The role of phospholipase C in pollen tube growth

by Dowd, Peter E.

Upon germination on a stigmatic surface, a pollen grain produces a tip-growing pollen tube that must grow down the style, locate the ovary and deliver the sperm to effect fertilization. Thus, pollen tube elongation represents regulated tip growth required for fertility. Tip growth is known to be driven by exocytosis localized to the growing apex, and one element thought to direct this secretory activity is a tip-focused Ca2+ gradient. Only a few regulators of tip growth have been identified in pollen tubes, e.g., the Rop family of G-proteins and their interacting proteins. Despite the role played by the pollen tube in plant reproduction, regulators that direct pollen tube growth and/or modulate the tip-focused Ca2+ gradient are not well characterized. Membrane lipid turnover is a regulatory theme in eukaryotic cells. For instance, the phosphatidylinositol-specific phospholipase Cs (PI-PLCs) process the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdInsP2) to produce diacyl glycerol, an activator of protein kinase C, and the Ca 2+mobilizing agent, inositol trisphosphate (InsP3). In pollen tubes, it has been proposed that PtdInsP2 compartmentalization mediated by PI-PLC is critical for the control of pollen tube elongation. Similarly, InsP3 has been proposed as a regulator of this process, as application of InsP3 has been shown to alter pollen tube growth. However, in all cases the molecular identity of the PLC(s) involved remains undetermined. The major goal of this thesis research is thus to isolate cDNA for a pollenexpressed PI-PLC of Petunia inflata that is involved in regulating pollen tube growth. A gene, named PetPLC1, was identified, and recombinant PetPLC1 produced in E. coli was found to have Ca2+-dependent PI-PLC activity expected of a membrane-bound PI-PLC. iv Expressing a catalytically inactive form of PetPLC1, named PetPLC1-H126A, in pollen tubes caused expansion of the apical Ca2+ gradient, disrupted the organization of the actin cytoskeleton, and delocalized growth at the tube tip. These effects were phenocopied by expressing the C2 domain of PetPLC1 alone in pollen tubes. Moreover, these phenotypes were suppressed by depolymerizing actin with low concentrations of Latrunculin B, suggesting that a critical site of action of PetPLC1 is in regulating actin structure at the growing tip. A GFP fusion of PetPLC1 expressed in pollen tubes were found to cycle on and off the apical plasma membrane in growing pollen tubes from a pool of vesicles that co-localized with the membrane dye, FM4-64. It is likely that PetPLC1 uses these vesicles as a way of locating to/from the apical membrane. A GFP fusion of PetPLC1-H126A, also localized to the apical plasma membrane until apical expansion of the pollen tube tip occurred. A GFP fusion to the PH (PtdInsP2)-binding domain of mammalian PLC?1 showed enrichment in apical regions depleted in PLC. Thus, PetPLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell. v