Ana M. Laxalt

Phospholipase D Activation Correlates with Microtubule Reorganization in Living Plant Cells

A phospholipase D (PLD) was shown recently to decorate microtubules in plant cells. Therefore, we used tobacco BY-2 cells expressing the microtubule reporter GFP-MAP4 to test whether PLD activation affects the organization of plant microtubules. Within 30 min of adding n-butanol, a potent activator of PLD, cortical microtubules were released from the plasma membrane and partially depolymerized, as visualized with four-dimensional confocal imaging. The isomers sec- and tert-butanol, which did not activate PLD, did not affect microtubule organization. The effect of treatment on PLD activation was monitored by the in vivo formation of phosphatidylbutanol, a specific reporter of PLD activity. Tobacco cells also were treated with mastoparan, xylanase, NaCl, and hypoosmotic stress as reported activators of PLD. We confirmed the reports and found that all treatments induced microtubule reorganization and PLD activation within the same time frame. PLD still was activated in microtubule-stabilized (taxol) and microtubule-depolymerized (oryzalin) situations, suggesting that PLD activation triggers microtubular reorganization and not vice versa. Exogenously applied water-soluble synthetic phosphatidic acid did not affect the microtubular cytoskeleton. Cell cycle studies revealed that n-butanol influenced not just interphase cortical microtubules but also those in the preprophase band and phragmoplast, but not those in the spindle structure. Cell growth and division were inhibited in the presence of n-butanol, whereas sec- and tert-butanol had no such effects. Using these novel insights, we propose a model for the mechanism by which PLD activation triggers microtubule reorganization in plant cells.

Phospholipid signalling in plant defence

Phospholipid-derived molecules are emerging as novel second messengers in plant defence signalling. Recent research has begun to reveal the signals produced by the enzymes phospholipase C, phospholipase D and phospholipase A2 and their putative downstream targets. These include the activation of a MAP kinase cascade and triggering of an oxidative burst by phosphatidic acid; the regulation of ion channels and proton pumps by lysophospholipids and free fatty acids; and the conversion of free fatty acids into bioactive octadecanoids such as jasmonic acid.

Multiple PLDs Required for High Salinity and Water Deficit Tolerance in Plants

High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plants response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plants response to high salinity and dehydration. A tomato (Lycopersicon esculentum) α-class PLD, LePLDα1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDα1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDα1 and AtPLDδ were found to be activated in response to salt stress. Moreover, pldα1 and pldδ single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth.

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 PLDβ1 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.

Nitric Oxide Triggers Phosphatidic Acid Accumulation via Phospholipase D during Auxin-Induced Adventitious Root Formation in Cucumber

Auxin and nitric oxide (NO) play fundamental roles throughout plant life. NO is a second messenger in auxin signal transduction leading to root developmental processes. The mechanisms triggered by auxin and NO that direct adventitious root (AR) formation are beginning to be unraveled. The goal of this work was to study phospholipid (PL) signaling during the auxin- and NO-induced AR formation in cucumber (Cucumis sativus) explants. Explants were labeled with 32P-inorganic phosphate and treated with the auxins indole-3-acetic acid or 1-naphthylacetic acid, or the NO donor S-nitroso N-acetyl penicillamine, in the presence or absence of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. PLs were separated by thin-layer chromatography and quantified. We report that the signaling PLs phosphatidic acid (PA), phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulated within 1 min after auxin or NO treatment. Both auxin and NO evoked similar and transient time course responses, since signaling PLs returned to control levels after 20 or 30 min of treatment. The results indicate that auxin relies on NO in inducing PA, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulation. Furthermore, we demonstrate that auxin and NO trigger PA formation via phospholipase D (PLD) activity. Explants treated for 10 min with auxin or NO displayed a 200% increase in AR number compared with control explants. In addition, PLD activity was required for the auxin- and NO-induced AR formation. Finally, exogenously applied PA increased up to 300% the number of ARs. Altogether, our data support the idea that PLD-derived PA is an early signaling event during AR formation induced by auxin and NO in cucumber explants.

Accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase RNA under biological stress conditions and elicitor treatments in potato

Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whether the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. This increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts sinergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.

Extracellular ATP induces nitric oxide production in tomato cell suspensions

In plant as well as in animal systems, extracellular ATP (eATP) regulates a broad range of physiological processes. In animals, downstream signaling events regulated by eATP have been described before. Among the second messengers reported to be involved in eATP signaling, there are inositol

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.

Phospholipase Dδ is involved in nitric oxide-induced stomatal closure

Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H2O2 and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H2O2 levels as the wild type in response to ABA. However, ABA- or H2O2-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H2O2 in ABA-induced stomatal closure.

Phosphatidic acid formation is required for extracellular ATP‐mediated nitric oxide production in suspension‐cultured tomato cells

*In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca(2+), nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown. *In vivo phospholipid signaling upon extracellular ATP treatment was studied in (32)P(i)-labeled suspension-cultured tomato (Solanum lycopersicum) cells. *Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mM induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase. *Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation.