Evidence for disproportionate replication of ribosomal RNA genes during endosperm development

Corn endosperm development represents a complex series of genetic events with rapid physiological and morphological changes. These changes are especially striking during the period 12-18 days post-pollination. During this time, endosperm mitoses cease except in the peripheral area, starch and protein synthesis escalates, nuclear and nucleolar volumes increase, and cellular DNA amounts increase in a dramatic fashion. Results reported here indicate that rDNA amounts are not constant during endosperm development; the proportion of rDNA increases during this 12-18 days post-pollination period but subsequently decreases to near initial levels.

In 1977, 1978, 1980, and 1981, plants of the single cross Wf9 x B37 were self-pollinated and the developing ears collected periodically, frozen, and stored at -20 C. In 1978, inbreds Wf9, B37, and A619 were sampled for comparative purposes. The Wf9 material was not usable due to poor development. DNA isolation, rRNA labeling, and DNA/rRNA hybridization procedures were varied over the years in an attempt to determine whether the unexpected results were a function of the procedure or represented a developmental phenomenon. Filter saturation DNA/rRNA hybridization procedures published by Phillips et al. (Chromosoma 36:79-88, 1971) were used in 1977 and 1978. In 1980 and 1981, DNA and rRNA were isolated by procedures described by Mascia et al. (Gene 15:7-20, 1981). In 1980 we employed an aqueous DNA/rRNA hybridization technique published by Casey and Davidson (Nucleic Acids Res. 4:1539-1552, 1977). The 1978 hybridization procedure was followed in 1981. 3H-rRNA of specific activity 3-4 x 103 cpm/ug rRNA was isolated from 6-day-old seedlings of inbred A188 for the 1977 and 1978 experiments. 32p-end-labeled rRNA used for the 1980 and 1981 experiments was extracted from Black Mexican Sweet corn suspension cultures; this RNA was kindly provided by Brenda Hunter in 1981. The specific activity was 3 x 104 to 6 x 105 cpm/ug rRNA. Only the hybridization percent can be reported; rRNA gene multiplicities cannot be estimated because the DNA content of endosperm cells changes during development.

Unexpectedly, the relative amount of rDNA was found to change during endosperm development. A hybridization peak occurred for all three lines (Wf9 x B37, B37, and A619); the peak was apparent in each of the four years for Wf9 x B37. Maximum hybridization values for Wf9 x B37 were 0.49%, 0.46% and 0.74% for 1977, 1978, 1980, and 1981 respectively. The reason for a higher maximum hybridization value in 1981 is not known. The time of occurrence of the maximum hybridization value ranges from 12 to 18 days post-pollination with an average of 14-15 days. The variation may be due to varying environmental conditions in the different years.

Our hypothesis is that when endosperm cells cease dividing they continue to synthesize DNA and the rDNA is preferentially replicated, at least to some degree. As other parts of the genome continue to replicate, the proportion of rDNA is reduced. DNA synthesis during corn endosperm development might be analogous to Drosophila salivary gland polytenization in that different portions of the chromosomes may be replicated to different degrees. If selected regions are preferentially replicated, the multiplicity of those DNA sequences could be extremely high.

Figure 1.

R. L. Phillips, A. S. Wang and P. N. Mascia


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