The Genetic Basis of Constitutive and Herbivore-Induced ESP-Independent Nitrile Formation in Arabidopsis    

Abstract

Glucosinolates are a group of thioglucosides that are components of an activated chemical defense found in the Brassicales. Plant tissue damage results in hydrolysis of glucosinolates by endogenous thioglucosidases known as myrosinases. Spontaneous rearrangement of the aglucone yields reactive isothiocyanates that are toxic to many organisms. In the presence of specifier proteins, alternative products, namely epithionitriles, simple nitriles, and thiocyanates with different biological activities, are formed at the expense of isothiocyanates. Recently, simple nitriles were recognized to serve distinct functions in plant-insect interactions. Here, we show that simple nitrile formation in Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 rosette leaves increases in response to herbivory and that this increase is independent of the known epithiospecifier protein (ESP). We combined phylogenetic analysis, a screen of Arabidopsis mutants, recombinant protein characterization, and expression quantitative trait locus mapping to identify a gene encoding a nitrile-specifier protein (NSP) responsible for constitutive and herbivore-induced simple nitrile formation in Columbia-0 rosette leaves. AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. Four of these homologues possess one or two lectin-like jacalin domains, which share a common ancestry with the jacalin domains of the putative Arabidopsis myrosinase-binding proteins MBP1 and MBP2. A sixth ESP homologue lacked specifier activity and likely represents the ancestor of the gene family with a different biochemical function. By illuminating the genetic and biochemical bases of simple nitrile formation, our study provides new insights into the evolution of metabolic diversity in a complex plant defense system.

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

“…The proteins that had accumulated to higher levels at the end of the day and that were reduced at the end of the night include NITRILE SPECIFIER PROTEIN 5 (NSP5 (AT5G48180). NSP5 is a protein in the glucosinolate pathway that catalyzes the hydrolysis of aglucone into nitriles and therefore prevents the spontaneous rearrangement of aglucone to the reactive isothiocyanate (83). Both isothiocyanates and nitriles seem to play a role in the biotic defense response and glucosinolates have also been shown to form a link between secondary metabolism and the circadian clock (84).…”

MODIFIED VACUOLE PHENOTYPE1 Is an Arabidopsis Myrosinase-Associated Protein Involved in Endomembrane Protein Trafficking

“…lack of epithionitrile specifier activity and hence epithionitriles within the Columbia-0 (Col-0) ecotype (Zhang et al, 2006;Burow et al, 2009). However, the mvp1-1 mutation caused an increase in the amount of nitrile formed (Fig.…”

Advancing Genetic Theory and Application by Metabolic Quantitative Trait Loci Analysis

“…The cloning of metabolic QTL, largely for secondary metabolites, has revealed several examples of neofunctionalization where duplicated genes have evolved independent biochemical functions (Kliebenstein et al, 2001a;Lambrix et al, 2001;de Meijer et al, 2003;Benderoth et al, 2006;Schnee et al, 2006;Burow et al, 2009). For example, a tandem duplication generated two genes in glucosinolate metabolism, AOP2 and AOP3, that evolved different biochemical reactions using the same substrate (Kliebenstein et al, 2001a).…”

“…These analyses can be extended to identify the genes controlling the biochemical reaction in specific tissues (Sergeeva et al, 2006). However, more detailed metabolic QTL experiments are beginning to show that gene duplicates diverging by tissue expression pattern may represent only the simplest scenario, while subfunctionalization can generate complex expression patterns that integrate development, ontogeny, and environment such that duplicated genes may show highly conditional effects Burow et al, 2009). In one example, the main metabolic QTL controlling secondary metabolite-mediated insect resistance within Arabidopsis, ESP and ESM1, result from two successive whole-genome duplications generating four highly similar genomic regions .…”

“…In one example, the main metabolic QTL controlling secondary metabolite-mediated insect resistance within Arabidopsis, ESP and ESM1, result from two successive whole-genome duplications generating four highly similar genomic regions . Further endoreduplication and gene loss accompanied by promoter divergence has generated QTL conditional on tissue, ontogeny, and environmental influences (Lambrix et al, 2001;Zhang et al, 2006;Burow et al, 2009). Gene duplication, followed by sequence changes that altered expression patterns of duplicated genes, has apparently given the plant more precise control over its metabolic defenses.…”