Potato tubers exhibit both homolytic and heterolytic

Lipids and Signalling: Oxylipins 2

enoic acid (a-ketol) is an endogenous flowering factor in Lemna paucicostata.

Discussion T h e results of the present work demonstrate that the LOX pathway in tulip bulbs and leaves operates via the 9-LOX and AOS activities. Tulip is the second plant species known to possess predominantly 9-LOX activity in its leaves, along with recently characterized potato leaves [6]. It seems logical to assume that ketols, being the predominant LOX products in such tissues as tulip bulbs and maize seeds, must have their own physiological importance. As found recently, the y-ketol (1 1E)-lO-oxo-l3-hydroxy-1 O-octadecenoic acid and a related macrolactone, (1 1E)-100x0-1 1-octadecen-l3-olide, isolated from maize seeds, exhibit strong cytotostatic activity [ l ] . One might propose, in connection with this, that reduction of y-ketols and related oxylipins by yketol reductase may have physiological importance as an intracellular mechanism for detoxification of these metabolites. As reported recently [7], (1 2Z,15Z)-9-hydroxy-lO-oxo-12,1S-octadecadi-

This work was supported by grant 00-04-4822I from the Russian Foundation of Basic Research and grant 00- 15-97904 (program for the support of leading scientific schools).

References I Grechkin, A. N. ( I 998) Prog. Lipid Res. 37, 3 17-352 2 Harnberg. M. and Gardner, H. W. ( 1992) Biochirn. Biophys. Ada 1165, 1-18 3 Grechkin, A. N., Kurarnshin, R. A,, Safonova, E.Y., Latypov, S. K. and Ilyasov, A. V. ( I 99 I ) Biochim. Biophys. Acta 1086, 3 17-325 4 Grechkin, A. N., Kurarnshin, R. A. and Tarchevsky, I. A. ( I 99 I ) Bioorgan. Khirn. 17,997-998 5 Vick B. A. and Zirnmerman, D. C. ( I 983) Biochern. Biophys. Res. Cornrnun. I I I, 4701177 6 Harnberg, M. ( 1999) Lipids 34, I I 3 I - I I42 7 Yokoyarna, M., Yarnaguchi, S., Inornata, S., Kornatsu, K., Yoshida, S., lida, T., Yokokawa, Y., Yarnaguchi, M., Kaihara. S. and Takirnoto, A. (2000) Plant Cell Physiol. 4 I, I 10-I I3 Received 7 August 2000

Potato tubers exhibit both homolytic and heterolytic hydroperoxidefatty acid-cleaving activities M.-L. Fauconnier"', J. Delcarte*, P. Hoyaux*, P. du Jardint and M. Marlier* "Unite de Chimie Generale et Organique, Faculte Univenitaire des Sciences Agronomiques de Gembloux, Passage des Deportes, 2-6-5030 Gembloux, Belgium, and tUnite de Biologie Vegetale, Faculte Universitaire des Sciences Agronomiques de Gembloux, Passage des Deportes, 2-6-5030 Gembloux, Belgium

Abstract

Introduction

T h e action of a crude potato-tuber extract on 9and 13-hydroperoxides of linoleic and linolenic acids was investigated. HPLC analysis revealed that 5O0/, of the 9-hydroperoxide isomers and almost all the 13-hydroperoxide isomers were rapidly enzymically metabolized. No degradation of fatty acid hydroperoxides was observed with a thermally denatured enzymic extract. G C - M S identification of the volatiles formed by the reaction revealed that no volatiles were detected from the 9-hydroperoxide isomers, whereas 13-hydroperoxide of linolenic acid was cleaved into (Z)-3-hexenal, pentenols or dimers of pentene.

Depending on botanical origin and on reaction conditions, plant lipoxygenases can form variable amounts of 13- or 9-hydroperoxides of linoleic or linolenic acid. Fatty acid hydroperoxides can be degraded further in a variety of compounds implicated in essential physiological roles in plants (jasmonic acid, traumatin) or responsible for the characteristic green note odour of plants and fruits (C, or C, aldehydes and alcohols) [l]. Four main fatty acid hydroperoxide-decomposing activities are described : allene oxide synthase catalyses the conversion of fatty acid hydroperoxides into allene oxides, precursors of a- and y-ketols and of jasmonic acid (from 13hydroperoxide of linolenic acid) [2]. Peroxigenase and epoxigenase cause epoxidation of fatty acid hydroperoxides [3,4]. Divinyl ether synthase transforms fatty acid hydroperoxides into fatty acid divinyl ethers [5,6]. Finally, hydroperoxide lyase cleaves fatty acid hydroperoxides into alde-

~

~

Key words fatty acid hydroperoxide hydroperoxide lyase, Iipoxygenase,Solonurn tuberosum L 'To whom correspondence should be addressed (e-mail fauconnier rnl(4fsagx ac be)

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0 2000 Biochemical Society

Biochemical Society Transactions 2000 Volume 28, part 6

under the conditions described above but omitting the detergent Triton X-100. T h e reaction of this extract with 13-hydroperoxide of linolenic acid formed dimers of pentene, 2-penten-1 -01 and 1-penten-3-01 but no (Z)-3-hexenal.

hydes, alkanes or alcohols and 0x0-acids. We investigated here the conversion of fatty acid hydroperoxides by a crude extract of potato tubers.

Results

Discussion

9- and 13-hydroperoxides of linoleic and linolenic acid were synthesized using respectively potato and soya bean lipoxygenases. T h e fatty acid hydroperoxides were extracted and purified using a C,, microcolumn. T h e purity, controlled by H P L C analysis, was higher than 95 yo for the four fatty acid hydroperoxides synthesized. Potato tubers (Bintje) were crushed in a Waring blender with a buffer containing 0.2% (w/v) Triton X-100. T h e extract was agitated on ice and centrifuged. T h e supernatant was used as the crude extract for the experiments. Crude extracts were incubated for 15 min at room temperature with, respectively, the 13- and 9-hydroperoxides of linoleic and linolenic acid. An aliquot was taken for direct H P L C analysis to determine the amount of hydroperoxides consumed. T h e 13-hydroperoxides of linoleic and linolenic acid were almost completely degraded by the potato extract, whereas 50% of the 9-hydroperoxides of linoleic and linolenic acid remain in the reaction medium after the reaction. T h e enzymic nature of the reaction was confirmed by performing the experiments in the same conditions with the crude extract thermally denatured. In those conditions, no decomposition of fatty acid hydroperoxides was observed. Volatile compounds potentially present after reaction were extracted by diethyl ether. T h e extract was submitted to GC-MS analysis for identification. T h e extract obtained from the reaction of 9-hydroperoxides of linoleic and linolenic acid with the crude extract contained no volatile compounds under our experimental conditions. On the other hand, the following compounds were identified in the extract resulting from the action of potato extract on 13-hydroperoxide of linolenic acid : (Z)-3-hexenal, dimers of pentene (seven isomers), 2-penten-1 -01 and 1-penten-3-01. In the extract obtained with 13-hydroperoxide of linoleic acid, hexanal and pentan-1-01 were identified. No volatile compounds were identified when a thermally denatured extract was used. T h e identification of volatile compounds was realized on the basis of mass spectrum and by comparison with original molecules when commercially available. A crude extract of potato tubers was prepared

T h e results presented here show that a crude potato-tuber extract can metabolize the four isomers of fatty acid hydroperoxide; the 13-isomers are completely transformed while the 9-isomers are only partially degraded. This last result is astonishing because in potato tubers, 95% of isomers formed by lipoxygenase are 9-isomers [7]. T h e volatile compounds formed from the 13isomers are typical of two different enzymic mechanisms. T h e first one is due to hydroperoxide lyase and furnishes (Z)-3-hexenal and hexanal, respectively, from 13-hydroperoxides of linolenic and linoleic acid. Hydroperoxide lyase is a membrane-bound enzyme and it is logical that its activity is not found in an extract obtained without detergent [8]. T h e formation of dimers of pentene and of C, alcohols is due to homolytic cleavage of fatty acid hydroperoxides, but the enzymes responsible for the reaction were not clearly identified. T h e homolytic cleavage is usually described in mushrooms and in algae but the products have also been identified in olive oil 191 and in soya bean [10,11]. In soya bean, two hypotheses are proposed : the cleavage is either due to a soluble homolytic hydroperoxide lyase [ l l ] or is a secondary reaction of lipoxygenase [ 101. Further investigations are being undertaken to determine if lipoxygenase is responsible for the homolytic cleavage or if another enzyme is implicated. T h e identification of non-volatile products formed from 9-hydroperoxides is also in progress : ketols and divinyl ether fatty acids are potential metabolites formed respectively by allene oxide synthase and divinyl ether synthase.


M.-L.F. and 1.0. are respectively senior research assistant and research assistant of the Fonds National de la Recherches Scientifiques de Belgique. P.H. is a research assistant of the Fonds pour la formation a la recherche dans I'lndustries et dans I 'Agriculture.

References Gardner, H. W. ( I99 I ) Biochim. Biophys. Acta 1084,

22 1-239 Song, W. C., Funk C. D. and Brash, A. R. ( I 993) Proc. Natl. Acad. Sci. U.S.A. 90,85 19-8523 Blee, E. and Schuber, F. (1990) J. Biol. Chem. 265,

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Hamberg, M. and Hamberg, G. ( 1990) Arch. Biochern. Biophys. 2 8 3 , 4 0 9 4 I 6 Grechkin, A. N., Ilyasov, A. V. and Hamberg, M. ( I 997) Eur. J. Biochem. 245, 137- I42 Weber, H.. Chetelat, A,, Caldelari, D. and Farmer, E. E. ( I 999) Plant Cell I I, 485493 Mulliez, E., Leblanc,J. P., Girerd, J. 1.. Rigaud, M. and Chottard. J. C. ( 1987) Biochern. Biophys. Acta 9 16, I 3-23 Fauconnier, M. L. and Marlier, M. ( I 997) Grasas Aceites 48, 30-37

9 Angerosa, F., Camera, L., d'Alessandro, N. and Mellerio, G. ( I 998) J. Agric. Food Chem. 46, 648-653 10 Salch, Y.P., Grove, M. J., Takarnura, H. and Gardner, H. W ( I 995) Plant Physiol. 108, I2 I I -I 2 I 8 I I Kondo, Y., Hashidoko, Y. and Mizutani, J. ( I 995) Biochim. Biophys. Acta 1255, 9- I5

Received 9 June 2000

Characterization of a Euphorbia lagascae epoxide hydrolase gene that is induced early during germination J. Edqvist' and 1. Farbos Department of Plant Biology, SLU, Box 7080,750 07 Uppsala, Sweden

Abstract

seems likely that the degradation requires the activity of an epoxide hydrolase (EH). EHs are a group of functionally related enzymes that catalyse the cofactor-independent hydrolysis of epoxides to their corresponding vicinial diols by the addition of a water molecule. Little is known about the actual function of EHs in plants, although they have been suggested to be involved in the biosynthesis of cutin. In potato it was shown that accumulation of the E H transcript is induced after wounding, and also in response to methyl jasmonate [ l ] . In the case of Arabidopsis thaliana, the E H transcript was found to accumulate in young plants after treatment with auxin [ 2 ] . T h e tobacco E H gene was shown recently to be activated during tobacco mosaic virus infection [3]. In contrast to the A . thaliana EH, transcription of the tobacco gene did not respond to treatment with auxin. Based on these limited data, plant EHs appear to play a role in responding to environmental stresses. Here we report the cloning and characterization of an E H gene from E . lagascae. T h e structure of the gene is unusual in that it lacks introns. A detailed investigation of the transcription pattern of the E H gene shows that the gene is induced during germination. We have used in situ hybridization to identify in which tissues the gene is expressed during germination. We speculate that this E H enzyme is involved in the catabolism of epoxidated fatty acids during germination of E . lagascae seeds.

In Euphorbia lagascae the major fatty acid in triacylglycerol is the epoxidated fatty acid vernolic acid (cis- 12-epoxyoctadeca-cis-9-enoicacid). T h e enzymic reactions occurring during the catabolism of epoxidated fatty acids during germination are not known, but it seems likely that the degradation requires the activity of an epoxide hydrolase. Epoxide hydrolases are a group of functionally related enzymes that catalyse the cofactorindependent hydrolysis of epoxides to their corresponding vicinal diols by the addition of a water molecule. Here we report the cloning and characterization of an epoxide hydrolase gene from E . lagascae. T h e structure of the gene is unusual since it lacks introns. A detailed investigation of the transcription pattern of the epoxide hydrolase gene shows that the gene is induced during germination. We have used in situ hybridization to identify in which tissues the gene is expressed during germination. We speculate that this epoxide hydrolase enzyme is involved in the catabolism of epoxidated fatty acids during germination of E . lagascae seeds.

Introduction In Euphorbia lagascae the major fatty acid in triacylglycerol is the epoxidated fatty acid vernolic acid (cis-12-epoxyoctadeca-cis-9-enoicacid). T h e enzymic reactions of epoxidated fatty acid catabolism during germination are not known, but it

Results and discussion Key words expressed sequence tag, gene regulation, metabolism, oxyipin Abbreviation used EH. epoxide hydrolase 'To whom correspondence should be addressed (e-mail JohanEdqvist@vbiol slu se)

Cloning and sequencing of a cDNA encoding EH A cDNA library was constructed of mRNA isolated from germinating seeds of E . lagascae.

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