The immunocytochemical localization of transgenically-expressed proteins in plants

Abstract

A range of tissue preparation and immunocytochemical localization techniques were assessed for their possible application in the study of the transgenic expression of the legA gene, encoding Pisum sativum legumin in transformed Nicotiana plumbagimfolia seeds. Immuno-gold localization methods were developed at the EM level, using sections of L.R. White embedded seeds. An endogenous legumin-like protein in the Nicotiana plumbagtmfolia seeds was recognised by antibodies raised against Pisum legumin. In order to determine the temporal and spatial distribution of legA encoded legumin in the transformed seeds, it was necessary to develop a suitable means of discriminating between Pisum legumin and the endogenous protein. Cross reacting antibodies were precipitated out of a polyclonal antiserum by pre treatment with an extract from non-transformed Nicotiana plumbaginifolia seeds and the remaining antibodies were then used to achieve specific localization of Pisum legumin. A method was also developed for the localization of the endogenous legumin-like protein in the non-transformed seeds, using a non-pretreated antibody preparation. Both proteins were detected in the protein bodies of transformed Nicotiana plumbaginifolia seeds, but their temporal and spatial distributions were different. The endogenous protein was abundant in all seed tissues, whenever and wherever there were protein bodies present. Pisum legumin was present at a detectable level in the older developing and mature transformed seeds, principally in the cotyledons and adjacent embryo axis and to a lesser extent in the endosperm. These results clearly demonstrate that the legA gene is being deferentially expressed, in a manner which is different to the expression of the endogenous legumin-like protein. This suggests that its differential expression is under the control of the non-coding regions of the legA gene, rather than being determined by endogenous Nicotiana control elements. The significance of these results is discussed in relation to those achieved in other transgenic situations. The use-fullness of the methods and their potential future applications are considered.