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United States Department of Agriculture

Agricultural Research Service

Title: Expression of the Brassica Napus Fad3 Gene in Yeast: Increased Production of Linolenic Acid at Lower Growth Temperatures Is Due to a Post Transcriptional Increase in Steady-State Amount of Desaturase Enzyme

Authors
item Dyer, John
item Chapital, Dorselyn
item Cary, Jeffrey
item Pepperman Jr, Armand

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 8, 2001
Publication Date: N/A

Interpretive Summary: Fatty acids are the major components of vegetable oils. They may be saturated or unsaturated, depending on the plant that they were derived from. We are interested in learning how plants create unsaturated fatty acids, since oils that contain high amounts of unsaturated fatty acids can be used in certain industrial applications. Fatty acids are also a major component of cellular membranes, which surround all of the cells in the plant. The fatty acid composition is often a mixture of saturated and unsaturated fatty acids, however exposure of plants to chilling temperatures results in a substantial increase in amount of unsaturated fatty acids. How this protective increase occurs is not known. A family of enzymes called fatty acid desaturases makes unsaturated fatty acids. We developed a system that would allow us to study the function of plant fatty acid desaturases by expressing the plant desaturase genes in yeast. Yeast have a very simple fatty acid composition that allowed us to make very careful measurements of the plant enzyme activity. During the course of these studies, we observed that yeast cells grown at cooler temperatures contained much higher amounts of the unsaturated fatty acids produced by the plant enzyme, similar to the effect observed in plants. By using a variety of experimental techniques, we determined that this increase was due to a substantial increase in amount of the plant desaturase enzyme in yeast cells. This information provides important insight to how these enzymes might be regulated by temperature in plants. This information will be of benefit to other scientists.

Technical Abstract: Plants respond to chilling exposure by increasing the relative proportion of polyunsaturated fatty acids in their lipids. However, unlike the response in other organisms, plant fatty acid desaturase genes are not upregulated during this process. We expressed the Brassica napus FAD3 gene, which encodes an enzyme for synthesis of linolenic acid, in Saccharomyces cerevisiae under control of the galactose-inducible GAL1-10 promoter. Small amounts of linolenic acid were detected in yeast lipids, and the amount could be significantly increased by growth of cells at cooler temperatures. Characterization of the endogenous homeoviscous response of wild-type yeast to chilling revealed an increase in the ratio of C16/C18 fatty acids, rather than increase in degree of unsaturation. Measurement of the steady-state amount of Fad3 protein using an epitope- tagged version of the enzyme revealed a 10-fold increase in amount of desaturase in yeast cells grown at cooler temperatures. The amount of desaturase detected at each temperature directly correlated with amount of linolenic acid present in yeast lipids. Northern blotting revealed that the amount of FAD3 mRNA did not change appreciably at cooler temperatures, indicating that the increase in steady-state amount of desaturase protein was a post-transcriptional event. Implications for homeoviscous adaptation in plants are discussed.

Last Modified: 7/28/2014
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