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Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defense signaling cascades by binding to proteins. NPR1 is a transcriptional coactivator and is a key target of SA binding. Many other proteins have been shown to bind SA. Among these proteins are important enzymes of primary metabolism. Here, we describe that the A1 isomer of chloroplast glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA, as shown in surface plasmon resonance experiments. Additionally, we show that SA inhibits its GAPDH activity in vitro. To gain an insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein–ligand complex. The molecular docking analysis led to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket, a surface cavity around Asn35, would efficiently bind SA in the presence of a solvent. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the binding of NADP+ to GAPA1. NADH inhibited, in a dose-response manner, the binding of SA to GAPA1, validating our data. The use of the methodology to study SA binding to other proteins will be discussed at the end of the talk.

Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA&rsquo, s ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein&ndash, ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket&mdash, a surface cavity around Asn35&mdash, would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.

We show that both abscisic acid (ABA) and salicylic acid (SA) inhibit in vivo basal PI-PLC activity in Arabidopsis suspension cells. However, SA also activates in vivo phosphatidylinositol phosphorylation, which ABA does not. The transcriptome response of Arabidopsis suspension cells to a 4 h ABA treatment was compared to that of cells treated with U73122 or wortmannin, inhibitors of phospholipase C (PLC) and of the phosphatidylinositol-4-kinase that provides substrates to PLC, respectively. A clear over representation of genes for which ABA and these inhibitors have similar effects can be observed. Furthermore, 28% of ABA-induced genes were also induced by SA, while 40% of ABA-repressed genes were also repressed by SA. A similarity search in publically accessible transcriptome data confirms that those genes are also similarly regulated in response to ABA and SA in seedlings. We have shown that some of the SA responsive genes responded via an increase in phosphorylated phosphatidylinositol (phosphoinositides), while others did so via the activation of a phospholipase D pathway, and that the SA response of other genes was mimicked by the inhibition of PI-PLC. Interestingly, the pool of genes similarly regulated by SA and ABA is impoverished in genes responsive to SA via phosphoinositides but is enriched in those for which the response to SA is mimicked by the inhibition of PI-PLC. The increase in phosphoinositide appears to be an important process explaining the SA-specific response, while inhibition of a basal PI-PLC might contribute to the common ABA and SA transcriptomic responses.

Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional

Phosphatidic acid (PA) is a simple phospholipid observed in most organisms. PA acts as a key metabolic intermediate and a second messenger that regulates many cell activities. In plants, PA is involved in numerous cell responses induced by hormones, stress inputs and developmental processes. Interestingly, PA production can be triggered by opposite stressors, such as cold and heat, or by hormones that are considered to be antagonistic, such as abscisic acid and salicylic acid. This property questions the specificity of the responses controlled by PA. Are there generic responses to PA, meaning that cell regulation triggered by PA would be always the same, even in opposite physiological situations? Alternatively, do the responses to PA differ according to the physiological context within the cells? If so, the mechanisms that regulate the divergence of PA-controlled reactions are poorly defined. This review summarizes the latest opinions on how PA signalling is directed in plant cells and examines the intrinsic properties of PA that enable its regulatory diversity. We propose a concept whereby PA regulatory messages are perceived as complex "signatures" that take into account their production site, the availability of target proteins and the relevant cellular environments.

Salicylic acid (SA) is a small phenolic compound whose therapeutic properties on human health have long been described. In plants, it is a key phytohormone that controls many physiological processes. In the last 20 years, great attention has been paid to its role in plant pathogen defence. The synthesis of SA is indeed one of the crucial ways a plant reacts to a biotic attack. SA is involved in both local and systemic resistance. SA metabolism (biosynthesis, conjugation and accumulation) and the signalling pathways that control SA levels are described here. The transcription factor NPR1 is an established cornerstone of SA signalling. Yet, both NPR1-dependent and NPR1-independent signalling pathways have been described, but very little is known about the effectors of the NPR1-independent pathway. We present work using different SA over-accumulating mutants as tools for studying the control of SA biosynthesis and the downstream effects of SA. Recently, new evidence has been provided concerning SA perception. Interestingly, NPR1 was proposed to be a SA-receptor, as were proteins involved in the control of NPR1 turnover. The different proteins involved in SA metabolism, in the regulation of SA levels and in the response to SA can define a “SA signalling module”. It is possible to use the genes encoding members of this module as indicators to identify stress situations where SA signalling is activated. This is illustrated for different biotic and abiotic stress responses.

Phosphoinositides are minor constituents of eukaryotic membranes but participate in a wide range of cellular processes. The most abundant and best characterized phosphoinositide species are phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and its main precursor, phosphatidylinositol 4-phosphate (PI4P). PI4P and PI(4,5)P₂ regulate various structural and developmental functions but are also centrally involved in a plethora of signal transduction pathways in all eukaryotic models. They are not only precursors of second messengers but also directly interact with many protein effectors, thus regulating their localisation and/or activity. Furthermore, the discovery of independent PI(4,5)P₂ signalling functions in the nucleus of mammalian cells have open a new perspective in the field. Striking similarities between mammalian, yeast and higher plant phosphoinositide signalling are noticeable, revealing early appearance and evolutionary conservation of this intracellular language. However, major differences have also been highlighted over the years, suggesting that organisms may have evolved different PI4P and PI(4,5)P₂ functions over the course of eukaryotic diversification. Comparative studies of the different eukaryotic models is thus crucial for a comprehensive view of this fascinating signalling system. The present review aims to emphasize convergences and divergences between eukaryotic kingdoms in the mechanisms underlying PI4P and PI(4,5)P₂ roles in signal transduction, in response to extracellular stimuli.

In response to environmental stresses, or during development, plant cells will produce lipids that will act as intracellular mediators (Janda et al., 2013; Ruelland et al., 2015). Glycerophospholipid and/or sphingolipid second messengers resulting from the action of lipid metabolizing enzymes (e.g., lipid-kinases or lipases) are commonly found within cells. The articles published within this Research Topics clearly illustrate the trends of this research field that we can sum up as follows.

SPE IPM UB INRA SUPDAT; International audience; Cryptogein is a 10 kDa-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 NADPH oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) isoform D. Using radioactive [33P]-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 due to 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 line, 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.

Confronted to changes in temperatures, plants readjust their biochemical makeup to adapt and survive. The fact that temperature changes can induce cellular responses indicates that temperature is sensed and that the temperature signal is transduced into the cell. While the signalling pathways triggered temperature changes are well described, the way plants sense temperature is often considered as elusive. This review is focused on the mechanisms by which plants sense temperature. We show that plants have no internal thermometer as such, but that the very alterations in cellular equilibria triggered by temperature changes act as networked thermostats to sense heat and cold. Amongst these temperature-sensitive devices, we identified membrane fluidity, protein conformation, cytoskeleton depolymerization, and metabolic reactions. Besides, other molecular switches are proposed. A model of the temperature sensing “machinery” is proposed. Finally, we discuss the specificities of temperature sensing, showing that signalling events can feed-back perception steps.

International audience; Diacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol into phosphatidic acid. To fulfill their role in many signalling processes, DGKs must be located at, or in, membranes. Most mammalian DGKs are cytosolic and are recruited to membranes upon stimulation, except for e type DGKs that are permanently membrane-associated through a hydrophobic segment. Nothing is known about the mechanism(s) involved in the membrane localization of plant DGKs. By fusion to fluorescent proteins, we show that two DGKs from cluster I in Arabidopsis thaliana possess amino-terminal hydrophobic segments that are sufficient to address them to endoplasmic reticulum membranes.

Salicylic acid (SA) has a central role in defense against pathogen attack. In addition, its role in such diverse processes as germination, flowering, senescence, and thermotolerance acquisition has been documented. However, little is known about the early signaling events triggered by SA. Using Arabidopsis (Arabidopsis thaliana) suspension cells as a model, it was possible to show by in vivo metabolic phospholipid labeling with 33Pi that SA addition induced a rapid and early (in few minutes) decrease in a pool of phosphatidylinositol (PI). This decrease paralleled an increase in PI 4-phosphate and PI 4,5-bisphosphate. These changes could be inhibited by two different inhibitors of type III PI 4-kinases, phenylarsine oxide and 30 μ m wortmannin; no inhibitory effect was seen with 1 μ m wortmannin, a concentration inhibiting PI 3-kinases but not PI 4-kinases. We therefore undertook a study of the effects of wortmannin on SA-responsive transcriptomes. Using the Complete Arabidopsis Transcriptome MicroArray chip, we could identify 774 genes differentially expressed upon SA treatment. Strikingly, among these genes, the response to SA of 112 of them was inhibited by 30 μ m wortmannin, but not by 1 μ m wortmannin.

In plants, a temperature downshift represents a major stress that will lead to the induction or repression of many genes. Therefore, the cold signal has to be perceived and transmitted to the nucleus. In response to a cold exposure, we have shown that the phospholipase D (PLD) and the phospholipase C (PLC)/diacylglycerol kinase pathways are simultaneously activated. The role of these pathways in the cold response has been investigated by analyzing the transcriptome of cold-treated Arabidopsis (Arabidopsis thaliana) suspension cells in the presence of U73122 or ethanol, inhibitors of the PLC/diacylglycerol kinase pathway and of the phosphatidic acid produced by PLD, respectively. This approach showed that the expression of many genes was modified by the cold response in the presence of such agents. The cold responses of most of the genes were repressed, thus correlating with the inhibitory effect of U73122 or ethanol. We were thus able to identify 58 genes that were regulated by temperature downshift via PLC activity and 87 genes regulated by temperature downshift via PLD-produced phosphatidic acid. Interestingly, each inhibitor appeared to affect different cold response genes. These results support the idea that both the PLC and PLD pathways are upstream of two different signaling pathways that lead to the activation of the cold response. The connection of these pathways with the CBF pathway, currently the most understood genetic system playing a role in cold acclimation, is discussed.

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.

International audience; Phosphoinositide-specific phospholipase C (PI-PLC) cleaves, in a Ca 2þ-dependent manner, phosphati-dylinositol-4,5-bisphosphate (PI-4,5-P 2) into diacylglycerol (DAG) and inositol triphosphate (IP 3). PI-PLCs are multidomain proteins that are structurally related to the PI-PLCzs, the simplest animal PI-PLCs. Like these animal counterparts, they are only composed of EF-hand, X/Y and C2 domains. However, plant PI-PLCs do not have a conventional EF-hand domain since they are often truncated, while some PI-PLCs have no EF-hand domain at all. Despite this simple structure, plant PI-PLCs are involved in many essential plant processes, either associated with development or in response to environmental stresses. The action of PI-PLCs relies on the mediators they produce. In plants, IP 3 does not seem to be the sole active soluble molecule. Inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) also transmit signals, thus highlighting the importance of coupling PI-PLC action with inositol-phosphate kinases and phosphatases. PI-PLCs also produce a lipid molecule, but plant PI-PLC pathways show a peculiarity in that the active lipid does not appear to be DAG but its phosphorylated form, phosphatidic acid (PA). Besides, PI-PLCs can also act by altering their substrate levels. Taken together, plant PI-PLCs show functional differences when compared to their animal counterparts. However, they act on similar general signalling pathways including calcium homeostasis and cell phosphoproteome. Several important questions remain unanswered. The cross-talk between the soluble and lipid mediators generated by plant PI-PLCs is not understood and how the coupling between PI-PLCs and inositol-kinases or DAG-kinases is carried out remains to be established.

International audience; Phospholipids have recently been found to be integral elements of hormone signalling pathways. An Arabidopsis thaliana double mutant in two type III phosphatidylinositol-4-kinases (PI4Ks), pi4kIII beta 1 beta 2, displays a stunted rosette growth. The causal link between PI4K activity and growth is unknown. Using microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and multiple phytohormone analysis by LC-MS we investigated the mechanism responsible for the pi4kIII beta 1 beta 2 phenotype. The pi4kIII beta 1 beta 2 mutant accumulated a high concentration of salicylic acid (SA), constitutively expressed SA marker genes including PR-1, and was more resistant to Pseudomonas syringae. pi4kIII beta 1 beta 2 was crossed with SA signalling mutants eds1 and npr1 and SA biosynthesis mutant sid2 and NahG. The dwarf phenotype of pi4kIII beta 1 beta 2 rosettes was suppressed in all four triple mutants. Whereas eds1 pi4kIII beta 1 beta 2, sid2 pi4kIII beta 1 beta 2 and NahG pi4kIII beta 1 beta 2 had similar amounts of SA as the wild-type (WT), npr1pi4kIII beta 1 beta 2 had more SA than pi4kIII beta 1 beta 2 despite being less dwarfed. This indicates that PI4KIII beta 1 and PI4KIII beta 2 are genetically upstream of EDS1 and need functional SA biosynthesis and perception through NPR1 to express the dwarf phenotype. The slow root growth phenotype of pi4kIII beta 1 beta 2 was not suppressed in any of the triple mutants. The pi4kIII beta 1 beta 2 mutations together cause constitutive activation of SA signalling that is responsible for the dwarf rosette phenotype but not for the short root phenotype.

The chloroplastic NADP malate dehydrogenase is completely inactive in its oxidized form and is activated by thiol/disulfide interchange with reduced thioredoxin. To elucidate the molecular mechanism underlying the absence of activity of the oxidized enzyme, we used site-directed mutagenesis to delete or substitute the two most C-terminal residues (C-terminal Val, penultimate Glu, both bearing negative charges). We also combined these mutations with the elimination of one or both of the possible regulatory N-terminal disulfides by mutating the corresponding cysteines. Proteins mutated at the C-terminal residues had no activity in the oxidized form but were partially inhibited when pretreated with the histidine-specific reagent diethyl pyrocarbonate before activation, showing that the active site was partially accessible. Proteins missing both N-terminal regulatory disulfides reached almost full activity without activation upon elimination of the negative charge of the penultimate Glu. These results strongly support a model where the C-terminal extension is docked into the active site through a negatively charged residue, acting as an internal inhibitor. They show also that the reduction of both N-terminal bridges is necessary to release the C-terminal extension from the active site. This is the first report for a thiol-activated enzyme of a regulatory mechanism resembling the well known intrasteric inhibition of protein kinases.

The light-dependent mechanisms responsible for the regulation of two enzymes of the C4 photosynthetic cycle, phosphoenolpyruvate carboxylase and NADP-dependent malate dehydrogenase in Sorghum are presented as an illustration of adaptative mechanisms of plants to light. Emphasis is put on the organization of the signaltransduction chains which upregulate both enzymes at transcriptional and post-translational levels. For both enzymes, small gene families include a photosynthesis-related gene, the expression of which is light-triggered. The light-controlled phosphorylation of C4 PEPC implies cross-talk between photosynthetic cell types, cytosolic pH and calcium changes, and the upregulation of a specific protein-serine kinase. This post-translational modification strongly influences its regulation by photosynthesis-related metabolites. NADP-MDH activity is controlled by a chloroplastic redox cascade involving thioredoxin as the ultimate relay. The significance of these regulatory circuits is discussed in relation with the flexibility of the metabolism in the context of the C4 photosynthesis.

The role of the invariant Trp residue at the redox site of thioredoxins was investigated by site-directed mutagenesis of a Chlamydomonas reinhardtii thioredoxin h. Though being still redox active with NADPH-thioredoxin reductase and chemical substrates [dithiothreitol and 5,5'-dithio-bis(2-nitrobenzoic acid)] the Trp35-->Ala-mutated protein completely lost the capacity to activate the thiol-regulated NADPH-dependent malate dehydrogenase. However, it was able to activate a mutant malate dehydrogenase where only the most exposed disulfide was retained. The pH dependence of the redox-site Cys beta 1H/13C-NMR frequencies of the wild-type and mutated proteins, in both the reduced and oxidised states, were compared over the pH range 5.8-10. The mutation does not affect the conserved buried Asp30, which titrates with a pKa of 7.5 in the oxidised proteins in agreement with previous studies. However, for the reduced forms of the proteins, the pH dependence of resonances of both Cys was strongly affected by the mutation. In the case of the wild-type thioredoxin, two apparent pKa values were found around 7.0 and 9.5 and could be assigned to the titration of Cys36 and Cys39 thiol, respectively, similar to the case of Escherichia coli thioredoxin. For the mutated thioredoxin a single pKa was found around 8.3. This result can be interpreted as a single pKa of either Cys36 or Cys39 or both. While the mutation clearly affects ionisations, the measured redox potentials of the active-site Cys pair are not significantly affected by the Trp35-->Ala mutation. Possible roles of an aromatic side chain on the reactivity of the catalytic Cys residues in thioredoxins are proposed.

International audience; Phosphoglycerolipids are essential structural constituents of membranes and some also have important cell signalling roles. In this review, we focus on phos-phoglycerolipids that are mediators in hormone signal transduction in plants. We first describe the structures of the main signalling phosphoglycerolipids and the metabolic pathways that generate them, namely the phospholi-pase and lipid kinase pathways. In silico analysis of Arabidopsis transcriptome data provides evidence that the genes encoding the enzymes of these pathways are trans-criptionally regulated in responses to hormones, suggesting some link with hormone signal transduction. The involvement of phosphoglycerolipid signalling in the early responses to abscisic acid, salicylic acid and auxins is then detailed. One of the most important signalling lipids in plants is phosphatidic acid. It can activate or inactivate protein kinases and/or protein phosphatases involved in hormone signalling. It can also activate NADPH oxidase leading to the production of reactive oxygen species. We will interrogate the mechanisms that allow the activation/ deactivation of the lipid pathways, in particular the roles of G proteins and calcium. Mediating lipids thus appear as master players of cell signalling, modulating, if not controlling , major transducing steps of hormone signals.

International audience; 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.

International audience; Phosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 μM wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation.

International audience; Non-specific phospholipases C (NPCs) were discovered as a novel type of plant phospholipid-cleaving enzyme homologous to bacterial phosphatidylcholine-specific phospholipases C and responsible for lipid conversion during phosphate-limiting conditions. The six-gene family was established in Arabidopsis, and growing evidence suggests the involvement of two articles NPCs in biotic and abiotic stress responses as well as phytohormone actions. In addition, the diacylglycerol produced via NPCs is postulated to participate in membrane remodelling, general lipid metabolism and cross-talk with other phospholipid signalling systems in plants. This review summarises information concerning this new plant protein family and focusses on its sequence analysis, biochemical properties, cellular and tissue distribution and physiological functions. Possible modes of action are also discussed.

Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first step in the synthesis of phosphoinositide pools hydrolysed by phosphoinositide-dependent phospholipase C (PI-PLC) and thus constitute a potential key regulation point of this pathway. Twelve putative PI4K isoforms, divided as type-II (AtPI4KIIγ1- 8) and type-III PI4Ks (AtPI4KIIIα1- 2 and AtPI4KIIIβ1- 2), have been identified in Arabidopsis genome. By a combination of pharmalogical and genetic approaches we recently evidenced that AtPI4KIIIβ1 and AtPI4KIIIβ2 contribute to supply PI-PLC with substrate and that AtPI4KIIIα1 is probably also involved in this process. Given the current knowledge on PI-PLC and type-III PI4Ks localization in plant cells it raises the question whether type-III PI4Ks produce phosphatidylinositol 4-phosphate at the site of its consumption by the PI-PLC pathway. We therefore discuss the spatial organization of substrate supply to PI-PLC in plant cells with reference to recent data evidenced in mammalian cells.

Phosphatidylinositol-4-phosphate (PtdIns4P) is the most abundant phosphoinositide in plants and the precursor of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)]. This lipid is the substrate of phosphoinositide-dependent phospholipase C (PI-PLC) that produces diacylglycerol (DAG) which can be phosphorylated to phosphatidic acid (PtdOH). In plants, it has been suggested that PtdIns4P may also be a direct substrate of PI-PLC. Whether PtdIns4P is the precursor of PtdIns(4,5)P(2) or a substrate of PI-PLC, its production by phosphatidylinositol-4-kinases (PI4Ks) is the first step in generating the phosphoinositides hydrolyzed by PI-PLC. PI4Ks can be divided into type-II and type-III. In plants, the identity of the PI4K upstream of PI-PLC is unknown. In Arabidopsis, cold triggers PI-PLC activation, resulting in PtdOH production which is paralleled by decreases in PtdIns4P and PtdIns(4,5)P(2). In suspension cells, both the PtdIns4P decrease and the PtdOH increase in response to cold were impaired by 30 μM wortmannin, a type-III PI4K inhibitor. Type-III PI4Ks include AtPI4KIIIα1, β1 and β2 isoforms. In this work we show that PtdOH resulting from the PI-PLC pathway is significantly lowered in a pi4kIIIβ1β2 double mutant exposed to cold stress. Such a decrease was not detected in single pi4kIIIβ1 and pi4kIIIβ2 mutants, indicating that AtPI4KIIIβ1 and AtPI4KIIIβ2 can both act upstream of the PI-PLC. Although several short-term to long-term responses to cold were unchanged in pi4kIIIβ1β2, cold induction of several genes was impaired in the double mutant and its germination was hypersensitive to chilling. We also provide evidence that de novo synthesis of PtdIns4P by PI4Ks occurs in parallel to PI-PLC activation.

International audience; 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 (SA) plays a central role in defense against pathogen attack, as well as in germination, flowering, senescence, and the acquisition of thermotolerance. In this report we investigate the involvement of phospholipase D (PLD) in the SA signaling pathway. In presence of exogenous primary alcohols, the production of phosphatidic acid by PLD is diverted toward the formation of phosphatidylalcohols through a reaction called transphosphatidylation. By in vivo metabolic phospholipid labeling with 33Pi, PLD activity was found to be induced 45 min after addition of SA. We show that incubation of Arabidopsis (Arabidopsis thaliana) cell suspensions with primary alcohols inhibited the induction of two SA-responsive genes, PATHOGENESIS-RELATED1 and WRKY38, in a dose-dependent manner. This inhibitory effect was more pronounced when the primary alcohols were more hydrophobic. Secondary or tertiary alcohols had no inhibitory effect. These results provide compelling arguments for PLD activity being upstream of the induction of these genes by SA. A subsequent study of n-butanol effects on the SA-responsive transcriptome identified 1,327 genes differentially expressed upon SA treatment. Strikingly, the SA response of 380 of these genes was inhibited by n-butanol but not by tert-butanol. A detailed analysis of the regulation of these genes showed that PLD could act both positively and negatively, either on gene induction or gene repression. The overlap with the previously described phosphatidylinositol-4-kinase pathway is discussed.

Exposure to low temperatures is one of the most important plant abiotic stress factors. In this review we describe the damages that chilling and/or freezing temperatures can cause to plant cells. Confronted to these damages, some plants are able to adapt through mechanisms based on protein synthesis, membrane composition changes, and activation of active oxygen scavenging systems. These adaptive mechanisms rely in part on gene induction. The best understood genetic pathway leading to gene induction upon a temperature downshift is based on C-repeat-binding factors (CBF) activating promoters through the C-repeat (CRT) cis-element. Such activation of transcription factors suggests that cold, as a signal, has been transduced into the cells. Calcium entry is a major signalling event occurring immediately after a temperature downshift. The increase in cytosolic calcium will activate many enzymes, such as phospholipases and calcium dependent-protein kinases. A MAP-kinase module has been shown to be involved in the cold response. Ultimately, the activation of those signalling pathways leads to changes to the transcriptome. In this review we have focused on the genetic and signalling pathways activated early after cold exposure. Much of the data cited is from the model plant Arabidopsis but when possible evidence from other plants is presented.

"Phosphoinositide" refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active "per se," by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression "per se." However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides "per se." Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones.

The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.

During thioredoxin-mediated activation of chloroplastic NADP-malate dehydrogenase, a homodimeric enzyme, the interaction between subunits is known to be loosened but maintained. A modeling of the 3D structure of the protein identified Asp-101 as being potentially involved in the association between subunits through an electrostatic interaction. Indeed, upon site-directed substitution of Asp-101 by an asparagine, the mutated enzyme behaved mainly as a monomer. The mutation strongly affected the catalytical efficiency of the enzyme. The now available 3D structure of the enzyme shows that Asp-101 is protruding at the dimer interface, interacting with Arg-268 of the neighbouring subunit.

Oxidation-reduction midpoint potentials (E(m)) have been measured for the thioredoxin-dependent, reductive activation of sorghum nicotinamide adenine dinucleotide phosphate- (NADP-) dependent malate dehydrogenase (MDH) in the wild-type enzyme and in a number of site-specific mutants. The E(m) value associated with activation of the wild-type enzyme, -330 mV at pH 7.0, can be attributed to the E(m) of the C365/C377 disulfide present in the C-terminal region of the enzyme. The C24/C29 disulfide, located in the N-terminal region of the enzyme and the only other disulfide present in oxidized, wild-type MDH, has a E(m) value of -280 mV at pH 7.0. A third regulatory disulfide, C24/C207, that is absent in the oxidized enzyme but is thought to be formed during the activation process, has an E(m) value at pH 7.0 of -310 mV. E(m) vs pH profiles suggest pK(a) values for the more acidic cysteine involved in the formation of each of these disulfides of 8.5 for C24/C29; 8.1 for C24/C207; and 8.7 for C365/C377. The results of this study show that the N-terminal disulfide formed between C24 and C29 has a more positive E(m) value than the two other disulfides and is thus is likely to be the "preregulatory disulfide" postulated to function in activating the enzyme.

Studies on redox signaling and light regulation of chloroplast enzymes have highlighted the importance of the ferredoxin–thioredoxin thiol–disulfide interchange cascade. Recent research has focused on the intramolecular mechanism by which the reduction status of a chloroplast enzyme affects its catalytic properties, and site-directed mutagenesis has been used to identify the regulatory cysteines involved. For some of the thiol-regulated enzymes, structure–function studies have revealed that the complex conformational changes that occur might be associated with disulfide isomerization and auto-inhibition. Transgenic approaches indicate that this regulation constitutes a rapid means to adjust enzyme activity to metabolic needs.

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.

In addition to being submitted to thiol-regulation by reduced thioredoxin, the chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH: EC. 1.1.1.82) is regulated by the redox state of its cofactor. Indeed, the oxidized cofactor NADP inhibits the reductive activation of the enzyme. This feature, first reported 15 years ago [1, 2] is still poorly understood. The activity of the fully activated enzyme is not inhibited by NADP, thus the effect on activation must be linked to a subtle difference between the conformation of the acitve vs. inactive enzyme. NAD does not inhibit significantly: the specificity of the inhibition parallels the specificity of the enzymatic reaction. Modeling of the 3-D structure of NADP-MDH based on the coordinates of the permanently active NAD-dependent porcine heart enzyme [3] showed only one coenzyme binding site, accessible from the outside. The accessibility of the coenzyme binding site in the oxidized enzyme is also substantiated by the fact that triazine-dye affinity columns mimicking the structure of the cofactor (matrex red A) can be used to purify the enzyme. The hypothesis put forward in relation with the model was that the oxidized cofactor would bind in a wrong orientation [3]. In the course of our investigation on the mechanism of the reductive activation process of the enzyme by thiol/disulfide interchange with reduced thioredoxin, we created a number of partially deregulated mutants where selected disulfides were suppressed by site-directed mutagenesis by exchanging cysteines for serines or alanines [3,4]. The proteins mutated at the C-terminal cysteines 365 and/or 377, quite unexpectedly, lost their sensitivity to inhibition by NADP. This result suggested that in some way, the C-terminal end of the enzyme was implicated in the inhibition effect. In the present paper, the effect of NADP on NADP-MDH activation has been further investigated by additional site-directed mutagenesis experiments targeting either the last two C-terminal residues, or residues supposed to be implicated in the cofactor specificity. A tentative explanation is proposed for the observed effects: the positively charged oxidized cofactor would intaract with the negatively charged C-terminus of the enzyme, thus enhancing the locking of the active site.

International audience; The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thi-oredoxins. Previous experiments showed that four cys-teines located in specific N-and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation ki-netics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility , mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cys-teines were produced and purified. The C29S/C365A/ C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity , leading to the hypothesis that a Cys 24-Cys 207 disul-fide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.

The chlorplast NADP-malate dehydrogenase is activated through the reduction of two different disulfides per subunit. The activated enzyme, as well as a permanently active mutant where all four regulatory cysteines were replaced are still sensitive to thiol reagents. This observation suggested the presence of an additional important cysteine at the active site. In an attempt to identify that cysteine, site-directed mutagenesis was performed on the cDNA encoding sorghum leaf NADP-malate dehydrogenase. The replacement of Cys-175 by an alanine yielded an enzyme whose sensitivity to thiol reagents was markedly decreased whereas its catalytic activity was enhanced. This finding suggests that Cys-175 has no catalytic function but is located close to the active site.

Membrane rigidification could be the first step of cold perception in poikilotherms. We have investigated its implication in diacylglycerol kinase (DAGK) activation by cold stress in suspension cells from Arabidopsis mutants altered in desaturase activities. By lateral diffusion assay, we showed that plasma membrane rigidification with temperature decrease was steeper in cells deficient in oleate desaturase than in wild type cells and in cells overexpressing linoleate desaturase. The threshold for the activation of the DAGK pathway in each type of cells correlated with this order of rigidification rate, suggesting that cold induced-membrane rigidification is upstream of DAGK pathway activation.