Identification of the Arabidopsis palmitoyl-monogalactosyldiacylglycerol delta7-desaturase gene FAD5, and effects of plastidial retargeting of Arabidopsis desaturases on the fad5 mutant phenotype.
Hexadeca 7,10,13-trienoic acid (16:3Delta(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 Delta7-desaturase FAD5, and is characterized biochemically by the absence of 16:3Delta(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 16:3Delta(7,10,13) and restored leaf chlorophyll content. Arabidopsis carrying a T-DNA insertion in At3g15870 had wild-type levels of both 16:3Delta(7,10,13) and chlorophyll. Together, these data formally prove that At3g15850 is FAD5. Interestingly, the fad5 phenotype was partially complemented when extraplastidial Delta9-desaturases of the Arabidopsis desaturase (ADS) family were expressed as fusions with a plastidial transit peptide. Tight correlation between leaf 16:3Delta(7,10,13) levels and chlorophyll content suggests a role for plastidial fatty acid desaturases in thylakoid formation.
Heilmann
Ingo
I
Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.
Mekhedov
Sergei
S
King
Barbara
B
Browse
John
J
Shanklin
John
J
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins; D002734:Chlorophyll / Q000378:metabolism; D018389:Codon, Nonsense; D044943:Fatty Acid Desaturases / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D005810:Multigene Family; D010641:Phenotype; D010802:Phylogeny; D018515:Plant Leaves / Q000378:metabolism; D018087:Plastids / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
D029681:Arabidopsis Proteins; D018389:Codon, Nonsense; D002734:Chlorophyll; D044943:Fatty Acid Desaturases; C496082:palmitoyl-monogalactosyldiacylglycerol delta(7)-desaturase, Arabidopsis