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.

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

“…Turnover-Previous analyses implicated the ERAD pathway in the degradation of mammalian SCD (71), plant Fad2 (15), and yeast SCD (29), and here we have demonstrated that Fad3 proteins are also degraded by a UPS-dependent process in both yeast (Fig. 7) and plant (Fig.…”

“…3C), lysine residues were shown to be important for determining SCD half-life, with substitution of a single lysine residue within an N-terminal sequence resulting in a significant increase in protein half-life (70). The ER-localized Fad2 enzymes of plants have been also shown to contain degradation signals within their N termini (15). Unlike Fad3, however, the plastidial Fad8 enzyme has been shown to contain a distinct degradation signal within the last 44 amino acids of the protein sequence (16), with the difference in positioning of these degradation signals in Fad8 and Fad3 possibly being a reflection of unique aspects of their protein biogenesis.…”

“…For instance, PUFA content in plant cells generally increases as temperature decreases, but amounts of mRNA for associated FAD genes often do not change appreciably at cooler temperatures. Furthermore, expression of either FAD2 genes in yeast cells (14,15) or FAD7/FAD8 chimeric genes in transgenic Arabidopsis (16) reveals that Fad2 and Fad8 enzymes are shortlived proteins for which abundance is modulated by temperature. As such, one of the mechanisms for Fad regulation in plant cells appears to involve the modulation of Fad protein half-life in response to temperature.…”

Understanding the Biochemical Basis of Temperature-Induced Lipid Pathway Adjustments in Plants

“…In wheat, FAD2 was induced at low temperature and also correlated with the enhanced eukaryotic pathway. In developing seeds, an induction of FAD2-1 and FAD2-2 expression in soybean (Glycine max) at high temperature has been reported previously (Heppard et al, 1996), but that was rationalized in the context of posttranscriptional regulation of enhanced degradation of the soybean FAD2-1 (Tang et al, 2005). However, it is also possible that the induction of FAD2 was associated with a higher lipid pathway activity through the eukaryotic pathway in developing seeds.…”

FAD2 and FAD3 Desaturases Form Heterodimers That Facilitate Metabolic Channeling in Vivo

“…The ER-localized FAD3 is regulated extensively by both temperature-dependent changes in translational efficiency and modulation of protein half-life (27,28). Furthermore, expression of FAD2 in yeast cells reveals a short half-life, and its abundance is also modulated by temperature, although the details of the turnover mechanism are currently unknown (29,30).…”