Teun Munnik

Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase

Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using 32P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid 32P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e., (1) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), (2) via phospholipase D hydrolysis of structural phospholipids, or (3) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential 32P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid 32P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of 32P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in 32P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in 32P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1, and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

PHOSPHOLIPID SIGNALLING IN PLANTS

book, 1996, p. 61. w x 424 H. Pfaffmann, E. Hartmann, A.O. Brightman, D.J. Morre, Ž . Plant Physiol. 85 1987 1151–1155. w x 425 C. Pical, A.S. Sandelius, P.M. Melin, M. Sommarin, Plant Ž . Physiol. 10

Phosphatidic acid : an emerging plant lipid second messenger

Evidence is accumulating that phosphatidic acid is a second messenger. Its level increases within minutes of a wide variety of stress treatments including ethylene, wounding, pathogen elicitors, osmotic and oxidative stress, and abscisic acid. Enhanced signal levels are rapidly attenuated by phosphorylating phosphatidic acid to diacylglycerol pyrophosphate. Phosphatidic acid is the product of two signalling pathways, those of phospholipases C and D, the former in combination with diacylglycerol kinase. Families of these genes are now being cloned from plants. Several downstream targets of phosphatidic acid have been identified, including protein kinases and ion channels.

A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis

Here we report on a lipid‐signalling pathway in plants that is downstream of phosphatidic acid and involves the Arabidopsis protein kinase, AGC2‐1, regulated by the 3′‐phosphoinositide‐dependent kinase‐1 (AtPDK1). AGC2‐1 specifically interacts with AtPDK1 through a conserved C‐terminal hydrophobic motif that leads to its phosphorylation and activation, whereas inhibition of AtPDK1 expression by RNA interference abolishes AGC2‐1 activity. Phosphatidic acid specifically binds to AtPDK1 and stimulates AGC2‐1 in an AtPDK1‐dependent manner. AtPDK1 is ubiquitously expressed in all plant tissues, whereas expression of AGC2‐1 is abundant in fast‐growing organs and dividing cells, and activated during re‐entry of cells into the cell cycle after sugar starvation‐induced G1‐phase arrest. Plant hormones, auxin and cytokinin, synergistically activate the AtPDK1‐regulated AGC2‐1 kinase, indicative of a role in growth and cell division. Cellular localisation of GFP‐AGC2‐1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The agc2‐1 knockout mutation results in a reduction of root hair length, suggesting a role for AGC2‐1 in root hair growth and development.

Phospholipase D Activation Correlates with Microtubule Reorganization in Living Plant Cells

A phospholipase D (PLD) was shown recently to decorate microtubules in plant cells. Therefore, we used tobacco BY-2 cells expressing the microtubule reporter GFP-MAP4 to test whether PLD activation affects the organization of plant microtubules. Within 30 min of adding n-butanol, a potent activator of PLD, cortical microtubules were released from the plasma membrane and partially depolymerized, as visualized with four-dimensional confocal imaging. The isomers sec- and tert-butanol, which did not activate PLD, did not affect microtubule organization. The effect of treatment on PLD activation was monitored by the in vivo formation of phosphatidylbutanol, a specific reporter of PLD activity. Tobacco cells also were treated with mastoparan, xylanase, NaCl, and hypoosmotic stress as reported activators of PLD. We confirmed the reports and found that all treatments induced microtubule reorganization and PLD activation within the same time frame. PLD still was activated in microtubule-stabilized (taxol) and microtubule-depolymerized (oryzalin) situations, suggesting that PLD activation triggers microtubular reorganization and not vice versa. Exogenously applied water-soluble synthetic phosphatidic acid did not affect the microtubular cytoskeleton. Cell cycle studies revealed that n-butanol influenced not just interphase cortical microtubules but also those in the preprophase band and phragmoplast, but not those in the spindle structure. Cell growth and division were inhibited in the presence of n-butanol, whereas sec- and tert-butanol had no such effects. Using these novel insights, we propose a model for the mechanism by which PLD activation triggers microtubule reorganization in plant cells.

Water Deficit Triggers Phospholipase D Activity in the Resurrection Plant Craterostigma plantagineum

Phospholipids play an important role in many signaling pathways in animal cells. Signaling cascades are triggered by the activation of phospholipid cleaving enzymes such as phospholipases C, D (PLD), and A2. Their activities result in the formation of second messengers and amplification of the initial signal. In this study, we provide experimental evidence that PLD is involved in the early events of dehydration in the resurrection plant Craterostigma plantagineum. The enzymatic activity of the PLD protein was activated within minutes after the onset of dehydration, and although it was not inducible by abscisic acid, PLD activity did increase in response to mastoparan, which suggests a role for heterotrimeric G proteins in PLD regulation. Two cDNA clones encoding PLDs, CpPLD-1 and CpPLD-2, were isolated. The CpPLD-1 transcript was constitutively expressed, whereas CpPLD-2 was induced by dehydration and abscisic acid. Immunological studies revealed changes in the subcellular localization of the PLD protein in response to dehydration. Taken together, the data on enzymatic activity as well as transcript and protein distributions allowed us to propose a role for PLD in the events leading to desiccation tolerance in C. plantagineum.

Phospholipid signalling in plant defence

Phospholipid-derived molecules are emerging as novel second messengers in plant defence signalling. Recent research has begun to reveal the signals produced by the enzymes phospholipase C, phospholipase D and phospholipase A2 and their putative downstream targets. These include the activation of a MAP kinase cascade and triggering of an oxidative burst by phosphatidic acid; the regulation of ion channels and proton pumps by lysophospholipids and free fatty acids; and the conversion of free fatty acids into bioactive octadecanoids such as jasmonic acid.

Plant phospholipid signaling: “in a nutshell”

Since the discovery of the phosphoinositide/phospholipase C (PI/PLC) system in animal systems, we know that phospholipids are much more then just structural components of biological membranes. In the beginning, this idea was fairly straightforward. Receptor stimulation activates PLC, which hydrolyses phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)P2] into two second messengers: inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DG). While InsP3 difuses into the cytosol and triggers the release of calcium from an internal store via ligand-gated calcium channels, DG remains in the membrane where it recruits and activates members of the PKC family. The increase in calcium, together with the change in phosphorylation status, (in)activates a variety of protein targets, leading to a massive reprogramming, allowing the cell to appropriately respond to the extracellular stimulus. Later, it became obvious that not just PLC, but a variety of other phospholipid-metabolizing enzymes were activated, including phospholipase A, phospholipase D, and PI 3-kinase. More recently, it has become apparent that PtdIns4P and PtdIns(4,5)P2 are not just signal precursors but can also function as signaling molecules themselves. While plants contain most of the components described above, and evidence for their role in cell signaling is progressively increasing, major differences between plants and the mammalian paradigms exist. Below, these are described “in a nutshell.”

The Arabidopsis Phosphatidylinositol Phosphate 5-Kinase PIP5K3 Is a Key Regulator of Root Hair Tip Growth

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] functions as a site-specific signal on membranes to promote cytoskeletal reorganization and membrane trafficking. Localization of PtdIns(4,5)P2 to apices of growing root hairs and pollen tubes suggests that it plays an important role in tip growth. However, its regulation and mode of action remain unclear. We found that Arabidopsis thaliana PIP5K3 (for Phosphatidylinositol Phosphate 5-Kinase 3) encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P2, that is preferentially expressed in growing root hairs. T-DNA insertion mutations that substantially reduced the expression of PIP5K3 caused significantly shorter root hairs than in the wild type. By contrast, overexpression caused longer root hairs and multiple protruding sites on a single trichoblast. A yellow fluorescent protein (YFP) fusion of PIP5K3, driven by the PIP5K3 promoter, complemented the short-root-hair phenotype. PIP5K3-YFP localized to the plasma membrane and cytoplasmic space of elongating root hair apices, to growing root hair bulges, and, notably, to sites about to form root hair bulges. The signal was greatest in rapidly growing root hairs and quickly disappeared when elongation ceased. These results provide evidence that PIP5K3 is involved in localizing PtdIns(4,5)P2 to the elongating root hair apex and is a key regulator of the machinery that initiates and promotes root hair tip growth.

An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be natures preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.