Developmental co-variation of RNA editing extent of plastid editing sites exhibiting similar cis-elements

Abstract

In tobacco, 30 of 34 sites in chloroplast transcripts that undergo C-to-U RNA editing can be grouped into clusters of 2-5 sites based on sequence similarities immediately 5' to the edited C. According to a previous transgenic analysis, overexpression of transcripts representing one cluster member results in reduction in editing of all cluster members, suggesting that members of an individual cluster share a trans-factor that is present in limiting amounts. To compare leaves and roots, we quantified the editing extent at 34 sites in wild-type tobacco and at three sites in spinach and Arabidopsis. We observed that transcripts of most NADH dehydrogenase subunits are edited inefficiently in roots. With few exceptions, members of the same editing site cluster co-varied in editing extent in chloroplasts versus non-green root plastids, with members of most clusters uniformly exhibiting either a high or low editing extent in roots. The start codon of the ndhD transcript must be created by editing, but the C target is edited inefficiently in roots, and no NDH-D protein could be detected upon immunoblotting. Our data are consistent with the hypothesis that cluster-specific trans-factors exist and that some are less abundant in roots, limiting the editing extent of certain sites in root plastids.

Figure 1

The three recently discovered editing sites of tobacco, rps2-1, ndhD-3 and ndhD-4, can be grouped into clusters. Bold letters represent conserved nucleotides between members of the cluster. Gaps (–) were introduced to show similarities. C, C target of editing.

Figure 2

Editing extent of the 34 sites in young leaf chloroplasts of mature tobacco plants and in root plastids of 1-month-old tobacco plants. The percentage of edited transcripts was determined on PPE products by quantifying the radioactivity associated with edited and unedited sites using ImageQuant software (Molecular Dynamics). The x-axis represents the 34 editing sites listed in Table 2.

Figure 3

PPE assays showing the different editing extents of the four sites located within ndhD transcripts in leaves (L) and roots (R) of tobacco. PPE was performed on site-specific RT–PCR products, from radiolabeled ndhD-1(G), ndhD-2(G), ndhD-3(G) and ndhD-4(T). The primer extension was poisoned by ddNTP incorporation, ddGTP for ndhD-1, -2 and -3 and ddTTP for ndhD-4. PPE products were resolved on 12% sequencing gels, which were then exposed on a phosphorimager screen. 0, PPE without template indicating the size of the radiolabeled oligonucleotide; g, PPE with a cloned (genomic) unedited PCR product; c, PPE with a cloned edited RT–PCR product; L, PPE made from leaf extracts of mature tobacco plants; R, PPE made from root extracts of young tobacco; E, PPE product corresponding to the edited transcript; NE, PPE product corresponding to the unedited transcript.

Figure 4

Editing efficiencies of 30 editing sites in tobacco leaves and roots that can be grouped according to cis-element cluster. The first two clusters were shown to cross-compete in transgenic chloroplasts (22).

Figure 5

Putative cis-elements conserved in the upstream sequences of sites in ndhF-2 and atpA-2 clusters. Bold letters represent conserved nucleotides between members of the cluster, and lower case letters indicate nucleotide polymorphism within a putative cis-element. Gaps (–) were introduced to show similarities. C, C target of editing.

Figure 6

The NDH-D polypeptide is not detected in tobacco root plastids. Immunoblot analysis of total protein from leaf (10 µg) and root (40 µg) using an anti-NDH-D antibody. The calculated molecular mass of NDH-D is 56.3 kDa (MWCALC, Infobiogen). The migration of BioRad pre-stained standard carbonic anhydrase is shown on the left.

Figure 7

NdhB-3 shares sequence similarities in its upstream region with other editing sites in maize (Zm, Zea mays; Nt, Nicotiana tabacum). (A) Sequence alignment of the ndhF-2 cluster in tobacco and the putative ndhB-3 cluster in maize. (B) Comparison of sequences in maize and tobacco ndhB-3, edited in both species, illustrating that the two sites contain both the tobacco and maize conserved elements. (C) Sequence comparison between sites edited in tobacco and the homologous sites in maize, which carry a genomic T. (D) Sequence comparison between sites edited in maize and the homologous sites in tobacco, which carry a genomic T. Bold letters represent conserved nucleotides between members of the cluster, and lower case letters indicate nucleotide polymorphisms. Gaps (–) were introduced to show similarities. C, C target of editing; U, location of the editing sites in the other species.