Localization of the Arabidopsis Oleoyl Δ12 Desaturase FAD2.
The amount of unsaturated phospholipids in plant cell membranes is crucial for the optimal function of these membranes, especially in processes of secretion and endocytosis as well as in the homeostasis of integral membrane proteins. In Arabidopsis, the oleoyl-Δ12-desaturase FAD2 is responsible for the conversion of oleic acid (18:1Δ9) to linoleic acid (18:2Δ9,12) and thus for the further desaturation of the fatty acid. Despite its fundamental importance for plant physiology, little information was available on the distribution of the enzyme in the cell. In collaboration with the IPB, MLU scientists have now determined the subcellular localization of fluorescent FAD2 fusions in leaves and roots of Arabidopsis plants as well as in protoplasts.
Based on the hypothesis that FAD2 modifies phospholipid-associated fatty acids near the endoplasmic reticulum (ER), the scientists determined the distribution patterns of functional FAD2 fusions using in vivo confocal imaging, quantitative image analysis, and a panel of co-expressed organelle-specific fluorescent markers. High-resolution fluorescence microscopy revealed that the FAD2 fusions are not directly localized to the ER, but form a ring-shaped pattern around the adjacent pre-cis-Golgi stacks. This clustered localization of the desaturase is based on the presence of an unusual retention signal at the C-terminus. Deletions or substitutions in this region of the protein led to a loose distribution of FAD2 fusions in the ER-associated region. The desaturation of oleic acid apparently takes place in the pre-cis-Golgi stacks at the exit of the ER, from where the product, linoleic acid, is distributed to the surrounding (plasma) membranes by secretory mechanisms.
Localizing FADs is a technically challenging endeavor, as the topology of these membrane-integrated enzymes must be taken into account. The fluorescent tags must not interfere with the catalytic center of the enzyme, its N-terminal signal peptide, or the C-terminal retention sequence. Therefore, the physiological functionality of the FAD2 fusion constructs was tested both in yeast and by complementation of Arabidopsis mutants before starting imaging analyses.
The pathways of fatty acid desaturation in plants are of great interest for the industrial production of seed oil. According to previous research, the spatial distribution and molecular interaction of the enzymes involved play an important role in the formation of modified fatty acids in these oils. In the vegetative part of the plant, FAD2, as an important regulator of membrane lipid homeostasis, also appears to contribute to stress tolerance, e.g. to high salt concentrations.