Biosynthesis of the Cyanogenic Glucoside Dhurrin in Seedlings of Sorghum bicolor (L.) Moench and Partial Purification of the Enzyme System Involved

Abstract

The cyanogenic glucoside dhurrin is rapidly synthesized in etiolated seedlings of Sorghum bicolor (L.) Moench. The dhurrin content of the seedlings increases sigmoidally with the germination time. Shoots of 10 centimeters height contain 850 nanomoles of dhurrin per shoot corresponding to 6% of the dry weight. The biosynthetic activity sharply rises upon germination and reaches a maximum level of 10 nanomoles dhurrin/(hour x shoot) after 48 hours when the shoots are 3 centimeters high. This maximum level is followed by a sharp decline in activity when germination time exceeds 65 hours. Dhurrin and the dhurrinsynthesizing enzyme system are primarily located in the upper part of the etiolated shoot where both are evenly distributed between the coleoptile, the primary leaves and the upper 0.5 centimeter of the first intemode including the shoot apex. Dhurrin constitutes 30% of the dry weight of the upper 1.2 centimeter of 10 centimeter high shoots. The seed and root contain neither dhurrin nor the dhurrin-synthesizing enzyme system. The codistribution of dhurrin and the enzyme system throughout the seedling indicates that production and storage sites are located within the same cell. Purification of the dhurrin-synthesizing enzyme by gel filtration or by sucrose gradient centrifugations results in a tenfold increase in specific activity. Further purification is accompanied by a decline in specific activity due to loss of essential components as demonstrated by reconstitution experiments. linamarin and lotaustralin and plants homozygous for the ac allele are acyanogenic (5). Traditional breeding has produced 'low cyanide' cultivars of cassava ( 11) and sorghum (12), but so far it has not been possible to obtain totally acyanogenic varieties. The polymorphism of cyanogenesis in white clover (5,14) demonstrates that the cyanogenic character is not of vital importance for normal growth and development of each individual cyanogenic plant. It may therefore be possible to convert a cyanogenic plant like cassava into an acyanogenic form by application of the techniques of molecular biology. A prerequisite for such experiments would be the identification and characterization of the enzymes involved in the biosynthesis of cyanogenic glucosides.We have chosen sorghum as the model plant for our studies. (1). Studies involving labeled precursors and trapping experiments have shown that N-hydroxytyrosine, p-hydroxyphenylacetaldoxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile are intermediates in the pathway (Fig. 1) (20,22,24,27). A microsomal preparation isolated from etiolated seedlings of sorghum catalyzes the conversion of L-tyrosine into p-hydroxymandelonitrile, i.e. all but the last step in the pathway (20,22). The dhurrin-synthesizing enzyme system present in the microsomal preparation constitutes a highly organized enzyme system exhibiting catalytic facilitation and thereby providing an efficient mechanism for channeling the flow of carbon from tyrosine into p-hydroxymandelonitrile (23). The latter ...

Effect of sorghum seedlings, and previous crop, on soil fluorescent Pseudomonas spp.

“…Phytotoxic compounds in sorghum stalks and leaves, such as phenolics or cyanogenic glucosides, may have contributed to this allelopathic effect (Halkier and Møller 1989;Sène et al 2001). In the present study, growth chamber assays were used to determine whether cycling of sorghum seedlings or cycling of wheat in soil previously cropped with sorghum genotypes would effect seedling growth.…”

Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism

“…Dhurrin content reaches maximum in 2-day-old dark grown sorghum seedlings after which time degradation proceeds faster than de novo synthesis (30). Analyses of 2-and 4-day-old seedlings by LC-MS showed the presence of a component of m/z 359, the intensity of which was three times higher in 4-day-old compared with 2-day-old seedlings.…”

“…6). This compound has a high structural similarity to the cyanogenic glucoside dhurrin, which is present in high amounts in young seedlings of S. bicolor (30)(31)(32)(33). However, dhurrin is not a direct substrate for the SbNIT4A/B2 complex (data not shown).…”

Prunasin Hydrolases during Fruit Development in Sweet and Bitter Almonds

“…Following incubation (10 min, 30°C, 300 rpm shaking) in a closed Eppendorf tube to avoid loss of volatile HCN formed, the enzyme reaction was stopped by freezing the samples in liquid nitrogen and adding NaOH (40 mL of 6 M) to the frozen sample. After thawing, the samples were left at room temperature (20 min), and a 60-mL aliquot was used to for colorimetric determination of HCN content (580-to 750-nm scan; UV/VIS spectrometer, Lambda 800 [Perkin-Elmer]; Halkier and Møller, 1989) by the addition of 100% glacial HOAc (12.5 mL) immediately followed by 50 mL of reagent A (50 mg of succinimide and 125 mg of N-chlorosuccinimide in 50 mL of water) and 50 mL of reagent B (3 g of barbituric acid and 15 mL of pyridine in 35 mL of water). After thorough mixing and incubation (5 min), the cyanide content was determined spectrophotometrically.…”