Glucosinolates are a class of thioglycosides found predominantly in plants of the order Brassicales whose function in anti-herbivore defense has been attributed to the products formed by myrosinase-catalyzed hydrolysis upon plant tissue damage. The most common type of hydrolysis products, the isothiocyanates, are toxic to a wide range of organisms. Depending on the glucosinolate side-chain structure and the presence of certain protein factors, other types of hydrolysis products, such as simple nitriles, epithionitriles and organic thiocyanates, can be formed whose biological functions are not well understood. Of the proteins controlling glucosinolate hydrolysis, only epithiospecifier proteins (ESPs) that promote the formation of simple nitriles and epithionitriles have been identified on a molecular level. We investigated glucosinolate hydrolysis in Lepidium sativum and identified a thiocyanate-forming protein (TFP) that shares 63-68% amino acid sequence identity with known ESPs and up to 55% identity with myrosinase-binding proteins from Arabidopsis thaliana, but differs from ESPs in its biochemistry. TFP does not only catalyze thiocyanate and simple nitrile formation from benzylglucosinolate but also the formation of simple nitriles and epithionitriles from aliphatic glucosinolates. Analyses of glucosinolate hydrolysis products in L. sativum autolysates and TFP transcript accumulation revealed an organ-specific regulation of thiocyanate formation. The identification of TFP defines a new family of proteins that control glucosinolate hydrolysis and challenges the previously proposed reaction mechanism of epithionitrile formation. As a protein that promotes the formation of a wide variety of hydrolysis products, its identification provides an important tool for further elucidating the mechanisms of glucosinolate hydrolysis as well as the ecological role and the evolutionary origin of the glucosinolate-myrosinase system.
"All specifier proteins that have been studied with respect to their Fe 2? dependency so far had an increased activity (i.e. higher ratio of non-isothiocyanate:isothiocyanate product formation) in assays with addition of Fe 2? than in assays without added Fe 2? (Burow et al. 2009; Kong et al. 2012; Kuchernig et al. 2011; Wittstock and Burow 2007) and in case of purified recombinant AtESP, a strict dependency on Fe 2? has been demonstrated (Burow et al. 2006). Moreover, simple nitrile formation upon glucosinolate hydrolysis can also be caused by Fe 2? (usually [0.01 mM) in the absence of specifier proteins (Burow et al. 2009; Kong et al. 2012; Wittstock and Burow 2007). In case of purified recombinant AtESP, it has been possible to completely block specifier protein activity by addition of EDTA (2 mM) while AtNSP1 (At3g16400) retained some of its activity in the presence of 10 mM EDTA (Burow et al. 2006, 2009). "
[Show abstract][Hide abstract] ABSTRACT: As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism.
"To date, nine plant specifier proteins with different substrate and product specificities have been identified at the molecular level and characterized biochemically, namely the epithiospecifier proteins (ESPs) from Arabidopsis thaliana and Brassica oleracea, the thiocyanate-forming proteins (TFPs) from Lepidium sativum and Thlaspi arvense and five nitrile-specifier proteins (NSPs) from A. thaliana. Typically, specifier proteins of different types share 50–80% amino acid sequence identity. "
[Show abstract][Hide abstract] ABSTRACT: Background
The glucosinolate-myrosinase system is an activated chemical defense system found in plants of the Brassicales order. Glucosinolates are stored separately from their hydrolytic enzymes, the myrosinases, in plant tissues. Upon tissue damage, e.g. by herbivory, glucosinolates and myrosinases get mixed and glucosinolates are broken down to an array of biologically active compounds of which isothiocyanates are toxic to a wide range of organisms. Specifier proteins occur in some, but not all glucosinolate-containing plants and promote the formation of biologically active non-isothiocyanate products upon myrosinase-catalyzed glucosinolate breakdown.
Based on a phytochemical screening among representatives of the Brassicales order, we selected candidate species for identification of specifier protein cDNAs. We identified ten specifier proteins from a range of species of the Brassicaceae and assigned each of them to one of the three specifier protein types (NSP, nitrile-specifier protein, ESP, epithiospecifier protein, TFP, thiocyanate-forming protein) after heterologous expression in Escherichia coli. Together with nine known specifier proteins and three putative specifier proteins found in databases, we subjected the newly identified specifier proteins to phylogenetic analyses. Specifier proteins formed three major clusters, named AtNSP5-cluster, AtNSP1-cluster, and ESP/TFP cluster. Within the ESP/TFP cluster, specifier proteins grouped according to the Brassicaceae lineage they were identified from. Non-synonymous vs. synonymous substitution rate ratios suggested purifying selection to act on specifier protein genes.
Among specifier proteins, NSPs represent the ancestral activity. The data support a monophyletic origin of ESPs from NSPs. The split between NSPs and ESPs/TFPs happened before the radiation of the core Brassicaceae. Future analyses have to show if TFP activity evolved from ESPs at least twice independently in different Brassicaceae lineages as suggested by the phylogeny. The ability to form non-isothiocyanate products by specifier protein activity may provide plants with a selective advantage. The evolution of specifier proteins in the Brassicaceae demonstrates the plasticity of secondary metabolism within an activated plant defense system.
"NSPs promote simple nitrile formation at physiological pH values, but do not catalyse epithionitrile or thiocyanate formation (Burow et al., 2009; Kissen and Bones, 2009; Wittstock and Burow, 2010). In the presence of myrosinase and TFP, thiocyanates are formed with only the following glucosinolates: allylglucosinolate, benzylglucosinolate, and 4-methylthiobutylglucosinolate (Burow et al., 2007a; Wittstock and Burow, 2010). The Arabidopisis myrosinases have been well characterized , with TGG1, TGG4, and TGG5 showing activation in the range of 1–5 mM ascorbic acid in vitro, though higher concentrations inhibited enzyme activity (Andréasson et al., 2009; Wittstock and Burow, 2010). "
[Show abstract][Hide abstract] ABSTRACT: Oilseed rape and other crop plants of the family Brassicaceae contain a unique defence system known as the glucosinolate-myrosinase system or the 'mustard oil bomb'. The 'mustard oil bomb' which includes myrosinase and glucosinolates is triggered by abiotic and biotic stress, resulting in the formation of toxic products such as nitriles and isothiocyanates. Myrosinase is present in specialist cells known as 'myrosin cells' and can also be known as toxic mines. The myrosin cell idioblasts of Brassica napus were genetically reprogrammed to undergo controlled cell death (ablation) during seed development. These myrosin cell-free plants have been named MINELESS as they lack toxic mines. This has led to the production of oilseed rape with a significant reduction both in myrosinase levels and in the hydrolysis of glucosinolates. Even though the myrosinase activity in MINELESS was very low compared with the wild type, variation was observed. This variability was overcome by producing homozygous seeds. A microspore culture technique involving non-fertile haploid MINELESS plants was developed and these plants were treated with colchicine to produce double haploid MINELESS plants with full fertility. Double haploid MINELESS plants had significantly reduced myrosinase levels and glucosinolate hydrolysis products. Wild-type and MINELESS plants exhibited significant differences in growth parameters such as plant height, leaf traits, matter accumulation, and yield parameters. The growth and developmental pattern of MINELESS plants was relatively slow compared with the wild type. The characteristics of the pure double haploid MINELESS plant are described and its importance for future biochemical, agricultural, dietary, functional genomics, and plant defence studies is discussed.
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