Functional analysis of phospholipase Dδ family in tobacco pollen tubes

Abstract

Phosphatidic acid (PA), important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD) but the isoforms responsible for production of plasma membrane PA were not identified yet and their functional roles remain unknown. Following genome-wide bioinformatic analysis of PLD family in tobacco, we focused on the pollen-overrepresented PLDδ class. Combining live-cell imaging, gene overexpression or knock-down, lipid-binding and structural bioinformatics, we characterized 5 NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane-bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Knock-down, overexpression and in vivo PA level analyses revealed isofom PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. PA promotes binding of PLDδ3 to the PM, thus creating a positive feedback loop, where PA accumulation leads to the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes, that are crucial for plant cell tip growth.