Detection of spatially- and stage-specific proteins in extracts from single embryos of the domesticated carrot

Single embryos, representing each of four distinct morphological stages, were selected from cultures of the domesticated carrot for analysis of total [35S]methionine-labelled proteins. Following exposure to radiolabel for 12 to 18h, embryos were individually disrupted in a 3mm diameter, precisely-matched, plastic mortar and pestle. Radiolabelled proteins extracted by this procedure were separated by two-dimensional electrophoresis procedures, consisting of isoelectric focusing in 1mm tubes, followed by SDS-PAGE in a small slab gel. Comparisons of autoradiographs of these gels revealed that the levels of a number of proteins were modulated during the conversion of disordered callus cells into maturing embryos. In addition, miniature surgical techniques were used to separate the apex (cotyledon end) from the base (root end) of late-stage embryos, for extraction of proteins and analysis of spatial differences in protein distribution. About five proteins in extracts from each section were observed to be synthesized at different rates in the two halves, indicating that there are molecular correlates for early polarized growth. About half of the proteins, whose appearances were unique to apical and basal sections of embryos, were also observed to fluctuate in comparisons of autoradiographs of two-dimensional protein separations from embryos at different developmental stages.