Search

The Arabidopsis

International audience; Background and aims The rhizosphere microbiome has been shown to contribute to nutrient acquisition, protection against biotic and abiotic stresses and, ultimately, to changes in the development and physiology of plants. Here, using a controlled natural selection approach, we followed the microbial dynamics in the soil of Arabidopsis thaliana plants infected with the foliar pathogen Pseudomonas syringae DC3000 (Pst). Methods Plants were iteratively cultivated on a pasteurised soil inoculated with the soil microbial community of the previous iteration isolated from the rhizosphere of plants infected with Pst (pst-line) or not (mock-line). Modification of soil microbial communities was assessed through an amplicon-based metagenomic analysis targeting bacterial and fungal diversity. Plant fitness and transcript abundance of stress hormone related genes were also analysed. Results At the tenth and eleventh iterations respectively, we observed a reduction in disease severity of 81% and 85% in pst-lines as compared to mock-lines. These changes were associated with (i) an early induction of defence mechanisms mediated by salicylic acid, in pst-line as compared to mock-line, shown by the decrease in transcript abundance of salicylic acid related genes, whereas jasmonic acid, ethylene or abscisic acid related genes remained unchanged and (ii) a shift in soil bacterial, and not in fungal, composition. Conclusions Our study suggests that these changes in soil bacterial composition are mediated by plant-soil feedback in response to Pst and resulted in an activation of SA-related immune response in the plant. This supports the concept of applying plant-soil feedbacks to enhance soil suppressiveness against foliar pathogens.

Flagellin perception is a keystone of pattern-triggered immunity in plants. The recognition of this protein by a plasma membrane (PM) receptor complex is the beginning of a signaling cascade that includes protein phosphorylation and the production of reactive oxygen species (ROS). In both Arabidopsis (Arabidopsis thaliana) seedlings and suspension cells, we found that treatment with flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, caused a rapid and transient decrease in the level of phosphatidylinositol (PI) 4,5-bisphosphate along with a parallel increase in phosphatidic acid (PA). In suspension cells, inhibitors of either phosphoinositide-dependent phospholipases C (PLC) or diacylglycerol kinases (DGKs) inhibited flg22-triggered PA production and the oxidative burst. In response to flg22, receptor-like kinase-deficient fls2, bak1, and bik1 mutants (FLAGELLIN SENSITIVE 2, BRASSINOSTEROID INSENSITIVE 1-associated kinase 1, and BOTRYTIS-INDUCED KINASE 1, respectively) produced less PA than wild-type (WT) plants, whereas this response did not differ in NADPH oxidase-deficient rbohD (RESPIRATORY BURST OXIDASE HOMOLOG D) plants. Among the DGK-deficient lines tested, the dgk5.1 mutant produced less PA and less ROS after flg22 treatment compared with WT seedlings. In response to flg22, dgk5.1 plants showed lower callose accumulation and impaired resistance to Pseudomonas syringae pv. tomato DC3000 hrcC-. Transcriptomics revealed that the basal expression of defense-related genes was altered in dgk5.1 seedlings compared with the WT. A GFP-DGK5 fusion protein localized to the PM, where RBOHD and PLC2 (proteins involved in plant immunity) are also located. The role of DGK5 and its enzymatic activity in flagellin signaling and fine-tuning of early immune responses in plant–microbe interactions is discussed.

Salicylic acid (SA) is a plant hormone almost exclusively associated with the promotion of immunity. It is also known that SA has a negative impact on plant growth, yet only limited efforts have been dedicated to explain this facet of SA action. In this review, we focus on SA-related reduced growth and discuss whether it is a regulated process and if the role of SA in immunity imperatively comes with growth suppression. We highlight molecular targets of SA that interfere with growth and describe scenarios where SA can improve plant immunity without a growth penalty.

Background and Aims We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kβ1β2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kβ1β2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kβ1 and PI4Kβ2. Methods To achieve this we used the sid2pi4kβ1β2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kβ1β2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. Key Results We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kβ1β2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kβ1β2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. Conclusions Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kβ1β2 triple mutant revealed the role of PI4Kβ1/β2 per se, thus showing the importance of these enzymes in plant defence responses.

A secreted peptide, a member of a Brassicaceae-specific gene family, acts on pathogen defense response and root development through the phospholipid pathway and ROS regulation., Small secreted peptides are important players in plant development and stress response. Using a targeted in silico approach, we identified a family of 14 Arabidopsis genes encoding precursors of serine-rich endogenous peptides (PROSCOOP). Transcriptomic analyses revealed that one member of this family, PROSCOOP12, is involved in processes linked to biotic and oxidative stress as well as root growth. Plants defective in this gene were less susceptible to Erwinia amylovora infection and showed an enhanced root growth phenotype. In PROSCOOP12 we identified a conserved motif potentially coding for a small secreted peptide. Exogenous application of synthetic SCOOP12 peptide induces various defense responses in Arabidopsis. Our findings show that SCOOP12 has numerous properties of phytocytokines, activates the phospholipid signaling pathway, regulates reactive oxygen species response, and is perceived in a BAK1 co-receptor-dependent manner.

International audience; Brassinosteroids (BRs) are plant hormones of steroid nature, regulating various developmental and adaptive processes. The perception, transport, and signaling of BRs are actively studied nowadays via a wide range of biochemical and genetic tools. However, most of the knowledge about BRs intracellular localization and turnover relies on the visualization of the receptors or cellular compartments using dyes or fluorescent protein fusions. We have previously synthesized a conjugate of epibrassinolide with green fluorescent dye BODIPY (eBL-BODIPY). Here we present a detailed assessment of the compound bioactivity and its suitability as probe for in vivo visualization of BRs. We show that eBL-BODIPY rapidly penetrates epidermal cells of Arabidopsis thaliana roots and after long exposure causes physiological and transcriptomic responses similar to the natural hormone.

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.

International audience; Actin cytoskeleton is indispensable for plant cell integrity. Besides, increasing evidences illustrate its crucial role in plant responses to environment, including defence against pathogens. Recently, we have demonstrated that pre-treatment with actin disrupting drugs latrunculin B (latB) and cytochalasin E can enhance plant resistance against bacterial and fungal pathogens via activation of salicylic acid (SA) pathway. Here, we show that actin depolymerization in Arabidopsis thaliana seedlings not only triggers SA biosynthesis by ICS1, but also induces callose deposition via callose synthase PMR4. This effect is SA-independent since still present in mutants that do not accumulate SA. LatB also triggers the expression of several defence related genes. We could distinguish genes, induced in a SA-dependent manner (PR1, WRKY38, ICS1) and those that are SA-independent (PR2, PAD4, BAP1). As actin cytoskeleton is tightly connected with membrane trafficking, we assayed the effect of latB on mutant plants invalidated in phosphatidylinositol 4-kinase beta1 and beta2 (PI4Kβ1β2). Deficiency in PI4Kβ1β2 enhanced latB-triggered actin filaments depolymerisation. Yet, it did not lead to a stronger callose deposition or SA biosynthesis in response to latB. Surprisingly, introduction of NahG construct or pad4 mutation in pi4kß1ß2 background had much lower effect on SA induction and SA-dependent gene expression changes than in wild type. We can thus conclude that actin disruption itself triggers immune-like responses: there is an induction of SA via PAD4 coupled to ICS1; it leads to the induction of PR1 and WRKY38, and this requires a functional PI4Kβ1β2 to be properly regulated. However, an alternative, SA-independent, pathway also exists that leads to the enhanced expression of PR2 and to callose accumulation.

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.

a b s t r a c t Despite the fact that all plants follow strict developmental programmes, they also have intrinsic mech- anisms to monitor the environment and activate appropriate responses at the (sub-)cellular level, facilitating adaptation to abiotic and biotic fluctuations. The functionality of plant adaptive systems always relies on the sum of signalling machineries that control their transition from the resting state. Phosphoglycerolipids play a role in such signalling mechanisms. These structural components of cell membranes can be converted into multiple bioactive lipids, but also into soluble molecules. Together they shape cell metabolism via binding to downstream protein targets, thus affecting enzymatic activi- ties, vesicle trafficking and ion fluxes. The conversion of lipids is catalysed by the hydrolytic activity of phospholipases and by the action of lipid-kinases and lipid-phosphatases. These activities are strictly regulated in plant cells and are highly reactive to various environmental signals. While phospholipases have been shown to be essential for plant growth and adaptability, many aspects of phosphoglyc- erolipid signalling at the molecular level remain unknown. Here, we summarise the latest concepts and challenges associated with phosphoglycerolipid signalling in relation to environmental responses in plants.

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.

Xerophyta humilis is a poikilochlorophyllous monocot resurrection plant used as a model to study vegetative desiccation tolerance. Dehydration imposes tension and ultimate loss of integrity of membranes in desiccation sensitive species. We investigated the predominant molecular species of glycerolipids present in root and leaf tissues, using multiple reaction monitoring mass spectrometry, and then analysed changes therein during dehydration and subsequent rehydration of whole plants. The presence of fatty acids with long carbon chains and with odd numbers of carbons were detected and confirmed by gas chromatography. Dehydration of both leaves and roots resulted in an increase in species containing polyunsaturated fatty acids and a decrease in disaturated species. Upon rehydration, lipid saturation was reversed, with this being initiated immediately upon watering in roots but only 12-24 hr later in leaves. Relative levels of species with short-chained odd-numbered saturated fatty acids decreased during dehydration and increased during rehydration, whereas the reverse trend was observed for long-chained fatty acids. X. humilis has a unique lipid composition, this report being one of the few to demonstrate the presence of odd-numbered fatty acids in plant phosphoglycerolipids.

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.

International audience; Gravity is one of the environmental cues that direct plant growth and development. Recent investigations of different gravity signalling pathways have added complexity to how we think gravity is perceived. Particular cells within specific organs or tissues perceive gravity stimulus. Many downstream signalling events transmit the perceived information into subcellular, biochemical, and genomic responses. They are rapid, non-genomic, regulatory, and cell-specific. The chain of events may pass by signalling lipids, the cytoskeleton, intracellular calcium levels, protein phosphorylation-dependent pathways, proteome changes, membrane transport, vacuolar biogenesis mechanisms, or nuclear events. These events culminate in changes in gene expression and auxin lateral redistribution in gravity response sites. The possible integration of these signalling events with amyloplast movements or with other perception mechanisms is discussed. Further investigation is needed to understand how plants coordinate mechanisms and signals to sense this important physical factor.

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.

Growth is the best visible sign of plant comfort. If plants are under stress, a difference in growth with control conditions can indicate that something is going wrong (or better). Phytohormones such as auxin, cytokinins, brassinosteroids or giberellins, are important growth regulators and their role in plant growth was extensively studied. On the other hand the role of salicylic acid (SA), a phytohormone commonly connected with plant defense responses, in plant growth is under-rated. However, studies with SA-overaccumulating mutants directly showed an influence of SA on plant growth. Recently we characterized an Arabidopsis SA-overaccumulating mutant impaired in phosphatidylinositol-4-kinases (pi4kIIIβ1β2). This mutant is dwarf. The crossing with mutants impaired in SA signaling revealed that pi4kIIIβ1β2 stunted rosette is due to high SA, while the short root length is not. This brings into evidence that upper and lower parts of the plants, even though they may share common phenotypes, are differently regulated.

We recently showed that—in Arabidopsis thaliana suspension cells—phosphoinositide dependent-phospholipase C (PI-PLC) and diacylglycerol kinase (DGK) negatively regulated the basal expression of most DREB2 genes. DREB2 genes encode transcription factors that bind to Drought Responsive Elements (DRE). Those elements are also bound by DREB1 factors. While DREB2 factors are mostly involved in drought and heat responses, DREB1s are induced in the response to chilling. We here show that the pharmacological inhibition of PI-PLC or DGK leads to the basal induction of DREB1 genes. However, the induction is much less marked for the DREB1 genes than that of DREB2A, a member of the DREB2 family. This illustrates that DREB1 and DREB2 genes, while having the same targets, are not submitted to the same transcription regulation, and that lipid signaling might in part explain these differences in the regulation of the DREB genes.

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; Due to the increasing demand for biofuel production, it is an important goal to optimize the seed productivity and quality of oilseed plants even in adverse conditions. Acting on signalling mechanisms might provide means to attain such goals. In this study, we were interested in the effect of a brassinosteroid hormone 24-epibrassino-lide (24-EBR) on Brassica napus cultivated in salt stress condition. We show that salt stress leads to a 60 % decrease in seed production in B. napus. This is accompanied by a 50 % decrease in seed oil content. Treatment with 24-EBR had no effect on seed and oil productivity in control plants. However, it could rescue half of the seed production and all the oil production in salt-treated plants. The fatty acid composition of seed oil in B. napus was selectively affected by salt stress, 24-EBR or combined treatment. Besides these long-term actions of 24-EBR, we have also investigated its short-term actions in cell signalling. We did so by in vivo labelling of plantlets with fluorescently labelled phospha-tidylcholine. A treatment of 2 h with 24-EBR was sufficient to induce a substantial increase in the content of diacyl-glycerol and phosphatidic acid, two lipid mediators. Non-specific phospholipases C and phospholipases D are involved in these increases. Therefore, brassinosteroid treatments appear as promising way to gain oil productivity when plants have to grow in unfavourable conditions such as salt stress. The link between long-term actions and short-term signalling of 24-EBR is discussed.

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.

Unfortunately, the original publication of this article contains errors. In table 1, the phrase “Localized under amyloplasts” should be changed to “Associated with amy l o p l a s t s ” . I n “P r o t e i n Pho s p h o r y l a t i o n / dephosphorylation” section, the citation “Clore et al., 2003” should be deleted from the complex (Clore et al., 2003; Liu et al., 2009). In the middle of the same paragraph, the citation “Clore et al., 2003” should be deleted from the complex (Chang, Kaufman, 2000; Clore et al., 2003), and the citation (Clore at al., 2003) at the end of the same paragraph should be also changed to (Chang et al., 2003) and lost citation “Chang SC, Cho MH, Kim SK, Lee JS, Kirakosyan A, Kaufman PB (2003) Changes in phosphorylation of 50 and 53 kDa soluble proteins in graviresponding oat (Avena sativa) shoots Journal of Experimental Botany 54:1013-1022” should be added to the list of references. In “Protein Phosphorylation/dephosphorylation” section, at the beginning of the first paragraph in sentence “The relatively long time (75 min) needed for the MAP kinase activity to rise suggests that it only acts after the onset of gene expression or as the result of auxin transport/action” the phrase “or as the result of auxin transport/action” should be deleted. In section “Phosphoinositide-specific phospholipase C” at the beginning of the second paragraph the sentence “The increase in IP3 in response to gravity is independent on auxin transport” should be changed to “Changes in InsP3 may precede auxin redistribution”. In the sentence "Results of pharmacological studies show that IP3 action in gravity response is calcium mediated and the second phase of calcium increase is dependent on PI-PLC production of IP3 (Perera et al. 2001; Plieth and Trewavas 2002; Toyota et al. 2013a) at the end of the 3 paragraph the “Perera et al. 2001” citation should be changed to “Perera et al. 2006”. In table 3, in the line containing an information about exogenous application of H2O2 to the lower side of pulvinus the word “no” should be replaced with “+” and description “no, No effect” under the table should be deleted. The online version of the original article can be found at http://dx.doi.org/ 10.1007/s00709-015-0859-5.