Biotechnological Aspects : Fatty Acids reticulum. Green fluorescent protein-AtACH5 and -AtACH4 fusion proteins were localized in the endoplasmic reticulum in onion epidermal cells (results not shown ; work conducted by Dr Gert-Jan de Boer in Dr Chris Somerville ’s lab- oratory at Stanford University, Stanford, CA, U.S.A.). Like the first family of ACHs, these proteins have no known physiological role. AtACH5 has been successfully overexpressed in E. coli, and extracts of these cells show increased ACH activity over cells carrying only an empty expression vector (results not shown). Arabidopsis plants with mutations in the AtACH5 gene have no visible phenotype, either morphologically or at the lipid level. However, since lipid synthesis happens largely in the endoplasmic reticulum, these proteins have the potential to regulate synthesis by dictating which types of fatty acyl- CoA are admitted to the pathway. Future experiments, including characteriz- ation of mutants and biochemical studies of puri- fied enzymes, will help determine whether ACHs Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana M. Smith 1 , H. Moon and L. Kunst Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada Abstract Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) nfi3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Δ12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity. Key words : castor, hydroxylase, ricinoleic acid, yeast. Abbreviations used : ER, endoplasmic reticulum ; FAME, fatty acid methyl ester. 1 To whom correspondence should be addressed (e-mail msmith!mail.botany.ubc.ca). play a role in regulating the characteristics of the lipids produced in plant cells. In addition, this work will help advance understanding of the role of ACHs in other organisms, where their true physiological role remains to be discovered. References 1 Naggert, J., Narasimhan, M. L., DeVeaux, L., Cho, H., Randhawa, Z. I., Cronan, Jr, J. E., Green, B. N. and Smith, S. (1991) J. Biol. Chem. 266, 11040–11050 2 Watanabe, H., Shiratori, T., Wshoji, H., Miyatake, S., Okazaki, Y., Ikuta, K., Sato, T. and Saito, T. (1997) Biochem. Biophys. Res. Commun. 238, 234–239 3 Jones, J. M., Nau, K., Geraghty, M. T., Erdmann, R. and Gould, S. J. (1999) J. Biol. Chem. 274, 9216–9223 4 Poupon, V., Be ’ gue, B., Gagnon, J., Dautry-Varsat, A., Cerf- Bensussan, N. and Benmerah, A. (1999) J. Biol. Chem. 274, 19188–19194 5 Li, J., Derewenda, U., Dauter, Z., Smith, S. and Derewenda, Z. S. (2000) Nat. Struct. Biol. 7, 555–559 Received 7 August 2000 Introduction Hydroxy fatty acids accumulate in the seeds of a small number of plant species. Of these, castor bean (Ricinus communis) is the most well known, producing a seed oil containing 85–90 % ricinoleic acid (18:1-OH). This fatty acid is a valuable raw material and is used in a variety of applications ranging from the manufacture of Nylon and speciality lubricants to paints and cosmetics. Cas- tor oil is the only significant commercial source of hydroxy fatty acids but is less than ideal as the seeds contain potent toxins and allergens. There is considerable interest in finding an alternative supply of hydroxy fatty acids, either from another species of plant (such as members of the Les- querella genus), or by the genetic engineering of an existing crop species. In castor, 18:1-OH is formed by the direct Δ12-hydroxylation of oleic acid (18 : 1) esterified to position sn-2 of phosphatidylcholine [1]. The reaction is catalysed by a protein almost identical to an endoplasmic reticulum (ER) Δ12-desaturase. A cDNA encoding the castor Δ12-oleate hydroxyl- ase has been isolated [2,3] and a similar sequence, # 2000 Biochemical Society 947