Identification of corresponding genes for three low-a-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed Toyoaki Anai * , Tomoko Yamada, Takehito Kinoshita 1 , Shaikh M. Rahman 2 , Yutaka Takagi Laboratory of Plant Breeding, Faculty of Agriculture, Saga University, Honjyo-machi 1, Saga 840-8502, Japan Received 2 December 2004; received in revised form 25 February 2005; accepted 25 February 2005 Available online 17 March 2005 Abstract In order to keep the quality of vegetable oil stable, it is important to reduce the a-linolenic acid content. Previously, we developed three low-a-linonenic acid soybean mutants, ‘J18’, ‘M5’, and ‘M24’. In this study, we obtained and characterized four cDNAs, GmFAD3-1a, GmFAD3-1b, GmFAD3-2a, and GmFAD3-2b, encoding microsomal v-3 fatty acid desaturase from soybean developing seeds. The GmFAD3- 1b transcript does not accumulate in the ‘J18’ mutant, whereas ‘M5’ and ‘M24’ contain a 19-base pair and a single-base pair deletion in the coding region of the GmFAD3-1b and GmFAD3-1a genes, respectively. Furthermore, heterologous expression of these two mutant genes in Saccharomyces cerevisiae revealed that the COOH-terminal region of both mutant gene products is essential for their enzymatic activity. In this report, we also discuss the contribution of each microsomal v-3 fatty acid desaturase gene for a-linolenic acid biosynthesis in soybean seeds. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Soybean mutant; a-Linolenic acid; Microsomal v-3 fatty acid desaturase; Frame shift; Yeast expression system; FAD3 1. Introduction Polyunsaturated fatty acids are essential nutritional factors of humans and are mainly supplied from vegetable oils, but they easily oxidize and degrade in the course of a heat process or long-term storage. Especially, trienoic a- linolenic acid is the most unstable component of vegetable oil, including soybean oil [1,2]. The enzymatic aspect of polyunsaturated fatty acid synthesis has been well char- acterized. A higher plant has two distinct polyunsaturated fatty acid biosynthesis pathways, a microsomal pathway and a plastidal pathway. The microsomal pathway is the most important for the fatty acid production of storage oil in developing seeds. The key enzyme of a-linolenic acid production, microsomal v-3 fatty acid desaturase, catalyzes the introduction of the third double bond into the v-3 position of linoleic acid [3]. Hence, we and other groups developed soybean germplasms with low-a-linolenic acid content by induced mutation [4–9]. Three independent loci are involved in the a-linolenic acid level of soybean seed. Combining these loci, Ross et al. developed the 1%-linolenate line and characterized its agronomic and seed traits [9]. Previously, we isolated three low-a-linolenic acid soybean mutants, ‘J18’, ‘M5’, and ‘M24’, from an X ray-irradiated seed population. The mutant alleles in ‘M5’ and ‘M24’ were designated as fan and fanx a , respectively [7,8]. The ‘J18’ was developed from another population of X ray-irradiated seed and was allelic to fan (unpublished data). The a- linolenic acid contents of ‘J18’, ‘M5’, and ‘M24’ and the www.elsevier.com/locate/plantsci Plant Science 168 (2005) 1615–1623 * Corresponding author. Tel.: +81 952 28 8725; fax: +81 952 28 8725. E-mail address: anai@cc.saga-u.ac.jp (T. Anai). 1 Saga Prefectural Agricultural Research Center, Kawasoe-machi 1088, Saga 840-2205, Japan. 2 Biotechnology Division, Fukuoka Agricultural Research Center, Yoshiki 587, Chikushino, Fukuoka 818-8549, Japan. 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.02.016