Mitochondrial Fatty Acid Synthase Utilizes Multiple Acyl Carrier Protein Isoforms

Abstract

Acyl carrier protein (ACP) is a highly conserved cofactor protein that is required by Type II fatty acid synthases (FASs). Here, we demonstrate that up to three mitochondrial ACP (mtACP) isoforms support the Arabidopsis (Arabidopsis thaliana) mitochondrially localized Type II FAS. The physiological importance of the three mtACPs was evaluated by characterizing the single, double, and triple mutants. The mtACP1 (At2g44620), mtACP2 (At1g65290), and mtACP3 (At5g47630) single mutants showed no discernible morphological growth phenotype. Functional redundancy among the three mtACPs was indicated by the embryo-lethal phenotype associated with simultaneous loss of all three mtACP genes. Characterization of all double mutant combinations revealed that although the mtacp1 mtacp3 and mtacp2 mtacp3 double mutant combinations showed no observable growth defect, the mtacp1 mtacp2 double mutant was viable but displayed delayed growth, reduced levels of posttranslationally lipoylated mitochondrial proteins, hyperaccumulation of photorespiratory Gly, and reduced accumulation of many intermediates in central metabolism. These alterations were partially reversed when the mtacp1 mtacp2 double mutant plants were grown in a nonphotorespiratory condition (i.e. 1% CO₂ atmosphere) or in the presence of 2% Suc. In summary, mtACP, as a key component of mitochondrial fatty acid biosynthesis, is important in generating the fatty acid precursor of lipoic acid biosynthesis. Thus, the incomplete lipoylation of mitochondrial proteins in mtacp mutants, particularly Gly decarboxylase, affects the recovery of photorespiratory carbon, and this appears to be critical during embryogenesis.

Figure 1.

Phylogenetic analysis of ACPs. Plant ACP protein sequences were aligned using the ClustalW program and depicted as a rooted phylogenetic tree using the neighbor-joining method in MEGAX. The protein sequence of Escherichia coli ACP was chosen as an outgroup. The numbers at each node represent the bootstrap values using 1,000 replicates. The protein IDs for ACPs are listed in Supplemental Dataset S1.

Figure 2.

Subcellular localization of Arabidopsis mtACP1, mtACP2, and mtACP3. Each row shows images obtained from wild-type nontransgenic plants or from plants carrying the indicated GFP fusion transgene. Fluorescence signals from roots were monitored by confocal laser scanning microscopy using GFP fluorescence (column 1) and MitoTracker fluorescence (column 2). The resulting images were overlaid (column 3) to indicate the colocalization of GFP with the MitoTracker signal.

Figure 3.

Expression profile of mtACP in different Arabidopsis tissues. Data represent the expression level of the individual mtACP isoform mRNA in different tissues of wild-type Arabidopsis plants as quantified by RT-qPCR. The expression levels were normalized against the level of the reference gene, UQB10. Values are means ± se (n = 5).

Figure 4.

Morphological phenotypes of mtacp single and double mutants. Images were taken of plants at 16 d and 40 d post imbibition. WT, Wild type.

Figure 5.

Seed development phenotypes of mtacp mutants. Images of siliques developing on selfed progeny of the indicated genotypes. One side of the ovary wall was removed from the fully elongated, fresh silique to reveal the developing seeds. The abnormal seeds are brown or white. Scale bars = 1 mm. WT, Wild type.

Figure 6.

Protein lipoylation status in mtacp1 mtacp2 double mutant plants. A, Coomassie Brilliant Blue-stained SDS-PAGE analysis of protein extracts prepared from leaves of the indicated genotypes. M, Protein molecular weight markers; WT, wild type. B, Immunoblot analysis using antilipoic acid antibody to identify the lipoylation status of the H subunit of GDC, the E2a and E2b subunits of PDH, and the E2b subunit of KGDH. The accumulation of the H protein subunit was determined in parallel using anti H-protein antibodies.

Figure 7.

Morphological and metabolic phenotypes of the mtacp1 mtacp2 double mutant at 16 d postimbibition. A, Alterations in the metabolome of the whole seedlings of the mtacp1 mtacp2 double mutant as compared to the wild-type (WT) seedlings grown in either an ambient atmosphere, an atmosphere containing 1% CO₂, or on media containing 2% Suc. The x axes represent the fold change (on a log-base 2 scale) of the relative abundance of each metabolite in the double mutant versus the wild-type plants. The colored data points above the horizontal dashed gray line indicate statistically significant changes in metabolite levels (P < 0.05, n = 5; false discovery rate-adjusted Student’s t test). The data points in each plot represent 59 metabolites that were chemically identified (listed in Supplemental Dataset S2); these include amino acids (red data points), organic acids (purple data points), sugars (black data points), and lipids (blue data points). B, Morphological phenotypes of wild-type and mutant seedlings grown in ambient air, 1% CO₂ atmosphere, or 2% Suc-containing media.