Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation

Abstract

The endoplasmic reticulum-associated oleate desaturase FAD2 (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine Delta12-desaturase) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic 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 led to the identification of two domains that appear to be important in 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 downregulate enzyme activity. Collectively, the results show that post-translational regulatory mechanisms are likely to play an important role in modulating FAD2-1 enzyme activities.