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.     

Cytidinediphosphate diacylglycerol synthase—Mediated phosphatidic acid metabolism is crucial for early embryonic development of Arabidopsis

Embryonic development is a key developmental event in plant sexual reproduction; however, regulatory networks of plant early embryonic development, particularly the effects and functional mechanisms of phospholipid molecules are still unknown due to the limitation of sample collection and analysis. We innovatively applied the microspore-derived in vitro embryogenesis of Brassica napus and revealed the dynamics of phospholipid molecules, especially phosphatidic acid (PA, an important second messenger that plays an important role in plant growth, development, and stress responses), at different embryonic developmental stages by using a lipidomics approach. Further analysis of Arabidopsis mutants deficiency of CDS1 and CDS2 (cytidinediphosphate diacylglycerol synthase, key protein in PA metabolism) revealed the delayed embryonic development from the proembryo stage, indicating the crucial effect of CDS and PA metabolism in early embryonic development. Decreased auxin level and disturbed polar localization of auxin efflux carrier PIN1 implicate that CDS-mediated PA metabolism may regulate early embryogenesis through modulating auxin transport and distribution. These results demonstrate the dynamics and importance of phospholipid molecules during embryo development, and provide informative clues to elucidate the regulatory network of embryogenesis.     

Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.     

Combined metabolomic and genetic approaches reveal a link between the polyamine pathway and albumin 2 in developing pea seeds

Several legume seed proteins that are potentially allergenic, poorly digested by farm animals, and/or have undesirable functional properties, have been described. One of these is the albumin protein in pea (Pisum sativum) called PA2. A naturally occurring mutant line that lacks PA2 has been exploited in studies to determine the biological function of this nonstorage protein in seed development. The mutant, which has a small seed, a tall plant phenotype, and lacks most of the PA2-encoding genes, has been crossed with a standard cultivar, 'Birte,' which contains PA2 to give rise to a recombinant inbred (RI) population. An F(3) line carrying the mutation and having a short plant phenotype has been used to generate backcross (BC) lines with 'Birte.' Despite having a lower albumin content, seeds from the mutant parent and RI lines lacking PA2 have an equivalent or higher seed nitrogen content. Metabolite profiling of seeds revealed major differences in amino acid composition and polyamine content in the two parent lines. This was investigated further in BC lines, where the effects of differences in seed size and plant height between the two parents were eliminated. Here, differences in polyamine synthesis were maintained as was a difference in total seed protein between the BC line lacking PA2 and 'Birte.' Analysis of enzyme activities in the pathways of polyamine synthesis revealed that the differences in spermidine content were attributable to changes in the overall activities of spermidine synthase and arginine decarboxylase. Although the genes encoding spermidine synthase and PA2 both localized to the pea linkage group I, the two loci were shown not to be closely linked and to have recombined in the BC lines. A distinct locus on linkage group III contains a gene that is related to PA2 but expressed predominantly in flowers. The results provide evidence for a role of PA2 in regulating polyamine metabolism, which has important functions in development, metabolism, and stress responses in plants.     

A salt stress-activated mitogen-activated protein kinase in soybean is regulated by phosphatidic acid in early stages of the stress response

Salt stress inhibits plant growth and development and plants activate kinase-dependent survival pathways in response to salt stress. However, the role of soybean mitogenactivated protein kinases (MAPKs) in salt stress response has yet to be characterized. In this study, we found that salt stress activates Glycine max MAP kinase 1 (GMK1), a soybean MAPK. The activity of GMK1 induced with increasing salt concentrations, up to 300 mM NaCl, after 5 min of the treatment and was regulated by post-translational modification. We found that mastoparan, a heteromeric G-protein activator, also activated GMK1, and that n-butanol, a phospholipase D inhibitor, and neomycin, a phospholipase C inhibitor, inhibited its activity. Moreover, GMK1 activity was reduced by suramin, a heteromeric G-protein inhibitor, and by two inhibitors of phosphatidic acid (PA) generation after 5 min of 300 mM NaCl treatment. Endogenous PA levels were highest 5 min after induction of salt stress, and exogenous PA directly activated GMK1. From these data, we propose that salt stress signaling is transduced from heteromeric G-protein to GMK1 via phospholipases in the early stages of the response to salt stress in soybean.     

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.     

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.     

Phospholipids as Plant Growth Regulators

In this paper the potential to use phospholipids and lysophospholipids as plant growth regulators is discussed. Recent evidence shows that phospholipids and phospholipases play an important signalling role in the normal course of plant development and in the response of plants to abiotic and biotic stress. It is apparent that phospholipase A (PLA), C (PLC) and D (PLD), lysophospholipids, and phosphatidic acid (PA) are key components of plant lipid signalling pathways. By comparison, there is very little information available on the effect of exogenously applied phospholipids on plant growth and development. This paper serves to introduce phospholipids as a novel class of plant growth regulator for use in commercial plant production. The biochemistry and physiology of phospholipids is discussed in relation to studies in which phospholipids and lysophospholipids have been applied to plants and plant parts. Implicit in the observations is that phospholipids impact the hypersensitive response and systemic acquired resistance in plants to improve crop performance and product quality. Based on published data, a scheme outlining a possible mode of action of exogenously applied phospholipids is proposed.     

一氧化氮在植物生长发育和抗逆过程中的作用研究进展

NO不仅在植物的抗病过程中发挥重要作用,同时也参与植物生长、发育和对干旱、高盐、高温、低温等非生物胁迫的响应等过程。该文对近年来国内外有关NO在植物生长、发育、非生物胁迫抗性过程中的作用及其与植物激素之间的互作关系等方面的研究进展进行综述,为相关研究提供信息和资料。     

The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice

We are exploiting the broad host range of the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 to elucidate the molecular basis of bacterial virulence in plants, nematodes, insects and mice. In this report, we characterize the role that two PA14 gene products, MucD and AlgD, play in virulence. MucD is orthologous to the Escherichia coli periplasmic protease and chaperone DegP. DegP homologues are known virulence factors that play a protective role in stress responses in various species. AlgD is an enzyme involved in the biosynthesis of the exopolysaccharide alginate, which is hyperinduced in mucD mutants. A PA14 mucD mutant was significantly impaired in its ability to cause disease in Arabidopsis thaliana and mice and to kill the nematode Caenorhabditis elegans. Moreover, MucD was found to be required for the production of an extracellular toxin involved in C. elegans killing. In contrast, a PA14 algD mutant was not impaired in virulence in plants, nematodes or mice. A mucDalgD double mutant had the same phenotype as the mucD single mutant in the plant and nematode pathogenesis models. However, the mucDalgD double mutant was synergistically reduced in virulence in mice, suggesting that alginate can partially compensate for the loss of MucD function in mouse pathogenesis.     

Downregulation of sirtuin 3 by palmitic acid increases the oxidative stress,impairment of mitochondrial function,and apoptosis in liver cells

Elevated levels of saturated fatty acids show a strong cytotoxic effect in liver cells. Sirtuin 3 (SIRT3), a mitochondrially localized member of NAD⁺‐dependent deacetylase has been shown to protect hepatocytes against the oxidative stress. The role of SIRT3 on the cytotoxicity caused by fatty acids in liver cells is not fully understood. The aim of this study was to evaluate the expression level of SIRT3, oxidative stress, and mitochondrial impairments in human hepatoma HepG2 cells exposed to palmitic acid (PA). Our results showed that PA treatment caused the deposition of lipid droplets and resulted in an increased expression of tumor necrosis factor‐α in a dose‐dependent manner. Excessive accumulation of PA induces the reactive oxygen species formation and apoptosis while dissipating the mitochondrial transmembrane potential. The level of SIRT3 expression in both nuclear and mitochondrial fractions in HepG2 cells was decreased with the increase in PA concentrations. However, in the cytosolic fraction, the SIRT3 was undetectable. In conclusion, our results showed that PA caused an increase in inflammation and oxidative stress in HepG2 cells. The exposure of PA also resulted in the decline in transmembrane potential and an increase in apoptosis. The underexpression of nuclear and mitochondrial SIRT3 by PA suggests that the PA target the process that regulates the stress‐related gene expression and mitochondrial functions.     

Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses

Nitric oxide (NO), polyamines (PAs), diamine oxidases (DAO) and polyamine oxidases (PAO) play important roles in wide spectrum of physiological processes such as germination, root development, flowering and senescence and in defence responses against abiotic and biotic stress conditions. This functional overlapping suggests interaction of NO and PA in signalling cascades. Exogenous application of PAs putrescine, spermidine and spermine to Arabidopsis seedlings induced NO production as observed by fluorimetry and fluorescence microscopy using the NO-binding fluorophores DAF-2 and DAR-4M. The observed NO release induced by 1 mM spermine treatment in the Arabidopsis seedlings was very rapid without apparent lag phase. These observations pave a new insight into PA-mediated signalling and NO as a potential mediator of PA actions. When comparing the functions of NO and PA in plant development and abiotic and biotic stresses common to both signalling components it can be speculated that NO may be a link between PA-mediated stress responses filing a gap between many known physiological effects of PAs and amelioration of stresses. NO production indicated by PAs could be mediated either by H₂O₂, one reaction product of oxidation of PAs by DAO and PAO, or by unknown mechanisms involving PAs, DAO and PAO.     

Does polyamine catabolism influence root development and xylem differentiation under stress conditions?

Amine oxidases (AOs) oxidize polyamines (PAs) to aldehydes, simultaneously producing the removed amine moiety and hydrogen peroxide (H₂O₂). AOs, which include copper-containing amine oxidases (CuAOs) and flavin-containing amine oxidases (PAOs), are stress-inducible enzymes involved in both PA homeostasis and H₂O₂ production. Here, we suggest that H₂O₂ derived from PAO-mediated PA catabolism has a role in inducing root xylem differentiation during plant stress responses, whereas its involvement in this event during plant development under physiological conditions is not suitably supported by the currently available data. Moreover, we show that spermidine (Spd) supply leads to a higher induction of cell death in wild-type (WT) tobacco (Nicotiana tabacum) plants as compared to tobacco plants over-expressing maize (Zea mays) PAO (S-ZmPAO) in the cell wall, in apparent contradiction with the already reported results obtained by the analysis of the corresponding WT and S-ZmPAO Spd-untreated plants. Considering this last observation, we propose that PAs  diversely affect plant development and stress responses depending on the expression levels of AOs, which in turn may lead to different plant responses by altering the PAs/H₂O₂ balance.     

Plant Polyamines in Reproductive Activity and Response to Abiotic Stress*

In this review we will focus on two areas in which the accumulated evidence for a critical physiological role of polyamines is becoming compelling, i.e. reproductive activity and response to abiotic stress. Regarding reproductive behavior, it seems clear that polyamines are members of the array of internal compounds required for flower initiation, normal floral organ morphogenesis, fruit growth and fruit ripening in particular plant species. Abiotic stresses such as osmotic stress can “turn on” arginine decarboxylase (ADC) genes, resulting in a rapid increase in their mRNA levels. Localization of ADC enzyme in the chloroplast suggests a role of PAs in the maintenance of photosynthetic activity during senescence responses induced by osmotic stress. We envisage that the use of transgenic plants and improved molecular probes will resolve in the near future the physiological significance of stress-induced changes in PA metabolism as well as the role of these compounds in reproductive activity.     

Plant PA signaling via diacylglycerol kinase

Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plant's response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential ³²P-radiolabelling protocol can be used. Based on this, and more recently on reverse-genetic approaches, DGK has taken center stage, next to PLD, as a generator of PA in biotic and abiotic stress responses. The DAG substrate is generally thought to be derived from PI-PLC activity. The model plant system Arabidopsis thaliana has 7 DGK isozymes, two of which, AtDGK1 and AtDGK2, resemble mammalian DGK?, containing a conserved kinase domain, a transmembrane domain and two C1 domains. The other ones have a much simpler structure, lacking the C1 domains, not matched in animals. Several protein targets have now been discovered that bind PA. Whether the PA molecules engaged in these interactions come from PLD or DGK remains to be elucidated.