Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts

Although the activation of phospholipase A₂ (PLA₂) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA₂ in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA₂ activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA₂ activity in ras-transformed cells compared to the corresponding palmitatestearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA₂ activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA₂ in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells.     

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root

Phospholipase A₂ (PLA₂), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA₂s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA₂α, a PLA₂ isoform, colocalized with the Golgi marker. Impairments of PLA₂ function by PLA₂α mutation, PLA₂-RNA interference (RNAi), or PLA₂ inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA₂ hydrolytic product) restored the PM localization of PINs in the pla₂α mutant and the ONO-RS-082–treated seedling. Suppression of PLA₂ activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA₂ inhibitor treatments, PLA₂α mutation, or PLA₂-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA₂, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.     

Host Specialization among Vegetative Compatibility Groups of Verticillium dahliae in Relation to Verticillium longisporum

A collection of 24 isolates of Verticillium dahliae and 10 isolates of Verticillium longisporum originating from nine different host plants and from several geographic regions was tested for host specificity on 11 economically important crops such as potato, tomato, strawberry, linseed, three legumes and four Brassica species. In order to reveal host specificity the potential of each isolate to induce disease and affect plant yield was recorded for all isolate–host combinations. The collected data were statistically processed by means of a cluster analysis. As a result, the host range of individual isolates was found to be more dependent on the vegetative compatibility group (VCG) of the isolate than on its original host plant provenance. Twenty‐two out of 24 V. dahliae isolates belonged to either VCG 2B or 4B. VCG 2B isolates showed specificity for legumes, strawberry, potato and linseed, whereas VCG 4B was specifically virulent on potato, strawberry and linseed. Subgroups within VCG 2B and 4B almost lacking any host preference were designated 2B* and 4B*. Three isolates from VCG 2B*, however, severely attacked tomato which is a host outside the authentic host range of VCG 2B. The pathogenicity of V. longisporum isolates was restricted to cruciferous hosts. Conversely, cruciferous plants were not affected by isolates from VCGs 2B and 4B of V. dahliae. This lack of cross‐infectivity of certain subpopulations of V. dahliae and of V. longisporum may be useful in the management of this soil‐borne wilt disease.     

Characterisation of a plasma membrane-associated phospholipase A2 activity increased in response to cold acclimation

We here demonstrate the presence of a plasma membrane-associated phospholipase A₂ (EC 3.1.1.4; PLA₂) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA₂ activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca²⁺, with full enzyme activity at 10 μmol/L Ca²⁺. The Ca²⁺-dependent PLA₂ activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca²⁺-dependent PLA₂ activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA₂ activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA₂ in cold acclimation in plants.     

Activation of fluoride-stimulated adenylate cyclase by phospholipase A2 in the caudate nucleus of the rat brain

Phospholipase A₂ (PLA₂) increases adenylate cyclase (AC) activity in the rat caudate nucleus in a dose-dependent manner. After maximal stimulation by fluoride, PLA₂ treatment further increases AC activity 2.4 fold. Adenylate cyclase activity is maximal after 45% hydrolysis of the phospholipids. Of the products of PLA₂ treatment only lysophosphatidylcholine (LPC) produces such an increase in AC activity. In contrast to PLA₂ treatment, LPC solubilizes the enzyme, decreases the Km value for ATP, and requires much larger amounts of LPC than that produced by lipase treatment. After maximal stimulation with fluoride and PLA₂, removal of most of the LPC does not reduce the activity of adenylate cyclase. These findings suggest that removal of membrane lipid rather than generation of LPC is responsible for the activation of brain adenylate cyclase by phospholipase A₂.     

Involvement of phospholipase A<Subscript>2</Subscript> in the response of <Emphasis Type="Italic">Solanum</Emphasis> species to an elicitor from <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Changes in activity of phospholipase A₂ (PLA₂), a key enzyme in lipid metabolism and signal network in defence mechanisms, were investigated in Solanum species and Phytophthora infestans interaction. We have compared PLA₂ activity in response to an elicitor, a culture filtrate (CF) derived from P. infestans, in non-host resistant Solanum nigrum var. gigantea, field resistant S. tuberosum cv Bzura and susceptible S. tuberosum clone H-8105. To elucidate the contribution of specific forms of PLA₂ to plant defence mechanism reasonably selective PLA₂ inhibitors, haloenol lactone suicide substrate (HELSS) and p-bromophenacyl bromide (BPB), which discriminate between Ca⁺²-independent PLA₂ (iPLA₂) and Ca⁺²-dependent secretory PLA₂ (sPLA₂), were used. The in vivo and in vitro effects of the inhibitors on PLA₂ activity and on generation of reactive oxygen species (ROS) induced by CF in the studied plants were assayed. We found that PLA₂ activity increased in response to CF treatment, displaying various kinetics and intensity depending on the resistance status of a given genotype. Differences among the genotypes in the effects of each inhibitor on CF-induced PLA₂ activity and on ROS production may reflect the diversity of PLA₂ isoforms in plants. Contrary to BPB, the inhibitory effect of HELSS was observable mainly on CF-induced PLA₂ activity, which suggests that iPLA₂ participates in signal transduction in defence reactions. Various effects of the two inhibitors on PLA₂ activity and ROS production suggest different contribution of sPLA₂ and iPLA₂ to modulation of defence reactions in the interaction between Solanum genotypes and P. infestans.     

Differential resistance of oilseed rape cultivars (Brassica napus ssp. oleifera) to Verticillium longisporum infection is affected by rhizosphere colonisation with antagonistic bacteria, Serratia plymuthica and Pseudomonas chlororaphis

The effect of a seed treatment with the antagonistic bacteria Serratia plymuthica (strain HRO-C48) and/or Pseudomonas chlororaphis (strain MA 342) on the infection of oilseed rape with Verticillium longisporum was assessed with ten different cultivars. Soil was inoculated with microsclerotia and mycelium of a V. longisporum culture. Seeds were treated with rifampicin-resistant antagonistic bacteria at a rate of log₁₀ 6–7 cells per seed. Resistance against V. longisporum infection did not differ between cultivars and was generally low. A significant disease reduction recorded as area under disease progress curve (AUDPC) was obtained with both antagonistic rhizobacteria with no significant difference between the treatments. Percent of healthy plants was approximately 70% in all bacterial treatments. Significant differences were observed between the cultivars ranging from 46.5% (cultivar Titan) to 72.6% (Trabant). The combined use of both bacteria could not provide additional control effects. The bacterial density in the rhizosphere was not related to the control effect, but increased by log₁₀ 2 on infection with V. longisporum. Growth promotion effects were also not related to the control effect. At present, neither the application of chemical fungicides nor breeding for resistance against V. longisporum in oilseed rape can provide a solution for this increasingly problematic plant pathogen. The present results now open perspectives to control V. longisporum in oilseed rape by making use of cultivars, which express resistance against this pathogen on interaction with the antagonistic rhizobacteria S. plymuthica or P. chlororaphis.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Enzymatic degradation of polar lipids in Vigna unguiculata leaves and influence of drought stress

¹⁴C-labelled polar lipids (monogalactosyl-diacylglycerol [MGDG], digalactosyl-diacylglycerol [DGDG], phosphatidylcholine [PC] and phosphatidylglycerol [PG]), purified from Vigna unguiculata leaves, were used as substrates to study the lipolytic activities of Vigna unguiculata leaf extracts. Analysis of the radioactive degradation products revealed the presence of at least three enzyme activities contributing to the hydrolysis of the four main leaf membrane lipids: Lipolytic acyl hydrolase (LAH) activities responsible for the deacylation of galactolipids and phospholipids, phospholipase D (PLD, EC 3.1.4.4) activity which gives rise to phosphatidic acid, and as suggested by the presence of diacylglycerols in minor quantities after phospholipid hydrolysis, phosphatidate phosphohydrolase (PAP, EC 3.1.3.4) and/or phospholipase C (PLC, EC 3.1.4.3.) activity. Under the conditions described in the present paper, the presence of phospholipase A (PLA₁, EC 3.1.1.3 and PLA₂, EC 3.1.1.4) activities remains hypothetical, due to the absence of lysophospholipids. LAH and PLD were partially soluble and partially associated with the membranes. When Vigna unguiculata plants were submitted to drought, the enzymatic degradation of galactolipids and phospholipids increased. The stimulation of lipolytic activities was greater in the drought-sensitive cultivar of Vigna unguiculata (cv. 1183) than in the drought-tolerant (cv. EPACE-1) one. In cv. 1183, MGDG- and DGDG-LAH activities in the membrane fractions were dramatically stimulated at a rather moderate water deficit (?0.75 MPa). A sharp increase in membrane phospholipolytic activities was also observed at mild drought stress (?1.2 MPa). In contrast, in cv. EPACE-1, the stimulation of lipolytic activities was less drastic and occurred at lower leaf water potentials (below ?1.2 MPa for galactolipases, and below ?1.4 MPa for phospholipases). Our results confirm the presence in leaves of higher plants of a very active LAH acting on galactolipids, whereas PLD is the main enzyme responsible for the degradation of phospholipids, particularly when plants are submitted to drought stress. The differences in stimulation of lipolytic activities between the two Vigna cultivars was in accordance with the different levels of membrane lipid degradation shown previously and could explain their different capacity to sustain drought.     

Phospholipase A2 in astrocytes

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A₂ (PLA ₂) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA ₂ are expressed in astrocytes: group IV calcium-dependent cytosolic PLA ₂ (cPLA₂), group VI calcium-independent PLA ₂ (iPLA₂), and group II secretory PLA ₂ (sPLA₂). Upregulation of PLA ₂ in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA ₂ activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.     

Release of GPI-Anchored Zn2+-Glycerophosphocholine Cholinephosphodiesterase as an Amphiphilic Form from Bovine Brain Membranes by Bee Venom Phospholipase A2

Enzymatic release of Zn²⁺-glycerophosphocholine (GPC)cholinephosphodiesterase, as an amphiphilic form, from bovine brain membranes was examined. Of various membrane hydrolases, bee PLA₂ was the most effective in the release of the GPC cholinephosphodiesterase (amphiphilic form, 63–70%) from membrane. Compared to pancreatic PLA₂, bee PLA₂ was more efficient in the release of GPC cholinephosphodiesterase. In pH-dependent release of GPl-anchored phosphodiesterase, there was a similar pH-release profile between PLA₂-mediated release and spontaneous one, implying the involvement of membrane disruption in the PLA₂ action. The PLA₂-mediated release showed a limited time-dependence (until 45 min) and a limited dose dependence (up to 3 units / ml), characteristic of a receptor-type binding. An ionic binding of PLA₂ to membrane may be alluded from the interfering effect of anionic phospholipids on the PLA₂ action. In support of an interaction between PLA₂ and membrane glycoproteins, the PLA₂ action was found to be blocked by lectins, wheat germ agglutinin or concanavalin A. In combination with detergent, the PLA₂-mediated release was found to be enhanced synergistically by saponin, a cholesterol-complexing agent. Meanwhile, an additive interaction between PLA₂ and lysolecithin suggests that PLA₂ action is independent of lysolecithin. It is suggested that the binding of PLA₂ to specific sites of membranes, probably rich in GPI-anchored glycoproteins, may be related to the facilitated release of GPI-anchored proteins as amphiphilic form.     

Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape

Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A₂ (PLA₂s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA₂ in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA₂ activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA₂ activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.     

Species of plants and associated arbuscular mycorrhizal fungi mediate mycorrhizal responses to CO2 enrichment

It has been suggested that enrichment of atmospheric CO₂ should alter mycorrhizal function by simultaneously increasing nutrient‐uptake benefits and decreasing net C costs for host plants. However, this hypothesis has not been sufficiently tested. We conducted three experiments to examine the impacts of CO₂ enrichment on the function of different combinations of plants and arbuscular mycorrhizal (AM) fungi grown under high and low soil nutrient availability. Across the three experiments, AM function was measured in 14 plant species, including forbs, C₃ and C₄ grasses, and plant species that are typically nonmycorrhizal. Five different AM fungal communities were used for inoculum, including mixtures of Glomus spp. and mixtures of Gigasporaceae (i.e. Gigaspora and Scutellospora spp.). Our results do not support the hypothesis that CO₂ enrichment should consistently increase plant growth benefits from AM fungi, but rather, we found CO₂ enrichment frequently reduced AM benefits. Furthermore, we did not find consistent evidence that enrichment of soil nutrients increases plant growth responses to CO₂ enrichment and decreases plant growth responses to AM fungi. Our results show that the strength of AM mutualisms vary significantly among fungal and plant taxa, and that CO₂ levels further mediate AM function. In general, when CO₂ enrichment interacted with AM fungal taxa to affect host plant dry weight, it increased the beneficial effects of Gigasporaceae and reduced the benefits of Glomus spp. Future studies are necessary to assess the importance of temperature, irradiance, and ambient soil fertility in this response. We conclude that the affects of CO₂ enrichment on AM function varies with plant and fungal taxa, and when making predictions about mycorrhizal function, it is unwise to generalize findings based on a narrow range of plant hosts, AM fungi, and environmental conditions.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom

An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24 h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.     

Multifunctional role of a fungal pathogen-secreted laccase 2 in evasion of insect immune defense

Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response-generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph-derived in vivo fungal hyphal bodies or be secreted. Targeted gene-knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen-secreted laccase 2 in evasion of insect immune defenses.     

Phospholipase A2 in hemocytes of the tobacco hornworm,Manduca sexta

On the hypothesis that prostaglandins and other eicosanoids mediate nodulation responses to bacterial infections in insects, we describe an intracellular phospholipase A₂ (PLA₂) in homogenates prepared from hemocytes collected from the tobacco hornworm, Manduca sexta. PLA₂ hydrolyzes fatty acids from the sn-2 position of phospholipids. Some PLA₂s are thought to be the first and rate-limiting step in biosynthesis of prostaglandins and other eicosanoids. The hemocyte PLA₂ activity was sensitive to hemocyte homogenate protein concentration (up to 250 μg protein/reaction), pH (optimal activity at pH 8.0), and the presence of a Ca²⁺ chelator. Like PLA₂s from mammalian sources, the hemocyte PLA₂ was inhibited by the phospholipid analog oleyoxyethyl phosphorylcholine. Whereas most intracellular PLA₂s require Ca²⁺ for catalytic activity, some PLA₂s, including the hemocyte enzyme, are Ca²⁺-independent. The hemocyte PLA₂ exhibited a preference for arachidonyl-associated substrate over palmitoyl-associated substrate. These findings show that M. sexta hemocytes express a PLA₂ that shows a marked preference for hydrolyzing arachidonic acid from phospholipids. The biological significance of this enzyme relates to cellular immune responses to bacterial infections. The hemocyte PLA₂ may be the first biochemical step in synthesis of the eicosanoids that mediate cellular immunity in insects. © 1996 Wiley-Liss, Inc.