Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana

Abstract

Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30 -45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinasecatalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.Brassicaceae plants such as oilseed rape (Brassica napus), turnip (Brassica rapa), and white mustard (Sinapis alba) as well as the model plant Arabidopsis thaliana contain a group of secondary metabolites known as glucosinolates (GSLs) 2

The Seed Composition of Arabidopsis Mutants for the Group 3 Sulfate Transporters Indicates a Role in Sulfate Translocation within Developing Seeds

“…The second is the nitrile-specifier protein NSP2 (spot 49 in Figs. 5 and 7A) involved in the hydrolysis of glucosinolates to nitrile, sulfur, and sulfate (Wittstock and Halkier, 2002;Grubb and Abel, 2006;Kissen and Bones, 2009), which suggests an increased catabolism of these compounds in sultr3;4 developing seeds. Glucosinolates are transported from all parts of the plant into developing seeds, and most of the glucosinolates contained in oilseeds are located in the embryo (Josefsson, 1970;Gijzen et al, 1989).…”

Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination

“…In addition, nitrile-specifier protein 2 is abundant in both dry and germinating Arabidopsis seeds and is also progressively translated (supplemental Tables S1 and S7). Through in vitro assays on mature seed extracts, nitrile-specifier protein 2 was shown to preferentially promote nitrile production upon glucosinolate breakdown, thereby allowing efficient remobilization of the sulfur and nitrogen contents (106). Thus, glucosinolate degradation during Arabidopsis seed germination could represent a significant source of sulfur and nitrogen, thereby fueling the sulfur amino acid metabolism essential for germination (107,108).…”

Engineering Glucosinolates in Plants: Current Knowledge and Potential Uses

“…ITCs of allyl and 3-butenylglucosinolates were more attractive than those of simple nitriles and epithionitriles in P. rapae [59]. Kissen and Bones [60] also found that the introduction of Brassica nigra GSL-ALK gene into Arabidopsis ecotype col-5 plants (normally producing methyl sulfinyl alkylGSL, GSL-ALK gene) leads to the conversion of 4-methyl sulfinyl butyl GSL in to 3-butenylGSL which, in turn, gets degraded into 3-butenylisothiocyanate upon infestation with B. brassicae. Further, these infested plants were shown to be more attractive to Diaeretiella rapae (Hymenaptera: Braconidae) than uninfested plants.…”