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RESEARCH
My lab is interested in the role of phospholipids in plant cell Signalling and Development. Especially, phosphatidic acid (PA), DGPP and polyphosphoinositides, like PtdInsP and PtdInsP2, have our interest. These molecules are present at very low concentrations in cellular membranes and rountinely missed by HPLC and MS analyses, simply because structural lipids, such as PC, PE, PI and PG, make up the mass of the membrane. Nonetheless, these minor signalling lipids can easily be picked up by 32P-labelling (see TECHNIQUES), because their turnover is much faster than structural phospholipids and because they are directly labelled by ATP via specific lipid kinases. The key players involved in their metabolism are depicted in a 'simple' cartoon. In short, this involves the so-called PLC-and PLD signalling cascades. They both generate PAbut this occurs, very likely, at different locations and has different functions (see ref 27, 45, 64 68)
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Plant Stress
Plants cannot run away! Instead, over millions of years of evolution, they have 'developed' smart strategies to be able to quickly respond and adapt to sudden environmental changes. The 'stress' plants usually encounters can be divided in biotic (i.e. pathogens, herbivores) and abiotic stress (i.e. temperature, UV radiation, oxidative stress, water stress). Our main interest is to study the role of phospholipid signals during temperature stress (cold, heat), water stress (drought, salinity and hypoosmotic stress) and during plant-pathogen interactions (see below).
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32P-labelling and TLC
Phospholipids are routinely labelled in vivo using 32P-orthophosphate. In general, cells-, seedlings- or leaf discs are used and labelling times vary from min to hrs or even O/N, depending on the type of experiment (e.g. turnover, mass levels).To monitor PLD, transphosphatidylation assays can be conducted using primary alcohols (ref 9, 18). Shown is an example using the green alga Chlamydomonas. Cells were labelled for 4 hrs and then stimulated for 5 min in the presence of a low concentration of alcohol that the cells don't mind. Lipids were then extracted and chromatographed using a Ethylacetate TLC system that separates phosphatidylalcohols from other phospholipids (panel a). Autoradiography visualises their positions while PhosphoImaging is used to quantify the individual spots. In this way, in vivo activation of PLD with mastoparan was shown, resulting in increased phosphatidylalcohol spots.
Using a similar protocol for time-course experiments, the timing and duration of PLD stimulation can be established. However, samples can also be used to visualise the sequential activities of PLC, DAG kinase and PA kinase. The lipids are now chromatographed in an Alkaline TLC system that separates most phospholipids (panel b). Note the dramatic hydrolysis of PtdInsP2 within 15 sec to produce DAG that is immediately converted to PA by DAG kinase. PI- and PIP-kinase activities also increase to maintain PtdInsP2 levels. Since PLC is down regulated before these lipid kinases are, radioactive PtdInsP and PtdInsP2 are transiently over-produced. In the same time frame, the PA signal is attenuated by PA kinase producing DGPP. Figure A and B are adapted from Ref. 27
see Ref 27 |
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Differential labelling protocol - distinguishing between DGK- and PLD generated PA
Chlamydomonas cells were metabolically prelabelled with 32P-orthophosphate for the times indicated and subsequently treated for 1 min with either buffer ( control; panel A, B) or 1 µM mastoparan ( stimulated; panel C, D), both in the presence of 0.1 % n-butanol. Lipids were then extracted, split into half, and either seperated on EtAc TLC (A, C), to separate the PLD-catalyzed phosphatidylbutanol (PBut), or Alkaline TLC (B, D), to visualize the rest of the phospholipids, including PA and its phosphorylated product, diacylglycerolpyrophosphate (DGPP). The point is that all cells are treated for the same time period (1 min) but that the PBut will only be radioactive when its substrate (i.e. PE) becomes labelled (>40min). The same holds of course for the PA that would be coming through the PLD pathway. In contrast, when PA is generated via DGK, then the label comes from ATP and since this is labelled within seconds, a massive radioactive PA response can be witnessed if a DGK was involved. Since PA kinase also uses ATP, a similar response in DGPP can be picked up (panel D). Since the specific radioactivity of 32 P-ATP decreases in time, the 1-min response decreases concomitantly. The combined assay is a relative measure and evidence for PA coming through a DGK and/or PLD pathway. See details in Arisz et al., 2009 (Ref 68.
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Visualizing Lipid Signalling using Lipid Biosensors
Using GFP fusions of various lipid-binding domains and expressing them stably in tobacco BY-2 cells and Arabidopsis seedlings, we have been able to topographically visualize where certain lipids are localised, and where they are generated. Currently, we have biosensors for PtdIns3P (ref 28), PtdIns4P (ref 62), PtdIns(4,5)P2 (ref 55) and DAG (in prep). We are working on PA sensors. Dr. Joop Vermeer, Ringo van Wijk and Wendy Roels are responsible for this project, which occurs in close collaboration with the Lab of Dorus Gadella.
Vermeer et al. (2006) - PI3P
van Leeuwen et al. (2007) - PI(4,5)P2
Vermeer et al. (2009) - PI4P |
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Arabidopsis T-DNA insertion mutants
Using T-DNA insertion mutants, we are trying to provide furtehr evidence for the participation of PLC, PLD and DGK in signalling and developmental pathways. In Arabidopsis, there are 9 PLC, 12 PLD and 7 DGK genes. In addition, there are 11 PIP5K, 4 PI4K, 1 PI3K and multiple PA- and PPI phosphatase genes.
Below, an example of Reduced salt tolerance in Arabidopsis PLD mutants is shown. Seeds from wild-type (Col-0, black circles),pldα1 (open squares), pldδ (open triangles) or pldα1/pldδ double (open diamonds) knock-out mutant lines were sown on agar plates and grown vertically in a growth chamber. After 3 days seedlings were transferred to fresh plates supplementedwith0, 75 or 150 mM NaCl. Plates were scanned after 8 days (a) and primary root growth was followed and averaged ± SE during 4 days after transfer (b; n=12-16; a representative experiment is presented). In general, this work involve(s/d) Joop Vermeer, Ringo van Wijk, Wendy Roels, Saskia van Wees, Bastiaan Bargmann, Bas van Schooten, Steven Arisz and Christa Testerink.
Bargmann et al. (2009) PLD - osmotic stress
Bargmann et al. (2009) PLD - wounding |
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PLANT STRESS AND DEVELOPMENT
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Role of Phospholipids in Membrane Trafficking
..... text to be written
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Temperature Stress
Heat stress induces an array of physiological adjustments that facilitate continued homeostasis and survival during periods of elevated temperatures. Recently, we found that within minutes of a sudden temperature increase, plants deploy specific phospholipid-based signalling pathways to specific intracellular locations: a PLD and a phosphatidylinositolphosphate kinase (PIPK) are activated, and PA and PIP2 rapidly accumulate. Using our PIP2-specific lipid biosensor, we could show that the heat-induced PIP2 is localized to the plasma membrane, the nuclear envelope, nucleolus and punctate cytoplasmic structures (see pict below). Increases in the steady-state levels of PA and PIP2 occured within several min of temperature increases from ambient levels of 20-25°C to 35°C and above. Similar patterns were observed in heat stressed Arabidopsis seedlings and rice leaves. The PA which accumulates in response to heat stress results in large part from the activation of PLD rather than the sequential action of PLC and DGK. 32P-Pulse-labelling analysis reveals that the PIP2 response is due to the activation of a PIPK rather than the inhibition of a lipase or PIP2 phosphatase. This ms has just been accepted for publication in the PlantJ. where results are discussed in the context of the diverse cellular roles PIP2 and PA play, including regulation of ion channels and the cytoskeleton (see ref 64, 69).
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Osmotic Stress
Osmotic stress involves salinity, drought but also hypotonic stress. We are investigating which phospholipid signalling pathways are activated in response to these osmotic stresses. Using 32P-labelling, various PA and PPI responses have been uncovered (see refs 18, 21, 26, 29, 30, 40, 55). Currently, we are using the lipid biosensors to find out where these lipids are generated (ref 55), and T-DNA insertion mutants to investigate which genes are involved (ref 63; Arisz et al; Van Schooten et al., In prep).
Darwish et al. (2009)
Bargmann et al. (2009) |
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Plant Defence
PLC and PLD signalling cascades can individually generate PA, an important eukaryotic lipid second messenger. PLC generates it indirectly, via the hydrolysis of PI(4,5)P2 and the subsequent phosphorylation of diacylglycerol (DAG) into PA via DAG kinase (DGK). PLD generates PA directly by hydrolyzing structural pospholipids such as phosphatidylcholine. Earlier, we have provided evidence for the role of PA in plant defence using elicitor-challenged cell suspensions of tomato, parsley and alfalfa. Currently, we are adressing PA's role in the model system Arabidopsis thaliana.
Arabidopsis contains 9 PLCs, 7 DGKs and 12 PLDs. To identify the genes involved in plant defence and to characterise their individual functions, T-DNA insertion lines for most of these genes were collected. Plants were then analysed for their disease resistance and sensitivity using virulent and avirulent strains of the bacterial pathogen, Pseudomonas syringae and its natural pathogen Hyaloperonospora parasitica, the causal agent of downy mildew. A DGK gene was found to be required for full resistance against virulent Pseudomonas and H. parasitica while two PLD genes were found to be involved in resistance against avirulent Pseudomonas strains.
Van der Luit et al. (2000)
Laxalt&Munnik (2002)
Den Hartog et al. (2003)
De Jong et al. (2004)
Bargmann et al. (2006) |
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Pollen Tube Growth
Work from Dr. Laura Zonia is focussing on one of the fastest growing cellsof this planet, tobacco pollen tubes. The goal of this research is to identify key information cascades that control pollen tube growth, and to understand how these networks link to thebiomechanics driving cell elongation. We have identified several key cascades, including actin cytoskeleton, ion fluxes and phospholipid signals. Other workers have identified other cascades important for pollen tube growth, including GTPases, protein kinases, and processes involved in cell wall synthesis. When we searched for common themes across these information cascades, osmoregulation and cell volume status emerged as a key component. Further work indicated that indeed many of these networks converge with processes involved in cellular osmoregulation. Our most recent work shows that transcellular hydrodynamic flux drives pollen tube growth and modulates the rates of exocytosis and endocytosis.
Zonia&Munnik TiPS 2009
Zonia&Munnik, 2008 |
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Time-lapse images of tobacco pollen tubes double-labelled with FM 1-43 (green) and FM 4-64 (red) to identify sites of endocytosis and exocytosis and visualize membrane trafficking patterns. The first 3 images are from a pollen tube undergoing normal growth. The next3 images are from a pollen tube undergoinghypertonic stress, which stimulates endocytic membrane retrieval at the apex and inhibits exocytosis. The last 2 images are from a pollen tube undergoing hypotonic stress, which stimulates exocytosis and growth and attenuates endocytosis. Together with previous work (Zonia and Munnik, 2007), these data reveal that transcellular hydrodynamic flux is a key integrator ofpollen tube growth, providing a motive force for cell elongation and regulating the rates of membrane insertion(exocytosis) and retrieval (endocytosis). (see refs 40, 49, 51, 53,57,60, 66)
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 Diacylglycerol Kinase (DGK)
Accumulating evidence suggests that PA plays a pivotal role in the plant's response to environmental signals. Besides PLD activity, PA can also be generated by DGK. To establish which metabolic route is activated, a differential 32P-radiolabelling protocol can be used. Based on this, and more recently on reverse-genetic approaches, DGKhas taken center stage, next to PLD, as a generator of PA in biotic and abiotic stress responses. The DAG substrate is generally thought to be derived from PI-PLC activity. The model plant system Arabidopsis thaliana has 7 DGK isozymes, two of which, AtDGK1 and AtDGK2, resemble mammalian DGKepsilon, containing a conserved kinase domain, a transmembrane domain and two C1 domains. The other ones have a much simpler structure, lacking the C1 domains, not matched in animals. Several protein targets have now been discovered that bind PA. Whether the PA molecules engaged in these interactions come from PLD or DGK remains to be elucidated.
DGK review BBA 2009 |
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