Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 29, 2013
Publication Date: October 11, 2013
Citation: Cao, H., Shockey, J.M., Klasson, K.T., Chapital, D.C., Mason, C.B., Scheffler, B.E. 2013. Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues. PLoS ONE 8(10):e76946. doi: 10.1371/journal.pone.0076946. Interpretive Summary: Triacylglycerols (TAGs) are the primary form of energy storage in eukaryotes. They also serve as a reservoir of fatty acids for membrane biogenesis of the cells and lead to obesity when excessively accumulated in adipose tissues. Understanding plant TAG biosynthesis will help to create new oilseed crops with value-added properties. Our project focuses on engineering oilseed crops to produce industrially useful oils. One such oil is the drying oil found in the seeds of tung tree (Vernicia fordii). Many questions related to DGAT expression and function in tung tree are yet to be answered. For example, does the putative soluble DGAT3 isoform exist in tung tree? How many isoforms of DGATs are expressed in tung seeds? What are the relative expression levels of these isoforms in tung seeds? How do their expression patterns coordinate with tung oil biosynthesis in the seeds? In this study, we identified tung DGAT3 and used TaqMan qPCR assay to evaluate the relative abundance and tissue distribution of the three tung DGAT mRNAs in the seeds, leaves, and flowers of tung tree. Northern blotting was used to confirm the expression pattern of DGATs in tung tissues. Finally, we raised antibodies against recombinant DGAT2 protein and used them to detect endogenous DGAT2 in developing tung seeds. Our results demonstrate that 1) a DGAT3 gene is present in tung tree; 2) all three isoforms of DGAT family genes are expressed in developing seeds, leaves, and flowers; 3) DGAT2 is firmly established as the major DGAT mRNA in tung seeds; 4) DGAT3 gene is likely plays a significant role in TAG metabolism in tung leaves, flowers, and immature seeds prior to active tung oil biosynthesis; and 5) endogenous DGAT2 protein was detected in developing tung seeds using DGAT2 protein-specific antibodies raised against recombinant DGAT2 protein. These results suggest that DGAT2 protein is probably the major contributor to tung oil biosynthesis in the seeds. Therefore, DGAT2 should be a major focus of metabolic engineering efforts targeting drying oil production in transgenic organisms.
Technical Abstract: Diacylglycerol acyltransferases (DGAT) are responsible for the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes, including DGAT1 and DGAT2 of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. In this study, we cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Quantitative real-time PCR, along with northern and western blotting, were used to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves, and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers, and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.