Evidence for Sulfhydryl Involvement in Regulation of Phytoalexin Accumulation in Trifolium repens Callus Tissue Cultures

Abstract

White clover (Trifolium repens L.) callus tissue cultures accumulated the phytoalexin medicarpin after treatment with sulfhydryl reagents. After 24-hour exposures to sulfhydryl reagents, maximum obtainable levels of medicarpin, determined by high performance liquid chromatography analysis, were found with 50 millimolar N-ethyl maleimide, 25 millimolar HgCl(2), 2 millimolar p-chloromercuribenzoic acid, and 0.5 millimolar iodoacetamide. Increased medicarpin levels were also observed in callus treated with p-chloromercuribenzene sulfonic acid, but the highest concentration tested (11.8 millimolar) did not produce the maximum response. After sulfhydryl treatment, medicarpin levels were unchanged for 4 to 6 hours, but steadily increased thereafter with maximum accumulation occurring by 48 to 50 hours for p-chloromercuribenzoic acid, p-chloromercuribenzene sulfonic acid, and HgCl(2) treated callus. Medicarpin levels did not increase in iodoacetamide-treated callus until 8 hours after sulfhydryl exposure, and medicarpin levels were still increasing linearly after 50 hours. Three other metabolic inhibitors, KCN, NaF, and Na(3)AsO(4), did not exhibit elicitor activity, indicating cell death was not a factor in the response. Pretreatment of callus with 20 millimolar dithiothreitol followed by 40 millimolar N-ethyl maleimide did not produce the phytoalexin response. Preincubation with dithiothreitol also prevented elicitor activity of HgCl(2) and p-chloromercuribenzene sulfonic acid. These results suggested that dithiothreitol pretreatment somehow prevented sulfhydryl groups within the cell from reacting with the test compounds. These experiments established that the integrity of sulfhydryl groups is important in regulating phytoalexin accumulation in callus cells.