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Title: TUNG TREE DGAT1 AND DGAT2 HAVE NONREDUDANT FUNCTIONS IN TRIACYLGLYCEROL BIOSYNTHESIS AND ARE LOCALIZED TO DIFFERENT SUBDOMAINS OF THE ENDOPLASMIC RETICULUM

Author
item Shockey, Jay
item GIDDA, SATINDER - UNIVERSITY OF GUELPH
item Chapital, Dorselyn
item Kuan, Jui-Chang
item DHANOA, PREETINDER - UNIV. OF GUELPH
item Bland, John
item ROTHSTEIN, STEVEN - UNIV. OF GUELPH
item MULLEN, ROBERT - UNIV. OF GUELPH
item Dyer, John

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/25/2006
Publication Date: 8/18/2006
Citation: Shockey, J.M., Gidda, S., Chapital, D.C., Kuan, J.W., Dhanoa, P.K., Bland, J.M., Rothstein, S.J., Mullen, R.T., Dyer, J.M. 2006. Tung tree DGAT1 and DGAT2 have nonredudant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. The Plant Cell 18:2294-2313.

Interpretive Summary: The seeds of the tung tree produce large amounts of oil that contain a unique type of fatty acid, called eleostearic acid. The presence of this fatty acid gives this oil attractive properties as a drying oil, making it useful as a varnish-like coating for products like furniture and computer chips, and as a component in various plastics and other polymers. Unfortunately, in the United States, the tung tree is poorly suited for growth outside of the Gulf South, which limits the total acreage that can be planted, which in turn limits the amount of oil that can be produced domestically. As an alternative, we propose to produce tung-like oils in microbes and/or other domesticated oilseed crops by cloning the genes that are responsible for tung oil synthesis and introducing them into the other organisms. This process involves at least five and possibly as many as eight different enzymes. Previous research indicates that one of the most important enzymes in this process is called diacylglycerol acyltransferase (DGAT), which carries out the last step in the oil synthetic pathway. Plants and other higher organisms contain at least two different types of DGAT genes, typically named DGAT1 and DGAT2. In this paper, we have cloned both types of DGAT genes from tung, and have carried out a series of experiments to compare the properties of DGAT1 and DGAT2, as a first step in determining whether one enzyme type, or both, play an important role in production of oils rich in eleostearic acid. The enzymes are significantly different in many respects. The RNA products that carry the information for production of the enzymes are produced at different levels in different parts of the plant, especially in developing seeds, where DGAT2 is turned on to much higher levels than is DGAT1. When the enzymes are produced in yeast which are being fed the fatty acid building blocks of tung oil, DGAT2 produces a pool of oils that is highly enriched in eleostearic acid, compared to DGAT1. And finally, when these two enzymes are produced in individual plant cell cultures, both are targeted to the ER, the section of cells that is known to be the site of oil production. However, close inspection of the cells reveals that these two enzymes are targeted to distinct, non-overlapping portions within the ER, which suggests that DGAT1 and DGAT2 may produce different types of oil to be used for different purposes in the plant. Our hypothesis is that DGAT2 appears to play a bigger role in eleostearic acid-containing oils and is therefore the enzyme that will pursue more aggressively in our research.

Technical Abstract: The seeds from the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) that contain approximately 80% eleostearic acid, a novel conjugated fatty acid that is used in numerous industrial products. We are interested in understanding the biochemical, physiological, and cellular properties of all the enzymes that participate in TAG biosynthesis. In the current study we focus on diacylglycerol acyltransferases (DGATs), the enzymes that catalyze the committed step in oil synthesis in tung and other oilseeds. We have isolated the genes and cDNAs encoding both type-1 and type-2 DGATs from developing tung seeds. Both genes were differentially expressed during seed development, although DGAT2 was more strongly induced, coincident with the onset of oil and eleostearic acid synthesis. Compared to DGAT1, DGAT2 also showed a stronger preference for eleostearate-containing substrates; producing five-fold more trieleostearin, the main triacylglycerol component of tung oil. Immunofluorescence microscopy revealed that both enzymes are targeted to the endoplasmic reticulum membrane, but each enzyme was specifically targeted to a distinct subdomain within this organelle. These data collectively suggest that each type of DGAT has evolved to fulfill different roles during oil seed biosynthesis perhaps by synthesizing different pools of TAGs within the ER.