Malonyl-CoA synthetase, encoded by ACYL ACTIVATING ENZYME13, is essential for growth and development of Arabidopsis

Abstract

Malonyl-CoA is the precursor for fatty acid synthesis and elongation. It is also one of the building blocks for the biosynthesis of some phytoalexins, flavonoids, and many malonylated compounds. In plants as well as in animals, malonyl-CoA is almost exclusively derived from acetyl-CoA by acetyl-CoA carboxylase (EC 6.4.1.2). However, previous studies have suggested that malonyl-CoA may also be made directly from malonic acid by malonyl-CoA synthetase (EC 6.2.1.14). Here, we report the cloning of a eukaryotic malonyl-CoA synthetase gene, Acyl Activating Enzyme13 (AAE13; At3g16170), from Arabidopsis thaliana. Recombinant AAE13 protein showed high activity against malonic acid (K(m) = 529.4 ± 98.5 μM; V(m) = 24.0 ± 2.7 μmol/mg/min) but little or no activity against other dicarboxylic or fatty acids tested. Exogenous malonic acid was toxic to Arabidopsis seedlings and caused accumulation of malonic and succinic acids in the seedlings. aae13 null mutants also grew poorly and accumulated malonic and succinic acids. These defects were complemented by an AAE13 transgene or by a bacterial malonyl-CoA synthetase gene under control of the AAE13 promoter. Our results demonstrate that the malonyl-CoA synthetase encoded by AAE13 is essential for healthy growth and development, probably because it is required for the detoxification of malonate.

Figure 1.

Biochemical Characterization of Recombinant AAE13 Protein. (A) SDS-PAGE gel with Coomassie blue–stained His-AAE13 protein, following purification by nickel-affinity chromatography. Size of molecular mass markers (MWM; kD) are indicated at left. (B) and (C) HPLC-based assay for malonyl-CoA synthetase activity of recombinant His-AAE13. The control in (B) contained boiled enzyme. Peak 1 is free CoA with absorption peak at 257.5 nm (inset). Active enzyme in (C) produces malonyl-CoA (peak 2) with absorption peak at 256.3 (inset). Assays were incubated for 10 min as described in Methods. (D) The optimum pH for His-AAE13 against malonic acid; 200 mM potassium phosphate buffer at pH 6.0 to 7.5 and 100 mM Tris-HCl buffer at pH 7.5 to 9.0 were used. The experiment was repeated twice with similar results. (E) Kinetic analysis of AAE13 activity with malonic acid. For the determination of K_m and V_max, the malonic acid concentration in the assay mixtures varied between 62.5 and 625 μM. Experiments were performed at least in triplicate. Values for K_m and V_max were obtained by nonlinear regression to the Michaelis-Menten equation. [See online article for color version of this figure.]

Figure 2.

Alignment of Characterized AAE13 Homologs. Malonyl-CoA synthetases from Arabodopsis (AtAAE13), human (Hs ACSF3), B. japonicum (Bradyrhizobium), and Rhizobium leguminosarum (Rhizobium) were aligned using Vector NTI. Dark-gray shading indicates identical residues; light-gray shading indicates conserved substitutions. The underlined sequence is the conserved 12–amino acid AMP binding motif (PS00455).

Figure 3.

Expression and Activity of AAE13 in Wild-Type Arabidopsis. (A) Subcellular localization of AAE13. Arabidopsis leaf-mesophyll protoplasts transfected with Pro35S:GFP (left), Pro35S:GFP-AAE13 (center), or Pro35S:AAE13-GFP (right) constructs. Green fluorescence signals were observed using confocal microscopy with excitation at 488 nm and detection at 505 nm. (Arrows indicate nuclei; bars = 20 μm). (B) Malonyl-CoA synthetase activities in different tissues of the wild type determined by the HPLC-based assay. (C) Organ-specific expression data of AAE13 retrieved from the Genevestigator database (www.genevestigator.ethz.ch/). (D) Free malonic acid levels in different tissues of wild-type Arabidopsis as quantified by GC-MS. Values represent mean ± sd (n = 3).

Figure 4.

Effect of Malonic Acid on the Growth and Metabolism of Arabidopsis Seedlings. (A) Arabidopsis seedlings grown on agar plates supplemented with different concentrations of malonic acid. The pictures were taken 7 d after germination. (B) GC-MS chromatograms of water-soluble constituents extracted from shoots of seedlings grown without malonic acid (top) or with 5 mM malonic acid. Peaks 1 to 6 correspond to malonic acid, Val, Pro, Gly, succinic acid, and glyceric acid, respectively. (C) Contents of malonic acid and succinic acid in the shoots of Arabidopsis seedlings grown on different concentrations of malonic acid. Data are means ± sd (n = 3). [See online article for color version of this figure.]

Figure 5.

Molecular Characterization of the aae13-1 Mutant. (A) Diagram of the aae13-1 locus, showing locations of the T-DNA insert and the primers used for analysis. Black boxes, exons; lines, introns. (B) Identification of the mutant by genomic PCR using gene-specific primers, P1 and P2, and the T-DNA left border primer, LBa1 (left panel). RT-PCR (right panel) indicates that the aae13-1 mutant lacks the full-length AAE13 transcript, detected by P3+P4 and P3+P5, but contains a truncated and chimeric transcript detected by P3+P5 and P3+P6. A repeat experiment gave similar results. Het, heterozygote; Homo, homozygote; WT, wild type; WT geno, wild-type genomic DNA. (C) Comparison of the C-terminal amino acid sequence of AAE13 with that of a truncated and chimeric aae13-1 protein. The amino acid sequence in gray was derived from the T-DNA.

Figure 6.

Phenotypic Characterization of the aae13-1 Mutant. (A) Phenotypes of wild-type (WT) and aae13-1 plants after 12 d (top) and 48 d (bottom) growth on agar medium supplemented with 1% Suc. For aae13-1 at 48 d, the inset shows an alternative view of the apical region. (B) Severely reduced growth and development of aae13-1 plants on soil after 23 d (top) and 39 d (bottom). The plant aae13-1 #1 is one of the few that matured and eventually set seed. (C) Comparison of flowers, pollen, and siliques of aae13-1 #1 (right in each panel) and the wild type. (D) Concentrations of malonic acid and succinic acid measured in 12-d-old seedlings of the wild type and aae13-1. In the wild type, concentrations were below the detection limit of ~2 μg/g fresh weight. Data are means ± sd of three independent measurements.

Figure 7.

Complementation of the aae13-1 Mutant. (A) Identification of aae13-1 plants expressing the malonyl-CoA synthetase from B. japonicum under control of the AAE13 promoter (construct pBjMCS). Genomic PCR (left panel) with primers P1, P2, and LBa1 confirm absence of the wild-type (WT) allele. RT-PCR (right panel) with primers BP1+BP2, specific for the coding sequence in Bj MCS, confirm expression of the bacterial gene. Three independent complemented lines were analyzed, with similar results. (B) The Bj MCS transgene restores growth of the aae13-1 mutant (right) to the wild type. (C) Genotyping of T1 progeny of an AAE13/aae13-1 heterozygous plant transformed with a AAE13 cDNA under control of the AAE13 promoter. Healthy, BASTA-resistant T1 plants were genotyped using primers P1, P2, and LBa1. Absence of the band at 0.9 kb, derived from the endogenous AAE13 allele, indicates that plants #15 and #21 are aae13-1 homozygotes. The band at 0.5 kb is derived from the cDNA sequence of the transgene. (The 0.4-kb band derived from the aae13-1 allele migrates slightly below this.) (D) Growth and development of homozygous aae13-1 plants carrying the transgene (plants #15 and #21) are similar to wild-type (plant #16) and heterozygous (plant #22) siblings.

Figure 8.

Characterization of Plants Overexpressing AAE13. (A) Phenotype of wild-type (left) and plants homozygous for the Pro35S:AAE13 transgene (line #6; right). (B) and (C) Malonyl-CoA synthetase activity (B) and malonic acid content (C) in rosette (L) and flower (F) tissues of 6-week-old wild-type (WT) and Pro35S:AAE13 transgenic (OX) plants. Data are means ± sd (n = 3). (D) Wild-type and Pro35S:AAE13 (OX) seedlings grown on agar plates supplemented with different concentrations of malonic acid. The pictures were taken 7 d after germination.