Nuclear volume, nucleolar volume and rDNA changes during endosperm development

Hybrid Wf9 x B37 plants were self-pollinated and the developing endosperm periodically collected, fixed overnight in 3 parts 95% alcohol and 1 part glacial acetic acid, and then stored at 4 C. A small piece of tissue was dissected from the central portion of the endosperm and squashed in propionic carmine. Nuclear and nucleolar areas were obtained using a Zeiss Multiple Operations Processor (MOP-3). Nuclear and nucleolar volumes were obtained from the following formulas: V = 4/3 pr3, r = (area/p)1/2.

Mitoses were not observed on or after 16 days post-pollination (Table 1). Nuclei larger than 3,000 µ3 were not observed undergoing mitosis. The frequency of telophase was much higher than other mitotic stages in 2 to 8 days post-pollination endosperm indicating that telophase is unusually long in young endosperm tissue.

Number of nucleoli per nucleus at different developmental times is presented in Table 2. Diffuse nucleoli were observed in the majority of the nuclei at the earlier stages of endosperm development, namely 2 to 8 days post-pollination. A single nucleolus/cell was found to be the predominant type at later stages. Diffuse nucleoli were detected only in telophase nuclei suggesting that the telophase nucleolus requires considerable time to become organized in endosperm tissue.

The nuclear volume increases 244-fold, from 368 µ3 to 90,000 µ3 at 2 to 22 days post-pollination (Fig. 1). The most substantial increase was observed between 16 and 22 days post-pollination even though no mitoses were observed during this period.

Nuclei and nucleoli of interphase endosperm cells 12 days post-pollination were measured (Fig. 2). The nucleolar volume ranges from 50 µ3 to 1900 µ3. Nuclear and nucleolar volumes are closely related.

In situ hybridization was carried out with the 12 days post-pollination endosperm interphase nuclei. The nuclei were hybridized with 125I-rRNA (sp. act. 5x107 dpm/ug) and exposed for 7 days at 4 C. Silver grain number was correlated with nuclear and nucleolar volume (Figs. 2 and 3). The average number of silver grains per nucleolus ranged from 10 to 175. The observations suggest that the amount of rDNA increases with increases in nuclear and nucleolar volume. The silver grains were scattered over the entire nucleolus and were not localized over a particular region. This is different from the situation found in pachytene cells of the microspore mother cell, in which the silver grains are localized only over the NOR. The result indicates that the endosperm nucleolus contains dispersed rDNA, and agrees with our previous report that the amount of rDNA and nucleolar size are correlated (Chromosoma 36:79-88).

Table 1.

Table 2.

Figure 1.

Figure 2.

Figure 3.

R. L. Phillips and A. S. Wang

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