ISSN 0095 4527, Cytology and Genetics, 2012, Vol. 46, No. 3, pp. 131–135. © Allerton Press, Inc., 2012. Original Russian Text © I.V. Pokotylo, S.V. Kretinin, V.S. Kravets, 2012, published in Tsitologiya i Genetika, 2012, Vol. 46, No. 3, pp. 3–9.
Role of Phospholipase D in Metabolic Reactions of Transgenic Tobacco cax1 Cells under the Influence of Salt Stress I. V. Pokotylo, S. V. Kretinin, and V. S. Kravets Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kiev e mail: [email protected] Received February 15, 2011
Abstract—This work was aimed at investigating the primary reactions of plant cell metabolism in response to salt stress. It was found that the phospholipase D regulatory enzyme is activated in wild type and transgenic cax1 tobacco plants during the early stages of the influence of salt stress. We have shown that a disturbance in the intracellular homeostasis of calcium ions and oppression of phospholipase D activity decrease the resis tance of tobacco plants under the influence of salinity and also indicate the involvement of such systems in signaling during stress adaptation of plants. DOI: 10.3103/S0095452712030085
INTRODUCTION stress factors [10, 11]. It was found that many regula 2+ The prevalence and extent of the adverse influence tory enzymes of plant cells are Ca dependent, of salt stress determine its positioning as one of the key including the majority of plant PLD isoforms contain growth limiting factors. In the majority of plants, sen ing C2 domains [12], as well as protein SOS3 [13]—a sitiveness to its effects is maintained throughout the component of the key cellular adaptation system salt whole ontogenesis and contributes to a significant overly sensitive (SOS) of plants, which is activated by the action of salt stress and restricts the flow of toxic inhibition of metabolic processes. However, plant 2+ organisms are able to directly respond to changing ions Na into the cytosol [2]. Interestingly, phospha tidic acid, being a product of PLD, promotes the acti environmental conditions and to activate compensa + + tory mechanisms at the biochemical level [1, 2]. vation of vacuolar Na /H antiporter SOS1—an effector component of the SOS system [14], which Among the considered mediators of stress signal may indicate the integration of signaling systems in reception, one of the leading places is held by phos plant cells during stress signaling. pholipases—enzymes that can provide the production Thus, today the proposed regulatory interaction of signaling molecules during hydrolysis of polar phos 2+ pholipids of membranes [3]. Several isoforms of phos between plant phospholipase enzymes and Ca ions, pholipase D (PLD) of plants are characterized by as well as their direct involvement in cellular regula localization in the plasmalemma [4], where the initial tion, is an important mechanism for the development contact of biological processes of the cytosol with of a protective response under stressful conditions. In changeable external environment is mediated. It is this paper, using a model of transgenic plants, we ana lyzed the characteristics of involvement of PLD and assumed that the interaction of proteins with receptor 2+ like properties, activator G proteins and phospholi intracellular transporters of Ca ions in the regulation pases may be one of the mechanisms capable of pro of metabolism under the conditions of salt stress and viding the initiation of signal transduction and activa their role in the development of adaptive responses of tion of defense reactions in plants in response to the plants. influence of stress factors [5, 6]. In support of this view, it was shown that activation of PLD and an increase in MATERIALS AND METHODS production of phosphatidic acid and other second mes sengers of a lipid nature are observed at the early stages We used tobacco plants Nicotiana tabacum KY 160, of a series of abiotic [7] and biotic stresses [8], as well as as well as transgenic tobacco plants cax1 that express in response to the influence of hormones [9]. the coding area of the H+/Ca2+ gene of a vacuolar At the same time, calcium ions in plant cells are antiporter of Arabidopsis CAX1 (Cation Exchanger 1, also known as important signaling agents, and the At2g38170), with the use of the promoter of cauli characteristics of their intracellular compartmental flower mosaic virus [15]. ization and concentration gradients directly modulate The PLD activity in vivo was determined by a the course of a number of metabolic processes under transphosphatidylation reaction of P33 labeled lipids normal growth conditions and under the influence of from cuttings of tobacco leaves in the presence of 0.8%
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for studying the negative influence of stress and possi 0.20 bilities of its compensation by biotechnological meth ods. Salt stress caused an increase in the concentration 0.15 of malondialdehyde (MDA) in the tissues of tobacco leaves, and its content exceeded the physiological level more than twice after a day’s exposure of the plants to 0.10 a growth medium containing 200 mM of NaCl (Fig. 1). It is known that MDA is a marker of degradation of poly 0.05 unsaturated lipids, and accumulation indicates the damaging of cell membranes and development of the state of oxidative stress. Thus, the obtained results may 0 indicate the rapid inhibition of metabolism in tobacco 0 24 72 120 plants under salinity conditions and suggest the need WT n butanol , n butanol caxl caxl for rapid induction of protection reactions of an Fig. 1. Accumulation of MDA in tissues of tobacco leaves organism under the influence of a stressor. under salt stress: vertically, MDA concentration, µM/mg In this regard, we have established the presence of of wet weight; horizontally, salt stress influence, days. significant activation of phospholipase D in vivo in tobacco plants at the early stages of the influence of n butanol in an incubation medium [9] in response to salt stress (Fig. 2), which was previously shown for short term salt stress in the form of 50 mM of NaCl. plants of arabidopsis [20] and rice [21]. An increase in Phospholipids were separated on silica gel plates for the production of phosphatidyl butanol as a result of a thin layer chromatography in the organic phase of the transphosphatidylation reaction, which is an indicator system ethyl acetate : isooctane : formic acid : water of PLD activation, was observed 10 min after exposure (12 : 2 : 3 : 10 vol./vol.). In order to identify the prod to 50 mM of NaCl. This may indicate the direct ucts, we used lipid standards (Sigma, United States). involvement of PLD, as well as of phosphatidic acid as The labeled phospholipids were visualized by autorad a lipophilic second messenger, in the initiation of sig iography on an X ray film. The quantification of the naling aimed at the activation of compensatory activity of samples of phospholipids was determined responses under the conditions of salinity. on a LKB Wallac Rack Beta 1219 liquid scintillation In order to investigate the characteristics of realiza counter. tion of plant protective reactions under stress condi To assess the level of adaptation of tobacco plants to tions, in the present work, we used transgenic tobacco the action of salt stress, simulated in a Hoagland– plants cax1, characterized by overexpression of the Arnon medium with 200 mM of NaCl, we determined CAX1 arabidopsis gene. CAX antiporters provide low in leaf tissues the content of malondialdehyde (MDA) affinity transport of cytosolic Ca2+ ions in the vacu and the activity of superoxide dismutase (SOD) and ole—the compartment accumulating Ca2+ ions in catalase antioxidant enzymes. The intensity of lipid mature plant cells [22]. peroxidation was assessed by the level of MDA in Previously, it was found that transgenic tobacco accordance with the method in [16]. The supernatant plants cax1 are characterized by depletion of the cyto absorption was measured at 532 nm using the coeffi 2+ –1 solic pool of Ca ions (which causes a violation of cient of millimolar extinction of MDA 155/mM cm their total cellular homeostasis and signaling func when calculating the concentration. The SOD activity tions), as well as increased sensitivity to the action of was determined by analyzing the decrease in the inten cold shock [15]. Expression of the CAX1 gene of arabi sity of photochemical reduction of nitro blue tetrazo dopsis disrupted the compartmentalization of Ca2+ lium [17]. ions in tomatoes [23], while for the halophytic plant The catalase activity was determined by the reduc Suaeda salsa the involvement of the SsCAX1 anti tion in quantity of the colored complex of ammonium porter in the regulation of Ca2+ signaling under salt molybdate that reacts with hydrogen peroxide with the stress was demonstrated [24]. As a result of the con use of the millimolar extinction coefficient of H2O2 ducted studies, we have found that in transgenic 2.22 ×10–3 mM–1 cm–1 [18]. tobacco plants cax1 under the influence of salt stress The specific enzyme activity was determined by activation of PLD is also observed (Fig. 2). Despite the analyzing soluble proteins in the extracts studied by fact that plant PLD is known as Ca2+ dependent the method of Bradford [19] and expressed in units of enzyme [12], activation of PLD under stressful condi enzymatic activity (μM/min · L)/mg of protein. tions can apparently take place in the presence of a deficit in the level of cytosolic Ca2+ ions. This may indicate the presence of intracellular mechanisms of RESULTS AND DISCUSSION local maintenance of the concentration of calcium Tobacco plants are glycophyte organisms sensitive ions in membrane microdomains and lipid rafts, to the effects of salinity and are widely used as objects which is required for the initiation of signaling at a
CYTOLOGY AND GENETICS Vol. 46 No. 3 2012 ROLE OF PHOSPHOLIPASE D IN METABOLIC REACTIONS 133 change in environmental conditions. These results may (a) also indicate the possibility of prevailing participation of calcium independent PLD isoforms [25] in the pro cesses of signal transduction induced by salt stress, as well as isoforms of PLD β and γ, which can be active in the presence of micromolar concentrations of Ca2+ ions. Pbut An analysis of transgenic tobacco plants cax1 revealed that a dysfunction of the metabolism and sig naling functions of Ca2+ ions in the studied plants led to a reduction in their overall resistance to salinity. The cax1 plants after 120 h of exposure to salt stress in the form of 200 mM of NaCl were characterized by an Pa increased MDA content (more than 25%) in their tis sues in comparison with wild type plants under iden tical conditions (Fig. 1). An assessment of the level of plant adaptation and the degree of the negative influ ence of salt stress was performed by determining the activity of cellular antioxidant enzymes. It is known that the growth of plants under salinity causes inhibi tion of photosynthesis and excessive reduction of the Sphl electron transport chain in chloroplasts and mito chondria. This contributes to a multiple increase in 123456 the production of reactive oxygen species (ROS’s). (b) The resulting accumulation of ROS’s in plant cells is 1800 capable of producing toxic effects, including lipid per oxidation and oxidative damage to DNA and proteins. The ability of cells to effectively suppress the excessive accumulation of ROS’s under the conditions of salt 1600 stress is one of the determining factors of adaptation of plants [26]. Furthermore, induction of antioxidant 300 enzymes is considered to be a promising method for increasing plant resistance to the influence of stresses 200 [27]. As a result of the conducted studies, it was found that under the conditions of salt stress enzymes of 100 antioxidant protection, including superoxide dismu tase (Fig. 3) and catalase (Fig. 4), are activated in 0 tobacco plants. At the same time, the transgenic cax1 123456 plants were characterized by a significant relative Pbut Pa decrease in catalase activity after 120 h of exposure to salt stress compared to wild type plants. The relative decrease in the activity of superoxide dismutase in the Fig. 2. Activation of PLD under the influence of salt stress: transgenic cax1 plants was less pronounced and (a) autoradiogram of chromatogram of P33 labeled lipids amounted to about 15% after 72 h of exposure to the from tobacco plants; (b) scintillation count of phosphati stressor. The experimental data obtained may indicate dyl butanol and phosphatidic acid zones in the autoradio the importance of maintaining a physiologically sig gram. Vertically, the amount of radiolabeled products, dis 2+ integrations in 1 min; horizontally, 1, wild type plants nificant level of Ca ions in the cytosol and the possi (control); 2, plants cax1 (control); 3, wild type plants (salt ble involvement of calcium signaling in the formation stress, 10 min); 4, plants cax1 (salt stress, 10 min); 5, wild of protective reactions of metabolism under the salin type plants (salt stress, 20 min); 6, plants cax1 (salt stress, ity conditions. 20 min); Pbut is phosphatidyl butanol, Pa is phosphatidic acid, and S is structural phospholipids. Application of the inhibitor of PLD activity phl n butanol [28] in the present study allowed us to deter mine the role of such phospholipase enzymes in the tase activity in the tissues of wild type tobacco leaves development of defense reactions of a plant organism by 30% (Fig. 3). In addition, the relative decrease in under stressful conditions. It was established that catalase activity was more significant and amounted to exposure to 50 mM of n butanol led to increased con centrations of MDA in the leaves of tobacco plants about 60% of its activity in the control plants (Fig. 4). under salt stress (Fig. 1). Inhibition of PLD activity On the basis of the results obtained, we can assume also stipulated a relative decrease in superoxide dismu that the regulatory PLD enzyme, as well as lipid mes
CYTOLOGY AND GENETICS Vol. 46 No. 3 2012 134 POKOTYLO et al.
250 2000 1750 200 1500 1250 150 1000
100 750 500 50 250 0 0 02472120 02472120 WT n butanol caxl caxl, n butanol WT n butanol caxl caxl, n butanol Fig. 3. Activation of SOD in tissues of tobacco leaves under Fig. 4. Activation of catalase in tissues of tobacco leaves salt stress: vertically, activity of SOD, act. units/mg of pro under salt stress: vertically, activity of catalase, act. units/mg tein; horizontally, salt stress influence, days. of protein; horizontally, salt stress influence, days. sengers, which products it provides, participates in the REFERENCES development of protective reactions of an organism, 1. Radyukina, N., Ivanov, Y., Kartashov, A., providing the activation of antioxidant protection Shevyakova, N., Rakitin, V., Khryanin, V., and Kuz enzymes, and inhibition of PLD functions determines netsov, V., Inducible and Constitutive Mechanisms of increased sensitivity of plants to salt stress. Salt Stress Resistance in Geum urbanum L, Rus. J. Plant Physiol., 2007, vol. 54, no. 5, pp. 612–618. In addition, we found that inhibition of PLD activ 2. Qiu, Q.S., Guo, Y., Quintero, F.J., Pardo, J.M., Schu ity in transgenic plants cax1 determines the additive maker, K.S., and Zhu, J.K., Regulation of Vacuolar inhibition of adaptive reactions of an organism under Na+/H+ Exchange in Arabidopsis thaliana by the Salt the conditions of salt stress and leads to a more than Overly Sensitive (SOS) Pathway, J. Biol. Chem., 2004, two fold increase in the MDA content in the tissues of vol. 279, no. 1, pp. 207–215. leaves compared to wild type plants (Fig. 1). It was 3. Ruy, S. and Wang, X., Activation of Phospholipase D shown that tobacco plants cax1 under the influence of and the Possible Mechanism of Activation in Wound Induced Lipid Hydrolysis in Castor Bean Leaves, Bio n butanol were additionally characterized by a signifi chim. Biophys. Acta, 1996, vol. 1303, pp. 243–250. cant reduction in the activity of catalase (Fig. 4) and 4. Novotna, Z., Martinec, J., Profotova, B., Zdarova, S., SOD (Fig. 3) antioxidant enzymes, which indicates Kader, J.C., and Valentova, O., In vitro Distribution the inability of a tobacco plant to activate protection and Characterization of Membrane Associated pld and reactions of metabolism under the conditions of non pi plc in Brassica napus, J. Exp. Bot., 2003, vol. 54, functionality of cellular signaling systems involving no. 383, pp. 691–698. PLD and Ca2+ ions. 5. Ritchie, S. and Gilroy, S., Abscisic Acid Stimulation of Phospholipase D in the Barley Aleurone Is G Protein Thus, as a result of the conducted studies, it was Mediated and Localized to the Plasma Membrane, found that at the early stages of the influence of salt Plant Physiol., 2000, no. 2, pp. 693–702. stress wild type tobacco plants, as well as transgenic 6. Munnik, T., Arisz, A., de Vrije, T., and Musgrave, A., G tobacco plants cax1, exhibit activation of PLD. In Protein Activation Stimulates Phospholipase D Signal addition, experimental data indicate the likely discrete ing in Plants, Plant Cell, 1995, vol. 7, pp. 2197–2210. involvement of both PLD and Ca2+ ions in metabolic 7. Li, W., Li, M., Zhang, W., Welti, R., and Wang, X., The adaptation processes of tobacco plants under salt Plasma Membrane Bound Phospholipase D[Delta] stress. Enhances Freezing Tolerance in Arabidopsis thaliana, Nat. Biotech., 2004, vol. 22, no. 4, pp. 427–433. 8. Andersson, M.X., Kourtchenko, O., Dangl, J.L., Mackey, D., and Ellerstrom, M., Phospholipase ACKNOLEDGMENTS Dependent Signalling during the AvrRpm1 and AvrRpt2 Induced Disease Resistance Responses in We are grateful to Professor K. Hirschi for provid Arabidopsis thaliana, Plant J., 2006, no. 6, pp. 947– ing seeds of tobacco plants cax1. The work was sup 959. ported by the National Academy of Sciences of 9. Kravets, V.S., Kolesnikov, Y.S., Kretynin, S.V., Get Ukraine (2.1.10.32 10. and TsNP 9.1 07). man, I.A., and Romanov, G.A., Rapid Activation of
CYTOLOGY AND GENETICS Vol. 46 No. 3 2012 ROLE OF PHOSPHOLIPASE D IN METABOLIC REACTIONS 135
Specific Phospholipase(S) D by Cytokinin in Amaran lizing the Principle of Protein Dye Binding, Anal. Bio thus Assay System, Physiol. Plant., 2010, vol. 138, no. 3, chem., 1976, nos. 1/2, pp. 248–254. pp. 249–255. 20. Bargmann, B.O.R., Laxalt, A.M., Riet, B.T., Van 10. Van der Meulen, R., Visser, K., and Wang, M., Effects Schooten, B., Merquiol, E., Testerink, C., Haring, M.A., of Modulation of Calcium Levels and Calcium Fluxes Bartels, D., and Munnik, T., Multiple PLDs Required on ABA Induced Gene Expression in Barley Aleurone, for High Salinity and Water Deficit Tolerance in Plants, Plant Sci., 1996, vol. 117, nos 1/2, pp. 75–82. Plant Cell Physiol., 2009, vol. 50, no. 1, pp. 78–89. 11. Monshausen, G.B., Bibikova, T.N., Weisenseel, M.H., 21. Darwish, E., Testerink, C., Khalil, M., El Shihy, O., and Gilroy, S., Ca2+ Regulates Reactive Oxygen Spe and Munnik, T., Phospholipid Signaling Responses in cies Production and pH during Mechanosensing in Salt Stressed Rice Leaves, Plant Cell Physiol., 2009, Arabidopsis Roots, Plant Cell Online, 2009, vol. 21, vol. 50, no. 5, pp. 986–997. no. 8, pp. 2341–2356. 22. Pottosin, I.I. and Schonknecht, G., Vacuolar Calcium 12. Zheng, L., Krishnamoorthi, R., Zolkiewski, M., and 2+ Channels, J. Exp. Bot., 2007, vol. 58, no. 7, pp. 1559– Wang, X., Distinct Ca Binding Properties of Novel 1569. C2 Domains of Plant Phospholipase D α and β, J. Biol. Chem., 2000, vol. 275, no. 26, pp. 19700–19706. 23. Chung, M.Y., Han, J.S., Giovannoni, J., Liu, Y., 13. Lin, H., Yang, Y., Quan, R., Mendoza, I., Wu, Y., Kim, C.K., Lim, K.B., and Chung, J.D., Modest Cal cium Increase in Tomatoes Expressing a Variant of Ara Du, W., Zhao, S., Schumaker, K.S., Pardo, J.M., and + Guo, Y., Phosphorylation of SOS3 Like Calcium bidopsis Cation/H Antiporter, Plant Biotech. Rep., Binding Protein by SOS2 Protein Kinase Stabilizes 2010, vol. 4, no. 1, pp. 15–21. Their Protein Complex and Regulates Salt Tolerance in 24. Han, N., Shao, Q., Bao, H., and Wang, B., Cloning Arabidopsis, Plant Cell, 2009, no. 5, pp. 1607–1619. and Characterization of a Ca2+/H+ Antiporter from 14. Yu, L., Nie, J., Cao, C., Jin, Y., Yan, M., Wang, F., Halophyte Suaeda salsa L, Plant Mol. Biol. Report., Liu, J., Xiao, Y., Liang, Y., and Zhang, W., Phospha 2010, vol., pp. 1–9. tidic Acid Mediates Salt Stress Response by Regulation 25. Qin, C. and Wang, X., The Arabidopsis Phospholipase of MPK6 in Arabidopsis thaliana, New Phytol., 2010, D Family. Characterization of a Calcium Independent vol. 188, no. 3, pp. 762–773. and Phosphatidylcholine Selective PLD1 with Dis 15. Hirschi, K.D., Expression of Arabidopsis CAX1 in tinct Regulatory Domains, Plant Physiol., 2002, Tobacco: Altered Calcium Homeostasis and Increased vol. 128, no. 3, pp. 1057–1068. Stress Sensitivity, Plant Cell, 1999, vol. 11, no. 11, 26. El Shabrawi, H., Kumar, B., Kaul, T., Reddy, M.K., pp. 2113–2122. Singla Pareek, S.L., and Sopory, S.K., Redox Homeo 16. Madhava, RaoK.V. and Sresty, T.V.S., Antioxidative stasis, Antioxidant Defense, and Methylglyoxal Detox Parameters in the Seedlings of Pigeon Pea (Cajanus ification as Markers for Salt Tolerance in Pokkali Rice, cajan (L.) Millspaugh) in Response to Zn and Ni Protoplasma, 2010, vol. 245, pp. 85–96. Stresses, Plant Sci., 2000, vol. 157, no. 1, pp. 113–128. 27. Joseph, B. and Jini, D., Development of Salt Stress 17. Van Rossum, M.W.P.C., Alberda, M., and Van der Tolerant Plants by Gene Manipulation of Antioxidant Plas, L.H.W., Role of Oxidative Damage in Tulip Bulb Enzymes, Asian J. Agricult. Res., 2011, vol. 5, no. 1, Scale Micropropagation, Plant Sci., 1997, vol. 130, pp. 17–27. no. 2, pp. 207–216. 28. Motes, C.M., Pechter, P., Yoo, C.M., Wang, Y.S., 18. Korolyuk, M., Ivanov, L., Maiorova, M., and Chapman, K.D., and Blancaflor, E.B., Differential Tokareva, V., Method of Determination of Catalase Effects of Two Phospholipase D Inhibitors, 1 Butanol Activity, Lab. Delo, 1988, vol. 1, pp. 16–19. and N Acylethanolamine, on in vivo Cytoskeletal 19. Bradford, M.M., A Rapid and Sensitive Method for the Organization and Arabidopsis Seedling Growth, Proto Quantitation of Microgram Quantities of Protein Uti plasma, 2005, vol. 226, nos. 3/4, pp. 109–123.
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