Functional analysis of phospholipase Dδ family in tobacco pollen tubes

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 PLDdelta class. Combining live-cell imaging, gene overexpression, lipid-binding and structural bioinformatics, we characterized 5 NtPLDdelta isoforms. Distinct PLDdelta isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane-bound PLDdeltas are tightly bound to PM, primarily via the central catalytic domain. Overexpression analyses suggested isoform PLDdelta3 as the most important member of the PLDdelta subfamily active in pollen tubes. Moreover only PLDdelta3 shows significant constitutive PLD activity in vivo and in turn, PA promotes binding of PLDdelta3 to the PM. This forms a positive feedback loop leading to PA accumulation and the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDdelta3 at the PM is important for maintaining the balance between various membrane trafficking processes, that are crucial for plant cell tip growth.