Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds

Abstract

In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and D-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two d-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.

Utilization of Amygdalin during Seedling Development of Prunus serotina

“…Nonetheless, this residual activity still exceeded 25 pmol glycoside hydrolyzed min-' g-' fresh wei;;ht. Because of its prevalence and protein body location, MDL is suspected to play an additional role as a storage protein and as such might be expected to be mobilized during germination (Swain et al, 1992b). However, this mobilization appai,ently occurs after the period surveyed here since cotyledonary MDL levels, unlike AH and PH levels, remained essentially unchanged throughout stages 1 to 4 (Fig.…”

“…Immunolocalization was undertaken essentially as described by Swain et al (1992b). In brief, stage l to 4 cotyledons were fixed, embedded in LR White mediuni, sectioned, and collected on glass microscope slides.…”

“…At maturity, each seed contained approximately 3 pmol of amygdalin, which is located within the cotyledonary parenchyma cells (Poulton and Li, 1994). By contrast, AH and PH are restricted to the procambium (Swain et al, 1992b). Thus, premature, large-scale cyanogenesis is prevented in undamaged seeds by tissue level compartmentalization.…”

Plant Cyanogenesis of Phaseolus lunatus and its Relevance for Herbivore–Plant Interaction: The Importance of Quantitative Data

“…Cyanogenic glycosides are separated in the intact plant from one or more specific b-glucosidases that are localized in the apoplast. In addition to separation on the cellular level, the cyanogenic component and the enzyme can be located in different tissues (Thayer and Conn, 1981;Frehner and Conn, 1987;Poulton, 1988;Swain et al, 1992). In case of injury, the b-glucosidase is brought into contact with its substrate.…”

“…In case of injury, the b-glucosidase is brought into contact with its substrate. By hydrolysis of the cyanogenic glycosides, a-hydroxynitriles are formed that are relatively unstable and dissociate either spontaneously or are enzymatically accelerated by an a-hydroxynitrile lyase into HCN and an aldehyde or a ketone (Conn, 1980;Hösel and Conn, 1982;Poulton, 1990;Poulton and Li, 1994;Swain et al, 1992). Hydrogen cyanide is toxic because of its affinity to the terminal cytochrome oxidase in the mitochondrial respiratory pathway and a number of other reactions (Solomonson, 1981;Brattsten et al, 1983).…”