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.

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Soybean [Glycine max (L.) Merr.] oil from commercial cultivars typically contains ca. 3% stearic acid (18:0). However, germplasm carrying different mutations at the locus governing stearic acid (Fas) may contain 3% to about 35% 18:0. Among these germplasm, a newly developed line, FAM94-41 (9% 18:0), carries a serendipitous natural mutation that is temporarily designated as the recessive fas nc allele, and the germplasm A6 (26% 18:0) carries the recessive fas a allele. Mendelian genetic analysis of progeny from FAM94-41 × A6 revealed that fas nc and fas a are allelic to each other and represent different mutations in the same structural gene. However, the gene products (enzymes) produced by these alleles are unknown. The observation that 18:0 concentrations among progeny from FAM94-41 × A6 increased primarily at the expense of unsaturated C 18 FA suggests that fas alleles may reduce either 18:0-acyl carrier protein (AcP) desaturase or 18:1-ACP thioesterase activity. However, it also is conceivable that elevated 18:0 concentrations may result from increased 3-keto-acyl-ACP synthetase (KAS) II activity. To test the latter possibility, a population was created that segregated for the fas nc and the fap 2 alleles (the latter of which is associated with reduced KAS-II activity). Mendelian genetic analysis showed that these alleles represent independent genes at different gene loci and interact in an additive genetic manner to increase the total saturate concentration in this population. Based on this finding, we speculate that fas alleles probably encode 18:0-ACP desaturase or 18:1-ACP thioesterase in soybeans.The stearic acid (18:0) concentration in soybeans typically averages about 3% of crude oil (1). However, this phenotypic trait may be genetically altered by mutations at gene loci, designated Fas, which typically result in elevated 18:0 concentrations. Nearly all of these variants have been induced by chemical or X-ray mutagenesis, with the exception of a newly developed line, FAM94-41. FAM94-41 (9% 18:0) is the only known soybean germplasm that carries a serendipitous natural mutation, presumably at Fas, which now is temporarily designated as the recessive fas nc allele. Prior to the discovery of fas nc , five germplasm lines were reported to carry homozygous recessive alleles that influenced 18:0 concentrations in soybean oil: fas a [A6 (2)], fas b [FA41545 (3)], fas [A81-606085 (3)], st 1 [KK-2 (4)], and or st 2 [M25 (4)]. As reported (3), fas a (30% 18:0), fas b (15% 18:0), and fas (19% 18:0) are allelic to each other and presumably represent different mutations in the same gene. In an F 3 population segregating for st 1 and st 2 , the observation of progeny with 18:0 concentrations above and below the respective parental means provides evidence for two independently inherited genes governing higher 18:0 concentrations in soybeans. The proposed st 1 st 1 st 2 st 2 genotype may elevate the 18:0 concentration beyond 35% in crude oil (4). However, it is unknown whether st 1 or st 2 is allelic to fas a , fas b , ...