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Arabidopsis tRNA Adenosine Deaminase Arginine Edits the
Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is
Essential for Efficient Chloroplast Translation
W
Etienne Delannoy,a Monique Le Ret,b Emmanuelle Faivre-Nitschke,b Gonzalo M. Estavillo,c Marc Bergdoll,b
Nicolas L. Taylor,a Barry J. Pogson,c Ian Small,a Patrice Imbault,b and José M. Gualbertoa,b,1
a Australian
Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, 6008 WA,
Australia
b Institut de Biologie Moléculaire des Plantes du Centre National de la Recherche Scientifique, Université de Strasbourg,
67084 Strasbourg Cedex, France
c Australian Research Council Centre of Excellence in Plant Energy Biology, School of Biology, Australian National University,
Canberra, 0200 ACT, Australia
RNA editing changes the coding/decoding information relayed by transcripts via nucleotide insertion, deletion, or
conversion. Editing of tRNA anticodons by deamination of adenine to inosine is used both by eukaryotes and prokaryotes
to expand the decoding capacity of individual tRNAs. This limits the number of tRNA species required for codon-anticodon
recognition. We have identified the Arabidopsis thaliana gene that codes for tRNA adenosine deaminase arginine (TADA), a
chloroplast tRNA editing protein specifically required for deamination of chloroplast (cp)-tRNAArg(ACG) to cp-tRNAArg(ICG).
Land plant TADAs have a C-terminal domain similar in sequence and predicted structure to prokaryotic tRNA deaminases
and also have very long N-terminal extensions of unknown origin and function. Biochemical and mutant complementation
studies showed that the C-terminal domain is sufficient for cognate tRNA deamination both in vitro and in planta. Disruption
of TADA has profound effects on chloroplast translation efficiency, leading to reduced yields of chloroplast-encoded
proteins and impaired photosynthetic function. By contrast, chloroplast transcripts accumulate to levels significantly above
those of wild-type plants. Nevertheless, absence of cp-tRNAArg(ICG) is compatible with plant survival, implying that two out
of three CGN codon recognition occurs in chloroplasts, though this mechanism is less efficient than wobble pairing.
INTRODUCTION
tRNAs are key components of gene expression in every living
organism. They serve as adaptor molecules helping to convert
nucleic acid–based information into chains of amino acids: each
RNA codon is recognized by the anticodon harbored by the
corresponding tRNA, according to the wobble rules proposed by
Francis Crick (Crick, 1966). However, several genetic systems,
such as organelles and some bacterial parasites, encode fewer
tRNAs than theoretically required for the translation of all codons
(Shinozaki et al., 1986; Osawa et al., 1992).
This lack can be compensated for by import of cytosolic tRNAs
as demonstrated for mitochondria of plants, fungi, and protozoa
(Salinas et al., 2008), but import is unlikely to explain all cases of
incomplete tRNA sets. For example, no import of cytosolic tRNA
has been demonstrated in chloroplasts or in human mitochondria (Lung et al., 2006), which according to wobble rules lack
1 Address
correspondence to [email protected].
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) are: Etienne Delannoy
[email protected] and José M. Gualberto (jose.gualberto@ibmp-ulp.
u-strasbg.fr).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.109.066654
complete tRNA sets. This suggests that exceptions to the
wobble rules must exist in some genetic systems.
Two mechanisms have been postulated to explain how translation occurs with a reduced tRNA set: two out of three and
superwobble decoding. Two out of three decoding postulates
that a tRNA pairing with only the first two codon bases (usually a
G and a C) can be sufficient for translation and that any base can
occur at the third (wobble) codon position (opposite position 34
of the tRNA; Figure 1). This mechanism is supported by several
examples (Lagerkvist, 1986). It has also been suggested to occur
in chloroplasts (Shinozaki et al., 1986) and supported by in vitro
experiments using wheat (Triticum aestivum) germ extracts and
bean (Phaseolus vulgaris) chloroplast tRNAs (Pfitzinger et al.,
1990). Further analysis showed that two out of three critically
depends on the stability of the second base pair formed between
the codon and anticodon and that this stability is influenced
by the structure of the tRNA anticodon loop (Lehmann and
Libchaber, 2008).
The so-called superwobble, or extended wobble, is a mechanism in which an unmodified uridine at the first anticodon
position (position 34 of the tRNA; Figure 1) can read all four
nucleotides at the wobble codon position. This mechanism
has also been suggested to occur in chloroplasts (Shinozaki
et al., 1986; Pfitzinger et al., 1990) and was demonstrated in
tobacco (Nicotiana tabacum) when one of the two chloroplast
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Figure 1. Representation of cp-tRNAArg(ICG) Binding to Its Corresponding Codon.
cp-tRNAArg(ICG) depicted as a typical cloverleaf structure. The numbering of the anticodon loop residues is according to the convention
recommended by Sprinzl et al. (1996). The modifications shown are
those identified in other cp-tRNAArg(ICG) that are expected to be
conserved in Arabidopsis (Francis et al., 1989; Pfitzinger et al. 1990). I,
inosine; N, A, C, U, or G; m6A, N6-methyl A; D, dehydro U; Gm, 2-Omethyl G; T, 5-methyl U; C, pseudo U.
(cp)-tRNAGly genes was deleted (Rogalski et al., 2008). In this
study, two out of three was ruled out for decoding of Gly codons,
but superwobble was also shown to be inefficient. In every case,
exceptions to wobble rules only concern the third codon position
(Figure 1), whereas proper Watson-Crick pairing at codon positions 1 and 2 is always required.
tRNAs are the most heavily modified RNA molecules, and
numerous examples of posttranscriptional modifications, notably at uridine 34 or purine 37, have been shown to expand the
ability of tRNAs to read additional codons (Agris et al., 2007).
Modifications at purine 37, outside the anticodon, are very
important to prevent hydrogen bonding within the anticodon
loop (Dao et al., 1994) and to increase base stacking (Grosjean
et al., 1998), strengthening the codon–anticodon interaction.
Deamination of adenosine 34 to inosine is a widespread modification expanding the set of codons read by a particular tRNA
(Figure 1). This deamination is performed by specific tRNA
adenosine deaminases (TADs or ADATs), which are essential in
prokaryotes and eukaryotes (Schaub and Keller, 2002). The
enzymes mediating tRNA adenosine deamination in bacteria and
yeast contain conserved motifs present in cytidine deaminases,
suggesting an evolutionary link between the two reactions. The
genomes of higher-plant chloroplasts encode two Arg tRNAs:
cp-tRNAArg(ACG), thought to be required for decoding the codons CGA/U/G/C (CGN), and cp-tRNAArg(UCU), thought to be
required to read the codons AGA/G. It has been shown that
cp-tRNAArg(ACG) is deaminated to cp-tRNA Arg(ICG) in bean
(Pfitzinger et al., 1990), which would be expected to allow
efficient translation of CGC and CGA codons. This tRNA modification was inherited from the cyanobacterial ancestor of chloroplasts and is well conserved in prokaryotes where it is essential
for cell survival (Wolf et al., 2002). However, as opposed to
prokaryotes, which translate the Arg CGG codon using a specific
tRNAArg(CCG), neither of the two cp-tRNAArg can form a wobble
pair with the CGG codon. The simplest explanation is that chloroplast CGG codons are translated by two out of three recognition by cp-tRNAArg(ICG) (Pfitzinger et al., 1990). However, this
mechanism was recently ruled out in tobacco chloroplasts for the
decoding of Gly codons (Rogalski et al., 2008), casting doubts on
the occurrence of this mechanism in vivo in chloroplasts.
In this work, we identified the Arabidopsis thaliana tRNA
adenosine deaminase arginine (TADA) gene that codes for a
deaminase responsible for the editing of the adenosine at the
wobble position of cp-tRNAArg(ACG). A mutation in TADA leads
to slower chloroplast translation, causing profound effects on
chloroplast function and plant development. However, as opposed to the case in prokaryotes, it is not lethal, adding weight to
the hypothesis that a two out of three mechanism can occur in
chloroplasts.
RESULTS
TADA Is a Nuclear Gene Encoding a Protein Containing a
Conserved Deaminase Domain
Adenosine-to inosine (A-to-I) editing of the wobble anticodon
position of several eukaryote and prokaryote tRNAs is catalyzed
by enzymes of the TAD/ADAT protein family (Schaub and Keller,
2002), which contain the conserved cytidine/deoxycytidylate
deaminase motif (InterPro: IPR002125). This motif comprises
three His and Cys residues involved in the coordination of a zinc
ion and an essential Glu residue that is required for the hydrolytic
reaction. The single chloroplast tRNA known to be edited by
A-to-I deamination is cp-tRNAArg(ACG), which has been directly
sequenced in the alga Codium fragile and in bean (Francis et al.,
1989; Pfitzinger et al., 1990). While searching for candidate
genes that might be implicated in chloroplast RNA editing, we
searched the Arabidopsis predicted proteome for sequences
containing the characteristic deaminase motif. Fifteen genes
were selected (see Supplemental Figure 1 online). Among them
eight correspond to the CDA family of cytidine deaminases and
were discounted because they lack the requirements for an
organellar RNA editing enzyme, namely, organellar targeting
sequences, affinity for RNA, deaminase activity on RNA substrates, and conservation in other plant species (Faivre-Nitschke
et al., 1999; S.E. Faivre-Nitschke and J.M. Gualberto, unpublished data). Of the remaining candidate genes, only two code
for proteins with predicted organellar targeting sequences:
At4g20960, which probably codes for the chloroplast riboflavin
tRNA Editing and Plastid Translation
deaminase; and At1g68720, which codes for a large protein
predicted by PREDOTAR (Small et al., 2004) and TargetP
(Emanuelsson et al., 2000) to be possibly plastid or mitochondrial
respectively. By sequence homology, we identified At1g68720
as likely to code for TADA.
The Arabidopsis TADA gene contains only three introns and
codes for a large protein of 1307 amino acids with a C-terminal
deaminase domain (Figure 2A). The recent identification of a
full-size cDNA (accession number AK117889) confirmed the
gene model. The presence of an in-frame stop codon in the
59-untranslated region unambiguously identified the initiation
codon. Most of the large 59-terminal exon codes a sequence
(1080 amino acids) that has no identifiable motif or similarities to
sequences outside the plant kingdom. A probable ortholog of
TADA is found in rice (Oryza sativa; Os06g0489500), which also
codes for a large protein (1590 amino acids). Surprisingly, the
predicted N-terminal regions of the Arabidopsis and rice proteins
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are poorly conserved (15% identity), while the region similar to
other deaminases is 65% identical (see Supplemental Figure 2
online). Additional plant homologs can be identified in the databases of genomic and EST sequences (see Supplemental Figure
3 online).
TADA Is Targeted to the Chloroplast
To determine the subcellular localization of the TADA protein,
several green fluorescent protein (GFP) fusions were prepared.
The corresponding plasmids were biolistically transformed into
Nicotiana benthamiana leaves, and the localization of the fluorescent fusion proteins observed by confocal microscopy. A
full-length TADA:GFP fusion showed no GFP fluorescence. However, the first 259 amino acids of the TADA protein efficiently
targeted GFP into chloroplasts (Figure 2B). We observed no
fluorescence in mitochondria, cytoplasm, or in the nucleus. Thus,
the TADA protein contains an N-terminal targeting sequence that
can unambiguously target the protein into chloroplasts.
TADA Is Required for the Specific Editing of
Chloroplast tRNAArg(ACG)
Figure 2. The Arabidopsis TADA Gene Codes for a Large Protein
Targeted to Chloroplasts.
(A) Structure of the Arabidopsis TADA gene. The coding sequences of
the exons are indicated by thick gray bars, and the transcribed region is
represented by the arrow. The T-DNA insertion sites in the mutants
described in this study are shown. The gene sequence selected for
expression of a hairpin RNA in the RNAi line Agri-140-69-2-CATMA1a58100 is represented by the black bar. Primers described in the
text are indicated by orange arrowheads.
(B) Localization of a protein-GFP fusion in epidermal cells of N.
benthamiana. 1, Visible image; 2, merged fluorescence and visible
images; green, GFP fluorescence; red, natural fluorescence of chloroplasts; yellow, colocalization of green and yellow fluorescence.
(C) RT-PCR analysis of TADA transcripts using primer P5 and P6 in four
independent tada-1 homozygous plants. C, negative control without
reverse transcriptase.
We obtained several T-DNA insertions lines for TADA. We could
only confirm T-DNA insertions for two lines. Line SALK_024767
has a T-DNA insertion whose left border is located just eight
nucleotides upstream of the predicted transcription initiation site
(Figure 2A). However, plants homozygous for the insertion
showed no decrease in TADA transcript abundance compared
with wild-type Columbia-0 (Col-0) plants and showed no visible
phenotype differences either. It is likely that promoters internal to
the T-DNA efficiently drive expression of the downstream gene
(Ulker et al., 2008). Line GK-119G08 contains a T-DNA insertion
that was mapped to the first exon of the gene, 1597 nucleotides
downstream of the initiation codon. The T-DNA insertion was
accompanied by the deletion of 25 nucleotides at the insertion
site. In homozygous plants, no transcript could be detected
spanning the region containing the T-DNA insertion (Figure 2C).
Thus, if a transcript is generated and translated from that locus,
the resulting truncated protein would lack most of the N-terminal
sequence, including the chloroplast targeting sequence. Line
GK-119G08 was therefore identified as mutant tada-1.
We have also characterized several lines transformed with an
RNA interference (RNAi) construct that we obtained from the
AGRIKOLA collection (Hilson et al., 2004). Plants from line Agri140-69-2-CATMA1a58100 accumulate no or very little amounts
of TADA mRNA (see Supplemental Figure 4B online).
We tested if TADA is involved in cp-tRNAArg(ACG) editing. The
tRNA was amplified by RT-PCR, and the cDNA was sequenced.
Inosine base pairs with cytosine during reverse transcription, so
A-to-I deamination leads to an apparent change in sequence
from A to G. In wild-type plants, the sequence profiles suggest
that there are almost equimolar amounts of edited and unedited
tRNA, as evaluated by the height of the A and G peaks at the
position corresponding to the anticodon wobble nucleotide
(Figure 3). However, no trace of cp-tRNAArg(ICG) could be found
in tada-1 plants. The same result was obtained with RNA
extracted from five independent homozygous plants. In RNAi
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Figure 3. The TADA Enzyme Is Specifically Required for Editing of cp-tRNAArg(ACG).
The sequences of the cp-tRNAArg(ACG) and of the indicated cytosolic tRNAs were amplified by RT-PCR, ligated to plasmid vector pGEM-T, and
reamplified using plasmid and tRNA-specific primers. Direct sequence of the uncloned PCR fragments show that cp-tRNAArg(ACG) is no longer edited
in the tada-1 mutant (presence of a G in the sequence at the position 34 [shaded] of the tRNA), while editing of cytosolic tRNAs is not affected. The
presence of T instead of A at position 37 (shaded) in the sequences of tRNAAla(ACG) shows that editing of this position into I that is further modified into
m1I is also not compromised in tada-1.
plants, amplification and sequencing of cp-tRNAArg(ACG) confirmed that silencing of TADA correlates with loss of tRNA
editing: in a plant where no TADA mRNA could be detected,
there was no evidence of tRNA editing, while in two plants
showing partial TADA suppression, a residual G is visible at the
position corresponding to the tRNA wobble nucleotide. Unaffected plants had equivalent amounts of edited and unedited
tRNA, as in wild-type plants (see Supplemental Figure 4C online).
Apart from chloroplast tRNAArg(ACG), several cytosolic Arabidopsis tRNAs are also expected to be edited by A-to-I deamination (http://www.inra.fr/internet/Produits/TAARSAT/table.html).
We therefore tested the hypothesis that TADA is also responsible for editing of cytosolic tRNAs. Primers were designed to
amplify cytosolic tRNAAla(AGC), tRNAVal(AAC), tRNAThr(AGU), and
tRNAArg(ACG). Our primers were designed to amplify only a
subpopulation of tRNAArg(ACG), for which there are several variants encoded by eight different genes in Arabidopsis. Both in the
wild type and in tada-1 we found evidence for efficient editing of
cytosolic tRNAAla(AGC), tRNAVal(AAC), and tRNAThr(AGU) (Figure
3). Surprisingly, we found no evidence that cytosolic tRNAArg(ACG)
is targeted for editing, either in wild-type Col-0 or in tada-1.
TADA is also not involved in editing of nucleotide A37 of
tRNAAla(AGC) which, in all eukaryotic systems studied, is edited
to I and further modified to m1I. In yeast and humans, TAD1 is the
deaminase involved (Gerber et al., 1998). In the sequence of the
RT-PCR products of tRNAAla(AGC), we find a T at position 37,
both in the wild type and tada-1, consistent with the presence
of m1I.
Thus, TADA seems to be exclusively involved in editing of the
prokaryote-type cp-tRNAArg(ACG), and other deaminases must
be responsible for editing of cytosolic tRNAs. No other chloroplast tRNA can be a substrate for TADA because no other
chloroplast tRNA has an A at the wobble position. Regarding
mitochondria, no tRNA adenosine deaminase is theoretically
required by the mitochondrial translation system, as there are no
mitochondrially encoded tRNAs with an A at the wobble position,
and all tRNAs that should require editing for function are
imported (presumably preedited) from the cytosol (Duchene
et al., 2008).
We also considered the hypothesis that TADA is responsible
for C-to-U editing of chloroplast and/or mitochondrial mRNAs
that, by analogy to animal APOBEC and AID proteins involved in
tRNA Editing and Plastid Translation
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C-to-U editing, might be catalyzed by enzymes containing the
same characteristic cytidine/deoxycytidylate deaminase domain
(Conticello, 2008). However, the plant TADA is apparently not
involved in organellar C-to-U editing; regions of the ndhB, ndhD,
rps12, and rps14 chloroplast transcripts containing 11 editing
sites were analyzed by RT-PCR and sequence, but no differences were found between mutant and wild-type plants (see
Supplemental Figure 5 online). We conclude that TADA is not
required for C-to-U chloroplast mRNA editing. We have also
tested if editing of mitochondrial transcripts is affected, but none
of 82 editing sites in the transcripts of ccmC, ccmFc, cox3, rps12,
nad3, and nad5 is impaired in editing in the tada-1 mutant (see
Supplemental Figure 6 online).
The Deaminase Domain of TADA Is Structurally Similar to
Other Prokaryotic TADA Enzymes
We constructed a homology model of the TADA deaminase
domain based on the crystal structure of Staphylococcus aureus
TadA (2B3J) in complex with RNA (Losey et al., 2006). This model
shows that most of the residues implicated in interactions with
the zinc ion and the ligand are conserved (Figure 4). In particular,
the interactions with base 34 of the tRNA are preserved (Figure
4B); Ile-26 is replaced by Val-1131 (as in Escherichia coli and
Aquifex aeolicus), while the three other amino acids involved are
absolutely conserved (Asn-1148, His-1159, and Ala-1160).
This allowed us to predict that the N-terminal domain, although
not built in our model, is not a hindrance to tRNA recognition,
being rather distant from the binding site (Figure 4A). A major
difference between bacterial TadAs and plant chloroplast TADAs
is an insertion of 16 residues that is present in all plant sequences
found in the databases (see Supplemental Figure 3 online),
except for the atypical Chlamydomonas reinhardtii protein that
also has no predicted N-terminal extension and targeting sequence. This insertion is also poorly conserved in sequence
among the different plant TADAs. Finally, our model also proposes a dimer of the same type as in the three bacterial TadA
structures known. The portions of the chain involved in contact
between the monomers are well conserved, with no sequence
insertions and little sequence variation, comparable to that
observed between the three known structures.
The Deaminase Domain of TADA Is Sufficient for the
Deamination of cp-tRNAArg(ACG) to cp-tRNAArg(ICG) Both in
Vitro and in Planta
We tested in vitro the activity of the C-terminal part of TADA on
the deamination of cp-tRNAArg(ACG). The sequence coding the
last 194 amino acids of TADA that comprises the deaminase domain (DN-TADA) was expressed in E. coli fused to an N-terminal
His-tag, and the activity of the purified soluble protein fraction
was tested on in vitro–synthesized cp-tRNAArg(ACG) labeled with
[32P]ATP. The presence of inosine was analyzed by thin layer
chromatography (TLC) as described (Grosjean et al., 2007). Onedimensional TLC showed a radioactive spot comigrating with
inosine monophosphate (IMP), as a consequence of adenosine
deaminase activity by DN-TADA (Figure 5A1, lane c). No activity
was detected in extracts prepared in the same conditions using
Figure 4. Model of the Arabidopsis TADA Protein Bound to tRNA
Substrate.
(A) Possible structure of an Arabidopsis TADA homodimer bound to two
cp-tRNAArg(ACG) substrates. Residues at the C terminus (Arg-1107) and
N terminus (Lys-1272) of the represented sequences are indicated. The
16–amino acid insertion (Gly-1225…Pro-1240) is shown in white.
(B) Detail of the active site showing the interactions with A34 of the tRNA
anticodon. The coordinated zinc ion is shown.
an unrelated construct cloned in the same expression vector,
used as control for contamination by bacterial TadA (Figure 5A1,
lane b). The identity of the IMP spot was further confirmed by
two-dimensional TLC analysis using different solvent systems
(Figure 5A2). To definitely confirm that the activity does not result
from bacterial TadA contamination, we also tested the activity of
DN-TADA purified to homogeneity in denaturing conditions and
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renatured in vitro. This protein fraction was less active than the
protein purified in native conditions but confirmed the deaminase
activity of DN-TADA on cp-tRNAArg(ACG) (Figure 5A3). We also
tested the activity of DN-TADA on other tRNA substrates,
namely, nuclear encoded cytosolic tRNAArg(ACG) and cytosolic
tRNAAla(AGC). Only cytosolic tRNAArg(ACG) was also recognized
as substrate by DN-TADA (Figure 5A1, lanes d and e, respectively).
We also tested the activity of DN-TADA in planta by fusing it to
the signal peptide of the chloroplast protein RECA1 (At1g79050)
and expressing it in tada-1 under the control of the 35S promoter.
DN-TADA restored the deamination of cp-tRNAArg(ACG) to cptRNAArg(ICG) (Figure 5B), confirming that TADA is responsible for
Figure 5. The C-Terminal Domain of TADA Is Sufficient for cp-tRNAArg
(ACG) Deamination in Vitro and in Planta.
(A) The C-terminal domain of Arabidopsis TADA (DN-TADA) was expressed in E. coli fused to an N-terminal His tag and tested for deamination activity on [32P]ATP-labeled tRNAs synthesized in vitro. The
reaction product was digested with nuclease P1 and the resulting
59-phosphate nucleotides analyzed by one-dimensional (1) or twodimensional TLC (2 and 3). (1) Deamination activity of DN-TADA on cptRNAArg(ACG) (lanes a to c), on cytosolic tRNAArg(ACG) and on cytosolic
tRNAAla(AGC). a, Control without protein; b, control for bacterial contamination; c to e, plus recombinant DN-TADA purified from the E. coli
soluble fraction. The position of migration of IMP is indicated (pI). (2)
Two-dimensional TLC analysis of DN-TADA activity on cp-tRNAArg(ACG).
The positions of the four mononucleotides visualized by UV shadowing
are indicated. (3) As in 2, with DN-TADA purified to homogeneity from the
insoluble fraction in denaturing conditions and refolded in vitro.
(B) Deamination of cp-tRNAArg(ACG) is restored to wild-type levels in
tada-1 complemented with a DN-TADA sequence fused to the chloroplast targeting sequence of RECA1.
this activity in Arabidopsis and that the large N-terminal extension is dispensable for function both in vitro and in planta.
A Lack of cp-tRNAArg(ICG) Impairs Photosynthesis
Homozygous tada-1 plants show severe phenotypes, including
delayed growth, pale-green leaves, and very poor fertility (Figure
6). Leaf cross sections do not show any histological differences
compared with young wild-type plants of the same size, and
there is no apparent difference in the number of chloroplasts per
cell (see Supplemental Figure 7 online). The pale-green color of
the mutant is therefore a consequence of reduced chlorophyll
content as confirmed by spectrophotometric quantification (see
Supplemental Table 1 online). Complementation of the tada-1
mutation by DN-TADA targeted to chloroplasts restored a wildtype phenotype (Figure 6), showing that it is the absence of cptRNAArg(ICG) that impairs plant growth. RNAi plants showed
similar phenotypes of slow growth rate and delay in flowering,
which correlated with the accumulation of TADA mRNA (see
Supplemental Figure 4A online).
Different photosynthetic parameters were derived from saturation pulse-induced fluorescence measurements to determine
the effects of the tada-1 mutation on photosynthesis and photoprotection (Kramer et al., 2004). The maximum quantum efficiency of photosystem II (PSII) photochemistry (Fv/Fm) of the
tada-1 mutant was 60% of that of Col-0 (0.45 6 0.039 versus
0.77 6 0.013, respectively). The tada-1 mutation also affected
other photosynthetic parameters, such as photochemical and
nonphotochemical quenching (NPQ) (Table 1). Measurement of
electron transport rates and chlorophyll fluorescence quenching
indicated that tada-1 plants dissipated more excitation energy by
NPQ than Col-0, as shown by the significantly 2.5-fold higher
light-dependent thermal dissipation component of NPQ(FNPQ)
and lower photochemical efficiency(FPSII) (Oxborough, 2004).
The coefficient of photochemical quenching, qL, or the fraction of
oxidized QA (degree of openness of PSII) was typical for Col-0
(0.59 6 0.035), while, by contrast, the values for tada-1 plants (0.0)
suggest that PSII is closed in the mutant (i.e., reduced QA pool).
A Lack of cp-tRNAArg(ICG) Has Profound Effects on the
Chloroplast Transcriptome and Translation
To understand the reasons for the sharp drop in photosynthesis
and chlorophyll content, we analyzed the accumulation of several chloroplast proteins by protein gel blots. In tada-1 total
protein extracts, we saw a marked decrease of all chloroplastencoded proteins tested (cytochrome f, cytochrome b6, and D1),
whereas most nucleus-encoded proteins were much less
affected (Figure 7). Coomassie blue staining of protein gels
also showed a decrease in RbcL accumulation. Interestingly,
nucleus-encoded PsaD and plastocyanin were also strongly
decreased in tada-1. These proteins are associated with the
electron transport chain, and their decrease could be a consequence of a deficiency in the assembly of functional photosystems, as shown by the fluorescence measurements. The
decrease in plastid-encoded proteins could be related to a
decrease in protein synthesis or stability. A pulse-labeling experiment showed that Met incorporation in RbcL is strongly
tRNA Editing and Plastid Translation
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Figure 6. The tada-1 Mutant Has a Phenotype of Slow Growth and Pale-Green, Round Leaves, Which Is Complemented by DN-TADA
(A) Difference in growth between 11-d-old tada-1 plants and wild-type plants grown in vitro.
(B) Difference in size, color, and leaf shape between tada-1 and wild-type plants. The mutant growth phenotype is complemented in tada-1 plants
expressing the DN-TADA protein.
reduced in tada-1, more than fivefold as determined by phosphor-imager quantification (Figure 8). This result does not exclude that the stability of plastid-encoded proteins is also
affected but clearly shows that protein synthesis is decreased
in tada-1 chloroplasts.
The decrease in the amounts of all chloroplast-encoded proteins tested as well as the decrease in RbcL synthesis suggests a
global slowdown of chloroplast protein synthesis. This could be
due to a decrease in chloroplast mRNA accumulation or translation. To discriminate between the two possibilities, we analyzed the accumulation of every chloroplast mRNA by
quantitative RT-PCR (qRT-PCR) (Figure 9). Surprisingly, the
tada-1 mutant overaccumulates most chloroplast transcripts
compared with the wild type. This is a consequence of the lack of
cp-tRNAArg(ICG), as expression of DN-TADA restores a transcription profile that does not significantly deviate from the wild
type. In tada-1, transcripts of petA, petB, and psbA (respectively
encoding cytochrome f, cytochrome b6, and protein D1) are 3.7,
2.5, and 1.7 times more abundant in tada-1 compared with the
wild type respectively, while the rbcL transcript is unchanged. In
all cases, there is no correlation between the decrease in protein
products and the accumulation of the corresponding transcripts
that remain at least at the same levels as in wild-type plants. This
further supports the hypothesis that tada-1 plants are impaired in
chloroplast translation.
Furthermore, RNAs transcribed by the nucleus-encoded RNA
polymerase, such as rpo, accD, and rpl transcripts, accumulate
more than those transcribed by the plastid-encoded RNA polymerase (PEP), such as rbcL, psa, and most psb transcripts
(Figure 9). Although the quantitative variations are different,
this pattern is similar to the chloroplast transcript profiles of
Arabidopsis PEP-deficient mutants, such as clb19 and ptac2
(Chateigner-Boutin et al., 2008), suggesting a partial inhibition of
PEP activity in the tada-1 mutant. This inhibition is consistent
with a decrease in chloroplast translation, as all four of the PEP
subunits are encoded in the plastid genome.
tada-1 Can Still Properly Synthesize Chloroplast Proteins
In the absence of cp-tRNAArg(ICG), if CGC and CGA codons
could not be recognized by unmodified cp-tRNAArg(ACG), plant
survival should depend on misreading of these codons by a
noncognate tRNA. This would result in incorporation of amino
acids other than Arg at positions coded by CGC or CGA. We
checked this possibility by studying the tryptic digest profile of
RbcL. Trypsin is a protease cleaving specifically after Arg or Lys
residues. If CGC and CGA codons are translated incorrectly,
peptides corresponding to cleavage at these particular sites
should no longer be detected. After fractionation of total tada-1
proteins by SDS-PAGE and trypsin digestion of the RbcL band,
we could unambiguously identify 31 peptides derived from
plastid-encoded proteins, namely, RbcL, AtpA, and AtpB (Table
2). Among these peptides, two correspond to cleavage at two out
of the eight potentially mistranslated Arg residues in RbcL, three
correspond to cleavage at three out of the eight potentially
mistranslated Arg residues in AtpB, and two correspond to
cleavage at two out of the eight potentially mistranslated Arg
residues in AtpA. Therefore, although we cannot exclude that
mistranslation occurs in tada-1 chloroplasts, our results show
that Arg residues are accurately incorporated at positions
corresponding to CGC and CGA codons.
Table 1. Photosynthetic Efficiency
Col-0
tada-1
FPSII
FNPQ
Ff,D
S
0.51 6 0.025
0.07 6 0.077
0.23 6 0.019
0.58 6 0.055
0.26 6 0.013
0.35 6 0.24
1.0
1.0
The photochemical efficiency, FPSII, light-dependent thermal dissipation
component of nonphotochemical quenching, FNPQ, the sum of fluorescence quenching and light-independent thermal dissipation, Ff,D, and
their respective sums, S, were determined at normal light. Four 3-weekold plants per line were measured.
8 of 14
The Plant Cell
Figure 7. Reduced Accumulation of Plastid-Encoded Proteins in tada-1.
Protein gel blots of total leaf proteins of wild-type Col-0 and tada-1 plants
were analyzed by immunodetection with antibodies recognizing plastidencoded and nucleus-encoded chloroplast proteins. Plants were grown
in vitro in agar plates without sucrose. PsbA, D1 protein; PetA, cytochrome f; PetB, cytochrome b6; PC, plastocyanine; PsaA, photosystem I
reaction center subunit A; PsaD, idem subunit D; PsbO, oxygen-evolving
enhancer protein 1; LHCII, 26-kD protein from light-harvesting complex
II; PCOR, protochlorophyllide oxidoreductase; RbcS, small subunit of
Rubisco; RbcL, large subunit of Rubisco.
high sequence similarity with bacterial TadA made three-dimensional structure modeling relatively straightforward. In the three
bacterial structures that have been determined (2B3J, 1Z3A, and
1WWR), the active site pocket is formed by residues from two
different chains. Therefore homodimerization is an absolute
requirement for the assembly of the biologically active unit. We
identified the positions putatively involved in dimer formation in
plant TADAs by comparing the accessible surface of the 2B3J
dimer and an isolated monomer (chain 2B3J:A). A third of the
residues in those positions is conserved compared with the three
known structures. Residues of chain B involved in tRNA binding
in pocket A are also well conserved (see Supplemental Figure 3
online): Arg-1176 and Arg-1200 are kept, and at position 1247 an
Arg is replaced by a Lys, preserving its basic nature. However,
positions 1178 and 1245 are less conserved: Glu is replaced by
Ala-1178 (although the E. coli protein has an Ile at this position),
and Asn is replaced by His-1245 in all plant sequences. Taken
together, despite these small differences, our model suggests
that the active form of plant TADA is likely to be a dimer, as in
2B3J, 1Z3A, or 1WWR.
We have shown that the TADA deaminase domain (DN-TADA)
is sufficient for the deamination of cp-tRNAArg(ACG) to cptRNAArg(ICG) both in vitro and in planta (Figure 5). Interestingly,
DN-TADA was also able to deaminate the cytosolic tRNAArg
(ACG) but not tRNAAla(AGC). Because chloroplast and cytosolic
tRNAArg(ACG) share little sequence identity, apart from the
anticodon loop bases, this suggests that the anticodon loop
nucleotides are necessary and sufficient for tRNA recognition by
TADA. Mutagenesis studies and the determination of TadA
structures showed that each one of the anticodon nucleotides
is required for the recognition of the tRNA by bacterial TadA (Wolf
et al., 2002; Kuratani et al., 2005; Losey et al., 2006; Luo and
Schramm, 2008). The bacterial TadA is probably the ancestor of
the eukaryotic ADAT2/TAD2 that, in yeast, forms a heterodimer
with ADAT3/TAD3 and catalyzes deamination of seven tRNA
species (Gerber and Keller, 1999). However, the bacterial TadA is
DISCUSSION
TADA Is a tRNAArg Adenosine Deaminase
The chloroplast translation system is derived from its cyanobacterial ancestor and retains predominant prokaryotic characteristics. The recruitment of proteins from the symbiont host has
had limited influence on the evolution of the plastid translation
system because a significant proportion of its components are
still encoded by the plastid genome. These include all rRNAs and
tRNAs. Chloroplasts have not acquired exogenous gene sequences during evolution and do not import external tRNAs like
plant mitochondria, which have evolved a translation system
able to cope with tRNAs that originate from three different
genomic compartments (Maréchal-Drouard et al., 1993). cptRNAs retain all the typical characteristics of prokaryotic tRNAs.
Among these is the editing of cp-tRNAArg(ACG) into cp-tRNAArg
(ICG), a deamination reaction that we show is catalyzed by the
TADA protein.
The Arabidopsis TADA gene encodes a large protein of which
only the C-terminal part resembles bacterial tRNA adenosine
deaminases. We modeled this C-terminal domain because the
Figure 8. Reduced Synthesis Rate of RbcL in tada-1.
Leaf discs of tada-1 and Col-0 were labeled with [35S]Met and incubated
for 10, 20, or 30 min in the presence of cycloheximide to inhibit cytosolic
translation. Total proteins were then fractionated by SDS-PAGE and
autoradiographed. The asterisk indicates the RbcL band.
tRNA Editing and Plastid Translation
9 of 14
Figure 9. Accumulation of Chloroplast Transcripts in tada-1 and DN-TADA.
Transcript abundances of all protein-encoding and rRNA genes of the chloroplast genome from tada-1 (black bars) and tada-1 complemented with DNTADA (gray bars) were compared with the wild type by qRT-PCR and normalized against a set of nuclear housekeeping genes. The genes are sorted
according to their physical location on the chloroplast chromosome. Error bars are standard deviations based on three biological replicates for tada-1
and technical triplicates for tada-1 complemented with DN-TADA. Asterisks mark transcripts for which the corresponding protein has been analyzed by
protein gel blots (Figure 7). Values are given in Supplemental Table 3 online.
unable to deaminate substrates of ADAT2/TAD2 in vitro, with the
exception of yeast tRNAArg. It is therefore not surprising that the
plant TADA, which is phylogenetically related to bacterial TadA,
is not involved in the deamination of other tRNAs. In vivo,
chloroplast targeting of TADA probably prevents it from deaminating the cytosolic tRNAArg(ACG). The functional homolog of
ADAT2/TAD2 in plants remains to be identified.
All higher-plant TADA sequences display large N-terminal
domains, whose possible function(s) remain mysterious. They
comprise the chloroplast targeting peptide, which in Arabi-
dopsis is contained in the first 259 amino acids (Figure 2B), but
the remaining N-terminal sequences contain no identifiable
motifs, have no similarities to non-plant proteins and are
poorly conserved within the plant kingdom (see Supplemental
Table 2 online). The Arabidopsis N-terminal domain is dispensable for the activity and specificity of TADA in vitro (Figure
5A). In vivo, the C-terminal domain alone is sufficient to restore
the deamination of cp-tRNAArg(ACG) to levels comparable to
the wild type (Figure 5B), reestablishing normal plant growth
(Figure 6).
Table 2. Tryptic Digest of RbcL from tada-1
RbcL
AtpA
AtpB
m/z Observed
z
Mr Calc
Sequence
Ion Score
511.2719
625.3409
626.8563
737.8978
978.0159
736.3841
1031.0219
+2
+3
+2
+2
+2
+2
+2
1020.524
1248.6714
1251.6935
1473.7787
1954.016
1470.7541
2060.0248
DTDILAAFR41
ESTLGFVDLLR350
VINALANPIDGR119
ASSVAQVVTSLQER216
(R109)IFNVLGEPVDNLGPVDTR127
VGLTALTMAEYFR261
GIYPAVDPLDSTSTMLQPR378
59
75
49
52
94
89
69
After mass spectrometry analysis of the RbcL band of tada-1, 31 peptides derived from RbcL, AtpB, and AtpA were unambiguously identified. Only
peptides corresponding to cleavage at potentially mistranslated Arg residues (in bold) are listed in this table. Ions score is 10*Log(P), where P is the
probability that the observed match is a random event. Ions scores >29 indicate identity or extensive homology (P < 0.05). Mr Calc, relative mass
calculated from the mass-to-charge (m/z) ratio. The full list of ions observed during this experiment is available in the Supplemental Data Set 1.
10 of 14
The Plant Cell
The Absence of cp-tRNAArg(ICG) Slows Down Chloroplast
Translation, Which Affects Photosynthesis
Contrary to the situation in bactweria, where TadA is essential for
growth, knockout of the Arabidopsis TADA gene is compatible
with plant survival. Nevertheless, the mutation has profound
effects on the efficiency of chloroplast translation (Figures 6 to 9).
In particular, the levels of key components of both photosystems
(protein D1 and PsaA) and of the thylakoidal electron transport
chain (cytochrome b6f complex subunit VI and cytochrome f) are
substantially lower. Although levels of LHCII, a PSII light-harvesting chlorophyll a/b binding protein of the antenna system
(Jansson, 1999), are not as affected as chloroplast-encoded
proteins, total chlorophyll content is only 40% of wild-type levels
in tada-1 plants (see Supplemental Table 1 online). The combination of inefficient energy transfer from the antenna complexes,
reduced QA pool, and impaired electron transport chain performance could result in a dramatically lower ratio of photons
absorbed by PSII being channeled through photochemical processes (Fv/Fm; Kramer et al., 2004) and the more than doubled
amount of nonphotochemical quenching, FNPQ, in the tada-1
plants (Table 1).
A priori, the reduced protein synthesis in tada-1 chloroplasts
could be due to any of the following defects: (1) reduced levels of
translatable mRNA, (2) reduced rates of translation initiation, (3)
reduced rates of protein synthesis (chain elongation), and (4)
increased rates of protein turnover. We excluded the first of these
possible explanations as we found that tada-1 mRNA levels are
at least equivalent to those found in wild-type plants. Given that
the primary defect in tada-1 plants is the loss of cp-tRNAArg(ICG),
the logical assumption is that chloroplast translation is impaired
at the elongation step.
When stalled at particular sites, ribosomes can dissociate and/
or induce cleavage of the mRNA (reviewed in Dreyfus, 2009).
However, slower elongation rates have been associated with the
accumulation of heavier polysomes both in yeast and mammals
(Hovland et al., 1999; Shenton et al., 2006; Sivan et al., 2007). As
a result, mRNAs densely covered with ribosomes might be
protected from degradation by nucleases, becoming more stable (Deana and Belasco, 2005; Dreyfus, 2009). This phenomenon, also proposed by Pfalz et al. (2009), could explain the
accumulation of plastid transcripts in tada-1, despite the expected reduction in PEP synthesis. A positive effect of reduced
chloroplast translation on chloroplast mRNA accumulation was
also found for the svr1 Arabidopsis mutant (Yu et al., 2008) but,
surprisingly, not in transplastomic tobacco plants impaired in
translation by deletion of one of the two cp-tRNAGly genes
(Rogalski et al., 2008). This could be because different compensatory mechanisms may be in place. However, in the latter study,
only three plastid mRNAs were monitored, including the rbcL
transcript whose steady state level is also not affected in tada-1.
Arg Codon Usage Is Biased in Arabidopsis Chloroplasts
The absence of cp-tRNAArg(ICG) in tada-1 plants theoretically
prevents translation of two out of the six Arg codons (CGA and
CGC) in Arabidopsis chloroplasts. However, the mutation is not
lethal, and Arg is still properly inserted at protein positions coded
by CGA and CGC codons (Table 2), implying that normal wobble
pairing rules can be infringed in chloroplasts. Even in wild-type
plants, neither cp-tRNAArg(ACG) nor cp-tRNAArg(ICG) can form a
wobble pair with CGG codons, which must be read by a nonstandard decoding mechanism. This might give a clue as to why a lack
of cp-tRNAArg(ICG) does not completely block plastid translation.
A detailed analysis of the codon usage of Arabidopsis chloroplast genes shows that the group formed by rbcL, psb, and
psa transcripts contains a significantly smaller proportion of
CGG codons than other chloroplast transcripts (see Supplemental Table 4 online; Z-test, P value < 0.01). rbcL and psbA
transcripts do not contain any CGG codons at all, suggesting
that this particular codon is not compatible with high translation
rates.
cp-tRNAArg(ACG) and cp-tRNAArg(ICG) have overlapping repertoires according to the wobble rules (both should read CGU
codons efficiently), which raises the question of why only ;50% of
cp-tRNAArg(ACG) is apparently deaminated. The Arg codon usage
shows a significant bias toward CGU codons compared with CGA
and CGC codons in highly translated mRNAs, such as those
encoding subunits of ribulose-1,5-bis-phosphate carboxylase/
oxygenase (Rubisco), PSI, and PSII (Z-test, P value < 0.001). This
might explain the retention of cp-tRNAArg(ACG), as it is expected to
be more efficient than cp-tRNAArg(ICG) at translating CGU codons.
These data therefore suggest that CGN codon usage of
chloroplast genes is under selection for optimal translation
efficiency, given the presence of both cp-tRNAArg(ACG) and
cp-tRNAArg(ICG), and that the preferred codon of the CGN series
for highly translated RNAs is CGU. This codon bias, which
minimizes the necessity for cp-tRNAArg(ICG), is undoubtedly a
major reason for why the tada-1 mutation is not lethal.
Two Out of Three Decoding Probably Occurs in Chloroplasts
A complete impairment of chloroplast translation is embryolethal in Arabidopsis and tobacco, as demonstrated by knockout
mutants either of chloroplast ribosomal proteins (Ahlert et al.,
2003; Rogalski et al., 2006; http://www.seedgenes.org), of aminoacyl-tRNA synthetases (Berg et al., 2005), or of some tRNAs
(Rogalski et al., 2008). Therefore, the lack of cp-tRNAArg(ICG) is
partially compensated for by some other mechanism in tada-1
chloroplasts. According to the superwobble rules, an unmodified
uridine at the first anticodon position (position 34 of the tRNA;
Figure 1) can read all four nucleotides at the wobble codon
position. The possibility for a uridine to accommodate any
nucleotide in the codon is restricted to position 34. For this
reason, the translation of any of the CGN Arg codons by cptRNAArg(UCU) is most unlikely because this would involve superwobbling to occur at both positions 34 and 36 of the tRNA.
Most probably, the translation of CGN codons in tada-1
chloroplasts occurs via two out of three decoding using
cp-tRNAArg(ACG). This tRNA lacks a uridine at position 34 required for superwobble but can form the strong Watson-Crick
pairing at positions 35 and 36 (Figure 1) required to stabilize
codon-anticodon pairing when there is a mismatch at the third
position (Lehmann and Libchaber, 2008). However, tobacco cptRNAGly(GCC) is apparently unable to decode by two out of three,
despite that positions 2 and 3 of the anticodon are cytidines
tRNA Editing and Plastid Translation
(Rogalski et al., 2008). This could be because, in cp-tRNAGly(GCC),
U32 and A38 can form a strong Watson-Crick pairing, whereas
C32 and A38 in cp-tRNAArg(ACG) can only form a single weak
hydrogen bond (Auffinger and Westhof, 1999). To allow two out
of three, the N32-N38 pair has to be non-Watson-Crick to allow
the flexibility of the anticodon loop required for proper stacking of
the bases (Lehmann and Libchaber, 2008).
These arguments are in favor of two out of three decoding
occurring in plastids, even in wild-type Arabidopsis plants, as the
Arg CGG codon cannot form a canonical wobble paring with
either cp-tRNAArg(ACG) or cp-tRNAArg(ICG). However, the decrease of translation activity in tada-1 clearly shows that cptRNAArg(ACG) cannot read all CGN Arg codons efficiently,
demonstrating that in this particular case, two out of three
pairing is much less effective than wobble pairing. From this
perspective, the deamination of cp-tRNAArg(ACG) to cptRNAArg(ICG) enhances plastidial translation by replacing inefficient two out of three by canonical wobble codon-anticodon
pairing.
METHODS
11 of 14
The activity of the protein was tested on in vitro–synthesized tRNAs
labeled with [32P]ATP. The cp-tRNAArg(ACG), cytosolic tRNAArg(ACG), and
cytosolic tRNAAla(AGC) sequences were amplified with primers P53+P54,
P55+P56, and P57+P58 directly fused to a T7 RNA polymerase promoter
including a BstNI site at the 39 terminus to correctly generate CCA 39-ends.
After BstNI digestion, PCR fragments were used as templates for transcription using the RiboMAX RNA production system (Promega) in the
presence of [a-32P]ATP. Incubation conditions were as described (Wolf
et al., 2002). Afterwards, the tRNA substrates were digested with nuclease
P1 to generate 59-monophosphate nucleosides that were analyzed by
one- or two-dimensional TLC as described (Grosjean et al., 2007).
Complementation of the tada-1 Mutant
The genomic fragment coding for the last 280 amino acids of TADA (DNTADA) was amplified with Gateway primers P47+P48 and recombined
into pDONR221 by BP cloning (Invitrogen). Similarly the signal peptide of
chloroplast protein RECA1 (At1g79050) was amplified with primers P49
and P50 and recombined by BP cloning into pDONRP4P1R. They were
fused into the binary vector pB7m24GW35S under the control of the 35S
promoter by LR cloning (Invitrogen). The segregation of the complemented
tada-1 mutant was checked as mentioned above, and the presence of the
DN-TADA construct was confirmed by PCR with primers pairs P51+P52 and
P53+P54 amplifying the 59 and 39 borders of the construct, respectively.
Plant Materials and Growth Conditions
Protein Gel Blots
All experiments used the Col-0 strain of Arabidopsis thaliana as genetic
background. Plants were grown on soil under long days (16 h light/8 h
dark) at 228C. For in vitro growth experiments, seeds were surface
sterilized and placed on Murashige and Skoog agar plates with or without
1% sucrose. Plates were incubated in the dark at 48C for 4 d to
synchronize germination and subsequently were in a controlled environment chamber (228C; 8 klux continuous light). T-DNA insertion lines were
obtained from the SALK (line SALK_024767) and Gabi-Kat collections
(line GK-119G08; Rosso et al., 2003). The RNAi line targeting TADA was
obtained from the AGRIKOLA project (line Agri-140-69-2-CATMA1a58100; Hilson et al., 2004).
Genotyping of mutant lines was performed by PCR using gene- and
T-DNA–specific primers. For SALK_024767, primers P1 and P3 were
used to amplify the wild-type fragment. The mutant allele was detected
with primer P3 and the T-DNA left border primer. For GK-119G08, primers
P5 and P6 were used to amplify wild-type sequence. The mutant allele
was amplified with primer P6 and the T-DNA left border primer. Expression of the mRNAs was tested by amplification of a cDNA fragment
spanning the insertion site of the T-DNA, using primers P5 and P6. Total
plant RNA and genomic DNA were extracted with TRIzol and DNAzol
(Invitrogen), respectively, as described (Zaegel et al., 2006).
Samples of 15 mg of proteins extracted from leaves of 3-week-old plants
were fractionated by SDS-PAGE and blotted onto Immobilon-P membranes
(Millipore). Antibodies against protein D1, PCOR, PsaD, plastocyanin, PsaA,
PsbO, and cytochrome b6 were purchased from Agrisera and used following the manufacturer’s recommendations. Antibodies against LHCII, Rubisco small subunit, and cytochrome f were a kind gift of Geraldine Bonnard
(Institut de Biologie Moléculaire des Plantes). Proteins recognized by the
antibodies were revealed by ECL (GE Healthcare) and autoradiography.
tRNA Deaminase Activity Test in Vitro
The C-terminal part of TADA (DN-TADA) containing the last 194 amino
acids, including the deaminase domain, was amplified with primers P8
+P10 and cloned in expression vector pRSET (Invitrogen) in frame with an
N-terminal His tag and expressed in Escherichia coli strain ROSETTA
(DE3) (Novagen). The soluble protein fraction was purified by zincchelating chromatography on Ni-NTA resin (Qiagen). The protein was
dialyzed against purification buffer (40 mM Tris-HCl and 300 mM NaCl)
containing 50% glycerol and stored at 2208C. Insoluble protein was
purified in denaturing conditions in the presence of 6 M urea and
renatured in vitro by stepwise dialysis during 3 d against the same buffer
containing decreasing concentrations of urea and increasing concentrations of glycerol plus 0.1% Triton and 0.1 mM ZnCl, until a final concentration of 50% glycerol and no urea. The renatured soluble protein was
virtually pure, as estimated by Coomassie Brilliant Blue staining.
Pulse Labeling of Chloroplast-Encoded Proteins
Pulse labeling experiments were conducted essentially as described
(Takahashi et al., 2007). Leaf discs of 19.6 mm2 from young leaves of 3
week-old plants were vacuum infiltrated for 30 s in 0.5 mL of 1 mM
KH2PO4 pH6.3, 0.1% Tween 20, 100 mCi of [35S]methionine (specific
activity >1000 Ci/mmol). Cycloheximide (100 mg/mL) was added to the
infiltration buffer to inhibit cytoplasmic translation. After infiltration, leaf
discs were washed in 10 mL water and floated in water plus 100 mg/mL
cycloheximide. Discs were exposed to light at 258C and immediately
frozen in nitrogen at the indicated times (10, 20 and 30 min, four leaf
discs for each time point). Total proteins were extracted, fractionated by
SDS-PAGE and blotted onto Immobilon-P membranes (Millipore) before
autoradiography. Quantifications were performed with a Fuji BAS 1000
Imager and MacBAS software version 2.1.
Analysis of RbcL by Mass Spectrometry
Total proteins from leaves of tada-1 were fractionated on a 12% SDS-PAGE
gel. After Coomassie Brilliant Blue staining, the band corresponding to
RbcL was excised, destained in acetonitrile 50% 10 mM NH4CO3, and
trypsin digested overnight. Peptides were extracted from the gel with 50%
acetonitrile and 0.5% TFA, dried by vacuum, and analyzed with an 1100
Series HPLC coupled with a 6510 Q-TOF mass spectrometer (Agilent).
Observation of GFP Fluorescence
For in vivo intracellular localization, the sequences encoding the fulllength and the first 259 amino acids of TADA were amplified with primers
12 of 14
The Plant Cell
P2+P10 and P2+P4 and cloned in plasmid pCK-GFP3A to express
protein:GFP fusions under the control of a 35S promoter as described
(Vermel et al., 2002). The resulting plasmids were Nicotiana benthamiana
leaves by bombardment, and images were obtained 24 h after transfection with a Zeiss LSM510 confocal microscope with 360 objectives.
The excitation wavelength for GFP detection was 488 nm. Chloroplasts
were visualized by the natural fluorescence of chlorophyll. Organelle
targeting predictions were determined with programs Predotar and
TargetP (http://urgi.versailles.inra.fr/predotar/predotar.html and www.
cbs.dtu.dk/services/TargetP/).
Analysis of Chloroplast Transcripts
tada-1 mutants were grown under continuous light in soil, and the analysis
of the steady state levels of chloroplast transcripts by qRT-PCR was
conducted on three independent biological replicates as described
(Chateigner-Boutin et al., 2008). Total Arabidopsis RNA was extracted
with the RNeasy plant mini kit (Qiagen), and genomic DNA was removed
using RNase-free DNase (DNA-free; Ambion). The absence of DNA was
confirmed by PCR prior to reverse transcription with random hexanucleotide primers and Superscript III reverse transcriptase (Invitrogen).
Real-time PCR analysis was conducted in 384-well plates with a LightCycler 480 (Roche) using primer pairs described by Chateigner-Boutin
et al. (2008) and the LightCycler 480 SYBR I Master mix (Roche). Primer
pairs actin2.8F + actin2.8R, Q18SF + Q18SR, UBC-1 + UBC-2, and YLS81 + YLS8-2 were used to measure the accumulation of nucleus-encoded
transcripts. The specificity of each primer pair was checked by sequencing the PCR product and subsequently by melting curve analysis. For
each primer pair and each comparison between mutant and wild-type
Col-0, a standard curve based on serial dilutions of the Col-0 cDNA was
included. The accumulation of each transcript was analyzed in triplicate
and, as the total set of primers was analyzed over more than one PCR
plate, primer pairs QC16S, QC23S, and QC18S were included in each
plate to correct for variations between plates. The raw data were analyzed
using the LightCycler 480 software release 1.5 (Roche) and crossing
points determined by second derivative maximum analysis. The accumulation of chloroplast transcripts was normalized by setting the average
ratio of nuclear transcripts to 1.
The chlorophyll content of 4-week-old tada-1 and wild-type plants was
determined according to Porra (2002) in 80% acetone and 2.5 mM
NaH2PO4, pH 7.8.
Modeling of TADA Structure
A search in the Protein Data Bank with the TADA predicted sequence was
performed with National Center for Biotechnology Information-BLAST.
Homologous proteins belonging to various families were identified:
cytosine, guanine, deoxycytidylate and cytidine deaminases, riboflavin
biosynthesis proteins, and tRNA adenosine deaminases. Three structures of this last family were found in the library: 1Z3A, 2B3J, and 1WWR,
corresponding to sequences from E. coli, Staphylococcus aureus, and
Aquifex aeolicus. Their respective scores and E-values are 127 and 2e29; 124 and 2e-28; and 100 and 2e-21. The 2B3J structure was chosen as
scaffold for modeling because although the 1Z3A and 2B3J structures are
almost identical (root mean square deviation of 1.2A; Losey et al., 2006),
the 1Z3A structure lacks the RNA molecule.
Sequences were aligned using ClustalX (Thompson et al., 1997), and the
model was built using Modeler (Sali and Blundell, 1993; http://salilab.org/
modeller/about_modeller.html). A large insertion of 17 residues (GGEGNGSEASEKPPPPV) in the Arabidopsis TADA sequence has no equivalent in
2B3J, 1Z3A, or 1WWR and therefore cannot be modeled accurately. No
constraint was imposed onto it, and it was modeled as a loop.
The tRNA fragments were modeled as rigid bodies after hand-editing to
adjust the sequence to the anticodon stem loop from cp-tRNAArg.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: SALK_024767; GK-119G08 (AL757232); Agrik line-140-69-2CATMA1a58100 (N205329 from the Nottingham Arabidopsis Stock Centre); and TADA, At1g68720 (NM_105544). Accession numbers for other
genes used in this study are listed in Supplemental Table 3 online.
Supplemental Data
The following materials are available in the online version of this article.
Analysis of tRNA and mRNA Editing
For analysis of tRNA editing by deamination, chloroplast cp-tRNAArg(ACG)
and cytosolic tRNAAla(AGC), tRNAVal(AAC), tRNAThr(AGT), tRNALeu(AAG),
tRNAIle(AAT), and tRNAArg(ACG) were amplified by RT-PCR with primers
P11+P12, P13+P14, P15+P16, P17+P18, P19+P20, P21+22, and P23+P24,
respectively. Because the amplified cDNA fragments are too small for
direct sequence analysis, they were ligated to plasmid vector pGEM-T
(Promega) to obtain DNA fragments long enough for sequencing. Ligated
DNA products were reamplified using universal sequence primer (vectorspecific) and the reverse tRNA primer. The uncloned cDNA sequences
were purified and directly sequenced using universal primer. A-to-I
deamination was revealed by the presence of G at the place of A because
reverse transcriptase will incorporate a C in place of an I.
Amplification and direct sequence of chloroplast and mitochondrial
gene transcripts were done using primers P25 to P46 (see Supplemental
Table 5 online).
Fluorescence Analysis and Chlorophyll Content
Supplemental Figure 1. Cytidine/Deoxycytidylate Deaminase Motif
Found in Several Arabidopsis Predicted Proteins.
Supplemental Figure 2. Alignment of the Arabidopsis, Rice, and
E. coli TADA Proteins Highlights the Poor Conservation of the Large
N-Terminal Domains of the Plant TADA.
Supplemental Figure 3. Alignment of the C-Terminal Active Domains
of Plant TADA Sequences Found in Genomic and EST Databases
Compared with Bacterial TADAs.
Supplemental Figure 4. RNAi Plants Deficient in TADA Expression
Are Also Affected in cp-tRNAArg Editing.
Supplemental Figure 5. TADA Is Not Involved in Chloroplast C-to-U
Editing.
Supplemental Figure 6. TADA Is Not Involved in Mitochondrial C-to-U
Editing.
Supplemental Figure 7. tada-1 Plants Have Normal Leaf Structure
and Equivalent Number of Chloroplasts per Cell as Wild-Type Plants.
Supplemental Table 1. Chlorophyll Content of tada-1 Plants.
Fluorescence induction kinetics at room temperature (228C) were measured using a pulse amplitude modulation fluorometer (PAM101; H. Walz)
as described (Giraud et al., 2008) on 3-week-old seedlings grown on soil at
228C under continuous light at 100 mmol m22 s21. Fluorescence measurements were made on four plants for each of the Col-0 and tada-1 lines.
Supplemental Table 2. Similarities between Plant TADA N-Terminal
and C-Terminal Domains.
Supplemental Table 3. Accumulation of Chloroplast Transcripts in
tada-1 and tada-1 Complemented with DN-TADA Compared with Col-0.
tRNA Editing and Plastid Translation
Supplemental Table 4. Frequencies of the CGN Codons in Arabidopsis Chloroplast-Encoded Proteins.
Supplemental Table 5. Primers Used in This Study.
Supplemental Data Set 1. online. Mass Spectrometry Analysis of
the RbcL Band in tada-1.
ACKNOWLEDGMENTS
This work was supported by the Centre National de la Recherche
Scientifique, the French Ministry of Research, the Australian Research
Council Centre of Excellence in Plant Energy Biology (Grant
CEO561495), the State Government of Western Australia, and by an
Australian Research Council Linkage International grant (LX0776042).
We thank Geraldine Bonnard for antibodies and to Laurence MaréchalDrouard for helpful discussions.
Received February 28, 2009; revised June 12, 2009; accepted June 26,
2009; published July 14, 2009.
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Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast
tRNA Arg(ACG) and Is Essential for Efficient Chloroplast Translation
Etienne Delannoy, Monique Le Ret, Emmanuelle Faivre-Nitschke, Gonzalo M. Estavillo, Marc
Bergdoll, Nicolas L. Taylor, Barry J. Pogson, Ian Small, Patrice Imbault and José M. Gualberto
Plant Cell; originally published online July 14, 2009;
DOI 10.1105/tpc.109.066654
This information is current as of June 14, 2017
Supplemental Data
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