Leakage of Dhurrin and p -Hydroxybenzaldehyde from Young Sorghum Shoots Immersed in Various Solvents

Abstract

Spectral scanning was used to provide estimates of the leakge of the cyanogenic glucoside, dhurrin (p-hydroxy4Sj-mandelonitrle-D-gluco-side) and its metabolite, p-hydroxybenzaldehyde (p-HB), from young light-grown shoots of Atlas sorghum (Sorghum bicolor [L.] Moench) when these shoots were immersed in water, toluene, chloroform or mixtures of water and toluene or water and chloroform. Minimal leakage of dhurrin and virtually no leakage of p-HB occurred with water as the solvent. The 0.5% concentration (v/v) of both toluene and chloroform was more effective than either the 1.0 or 2.0% concentrations in effecting leakage of the two solutes. With either 0.5% toluene or 0.5% chloroform as the solvent, 80 to 90% of the total dhurrin was extracted from shoots in a 3-hour period. Breakdown of dhurrin during extraction was much more extensive with 0.5% chloroform than with 0.5% toluene. Some loss of p-HB occurred during 3-or 6-hour extractions in the water-organic solvent mixtures; spectnral and chromatogrphic evidence suggested partial conversion ofp-HB top-hydroxybenzoic acid. With undiluted toluene or chloroform as solvents, extracts contained appreciable amounts of free p-HB but essentially no dhurrin. These solvents were less effective than the water-organic solvent mixtures in extracting the solutes from the shoot issue. Weimberg et al. (6) observed that low mol wt solutes (sugars, amino acids, and inorganic ions) readily leaked from roots and leaves of young sorghum (Sorghum bicolor [L.] Moench) plants when the tissues were immersed in water containing a small amount (0.2 to 2% by volume) of toluene. With 0.5% toluene at 25C, quasiquantitative leakage of the small mol wt solutes occurred in 3 h, but macromolecules were retained in the plant tissues. The authors concluded that toluene was able to diffuse rapidly into the plant cells and to render the membranes permeable to small molecules and ions. Sorghum leaves, especially those of young seedlings, are characterized by the presence of the cyanogenic glucoside, dhurrin (p-hydroxy-[SJ-mandelonitrile-#-D-glucoside), which is localized in the vacuoles of epidermal cells (4). The enzymes, dhurrin ,8-glucosidase and hydroxynitrile lyase, catalyze the breakdown of Project 12-1 14. dhurrin to HCN, glucose, and p-HB;2 these enzymes occur primarily in the mesophyll cells (4) and are thus separated from dhurrin in intact leaves. The leakage of dhurrin and its metabo-lite, p-HB, from young sorghum shoots immersed in water, toluene, chloroform, or mixtures of water and toluene or water and chloroform was investigated in the experiments reported in this paper. MATERIALS AND METHODS Seeds of the forage sorghum cultivar, Atlas, were planted in Hoagland solution-saturated vermiculite and were incubated at 27°C under continuous cool-white fluorescent light (about 150 ,uE * m2. s-'). Water was added as needed to keep the vermiculite moist. Seven d after the seeds were planted, shoots were excised at the cotyledonary node and were sorted at random into groups of 25 or 30 shoots. Each group was weighed; the fresh weight per shoot for all experiments was 63 ± 1 mg (mean ± SE). Groups of shoots were then assigned to treatments which were imposed in 125-ml Erlenmeyer flasks. Shoots were cut transversely into two pieces to facilitate their introduction into the flasks. Solvents (100 ml except as noted) were then added to the flasks. For those flasks receiving mixtures of water and toluene or chloroform, 100 ml of water was first added to the tissue in the flask, then the organic solvents were added. As in the report of Weimberg et al. (6), solvents consisting of 0.5, 1.0, or 2.0 ml of toluene or chloroform in 100 ml ofwater are referred to as 0.5, 1.0, or 2.0% toluene or chloroform, respectively. Filled flasks were immediately placed on a reciprocating shaker (about 100 cycles per min, 5-cm stroke) where they were incubated at room temperature (about 27C). One-ml samples were removed at specified times for spectal scanning. For those treatments involving pure tolu-ene or chloroform, 1.0 ml of water was added to each 1.0-ml sample, and the organic solvent was then evaporated by placing the tube in a water bath at about 55°C and directing a gentle stream of air on the liquid while constantly rotating the tube. Evaporation was stopped when the organic phase appeared to be completely vaporized. Each 1-ml sample was diluted with 9 ml 0.1 N NaOH and immediately scanned from 400 to 240 nm with a Perkin-Elmer Lambda 3 spectrophotometer equipped with a Model 561 recorder.3 The basic solutions were then held at room temperature for 3 h to permit hydrolysis of any dhurrin present 2 Abbreviations: p-HB, p-hydroxybenzaldehyde; p-HBA, p-hydroxy-benzoic acid; A., molar absorbance.