From amino acid to glucosinolate biosynthesis: protein sequence changes in the evolution of methylthioalkylmalate synthase in Arabidopsis

Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the k(cat)/K(m) for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism.     

Mathematical modelling of aliphatic glucosinolate chain length distribution in Arabidopsis thaliana leaves

Aliphatic glucosinolates are a major class of defensive secondary metabolites in plants that are mostly derived from methionine. Occurring in different chain lengths, they show a structural diversity arising from the variable number of chain elongation cycles taking place during their biosynthesis. The key enzymes in determining glucosinolate chain length are the methylthioalkylmalate (MAM) synthases, MAM1 and MAM3, with MAM3 showing a broader substrate specificity than MAM1. A comparison of the measurements of wild type and MAM1 knockout mutant plants shows the following distinct changes in glucosinolate chain length profiles:

Biosynthesis of methionine-derived glucosinolates in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>: recombinant expression and characterization of methylthioalkylmalate synthase,the condensing enzyme of the chain-elongation cycle

The major class of glucosinolates in Arabidopsis thaliana (L.) Heynh. are biosynthesized from methionine involving a three-step chain-elongation cycle. Each passage through the cycle results in the net addition of a single methylene group, with up to six cycles of elongation occurring in A. thaliana. The first reaction of the cycle is catalyzed by a methylthioalkylmalate synthase (MAMS), which condenses a -methylthio-2-oxoalkanoic acid with acetyl-CoA. Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. The Columbia ecotype is dominated by compounds that have undergone only two elongation cycles. The A. thaliana MAM1 protein exhibits basic sequence similarity to other previously described enzymes catalyzing the condensation of 2-oxo acids and acetyl-CoA, such as isopropylmalate synthase (EC 2.3.3.13), an enzyme of leucine biosynthesis, and homocitrate synthase (EC 2.3.3.14). It also shares similar properties with them, including the catalytic requirements for a divalent metal ion and an adenine nucleotide. However, the MAM1 protein does not show activity with the substrates of any of these other enzymes, and was chromatographically separable from isopropylmalate synthase in extracts of A. thaliana. Thus, MAM1 is exclusively an enzyme of secondary metabolism, distinct from primary metabolic enzymes catalyzing similar reactions.Abbreviations IPMS Isopropylmalate synthase - MAM Methylthioalkylmalate - MAMS Methylthioalkylmalate synthase     

Interaction of glucosinolate content of Arabidopsis thaliana mutant lines and feeding and oviposition by generalist and specialist lepidopterans

The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), is an insect specialized on glucosinolate-containing Brassicaceae that uses glucosinolates in host-plant recognition. We used wild-type and mutants of Arabidopsis thaliana (L.) Heynh. (Brassicaceae) to investigate the interaction between plant glucosinolate and myrosinase content and herbivory by larvae of the generalist Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) and the specialist P. xylostella. We also measured glucosinolate changes as a result of herbivory by these larvae to investigate whether herbivory and glucosinolate induction had an effect on oviposition preference by P. xylostella. Feeding by H. armigera and P. xylostella larvae was 2.1 and 2.5 times less, respectively, on apk1 apk2 plants (with almost no aliphatic glucosinolates) than on wild-type plants. However, there were no differences in feeding by H. armigera and P. xylostella larvae on wild-type, gsm1 (different concentrations of aliphatic glucosinolates compared to wild-type plants), and tgg1 tgg2 plants (lacking major myrosinases). Glucosinolate induction (up to twofold) as a result of herbivory occurred in some cases, depending on both the plant line and the herbivore. For H. armigera, induction, when observed, was noted mostly for indolic glucosinolates, while for P. xylostella, induction was observed in both aliphatic and indolic glucosinolates, but not in all plant lines. For H. armigera, glucosinolate induction, when observed, resulted in an increase of glucosinolate content, while for P. xylostella, induction resulted in both a decrease and an increase in glucosinolate content. Two-choice tests with wild-type and mutant plants were conducted with larvae and ovipositing moths. There were no significant differences in preference of larvae and ovipositing moths between wild-type and gsm1 mutants and between wild-type and tgg1 tgg2 mutants. However, both larvae and ovipositing moths preferred wild-type over apk1 apk2 mutants. Two-choice oviposition tests were also conducted with P. xylostella moths comparing undamaged plants to plants being attacked by larvae of either P. xylostella or H. armigera. Oviposition preference by P. xylostella was unaffected as a result of larval plant damage, even in the cases where herbivory resulted in glucosinolate induction.     

Expression of a Brassica Isopropylmalate Synthase Gene in Arabidopsis Perturbs Both Glucosinolate and Amino Acid Metabolism

Isopropylmalate synthase (IPMS) is a key enzyme in the biosynthesis of the essential amino acid leucine, and thus primary metabolism. In Arabidopsis, the functionally similar enzyme, methythiolalkylmalate synthase (MAM), is an important enzyme in the elongation of methionine prior to glucosinolate (GSL) biosynthesis, as part of secondary metabolism. We describe the cloning of an IPMS gene from Brassica, BatIMS, and its functional characterisation by heterologous expression in E. coli and Arabidopsis. Over expression of BatIMS in Arabidopsis resulted in plants with an aberrant phenotype, reminiscent of mutants in GSL biosynthesis. Metabolite analyses showed that these plants had both perturbed amino acid metabolism and enhanced levels of GSLs. Microarray profiling showed that BatIMS over expression caused up regulation of the genes for methionine-derived GSL biosynthesis, and down regulation of genes involved in leucine catabolism, in addition to perturbed expression of genes involved in auxin and ethylene metabolism. The results illustrate the cross talk that can occur between primary and secondary metabolism within transgenic plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The role of glucosinolates and the jasmonic acid pathway in resistance of Arabidopsis thaliana against molluscan herbivores

Although slugs and snails play important roles in terrestrial ecosystems and cause considerable damage on a variety of crop plants, knowledge about the mechanisms of plant immunity to molluscs is limited. We found slugs to be natural herbivores of Arabidopsis thaliana and therefore investigated possible resistance mechanisms of this species against several molluscan herbivores. Treating wounded leaves with the mucus residue (‘slime trail’) of the Spanish slug Arion lusitanicus increased wound‐induced jasmonate levels, suggesting the presence of defence elicitors in the mucus. Plants deficient in jasmonate biosynthesis and signalling suffered more damage by molluscan herbivores in the laboratory and in the field, demonstrating that JA‐mediated defences protect A. thaliana against slugs and snails. Furthermore, experiments using A. thaliana mutants with altered levels of specific glucosinolate classes revealed the importance of aliphatic glucosinolates in defending leaves and reproductive structures against molluscs. The presence in mollusc faeces of known and novel metabolites arising from glutathione conjugation with glucosinolate hydrolysis products suggests that molluscan herbivores actively detoxify glucosinolates. Higher levels of aliphatic glucosinolates were found in plants during the night compared to the day, which correlated well with the nocturnal activity rhythms of slugs and snails. Our data highlight the function of well‐known antiherbivore defence pathways in resistance against slugs and snails and suggest an important role for the diurnal regulation of defence metabolites against nocturnal molluscan herbivores.     

Plants and tortoises: mutations in the Arabidopsis jasmonate pathway increase feeding in a vertebrate herbivore

Photosynthetic tissues, the major food source of many invertebrates and vertebrates, are well defended. Many defence traits in leaves are controlled via the jasmonate signalling pathway in which jasmonate acts as a hormone by binding to a receptor to activate responses that lead to increased resistance to invertebrate folivores. We predicted that mutations in jasmonate synthesis might also increase the vulnerability of leaves to vertebrate folivores and tested this hypothesis using the Eastern Hermann’s tortoise (Eurotestudo boettgeri) and an Arabidopsis thaliana (Brassicaceae) allene oxide synthase (aos) mutant unable to synthesize jasmonate. Tortoises preferred the aos mutant over the wild type (WT). Based on these results, we then investigated the effect of mutating jasmonate perception using a segregating population of the recessive A. thaliana jasmonate receptor mutant coronatine insensitive1‐1 (coi1‐1). Genotyping of these plants after tortoise feeding revealed that the homozygous coi1‐1 receptor mutant was consumed more readily than the heterozygous mutant or the WT. Therefore, the plant’s ability to synthesize or perceive jasmonate reduces feeding by a vertebrate herbivore. We also tested whether or not tortoise feeding behaviour was influenced by glucosinolates, the principal defence chemicals in Arabidopsis leaves with known roles in defence against many generalist insects. However, in contrast to what has been observed with such insects, leaves in which the levels of these compounds were reduced genetically were consumed at a similar rate to those of the WT.     

Multifunctional flavonoid dioxygenases: Flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L.

Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are “multifunctional” enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.     

Interactions between glucosinolate- and myrosinase-containing plants and the sawfly Athalia rosae

Several insects have specialised on using Brassicaceae as host plants. Therefore, they evolved metabolic pathways to cope with the defensive glucosinolate–myrosinase system of their diet. Larvae of the turnip sawfly, Athalia rosae L. (Hymenoptera: Tenthredinidae), incorporate various glucosinolates from their hosts into their haemolymph. The ability to sequester these metabolites makes A. rosae a useful model system to study mechanisms of glucosinolate metabolism in this species compared to other specialists, and to study effects of sawfly feeding on levels of glucosinolates and their hydrolysing enzymes in plants. The levels of plant metabolites might in turn directly affect the performance of the insect. On the one hand, costs for glucosinolate uptake and avoidance of myrosinase activity were postulated. On the other hand, sequestration of glucosinolates can be part of the insect’s defence against several predators. Here, the findings on glucosinolate metabolic pathways are compared between different herbivores and the sawfly. The impact of different glucosinolate levels and myrosinase activities on the performance of A. rosae is discussed. Furthermore, effects of feeding by A. rosae larvae on the chemical composition and enzyme activities of various Brassicaceae species are summarised. Induction patterns vary not only between different plant species and cultivars but also due to the inducing agent. Finally, the plant–herbivore interactions are discussed with regard to the sawflies’ defence abilities against different carnivore guilds.     

Do metal-rich plants deter herbivores? A field test of the defence hypothesis

Some plant species growing on metalliferous soils are able to accumulate heavy metals in their shoots up to very high concentrations, but the selective advantage of this behaviour is still unknown. The most popular hypothesis, that metals protect plants against herbivores, has been tested several times in laboratory conditions, with contradictory results. We carried out the first large-scale test of the defence hypothesis in eight natural populations of the model Zn hyperaccumulator Thlaspi caerulescens J. and C. Presl (Brassicaceae). In two climatic regions (temperate, Belgium–Luxembourg, and Mediterranean, southern France), we worked in metalliferous and in normal, uncontaminated environments, with plants spanning a wide range of Zn concentrations. We also examined the importance of glucosinolates (main secondary metabolites of Brassicaceae) as antiherbivore defences. When exposed to natural herbivore populations, T. caerulescens suffered lower herbivory pressures in metal-enriched soils than in normal soils, both in Belgium–Luxembourg and in southern France. The trapping of gastropods shows an overall lower population density in metalliferous compared to normal environments, which suggests that herbivory pressure from gastropods is lower on metalliferous soils. In addition, foliar concentration of glucosinolates was constitutively lower in all populations from metal-enriched soils, suggesting that these have evolved towards lower investment in organic defences in response to lower herbivory pressure. The Zn concentration of plants had a protective role only for Belgian metallicolous plants when transplanted in normal soils of Luxembourg. These results do not support the hypothesis that Zn plays a key role in the protection of T. caerulescens against enemies. In contrast, glucosinolates appear to be directly involved in the defence of this hyperaccumulator against herbivores.     

Insect Attraction versus Plant Defense: Young Leaves High in Glucosinolates Stimulate Oviposition by a Specialist Herbivore despite Poor Larval Survival due to High Saponin Content

Glucosinolates are plant secondary metabolites used in plant defense. For insects specialized on Brassicaceae, such as the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), glucosinolates act as “fingerprints” that are essential in host plant recognition. Some plants in the genus Barbarea (Brassicaceae) contain, besides glucosinolates, saponins that act as feeding deterrents for P. xylostella larvae, preventing their survival on the plant. Two-choice oviposition tests were conducted to study the preference of P. xylostella among Barbarea leaves of different size within the same plant. P. xylostella laid more eggs per leaf area on younger leaves compared to older ones. Higher concentrations of glucosinolates and saponins were found in younger leaves than in older ones. In 4-week-old plants, saponins were present in true leaves, while cotyledons contained little or no saponins. When analyzing the whole foliage of the plant, the content of glucosinolates and saponins also varied significantly in comparisons among plants that were 4, 8, and 12 weeks old. In Barbarea plants and leaves of different ages, there was a positive correlation between glucosinolate and saponin levels. This research shows that, in Barbarea plants, ontogenetical changes in glucosinolate and saponin content affect both attraction and resistance to P. xylostella. Co-occurrence of a high content of glucosinolates and saponins in the Barbarea leaves that are most valuable for the plant, but are also the most attractive to P. xylostella, provides protection against this specialist herbivore, which oviposition behavior on Barbarea seems to be an evolutionary mistake.     

A Gene Controlling Variation in Arabidopsis Glucosinolate Composition Is Part of the Methionine Chain Elongation Pathway

Arabidopsis and other Brassicaceae produce an enormous diversity of aliphatic glucosinolates, a group of methionine (Met)-derived plant secondary compounds containing a beta-thio-glucose moiety, a sulfonated oxime, and a variable side chain. We fine-scale mapped GSL-ELONG, a locus controlling variation in the side-chain length of aliphatic glucosinolates. Within this locus, a polymorphic gene was identified that determines whether Met is extended predominantly by either one or by two methylene groups to produce aliphatic glucosinolates with either three- or four-carbon side chains. Two allelic mutants deficient in four-carbon side-chain glucosinolates were shown to contain independent missense mutations within this gene. In cell-free enzyme assays, a heterologously expressed cDNA from this locus was capable of condensing 2-oxo-4-methylthiobutanoic acid with acetyl-coenzyme A, the initial reaction in Met chain elongation. The gene methylthioalkylmalate synthase1 (MAM1) is a member of a gene family sharing approximately 60% amino acid sequence similarity with 2-isopropylmalate synthase, an enzyme of leucine biosynthesis that condenses 2-oxo-3-methylbutanoate with acetyl-coenzyme A.     

Metabolism of glucosinolate-derived isothiocyanates to glutathione conjugates in generalist lepidopteran herbivores

The defensive properties of the glucosinolate-myrosinase system in plants of the order Brassicales have been attributed to the formation of toxic isothiocyanates generated upon tissue damage. Lepidopteran herbivores specialised on brassicaceous plants have been shown to possess biochemical mechanisms preventing the formation of isothiocyanates. Yet, no such mechanisms are known for generalist lepidopterans which also occasionally but successfully feed on plants of the Brassicales. After feeding on Arabidopsis thaliana plants, faeces of Spodoptera littoralis larvae contained glutathione conjugate derivatives (cysteinylglycine- and cysteinyl-isothiocyanate-conjugates) of the plant's major glucosinolate hydrolysis product, 4-methylsulfinylbutyl isothiocyanate. When caterpillars fed on leaves of A. thaliana containing [¹⁴C]₄-methylsulfinylbutyl glucosinolate, more than half of the ingested radioactivity was excreted as the unmetabolised corresponding isothiocyanate, and only 11% as glutathione conjugate derivatives. However, these conjugates were demonstrated to be the major metabolites of isothiocyanates in S. littoralis, and their abundance was shown to correlate with the amount of isothiocyanates ingested. Analysis of larval faeces from several species of generalist lepidopterans (Spodoptera exigua, S. littoralis, Mamestra brassicae, Trichoplusia ni and Helicoverpa armigera) fed on different Brassicaceae revealed that glutathione conjugates arise from a variety of aliphatic and aromatic isothiocyanates derived from dietary glucosinolates.     

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC_Cs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC_Cs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C₄ and C₅) or MCL (C₆) substrates substantiates the fundamental classification of PHA synthases.     

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

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.     

Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids

Characteristic of cruciferous plants is the synthesis of nitrogen- and sulfur-rich compounds, such as glucosinolates and indole alkaloids. The intact glucosinolates have limited biological activity, but give rise to an array of bio-active breakdown products when hydrolysed by endogenous β-thioglucosidases (myrosinases) upon tissue disruption. Both glucosinolates and indole alkaloids constitute an important part of the defence of plants against herbivores and pathogens, with the difference that a basal level of glucosinolates is ever-present in the plant whereas indole alkaloids are true phytoalexins that are de novo synthesised upon pathogen attack. With the completion of the genome sequence of the model plant, Arabidopsis thaliana, which is a crucifer, many genes involved in the biosynthesis of glucosinolates and indole alkaloids have been identified and cytochromes P450 are key players in these pathways. In the present review, we will focus on the cytochromes P450 in the biosynthesis of both groups of compounds. Their functional roles and regulation will be discussed.