Molecular Biology Reports

https://doi.org/10.1007/s11033-021-06609-1

ORIGINAL ARTICLE

The first report of RNA U to C or G editing in the mitochondrial NADH

dehydrogenase subunit 5 (Nad5) transcript of wild barley
Ahmed M. Ramadan1,2   · Afnan A. Alnufaei1 · Thana K. Khan1 · Hani M. Ali1 · Hala F. Eissa2,3 · Sabah M. Hassan1,4

Received: 13 April 2021 / Accepted: 29 July 2021

© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Background  Nad dehydrogenase complex in mtDNA has a significant role in cellular respiration. One of the largest subunits
in the complex is subunit 5 (Nad5).
Methods and results  Four cDNAs of the Hordeum vulgare subsp. spontaneum nad5 gene have been characterized and sub-
jected to four phases of 0.5 M salinity, at 0 h (control, accession no. MT235236), after 2 h (acc. no. MT235237), after 12 h
(acc. no. MT235238) and after 24 h (acc. no. MT235239). Utilizing raw data from RNA-seq, ten RNA editing sites were
reported. Seven sites have common editing from C to U in positions (C1490, C1859, C1895, C1900, C1901, C1916, C1918).
A rare editing event U to C was detected in two positions (U1650 and U1652) and a novel editing event U to G was for the
first time in positions nad5-U231. The highest editing level was shown in 2 and 12 h after salinity exposure. After 24 h,
these edits were disrupted, possibly due to the launch of the programed cell death mechanism. However, the RNA editing
in positions U1650, U1652 and U231 was fixed at all exposure times.
Conclusions  Although study clarified the role of salinity stress in nad5 RNA editing sites, the main achievements are first
report of U to G RNA editing in plants at position U231 and first report of U to C editing in the nad5 gene at U1650 and
U1652.

Keywords  RNA editing · Novel type · Nad5 · Wild barley

Introduction widespread form of RNA editing in animals, which by the

RNA editing is considered as a sort of post-transcriptional
modification, which includes deletions, insertions, and
nucleotide alterations. RNA splicing, capping and poly-
adenylation are not included [1, 2]. This mechanism was
discovered for the first time in Trypanosoma mitochondria
[3]. DNA modification of Adenosine (A) to inosine (I) is the
* Ahmed M. Ramadan
ahmedramadan782@yahoo.com; aamara@kau.edu.sa
1
Department of Biological Sciences, Faculty of Science, King
Abdulaziz University (KAU), PO Box 80141, Jeddah 21589,
Saudi Arabia
2
Department of Plant Molecular Biology, Agricultural Genetic
Engineering Research Institute (AGERI), Agriculture
Research Center (ARC), Giza, Egypt
3
College of Biotechnology, Misr University for Science
and Technology (MUST), 6th October City, Egypt
4
Department of Genetics, Faculty of Agriculture, Ain Shams
University, Cairo, Egypt
translation machinery is interpreted as guanosine (G) of the
subsequent RNA, and such a process is attained by deami-
nase activity [1]. Another type of this mechanism, which
was found in the apolipoprotein-B gene and obtained by the
cytidin deaminase (apobec/ACF complex) editosome, is a
cytidine-to-uridine (C-to-U) modification [4]. RNA editing
in the plant kingdom is common in plastid and mitochondrial
transcripts, where editing of cytidine to uridine nucleotide is
observed in transcripts and results from the action of nuclear
pentatricopeptide repeat (PPR) proteins [5, 6]. Arabidopsis
thaliana was reported to have 34 chloroplast sites [7] and
619 in mitochondrial [8]. RNA editing includes non-coding
regions like introns, tRNAs and microRNAs as well as cod-
ing sequences [9, 10]. In the coding sequence, most cases of
RNA editing with combined biological functions take place
in either the first or second position of codons [11]. Many
factors affect RNA editing, such as developmental stages,
tissue types, environmental factors, and ecotypes [8, 12].
In several studies, RNA editing of terrestrial mitochondrial
plant transcripts, including Cytb, NadI, CoxII and CoxIII, has

13
Vol.:(0123456789)

been investigated [13]. RNA editing in the nad5 transcript
involves cytidine modification to uridines [14]. A reverse
change was identified in the genes of rps1, nad5, atp1 and
rpl2, which in some ferns alters genomic T to RNA C [15].
The initiating enzyme of the transfer electron chain is
Complex I, which includes the NADH dehydrogenase subu-
nit 5 (nad5). The critical role of this subunit is shown in
the connection between RNA editing in the Nad gene and
tolerance of abiotic stress [14, 16]. Next-generation sequenc-
ing (NGS) offers a speedy and cost-efficient tool for finding
organelle RNA editing sites [8]. In order to reliably RNA
editing detection in tissues or low-level editing, NGS tech-
nology is used to achieve these research objectives. In con-
trast, NGS and bioinformatics tools have helped scientists in
discovering several new Arabidopsis editing sites [7, 8, 17].
In the current study, RNA-seq data from NGS Illumina
2000 has been used to explore the editing sites of the wild
barley nad5 gene as well as the effect of salinity on editing
behavior in this gene.
Materials and methods
RNA sequencing data
The RNA-seq data of Hordeum vulgare subsp. spontaneum
leaves was collected from the NCBI. All samples in this
%RNA editing = 2(Ct mean of T variant−Ct mean of C variant)∕{2(Ct mean of T variant−Ct mean of C variant) + 1} × 100
data were subjected to 500 mM NaCl as a salinity treatment
for different periods of exposure. The data was used as a
control (SRR1028012 and SRR1028011), for a 2-h expo-
sure (SRR1055527, SRR1049592 and SRR1049587), 12-h
exposure (SRR1049626, SRR1049609 and SRR1049570),
and a 24-h exposure (SRR1049655, SRR1049648 and
SRR1049643) [18].
Analysis of RNA editing
CLC Genomic Workbench 3.6.5 (CLC Bio, Qiagen, Den-
mark) was used for the determination of RNA editing sites
as modified by Ishii et al. [19]. Mapping parameters were
changed for the purpose of the elimination of undesirable
reads, with a similarity of 0.98 and a length fraction of 0.98.
Mitochondrial reads of the nad5 gene of Hordeum vulgare
subsp. spontaneum were mapped (Accession no. AP017300)
as the template [20]. The lowest fraction of similarity was
80%, while the lowest length of the fraction read was 50%.
The framework of nucleotide editing is a 5% low-frequency
variant, with minimum coverage set to 20, minimum count
13
Molecular Biology Reports
set to 4 and minimum frequency set to 5%. Later, the RNA
editing sites, total read counts and coverage depth were iden-
tified. The frequency of each conversion site between all
salt exposure periods was estimated according to the reads
number for altered nucleotides divided by total reads [21].
Deduced amino acids analysis of Nad5 gene
CLC genomic workbench 3.6.5 tool was used for RNA edit-
ing sites identification by analyzing the genomic nad5 gene
and cDNAs of H. vulgare subsp. spontaneum. Also, predict-
ing the changes in secondary and 3D structure proteins has
been done by the same program.
Validation of RNA editing sites
Validation of predicted editing sites from RNA-seq data was
done using the leaves of each salinity treatment (in biologi-
cal triplicates) according to Bahieldin et al. [18]. In all sam-
ples, RNA extraction was done by Qiazol (Qiagen, Cat No.
79306). All reactions of cDNA synthesis include 1 μg of
total RNA, and 1 μM poly dT oligonucleotide for each one
(Biolegio, Nijmegen, Netherlands). The system which was
applied to achieve the qRT-PCR reaction was the Mx3005P
qPCR system (Stratagene) according to [22] using primers
designed by PRIMER 3 (Koressaar et al. 2018) (Table 1).
For normalization purpose, α‐tubulin gene was used [23].
Validation of T sites (position 231, 1650 and 1859)
on DNA levels
To trust the editing sites of the previous DNA sequence of
nad5 [20] specially in U editing sites, DNA is ectracted from
wild barley leaves using DNeasy Plant Pro Kit and regular
PCR is done using REDTaq® ReadyMix™ PCR with for-
ward primers (including edited and original nucleotide) for
positions 231, 1650 and 1859 (Table 1), and reverse (AAA​
ACCG ​ GCT ​ ACG
​ GAA ​ CTA ​ CC) for position 231, (GGAA ​ TT​
GGC​CCA​AAA​ATT​GG) for position 1650 and (GCT​CGT​
TGG​AAT​TGA​TCC​GC) for position 1859. PCR conditions
were denaturation at 94 °C for 4 min, 35 cycles of 94 °C/
min, 60 °C/30 s and 72 °C for /min, and 72 °C for 5 min.
Statistical analysis
The analysis of variance (ANOVA) test was used for ana-
lyzing data by SPSS program. Using Tukey’s HSD, several
comparisons were made [24].
Molecular Biology Reports

Table 1  Primers were designed Position F 5′ → 3′ R 5′ → 3′

as two forward specific primers
with different 3′ end (original
and substitute nucleotide) and
one reverse primer site except
for positions 1916 and 1918,
two specific reverse and one
forward
231 ATG​CTT​CTT​GGG​GCT​TCT​TG
ATG​CTT​CTT​GGG​GCT​TCT​TT
1490 CAA​TTT​TTG​GGC​CAA​TTC​CCT
CAA​TTT​TTG​GGC​CAA​TTC​CCC​
1650 GAG​CCT​TTC​AAA​CTA​GTA​CC
GAG​CCT​TTC​AAA​CTA​GTA​CT
1652 GCC​TTT​CAA​ACT​AGT​ACT​TC
GCC​TTT​CAA​ACT​AGT​ACT​TT
1859 AGT​CAA​CTT​CAA​AGT​GGA​TCT
AGT​CAA​CTT​CAA​AGT​GGA​TC
1895 TTT​GCA​ATG​TTA​CTT​GGT​TT
TTT​GCA​ATG​TTA​CTT​GGT​TC
1900, 1901 ATG​TTA​CTT​GGT​TCA​ACT​TT
ATG​TTA​CTT​GGT​TCA​ACT​CC
1916 TCG​GAT​ATT​CAG​TCT​CAT​TC
1918 TCG​GAT​ATT​CAG​TCT​CAT​TC
α‐Tubulin TCC​ATG​ATG​GCC​AAG​TGT​GA
GTA​AAA​ATG​GAT​AAA​TAA​CACA​
GTA​CTA​GTT​TGA​AAG​GCT​C
GTG​TAC​GAG​ATA​CCA​TAA​GG
GTG​TAC​GAG​ATA​CCA​TAA​GG
TCA​CTA​ACA​AAA​TGA​AAG​AC
TCA​CTA​ACA​AAA​TGA​AAG​AC
CTC​TTG​ACT​TGA​CTT​ATT​AA
AGA​GAG​TCC​CAC​ATA​CGA​A
AGA​GAG​TCC​CAC​ATA​CGA​G
ATA​GAG​AGT​CCC​ACA​TAC​A
ATA​GAG​AGT​CCC​ACA​TAC​G
GAC​ATC​CCC​ACG​GTA​CAT​GAG​

Results and discussion Conserved domain analysis

Identification of wild barley Nad5 CDS At the protein level, the current impact of RNA editing on
the function of NADH subunit 5 (nad 5) Domain analysis
Hordeum vulgare subsp. spontaneum genomic and cDNA and CDD accession number CL0425 (Fig. S2) must be
for the nad5 gene were recovered for control (acc. no. measured.
MT235236), after 2 h (acc. no. MT235237), after 12 h (acc.
no. MT235238) and after 24  h (acc. no. MT235239) of Secondary and 3D structure of Nad5 protein
salt stress. To identify nad5 gene transcripts, 105,526,154
pair-end RNA reads were used at 0  h (control), while Secondary structure protein revealed changes in quantity and
159,631,812 for 2 h, 161,793,598 for 12 h and 159,631,812 length of alpha helix and beta sheets (Fig. S3). However, no
for 24 h. Nad5 gene (acc. no. AP017300) reference was used changes were observed in 3D structure (Fig. 4).
for mapping nad5 transcripts.
Nad5 gene editing validation
RNA editing and amino acid modifications To confirm editing sites and clarity on the value of the RNA-
seq tool in characterization, the editing sites were validated
Through comparing the nad5 genomic sequence and the
by RT-qPCR. Nad5 editing positions (U231, U1650, U1652,
sequence of the four treatments cDNA (Fig. S1), C to U
C1490, C1859, C1895, C1900, C1901, C1916, and C1918)
editing was shown in seven sites (C1490, C1859, C1895,
were quantified and measured at four exposure intervals
C1900, C1901, C1916, C1918), two U to C (U1650 and
(Fig. 2). Also, positions (U231, U1650, U1652) were con-
U1652) and one U to G (U231). All of them are conserved
firmed on DNA level using original and edited nucleoide
through 2- and 12-h treatments. However, RNA editing
in 3′ end to ensure that the nucleotides are correct on DNA
was significantly recorded in the control in just three sites
level as well as published sequence [20] (Fig. 3).
(U1650, U1652 and U231) and in four sites with 24-h treat-
ment (C1490, U1650, U1652 and U231). The editing ratio
is salinity and exposure time dependent (Fig. 1, Table 2).
Moreover, two editing sites in the same codon were found
(C1900 and C1901).

13

120
** **
100 **
**
80
RNA eding %
60
40
20
0
UUU(F)-UUG CCC (P)-CCU ACU (T)-AUC
(L) , U231 (T) C1490 (T) U1650
Fig. 1  Differential and efficiency of nad5 RNA editing, compar-
ing with control, using reads derived from total RNA-seq. (C or U)
nucleotide position, (F) Phenylalanine, (W) Tryptophan, (P) Proline,
Discussion
RNA editing process was discovered in the majority of
organisms, mainly in mitochondria [5, 8] and chloroplasts
[7, 25]. This process has a significant role in the expres-
sion of functional proteins. The connection between abiotic
stress and RNA editing has been reported in many stud-
ies [8, 16]. Complex I (NADH dehydrogenase) subunits,
including nad5 subunit, are involved in electron transport
chain in mitochondria. Disruption in RNA editing of com-
plex I subunits increases the concentrations of free oxygen
(ROS), which contributes to increased sensitivity to drought
stress in Arabidopsis [16]. Depending on the species, RNA
editing that occurs in the nad5 gene may differ, with 31
editing sites in Arabidopsis, 44 sites in onion plant, 30 in
Cucumber, 33 in wild carrot, 33 in Soybean, and 29 in sun-
flower [11]. Before studying RNA editing by bioinformatics
tools, we should exclude any effect of heteroplasmy [26]
on nucleotide change, which may confuse the RNA editing
analysis (Table. 2). In the current study, 10 RNA editing
sites were reported (nucleotide nos. C1490, C1859, C1895,
C1900, C1901, C1916, C1918, U231, U1650 and U1652)
in Hordeum vulgare subsp. spontaneum. All editing sites
were found in different ratios (except for U231, U1650 and
U1652) between all four exposure times with high frequency
(Fig. 1 and Table 2). All editing sites were validated with
real-time PCR (Fig. 2).
Several observations were reported in the current study.
Unexpectedly, the U to G editing type in position U231 was
observed at all exposure time at the same ratio. To exclude
any sequencing errors, we reconfirmed T nucleotide in
13
Molecular Biology Reports
control 2H 12H 24H
** ** ** ** ** ** ** ** ** **
**
UUU (F)- UCU (S)- UCA (S)-UUA CCA (P)-UUA UCU (S)- CGU (S)-UGU
UCU(S) UUU (F) (L) C1895 (L) C1900, UUU (F) (L) C1918
U1652 C1859 1901 C1916
Codon cha nge & nucleode
(T) Threonine, (S) Serine, (L) Leucine. Data are expressed as means
with ± SD (black bars) of three biological replicates. **Indicate sig-
nificant difference between treatments P < 0.01
nad5-231on DNA level by PCR (Fig. 3). This phenomenon
was reported here for the first time in plants. This editing
type raises several questions, the most important of which
are how uracil is chemically converted into guanine and
what the enzymes are involved in this process. Also, when
comparing nad5-U231 with most plants nad5 (Fig. S4), we
observed that this site is G231 in all plants except H. vul-
gare and Triticum aestivum. This indicates that this type of
RNA editing may aim to restore the conserved protein, in
other words, this indicates that RNA editing in U231 came
as a result of evolution to correct a mutation that may have
occurred in the past. Furthermore, the fixed ratio of U231 as
well as U1650 and U1652 across all exposure time indicates
the vital function of this editing in this gene.
The second observation is the first report of RNA edit-
ing from U to C in the nad5 gene. This type of editing was
reported before in coxI and cob genes [27] but not reported
in nad5. In the third observation, two editing sites are
involved in one codon (C1900 and C1901), but many reports
have previously investigated this phenomenon [11, 28, 29].
Still, there is no amino acid modification, even though RNA
editing occurs in U1650 (threonine). However, this observa-
tion was examined in previous studies [11, 28], and there
was no explanation for why RNA editing is vital in plants
devoid of any amino acid modification. However, it is sug-
gested that under stress, the tRNA becomes incapable of
supplying an additional amount of the amino acid from this
codon. So, as an instant act, the cell must alter some sites
by editing to call additional tRNA to deliver more of a par-
ticular amino acid [30].
 Molecular Biology Reports

Table 2  Nad5 editing events using reads derived from total RNA-seq. as well as DNA- seq (to exclude heteroplasmy)
Position Codon Heteroplasmy Control 2H 12 h 24 h
change
Total Edited  ~ % Edit- Total Edited  ~ % Edit- Total Edited  ~ % Edit- Total Edited  ~ % Edit- Total Edited  ~ % Edit-
coverage cover- ing coverage coverage ing coverage coverage ing coverage coverage ing coverage coverage ing
age

U231 UUU (F)- 200 0 0 425 425 1900-04- 326 326 100 230 230 100 253 253 100
UGG(W) 09
C1490 CCC (P)- 250 0 0 500 165 33 321 321 100 213 213 100 293 266 91
CUC(L)
U1650 ACU (T)- 233 0 0 460 322 70 425 227 70 290 203 70 265 159 60
ACC(T)
U1652 UUU (F)- 213 0 0 523 366 70 450 450 100 300 210 70 267 160 60
UCU(S)
C1859 UCU (S)- 210 0 0 486 0 0 466 450 100 258 258 100 284 85 30
UUU(F)
C1895 UCA (S)- 200 0 0 635 0 0 411 411 100 262 262 100 259 0 0
UUA(L)
1900 CCA (P)- 260 0 0 526 0 0 361 361 100 265 265 100 321 0 0
UCA(L)
C1901 CCA (P)- 260 0 0 525 0 0 301 301 100 267 267 100 315 0 0
CUA (L)
C1916 UCU (S)- 235 0 0 592 0 0 305 305 100 283 283 100 295 0 0
UUU (F)
C1918 CGU (R)- 235 0 0 498 0 0 354 354 100 291 291 100 264 0 0
UGU (C)

13
 Molecular Biology Reports

120
control 2H 12H 24H
**
** ** **
100
** ** ** ** ** ** **
**
80
RNA EDITING %

60

40
**
20

0
U231 C1490 U1650 U1652 C1859 C1895 C1900, C1916 C1918
1901
EDITING SITES

Fig. 2  qRT-PCR Confirmation of H. vulgare subsp. spontaneum salinity exposure, 24 h after salinity exposure). Data are expressed as
editing sites predicted by CLC genomic workbench in different four means with ± SD (black bars) of three biological replicates. **Indi-
times of salinity stress (control, 2 h after salinity exposure, 12 h after cate significant difference between treatments P < 0.01

Fig. 3  PCR analysis for nad5 M 1 2 3 4 5 6

DNA in positions 231, 1650
and 1652. (M) 100 bp ladder
bioron™. (1, 4) nad5-1650
using original and alternate
nucleotide in 3′ forward
primers, respectively. (2, 5)
nad5-1652 using original
and alternate nucleotide in 3′
forward primers, respectively.
(3, 6) nad5-231 using original
and alternate nucleotide in 3′
forward primers, respective

200 bp

Two unusual phenomena were observed here. Changes in
quantity and length of alpha sheets and beta helix of protein
secondary structure depend on RNA editing (Fig. 4) without
any significant effect on the 3D structure of protein, although
the change in 3D structure depends on RNA editing and was
reported [30].
These findings differ from the majority of studies which
reported the high editing site frequency in the nad5 gene
under normal conditions, like in Arabidopsis, Allium cepa,
Cucumis sativus, and Helianthus annus [11]. This obser-
vation indicates that some wild cereal plants have unusual
respiratory needs in comparison to cultured plants, which
signifies their productivity and shows most energy is gen-
erated to cope with the stress induced by the environment
more than by any other traits.
Most of the editing sites ratio has risen after 2 and 12 h
after salinity exposure (Table 2). A rise in RNA editing is
known to be essential for plant potency with acclimation
needs to environmental stress [31]. This truth confirms our
outcomes and indicates that RNA editing has a significant
say in respiration performance in the cell to provide plants
with sufficient molecules of ATP to deal with salt stress.
However, the downfall in RNA editing frequency after 24 h
of salt exposure indicates that the plant has lost tolerance
due to the extreme salinity and may initiate programmed cell
death. Many studies in plants have supported this assump-
tion [32–36], yeasts [37] and algae [38].

13
Molecular Biology Reports
Fig. 4  Predicted 3D nad5 protein at different time salt exposure as well as non-edited protein. Non-edited protein (a), control (b), 2 h after salt
exposure (c), 12 h after salt exposure (d), 24 h after salt exposure (e)
Conclusion
Two achievements occurred in this study, the first report of
U to G RNA editing in plants at position U231, which needs
more study to know what the enzymes are involved in this
type of editing. Also, the first report of U to C editing in the
nad5 gene at two positions, U1650 and U1652. The study
clarified that salinity stress alters nad5 RNA editing sites
significantly. Salinity exposure for 2 and 12 h showed the
maximum editing. But these edits were interrupted after 24 h
possibly due to the start of programmed cell death.
Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11033-0​ 21-0​ 6609-1.
Acknowledgements  This project was funded by the Deanship of Sci-
entific Research (DSR) at King Abdulaziz University, Jeddah, under
Grant No. G- 91-130-1441. The authors, therefore, acknowledge and
thank DSR’s technical and financial support.
Funding  This project was funded by the Deanship of Scientific
Research (DSR) at King Abdulaziz University, Jeddah, under Grant
No. G- 91-130-1441.
Data availability  All data generated or analyzed during this study is
included in this published article [and its supplementary information
files].
Declarations 
Conflict of interest  The authors declare that they have no competing
interests.
Informed consent  All the authors have consent for publication.
Research involving human and/or animal participants  This article does
not contain any studies with human participants or animals performed
by the authors.
References
1. Nishikura K (2006) Editor meets silencer: crosstalk between
RNA editing and RNA interference. Nat Rev Mol Cell Biol
7(12):919–931
2. Farajollahi S, Maas S (2010) Molecular diversity through RNA
editing: a balancing act. Trends Genet 26(5):221–230
3. Benne R, Van den Burg J, Brakenhoff JP, Sloof P, Van Boom JH,
Tromp MC (1986) Major transcript of the frame shifted coxII gene
from trypanosome mitochondria contains four nucleotides that are
not encoded in the DNA. Cell 46(6):819–826
13

4. Maris C, Masse J, Chester A, Navaratnam N, Allain FH (2005)
Structure of the apoB mRNA stem-loop and its interaction with
the C to U editing APOBEC1 complementary factor. RNA
11(2):173–186
5. Castandet B, Araya A (2011) RNA editing in plant organelles.
Why make it easy? Biochemistry (Mosc) 76(8):924–931
6. Takenaka M, Jörg A, Burger M, Haag S (2019) RNA editing
mutants as surrogates for mitochondrial SNP mutants. Plant
Physiol Biochem 135:310–321
7. Ruwe H, Castandet B, Schmitz-Linneweber C, Stern DB (2013)
Arabidopsis chloroplast quantitative editotype. FEBS Lett
587:1429–1433
8. Bentolila S, Oh J, Hanson MR, Bukowski R (2013) Comprehen-
sive high-resolution analysis of the role of an Arabidopsis gene
family in RNA editing. PLoS Genet 9:e1003584
9. Licht K, Jantsch MF (2016) Rapid and dynamic transcriptome
regulation by RNA editing and RNA modifications. J Cell Biol
213(1):15–22
10. Picardi E, D’Erchia AM, Gallo A, Montalvo A, Pesole G (2014)
Uncovering RNA editing sites in long non-coding RNAs. Front
Bioeng Biotechnol 2:64
11. Edera AA, Gandini CL, Sanchez-Puerta MV (2018) Towards a
comprehensive picture of C-to-U RNA editing sites in angiosperm
mitochondria. Plant Mol Biol 97(3):215–231
12. Tseng CC, Lee CJ, Chung YT, Sung TY, Hsieh MH (2013)
Differential regulation of Arabidopsis plastid gene expression
and RNA editing in non-photosynthetic tissues. Plant Mol Biol
82(4–5):375–392
13. Takenaka M, Verbitskiy D, van der Merwe JA, Zehrmann A,
Brennicke A (2008) The process of RNA editing in plant mito-
chondria. Mitochondrion 8(1):35–46
14. Wang C, Chen X, Li H, Song W (2007) RNA editing analysis
of mitochondrial nad3/rps12 genes in cytoplasmic male sterility
and male-fertile cauliflower (Brassica oleracea var. botrytis) by
cDNA-SSCP. Bot Stud 48(1):13–23
15. Knie N, Grewe F, Fischer S, Knoop V (2016) Reverse U-to-C
editing exceeds C-to-U RNA editing in some ferns—a monilo-
phyte-wide comparison of chloroplast and mitochondrial RNA
editing suggests independent evolution of the two processes in
both organelles. BMC Evol Biol 16:134
16. Yuan H, Liu D (2012) Functional disruption of the pentatrico-
peptide protein SLG1affects mitochondrial RNA editing, plant
development, and responses to abiotic stresses in Arabidopsis.
Plant J 70:432–444
17. Lo Giudice C, Hernández I, Ceci LR, Pesole G, Picardi E (2019)
RNA editing in plants: a comprehensive survey of bioinformatics
tools and databases. Plant Physiol Biochem 137:53–61
18. Bahieldin A, Atef A, Sabir JS, Gadalla NO, Edris S, Alzohairy
AM, Radhwan NA, Baeshen MN, Ramadan AM, Eissa HF et al
(2015) RNA-seq analysis of the wild barley (H. spontaneum) leaf
transcriptome under salt stress. C R Biol 338(5):285–297
19. Ishii S, Suzuki S, Norden-Krichmar TM, Tenney A, Chain PS,
Scholz MB, Nealson KH, Bretschger O (2013) A novel metatran-
scriptomic approach to identify gene expression dynamics during
extracellular electron transfer. Nat Commun 4:1601
20. Hisano H, Tsujimura M, Yoshida H, Terachi T, Sato K (2016)
Mitochondrial genome sequences from wild and cultivated barley
(Hordeum vulgare). BMC Genomics 17(1):824
21. Chen TC, Liu YC, Wang X, Wu CH, Huang CH, Chang CC
(2017) Whole plastid transcriptomes reveal abundant RNA edit-
ing sites and differential editing status in Phalaenopsis aphrodite
13
Molecular Biology Reports
subsp. formosana. Bot Stud 58(1):38. https://​doi.​org/​10.​1186/​
s40529-​017-​0193-7
22. Wang M, Cui L, Feng K, Deng P, Du X, Wan F, Weining S, Nie X
(2015) Comparative analysis of Asteraceae chloroplast genomes:
structural organization, RNA editing and evolution. Plant Mol
Biol Rep 33(5):1526–1538
23. Rodrigues NF, Christoff AP, da Fonseca GC, Kulcheski FR, Mar-
gis R (2017) Unveiling chloroplast RNA editing events using next
generation small RNA sequencing data. Front Plant Sci 8:1686
24. Tukey J (1949) Comparing individual means in the analysis of
variance. Biometrics 5(2):99–114
25. Takenaka M, Zehrmann A, Verbitskiy D, Härtel B, Brennicke A
(2013) RNA editing in plants and its evolution. Annu Rev Genet
47:335–352
26. Rania MM, Areej AS, Thana KK, Hani MA, Ahmed MR (2019)
Single nucleotide polymorphism analysis in plastomes of eight
Catharanthus roseus cultivars. Biotechnol Biotechnol Equip
33(1):419–428. https://​doi.​org/​10.​1080/​13102​818.​2019.​15796​71
27. Schuster W, Hiesel R, Wissinger B, Brennicke A (1990) RNA edit-
ing in the cytochrome b locus of the higher plant Oenothera ber-
teriana includes a U-to-C transition. Mol Cell Biol 10(5):2428–
2431. https://​doi.​org/​10.​1128/​mcb.​10.5.​2428
28. Hajrah NH, Obaid AY, Atef A, Ramadan AM, Arasappan D, Nel-
son CA, Edris S, Mutwakil MZ, Alhebshi A, Gadalla NO et al
(2017) Transcriptomic analysis of salt stress responsive genes in
Rhazya stricta. PLoS ONE 12(5):e0177589
29. Xiao H, Zhang Q, Qin X, Xu Y, Ni C, Huang J, Zhu L, Zhong F,
Liu W, Yao G, Zhu Y, Hu J (2018) Rice PPS1 encodes a DYW
motif-containing pentatricopeptide repeat protein required for five
consecutive RNA-editing sites of nad3 in mitochondria. New Phy-
tol 220:878–892
30. Ramadan AM (2020) Salinity effects on nad3 gene RNA editing of
wild barley mitochondria. Mol Biol Rep 47(5):3857–3865. https://​
doi.​org/​10.​1007/​s11033-​020-​05475-7
31. Ramadan AM (2014) RNA editing in Calotropis procera mito-
chondrial nadh-dehydrogenase subunit 3 gene. Egypt J Genet
Cytol 43:353–364
32. He P, Xiao G, Liu H, Zhang L, Zhao L, Tang M, Huang S, An Y,
Yu J (2018) Two pivotal RNA editing sites in the mitochondrial
atp1mRNA are required for ATP synthase to produce sufficient
ATP for cotton fiber cell elongation. New Phytol 218(1):167–182
33. Katsuhara M (1997) Apoptosis-like cell death in barley roots
under salt stress. Plant Cell Physiol 38:1091–1093
34. Katsuhara M, Shibasaka M (2000) Cell death and growth recovery
of barley after transient salt stress. J Plant Res 113:239–243
35. Lin J, Wang Y, Wang G (2005) Salt stress-induced programmed
cell death via Ca2+-mediated mitochondrial permeability transi-
tion in tobacco protoplasts. Plant Growth Regul 45:243–250
36. Li JY, Jiang AL, Zhang W (2007) Salt stress-induced programmed
cell death in rice root tip cells. J Integr Biol 49:481–486
37. Li JY, Jiang AL, Chen HY, Wang Y, Wang Y, Zhang W (2007)
Lanthanum prevents salt stress-induced programmed cell death
in rice root tip cells by controlling early induction events. J Integr
Biol 49:1024–1031
38. Huh GH, Damsz B, Matsumoto TK, Reddy MP, Rus AM, Ibeas JI,
Narasimhan ML, Bressan RA, Hasegawa PM (2002) Salt causes
ion disequilibrium-induced programmed cell death in yeast and
plants. Plant J 29:649–659
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.