Presence of oligosaccharides in seed-coat mucilage of Lepidium sativum : role in allelopathy
Lepidimoide is a naturally occurring disaccharide reported to be an oligosaccharin, i.e. to exhibit ‘hormone-like’ biological activity. It was found in cress (Lepidium sativum) root exudates and exerts apparently allelopathic effects on neighbouring Amaranthus seedlings. In the present study the effect of cress root exudates on hypocotyl and root length of Amaranthus caudatus and Lactuca sativa was studied. The seedlings of both species grown with Lepidium sativum seedlings had longer hypocotyls and shorter roots as compared to the control. In this study I found an active principle with biological effects similar to those of lepidimoide to be more abundant in cress seed-coat mucilage than in root exudates. The active principle peaked 24 hours after seed soaking, and thereafter plateaued. I also for the first time confidently proved that the bioactive compound(s) were exuded by cress and were not microbial digestion products or seed treatment chemicals. Quantitative tests of cress root exudates and cress seed-coat mucilage showed the presence of hexoses, pentoses, uronic acids and unsaturated uronic acid. The presence of unsaturated uronic acid might be of interest because the known structure of lepidimoide includes an unsaturated uronic acid. Active principle from mucilage was partitioned into the aqueous phase when shaken with ethyl acetate at pH 2, 6.5 and 12, showing it to be hydrophilic, unlike auxins and gibberellins. The mucilage was also heated at 130°C for 48 h and severe heating did not affect its biological activity, suggesting that if the compound is lepidimoide then it is heat-resistant. In an attempt to test whether the compound is of high or low Mr, the mucilage was partitioned into 75% ethanol-precipitated and non-precipitated fractions. The biological activity in the non-precipitated fraction was very high, and was further separated by gel-permeation chromatography (GPC). GPC on Bio-Gel P-10 and P-2 suggested that the active principle had Mr ~500–750, compatible with oligosaccharide(s), suggesting that a particular oligosaccharide may be the active principle. TLC separation of bioactive fractions from P-2 showed that the bioactive compound migrated between GalA and Gal but co-migrated with sucrose; however, paper chromatography separation proved that the compound is not sucrose and might be a different disaccharide (lepidimoide). From the structure of lepidimoide, Fry et al. (1993) proposed that lepidimoide is formed by the lyase-catalysed cleavage of a pectic polysaccharide, rhamnogalacturonan-I (RG-I). So I tried to prepare lepidimoide or lepidimoide-like compounds by the action of RG-I lyase from Pichia pastoris on purified potato RG-I. The lyase showed its activity but the digest did not demonstrate biological activity, which might be due to presence of tris-HCl buffer in the solution. An attempt was also made to prepare lepidimoide by methyl esterification and -elimination of purified potato RG-I but again the product did not show any biological activity, which might be due to presence of borate buffer in the solution. This part of research might be useful for future work on preparation of lepidimoide and lepidimoide-like compounds.