American Journal of Plant Sciences, 2011, 2, 535-538
doi:10.4236/ajps.2011.24063 Published Online October 2011 (http://www.SciRP.org/journal/ajps)
535
The Post-Transcriptional mRNA Editing Analysis
of cox3 Mitochondrial Gene in Fern Asplenium
nidus Reveals Important Features
Sebastian Panarese, Guglielmo Rainaldi
Department of Biochemistry and Molecular Biology, University of BARI “Aldo Moro”, Bari, Italy.
Received July 18th, 2011; revised August 20th, 2011; accepted September 5th, 2011.
ABSTRACT
In the mitochondria and chloroplasts of flowering plants (angiosperms), transcripts of protein-coding genes are altered
after synthesis so that their final primary nucleotide sequence differs from that of the corresponding DNA sequence.
This posttranscriptional mRNA editing consists almost exclusively of C-to-U substitutions (direct) and less frequently of
U-to-C substitution (reverse). Editing occurs predominantly within coding regions, mostly at isolated C residues, and
usually at first or second positions of codons, thereby almost always changing the amino acid fro m that specified by the
unedited codon. Editing may also create initiation and termination codons. The effect of C-to-U RNA editing in plants is
to make proteins encoded by plant organelles more similar in sequence to their non plant homologs, then specific C-to-
U editing events are essential for the production of functional plant mitochondrial proteins. Our attention has been
devoted to the study of the mRNA editing in cox3 mitochondrial gene of fern Asplenium nidus. This study reveals the
extreme importance of both C-to -U and U-to-C substitutions for protein expression .
Keywords: mt DNA, Plant, Monilophytes, Aspleniu m ni d us
1. Introduction
Mitochondrial genomes of land plants have been fully
sequenced and characterized in several species belonging
to the Briophytes (Marchantia polymorpha [1] and Phy-
scomitrella patens [2]) and Spermatophytes (Arabidopsis
thaliana [3], Beta vulga ris [4], Oryza sativa [5], Brassica
napus [6], Zea mays [7], Nicotiana tabacum [8] and
Triticum aestivum [9]).
The comparison of organization, structure and expres-
sion between Spermatophyte mitochondrial genomes
reveals several homogeneous features which can be sum-
marized as follows: i) the presence of repeated sequences,
ii) a heterogeneous structure, iii) the presence of DNA
segments of extra mitochondrial origin (mainly chloro-
plastic) carrying in some cases active genes (usually for
tRNAs) [10-12], iv) the editing of transcription products
of structural genes, v) an incomplete set of tRNA genes.
To gain more knowledge on the mitochondrial bio-
genesis of Monilophytes, in particular respect to editing
process, we chose plants of a filicales family, the fern
Asplenium nidus, available at the Botanical Garden of the
University of Bari.
In our previous investigations [13] we verified high
level of editing process in filicales with to respect Sper-
matophytes plants. In our system, the editing analysis of
cox3 mitochondrial gene confirms this hypothesis.
2. Materials and Methods
2.1. Sources of Mitochondrial DNA
Two alternative procedures for the isolation of organelles
have been developed depending on the tissue used as
starting material: roots or leaves. In the former the soil
contained in the thick network of roots was removed by
hand and washed in distilled water. This step was fol-
lowed by drying the roots on filter paper and weighing
and wrapping them with a double layer of sterile gauze.
After washing several times with sterile water, the roots
were suspended in sterile buffer (Mannitol 0,4 M, Mops
25 mM, EGTA 1 mM, PVP 1% pH 7,8) and homogeni-
zed in a blender with five hits every five seconds at me-
dium speed.
Fractionation, lysis of organelles and DNA extraction
were as reported by Hanson [14].
The isolation of organelles and DNA from green

The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene
536 in Fern Asplenium nidus Reveals Important Features
leaves (leaf-procedure) was carried out using the same
protocol with few modifications [14].
The mitochondrial DNA isolated by the two alterna-
tive procedures showed significant different levels of
chloroplast contamination, as judged from the PCR am-
plification of highly conserved chloroplast regions [15].
Using as a template the mtDNA prepared from the
roots, where the copy number of plastid DNA are re-
duced [16], no amplified products could be detected. The
results of these experiments are reported in Figure 1.
The leaf procedure was used mainly for the isolation
of total RNA employed for reverse transcription and
cDNA synthesis and for the investigation concerning
editing of co x 3 gene transcripts.
2.2. Total RNA Extraction from A. Nidus
RNeasy Plant Mini Kit (Qiagen) was used for the purifi-
cation of total RNA from fresh or frozen plant tissues.
An accurate photometric detection of starting material
was essential in order to obtain optimal RNA yield and
purity. A maximum amount of 100 mg plant material
was generally processed.
2.3. RT-PCR
RT-PCR experiments were carried out in a single step by
means of SuperScript One-Step RT-PCR with Platinum
Taq. Components for both cDNA synthesis and PCR
1 2 3 4 5 6 7 8
Figure 1. Multiple test of amplification performed on nu-
cleic acids extracted with the two different procedures. The
panels show two groups of three amplifications carried out
with the same pairs of universal primers (BA48557-A49291,
BA49317-A49855, BA49873-A50272, [14] for the detection
of cpDNA in the nucleic acid fractions isolated by the two
procedures. Lanes 1-3: template isolated by the leaf proce-
dure. 5-7: template (arrows) isolated by the root procedure.
4 and 8: 1 Kb DNA ladder (3000, 2000, 1500, 1200, 1031,
900, 800, 700, 600, 500, 400, 300, 200, 100 bp).
were combined in a single tube, using gene-specific
primers and target RNAs from total RNA. Reverse tran-
scription automatically followed PCR cycling without
additional steps. The system consisted of two major
components: RT/Platinum Taq Mix and 2X Reaction
Mix where the RT/Platinum Taq Mix contained a mix-
ture of SuperScript II Reverse Transcriptase and Plati-
num Taq DNA Polymerase for optimal cDNA synthesis
and PCR amplification respectively.
The following cycling conditions were established:
cDNA synthesis pre-denaturation (1X) at 50˚C for 15-30’;
94˚C for 2’; PCR amplification (40X) at 94˚C for 20”;
50˚C - 60˚C for 30”; 68˚C - 72˚C for 1’/kb; final exten-
sion (1X) at 72˚C for 7’.
2.4. DNA Cloning in Ta Vectors
The cloning of dsDNA fragments was carried out using
pGEM-T Easy Vector Systems and following the manu-
facturer instructions.
2.5. DNA Sequencing and Determination of
Editing Rates
Sequencing of individual cDNA of 23 clones was per-
formed automatically with the Big Dye1 Terminator Cy-
cle Sequencing Kit (Applied Biosyste ms).
ClustalW program was used to determine editing sites,
aligning DNA and cDNA sequences.
Sequence of cox3 gene were deposited in GeneBank
database (accession number: FR669448).
3. Results and Discussion
3.1. Analysis of Editing Sites on Cox3
Transcripts
The Figure 2 shows the comparison of mitochondrial
sequence of cox3 gene with the corresponding cDNA.
Table 1 shows editing data about some cox3 mitochon-
drial genes of diffe rent plant species.
The analysis of results obtained in this investigation
shows some specific features for the A. nidus mitochon-
Table 1. Comparison of editing value in cox3 mitochondrial
transcripts betw een some land plants.
Editing C-UEditing U-C Site Tot % Gene bp
P. patens 1 1 0.1798
B. vulgaris 4 4 0.5798
B. napus 7 7 0.9798
A. thaliana 8 8 1.0798
T. aestivum 12 1 13 1.6798
O. sativa 1 1 0.1843
A. nidus 19 13 32 4.0798

The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene 537
in Fern Asplenium nidus Reveals Important Features
Figure 2. Alignment of genome sequence (upper) with
cDNA counterpart of cox3 mitochondrial gene of A. nidus.
Nucleotides underlined correspond to sequencing primers.
drial cox 3 gene:
1) for the transcript of the gene, direct editing events
(19 C-U; 2,3%) and reverse editing events (13 U-C; 1,7%)
are almost equivalent;
2) the editing events have a high relevance: they sup-
press four stop codons within the cox3 gene;
3) 25 amino acids changes and 4 suppressions of stop
codons (3 TAA-1 TAG) are found. These stop codons
are corrected by U to C editing to sense codons of the
amino acid Q (Figure 3);
4) in particularly critical positions, editing events of
both types generate transcripts which can be translated as
functional proteins;
5) the initial codon of cox3 is an ACG codon wh ich is
Figure 3. Amino acids changes after editing events in A.
nidus cox3 mitochondrial transcript.
not changed to AUG by a direct editing event.
This observation can be considered in agreement with
the finding of Dong F. G. [17] who found that the radish
(Raphanus sativus L.) mitochondrial cox2 gene contains
an ACG at the predicted translation initiation site which
is not converted to AUG codon in the mRNA, although
15 other RNA editing sites were identified. So our find-
ing confirms that in plant mitochondria ACG codon can
be utilized as initiation codon;
6) cox3 editing events have a higher frequency on A.
nidus transcripts compared with the same genes studied
on other angiosperm genomes (Table 1);
7) reverse editing events (U-C) are considerably more
frequent in cox3 transcripts of A. nidus than in other
plant species, where they are nearly absent (Table 1);
8) as shown in Figure 4, for 17 codons changes occur
in first position, for 13 codons in second positio n and for
only one codon in third position.
Figure 4. Distribution of editing events across codon posi-
tions.

The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene
in Fern Asplenium nidus Reveals Important Features
538
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