Igor Pokotylo
email: pokotylo@bpci.kiev.ua

Yaroslav Kolesnikov
email: kolesnikov@bpci.kiev.ua

Volodymyr Kravets
email: kravets@bpci.kiev.ua

Alain Zachowski
email: alain.zachowski@upmc.fr

Eric Ruelland
email: eric.ruelland@upmc.fr

Plant phosphoinositide-dependent phospholipases C: variations around a canonical theme

Keywords: DGK, DAG-kinase, IP3, inositol triphosphate, IP5, inositol pentakisphosphate, IP6, inositol hexakisphosphate, Phospholipase C, Lipid signalling, Phosphoinositides, Inositol-phosphate, Phosphatidic acid

Phosphoinositide-specific phospholipase C (PI-PLC) cleaves, in a Ca 2+ -dependent manner, phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) into diacylglycerol (DAG) and inositol triphosphate (IP3). PI-PLCs are multidomain proteins that are structurally related to the PI-PLCζs, 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 rely on the mediators they produce. In plants, IP3 does not seem to be the sole active soluble molecule. Inositol pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) 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.

Introduction

Phosphoinositide-specific phospholipases C (PI-PLCs) are essential enzymes that cleave, in a Ca 2+ -dependent manner, membrane phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) to produce two second messengers: i) a lipid, diacylglycerol (DAG), and ii) a soluble molecule, inositol 1,4,5triphosphate (IP3). PI-PLCs have been identified in many eukaryotes from yeast to mammals [START_REF] Kadamur | Mammalian Phospholipase C[END_REF][START_REF] Yoko-O | The putative phosphoinositidespecific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth[END_REF] while simplified PI-PLCs are also present in bacteria [START_REF] Wei | Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors[END_REF], indicating the probable common evolutionary origin of all PI-PLCs.

In animals, the role and regulation of PI-PLC isozymes are well established [START_REF] Kadamur | Mammalian Phospholipase C[END_REF]. The canonical model states that PI-PLC action is linked to the activation of protein kinase C (PKC) by DAG and to an intracellular Ca 2+ release mediated by IP3-sensitive channels (IP3 receptors). The mode of action of plant PI-PLCs must be different from the animal model since the amount of PI-4,5-P2 is lower in plant membranes [START_REF] Delage | Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: convergences and divergences among eukaryotic kingdoms[END_REF], plants apparently lack conventional IP3 receptors, and no plant PKC orthologs have been identified to date. However, clear roles (that we will detail) have been assigned to plant PI-PLCs in response to environmental stresses and during development. Despite the conclusive data concerning the role(s) of PI-PLCs in the plant cell, little is known about their in planta regulation. In this review we focus on the latest advances in plant PI-PLCs including their biochemical characteristics that precondition their role as regulators of many plant physiological reactions, their regulation and their modes of action. We will show that, as for animal systems, there is a tight connection between PI-PLC and Ca 2+ -signalling while the important mediator for this action might not be IP3, but inositol pentakisphosphate (IP5) or inositol hexakisphosphate (IP6). Finally, we will describe and discuss the active contribution of plant PI-PLCs to phosphatidic acid (PA) production in concert with diacylglycerol kinases (DGK). Indeed, PA is probably the lipid mediator produced by plant PI-PLC pathways, and protein targets of PA have been identified. Whether the lipid and soluble mediators act on separate signalling pathways or control the same pathways, synergistically or antagonistically, is an exciting ongoing field of investigation.

Structure of PI-PLCs

In every PI-PLC, the catalytic X and Y domains are flanked by regulatory sequences. In animals, PLCζ is the structurally simplest isoform, with the catalytic domains being flanked by an N-terminal ЕF-hand domain and a C-terminal С2 domain [START_REF] Kadamur | Mammalian Phospholipase C[END_REF] (Fig. 1). The other mammalian PI-PLCs have additional domains. They all possess an N-terminal pleckstrin homology (РН) domain, involved in membrane targeting and protein binding [START_REF] Singh | Molecular modeling of the membrane targeting of phospholipase C pleckstrin homology domains[END_REF][START_REF] Fujii | of phospholipase C-δ1 regulates its enzymatic activity[END_REF] while additional domains depend on specific isoforms. For instance, a C-terminal PDZ-domain (Post synaptic density-95, Drosophila disc large tumor suppressor, and Zonula occludens-1 protein) with a protein binding motif required for supramolecular complex formation is present in the mammalian PLCβ [START_REF] Kim | PDZ domain-containing 1 (PDZK1) protein regulates phospholipase C-β3 (PLC-β3)-specific activation of somatostatin by forming a ternary complex with PLC-β3 and somatostatin receptors[END_REF]. All plant PI-PLCs are structurally related to the PLCζ isoform [START_REF] Wang | Lipid signaling[END_REF], as they are formed by the succession of EF-hand, X/Y and C2 domains.

X and Y domains

Catalytic activity of all PI-PLCs is believed to strictly rely on the X/Y domains. It was demonstrated that single aminoacid substitution in X-domain abolished PLC catalityc activity [START_REF] Dowd | Petunia phospholipase C1 is involved in pollen tube growth[END_REF]. Many residues are highly conserved in these domains of all eukaryotic PI-PLCs and they are involved in substrate binding and catalysis. For example, in animal PLCδ1, Lys 438 , Lys 440 , Ser 522 and Arg 549 are involved in interacting with the 4-and 5-phosphates of the substrate headgroup [START_REF] Ellis | Catalytic domain of phosphoinositide-specific phospholipase C (PLC): Mutational analysis of residues within the active site and hydrophobic ridge of PLCδ1[END_REF] and the positive charge of Arg 549 has been shown to be required for the preferential hydrolysis of PI-4,5-P2 over PI [START_REF] Wang | Positive charge at position 549 is essential for phosphatidylinositol 4,5-bisphosphate-hydrolyzing but not phosphatidylinositol-hydrolyzing activities of human phospholipase C δ1[END_REF]. Two conserved His residues in the X domain participate in the mixed acid/base-catalyzed reaction of phosphoinositide hydrolysis: one stabilizes the pentavalent phosphoryl transition state, and the other protonates and disjoints DAG and, being a nucleophile, attacks a 1,2-cyclicinositolphosphate intermediate [START_REF] Essen | Crystal structure of a mammalian phosphoinositide-specific phospholipase C delta[END_REF]. These His residues are conserved in plant PI-PLCs and correspond to His 126 and His 169 of the X domain of mung bean enzyme [START_REF] Kim | The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.)[END_REF]. Substitution of a conserved Ser to Asn in the Y region of the active site of Physcomitrella patens PLC2 correlates with a reduced catalytic activity in comparison with the PLC1 isoform [START_REF] Mikami | Isolation of cDNAs encoding typical and novel types of phosphoinositide-specific phospholipase C from the moss Physcomitrella patens[END_REF]. However, whether this Ser corresponds to Ser 522 in animal PLCδ1 and therefore has a similar role in substrate interaction remains to be shown. Interestingly, Arabidpsis thaliana AtPLC8 and AtPLC9, that are the most divergent proteins compared to other AtPLCs, have long deletions in the Y region [START_REF] Tasma | Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana[END_REF]. Consequently their catalytic activity is questionable. However, a distinct role of AtPLC9 in plant stress responses has been demonstrated using a plc9 mutant that displays a thermosensitive phenotype [START_REF] Zheng | Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis[END_REF].

EF-hand domain

The conventional EF-hand domain consists of four helix-loop-helix folding motifs and is often characteristic of calcium-binding proteins [START_REF] Kumar | In silico prediction and analysis of Caenorhabditis EF-hand containing proteins[END_REF]. In mammalian PI-PLCs, the EF-hand acts as an allosteric regulatory domain that binds calcium and lipids, stabilizes PI-PLC structure and assists in active site formation [START_REF] Kobayashi | Spectroscopic characterization of the EF-hand domain of phospholipase C δ1: Identification of a lipid interacting domain[END_REF]. Moreover, in PLCβ3, the EF-hand participates in the formation of an active complex with Gα protein [START_REF] Waldo | Kinetic scaffolding mediated by a phospholipase C-β and Gq signaling complex[END_REF], while in PLCγ, it facilitates binding to tyrosine kinases [START_REF] Essen | A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-δ1[END_REF]. These observations plead for a major role of the EF-hand as a structural determinant of PI-PLCs. However, plant PI-PLCs have no full length EF-hand domain since most of them have a truncated EF-hand consisting of only two helix-loop-helix motifs [START_REF] Essen | Crystal structure of a mammalian phosphoinositide-specific phospholipase C delta[END_REF][START_REF] Otterhag | N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana[END_REF] while several plant PI-PLCs have no N-terminal EF-hand, such as AtPLC2 [START_REF] Hirayama | AtPLC2, a gene encoding phosphoinositidespecific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana[END_REF]. Soybean PI-PLC1 contains an additional putative EF-hand type calciumbinding motif located in between the X and Y domains, while the N-terminal EF-hand is truncated [START_REF] Shi | Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean[END_REF]. Because the EF-hand mediates the calcium-dependent activation of PLCζ [START_REF] Kouchi | The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Cζ[END_REF], it is tempting to postulate a similar role in plant PI-PLCs. Whether a truncated EF-hand can still carry out this function is unknown. Interestingly, PI-PLCs with no N-terminal EF-hand still show a catalytic activity [START_REF] Kopka | Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato[END_REF]. Clearly the importance of the EF-hand domain is, as yet, not understood for plant PI-PLCs.

C2 domain

All identified plant PI-PLCs contain a C2 domain. This domain is described to bind phospholipid, and calcium may positively participate in this process [START_REF] Kouchi | The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Cζ[END_REF]. In potato and rice PI-PLCs, specific hydrophobic residues [START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF] and the polybasic region K-(K,R)-T-K [START_REF] Kopka | Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato[END_REF] in the C2 domain possibly mediate C2 domain binding to anionic phospholipids. In some plants, the С2 domain is sufficient to address PI-PLCs to the membranes [START_REF] Dowd | Petunia phospholipase C1 is involved in pollen tube growth[END_REF][START_REF] Kim | The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.)[END_REF][START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF] while in other cases, the C2 domain is involved in membrane targeting only when the EF-hand is present [START_REF] Helling | Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling[END_REF].

Other structural determinants

A highly hydrophilic and extremely divergent linker region between the X and Y domains is also believed to be essential and to play different roles in different PI-PLCs. In animals, the X-Y linker has an autoinhibitory function in all PI-PLCs, except PLCζ [START_REF] Hicks | General and versatile autoinhibition of PLC isozymes[END_REF]. In PLCζ, the linker is rather positively charged and it is required for PI-4,5-P 2 binding [START_REF] Nomikos | Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region[END_REF]. In contrast, plant PI-PLCs contain a linker region containing a high percentage of acidic residues that are presumed to be exposed at the surface of the folded protein [START_REF] Hirayama | AtPLC2, a gene encoding phosphoinositidespecific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana[END_REF]. To date, the role of the linker in plant PI-PLC activity remains to be identified.

A sequence with high homology to the G protein coupled-receptor motif has been found in the N-terminus region of Brassica napus PI-PLC2 [START_REF] Das | Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1)comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B. napus[END_REF] as well as in AtPLC2 [START_REF] Hirayama | AtPLC2, a gene encoding phosphoinositidespecific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana[END_REF]. Its function is unknown.

The Cys 7 residue is essential for disulphide bond formation resulting in homodimerization of Chlamydomonas reinhardtii PI-PLC and a lower lipid affinity [START_REF] Awasthi | Disulphide bridges of phospholipase C of Chlamydomonas reinhardtii modulates lipid interaction and dimer stability[END_REF]. A Cys residue is present in the first 10 residues of most plant PI-PLCs but whether it is involved in homodimerization remains to be shown. Homodimerization via uncharacterized mechanisms have been demonstrated previously for putative animal and bacterial PLCs [START_REF] Shao | Dimer structure of an interfacially impaired phosphatidylinositol-specific phospholipase C[END_REF][START_REF] Singer | A unique fold of phospholipase C-beta mediates dimerization and interaction with Galphaq[END_REF].

PI-PLC-like proteins

Several PI-PLC-like proteins lacking one or several canonical PI-PLC domains are found in plants. A Medicago truncatula DNF2 protein containing only the PI-PLC X domain, and thus closer to bacterial PI-PLCs than to eukarotic PI-PLCs, has been shown to be involved in the control of symbiotic relations with Sinorhizobium meliloti [START_REF] Bourcy | Medicago truncatula DNF2 is a PI-PLC-XD-containing protein required for bacteroid persistence and prevention of nodule early senescence and defense-like reactions[END_REF]. Whether this protein possesses a PI-PLC catalytic activity or only interacts with PLC substrates remains to be shown. Putative PI-PLC proteins with a similar structure are also found in Arabidopsis, maize and rice (supplemental table 1).

Taken together, these data indicate that plant PI-PLCs possess a relatively simple structure when compared to animal PI-PLCs, they contain several essential domains -namely the X/Y catalytic domains, a C2 domain and truncated EF-hand domains. Regardless of these limited structural features, plant PI-PLC are regulatory targets that have important functions within plant cells. Inconstant sequence features of plant PI-PLC such as signal peptide sequence (SP) [START_REF] Vossen | Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance[END_REF] and sequence with homology to G-protein coupled receptor (GPCR) [START_REF] Das | Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1)comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B. napus[END_REF] are indicated. Putative sites of plant PI-PLC regulation are marked: Ca 2+ binds to plant PI-PLC both within the C2 domain and X-Y linker region [START_REF] Hirayama | AtPLC2, a gene encoding phosphoinositidespecific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana[END_REF][START_REF] Shi | Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean[END_REF][START_REF] Kopka | Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato[END_REF][START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF]. Interactions with Gα protein [START_REF] Misra | Heterotrimeric G-protein complex and Gprotein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C[END_REF], membrane lipids [START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF] and NtC7 in tobacco [START_REF] Nakamura | A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants[END_REF] have been observed at the C2 domain. At the N-terminus, plant PI-PLC may undergo dimerization [START_REF] Awasthi | Disulphide bridges of phospholipase C of Chlamydomonas reinhardtii modulates lipid interaction and dimer stability[END_REF] or associate with membranes [START_REF] Otterhag | N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana[END_REF]. Site of phosphorylation detected between X and Y domains of PI-PLC [START_REF] Nühse | Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses[END_REF]. Also indicated are typical direct and indirect products of phosphoinositide hydrolysis by PI-PLC and plant physiological processes dependent on PI-PLC activity. IP3, inositol trisphosphate; IP6, inositol hexakisphosphate; DAG, diacylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol, PIP2, Phosphatidylinositol 4,5-bisphosphate.

Phylogenetic considerations

Due to the high level of structural identity, all PI-PLCs might share a common ancestor. Throughout evolution, PI-PLCs have evolved into distinct groups that are now represented as isozymes within multigene families. We have conducted a phylogenetic analysis of a number of plant PI-PLC enzymes (Fig. 2). They are usually grouped according to taxonomical traits. Algae PI-PLC sequences are more divergent from those of other plant species and more related to human and yeast PI-PLCs. Higher plant PI-PLCs are separated from human and yeast PI-PLC proteins which group together. Grouping of PI-PLCs from non-vascular plants -lycophytes and moss -was also observed, suggesting that PI-PLC sequences further independently multiplied and evolved after the separation of mosses, lycophytes, and vascular plants. All but one of monocot PI-PLCs were found to be grouped together. Multiple independent intraspecies duplications must have occurred, leading to PI-PLC family expansion in each species. For example, in Arabidopsis, genes AtPLC8 and AtPLC9 occur in a tandem array on chromosome 3 and may represent a relatively recent local duplication. Progenitors of AtPLC1/AtPLC3 possibly arose from a single gene by a duplication event on chromosome 5, with a subsequent duplication and relocation of AtPLC3 to chromosome 4 [START_REF] Tasma | Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana[END_REF][START_REF] Hunt | Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms[END_REF]. Highly similar AtPLC1, AtPLC4 and AtPLC5 genes are organized in tandem in a small DNA region of chromosome 5 possibly due to recent gene duplication events [START_REF] Hunt | Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms[END_REF]. Conserved blocks in PI-PLC-X, PI-PLC-Y and C2 domains from 53 PI-PLC sequences were subjected to phylogenetic analysis. An unrooted phylogenetic tree was created using the maximum likelihood method in PhyMl (WAG substitution model). Numbers at nodes represent values of neighbour-joining bootstrap support obtained using the aLRT (approximate likelihood-ratio test) method. Branches below 50% were collapsed. The scale bar represents the number of substitutions per site. Unclassified proteins were named according to maximum similarity with the respective PI-PLC isozyme of Arabidopsis. Species abbreviations: At, Arabidopsis.

Regulation of plant PI-PLCs

Due to their intrinsic role as essential regulators of metabolism, PI-PLCs constantly undergo a fine tuning of activity mediated by effectors, regulatory domains and intracellular localization.

Localization

PI-PLCs have no predicted transmembrane domains (TMHMM Server v. 2.0) and there are no current experimental data suggesting palmitoylation of plant PI-PLCs. However, PI-PLC activity strictly requires either transient or permanent association with cell membranes where phosphoinositides are located.

In plants, PI-PLC activities were traditionally studied as either cytosolic or membraneassociated, with differences in preferred substrates and Ca 2+ requirements [START_REF] Shi | Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean[END_REF]. Yet these two PI-PLC fractions may represent pools of the same proteins since a specific antibody raised against the N-terminal domain of AtPLC4 reacted with a 68 kDa protein both in the plasma membrane and cytosolic fractions [START_REF] Cao | Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits[END_REF]. Whether a mechanism of stress-induced translocation towards membranes, as shown for phospholipase D, is applicable to PI-PLC is currently unclear.

PI-PLC proteins apparently employ several means for being targeted to membranes. Critical roles of EF-hand [START_REF] Helling | Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling[END_REF] and C2 domains [START_REF] Kim | The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.)[END_REF][START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF] in Ca 2+ -regulated membrane-targeting has been demonstrated in plants. PI-4,5-P2 also appears to be important for PI-PLC membrane targeting. In mammals, the PH domain directs PLC activation and high affinity association with PI-4,5-P2 in membranes [START_REF] Lomasney | Phosphatidylinositol 4,5-bisphosphate binding to the pleckstrin homology domain of phospholipase C-δ1 enhances enzyme activity[END_REF]. A role of the X-Y linker region in the binding of PLCζ, that lacks the PH domain, to PI-4,5-P2, has been suggested [START_REF] Nomikos | Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region[END_REF]. Plant PI-PLCs also lack the PH domain, and their X-Y linker is negatively charged, contrary to that of PLCζ, and plant cell membranes are poor in PI-4,5-P2 when compared to animal membranes [START_REF] Van Leeuwen | Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings[END_REF]. Therefore, a PI-4,5-P2-dependent mechanism of plant PLC membrane targeting still requires clarification.

So, to which membranes are PLCs associated? Most data point to a plasma membrane localization, based on studies with fluorescent fusion proteins [START_REF] Dowd | Petunia phospholipase C1 is involved in pollen tube growth[END_REF][START_REF] Helling | Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling[END_REF][START_REF] Liu | Contributions of PIP2-specific-phospholipase C and free salicylic acid to heat acclimation-induced thermotolerance in pea leaves[END_REF] or tandem mass spectrometry studies [START_REF] Benschop | Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis[END_REF][START_REF] Elmore | Quantitative proteomics reveals dynamic changes in the plasma membrane during Arabidopsis immune signaling[END_REF][START_REF] Li | Comparison of plasma membrane proteomic changes of Arabidopsis suspension-cultured cells (T87 Line) after cold and ABA treatment in association with freezing tolerance development[END_REF][START_REF] Mitra | An efficient organic solvent based extraction method for the proteomic analysis of Arabidopsis plasma membranes[END_REF]. However, PI-PLC has been observed with the endoplasmic reticulum of wheat [START_REF] Khalil | Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositidespecific phospholipase C, PI-PLC1[END_REF] and barley [START_REF] Johnston | Heat shock-induced changes in lipid and protein metabolism in the endoplasmic reticulum of barley aleurone layers[END_REF]. Interestingly, PI-4,5-P2 is found only in plasma membranes [START_REF] Delage | Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: convergences and divergences among eukaryotic kingdoms[END_REF], thus raising the question as to the PI-PLC substrate in other membranes.

Interestingly, several studies indicate the presence of cytosol-located PI-PLCs. NtPLCδ1 was shown to be cytoplasmic in quiescent tobacco BY2 cells [START_REF] Tripathy | Characterization and functional validation of tobacco PLC delta for abiotic stress tolerance[END_REF] and a strong association to the actin cytoskeleton of a soluble protein that reacted with anti-bovine PLCβ1 polyclonal antibodies was demonstrated in oat roots [START_REF] Huang | Phosphoinositide-specific phospholipase C in oat roots: association with the actin cytoskeleton[END_REF].

Less data are available concerning the tissue localization of PI-PLCs. Genevestigator data [START_REF] Hruz | Genevestigator V3: A reference expression database for the meta-analysis of transcriptomes[END_REF] points out that the root and pollen are organs with high PLC transcript levels (Fig. 3A,3B). GUS expression under the control of the AtPLC1 promoter was detected in petioles and vascular tissues. AtPLC5::GUS was detected in guard cells, roots and vascular cells. AtPLC4::GUS was detected in pollen and certain floral organs (fig. 3C) [START_REF] Hunt | Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms[END_REF]. A,B) Microarray data were collected using Genevestigator interface [START_REF] Hruz | Genevestigator V3: A reference expression database for the meta-analysis of transcriptomes[END_REF]. In (A) color saturation represents the absolute level of transcript abundance. In (B) the relative transcript prevalence is given as percentage of the level in the organ where the isoform is expressed at maximum level. (C) Organs and tissues where Arabidopsis PI-PLC isoforms are mainly expressed as shown by real-time PCR or promoter activity studies [START_REF] Tasma | Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana[END_REF][START_REF] Hunt | Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms[END_REF]. In (C), the expression of PI-PLC6, PI-PLC8 and PI-PLC9 of Arabidopsis can be distinguished.

Ca 2+ dependency

All Arabidopsis PI-PLCs, with the possible exception of AtPLC4 [START_REF] Hunt | Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms[END_REF], strictly require Ca 2+ for their activity. Our current knowledge provides clues for both Ca 2+ -driven activation and membrane targeting of plant PI-PLC [START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF]. Ca 2+ requirement is often linked to substrate preference. PI-PLCs from Cupressus lusitanica [START_REF] Zhao | Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures[END_REF] and Physcomitrella patens [START_REF] Mikami | Isolation of cDNAs encoding typical and novel types of phosphoinositide-specific phospholipase C from the moss Physcomitrella patens[END_REF] can use non-phosphorylated PI as a substrate at high Ca 2+ concentrations. Not only do PI-PLCs depend on Ca 2+ , but in mammals they also actively contribute to its liberation from intracellular stores (via IP3 receptors), thus making them essential enzymes of Ca 2+ turnover. Although no IP3 receptor has been genetically characterized in plants, there is evidence for IP3-induced Ca 2+ release, suggesting the presence of a conserved mechanism of Ca 2+ regulation (see 9.3).

Post translational modifications

4.3.1. Phosphorylation. In animals, regulation of PLCγ activity by phosphorylation of tyrosine residues of X/Y linkers within the catalytic domain has been reported [START_REF] Gresset | Mechanism of phosphorylation-induced activation of phospholipase C-γ isozymes[END_REF]. In plants, phosphorylation of PI-PLCs has also been shown. Several phosphorylation sites were identified in AtPLCs by mass spectrometry peptide analyses [START_REF] Durek | PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update[END_REF](Table 1), some of them located within functional domains, such as the phosphorylation of Thr 29 within an EF-hand like motif of AtPLC2, and phosphorylation of either Ser 346 or Ser 348 in the Y domain [START_REF] Whiteman | Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana[END_REF][START_REF] Nühse | Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database[END_REF][START_REF] Chen | Comparative analysis of phytohormone-responsive phosphoproteins in Arabidopsis thaliana using TiO2-phosphopeptide enrichment and mass accuracy precursor alignment[END_REF] while AtPLC7 contains adjacent phosphorylation sites at Thr 169 and Ser 170 in the X catalytic domain [START_REF] Nühse | Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database[END_REF].

Palmitoylation.

A covalent attachment of a fatty acid residue (S-acylation) is a common post-translational control of various protein properties [START_REF] Hemsley | Multiple roles for protein palmitoylation in plants[END_REF]. Protein lipid modifications facilitate membrane targeting and interaction with negatively charged head groups of lipids such as PI-4,5-P2. Currently, there are no experimental data concerning palmitoylation of plant PI-PLCs. However, AtPLC1 and AtPLC3 have several high-probability putative palmitoylation sites at cysteine residues (Table 2) [START_REF] Ren | CSS-Palm 2.0: an updated software for palmitoylation sites prediction[END_REF].

Ubiquitination.

To our knowledge, no experimental data are available regarding ubiquitination of plant PI-PLCs. However, computational predictions suggest the presence of putative ubiquitination sites in AtPLC1, AtPLC2, AtPLC5, AtPLC6, AtPLC7 and AtPLC8 (BDM-PUB) (Table 2). 4.3.4. SUMOylation. Attachment of small ubiquitin-like modifiers (SUMOylation) to specific lysine residues in protein targets is another conserved mechanism of protein post-translational regulation [START_REF] Mazur | Global SUMO proteome responses guide gene regulation, mRNA biogenesis, and plant stress responses[END_REF]. Unlike the related ubiquitination, SUMOylated proteins are not directed for degradation and instead SUMOylation affects protein stability, activity and localization. Limited data are available regarding bona fide SUMOylation of plant PI-PLCs. SUMOylation of PLC8 was detected in heat-treated transgenic Arabidopsis plants overexpressing AtSUMO1 [START_REF] Park | Identification and molecular properties of SUMO-binding proteins in Arabidopsis[END_REF]. In addition, numerous putative SUMOylation sites were detected in PI-PLCs of Arabidopsis, and AtPLC2, AtPLC5, AtPLC6 and AtPLC8 have conventional tetrapeptide Ψ-K-x-E motifs while AtPLC7 has a good probability for the presence of a non-consensual SUMOylation site [START_REF] Ren | Systematic study of protein sumoylation: Development of a site-specific predictor of SUMOsp 2.0[END_REF]. These predictions (Table 2) now require experimental proof. 4.3.5. Other modifications. In plants, treatment with the NO-donor S-nitroso-Nacetylpenicilamine induced PA formation both by PI-PLC and phospholipase D (PLD) [START_REF] Distéfano | Nitric oxide-induced phosphatidic acid accumulation: a role for phospholipases C and D in stomatal closure[END_REF] suggesting a possible mechanism of PI-PLC activity regulation via nitrosylation or nitration of cysteine/tyrosine residues. Whether PI-PLC is the direct target of nitrosylation is not known.

Protein-protein interactions

In mammalian cells, PI-PLCs interact with a range of regulatory elements including G-proteins [START_REF] Gutman | Differential regulation of phospholipase C-β2 activity and membrane interaction by Gαq, Gβ1γ2, and Rac2[END_REF], tyrosine kinases [START_REF] Zhang | Focal adhesion kinase promotes phospholipase C-γ1 activity[END_REF] and others [START_REF] Aisiku | Identification of a novel binding partner of phospholipase Cβ1: Translin-Associated Factor X[END_REF]. Such interactions are thought to provide basic mechanisms of PI-PLC activity regulation and coordination with other cell effectors. Less is known about PI-PLC protein partners in plant cells. Current data point out a possible interaction with a G-protein coupled receptor system. PI-PLC activity was increased by cholera toxin (a G-protein agonist) and inhibited by pertussis toxin (a G-protein antagonist) in Lilium daviddi pollen protoplasts [START_REF] Pan | Characterization of phosphatidylinositol-specific phospholipase C (PI-PLC) from Lilium daviddi pollen[END_REF]. PI-PLC activity from the cytosolic fraction of Phaseolus vulgaris root nodules was stimulated in vitro by mastoparan, a G-protein agonist [START_REF] De Los Santos-Briones | GTPγS antagonizes the mastoparan-induced in vitro activity of PIP2-phospholipase C from symbiotic root nodules of Phaseolus vulgaris[END_REF]. Yeast two-hybrid experiments provided evidence for an interaction between PI-PLC and some G-protein subunits in Lilium daviddi [START_REF] Sun | The physical interaction between LdPLCs and Arabidopsis G beta in a yeast twohybrid system[END_REF] and Pisum sativum [START_REF] Misra | Heterotrimeric G-protein complex and Gprotein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C[END_REF]. Interestingly, Gα1, but not Gβ, is able to bind to the C2 domain of PLCδ from Pisum sativum [START_REF] Misra | Heterotrimeric G-protein complex and Gprotein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C[END_REF]. PI-PLC1 from wheat (Triticum aestivum) was reported to interact with the Ga3 Gα subunit when expressed in tobacco epidermal leaf tissues [START_REF] Khalil | Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositidespecific phospholipase C, PI-PLC1[END_REF].

Other proteins were also found to interact with PLCs. An example is Nicotiana tabacum NtC7 receptor-like protein implicated in plant reaction to wounding and osmotic stress resistance, which interacts with the C2 domain of NtPI-PLC in yeast two-hybrid screening and in planta when assayed by bimolecular fluorescence complementation [START_REF] Nakamura | A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants[END_REF]. Both proteins were detected in the plasmalemma when expressed in onion epidermal cell layers. Authors postulated that NtC7, that contains a C-terminal transmembrane domain, may drive PI-PLC plasma membrane targeting [START_REF] Nakamura | A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants[END_REF]. No protein encoded by the Arabidopsis genome shows a significant homology to NtC7.

High throughput screening for protein-protein interactions using the split-ubiquitin method yielded additional putative PI-PLC protein partners [START_REF] Lalonde | Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations[END_REF]. The roles of AtPLC protein partners (Table 1) could give indications concerning the role(s) of PI-PLCs. As an example, AtPLC1 interacted with cyclic nucleotide-gated channel 13 protein, a putative cyclic nucleotide and calmodulin-regulated ion channel that affects Ca 2+ cell signalling [START_REF] Ma | Ca 2+ conduction by plant cyclic nucleotide gated channels and associated signaling components in pathogen defense signal transduction cascades[END_REF]. AtPLC7 cross-reacted with diacylglycerol kinase 2 (DGK2), thus showing that they interact not only functionally but also physically in DAG-and PAdependent lipid signalling [START_REF] Arisz | Plant PA signaling via diacylglycerol kinase[END_REF]. Interactions between PI-PLC and protein kinases have also been reported (Table 1). Such interactions may result in the phosphorylation of PI-PLC or in regulating the phosphorylation of other protein partners. However, the physiological significance of most of the observed protein-protein interactions remains to be elucidated.

Plant PI-PLC expression and co expression web

The role of plant PI-PLCs can be assessed using transcriptome data. Expression studies of individual PI-PLC genes from different species have been performed but for this review we focus on transcriptome-wide analyses, in response to hormone treatment or environmental stresses (Fig. 4). For species where expression data of different isoforms are available, the expression pattern is usually chracterized by a multidirectional regulation of the different isoforms. This is the case during elicitor and heat treatments for the PI-PLC genes of wheat and Arabidopsis. This kind of data can be interpreted as an evidence that PI-PLC genes in families are not necessarily redundant, and that some have specific and distinct roles, therefore have distinct expression regulation. Yet for some elicitations, all PI-PLC genes of a same species are regulated in the same way, as seen for cold treatment, abscisic acid (ABA shoots), drought, salt stress and wounding in Arabidopsis. These treatments, in the organ(s) considered, induce the expression of some PLC genes, while others are not affected. This is in agreement with a PI-PLC role in response to cold and osmotic stresses (see below).

It is conceivable that co-expression of genes may signify their functional association. A coexpression study was performed using the Genevestigator interface [START_REF] Hruz | Genevestigator V3: A reference expression database for the meta-analysis of transcriptomes[END_REF](Table 1.) Several AtPLCs coexpress with genes implicated in plant stress and hormonal responses. AtPLC1 was found to be coexpressed with an allene oxide synthase (At5g42650) involved in the jasmonic acid (JA) biosynthetic pathway [START_REF] Park | Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts[END_REF] and with a member of the calcium-dependent phosphotriesterase superfamily (At3g51450) implicated in plant responses to salicylic acid (SA), methyl jasmonate, wounding or pathogen inoculation [START_REF] Sohani | Phylogenetic and transcriptional analysis of a strictosidine synthase-like gene family in Arabidopsis thaliana reveals involvement in plant defence responses[END_REF]. AtPLC4 co-expressed with At5g63990 encoding a 3'(2'),5'-bisphosphate nucleotidase/inositol polyphosphate 1-phosphatase, with a possible role during cold stress [START_REF] B.-H. Lee | The Arabidopsis cold-responsive transcriptome and its regulation by ICE1[END_REF] and functionally related to the stress-responsive FRY1 gene implicated in the attenuation of the IP3 signal [START_REF] Xiong | FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis[END_REF]. Due to their high similarity, AtPLC8 and AtPLC9 expression cannot be distinguished in Arabidopsis ATH1 22k arrays. AtPLC8/9 co-expressed with At4g16950 and At4g19530, both genes involved in plant immunity encoding NB-LRR receptor-like proteins [START_REF] Bailey | Molecular cloning of ATR5Emoy2 from Hyaloperonospora arabidopsidis, an avirulence determinant that triggers RPP5-mediated defense in Arabidopsis[END_REF][START_REF] Swiderski | The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction[END_REF]. AtPLC5 co-expressed with another biotic-stress protein, an SOS2-like Protein Kinase 5 (At2g30360) that is involved in controlling several plant activities and shown to directly phosphorylate NPR1 -a critical component of plant immunity [START_REF] Xie | PKS5, a SNF1-related kinase, interacts with and phosphorylates NPR1, and modulates expression of WRKY38 and WRKY62[END_REF]. AtPLC3 appears to co-express with type I metacaspase (At5g64240) that is known to be involved in the control of cell death [START_REF] Coll | Arabidopsis Type I metacaspases control cell death[END_REF] and AtPLC7 co-expresses with a member of the phosphate starvation-induced glycerol-3-phosphate permease gene family (At4g17550) possibly implicated in phosphorus transport during phosphate starvation [START_REF] Ramaiah | Characterization of the phosphate starvation-induced gycerol-3-phosphate permease gene family in Arabidopsis[END_REF]. Interestingly, AtPLC4 significantly co-expressed with AtPLC5, indicating the possible redundancy of these 2 isoforms. Transcriptional analysis of plant PI-PLC gene expression was performed in silico using Genevestigator (https://www.genevestigator.com/). Shown here are cumulative representative data concerning changes in PI-PLC gene expression in different growth conditions. When several expression data points with different time or different dose of treatment were available, the one with the most apparent and consistent changes in PI-PLC expression was chosen. ABA, abscisic acid; CK, cytokinins; Bl, brassinolide; GA, gibberelic acid; IAA, indolacetic acid; ET, ethylene; MeJa, methyl jasmonate; SA, salycilic acid. Species abbreviations: At, Arabidopsis thaliana; Si, Solanum licopersicum; Os, Oryza sativa; Zm, Zea mays; Hv, Hordeum vulgare; Ta, Triticum aestivum, Gm, Glycine max.

PI-PLCs in plant development

PI-PLCs are often considered to be stress-activated enzymes. However, they also play a role in regulating growth and development-related processes both in animals [START_REF] Jing | Phospholipase C gamma-1 is required for granulocyte maturation in zebrafish[END_REF] and plants [START_REF] Khodakovskaya | Winter Sederoff, Increasing inositol (1,4,5)-trisphosphate metabolism affects drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increases in tomato[END_REF]. The role of PI-PLC in plant development seems to be multifaceted. For instance, over-expression of BnPI-PLC2 caused both an early shift from vegetative to reproductive phases, and shorter maturation periods, together with alterations in hormonal distribution patterns in plant tissues [START_REF] Georges | Over-expression of Brassica napus phosphatidylinositol-phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation[END_REF]. IP3 is involved in the differentiation of xylem vessels [START_REF] Zhang | Involvement of phosphoinositide turnover in tracheary element differentiation in Zinnia elegans L. cells[END_REF] and stolon-to-tuber transition [START_REF] Cenzano | Phospholipid and phospholipase changes by jasmonic acid during stolon to tuber transition of potato[END_REF]. An influence of PI-PLC was also noted during asymmetric cell divisions that produce stomatal complexes in Zea mays [START_REF] Apostolakos | The involvement of phospholipases C and D in the asymmetric division of subsidiary cell mother cells of Zea mays[END_REF]. PI-PLCs also seem to participate in cell cycle progression in tobacco, through DNA synthesis control [START_REF] Apone | The G-protein-coupled receptor GCR1 regulates DNA synthesis through activation of phosphatidylinositol-specific phospholipase C[END_REF].

Involvement of PI-PLCs in plant development has been well studied during polarized pollen growth. Such an asymmetric cell expansion is known to rely on several events including calcium signalling, vesicular trafficking and cytoskeleton rearrangements [START_REF] Cole | Polarized growth: maintaining focus on the tip[END_REF].

In the elongating pollen tube, PI-PLC accumulates in the plasma membrane specifically at the flanks of the tip, but not at the very apex. On the contrary, PI-4,5-P2 exclusively accumulates at the apex of the pollen tube [START_REF] Dowd | Petunia phospholipase C1 is involved in pollen tube growth[END_REF][START_REF] Helling | Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling[END_REF]. It was shown that the PI-PLC inhibitor U73122 strongly inhibited pollen tube growth and led to swollen tips, thus indicating that expansion is no longer polarized [START_REF] Helling | Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling[END_REF]. The same effect was produced when a PI-PLC inactive form, that competes with the native protein for the localization in the membrane, is expressed in the pollen tube [START_REF] Dowd | Petunia phospholipase C1 is involved in pollen tube growth[END_REF]. These effects (reduction of growth and swelling) correlate with the spreading of PI-4,5-P2 to the flanks of the tip. Therefore PI-PLC has an active role to create and maintain a PI-4,5-P2 gradient in the pollen tip, between the apical and lateral membranes, and this gradient is necessary for polarized growth [START_REF] Dowd | Petunia phospholipase C1 is involved in pollen tube growth[END_REF][START_REF] Helling | Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling[END_REF][START_REF] Sousa | Arabidopsis phosphatidylinositol-4-monophosphate 5-kinase 4 regulates pollen tube growth and polarity by modulating membrane recycling[END_REF]. The role of PI-4,5-P2 might be related to the control of actin cytoskeleton dynamics, of membrane trafficking including clathrin-dependent endocytosis, and to the control of apical pectin deposition [START_REF] Ischebeck | Phosphatidylinositol-4,5bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum[END_REF][START_REF] Zhao | Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis and tobacco[END_REF][START_REF] Ischebeck | Type B phosphatidylinositol-4-phosphate 5-kinases mediate Arabidopsis and Nicotiana tabacum pollen tube growth by regulating apical pectin secretion[END_REF]. In root hair tips, PI-4,5-P2 is also important for efficient growth. Arabidopsis mutants deficient in phosphatidylinositol-4-phosphate 5-kinase gene -enzyme producing PI-4,5-P2 -were significantly impaired in root hair development [START_REF] Kusano | The Arabidopsis phosphatidylinositol phosphate 5-Kinase PIP5K3 is a key regulator of root hair tip growth[END_REF][START_REF] Stenzel | The type B phosphatidylinositol-4-phosphate 5-kinase 3 is essential for root hair formation in Arabidopsis thaliana[END_REF].

PI-PLCs in abiotic stress

Plants are sessile organisms that require specific mechanisms in order to resist or adapt to undesirable environmental conditions, such as increased soil salt concentration, high or low temperature, and water shortage.

Osmotic stress.

The role of PI-PLC in the production of polyphosphoinositides and their role during osmoticstress induced cell signalling has been discussed in detail by Munnik et al [START_REF] Munnik | Osmotic stress-induced phosphoinositide and inositol phosphate signalling in plants[END_REF]. Different salts (NaCl, KCl) and osmotic stress inducers (mannitol, sorbitol and mannose) [START_REF] Dewald | Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed Arabidopsis[END_REF][START_REF] Im | Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum[END_REF][START_REF] Mosblech | Phosphoinositide and inositolpolyphosphate signalling in defense responses of Arabidopsis thaliana challenged by mechanical wounding[END_REF][START_REF] Heilmann | Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in Galdieria sulphuraria[END_REF] evoke a rapid (often within seconds) increase in IP 3 levels. This increase has a positive role regarding osmotic stress resistance as supported by genetic studies of inositol polyphosphate kinases [START_REF] Yang | Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana[END_REF][START_REF] Zhu | Molecular characterization of ThIPK2, an inositol polyphosphate kinase gene homolog from Thellungiella halophila, and its heterologous expression to improve abiotic stress tolerance in Brassica napus[END_REF] and phosphatases [START_REF] Xiong | FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis[END_REF][START_REF] Perera | Transgenic Arabidopsis plants expressing the Type 1 inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling[END_REF]. Apparently not all PI-PLCs participate in stress signalling as PA, PI-4-P or PI-4,5-P2 levels were found to be identical in wild type and in a AtPLC3/AtPLC6/AtPLC9 triple mutant in response to salts [START_REF] Van Schooten | Dissecting Arabidopsis phospholipid signaling using reverse genetics Faculty of Science[END_REF].

The increase in IP3 production during osmotic-stress is accompanied by a rise of PI-4,5-P2 levels [START_REF] Dewald | Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed Arabidopsis[END_REF][START_REF] Darwish | Phospholipid signaling responses in saltstressed rice leaves[END_REF]. This may suggest that PI-PLC functions simultaneously with phosphoinositide kinases in stress signalling. Whether this PI-4,5-P2 is produced to serve as a PI-PLC substrate [START_REF] Delage | Eat in or take away? How phosphatidylinositol 4kinases feed the phospholipase C pathway with substrate[END_REF] or as a specific signalling molecules per se [START_REF] Delage | Arabidopsis Type-III phosphatidylinositol 4-kinases β1 and β2 are upstream of the phospholipase C pathway triggered by cold exposure[END_REF] is not known.

PI-PLC activation in response to osmotic stress may be dependent on activation of a specific receptor or an unspecific destabilization of cellular compartments resulting in a calcium increase [START_REF] Parre | Calcium signaling via phospholipase C is essential for proline accumulation upon ionic but not nonionic hyperosmotic stresses in Arabidopsis[END_REF]. Interestingly, the rapid PLC activation in response to hyperosmotic conditions caused by mannitol in wheat roots was shown to be the result of microtubule destabilization [START_REF] Komis | Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum[END_REF].

Rapid calcium release into the cytosol in response to salt and osmotic stress observed in Arabidopsis root tips [START_REF] Dewald | Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed Arabidopsis[END_REF], seedlings [START_REF] Perera | Transgenic Arabidopsis plants expressing the Type 1 inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling[END_REF] and tobacco cells [START_REF] Cessna | The externally derived portion of the hyperosmotic shock-activated cytosolic calcium pulse mediates adaptation to ionic stress in suspension-cultured tobacco cells[END_REF] was shown to be dependent on PI-PLC. PI-PLCs affect several cellular processes in hyperosmotic stress conditions including microtubule polymer recovery in plasmolyzed wheat root cells [START_REF] Komis | Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum[END_REF], rapid MAPK activation and ROS generation in soybean [START_REF] Im | Soybean MAPK, GMK1 Is dually regulated by phosphatidic acid and hydrogen peroxide and translocated to nucleus during salt stress[END_REF], release of Tubby transcriptional factors from the plasma membrane [START_REF] Reitz | The subcellular localization of Tubby-like proteins and participation in stress signaling and root colonization by the mutualist Piriformospora indica[END_REF] and the control of phosphoenolpyruvate carboxylase kinase 1 gene expression in sorghum [START_REF] Monreal | Factors involved in the rise of phosphoenolpyruvate carboxylase-kinase activity caused by salinity in sorghum leaves[END_REF].

Abscisic acid (ABA) is one of the key plant stress hormones that accumulates upon stress exposure and it controls many plant defence reactions [START_REF] Fujita | ABA-mediated transcriptional regulation in response to osmotic stress in plants[END_REF]. In plants, PI-PLC seems to be actively involved in ABA-dependent signalling. As an example, exogenous ABA treatment evoked IP3 accumulation in Arabidopsis seedlings [START_REF] Xiong | FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis[END_REF]. More importantly, in Commelina communis ABAsensitivity of guard-cells that control leaf transpiration was impaired by the addition of heparin -an IP3 antagonist [START_REF] Mills | The effects of manipulating phospholipase C on guard cell ABA-signalling[END_REF]. Previously, inhibitor studies demonstrated that the regulatory role of PI-PLC on guard-cell movements is likely to be implemented via calcium oscillations [START_REF] Staxén | Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C[END_REF].

Proline is an osmolyte that accumulates in plants in order to alleviate osmotic stress action. In Arabidopsis, PI-PLC, IP3 and IP3-gated calcium release regulate transcriptional and posttranscriptional events which subsequently lead to proline accumulation in response to ionic, but not to non-ionic hyperosmotic stress [START_REF] Parre | Calcium signaling via phospholipase C is essential for proline accumulation upon ionic but not nonionic hyperosmotic stresses in Arabidopsis[END_REF]. Gene expression changes are also known to be mediated by PI-PLC in response to dehydration [START_REF] Perera | Transgenic Arabidopsis plants expressing the Type 1 inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling[END_REF][START_REF] Takahashi | Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture[END_REF]. However, an artificial decrease in IP3 levels by expressing inositol polyphosphate phosphatase increased drought tolerance [START_REF] Perera | Transgenic Arabidopsis plants expressing the Type 1 inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling[END_REF]. This discrepancy may be in part explained by pharmacological studies conducted with the halophyte plant Thellungiella halophita. In this species, in the absence of stress and in response to moderate salt stress (200 mM), PI-PLC negatively regulated proline accumulation and expression of genes coding proline metabolism enzymes. However, during severe salt and osmotic stress conditions, PI-PLC positively affected the osmolyte level [START_REF] Ghars | Phospholipases C and D modulate proline accumulation in Thellungiella halophila/salsuginea differently according to the severity of salt or hyperosmotic stress[END_REF]. This supports a notion about divergence in metabolism responses to osmotic stress evoked by PI-PLC depending on stress severity or plant robustness.

Heat stress.

Heat exposure increased PI-PLC activity in pea membranes with a maximum at 40 min [START_REF] Ruelland | How plants sense temperature[END_REF]. This observation was consistent with the observed PI-PLC protein accumulation in heat-treated plants [START_REF] Liu | Contributions of PIP2-specific-phospholipase C and free salicylic acid to heat acclimation-induced thermotolerance in pea leaves[END_REF]. In Arabidopsis, rapid (within minutes) and substantial IP3 accumulation in response to heat stress was reported [START_REF] Liu | Primary evidence for involvement of IP3 in heat-shock signal transduction in Arabidopsis[END_REF]. The heat-activated AtPLC9 activity was required for intracellular calcium accumulation, AtHsp promoter activity, stress-dependent gene expression and heat acclimation [START_REF] Zheng | Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis[END_REF][START_REF] Liu | Primary evidence for involvement of IP3 in heat-shock signal transduction in Arabidopsis[END_REF].

Cold stress.

Despite its opposite physical nature, cold stress induces signal transduction in cells that also involves PI-PLC pathway(s). Rapid and transient IP3 accumulation with a simultaneous decrease in PI-4-P and PI-4,5-P2 levels was observed in winter wheat tissues [START_REF] Bucolova | A possible role of phosphatidylinositols in transduction of temperature signal in winter wheat[END_REF], Arabidopsis suspension cells [START_REF] Ruelland | Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells[END_REF] and oilseed rape leaves [START_REF] Smoleńska-Sym | Phosphatidylinositol metabolism in low temperature-affected winter oilseed rape leaves[END_REF] subjected to cold stress. Later, mutant analyses of inositol polyphosphate kinases [START_REF] Heilmann | Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in Galdieria sulphuraria[END_REF] and phosphatases [START_REF] Xiong | FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis[END_REF][START_REF] Williams | Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9 lead to overaccumulation of PtdIns(4,5)P2 and constitutive expression of the stress-response pathway[END_REF] validated the role of IP3 in cold tolerance. Cold-induced activation of PI-PLCs is dependent on calcium entry into the cells [START_REF] Ruelland | Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells[END_REF] while substrates for PI-PLCs are supplied by type III-phosphatidylinositol 4-kinases [START_REF] Delage | Arabidopsis Type-III phosphatidylinositol 4-kinases β1 and β2 are upstream of the phospholipase C pathway triggered by cold exposure[END_REF]. Analysis of Arabidopsis mutants altered in fatty acid desaturase activity suggests that cold-induced membrane rigidification is upstream of PI-PLC activation [START_REF] Vaultier | Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells[END_REF], leading to the model where cold would rigidify membranes, leading to calcium entry and then to PI-PLC activity. To date, main downstream components of cold-induced PI-PLC activation are poorly studied. A plant expressing the mammalian type I inositol polyphosphate 5-phosphatase (that cleaves IP3) accumulates 30% less calcium in response to cold [START_REF] Perera | Transgenic Arabidopsis plants expressing the Type 1 inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling[END_REF]. In addition, pharmacological studies have suggested that phosphoinositide metabolism may be upstream of microtubule depolymerization in response to cold stress [START_REF] Bartolo | Lithium decreases cold-induced microtubule depolymerization in mesophyll cells of spinach[END_REF] while expression of a subset of genes regulated by low temperature was dependent on PI-PLC activity [START_REF] Vergnolle | The cold-induced early activation of phospholipase C and D pathways determines the response of two distinct clusters of genes in Arabidopsis cell suspensions[END_REF]. It should be noted that cold stress also induces the expression of PI-PLC genes in B. napus [START_REF] Das | Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1)comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B. napus[END_REF], Zea mays [START_REF] Zhai | Characterization of a novel phosphoinositide-specific phospholipase C from Zea mays and its expression in Escherichia coli[END_REF][START_REF] Sui | Cloning and expression analysis of some genes involved in the phosphoinositide and phospholipid signaling pathways from maize (Zea mays L.)[END_REF], winter wheat [START_REF] Skinner | Phospholipid acyl chain and phospholipase dynamics during cold acclimation of winter wheat[END_REF] and Arabidopsis [START_REF] Tasma | Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana[END_REF], thus supporting a role of PI-PLC in cold acclimation.

Heavy metal stress.

Toxic metals can severely inhibit plant cell metabolism by affecting many main enzymatic reactions. Ni 2+ , Zn 2+ and especially Cu 2+ decreased in vitro PI-PLC activity in both membrane and soluble fractions of Catharanthus roseus roots [START_REF] Piña-Chable | Effect of different inhibitors on phospholipase C activity in Catharanthus roseus transformed roots[END_REF]. However, in another study, copper excess rapidly increased in vivo DAG accumulation in roots of B. napus that could reflect PI-PLC activation [START_REF] Russo | Brassica napus subjected to copper excess: Phospholipases C and D and glutathione system in signalling[END_REF]. Moreover, copper-induced release of intracellular calcium in marine alga Ulvia compressa seems to be dependent on PI-PLC activity [START_REF] González | Copperinduced intracellular calcium release requires extracellular calcium entry and activation of L-type voltagedependent calcium channels in Ulva compressa[END_REF].

Aluminium is currently the best studied toxic metal in terms of affecting plant PI-PLC activity. In Coffea arabica suspension cells, aluminium treatment induced a rapid PI-PLC activation and IP3 accumulation [START_REF] Martínez-Estévez | Aluminium differentially modifies lipid metabolism from the phosphoinositide pathway in Coffea arabica cells[END_REF]. However, a prolonged exposure led to inhibition of PI-PLC [START_REF] Yakimova | Signal transduction events in aluminuminduced cell death in tomato suspension cells[END_REF][START_REF] Ramos-Díaz | Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway[END_REF]. Aluminium may block PI-4,5-P2 hydrolysis by binding to it or substituting calcium bound to liposomal lipids [START_REF] Kopka | Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato[END_REF]. The mechanisms involved in the rapid activation of PI-PLC by aluminium remain to be revealed.

Other stresses.

Several other stress signals appear to be transmitted in plant cells by a PI-PLC pathway. Hypoxia induces a rapid G-protein-dependent IP3 accumulation in rice roots. This PLC activation is further transmitted by IP3-sensitive calcium channels, calcium and calmodulin that are required for gamma-aminobutyric acid accumulation and cellular potassium loss during anaerobic stress [START_REF] Reggiani | Evidence for the involvement of phospholipase C in the anaerobic signal transduction[END_REF]. Interestingly, in Peganum harmala calli, extremely low frequency electromagnetic fields rapidly reduced PI-4,5-P2 levels [START_REF] Piacentini | Phospholipase Cdependent phosphoinositide breakdown induced by ELF-EMF in Peganum harmala calli[END_REF]. This effect was blocked by PI-PLC inhibitors, indicating that membranes may be the primary site of electromagnetic stimulus perception that induces PI-PLC activation.

PI-PLCs in biotic stress

Plants possess unique defence mechanisms that enable a single cell to fulfil the role of an integrated immune system. Two defence strategies are usually employed during plant-pathogen interactions [START_REF] Spoel | How do plants achieve immunity? Defence without specialized immune cells[END_REF]. Upon contact with a pathogen, plants are able to rapidly develop a hypersensitive response (HR) leading to cell death at the point of infection and thus restricting pathogen propagation. Alternatively plants can induce systemic acquired resistance (SAR) or induced systemic resistance (ISR) by activation of defence genes and production of antimicrobial metabolites that limit pathogen growth at the whole plant level. Phospholipases and phospholipid-derived molecules are recognized to be intrinsic components of both defence strategies [START_REF] Canonne | Phospholipases in action during plant defense signaling[END_REF].

PI-PLC-derived molecules seem to be involved in plant defence reactions. DAG rapidly accumulated in rice cells following exposure to the elicitor N-acetylchitooligosaccharide [START_REF] Yamaguchi | Activation of phospholipases by N-acetylchitooligosaccharide elicitor in suspension-cultured rice cells mediates reactive oxygen generation[END_REF] while rapid and transient accumulation of IP3 was observed in elicited Cupressus lusitanica cells [START_REF] Zhao | Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures[END_REF]. PI-PLC inhibitor U73122 reduced accumulation of phytoalexin and ROS production in tobacco cells elicited with riboflavin [START_REF] Wang | Involvement of phospholipases C and D in the defence responses of riboflavin-treated tobacco cells[END_REF] and in transgenic tobacco cells elicited during a simulated Cf-4/Avr4 interaction [START_REF] De Jong | Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction[END_REF]. U73122 also reduced ROS production in tomato cells elicited with chitosan [START_REF] Raho | Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide[END_REF].

Transcriptional activation of PI-PLC genes is common during biotic stress conditions. In tomato, several PI-PLC gene family members were identified as crucial components of plant defence systems [START_REF] Vossen | Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance[END_REF]. Prominent roles were ascribed to tomato SlPLC4 and SlPLC6 genes that are induced under biotic stress and differentially control the onset of HR and plant resistance against Cladosporium fulvum, Verticillium dahlia and Pseudomonas syringae pathogens.

Despite numerous biochemical evidences, the roles of the molecules produced by PI-PLC in mediation of defence reactions are not entirely clear. The addition of synthetic DAG induced quick ROS accumulation in rice cells [START_REF] Yamaguchi | Activation of phospholipases by N-acetylchitooligosaccharide elicitor in suspension-cultured rice cells mediates reactive oxygen generation[END_REF] and activated expression of defence-related genes [START_REF] Yamaguchi | Elicitor-induced activation of phospholipases plays an important role for the induction of defense responses in suspension-cultured rice cells[END_REF]. It was also shown that phorbol myristate acetate, an analogue of DAG that cannot be converted to PA, mimicked the effects of chitosan elicitor and induced anthraquinone accumulation in cultured Rubia tinctorum cells [START_REF] Vasconsuelo | Involvement of the PLC/PKC pathway in chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures[END_REF], suggesting that DAG can act not only as a PA precursor, but can also have a role per se during plant immune signalling.

Arabidopsis mutants expressing a mammalian type I inositol polyphosphate 5-phosphatase, thus characterized by low levels of IP 3 and IP 6 , had a reduced cytosolic Ca 2+ increase in response to flagellin [START_REF] Ma | Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca 2+ elevation and downstream immune signaling in plants[END_REF]. Therefore, it is possible that PI-PLC can regulate plant defence reactions by bringing about changes to cytosolic Ca 2+ that are perceived by Ca 2+ dependent protein kinases [START_REF] Gao | Bifurcation of Arabidopsis NLR immune signaling via Ca 2+ -dependent protein kinases[END_REF].

Symbiotic relations are special cases of plant-bacteria interactions mediated by a special class of molecules named nodulation factors (Nod) recognized by special plant receptor kinases. An essential role of PI-PLC, PLD and their corresponding lipid products, DAG and PA, in controlling downstream responses of Medicago truncatula root hair cells during Nod factor-induced signalling has been convincingly revealed [START_REF] Charron | Pharmacological evidence that multiple phospholipid signaling pathways link rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca 2+ spiking and specific ENOD gene expression[END_REF]. Interestingly, the symbiotic relationship between Medicago truncatula and Sinorhizobium meliloti was shown to be controlled by DNF2 protein that contains the X domain of PI-PLC [START_REF] Bourcy | Medicago truncatula DNF2 is a PI-PLC-XD-containing protein required for bacteroid persistence and prevention of nodule early senescence and defense-like reactions[END_REF]. The other conventional domains of PI-PLC are absent in DNF2. The authors speculated that DNF2 may bind, but not cleave, phosphoinositides, thus preventing their hydrolysis by PI-PLC and the onset of defence reactions that may result in bacteroid degradation.

Intriguingly, plant pathogens also frequently make use of PI-PLC signalling. Despite having biochemical properties similar to those of plant PI-PLCs, bacterial PI-PLC activity is often a prerequisite for pathogenicity [START_REF] Poussin | The ability of Listeria monocytogenes PI-PLC to facilitate escape from the macrophage phagosome is dependent on host PKCβ[END_REF]. It is thought that they act to either suppress or overwhelm plant innate defence mechanisms or to lyse cells. This indicates the requirement for a precise balancing of defence mechanisms in vivo, granting either plant resistance or disease. Such knowledge is important for the future biotechnological development of disease-resistant plants.

In summary, plant PI-PLC plays an important role in signal transduction in response to different stresses, representing a key hub within the complex network of cellular regulatory systems. However, the regulation of PI-PLCs, the involvement of specific PI-PLC genes, the mechanistic action of generated second messengers in abiotic and biotic stress signalling and responses require further investigation.

PI-PLC modes of action in plant cells

Taking into account the multitude of signalling events involving PI-PLCs, it is of great interest to know which downstream events convey PI-PLC signals. PI-PLC signalling in plants can be associated with changes in the cellular concentration of several molecules; either direct (DAG, IP 3 ) or indirect (PA) products [START_REF] Wang | Plant Phospholipases: An Overview[END_REF], and phosphoinositide substrates [START_REF] Boss | Phosphoinositide Signaling[END_REF].

Diacylglycerol

In animals, DAG serves as a classical second messenger that activates protein kinase C (PKC) by binding to its C1 domain [START_REF] Rahman | Identification of the activator-binding residues in the second cysteine-rich regulatory domain of protein kinase Cθ (PKCθ)[END_REF]. However, no homologous PKC genes have been identified in plants. In addition, DAG has not been shown to activate any purified plant protein kinase. Although different effects of the DAG analogue phorbol myristate acetate have been described [START_REF] Chandok | Phorbol myristate acetate replaces phytochrome-mediated stimulation of nitrate reductase in maize[END_REF][START_REF] Ishioka | Adventitious bud induction by protein kinase C activators in Torenia stem segments[END_REF], their relation to PI-PLC activity and DAG action remains to be established. Thus, the canonical role of DAG as a protein kinase activator in plants is controversial. On the other hand, many different C1 domain-containing proteins are encoded by Arabidopsis [START_REF] Janda | Phosphoglycerolipids are master players in plant hormone signal transduction[END_REF], rice and other plant genomes. C1 domain containing-proteins are not limited to predicted protein kinase activities, one such protein was shown to possess a transactivation activity in wheat [START_REF] Zhao | The role of TaCHP in salt stress responsive pathways[END_REF], but the role of DAG has yet to be investigated. Taking into account that DAG is also a precursor of galactolipids, structural phospholipids and storage lipids, only specifically localized, tightly controlled and transient DAG accumulation may fulfil signalling functions in plants. Determination of such DAG signalling levels in plants is still a challenge and complicated by the observation that basal DAG levels differ between plant organs [START_REF] Nakamura | The diacylglycerol forming pathways differ among floral organs of Petunia hybrida[END_REF]. 9.2 Phosphatidic acid. DAG, a direct product of PI-PLC [START_REF] Pokotylo | The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling[END_REF], can be phosphorylated to PA by diacylglycerol kinases (DGK) [START_REF] Sueldo | Phosphatidic acid formation is required for extracellular ATP-mediated nitric oxide production in suspension-cultured tomato cells[END_REF][START_REF] Arisz | Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase[END_REF]. PA can also be produced by the action of phospholipases D on structural phospholipids. The coupling of DGK with PI-PLC, leading to PA synthesis, has been established in response to salt in rice [START_REF] Darwish | Phospholipid signaling responses in saltstressed rice leaves[END_REF], to cold in Arabidopsis cells [START_REF] Ruelland | Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells[END_REF][START_REF] Vergnolle | The cold-induced early activation of phospholipase C and D pathways determines the response of two distinct clusters of genes in Arabidopsis cell suspensions[END_REF], seedlings [START_REF] Delage | Arabidopsis Type-III phosphatidylinositol 4-kinases β1 and β2 are upstream of the phospholipase C pathway triggered by cold exposure[END_REF] and adult leaves [START_REF] Arisz | Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase[END_REF], and to shear stress in Taxus cuspidata cells [START_REF] P.-P. Han | Lipidomic analysis reveals activation of phospholipid signaling in mechanotransduction of Taxus cuspidata cells in response to shear stress[END_REF].

In plants, as in animals, PA is a well-known second messenger [START_REF] Testerink | Molecular, cellular, and physiological responses to phosphatidic acid formation in plants[END_REF]. In Arabidopsis, PA binds and mediates membrane recruitment of glyceraldehyde 3-phosphate dehydrogenase, clathrin heavy chain proteins and sucrose non-fermenting-1-related protein kinase 2 during salt stress [START_REF] Mcloughlin | The Snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress[END_REF][START_REF] Mcloughlin | Identification of novel candidate phosphatidic acid-binding proteins involved in the salt-stress response of Arabidopsis thaliana roots[END_REF]. PA activates calcium-dependent protein kinase [START_REF] Dixit | Phospholipid mediated activation of calcium dependent protein kinase 1 (CaCDPK1) from chickpea: a new paradigm of regulation[END_REF], MAP kinase GMK1 [START_REF] Im | Soybean MAPK, GMK1 Is dually regulated by phosphatidic acid and hydrogen peroxide and translocated to nucleus during salt stress[END_REF] and monogalactosyldiacylglycerol synthase [START_REF] Rocha | Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies[END_REF]. In contrast, inhibition of actin-capping proteins [START_REF] Pleskot | Structural insights into the inhibition of actin-capping protein by interactions with phosphatidic acid and phosphatidylinositol (4,5)-bisphosphate[END_REF] and protein phosphatase 1 [START_REF] Takemiya | Phosphatidic acid inhibits blue light-induced stomatal opening via inhibition of protein phosphatase 1[END_REF] by PA has been reported. PA has also been suggested to play a role in ATPase interactions with 14-3-3 proteins, causing inhibition of ATPase activity [START_REF] Camoni | Binding of phosphatidic acid to 14-3-3 proteins hampers their ability to activate the plant plasma membrane H + -ATPase[END_REF], and induced proteolytic cleavage of bound glyceraldehyde 3-phosphate dehydrogenase [START_REF] Kim | Phosphatidic acid binds to cytosolic glyceraldehyde-3-phosphase dehydrogenase and promotes its cleavage in Arabidopsis[END_REF].

The fact that PA is an active mediator in the PLC/DGK pathway is reinforced considering the effect of DGK inhibition or over-expression. Overexpression of rice OsBIDK1 (a DGK) in tobacco conferred amplified resistance against tobacco mosaic virus and Phytophthora parasitica var. nicotianae pathogen [START_REF] Zhang | Overexpression of a rice diacylglycerol kinase gene OsBIDK1 enhances disease resistance in transgenic tobacco[END_REF]. Application of R59022, a DGK inhibitor, hampered defensive ROS generation in xylanase-induced ROS production in tomato cells [START_REF] Laxalt | Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells[END_REF]. In another study, PI-PLC inhibitor U73122 reduced ROS production in tomato cells elicited with chitosan [START_REF] Raho | Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide[END_REF] and the authors assumed that this was dependent on NO-induced PLC/DGK generation of PA. DGK inhibitor R59022 reduced primary root elongation and plant growth in Arabidopsis [START_REF] Gómez-Merino | Arabidopsis AtDGK7, the smallest member of plant diacylglycerol Kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration: The DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development[END_REF] and inhibited tobacco pollen tube growth [START_REF] Pleskot | Turnover of phosphatidic acid through distinct signalling pathways affects multiple aspects of tobacco pollen tube tip growth[END_REF].

Soluble inositol-phosphates.

PI-PLC action in plants is thought to be tightly related to polyphosphoinositide signalling [START_REF] Munnik | Green light for polyphosphoinositide signals in plants[END_REF]. Previously, many effects of soluble phosphorylated inositols produced by PI-PLC have been studied in relation to calcium signalling [START_REF] Batistič | Analysis of calcium signaling pathways in plants[END_REF]. In animals, a role of IP3 in the regulation of Ca 2+ fluxes by binding to IP3 receptors (Ca 2+ channel) located mainly on the ER is dogmatic [START_REF] Rossi | Analysis of IP3 receptors in and out of cells[END_REF][START_REF] Zhang | Inositol 1,4,5-trisphosphate receptor subtype-specific regulation of calcium oscillations[END_REF]. The physiological role of IP3 is also obvious in plants however it is most likely mediated by different mechanisms. Early findings concerning IP3 influence on cell calcium oscillations are thoroughly summarized in the review by Krinke et al [180]. More recent studies revealed that IP3 is also implicated in regulation of diurnal cytosolic Ca 2+ oscillations induced by exogenous calcium [START_REF] Tang | Coupling diurnal cytosolic Ca 2+ oscillations to the CAS-IP3 pathway in Arabidopsis[END_REF] and Ca 2+ -dependent proline accumulation in salt-stressed Arabidopsis [START_REF] Parre | Calcium signaling via phospholipase C is essential for proline accumulation upon ionic but not nonionic hyperosmotic stresses in Arabidopsis[END_REF]. Apart from PI-PLC, several other enzymes are involved in the control of IP3 abundance and induced calcium signalling. Studies of the supo1 mutant of Arabidopsis defective in inositol polyphosphate 1-phosphatase have shown that regulation of auxin distribution through PIN transporters is mediated by cytosolic calcium release controlled by IP3 [START_REF] Zhang | Inositol trisphosphate-induced Ca 2+ signaling modulates auxin transport and PIN polarity[END_REF]. It was also shown that elevated IP3 levels in Arabidopsis inositol polyphosphate 5-phosphatase mutants resulted in elevated concentrations and altered distributions of cytosolic Ca 2+ in developing pollen [START_REF] Wang | Inositol polyphosphate 5-phosphatase-controlled Ins(1,4,5)P3/Ca 2+ is crucial for maintaining pollen dormancy and regulating early germination of pollen[END_REF]. However, there is no direct genetic or molecular evidence for the existence of plant IP3 receptors. If such a receptor exists, it apparently does not share sequence homology with the analogous animal proteins, and most likely represents a multidomain heteromeric complex [180]. Nevertheless, several studies have provided indirect evidence of a receptor-like IP3-binding protein or complex in plant cells. Indeed, IP3 had a high affinity towards reticulum-enriched membrane fractions prepared from Chenopodium rubrum leaves [START_REF] Martinec | Subcellular localization of a high affinity binding site for d-myo-Inositol 1,4,5-trisphosphate from Chenopodium rubrum[END_REF]. While, 2-aminoethoxydiphenylborate, an inhibitor of IP3-mediated calcium release described in animals, was able to reduce cytosolic Ca 2+ spiking in M. truncatula root hairs induced by Nod factors [START_REF] Engstrom | Pharmacological analysis of Nod factor-induced calcium spiking in Medicago truncatula. Evidence for the requirement of Type IIA calcium pumps and phosphoinositide signaling[END_REF].

It is also apparent that the regulatory role of soluble PI-PLC products in plants is not limited to IP3 production. Products of further IP3 phosphorylation, inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5) and inositol hexakisphosphate (IP6), seem to be equally important. According to several studies, phosphorylated products of IP 3 produced via inositol-phosphate kinase activity fulfil their own signalling role in plants. IP6 (also named phytate) is relatively abundant in plant cells and sometimes regarded as a phosphorus storage molecule. However its signalling role has been demonstrated since it triggered intracellular calcium release after ABA addition in patchclamped guard cell protoplasts of Vicia faba [START_REF] Lemtiri-Chlieh | Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells[END_REF] and regulated potassium-inward rectifying channel conductance [START_REF] Lemtiri-Chlieh | Inositol hexakisphosphate is a physiological signal regulating the K + -inward rectifying conductance in guard cells[END_REF]. The role of IP6 in plant resistance was also demonstrated [START_REF] Murphy | A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens[END_REF] with authors suggesting that low levels of IP6 in mutant plants lacking myo-inositol phosphate synthase or IP5-2-kinase genes corresponded to a higher sensitivity to infection by different groups of pathogens attributed to impaired SA accumulation. Other genetic studies indicate that components of a phosphoinositide signalling pathway are involved in the induction of wounding-inducible defence gene expression and resistance to herbivores [START_REF] Mosblech | Phosphoinositide and inositolpolyphosphate signalling in defense responses of Arabidopsis thaliana challenged by mechanical wounding[END_REF]. In turn, tolerance to oxidative stress may be mediated in part by products of IP3 phosphorylation [START_REF] Yang | Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana[END_REF][START_REF] Zhu | Molecular characterization of ThIPK2, an inositol polyphosphate kinase gene homolog from Thellungiella halophila, and its heterologous expression to improve abiotic stress tolerance in Brassica napus[END_REF].

IP4 and IP5, but not IP3, have also been reported to be potent activators of the COI-JAZ coreceptor interaction with coronatine, an analogue of JA [START_REF] Sheard | Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor[END_REF]. Beyond JA, the metabolism of soluble inositol polyphosphates might also affect auxin signalling through modulation of the level of the IP6 ligand of auxin receptor TIR1 [START_REF] Tan | Mechanism of auxin perception by the TIR1 ubiquitin ligase[END_REF] but how this affects auxin response remains unclear. Moreover, whether other hormone receptors are affected by these metabolites has not yet been described. 9.4 Phosphoinositide level. PI-PLC activity will not only result in the production of molecules. It will also lead to a decrease in its substrates, the PI-4,5-P2. Because PI-4,5-P2 and its precursor PI-4-P, can bind to proteins, thus modifying their localization and/or activity [START_REF] Delage | Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: convergences and divergences among eukaryotic kingdoms[END_REF], theoretically PI-PLC action could be transduced through the modification of the level of these molecules, the phosphoinositides. Does it really occur in plant cells? It seems that it is indeed the role of PI-PLCs in the pollen tube. In this organ, inhibiting PI-PLC [START_REF] Dowd | Petunia phospholipase C1 is involved in pollen tube growth[END_REF][START_REF] Helling | Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling[END_REF], or overexpressing PI-4-P-5-kinases [START_REF] Ischebeck | Phosphatidylinositol-4,5bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum[END_REF][START_REF] Stenzel | Variable regions of PI4P 5-Kinases direct PtdIns(4,5)P(2) toward alternative regulatory functions in Tobacco pollen tubes[END_REF], that produce PI-4,5-P2, result in the same phenotype, the swelling of the pollen tip (which corresponds to a loss of polarized growth). The common action of inhibiting PI-PLC and of overexpressing PI-4-P-5-kinases is to lead to an increase in PI-4,5-P2. As already explained, the strict control of the level of PI-4,5-P2, in a particular pollen tube zone, is necessary for pollen polarized growth. PI-PLC has a major role in this process.

Conclusion and open questions

In plants, PI-PLCs have major roles in responses to environmental stresses and in plant development. Despite the importance of these roles, the functioning of the PI-PLC pathway is far from being fully understood.

To function, PI-PLCs must be coupled to the enzymes that produce the substrates, i.e. the phosphoinositides. It is important to understand if stimulated PI-PLCs use the phosphoinositide pool already present in plant membranes before the activating stress, or if a de novo phosphoinositide synthesis, occurring concomitantly with PI-PLC activity, participates in providing the substrate used by PI-PLC. Our data show that during a cold stress, de novo synthesis by PI-4 kinases participates in substrate providing [START_REF] Delage | Arabidopsis Type-III phosphatidylinositol 4-kinases β1 and β2 are upstream of the phospholipase C pathway triggered by cold exposure[END_REF] but whether this can be generalised to other activation situations is not known. Besides, the phosphorylation of PI into PI-4-P by PI-4-kinases takes place most probably in endomembranes. How the de novo synthesis of PI-4-P is compatible with PI-4,5-P2 production and subsequent hydrolysis by PI-PLC in the plasma membrane is another intriguing question [START_REF] Delage | Eat in or take away? How phosphatidylinositol 4kinases feed the phospholipase C pathway with substrate[END_REF].

We have also seen that most PI-PLC actions do not rely on the direct enzyme products, but on their phosphorylation products, such as IP5/IP6 or PA. Therefore a functional coupling between PI-PLC and DGK and inositol-phosphate-kinase exists. Does this coupling require a physical interaction between these enzymes? And if yes, which isoforms are involved? The data discussed above suggesting an interaction between Arabidopsis PLC7 and DGK2 support the existence of physical coupling but it needs more characterization, including structural characterization. Does this mean that PI-PLC is part of a supramolecular complex, a so-called signalosome or signalling platform that comprise PI-PLC, the kinases necessary for the phosphorylation of the products, together with the proteins that are the targets of these phosphorylated products? The existence of such a platform is an exciting hypothesis to investigate.

The roles of IP5/IP6 or PA in the PI-PLC transducing pathway seem clearly established. Does this mean that PI-PLC action is not mediated also by other molecules? First, the potential of DAG as an active molecule cannot be ruled out. At least the capacity of plant C1 domain-bearing proteins to actually bind DAG should be considered. Moreover, proteins can certainly bind DAG through nonconserved motifs. Besides, PI-4-P and PI-4,5-P2 are active molecules per se [START_REF] Delage | Signal transduction pathways involving phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: convergences and divergences among eukaryotic kingdoms[END_REF] and therefore PI-PLC could also affect cells by diminishing phosphoinositide levels. Such a regulatory role occurs during polarized growth. Does it also intervene in response to environmental stresses and/or hormones.

Calcium metabolism clearly appears to be the main cellular process controlled by PI-PLC via soluble inositol-phosphates and their action on calcium channels. But it is also possible that PI-PLCs influence Ca 2+ signalling indirectly by affecting Ca 2+ -sensitive cell targets. Indeed, recently it has been shown that the activity of stress-induced calcium-dependent protein kinase CaCDPK1 from chickpea, implicated in salt-stress responses, is regulated both by PA and, less efficiently, by DAG [START_REF] Dixit | Phospholipid mediated activation of calcium dependent protein kinase 1 (CaCDPK1) from chickpea: a new paradigm of regulation[END_REF]. Furthermore, PCaP2 protein located predominantly in the plasma membrane of Arabidopsis root hair cells was shown to interact with PI-4,5-P2 [START_REF] Kato | An Arabidopsis hydrophilic Ca 2+ -binding protein with a PEVK-rich domain, PCaP2, is associated with the plasma membrane and interacts with calmodulin and phosphatidylinositol phosphates[END_REF]. This raises the of how the different active molecules generated or controlled directly by PI-PLC (DAG/PA, inositol-phosphates and phosphoinostides) act together. Is there necessarily a synergetic action between lipid and soluble messengers, or can antagonistic actions be envisaged?

Finally, more studies are necessary to understand PI-PLC signalling since the actual in planta substrate is still undetermined. PI-4-P is much more abundant in plant cell membranes than PI-4,5-P2, and the PI-4,5-P2 vs. PI-4-P ratio is 10-fold higher in animals that in plants. Taking into account that PI-PLC can use PI-4-P as a substrate in vitro raises the possibility of this monophosphorylated PI to be an in vivo PI-PLC substrate. When U73122, a PI-PLC inhibitor, is added to plant cells, PIP2 level increases [START_REF] Van Leeuwen | Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings[END_REF] thus indicating that PI-4,5-P2 is an in vivo PI-PLC substrate, but this does not rule out that PI-4-P also has this role. Besides, it was shown that, in vitro, tomato SlPLC4 and SlPLC6 do not hydrolyse PI-4,5-P2 but PI [START_REF] Vossen | Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance[END_REF].

In conclusion, plant PI-PLCs are amongst the structurally simplest eukaryotic PI-PLCs. Like the mammalian PLCζs, they are only composed of EF-hand, X/Y and C2 domains and they do not possess a PH domain. Nevertheless, they are targets of many regulatory processes that require further characterization, including the role of protein-protein interactions in the control of plant PI-PLC activity. Even though significant differences with the mammalian canonical PI-PLC transduction module exist, including the importance of phosphorylated forms of the direct PI-PLC products as mediators, the cellular effects controlled by PI-PLCs are similar and include calcium signatures and cell phosphoproteome.

Fig. 1 .

 1 Fig. 1. Domain analysis and physiological role of plant PI-PLCs. Simplified representative polypeptide sequences of Arabidopsis thaliana PLC1, Homo sapiens PLCζ1 and Bacillus cereus PLCa with marked domains according to Pham (http://pfam.sanger.ac.uk). The spatial domain location in the sequence is relative.Inconstant sequence features of plant PI-PLC such as signal peptide sequence (SP)[START_REF] Vossen | Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance[END_REF] and sequence with homology to G-protein coupled receptor (GPCR)[START_REF] Das | Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1)comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B. napus[END_REF] are indicated. Putative sites of plant PI-PLC regulation are marked: Ca 2+ binds to plant PI-PLC both within the C2 domain and X-Y linker region[START_REF] Hirayama | AtPLC2, a gene encoding phosphoinositidespecific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana[END_REF][START_REF] Shi | Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean[END_REF][START_REF] Kopka | Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato[END_REF][START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF]. Interactions with Gα protein[START_REF] Misra | Heterotrimeric G-protein complex and Gprotein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C[END_REF], membrane lipids[START_REF] Rupwate | C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C[END_REF] and NtC7 in tobacco[START_REF] Nakamura | A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants[END_REF] have been observed at the C2 domain. At the N-terminus, plant PI-PLC may undergo dimerization[START_REF] Awasthi | Disulphide bridges of phospholipase C of Chlamydomonas reinhardtii modulates lipid interaction and dimer stability[END_REF] or associate with membranes[START_REF] Otterhag | N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana[END_REF]. Site of phosphorylation detected between X and Y domains of PI-PLC[START_REF] Nühse | Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses[END_REF]. Also indicated are typical direct and indirect products of phosphoinositide hydrolysis by PI-PLC and plant physiological processes dependent on PI-PLC activity. IP3, inositol trisphosphate; IP6, inositol hexakisphosphate; DAG, diacylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol, PIP2, Phosphatidylinositol 4,5-bisphosphate.

Fig. 2 .

 2 Fig. 2. Phylogenetic analysis of selected plant, mammal and yeast PI-PLCs. Sequences of PI-PLCs were obtained from NCBI and UniProtKB and aligned using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/).Conserved blocks in PI-PLC-X, PI-PLC-Y and C2 domains from 53 PI-PLC sequences were subjected to phylogenetic analysis. An unrooted phylogenetic tree was created using the maximum likelihood method in PhyMl (WAG substitution model). Numbers at nodes represent values of neighbour-joining bootstrap support obtained using the aLRT (approximate likelihood-ratio test) method. Branches below 50% were collapsed. The scale bar represents the number of substitutions per site. Unclassified proteins were named according to maximum similarity with the respective PI-PLC isozyme of Arabidopsis. Species abbreviations: At, Arabidopsis. thaliana; Gm, Glycine max; Os, Oryza sativa; Pp, Physcomitrella patens; Ps, Picea sitchensis; Nt, Nicotiana

Fig. 3 .

 3 Fig.3. Organ-specific expression of Arabidopsis PI-PLCs. Each PI-PLC isoform is color-coded. (A,B) Microarray data were collected using Genevestigator interface[START_REF] Hruz | Genevestigator V3: A reference expression database for the meta-analysis of transcriptomes[END_REF]. In (A) color saturation represents the absolute level of transcript abundance. In (B) the relative transcript prevalence is given as percentage of the level in the organ where the isoform is expressed at maximum level. (C) Organs and tissues where Arabidopsis PI-PLC isoforms are mainly expressed as shown by real-time PCR or promoter activity studies[START_REF] Tasma | Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana[END_REF][START_REF] Hunt | Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms[END_REF]. In (C), the expression of PI-PLC6, PI-PLC8 and PI-PLC9 of Arabidopsis can be distinguished.

Fig. 4 .

 4 Fig. 4. Changes in plant PI-PLC gene expression in response to hormone treatments and during stresses.Transcriptional analysis of plant PI-PLC gene expression was performed in silico using Genevestigator (https://www.genevestigator.com/). Shown here are cumulative representative data concerning changes in PI-PLC gene expression in different growth conditions. When several expression data points with different time or different dose of treatment were available, the one with the most apparent and consistent changes

  Nicotiana tabacum; Sl, Solanum licopersicum; Ld, Lilium davidii; Sb, Sorghum bicolor; Pi, Petunia integrifolia; St. Solanum tuberosum; Bn, Brassica napus; Sm, Selaginella moellendorffii; Mp, Micromonas pusilla; Ot, Ostreococcus tauri; Hs, Homo sapiens; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Zm, Zea mays.

thaliana; Gm, Glycine max; Os, Oryza sativa; Pp, Physcomitrella patens; Ps, Picea sitchensis; Nt,

Table 1

 1 Biochemical properties and localization of PI-PLCs identified in Arabidopsis. PM, plasma membrane. *identified experimentally[START_REF] Cao | Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits[END_REF][START_REF] Lalonde | Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations[END_REF], **MS/MS[40- 43], *** in silico analysis[START_REF] Hruz | Genevestigator V3: A reference expression database for the meta-analysis of transcriptomes[END_REF]

			Ca 2+
	Name	AGI	dependency	Cell Localization **	Protein-protein interaction*	Co-expression***
			*
	AtPLC1	At5g58670	+	PM	At4g01010	At5g42650, At3g51450
					At4g00430, At5g02620, At4g31340, At5g27370,	At1g06460,
	AtPLC2	At3g08510	+	PM	At5g64870	At3g45780,
						At4g04340
						At5g02600,
	AtPLC3	At4g38530	+	PM		At1g61660,
						At5g64240
	AtPLC4	At5g58700	+(-)	PM		At5g63990, At5g58690
	AtPLC5	At5g58690	+	?		At5g58700
	AtPLC6	At2g40116	?	?
	AtPLC7	At3g55940	?	PM	At5g63770	At4g17550
	AtPLC8	At3g47290	?	?
	AtPLC9	At3g47220	?	?	At1g12840, At2g22425, At3g08040, At3g12180 At3g48890, At4g30850, At5g47180, At1g21240 At4g20790, At5g59650,At1g31812, At2g26180	At4g16950, At4g19530
					At2g41400, At3g25805,At5g37050

Table 2

 2 Post-translational modifications of Arabidopsis PI-PLCs identified in Arabidopsis. Phosphorylation sites are experimentally obtained[START_REF] Durek | PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update[END_REF] while the other modifications are putative, as predicted using SUMOsp 2.0, CSS-Palm 3.0 and BDM-PUB software[START_REF] Ren | CSS-Palm 2.0: an updated software for palmitoylation sites prediction[END_REF][START_REF] Ren | Systematic study of protein sumoylation: Development of a site-specific predictor of SUMOsp 2.0[END_REF]

	Name	AGI	Phosphorylation	SUMOylation	Palmitoylation Ubiquitination
			sites
	AtPLC1	At5g58670	S 260		Cys 8 , Cys 10 ,	Lys 2
					Cys 11 , Cys 226
	AtPLC2	At3g08510	S 280 , S 286 , T 29 ,	Lys 577	Cys 9 , Cys 11	Lys 3 , Lys 358
			S 346/ S 348
	AtPLC3	At4g38530			Cys 8 , Cys 10 ,
					Cys 11 , Cys 313
	AtPLC4	At5g58700	S 280		Cys 14 , Cys 439
	AtPLC5	At5g58690		Lys 2 , Lys 295 ,	Cys 13 , Cys 14	Lys 264 , Lys 266 ,
				Lys 497		Lys 275 , Lys 448
	AtPLC6	At2g40116		Lys 6 Lys 364		Lys 3
	AtPLC7	At3g55940	T 169 , S 170 ,	Lys 60	Cys 9 , Cys 11	Lys 3 , Lys 364
			T 19 /S 21 , S 287 /S 293
	AtPLC8	At3g47290		Lys 202	Cys 519	Lys 60
	AtPLC9	At3g47220			Cys 519

ACKNOWLEDGEMENTS

This work was supported by grants from NAS of Ukraine (9.1-06-13 and 8-13) and from SFFR of Ukraine (F41.4/042-2012). The authors acknowledge funding from the French Agence Nationale de la Recherche Programme Blanc PANACEA NT09_517917 (contract to ER). We would like to thank Michael Hodges (Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Université Paris Sud, France) for carefully reading the manuscript.

Supplementary table 1

Plant PI-PLC-X domain containing proteins

Species