Exogenous Nitro-Oleic Acid Treatment Inhibits Primary Root Growth by Reducing the Mitosis in the Meristem in Arabidopsis thaliana

Nitric oxide (NO) is a second messenger that regulates a broad range of physiological processes in plants. NO-derived molecules called reactive nitrogen species (RNS) can react with unsaturated fatty acids generating nitrated fatty acids (NO2-FA). NO2-FA work as signaling molecules in mammals where production and targets have been described under different stress conditions. Recently, NO2-FAs were detected in plants, however their role(s) on plant physiological processes is still poorly known. Although in this work NO2-OA has not been detected in any Arabidopsis seedling tissue, here we show that exogenous application of nitro-oleic acid (NO2-OA) inhibits Arabidopsis primary root growth; this inhibition is not likely due to nitric oxide (NO) production or impaired auxin or cytokinin root responses. Deep analyses showed that roots incubated with NO2-OA had a lower cell number in the division area. Although this NO2-FA did not affect the hormonal signaling mechanisms maintaining the stem cell niche, plants incubated with NO2-OA showed a reduction of cell division in the meristematic area. Therefore, this work shows that the exogenous application of NO2-OA inhibits mitotic processes subsequently reducing primary root growth.Fil: Di Fino, Luciano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Cerrudo, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Salvatore, Sonia Rosana. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Schopfer, Francisco Jose. University of Pittsburgh at Johnstown; Estados UnidosFil: Garcia-Mata, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

The tomato phosphatidylinositol-phospholipase C2 (SlPLC2) is required for defense gene induction by the fungal elicitor xylanase

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated upon pathogen attack. We have previously shown that the fungal elicitor xylanase rapidly induces nitric oxide (NO), which is required for PI-PLCs activity and downstream defense responses in tomato cell suspensions. Here, we show that all six SlPLC genes are expressed in tomato cell suspensions. Treatment of the cells with xylanase induces an early increase in SlPLC5 transcript levels, followed by a raise of the amount of SlPLC2 transcripts. The production of NO is required to augment SlPLC5 transcript levels in xylanase-treated tomato cells. Xylanase also induces SlPLC2 and SlPLC5 transcript levels in planta. We knocked-down the expression of SlPLC2 and SlPLC5 by virus-induced gene silencing. We found that SlPLC2 is required for xylanase-induced expression of the defense-related genes PR1 and HSR203J.Fil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Ramírez, Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Abd El Haliem, Ahmed. Wageningen University; Países BajosFil: Vossen, Jack H.. Wageningen University; Países BajosFil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Joosten, Matthieu H. A. J.. Wageningen University; Países BajosFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Hydrogen sulfide increases production of NADPH oxidase-dependent hydrogen peroxide and phospholipase D-derived phosphatidic acid in guard cell signaling

Hydrogen sulfide (H2S) is an important gaseous signaling molecule in plants that participates in stress responses and development. L-Cys desulfhydrase 1, one of the enzymatic sources of H2S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H2S in stomatal closure and the interplay between H2S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H2O2) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis (Arabidopsis thaliana). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for H2S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H2S stimulates H2O2 production in Arabidopsis guard cells. Additionally, we observed an interplay between H2S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H2S-induced stomatal closure, and most of the H2S-dependent H2O2 production required the activity of PLDα1. Finally, we showed that H2S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H2S in the signaling network that controls stomatal closure, and suggest that H2S regulates NADPH oxidase and PLD activity in guard cells.Fil: Scuffi, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Nietzel, Thomas. Westfalische Wilhelms Universitat; Alemania. Universitat Bonn; AlemaniaFil: Di Fino, Luciano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Meyer, Andreas J.. Universitat Bonn; AlemaniaFil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Schwarzlände, Markus. Westfalische Wilhelms Universitat; Alemania. Universitat Bonn; AlemaniaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Garcia-Mata, Carlos. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Nitro-oleic acid triggers ROS production via NADPH oxidase activation in plants: A pharmacological approach

Nitrated fatty acids (NO2-FAs) are important signaling molecules in mammals. NO2-FAs are formed by the addition reaction of nitric oxide- and nitrite-derived nitrogen dioxide with unsaturated fatty acid double bonds. The study of NO2-FAs in plant systems constitutes an interesting and emerging area. The presence of NO2-FA has been reported in olives, peas, rice and Arabidopsis. To gain a better understanding of the role of NO2-FA on plant physiology, we analyzed the effects of exogenous application of nitro-oleic acid (NO2-OA). In tomato cell suspensions we found that NO2-OA induced reactive oxygen species (ROS) production in a dose-dependent manner via activation of NADPH oxidases, a mechanism that requires calcium entry from the extracellular compartment and protein kinase activation. In tomato and Arabidopsis leaves, NO2-OA treatments induced two waves of ROS production, resembling plant defense responses. Arabidopsis NADPH oxidase mutants showed that NADPH isoform D (RBOHD) was required for NO2-OA-induced ROS production. In addition, on Arabidopsis isolated epidermis, NO2-OA induced stomatal closure via RBOHD and F. Altogether, these results indicate that NO2-OA triggers NADPH oxidase activation revealing a new signaling role in plants.Fil: Arruebarrena Di Palma, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Di Fino, Luciano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Salvatore, Sonia Rosana. University of Pittsburgh; Estados UnidosFil: D'Ambrosio, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Garcia-Mata, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Schopfer, Francisco Jose. University of Pittsburgh; Estados UnidosFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Nitro-Oleic Acid Induced Reactive Oxygen Species Formation and Plant Defense Signaling in Tomato Cell Suspensions

Nitrated fatty acids (NO2-FAs) are formed by the addition reaction of nitric oxide- and nitrite-derived nitrogen dioxide with unsaturated fatty acids. Nitrated fatty acids act as signaling molecules in mammals through the formation of covalent adducts with cellular thiols. The study of NO2-FAs in plant systems constitutes an interesting and emerging area. The presence of NO2-FA has been reported in olives, peas, rice and in Arabidopsis. To gain a better understanding of the role of NO2-FA on plant physiology, we analyzed the effects of exogenous application of nitro-oleic acid (NO2-OA) to tomato cell cultures. We found that NO2-OA induced reactive oxygen species (ROS) production in a dose-dependent manner via activation of NADPH oxidases, which requires calcium entry from the extracellular compartment and protein kinase activation, a mechanism that resembles the plant defense responses. NO2-OA-induced ROS production, expression of plant defense genes and led to cell death. The mechanism of action of NO2-OA involves a reduction in the glutathione cellular pool and covalently addition reactions with protein thiols and reduced glutathione. Altogether, these results indicate that NO2-OA triggers responses associated with plant defense, revealing its possible role as a signal molecule in biotic stress.Fil: Arruebarrena Di Palma, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Di Fino, Luciano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Salvatore, Sonia Rosana. University of Pittsburgh; Estados UnidosFil: D'ambrosio, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Gergoff Grozeff, Gustavo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Garcia-Mata, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Schopfer, Francisco Jose. University of Pittsburgh; Estados UnidosFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Role for arabidopsis PLC7 in stomatal movement, seed mucilage attachment, and leaf serration

Phospholipase C (PLC) has been suggested to play important roles in plant stress and development. To increase our understanding of PLC signaling in plants, we have started to analyze knock-out (KO), knock-down (KD) and overexpression mutants of Arabidopsis thaliana, which contains nine PLCs. Earlier, we characterized PLC2, PLC3 and PLC5. Here, the role of PLC7 is functionally addressed. Promoter-GUS analyses revealed that PLC7 is specifically expressed in the phloem of roots, leaves and flowers, and is also present in trichomes and hydathodes. Two T-DNA insertion mutants were obtained, i.e., plc7-3 being a KO- and plc7-4 a KD line. In contrast to earlier characterized phloem-expressed PLC mutants, i.e., plc3 and plc5, no defects in primary- or lateral root development were found for plc7 mutants. Like plc3 mutants, they were less sensitive to ABA during stomatal closure. Double-knockout plc3 plc7 lines were lethal, but plc5 plc7 (plc5/7) double mutants were viable, and revealed several new phenotypes, not observed earlier in the single mutants. These include a defect in seed mucilage, enhanced leaf serration, and an increased tolerance to drought. Overexpression of PLC7 enhanced drought tolerance too, similar to what was earlier found for PLC3-and PLC5 overexpression. In vivo 32Pi-labeling of seedlings and treatment with sorbitol to mimic drought stress, revealed stronger PIP2 responses in both drought-tolerant plc5/7 and PLC7-OE mutants. Together, these results show novel functions for PLC in plant stress and development. Potential molecular mechanisms are discussed.Fil: van Wijk, Ringo. University of Amsterdam; Países BajosFil: Zhang, Qianqian. University of Amsterdam; Países BajosFil: Zarza, Xavier. University of Amsterdam; Países BajosFil: Lamers, Mart. University of Amsterdam; Países BajosFil: Reyes Marquez, Francisca. Wageningen University and Research, Wageningen; Países BajosFil: Guardia, Aisha Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Scuffi, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Garcia-Mata, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Ligterink, Wilco. Wageningen University and Research, Wageningen; Países BajosFil: Haring, Michel A.. University of Amsterdam; Países BajosFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Munnik, Teun. University of Amsterdam; Países Bajo

Mechanisms of Xylanase-induced Nitric Oxide and Phosphatidic Acid Production in Tomato Cells

The second messenger nitric oxide (NO), phosphatidic acid (PA) and reactive oxygen species (ROS) are involved in the plant defense response during plant–pathogen interactions. NO has been shown to participate in PA production in response to the pathogen-associated molecular pattern xylanase in tomato cell suspensions. Defense responses downstream of PA include ROS production. The goal of this work was to study the signaling mechanisms involved in PA production during the defense responses triggered by xylanase and mediated by NO in the suspension-cultured tomato cells. We analyzed the participation of protein kinases, guanylate cyclase and the NO-mediated posttranslational modification S-nitrosylation, by means of pharmacology and biochemistry. We showed that NO, PA and ROS levels are significantly diminished by treatment with the general protein kinase inhibitor staurosporine. This indicates that xylanase-induced protein phosphorylation events might be the important components leading to NO formation, and hence for the downstream regulation of PA and ROS levels. When assayed, a guanylate cyclase inhibitor or a cGMP analog did not alter the PA accumulation. These results suggest that a cGMP-mediated pathway is not involved in xylanase-induced PA formation. Finally, the inhibition of protein S-nitrosylation did not affect NO formation but compromised PA and ROS production. Data collectively indicate that upon xylanase perception, cells activate a protein kinase pathway required for NO formation and that, S-nitrosylation-dependent mechanisms are involved in downstream signaling leading to PA and ROS.Fil: Lanteri, Maria Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentin

H2S como un nuevo componente de la señalización estomática durante el ataque de patógenos

Hydrogen sulfide (H2S) is a new gasotransmitter involved in several processes in plants. It is endogenously produced in part, by the cytosolic enzyme L-cysteine desulfhydrase 1 (DES1) which degrades L-cysteine into H2S, piruvate and ammonia. It is known that participates actively during stomatal closure induced by different stimuli. Stomata are pores surrounded by a pair of cells, guard cells, through which plants regulate the gaseous exchange with the environment and the loss of water by evapotranspiration. Its regulation is important to maintain carbon and water homeostasis and also for plant defense since pathogen can enter into the plant through them.In our lab we demonstrated, using Arabidopsis thaliana epidermal peels, that H2S and DES1 participate in bacterial elicitor flg22-induced stomatal closure. This signaling network includes other components like NADPH oxidase RBOHD and phosholipase C2 (PLC2).Here we move forward studying the role of H2S during flg22-induced stomatal closure. We made use of wild type Arabidopsis plants to evaluate pathogen-response genes expression specifically in guard cells upon GYY (a H2S donor) treatments and DES1 expression under flg22 treatment. Furthermore we analyzed MAPK activity in response to flg22 and Pst DC3000 hrcC- infection in Arabidopsis des1 mutant plants.Fil: Scuffi, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Garcia-Mata, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaXXXII Reunión Argentina y XVI Congreso Latinoamericano de Fisiología VegetalCordobaArgentinaSociedad Argentina de Fisiología Vegeta

Phosphatidylinositol 4-phosphate accumulates extracellularly upon xylanase treatment in tomato cell suspensions

Various phosphoinositides have been implicated in plant defence signalling. Until now, such molecules have been exclusively related to intracellular signalling. Here, evidence is provided for the detection of extracellular phosphatidylinositol 4-phosphate (PI4P) in tomato cell suspensions. We have analysed and compared the intracellular and extracellular phospholipid profiles of [(32)P(i)]-prelabelled tomato cells, challenged with the fungal elicitor xylanase. These phospholipid patterns were found to be different, being phosphatidylinositol phosphate (PIP) the most abundant phospholipid in the extracellular medium. Moreover, while cells responded with a typical increase in phosphatidic acid and a decrease in intracellular PIP upon xylanase treatment, extracellular PIP level increased in a time- and dose-dependent manner. Using two experimental approaches, the extracellular PIP isoform was identified as PI4P. Addition of PI4P to tomato cell suspensions triggered the same defence responses as those induced by xylanase treatment. These include production of reactive oxygen species, accumulation of defence-related gene transcripts and induction of cell death. We demonstrate that extracellular PI4P is accumulated in xylanase-elicited cells and that exogenous application of PI4P mimics xylanase effects, suggesting its putative role as an intercellular signalling molecule.Fil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Testerink, Christa. University of Amsterdam; Países BajosFil: Munnik, Teun. University of Amsterdam; Países BajosFil: de la Canal, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin