University of Minnesota

Maize plastid genome BamHI 14 fragment has conserved intron-containing open reading frame

--Jaakko Kangasjarvi, Dennis E. Mathews and Burle G. Gengenbach

In previous studies (MNL 63:105, 1989) we have found that the plastid transcript accumulation pattern differs between chloroplasts and amyloplasts. A 2.4 kb transcript from the plastid BamHI 14 fragment accumulated progressively during endosperm development and was more abundant in older endosperm than in leaf total RNA. No known genes have been identified on maize BamHI 14, but the corresponding region of three other species has conserved intron-containing open reading frames (ORF170 in rice). No function is known or has been proposed for the hypothetical protein. Our subsequent PCR analysis (MNL 64:102, 1990) suggested the presence and expression of a similar intron-containing open reading frame in the maize plastid genome, located between psaA and rps4. We have now sequenced the maize plastid genome BamHI 14 fragment and the cDNA of the fully spliced putative ORF to verify the existence of the similar ORF in maize and to establish the splicing sites. We also determined its pattern of expression by PCR amplification of total and polysomal RNA isolated from various tissues and endosperm developmental stages.

BamHI 14 fragment was subcloned from a cosmid clone. Mature ORF170 was cloned with PCR from reverse transcribed leaf total RNA using ORF170 specific primers. Both were sequenced using the dideoxy procedure. Total RNA was isolated from 7 d old shoots and roots, 8-12 DAP endosperm and developing leaves. Polysomes were isolated from developing endosperm, roots and seedling leaves. All the RNAs were treated with RNase-free DNase. cDNA was synthesized using random primers and amplified with PCR. Cloned BamHI 14 fragment and mature ORF170 were used as specific controls for the full length transcript and fully spliced transcript, respectively.

The nucleotide sequence of maize plastid BamHI 14 fragment (3.1 kb) revealed the expected open reading frame containing three exons and two introns, as found in other species where the homologous region has been sequenced. 5' and 3' ends of the introns contained splicing sequences conserved in plastids and the cDNA verified the expected splicing sites. As in other species, maize exons I and II start with ATG and have an in-frame stop codon inside the introns.

ORF170 is expressed in maize chloroplasts and amyloplasts, but the developmental processing of the full length transcript is different in these two plastid types. The fully processed transcript was present at the early stages of endosperm development during the time of differentiation of proplastids to amyloplasts. When RNA from 8-12 DAP endosperm was used in the cDNA synthesis, there was a proportional increase in the amount of unspliced transcript (1.9 kb) compared to mature message at older stages; the abundance of the intermediate processing product (1.2 kb, one intron removed) decreased. The decrease in the abundance of the partially processed form and the accumulation of the unspliced form coincided with differentiation and the onset of starch accumulation. Transcription of ORF170 continued through development of endosperm at least up to 16 DAP and the regulation of expression appeared to be at the level of transcript processing as has been suggested for plastid intron-containing genes. In contrast to endosperm, the completely processed transcript (0.45 kb) was the most abundant amplified product for root, old and young leaf and seedling RNA of maize. The splicing pattern of ORF170 transcripts did not change in subsequently older leaves and two intermediate processing products were observed which was different from the pattern found in endosperm.

Total polysomal RNA from various tissues contained transcripts from the BamHI 14 region. Only the mature transcript was detected in roots, shoots and 8 DAP endosperm. Polysomal RNA from 10 DAP endosperm contained the unspliced transcript and at 13 DAP both were present. The intermediate processing product was not detectable in any of the polysome samples. The presence of the transcripts from the BamHI 14 fragment in polysomal RNA suggests that the gene is being translated in all the tissues and endosperm developmental stages studied. In leaves, roots and 8 DAP endosperm this was as expected, because the mature transcript was most abundant in the total RNA. The association of the unspliced transcript with polysomes at 10 and 13 DAP is surprising; however exons 1 and 2 are open reading frames and thus could be translated separately. This possibility is under study. We are currently making antibodies against the ORF170 protein expressed from the cloned cDNA in E. coli to determine whether a corresponding protein is actually synthesized in plastids.

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