We have initiated a study to characterize the amyloplast genome and to determine whether plastid genes are expressed specifically during amyloplast biogenesis in the endosperm. Five overlapping cosmid clones (from D.F. Lonsdale) representing the complete plastid genome were used in Southern hybridization analyses of total cellular DNA from 16-day-old endosperm of inbred A188. Sequences homologous to plastid DNA were present in the total DNA of endosperm tissue. When endosperm DNA was digested with BamHI, PstI or XhoI, the hybridization patterns for individual cosmid probes were identical to those of total DNA from green leaf tissue. Indistinguishable patterns also were obtained when one of the cosmids was hybridized to leaf and endosperm DNA digested with a 4-base-pair cutter, MspI. We conclude that the amyloplast genome in the early stages of grain filling is very similar and most likely identical to the genome in differentiated chloroplasts.
The amount of DNA per nucleus in endosperm cells of A188 has consistently been shown to increase about 30-fold between day 14 and day 20 post-pollination (R.V. Kowles and R.L. Phillips, Proc. Natl. Acad. Sci. 82:7010, 1985). To determine whether the plastid genome copy number also increases during this period, the ratio of two plastid genes (Cytochrome f and P700) to a representative nuclear gene (rRNA gene) was measured. Total cell DNA was labeled with 32P-CTP by random primer labeling, then hybridized to a predetermined excess of a cloned plastid gene or cloned nuclear rDNA bound to a nitrocellulose filter, and the amount of radioactivity bound was quantitated by scintillation spectroscopy. Under these conditions the amount of hybridization to the specific clone was assumed to be proportional to the amount of that sequence in the total DNA sample.
We found that plastid DNA also increases during endosperm development. For example, the ratio of the plastid cytochrome f gene to nuclear rDNA increased about 2-fold from day 10 to day 16 post-pollination and then dropped to about a 35% increase by day 30. Even though the amount of nuclear DNA per nucleus was not measured in these samples, it is apparent that the amount of plastid DNA keeps pace with or even exceeds the increase in nuclear DNA during endosperm development.
Andrew J. McCullough and Burle G. Gengenbach
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