Peroxisomal ATP-binding cassette transporter COMATOSE and the multifunctional protein abnormal INFLORESCENCE MERISTEM are required for the production of benzoylated metabolites in Arabidopsis seeds

Abstract

Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens, and symbionts and are potentially important in plant development. Peroxisomal β-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ATP-binding cassette transporter COMATOSE is required for the accumulation of benzoylated secondary metabolites in Arabidopsis (Arabidopsis thaliana) seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for the accumulation of these compounds, and MULTIFUNCTIONAL PROTEIN2 contributes to the synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl coenzyme A thiolases, KAT2 plays the primary role in BA synthesis. Thus, BA biosynthesis in Arabidopsis employs the same core set of β-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid.

Figure 1.

Analysis of glucosinolate content in seeds of β-oxidation mutants. Structures of specific glucosinolates are depicted. A, 3BZO (CAS no. 80667-69-2). B, 4BZO (CAS no. 75331-11-2). C, 4MTB, 4-Methylthiobutyl glucosinolate (CAS no. 21973-56-8), the most abundant nonbenzoylated glucosinolate. D, Total glucosinolates. Values (μmol g⁻¹ seed) are averages of eight individual parent plants for each genotype. Error bars represent sd (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA). Plants were grown under long-day conditions, except those marked with asterisks after their name, which were grown in short days. The different shading identifies analyses of mutants and the wild types that were grown in separate batches and processed in separate analytical runs. The full data set including other glucosinolates is provided in Supplemental Table S2, and β-oxidation mutant details are provided in Supplemental Table S1.

Figure 2.

Phenolic choline esters content in seeds of β-oxidation mutants. Compounds were identified based on the data of Böttcher et al. (2009). A to C, BA-containing choline esters HBAC1 (CAS no. 1050631-62-3), HBAC2 (CAS no. 1050631-92-9), and HBAC3 (CAS no. 1050631-96-3). D, Sinapoyl choline (CAS no. 18696-26-9). Structures are provided for the specific metabolites. Values are averages from eight individual parent plants for each genotype. Error bars represent sd (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA). The data are presented as peak areas (of extracted ion traces) × 10⁻⁶ mg⁻¹ seed sample but are not further normalized, as described in “Materials and Methods.” As the analyses are only strictly comparable among samples run in the same batch, different types of shading identify the batches of mutants and wild types grown and analyzed together. Plants were grown under long-day conditions, except those marked with asterisks after their name, which were grown in short days. The full data set including other sinapoyl choline esters is provided in Supplemental Table S3, and β-oxidation mutant details are provided in Supplemental Table S1. E, Structures of other analyzed phenolic choline esters: (i) 5-hydroxyferuloylcholine (CAS no. 1191425-45-2); (ii) feruloylcholine-5-8′ cross-coupled to coniferyl alcohol (CAS no.1050631-58-7); (iii) sinapoylcholine-4-O-8′ coupled to coniferyl alcohol (CAS no. 1050631-54-3).

Figure 3.

Analysis of SA content in seeds of β-oxidation mutants. Values are μg g⁻¹ seed and the average of seeds collected from eight individual parent plants for each genotype. Error bars represent sd (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA). Plants were grown under long-day conditions, except those marked with asterisks after their name, which were grown in short days. The different shading identifies analyses of mutants and wild types that were grown in separate batches and processed in separate analytical runs.

Figure 4.

A, Expression of key genes of BA biosynthesis during seed development. Absolute expression values were obtained from the Electronic Fluorescent Pictograph browser (http://bar.utoronto.ca/efp_arabidopsis/cgi-bin/efpWeb.cgi) and log₂ transformed. Coexpressed β-oxidation genes are shown in blue; other β-oxidation genes and AAO4 are in black, and BZO1 is in red. B, General model of peroxisomal BA production and its relation to BG production. Metabolites detected and quantified in this study are highlighted in boxes, and β-oxidation enzymes shown to play a role in BA biosynthesis are printed blue or red as in A. Multiple arrows between metabolites indicate multiple enzymatic steps. The scheme is modeled after Lee et al. (2012) and Sønderby et al. (2010).