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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Identification of the Arabidopsis Palmitoyl-Monogalactosyldiacylglycerol 7-Desaturase Gene FAD5, and Effects of Plastidial Retargeting of Arabidopsis Desaturases on the fad5 Mutant Phenotype¹

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973 (I.H., J.S.); Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824 (S.M.); and Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164 (B.K., J.B.)

Hexadeca 7,10,13-trienoic acid (16:3^7,10,13) is one of the most abundant fatty acids in Arabidopsis (Arabidopsis thaliana) and a functional component of thylakoid membranes, where it is found as an sn-2 ester of monogalactosyldiacylglycerol. The Arabidopsis fad5 mutant lacks activity of the plastidial palmitoyl-monogalactosyldiacylglycerol 7-desaturase FAD5, and is characterized biochemically by the absence of 16:3^7,10,13 and physiologically by reduced chlorophyll content and a reduced recovery rate after photoinhibition. While the fad5 mutation has been mapped, the FAD5 gene was not unambiguously identified, and a formal functional characterization by complementation of fad5 mutant phenotypes has not been reported. Two candidate genes (At3g15850 and At3g15870) predicted to encode plastid-targeted desaturases at the fad5 chromosomal locus were cloned from fad5 plants and sequenced. A nonsense mutation changing codon TGG (Trp-98) into TGA (stop) was identified in At3g15850 (ADS3), whereas the fad5 At3g15870 allele was identical to wild type (after correction of a sequencing error in the published wild-type genomic At3g15870 sequence). Expression of a genomic clone or cDNA for wild-type At3g15850 conferred on fad5 plants the ability to synthesize 16:3^7,10,13 and restored leaf chlorophyll content. Arabidopsis carrying a T-DNA insertion in At3g15870 had wild-type levels of both 16:3^7,10,13 and chlorophyll. Together, these data formally prove that At3g15850 is FAD5. Interestingly, the fad5 phenotype was partially complemented when extraplastidial 9-desaturases of the Arabidopsis desaturase (ADS) family were expressed as fusions with a plastidial transit peptide. Tight correlation between leaf 16:3^7,10,13 levels and chlorophyll content suggests a role for plastidial fatty acid desaturases in thylakoid formation.

² Present address: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894.

³ Present address: Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94143.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.052951.

^* Corresponding author; e-mail shanklin{at}bnl.gov; fax 631–344–3407.

Received September 7, 2004; returned for revision October 15, 2004; accepted October 15, 2004.

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