Phospholipases in action during plant defense signaling

Abstract

Eukaryotic organisms rely on intricate signaling networks to connect recognition of microbes with the activation of efficient defense reactions. Accumulating evidence indicates that phospholipids are more than mere structural components of biological membranes. Indeed, phospholipid-based signal transduction is widely used in plant cells to relay perception of extracellular signals. Upon perception of the invading microbe, several phospholipid hydrolyzing enzymes are activated that contribute to the establishment of an appropriate defense response. Activation of phospholipases is at the origin of the production of important defense signaling molecules, such as oxylipins and jasmonates, as well as the potent second messenger phosphatidic acid (PA), which has been shown to modulate the activity of a variety of proteins involved in defense signaling. Here, we provide an overview of recent reports describing the different plant phospholipase pathways that are activated during the establishment of plant defense reactions in response to pathogen attack.

In plant cells, perception of pathogenic microbes largely relies on transmembrane pattern recognition receptors that specifically recognize highly conserved pathogen-derived molecules called PAMPs/MAMPs (pathogen-/microbial-associated molecular patterns), such as bacterial flagellin.¹ PAMP recognition by the plant leads to basal defense responses. A second layer of defense is based on the recognition of specific pathogen-derived molecules, called effectors, primarily by an additional class of plant cytoplasmic receptor proteins [nucleotide-binding leucine-rich repeat (NB-LRR) proteins] but also by protein receptors predicted to be located at the plasma membrane [receptor-like proteins (RLPs) and receptor-like kinases (RLKs)]. This recognition leads to the activation of plant immune responses that are frequently associated to the development of hypersensitive cell death (HR) at the inoculation site, which has been shown to contribute to plant resistance.²

The activation of plant immunity involves a variety of early signaling events, including rapid accumulation of reactive oxygen species (ROS), changes in cellular ion fluxes, activation of protein kinase cascades, changes in gene expression and production of stress-related hormones.^3,4 During recent years, a substantial number of reports have also shown the importance of lipids and lipid-related molecules, including glycerolipids, sphingolipids, fatty acids, oxylipins, jasmonates and sterols, in the regulation of plant defense responses.⁵

Phospholipids are more than structural components in biological membranes. Indeed, evidence that phospholipases and phospholipid-derived molecules are involved in plant signaling, and more particularly in plant immunity, is rapidly accumulating.^6,7 In plants, phosphatidic acid (PA) can be produced from phospholipids by phospholipase D (PLD) enzymes or from diacylyglycerol (DAG) by DAG kinases (DGKs) in the phospholipase C (PLC) pathway. PA is a potent secondary signal messenger molecule that modulates the activity of kinases, phosphatases, phospholipases and proteins involved in membrane-trafficking, Ca²⁺ signaling and the oxidative burst.^8,9 In addition, a growing body of evidence indicates that phospholipase A (PLA) [and related molecules such as lysophospholipids (LPLs) and free fatty acids (FFAs)] and phospholipase C (PLC) (and its related molecules DAG and DGK) play important roles in the control of the plant defense response to the attack by invading pathogens.⁷

Here, we review the recent advances in understanding phospholipase-mediated signaling and its importance in the control of plant immune responses.

Phospholipase A

Members of the phospholipase A (PLA) superfamily catalyze the hydrolysis of membrane phospholipids at the sn-1 (PLA₁) or sn-2 position (PLA₂) of the glycerol backbone to produce free fatty acids (FFAs) and lysophospholipids (LPLs) (Fig. 1). In contrast to the case of animal and yeast cells, the major components of plant cell membranes are not phospholipids but galactolipids. In agreement with this observation, plants encode enzymes that are able to hydrolyze galactolipids. However, most of these enzymes, which are called LAHs (Lipid Acyl Hydrolases), are also able to act on phospholipids, but not on triacylglycerol (TAG). In Arabidopsis, PLAs can be classified in three main groups: (1) the patatin-like proteins, which present non-specific LAH activity and are able to hydrolyze both at sn-1 and sn-2 positions of phospholipids and galactolipids, (2) the DAD (Defective in Anther Dehiscence)-like proteins with LAH and TAG lipase activity and (3) the secretory PLA₂s that specifically hydrolyze phospholipids at their sn-2 position. For most plant PLAs, substrate specificities and cleavage positions are still poorly characterized.

Figure 1.

Schematic representation of phospholipid structure and the sites of cleavage by phospholipases. (A) General structure of a phospholipid, with two fatty acyl chains linked to a glycerol backbone, at the sn-1 and sn-2 positions, and a phosphate group creating the «phosphatidyl» moiety to which a variable head group is attached. The sites of phospholipase activity are indicated by arrows. Lipid acyl hydrolases (LAHs) are able to hydrolyze phospholipids at sn-1 and sn-2 positions. (B) Possible head groups and resulting phospholipids are indicated: PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D; Fatty Acid 1, fatty acid in sn-1 position; Fatty Acid 2, fatty acid in sn-2 position; P, phosphate group.

In Arabidopsis, protein complexes containing EDS1 (Enhanced Disease Susceptibility1), PAD4 (Phytoalexin Deficient4) and SAG101 (Senescence-Associated Gene101) constitute an essential regulatory node that mediates both effector-triggered activation of several TIR-NB-LRR proteins and basal defense responses.¹⁰ Interestingly, EDS1, PAD4 and, less convincingly, SAG101 have homology to LAHs. However, no enzymatic activity has been reported to date for any of these proteins and it has been thus suggested that, as described for other signaling proteins, they may play a structural rather than an enzymatic role during plant immunity.¹⁰

LAHs have been mainly linked to plant immunity through their role in oxylipin and jasmonic acid (JA) biosynthesis, molecules with well-studied effects on plant defense responses.^11,12 Oxylipins and jasmonates are synthesized from FFAs liberated from membrane phospholipids or galactolipids by deacylating enzymes, and oxidized by a lipoxygenase (LOX) or α-dioxygenase (α-DOX).^11,13 Besides their role in oxylipin or JA biosynthesis, induction of PLA₂ activity during pathogen defense has also been correlated with ROS production.¹⁴ In addition, in elicitor-treated cells of Californian poppy, lysophosphatidylcholine (LPC), which is transiently generated by PLA₂, was proposed to activate a vacuolar H⁺/Na⁺ antiporter to decrease the cytosolic pH and activate phytoalexin biosynthesis.^15,16

Patatins.

Patatins were first described as vacuolar storage proteins in potato tubers.¹⁷ Patatin-like proteins (pPLAs) are present in many plant species and have been implicated in plant defense signaling through JA or oxylipin accumulation. For example, Arabidopsis plants mutated in pPLAI, encoding a protein with a patatin-type catalytic domain, showed increased resistance to inoculation by the necrotrophic pathogen Botrytis cinerea. These plants also displayed reduced basal levels of JA, but the pathogen-induced production of JA was not modified.¹⁸ How the decrease in JA basal levels is linked to enhanced resistance to Botrytis is still unclear.

The expression of an additional member of the Arabidopsis pPLA family, pPLA-IIα/PLP2, is induced after infection with B. cinerea or Pseudomonas syringae pv. tomato.¹⁹ Altered expression of PLP2 in Arabidopsis modifies plant susceptibility to Botrytis and Pseudomonas and to treatment with paraquat, a cell death-promoting molecule. Indeed, PLP2 expression favored the development of necrotizing pathogens, and increased resistance to the obligate pathogen cucumber mosaic virus.^19,20 In response to Botrytis infection, PLP2 expression induces late accumulation of oxylipins through the α-DOX pathway,²⁰ which are thought to limit HR spreading. PLP2 would thus participate to the execution of plant cell death through non-specific hydrolysis of membrane lipids followed by cell death spreading controlled by oxylipins. In this scenario, PLP2 activity may be exploited by pathogens with different lifestyles to facilitate host colonization.

The expression of three tobacco patatin-like proteins (NtPAT1–3) was rapidly induced, preceding JA accumulation, during the HR triggered in response to tobacco mosaic virus (TMV).²¹ These enzymes display PLA₂ activity and may contribute to FFA production for JA biosynthesis. In the elicitor-infiltrated zone, NtPAT PLA₂ activity was rapidly induced before JA accumulation and cell death appearance.²² Activation of NtPAT1–3 and 9-LOX was detected in cryptogein-elicited tobacco leaves²³ and a patatin-like and a 9-LOX protein were co-expressed in cotton leaves following infection with Xanthomonas campestris.²⁴ Taken together, these results suggest that patatins may contribute to oxylipin biosynthesis though their metabolic association with LOXs (Fig. 2).

Figure 2.

Model of phospholipase signaling in plant defense responses. Pathogen recognition induces the activation of phospholipase-mediated signaling pathways. Phospholipase A (PLA) hydrolyzes membrane phospholipids (PL) to produce free fatty acids (FFA) and lysophospholipids (LPL). PLAs are classified in three main groups: (1) the patatin-like proteins, responsible for the production of oxilipins and jasmonates, (2) the DAD (Defective in Anther Dehiscence)-like proteins with, to date, no well-established role in plant immunity and (3) the secretory PLA₂s. Phospholipids from the plasma membrane, as well as from various intracellular membranes (mitochondria, Golgi, chloroplast…), are potential substrates for PLA activity. EDS1, PAD4 and SAG101 are essential immunity components that shuttle across the nuclear envelope to regulate defense responses. Despite their sequence homology to LAHs, the fact that no enzymatic activity has been demonstrated to date for these proteins suggests that their LAH domain may play a structural role rather than an enzymatic role. Similarly, a non-enzymatic function has been shown for AtsPLA₂-α, a negative regulator of plant defense that is translocated into the nucleus in the presence of the transcription factor AtMYB30. Phospholipase C (PLC) is a plasma membrane protein that hydrolyzes phosphatidylinositol (PI), phosphatidylinositol bisphosphate (PIP₂) or phosphatidylinositol phosphate (PI₄P) to produce inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃, which may be converted to IP₆, diffuses in the cytosol where it releases Ca²⁺ from internal stores whereas DAG is rapidly converted to phosphatidic acid (PA) through the action of diacylglycerol kinase (DGK). Increased PA levels modulate additional signaling components, which are indicated in the figure. PA signaling may be attenuated via its phosphorylation to diacylglycerol pyrophosphate (DGPP) by a PA kinase (PAK). Finally, PLD also generates PA through the hydrolysis of structural phospholipids, such as phosphatidylcholine (PC) or phosphatidyl-ethanolamine (PE). PLDs present different subcellular localizations and may thus use phospholipids from the plasma membrane or from intracellular membranes.

DAD-like proteins.

DAD1 encodes a chloroplastic PLA₁ that initiates JA synthesis in stamens after release of free linolenic acid from membrane phospholipids. The dad1 mutant is thus impaired in pollen development and anther dehiscence.²⁵ No clear evidence that DAD1-like proteins play a role in the regulation of plant immunity has been reported to date, although a recent report indicates that expression of the DAD-like LAHs PLA₁-Iγ1 and PLA₁-Iγ2 was induced after infection with B. cinerea and P. syringae, while PLA₁-III was only induced after treatment with Botrytis.²⁶ However, following Botrytis or Pseudomonas infection, knock-out lines in these three DAD1-like genes exhibited similar resistance level and JA profiles, compared to wild-type plants.

Secretory PLA2s.

The Arabidopsis secretory PLA₂ family (AtsPLA₂) consists of four isoforms denoted AtsPLA₂-α, -β, -γ and -δ.²⁷ Although animal sPLA₂s have been previously implicated in host defense,²⁸ evidence of the role of sPLA₂s in plant immunity is more limited. AtsPLA₂-α, which controls auxin transport protein trafficking to the plasma membrane,²⁹ has been recently involved in non-enzymatic control of plant defense. Consistent with the presence of a signal peptide in its N-terminus, AtsPLA₂-α was localized intracellularly in Golgi-associated vesicles and later secreted to the extracellular space.^29,30 However, in the presence of the transcription factor AtMYB30, a positive regulator of Arabidopsis defense reactions, AtsPLA₂-α is partially relocalized to the nucleus, where both proteins interact. This protein interaction leads to repression of the AtMYB30-mediated transcriptional activity and negative regulation of plant HR and defense responses (Fig. 2).³⁰ The regulation of plant defense mediated by AtsPLA₂-α is independent of its enzymatic activity, suggesting that PLA functions may extend beyond their lipid hydrolyzing activity, as previously described for several animal sPLAs.²⁸

Phospholipase D

Phospholipases D (PLDs) catalyze the hydrolysis of structural phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), to produce phosphatidic acid (PA) and the respective headgroup (Fig. 1). PA, which can also be produced via the PLC pathway (see below), has emerged as a crucial second messenger in the regulation of numerous cellular functions, including the signaling pathways associated to plant defense (Fig. 2).⁷ While humans present two PLD enzymes, and yeast only one, PLDs are overrepresented in plants. For example, Arabidopsis and rice encode 12 and 17 PLDs, respectively.^31,32 Plant PLDs have been classified in 6 classes (α, β, γ, δ, ε and ζ), depending on their sequence homology and in vitro enzymatic activity.³¹

PLD activity has been associated to a variety of stress responses in plants.⁶ Activation of PLD activity during plant defense was first described in rice, after infection by Xanthomonas oryzae.³³ Interestingly, PLD was recruited to the plasma membrane at the point where bacteria attack the cell. Elicitor treatment of tomato,^34,35 tobacco³⁶ or rice cells³⁷ were also found to induce PLD activity. In Arabidopsis, expression of the α, β and γ class of PLD genes is induced after infiltration by both virulent and avirulent strains of P. syringae.³⁸ After recognition of the P. syringae avirulence proteins AvrRpm1 and AvrRpt2 in Arabidopsis, a biphasic accumulation of PA was observed. The first wave of PA was attributed to the PLC/DGK pathway (see below) whereas a later and higher peak of PA was found to be due to PLD activity.³⁹

The effects of PLD activation during plant-pathogen interactions are varied. Indeed, PA has been shown to induce ROS production^40,41 and activate defense-related³⁷ or ethylene-responsive genes.⁴² PLDs also participate in salicylic acid-dependent signaling.⁴³ In contrast, LePLDβ1 is induced after elicitor treatment in tomato and is a negative regulator of ROS production.⁴⁴ Similarly, OsPLDβ1-knockdown plants spontaneously activate defense responses, suggesting that OsPLDβ1 would function as a negative regulator of disease resistance.⁴⁵ Several hypotheses have been formulated to explain the differential regulation of plant defense displayed by distinct PLD family members. PLDs may be localized in different subcellular compartments^46,47 and, depending on their subcellular localization, they may generate different molecular species of PA,⁴⁸ probably modifying the interactions between PA and its target proteins. In addition, PLDs display different biochemical properties with regard to their requirement for Ca²⁺ or substrate phospholipids, for instance.⁴⁹

An interesting link has been made in Arabidopsis between PLA₂ and PLD activities, during the regulation of plant defense. In Arabidopsis plants of the Pi-0 ecotype infected with P. syringae expressing the AvrBst effector, PA is produced through the PLD pathway, which is necessary for the establishment of resistance and HR.⁵⁰ This resistance phenotype displayed by Pi-0 plants is due to a loss of function mutation in SOBER1, a conserved α/β hydrolase with PLA₂ activity.⁵¹ Conversely, in the susceptible Col-0 Arabidopsis ecotype, SOBER1 PLA₂ activity would compete with PLD for substrate availability, thereby reducing PA accumulation in response to AvrBst elicitation. These findings underline the importance of PA in the regulation of plant defense signaling.

Phospholipase C

Plant PLC is a plasma membrane protein and its activity requires calcium. Depending on the calcium concentration, PLC is able to use different substrates. At low (micromolar) Ca²⁺ concentrations, both phosphatidylinositol-4-phosphate (PI₄P) and phosphatidylinositol4,5-bisphosphate (PIP₂) are hydrolyzed, whereas at higher (millimolar) Ca²⁺ concentrations, PLC preferentially uses phosphatidylinositol (PI). As PLC was first reported to hydrolyze PIP₂ both in vitro and in vivo, it was assumed that PLC uses predominantly PIP₂ as substrate. However, plant cell membranes have no or very little PIP₂. Conversely, there is plenty of PI₄P in the plasma membrane, with at least one additional pool occurring in the Golgi. Therefore, it has been proposed that PI₄P is very likely the in vivo substrate of PLC.⁶ Hydrolysis of PLC substrates leads to the production of inositol 1,4,5-trisphosphate (IP₃) and DAG (Figs. 1 and 2). While DAG remains in the plasma membrane, IP₃ diffuses into the cytosol, where it induces the release of Ca²⁺ from intracellular store(s), most probably from the vacuole.⁷ IP₃ may be rapidly converted into IP₆, which is also able to trigger an increase in cytosolic Ca²⁺ levels. In animal cells, DAG activates members of the protein kinase C (PKC) superfamily. However, in plants, which lack PKC, DAG is rapidly phosphorylated to PA through the action of the DGK (Fig. 2).^34,52 One of the main features of a second messenger signal is its transient nature. PA may thus be phosphorylated into diacylglycerol pyrophosphate (DGPP) by the PA kinase (PAK). Whether the formation of DGPP is just a mechanism for attenuating PA signaling or whether it also acts as a signaling molecule remains to be elucidated.⁶ Although eukaryotic PLCs have been classified in six subfamilies, all plant PLCs belong to the PLCζ class. The Arabidopsis genome contains nine PLCs, three of which have been characterized,^53–55 and seven DGK genes.

The PLC pathway responds to a variety of stress-induced signals from specific membrane receptors, which perceive signaling molecules such as hormones or pathogen elicitors. In particular, PAMP recognition triggers the activation of the PLC pathway, supporting the idea that PLC-mediated signaling is involved in basal defense responses.^34,56 Expression of the rice gene OsPLC1 was shown to be induced by diverse chemical and biological inducers of plant defense pathways and during the incompatible interaction between rice and the pathogen fungus Magnaporthe grisea, suggesting that OsPLC1 might be involved in the activation of the signaling pathway leading to disease resistance in rice.⁵⁷ As mentioned above, recognition of the Pseudomonas avirulence proteins AvrRpm1 or AvrRpt2 induced a biphasic accumulation of PA, with the first wave attributed to PLC/DGK activities.³⁹ In tomato (Solanum lycopersicum) cell cultures expressing the cognate Cf-4 resistance gene, the race-specific elicitor Avr4 from the pathogenic fungus Cladosporium fulvum rapidly induces accumulation of PA, via the sequential activity of PLC and DGK.⁵² Intriguingly, accumulation of PA is associated with ROS production, potentially via direct activation of an NADPH oxidase, contributing to the defense response.^52,58 More recently, the tomato PLC isoform, SlPLC4 was shown to be required for full Cf-4/Avr4 recognition.⁵⁹ Interestingly, an additional tomato PLC isoform, SlPLC6, is not involved in the establishment of the Cf-4/Avr4-mediated HR but rather in general plant defense responses against various pathogens. Therefore, a differential requirement of PLC isoforms in plant immune responses has been proposed.⁵⁹ In agreement with this hypothesis, Arabidopsis PLC proteins display differential organ expression and it has been reported that most of the PLC genes are induced in response to diverse environmental stimuli and, particularly, during plant defense responses.⁶⁰ Similarly, expression of DGKs is induced during different environmental stresses including microbial elicitation, suggesting that DGK, as a generator of PA, has an important role in plant basal resistance.^61,62

It is noteworthy that, beyond the previously described effect of PA in inducing ROS production,^40,41,52 PA also mediates the induction of additional pathogen-induced signaling components (Fig 2). For example, CTR1, a protein kinase known to repress plant ethylene responses triggered by biotic and abiotic stress stimuli, is negatively regulated by PA in vitro. Therefore, stress-induced PA formation might trigger downstream ethylene responses via its interaction with CTR1. Accumulation of stressinduced PA would thus represent an alternative way to induce ethylene-mediating resistance responses in the absence of ethylene.⁶³ Additionally, the Arabidopsis protein kinase AtPDK1 is activated through PA binding, triggering the subsequent activation of the kinase AtAGC2-1.⁶⁴ AtAGC2-1 is also known as OXI1, a kinase that mediates oxidative burst signaling and acts upstream of a MAP kinase cascade involved in basal resistance against Hyaloperonospora arabidopsidis.⁶⁵ This observation strongly suggests that PA may contribute to the activation of plant basal defense through MAP kinase signaling.

Conclusions and Perspectives

Significant advances have been made during the last decade in elucidating phospholipid-based signaling in plants. Molecular, biochemical and genetic studies have allowed uncovering the important role played by lipids in the intricate signaling network triggered in plant cells after pathogen recognition. More particularly, a growing body of data indicates that phospholipases and phospholipid-derived molecules play crucial roles in the modulation of plant defense responses through their action on the generation of ROS, activation of protein kinases, Ca²⁺ signaling, hormone production and defense-related gene activation, for example.

Despite the technical difficulties associated to measuring and manipulating transient second messenger signaling components, the role of some phospholipase-derived molecules in plant defense, in particular PA, is now well established. The reported identification of PA targets⁶⁶ should allow a better understanding of its function during plant-pathogen interactions. In addition, the identification of LAHs with non-enzymatic functions^10,30 and their importance for the regulation of plant defense are also intriguing findings that need to be better understood in the future.

What are the upstream signal(s) that trigger activation of phospholipases following pathogen recognition? How is phospholipase activity regulated? What are the cellular targets that are activated by phospholipases and phospholipid-derived molecules? What are the molecular mechanisms that allow integrating phospholipase activities in the complex signaling pathways that lead to the establishment of plant defense? Are pathogens able to manipulate host phospholipid signaling for their own benefit and, if so, what are the mechanisms involved? Providing answers to these and other questions remains an important challenge for future research.