NSP-Dependent Simple Nitrile Formation Dominates upon Breakdown of Major Aliphatic Glucosinolates in Roots, Seeds, and Seedlings of Arabidopsis thaliana Columbia-0.

Ute Wittstock,Kathrin Meier,Friederike Dörr,Beena M Ravindran

doi:10.3389/fpls.2016.01821

Ute Wittstock, Kathrin Meier + Show 2 more

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https://doi.org/10.3389/fpls.2016.01821

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Abstract

One of the best-studied plant defense systems, the glucosinolate-myrosinase system of the Brassicales, is composed of thioglucosides known as glucosinolates and their hydrolytic enzymes, the myrosinases. Tissue disruption brings these components together, and bioactive products are formed as a consequence of myrosinase-catalyzed glucosinolate hydrolysis. Among these products, isothiocyanates have attracted most interest as chemical plant defenses against herbivores and pathogens and health-promoting compounds in the human diet. Previous research has identified specifier proteins whose presence results in the formation of alternative product types, e.g., nitriles, at the expense of isothiocyanates. The biological roles of specifier proteins and alternative breakdown products are poorly understood. Here, we assessed glucosinolate breakdown product profiles obtained upon maceration of roots, seedlings and seeds of Arabidopsis thaliana Columbia-0. We identified simple nitriles as the predominant breakdown products of the major endogenous aliphatic glucosinolates in root, seed, and seedling homogenates. In agreement with this finding, genes encoding nitrile-specifier proteins (NSPs) are expressed in roots, seeds, and seedlings. Analysis of glucosinolate breakdown in mutants with T-DNA insertions in any of the five NSP genes demonstrated, that simple nitrile formation upon tissue disruption depended almost entirely on NSP2 in seeds and mainly on NSP1 in seedlings. In roots, about 70–80% of the nitrile-forming activity was due to NSP1 and NSP3. Thus, glucosinolate breakdown product profiles are organ-specifically regulated in A. thaliana Col-0, and high proportions of simple nitriles are formed in some parts of the plant. This should be considered in future studies on biological roles of the glucosinolate-myrosinase system.

Highlights

Arabidopsis thaliana (Brassicaceae) lends itself as a model to study biological roles of glucosinolates, a group of amino acidderived thioglucosides present in the Brassicales (Figure 1A) (Halkier and Gershenzon, 2006; Kopriva, 2016), due to the wealth of information on glucosinolate metabolism in this species and the relative ease with which its metabolism can be manipulated
NSP1 had the by far highest expression levels regardless of the organ/stage analyzed while NSP4 was expressed at 70- to 100-fold lower levels and NSP3 expression proved to be largely root-specific (Figure 2C; Supplementary Figure 4)
We expected that NSP2 determines glucosinolate breakdown product profiles in seed homogenates, while mostly NSP1 and NSP3 contribute to simple nitrile formation in roots

Summary

Introduction

Arabidopsis thaliana (Brassicaceae) lends itself as a model to study biological roles of glucosinolates, a group of amino acidderived thioglucosides present in the Brassicales (Figure 1A) (Halkier and Gershenzon, 2006; Kopriva, 2016), due to the wealth of information on glucosinolate metabolism in this species and the relative ease with which its metabolism can be manipulated. Depending on the plant species, organ and the glucosinolate structure, other products such as simple nitriles, epithionitriles, and organic thiocyanates can be formed upon glucosinolate hydrolysis, and the biological roles of these products are much less understood (Wittstock and Burow, 2007). In A. thaliana and many other species of the Brassicaceae, formation of non-isothiocyanate products depends on so-called specifier proteins, namely epithiospecifier protein (ESP) and nitrilespecifier proteins (NSPs), as well as on additional factors such as the epithiospecifier modifier gene (ESM1) (Lambrix et al, 2001; Zhang et al, 2006; Burow et al, 2009; Kissen and Bones, 2009; Kuchernig et al, 2012). It can be difficult to discriminate between contributions of ESP and NSP, as simple nitriles of nonalkenyl glucosinolates can be generated by ESP (Burow et al, 2006a,b)

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