Molecular cloning and characterization of genes encoding two microsomal oleate desaturases (FAD2) from olive M. Luisa Herna ´ndez, Manuel Mancha, Jose ´ M. Martı ´nez-Rivas * Instituto de la Grasa (CSIC), Apartado 1078, E- 41080 Sevilla, Spain Received 10 March 2005; received in revised form 1 April 2005 Available online 17 May 2005 Abstract Two different cDNA sequences, designated OepFAD2-1 and OepFAD2-2, encoding two microsomal oleate desaturases (FAD2) have been isolated from olive (Olea europaea cv. Picual) using a PCR approach. Both deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and possess a C-terminal endoplasmic reticulum retention signal. Phylogenetic analysis shows that OepFAD2-1 and OepFAD2-2 are grouped with other plant FAD2 sequences. Functional expression of the corresponding FAD2 cDNAs in yeast confirmed that they encode microsomal oleate desaturases. Genomic South- ern blot analysis is consistent with the presence of at least two copies of each OepFAD2 gene in the olive genome. OepFAD2-1 tran- script was strongly detected in very young seeds and in leaves, showing low levels in mesocarps, while the transcript of the OepFAD2-2 gene was moderately expressed in developing seeds, ripening mesocarp and leaves. These expression data suggest dif- ferential functions for the two olive microsomal oleate desaturase genes, with FAD2-1 possibly responsible for the desaturation of reserve lipids in the young seed, while FAD2-2 may be mainly involved in storage lipid desaturation in the mature seeds and the mesocarp. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Olea europaea; Oleaceae; Olive; Gene expression; Microsomal oleate desaturase; FAD2; Oleic acid; Linoleic acid 1. Introduction Plant lipids contain polyunsaturated fatty acids, mainly linoleic and a-linolenic acids, which play crucial roles in plant metabolism as storage compounds mainly in the form of triacylglycerols (TAG), as structural com- ponents of membrane lipids, and as precursors of signal- ling molecules involved in plant development and stress response (Ohlrogge and Browse, 1995; Weber, 2002). Linoleic acid, together with oleic acid, is a major fatty acid in vegetable oils and its content greatly affects tech- nological properties such as their oxidative stability (Ma ´rquez-Ruı ´z et al., 1999) and nutritional characteris- tics (Cunnane, 2003). In higher plants, the fatty acid biosynthesis is cata- lyzed in the plastid by a type II (dissociable) fatty acid synthase, leading primarily to the synthesis of palmi- toyl-ACP and stearoyl-ACP by successive additions of two carbon atoms from acetyl-CoA (Harwood, 1996). Still in the plastid, most of the stearoyl-ACP is desatu- rated to oleoyl-ACP by the soluble stearoyl-ACP desat- urase. This oleic acid, which is the main product of the plastidial fatty acid synthesis, is largely activated to oleoyl-CoA and exported to the cytosol, where is incor- porated into glycerolipids and can be further desatu- rated to linoleic acid by the microsomal oleate desaturase (FAD2). This enzyme is located in the endoplasmic reticulum (ER), use phospholipids as acyl 0031-9422/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.04.004 * Corresponding author. Tel.: +34 954 611550; fax: +34 954 616790. E-mail address: mrivas@cica.es (J.M. Martı ´nez-Rivas). www.elsevier.com/locate/phytochem Phytochemistry 66 (2005) 1417–1426 PHYTOCHEMISTRY