Arabidopsis tRNA Adenosine Deaminase Arginine Edits theWobble Nucleotide of Chloroplast tRNAArg(ACG) and IsEssential 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 Jose M. Gualbertoa,b,1

a Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, 6008 WA,

Australiab Institut de Biologie Moleculaire des Plantes du Centre National de la Recherche Scientifique, Universite de Strasbourg,

67084 Strasbourg Cedex, Francec 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 mitochon-

dria (Lung et al., 2006), which according to wobble rules lack

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 trans-

lation 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 mech-

anism 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

1 Address correspondence to jose.gualberto@ibmp-ulp.u-strasbg.fr.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Etienne Delannoy(edelanno@gmail.com) and Jose M. Gualberto (jose.gualberto@ibmp-ulp.u-strasbg.fr).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.066654

The Plant Cell, Vol. 21: 2058–2071, July 2009, www.plantcell.org ã 2009 American Society of Plant Biologists

(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 posi-

tions 1 and 2 is always required.

tRNAs are the most heavily modified RNA molecules, and

numerous examples of posttranscriptional modifications, nota-

bly 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 mod-

ification 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

enzymesmediating 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 co-

dons 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-tRNAArg(ICG) in bean

(Pfitzinger et al., 1990), which would be expected to allow

efficient translation of CGC and CGA codons. This tRNA mod-

ification was inherited from the cyanobacterial ancestor of chlo-

roplasts and is well conserved in prokaryoteswhere 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 chlo-

roplast CGG codons are translated by two out of three recog-

nition by cp-tRNAArg(ICG) (Pfitzinger et al., 1990). However, this

mechanismwas 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 op-

posed 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 sub-

strates, and conservation in other plant species (Faivre-Nitschke

et al., 1999; S.E. Faivre-Nitschke and J.M. Gualberto, unpub-

lished data). Of the remaining candidate genes, only two code

for proteins with predicted organellar targeting sequences:

At4g20960, which probably codes for the chloroplast riboflavin

Figure 1. Representation of cp-tRNAArg(ICG) Binding to Its Correspond-

ing Codon.

cp-tRNAArg(ICG) depicted as a typical cloverleaf structure. The number-

ing 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-O-

methyl G; T, 5-methyl U; C, pseudo U.

tRNA Editing and Plastid Translation 2059

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 ormitochondrial

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 theArabidopsis and rice proteins

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 data-

bases 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 fluo-

rescent fusion proteins observed by confocal microscopy. A

full-length TADA:GFP fusion showed noGFPfluorescence. How-

ever, the first 259 amino acids of the TADA protein efficiently

targeted GFP into chloroplasts (Figure 2B). We observed no

fluorescence inmitochondria, cytoplasm, or in the nucleus. Thus,

the TADA protein contains anN-terminal targeting sequence that

can unambiguously target the protein into chloroplasts.

TADA Is Required for the Specific Editing of

Chloroplast tRNAArg(ACG)

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 Agri-

140-69-2-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

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-CAT-

MA1a58100 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 chloro-

plasts; 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.

2060 The Plant Cell

plants, amplification and sequencing of cp-tRNAArg(ACG) con-

firmed 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. Unaf-

fected 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 Arabi-

dopsis tRNAs are also expected to be edited by A-to-I deamina-

tion (http://www.inra.fr/internet/Produits/TAARSAT/table.html).

We therefore tested the hypothesis that TADA is also responsi-

ble 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 var-

iants 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 foundnoevidence 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 tom1I. 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 chloro-

plast 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 tRNAswith an A at thewobble 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

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.

tRNA Editing and Plastid Translation 2061

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 differ-

ences 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). Thismodel

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 theN-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 se-

quence. This insertion is also poorly conserved in sequence

among the different plant TADAs. Finally, our model also pro-

poses 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

Deaminationofcp-tRNAArg(ACG) tocp-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 do-

main (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) labeledwith

[32P]ATP. The presence of inosine was analyzed by thin layer

chromatography (TLC) as described (Grosjean et al., 2007). One-

dimensional 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

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

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.

2062 The Plant Cell

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, respec-

tively).

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

cp-tRNAArg(ICG) (Figure 5B), confirming that TADA is responsible

for 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 wild-

type phenotype (Figure 6), showing that it is the absence of cp-

tRNAArg(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 satu-

ration pulse-induced fluorescence measurements to determine

the effects of the tada-1mutation on photosynthesis and photo-

protection (Kramer et al., 2004). The maximum quantum effi-

ciency 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 dissipatedmore 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

(0.596 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 sev-

eral chloroplast proteins by protein gel blots. In tada-1 total

protein extracts, we saw a marked decrease of all chloroplast-

encoded 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 conse-

quence of a deficiency in the assembly of functional photosys-

tems, 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 ex-

periment showed that Met incorporation in RbcL is strongly

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 ex-

pressed in E. coli fused to an N-terminal His tag and tested for deam-

ination 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 two-

dimensional TLC (2 and 3). (1) Deamination activity of DN-TADA on cp-

tRNAArg(ACG) (lanes a to c), on cytosolic tRNAArg(ACG) and on cytosolic

tRNAAla(AGC). a, Control without protein; b, control for bacterial con-

tamination; 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 chloro-

plast targeting sequence of RECA1.

tRNA Editing and Plastid Translation 2063

reduced in tada-1, more than fivefold as determined by phos-

phor-imager quantification (Figure 8). This result does not ex-

clude 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 pro-

teins tested aswell 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 trans-

lation. To discriminate between the two possibilities, we ana-

lyzed 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 tran-

scription 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 poly-

merase (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 twoout

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.

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.

Table 1. Photosynthetic Efficiency

FPSII FNPQ Ff,D S

Col-0 0.51 6 0.025 0.23 6 0.019 0.26 6 0.013 1.0

tada-1 0.07 6 0.077 0.58 6 0.055 0.35 6 0.24 1.0

The photochemical efficiency, FPSII, light-dependent thermal dissipation

component of nonphotochemical quenching, FNPQ, the sum of fluores-

cence quenching and light-independent thermal dissipation, Ff,D, and

their respective sums, S, were determined at normal light. Four 3-week-

old plants per line were measured.

2064 The Plant Cell

DISCUSSION

TADA Is a tRNAArg Adenosine Deaminase

The chloroplast translation system is derived from its cyanobac-

terial ancestor and retains predominant prokaryotic character-

istics. 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 se-

quences 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 (Marechal-Drouard et al., 1993). cp-

tRNAs 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

high sequence similarity with bacterial TadA made three-dimen-

sional 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 comparedwith 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 cp-

tRNAArg(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 tRNAby 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 andKeller, 1999). However, the bacterial TadA is

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 plastid-

encoded and nucleus-encoded chloroplast proteins. Plants were grown

in vitro in agar plates without sucrose. PsbA, D1 protein; PetA, cyto-

chrome 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.

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 2065

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 deam-

inating 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 dis-

pensable 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).

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 DN-

TADA (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.

Table 2. Tryptic Digest of RbcL from tada-1

m/z Observed z Mr Calc Sequence Ion Score

RbcL 511.2719 +2 1020.524 DTDILAAFR41 59

625.3409 +3 1248.6714 ESTLGFVDLLR350 75

AtpA 626.8563 +2 1251.6935 VINALANPIDGR119 49

737.8978 +2 1473.7787 ASSVAQVVTSLQER216 52

AtpB 978.0159 +2 1954.016 (R109)IFNVLGEPVDNLGPVDTR127 94

736.3841 +2 1470.7541 VGLTALTMAEYFR261 89

1031.0219 +2 2060.0248 GIYPAVDPLDSTSTMLQPR378 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.

2066 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-harvest-

ing 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 combi-

nation of inefficient energy transfer from the antenna complexes,

reduced QA pool, and impaired electron transport chain perfor-

mance could result in a dramatically lower ratio of photons

absorbed by PSII being channeled through photochemical pro-

cesses (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 sta-

ble (Deana and Belasco, 2005; Dreyfus, 2009). This phenome-

non, also proposed by Pfalz et al. (2009), could explain the

accumulation of plastid transcripts in tada-1, despite the ex-

pected 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 compen-

satorymechanismsmay 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 nonstan-

dard decodingmechanism. Thismight 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 chlo-

roplast 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 Supple-

mental 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 reper-

toires according to the wobble rules (both should read CGU

codons efficiently), which raises the question ofwhy only;50%of

cp-tRNAArg(ACG) is apparently deaminated. The Arg codon usage

shows a significant bias toward CGU codons comparedwith 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 retentionof cp-tRNAArg(ACG), as it is expected to

bemoreefficient than cp-tRNAArg(ICG) at translatingCGUcodons.

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.

TwoOutofThreeDecodingProbablyOccurs inChloroplasts

A complete impairment of chloroplast translation is embryo-

lethal 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 ami-

noacyl-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 cp-

tRNAArg(UCU) is most unlikely because this would involve super-

wobbling 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 re-

quired 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 cp-

tRNAGly(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 2067

(Rogalski et al., 2008). This could bebecause, 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-typeArabidopsis plants, as the

Arg CGG codon cannot form a canonical wobble paring with

either cp-tRNAArg(ACG) or cp-tRNAArg(ICG). However, the de-

crease of translation activity in tada-1 clearly shows that cp-

tRNAArg(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 cp-

tRNAArg(ICG) enhances plastidial translation by replacing ineffi-

cient two out of three by canonical wobble codon-anticodon

pairing.

METHODS

Plant Materials and Growth Conditions

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 onMurashige 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 environ-

ment 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-CAT-

MA1a58100; 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. ForGK-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. Expres-

sion 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).

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 framewith an

N-terminal His tag and expressed in Escherichia coli strain ROSETTA

(DE3) (Novagen). The soluble protein fraction was purified by zinc-

chelating 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 concentra-

tions of glycerol plus 0.1% Triton and 0.1 mM ZnCl, until a final concen-

tration of 50% glycerol and no urea. The renatured soluble protein was

virtually pure, as estimated by Coomassie Brilliant Blue staining.

The activity of the protein was tested on in vitro–synthesized tRNAs

labeledwith [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 aBstNI site at the 39 terminus to correctly generateCCA39-ends.

After BstNI digestion, PCR fragments were used as templates for tran-

scription 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 substrateswere digestedwith 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-1Mutant

The genomic fragment coding for the last 280 amino acids of TADA (DN-

TADA) 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-TADAconstructwas confirmedbyPCRwith primers pairs P51+P52 and

P53+P54 amplifying the 59 and 39 borders of the construct, respectively.

Protein Gel Blots

Samples of 15 mg of proteins extracted from leaves of 3-week-old plants

were fractionatedbySDS-PAGEandblottedonto Immobilon-Pmembranes

(Millipore). Antibodies against proteinD1,PCOR,PsaD, plastocyanin, PsaA,

PsbO, and cytochrome b6 were purchased from Agrisera and used follow-

ing the manufacturer’s recommendations. Antibodies against LHCII, Ru-

bisco small subunit, and cytochrome fwere a kind gift of Geraldine Bonnard

(Institut de Biologie Moleculaire des Plantes). Proteins recognized by the

antibodies were revealed by ECL (GE Healthcare) and autoradiography.

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 leavesof tada-1were fractionatedona12%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 full-

length and the first 259 amino acids of TADA were amplified with primers

2068 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 transfec-

tion 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-1mutantswere 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 hexanu-

cleotide primers and Superscript III reverse transcriptase (Invitrogen).

Real-time PCR analysis was conducted in 384-well plates with a Light-

Cycler 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, andYLS8-

1 + YLS8-2 were used to measure the accumulation of nucleus-encoded

transcripts. The specificity of each primer pair was checked by sequenc-

ing 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 rawdatawere analyzed

using the LightCycler 480 software release 1.5 (Roche) and crossing

points determined by second derivative maximum analysis. The accu-

mulation of chloroplast transcripts was normalized by setting the average

ratio of nuclear transcripts to 1.

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, andP23+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 (vector-

specific) 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

Fluorescence induction kinetics at room temperature (228C) were mea-

sured 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 measure-

ments were made on four plants for each of the Col-0 and tada-1 lines.

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 struc-

tures 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 2e-

29; 124 and 2e-28; and 100 and 2e-21. The 2B3J structure was chosen as

scaffold formodeling 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.

Sequenceswere 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 insertionof 17 residues (GGEGNG-

SEASEKPPPPV) 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 fragmentsweremodeled 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-140-69-2-

CATMA1a58100 (N205329 from the Nottingham Arabidopsis Stock Cen-

tre); 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.

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.

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 2069

Supplemental Table 4. Frequencies of the CGN Codons in Arabi-

dopsis Chloroplast-Encoded Proteins.

Supplemental Table 5. Primers Used in This Study.

Supplemental Data Set 1. 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 Marechal-

Drouard for helpful discussions.

Received February 28, 2009; revised June 12, 2009; accepted June 26,

2009; published July 14, 2009.

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tRNA Editing and Plastid Translation 2071

DOI 10.1105/tpc.109.066654; originally published online July 14, 2009; 2009;21;2058-2071Plant Cell

Bergdoll, Nicolas L. Taylor, Barry J. Pogson, Ian Small, Patrice Imbault and José M. GualbertoEtienne Delannoy, Monique Le Ret, Emmanuelle Faivre-Nitschke, Gonzalo M. Estavillo, Marc

(ACG) and Is Essential for Efficient Chloroplast TranslationArgtRNA tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of ChloroplastArabidopsis

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References /content/21/7/2058.full.html#ref-list-1

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