Catherine Cantrel

Activation of Phospholipases C and D Is an Early Response to a Cold Exposure in Arabidopsis Suspension Cells

The signaling events generated by a cold exposure are poorly known in plants. We were interested in checking the possible activation of enzymes of the phosphoinositide signaling pathway in response to a temperature drop. In Arabidopsis suspension cells labeled with33PO4 3−, a cold treatment induces a rapid increase of phosphatidic acid (PtdOH) content. This production was due to the simultaneous activation of phospholipase C (through diacylglycerol kinase activity) and phospholipase D, as monitored by the production of inositol triphosphate and of transphosphatidylation product, respectively. Moreover, inhibitors of the phosphoinositide pathway and of diacylglycerol kinase reduced PtdOH production. Enzyme activation occurred immediately after cells were transferred to low temperature. The respective contribution of both kind of phospholipases in cold-induced production of PtdOH could be estimated. We created conditions where phospholipids were labeled with33PO4 3−, but with ATP being nonradioactive. In such conditions, the apparition of radioactive PtdOH reflected PLD activity. Thus, we demonstrated that during a cold stress, phospholipase D activity accounted for 20% of PtdOH production. The analysis of composition in fatty acids of cold-produced PtdOH compared with that of different phospholipids confirmed that cold-induced PtdOH more likely derived mainly from phosphoinositides. The addition of chemical reagents modifying calcium availability inhibited the formation of PtdOH, showing that the cold-induced activation of phospholipase pathways is dependent on a calcium entry.

Nitric oxide participates in cold‐responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana

Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.

Insights into the Role of Specific Lipids in the Formation and Delivery of Lipid Microdomains to the Plasma Membrane of Plant Cells

The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual Δ5-sterols are replaced by 9β,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that Δ5-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.

Acyl Chains of Phospholipase D Transphosphatidylation Products in Arabidopsis Cells: A Study Using Multiple Reaction Monitoring Mass Spectrometry

Background Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor. Methodology/Principal findings Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates. Conclusions MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.

Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome

Basal phosphoinositide-dependent phospholipase C (PI-PLC) activity controls gene expression in Arabidopsis suspension cells and seedlings. PI-PLC catalyzes the production of phosphorylated inositol and diacylglycerol (DAG) from phosphoinositides. It is not known how PI-PLC regulates the transcriptome although the action of DAG-kinase (DGK) on DAG immediately downstream from PI-PLC is responsible for some of the regulation. We previously established a list of genes whose expression is affected in the presence of PI-PLC inhibitors. Here this list of genes was used as a signature in similarity searches of curated plant hormone response transcriptome data. The strongest correlations obtained with the inhibited PI-PLC signature were with salicylic acid (SA) treatments. We confirm here that in Arabidopsis suspension cells SA treatment leads to an increase in phosphoinositides, then demonstrate that SA leads to a significant 20% decrease in phosphatidic acid, indicative of a decrease in PI-PLC products. Previous sets of microarray data were re-assessed. The SA response of one set of genes was dependent on phosphoinositides. Alterations in the levels of a second set of genes, mostly SA-repressed genes, could be related to decreases in PI-PLC products that occur in response to SA action. Together, the two groups of genes comprise at least 40% of all SA-responsive genes. Overall these two groups of genes are distinct in the functional categories of the proteins they encode, their promoter cis-elements and their regulation by DGK or phospholipase D. SA-regulated genes dependent on phosphoinositides are typical SA response genes while those with an SA response that is possibly dependent on PI-PLC products are less SA-specific. We propose a model in which SA inhibits PI-PLC activity and alters levels of PI-PLC products and substrates, thereby regulating gene expression divergently.

Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein

Cryptogein is a 10u2009kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [33 P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

Occurrence of an acyl-CoA:1-acylglycerophosphorylcholine acyltransferase in plant mitochondria

In the presence of oleoyl‐CoA, purified and intact mitochondria from potato tuber formed phosphatidylcholine from labeled lysophosphatidylcholine. The labeled oleoyl moiety of the acyl‐CoA was also incorporated in the absence of exogenous lysolipids, such incorporation being largely increased by the addition of exogenous lysophosphatidylcholine. In the presence of various other lysophospholipids, no synthesis of the corresponding phospholipids was observed, suggesting a high specificity of the acyltransferase towards the acyl acceptor. This enzyme was chiefly located in the outer membrane of mitochondria. These results indicate that any acylglycerophosphorylcholine transferred from the endoplasmic reticulum to mitochondria may be acylated to phosphatidylcholine.

Multiple reaction monitoring mass spectrometry is a powerful tool to study glycerolipid composition in plants with different level of desaturase activity

In plants, two lipid desaturation pathways exist. A so-called prokaryotic pathway is active in plastids and responsible for unsaturation of 16 carbon fatty acids. An eukaryotic one, in the endoplasmic reticulum, acts on 18 carbon fatty acids. Desaturase activities are affected in stressed plants, and conversely, they have an impact on the capability of plants to adapt to stress. So knowing lipid unsaturation is important for physiological studies. Analysis of lipids by mass spectrometry, in the multiple reaction mode, gives access to the molecular species present in each membrane lipid class. We illustrate the powerfulness of this technique by applying it to phospholipids and galactolipids extracted from plants where the desaturation pathways are present at variable level.

Over-expression of the Anti-apoptotic Protein Bcl-2 Affects Membrane Lipid Composition in HL-60 Cells

We studied modifications induced at the membrane lipid level by over-expression of the anti-apoptotic protein Bcl-2. When total cell phospholipids were analyzed, the transformation led to a moderate decrease in poly-unsaturated fatty acids, compensated by an increase in mono-unsaturated species. At the mitochondrial membrane level, the changes were more important and occurred in saturated and dimethyl acetal fatty acids, which became more abundant, while unsaturated fatty acid content diminished. This indicates a decline in oxidation-sensitive fatty acids (unsaturated species) together with a gain in oxidation-insensitive saturated fatty acids and in plasmalogen (as detected by dimethyl acetal fatty acids) considered as oxygen species scavengers. Theses changes, combined with the protective role of Bcl-2 against oxidation due to its effect on the redox potential, should protect cells from apoptosis starting in mitochondria.

Effect of High Temperatures on Oxygen Evolving Activity and Lipid Composition of Isolated Chloroplasts from Spinach and Jojoba Leaves

The effects of temperature stress on chloroplasts were investigated by comparison of spinach (Spinacia oleraoea L.), a temperate plant, and jojoba (Simmondsia chinensis (Link) Schneider), a desert-adapted plant.