Plant phospholipid signaling: “in a nutshell”

Abstract

Since the discovery of the phosphoinositide/phospholipase C (PI/PLC) system in animal systems, we know that phospholipids are much more then just structural components of biological membranes. In the beginning, this idea was fairly straightforward. Receptor stimulation activates PLC, which hydrolyses phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)P₂] into two second messengers: inositol 1,4,5-trisphosphate (InsP₃) and diacylglycerol (DG). While InsP₃ difuses into the cytosol and triggers the release of calcium from an internal store via ligand-gated calcium channels, DG remains in the membrane where it recruits and activates members of the PKC family. The increase in calcium, together with the change in phosphorylation status, (in)activates a variety of protein targets, leading to a massive reprogramming, allowing the cell to appropriately respond to the extracellular stimulus. Later, it became obvious that not just PLC, but a variety of other phospholipid-metabolizing enzymes were activated, including phospholipase A, phospholipase D, and PI 3-kinase. More recently, it has become apparent that PtdIns4P and PtdIns(4,5)P₂ are not just signal precursors but can also function as signaling molecules themselves. While plants contain most of the components described above, and evidence for their role in cell signaling is progressively increasing, major differences between plants and the mammalian paradigms exist. Below, these are described “in a nutshell.”

PLC SIGNALING

If we did not know about the existence of the PI/PLC system from animals and only had the data from plants today (1–5), then we would have never come up with a signaling system as depicted in Fig. 1. The reasons for this are briefly summarized below.

Fig. 1.

Plant PI/PLC signaling: Differences and similarities to the mammalian paradigm. Higher plants lack both InsP₃ receptor, a ligand-gated Ca²⁺ channel, and PKC; hence, these are striked-out (X). Instead, plants seem to use their phosphorylated products, InsP₆ and PA, as signaling molecules. PA can also be generated by PLD and is attenuated by PA kinase (PAK), a novel lipid kinase that is absent from mammalian cells. PAK generates diacylglycerolpyrophosphate (DGPP), which might function as a signaling molecule itself (19). Plant PLCs belong to the PLCζ subfamily. It is not known how they are regulated but not through heterotrimeric G-proteins (G), which is therefore striked out. Plant PtdIns(4,5)P₂ (PIP₂) quantities are extremely low, and plant PLCs lack the PH domain (Fig. 2). Instead, PtdIns4P (PIP) is a better candidate to be the in vivo PLC substrate. The resulting InsP₂ can be phosphoryated to InsP₆ via two dual-specificity inositolpolyphosphate kinases (IPK), while DG is phosphorylated to PA via DGK. Evidence is also emerging that PtdIns4P and PtdIns(4,5)P₂ themselves function as signaling molecules, involving membrane trafficking, organization of the cytoskeleton, and regulation of ion channels. In such a scenario, PLC would function as an attenuator of PIP and PIP₂ signaling. Solid arrows indicate metabolic conversions. Dashed arrows represent mechanisms of regulation.

No inositol 1,4,5-trisphosphate receptor nor PKC

Several plant genomes have been sequenced nowadays, including Arabidopsis, rice, and poplar, and many expressed sequence tag libraries of various higher plant species are available, but none of them seem to encode an InsP₃ receptor (6). An exception is Chlamydomonas, a unicellular green algae with two flagella, where an InsP₃ receptor has been identified. Other ciliated organisms, such as Paramecium, also contain an InsP₃ receptor. Apparently, higher plants have lost this in evolution (6). Similarly, the most important diacylglycerol (DG) target, PKC, is lacking from plant genomes, including lower plants. There are numerous articles describing an effect of “PKC-specific” inhibitors, but these most likely reflect protein kinases that are not present in mammalians. These include calcium/calmodulin-dependent protein kinase (CDPK), calcineurin B-like proteins that interact specifically with a group of CBL-interacting protein kinases (CIPK), and AGC kinases (with similarities to PKA, PKC, and PKG) (7). CDPKs and CIPKs respond to calcium, while some members of the CDPK and AGC family may be regulated by phosphoinositides and/or phosphatidic acid (PA; see below). Although we were hopeful before the genomic era (3), we have to accept now that plants lack PKC.

Low on PtdInsP₂

Labeling experiments using ³²P_i or ³H-Inositol, but also recent PtdInsP₂ biosensor (GFPPH_PLCδ1) studies, have shown that plant cells contain extremely low amounts of PtdInsP₂ (8–10). In contrast, PtdIns4P levels seem to be normal, i.e., similar to those observed in animals. Typical ³²P-ratios between PtdInsP and PtdInsP₂, measured in many different plant cells and tissues, reveal 30- to 100-fold lower PtdInsP₂ levels than PtdInsP. In animals and Chlamydomonas, the PtdInsP:PtdInsP₂ ratio is usually close to 1 (1–3).

Plant PLCs belong to the PLCζ class

Eukaryotic PI/PLCs have been classified into β, γ, δ, ɛ, η, and ζ subfamilies (Fig. 2). While mammalian cells contain all six isoforms (13 in total), plants exhibit only one class of PLCs. Originally, these were classified as PLCδ isoforms (4); however, after the recent discovery of the sperm-specific mammalian PLCζ, which lacks the typical Pleckstrin homology (PH) domain, it became clear that plant PLCs belong to the PLCζ class (Fig. 2) (12).

Fig. 2.

Domain structure and organization of PI/PLC isozymes. Plant PLCs belong to the most simple group, the PLCζs. PLCη undergoes alternative splicing, generating variable C termini with a PDZ binding motif being only present in the longer forms. Adapted from (11).

The ζ-isoform represents the most simple PI/PLC, consisting of the minimal core structure: the catalytic X- and Y-domain, an EF-hand domain, and a C2 lipid binding domain (Fig. 2). Additional subfamilies contain the PH domain and various other conserved sequence regions, allowing PLCβs to be regulated by trimeric G-proteins, PLCγs by tyrosine kinases, and PLCɛs by trimeric G-proteins and Ras. It is still not clear how PLCδ, -η, and -ζ isoforms are regulated, but this may involve calcium, especially for PLCη (Fig. 2) (11).

Plant PLC activity indeed requires calcium. At low micromolar Ca²⁺ concentrations, both PtdIns4P and PtdIns(4,5)P₂ are hydrolyzed and at millimolar concentrations, PLC also uses PtdIns as substrate (3). In vivo, PLC has always been assumed to hydrolyze PtdIns(4,5)P₂. However, plants have no, or very little, PtdInsP₂ in their membranes, and an InsP₃ receptor is lacking, so wouldn't it make more sense to propose PtdIns4P as the in vivo substrate? In vitro, PtdIns4P is equally well hydrolyzed as PtdIns(4,5)P₂, and in vivo, PtdIns4P turnover and quantities are much more in agreement with the PA responses resulting from DG phosphorylation than PtdIns(4,5)P₂. Moreover, plant PLCs lack the PH domain and are thus unlikely to find the few molecules of PtdIns(4,5)P₂. Recent studies using a PtdIns4P biosensor indicate that there is plenty of PtdIns4P in the plasma membrane, with at least one additional pool occurring at the Golgi (8).

InsP₆ rather than InsP₃

When microinjected, or released via photoactivation of a caged variant, InsP₃ was shown to release Ca²⁺ from an intracellular store in the early 90s. Obviously, this fitted the paradigm, so the plant PI system was a fact, even though Robin Irvine was still skeptical (13). It now seems he was right! Recent work from Brearley's lab indicates that the Ca²⁺ release is actually caused by InsP₆ (14). InsP₆ was shown to release Ca²⁺ at a 10-fold lower concentration than InsP₃, and when InsP₃ was microinjected, it was rapidly converted into InsP₆. Also, the hormonal stimulation via abscisic acid (ABA) (to which it was linked) was shown to generate an InsP₆ response rather than InsP₃ (14).

In yeast, InsP₆ is not related to Ca²⁺ signaling but directly regulates gene transcription and mRNA export from the nucleus. This pathway involves a PLC and two inositol polyphosphate multikinases, which can stepwise phosphorylate InsP₃ to InsP₆ (15). Could this reflect the pathway that is operational in plants too? PLCζ could hydrolyze PtdIns4P to produce Ins(1,4)P₂, which would then be sequentially phosphorylated by similar inositol dual-specificity polyphosphate multikinases (IPK). Two such Arabidopsis IPK genes, AtIPKβ1 and AtIPKβ2, have recently been identified (Table 1) (16).

TABLE 1.

Phospholipid signaling KO mutants in Arabidopsis

Class	Arabidopsis Gene	Enzymatic Activity (in Vitro)	Phenotype	Refs.
PLA	AtPLAI	Acylhydrolase (sn-1 and sn-2) preferring galactolipids over phospholipids	Less resistant to necrotrophic fungus Botrytis cinerea	(35)
	AtDAD1	sn1-acylhydrolase in JA pathway	Anther dehiscence, pollen maturation, and flower opening	(41)
PLC	AtPLC	PI/PLC	Lateral root growth	–a
PLD	AtPLDα1	Phospholipase D	Reduced ABA responses; enhanced seed quality	(55, 56)
	AtPLDα3	Phospholipase D	Reduced salt tolerance	(57)
	AtPLDδ	Phospholipase D	Increased sensitivity to oxidative stress; reduced freezing tolerance	(58, 59)
	AtPLDα1/PLDδ	Phospholipase D	Reduced salt and osmotic stress tolerance	(60)
	AtPLDζ2	Phospholipase D	Reduced auxin sensitivity; increased sensitivity to Pi starvation	(61, 62)
	AtPLDζ1/2	Phospholipase D	Increased sensitivity to Pi starvation	(63)
PI3K	AtVPS34	PI 3-kinase	Lethal; antisense plants are severely affected in growth and development	(43–45)
PI4K	AtPI4Kβ1	PI 4-kinase	AtPI4Kβ1/β2 double mutants display distorted root hair development	(24)
	AtPI4Kβ2	PI 4-kinase
PIP5K	AtPIP5K3	PI4P 5 kinase	Shorter root hairs	(22, 23)
3PTase	AtPTEN1	Dual specificity 3-PTase and Tyr phosphatase	Pollen development (RNA interference) (pollen cell death after mitogenesis)	(54)
4PTase	RHD4, AtSAC7	PtdIns4P 4-phosphatase	Bulging root hairs	(25)
5PTase	FRA7 AtSAC1	PtdIns(3,5)P2 5-phosphatase	Alterations in actin cytoskeleton organization and reduced cell wall thickening	(53)
	AtSAC9	Ins(1,4,5)P3- and PtdIns(4,5)P2 5-phosphatase	Reduced growth, hyponastic, purple stress leaves	(64)
	At5Ptase1 and 2	Type I inositol polyphosphate 5-phosphatase toward Ins(1,4,5)P3	Germination and seedling development in double mutant	(65)
	FRA3	Type II inositol poly-phosphate 5-phosphatase; PtdIns(4,5)P2 > Ins(1,4,5)P3	Reduction in secondary wall thickness and stem strength, alterated actin deposition in fiber cells	(66)
	At5Ptase13	Type I inositol polyphosphate 5-phosphatase toward Ins(1,4,5)P3	Altered auxin levels, blue light signaling	(67, 68)
	MRH3 (At5Ptase5)	Inositol polyphosphate 5-phosphatase	Root hair initiation	(69)
	CVP2 (At5Ptase6)	Inositol polyphosphate 5-phosphatase	Vascular patterning cotyledon	(70)
IPK	AtIPK1/AtIPK2β	Inositolpolyphospate kinase	InsP6 levels down, Pi sensing, and root hair growth	(16)

^aUnpublished observations.

Plants are well known for their phytate (InsP₆) content in seeds, which mainly reflects the mechanism to store huge amounts of phosphate and inositol required for germination. Nonetheless, InsP₆ may play a completely different role during plant development and in response to agonists, similar to the one that is emerging in mammalian and yeast fields (15). An exciting example of such may be the unexpected discovery of InsP₆ in the crystal structure of the auxin (a plant hormone) receptor, TIR1 where it is structurally required for auxin binding and receptor function (17). The major question now is, does this reflect InsP₆ signaling and does it require a PLC-mediated pathway?

PA signaling rather than DG

A convincing role for DG as a plant signaling molecule has never really been reported. Though it cannot be excluded, the lack of evidence over the last 20 years, together with the absence of its primary target PKC, leaves very little ground to put it into the plant PI/PLC model today (Fig. 1). As a precursor for glycolipids, storage lipids, and the major structural phospholipids, together accounting for ∼90% of all plant lipids, DG does not seem to be the most favorable molecule as a membrane-localized-second messenger either. Instead, we see that the PLC-generated DG is rapidly phosphorylated to PA by DG kinase (DGK) and that PA has typically emerged as the plant's second messenger (5, 18, 28). Over the years, a number of biotic (pathogens) and abiotic (e.g., temperature, osmotic) stress signals have been shown to activate this pathway; meanwhile, a number of plant PA targets have been identified (5). Arabidopsis contains seven DGK genes, which are differentially expressed throughout the plant and in response to stress, but knockout mutants have not resulted in a phenotype yet (unpublished observations), indicating a high degree of redundancy. PA can also be generated via the PLD pathway. More about PLD, PA signaling, and downstream targets can be found below.

PAK and DGPP

What is also different from mammalian systems is that PA can be phosphorylated into DGPP by a PA kinase (PAK) (Fig. 1). The gene encoding this novel lipid kinase is still unknown, but the enzyme seems to be present in every tissue and is enriched in plasma membrane fractions. The idea is that PAK attenuates PA signaling, but DGPP could also be a signaling molecule itself (19).

Inositol lipids and phosphates

Apart from being signaling precursors, it is evident that PtdIns4P and PtdIns(4,5)P₂ can also function as signaling molecules themselves. Both lipids are involved in polar growth of pollen tubes and root hairs and during cell plate formation, as shown by lipid biosensors, but also judged from the recent phenotypes of PI 4-kinase, PtdInsP 5-kinase and PtdIns4P phosphatase mutants (Table 1) (8, 20–25). Similarly, several cell wall assembly mutants have been identified that may reflect defects in vesicular trafficking (fragile fibers; FRA mutants) and vascular patterning (Table 1). These include several 5-phosphatase mutants, of which it is not always clear whether this affects the lipid or the inositolphosphate, nonetheless emphasizing their importance.

Transgenic plants constitutively overexpressing a (human) InsP₃ 5-phosphatase have been reported to have various phenotypes. While these have been interpreted as “attenuation of InsP₃ signaling” (26), they might very well reflect defects in InsP₆ or raffinose family oligosacharide metabolism. The latter requires inositol as a precursor and functions to protect cellular structures during desiccation and as carbon reserves for early germination (27).

PHOSPHOLIPASE D

As mentioned above, PA is emerging as an important plant lipid second messenger. PA is rapidly and transiently generated in response to a variety of biotic and abiotic stresses, either via the PLC/DGK pathway as discussed above, or directly via PLD.

This enzyme catalyzes the hydrolysis of structural lipids, like PC and PE, to produce PA and the respective headgroup. Using differential ³²P₁-labeling techniques and PLD-specific transphosphatidylation assays, it is possible to distinguish between both PA-generating pathways (28). In this way, a variety of environmental cues have been shown to activate the PLD pathway, including plant defense elicitors, cold, wounding, heat, oxidative stress, and osmotic stress (5, 29, 30).

Plants are true PLD champions. While humans only contain 2 PLD genes, and yeast 1 (SPO14), Arabidopsis contains 12 PLD genes and rice even 17 (29–31). Plant PLDs can be classified into 2 groups based on their lipid binding domains. Those with a combined PX and PH domain belong to the PLDζ class and are homologous to the mammalian and yeast PLDs. The others, representing the majority of plant PLDs, belong to the C2 class, containing a C2 (calcium and lipid binding) domain. Arabidopsis has 10 C2-PLDs, PLDα1-3, β1-2, γ1-3, δ, and ɛ, and two PX-PH-PLDs, PLDζ1-2.

Using T-DNA insertion knockout mutants, individual PLDs have been linked to specific plant responses, including ABA signaling, osmotic stress, reactive oxygen species (ROS), freezing, auxin, Pi starvation, and root and root hair development (Table 1).

PA properties and its targets

PA formation has a profound effect on membrane curvature and surface charge. Its small anionic phosphomonoester headgroup resides very close to the hydrophobic interior of the lipid bilayer, which is different from other anionic phospholipids. Moreover, hydrogen bonding increases the negative charge of PA, explaining why it can form strong interactions with target proteins, which has recently been proposed as the electrostatic/hydrogen bond switch model (32). The combined effects are likely to be crucial for specific PA responses (33). Major progress has been made in identifying molecular PA targets. Like in mammalian cells, these include protein kinases, phosphatases, and proteins involved in membrane trafficking and the organization of the cytoskeleton. Both activation of positive regulators and inhibition of negative regulators have been reported (5, 18). Examples include PDK1, mediating responses to ROS in root hair development and pathogens, CTR1, a crucial protein kinase in ethylene signaling, ABI1, a protein phosphatase in ABA signaling, AtCP, an actin capping protein, and AGD7, an ArfGAP (32). Though several PA binding motifs have been recognized (5, 34), a general PA binding domain still remains obscure.

PHOSPHOLIPASE A

PLA catalyzes the hydrolysis of phospholipids into lysophospholipids and free fatty acids, either at the sn-1 (PLA₁) or 2-position (PLA₂) of the glycerol backbone, or both (PLB). Plants contain numerous PLAs. In Arabidopsis, three different families can be distinguished: four small secretory sPLAs, 10 patatin-like pPLAs, and 14 lipase-like PLA₁s. For most, it is not clear what their substrate is or which position they hydrolyze; some exhibit acyltransferase or acylhydrolyse activity, sometimes even toward nonphospholipids, such as galactolipids (35). As such, it is not always clear whether effects reflect general lipid metabolism or signaling. Nonetheless, evidence is increasing for PLA's involvement in disease resistance, auxin, and light (Table 1; 35–39).

Similar to the eicosanoid (C20) pathway in animals, plants exhibit an octadecanoid (C18) pathway, playing an important role in, for example, the plant's defense against pathogens and herbivores, in particular, jasmonic acid (JA) and its volatile, methyl JA (40). JA is also important in flower development. The latter involves DAD (for defective in anther dehishence), a gene encoding a PLA₁ (41).

Lysophopholipids have also been also implicated in cell signaling (3). In particular, lysophosphatidylcholine, which is proposed to activate a vacuolar H⁺/Na⁺ antiporter to regulate the cytosolic pH in response to a pathogenic elicitor (36, 42).

PI3 KINASE

Arabidopsis only contains one PI3K, which is Vps34p-like (type III). Silencing AtVPS43 causes severe defects in development, while knockout mutants are lethal, indicating an important function (43–45). About 5–15% of the PtdInsP pool in plants is PtdIns3P, with the majority (∼80%) being PtdIns4P and containing a few percent of PtdIns5P (46). PtdIns3P has been imaged in living cells by expressing the PtdIns3P biosensor YFP-2xFYVE, revealing predominant labeling of late endosomes, multivesicular bodies, and prevacuolar membranes (9). Only ∼20% of the early endosomes were labeled. The YFP-2xFYVE labeling pattern is sensitive to wortmannin, as all labeled structures disappeared within 20 min of treatment, with all fluorescence reappearing in the nucleus. HPLC-headgroup analyses revealed that the FYVE-overexpressing cells contained double amounts of PtdIns3P. Apparently, cells sense free PtdIns3P levels, and since overexpression of FYVE leads to preoccupation of PtdIns3P, competing with endogenous targets, cells simply make more. This probably also explains why there is no apparent phenotype in cell suspensions or Arabidopsis seedlings that constitutively express the sensor (9), although overexpression behind a root-hair-specific promotor did have a dose-dependent effect on root hair elongation (47). Wortmannin and LY294002 inhibit root hair growth. The same inhibitors have been used to imply the involvement of PtdIns3P in the production of ROS and actin dynamics (43, 47, 48). In Arabidopsis, 11 proteins with a PX and 16 with a FYVE domain have been predicted (49).

Other D3-PPI

Plants contain small amounts of PtdIns(3,5)P₂ but lack PtdInsP₃. Earlier, the presence of PtdIns(3,4)P₂ had been reported, but this was before the discovery of PtdIns(3,5)P₂ and has not been reproduced so far (50). Like yeast, plants make PtdIns(3,5)P₂ in response to osmotic stress (50). Yeast PtdIns(3,5)P₂ is involved in the retrograde trafficking between organelles and the endocytic/lysosomal system and made by a PtdIns3P 5-kinase called Fab1p (51). FAB1 mutants (for formation of aploid and binucleate cells) have enlarged vacuoles that do not acidify correctly and have nuclear segregation defects. Arabidopsis contains 4 putative FAB genes (1). Proposed effectors for PtdIns(3,5)P₂ include the PROPPIN family of seven-bladed β-propellers (51) of which Arabidopsis homologs are present.

Degradation of PtdIns(3,5)P₂ occurs through 3- or 5-phosphatases. In vitro, At5PTase11 and AtSAC1/FRA7 have been shown to dephosphorylate PtdIns(3,5)P₂ at the 5-position (52). AtSAC1/FRA7 mutants have defects in the organization of the actin cytoskeleton and exhibit reduced cell wall thickening (53). Whether PtdIns(3,5)P₂ is the (only) substrate in vivo remains to be shown. In total, Arabidopsis contains 15 At5PTase and 9 SAC genes (80–89). The SAC (for suppressor of actin) family of phosphatases contains both 4- and 5-specific phosphatases (Table 1).

3-Phosphatases

Arabidopsis contains three homologs of PTEN (54) and two potential MTM (myotubularin-type phosphatase) genes that are predicted to be catalytically active. AtPTEN1 is a dual-specificity phosphatase which, in vitro, has phosphatase activity toward phosphotyrosine and PtdInsP₃. It is exclusively found in pollen grains and expressed during the later stages of development. Knockout mutants are lethal, and RNA interference suppression results in cell death after mitosis, indicating that this gene is essential for pollen tube development (54).

Abbreviations

• ABA, abscisic acid

• CDPK, calcium/calmodulin-dependent protein kinase

• DG, diacylglycerol

• DGK, diacylglycerol kinase

• DGPP, diacylglycerolpyrophosphate

• InsP₃, inositol 1,4,5-trisphosphate

• IPK, inositol dual-specificity polyphosphate multikinase

• JA, jasmonic acid

• PA, phosphatidic acid

• PAK, phosphatidic acid kinase

• PH, Pleckstrin homology domain

• PI/PLC, phosphoinositide/phospholipase C

• PLA, phospholipase A

• PLD, phospholipase D

• PtdInsP₂, phosphatidylinositol4,5-bisphosphate

• ROS, reactive oxygen species