Abstract
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.
Highlights
The pollen grain of flowering plants germinates on a receptive stigma and develops a pollen tube that grows through the pistil toward the ovules, serving as a courier that delivers the sperm cells to the embryo sac
THE EVOLUTION OF KEY Phosphatidic acid (PA) SIGNALING GENES SHOWS A HIGH DIVERSITY IN THE PLANT KINGDOM In order to elucidate the pattern of PA signaling in pollen tubes, we analyzed the evolution and gene expression of phospholipases D (PLD), diacylglycerol kinases (DGKs), and lipid phosphate phosphatases (LPPs), focusing on multicellular “higher” plants with cells that exhibit tip growth
EXPRESSION ANALYSIS OF ARABIDOPSIS GENES ENCODING PLDs, DGKs, AND LPPs Given that our work focuses on the role of PA in the regulation of pollen tube growth, we were interested in the expression levels of genes coding for PLD, DGK, and LPP in various plant organs as well as pollen
Summary
The pollen grain of flowering plants germinates on a receptive stigma and develops a pollen tube that grows through the pistil toward the ovules, serving as a courier that delivers the sperm cells to the embryo sac. Pollen tubes perform a specialized type of cell expansion called “tip growth” in which all growth occurs in a single region, the apical part of the tip-growing cell. Other cell types such as root hairs, fungal hyphae, and neuronal cells likewise elongate by tip growth, sharing conserved mechanisms that bring about this type of growth. Lipid signaling employs various messenger molecules that carry out communication between the plasma membrane, the endomembrane compartments, and the cytoplasm. This communication system is based on specific lipids as messengers and enzymes responsible for their production and degradation. In addition to PPIs, the simplest phospholipid, phosphatidic acid (PA), has emerged as an important signaling molecule that plays a fundamental role in a variety of biotic and abiotic stresses such as wounding, pathogen attack, salinity, drought, and cold
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