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

Effect of glucosinolate profile modifications in Arabidopsis thaliana (L.) on the performance of different specialist Lepidoptera

Die Glucosinolate (GS) sind charakteristische sekundäre Pflanzeninhaltsstoffe, vorkommend in der Gruppe der Brassicaceae und anderen Familien der Ordnung Brassicales (Halkier & Gershenzon 2006). Bisher sind mehr als 120 verschiedene GS beschrieben, welche eine gemeinsame Grundstruktur mit variablem Seitenkettenrest kennzeichnet (Fahey et al. 2001). Je nach chemischer Natur der Seitenkette werden die GS in aliphatische, aromatische und Indolyl-GS unterteilt. Alle GS-enthaltenden Pflanzen besitzen zusätzlich räumlich getrennt von den GS hydrolysierende Enzyme, so genannte Myrosinasen. Erst nach Zellbeschädigung kommen die beiden Komponenten in Kontakt zueinander und weitere biologisch aktive Verbindungen wie z. B. Isothiocyanate und Nitrile werden freigesetzt (Rask et al. 2000). Das GS-Myrosinase-System ist ein effektives Abwehrsystem insbesondere gegenüber generalistischen Insekten, Pathogenen und Bakterien, allerdings dienen vielen spezialisierten Insekten diese Stoffe zur Wirtspflanzenfindung und -akzeptanz (Renwick 2002, Halkier & Gershenzon 2006). Die Modellpflanze Arabidopsis thaliana L. enthält als Vertreter der Brassicaceae GS als Fraßabwehrstoffe. In A. thaliana als auch in Brassica ist das aliphatische GS-Muster sehr variabel, wohingegen die Indolyl-GS weit verbreitet sind (Kliebenstein 2001, Li & Quiros 2002). Allerdings fehlen Studien zur Funktion dieser GS-Klassen innerhalb der Pflanzenresistenz gegenüber Phytophagenfraß. Deshalb wurden zwei A. thaliana -Mutanten mit verändertem aliphatischen bzw. Indolyl-GS-Profil im Vergleich zu Columbia WT auf die Wirtspflanzeneignung für drei verschieden spezialisierte Lepidoptera-Arten getestet.Plants have developed diverse, complex defense mechanisms for dealing with their enemies. Members of the Brassicaceae family use the glucosinolate(GS)-myrosinase system to deal with their enemies. According to their precursor amino acid the GS are classified in aliphatic, aromatic, and indolyl GS. Indolyl GS are widely distributed in Arabidopsis thaliana (L.) ecotypes and Brassica ssp., but the presence of aliphatic GS is highly variable. The impact of certain GS classes on plant resistance to insects is not yet discovered. Therefore, we studied the host-plant suitability of A. thaliana Columbia GS mutants to different Lepidopteran species compared to the wild-type. Two specialist species, Pieris rapae L. and Pieris brassicae L. and one generalist species Spodoptera exigua (Hübner) were selected for the feeding studies. As mutants we used mam3+ with reduced aliphatic GS levels compared to Columbia wild-type and cyp79B2-cyp79B3- which is characterized by the complete loss of indolyl GS. The weight gain of the generalist S. exigua within three days was significantly higher on mutant lines of mam3+ and cyp79B2-cyp79B3- than on Columbia wild-type. In contrast, the performance of the specialist species P. rapae and P. brassicae was not different on the genotypes. The reason for different host-plant suitability of mutants for the generalist and specialist insects is discussed

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

Glucosinolate biosynthesis from amino acids

Glucosinolates are natural plant products that are derived from aliphatic and aromatic amino acids by certain plants. Glucosinolates may be metabolized to a variety of potentially toxic products via myrosinase-catalysed hydrolysis. The enzyme and substrates are sequestered in different cell compartments and come into contact only upon tissue disruption. Glucosinolate-derived breakdown products are biologically active and influence interactions with insects and microbes as well as human health. In addition, indole glucosinolates contribute to the innate immune system of plants via the regulation of callose deposition in response to pathogen infection. Transcriptional regulation of glucosinolate biosynthesis is an important area of research, as is the spatial distribution of glucosinolates using imaging technique

A Rootstock Provides Water Conservation for a Grafted Commercial Tomato (<i>Solanum lycopersicum</i> L.) Line in Response to Mild-Drought Conditions: A Focus on Vegetative Growth and Photosynthetic Parameters

<div><p>The development of water stress resistant lines of commercial tomato by breeding or genetic engineering is possible, but will take considerable time before commercial varieties are available for production. However, grafting commercial tomato lines on drought resistant rootstock may produce drought tolerant commercial tomato lines much more rapidly. Due to changing climates and the need for commercial production of vegetables in low quality fields there is an urgent need for stress tolerant commercial lines of vegetables such as tomato. In previous observations we identified a scion root stock combination (‘BHN 602’ scion grafted onto ‘Jjak Kkung’ rootstock hereafter identified as 602/Jjak) that had a qualitative drought-tolerance phenotype when compared to the non-grafted line. Based on this initial observation, we studied photosynthesis and vegetative above-ground growth during mild-drought for the 602/Jjak compared with another scion-rootstock combination (‘BHN 602’ scion grafted onto ‘Cheong Gang’ rootstock hereafter identified as 602/Cheong) and a non-grafted control. Overall above ground vegetative growth was significantly lower for 602/Jjak in comparison to the other plant lines. Moreover, water potential reduction in response to mild drought was significantly less for 602/Jjak, yet stomatal conductance of all plant-lines were equally inhibited by mild-drought. Light saturated photosynthesis of 602/Jjak was less affected by low water potential than the other two lines as was the % reduction in mesophyll conductance. Therefore, the Jjak Kkung rootstock caused aboveground growth reduction, water conservation and increased photosynthetic tolerance of mild drought. These data show that different rootstocks can change the photosynthetic responses to drought of a high yielding, commercial tomato line. Also, this rapid discovery of one scion-rootstock combination that provided mild-drought tolerance suggests that screening more scion-rootstock combination for stress tolerance may rapidly yield commercially viable, stress tolerant lines of tomato.</p></div

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

The total number of mature leaves (A, B, C) and the total plant length (D, E, F) for three different lines of tomato grown with daily watering (control) or daily watering until water was withheld between day 20 and day 28 (mild-drought treatment).

<p>602 = ‘BHN 602’; 602/Cheong  =  refers to line ‘BHN 602’ scion grafted onto ‘Cheong Gang’ rootstock; 602/Jjak refers to line ‘BHN 602’ scion grafted onto ‘Jjak Kkung’ rootstock. Error bars represent two standard errors on each side of the mean. *  =  significant difference between control and mild-drought treated plants within a plant type (Student's t-test; p<0.05). δ  =  significant difference between 602/Jjak control plants and 602 control plants (Student's t-test; p<0.05).</p