Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/23/2005
Publication Date: 4/25/2005
Citation: Shockey, J.M., Dhanoa, P.K., Dupuy, T., Chapital, D.C., Mullen, R.T., and Dyer, J.M. 2005. Cloning, functional analysis, and subcellular localization of two isoforms of NADH:cytochrome b5 reductase from developing seeds of tung (Vernicia fordii). Plant Science. 169(2):375-385. Interpretive Summary: The seeds of the tung tree produce oil that is rich in eleostearic acid, a fatty acid whose chemical structure imparts excellent drying properties to the oil. While once widely cultivated in the Gulf South region of the United States, hurricane damage has nearly eliminated the tung orchards. However, the demand for tung oil in domestic industry persists, leading to the need to import tung oil from other countries. The research described in this manuscript is part of an ongoing project dedicated to the production of tung-like drying oils in common baker's yeast, which can be grown in large amounts in a factory setting. Earlier work in this project has led to the production of low levels of eleostearic acid in the oil of yeast, but this level needs to be increased substantially to make industrial processing cost-effective. One approach to achieving this increase is to identify other genes from tung that participate in the production of oil in the seeds. These genes will be included in the yeast system, as a means of constructing all necessary tung proteins in an 'assembly line' that will more effectively funnel common fatty acids found in the yeast cells into eleostearic acid. Two genes abbreviated CBR1A and CBR1B have been isolated from developing tung seeds and tested for the ability to produce reduced cytochrome b5, the necessary form of a carrier protein that provides an essential compound to FADX, the enzyme that actually produces eleostearic acid. These two genes are the first known CBR genes cloned from an exotic plant species such as tung that produces industrially useful fatty acids. After expression in yeast, these genes will provide valuable information regarding the need for CBR genes in any such endeavor to produce novel oils in yeast or transgenic plants. As a part of the larger, longer term goal of producing tung-like drying oils in yeast or other organisms, this work will help U.S. agriculture obtain a domestic source of high-quality drying oils, thus eliminating the need to depend on importation of inferior oils that tend to vary greatly in their quality and price.
Technical Abstract: Two genes and the corresponding cDNAs for NADH:cytochrome b5 reductase (VfCBR1A and VfCBR1B) from tung (Vernicia fordii) were cloned and characterized. After overexpression in Saccharomyces cerevisiae, VfCBR1A was enzymatically active, and like its Arabidopsis homolog, displayed strict specificity for NADH as the reductant. Both tung genes are expressed at similar levels in various tissues throughout the plant, but differ substantially in their genomic architecture. Phylogenetic comparisons of many cloned and putative CBR genes from higher plants and yeast revealed two general classes of sequences. The separation of the classes likely reflects differences in the subcellular targeting of the two types of proteins. Immunofluorescence analyses of tobacco BY2 cells containing transiently expressed tung CBR1A, CBR1B, or Arabidopsis CBR revealed definitive targeting of the proteins to the endoplasmic reticulum while a previously uncharacterized Arabidopsis CBR protein was targeted specifically to mitochondria. The subcellular localization and biochemical properties of the tung enzymes are consistent with a potential role of these enzymes in fatty acid desaturation and conjugation.