A survey has been made of the occurrence and distribution of three enzymes which metabolize cyanide in a variety of higher plants including both cyanogenic and non-cyanogenic species. The enzymes investigated were f8-cyanoalanine synthase, rhodanese and formamide hydrolyase. fiCyanoalanine synthase was found to be present in every higher plant tested whereas rhodanese was found to occur far less commonly in plants. Formamide hydrolyase activity was not detected in any of the higher plants tested.In addition, quantitative analyses have been made of the potential hydrogen cyanide content of each plant investigated. A general trend was apparent between the hydrogen cyanide potential and cyanide metabolizing activity, in that the higher the hydrogen cyanide potential, in general, the higher the cyanide metabolizing activity.Many plants have the ability to produce HCN; more than 2,000 species have been demonstrated to be cyanogenic. The mechanism for the production of HCN, in most species, is the degradation of cyanogenic glycosides (7). The present study investigates the occurrence and distribution of cyanide metabolizing enzymes in a variety of higher plants, including both cyanogenic and noncyanogenic species. This information in turn is compared with the HCN potential of each plant studied. The HCN potential is a reflection of the concentration of cyanogenic glycosides in the plant which, upon degradation, leads to the release of HCN.The enzymes selected for investigation were fi-cyanoalanine synthase (EC 4.4.1.9), rhodanese (EC 2.8.1.1), and FHL3 (EC 4.2.1.66). The reactions they catalyze are shown in equations 1-3, respectively:The enzyme 83-cyanoalanine synthase has been shown to be present in several plant species (1,9) and in some bacteria (4,8 The crude homogenate, from both methods, was squeezed through cheesecloth and the filtrate centrifuged at 12,000g for 10 min. The resulting supernatant liquid (hereafter termed the enzyme extract) was used for enzyme assays. All steps in the preparation of the enzyme extract were carried out below 4 C. ENZYME ASSAYS All substrate solutions were prepared immediately prior to the enzyme assay. Enzyme activities were determined in every instance by reference to the appropriate boiled control. Controls containing no substrate(s) and controls containing no enzyme extract were also performed.
The tissue distributions of dhurrin lphydroxy-(S)-mandelonitrhie8-Dplucosidel and ofenzymes involved in its metaboUsm have been investigated in leaf blades of light-grown Sorghu bicolor seedlngs. Enzymk digestion of these leaves using celulase has enabled preparations of epidermal and mesophyli protoplasts and bundle sheath strands to be isolated with only minor cross-contamination. Dhurrin was located entirely in the epidermal layers of the leaf blade, whereas the two enzymes responsible for its catabolism, namely dhurrin ,-glucosidase and hydroxynitrile lyase, resided almost exclusively in the mesophyli tissue. The final enzyme of dhurrin biosynthesis, uridine diphosphate glucose;p-hydroxymandelonitrile glucosyltransferase, was found in both mesophylH (32% of the total activity of the leaf blade) and epidermal (68%) tissues. The bundle sheath strands did not contain significant amounts of dhurrin or of these enzymes. It was concluded that the separation of dhurrin and its catabolic enzymes in different tissues prevents its large scale hydrolysis under normal physiological conditions. The well documented production of HCN (cyanogenesis), which occurs rapidly on crushing Sorghum leaves, would be expected to proceed when the contents of the ruptured epidermal and mesophyll cells are allowed to mix.
Large numbers of vacuoles (106-107) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic gluncoside dhurrin. Leaves from Ught-grown seedings were incubated for 4 hours in 1.5% cealysin and 0.5% macerase to yield mesophyl protoplats which then were recovered by cent ation, quantitated by a hemocy-tometer, and assayed for cyanogenic glucosides. Mature vacooles, released from the protophats by osmotic shock, were purified on a discontinuous Ficoli gradient and monitored for intactness by their ability to maintain a slghtly add interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 pmoles of cyanogenic glucouide dhurrin per 107 protop_as or 107 vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum. The isolation of organelles may provide a convenient procedure for investigating the localization of secondary plant products. Until recently, however, the largest organelle in plant cells, the vacuole, has been available for study only in small numbers (4), in an immature form (10), or from unicellular organisms (6, 11). Wagner and Siegelman (13) and Lorz et al. (8) have described a technique for the isolation of large numbers of vacuoles which works well in petals of several species and the leaves of a few. However, the procedure requires long periods of incubation (12-24 hr) of the tissue involved in a digesting solution for the release of the protoplasts. Moreover, the procedure as described was not effective in obtaining large numbers of vacuoles from Sorghum. Leigh and Branton (7) have circumvented the long incubation of the tissue by using a mechanical slicer for the isolation of vacuoles. This technique works effectively on firmer tissues such as red beet root tissue; however, their technique would have limited use on leaf tissue. This paper describes a technique which combines a relatively short incubation period (4 hr) for preparation of the protoplasts with a Ficoll discontinuous gradient to produce large numbers of vacuoles. The technique has been utilized to study the localization of the cyanogenic glucoside of Sorghum. MATERIALS AND METHODS Plant Material. Six-day-old light-grown shoots of Sorghum bicolor (Linn) Moench, variety Redland x Greenleaf, were harvested 1 cm above the caryopsis after germination at 24 C on water-saturated vermiculite under a 14-hr photoperiod. Isolation of Vacuoles. The expanded leaves of approximately 3 g of tissue were excised above the caryopsis, abraded with 150 grit carborundum using a small artist brush after the technique of Beier and Bruening (2), rinsed in distilled H20, and incubated for 4 hr in 36 ml of 0.5 M mannitol, 25 mm K-phosphate-citrate buffer (pH 5.5), containing 5 mg/ml macer-ase and 15 mg/ml cellulysin. The incubation was carried out at 37 C in a shaking water bath at 18 oscillations/mi...
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