Progress in Research 52 (2013) 62–79

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Progress in Lipid Research

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Review The plant non-specific C gene family. Novel competitors in lipid signalling

Igor Pokotylo a,b,Prˇemysl Pejchar b, Martin Potocky´ b, Daniela Kocourková b, Zuzana Krcˇková b, ⇑ Eric Ruelland c, Volodymyr Kravets a, Jan Martinec b, a Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine b Institute of Experimental Botany, v. v. i., Academy of Sciences of the Czech Republic, Prague, Czech Republic c UPMC Univ Paris 06, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, EAC7180 CNRS, Paris, France article info abstract

Article history: Non-specific C (NPCs) were discovered as a novel type of plant phospholipid-cleaving Received 27 April 2012 homologous to bacterial -specific phospholipases C and responsible for lipid Received in revised form 25 September 2012 conversion during phosphate-limiting conditions. The six-gene family was established in Arabidopsis, and Accepted 25 September 2012 growing evidence suggests the involvement of two articles NPCs in biotic and abiotic stress responses as Available online 23 October 2012 well as phytohormone actions. In addition, the diacylglycerol produced via NPCs is postulated to partic- ipate in membrane remodelling, general lipid and cross-talk with other phospholipid signal- Keywords: ling systems in plants. This review summarises information concerning this new plant and Plant nonspecific focusses on its sequence analysis, biochemical properties, cellular and tissue distribution and physiolog- Phosphatidylcholine-specific phospholipase C Diacylglycerol ical functions. Possible modes of action are also discussed. Cell signaling Ó 2012 Elsevier Ltd. All rights reserved. Metabolism regulation

Contents

1. Introduction ...... 63 2. Bacterial phosphatidylcholine-specific phospholipase C ...... 64 3. Animal phosphatidylcholine-specific phospholipase C ...... 64 4. Plant non-specific phospholipase C (NPC) ...... 65 4.1. In silico analysis of NPC gene family in plants...... 65 4.1.1. 3D model of plant NPC...... 68 4.2. Biochemical properties of NPC ...... 68 4.3. Intracellular distribution...... 69 4.4. Tissue distribution...... 69 4.5. Physiological function of NPC...... 70 4.5.1. Phosphate deficiency ...... 70 4.5.2. Role in abiotic stress ...... 71 4.5.3. Role in biotic stress ...... 71 4.5.4. Role in hormonal regulation ...... 72

Abbreviations: ABA, abscisic acid; BL, 24-epibrassinolide; BY-2, Bright Yellow 2; BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D609, tricyclodecan-9-yl- xanthogenate; DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; DGK, diacylglycerol kinase; EDTA, ethylenediaminetetraacetic acid; GUS, b-glucuronidase; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; MAG, monoacylglycerol; MGDG, monogalactosyldiacylglycerol; MeJA, methyl jasmonate; NPC, non-specific phospholipase C; PA, phosphatidic acid; PAP, phosphatidic acid ; PC, phosphatidylcholine; PC-PLC, phosphatidylcholine-specific phospholipase C; PE, phosphatidylethanolamine; PIP2, phosphatidylinositol 4,5-bisphosphate; PI-PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; PL, phospholipid; PLD, ; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; ROS, reactive oxygen species; SA, salicylic acid; SQDG, sulphoquinovosyldiacylglycerol; TDTMA, tetradecyltrimethylammonium bromide. ⇑ Corresponding author. Address: Institute of Experimental Botany, v. v. i., Rozvojová 263, 165 02 Prague 6 – Lysolaje, Czech Republic. Tel.: +420 225 106 416; fax: +420 225 106 456. E-mail address: [email protected] (J. Martinec).

0163-7827/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plipres.2012.09.001 I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 63

4.6. Possible mode of action ...... 72 4.6.1. ...... 72 4.6.2. Signal transduction...... 73 4.6.3. Membrane remodelling ...... 75 5. Conclusions and perspective ...... 75 Acknowledgements ...... 75 References ...... 75

1. Introduction in cell signalling and regulation in general remains far less under- stood. PC-hydrolysing phospholipases were first discovered under Phospholipases (Fig. 1) are now well recognised as key compo- the name ‘‘ C’’ in [5] and later established as nents of the regulatory systems of cellular growth and develop- important bacterially secreted pathogenicity factors performing ment in living organisms. They give rise to an array of second host membrane lysis and defence signalling interference [6]. PC- messenger molecules and lipid derivatives and are implicated in PLC activity was also identified in fungi [7] and was acknowledged both metabolism and intracellular signalling. Recent research pro- to be an essential source of phospholipid-derived signal molecules gress has made it possible to convincingly reveal an important role in animal cells [8,9]. It was demonstrated that transient increases for phospholipases in mediation of stress responses directed to in DAG production occur in cellular membranes in response to var- provide acclimation to ever-changing environmental conditions. ious stimuli [10,11], whereas attributed PC-PLC activity appears to Phospholipases C (PLC) are able to cleave membrane phospho- be involved in a number of intracellular regulatory events (see sec- , facilitating release of water-soluble phosphorylated head- tion 3). However, the current lack of molecular and genetic charac- groups from hydrophobic diacylglycerols (DAG). PLCs in living terisation of PC-PLCs in animals hampers the progress of research. systems can generally be divided into phosphatidylinositol-specific Although putative PC-PLC activity in plants was observed as phospholipases C (PI-PLC) and phosphatidylcholine-specific phos- early as 1955 [12], PC-PLC functions in plants have long remained pholipases C (PC-PLC) according to specificity range. vague and their role elusive. Some supporting clues for PC-hydro-

The role of specific phosphatidylinositol 4,5-bisphosphate (PIP2) lysing PLC occurrence were identified in plant organs and tissues cleaving PI-PLCs in cell metabolism regulation is fairly well studied such as peanut seeds [13], rice grains [14], tomatoes [15] and oth- in various organisms. Thus, multidomain animal PI-PLCs are G-pro- ers [16]. However, this data lacked sufficient enzymatic character- tein activated that are notably responsible for intracellu- isation [17]. Eventually, in 2002, fluorescently labelled PC was lar calcium level regulation and protein kinase C (PKC) activation shown to be directly cleaved to produce DAG in parsley and tobac- [1]. Despite a lack of identified inositol 1,4,5-trisphosphate recep- co cells, suggesting the presence of NPC (PC-PLC) as a novel type of tors in plants [2], PI-PLCs are unquestionably implicated in the reg- phospholipase in plants with putative signalling functionality [18]. ulation of growth, development and stress responses in Arabidopsis Later, six NPC genes were identified in the Arabidopsis genome and other plant species [3]. In turn, bacterial PI-PLCs belong to se- based on sequence similarities with bacterial PC-PLCs [19]; nine creted pathogenicity factors that typically confer virulence [4]. NPC genes were identified in soybean plants [20]. DAG production PC-PLCs, in plants also known as non-specific PLCs (NPC), are via PC hydrolysis was also observed in Petunia hybrida, suggesting characterised by broader substrate ranges that include abundant the omnipresence of NPCs in the plant kingdom [21]. A subsequent phosphatidylcholine (PC) (discovered in 1847 by a French chemist increase in research interest in dissection of NPC’s roles in plants Theodore Nicolas Gobley as ‘‘’’ molecules), while their role over the recent years has promoted the identification of NPC

Fig. 1. Phospholipase C- and phospholipase D-dependent signalling in plants. A schematic diagram depicts contemporary model of metabolism regulation carried out by plant cell phospholipases. Various augmenting signalling pathways are shown, demonstrating synergistic interactions between phospholipases and lipid second messenger molecules in excitation of cell responses. PIP2, phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; NPC, non-specific phospholipase C; PI-PLC, phosphatidyl- inositol-specific phospholipase C; PLD, phospholipase D; Pchol, ; DAG, diacylglycerol; PA, phosphatidic acid; DGPP, diacylglycerol pyrophosphate; IP3/IP6, inositol 1,4,5-trisphosphate/inositol hexakisphosphate; PKC, protein kinase C; DGK, diacylglycerol kinase; PAP, phosphatidic ; PAK, phosphatidic acid kinase; DGPPP, diacylglycerol pyrophosphate phosphatase; ROS, reactive oxygen species; PAB, PA-binding proteins. 64 I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 involvement in regulation of diverse cellular processes including of Pseudomonas cepacia PC-PLC is 72 kDa [39]. Non-toxic PC-PLCs root development and brassinosteroid hormone signalling [22], have not responded to in vitro treatment with bivalent ions [40], Al stress signalling [23], abscisic acid (ABA) sensing, and tolerance and EDTA chelator was reported to significantly stimulate the to hyperosmotic and salt stresses [24,25]. However, the NPC3 pro- activity of PC-PLC from L. pneumophila [29]. The PlcHR2 phospholi- tein of Arabidopsis was shown to perform lysophosphatidic acid pase C found in Pseudomonas species was also shown to be Ca2+- (LPA) phosphatase activity, producing monoacylglycerol (MAG) independent [40]. Interestingly, Gram-negative Pseudomonas aeru- rather than DAG, which may hint at a possible multivalent func- ginosa produced several types of PC-PLCs [41] that differ in sub- tionality for NPCs [26]. Taken together, this evidence denotes NPCs strate specificity and toxic properties. Thus, PLC-H identified in P. in plant species and suggests their putative role in plant metabo- aeruginosa was haemolytic [42] and possessed syn- lism regulation. thase activity [43]. It is apparent that PC-PLCs from Gram-positive and Gram-neg- ative bacteria have evolved independently. Intriguingly, non-toxic 2. Bacterial phosphatidylcholine-specific phospholipase C bacterial PC-PLCs, including PlcB from Mycobacterium tuberculosis and PlcN from Ralstonia solanacearum clearly show a resemblance Since the discovery of PC-PLC specific activity in Clostridium to putative plant NPCs [19], indicating not only a common evolu- perfringens (formerly known as C. welchii) toxin in 1941 [5], various tionary history but also putative cell functions unrelated to PC-PLC genes and corresponding enzymes have been identified in straightforward membrane lysis. Some data also indicate that Gram-positive bacteria, including Bacillus cereus, Listeria monocyt- NPC may belong to a superfamily of phosphatase/phospholipase ogenes, Clostridium species [6] and, more recently, in Gram- proteins related to the acid phosphatase AcpA from Francisella negative bacteria , such as Pseudomonas species [27], Burkholderia tularensis [44]. pseudomallei [28], Legionella pneumophila [29]. All identified At the same time, bacteria apparently contain several evolution- bacterial PC-PLCs fall into two distinct groups of sequentially ary divergent types of PC-PLC, such as NaCl-stimulated enzyme unrelated enzymes. purified from a marine streptomycete [45], or membrane-bound PC-PLCs from the first group are predominantly found in Gram- PC-PLC protein with a broad pH optimum identified in Ureaplasma positive bacteria and were identified as potent toxins with haemo- urealyticum, which lacks cell walls [46]. lytic properties related to the Clostridium perfringens a-toxin. Such Alongside additional data available on PC-PLC pathogenic prop- toxic PC-PLCs represent single polypeptide enzymes that require erties, it is surprising that to date little is known about the physi- ions for activation and are reversibly inactivated by EDTA or ological functions of non-toxic PC-PLCs. They are thought to be o-phenanthroline metal chelators [6]. Several toxic PC-PLCs have involved in lipid remodelling during phosphate-limiting been shown to also require Ca2+ or Mg2+ ions for their activation conditions, as has been shown for the intracellular PC-PLC of Sino- [30]. Some genes encoding this zinc-metallophospholipase C have rhizobium meliloti [47]. Regulation of some PC-PLC genes by exog- been isolated and characterised [31]. The molecular mass of the enous phosphate levels also supports this hypothesis [6]. enzymes lies within the range of 28–34 kDa [6]. Amino acid com- position of these PC-PLCs revealed the presence of a signal se- quence denoting protein recruitment into secretory systems. 3. Animal phosphatidylcholine-specific phospholipase C Toxic bacterial PC-PLCs have wide-ranging substrate specificity that, apart from phosphatidylcholine, includes phosphatidyletha- PC hydrolysis is now recognised as a constitutive element of cell nolamine (PE), phosphatidylserine (PS), sphingomyelin and other signalling and metabolism regulation in animals [8,9], while PC- lipids [6]. The molecular structure of the well-studied C. perfringens cleaving PLC appears to be a key agent in the generation of second PC-PLC toxin (a-toxin) revealed a two-domain protein. The N-ter- messengers [48,49]. minal domain contains the phospholipase C , which also Animal PC-PLC genes apparently show no sequence similarities incorporates zinc ions. The C-terminal C2-like PLAT (Polycystin-1, with other known PC-PLCs in diverse living organisms, which Lipoxygenase, Alpha-Toxin) domain was found to be similar to li- would explain why no animal PC-PLC genes have yet been cloned pid binding domains in eukaryotes [32] and appears to be respon- or characterised. However, considerable effort has been invested sible for binding membrane phospholipids in a calcium-dependent into PC-PLC functional characterisation, and it is now evident that manner [33]. Interestingly, the N-terminal domain of a-toxin re- PC-PLC is directly involved in the control of cell proliferation and tained PC-PLC activity when expressed in , but differentiation, as well as a number of other cell functions [50,51]. lacked haemolytic and sphingomyelinase activities that are sup- Until now, PC-PLC activity has been detected in multiple animal posedly granted by a lipoxygenase-like C-terminal domain [34]. sources including canine myocardium [52], acrosomes of bull sem- Toxic bacterial PC-PLCs have been extensively studied as pathoge- inal plasma [53], human vascular endothelial cells [54] and chicken nicity factors responsible for host membrane lysis. In addition, PC- blastodisc [55]. Interestingly, cytosolic 66 kDa PC-PLC identified PLCs can interfere with eukaryotic cellular signalling and take con- using antibacterial PC-PLC antibody in mouse NIH-3T3 fibroblasts trol of host immune responses [35]. For instance, secreted PC-PLC [56] and NIH-3T3 H-ras transformants [57] was rapidly translocated from C. perfringens plays a role in the aggregation of blood platelets to the plasma membrane upon mitogen-mediated cell receptor [36] and was shown to inhibit defensive superoxide generation in stimulation. A similar pattern was observed with PC-PLC activity human polymorphonuclear leukocytes by interacting with mem- predominantly localised to the plasma membrane in oncogenically brane components of NADPH oxidase [37]. However, some PC- transformed human epithelial ovarian cells [58] and NK cytolytic PLC from Gram-positive bacteria can have functions unrelated to cells, where PC-PLC may contribute to lysis of target cells [59,60]. those of typical toxic enzymes. Secreted PC-PLC from B. cereus is in- To date, the regulatory role of PC-PLC has been largely attrib- volved in the defence mechanism of bacteria to phagocytosis [38]. uted to DAG production. In animals, DAG can typically arise via

Furthermore, some bacterial PC-PLCs may be used as vaccines PC or PIP2 hydrolysis, or by phosphatidic acid (PA) dephosphoryla- against diseases such as gangrene [36]. tion. DAG has multivalent functionality that includes alteration of The second group of PC-PLCs is primarily produced by Gram- membrane curvature and properties, orchestration of lipid metab- negative bacteria. These enzymes do not contain zinc ions and typ- olism, and participation in lipid-mediated signalling [61,62]. ically lack haemolytic properties. Their molecular weight differs Among the most important features of DAG is its ability to bind several-fold from that of the toxic enzymes. For example, the Mw to the C1 domain [63] and facilitate the activation of PKC– an I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 65 enzyme known for its immensely wide cellular functionality in lymphocytes [79]. It is interesting that in animal cells, [64,65]. DAG-binding C1 domains have also been identified in a kinases are also responsible for phosphocholine production number of other proteins with diverse regulatory functions. For in- through choline phosphorylation in the presence of ATP and mag- stance, a DAG-mimicking phorbol induced a PKC-indepen- nesium [80]. Choline kinases have been shown to be involved in dent exocytosis in rat PC12 cells mediated by a RasGRP3 protein cell division and growth regulation [80], and play significant role that contains a C1 domain [66]. in regulation of both normal human mammary epithelial cell pro- Initial reports indicated that PC-derived DAG may be ineffective liferation and breast tumour progression [81]. for PKC activation [67], but it was later shown that B. cereus derived Several other putative functions of PC-PLCs affecting animal cell PC-PLC not PI-PLC, inhibited the formation of cAMP by adenylate metabolism are now being considered. Understanding of the spec- cyclase, which is negatively regulated by PKC in Swiss 3T3 fibro- ificity of PC-PLC activity and the role of phospholipid-derived sig- blasts [68]. In addition, the regulatory function of melatonin hor- nalling substances in animal cells is impeded by a lack of PC-PLC mone in rat retinal ganglion cells was mediated by PKC activated sequence and structural information. exclusively via PC-PLC action [69]. A specific role for PC-PLC in the regulation of leukemic myeloid cell proliferation via MAP kinase cascade stimulation and NF-jB activation has also been reported to 4. Plant non-specific phospholipase C (NPC) be PKC-dependent [70]. It is now thought that PC-derived DAG is responsible for activation of PKC isoforms dependent solely on 4.1. In silico analysis of NPC gene family in plants DAG, namely PKC-d, PKC-e, PKC-g and PKC-h [71]. Alternatively, PC-PLC may provide a sustained DAG source that can assist in the Initial reports on plant NPCs suggested that they were not re- activation of conventional type PKCs that depend both on DAG lated to any other known plant phospholipase family. Previously, 2+ and Ca ions (PKC-a, PKC-bI, PKC-bII, PKC-c) and are first initiated multiple sequence alignments of Arabidopsis NPCs with PLC from by DAG produced via short-lived PIP2 hydrolysis [72]. M. tuberculosis revealed three conserved regions unrelated to DAG produced by PC-PLC may also be phosphorylated to PA by known domains of plant phospholipases [19]. Although the NPC diacylglycerol kinases (DGK) [73,74] and act as a contributor to PA- family clearly arose early in evolution, bacterial, plant and inverte- dependent cell metabolism regulation in addition to phospholipase brate lineages have each developed distinct NPC sequence features. D (PLD) [75]. The reverse dephosphorylation of PA may also play a Genomes of higher plants typically contain several genes coding regulatory role. Thus, PA-derived DAG has been shown to be for putative NPCs proteins consisting of 510–540 amino acid resi- important for vesicle and tubule formation in the Golgi apparatus dues. NPC proteins contain a central phosphoesterase domain of HeLa cells [76]. (Fig. 2) that is typically present in enzymes with activity, In animals, the regulatory role of phosphocholine, which is con- such as NPCs and acid . No motifs known from other currently produced alongside DAG during PC hydrolysis by PC-PLC, plant lipid signalling proteins (i.e., C2, XY, EF, PH, PX, ENTH, FYVE has also been discussed. Phosphocholine is involved in many long- domains) are present. Generally, plant NPCs show a high level of term cellular responses such as activation of proliferation/differen- similarity, especially in the phosphoesterase domain (Fig. 2). Close tiation and cell transformation promotion [77]. Moreover, phos- comparison with Gram-negative bacterial PC-PLCs reveals four phocholine induces DNA synthesis and mitogenesis in mouse motifs with invariable residues that are likely crucial for the PC- fibroblasts [78] and acts as a specific receptor molecule for perforin PLC catalytic activity (Figs. 2 and 4). The majority of plant NPCs

Fig. 2. Schematic alignment of Arabidopsis NPCs with known PC-PLCs from P. aeruginosa and M. tuberculosis [42] and acid phosphatase from F. tularensis [44]. The schema is based on an excerpt from large multiple alignment that contained 83 plant, bacterial, fungal and protist sequences and was constructed using Mafft [208]. The presence of putative N-terminal signal peptide, phosphoesterase domain (Pfam entry PF04185), domain of unknown function 756 (Pfam entry PF05506), conserved regions, putative active site residues and conserved active site-stabilising ion pairs is indicated. Various shades of blue indicate conserved amino acid residues (light blue – less conserved, deep blue – most conserved). At, A. thaliana; Ft, F. tularensis; Pa, P. aeruginosa; Mt, M. tuberculosis; AcpA, acid phosphatase A, NPC, non-specific phospholipase C, plcN, non-haemolytic phospholipase C. See text for more details. 66 I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79

Fig. 3. Phylogenetic analysis of plant NPC. Multiple alignment of NPC sequences obtained from the NCBI database, Doe Joint Genome Institute and Genoscope-Centre National de Séquençage, was created using Mafft v6.238b in L-INS-I mode [208] and then edited with Jalview [209]. The conserved blocks were concatenated to give a final matrix with 428 positions from 47 NPC-like sequences that were then subjected to phylogenetic analysis. A phylogenetic tree was constructed using maximum likelihood method from NPC amino acid sequences using PhyML (WAG model + C +I) [210]. Numbers at nodes represent values of neighbour-joining bootstrap support obtained using aLRT (approximate likelihood-ratio test) method. Circles at the nodes represent 100% support, and missing values indicate support below 75%. Branches collapsed where the support was below 50%. Non-plant PC-PLC homologues were used as a phylogenetic tree root. The scale bar represents 0.2 substitutions/site. Species abbreviations: At, A. thaliana; Gm, G. max; Os, O. sativa; Pp, P. patens; Ps, P. sitchensis; Pt, P. trichocarpa; Sb, Sorghum bicolor; Sm, S. moellendorffii; Vv, V. vinifera; NPC, non-specific phospholipase C. I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 67

Fig. 4. 3D structure of plant NPC. 3D model of plant NPC structure was predicted using AtNPC2 sequence as a template. Final model was designed based on published structures of acid phosphatase from bacteria F. tularensis (PDB code 2DG1, [44]) and human estrone sulphatase (PDB code 1P49, [211]) using MODELLER 9v7 software [212]. Seventy generated structures were then evaluated with Prosa (https://prosa.services.came.sbg.ac.at/prosa.php) and WhatIf (http://swift.cmbi.ru.nl/servers/html/index.html) algorithms, and the best model is shown. The pair of images is rotated 90° around y-axis. Side chains are shown for putative active site residues (orange) and a structure- stabilising ion pair (cyan). Electrostatic potential was mapped onto AtNPC2 ranging from 5 (red) to + 5 (blue) kbT/ec. See the main text for details.

contain a putative signal peptide at the N-terminus that is followed plants, we performed a phylogenetic analysis of NPC protein se- by a short variable region and a highly conserved domain contain- quences from several evolutionarily distinct plant species (Fig. 3). ing invariable ENRSFDxxxG motifs at the beginning of the phos- In addition to dicot species like poplar (Populus trichocarpa), grape- phoesterase domain. The putative signal peptide is missing in vine (Vitis vinifera) and soya (Glycine max), we also analysed the Arabidopsis NPC3, NPC4 and NPC5. Two other invariable motifs, genomes of the monocotyledons rice (Oryza sativa) and sorghum TxPNR and DExxGxxDHV, are found in the middle of the domain (Sorghum bicolor), sitka spruce (Picea sitchensis)—representing followed by a GxRVPxxxxxP region that closes the phosphoesterase gymnosperms—and moss Physcomitrella patens and lycophyte domain. Interestingly, the 50–100 amino acids at the C-terminus Selaginella moellendorffii—representing evolutionarily ancient form the most divergent part of NPC sequences, with distinct plants. Phylogenetic analysis of plant NPC sequences suggested lengths and sequence conservation among NPC subfamilies. This that the common ancestor of all seed plants already had at least may be the part of the molecule responsible for the functional dif- one NPC1-, NPC2- and NPC6-like gene. Interestingly, the NPC3–5 ferences of various NPC isoforms through facilitating interactions subfamily was not identified in spruce (and other available gymno- with other proteins or defining protein localization. sperms sequences, data not shown). As no complete gymnosperm To gain insight into the evolution of plant NPCs and determine genome sequence is available and spruce NPC analysis was based whether NPC diversity seen in Arabidopsis is conserved across on EST sequences, one cannot yet exclude the presence of this 68 I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 subfamily in gymnosperms. Alternatively, it is possible that NPC3– 4.2. Biochemical properties of NPC 5 subfamily is present only in angiosperms and emerged after the separation of gymnosperm and angiosperm ancestors, possibly NPC3, NPC4 and NPC5 from Arabidopsis thaliana have been from NPC2 clade (as indicated by phylogeny). Surprisingly, Physc- cloned and partly characterised biochemically (Table 1). Recombi- omitrella and Selaginella were found to contain only NPC1-like nant NPC4 expressed in E. coli showed activity towards PC and PE, genes. Two evolutionary simple explanations are possible: either while the ability to cleave PA and PIP2 substrates was negligible the common ancestor of land plants contained NPC1, NPC2 and [19]. NPC4 also demonstrated moderate hydrolytic activity to- NPC6 subfamilies and NPC2 with NPC6 gradually disappeared dur- wards PS and hydrolyses phosphatidylglycerol to a small extent ing evolution towards lycophytes and brychophytes, or the NPC1 [25]. NPC4 activity was slightly elevated in the presence of 2 mM subfamily is the ancestral NPC, which subsequently gave rise to EGTA chelator [19]. all other NPC types (this scenario is not supported by the phyloge- Recombinant NPC5 protein expressed in E. coli was able to netic analysis). Both Selaginella and Physcomitrella NPCs further cleave PC and PE to produce DAG. However, the hydrolytic activity multiplied independently after the separation of mosses, lyco- of NPC5 was more than 40-fold lower than that of NPC4 [82]. phytes, and seed plants. Rapid diversification of NPCs is evident Purified recombinant NPC3 protein demonstrated specific LPA from the evolution of angiosperm orthologs, where multiple dupli- phosphatase activity, resulting in MAG production [26]. LPA phos- cations within all NPC subfamilies are frequently observed. In con- phatases are known as important regulatory enzymes in animals trast, no NPCs were found in green algae, suggesting that the NPC [83], but their importance in plants has not yet been ascertained. family may have been lost throughout algae evolution. The enzyme was marginally stimulated by low concentrations of the non-ionic detergent Triton X-100, but treatment with CHAPS 4.1.1. 3D model of plant NPC or NP40 detergents resulted in inhibition of substrate conversion. Although no experimental three-dimensional structure of a LPA phosphatase activity was also reduced in the presence of lyso- plant NPC is available, the homology of plant NPCs with the acid phosphatidylcholine (LPC) or lysophosphatidylethanolamine (LPE), phosphatase (AcpA) family enabled us to construct a 3D model but it was not affected by PA, PC, MAG or DAG. Phosphatase activ- 2+ for Arabidopsis NPC employing recently published structure of ity of NPC3 did not require the presence of Ca or other bivalent AcpA from the bacterium F. tularensis [44]. Analysis of the AtNPC2 ions and was independent of fatty acid variations in the LPA sub- 3D model (Fig. 4) showed that, similarly to the bacterial AcpA, the strate. NPC3 was also unresponsive to treatment with non-specific backbone of plant PC-PLC is formed by a beta sheet (composed of 7 sodium o-vanadate and sodium fluoride phosphatase inhibitors. beta structures) embedded inside the protein and surrounded by The authors [26] additionally showed that the purified enzyme several alpha helixes (6 in FtAcpA, 7 in plant NPCs). Importantly, did not hydrolyse sphingosine-1-phosphate, diacylglycerol pyro- the majority of amino acid residues forming the active site of the phosphate, glycerol-3-phosphate, LPC, LPE or PA. More impor- bacterial AcpA are conserved, including the residues that bind a tantly, NPC3 lacked the ability to hydrolyse PC, PE or PS [26], still-unidentified metal cation. This is also true for the ion pair of indicating that the NPC3 gene may be paralogous to other plant aspartate-arginine, which probably acts as a stabiliser of the active NPC genes that code for phospholipase C-type enzymes. site topology. Altogether, this evidence suggests that for eukaryotic The studies of plant NPC activity in vitro revealed that in micro- PC-PLCs, the bacterial PC-PLCs and homologous acid phosphatases, somal fractions prepared from BY-2 cells, PC hydrolysis was inhib- the common ping-pong reaction mechanism, with an intermediate ited by Al at high concentrations (P100 lM AlCl3) [23]. in which the substrate phosphate group is covalently bound to Specific inhibitors serve as important tools for revealing the role nucleophilic amino acid from the active site, is likely to be main- of the studied proteins. No specific inhibitor has been described for tained. By contrast, none of the four cysteines, which form two NPCs, although both animal PC-PLC-directed inhibitors [84,85] and disulphide bridges stabilising the AcpA molecule, are present in bacterial PC-PLC-directed [86] inhibitors have been identified. plant NPCs. The predicted model of AtNPC2 also indicates that Tricyclodecan-9-yl-xanthogenate, commonly known as D609, the variable C-terminal domain forms a ‘‘cap’’ that covers part of has been widely studied as a specific inhibitor of PC-PLC activity the molecule from the proposed active centre and thus does not in animals, exhibiting a variety of biological effects including anti- participate directly to the catalysis. This finding is consistent with viral, antitumoural, and anti–inflammatory influence [87]. How- the sequence analysis of the NPC proteins. Interestingly, the active ever, it is also a potent antioxidant [88] and inhibitor of group IV site of plant NPCs forms a relatively strongly negatively charged cytosolic [89]. This indicates that at least some pocket that may be involved in phospholipid substrate binding observed functions may be attributed to side effects of the influ- (Fig. 4). ence of D609 on PC-PLC activity. Interestingly, D609 also inhibits

Table 1 Biochemical properties, physiological properties and expression patterns of NPC, characteristic of identified NPC-coding genes and corresponding to gene products in A. thaliana. Putative roles for NPCs to particular cell reactions are also provided. Data were obtained by in silico analysis and from mentioned sources. aa, amino acid; ABA, abscisic acid; BL, 24-epibrassinolide; LPA, lysophosphatidic acid; NPC, non-specific phospholipase C; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.

Name Gene locus Cloned Number Calculated Substrate Localisation Predominant Activation/stimulus expression Reference b of aa Mw (kDa) tissue expression NPC1 At1g07230 – 533 60.0 a Endomembranesb Siliques a [25] NPC2 At2g26870 – 514 57.6 a Endomembranesb Siliques, roots a [25] NPC3 At3g03520 + 523 59.1 LPA Tonoplast Leaves, roots BL signalling, auxin, cytokinin [22,25,26,93] NPC4 At3g03530 + 538 60.7 PC, PE Plasma Old leaves, roots Phosphate deficiency, ABA, salt stress, [19,22,24,25] (PS) membrane BL signalling, auxin, cytokinin NPC5 At3g03540 + 521 59.0 PC, PE Cytosol Inflorescences Phosphate deficiency [22,25,82] NPC6 At3g48610 – 520 57.9 a Mitochondria, Siliques, roots a [25] plastidsb

a Experimentally unidentified. b Based on sequence analysis and prediction programmes (The Arabidopsis Information Resource (TAIR), www.arabidopsis.org; Cell eFP browser, http://bar.utoronto.ca/ cell_efp/cgi-bin/cell_efp.cgi). I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 69 some bacterial PC-PLC enzymes, indicating that animal and bacte- 4.4. Tissue distribution rial PC-PLC may share some structural similarities [90]. For in- stance, PC-PLC from Gram-negative Pseudomonas fluorescens Identification of the levels and spatial properties of NPC expres- reacted to animal D609 PC-PLC inhibitor treatment [27]. NPC activ- sion in tissues and organs of Arabidopsis may provide an effective ity in BY-2 cells was not altered after treatment with 20 M of D609 means to further disclose their role in plant growth and develop- animal PC-PLC inhibitor [23], indicating that plant NPC may ment. NPC genes are characterised by changeable level of expres- possess regulatory properties different from those of animal or sion, with a somewhat pronounced intra-organismal expression bacterial PC-PLC. pattern. Average signal percentiles of the corresponding mRNAs Taking into consideration the identified sequence similarities in single channel arrays range from 25 for NPC4 to 71.4 for NPC1 between plant NPC and bacterial PC-PLC, TDTMA-type (tetrade- and 77.2 for NPC6 (The Arabidopsis Information Resource (TAIR), cyltrimethylammonium bromide) inhibitors of bacterial enzyme www.arabidopsis.org), consistent with the differentiated expres- may also be effective in repressing NPC activity [86]. sion potential of other phospholipases known to be implicated in cell signalling, including PI-PLC (percentile ranges 42–90) and 4.3. Intracellular distribution PLD (percentile ranges 26–94). More importantly, generally low expression potential may suggest an inducible expression and Localisation data may provide additional clues into the func- putative signalling role for NPC2 (51.6) NPC3 (51.5), NPC4 and tional role of NPC in plant cells. In terms of subcellular localisation, NPC5 (24.8), which have been experimentally defined for NPC3 plant phospholipases can be typically divided into cytosolic and and NPC4 [22,24,25]. membrane-bound enzymes [17,91]. Peters et al. have provided a tissue-specific analysis of NPCs NPC activity has been experimentally detected in the cellular expression in Arabidopsis [25]. Experiments exploiting a b-glucu- membranes of stamens and pistils of Petunia hybrida [21] and the ronidase (GUS) reporter system demonstrated that PNPC3:GUS and plasma membrane fraction prepared from BY-2 suspension cell PNPC4:GUS constructs localised to root tips, cotyledons and outer cultures [23]. A similar pattern was observed in oat roots, where leaf margins. Additionally, in floral organs, PNPC3:GUS and PNPC4:GUS a phospholipase that reacted with NPC4-raised antibody was found constructs were expressed predominantly in pollen sac tissues of in the plasma membrane [92]. Analysis of subcellular fractions young anthers [22]. In another study, PNPC4:GUS constructs were from Arabidopsis leaves using polyclonal anti-NPC4 antibody re- expressed in ageing leaves with low levels of expression in roots, vealed that NPC4 is located in the microsomal membrane fraction stems, siliques and flowers [95]. GUS staining additionally allowed and not the plastids [19]. Further analysis demonstrated that NPC4 determination of NPC2 gene expression patterns, which were local- is localised specifically to the plasma membrane [19], as confirmed ised to the meristematic zone, elongation zone and vascular tissue by analysis of the NPC4-GFP construct [82] (Fig. 5). NPC3 was of maturation zone of root tips and to developing leaves, with ob- experimentally identified to be present in the tonoplast, as shown served deterioration with leaf maturation [96] (Fig. 6A). by one-dimensional SDS–PAGE analysis of a tonoplast-enriched Analysis of array data (Arabidopsis eFP Browser, http://bar.uto- fraction followed by nano-LC MS/MS [93]. Unfortunately, the ronto.ca; Genevestigator, www.genevestigator.com) allowed the determinants for NPC4 and NPC3 membrane localisation are cur- identification of significantly amplified expression of all NPC genes rently unknown and may include palmitoylation, myristoylation, in developing seeds, notably in chalazal endosperm and seed coat – GPI-anchoring among other factors. regions known to be among primary lipid storage and metabolism NPC5-GFP constructs localised to the cytosol in Arabidopsis [82]. sites (Fig. 6B and C) [97]. Elevated expression of NPCs was also de- Because phospholipases convert hydrophobic substrates, it is pos- tected in roots, floral organs, siliques and leaves at various stages of sible that cytosolic phospholipases are translocated to membranes development. In addition, expression of NPC1 was increased in root upon stimulation, as shown for PLDa in Arabidopsis [94]. columella cells, stem epidermis, xylem and cork, as well as in guard The subcellular distribution of other NPCs is uncertain but has cells and trichomes. Expression of NPC1 was low in germinating been predicted to localise to endomembranes despite a lack of and mature pollen. NPC2 was upregulated in siliques and during identified transmembrane domains (Cell eFP Browser, http:// early stages of seed development in the meristem of developing bar.utoronto.ca; http://www.cbs.dtu.dk/services/TMHMM). embryos and had relatively low expression in roots and leaves of

Fig. 5. NPC intracellular distribution pattern in A. thaliana. Schematic representation of identified and putative sites of NPC expression in Arabidopsis cells are depicted by corresponding coloured circles. Main cell organelles and compartments are indicated. Intracellular expression of AtNPC4, AtNPC5 and AtNPC3 was observed in plasma membrane, cytosol and tonoplast, respectively [19,82,93]. Expression patterns of AtNPC1, AtNPC2 and AtNPC6 remain uncertain. However, signal peptide sequences identified in above-mentioned NPCs and in silico sequence analysis have provided clues for AtNPC1 and AtNPC2 localisation to endomembranes, while AtNPC6 was predicted to localise to plastids and mitochondria. NPC, non-specific phospholipase C. Results were obtained experimentally. 70 I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79

Fig. 6. Expression pattern of A. thaliana NPC genes in planta. Diagrams show that colour-coded NPC genes are expressed divergently in Arabidopsis. Experimental data (A) for NPC tissue expression were provided by quantitative PCR and GUS staining [22,25,95,96]. The expression levels of individual NPC genes are compared within the whole plant; circle size represents relative gene abundance. However, the combined quantities of transcripts of all NPC genes are not comparable in this figure. Analyses of Arabidopsis Affymetrix 22 K array data for NPC expression in tissues of Arabidopsis (B) and throughout ontogeny (C) were performed via Genevestigator (http://www.genevestigator.com). Developmental stages of Arabidopsis are abbreviated as follows: G, germinated seed; S, seedling; yR, young rosette; dR, developed rosette; B, bolting; yF, young flower; dF, developed flower; FS, flowers and siliques; S, siliques. NPC, non-specific phospholipase C.

Arabidopsis. NPC3 was amply expressed in endodermis, phloem changes in NPC activity can be attributed to stimulation of presyn- pole and phloem companion cells in root elongational and matura- thesised enzymes by posttranslational modification and/or intra- tional zones and root stele, as well as in cork, cambium and xylem cellular translocation. Therefore, there is a physiological role for of the stem. NPC3 expression was also relatively active in dry seeds NPC in plant cells, and NPC should be added to the list of phospho- and early germinating seeds. Additionally, NPC4 expression was involved in signal transduction. detected in root xylem and developing floral organs. NPC4 also has low expression levels in germinating pollen. NPC6 expression 4.5.1. Phosphate deficiency has seemingly uniform distribution across Arabidopsis organs, but During phosphate shortages, organic phosphate can be mobi- was notably higher in seedling hypocotyls and lower in seedling lised from membrane phospholipids, which incorporate up to a roots and germinating pollen. Interestingly, NPC6 was expressed third of total phosphorus in plant cells. NPCs are able to facilitate significantly more in young leaves than in old leaves of Arabidopsis such mobilisation by separating the phosphate-containing head and was intensively expressed in shoot apical meristem, leaf pri- groups from phosphatidylcholine or other phospholipids. Concur- mordial and floral organs. It is important to mention that NPC5 rently produced DAG can serve as a precursor to production of and NPC4 expression data were indistinguishable in the Arabidopsis substituting sulpholipids [99] and [100], allowing ATH1 22k genome array. However, because expression levels for retention of membrane integrity and other crucial properties. In NPC5 were estimated to be significantly lower than those of NPC4 2003, it was shown that under phosphate deprivation, transient in- in a majority of Arabidopsis tissues, these results may be applicable creases in PC content in Arabidopsis followed rapid PC decline to NPC4 to some extent [25]. accompanied by DAG accumulation, suggesting NPC activation Proteomics data analysis (Proteomics identifications database, [101]. Later, the NPC4 gene was strongly expressed in Arabidopsis http://www.ebi.ac.uk/pride) has confirmed that NPC proteins, plants experiencing lack of phosphate [19]. NPC4 T-DNA insertion excluding NPC5, are present in the roots of Arabidopsis. NPC1, mutant lines in similar conditions demonstrated a significant NPC2 and NPC6 were also found in leaves, NPC3 and NPC4 in seeds reduction in NPC activity. Based on these results, Nakamura et al. and NPC1 and NPC2 in the floral organs of Arabidopsis plants. NPC5 [19] postulated that PC hydrolysis by NPC4 plays an important role protein was present in leaves subjected to mannitol-induced in securing supplies of inorganic phosphate and DAG from mem- drought stress. brane-localised phospholipids. Liberation of phosphate serves cel- lular metabolism, while DAG contributes to galactolipid synthesis, 4.5. Physiological function of NPC for example digalactosyldiacylglycerol (DGDG). However, the galactolipid composition of the npc4 mutants was unchanged, leav- NPC is relevant to various plant metabolic responses to both ing the role of NPC4 questionable [19]. environmental and organismal stimuli. Long-term changes in Thus, NPC5 also appears to be essential for normal growth and NPC activity and gene expression were observed under biotic and galactolipid accumulation during phosphate deficiency [82]. NPC5 abiotic stresses, phosphate deficiency and certain hormone treat- expression was increased and T-DNA mutant npc5 displayed re- ments (auxins, cytokinins, ABA). In contrast, rapid changes in duced DGDG accumulation during phosphate deficiency. Analysis NPC activity and DAG production were observed in plant cells in of acyl chains in DGDG molecules revealed that NPC5 affects response to Al, brassinosteroids and elicitors. These responses oc- mainly eukaryotic-type galactolipid synthesis that takes place out- cur faster than de novo protein synthesis under the control of fast side of plastids [82]. Another study suggested specific activation of promoters, which take approximately 30–45 min [98]. Thus, the NPC5 gene in roots of Arabidopsis induced by Pi deficiency I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 71

[102]. Interestingly, while expression levels of NPC4/5 genes were 4.5.3. Role in biotic stress significantly induced during phosphate starvation, recovery of The role of phospholipases in plant defence reactions has been phosphate uptake resulted in rapid inhibition of their transcription demonstrated repeatedly [115,116], giving evidence for a promi- [103]. In addition, activation of NPC activity during phosphate-lim- nent role for lipid second messengers. The first evidence of possi- iting conditions was impaired in mutants defective for IAA14 and ble NPC function in signal transduction during plant defence ARF7/19 transcription factors, suggesting the involvement of aux- responses was provided by Scherer et al. [18] who studied the ef- in-mediated signalling pathways [104].Annpc3 knockout mutant fect of glycoprotein elicitors from Phytophthora sojae and cryptog- demonstrated weak impairment of lateral root growth induced ein elicitors isolated from Phytophthora cryptogea in cell lines of by phosphate deficiency [22]. It is important to note that phospho- parsley (Petroselinum crispum) and tobacco (Nicotiana benthami- lipid conversion to phosphate-lacking lipids can also be accom- ana), respectively. Adding synthetic PC-bearing fluorescent tags plished via the combined action of PLD and phosphatidate on both fatty acid residues (bis-BODIPY-PC) to the cell cultures phosphatase (PAP) [105]. Thus, antibodies raised against the C-ter- demonstrated a rapid decline in fluorescently labelled DAG pro- minus of NPC4 interacted with a protein exhibiting PLD-like activ- duction, suggesting inhibition of NPC activity. A decrease in DAG ity induced in phosphate-defficient oat [92]. Nevertheless, plant level also occurred after treatment with mastoparan, a peptide NPCs are unquestionably appearing among key enzymes responsi- capable of G-proteins activation. The observed level of fluores- ble for lipid remodelling during phosphate-limited conditions. cently labelled PA was insignificant, indicating that the fluores- cent DAG originated predominantly through direct action of NPC and not due to activation of PLD and PAP [18]. By contrast, the 4.5.2. Role in abiotic stress expression of the gene homologous to AtNPC5 was increased 4- Phospholipid signalling plays a role in the activation of defence fold in Citrus sinensis plants following infection with Candidatus reactions in osmotic and salt stress conditions. Rapid accumulation Liberibacter asiaticus [117]. of PC–derived DAG was observed in plasma membranes of Dunal- DNA microarray data available on the web (Arabidopsis eFP iella salina following hypo-osmotic shock [106]. The expression le- Browser, http://bar.utoronto.ca; Genevestigator, www.genevesti- vel of NPC4 increased 12-fold in the roots of Arabidopsis after 3 to gator.com) demonstrate that the NPC gene family, notably NPC6, 6 h of 100 mM NaCl treatment [24]. The authors also demonstrated is predominantly downregulated following inoculation with vari- that DAG production via NPC activity increased in a time- and ous strains of Pseudomonas syringae and Pseudomonas parasitica, dose- dependent manner in salt-stressed Arabidopsis seedlings. representing both compatible and incompatible plant-pathogen More importantly, unlike WT plants, npc4 knockout mutants were interactions. NPC6 was also significantly repressed after flg22 characterised by a reduced germination rate when sown on media treatment. However, NPC3 and NPC4 did demonstrate a positive re- containing 150 mM NaCl [24]. In another study [25], npc4 plants sponse to Botrytis cinerea, Golovinomyces orontii, Pseudomonas were also shown to have reduced germination and overall viability syringae and Phytophthora infestans treatment. In addition, NPC1 under salt and drought stress conditions. Unlike WT plants, mu- and NPC4 reacted to bacteria-derived elicitors, namely flg22 and tants overexpressing NPC4 were characterised by a higher germi- HprZ, suggesting a probable bivalent function for NPCs during nation level and maintained a greater root length and dry weight stress responses and the ability to perceive dissimilar elicitors. under both salt stress and hyperosmosis [25]. Based on experimen- Interestingly, NPC3 expression was elevated during Bemisia tabaci tal data, both groups of authors suggested that NPC4 participates in whitefly infection and after methyl jasmonate (MeJA) treatment triggering plant salt stress responses likely via ABA-dependent in Arabidopsis ler and penta mutants, suggesting the possibility that mechanisms (for details, see section 4.5.4.). NPC3 is involved in defence reactions against insect pests. Aluminium toxicity can cause rapid inhibition of root growth The putative role of NPC in defence responses may be attributed mediated by depolarisation of the plasma membranes [107,108], to DAG production, its conversion to PA via DGK, or other lipid sec- disruption of ion fluxes [109], and calcium homeostasis [110] as ond messengers. Synthetic DAG was able to activate defensive 6- well as by affecting the cytoskeleton [111]. Certain phospholipases methoxymellein phytoalexin production in carrot cells [118]. and lipid intermediates are known to perform signalling or meta- DAG and PA also control the generation of reactive oxygen species bolic roles during Al stress [112,113]. By studying the metabolic (ROS) and the induction of elicitor-responsive genes in rice cells pathways involved in the formation and degradation of DAG, it [119]. Treatment with N,N‘,N‘‘,N‘‘‘-tetraacetylchitotrehalose and has been shown that decreases in DAG accumulation in Al-treated flg22 elicitors promoted accumulation of PA in tomato cell culture tobacco BY-2 cells depend on NPC activity [23]. The authors dem- that was not associated with PLD activation [120], giving evidence onstrated that a reduction in DAG production was not due to a de- for the role of DAG/DGK in specific plant defence reactions. DGK- crease in cell viability and that exogenously applied DAG was able assisted DAG transformation may supplement PA production, to restore Al-inhibited growth of tobacco pollen tubes, indicating which was shown to directly regulate defensive ROS generation an essential role for DAG during Al stress [23]. by NADPH oxidase in Arabidopsis [121] and to be an early step in The expression level of NPC3 was increased 14.6-fold after 2 h in plant response to wounding [122]. Contribution of DAG/DGK to Arabidopsis seedlings subjected to 37 °C heat stress [114], suggest- PA production was also observed during the reaction of suspen- ing a role for NPCs in thermotolerance. Gene expression arrays also sion-cultured alfalfa cells to chitotetraose and xylanase elicitors provided clues into the behaviour of NPC1, NPC3 and NPC4(5) in re- [123], the early NO-dependent PA production in tomato cells elic- sponse to drought stress, heat stress, cold stress, hypoxia and os- ited with xylanase [124] and the avirulent interaction of tobacco motic stress responses—conditions that may require membrane cells transformed with the Cladosporium fulvum Avr gene readjustment and stress signalling performed by NPCs. Addition- [125]. Moreover, expression of the rice defence-responsive Os- ally, NPC1 reacted to chemical-induced genotoxic and oxidative BIDK1 DGK gene in tobacco has increased its resistance to tobacco stress. Expression of some NPCs was also altered following changes mosaic virus and Phytophthora parasitica var. nicotianae [126], sug- in the plant illumination regime, indicating putative allocation to gesting a function for DGK in lipid signalling pathways. control of photosynthetic membranes. Conversely, NPC2 did not Putative inclusion of plant NPC and other DAG production/con- significantly react to abiotic stress, while expression of NPC6 was version enzymes in defence signalling to elicited cells and probable inhibited during osmotic and cold stress conditions (Arabidopsis roles in other signalling pathways have been postulated. Moreover, eFP Browser, http://bar.utoronto.ca; Genevestigator, http:// it is becoming evident that NPC falls into a dualistic pattern www.genevestigator.com). common for other stress-responsive elements, demonstrating 72 I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 repression or activation depending on the nature of the stimulus Under specific conditions, NPC4 expression was also positively and its spatial and temporal characteristics. responsive to ABA, MeJA, BL or SA treatment. NPC1 and NPC6 were also activated under the influence of zeatin. 4.5.4. Role in hormonal regulation Growing evidence indicates a response from NPC to hormonal The functions of plant hormones are mediated by a number of treatment. Localisation of some NPC isoforms in the plasma mem- signalling agents including phospholipases C and D. At the same brane suggests a role for NPC in early stages of hormonal percep- time, the direct actions of the novel NPCs on hormonal regulation tion. In addition, expression patterns of NPC matching sites of remain unclear. NPC activity was rapidly induced in a BY-2 cell cul- intensive growth and hormone accumulation suggest that NPC ture upon treatment with 24-epibrassinolide (BL) in situ [22]. The participation in intracellular amplification of hormonal signals is authors also found that npc3 and npc4 knockout mutants of Arabid- essential both for normal growth and stress reactions. opsis exhibited impaired sensitivity to BL and altered expression of TCH4 and LRX2 BL-responsive genes, in addition to reduced pri- 4.6. Possible mode of action mary root length and lateral root number. Sensitivity to 1-NAA auxin was sustained in NPC-deficient plants, but expression of The regulatory role of NPC in plants is mediated via diacylglyc- IAA19 and IAA20 genes involved in auxin signalling was decreased erol production [128]. Diacylglycerol molecular species consist of in npc3. In addition, auxins, cytokinins and brassinosteroids af- two fatty acid moieties bonded to a glycerol by ester linkages to fected the expression level of NPCs as assessed by semiquantitative form highly non-polar acylglycerols conferring distinct properties PCR [22]. The NPC4 transcript level increased after 3 h of zeatin as lipid intermediates, membrane components and second mes- treatment and 24 h of brassinolide treatment. However, only min- sengers. Impaired DAG generation or turnover has severe effects or changes in the expression levels of the other NPC genes were ob- on development and growth in most living organisms [129–133]. served [22].The expression of PNPC3:GUS and PNPC4:GUS constructs In recent years, great progress in the understanding of DAG metab- after treatment with 1-NAA or brassinolide was intensified in root olism in human cells has been made at the molecular level. Dereg- tips, cotyledons, leaf margins and floral organs, similar to the ulation of DAG metabolism has been linked to the pathophysiology expression of the synthetic DR5:GUS auxin reporter [127], which of several human diseases including cancer, diabetes, immune sys- suggests a functional connection of auxins, brassinosteroids and tem disorders and Alzheimer’s disease, as well as the disruption of NPC that is important for root cell division and expansion. The organ development and cell growth [62]. It is currently thought expression pattern of the PNPC2:GUS construct also correlated with that role of DAG in plant cells is restrained to participation in lipid auxin-rich root zones. However, an npc2 mutant was not aberrant turnover and endomembrane remodelling. However, emerging re- in root formation density and length when treated with IAA [96]. search data indicate a complementary role for DAG in cell regula- A role for NPC in ABA-mediated responses in Arabidopsis has tion and signalling. also been disclosed. It was shown that expression of NPC4 was in- duced by ABA treatment, but not by salicylic acid (SA) or MeJA 4.6.1. Lipid metabolism treatment [95]. More importantly, npc4 mutant plants that accu- In plants, NPC is responsible for the production of both DAG and mulated higher levels of ABA in seeds were less sensitive to ABA phosphocholine that enter cell metabolic pathways. While the treatment, resulting in abnormal germination, growth and stomata known metabolic role of phosphocholine is rather trivial in plants, movement [25]. Expression levels of ABA-responsive genes (ABI1, DAG is known as a key intermediate in generic lipid metabolism ABI2, RAB18, PP2CA, OST1, RD29B, ERA1 and SOT12) were also signif- (Fig. 7). In plant cells, DAG can arise either metabolically from glyc- icantly altered in npc4 mutants under ABA treatment and under erol-3-phosphate or through membrane polar lipid cleavage by li- salt stress treatment that concurrently evoke ABA signalling pase enzymes [62]. Due to its hydrophobicity, transiently [24,25]. NPC4-overexpressing Arabidopsis plants demonstrated in- generated DAG typically remains bound to cellular membranes creased sensitivity to ABA inhibition of seed germination, accom- where numerous supplementary enzymatic systems are employed panied by induced salt and osmotic stress tolerance, giving to precisely control its turnover [61]. Depending on its origin and strong evidence for a role for NPC in ABA-mediated plant stress re- cellular localisation, the fatty acid composition of DAG may vary sponses. Interestingly, npc4 mutants accumulated lower DAG lev- and form distinct C18/C16 and C18/C18 molecular species. DAG els in leaves under normal growth conditions and under ABA- may give rise to diverse lipid species that, in turn, are of crucial treatment, although the composition of DAG molecular species, importance for orchestration of cell metabolism and physiological membrane phospholipids and galactolipids was unchanged. The reactions. In plants, DAG predominantly functions as a precursor of phenotype of ABA-insensitive npc4 mutants was restored by appli- glycerolipid metabolism. However, diverse cellular DAG pools also cation of both synthetic DAG and PA. Moreover, when DAG was ap- participate in the synthesis of membrane glycolipids, sulpholipids plied with diacylglycerol kinase inhibitor 1 in the presence of ABA, and other lipid types, as well as in biosynthesis of storage triacyl- DAG was no longer able to fully restore the phenotype. This evi- glycerols. In the ER, DAG acts as a substrate for both PC and PE syn- dence suggests that NPC-derived DAG production influences ABA thesis; while in the plastid envelope, DAG is converted into signalling and is also important in DAG/PA conversion. Based on monogalactosyldiacylglycerol (MGDG) and sulpholipids (for re- these results [25], the authors proposed a working model for the view see [134,135]). DAG can also be converted in two steps to function of NPC4 and derived DAG in mediating the response of cytidinediphosphate-diacylglycerol, which is important for photo- Arabidopsis to ABA. Under normal conditions, NPC4 contributes to synthetic processes and biosynthesis of phosphatidylinositol, the basal production of DAG that promotes stomatal opening. Un- phosphatidylglycerol and cardiolipin [136]. In the capacity of der stress conditions, ABA levels increase and consequently induce DAG-derived products, galactolipids are essential for maintaining NPC4 to produce DAG. Stress-induced DAG is phosphorylated by plastid thylakoid membrane composition and the direction of pho- DGK to PA, which promotes ABA-promoted stomatal closure. tosynthesis [137], while sulphoquinovosyldiacylglycerol (SQDG) DNA microarray data (Arabidopsis eFP Browser http://bar.uto- sulpholipids are specifically implicated in photosystem II regula- ronto.ca; Genevestigator www.genevestigator.com) indicate that tion and stabilisation [138]. MGDG also appears to be important brassinolide evokes inhibition of NPC1 and NPC4 expression during pollen germination and pollen tube growth, as well as un- accompanied by activation of NPC6. ABA treatment resulted in a der phosphate-limiting conditions [139]. somewhat reversed pattern, with down-regulation of NPC6 expres- There is direct evidence that NPC affects DAG production and sion and up-regulation of NPC3/NPC4 expression in Arabidopsis. the balance of other lipids in plants. Specifically, npc4 mutants I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 73

Fig. 7. Possible mode of NPC action in plant cells. Several putative regulatory pathways are shown for plant NPCs, including cell signalling with impacts on lipid metabolism, cell membranes and membrane-associated processes. NPC can enzymatically give rise to both DAG and PChol. The cellular role of the latter has been called into question. By contrast, there are a number of potential functions in plant cells ascribed to DAG. DAG can affect cell signalling either by direct binding to proteins/PK or via conversion to PA. DAG as a substrate can provide means for the biosynthesis of various lipid species, including those required for stress adaptation. Finally, as a highly hydrophobic molecule, DAG can significantly affect properties of cell membranes as sites of crucial cell activity. Non-enzymatic functions of NPC are also considered and include putative protein– protein interactions that may affect the activity of either target or interaction proteins. DAG, diacylglycerol; DGK, diacylglycerol kinase; NPC, non-specific phospholipase C; PA, phosphatidic acid; PC, phosphatidylcholine; Pchol, phosphocholine; PK, protein kinase. accumulate less DAG in leaves [25]. The production of DAG was been identified in plant cells [147]. Munnik and Meijer [148,149] also reduced in npc4 mutants during phosphate-limiting condi- presented evidence that DAG, a product of PIP2 hydrolysis, is rap- tions in Arabidopsis [19], while such plants accumulated fewer idly phosphorylated to PA, which plays an active role in plant MGDG and DGDG glycolipids and marginally more SQDG. How- signalling processes. The question remains as to how important ever, the overall profile of DAG molecular species and the content the contribution of NPC is to the formation of these signalling of other lipids did not significantly change in npc4 under normal molecules alongside metabolic DAG biosynthesis and DAG growth conditions [25]. In vitro activity of NPC5 was considerably production via PI-PLC and PLD/PAP. It also appears that in some lower than for NPC4; however, NPC5 seems to be responsible for plant systems, DAG is likely to act as a signalling molecule per se. mainstream production of DGDG in leaves during phosphate depri- Thus, synthetic DAGs were shown to elicit ion flux in protoplasts vation [82] (for details see section 4.5.1). Galactolipid content and of patch-clamped guard cells of Vicia faba and promote the opening levels of main membrane phospholipids were unchanged in npc5, of intact stomata of Commelina communis, which may again point although some changes were observed in the fatty acid composi- to their possible recruitment to ABA signalling [150]. Additionally, tion of galactolipids [82]. Atlpp2–2 knockout mutants of Arabidopsis with depressed lipid Interestingly, some DAG-derived lipid species appear to be phosphate phosphatase activity, responsible for DAG production implicated in plant hormone perception and stress tolerance. Thus, via PA dephosphorylation, demonstrated impaired ABA sensitivity Arabidopsis mutants deficient in DGDG synthase that utilises DAG accompanied by excess PA accumulation [151]. However, the as a substrate showed impaired thermotolerance [140], Arabidopsis activity of DGK, which is responsible for reverse DAG?PA conver- plants lacking diacylglycerol acyltransferase showed increased sion, was also reported to be elevated under ABA treatment in the sensitivity to ABA and osmotic stress during germination [141]. same study, suggesting an interconnection of lipid messengers. DAG-derived SQDG lipids appear to be important for plant salt DAG was also able to mediate membrane polarisation via endo- stress acclimation [142]. DAG can also be converted to PA by cytic recycling required for tobacco pollen tube polar growth DGK and participate in jasmonic acid biosynthesis via activ- [152] and is implicated in the perception of light irradiation that ity [143]. is important for circadian clock functioning and leaflet movements From the other hand, phosphocholine produced by NPC may of Samanea saman plants [153]. Indirect evidence exists for the give rise to free choline and contribute to biosynthesis of the osmo- involvement of DAG species in the regulation of cell-cycle progres- protectants glycine betaine [144] and choline-O-sulphate [145]. sion in stamen hair cells of Tradescantia virginiana [154]. Such data indicate that DAG may act as a signalling molecule in plant cells, 4.6.2. Signal transduction but the molecular nature of such signalling cascades awaits The signalling role of DAG is not obvious in plants and is now elucidation. It is important to mention that PI-PLC-derived DAG being discussed [146]. So far, no DAG-responsive proteins have and NPC-derived DAG are likely to differ in fatty acid composition 74 I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 and may have different roles in plant metabolism regulation ing resemblance to animal PKC in their structural or biochemical [155]. properties. Thus, cloned plant transcripts of bean PVPK-1- and rice of phorbol—plant-derived tetracyclic diterpenoid com- G11A-encoding protein kinases were characterised by their rela- pounds—have long been known as DAG-mimicking molecules that, tionship to the catalytic domain of PKC but differed in the structure in animals, are able to excite distinct intracellular responses [156]. of their regulatory domains [171], and the phospholipid-activated More than 30 different mammalian proteins, including protein ki- protein kinase of Amaranthus tricolor cross-reacted with antiserum nase C [157] and D [158], have been shown to bind both DAG and raised against the regulatory sub-unit of PKC from bovine brain phorbol esters. Both have also shown functional similarity in regu- [172,173]. Similarly, PKC-like enzymes from Marchantia polymor- lation of cell growth and differentiation, tumour progression and pha thalli interacted with PKC-specific fluorescent tags and reacted other processes [159]. In plants, the commonly used DAG substi- to spermine treatment during programmed cell death [174]. tute phorbol 12-myristate 13-acetate (PMA) was involved in The activity of PKC-type enzymes involved in nitrate reductase ROS-dependent regulation of tobacco defence hsr203J defence gene gene expression in maize was regulated by Ca2+ and PMA, phos- expression [160], intensified 6-methoxymellein phytoalexin pro- pholipids or synthetic analogues of diacylglycerol [175]. Earlier, duction in carrot cells [118] and had a flowering-promoting effect the same authors showed that PKC-like kinase activity in maize in Lemna aequinoctialis and Lemna gibba plants [161]. Additionally, was stimulated by PMA in the presence of PS and calcium and PMA elicited transient activation of a 45-kDa protein with proper- was precipitated by animal PKC antibodies [176]. Purified protein ties of wound-induced MAP kinase in tobacco cell suspension cul- kinase from Brassica campestris showed typical characteristics of tures [162], and both PMA and DAG in suspended Rubus cells conventional PKC while responding to Ca2+, diacylglycerol, phos- affected the activity of laminarinase, an enzyme that can lyse fun- pholipids or phorbol esters [177], and PMA treatment stimulated gal cells and induce oligosaccharide production important for sig- PKC-like protein kinase in Brassica juncea [178]. The latter enzyme nalling in pathogenic invasion [163]. PMA also induced was purified to homogeneity and characterised as calcium-depen- benzophenanthridine alkaloid accumulation in cell suspension cul- dent and oleylacetylglycerol- and PMA-activated. Calcium-depen- tures of Sanguinaria canadensis [164] and mediated the hypersensi- dent protein kinase from rice membranes was able to tive response in lemon seedlings by inducing phenylalanine phosphorylate MARCKS (myristoylated alanine-rich C-kinase sub- ammonia- activity and synthesis of scoparone [165]. Sato strate) peptide, a highly specific substrate for animal PKC [179]. et al. [166] showed that the A. thaliana K+ KAT1 channel implicated In wheat cells, protein kinase activity was stimulated by concur- in stomata functioning was inhibited by PMA when expressed in rent treatment with phospholipids and PMA/synthetic DAGs frog oocytes. Thus, PMA/DAG is likely involved in biotic/abiotic [180]. In this regard, attention is drawn towards the disclosure of stress responses either directly or through the activation of plant a possible role of plant PKC-like proteins in cell regulation. A func- protein kinases or PKC-analogous proteins. tional homolog of mammalian PKC was shown to mediate defence Indeed, PKC is the principal target of DAG regulation in animal responses in the elicitor-induced potato plants [181]. Pea DNA cells [167]. PKC enzymes belong to a ubiquitous serine/threonine topoisomerase I was activated after phosphorylation either by ani- kinase family, which are strongly activated by DAG molecules ow- mal-derived PKC or by endogenous plant PKC-like phorbol-ester- ing to the presence of C1 DAG/phospholipid-binding sites in the N- binding enzyme localised in nuclei [182]. Moreover, treatment terminal regulatory domain. with animal PKC in vitro promoted the activity of stress-inducible There is no direct evidence for the existence of plant protein ki- pea DNA helicase 47 [183]. Treatment with the NPC-15437 specific nases that incorporate C1 domains. However, putative DAG-bind- inhibitor of animal PKC repressed calcium-dependent intracellular ing sites rich in cysteines and histidines were predicted in responses induced by ergosterol and cryptogein elicitors in tobacco numerous A. thaliana proteins, including 104 (Protein kinase C-like, suspension cells [184]. PKC-like enzyme, alongside PI-PLC, was also phorbol ester/diacylglycerol binding domain - IPR002219), 224 implicated in anthraquinone formation in Rubia tinctorum under (C1-like domain - IPR011424), and 208 (DC1 – plant-specific chitosan treatment, while PMA alone was capable of mimicking ‘‘divergent C1’’ domain - IPR004146) (InterPro database, http:// the effects of the chitosan elicitor [185]. www.ebi.ac.uk/, Pokotylo, unpublished results). Among Independently, the regulatory role of NPC-derived DAG can be them, DGKs and proteins containing zinc-finger and PHD-finger mediated through conversion to PA via the action of DGK. PA is cur- (Plant Homeo Domain) motifs were found, suggesting putative reg- rently known as an irreplaceable signalling agent in all living organ- ulatory functions via binding to DNA, RNA, or other proteins. isms, while the number of PA-binding regulatory proteins/protein Intriguingly, C1-like domain-containing histone-lysine N-methyl- kinases are crucially involved in the regulation of plant metabolism ATX1 of Arabidopsis functions both in ABA–dependent (for review see [128]). In turn, DGKs belong to a conserved lipid ki- and ABA–independent osmotic stress response [168]. However, nase family responsible for ATP-mediated DAG phosphorylation the majority of the putative DAG-binding proteins remain [155]. DGKs are ubiquitously found in eukaryotes, where the role uncharacterised. of DGK is attributed to attenuation of DAG levels and potentiation In addition to Arabidopsis, DAG-binding domains were also of PA production, thus interconnecting lipid metabolism with sig- identified in stress-responsive NtDC1A and NtDC1B proteins from nalling [186]. DGKs identified in plant systems are thought to local- tobacco [169] and TaCHP protein from wheat [170]. DAG-binding ise to cell membranes [187] and are generally classified into three sites were found in, for example, nucleotide sequences of histone phylogenetic groups based on the presence of functional domains methyltransferases from barley and P. patens and in putative [155,188]. Interestingly, alternatively spliced variants of tomato nucleoredoxin from Ricinus communis (European Nucleotide Ar- LeCBDGK genes revealed a calmodulin-binding DGK [189]. How- chive, http://www.ebi.ac.uk/ena, Pokotylo, unpublished results), ever, the general functions and properties of intracellular regulation giving evidence for a role in gene regulation and signalling. of plant DGKs remain largely unclear. In animals, PIP2 is considered A multiplicity of functions has been ascribed to DAG-binding to be a precursor of DAG production involved in DGK-assisted con- PKC in animal cells. It is established that PKCs are implicated in version and activation of PKC enzymes that has so far been elusive intracellular signalling and regulation of immune responses, recep- in plants. However, PIP2 levels in plants are as much as 100-fold tor desensitisation, and transcription, as well as cell proliferation lower than in mammalian cells [190]. Thus, DAG molecules in- and differentiation (for review see [71,167]). At the same time, no volved in DGK-assisted turnover may arise from other sources, PKC sequence homologues were detected in Arabidopsis or other including NPC-mediated PC hydrolysis and phosphatidylinositol plants, although a number of identified plant kinases exhibit a strik- 4-phosphate cleavage by PI-PLC. I. Pokotylo et al. / Progress in Lipid Research 52 (2013) 62–79 75

It is now established that in plants DGKs may provide intracellu- bacterial PC-PLC facilitates fusion of PC/PS/ containing lar amplification of PA signalling, either supplementing or substi- vesicles [205], while 1,2-isomers of diacylglycerol, but not 1,3-iso- tuting PA molecular species generated by PLD. The importance of mers, increase calcium-induced fusion of PS vesicles in vitro [206], DGK-mediated conversion of DAG to PA in ABA signalling was implicating NPC in various cell physiological activities including clearly shown [25]. DGK were shown to be responsible for PA pro- exocytosis, endocytosis, membrane build-up and cell division. duction in cold-stressed Arabidopsis cells [191]. Moreover, some DAG produced by PI-PLC [152] and possibly by NPC [23] pre- data suggest that the PA pool originating from DAG functions with- sumably undergoes endocytic recycling in membranes important in the initial signalling phase of biphasic PA accumulation in stim- for tobacco pollen tube growth. The role of NPC and DAG in cell ulus-triggered cells [124]. Similarly to animal systems, where DGKs growth can be demonstrated in pollen tubes. Under physiological are often regarded as DAG signal modulators rather than as media- conditions, pollen tube growth requires the transport of large tors of PA signalling [192], DGK in plants may possess complemen- quantities of material for the rapidly emerging membrane, and tary intracellular functionality not attributed to PA formation while an accumulation of DAG was indeed observed [152]. By contrast, modulating DAG, thereby implicating it in both signalling and lipid in the presence of Al3+, DAG production was reduced and pollen biosynthesis. It was shown that overexpression of the rice DGK tube growth was inhibited [23]. gene OsBIDK1 activated under elicitor treatment enhanced disease In membranes, DAG can modify lipid-protein interactions and resistance in transgenic tobacco [126]. Treatment with R59022 alter exposure of surface membrane receptors [62]. Interestingly, DGK inhibitor increased phytoalexin accumulation induced by fun- it was recently reviewed that DAG can modify activity of the very gal elicitor in pea epicotyl tissues [193]. R59022 also reduced root PLC enzymes that grant its production [207]. Asymmetrical DAG elongation and depressed growth of Arabidopsis seedlings [133], distribution in the membrane may additionally facilitate formation suggesting a role for DGKs in both stress responses and develop- of membrane domains and functional lipid rafts [198]. mental processes in plants. Thus, while both DAG, originated from PC cleavage by NPC, and PA are considered to be important lipid-de- 5. Conclusions and perspective rived second messengers, DGKs appear to be among the main reg- ulatory elements that provide coordination of their functions. Here, we present collective evidence for plant NPC enzymes as Plant NPCs may also affect cell function via protein–protein novel agents involved in the control of cell physiological functions. interactions independently of their primary enzymatic activity. An NPC regulatory role implemented via DAG production affects NPC3 was shown to interact with PIN4 auxin efflux carrier protein metabolic regulation, biotic and abiotic stress responses and hor- (Interactomics Web, http://interactomics2.stanford.edu) which is mone sensing. The available data provide support for including involved in auxin transport to developing roots [194], although NPC gene family as key agents involved in cell signalling alongside the significance of this interaction remains to be elucidated. other plant phospholipase enzymes. Despite the significance of The particular signalling role of phosphocholine produced DGK in cell signalling, the regulatory role of DAG produced by either metabolically or via PC cleavage is currently neglected in NPC may also be uncoupled from DGK and be accomplished via plants. Nevertheless, the phosphocholine moiety has been shown other putative DAG responsive elements in plant cells. However, to be important in the excitation of defence responses in cultured further studies are required for elucidation of the molecular prop- rice cells by glycosyl inositol-phosphoceramides from the phyto- erties of NPC action, the implication of NPC in as yet unexplored pathogenic fungus Trichoderma viride [195]. Choline kinases have growth and developmental processes, and the putative interaction also been significantly activated in salt-stressed [196] and heat- of NPC with other signalling systems in plant cells. stressed Arabidopsis [197]. Much progress has been made in the general understanding of NPC functioning. A wealth of information on the expression of 4.6.3. Membrane remodelling the NPC gene family in different plant species is now available, DAG species are principal membrane constituents that have a but much remains to be elucidated about the nature of the ex- role in membrane biogenesis, remodelling and behaviour. Cur- tended role of NPC in cell regulation and signalling. There is also rently, DAG is known to have a major function as a multivalent a large void in the understanding of the molecular properties of modulator of various membrane-related cellular activities such NPC action. These and similar questions will most likely be an- as vesicular transport, endocytosis, secretion, and others swered using novel experimental approaches, including advanced [61,62,135,198,199]. genetic manipulations and biophysical methods for studying mem- Plants are typically characterised by somewhat increased mem- brane-associated processes. brane DAG content compared with that in animals, although its We should also mention a possible future use for NPC as a target overall level remains low in the membranes. Depending on the of genetically altered plants resistant to disease or stress. Initial stimulus, the membranes of animal cells contain approximately evidence has been already demonstrated for the role of NPC in 0.2 to 1.4 nmol of DAG per 100 nmol of phospholipids (PL) [200– resistance to salt stress. 202]. DAG content measured in the alga Dunaliella salina was 4.2 nM per 100 nM PL [106] and in the tree Samanea saman varied Acknowledgements from 3.5 nM/100 nM PL in unstimulated tissues to 4.2 nM/ 100 nM PL following exposure to light [153], suggesting relatively This work was supported by Grants No. P501/12/1942 and high importance for membrane stabilisation in plants. P501/12/P950 from the Czech Science Foundation, Grant No. In animals, DAG levels directly affect cytoskeletal reorganisation, ME09108 of the Czech Ministry of Education, Youth and Sports, membrane trafficking and exocytosis [147]. Local DAG accumula- Grant No. IAA601110916 from the Grant Agency of the Academy tion can perturb phospholipid planar bilayers, promote the forma- of Sciences of the Czech Republic and Grants No. F40.4/081 and tion of non-lamellar membrane phases and influence membrane F41.4/041 from the State Fund for Fundamental Researches of curvature [61,62]. Szule et al. 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