Acyl-CoA elongase expression during seed development in Brassica napus Juliette Puyaubert a , Bertrand Garbay a , Patricia Costaglioli a;b , Wilfrid Dieryck a;b , Thomas J. Roscoe c , Michel Renard d , Claude Cassagne a;b , Rene ¨ Lessire a ; * a Laboratoire de Biogene ©se Membranaire, CNRS UMR 5544, Universite ¨ V. Segalen Bordeaux 2, 146, Rue Le ¨o Saignat, 33076 Bordeaux Cedex, France b Ecole Supe ¨rieure de Technologie des Biomole ¨cules de Bordeaux, Universite ¨ V. Segalen Bordeaux 2, 146, Rue Le ¨o Saignat, 33076 Bordeaux Cedex, France c Laboratoire de Ge ¨nome et De ¨veloppement des Plantes, CNRS UMR 5096, Universite ¨ de Perpignan, 52, Avenue de Villeneuve, 66860 Perpignan Cedex, France d UMR INRA-ENSAR, Ame ¨lioration des Plantes et Biotechnologies Ve ¨ge ¨tales, B.P. 35327, 33653 Le Rheu, France Received 8 March 2001; received in revised form 29 June 2001; accepted 9 July 2001 Abstract The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn- FAE1 gene expression was studied during seed development using two different cultivars : Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars ; (ii) that they are coordinately regulated ; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl- CoA synthase activity. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Low erucic acid rapeseed; Seed development; Lipid synthesis; Very long chain fatty acid; 3-Ketoacyl-CoA synthase; Fatty acid elongation 1 ; Brassica napus 1. Introduction In many higher plants, C16:1 and C18:1 fatty acids are the major components of the seed storage triacylglycerols (TAG). High erucic acid rapeseed (HEAR) oil is di¡erent since erucic acid (C22:1, v13) represents from 45 to 60% of the total fatty 1388-1981 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII:S1388-1981(01)00152-4 Abbreviations : HEAR, high erucic acid rapeseed ; LEAR, low erucic acid rapeseed ; VLCMFA, very long chain monounsatu- rated fatty acid(s); LPAAT, lysophosphatidic acid acyltransfer- ase ; TAG, triacylglycerol(s) ; WAP, week after pollination * Corresponding author. Fax : +33-5-56-51-83-61. E-mail address : rene.lessire@biomemb.u-bordeaux2.fr (R. Lessire). Biochimica et Biophysica Acta 1533 (2001) 141^152 www.bba-direct.com