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40 Pag., 8 Fig. The definitive version is available at: http://jxb.oxfordjournals.org/ ; 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. ; This work was supported by the Aragón Government (PIP 140/2008) and the Spanish Ministry of Science and Innovation (Grants BFU2005-07422-CO2-01 and AGL2008-00733). VA was the recipient of a predoctoral fellowship from the Aragón Government. BL was the recipient of a predoctoral fellowship from the JAE-CSIC Program. ; Peer reviewed

ω3 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 ω3 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. ; This work was supported by the Aragón Government (grant no. PIP 140/2005) and the Ministry of Education and Culture of Spain (grant nos. BFU2005–07422–C02–01, AGL2005–05104, BFU2005–01094, and PIP2006–120). R.C. and V.A. were recipients of a predoctoral fellowship from the Aragón Government. ; Peer reviewed