Plastid transcript accumulation in developing endosperm

--Andrew McCullough and Burle Gengenbach

Amyloplasts in storage organs such as the endosperm of maize kernels are considered to be differentiated plastids that do not carry out photosynthetic activities, but rather accumulate starch. This study was initiated to characterize the plastid genome in differentiated amyloplasts and to determine whether plastid genes are expressed specifically during amyloplast biogenesis in maize endosperm. We have previously reported (McCullough and Gengenbach, Genome 30, suppl. 1:315, 1988) that the plastid genome is present in total DNA prepared from maize endosperm tissue and that the abundance of plastid DNA increases relative to nuclear rDNA during early endosperm development. Nuclease protection assays with total endosperm RNA and cosmid clones of plastid DNA showed at least part of most plastid BamHI fragments were protected by RNA isolated from 16 day post-pollination (dpp) endosperm.

Restriction fragments representing most of the plastid genome recently were used as probes in Northern hybridizations. We detected fewer RNA species homologous to plastid DNA in endosperm than in leaves. As a proportion of the total RNA, the abundance of most plastid transcripts was lower in endosperm than in leaves. We have, however, identified probes for which the corresponding transcripts accumulate to a higher abundance in 16 dpp endosperm than in shoots, leaves, or roots. One of these probes, BamHI fragment 23, contains the ribosomal protein (rp) s12-s7 operon. This operon contains exon II, intron II and exon III of rps12 and the uninterrupted rps7 gene. Exon I and intron I of rps12 map several kilobases from BamHI 23 and maturation of rps12 mRNA is thought to involve trans-splicing. In Northern analysis, the BamHI 23 probe detected two preferentially accumulating transcripts in 16 dpp endosperm RNA. One of these transcripts was not detected in leaf, etiolated shoot, or root RNA. The second was much more abundant in the endosperm RNA than in RNA from other tissues; it also was detected in endosperm RNA by a probe containing exon I of rps12 (BamHI fragment 10) suggesting that trans-splicing occurs in endosperm plastids.

Probes specific for the rps7 coding region and exon II and intron II of rps12 were constructed. Subsequent Northern analyses revealed that the transcripts preferentially accumulating in 16 dpp endosperm contained homology to all three probes, suggesting that these transcripts were unprocessed, polycistronic RNA molecules. RNA isolated from 5, 8, 12, 16, and 20 dpp endosperms was probed with BamHI 23 which revealed a progressive accumulation of these unprocessed transcripts over the 5-12 dpp period. The levels of other, but unrelated, plastid transcripts homologous to these probes remained constant during this period. Experiments are in progress to further characterize the structure and accumulation pattern of these and other transcripts which accumulate preferentially in endosperm plastids. Our results agree with those of Dang and Gruissem (EMBO J. 7:3301-8, 1988) which strongly suggest that post-transcriptional rather than transcriptional events play key roles in plastid gene regulation.

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