As reported last year (MGCNL 58:136, 1984), 1D SDS PAGE separations of protein extracted from maize embryos enabled the detection of qualitative and quantitative differences in newly synthesized polypeptides occurring as a function of both development and genotype. In order to better resolve some of these differences, 2D IEF-SDS PAGE was performed.
Included in this study were two inbred cultivars, Oh43 and M14 and their reciprocal hybrids. Embryos were selected at 17 and 20 days post-pollination and subsequently at 5 day intervals to 40 days post-pollination. Labelling of embryos in situ with 35S-methionine was performed as described by Kriz (MGCNL 56:14-15, 1982).
Between 150 and 300 polypeptides were resolved routinely following fluorographic analysis of 2D gels. Initial attempts to quantitate some of these data have revealed an apparent increase of 20-30% in the number of polypeptides synthesized at 30 days over that at 20 days post-pollination. Conversely, the number of polypeptides synthesized at 40 days decreased by 15% from that observed at 30 days post-pollination. A shift in the distribution of newly synthesized polypeptides was also evident, in that a larger proportion of low molecular weight polypeptides (below ~30 Kd) were observed during the latter stages of development, between 30 and 40 days post-pollination.
Comparisons of 2D patterns obtained from different inbreds have suggested that at least 5% of the polypeptides synthesized may be genotypically unique. A prominent example of this was observed in Oh43 where a pair of spots of approximate pI 5 and molecular weight 69 Kd could be detected throughout the period of study but at no time were evident in M14. Also in M14 a group of spots migrating at approximately pI 4 and molecular weight 76 Kd were observed at all stages after 25 days, but could not be detected in Oh43 prior to 35 days post-pollination. In the latter case this may indicate that regulation of the synthesis of these polypeptides is timed differently in the two cultivars. All of the polypeptides synthesized by the inbred parents were detected in both reciprocal hybrids.
A number of polypeptides appeared to vary quantitatively with respect to relative amounts synthesized across both development and genotype. In those cases where the inbreds showed qualitative differences, these were reflected quantitatively in the hybrids, such that the quantity synthesized appeared to be most influenced by the maternal parent. These findings suggest a possible allele dosage effect in the case of the specific peptides studied.
The utilization of computerized image processing and statistical analysis are presently being developed to facilitate the collection, manipulation and interpretation of data obtained from 2D electrophoretic separations.
J. G. Boothe and D. B. Walden
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