MAM3 catalyzes the formation of all aliphatic glucosinolate chain lengths in Arabidopsis

Abstract

Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of omega-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.

Figure 1.

Scheme of Met-derived glucosinolate biosynthesis in Arabidopsis. This process can be divided into the Met chain elongation cycle (I) and the biosynthesis of the core glucosinolate structure (II). The parent methylthioalkyl glucosinolates can undergo further side chain modifications.

Figure 2.

Immunocytochemical localization of MAM3 protein in Arabidopsis leaves. Cross section of leaves were probed with anti-MAM3 antibody (A and C) or with preimmune serum (E) followed by a fluorescence-labeled secondary antibody. A strong green fluorescence label within chloroplasts in A and C is indicative of the MAM3 protein. Chloroplasts are defined by positive 4,6-diamidino-2-phenylindole staining (B) of the same section as shown in A and by starch granules visualized in D by the differential interference contrast image of C. Negative control performed by treatment with preimmune serum did not exhibit any label (E). Bars = 20 μm in A, B, E, and 5 μm in C and D.

Figure 3.

Glucosinolate profile of leaves (A) and seeds (B) of MAM3 mutants compared to Col-0 wild type. Glucosinolates were purified as desulfoglucosinolates, fractionated by reverse-phase HPLC, and individually identified and quantified. Individual Met-derived glucosinolates are grouped according to their chain length (no. of methylene carbons in the R group, C₂–C₈), while indole glucosinolates are depicted separately. Data show the means and ses of three replicate samples.

Figure 4.

Glucosinolate profile of leaves (A) and seeds (B) of the MAM1 insertion mutant gsm1-3 compared to Col-0 wild type. Aliphatic glucosinolates sum up to 22.4/87.2 μmol g⁻¹ dry weight (leaf/seed) in wild type and 27.3/96.9 μmol g⁻¹ dry weight in the mutant. Glucosinolates were isolated, identified, and quantified as explained in the Figure 3 legend.

Figure 5.

Steady-state mRNA transcript levels of MAM1 and MAM3 in tissues of Col-0 wild-type and MAM3 mutant lines determined by RT-PCR. DNA fragments for individual transcripts of ACT8 (actin), MAM1, and MAM3 were generated by PCR amplification of products from RT activity primed by oligo(dT) hybridization to the RNA. The PCR products are shown after separation on 1% agarose gels stained with ethidium bromide. Tissue source: 1, roots; 2, mature leaves; 3, expanding leaves; 4, flowers; and 5, siliques.