Turnover of Phosphatidic Acid through Distinct Signaling Pathways Affects Multiple Aspects of Pollen Tube Growth in Tobacco.

Phosphatidic acid (PA) is an important intermediate in membrane lipid metabolism that acts as a key component of signaling networks, regulating the spatio-temporal dynamics of the endomembrane system and the cytoskeleton. Using tobacco pollen tubes as a model, we addressed the signaling effects of PA by probing the functions of three most relevant enzymes that regulate the production and degradation of PA, namely, phospholipases D (PLD), diacylglycerol kinases (DGKs), and lipid phosphate phosphatases (LPPs). Phylogenetic analysis indicated a highly dynamic evolution of all three lipid-modifying enzymes in land plants, with many clade-specific duplications or losses and massive diversification of the C2-PLD family. In silico transcriptomic survey revealed increased levels of expression of all three PA-regulatory genes in pollen development (particularly the DGKs). Using specific inhibitors we were able to distinguish the contributions of PLDs, DGKs, and LPPs into PA-regulated processes. Thus, suppressing PA production by inhibiting either PLD or DGK activity compromised membrane trafficking except early endocytosis, disrupted tip-localized deposition of cell wall material, especially pectins, and inhibited pollen tube growth. Conversely, suppressing PA degradation by inhibiting LPP activity using any of three different inhibitors significantly stimulated pollen tube growth, and similar effect was achieved by suppressing the expression of tobacco pollen LPP4 using antisense knock-down. Interestingly, inhibiting specifically DGK changed vacuolar dynamics and the morphology of pollen tubes, whereas inhibiting specifically PLD disrupted the actin cytoskeleton. Overall, our results demonstrate the critical importance of all three types of enzymes involved in PA production and degradation, with strikingly different roles of PA produced by the PLD and DGK pathways, in pollen tube growth.