The x3 fatty-acid desaturases: FAD7 and FAD8 (plastid) and FAD3 (reticular) are responsible for trienoic fatty-acid (TA) production in plants. The expression of these enzymes seemed to be regulated differently in response to light. Darkness leads to a decrease in total TA level. Under such conditions, FAD3 and FAD8 transcript levels were undetectable but increased after re-illumination concomitant with TA levels, indicating a transcriptional control. On the contrary, FAD7 transcript levels were similar to illuminated control cells, suggesting the presence of a post-transcriptional control mechanism. Furthermore, FAD7 mRNA stability increased dramatically in darkness. Analysis of FAD7 protein accumulation using specific antibodies revealed that FAD7 was very stable whatever the light or darkness conditions. These results indicate that FAD7 enzyme availability is not limiting for 18:3 production in darkness. Our data point to an additional post-translational regulatory mechanism that controls the activity of FAD7 in response to light.

The FAD7 gene encodes a ω3 fatty acid desaturase which catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts. A novel GmFAD7 gene (named GmFAD7-2) has been identified in soybean, with high homology to the previously annotated GmFAD7 gene. Genomic sequencing analysis together with searches at the soybean genome database further confirmed that both GmFAD7 genes were located in two different loci within the soybean genome, suggesting that the soybean ω3 plastidial desaturase FAD7 is encoded by two different paralogous genes. Both GmFAD7-1 and GmFAD7-2 genes were expressed in all soybean tissues examined, displaying their highest mRNA accumulation in leaves. This expression profile contrasted with GmFAD3A and GmFAD3B mRNA accumulation, which was very low in this tissue. These results suggested a concerted control of plastidial and reticular ω3 desaturase gene expression in soybean mature leaves. Analysis of GmFAD7 protein distribution in different soybean tissues showed that, in mature leaves, two bands were detected, coincident with the higher expression level of both GmFAD7 genes and the highest 18:3 fatty acid accumulation. By contrast, in seeds, where FAD7 activity is low, specific GmFAD7 protein conformations were observed. These GmFAD7 protein conformations were affected in vitro by changes in the redox conditions of thiol groups and iron availability. These results suggest the existence of tissue-specific post-translational regulatory mechanisms affecting the distribution and conformation of the FAD7 enzymes related with the control of its activity.

v3 fatty acid desaturases are the enzymes responsible for the synthesis of trienoic fatty acids in plants. These enzymes have been mainly investigated using molecular, biochemical, and genetic approaches but very little is known about their subcellular distribution in plant cells. In this work, the precise subcellular localization of the v3 desaturase FAD7 was elucidated by immunofluorescence and immunogold labeling using a monospecific GmFAD7 polyclonal antibody in soybean (Glycine max) photoautotrophic cell suspension cultures. Confocal analysis revealed the localization of the GmFAD7 protein within the chloroplast; i.e. signals from FAD7 and chlorophyll autofluorescence showed specific colocalization. Immunogold labeling was pursued on cryofixed and freeze-substituted samples for convenient preservation of antigenicity and ultrastructure of membrane subcompartments. Our data revealed that the FAD7 protein was preferentially localized in the thylakoid membranes. Biochemical fractionation of purified chloroplasts and western analysis of the subfractions further confirmed these results. These findings suggest that not only the envelope, but also the thylakoid membranes could be sites of lipid desaturation in higher plants.Trienoic (TA) and dienoic fatty acids represent as much as 70% of total fatty acids from leaf or root lipids (Douce et al., 1990). They influence the function of biological membranes by maintaining their appropriate fluidity. Apart from this key role in cell function, TAs also serve as precursors of important plant hormones like jasmonates (JAs) that are involved in defense signaling against pathogen attack, the wound response, and plant development and adaptation to environmental stress (for review, see Iba, 2002;Schaller et al., 2005). Desaturase activity responsible for polyunsaturated fatty acid production was initially detected in microsomal and plastid preparations of various plant tissues (Ohlrogge and Browse, 1995). However, the difficulties in purifying these enzymes by biochemical methods hampered their identification. The molecular characterization of a collection of Arabidopsis (Arabidopsis thaliana) mutants defective in membrane lipid unsaturation allowed for the identification of desaturase genes, providing specific knowledge about their number, substrate specificity, and predicted location (Wallis and Browse, 2002). Thus, TAs are synthesized from dienoics by the activity of several v3 desaturases that are localized in two different cell compartments: FAD3 is specific of the endoplasmic reticulum while FAD7 and its cold-inducible isozyme, FAD8, are plastid specific (Wallis and Browse, 2002). However, with the exception of one plastidial enzyme, the stearoyl acyl-carrier protein (ACP) desaturase FAB2, which is the only soluble desaturase isolated up to now, no detailed studies investigating the activity and precise localization of these enzymes were performed mainly due to the difficulties in developing purification methods, the absence of specific antibodies, or in performing enzy...