Identification of the Arabidopsis Palmitoyl-Monogalactosyldiacylglycerol Δ7-Desaturase Gene FAD5, and Effects of Plastidial Retargeting of Arabidopsis Desaturases on the fad5 Mutant Phenotype

Abstract

Hexadeca 7,10,10,13 ) 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 D7-desaturase FAD5, and is characterized biochemically by the absence of 16:3D 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 16:3D 7,10,13 and restored leaf chlorophyll content. Arabidopsis carrying a T-DNA insertion in At3g15870 had wild-type levels of both 16:3D 7,10,13 and chlorophyll. Together, these data formally prove that At3g15850 is FAD5. Interestingly, the fad5 phenotype was partially complemented when extraplastidial D9-desaturases of the Arabidopsis desaturase (ADS) family were expressed as fusions with a plastidial transit peptide. Tight correlation between leaf 16:3D 7,10,13 levels and chlorophyll content suggests a role for plastidial fatty acid desaturases in thylakoid formation.Plants that accumulate hexadeca 7,10,13-trienoic acid (16:3D 7,10,13 ) are termed 16:3 plants and include rape (Brassica napus), spinach (Spinacia oleracea), and Arabidopsis (Arabidopsis thaliana). In these plants, 16:3D 7,10,13 can comprise up to 30% of the total fatty acid (Browse et al., 1986), making 16:3D 7,10,13 the second-most-abundant fatty acid after a-linolenic acid (18:3D 9,12,15 ). Pioneering work by Siebertz and Heinz established that 16:3D 7,10,13 is confined to the sn-2 position of monogalactosyldiacylglycerol (MGDG) and results from a sequence of desaturation steps starting with 16:0 (Siebertz and Heinz, 1977). In Arabidopsis, a three-step desaturation pathway is defined by mutants in FAD5 (Kunst et al., 1989), FAD6 (Falcone et al., 1994), and FAD7 (Iba et al., 1993) or FAD8 (McConn et al., 1994, genes that encode desaturases that sequentially introduce the D7, D10, and D13 double bonds, respectively. Plants in which the initial FAD5 D7-desaturation step was compromised and that were devoid of unsaturated 16-carbon fatty acids on MGDG were identified in an ethyl methanesulfonate mutant screen (Kunst et al., 1989). Aside from the absence of 16:3D 7,10,13 from their membrane lipids and a moderate...

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“…However, in S1 and S7, 16:3 MGD is largely replaced by 18:3 (data not shown), and our understanding of the biophysics of these two fatty acids does not provide any rationale for a substantial change in molecular or membrane properties. Furthermore, available evidence indicates that decreased 16:3 is correlated with lower leaf chlorophyll content (Heilmann et al, 2004) and reduced photosynthetic performance at low temperature (Routaboul et al, 2000). By contrast, the fad5-2 mutation reduces overall unsaturation both in the individual chloroplast lipids (Table III) and in the overall leaf fatty acid profile (Table II).…”

“…4). The fad5-1 allele encodes a Trp-98 stop mutation and is thus likely to be a null allele (Heilmann et al, 2004).…”

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