In vivo substrates and the contribution of the common phospholipase D, PLDalpha, to wound-induced metabolism of lipids in Arabidopsis

The common plant phospholipase D (PLD), PLDalpha, has been proposed to be involved in wound-induced production of jasmonic acid. To better understand the role(s) of PLDalpha in the wound response, detailed lipid analysis was carried out to determine the in vivo substrates and the contribution of PLDalpha in wound-induced lipid metabolism in Arabidopsis thaliana. Mechanical wounding of Arabidopsis leaves resulted in significantly less hydrolysis of phosphatidylcholine (PC) in PLDalpha-deficient than in wild-type plants. Hydrolysis of phosphatidylethanolamine, phosphatidylglycerol (PG), and phosphatidylinositol within 30 min of wounding was not significantly different in PLDalpha-deficient and wild-type leaves. Phosphatidic acid (PA) levels increased rapidly in wild-type and, to a lesser extent, in PLDalpha-deficient plants. The acyl composition of the PA generated by wounding suggests that the major in vivo substrate of PLD in wild-type leaves was PC, and that PG hydrolysis accounted for 10-15% of the wound-induced PA in wild-type leaves. Comparison of the acyl compositions of the wound-induced PA of wild-type and PLDalpha-deficient leaves indicated that PLDalpha hydrolyzed PG more readily than other PLD isoforms did. Wounding produced substantial increases in free linoleic and linolenic acids in wild-type plants, whereas PLDalpha-deficient plants showed only a slight increase in linoleic acid and no significant increase in linolenic acid. These results demonstrate that PLDalpha and at least one other PLD isoform, as well as other hydrolytic enzymes, are active in mechanically wounded Arabidopsis leaves, and PLDalpha is involved in wound-induced metabolism of polyunsaturated fatty acids.     

Suppression of phospholipase Dalpha1 induces freezing tolerance in Arabidopsis: response of cold-responsive genes and osmolyte accumulation

Phospholipase D (PLD; EC 3.1.4.4) plays an important role in membrane lipid hydrolysis and in mediation of plant responses to a wide range of stresses. PLDalpha1 abrogation through antisense suppression in Arabidopsis thaliana resulted in a significant increase in freezing tolerance of both non-acclimated and cold-acclimated plants. Although non-acclimated PLDalpha1-deficient plants did not show the activation of cold-responsive C-repeat/dehydration-responsive element binding factors (CBFs) and their target genes (COR47 and COR78), they did accumulate osmolytes to much higher levels than did the non-acclimated wild-type plants. However, a stronger expression of COR47 and COR78 in response to cold acclimation and to especially freezing was observed in PLDalpha1-deficient plants. Furthermore, a slower activation of CBF1 was observed in response to cold acclimation in these plants compared to the wild-type plants. Typically, cold acclimation resulted in a higher accumulation of osmolytes in PLDalpha1-deficient plants than in wild-type plants. Inhibition of PLD activity by using lysophosphatidylethanolamine (LPE) also increased freezing tolerance of Arabidopsis, albeit to a lesser extent than did the PLD antisense suppression. Exogenous LPE induced expression of COR15a and COR47 in the absence of cold stimulus. These results suggest that PLDalpha1 plays a key role in freezing tolerance of Arabidopsis by modulating the cold-responsive genes and accumulation of osmolytes.     

Nonspecific Phospholipase C NPC4 Promotes Responses to Abscisic Acid and Tolerance to Hyperosmotic Stress in Arabidopsis

Diacyglycerol (DAG) is an important class of cellular lipid messengers, but its function in plants remains elusive. Here, we show that knockout of the Arabidopsis thaliana nonspecific phospholipase C (NPC4) results in a decrease in DAG levels and compromises plant response to abscisic acid (ABA) and hyperosmotic stresses. NPC4 hydrolyzes various phospholipids in a calcium-independent manner, producing DAG and a phosphorylated head group. NPC4 knockout (KO) plants display decreased ABA sensitivity in seed germination, root elongation, and stomatal movement and had decreased tolerance to high salinity and water deficiency. Overexpression of NPC4 renders plants more sensitive to ABA and more tolerant to hyperosmotic stress than wild-type plants. Addition of a short-chain DAG or a short-chain phosphatidic acid (PA) restores the ABA response of NPC4-KO to that of the wild type, but the addition of DAG together with a DAG kinase inhibitor does not result in a wild-type phenotype. These data suggest that NPC4-produced DAG is converted to PA and that NPC4 and its derived lipids positively modulate ABA response and promote plant tolerance to drought and salt stresses.     

Quantitative profiling of polar glycerolipid species from organs of wild-type Arabidopsis and a phospholipase Dalpha1 knockout mutant

Lipid profiling is a targeted metabolomics platform that provides a comprehensive analysis of lipid species with high sensitivity. Profiling based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides quantitative data and is adaptable to high throughput analyses. Here we report the profiling of 140 apparent molecular species of polar glycerolipids in Arabidopsis leaves, flower stalks, flowers, siliques, roots, and seeds. Considerable differences in lipid species occur among these organs, providing insights into the different lipid metabolic activities in a specific organ. In addition, comparative profiling between wild-type and a knockout mutant pldalpha1 (locus ID: AT3G15730) provides insight into the metabolic function of phospholipase D (PLD) in different organs. PLDalpha1 contributes significantly to phosphatidic acid (PA) levels in roots, seeds, flowers, and flower stalks, but little to basal PA levels in siliques and leaves. In seeds of the pldalpha1 mutant plants, levels of PA, lysophosphatidylcholine, and lysophosphatidylethanolamine were significantly lower than those of wild-type seeds, suggesting a role for PLDalpha1 in membrane lipid degradation in seeds.     

Patatin-related phospholipase pPLAIIIβ-induced changes in lipid metabolism alter cellulose content and cell elongation in Arabidopsis

The release of fatty acids from membrane lipids has been implicated in various plant processes, and the patatin-related phospholipases (pPLAs) constitute a major enzyme family that catalyzes fatty acid release. The Arabidopsis thaliana pPLA family has 10 members that are classified into three groups. Group 3 pPLAIII has four members but lacks the canonical lipase/esterase consensus catalytic sequences, and their enzymatic activity and cellular functions have not been delineated. Here, we show that pPLAIIIβ hydrolyzes phospholipids and galactolipids and additionally has acyl-CoA thioesterase activity. Alterations of pPLAIIIβ result in changes in lipid levels and composition. pPLAIIIβ-KO plants have longer leaves, petioles, hypocotyls, primary roots, and root hairs than wild-type plants, whereas pPLAIIIβ-OE plants exhibit the opposite phenotype. In addition, pPLAIIIβ-OE plants have significantly lower cellulose content and mechanical strength than wild-type plants. Root growth of pPLAIIIβ-KO plants is less sensitive to treatment with free fatty acids, the enzymatic products of pPLAIIIβ, than wild-type plants; root growth of pPLAIIIβ-OE plants is more sensitive. These data suggest that alteration of pPLAIIIβ expression and the resulting lipid changes alter cellulose content and cell elongation in Arabidopsis.     

儿茶素诱导的拟南芥根细胞膜脂变化

儿茶素是一种可以短时间内杀死植物细胞的植物毒素,由于具有强的植物毒性,儿茶素是开发除草剂的理想化合物,它可以诱导植物根系统的死亡。为了研究植物根细胞膜脂对化学胁迫的响应规律,我们运用高通量的脂类组学方法检测了拟南芥根中膜脂分子的组成,比较了儿茶素处理下拟南芥野生型（WS）及磷脂酶Dδ缺失突变体（PLDδ KO）根中膜脂分子的组成情况、膜脂含量、双键指数及碳链长度值。结果发现,儿茶素处理拟南芥根90min后,二半乳糖基二酰甘油（DGDG）、单半乳糖基二酰甘油（MGDG）、磷脂酰甘油（PG）、磷脂酰胆碱（PC）及磷脂酰肌醇（PI）的总含量在WS与PLDδ KO植株根中都显著下降,磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）在WS中下降,在PLDδ KO中上升。儿茶素处理导致PLDδ KO植株的PC/PE比值显著下降,WS植株PS碳链长度显著增加。上述结果说明儿茶素处理后,磷脂酶Dδ缺失突变体膜不稳定性增加,PLDδ KO植株对儿茶素胁迫更加敏感。     

The oleate-stimulated phospholipase D, PLDdelta, and phosphatidic acid decrease H2O2-induced cell death in Arabidopsis

Hydrolysis of common membrane phospholipids occurs in response to various environmental stresses, but the control and cellular function of this hydrolysis are not fully understood. Hydrogen peroxide (H2O2) is a pivotal signaling molecule involved in various stress responses. Here, we show that the plasma membrane-bound phospholipase D, PLDdelta, is activated in response to H2O2 and that the resulting phosphatidic acid (PA) functions to decrease H2O2-promoted programmed cell death. The Arabidopsis genome has 12 PLD genes, and knockout of PLDdelta abolishes specifically the oleate-stimulated PLD activity. H2O2 treatment of Arabidopsis cells activates PLD enzyme activity, and ablation of PLDdelta abolishes that activation. PLDdelta-null cells display increased sensitivity to H2O2-induced cell death. The addition of PA to PLDdelta-null cells mitigates the H2O2 effect, whereas suppression of the H2O2-induced PA formation in wild-type cells increases the effect. PLDdelta-ablated plants exhibit increased susceptibility to stress. These results demonstrate that activation of oleate-stimulated PLDdelta constitutes an important step in the plant response to H2O2 and increasing plant stress tolerance.     

Regulation of plant water loss by manipulating the expression of phospholipase Dα

Phospholipase D (PLD) has been implicated in various processes, including signal transduction, membrane trafficking, and membrane degradation. Multiple forms of PLD with distinct biochemical properties have been described in the cell. In Arabidopsis, PLDalpha and PLDgamma, but not PLDbeta, were detected in guard cells, and antisense suppression resulted in a specific loss of PLDalpha. The abrogation of PLDalpha rendered plants less sensitive to abscisic acid and impaired stomatal closure induced by water deficits. PLDalpha-depleted plants exhibited accelerated transpirational water loss and a decreased ability to tolerate drought stress. Overexpression of PLDalpha enhanced the leaf's sensitivity to abscisic acid. These findings provide molecular and physiological evidence that PLDalpha plays a crucial role in regulating stomatal movement and plant-water status.     

儿茶素诱导的拟南芥根细胞膜脂变化

儿茶素是一种可以短时间内杀死植物细胞的植物毒素,由于具有强的植物毒性,儿茶素是开发除草剂的理想化合物,它可以诱导植物根系统的死亡.为了研究植物根细胞膜脂对化学胁迫的响应规律,我们运用高通量的脂类组学方法检测了拟南芥根中膜脂分子的组成,比较了儿茶素处理下拟南芥野生型(WS)及磷脂酶Dδ缺失突变体( PLDδ-KO)根中膜脂分子的组成情况、膜脂含量、双键指数及碳链长度值.结果发现,儿茶素处理拟南芥根90 min后,二半乳糖基二酰甘油(DGDG)、单半乳糖基二酰甘油(MGDG)、磷脂酰甘油(PG)、磷脂酰胆碱(PC)及磷脂酰肌醇(PI)的总含量在WS与PLDδ-KO植株根中都显著下降,磷脂酰乙醇胺(PE)和磷脂酰丝氨酸(PS)在WS中下降,在PLDδ-KO中上升.儿茶素处理导致PLDδ-KO植株的PC/PE比值显著下降,WS植株PS碳链长度显著增加.上述结果说明儿茶素处理后,磷脂酶Dδ缺失突变体膜不稳定性增加,PLDδ-KO植株对儿茶素胁迫更加敏感.     

Phospholipase D- and phosphatidic acid-mediated signaling in plants

The phospholipase D (PLD) family in higher plants is composed of multiple members, and each of the Arabidopsis PLDs characterized displays distinguishable properties in activity regulation and/or lipid preferences. The molecular and biochemical heterogeneities of the plant PLDs play important roles in the timing, location, and amount of phosphatidic acid (PA) produced. PLD-catalyzed production of PA has been shown to play important roles in plant growth, development, and response to various stresses, including drought, salinity, freezing, and nutrient deficiency. PLD and PA affect cellular processes through different modes of action, including direct target protein binding and biophysical effects on cell membranes. Improved knowledge on the mechanism by which specific PLDs and PA mediate given plant responses will facilitate the understanding of the molecular processes that connect the stimulus perception on membranes to intracellular actions and physiological responses.     

Suppression of phospholipase Dγs confers increased aluminum resistance in Arabidopsis thaliana

Aluminum (Al) toxicity is the major stress in acidic soil that comprises about 50% of the world's arable land. The complex molecular mechanisms of Al toxicity have yet to be fully determined. As a barrier to Al entrance, plant cell membranes play essential roles in plant interaction with Al, and lipid composition and membrane integrity change significantly under Al stress. Here, we show that phospholipase Dγs (PLDγs) are induced by Al stress and contribute to Al-induced membrane lipid alterations. RNAi suppression of PLDγ resulted in a decrease in both PLDγ1 and PLDγ2 expression and an increase in Al resistance. Genetic disruption of PLDγ1 also led to an increased tolerance to Al while knockout of PLDγ2 did not. Both RNAi-suppressed and pldγ1-1 mutants displayed better root growth than wild-type under Al stress conditions, and PLDγ1-deficient plants had less accumulation of callose, less oxidative damage, and less lipid peroxidation compared to wild-type plants. Most phospholipids and glycolipids were altered in response to Al treatment of wild-type plants, whereas fewer changes in lipids occurred in response to Al stress in PLDγ mutant lines. Our results suggest that PLDγs play a role in membrane lipid modulation under Al stress and that high activities of PLDγs negatively modulate plant tolerance to Al.     

Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.     

Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis

Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLDalpha, PLDbeta, and PLDgamma differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD, PLDalpha, decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein. Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLDalpha attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLDalpha-suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA.     

Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate

In mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane trafficking and defence responses. In plant cells a signalling role for PLD and PA is also emerging. Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger. Here we report that increases in PA and its conversion to DGPP are common stress responses to water deficit. Increases occur within minutes of treatment and are dependent on the level of stress. Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1-butanol, and part is due to diacylglycerol kinase activity as monitored via 32P-PA formation in a differential labelling protocol. Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cell-suspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum. These results provide further evidence that PLD and PA play a role in plant signalling, and provide the first demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.     

Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dδ Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation

Senescence is the last phase of the plant life cycle and has an important role in plant development. Degradation of membrane lipids is an essential process during leaf senescence. Several studies have reported fundamental changes in membrane lipids and phospholipase D (PLD) activity as leaves senesce. Suppression of phospholipase Dα1 (PLDα1) retards abscisic acid (ABA)-promoted senescence. However, given the absence of studies that have profiled changes in the compositions of membrane lipid molecules during leaf senescence, there is no direct evidence that PLD affects lipid composition during the process. Here, we show that application of n-butanol, an inhibitor of PLD, and N-Acylethanolamine (NAE) 12∶0, a specific inhibitor of PLDα1, retarded ABA-promoted senescence to different extents. Furthermore, phospholipase Dδ (PLDδ) was induced in leaves treated with ABA, and suppression of PLDδ retarded ABA-promoted senescence in Arabidopsis. Lipid profiling revealed that detachment-induced senescence had different effects on plastidic and extraplastidic lipids. The accelerated degradation of plastidic lipids during ABA-induced senescence in wild-type plants was attenuated in PLDδ-knockout (PLDδ-KO) plants. Dramatic increases in phosphatidic acid (PA) and decreases in phosphatidylcholine (PC) during ABA-induced senescence were also suppressed in PLDδ-KO plants. Our results suggest that PLDδ-mediated hydrolysis of PC to PA plays a positive role in ABA-promoted senescence. The attenuation of PA formation resulting from suppression of PLDδ blocks the degradation of membrane lipids, which retards ABA-promoted senescence.     

Phospholipase D in the signaling networks of plant response to abscisic acid and reactive oxygen species

Phospholipase D (PLD) has been implicated in different cellular processes in plant growth, development, and stress responses. Recent results have provided insights into the molecular mechanism by which PLD and its lipid product phosphatidic acid (PA) participate in cell signaling. Effector proteins that have been identified for PLD and PA in plants include a heterotrimeric G protein, protein phosphatase, and protein kinase. Evidence has been presented for a direct link from a PLD, PA, to a target protein in specific physiological processes. PLD and PA play multiple roles in the signaling networks of plant response to abscisic acid and reactive oxygen species.     

Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis

A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD alpha, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLD alpha-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLD alpha-deficient plants. These data suggest that PC, rather than PE and PG, is the major in vivo substrate of PLD alpha. The action of PLD alpha during freezing is of special interest because Arabidopsis plants that are deficient in PLD alpha have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLD alpha-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.     

Differential degradation of extraplastidic and plastidic lipids during freezing and post-freezing recovery in Arabidopsis thaliana

Changes in membrane lipid composition play important roles in plant adaptation to and survival after freezing. Plant response to cold and freezing involves three distinct phases: cold acclimation, freezing, and post-freezing recovery. Considerable progress has been made toward understanding lipid changes during cold acclimation and freezing, but little is known about lipid alteration during post-freezing recovery. We previously showed that phospholipase D (PLD) is involved in lipid hydrolysis and Arabidopsis thaliana freezing tolerance. This study was undertaken to determine how lipid species change during post-freezing recovery and to determine the effect of two PLDs, PLDalpha1 and PLDdelta, on lipid changes during post-freezing recovery. During post-freezing recovery, hydrolysis of plastidic lipids, monogalactosyldiacylglycerol and plastidic phosphatidylglycerol, is the most prominent change. In contrast, during freezing, hydrolysis of extraplastidic phospholipids, phosphatidylcholine and phosphatidylethanolamine, occurs. Suppression of PLDalpha1 decreased phospholipid hydrolysis and phosphatidic acid production in both the freezing and post-freezing phases, whereas ablation of PLDdelta increased lipid hydrolysis and phosphatidic acid production during post-freezing recovery. Thus, distinctly different changes in lipid hydrolysis occur in freezing and post-freezing recovery. The presence of PLDalpha1 correlates with phospholipid hydrolysis in both freezing and post-freezing phases, whereas the presence of PLDdelta correlates with reduced lipid hydrolysis during post-freezing recovery. These data suggest a negative role for PLDalpha1 and a positive role for PLDdelta in freezing tolerance.     

In vivo substrates and the contribution of the common phospholipase D,PLDα, to wound-induced metabolism of lipids in Arabidopsis

The common plant phospholipase D (PLD), PLDα, has been proposed to be involved in wound-induced production of jasmonic acid. To better understand the role(s) of PLDα in the wound response, detailed lipid analysis was carried out to determine the in vivo substrates and the contribution of PLDα in wound-induced lipid metabolism in Arabidopsis thaliana. Mechanical wounding of Arabidopsis leaves resulted in significantly less hydrolysis of phosphatidylcholine (PC) in PLDα-deficient than in wild-type plants. Hydrolysis of phosphatidylethanolamine, phosphatidylglycerol (PG), and phosphatidylinositol within 30 min of wounding was not significantly different in PLDα-deficient and wild-type leaves. Phosphatidic acid (PA) levels increased rapidly in wild-type and, to a lesser extent, in PLDα-deficient plants. The acyl composition of the PA generated by wounding suggests that the major in vivo substrate of PLD in wild-type leaves was PC, and that PG hydrolysis accounted for 10–15% of the wound-induced PA in wild-type leaves. Comparison of the acyl compositions of the wound-induced PA of wild-type and PLDα-deficient leaves indicated that PLDα hydrolyzed PG more readily than other PLD isoforms did. Wounding produced substantial increases in free linoleic and linolenic acids in wild-type plants, whereas PLDα-deficient plants showed only a slight increase in linoleic acid and no significant increase in linolenic acid. These results demonstrate that PLDα and at least one other PLD isoform, as well as other hydrolytic enzymes, are active in mechanically wounded Arabidopsis leaves, and PLDα is involved in wound-induced metabolism of polyunsaturated fatty acids.