Mark Andrew Smith

All fatty acids are not equal: discrimination in plant membrane lipids.

Plant membrane lipids are primarily composed of 16-carbon and 18-carbon fatty acids containing up to three double bonds. By contrast, the seed oils of many plant species contain fatty acids with significantly different structures. These unusual fatty acids sometimes accumulate to >90% of the total fatty acid content in the seed triacylglycerols, but are generally excluded from the membrane lipids of the plant, including those of the seed. The reasons for their exclusion and the mechanisms by which this is achieved are not completely understood. Here we discuss recent research that has given new insights into how plants prevent the accumulation of unusual fatty acids in membrane lipids, and how strict this censorship of membrane composition is. We also describe a transgenic experiment that resulted in an excessive buildup of unusual fatty acids in cellular membranes, and clearly illustrated that the control of membrane lipid composition is essential for normal plant growth and development.

Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana

Expression of a cDNA encoding the castor bean (Ricinus communis L.) oleate Δ12-hydroxylase in the developing seeds of Arabidopsis thaliana (L.) Heynh. results in the synthesis of four novel hydroxy fatty acids. These have been previously identified as ricinoleic acid (12-hydroxy-octadec-cis-9-enoic acid: 18:1-OH), densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid: 20:1-OH) and auricolic acid (14-hydroxy-eicos-cis-11,17-enoic acid: 20:2-OH). Using mutant lines of Arabidopsis that lack the activity of the FAE1 condensing enzyme or FAD3 ER Δ-15-desaturase, we have shown that these enzymes are required for the synthesis of C20 hydroxy fatty acids and polyunsaturated hydroxy fatty acids, respectively. Analysis of the seed fatty acid composition of transformed plants demonstrated a dramatic increase in oleic acid (18:1) levels and a decrease in linoleic acid (18:2) content correlating to the levels of hydroxy fatty acid present in the seed. Plants in which FAD2 (ER Δ12-desaturase) activity was absent showed a decrease in 18:1 content and a slight increase in 18:2 levels corresponding to hydroxy fatty acid content. Expression of the castor hydroxylase protein in yeast indicates that this enzyme has a low level of fatty acid Δ12-desaturase activity. Lipase catalysed 1,3-specific lipolysis of triacylglycerol from transformed plants demonstrated that ricinoleic acid is not excluded from the sn-2 position of triacylglycerol, but is the only hydroxy fatty acid present at this position.

Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana

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) n-3 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 Delta12-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.

A root-specific condensing enzyme from Lesquerella fendleri that elongates very-long-chain saturated fatty acids

The LfKCS45 gene with a high sequence similarity to known 3-ketoacyl-CoA synthases of the membrane-bound fatty acid elongase was isolated from Lesquerella fendleri. The LfKCS45 gene has a 1464 bp open reading frame without introns, and is predicted to encode a polypeptide of 487 amino acids with an estimated molecular mass of 54.6 kD. High-stringency DNA blot analysis indicated that there were no closely related genes to LfKCS45 in the L. fendleri genome. Analysis of the fatty acid composition of transformed yeast revealed that expression of the LfKCS45 protein results in the synthesis of two novel very-long-chain fatty acids identified as C28:0 and C30:0. LfKCS45 was found to be not active with acyl-CoA substrates C16 to C24 in length. Reverse transcription-PCR experiments showed that the LfKCS45 gene is expressed only in L. fendleri root tips. Histochemical assays for GUS activity in Arabidopsis transformed with the LfKCS45 promoter-GUS fusion construct confirmed this expression pattern and demonstrated that LfKCS45 transcription is restricted to the cells of the lateral root cap.

Production of Hydroxy Fatty Acids in Arabidopsis Thaliana

Castor bean (Ricinus communis) produces a seed oil that contains nearly 90% ricinoleic acid (12-hydroxyoctadeca-9-cis-enoic acid: 18:1-OH). This fatty acid is a valuable industrial raw material and is used in a wide variety of processes and products. In castor, 18:1-OH is synthesized by the 012-hydroxylation of oleate esterified to the sn-2 position of phosphatidylcholine (Bafor et al., 1991). The reaction is catalysed by an enzyme that is closely related to a FAD2 (ER-Δ12) desaturase (van de Loo et al., 1995). We have been using Arabidopsis lines, transformed with a gene construct encoding the castor hydroxylase, as a model system to study the synthesis and accumulation of hydroxy fatty acids in the seed.