Progress and Prospects of Research on the Role of Phosphatidic Acid in Response to Adverse Stress in Plants

Abstract

Lipid signaling plays a crucial role in how plants perceive and respond to environmental challenges. Among the various lipid mediators, phosphatidic acid (PA) serves as a key metabolic intermediate and second messenger that links membrane dynamics with stress signaling. It is produced rapidly through the coordinated actions of phospholipase C, phospholipase D and diacylglycerol kinase, and its transient accumulation enables plants to adjust defense and acclimation responses with remarkable precision. Recent studies have shown that PA participates in immune signaling, osmotic regulation, and redox control, functioning at the intersection of membrane remodeling and intracellular signal transduction. Through interactions with hormone signaling, calcium fluxes, and reactive oxygen species production, PA integrates multiple stress-responsive pathways, thereby helping to maintain physiological homeostasis under adverse conditions. This review summarizes current understanding of the biosynthetic regulation and signaling roles of PA, and discusses emerging perspectives that highlight its central role in plant immunity and stress adaptation.

1. Introduction

Plants endure various stressors during growth, including drought, high salinity, extreme temperatures, and pests and diseases. Global climate change is exacerbating these challenges, which severely impact plant growth, development, crop yield, and quality. In order to acclimatize to the environment [1], plants have evolved sophisticated signal transduction networks to sense and respond to external stress stimuli, and lipid signals regulate gene expression and activate acclimation processes in plants through dependent signaling cascades. Phosphatidic acid (PA) is the simplest membrane glycerophospholipid, consisting of a glycerol backbone, two hydrophobic fatty acid side chains, and a negatively charged phosphoric acid head [2]. The hydrophilic phosphate head and hydrophobic fatty acid chains confer its characteristic surfactant properties [2]. PA is a component of plant cell membranes and a key signalling intermediate in plant signalling processes [3,4], which is involved in the regulation of plant responses to various biotic and abiotic stresses [5]. In this review, we summarize how PA is involved in plant responses to biotic and abiotic stresses.

2. PA Biosynthesis, Metabolic Pathway

Lipids are important structural components of plants, and play an important role in plant cell growth, development and signal transduction. PA, a type of glycerophospholipid, accumulates mainly in cell membranes, acts as a key factor in the biosynthesis of various complex lipids and is additionally identified as a pivotal molecule in lipid signaling processes within plants [6,7,8,9,10].

2.1. Synthesis Pathway

PA, as a signaling molecule, is primarily produced by two distinct phospholipase pathways at the plasma membrane [6].

One of the phospholipase pathways is the phosphorylation of diacylglycerol (DAG) by diacylglycerol kinase (DGK) in the phospholipase C (PLC) pathway [11], where PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] to inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and DAG (Figure 1). Ins(1,4,5)P₃ diffuses into the cytoplasm while DAG remains in the membrane where it is rapidly phosphorylated to PA by DGK. This phosphorylation represents a major pathway for PA production, accounting for approximately 80% [12].

An alternative phospholipase-mediated route involves phospholipase D (PLD)-catalyzed hydrolysis of membrane phospholipids, particularly phosphatidylcholine (PC) and phosphatidylethanolamine (PE), yielding PA. This stress-inducible enzymatic activity selectively targets structural phospholipids at the membrane interface (Figure 1) [13]. The Arabidopsis PLD family has 12 members [14], most of which possess the C2 structural domain near the N-terminus, called the C2-PLD [16], and the C2 structural domain is required for the enzyme activation of PLD [14]. C2-PLDs vary in their substrate preferences and activation conditions, resulting in PAs with different acyl composition [17].

The PA family comprises various molecular species that play distinct and specific roles in signal transduction. The diversity of PA molecules stems from variations in fatty acid chain length and degree of unsaturation, which also governs their binding to target proteins, thereby activating entirely different signaling pathways [18]. PA synthesized via the PLD pathway tends to utilize PC as a substrate, producing unsaturated PAs that predominantly generate downstream stress signals such as oxidative bursts and general membrane remodeling. In contrast, the PLC pathway primarily employs DAG derived from PIP₂ as a substrate, yielding PAs with greater signaling specificity that are typically associated with precisely regulated processes like immune responses and cell proliferation [19]. The distinct substrate specificities of PLC and PLD pathways enable discrimination of PA origin through fatty acid profiling during signal transduction events [20].

2.2. Metabolic Pathway

PA plays a pivotal role in plant cells via its dynamic degradation and metabolic transformation. Acting as a central node in lipid turnover, PA connects membrane lipid metabolism with multiple signaling pathways. Its interconversion is tightly controlled by a series of lipid-modifying enzymes that determine both the duration and specificity of PA-mediated signaling. These enzymatic processes not only regulate the cellular pool of PA but also generate a spectrum of bioactive lipid intermediates that participate in membrane remodeling, signal relay, and stress adaptation [21].

Degradation of PA is accomplished by enzymes such as phospholipase A (PLA) and phosphatidic acid phosphatase (PAP). PLA releases free fatty acids (FFA) and forms lysophosphatidic acid (LPA) by hydrolyzing the ester bond at the sn-1 or sn-2 sites of PA (Figure 1) [14], which further affects the fluidity and stability of the cell membrane. This degradation process plays an important role in plant response to stress and the regulation of cell membrane stability [21,22]. PLA is activated during pathogen infection. The degradation products of PA are involved in regulating the immune response [23].

Within plant systems, PA undergoes phosphorylation through the action of PA kinase (PAK), resulting in its conversion to diacylglycerol pyrophosphate (DGPP) (Figure 1) [15]. Additionally, PA can be dephosphorylated by PAP to yield DAG and PI. DAG served as an intermediate in triacylglycerol (TAG) biosynthesis for energy reservoir formation (Figure 1), and as a regulatory component in membrane-associated processes, performing dual physiological functions [24]. The simultaneously generated PI serves as a core structural component of plant cell membranes [25]. These resultant lipid derivatives collectively orchestrate essential physiological mechanisms, including signal propagation pathways and the maintenance of membrane fluidity equilibrium in plant systems.

2.3. Cross-Talk Between Different Pathways Involved in PA Synthesis and Metabolism

In plant cells, the distinct pathways responsible for PA synthesis and metabolism do not operate in isolation but instead form a complex, interconnected regulatory network. This cross-talk is crucial for the cell’s precise control over the intensity, duration, spatial localization, and molecular diversity of PA signaling [26].

PLD hydrolyzes structural phospholipids (such as PC) to produce PA. PA here can be converted by PAP and generate DAG [27]. The resulting DAG can then serve as a substrate for DGK to be rephosphorylated into PA. Cells can rapidly adjust the levels of PA and DAG in membranes through this PA cycle without requiring de novo synthesis. This cycle also links the structural and signaling functions of phospholipids [28].

As mentioned earlier, phospholipids generated through different pathways undergo distinct downstream reactions, while also exhibiting synergistic effects where they can activate or inhibit each other. PLC hydrolyzes PIP₂ to produce IP₃, leading to elevated cytoplasmic Ca²⁺ concentrations that subsequently activate the PLD pathway, thereby amplifying lipid-derived signaling cascades to achieve coordinated responses [29,30]. ROS generated from PA or other pathways can oxidize and activate both PLD and DGK. This forms a positive feedback loop that is crucial for plant immunity and stress responses [31]. Concurrently, the synthesis of PA is subject to cross-pathway regulation. Under plant phosphorus starvation stress, the PLC pathway, PLD pathway, and lysophosphatidylserine–GDP–diphosphate pathway form a metabolic network [32]. This coordination ensures phospholipid homeostasis under phosphate limitation [32].

3. Effects of PA on Plant Membrane Systems

PA adopts a conical molecular geometry characterized by a compact phosphohead group, which carries a net charge of −1.5 to −2 under physiological pH, and two acyl chains -either saturated or unsaturated- that occupy a larger cross-sectional area [33]. This structural arrangement induces negative curvature stress in lipid bilayers, promoting outward membrane bending and facilitating processes such as membrane fusion and endocytic vesicle formation. PA-enriched regions reduce the energetic cost of membrane fission and cooperate with dynamic protein assemblies to sculpt membrane topology [26]. As negatively charged anionic phospholipids, the protonated state of PA’s phosphoric acid groups and their intermolecular interactions enable specific interactions with various lipids, significantly influencing membrane physical properties, phase behavior, and signal transduction functions [18].

Hydrogen bond-mediated interactions between PA and PE represent a fundamental mechanism regulating membrane structure. The primary amine group (–NH₃⁺) of PE serves as a potent hydrogen bond donor, forming strong intermolecular hydrogen bonds with the phosphomonoester headgroup of PA. Through this hydrogen bonding, PE withdraws electrons density from the oxygen atom of the phosphate group, destabilizing the second proton of PA and promoting its deprotonation, which results in a marked decrease in the pKa₂ value of PA [34,35]. Within PE bilayers, the pKa₂ of PA is substantially lower than that observed in phosphatidylcholine (PC) environments, leading to an increased negative charge density at physiological pH. Notably, PA-PE complexes act synergistically to induce negative membrane curvature, thereby facilitating both membrane fusion and fission processes. Molecular dynamics simulations further demonstrate that PE stabilizes the conical conformation of PA, enhancing its intrinsic negative curvature stress [34]. Simultaneously, the incorporation of cholesterol into PA-enriched membrane domains markedly increases the surface negative zeta potential, which promotes PA deprotonation and subsequently strengthens electrostatic interactions with PA-binding proteins [36].

Additionally, the charge state of PA is influenced by the local pH. Under acidic conditions, PA undergoes a reduction in net negative charge, accompanied by compaction of its head group and enhanced prominence of its conical molecular geometry. These structural changes promote preferential binding to divalent cations such as Ca²⁺ and Mg²⁺, effectively neutralizing the remaining negative charge [37]. This sensitivity to pH and the ability of PA to bind cations enables it to act as a molecular sensor, detecting changes in the local cellular microenvironment and translating these changes into alterations in membrane physical properties. Protonated PA exhibits reduced affinity for its binding protein Opi1, which translocates to the nucleus to inhibit phospholipid biosynthesis [37].

Beyond influencing membrane biophysical properties, PA also acts as an important signaling molecule linking changes in membrane status to intracellular pathways [16,38,39,40,41]. When PA accumulates, it can bind to a range of target proteins—including kinases, phosphatases, and other regulatory components—and modulate their activity [42]. PA further interacts with calcium and reactive oxygen species (ROS) signaling, providing additional layers of regulation [43,44,45]. Through these combined actions, PA helps convert external stimuli into appropriate physiological responses, forming a basis for the plant’s adaptation to the various biotic and abiotic stresses described in the following sections.

4. Role of PA in Abiotic Stresses

Environmental fluctuations induce diverse physiological acclimatization in plants, with predominant abiotic stressors comprising water deficit, salinity, and low-temperature conditions. Distinct classes of plant hormones elicit specialized signaling cascades to mediate these adaptive responses [46]. These abiotic stressors activate several molecular signalling pathways, among which the lipid signalling pathway driven by PA is the most prevalent pathway in the phenomenon of plant resistance to environmental stress [35,47].

4.1. PA Is Involved in the Regulation of Drought Stress in Plants

Drought stress inhibits plant development by affecting various physiological and biochemical processes. Lipid-derived signaling mediators exert essential regulatory functions in modulating plant water homeostasis, enhancing drought resistance, and facilitating prolonged aridity acclimation. Their dehydration-induced upregulation significantly influences these physiological processes [48]. The rice R3-MYB transcription factors OsTCL1 and OsTCL2 modulate germination capacity under water-deficit conditions. Mutant analyses reveal corresponding tcl1/tcl2 genotypes exhibit downregulated PLD gene expression profiles. Reduced PA biosynthesis impairs the rapid conversion of environmental stress signals into physiological responses, as PA acts as a second messenger, thereby impairing the precise regulation of seed germination timing and success under adverse conditions [49].

Stomatal transpiration constitutes the primary route of water loss in plants, making its regulation critical for maintaining hydration under water deficit conditions. PLD functions as a central coordinator of abiotic stress responses, orchestrating fundamental acclimatization processes at the molecular level. The PA produced by PLDα1 in Arabidopsis binds to the ABI1 protein phosphatase 2C to signal abscisic acid (ABA)-promoted stomatal closure [50]. The interaction between PLDα1 and the Gα subunit of heterotrimeric G-proteins facilitates abscisic acid-dependent suppression of stomatal aperture, thereby modulating hydraulic regulation in plants [50]. Heterologous expression of PbrPLD2 from Pyrus bretschneideri in both Arabidopsis and pear confers elevated drought resistance, manifested through optimized stomatal regulation and upregulation of stress-responsive genetic elements [51]. In perennial ryegrass (Lolium perenne), LpPLDδ3 displays distinct expression patterns among PLD gene family members, showing ABA-mediated downregulation while being upregulated under drought and thermal stress conditions. Heterologous expression of LpPLDδ3 in Arabidopsis enhances osmotic and thermotolerance phenotypes [52].

The PLC/DGK pathway significantly contributes to ABA-dependent drought responses via PA production [53]. In Arabidopsis, ABA-deficient 2 (ABA2) catalyzes ABA synthesis while paradoxically reducing stress tolerance. DGK5 and its lipid product PA suppress ABA biosynthesis through direct interaction with ABA2, consequently improving plant resilience to drought and high-salinity conditions (Figure 2) [54].

4.2. PA Is Involved in the Regulation of Plant Cold Stress

Cold stress mainly damages the cell membrane of plant tissue [56], resulting in cell dehydration and leaf wilting. Cellular homeostasis in plants depends fundamentally on precise protein composition and membrane fluidity regulation [57], rendering low-temperature responsiveness a physiologically pivotal acclimatization. Cold-induced membrane perturbations trigger calcium influx and elevate PA biosynthesis through coordinated PLD and PLC-DGK pathway activation (Figure 2) [51]. Exposure of Arabidopsis to non-lethal cold stress induced distinct alterations in phospholipid profiles, characterized by reduced PC, PE, and phosphatidylglycerol (PG) content alongside elevated PA and lysophospholipid accumulation [12].

The expression of the gene encoding PLDα1 in Arabidopsis was induced by cold. Knocking down this isoform had no significant effect on the accumulation of PA in response to cold, suggesting that multiple PLDs are involved in PA production [12]. Arabidopsis PLDδ mutant analysis revealed a 20% reduction in PA accumulation accompanied by diminished cold tolerance relative to wild-type counterparts. In contrast, transgenic overexpression lines exhibited improved freezing resistance, demonstrating PLDδ’s functional involvement in cold stress acclimatization [58].

Low-temperature conditions impair root system growth and functionality, compromising hydraulic and mineral acquisition [59]. Root-synthesized PA partially alleviates these cold-induced physiological constraints. Arbuscular Mycorrhizal (AM) symbiosis increases PA and Ca²⁺ signalling, as well as the expression of genes related to low (LT) and high temperature (HT) stress in perennial ryegrass roots. It also regulates antioxidant enzyme activities, reduces lipid peroxidation, and inhibited growth inhibition induced by LT and HT stresses [60]. During the recovery process from prolonged cold stress, researchers observed a significant increase in PLD activity in barley (Hordeum vulgare L.) roots. PA and PLD modulate mitochondrial proline dehydrogenase activity and proline accumulation through direct and indirect mechanisms, facilitating barley’s acclimatization to prolonged cold exposure [61]. Cold stimulation triggers immediate cold-responsive gene expression while concurrently inducing PtdInsP phosphorylation to yield PtdIns(4,5)P2. This phosphoinositide is subsequently cleaved to generate Ins(1,4,5)P3 and DAG, with DGK-mediated ATP-dependent DAG phosphorylation producing PA [62]. The PLC/DGK pathway was activated in Arabidopsis cell suspensions under cold stimulation, and the expression of AtDGK1 and AtDGK2 genes was up-regulated, and the expression of DGK genes was also upregulated in maize (Zea mays L.) roots and leaves within 30 min of cold stress [63,64]. The study by Tan et al. showed that the genes DGAT1, DGK2, DGK3 and DGK5 are all involved in the response to cold response in Arabidopsis [65]. Compared to the wild type, the DGAT1 mutant showed increased sensitivity to freezing. Lipid profiling revealed that freezing significantly increased the PA and DAG levels and decreased TAG levels in DGAT1 mutants. PA accumulation enhances nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function, elevating respiratory burst oxidase homolog D (RBOHD)-dependent H₂O₂ generation [65]. Arabidopsis lines with DGK2, DGK3, and DGK5 gene disruptions exhibited improved freezing tolerance concomitant with reduced PA biosynthesis [65].

4.3. PA Is Involved in the Regulation of Salt Stress in Plants

Drought and elevated salinity constitute primary sources of osmotic stress in plants. Salinity stress disrupts cellular osmotic equilibrium, leading to physiological dehydration and impaired hydraulic conductivity. Concurrently, ionic disequilibrium and oxidative damage emerge [66], collectively constraining plant development. Within this context, phospholipid-mediated signaling represents a critical regulatory mechanism for salt stress acclimatization [67], with stress-induced PA serving as a molecular transducer coordinating salinity response pathways. Under saline conditions, plants exhibit substantial PA accumulation with distinct tissue-specific distribution patterns [57,68], mediated through coordinated PLD and DGK enzymatic activities [69]. This lipid messenger modulates root development, as evidenced by impaired root elongation upon DGK functional disruption [70]. Biochemical studies reveal PA targets GAPDH in both barley [71] and Arabidopsis [72] roots during salt stress through the PLC-DGK pathway [73], while simultaneously activating SnRK2 protein kinase in Arabidopsis under similar conditions [74]. Some gene expression profiling studies have shown the important role of DGK genes in salt tolerance (Figure 2) [55]. Using the DGK inhibitor R59022 in Arabidopsis affected the activity and PA production of AtDGK2 and AtDGK7, and the altered growth and development of the plant confirmed this result [75].

In Populus euphratica, NADP-ME interacts with PePLDδ in Arabidopsis to stimulate PLD-mediated PA production and subsequent signaling cascades, thereby enhancing Na⁺ extrusion, suppressing ROS accumulation, and improving salinity tolerance [76]. Lipidomic profiling revealed substantial modifications in both compositional and structural features of membrane lipids within rice root cells following salt exposure. Quantitative lipid profiling demonstrated marked accumulation of predominant phospholipid species, notably PA, concomitant with modified membrane biophysical properties that enhanced salinity acclimatization in rice root systems [77]. PC treatment of Prunus persica seedlings enhanced cellular PLD activity, stimulating phospholipid degradation and subsequent PA accumulation. The resultant PA functioned as a lipid mediator, attenuating oxidative damage through suppression of salt-induced ROS and peroxide accumulation [78]. In addition, the MAPK signaling cascade demonstrates significant involvement in plant salinity acclimatization. Salt stress conditions induced PA binding to PpMPK6 in transgenic Arabidopsis overexpressing this kinase, resulting in salt-overly-sensitive 1 protein activation that enhanced Na⁺ extrusion capacity and improved salt tolerance phenotypes [79].

5. Role of PA in Biotic Stresses

Plants have evolved multifaceted strategies to counteract biotic challenges, particularly pathogenic invasions. Beyond constitutive physical deterrents like cuticular and cell wall barriers, plants employ intricate internal defense systems. Notably, even upon breaching of these structural defenses, immunological activation occurs through sophisticated intracellular recognition mechanisms [80]. Pattern-triggered immunity (PTI) constitutes the primary plant defense response, representing an evolutionarily conserved innate immune mechanism [81]. This nonspecific recognition system involves plasma membrane-localized pattern recognition receptors (PRRs) that detect MAMPs, subsequently initiating intracellular phosphorylation cascades and downstream signal transduction [82].

Effector-triggered immunity (ETI) represents a specialized defense mechanism activated through direct recognition of pathogen effectors by plant resistance proteins. This highly specific response involves intracellular NLR receptors containing nucleotide-binding and leucine-rich repeat domains, culminating in a localized hypersensitive reaction at infection sites [81]. Despite distinct activation mechanisms, pattern- and effector-triggered immunities converge on shared physiological responses. These conserved defense components encompass ROS generation, cytosolic calcium influx, pathogenesis-related protein synthesis, MAPK cascade induction, and phytohormone signaling-collectively constituting fundamental plant immune processes [83].

PA, a central glycerolipid metabolite, mediates oxidative burst responses during plant-pathogen interactions. NADPH oxidase-dependent ROS generation [84] involves PA binding to RBOHD, thereby modulating oxidative bursts during both PTI and ETI. Arabidopsis maintains PA homeostasis through DGK5 phosphorylation, which modulates ROS generation. This DGK5-dependent PA biosynthesis enhances plant defense against microbial pathogens, including both bacterial and fungal challenges [39]. In Arabidopsis DGK5 modulates both pattern- and effector-triggered immunity. The receptor-like cytoplasmic kinase BIK1, inducible by Botrytis cinerea and membrane-associated, interacts with multiple PRRs. Upon activation by PRR complexes, BIK1 phosphorylates RBOHD to control ROS generation and targets cyclic nucleotide-gated channels to elevate cytosolic calcium concentrations [85].

5.1. DGK5 Regulates PA to Influence Plant Immunity

DGK5 constitutes a critical component in flagellin-triggered immune responses and pathogen defense mechanisms [86], the PA generated through DGK5 activity facilitates NADPH oxidase activation, thereby initiating ROS-mediated signaling during biotic stress acclimatization (Figure 3) [87]. Transgenic tobacco expressing OsBIDK1 exhibits enhanced resistance to both tobacco mosaic virus and the oomycete pathogen Phytophthora parasitica var. nicotianae [88]. Pathogen challenge triggers coordinated upregulation of specific PLA2 isoforms and DGK activity, driving rapid PA biosynthesis during plant immune activation [89].

Current research identifies DGK5 as a BIK1-interacting protein, with flagellin-derived flg22 peptide inducing DGK5 dual phosphorylation that modulates immune signaling cascades in plants [86]. The receptor-like cytoplasmic kinase BIK1 phosphorylates DGK5 at Ser⁵⁰⁶ triggering rapid PA accumulation and immune activation. Conversely, MPK4-mediated Thr⁴⁴⁶ phosphorylation suppresses DGK5 enzymatic function and PA biosynthesis, resulting in downregulated defense responses (Figure 3). Opposing phosphorylation events mediated by BIK1 and MPK4 maintain cytosolic PA homeostasis through DGK5 regulation, thereby controlling ROS generation and coordinating two branches of plant immunity [90].

PA stabilizes RBOHD through suppression of vesicular degradation, amplifying chitin-triggered ROS generation. These oxidative bursts critically mediate pathogen defense responses [90]. DGK5 loss-of-function mutations disrupt diacylglycerol phosphorylation, compromising both PA biosynthesis and subsequent ROS production during chitin perception. Alternative splicing generates two DGK5 isoforms (α and β), with DGK5β containing a C-terminal calmodulin-binding domain absent in DGK5α. While dispensable for subcellular targeting, this domain facilitates PA biosynthesis and RBOHD stabilization. Consequently, DGK5β-derived PA preferentially associates with RBOHD [42]. The DGK5β isoform rescues the impaired PA biosynthesis, ROS generation, and Botrytis cinerea resistance observed in dgk5-1 mutants. Ser⁵⁰⁶ phosphorylation within DGK5β’s C-terminal calmodulin-binding domain enhances its catalytic activity, promoting PA production (Figure 3). Chitin perception initiates DGK5β phosphorylation, which stimulates PA biosynthesis while concurrently stabilizing RBOHD through vesicular degradation suppression, ultimately enhancing ROS production [42].

5.2. RBL1-Mediated Reprogramming of Lipid Metabolism Enhances Rice Immune Response by Regulating PA Accumulation

Sha et al. identified a 29-bp deletion in the RBL1 locus conferring broad-spectrum disease resistance while reducing crop productivity by roughly 95% [91]. Targeted genome editing generated the RBL1^Δ12 allele, which provides broad-spectrum pathogen resistance in rice while maintaining crop yield. The RBL1-encoded enzyme catalyzes PA conversion to cytidine diphosphate-diacylglycerol (CDP-DAG), representing a key step in phospholipid biosynthesis (Figure 3) [92]. RBL1 mutations substantially decreased PI and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] content while elevating PA accumulation. Lipidomic profiling demonstrated significantly increased PA and DAG levels in RBL1 mutants compared to japonica controls, accompanied by 71% and 49% reductions in PI and PG, respectively [91]. Certain phosphoinositides function as molecular determinants of disease susceptibility in plants [93]. Notably, PtdIns(4,5)P₂ accumulates in rice membrane compartments involved in pathogenic effector secretion and fungal colonization, implicating this lipid species in susceptibility mechanisms.

From a mutagenized Oryza sativa population displaying hypersensitive response-like lesions (a programmed cell death phenotype), Gong et al. identified a lesion-mimic mutant (LMM) exhibiting disease resistance [94]. Chemical complementation assays demonstrated that exogenous PI delayed lesion formation in RBL1 mutants, while PA accelerated this phenotype. Notably, diphenyliodonium chloride-mediated inhibition of RBOH activity similarly suppressed LMM development [94]. OsPAH2 overexpression in RBL1 mutants (OsPAH2::rbl1) reestablished normal PA concentrations without markedly affecting lesion formation frequency [95]. The modest attenuation of pathogenesis-related gene suppression implies PA elevation contributes minimally to immune enhancement in this genetic background [96].

5.3. PLD, PLA and PA Responses in Plant Biotic Stresses

PLD and PA play crucial roles in signal transduction cascades in plants subjected to biotic stress. In Arabidopsis, they accumulate at pathogen entry sites. Then, PA recruits different proteins, such as NADPH oxidase, which initiates PA-related defense signals and prevents pathogen penetration [96]. PA constitutes a principal lipid category exhibiting rapid responsiveness to Potato virus Y (PVY) during initial infection phases. In Nicotiana benthamiana, PLDα1 mediates PA accumulation following PVY challenge, demonstrating significant antiviral activity. The PVY-encoded 6K2 transmembrane protein physically associates with NbPLDa1, resulting in increased PA accumulation. This interaction additionally triggers MAPK cascade activation. Notably, exogenous PA application similarly induces MAPK signaling, suggesting a conserved mechanism of pathway stimulation [97]. PLDδ in Arabidopsis potentiates both basal immunity and non-host resistance against Blumeria graminis f. sp. Hordei (Bgh) through regulation of PA biosynthesis and subsequent signaling cascades [97]. PLDδ-deficient Arabidopsis exhibits delayed induction of chitin-responsive genes following stimulation [98]. Conversely, PLDα1 activity suppresses host resistance against Bgh [98].

PLA catalyzes phospholipid hydrolysis, yielding lysophospholipids that are subsequently converted to PA [14]. Pathogen infection enhances PLA activity, with the resultant PA serving as a critical mediator of immune activation, including antimicrobial biosynthesis and defense gene expression [99]. Specifically, PA directly interacts with and stimulates WIPK/SIPK kinase activity, triggering downstream defense gene activation and antimicrobial biosynthesis [97]. This signaling pathway represents a crucial plant defense strategy against pathogen invasion, with PA functioning as a lipid mediator that orchestrates diverse immunological processes.

5.4. PA and Actin Remodeling in Biotic Stress Response

The actin cytoskeleton undergoes dynamic reorganization during plant-microbe interactions, with PLD and PA serving as critical regulators of these structural changes and associated defense responses [100,101]. Fungal and oomycete invasion sites exhibit distinct actin bundle formation [102], with cytoskeletal reorganization facilitating essential defense processes including cytoplasmic streaming and directed mobilization of antimicrobial agents [103]. Pathogen challenge induces actin filament reorganization in plant cells [104], with both pathogenic and non-pathogenic bacteria eliciting transient actin bundling in Arabidopsis epidermal tissues [105]. The capping protein (CP) functions as a pervasive inhibitory factor modulating actin filament dynamics in plant systems. By associating with filament barbed termini, CP obstructs progressive polymerization, thereby enhancing filament stability [106]. Unlike other variants, the plant CP exhibits pronounced binding selectivity toward PA. The energetically active PA generated during immune responses suppresses CP function through direct interaction, destabilizing filament termini by inducing their dissociation [107]. Furthermore, exposure to n-butanol disrupts actin filament organization in plants by suppressing PLD function, leading to diminished PA concentrations. Conversely, elevated PA levels enhance actin filament bundling and reinforce the structural integrity of the actin network.

6. Role of PA in the Regulation of Phytohormones

Phytohormones are a chemically diverse group of signaling molecules synthesized during developmental processes and stress responses [108]. Their structural diversity encompasses adenine derivatives (cytokinins), terpenoid compounds (gibberellins, ABA), polyhydroxylated steroids (brassinosteroids), phenolic moieties (salicylic acid), and oxidized fatty acid derivatives (jasmonates) [109]. Phytohormones orchestrate diverse developmental and physiological processes through complex signaling networks. These regulatory pathways are modulated by lipid-derived messengers, with PA emerging as a key mediator of multiple hormonal signaling cascades [18,110].

Arabidopsis subjected to 4-h hormonal exposure exhibited differential expression of phospholipid biosynthetic genes [111]. Transcriptomic analysis demonstrated hormone-responsive modulation of phosphoglycerolipid pathway enzyme-encoding genes, implicating their protein products in hormonal signal transduction [111].

6.1. PA in Regulation of ABA

ABA is a hemiterpene plant hormone that plays a central role in plant stress responses, seed dormancy, and stomatal closure [9]. In addition to the aforementioned involvement of ABA in regulating osmotic stress including drought and high salinity, ABA deficiency also impairs root and stem growth in plants [112]. The accumulation of ABA in plant cells enhances the regulation of Ca²⁺ flux, which affects its role in defending cellular abscisic acid signalling through a specific mechanism [113]. In Arabidopsis, the primary ABA biosynthesis initiates with zeaxanthin epoxidation catalyzed by ABA1, generating xanthophyll intermediates subsequently converted to abscisic aldehyde via ABA2 dehydrogenase activity. Final ABA production requires sequential modification of this aldehyde by both ABA3 and abscisic aldehyde oxidase 3 enzymatic activities (Figure 2) [114]. Arabidopsis expresses AtLPP2 and AtLPP3, encoding lipidphosphatephosphatases, during seed germination. ABA treatment elevated both PA and DGPP accumulation in germinating seeds. The AtLPP2 insertion mutant exhibited enhanced ABA sensitivity during germination inhibition relative to wild-type plants, concomitant with elevated PA accumulation. This correlation implicates PA in ABA-mediated regulation of seed germination [115]. Similarly, in rice, the expression of α-amylase, a key step in germination, was inhibited when seeds were treated with PA [50]. PLD activity likely generates the PA pool mediating ABA-dependent germination suppression. Rice PLDβ1 loss-of-function mutants displayed attenuated ABA responses, manifesting as both reduced germination inhibition and diminished α-amylase suppression [116].

6.2. PA in Regulation of Auxin (IAA)

IAA governs plant morphogenesis via polar auxin transport mechanisms. The PIN-formed family proteins, serving as auxin efflux carriers, mediate this process through their asymmetric membrane localization, which critically determines IAA distribution and physiological activity [117]. PA coordinates with calcium signaling to modulate auxin transport through direct interaction with regulatory proteins. Specifically, PA activates calcium-dependent protein kinase CPK29, whose absence impairs PIN-mediated auxin efflux and subsequent IAA redistribution [118]. Meanwhile, PA helps plants maintain growth and development under adverse conditions by regulating IAA redistribution [119]. For example, under saline conditions, concurrent activation of PLDα1 and PLDδ generates PA that stimulates pinoid (PID) kinase activity. This PA-PID interaction enhances PIN2 phosphorylation, increasing its auxin efflux capacity and facilitating IAA redistribution in root apices to sustain growth during salt stress [119].

Disrupted PA-mediated auxin regulation impairs multiple aspects of plant morphogenesis. Arabidopsis phospholipase AtPI-PLC2 participates in auxin signal transduction pathways [120], with PLC2 knockout lines displaying characteristic auxin deficiency phenotypes: reduced primary root elongation, compromised gravitropic responses, and diminished root hair development. NO-mediated auxin signaling induces accumulation of PA, PI phosphate, and PI diphosphate in cucumber explants. This lipid remodeling, facilitated by PLD activity, promotes PA biosynthesis and subsequent adventitious root formation, mirroring the effects observed with exogenous PA application [121], Plant 3’-phosphoinositide-dependent kinase 1 functions as a central signaling node, integrating upstream PA cues to phosphorylate downstream AGC family kinases and modulate auxin efflux [122]. Arabidopsis PLDζ2-generated PA similarly mediates auxin responsiveness, evidenced by attenuated IAA sensitivity in both PLDζ2 knockout mutants and transgenic knockdown lines [123].

6.3. PA in Regulation of Salicylic Acid (SA)

SA orchestrates initial biotic stress responses and systemic acquired resistance establishment, triggering immune activation upon pathogen challenge [124]. PA enhances SA biosynthesis via stimulation of isochorismate synthase activity or regulation of phenylalanine ammonia-lyase-mediated pathways [125]. PA modulates SA signaling by regulating pivotal transcriptional regulators, including NPR1 [126]. Conversely, SA suppresses basal PI-specific PLC activity in Arabidopsis suspension cultures, leading to marked reduction in PA levels [127]. Concurrently, SA stimulates in vivo phosphatidylinositol phosphorylation [128]. In addition, modulates plant defense responses through self-regulatory inhibition at elevated concentrations [126]. By suppressing PA production, SA coordinates developmental processes with stress acclimatization, enabling optimal resource allocation between growth and defense mechanisms during pathogen infection [129].

6.4. PA in Regulation of Other Plant Hormones

Brassinosteroids (BR) are a crucial class of steroidal phytohormones that orchestrate fundamental aspects of plant morphogenesis and physiological development [130,131]. PA modulates BR signal transduction through direct interactions with core BR pathway components. PA potentially influences BR perception and signaling initiation by binding to either the BR receptor kinase BRI1 or its co-receptor BAK1 [132]. Under adverse conditions, BR is also able to help plants maintain growth and developmental homeostasis by inhibiting PA synthesis [132].

Ethylene (ET) signaling cascade plays a pivotal role in plant hypoxia acclimatization [133,134]. PA enhances ET signal transduction through direct interaction with con-insensitive 1 (CTR1), a negative regulator of this pathway. By suppressing CTR1 kinase activity, PA potentiates ET-mediated hypoxia responses in plants [135,136].

Jasmonic acid (JA), a class of oxylipin derivatives, serve as critical mediators of plant defense activation, morphogenesis, and environmental acclimatization [137]. These oxylipin derivatives arise from unsaturated fatty acid metabolism through sequential PLA and PLD enzymatic reactions. Phospholipase A-mediated cleavage of PA at sn-1 or sn-2 positions yields lysophosphatidic acid and α-linolenic acid [14]. which serves as the direct biosynthetic precursor for jasmonate production. A variety of membrane lipids release oxylipins in response to PLA and PLD under stress conditions [138], and mutations in Arabidopsis PLDα1 and PLDβ1 impair JA biosynthesis [139].

7. Outlook on the Future Research Direction of PA

7.1. Application and Limitations of Traditional Research Methods

Early research on PA relied on biochemical analysis methods such as thin-layer chromatography (TLC) to separate and identify phospholipid species, combined with radiolabeling technology to track the direction of PA anabolic substrates, but the limited resolution of TLC makes it difficult to accurately differentiate between structurally similar lipid molecules, and radiolabeling is cumbersome to perform and poses a safety risk [140]. Although enzyme activity assays can indirectly reflect the function of PA-related enzymes, it is difficult to monitor dynamic changes in enzyme activity in situ and in real time, and it is impossible to accurately interpret the spatial and temporal patterns of PA action in complex stress environments [141].

7.2. The Development and Breakthrough of New Modern Technologies

The rapid advancement of biotechnology and instrumental analysis has revitalized PA research through several cutting-edge technologies. Lipidomic technology using mass spectrometry (LC-MS/MS) can detect the lipid molecular panorama in different tissues of plants at different stages of stress with high sensitivity and high throughput, accurately quantify changes in PA and its metabolites, and combine with multivariate statistical analysis to reveal lipid metabolism characteristics closely related to stress resistance [142,143]. Gene editing technologies, such as CRISPR/Cas9, to achieve targeted knockout, mutation or overexpression of genes involved in PA production and metabolism, and direct analysis of gene functions in model plants and crops to elucidate their contribution to plant stress tolerance [144,145]. Fluorescent probe labelling and microscopic imaging technologies to develop fluorescent probes that specifically recognize PA and to visualize in real time the dynamic intracellular distribution of PA [146], its transporter and its interaction with target proteins, so as to visually represent the microscopic scenario of PA signalling in plant stress tolerance and to expand the understanding of PA at all levels, from the molecular to the cellular to the whole plant level. This will enhance our understanding of PA-mediated resistance mechanisms at the molecular, cellular, and whole-plant levels [18].

The PAleon sensor designed by Li et al. utilized FRET technology to achieve real-time monitoring of PA in living cells for the first time, revealing the second response characteristics of PA in the early stage of stress [67]. This innovative tool not only overcame the limitations of traditional methods, but also revealed for the first time the function of PA as a pH sensor, which provided a new theoretical basis for the study of plant adversity biology. Meanwhile, the integration of multi-omics techniques, such as lipomics, transcriptomics and proteins, helped to resolve the spatial and temporal distribution of PA and its role in plant immunity.

7.3. Future Research Directions and Challenges

As a star molecule in the field of plant resistance, PA runs through the whole process of plant acclimatization to adversity, from basic biosynthesis and metabolism to complex signalling and defense response, and the existing research results have laid a solid foundation for understanding the nature of plant resistance, but there are still many unknowns to be explored in the field of PA in plant resistance.

On the one hand, the mechanism of interactive dialog between PA and other signaling molecules (e.g., Ca²⁺, ROS, hormones, etc.) is not completely clear, and the mechanism of PA action in different organelles (e.g., mitochondrion, nucleus) is also spatio-temporally specific. We need to analyze the complex signaling network nodes with the help of multi-omics joint analysis and biophysical means to clarify the synergistic regulation mode and precisely manipulate the plant stress response. On the other hand, the molecular details of PA action need to be deeply excavated, and the regulatory mechanism of PA may involve synergistic or antagonistic effects under the conditions of compound adversity (e.g., drought and high temperature). Future studies need to probe deeply into the response mechanism of PA under multi-stress conditions to reveal its integrated regulatory role in plant adversity acclimatization. In addition, for practical applications, enhancing PA signaling pathway activity in crops through gene editing may bring about a new round of agricultural green revolution, and editing PA-related genes using CRISPR technology can breed new varieties with stronger adversity acclimatization ability, bring into play the great application potential of PA in crop stress tolerance improvement, and translate research results in the laboratory into practical applications. In terms of genetic engineering targets, regulating the expression of PLD isoforms or PA metabolizing enzymes can enhance plant tolerance to drought, salinity and low oxygen. With more PA-related studies, we expect to fully reveal the central position of this important signaling molecule in plant biology and its potential applications.