Complex Formation of Myrosinase Isoenzymes in Oilseed Rape Seeds Are Dependent on the Presence of Myrosinase-Binding Proteins

Abstract

The enzyme myrosinase (EC 3.2.3.1) degrades the secondary compounds glucosinolates upon wounding and serves as a defense to generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica napus) seeds. Immunhistochemical analysis of wild-type seeds showed that MBPs were present in most cells but not in the myrosin cells, indicating that the complex formation observed in extracts is initiated upon tissue disruption. To study the role of MBP in complex formation and defense, oilseed rape antisense plants lacking the seed MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses of 62 to 70 kD were present as free dimers from the antisense seed extract, whereas in the wild type, they formed complexes. In accordance with this, MBPs are necessary for myrosinase complex formation of the 62-to 70-kD myrosinases. The product formed from sinalbin hydrolysis by myrosinase was the same whether MBP was present or not. The performance of a common beetle generalist (Tenebrio molitor) fed with seeds, herbivory by flea beetles (Phyllotreta undulata) on cotyledons, or growth rate of the Brassica fungal pathogens Alternaria brassicae or Lepthosphaeria maculans in the presence of seed extracts were not affected by the down-regulation of MBP, leaving the physiological function of this protein family open.Specific ␤-thioglucosidases known as myrosinases (EC 3.2.3.1) are responsible for the degradation of glucosinolates, secondary metabolites found mainly in the members of the Brassicacea family (Chew, 1988; James and Rossiter, 1991; Louda and Mole, 1991). The myrosinases and glucosinolates are, to a large extent, preformed and most probably require disruption of the plant tissue to become activated. Myrosinase hydrolyses glucosinolates by releasing the Glc moiety. The remaining aglycone spontaneously rearranges into one of several toxic products, such as isothiocyanates, thiocyanates, and nitriles (for review, see Bones and Rossiter, 1996; Rask et al., 2000). The outcome of the degradation depends on the type of glucosinolate being degraded, the local milieu, and additional proteins such as the epithiospecifier protein (Chew, 1988; Louda and Mole, 1991; Lambrix et al., 2001).Several different myrosinase isoenzymes have been characterized in seeds, seedlings, and vegetative tissues of oilseed rape (Brassica napus; Lenman et al., 1993). More than 20 myrosinase genes seem to be present in oilseed rape (Xue et al., 1992), but few of these genes or cDNAs have been cloned as yet. Myrosinases expressed in seeds can be divided into three different subfamilies, denoted MA, MB, and MC. The three-dimensional structure of one myrosina...

Proteome Analysis ofArabidopsisLeaf Peroxisomes Reveals Novel Targeting Peptides, Metabolic Pathways, and Defense Mechanisms

“…Some of these defense-related proteins, such as BGL1, PYK10, and Brassica myrosinases, are reported to form polymers (Eriksson et al, 2002;Nagano et al, 2005;Lee et al, 2006). Likewise, nucleic acid metabolism-related proteins may be components of large protein complexes, such as ribosomes, exosomes, spliceosomes, or P-bodies (for review, see Eulalio et al, 2007).…”

Large‐scale identification of leaf senescence‐associated genes

“…The temporal expression pattern of the newly identi®ed HR-related gene, the lethal leaf spot homolog, clearly indicates its exclusive participation in the late stages of the senescence syndrome (Figure 2). The gene encoding myrosinase-binding protein (At1g52040), whose transcript is upregulated during senescence, has been implicated in defense responses (Eriksson et al, 2002). The enzyme myrosinase degrades the secondary compounds glucosinolates upon wounding and their products actively involved in defense against pests.…”

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