Electrophoretic study of storage protein mobilization and proteolytic activities in imbibed walnut kernels

Walnut kernels are dormant at maturity and therefore in order to germinate require moist-chilling or stratification, i.e. incubating imbibed seeds at cold (5ºC) for a certain period. It is assumed that reserve mobilization can not proceed in imbibed dormant seeds incubated at warm condition. In contrast to other dormant tree seeds, imbibing dormant walnut kernels showed storage protein mobilization irrespective of the temperature of incubation. However, there is no information on the nature of proteolytic processes at the protein and enzyme levels in imbibed walnut kernels. Persian walnut (Juglans regia L.) kernels were stratified at 5ºC for increasing periods up to 60 days. Thirty days stratified kernels displayed maximum germination percentage of 61% whereas only 23% of warm-incubated kernels germinated. Mobilization of total storage protein in kernels was faster under warm conditions when compared to stratification. Levels of soluble proteins remained unchanged following imbibition in both stratified and warm-incubated kernels. SDS-PAGE patterns of total proteins did not show any changes in walnut 19-23 and 32-35 kDa glutenins and 42-49 kDa vicilins under both temperature regimes. This technique however, revealed alterations in the polypeptide patterns of soluble protein fraction notably increased band intensity of 41-48 and 58 kDa in parallel with reduced band intensity of 18 kDa polypeptides. The increased levels of some polypeptides in the soluble fraction might have been resulted from the increased solubility of insoluble storage proteins, proteolysis or de novo protein synthesis, while reduced band intensity is likely to represent proteolytic processes. Using azocasein as a substrate to detect protease (s) involved in storage protein mobilization, a protease most active at pH 6.0 was found in extracts from walnut kernels. Since no significant differences were observed in measured protease activity between stratified and warm-incubated kernels, other proteases are supposed to be involved in the mobilization of walnut kernel storage proteins.