Identification and characterization of C-terminal alterations in Arabidopsis thaliana FATTY ACID DESATURASE 6 (FAD6)

Abstract

Lipid analysis of leaves from the Arabidopsis T-DNA insertion line Salk_109175C revealed a decreased ratio between two monogalactosydiacylglycerol molecular species, MGDG(18:3/16:3) and MGDG(18:3/18:3), as well as other alterations consistent with a disruption in chloroplast glycerolipid assembly. Salk_109175C has a confirmed insertion in the At5g64790 locus which did not co-segregate with the lipid phenotype in the F2 generation of a wild type (Col-0) x Salk_109175C cross. The mutation associated with the lipid alterations mapped to the At4g30950 locus, which encodes for the plastidial ω6 desaturase FATTY ACID DESATURASE 6 (FAD6). Sequencing revealed a splice site mutation between exons 9 and 10, which leads to the in-frame deletion of 13 amino acids near the C-terminal end of the protein. Sequence comparison across species revealed multiple amino acids within the deletion are conserved in all plants, and even cyanobacteria. We cloned and heterologously co-expressed several constructs of AtFAD6 with photosynthetic ferredoxin (AtFD2) under the control of a constitutive promoter (CDC11) or an inducible promoter (Gal1/10) in yeast, with the goal of determining which combination of gene and promoter results in the highest activity. The future of this project involves using this system to clone and express mutant fad6 constructs in yeast to evaluate the impact of conserved residues on enzyme function.