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Heat stress induces an array of physiological adjustments that facilitate continued homeostasis and survival during periods of elevated temperatures. Here, we report that within minutes of a sudden temperature increase, plants deploy specific phospholipids to specific intracellular locations: phospholipase D (PLD) and a phosphatidylinositolphosphate kinase (PIPK) are activated, and phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP(2)) rapidly accumulate, with the heat-induced PIP(2) localized to the plasma membrane, nuclear envelope, nucleolus and punctate cytoplasmic structures. Increases in the steady-state levels of PA and PIP(2) occur within several minutes of temperature increases from ambient levels of 20-25 degrees C to 35 degrees C and above. Similar patterns were observed in heat-stressed Arabidopsis seedlings and rice leaves. The PA that accumulates in response to temperature increases results in large part from the activation of PLD rather than the sequential action of phospholipase C and diacylglycerol kinase, the alternative pathway used to produce this lipid. Pulse-labelling analysis revealed that the PIP(2) response is due to the activation of a PIPK rather than inhibition of a lipase or a PIP(2) phosphatase. Inhibitor experiments suggest that the PIP(2) response requires signalling through a G-protein, as aluminium fluoride blocks heat-induced PIP(2) increases. These results are discussed in the context of the diverse cellular roles played by PIP(2) and PA, including regulation of ion channels and the cytoskeleton.

Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plant's response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential (32)P-radiolabelling protocol can be used. Based on this, and more recently on reverse-genetic approaches, DGK has 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.

Plants respond to wounding by means of a multitude of reactions, with the purpose of stifling herbivore assault. Phospholipase D (PLD) has previously been implicated in the wounding response. Arabidopsis (Arabidopsis thaliana) AtPLDalpha1 has been proposed to be activated in intact cells, and the phosphatidic acid (PA) it produces to serve as a precursor for jasmonic acid (JA) synthesis and to be required for wounding-induced gene expression. Independently, PLD activity has been reported to have a bearing on wounding-induced MAPK activation. However, which PLD isoforms are activated, where this activity takes place (in the wounded or non-wounded cells) and what exactly the consequences are is a question that has not been comprehensively addressed. Here, we show that PLD activity during the wounding response is restricted to the ruptured cells using (32)P(i)-labelled phospholipid analyses of Arabidopsis pld knock-out mutants and PLD-silenced tomato cell-suspension cultures. pldalpha1 knock-out lines have reduced wounding-induced PA production, and the remainder is completely eliminated in a pldalpha1/delta double knock-out line. Surprisingly, wounding-induced protein kinase activation, AtLOX2 gene expression and JA biosynthesis were not affected in these knock-out lines. Moreover, larvae of the Cabbage White butterfly (Pieris rapae) grew equally well on wild-type and the pld knock-out mutants.

The long-standing model of tip growth in pollen tubes considers that exocytosis and growth occur at the apex and that the pool of very small vesicles in the apical dome contains secretory (exocytic) vesicles. However, recent work on vesicle trafficking dynamics in tobacco pollen tubes shows that exocytosis occurs in the subapical region. Taking these and other new results into account, we set out to resolve specific problems that are endemic in current models and present a two-part ACE (apical cap extension)-H (hydrodynamics) growth model. The ACE model involves delivery and recycling of materials required for new cell synthesis and the H model involves mechanisms that integrate and regulate key cellular pathways and drive cell elongation during growth.

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."

High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plant's response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plant's response to high salinity and dehydration. A tomato (Lycopersicon esculentum) alpha-class PLD, LePLDalpha1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDalpha1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDalpha1 and AtPLDdelta were found to be activated in response to salt stress. Moreover, pldalpha1 and plddelta single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth.

Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4P) is the most abundant polyphosphoinositide. (32)Pi-labelling studies have shown that the turnover of PtdIns4P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4P, i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP-PH(FAPP1) was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP-PH(FAPP1) expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd-CFP, but not with the endocytic/pre-vacuolar marker GFP-AtRABF2b. Co-expression with the Ptdins3P biosensor YFP-2 x FYVE revealed totally different localization patterns. During cell division, YFP-PH(FAPP1) showed strong labelling of the cell plate, but PtdIns3P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana, a clear PtdIns4P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4P in tip growth.

Our recent work used novel methods to localize and track discrete vesicle populations in pollen tubes undergoing oscillatory growth. The results show that clathrin-dependent endocytosis occurs along the shank of the pollen tube, smooth vesicle endocytosis occurs at the tip, and exocytosis occurs in the subapical region. Here, growth of tobacco and lily pollen tubes is examined in greater temporal resolution using refraction-free high-resolution time-lapse differential interference contrast microscopy. Images were collected at 0.21 s intervals for 10 min, sequentially examined for millisecond details, compressed into video format and then examined for details of growth dynamics. The subapical growth zone is structurally fluid, with vesicle insertion into the plasma membrane, construction of new cell surface and cellular expansion. Incorporation of new membrane and wall materials causes localized disruption at the cell surface that precedes the start of the growth cycle by 3.44 +/- 0.39 s in tobacco, and 1.02 +/- 0.01 s in lily pollen tubes. Vesicle deposition increases after the start of the growth cycle and supports expansion of the growth zone. Growth reorientation involves a shift in the position and angle of the growth zone. In summary, these results support a new model of pollen tube growth.

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.1 PA was shown to inhibit (1) CTR1's protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.

Various phosphoinositides have been implicated in plant defence signalling. Until now, such molecules have been exclusively related to intracellular signalling. Here, evidence is provided for the detection of extracellular phosphatidylinositol 4-phosphate (PI4P) in tomato cell suspensions. We have analysed and compared the intracellular and extracellular phospholipid profiles of [(32)P(i)]-prelabelled tomato cells, challenged with the fungal elicitor xylanase. These phospholipid patterns were found to be different, being phosphatidylinositol phosphate (PIP) the most abundant phospholipid in the extracellular medium. Moreover, while cells responded with a typical increase in phosphatidic acid and a decrease in intracellular PIP upon xylanase treatment, extracellular PIP level increased in a time- and dose-dependent manner. Using two experimental approaches, the extracellular PIP isoform was identified as PI4P. Addition of PI4P to tomato cell suspensions triggered the same defence responses as those induced by xylanase treatment. These include production of reactive oxygen species, accumulation of defence-related gene transcripts and induction of cell death. We demonstrate that extracellular PI4P is accumulated in xylanase-elicited cells and that exogenous application of PI4P mimics xylanase effects, suggesting its putative role as an intercellular signalling molecule.

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.

Pollen tubes are one of the fastest growing eukaryotic cells. Rapid anisotropic growth is supported by highly active exocytosis and endocytosis at the plasma membrane, but the subcellular localization of these sites is unknown. To understand molecular processes involved in pollen tube growth, it is crucial to identify the sites of vesicle localization and trafficking. This report presents novel strategies to identify exocytic and endocytic vesicles and to visualize vesicle trafficking dynamics, using pulse-chase labelling with styryl FM dyes and refraction-free high-resolution time-lapse differential interference contrast microscopy. These experiments reveal that the apex is the site of endocytosis and membrane retrieval, while exocytosis occurs in the zone adjacent to the apical dome. Larger vesicles are internalized along the distal pollen tube. Discretely sized vesicles that differentially incorporate FM dyes accumulate in the apical, subapical, and distal regions. Previous work established that pollen tube growth is strongly correlated with hydrodynamic flux and cell volume status. In this report, it is shown that hydrodynamic flux can selectively increase exocytosis or endocytosis. Hypotonic treatment and cell swelling stimulated exocytosis and attenuated endocytosis, while hypertonic treatment and cell shrinking stimulated endocytosis and inhibited exocytosis. Manipulation of pollen tube apical volume and membrane remodelling enabled fine-mapping of plasma membrane dynamics and defined the boundary of the growth zone, which results from the orchestrated action of endocytosis at the apex and along the distal tube and exocytosis in the subapical region. This report provides crucial spatial and temporal details of vesicle trafficking and anisotropic growth.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLCdelta1 that specifically binds PtdIns(4,5)P(2). In this way, YFP-PH(PLCdelta1) has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P(2) is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PH(PLCdelta1) to try monitoring PtdIns(4,5)P(2)/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P(2). Confocal imaging revealed that most of the YFP-PH(PLCdelta1) fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PH(PLCdelta1) was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P(2), as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PH(PLCdelta1). Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PH(PLCdelta1) is a valuable plant PtdIns(4,5)P(2) reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P(2) in plant cell signalling are discussed.

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 nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.

H(2)O is one of the most essential molecules for cellular life. Cell volume, osmolality and hydrostatic pressure are tightly controlled by multiple signaling cascades and they drive crucial cellular functions ranging from exocytosis and growth to apoptosis. Ion fluxes and cell shape restructuring induce asymmetries in osmotic potential across the plasma membrane and lead to localized hydrodynamic flow. Cells have evolved fascinating strategies to harness the potential of hydrodynamic flow to perform crucial functions. Plants exploit hydrodynamics to drive processes including gas exchange, leaf positioning, nutrient acquisition and growth. This paradigm is extended by recent work that reveals an important role for hydrodynamics in pollen tube growth.

Aluminum (Al(3+)) has been recognized as a main toxic factor in crop production in acid lands. Phosphatidic acid (PA) is emerging as an important lipid signaling molecule and has been implicated in various stress-signaling pathways in plants. In this paper, we focus on how PA generation is affected by Al(3+) using Coffea arabica suspension cells. We pre-labeled cells with [(32)P]orthophosphate ((32)Pi) and assayed for (32)P-PA formation in response to Al(3+). Treating cells for 15 min with either AlCl(3) or Al(NO(3))(3) inhibited the formation of PA. In order to test how Al(3+) affected PA signaling, we used the peptide mastoparan-7 (mas-7), which is known as a very potent stimulator of PA formation. The Al(3+) inhibited mas-7 induction of PA response, both before and after Al(3+) incubation. The PA involved in signaling is generated by two distinct phospholipid signaling pathways, via phospholipase D (PLD; EC: 3.1.4.4) or via Phospholipase C (PLC; EC: 3.1.4.3), and diacylglycerol kinase (DGK; EC 2.7.1.107). By labeling with (32)Pi for short periods of time, we found that PA formation was inhibited almost 30% when the cells were incubated with AlCl(3) suggesting the involvement of the PLC/DGK pathway. Incubation of cells with PLC inhibitor, U73122, affected PA formation, like AlCl(3) did. PLD in vivo activation by mas-7 was reduced by Al(3+). These results suggest that PA formation was prevented through the inhibition of the PLC activity, and it provides the first evidence for the role of Al toxicity on PA production.

Phospholipase D (PLD) has been implicated in multiple plant stress responses. Its gene transcription and activity increase upon exposure to various stresses, and manipulation of PLD protein levels leads to altered stress tolerance. The plant PLD family is relatively large and heterogeneous, and different PLD isoforms are involved in separate stress responses. PLD and its product, phosphatidic acid, exert their effects by functioning in signal transduction cascades and by influencing the biophysical state of lipid membranes.

To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.

Phospholipase D (PLD) has been implicated in various cellular processes including membrane degradation, vesicular trafficking and signal transduction. Previously, we described a PLD gene family in tomato (Lycopersicon esculentum) and showed that expression of one of these genes, LePLDbeta1, was induced by treatment with the fungal elicitor xylanase. To further investigate the function of this PLD, a gene-specific RNAi construct was used to knock down levels of LePLDbeta1 transcript in suspension-cultured tomato cells. Silenced cells exhibited a strong decrease in xylanase-induced PLD activity and responded to xylanase treatment with a disproportionate oxidative burst. Furthermore, LePLDbeta1-silenced cell-suspension cultures were found to have increased polyphenol oxidase activity, to secrete less of the beta-d-xylosidase LeXYL2 and to secrete and express more of the xyloglucan-specific endoglucanase inhibitor protein XEGIP. Using an LePLDbeta1-green fluorescent protein (GFP) fusion protein for confocal laser scanning microscopy-mediated localization studies, untreated cells displayed a cytosolic localization, whereas treatment with xylanase induced relocalization to punctuate structures within the cytosol. Possible functions for PLDbeta in plant-pathogen interactions are discussed.

Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid that has been found in plants and yeast but not in higher animals. It is produced through phosphorylation of phosphatidic acid (PA) by the novel enzyme PA kinase (PAK). In plants, DGPP is virtually absent in non-stimulated cells but its concentration increases within minutes in response to various stimuli, including osmotic stress and pathogen attack, implying a role in stress signalling. DGPP is broken down by the enzyme DGPP phosphatase (DPP). DPP-encoding genes have been cloned from Arabidopsis thaliana and Saccharomyces cerevisiae (DPP1). In S. cerevisiae, the expression of DPP1 is regulated coordinately with the majority of genes encoding enzymes involved in phospholipid biosynthesis.

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H2O with 2H2O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing 2H2O with H2O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 mum distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180 degrees . Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.

Phosphatidic acid (PA) has only recently been identified as an important signaling molecule in both plants and animals. Nonetheless, it already promises to rival the importance of the classic second messengers Ca(2+) and cAMP. In plants, its formation is triggered in response to various biotic and abiotic stress factors, including pathogen infection, drought, salinity, wounding and cold. In general, PA signal production is fast (minutes) and transient. Recently, our understanding of the role of PA formation in stress responses as a result of phospholipases C and D activity has greatly increased. Moreover, the first protein targets of PA have been identified. Based on this recent work, potential mechanisms by which PA provokes downstream effects are emerging.

Phosphatidic acid (PA) is emerging as an important lipid signalling molecule. In plants, it is implicated in various stress-signalling pathways and is formed in response to wounding, osmotic stress, cold stress, pathogen elicitors, Nod factors, ethylene and abscisic acid. How PA exerts its effects is still unknown, mainly because of the lack of characterized PA targets. In an approach to isolate such targets we have used PA-affinity chromatography. Several PA-binding proteins were present in the soluble fraction of tomato and Arabidopsis cells. Using mass spectrometric analysis, several of these proteins, including Hsp90, 14-3-3 proteins, an SnRK2 serine/threonine protein kinase and the PP2A regulatory subunit RCN1 could be identified. As an example, the binding of one major PA-binding protein, phosphoenolpyruvate carboxylase (PEPC), was characterized further. Competition experiments with different phospholipids confirmed specificity for PA. Hypo-osmotic treatment of the cells increased the amount of PEPC that bound the PA beads without increasing the absolute amount of PEPC. This suggests that PEPC's affinity for PA had increased. The work shows that PA-affinity chromatography/mass spectrometry is an effective way to isolate and identify PA-binding proteins from plants.

Plant cells respond to different biotic and abiotic stresses by producing various uncommon phospholipids that are believed to play key roles in cell signalling. We can predict how they work because animal and yeast proteins have been shown to have specific lipid-binding domains, which act as docking sites. When such proteins are recruited to the membrane locations where these phospholipids are synthesized, the phospholipids activate them directly, by inducing a conformational change, or indirectly, by juxtaposing them with an activator protein. The same lipid-binding domains are present in Arabidopsis proteins. We believe that they represent an untapped well of information about plant lipid signalling.

The Cladosporium fulvum (Cf)-4 gene of tomato confers resistance to the fungus C. fulvum, expressing the corresponding avirulence (Avr)4 gene, which codes for an elicitor protein. Little is known about how such mechanisms work, but previous studies have shown that elicitor recognition activates Ca(2+) signalling and protein kinases, such as mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK). Here, we provide evidence that a new signalling component, the lipid second messenger phosphatidic acid (PA), is produced within a few minutes of AVR4/Cf-4 interaction. Using transgenic tobacco cells expressing the tomato Cf-4-resistance gene as a model system, phospholipid signalling pathways were studied by pre-labelling the cells with (32)P(i) and assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in (32)P-PA, together with its metabolic product diacylglycerol pyrophosphate (DGPP). AVR4 increased the levels of PA and DGPP in a Cf-4(+)-, time- and dose-dependent manner, while the non-matching elicitor AVR9 did not trigger any response. In general, PA signalling can be triggered by two different pathways: via phospholipase D (PLD), which generates PA directly by hydrolysing structural phospholipids like phosphatidylcholine (PC), or via PLC, which generates diacylglycerol (DAG) that is subsequently phosphorylated to PA by DAG kinase (DGK). To determine the origin of the AVR4-induced PA formation, a PLD-specific transphosphatidylation assay and a differential (32)P-labelling protocol were used. The results clearly demonstrated that most PA was produced via the phosphorylation of DAG. Neomycin and U73122, inhibitors of PLC activity, inhibited AVR4-induced PA accumulation, suggesting that the increase in DGK activity was because of increased PLC activity producing DAG. Lastly, evidence is provided that PLC signalling and, in particular, PA production could play a role in triggering responses, such as the AVR4-induced oxidative burst. For example, PLC inhibitors inhibited the oxidative burst, and when PA was added to cells, an oxidative burst was induced.

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.

Pollen tube cell volume changes rapidly in response to perturbation of the extracellular osmotic potential. This report shows that specific phospholipid signals are differentially stimulated or attenuated during osmotic perturbations. Hypo-osmotic stress induces rapid increases in phosphatidic acid (PA). This response occurs starting at the addition of 25% (v/v) water to the pollen tube cultures and peaks at 100% (v/v) water. Increased levels of PA were detected within 30 s and reached maximum by 15 to 30 min after treatment. The pollen tube apical region undergoes a 46% increase in cell volume after addition of 100% water (v/v), and there is an average 7-fold increase in PA. This PA increase appears to be generated by phospholipase D because concurrent transphosphatidylation of n-butanol results in an average 8-fold increase in phosphatidylbutanol. Hypo-osmotic stress also induces an average 2-fold decrease in phosphatidylinositol phosphate; however, there are no detectable changes in the levels of phosphatidylinositol bisphosphates. In contrast, salt-induced hyperosmotic stress from 50 to 400 mm NaCl inhibits phospholipase D activity, reduces the levels of PA, and induces increases in the levels of phosphatidylinositol bisphosphate isomers. The pollen tube apical region undergoes a 41% decrease in cell volume at 400 mm NaCl, and there is an average 2-fold increase in phosphatidylinositol 3,5-bisphosphate and 1.4-fold increase in phosphatidylinositol 4,5-bisphosphate. The phosphatidylinositol 3,5-bisphosphate increase is detected within 30 s and reaches maximum by 15 to 30 min after treatment. In summary, these results demonstrate that hypo-osmotic versus hyperosmotic perturbation and the resultant cell swelling or shrinking differentially activate specific phospholipid signaling pathways in tobacco (Nicotiana tabacum) pollen tubes.

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.

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.

Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp. bacteria to elicit Nod responses on their legume hosts. One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells. Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants. To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used to determine the contribution of each pathway to PA formation. In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment. However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor. No evidence was obtained for the activation of phospholipase A(2).

Phospholipids are emerging as novel second messengers in plant cells. They are rapidly formed in response to a variety of stimuli via the activation of lipid kinases or phospholipases. These lipid signals can activate enzymes or recruit proteins to membranes via distinct lipid-binding domains, where the local increase in concentration promotes interactions and downstream signaling. Here, the latest developments in phospholipid-based signaling are discussed, including the lipid kinases and phospholipases that are activated, the signals they produce, the domains that bind them, the downstream targets that contain them and the processes they control.

We show that differentiation of zoospores of the late blight pathogen Phytophthora infestans into cysts, a process called encystment, was triggered by both phosphatidic acid (PA) and the G-protein activator mastoparan. Mastoparan induced the accumulation of PA, indicating that encystment by mastoparan most likely acts through PA. Likewise, mechanical agitation of zoospores, which often is used to induce synchronized encystment, resulted in increased levels of PA. The levels of diacylglycerolpyrophosphate (DGPP), the phosphorylation product of PA, increased simultaneously. Also in cysts, sporangiospores, and mycelium, mastoparan induced increases in the levels of PA and DGPP. Using an in vivo assay for phospholipase D (PLD) activity, it was shown that the mastoparan-induced increase in PA was due to a stimulation of the activity of this enzyme. Phospholipase C in combination with diacylglycerol (DAG) kinase activity also can generate PA, but activation of these enzymes by mastoparan was not detected under conditions selected to highlight 32P-PA production via DAG kinase. Primary and secondary butanol, which, like mastoparan, have been reported to activate G-proteins, also stimulated PLD activity, whereas the inactive tertiary isomer did not. Similarly, encystment was induced by n- and sec-butanol but not by tert-butanol. Together, these results show that Phytophthora infestans contains a mastoparan- and butanol-inducible PLD pathway and strongly indicate that PLD is involved in zoospore encystment. The role of G-proteins in this process is discussed.

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.

Hyperosmotic stress induces the rapid formation of phosphatidic acid (PA) in Chlamydomonas moewusii via the activation of two signalling pathways: phospholipase D (PLD) and phospholipase C (PLC), the latter in combination with diacylglycerol kinase (DGK) (Munnik et al., 2000). A concomitant increase in cell Ca(2+) becomes manifest as deflagellation. When KCl was used as osmoticum we found that two concentration ranges activated deflagellation: one between 50 and 100 mm and another above 200 mm. Deflagellation in low KCl concentrations was complete within 30 sec whereas in high concentrations it took 5 min. PLC was not activated, as it was by high KCl concentrations that cause hyperosmotic stress. Moreover PLD was activated more strongly by low than by high KCl concentrations. Potassium was the most potent monovalent cation based on the induction of deflagellation and the formation of PA and PBut. During treatment, the external medium acidified, indicating an increase in H(+)-ATPase activity in order to re-establish the membrane potential. Activation of PLD and deflagellation at low KCl concentrations were abrogated by treatment with La(3+), Gd(3+) and EGTA, indicating the dependency on extracellular Ca(2+). This suggests that low concentrations of KCl depolarize the plasma membrane, resulting in the activation of H(+)-ATPases and opening voltage-dependent Ca(2+) +/- channels, observed as deflagellation and an increase in PLD activity.

Background and Aims: Patients suffering from inflammatory bowel disease show increased levels of the mast cell products histamine and tumour necrosis factor alpha (TNF-alpha). Treating these patients with antibodies against TNF-alpha diminishes the symptoms of diarrhoea. In this study, the effect of TNF-alpha on ion secretion induced by the mast cell mediator histamine in HT29cl.19A cells and mouse distal colon was investigated and the possible second messengers involved were studied., Methods: Electrophysiology of filter grown HT29cl.19A cells and isolated mouse distal colon was used to monitor the secretory response to histamine with and without prior exposure to TNF-alpha for 3-24 hours. Phospholipase D (PLD) activity and phosphatidic acid levels were analysed by 32P(i) labelling of HT29cl.19A cells., Results: In both experimental systems TNF-alpha was found to potentiate ion secretion induced by histamine. Phospholipid analysis of HT29cl.19A cells revealed that histamine activates the PLD pathway. Furthermore, TNF-alpha pretreated cells were found to have decreased phosphatidic acid levels, the intermediate product of the PLD pathway, which indicates upregulation of the enzyme phosphatidic acid phosphatase., Conclusions: The mast cell products TNF-alpha and histamine synergistically stimulate ion secretion in intestinal epithelium via upregulation of the PLD pathway.

Plant cells contain various phospholipase-based signaling pathways. In fact, their repertoire of phospholipase D (PLD) molecules far outnumbers those of mammalian and yeast cells. Munnik and Musgrave take a broad look at PLD function in animal, yeast, and plant cells, and suggest that a PLD-based connection between membranes and microtubules is a biological property worth considering across species.

Polyphosphoinositides play an important role in membrane trafficking and cell signalling. In plants, two PtdInsP isomers have been described, PtdIns3P and PtdIns4P. Here we report the identification of a third, PtdIns5P. Evidence is based on the conversion of the endogenous PtdInsP pool into PtdIns(4,5)P(2) by a specific PtdIns5P 4-OH kinase, and on in vivo (32)P-labelling studies coupled to HPLC head-group analysis. In Chlamydomonas, 3-8% of the PtdInsP pool was PtdIns5P, 10-15% was PtdIns3P and the rest was PtdIns4P. In seedlings of Vicia faba and suspension-cultured tomato cells, the level of PtdIns5P was about 18%, indicating that PtdIns5P is a general plant lipid that represents a significant proportion of the PtdInsP pool. Activating phospholipase C (PLC) signalling in Chlamydomonas cells with mastoparan increased the turnover of PtdIns(4,5)P(2) at the cost of PtdIns4P, but did not affect the level of PtdIns5P. This indicates that PtdIns(4,5)P(2) is synthesized from PtdIns4P rather than from PtdIns5P during PLC signalling. However, when cells were subjected to hyperosmotic stress, PtdIns5P levels rapidly increased, suggesting a role in osmotic-stress signalling. The potential pathways of PtdIns5P formation are discussed.

Plants are continuously exposed to all kinds of water stress such as drought and salinity. In order to survive and adapt, they have developed survival strategies that have been well studied, but little is known about the early mechanisms by which the osmotic stress is perceived and transduced into these responses. During the last few years, however, a variety of reports suggest that specific lipid and MAPK pathways are involved. This review briefly summarises them and presents a model showing that osmotic stress is transmitted by multiple signalling pathways.

Phospholipase D (PLD, EC 3.1.4.4.) has been implicated in a variety of plant processes, including signalling. In Arabidopsis thaliana a PLD gene family has been described and individual members classified into alpha-, beta- and gamma-classes. Here we describe a second PLD gene family in tomato (Lycopersicon esculentum) that includes three alpha- and two beta-classes. Different expression patterns in plant organs were observed for each PLD. In testing a variety of stress treatments on tomato cell suspensions, PLDbeta1 mRNA was found to rapidly and specifically accumulate in response to the fungal elicitor xylanase. The greatest increase was found 2 h after treatment with 100 microg m1(-1) xylanase (ninefold). In vivo PLD activity increased nearly threefold over a 1.5 h period of treatment. When the elicitor was injected into tomato leaves, PLDbeta1 mRNA accumulation peaked at 2 h (threefold increase), before decreasing to background levels within 72 h. Mutant, non-active xylanase was as effective as the active enzyme in eliciting a response, suggesting that xylanase itself, and not the products resulting from its activity, functioned as an elicitor. When chitotetraose was used as elicitor, no PLDbeta1 mRNA accumulation was observed, thus it is not a general response to elicitation. Together these data show that PLD genes are differentially regulated, reflecting potential differences in cellular function. The possibility that PLDbeta1 is a signalling enzyme is discussed.

In a previous study, it was found that exposure to tumor necrosis factor-alpha (TNF-alpha) potentiated the electrophysiological response to carbachol in a time-dependent and cycloheximide-sensitive manner. It was deduced that the potentiation could be due to protein kinase C activity because of increased 1,2-diacylglycerol. It was also observed that propranolol could decrease the electrophysiological response to carbachol (Oprins JC, Meijer HP, and Groot JA. Am J Physiol Cell Physiol 278: C463-C472, 2000). The aim of the present study was to investigate whether the phospholipase D (PLD) pathway plays a role in the carbachol response and the potentiating effect of TNF-alpha. The transphosphatidylation reaction in the presence of the primary alcohol 1-butanol [leading to stable phosphatidylbutanol (Pbut) formation] was used to measure activity of PLD. The phosphatidic acid (PA) levels were also measured. Muscarinic stimulation resulted in an increased formation of Pbut and PA. TNF-alpha decreased levels of PA.

Plant cells are continuously exposed to environmental stresses such as hyper-osmolarity, and have to respond in order to survive. When 32P-labelled Chlamydomonas moewusii cells were challenged with NaCl, the formation of a new radiolabelled phospholipid was stimulated, which was barely detectable before stimulation. The phospholipid was identified as lyso-phosphatidic acid (LPA), and was the only lyso-phospholipid to be accumulated. The increase in LPA was dose- and time-dependent. When other osmotically active compounds were used, the formation of LPA was also induced with similar kinetics, although salts were better inducers than non-salts. At least part of the LPA was generated by phospholipase A2 (PLA2) hydrolysing phosphatidic acid (PA). This claim is based on PA formation preceding LPA production, and PLA2 inhibitors decreasing the accumulation of LPA and promoting the conversion of PA to diacylglycerol pyrophosphate. The latter is another metabolic derivative of PA that is implicated in cell signalling. The involvement of multiple lipid-signalling pathways in hyperosmotic stress responses is discussed.

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

In mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane trafficking and defence responses. In plant cells a signalling role for PLD and PA is also emerging. Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger. Here we report that increases in PA and its conversion to DGPP are common stress responses to water deficit. Increases occur within minutes of treatment and are dependent on the level of stress. Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1-butanol, and part is due to diacylglycerol kinase activity as monitored via 32P-PA formation in a differential labelling protocol. Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cell-suspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum. These results provide further evidence that PLD and PA play a role in plant signalling, and provide the first demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.

Mastoparan induces Ca(2+)-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4, 5-trisphosphate (InsP(3); T. Munnik et al., 1998, Planta 207: 133-145). Even in the absence of extracellular Ca(2+), mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807-815; J.A.J. van Himbergen et al., 1999, J Exp Bot, in press) suggesting that InsP(3) mediates Ca(2+) release from intracellular stores. To test this hypothesis, cells were pre-loaded with (45)Ca(2+) and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 microM) induced release of intracellular (45)Ca(2+). Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of (32)P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense bodies (EDBs) are a major Ca(2+) store in C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity from (45)Ca(2+)-labelled cells, suggesting that (45)Ca(2+) monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was released from EDBs.

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 A(2). 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.

Plant growth is severely affected by hyper-osmotic salt conditions. Although a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mM NaCl). In contrast, at high salt concentrations (above 750 mM NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identified as SIMK, a member of the family of MAPKs (mitogen-activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear transloca- tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intra-cellular compartmentation, suggesting an intra-nuclear mechanism for the regulation of SIMK activity.

To study cell-cycle-related variations in wall permeability of Saccharomyces cerevisiae, two approaches were used. First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cells of increasing size. The subpopulations represented different stages of the cell cycle as judged by light microscopy. Cell wall porosity increased when these subpopulations became enriched with budded cells. Secondly, synchronous cultures were obtained by releasing MATa cells from alpha-factor induced G1-arrest. These cultures grew synchronously for at least two generations. The cell wall porosity increased sharply in these cultures, shortly before buds became visible and was maximal during the initial stages of bud growth. It decreased in cells which had completed nuclear migration and before abscission of the bud had occurred. The porosity reached its lowest value during abscission and in unbudded cells. We examined the incorporation of mannoproteins into the wall during the cell cycle. SDS-extractable mannoproteins were incorporated continuously. However, the incorporation of glucanase-extractable mannoproteins, which are known to affect cell wall porosity, showed cyclic oscillations and reached its maximum after nuclear migration. This coincided with a rapid decrease in cell wall porosity, indicating that glucanase-extractable mannoproteins might contribute to this decrease.

The cell wall porosity of batch-grown Saccharomyces cerevisiae was maximal in the early exponential phase and fell off rapidly to lower levels in later growth phases. Treatment of stationary-phase cells with alpha-mannosidase restored wall porosity to the level of cells in early exponential phase. When cells in the early exponential phase were treated with alpha-mannosidase, or tunicamycin, an inhibitor of N-glycosylation, even higher porosities were obtained. Mutants with truncated mannan side-chains in their wall proteins also had very porous walls. The importance of the mannan side-chains for wall porosity was also seen during sexual induction. Treatment with alpha pheromone, which leads to the formation of wall proteins with shorter mannan side-chains, enhanced wall porosity. Disulphide bridges also affect cell wall porosity. They were predominantly found in the glucanase-soluble wall proteins. Because the main part of the mannan side-chains is also found in this family of wall proteins, our results demonstrate that the glucanase-soluble mannoproteins limit cell wall porosity in yeast.

We have developed a new assay to determine relative cell wall porosity in yeasts, which is based on polycation-induced leakage of UV-absorbing compounds. Polycations with a small hydrodynamic radius as measured by gel filtration (poly-L-lysine) caused cell leakage independent of cell wall porosity whereas polycations with a large hydrodynamic radius (DEAE-dextrans) caused only limited cell leakage due to limited passage through the cell wall. This allowed the ratio between DEAE-dextran- and poly-L-lysine-induced cell leakage to be used as a measure of cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. Using this assay, we found that the composition of the growth medium affected cell wall porosity in S. cerevisiae. In addition, we could show that cell wall porosity is limited by the number of disulphide bridges in the wall and is dependent on cell turgor. It is argued that earlier methods to estimate cell wall porosity in S. cerevisiae resulted in large underestimations.