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Title: OLEATE DESATURASE ENZYMES OF SOYBEAN: EVIDENCE OF REGULATION THROUGH DIFFERENTIAL STABILITY AND PHOSPHORYLATION.

Author
item TANG, GUO-QING - NC STATE UNIVERSITY
item Novitzky, William
item GRIFFIN, CAROL - NC STATE UNIVERSITY
item Huber, Steven
item DEWEY, RALPH - NC STATE UNIVERSITY

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2005
Publication Date: 11/1/2005
Citation: Tang, G., Novitzky, W.P., Griffin, C., Huber, S.C., Dewey, R. 2005. Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation. Plant Journal. 44:433-446.

Interpretive Summary: The relative quantity of the various saturated and unsaturated fatty acids in soybean oil is a major determinant of quality and largely determines the commercial application of the oil. In soybean, the formation of polyunsaturated fatty acids (PUFAs) starts with the conversion of oleic acid to linoleic acid, which is catalyzed by the fatty acid desaturase enzyme known as FAD2. The fatty acid composition of soybean oil varies with genotype and also environment, with growth at elevated temperatures usually associated with a decrease in PUFA content of seed oil. However, little is known about the regulation of fatty acid desaturases that control PUFA content. We found that the soybean genome actually encodes two closely related, actively expressed FAD2-1 isoforms. Expression analyses in yeast of these two isoforms, designated FAD2-1A and FAD2-1B, unexpectedly revealed major differences in the stabilities of these two closely related enzymes, particularly at elevated temperatures. In addition, we obtained evidence that soybean FAD2-1 enzymes can be phosphorylated. Both enzyme phosphorylation and temperature-dependent regulation of protein turnover of FAD2-1 enzyme activities may be important during seed development. These results provide new insights into the mechanisms by which oleate desaturase enzymes may be regulated at the post-transcriptional level. Subtle differences in amino acid sequence between the closely related FAD2-1A and FAD2-1B isoforms proved to be responsible for substantial differences in the temperature-dependent stability of the enzymes when expressed in yeast. Furthermore, the capacity of the FAD2-1 enzymes to be phosphorylated provides an additional avenue of regulatory control. Understanding the mechanisms by which fatty acid composition is determined during seed development under varying environmental conditions brings us closer to the ultimate goal of regulating these processes in a directed, predictable manner.

Technical Abstract: The endoplasmic reticulum-associated oleate desaturase FAD2 (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase) is the key enzyme responsible for the production of linoleic acid in nonphotosynthetic tissues of plants. Little is known, however, concerning the post-transcriptional mechanisms that regulate the activity of this important enzyme. The soybean genome possesses two seed-specific isoforms of FAD2, designated FAD2-1A and FAD2-1B, which differ at only 24 amino acid residues. Expression studies in yeast revealed that the FAD2-1A isoform is more unstable than FAD2-1B, particularly when cultures were maintained at elevated growth temperatures. Analysis of chimeric FAD2-1 constructs lead to the identification of two domains that appear to be important in the mediating the temperature-dependent instability of the FAD2-1A isoform. The enhanced degradation of FAD2-1A at high growth temperatures was partially abrogated by treating the cultures with the 26S proteasome-specific inhibitor MG132 and by expressing the FAD2-1A cDNA in yeast strains devoid of certain ubiquitin conjugating activities, suggesting a role for ubiquitination and the 26S proteasome in protein turnover. In addition, phosphorylation state-specific antipeptide antibodies demonstrated that the Serine-185 of FAD2-1 sequences is phosphorylated during soybean seed development. Expression studies of phosphopeptide mimic mutations in yeast suggest that phosphorylation may down regulate enzyme activity. Collectively, the results show that post-translational regulatory mechanisms are likely to play an important role in modulating FAD2-1 enzyme activities.