An aeroponic culture system for the study of root herbivory on Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>Plant defense against herbivory has been studied primarily in aerial tissues. However, complex defense mechanisms have evolved in all parts of the plant to combat herbivore attack and these mechanisms are likely to differ in the aerial and subterranean environment. Research investigating defense responses belowground has been hindered by experimental difficulties associated with the accessibility and quality of root tissue and the lack of bioassays using model plants with altered defense profiles.</p> <p>Results</p> <p>We have developed an aeroponic culture system based on a calcined clay substrate that allows insect herbivores to feed on plant roots while providing easy recovery of the root tissue. The culture method was validated by a root-herbivore system developed for <it>Arabidopsis thaliana </it>and the herbivore <it>Bradysia </it>spp. (fungus gnat)<it>. Arabidopsis </it>root mass obtained from aeroponically grown plants was comparable to that from other culture systems, and the plants were morphologically normal. <it>Bradysia </it>larvae caused considerable root damage resulting in reduced root biomass and water absorption. After feeding on the aeroponically grown root tissue, the larvae pupated and emerged as adults. Root damage of mature plants cultivated in aeroponic substrate was compared to that of <it>Arabidopsis </it>seedlings grown in potting mix. Seedlings were notably more susceptible to <it>Bradysia </it>feeding than mature plants and showed decreased overall growth and survival rates.</p> <p>Conclusions</p> <p>A root-herbivore system consisting of <it>Arabidopsis thaliana </it>and larvae of the opportunistic herbivore <it>Bradysia </it>spp. has been established that mimics herbivory in the rhizosphere. <it>Bradysia </it>infestation of <it>Arabidopsis </it>grown in this culture system significantly affects plant performance. The culture method will allow simple profiling and <it>in vivo </it>functional analysis of root defenses such as chemical defense metabolites that are released in response to belowground insect attack.</p

De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication

Two Arabidopsis Genes (IPMS1 and IPMS2) Encode Isopropylmalate Synthase, the Branchpoint Step in the Biosynthesis of Leucine

Heterologous expression of the Arabidopsis (Arabidopsis thaliana) IPMS1 (At1g18500) and IPMS2 (At1g74040) cDNAs in Escherichia coli yields isopropylmalate synthases (IPMSs; EC 2.3.3.13). These enzymes catalyze the first dedicated step in leucine (Leu) biosynthesis, an aldol-type condensation of acetyl-coenzyme A (CoA) and 2-oxoisovalerate yielding isopropylmalate. Most biochemical properties of IPMS1 and IPMS2 are similar: broad pH optimum around pH 8.5, Mg(2+) as cofactor, feedback inhibition by Leu, K(m) for 2-oxoisovalerate of approximately 300 μm, and a V(max) of approximately 2 × 10(3) μmol min(−1) g(−1). However, IPMS1 and IPMS2 differ in their K(m) for acetyl-CoA (45 μm and 16 μm, respectively) and apparent quaternary structure (dimer and tetramer, respectively). A knockout insertion mutant for IPMS1 showed an increase in valine content but no changes in Leu content; two insertion mutants for IPMS2 did not show any changes in soluble amino acid content. Apparently, in planta each gene can adequately compensate for the absence of the other, consistent with available microarray and reverse transcription-polymerase chain reaction data that show that both genes are expressed in all organs at all developmental stages. Both encoded proteins accept 2-oxo acid substrates in vitro ranging in length from glyoxylate to 2-oxohexanoate, and catalyze at a low rate the condensation of acetyl-CoA and 4-methylthio-2-oxobutyrate, i.e. a reaction involved in glucosinolate chain elongation normally catalyzed by methylthioalkylmalate synthases. The evolutionary relationship between IPMS and methylthioalkylmalate synthase enzymes is discussed in view of their amino acid sequence identity (60%) and overlap in substrate specificity

MAM3 Catalyzes the Formation of All Aliphatic Glucosinolate Chain Lengths in Arabidopsis1[W][OA]

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 ω-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

De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication