First published online October 19, 2007; 10.1104/pp.107.109637
Plant Physiology 145:1336-1344 (2007)
© 2007 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
In Situ Molecular Identification of the Plastid  3 Fatty Acid Desaturase FAD7 from Soybean: Evidence of Thylakoid Membrane Localization1
Vanesa Andreu2,
Raquel Collados2,
Pilar S. Testillano,
María del Carmen Risueño,
Rafael Picorel and
Miguel Alfonso*
Department of Plant Nutrition, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, 50059 Zaragoza, Spain (V.A., R.C., R.P., M.A.); and Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain (P.S.T., M.d.C.R.)
 3 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 3 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.
1 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.
2 These authors contributed equally to the article.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for authors (www.plantphysiol.org) is: Miguel Alfonso (alfonso{at}eead.csic.es).
www.plantphysiol.org/cgi/doi/10.1104/pp.107.109637
* Corresponding author; e-mail alfonso{at}eead.csic.es.
Received September 24, 2007;
accepted October 4, 2007;
published October 19, 2007.
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