Fluorographic analysis of 2-D PAGE electropherograms coupled with the specific temperature-shift patterns (preceding contributions, this Newsletter) of known genotypes provides the technology for addressing a number of developmental and genetic problems. We currently are examining embryogenesis, seedling germination and flower development (see next contribution) employing inbreds and their reciprocal hybrids. We report the appearance of developmental shifts in the patterns of newly synthesized polypeptides obtained from maize embryos. The procedures adopted for this study involved the dissection under laboratory conditions of embryos resulting from controlled pollinations. The embryos were transferred to Murashige and Skoog medium and incubated at 27 C for 2 hrs in the presence of an isotopic probe (usually 35S-methionine). The subsequent protein extraction and electrophoretic techniques were identical to those reported in the preceding contributions to this Newsletter.
Results obtained for the incorporation of the labelled probe indicated that material 23 days post-pollination or older provided sufficient quantities of labelled protein in a single embryo to achieve a concentration of at least 200 cpm/ul in a homogenization volume of 150 ul. Material 27 days of age or older enabled a concentration of 500 cpm/ul to be achieved in the same volume. These concentrations were suitable for 1D and 2D fluorographic analysis, respectively. In each case, embryos younger than these required bulking of material to achieve the desired concentrations. Of the material from which sufficient quantities were obtained (17-31 days post-pollination), 2D fluorographic analysis routinely resolved over 100 peptides. These were classified into one of three categories: (1) those where the relative level of synthesis appeared unaltered throughout the period of study; (2) those that showed variations in the level of synthesis; (3) those where synthesis appeared restricted to particular time periods. Thus far we have been unsuccessful in obtaining sufficient material at such an early age as to recover only a few labelled spots in the 2-D fluorograms.
There appears to be a genetical component in addition to the developmental component, i.e., some inbreds differ from one another and some of the hybrids we have examined differ from their parental inbreds. We draw attention, however, to the inconclusiveness of these observations in the case of the embryos--until we can utilize an independent criterion, better than age and/or heat unit accumulation, we are reluctant to assign differences to the pedigree and ignore environmental variability.
In addition to these in vivo studies, total RNA derived from embryos of dried seeds was extracted and translated in a rabbit reticulocyte lysate system. The appearance of labelled bands on a 1D gel of the translation assay demonstrates the in vitro synthesis of proteins from this RNA, and thus the presence of stored mRNA in maize embryos. The time at which these messages were synthesized remains to be determined as does their ability to direct translation in vivo upon the onset of germination.
It is anticipated that these investigations will lead to a better understanding of the factors governing the regulation of gene expression in hybrids and their parental inbreds during early development.
J. Boothe, B. G. Atkinson and D. B. Walden
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