The toc132toc120 heterozygote mutant of Arabidopsis thaliana accumulates decreased levels of the major chloroplast lipids.

We used ESI-MS/MS to profile glycerolipids in a mutant of Arabidopsis thaliana that is null and heterozygous for the TOC132 and TOC120 genes, and is referred to as the toc132toc120± mutant. The goal was to assess the impact of a defective atToc132/120 receptor on the accumulation of chloroplast lipids. The mutant accumulated decreased amounts of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol (PG). In the cold-acclimated mutant, PG accumulated at the control levels. However, 34:4-PG (18:3/16:1Δ3trans) was significantly decreased, which indicates that the mutant was impaired in synthesis of the chloroplast-derived PG. Major molecular species of MGDG and DGDG were significantly decreased, which was indicative of the decreased levels of triunsaturated fatty acids in galactolipids. The cold-acclimated mutant accumulated increased levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), which indicate that defect in the atToc132/120 receptor did not impair the ER pathway of lipid synthesis. Both cold-acclimated wildtype and mutant plants accumulated increased levels of phosphatidic acid (PA). The increased levels of major molecular species of PA suggest that some pool of PA was derived from degradation of both the chloroplast and extra-chloroplast lipids. The cold-acclimated mutant had decreased double bond index (DBI) and increased acyl chain length (ACL), which was indicative of decreased membrane fluidity. However, a decrease in the ratio of MGDG to DGDG indicate that the mutant was capable of remodeling membrane lipids in response to low temperatures. We conclude that the defective Toc132/120 receptor resulted in decreased synthesis of chloroplast lipids and decreased membrane fluidity.

The TOC159 null mutant of Arabidopsis thaliana is impaired in the accumulation of plastid lipids and phosphatidylcholine.

We used electrospray ionization tandem mass spectrometry to profile glycerolipids in the TOC159 null mutant of Arabidopsis, which is referred to as plastid protein import 2, or ppi2. The goal was to evaluate the impact of a defective atToc159 receptor in the accumulation of plastid lipids. The ppi2 mutant is severely impaired in the accumulation of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol (PG), which are major components of the thylakoid membranes. Major molecular species of MGDG and DGDG are drastically decreased, which is consistent with our previous findings of decreased levels of hexadecatrienoic and linolenic acids. Under normal growth conditions, the ppi2 mutant accumulated significantly lower levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). In the cold-acclimated mutant, the amounts of PE and PI were similar to the wildtype level, which indicates that the ER pathway of lipid synthesis was functional in the mutant. The cold-acclimated ppi2 mutant accumulated increased amounts of phosphatidic acid (PA), which was mirrored by an increase in phospholipase Dα (PLDα) transcript levels. These data suggest that PLDα activity contributed to the accumulation of cold-induced PA in the ppi2 mutant. The accumulation of major molecular species in PA indicates that cold-induced PA originated from the degradation of both plastidial and extraplastidial lipids. Compared with the wildtype, the ppi2 mutant had a low double bond index and high acyl chain length, which is indicative of decreased membrane fluidity. Taken together, these data indicate that a defective atToc159 receptor severely impaired the plastid pathway of lipid synthesis, which negatively affected the synthesis and/or accumulation of PC.

Ozone-induced lipid changes in the wildtype and toc132toc120 heterozygote mutant of Arabidopsis thaliana.

We measured the fatty acids and lipid content in the wildtype and toc132toc120 heterozygote mutant of Arabidopsis thaliana that were exposed to elevated levels of ozone. The goal was to assess whether a defective atToc132/120 receptor would alter the mutant's response to ozone-induced stress. Increased malondialdehyde (MDA) levels were measured in wildtype plants that were exposed to ozone for 3 h and left in an ozone-free environment for 21 h. The increased levels of MDA were not positively correlated with changes in the levels of triunsaturated fatty acids from which MDA is derived. In both the wildtype and mutant plants, absolute amounts of the glycerolipids were not altered by ozone treatment. The untreated mutant, however, accumulated decreased levels of chloroplast lipids and triunsaturated fatty acids. In ozone-treated wildtype, the levels of 16:3 were significantly decreased and this was mirrored by decreased levels of TOC132 and FAD5 transcripts, and increased levels of SP1 E3 ligase transcripts. These data suggest a possible increase in protein ubiquitination under ozone-induced stress. In contrast, in ozone-treated mutant, the FAD5 transcripts accumulated at the control level. The untreated mutant, however, accumulated significantly increased levels of CAT1 and FAD7 transcripts, which indicates that a defective chloroplast receptor induced cellular stress. In ozone-treated wildtype, there was a small increase in 34:6-phosphatidic acid, which indicates that a small amount of the chloroplast-derived MGDG was degraded in response to ozone-induced stress. Overall, these data indicate that the wildtype and mutant responded differently in lipid composition and oxidation to ozone-induced stress.

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.

Labeling of major plant lipids and jasmonic acid using [1-(14C)] lauric acid.

A medium chain length fatty acid, [1-(14C)] lauric acid (12:0) was administered to the detached leaves of Artemisia and was incorporated into major lipids, including phospholipids and galactolipids. [1-(14C)]12:0 was elongated and desaturated into linolenic acid (18:3). In detached leaves of both Artemisia and Arabidopsis thaliana ecotype Columbia, radioactivity from [14C]18:3 was incorporated into jasmonic acid (JA) and methyl jasmonate (MJ). Higher amounts of [14C]JA were measured in Artemisia than Arabidopsis leaves. In Artemisia, [14C]JA was actively metabolized into [14C]MJ. Extracts prepared from the leaves of Artemisia, exhibited higher in vitro JA methyltransferase activity than those from Arabidopsis.

atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins.

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203-207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1-13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93-105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.