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.

The desert locust, Schistocerca gregaria, detoxifies the glucosinolates of Schouwia purpurea by desulfation.

Desert locusts (Schistocerca gregaria) occasionally feed on Schouwia purpurea, a plant that contains tenfold higher levels of glucosinolates than most other Brassicaceae. Whereas this unusually high level of glucosinolates is expected to be toxic and/or deterrent to most insects, locusts feed on the plant with no apparent ill effects. In this paper, we demonstrate that the desert locust, like larvae of the diamondback moth (Plutella xylostella), possesses a glucosinolate sulfatase in the gut that hydrolyzes glucosinolates to their corresponding desulfonated forms. These are no longer susceptible to cleavage by myrosinase, thus eliminating the formation of toxic glucosinolate hydrolysis products. Sulfatase is found throughout the desert locust gut and can catalyze the hydrolysis of all of the glucosinolates present in S. purpurea. The enzyme was detected in all larval stages of locusts as well as in both male and female adults feeding on this plant species. Glucosinolate sulfatase activity is induced tenfold when locusts are fed S. purpurea after being maintained on a glucosinolate-free diet, and activity declines when glucosinolates are removed from the locust diet. A detoxification system that is sensitive to the dietary levels of a plant toxin may minimize the physiological costs of toxin processing, especially for a generalist insect herbivore that encounters large variations in plant defense metabolites while feeding on different species.

Glucosinolate biosynthesis: demonstration and characterization of the condensing enzyme of the chain elongation cycle in Eruca sativa.

Glucosinolates are a group of sulfur-rich thioglucoside natural products common in the Brassicaceae and related plant families. The first phase in the formation of many glucosinolates involves the chain extension of the amino acid methionine. Additional methylene groups are inserted into the side chain of methionine by a three-step elongation cycle involving 2-oxo acid intermediates. This investigation demonstrated the first step of this chain elongation cycle in a partially-purified preparation from arugula (Eruca sativa). The 2-oxo acid derived from methionine, 4-methylthio-2-oxobutanoic acid, was shown to condense with acetyl-CoA to form 2-(2'-methylthioethyl)malate. The catalyst, designated as a 2-(omega-methylthioalkyl)malate synthase, belongs to a family of enzymes that mediate the condensation of acyl-CoAs with 2-oxo acids, including citrate synthase of the citric acid cycle, and 2-isopropylmalate synthase of leucine biosynthesis. The 2-(omega-methylthioalkyl)malate synthase studied here shares properties with other enzymes of this class, but appears chromatographically distinct and is found only in extracts of plant species producing glucosinolates from chain-elongated methionine derivatives. Although the principal glucosinolates of arugula are formed from methionine that has undergone two rounds of chain elongation to form dihomomethionine, studies with substrates and substrate analogs of different chain lengths showed that the isolated enzyme is responsible only for the condensation step of the first round of elongation.

Biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana: 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 omega-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.