Overexpression of the FAD3 Desaturase Gene in a Mutant of Arabidopsis

Abstract

A mutant of Arabidopsis contained increased levels of 18:3 fatty acids and correspondingly decreased levels of 18:2. The fatty acid phenotype was strongly expressed in root and seed tissues and this observation, together with other data, suggested that the mutation leads to increased activity of the endoplasmic reticulum 18:2 desaturase encoded by the FAD3 gene. Cel-blot analysis of RNA from wild-type and mutant plants established that FAD3 transcript levels were increased 80% in the mutant relative to the wild type. Cenetic analysis demonstrated a linkage between the new mutation and the fad3 locus. Linkage of the mutation to fad3 raises the possibility that the lesion is an alteration to the promoter or another regulatory region of the FAD3 gene, which results in increased transcription.A characteristic feature of the membranes in plant cells is their very high level of fatty acid unsaturation compared with membranes of other eukaryotes. Whereas glycerolipids from animal tissues typically contain an average of 2.0 to 2.5 double bonds/ molecule in the hydrocarbon chains (Harwood et al., 1986;Marsh, 1990), the number of double bonds/molecule in glycerolipids from higher plants is 3 to 3.4 from nonphotosynthetic tissues and 4.3 to 5.0 for chloroplasts (Harwood, 1980). The chloroplast membranes of plant cells are composed entirely of the uncharged galactolipids MGD and DGD, which make up about 75% of the total chloroplast thylakoid lipid content (Harwood, 1980). Polyunsaturated trienoic fatty acids (18:3 and 16:3) constitute up to 80% of the fatty acids in this organelle. This high level of membrane unsaturation is achieved by the synthesis of polyunsaturated a-linolenate (A9, 12, 15, and 18:3) and hexadecatrienoic acid (A7, 10, 13, and 16:3). The insertion of double bonds at the 12 and 15 positions in a-linolenate occurs in plants but not in other higher eukaryotes; therefore, 18:2 and 18:3 are two essential fatty acids in human nutrition.In plant cells lipid metabolism is initiated by the synthesis of 16:O-ACP and 18:O-ACP by the reactions of a type I1 fatty acid synthase (Ohlrogge and Browse, 1995). The 18: O-ACP is quickly and efficiently desaturated to 18:l-ACP

Insights into the Role of Specific Lipids in the Formation and Delivery of Lipid Microdomains to the Plasma Membrane of Plant Cells

“…We took advantage of two mutant Arabidopsis cell lines, fad2 and Fad3 1 , which are affected in their fatty acid desaturases. The 18:1 desaturase is shut down in the fad2 mutant (Okuley et al, 1994), resulting in an accumulation of 18:1 fatty acids (Caiveau et al, 2001), whereas the Fad3 1 mutant accumulates 18:3 fatty acids (Shah et al, 1997). Given the important role played by lipids with saturated fatty acyl chains in regulating the physicochemical properties of biological membranes, it was particularly interesting to check DRM formation in these two cell lines, which exhibit significant differences in the mean fluidity (defined as temperaturedependent lipid movements in the membrane) of their PM, compared to the PM of wild-type cells (Vaultier et al, 2006).…”

“…Because of their deficit in 18:1 desaturase (Okuley et al, 1994), fad2 cells accumulate fatty acyl chains enriched in 18:1 (Caiveau et al, 2001), whereas the Fad3 1 mutant accumulates 18:3 fatty acids (Shah et al, 1997).…”

“…Thus, the important changes in the fatty acid composition of fad2 and Fad3 1 membranes (Shah et al, 1997;Caiveau et al, 2001), and therefore their different and PM DRMs (small dots) represent the means 6 SD of four and seven replicates, respectively. B, Values for GA (black dots) and GA DRMs (white) represent the means 6 SD of three replicates.…”

bZIP67 Regulates the Omega-3 Fatty Acid Content of Arabidopsis Seed Oil by Activating FATTY ACID DESATURASE3

“…FAD3 encodes a microsomal 18:2 n6 desaturase that uses 18:2 n6 esterified to PC as its substrate and is required for the majority of 18:3 n3 produced in Arabidopsis seeds (Lemieux et al, 1990;Browse et al, 1993). The abundance of 18:3 n3 is known to be highly responsive to the level of FAD3 expression (Shah et al, 1997;Puttick et al, 2009;O'Neill et al, 2011) and FAD3 transcript abundance increases approximately fivefold during the maturation stage of embryo development (Winter et al, 2007). Reduced expression of FAD3 could therefore explain the higher level of 18:2 n6 and lower level of 18:3 n3 -containing species in both PC and TAG, in bzip67 (C) RT-PCR on RNA isolated from whole developing siliques of bzip67-1, bzip67-2 and wild-type (WT) plants using bZIP67 primers that amplify the fulllength cDNA (;1 kb).…”

Activity of Brassica napus and Perilla frutescens microsomal ω-3 desaturases expressed in yeast (Saccharomyces cerevisiae)

“…Desaturase enzymes in higher plants have an N-terminal domain related to cytochrome b5 (Sayanova et al, 1997). ω-3 desaturase enzyme is the product of the Fad3 gene (Shah et al, 1997). In Arabidopsis, the Fad7 locus encodes a chloroplast ω-3 desaturase (Iba et al, 1993;Horiguchi et al, 1996).…”