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Lipids and Signalling: Oxylipins 3 Extremely rapid effects of polyunsaturated fatty acids and N-acetylglucosamine on free-radical metabolism in cultured potato plant cells P. A. Kashulin’, M. N. Merzlyak, P. M. Zhiboedov and V. K. Zhirov Polar-Alpine Botanical Garden, Kola Science Centre, I 84230 Apatity, Faculty of Biology Moscow State University, I 19899 Moscow, Russia Abstract Materials and methods T h e effects of arachidonic and linoleic acids, separately and in co-operation with N-acetylglucosamine oligomers, on Solanum tuberosum plant suspension cell cultures were investigated in terms of the fluorescent oxygen-activated-speciessensitive dye 2‘,7‘-dichlorofluorescin diacetate. T h e inductors used triggered extremely rapid (within 2-10 min) generation of H,O, in the cells; the majority was expressed in cultures treated with combined polyunsaturated fatty acid and Nacetylglucosamine oligomers. T h e stimulation of free-radical generation may be related to defensive mechanisms modulating a plant-pathogenicmicro-organism interaction. In experiments, 14-day-old cell cultures of S. tuberosum L. var. T e m p in the steady-state phase of culture growth underwent 3 min of centrifugation at 2000 g and were resuspended in fresh Murashige and Scoog-based medium (pH 7.2) up to a density 2 x lo4 cell/ml. T h e native carbohydrate derivatives of surface glycoproteins were modelled by the N-acetyl-D-glucosamine trimer [(GlcNAc),] synthesized at the A. N. Bakh Institute of Biochemistry (Moscow, Russia). Linoleic and arachidonic acids (Sigma), either separately or in co-operation with (GlcNAc),, were considered as race-non-specific inductors for cells. Intracellular free-radical production was quantified in terms of the intracellular probe 2‘,7‘dichlorofluorescin diacetate (DCFscinDA). T h e quantity of fluorescent 2‘,7‘-dichlorofluoresceine (DCFscein), formed when DCFscinDA is oxidized in cells by hydrogen peroxide, depends directly on the size of the free-radical population in the dye microenvironment [2,3]. All fluorescent dyes were from Serva. T h e concentration of the acetone dye stock solution used in suspension cultures was 0.3 yo (v/v). Cell-free incubation medium was considered as a base fluorescence control. Accumulation of the fluorescent product DCFscein in cells was monitored with a Jasco-550 spectrofluorimeter at excitation and emission wavelengths of 495 and 523 nm, respectively. Data are given as means& S.E.M. Introduction T h e polyunsaturated fatty acids (PUFAs) and N acetylglucosamine polymers that are transported to the contact surfaces or constitute the surface structures of penetrating fungus hyphae are considered as potential elicitors of defensive responses in plants. Despite the accepted concept that different mechanisms underlie induction of defence reactions in plants mediated by specific carbohydrate and race-non-specific lipid derivatives, in many cases the early stages of both scenarios may be accompanied by cellular generation of active oxygen forms [l]. This work aimed to model the action of hydrocarbon and lipid derivatives on plants in suspension cultures of potato (Solanum tuberosum) cells. T h e system based on this species in combination with various races of potato blight fungus, Phytophthora infestans (Mont.) de Bari, or their components, is widely accepted as a model to study general plantpathogen relationships. Results and discussion T h e vitality of the cells, by analysis of intracellular esterase activity, was carried out using 2’,7’dichlorofluoresceine diacetate (DCFsceinDA) [2]. Addition of micromolar DCFsceinDA concentrations to the cell culture was followed by a green emission, qualitatively analogous to enzymic formation of DCFscein in incubation medium. This fluorescence showed the vitality of cells used, as the emission of derivatives of fluorescein is used routinely to mark living plant cells. T h e cells were capable of keeping the dye for no less then 2-3 h. Addition of DCFscinDA to the cell culture resulted in a slow elevation of fluorescence near 523 nm Key words: oxygen activated species, phytoimmunity, PUFA. Abbreviations used: PUFA. polyunsaturatedfatty acid; (GlcNAc),, N-acetyl-o-glucosamine trimer; DCFscein, 2’,7’-dichlorofluoresceine; DCFsceinDA, 2’,7’-dichlorofluoresceine diacetate; DCFscinDA, 2’,7’-dichlorofluorescin diacetate. ‘To whom correspondence should be addressed (e-mail [email protected]). 865 0 2000 Biochemical Society Biochemical Society Transactions (2000) Volume 28, part 6 Table I Comparative effects of arachidonic and linoleic acids on 5. tuberosum suspension cell cultures to produce DCFscein The fluorescence intensity at 523 nm reached in 4 min after addition of inductor, relative to the fluorescence of 0 0 I m M DCFscein in cellfree incubation medium is presented The cell density used was 2 x I O4 c e l l s h Values in parentheses are the numben of repeated expenments Fluorescence of DCFscein in cells (relative units) Inductor Inductor concentration (mM) . . . Arachidonic acid Linoleic acid 2.0 4.0 6.0 8.0 8. I +2.0 (5) 4.8f I .2 (5) 8.8f2.7 (5) 6.9f I .8 (5) 9.6f3.I (4) 7.8f2.3 (4) 9.853.2 (4) 8.2f2.8 (4) Table 2 The synergetic effects of N-acetylglucosamine and lipid inductors on production of DCFscein in S. tuberosum suspension cell culture Experimental details were as for Table I Values in parentheses are the numben of repeated expenments Fluorescence (relative units) ____ Fluorescence - - ~ Inductor-free cells (GlcNAc), ( I mM) (GlcNAc), ( I mM) followed by linoleic acid (2 mM) 5k0.4 (4) 4.2f I .6 (4) 20.6f2.8 (4) and was evidence of DCFscein formation from DCFscinDA. This relatively weak fluorescence (see Table 2) presumably reflects the basal level of free-radical generation, which was not limited by deacylation activity of intracellular esterases. Preincubation for 10 min of DCFscinDA-treated cell culture with (GlcNAc), or P U F A promoted the rapid rise of intracellular oxygen free radicals up to saturation levels. There were no significant differences found between the effects of arachidonic and linoleic acids (Table 1). At low concentration the effectiveness of arachidonic acid seemed higher, but the difference vanished with the rise in inductor concentration. T h e first recognition stages in plant-pathogen interaction may be followed by the involvement of PUFAs in this process. T o model this scenario the addition of (GlcNAc), and, consequently, PUFAs, was tested. T h e addition of arachidonic acid to cell cultures after 10 min of preincubation with DCFscinDA and a few minutes with (GlcNAc), resulted in a drastic and rapid ( < 2 min) increase in fluorescence near 523 nm, which amounted to 5-fold enhancement of the effect of (GlcNAc), alone (Table 2). Analogous action was induced by linoleic acid. T h e results presented show arachidonic 0 2000 Biochemical Society ~~ (GlcNAc), ( I mM) followed by arachidonic acid (2 mM) 23.6f4.1 (4) acid ' s capability of activating natural free-radical metabolism in suspension cell culture and reveal the synergism of PUFAs and (GlcNAc):, in the stimulation of H,O, generation. T h e generation of H,O, took place at the very onset of elicitor addition. This supports the available data that the activation of free-radical metabolism is an early stimulus-dependent defence reaction in plant cells. In such a short time it is hardly, if at all, possible for de novo protein synthesis to be responsible for the free-radical production. According to theoretical work on the question, H,O, formed under elicitor stimulation may be involved in the realization of oxidative o r second-messenger functions as an alternative receptor-linked plasmamembrane redox component in host plant cells [4]. T h e synergetic enhancement of free-radical metabolism by mutual effects of elicitors of differing chemical natures supports the evidence that the activity of arachidonic acid in induction of defence reactions in potato plants may be increased by p-glucan pretreatment [S-71. T h e co-operative and abrupt activation of free-radical metabolism by different inductors may underlie the mechanism of the hypersensitive immune reaction of potato plants infected with Phytophthora infestans fungus. 866 Lipids and Signalling: Oxylipins 3 5 Kurantr, M. J, and Zacharius, R. M. (198 I ) Physiol. Plant Pathol. 18, 69 -7 I 6 Maniara, G., Laine, R. and Kuc, J. A. ( 1984) Physiol. Plant Pathol. 24, 177- I 8 6 7 Preisig, C. L. and Kuc. J. A. ( 1985) Arch. Biochem. Biophys 236,379-389 References I Doke. N. and Chai. H. B. (I 987) in Molecular Determinants o f Plant Disease, pp. 235-25 I , Springer-Verlag,Tokyo 2 Ferrer, A. S.. Santema, j. S., Hilhont. R. and Viser, J, W. G. ( 1990) Anal. Biochem. 187, I 29- I 32 3 Hauglald. R. P. (1992) in Molecular Probes. Handbook of Fluorescent Probes and Research Chemicals, 5th edn, Molecular Probes, Eugene 4 Apostol, I., Heinstein, P. F. and Low, P. S.( I 989) Plant Physiol. 90, 109- I I 6 Received I 5 June 2000 o-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A I : possible involvement in plant defence F. Pinot"', M. Skrabst, V. Compagnon", J.-P. Salaun*, I. Benveniste", L. Schreibert and F. Durst" *IBMP-CNRS UPR406 Dept. d'Enzymologie Cellulaire et Moleculaire, 28 rue Goethe 67083 Strasbourg, France, and TLehrstuhl fur Botanik II, Julius Von Platz 3, D-97082 Wurzburg, Germany Abstract Introduction T h e C,, fatty acid derivatives 9,lO-epoxystearic acid and 9,lO-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94Al cloned from Vicia sativa. 'The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence o f N A D P H . After incubation of a synthetic raceniic mixture of 9,lOepoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 911,lOS enantiomers were incubated separately. We determined respective K,,, and YniLxvalues of 1.2 & 0.1 pM and 19.2 0.3 nmol/min per nmol of cytochrome P450 for the 9R,lOS enantiomer and of 5.9 0.1 p M and 20.2 & 1.O nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R, 10S, which is preferentially formed in V . sativa microsomes. Cutin analysis of V . sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C,, fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions. We previously showed that, in Vicia sativa microsomes, oleic acid is subjected to a cascade of reaction involving three different enzymes : an epoxygenase, an epoxide hydrolase and a cytochrome P45O-dependent o-hydroxylase [I]. Using oleic acid as a starting material, these enzymes are able to produce in nitro the major C,, cutin monomers. Inhibition studies suggested the presence of distinct fatty acid o-hydroxylases [2]. This was confirmed recently by the cloning of CYP94A1 [3] and CYP94A2 [4]. When expressed in yeast, CYP94A1 catalyses the w-hydroxylation of saturated and unsaturated fatty acids with chain lengths ranging from C,, to CIS.[3]. Treatment of etiolated V . satizm seedlings by the plant hormone methyl jasmonate led to an accumulation of transcripts coding for CYP94A1 and, concomitantly, to a stimulation of microsomal fatty acid whydroxylase activity [ S ] . Experimental CYP94A1 was expressed in Saccharomyces cerevisiae as described in [3]. Enzymic activities were determined by following the rate of metabolite formation by T L C [l]. Chiral analyses were performed by using pure synthetic 9R, 1 OSepoxystearate methyl ester as a standard. Briefly, radiolabelled enantiomers of 9,lO-epoxystearic acid were separated on H P L C (Waters, equipped with two 510 pumps, and a U6K injector from Waters) using a chiral column (Column Chiracel OB, 4.6 x 2 5 0 m m ; J. J . Baker Chemical Co.). Key words, cytochrome P450. epoxide. messenger. 'To whom correspondence should be addressed (e-mall franck.pinot(a)bota-u1p.u-strasbg.fr). 867 0 2000 Biochemical Society