The toc132toc120 heterozygote mutant of Arabidopsis thaliana accumulates reduced levels of hexadecatrienoic acid.

A null and heterozygous mutant for the Arabidopsis thaliana TOC132 and TOC120 genes accumulates increased levels of 16:0 and decreased 16:3, suggesting altered homeostasis in fatty acid synthesis. The FAD5 gene encodes a plastid desaturase that catalyzes the first step in the synthesis of 16:3 in monogalactosyldiacylglycerol (MGDG). In non-acclimated toc132toc120+/- mutant plants, the FAD5 gene was repressed and this correlated with decreased levels of 16:3. In cold-acclimated mutant however, the FAD5 gene was upregulated and there was a small increase in 16:3 levels relative to the non-acclimated mutant plants. The MGD1 gene was expressed at control levels and the mutant accumulated levels of MGDG that were similar to the wild type. In the mutant however, MGDG had decreased 16:3 levels, suggesting that the activity of FAD5 desaturase was compromised. In the mutant, the FAD2 and FAD3 genes were downregulated but levels of 18:3-PC were increased, suggesting posttranscriptional regulation for the ER-localized fatty acid desaturases. The Toc120 or Toc159 receptor is likely to compensate for a defective Toc132 receptor. In the cold-acclimated mutant, the TOC159 gene was repressed ca. 300-fold, whereas the TOC120 gene was repressed 7-fold relative to the non-acclimated wild type. Thus, the TOC159 gene is more sensitive to cold-stress and might not compensate for defect in the TOC132 gene under these conditions. Overall, these data show that a mutation in the TOC132 gene results in decreased 16:3 levels, indicating the need for an intact Toc132/Toc120 receptor, presumably to facilitate the import of the FAD5 preprotein into chloroplasts.

The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids.

We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2) accumulates normal levels of fatty acids, and transcripts of fatty acid desaturases and galactolipid synthesis enzymes. The ppi2-2 mutant accumulates decreased pigments and total fatty acid content. The MGD1 gene was downregulated and the mutant accumulates decreased levels of monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3-ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. Interestingly, glycerolipids in the mutant accumulate increased levels of 18:0. A gene that encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to bilayer forming plastid lipids was decreased in the cold-acclimated wild type but not in the ppi2-2 mutant. This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of 18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty acids. We conclude that expression of the MGD1 gene and synthesis of MGDG are tightly linked to plastid biogenesis.

A mutant of the Arabidopsis thaliana TOC159 gene accumulates reduced levels of linolenic acid and monogalactosyldiacylglycerol.

Previous studies have shown that a mutant of Arabidopsis that lacks the Toc159 receptor is impaired in chloroplast biogenesis. The mutant is referred as plastid protein import 2 or ppi2 and has an albino phenotype due to its inability to import the photosynthetic proteins. In this study, we measured fatty acid composition and transcript levels of plastid-localized fatty acid desaturases in the wild type and ppi2 mutant. The objective was to evaluate whether the Toc159 receptor was critical in the import of lipid-synthesizing enzymes. The ppi2 mutant accumulated decreased levels of oleic acid (18:1) and α-linolenic acid (18:3). The mutant accumulated drastically reduced amounts of the chloroplast lipid monogalactosyldiacylglycerol (MGDG), which contains more than 80% of 18:3. The expression of genes that encode stearoyl-ACP desaturase and MGD1 synthase were down-regulated in the ppi2 mutant, and this corresponded to decreased levels of 18:1 and MGDG, respectively. We conclude that in the ppi2 mutant the impaired synthesis of MGDG resulted in decreased amounts of 18:3. The mutant however, had a 30-fold increase in fad5 transcript levels; this increase was mirrored by a 16- to 50-fold accumulation of hexadecatrienoic acid (16:3), a fatty acid found exclusively in MGDG. Taken together, these data suggest that the Toc159 receptor is required in the import of stearoyl-ACP desaturase and MGD1 synthase into the chloroplasts. Since the expression of fad5 gene was up-regulated in the ppi2 mutant, we propose that fad5 desaturase is imported into plastids through the atToc132/atToc120 protein import pathway.