Temporal and Spatial Regulation of Cyanogenesis in Plants

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Rosaceous stone fruits (Prunus spp.) are among many commercially important crops causing cyanide poisoning in humans and livestock through their release of hydrogen cyanide from cyanogenic glycosides by endogenous enzymes. To understand better the biochemistry and physiology of plant cyanogenesis, the metobolism of the cyanoglycosides (R)-prunasin and (R)-amygdalin will be investigated in black cherry (Prunus serotina) using biochemical, immunocytochemical and molecular approaches. Levels of prunasin, amygdalin, two biosynthetic enzymes (UDPG:mandelonitrile glucosyltransferase (GT I) and UDPG:prunasin glucosyltransferase (GT II), and three catabolic enzymes (amygdalin hydrolase (AH), prunasin hydrolase (PH) and mandelonitrile lyase (MDL)) will be assayed during embryogenesis. The distribution of GT I and II and of amygdalin within seeds will be determined by immunocytochemistry and FT-IR microspectroscopy, respectively. Using previously raised polyclonal antibodies, an existing black cherry cDNA library will be screened to obtain AH, PH and MDL cDNA clones. These clones will be utilized to begin examining the temporal and spatial expression of these genes during embryogenesis by Northern blot analysis and in situ hybridization techniques. Whether germinating seedlings utilize amygdalin as a nitrogen source will be examined by measuring the levels and localization of the aforementioned cyanoglycosides and enzymes, along with the HCN detoxification enzyme Beta-cyanoalanine synthase, in various seedling parts.