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.

Leaf Lipid Alterations in Response to Heat Stress of Arabidopsis thaliana.

In response to elevated temperatures, plants alter the activities of enzymes that affect lipid composition. While it has long been known that plant leaf membrane lipids become less unsaturated in response to heat, other changes, including polygalactosylation of galactolipids, head group acylation of galactolipids, increases in phosphatidic acid and triacylglycerols, and formation of sterol glucosides and acyl sterol glucosides, have been observed more recently. In this work, by measuring lipid levels with mass spectrometry, we confirm the previously observed changes in Arabidopsis thaliana leaf lipids under three heat stress regimens. Additionally, in response to heat, increased oxidation of the fatty acyl chains of leaf galactolipids, sulfoquinovosyldiacylglycerols, and phosphatidylglycerols, and incorporation of oxidized acyl chains into acylated monogalactosyldiacylglycerols are shown. We also observed increased levels of digalactosylmonoacylglycerols and monogalactosylmonoacylglycerols. The hypothesis that a defect in sterol glycosylation would adversely affect regrowth of plants after a severe heat stress regimen was tested, but differences between wild-type and sterol glycosylation-defective plants were not detected.