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

Summary The endoplasmic reticulum‐associated oleate desaturase FAD2 (1‐acyl‐2‐oleoyl‐ sn ‐glycero‐3‐phosphocholine Δ12‐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.