Comparative evolutionary analysis of methylthioalkylmalate synthase genes and enzymes in the Capparales

JÜRGEN KROYMANN
Max Planck Institute for Chemical Ecology
Department of Genetics & Evolution
Winzerlaer Straße 10
Beutenberg Campus
D-07743 Jena
kroymann@ice.mpg.de
http://www.ice.mpg.de/tmo/home/home_en.htm

References
Pfalz M, Vogel H, Mitchell-Olds T, Kroymann J (2007) Mapping of QTL for resistance against the crucifer specialist herbivore Pieris brassicae in a new Arabidopsis inbred line population, Da(1)-12 × Ei-2. PLoS ONE DOI: 10.1371/journal.pone.0000578
In Arabidopsis thaliana and other crucifers, the glucosinolate-myrosinase system contributes to resistance against herbivory by generalist insects. As yet, it is unclear how crucifers defend themselves against crucifer-specialist insect herbivores. We analyzed natural variation for resistance against two crucifer specialist lepidopteran herbivores, Pieris brassicae and Plutella xylostella, among Arabidopsis thaliana accessions and in a new Arabidopsis recombinant inbred line (RIL) population generated from the parental accessions Da(1)-12 and Ei-2. This RIL population consists of 201 individual F₈ lines genotyped with 84 PCR-based markers. We identified six QTL for resistance against Pieris herbivory, but found only one weak QTL for Plutella resistance. To elucidate potential factors causing these resistance QTL, we investigated leaf hair (trichome) density, glucosinolates and myrosinase activity, traits known to influence herbivory by generalist insects. We identified several previously unknown QTL for these traits, some of which display a complex pattern of epistatic interactions. Although some trichome, glucosinolate or myrosinase QTL co-localize with Pieris QTL, none of these traits explained the resistance QTL convincingly, indicating that resistance against specialist insect herbivores is influenced by other traits than resistance against generalists.

Benderoth M, Textor S, Windsor AJ, Mitchell-Olds T, Gershenzon J, Kroymann J (2006) Positive selection driving diversification in plant secondary metabolism. Proc. Natl. Acad. Sci. USA 103, 9118-9123.
In Arabidopsis thaliana and related plants, glucosinolates are a major component in the blend of secondary metabolites and contribute to resistance against herbivorous insects. Methylthioalkylmalate synthases (MAM) encoded at the MAM gene cluster control an early step in the biosynthesis of glucosinolates and, therefore, are central to the diversification of glucosinolate metabolism. We sequenced bacterial artificial chromosomes containing the MAM cluster from several Arabidopsis relatives, conducted enzyme assays with heterologously expressed MAM genes, and analyzed MAM nucleotide variation patterns. Our results show that gene duplication, neofunctionalization, and positive selection provide the mechanism for biochemical adaptation in plant defense. These processes occur repeatedly in the history of the MAM gene family, indicating their fundamental importance for the evolution of plant metabolic diversity both within and among species.

Kliebenstein DJ, Kroymann J, Mitchell-Olds T (2005) The glucosinolate-myrosinase system in an ecological and evolutionary context. Curr. Opin. Plant Biol. 8, 264-271.
Functional analysis of natural variation in the model species Arabidopsis thaliana has enabled the cloning of many glucosinolate biosynthesis and hydrolysis genes. Variation in these genes is central to understanding the ecological role of the glucosinolate-myrosinase defense system, and allows us to dissect the evolutionary and ecological forces that shape polymorphism at underlying loci. These same genes are also variable in other crucifer species, suggesting the presence of recurring selection, possibly mediated by insects. By utilizing the genomic tools available in A. thaliana to investigate these loci fully, it might be possible to generate detailed evolutionary or ecological models to apply to other species.

Kroymann J, Mitchell-Olds T (2005) Epistasis and balanced polymorphism influencing complex trait variation. Nature 435, 95-98.
Complex traits such as human disease, growth rate, or crop yield are polygenic, or determined by the contributions from numerous genes in a quantitative manner. Although progress has been made in identifying major quantitative trait loci (QTL), experimental constraints have limited our knowledge of small-effect QTL, which may be responsible for a large proportion of trait variation. Here, we identified and dissected a one-centimorgan chromosome interval in Arabidopsis thaliana without regard to its effect on growth rate, and examined the signature of historical sequence polymorphism among Arabidopsis accessions. We found that the interval contained two growth rate QTL within 210 kilobases. Both QTL showed epistasis; that is, their phenotypic effects depended on the genetic background. This amount of complexity in such a small area suggests a highly polygenic architecture of quantitative variation, much more than previously documented. One QTL was limited to a single gene. The gene in question displayed a nucleotide signature indicative of balancing selection, and its phenotypic effects are reversed depending on genetic background. If this region typifies many complex trait loci, then non-neutral epistatic polymorphism may be an important contributor to genetic variation in complex traits.

Windsor AJ, Reichelt M, Figuth A, Svatoš A, Kroymann J, Kliebenstein DJ, Gershenzon J, Mitchell-Olds T (2005) Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae). Phytochemistry 66, 1321-1333.
Glucosinolates are biologically active secondary metabolites that display both intra- and interspecific variation in the order Brassicales. Glucosinolate profiles have not been interpreted within a phylogenic framework and little is known regarding the processes that influence the evolution of glucosinolate diversity at a macroevolutionary scale. We have analyzed leaf glucosinolate profiles from members of the Brassicaceae that have diverged from Arabidopsis thaliana within the last 15 million years and interpreted our findings relative to the phylogeny of this group. We identified several interspecific polymorphisms in glucosinolate composition. A majority of these polymorphisms are lineage-specific secondary losses of glucosinolate characters, but a gain-of-character polymorphism was also detected. The genetic basis of most observed polymorphisms appears to be regulatory. In the case of A. lyrata, geographic distribution is also shown to contribute to glucosinolate metabolic diversity. Further, we observed evidence of gene-flow between sympatric species, parallel evolution, and the existence of genetic constraints on the evolution of glucosinolates within the Brassicaceae.

Kroymann J, Mitchell-Olds T (2004) Function & evolution of an Arabidopsis insect resistance QTL. In: Tikhonovich I, Lugtenberg B, Provorov N (eds.). Biology of Molecular Plant-Microbe Interactions, Vol. 4. International Society of Molecular Plant-Microbe Interactions, APS Press, pp. 259-262.
Glucosinolates (mustard oil glycosides) are a diverse group of plant secondary compounds in cruciferous plants. They contribute to plant resistance against herbivorous insects and other pests, but they also influence the quality of agriculturally important crops. Their biosynthesis occurs in three independent stages, side chain elongation of a precursor amino acid, formation of the glucosinolate core structure, and side chain modification. Methylthioalkylmalate synthase (MAM) enzymes encoded at the GS-Elong locus determine the side chain length of methionine-derived glucosinolate structures. Composition of MAM genes is highly variable among Arabidopsis ecotypes caused by several insertion-deletion polymorphisms and by gene conversion events between gene copies arranged in tandem. Moreover, statistical methods of molecular population genetics suggest that one of the genes encoded at GS-Elong is subject to balancing selection. QTL fine mapping of near-isogenic Arabidopsis lines reveals that variation in glucosinolate profiles caused by polymorphic MAM genes at GS-Elong impacts plant resistance against generalist, but not specialist insect herbivores.

Kroymann J, Donnerhacke S, Schnabelrauch D, Mitchell-Olds T (2003) Evolutionary dynamics of an Arabidopsis insect resistance QTL. Proc. Natl. Acad. Sci. USA 100, 14587-14592.
Glucosinolate profiles differ among Arabidopsis thaliana ecotypes, caused by the composition of alleles at several glucosinolate biosynthetic loci. One of these, GS-Elong, harbors a family of methylthioalkylmalate synthase (MAM) genes that determine the side chain length of aliphatic glucosinolate structures. Fine mapping reveals that GS-Elong constitutes an insect resistance quantitative trait locus, caused by variation in glucosinolate profiles conferred by polymorphism of MAM alleles in this region. A sequence survey of randomly chosen ecotypes indicates that GSElong is highly variable among A. thaliana ecotypes: indel polymorphisms are frequent, as well as gene conversion events between gene copies arranged in tandem. Furthermore, statistical methods of molecular population genetics suggest that one of the genes, MAM2, is subject to balancing selection. This may be caused by ecological tradeoffs, i.e., by contrasting physiological effects of glucosinolates on generalist vs. specialist insects.

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