Effect of Freezing on the Hydrocyanic Acid Potential of Field-Grown Sorghum Tillers

Tissue disruption caused by freezing and thawing may contribute to rapid enzymatic breakdown of dhurrin [p-hydroxy-(S)mandelonitrile-p-D-glucoside], the cyanogenic glucoside of sorghum [Sorghum bicolor (L.) Moench], but published reports arenot in agreement as to the effects of freezing on the hydrocyanic acid potential (HCN-p) of field-grown sorghum leaves. Theseeffects were investigated in a series of experiments in the fan of 1983, a period during which weather conditions at Lincoln, NE permitted repeated sampling of frozen and nonfrozen leaves of field-grown sorghum tillers. Assays of these samples, both spectrophotometrically and with the cyanide electrode, indicated that the HCN-p of KS8 and N32 tiller leaves decreased after the tissue was frozen, but potentially dangerous levels remained in N32 tillers for at least 6 or 7 days after the leaves were frozen. Levels of HCN-p in KS8 tiller leaves were much lower than those in N32 leaves. p-Hydroxybenzoic acid, identified by its ultraviolet absorbance spectrum and chromatographic behavior, was found in extracts of frozen N32 tillers. Freezing and thawing of the tillers evidently led to partial breakdownof dhurrin to p-hydroxybenzaldehyde and subsequent oxidation of this aldehyde to p-hydroxybenzoic acid. (nonkilling, presumably) increased the hydrocyanic acid potential (HCN-p) of sorghum forage. Wattenbarger et al. (9) concluded that differing observations on the effects of frost and freezing on HCN-p in sorghum could perhaps be attributed to failure to distinguish between nonkilling frost and freezing. Like Harrington, they found that nonkilling frost usually increased HCN-p. Freezing, however, resulted in death of the plants and rapid loss in HCNp. At Lincoln, NE, weather conditions in the fall of 1983 allowed repeated sampling of frozen and nonfrozen leaf tissue from sorghum tillers. These samples were used in a series of experiments the objectives of which were a) to determine the effects of tissue freezing on HCN-p, and b) to identify a 280nm absorbing compound that was detected in extracts of some of the frozen leaves. MATERIALS AND METHODS Additional index words: Cyanogenesis, Dhurrin, p-Hydroxybenzaldehyde, p-Hydroxybenzoic acid, Sorghum bicolor (L.) Moench. KOJ IMA et al. (6) have shown that in leaves of sorghum [Sorghum bicolor (L.) Moench] seedlings the cyanogenic glucoside, dhurrin [p-hydroxy(Sl-mandelonitrile-ri-D-glucoside], is confined to epidermal tissue, but the enzymes responsible for dhurrin degradation, dhurrin l3-glucosidase and hydroxynitrile lyase, are localized' in mesophyll tissue. They concluded that physical separation of dhurrin from catabolic enzymes prevents degradation of dhurrin in intact leaves. However, mechanical disruption of the leaf tissue leads to mixing of the enzymes with dhurrin, with the result that the glucoside is broken down to glucose, p-hydroxybenzaldehyde (P-HB), and HeN. Tissue disruption can occur naturally in field-grown plants through freezing and thawing. Freezing and thawing of field-grown sorghum plants may result in dhurrin degradation if the enzymes of dhurrin catabolism are present in the plants and if the enzymes remain active following freezing and thawing. The need to allow time for dhurrin breakdown and HCN dissipation before previously frozen sorghum forage is fed to livestock has been recognized for some time. Harrington (4) recommended that at least 3 days should elapse between the time sorghum forage is exposed to a killing frost and the time the forage is fed to livestock. Harrington also reported that frost I Contribution from USDA-ARS and the Nebraska Agric. Exp. Stn., Lincoln 68583. Published as Paper no. 7425, Journal Series, Nebraska Agric. Exp. Stn. Received 19 Mar. 1984. The work reported was done under Nebraska Agric. Exp. Stn. Project 12-114. 2 George Holmes professor of agronomy; supervisory research geneticist, USDA-ARS, and professor of agronomy; associate professor of agricultural biochemistry; and research technologist in agronomy, respectively. Tillers from field-grown plants of the A-lines of KS8 (7) and N32 (8) sorghum were used in these experiments. As previously reported (5), whole shoots of week-old chambergrown seedlings of both lines had HCN-p values of about 2200 mg kg-I dry wt., but leaves of field-grown N32 plants approaching maturity were several fold higher in HCN-p than those of KS8. On 23 Sept. 1983 a freeze killed the upper leaves of the main stems and taller tillers of both KS8 and N32. The dead upper leaves formed a canopy that appeared to protect the younger tillers from freezing until 13 October when tips of many of the tiller leaves were frozen. A freeze that killed all tillers occurred on 11 November. Samples used in these experiments were harvested between 10 Oct. and 22 Nov. 1983. Tillers 30 to 50 em tall were harvested and immediately carried to the laboratory where all leaf tissue distal to the collar of the youngest leaf with a readily visible collar was excised. The leaves were wiped free of dust and debris and were cut into l-cm" pieces. Random samples of the tissue were then weighed for treatment and assay. Procedures for sample treatment and for the spectrophotometric assay of HCN-p have been described (3, 5). Briefly, samples were either autoclaved fresh (Treatment 1) or were dried 2 h at 75°C, ground, and incubated in water for 2 h at room temperature to provide extracts which were then autoclaved (Treatment 2) or hydrolyzed in base (Treatment 3). For Treatments 1 and 2, p-HB released from dhurrin by autoclaving was extracted with ether, the ether was evaporated, and the absorbance at 330 nm (the Amax ofP-HB) of the reconstituted ether phase served as the basis for calculation of HCN-p (5). For Treatment 3, the gain in A~~o between a spectral scan made immediately after the tissue extract was diluted in 0.1 M NaOH and a scan of the basic solution made about 3 h later, following dhurrin hydrolysis, provided values needed for calculation of HCN-p (5). For comparison with the spectrophotometric assays, HCN from 20 samples representing both KS8 and N32 was distilled into base as in the AOAC procedure for cyanide (1). 1183 Published in Crop Science (November-December 1984) v. 24, no. 6