Arabidopsis diacylglycerol acyltransferase1 mutants require fatty acid desaturation for normal seed development.

Diacylglycerol acyltransferase1 (DGAT1) is the major enzyme that synthesizes triacylglycerols (TAG) during Arabidopsis seed development. Mutant dgat1 seeds possess low oil content in addition to a high polyunsaturated fatty acid (PUFA) composition. Two genes encoding endoplasmic reticulum localized desaturase enzymes, fatty acid desaturase2 (FAD2) and fatty acid desaturase3 (FAD3), were upregulated in both dgat1-1 and dgat1-2 developing seeds. Crosses between both dgat1 mutant alleles and fad2-1 failed to generate plants homozygous for both dgat1 and fad2. Reciprocal crosses with wild-type plants demonstrated that both male and female dgat1 fad2 gametophytes were viable. Siliques from DGAT1/dgat1-1 fad2-1/fad2-1 and dgat1-1/dgat1-1 FAD2/fad2-1 possessed abnormal looking seeds that were arrested in the torpedo growth stage. Approximately 25% of the seeds exhibited this arrested phenotype, genetically consistent with them possessing the double homozygous dgat1 fad2 genotype. In contrast, double homozygous dgat1-1 fad3-2 mutant plants were viable. Seeds from these plants possessed higher levels of 18:2 while their fatty acid content was lower than dgat1 mutant controls. The results are consistent with a model where in the absence of DGAT1 activity, desaturation of fatty acids by FAD2 becomes essential to provide PUFA substrates for phospholipid:diacylglycerol acyltransferase (PDAT) to synthesize TAG. In a dgat1 fad2 mutant, seed development is aborted because TAG is unable to be synthesized by either DGAT1 or PDAT.

Lipidomic Analysis of Arabidopsis T-DNA Insertion Lines Leads to Identification and Characterization of C-Terminal Alterations in FATTY ACID DESATURASE 6.

Mass-spectrometry-based screening of lipid extracts of wounded and unwounded leaves from a collection of 364 Arabidopsis thaliana T-DNA insertion lines produced lipid profiles that were scored on the number and significance of their differences from the leaf lipid profiles of wild-type plants. The analysis identified Salk_109175C, which displayed alterations in leaf chloroplast glycerolipid composition, including a decreased ratio between two monogalactosyldiacylglycerol (MGDG) molecular species, MGDG(18:3/16:3) and MGDG(18:3/18:3). Salk_109175C has a confirmed insertion in the At5g64790 locus; the insertion did not co-segregate with the recessive lipid phenotype in the F2 generation of a wild-type (Columbia-0) × Salk_109175C cross. The altered lipid compositional phenotype mapped to the At4g30950 locus, which encodes the plastidial ω-6 desaturase FATTY ACID DESATURASE 6 (FAD6). Sequencing revealed a splice-site mutation, leading to the in-frame deletion of 13 amino acids near the C-terminal end of the 448 amino acid protein. Heterologous expression in yeast showed that this deletion eliminates desaturase activity and reduces protein stability. Sequence comparison across species revealed that several amino acids within the deletion are conserved in plants and cyanobacteria. Individual point mutations in four conserved residues resulted in 77-97% reductions in desaturase activity, while a construct with all four alanine substitutions lacked activity. The data suggest that the deleted region of FAD6, which is on the C-terminal side of the four putative transmembrane segments and the histidine boxes putatively involved in catalysis, is critical for FAD6 function.