes and are located in the ovaries, which are embedded in the female reproductive tissue- collectively referred to as the pistil. The male gametes are transported through a specialized cell called the pollen tube, which must penetrate and traverse through the transmitting tract, which can be dense with carbohydrates secreted from the surrounding cells, or open- depending on the species. Therefore, successful fertilization relies on the pollen tube’s ability to grow, successfully navigate to the ovules, and burst to release the sperm cells once inside the ovule. While these physiological events are well-understood, the molecular mechanisms underlying key processes are still unclear.

Sperm delivery relies on the pollen tube’s ability to maintain the structural integrity of its cell wall throughout the growth process; it must be rigid enough to penetrate the female tissue and prevent premature rupture, but also extensible at the tip to allow for elongation. This is regulated by a multitude of factors that control the structural integrity of the cell wall, which include the synthesis and trafficking of cell wall materials, secretion, and the proper assembly, organization, and modification of these materials once they are deposited into the extracellular space. Many factors have been identified, but many key players and their roles have yet to be discovered.

This dissertation describes how we identified new factors that regulate pollen tube growth by influencing the structural and mechanical properties of the cell wall, by using a combination of genetic, molecular, and bioinformatic approaches. We characterized the cell wall structural defects caused by loss of protein O-arabinosylation in Arabidopsis pollen tubes which primarily included loss of cell polarity, increased bursting frequency, and decreased elongation rates which, in combination, caused poor fertility. We examined the pollen tube cell wall structure and discovered that loss of protein O-arabinosylation was associated with changes in the composition of the cell wall and the organization of its components which have important roles in regulating the mechanical properties of the cell wall. Through a forward genetics screen, we identified mutations in key secretory genes- including members of the exocyst complex- that suppressed the effects caused by loss of protein O-arabinosylation and improved pollen tube growth and fertility, and we interrogated the link between the secretory pathway and cell wall structure. We also identified a mutation in a gene involved in phosphoinositide (PI) signaling, which appears to be suppressing the effects caused by loss of O-arabinosylation through another pathway. By characterizing the effects of this suppressor mutation and learning more about the gene involved, we have identified another novel factor that regulates tip growth and cell wall structure in Arabidopsis pollen tubes. Our findings described herein demonstrate how we have contributed to the overall knowledge of the plant development and reproduction field by addressing how protein O-arabinosylation, secretion, and PI signaling pathways combine to influence the structural and mechanical properties of the cell wall.