Journal of Experimental Botany, Vol. 52, No. 354, pp. 179-180,
January 2001
© 2001 Oxford University Press
Gene Notes |
Primary transcripts of ndhD of Liliaceae and Aloaceae require editing of the start and 20th codons
Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Alcalá, Alcalá de Henares, 28871-Madrid, Spain
Received 21 July 2000; Accepted 5 October 2000
Abstract
About 350 nucleotide sequences around the start codon of the plastid ndhD gene were determined in six species to investigate the requirement of C to U editing of the cryptic start codons. The comparison with other sequences in databanks showed that, in contrast to grasses and similarly to dicots, Liliaceae and Aloaceae require editing of the primary transcript to generate the AUG start codon. Primary transcripts of Liliaceae and Aloaceae also show a new editing site at the 59th nucleotide, which has not been described in other plants. In some cases, transcripts showed partial editing which suggests that the synthesis of the protein may be controlled at the level of transcript processing.
Key words: Aloaceae, editing, Liliaceae, ndh genes, plastid DNA.
The thylakoid NADH dehydrogenase complex seems to provide electrons to poise cyclic electron flow (Casano et al., 2000
) and is involved in the protection against photo-oxidative stress (Martín et al., 1996; Casano et al., 2000). Eleven polypeptides of this complex are encoded in the ndh genes located in the plastid DNA. Some transcripts of ndh genes are edited by changing C to U nucleotides (Del Campo et al., 2000) which usually renders codons for conserved amino acids (Maier et al., 1996) in the sequences of homologous polypeptides. In some dicots such as tobacco, spinach and snapdragon, editing creates an AUG start codon from a cryptic ACG start (editing site I) in the ndhD-encoded primary transcript (Neckermann et al., 1994). However, in monocots such as barley, rice and maize this position is corrected with the appropriate T at the genome level (Del Campo et al., 1998, 2000). This then assumed a distinction between dicots and monocots with respect to the restoration by editing of the start codon of the ndhD transcript. To test the limits of this assumption, the start codon sequence has been investigated in some monocot species different from the aforementioned grasses and, for comparison, in Arabidopsis thaliana. The restoration of a start codon by editing is reported here together with the finding of a new internal editing site in the ndhD transcripts of Liliaceae and Aloaceae.
The ndhD gene is at the 3' end of the ndhH-D operon (Martínez et al., 1997; Del Campo et al., 2000). This includes the ndh genes (in this order) H, A, I, G, E, and D and (surprisingly) the psaC gene (encoding the PsaC protein of photosystem I) that maps between ndhE and ndhD.
Both strands of around 350 nucleotide fragments were sequenced in an Applied Biosystem automatic sequencer by using PCR amplification products from plastid DNA of the monocots onion (Allium cepa, accession no. AJ278350), leek (Allium porrum, accession no. AJ278352), garlic (Allium sativum, accession no. AJ278351), Aloe (Aloe vera, accession no. AJ278353) and wild barley (Hordeum murinum, accession no. AJ278355) and of the dicot Arabidopsis thaliana (accession no. AJ278354). The fragments span 58 nucleotides of the 3' end of psaC gene, intergenic region of different length from one to other plant and around 185 nucleotides of the 5' end of ndhD gene. The primers (external to the fragment) were derived from the sequence of barley (Del Campo et al., 2000, accession no. AJ011848).
Except for wild barley, all species showed an ACG sequence at the start codon site as that reported in the dicots tobacco, spinach and snapdragon (Neckerman et al., 1994). Thus, it appears that Liliaceae and the closely related Aloe require C to U editing of the primary ndhD transcript to restore the start AUG codon. Wild barley, as other grasses (Del Campo et al., 1998), showed the appropriate ATG sequence at the start codon, while Arabidopsis showed the dicot ACG cryptic start sequence. It is usually assumed that editing arose to correct, at transcript level, inappropriate G-C pairs that appeared in the genome sequence of an old ancestor. Mutations in species derived from that ancestor have corrected at the genome level some of those G-C pairs making editing unnecessary (Freyer et al., 1995). The mutation that restored the appropriate ATG in plastid DNA did not affect all monocots, Liliaceae and Aloaceae being more closely related in this aspect to dicots than to other monocots. A comparison of the psaC–ndhD intergenic sequences reported here with those known of rice, maize, barley, tobacco, and spinach also indicates a greater closeness of the Liliaceae and Aloaceae with dicots (specially tobacco) than with grasses. These facts suggest that, in grasses, the correction at the level of the genome of the cryptic start codon of ndhD took place after the separation of the Liliaceae and Aloaceae from within the monocot main branch.
cDNA sequences corresponding to the determined genomic sequences were also investigated in Allium species, Aloe and Arabidopsis to look for edited transcripts. Transcripts of Arabidopsis, Aloe and garlic showed complete edition of the cryptic start codon to AUG. The onion transcript showed partial edition, as described for many dicots (Neckermann et al., 1994), while no edition was detected in the leek transcript. Both partial and no edition can arise by heterogeneity of the transcripts of the ndhH-D operon. Probably, there are many transcripts of different sizes including mRNA of ndhD, some of them edited and others non-edited. Frequently, identification of the edited transcripts requires transcript separation and/or the use of appropriate primer pairs for amplification (Del Campo et al., 2000). The variable extension of the start codon correction by editing in closely related plants suggests that the relative abundance of edited and unedited transcripts codons could vary depending on the physiological stage of the plant.
When comparing genomic with complementary DNA sequences a new editing site was identified (site II). The 59th nucleotide, which is C in the genomic sequence of Liliaceae and Aloaceae, is edited to U (T in the sequence) in the transcripts of these plants. This corresponds to the second nucleotide of the 20th codon. As Fig. 1 shows, in Zea mays, Hordeum vulgare, Hordeum murinum, Oriza sativa, Nicotiana tabaccum, Spinacia oleracea, and Arabidopsis thaliana genomic DNA this codon is TTA encoding a conserved Leu. However, in Allium cepa, Allium porrum, Allium sativa, and Aloe vera the corresponding genomic sequence is TCA which would code Ser. Editing of the 20th codon to TTA (UUA in mRNA) allows the synthesis of NDH-D polypeptide in Allium and Aloe with the conserved Leu at the 20th position. It is noteworthy that transcripts of Allium cepa and Allium porrum have completely edited site II while site I may remain unedited. This indicates a complex editing-processing step of ndhD-containing transcripts in Liliaceae as found for ndhA-containing transcripts in barley (Del Campo et al., 2000).
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It is noteworthy that Liliaceae are closer to dicots than to grasses with respect to editing site I, while grasses are closer to dicots than to Liliaceae with respect to site II (Fig. 1
). A similar divergence of closely related plants was found when comparing the editing sites of ndhB (Freyer et al., 1995). These facts probably indicate that the emergence and genomic correction of editing sites in plastids have taken place recently and within the same time scale.
It is probable that editing sites in plastid transcripts may be more frequent than described and extensive comparison of genomic and complementary DNA sequences are required to discover them (Freyer et al., 1997). This may be important to identify the conserved amino acids in the encoded protein and the editing that is probably required for regulatory functions such as that found in the untranslated region of RNA (Kudla and Bock, 1999).
Acknowledgments
This work has been supported by CAM, Comunidad Autónoma de Madrid, (Grant 07B/0011/1997). M López-Serrano hold a postdoctoral fellowship from Fundación Séneca-Centro de Coordinación de la Investigación, Murcia (Spain).
Notes
1 To whom correspondence should be addressed. Fax: +34 91 8855066. E-mail: bvmmm{at}bioveg.alcala.es 
References
Casano LM, Zapata JM, Martín M, Sabater B.2000. Chlororespiration and poising of cyclic electron transport: plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase. Journal of Biological Chemistry275, 942–948.
Del Campo EM, Albertazzi F, Freyer R, Maier M, Sabater B, Martín M.1998. Sequence and transcript editing of ndhB gene of Arabidopsis thaliana L. plastid (Accesion Nos AJ002490 and AJ002491) (PGR98-093). Plant Physiology117, 117–118.
Del Campo EM, Sabater B, Martín M.2000. Transcripts of the ndhH-D operon of barley plastids: possible role of unedited site III in splicing of the ndhA intron. Nucleic Acids Research28, 1092–1098.
Freyer R, Kiefer-Meyer M-C, Kössel H.1997. Occurrence of plastid RNA editing in all major lineages of land plants. Proceedings of the National Academy of Sciences, USA94, 6285–6290.
Freyer R, López C, Maier RM, Martín M, Sabater B, Kössel H.1995. Editing of the chloroplast ndhB encoded transcript shows divergence between closely related members of the grass family (Poaceae). Plant Molecular Biology29, 679–684.[Web of Science][Medline]
Kudla J, Bock R.1999. RNA editing in an untranslated region of the Ginkgo chloroplast genome. Gene234, 81–86.[Web of Science][Medline]
Maier RM, Zeltz P, Kössel H, Bonnard G, Gualberto JM, Grienenberger JM.1996. RNA editing in plant mitochondria and chloroplasts. Plant Molecular Biology32, 343–365.[Web of Science][Medline]
Martín M, Casano LM, Sabater B.1996. Identification of the product of ndhA gene as a thylakoid protein synthesized in response to photooxidatve treatment. Plant and Cell Physiology37, 293–298.
Martínez P, López C, Roldán M, Sabater B, Matín M.1997. Plastid DNA of five ecotypes of Arabidopsis thaliana: sequence of ndhG gene and maternal inheritance. Plant Science123, 113–122.
Neckermann K, Zeltz P, Igloi G, Kössel H, Maier R.1994. The role of RNA editing in conservation of start codons in chloroplast genomes. Gene146, 177–182.[Web of Science][Medline]
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