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The Plant Journal (2011) 66, 80–93 doi: 10.1111/j.1365-313X.2011.04490.x THE PLANT GENOME: AN EVOLUTIONARY VIEW ON STRUCTURE AND FUNCTION A global picture of tRNA genes in plant genomes Morgane Michaud, Valérie Cognat, Anne-Marie Duchêne* and Laurence Maréchal-Drouard* Institut de Biologie Moléculaire des Plantes, UPR 2357-CNRS, Université de Strasbourg, 12 rue du Général Zimmer, F-67084 Strasbourg Cedex, France Received 30 September 2010; accepted 4 January 2011. * For correspondence (fax +33 3 88 61 44 42; e-mail anne-marie.duchene@ibmp cnrs.unistra.fr or [email protected]). SUMMARY Although transfer RNA (tRNA) has a fundamental role in cell life, little is known about tRNA gene organization and expression on a genome-wide scale in eukaryotes, particularly plants. Here, we analyse the content and distribution of tRNA genes in five flowering plants and one green alga. The tRNA gene content is homogenous in plants, and is mostly correlated with genome size. The number of tRNA pseudogenes and organellar-like tRNA genes present in nuclear genomes varies greatly from one plant species to another. These pseudogenes or organellar-like genes appear to be generated or inserted randomly during evolution. Interestingly, we identified a new family of tRNA-related short interspersed nuclear elements (SINEs) in the Populus trichocarpa nuclear genome. In higher plants, intron-containing tRNA genes are rare, and correspond to genes coding for tRNATyr and tRNAMete. By contrast, in green algae, more than half of the tRNA genes contain an intron. This suggests divergent means of intron acquisition and the splicing process between green algae and land plants. Numerous tRNAs are co-transcribed in Chlamydomonas, but they are mostly transcribed as a single unit in flowering plants. The only exceptions are tRNAGly–snoRNA and tRNAMete–snoRNA cotranscripts in dicots and monocots, respectively. The internal or external motifs required for efficient transcription of tRNA genes by RNA polymerase III are well conserved among angiosperms. A brief analysis of the mitochondrial and plastidial tRNA gene populations is also provided. Keywords: alga, angiosperm, non-coding RNA, genome evolution, organelles, SINE. INTRODUCTION Transfer RNA (tRNA) is among the most ancestral RNA in the world. Its role in cell life is essential as it represents the interface between the genetic code and protein. In addition to its essential function in translation, several recent studies have shown that tRNA is a multi-functional molecule that is involved in many processes of cellular metabolism (Francklyn and Minajigi, 2010). Furthermore, tRNA-derived RNAs appear to be used in the RNA silencing pathway, and are a major source of short interspersed short interspersed nuclear elements (SINEs; Bermudez-Santana et al., 2010; Phizicky and Hopper, 2010). It is postulated that all tRNA genes (tDNAs) derive from an ancestral ‘proto-tRNA’ (Eigen et al., 1989). During evolution, a full set of tRNA genes was generated as the result of numerous mutation, duplication and re-organization events. However, little is known about the organization, distribution and expression of tRNA genes or tRNA-related sequences on a genome-wide scale in eukaryotes, and comparative studies are rare (Goodenbour 80 and Pan, 2006; McFarlane and Whitehall, 2009; BermudezSantana et al., 2010). With the completion of several plant genomes, we now have the opportunity to study their complete tRNA gene sets. Using the results of whole-genome sequencing projects, the software tRNAscan-SE, and the few experimental datasets available, we have analysed the genomic content and distribution of tRNA genes and tRNA-related genes in five flowering plants (Arabidopsis thaliana, Medicago truncatula, Populus trichocarpa, Oryza sativa and Brachypodium distachyon) and one green alga (Chlamydomonas reinhardtii). We first manually curated the lists of tDNAs extracted from each genome, correctly annotating pseudogenes as well as organellar-like tDNAs. As a result of this analysis, a new family of short interspersed tRNA-related SINEs was identified in P. trichocarpa. This enabled us to present here the most up-to-date set of ‘true’ tDNAs present within plant nuclear genomes. We then provide a ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd tRNA genes in plants 81 comprehensive comparative view of tRNA gene composition and organization. Emphasis is given to the 5¢ and 3¢ ciselements involved in nuclear tRNA gene expression. The existence of co-transcribed tRNAs and intron-containing tRNAs is also discussed. The content, composition and expression of tRNAs in mitochondrial and chloroplastic genomes are also assessed to provide a full picture of tRNA gene sets in these organisms. TRNA SEQUENCE DETECTION AND ANALYSIS All available tRNA gene sequences from five angiosperms (A. thaliana, At; M. truncatula, Mt; P. trichocarpa, Pt; O. sativa, Os; B. distachyon, Bd) and one green alga (C. reinhardtii, Cr) were used. These species were selected as they possess the most annotated genomes in terms of their tDNAs. When necessary, some data were also retrieved from two other species: the green alga Ostreococcus tauri and the moss Physcomitrella patens. The nuclear sequence data were obtained from the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/), the Medicago truncatula sequencing resources http://www.medicagohapmap. org/?genome and the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/) for the other plants. Although we recently annotated the Chlamydomonas tDNA sequences using version 3.0 of the genome release (Merchant et al., 2007; Cognat et al., 2008), version 4.0 is now available with an updated list of tDNAs. Organellar genome sequences were obtained from the Genbank database (http://www.ncbi.nlm.nih.gov/) or from the Organelle Genome database (http://gobase.bcm.umontreal.ca/). A full list of the accession numbers for the genes is given in Table S1. The existence of tRNAs was first predicted by using the highly efficient automated analysis program tRNAscan-SE version 1.21 (Lowe and Eddy, 1997; http:// lowelab.ucsc.edu/tRNAscan-SE/) using default parameters. However, to minimize the number of false positives, we also manually curated the lists of tDNAs extracted from each genome. By means of the manual curation, we correctly annotated a few genes or pseudogenes that were misannotated, correctly annotated numerous non-expressed mitochondrial or plastidial tDNA sequences inserted into the nuclear genome and recognized by tRNAscan-SE, re-annotated some tRNAs in terms of amino acid and codon recognition due to the presence of post-transcriptional anticodon modifications, and identified new tRNA-derived SINEs that could not be distinguished from true tRNAs by tRNAscan-SE. For manual curation, the following parameters were taken into account: constraints on the sequence and secondary structure of tDNAs, conserved upstream and downstream transcriptional sequence elements, and BLAST alignments (http://blast.ncbi.nlm.nih.gov/Blast.cgi). As an result of manual curation, a new family of 35 SINEs (Figure S1) was discovered in the poplar nuclear genome, although they are assigned as tRNALeu genes by tRNAscanSE and described as such in the genomic tRNA database (http://gtrnadb.ucsc.edu/; Chan and Lowe, 2009). This new family is described below. Another example of the benefits of manual curation is reduction of previous over-estimates of the number of elongator tRNAMet (tRNAMete) genes in rice (http://gtrnadb.ucsc.edu/). Some of these are either mitochondrial or plastidial tDNAs inserted into the nuclear genome, but they lack the regulatory transcription elements required for nuclear tDNA expression. Currently, there is no evidence regarding whether an organellar-like tDNA integrated into a plant nuclear genome can become functional. Moreover, some tRNAMete correspond to isoleucine tRNAs that have been wrongly annotated as methionine tRNA genes due to the presence of a CAT anticodon. The C at the first position of the anticodon is post-transcriptionally modified to a lysidine derivative, enabling the tRNA to be isoleucine-specific (Weber et al., 1990). Thus, using both tRNAscan-SE prediction software and manual curation, we have performed the most thorough survey on plant tRNA genes to date (summary in Table 1). Although a few ‘true’ or ‘false’ tRNA genes may inevitably have escaped our analysis, this probably represents a very tiny percentage of all Table 1 Overview of the number of tDNAs present on the six plant nuclear genomes studied here A. thaliana M. truncatula P. trichocarpa O. sativa B. distachyon C. reinhardtii All True Pseudo Organellar 637 691 652 723 615 329 599 452 562 516 479 256 14 148 38 14 30 2 24 (23m, 1p) 91 (33m, 58p) 17 (2m, 15p) 193 (24m, 166p, 3m/p) 106 (5m, 101p) 0 Others 35 71 Introns 81 20 30 27 30 161 ‘All’ indicates the number of tDNAs detected using tRNAscan-SE. ‘True’ indicates the number of tDNAs considered as likely to be expressed after manual curation (i.e. subtraction of pseudogenes, SINEs and organellar-like tDNAs). ‘Pseudo’ indicates the number of tDNAs considered as pseudogenes. ‘Organellar’ indicates the number of mitochondrial- or plastidial-like tDNAs inserted into plant nuclear genomes. The number of mitochondrial-like (m) or plastidial-like (p) tDNAs is given in parentheses. In O. sativa, ‘m/p’ indicates the number of tRNA gene sequences for which the origin (m or p) has not been unambiguously determined. ‘Others’ indicates the number of SINEs. ‘Introns’ indicates the number of expressed tDNAs containing an intron. ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 82 Morgane Michaud et al. For each of the six genomes included in our survey, we determined the number of tDNAs and tRNA pseudogenes as well as the number of organellar tRNA genes inserted into the nuclear genomes (Table 1). We cannot exclude the possibility that a few of the transferred organellar tRNA genes became functional once integrated into the nuclear genome, but so far none of them has been shown to be functional (Tian et al., 2007). In the absence of any experimental evidence, we therefore considered them as unexpressed genes and they were not considered as ‘true’ tDNAs. The number of true tDNAs does not greatly differ from one plant species to another for those that we analysed. In flowering plants, this number ranges between 500 and 600, whereas the green alga C. reinhardtii has only approximately 250. This limited variation in the green lineage has been reported previously, and contrasts with the situation in the animal kingdom where the number of nuclear tDNAs varies drastically (e.g. a few hundred in monkeys and apes, but more than 100 000 in cows and rats; Bermudez-Santana et al., 2010). With the exception of the nuclear genome of A. thaliana, we noted an expected correlation between the number of tDNAs and genome size (Figure 1a). This result is also apparent when looking at the number of tDNAs per chromosome (Figure 1b); a mean of 1.5 tDNAs are present per Mb per chromosome. In general, single tDNAs are mostly evenly distributed along chromosomes from all plants studied in this analysis. Furthermore, tRNA isoacceptor genes are also randomly dispersed on the various chromosomes (Figure S2). In general, tDNA clusters are rare in plant nuclear genomes, with only a few exceptions, notably in A. thaliana (Figure 1). In A. thaliana, chromosome 1 contains two large clusters of tDNAs due to extensive gene duplications (Theologis et al., 2000). One cluster contains 27 tandem tRNAPro genes, and the second contains 27 tandem repeats of tRNATyr–tRNATyr–tRNASer genes. These clusters are mainly responsible for the large number of tDNAs per Mb found for chromosome 1. The other four chromosomes also have a higher number of tDNAs per Mb (mean 4) compared to the other plant species. Overall, this explains the larger number of tRNAs in A. thaliana, and may reflect a stronger general tendency to gene duplication in this species compared to other plants. However, clustering does also occur in other plants, but not at the same level. For example, in Brachypodium, the four copies of the tRNAIle(TAT) gene are all present on chromosome 1. This suggests that, in some cases, the number of tDNAs implemented by gene duplication is facilitated within the Nb of tRNA genes NUCLEAR TRNA GENE ORGANIZATION (a) 700 600 500 400 300 200 100 0 At Bd Os Pt Mt Cr 0 200 400 Genome size (Mb) 600 (b) Nb of tRNA genes/Mb tRNA genes, and should not affect the interpretation of the data. The complete curated sets of extracted tRNA genes from the species used for the analyses are available at http:// www-ibmp.u-strasbg.fr/Drouard/tRNA.html. 10 8 6 4 2 0 0 1 2 3 4 5 6 7 8 Chromosome 9 10 11 12 Figure 1. Number of tDNAs on nuclear genomes of flowering plants. (a) Correlation between tDNA number and genome size in A. thaliana (At), M. truncatula (Mt), Populus trichocarpa (Pt), O. sativa (Os), B. distachyon (Bd) and C. reinhardtii (Cr). (b) Number of tDNAs per Mb in each chromosome. At, black diamond; Mt, grey triangle; Os, black square; Bd, cross. same chromosome. However, other constraints probably exist, as very large clusters were only observed in A. thaliana genome. In contrast to the overall correlation between tDNA gene content and genome size, there is no such correlation for the number of tRNA pseudogenes (Table 1). Over the course of evolution, mitochondrial (mt) and plastidial (pt) DNA fragments have become integrated into the nuclear genomes of flowering plants (Lin et al., 1999; Tian et al., 2007; and references therein). Following these DNA transfer events, variable amounts of tDNAs originating from the endosymbiotic organelles are found in nuclear genomes of higher plants (Table 1), and show between 95 and 100% sequence identity with their organellar counterparts. The number of organellar tDNA insertions varies greatly from one plant species to another (none in green algae, 17 in P. trichocarpa and 193 in O. sativa). With the exception of Arabidopsis, the number of plastidial tDNAs is higher than the number of mitochondrial ones. Mitochondrial tDNAs are mainly localized on a single chromosome, for example A. thaliana chromosome 2, which contains 75% of the mitochondrial genome, M. truncatula chromosome 1 and O. sativa chromosome 12, due to large but rare mitochondrial DNA fragment insertion. In contrast, plastidial tDNAs are mostly scattered across all chromosomes (Table S2) and are found as short (100–200 bp) or large DNA insertions (up to ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 tRNA genes in plants 83 10 000 bp). This suggests that most tDNA transfers occurred through a DNA-mediated pathway rather than an RNAmediated pathway. Moreover, two means of insertion of plastidial tDNAs into rice nuclear DNA have been proposed. Either the transfer is direct (from the plastidial DNA to the nuclear DNA), or indirect via the mitochondrial genome (i.e. first a transfer from the chloroplast to the mitochondria and then from the mitochondria to the nucleus; Tian et al., 2007). NUCLEAR TRNA ISOACCEPTOR AND ISODECODER GENES The anticodon triplet recognizes an mRNA codon and represents one of the main functional regions of a tRNA molecule. Plants use the universal genetic code in the three compartments in which translation occurs, and the 61 sense codons are linked to the 20 standard amino acids. For each amino acid, up to five tRNAs with distinct anticodons (tRNA isoacceptors) are required. The various tRNA isoacceptors charging the same amino acid constitute one family. As shown in Figure S3, the number of tRNA isoacceptors ranges from 45 to 47 in the six species studied. Among these species, 45 tRNA isoacceptors are evolutionary related. The presence of one or two supplementary tRNA isoacceptors in some plant species is either due to orphan tDNAs of unknown origin (in P. trichocarpa, O. sativa and B. distachyon), or the existence of a tRNASec gene in C. reinhardtii. Selenocysteine (Sec) is a rare amino acid that is found in several selenoproteins. It is recognized as the 21st amino acid in the genetic code, and its codeword, UGA, is normally (b) 45 40 35 30 25 20 15 10 5 0 y = 0.3646x + 6.0016 R 2 = 0.70195 Gly Leu Ala Arg Lys Met Phe Cys Asp Thr Ile Glu Gln Asn Trp His 0 Val 20 A. thaliana 40 60 80 50 Isoaccepting tRNA gene copy number (a) Isoaccepting tRNA gene copy number a stop codon (Rao et al., 2003). In eukaryotes, selenoproteins are found in animals but are absent in fungi or higher plants. However, Sec-containing proteins were found in the green algae C. reinhardtii (Novoselov et al., 2002). As incorporation of selenocysteine into proteins requires a specific tRNA designated tRNASec, such a tRNA is present in C. reinhardtii but is absent in higher plants. In various organisms (e.g. bacteria, yeast, Drosophila melanogaster or Caenorhabditis elegans), the number of tRNA genes gives a good approximation of the abundance of each tRNA isoacceptor, which, in turn, is correlated with the amino acid frequency (Duret, 2000). To study whether there is also such a gene-dosage effect in plants, we analysed the relationship between the number of tRNA genes charging one amino acid, and the frequency of the corresponding amino acid for four of the higher plants (a codon usage table is not available for B. distachyon) and for the green algae C. reinhardtii. A good correlation between the number of tRNA genes and the frequency of the amino acids was observed for all plants analysed (Figure 2 and Figures S4 and S5). This is in agreement with a similar analysis performed using the rice nuclear genome (Itoh et al., 2007). The best correlation obtained was for the green alga C. reinhardtii, which may reflect the lower complexity of its nuclear genome. In the case of A. thaliana, a weak correlation was obtained when all amino acids were considered (R2 = 0.16, Figure S5A). This is due to the existence of three tDNA families (Tyr, Ser and Pro) that are highly y = 0.4245x + 6.2807 R 2 = 0.70376 40 30 Val Met 20 Trp 10 0 100 0 Amino acid frequency/1000 Ala Gly Tyr Cys His 20 Pro Gln Ser Lys Ile Asn Phe Asp Thr O. sativa 40 60 80 100 Amino acid frequency/1000 (d) 30 20 10 A. thaliana 0 0 20 40 Codon frequency/1000 60 Isoaccepting tRNA gene copy number Isoaccepting tRNA gene copy number (c) Arg Leu Glu 30 20 10 O. sativa 0 0 20 40 60 Codon frequency/1000 Figure 2. tRNA isoacceptors and tRNA isodecoders in plant nuclear genomes. (a, b) Correlation between the number of tRNA gene copies specific for each amino acid and the frequency of occurrence of the same amino acid in A. thaliana (a) and O. sativa (b). In A. thaliana, three amino acids, Ser, Tyr and Pro, were excluded from this analysis. For additional information, see Figures S4 and S5. The frequency of amino acids was estimated by counting the number of corresponding codons available on the codon usage database (http://www.kazusa.or.jp/codon/). (c, d) Correlation between the number of tRNA gene copies specific for their cognate codons and the frequency of occurrence of the same codon(s) in A. thaliana (c) and O. sativa (d). The frequency of codons was estimated by counting the number of corresponding codons available on the codon usage database (http:// www.kazusa.or.jp/codon/). Graphs were produced using the data presented in Table S3. ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 84 Morgane Michaud et al. amplified on chromosome 1. When the three families are excluded from the analysis (Figure 2), or when the numbers of tDNA copies present on chromosome 1 are subtracted from the total number of tDNA copies for the corresponding amino acid (Figure S5B), the correlation significantly increases (R2 = 0.70). According to in vitro expression analysis, most of the tRNAs belonging to these three gene families are very probably expressed (Stange et al., 1991; Yukawa et al., 2007). However, two questions remain to be answered: do the numbers of tRNASer, tRNATyr or tRNAPro genes correlate with the abundance of the corresponding tRNAs, and/or are these tRNAs involved in other processes than translation in A. thaliana? Another question is whether the correlation observed between the number of tRNA genes and the frequency of the corresponding amino acid also holds for the number of tRNA isoacceptor genes and the codon usage. The decoding properties of individual tRNA species have not been studied in detail in higher plants. Furthermore, only a few sequences are available at the RNA level, restricting our knowledge on modified nucleotides present at the first position of the anticodon and codon/anticodon recognition. The influence of codon/anticodon interactions and modified bases on codon usage has been well studied in bacteria (Ran and Higgs, 2010) but not in plants, and only educated guesses can be made regarding which isoacceptor tRNAs read which codons. It is usually accepted that, in vivo, most tRNAs can translate more than one codon, and some codons can be translated by more than one isoacceptor tRNA. Nevertheless, assuming that a codon is preferentially read by only one tRNA isoacceptor (see Table S3), we analysed the relationship between the number of isoacceptor tRNA genes and the codon usage in A. thaliana and O. sativa. As shown in Figure 2, a very good and unexpected correlation exists for both plants. This suggests the existence of a tRNA isoacceptor gene-dosage effect with very simple rules to optimize translation. Full genome sequencing has allowed us to identify another level of complexity: that some differences are found elsewhere in the sequence of tRNA isoacceptors. Recently, these tRNAs were defined as tRNA isodecoders by Goodenbour and Pan (2006). For A. thaliana, the various sequences for tRNAs belonging to the same group of tRNA isoacceptor can be visualized at http://www.inra.fr/internet/Produits/TAARSAT/. The number of tRNA isodecoder genes (i.e. the number of distinct tRNA sequences) has been determined (Figure S3). In higher plants, this number varies between 147 and 194, and is of the same order of magnitude as in animals (Goodenbour and Pan, 2006). In the green alga C. reinhardtii, only 83 tRNA isodecoder genes were found. However, as shown in Figure S3, the number of tRNA isodecoders is correlated with the number of tRNA genes within a nuclear genome, and suggests that tRNA isodecoders derive from neutral evolutionary drift. In general, among each tRNA isoacceptor family, tRNA sequences are highly conserved within the same species but also between species. Interestingly, in higher plants, some tRNA isoacceptors show much more sequence fluctuation than others (i.e. their number of tRNA isodecoders is high). For example, of 50 plant tRNAPhe sequences, 48 are identical and only two show nucleotidic divergence (with 2 and 8 nucleotide differences, respectively, compared to the 48 other sequences). For tRNAGln(CTG), for which the number of tRNA isodecoders is higher, tRNA sequences are less conserved among the tRNA isoacceptor family (Figure S6). A simple explanation for these differences may be that the set of sequence determinants to ensure correct L-shaped structure and charging of a tRNA by a specific amino acid varies from one tRNA isoacceptor to the other. Each tRNA contains a few sets of nucleotides that are required for tRNA recognition by the cognate aminoacyltRNA synthetase. These identity elements are mostly located at the two distal extremities of tRNAs: the anticodon loop and the acceptor stem (Giege et al., 1998; Giege, 2008). It is generally assumed that similar aminoacylation rules are used in the various eukaryotes. Only a limited amount of data are available concerning such identity elements in plants. In most cases of aminoacylation, studies revealed that plant identity elements are similar to those found in other organisms (Carneiro et al., 1994; Dietrich et al., 1996; Giege et al., 1998; Ulmasov et al., 1998; Delage et al., 2003; Wu et al., 2007), but some differences were also observed (Salinas et al., 2005). For example, the G3.U70 base pair was identified as an identity element in Escherichia coli, Saccharomyces cerevisiae and Thermus thermophilus tRNAAla (Giege et al., 1998). In accordance with these results, mutation of G3.U70 to G3-C70 in A. thaliana tRNAAla inhibits its alanylation (Carneiro et al., 1994). In tRNAGly, the nucleotide at position 73 discriminates between prokaryotes (U73) and eukaryotes (A73). However, in plants, the mitochondrialencoded tRNAGly contains U73 and the three nuclearencoded tRNAGly have A73. Surprisingly, the active mitochondrial glycyl-tRNA synthetase (GlyRS), which is similar to prokaryotic GlyRSs, is able to glycylate both the mitochondrial-encoded and nuclear-encoded tRNAsGly (Salinas et al., 2005). An important question for the future is whether the differential sequence flexibility observed in the tRNA isoacceptor gene families only reflects selection pressure and/or whether some tRNA isodecoders perform distinct functions. Analysis of various biological samples coupled to microarrays as well as aminoacylation studies on tRNA isodecoders will enable such studies to be initiated. NUCLEAR TRNA GENE EXPRESSION Nuclear tDNAs are transcribed by RNA polymerase III (PolIII). Their promoters are typically PolIII type 2, and consist of conserved sequence elements within the transcribed region. ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 tRNA genes in plants 85 5¢-TGGCNNAGTGG-3¢ and 5¢-GGTTCGANNC-3¢ for A and B boxes, respectively. These sequences are highly conserved in A. thaliana tDNA sequences, with consensus sequences of 5¢-Tg/aGC/tNNAGTg/tG-3¢ and 5¢-G/aGTTCGANNC-3¢, respectively (Figure S7). The A box is found at positions 8–18, and the B box is 30–60 nucleotides downstream of the A box, depending on the presence of introns and the length of the variable loop in tRNAs. In most cases, the B box is located 13–22 nucleotides upstream of the end of the mature tRNA. These elements, called A and B boxes, are highly conserved in eukaryotes, partly because they encode the tRNA D and T loops (Galli et al., 1981; Hofstetter et al., 1981). Upstream elements were also found to be important for transcription of many tRNA genes. For example TATA box-like sequences were shown to greatly contribute to transcription efficiency in many organisms (Hamada et al., 2001; Schramm and Hernandez, 2002; Giuliodori et al., 2003; Dieci et al., 2006, 2007). In plants, analysis of the 50 nucleotides immediately upstream of 68 tDNAs has shown a high proportion of A and T nucleotides in this region, the presence of TATA-like motifs and the prevalence of CAA triplets (Choisne et al., 1998). AT-rich regions and TATA-like sequences appear to be important for a high level of expression, and CAA triplets correspond to transcription initiation sites (Ulmasov and Folk, 1995; Yukawa et al., 2000). We have therefore searched in more detail for such motifs in photosynthetic organisms. Frequency of AT and TATA-like elements upstream of tDNAs The 50 nucleotides upstream of tDNAs appear to be enriched in A and T residues compared to tDNA sequences. Moreover, there is a clear difference between flowering plants (approximately 70% AT in dicots and monocots) compared to the green alga C. reinhardtii (<50%; Figure 3a). However, these values should be viewed with caution because the AT content varies between genomes. For example, Chlamydomonas is only 36% AT-rich, compared with 64% in Arabidopsis. Consequently, the increase in the percentage of AT in A and B boxes The transcriptional internal control regions, called A and B boxes, are highly conserved among eukaryotes, particularly in the plant kingdom. The consensus sequences are (b) % 100 At (64%) Mt (67%) Pt (67%) Os (59%) Bd (54%) Cr (36%) 75 50 25 0 –50 –40 –30 –20 –10 Nucleotides position 0 0 5’ –50 –40 –30 –1 3’ –10 –20 (d) 60 30 50 25 % of sequences % of sequences (c) 1 Bits (a) 40 30 20 15 10 5 10 0 20 0 0 1 2 3 Number of CAA motifs 4 –15 –12 –9 C position –6 –3 Figure 3. Upstream region (from )50 to )1) of tDNAs. (a) AT frequency. The sequences were analysed in stretches of five nucleotides. On the x axis, 0 represents the sequence of mature tRNA from positions 1–5, )5 represents the upstream region from positions )1 to )5, and )50 represents the sequence from )46 to )50. The percentage in parentheses represents the AT percentage in the whole genome. (b) WEBLOGO analysis (http://weblogo.berkeley.edu/) of the upstream region of tDNAs in flowering plants. (c) Number of CAA triplets in flowering plants. (d) Position of the C residue in the most proximal CAA triplet. ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 86 Morgane Michaud et al. tDNA upstream regions compared to whole genomes is roughly the same in all species. In flowering plants, the percentage of AT is particularly high between positions )25 and )35, with a mean of 83% (Figure 3b). TATA-like sequences are found in this position upstream of many PolIII class III genes and some class II genes (Dieci et al., 2007). TATA-like sequences in direct orientation (as defined by PLACE; http://www.dna.affrc.go.jp/PLACE/signalup.html) are found in the region between )20 and )40 of 18% of upstream sequences in flowering plants, but are absent in the green alga. In this context, Chlamydomonas resembles animals, in which TATA boxes upstream of tDNAs do not occur frequently (Ulmasov and Folk, 1995). CAA motifs The )1 to )10 region is particularly rich in A residues but also in C residues (Figure 3b), and CAA triplets have been shown to be transcription initiation sites in tobacco (Nicotiana tabacum; Yukawa et al., 2000). In A. thaliana, 78% of tDNAs have one or more CAA motifs in the )1 to )50 region (Figure 3c), with the most proximal CAA motif starting at )6, )5 or )4 (Figure 3d). In agreement with this result, 65% of sequences in flowering plants have at least one CAA triplet in the )1 to )10 region, but this motif is only found in 13% of C. reinhardtii sequences. Downstream element PolIII terminates transcription at short runs of T residues (at least four; Dieci et al., 2007). The presence of short stretches of T residues downstream of plant tDNAs has been reported previously in Arabidopsis (http://www.inra.fr/internet/Produits/TAARSAT/). Its importance in PolIII transcription termination has been shown experimentally (Ulmasov and (a) Folk, 1995; Yukawa et al., 2000). In addition, the poly(U) tail is specifically recognized by the La protein (Wolin and Cedervall, 2002), and plays an important role in stabilizing the primary tRNA transcript. In C. reinhardtii, a short poly(T) tail is present 1–10 nucleotides downstream of the predicted 3¢ end on two-thirds of the tDNAs but is absent on the remaining third. In the latter case, lack of such a tail is probably due to the presence of polycistronic tRNAs, a situation that is usually not found in eukaryotes, in particular higher plants (Cognat et al., 2008). Indeed, in the five flowering plants studied here, a short run of T residues (4–15) has been found in the first 25 bp downstream of 90–97% of the tDNAs. In most other cases, such as several tRNATrp genes, the poly(T) stretch is found further downstream than the first 25 nucleotides. Finally, a few tRNA genes do not have any poly(T) domain in the near vicinity. They correspond to tRNAs that are co-transcribed with small nucleolar RNAs (snoRNAs). These snoRNAs contain a so-called C/D box, and play an essential role in guiding the methylation of ribosomal RNAs and a few other RNAs in eukaryotes. Families of di-cistronic tRNAGly–snoRNA genes and di-cistronic tRNAMete–snoRNA genes have been found on the Arabidopsis and rice nuclear genomes, respectively (Kruszka et al., 2003). We searched for di-cistronic tRNA–snoRNA genes in the five higher plants, and confirmed the widespread occurrence of these di-cistronic genes (Figures S8 and S9). Interestingly, the tRNAGly–snoRNA gene family was only identified in dicots (i.e. A. thaliana, M. truncatula and P. trichocarpa, with 9, 14 and 13 members, respectively) and the tRNAMete–snoRNA gene family was only identified in monocots (i.e. O. sativa and B. distachyon, with seven members in each plant; Figure 4). No other tDNAs associated to a snoRNA gene were retrieved using our criteria, i.e. tRNAGly Mt Pt At C (b) tRNAMete Os Bd D Figure 4. Sequence alignment of di-cistronic tRNA–snoRNA genes present in flowering plant species. (a) Sequence alignment of representative tRNAGly–snoRNA co-transcripts in A. thaliana, M. truncatula and P. trichocarpa. (b) Sequence alignment of representative tRNAMete–snoRNA co-transcripts in O. sativa and B. distachyon. The C and D conserved motifs of snoRNA sequences are indicated. Arrowheads indicate the putative 5¢ and 3¢ ends of snoRNA genes. Note the stretches of T residues present downstream of snoRNA gene sequences. ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 tRNA genes in plants 87 the presence of C/D boxes and poly(T)s downstream of the snoRNA gene. Whether the variants are all expressed and whether they are used to methylate the same rRNA sites requires experimental validation. Analysis of tRNA gene expression Although sequence elements required for tRNA gene expression in plants have been characterized (see above), only a handful of datasets are available regarding in vivo expression of plant tRNA genes. In C. reinhardtii, it has been shown that the most abundant tRNAs recognize the most frequent codons, whereas tRNAs of low abundance recognize rare codons (Vinogradova et al., 2009), but a complete analysis has not been performed. Similarly, a global expression profile on higher plant tRNAs is not yet available, although a considerable number of small non-coding RNA libraries have been obtained using deep sequencing technology. However, such libraries are usually restricted to 15–35 nucleotide long RNAs: classically full-length tRNAs are excluded from the analyses and only fragments of tRNAs are retrieved as by-products. In the near future, it would be of interest to use such deep sequencing approaches to perform a global analysis of tRNA expression from various plant tissues or under various environmental conditions. INTRON-CONTAINING TRNA GENES Only two families of higher plant nuclear-encoded tRNAs, tRNAMete and tRNATyr, have so far been reported to contain introns. In vitro maturation assays on mutated tRNATyr or tRNAMete have revealed the importance of the nucleotide sequence and also the pre-tRNA structure on the maturation (a) tRNAMete tRNATyr A A C C C C A A C G G C C G G C G C G C A U A C G C G C U A C G G C U A A U U CG UG U G A C UCGA U UGA UGGCC U A UGGCC CUCUC U A G C GCGA U U G G G ACUGG GAGAG ACUGG U GAGC G GCGC U C CUA UC U G C UC U C G GC A U U C U G U G U U GA A G G A U G A U G G C G C A C G A C G U C A UA U C U U AA G C GAU C GU U A U A G C C G G C C G U A G C A UA C A G A A U AG UC G AU .... ... . ... ... . ... .. ..... .... ... . ... ... . ... .. ..... efficiency of introns (Stange et al., 1992; Akama et al., 1997, 2000). In the predicted pre-tRNA structures, the 5¢ and 3¢ cleavage sites are present in single-stranded RNA regions separated by 3–5 bp (Figure 5a; Stange et al., 1991; Akama et al., 2000). These structural features have been shown to be important for in vitro intron splicing of tRNATyr and tRNAMete (Stange et al., 1992; Akama et al., 2000). Other experiments have demonstrated the deleterious effect of a cytidine residue at the 5¢ or 3¢ intron borders on intron maturation (Akama et al., 1997, 2000), and involvement of the D stem and the variable loop in pre-tRNATyr intron splicing (Stange et al., 1992). Our survey shows that all higher plant tRNAMete and tRNATyr genes contain an intronic sequence between positions 37 and 38 of their tRNA sequences. Analysis of the 185 tRNAMete and tRNATyr and their intronic sequences reveals: (i) striking similarities in the sequence and structure of the anticodon stem and the variable loop (Figure 5a), (ii) a variation in intron size (6–22 nucleotides), and (iii) limited sequence conservation in introns (Figure 5b). The intron sequences in the pre-tRNAMete family are more conserved than in the tRNATyr family. Sixty-two of the 65 tRNAMete introns have a GCT motif at the 5¢ end of the intron and a GAGT motif one nucleotide upstream of the 3¢ end of the intron. The GAG nucleotides are involved in the base pairing necessary to maintain the structure of the pre-tRNAMete, which is a prerequisite for intron splicing (Figure 5a; Akama et al., 2000). In the tRNATyr family, only a T at the first position of the intron and the G residue 2–4 nucleotides before the 3¢ end of the intron are conserved (Figure 5b). The T residue appears to be important for formation of pseudouridine at the second position of the anticodon (Akama et al., (b) tRNATyr At Mt Pt O s B d T AGACG . . . . . . . . . CAGA T T . T TCT T T AA . . . . . . . CAGAC . . T GGG T GA A A T CC T GA T GGC C . . T T G T T G . . . . . . . . . A GG T A . . T T TGAAG . . . . . . . . CAGA T T A T G tRNAMete At Mt Pt O s B d GC T A C . . . T GA GT G GC T T C . . . T GA GT A GC T T C . . . T GA GT T GC T A T . . . T GA GT T GC T T A A A GCGA GT G GC T GA GT Figure 5. Intron-containing tDNAs in flowering plant nuclear genomes. (a) Cloverleaf secondary structures of Arabidopsis intron-containing pre-tRNAMete and pre-tRNATyr. Intron cleavage sites are indicated by arrows. Anticodons are indicated by black dots. Nucleotides in the anticodon stem and variable loop region that are conserved between the tRNAMete and tRNATyr gene families are shown against a blue background. Nucleotides within intron sequences that are conserved within the tRNAMete or tRNATyr family are shown against green and grey backgrounds, respectively. (b) Alignments of the intron sequences present in pre-tRNATyr and pre-tRNAMete of flowering plants. For simplicity, only one representative sequence per nuclear genome is presented. Full sequence alignments are available upon request. The conserved nucleotides are underlined. ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 88 Morgane Michaud et al. 1997). Experimental analyses are required to determine the role of these conserved domains on intron splicing efficiency. In addition to the above intron-containing tRNAs, a putative tRNAAla in O. sativa and a putative tRNAThr in B. distachyon contain an intron between positions 37 and 38, but of a much larger size (129 and 132 nucleotides, respectively). These tRNA genes (Figure S10) are orphan genes and are not present in the other higher plant nuclear genomes. The B. distachyon tRNAThr gene is lacking one nucleotide in the anticodon stem. Nevertheless, the A and B internal boxes as well as the upstream CAA triplet and a downstream poly(T) stretch are present in both tDNAs. Whether these two tRNAs are expressed and functional is questionable and awaits experimental evidence. In green algae, the situation appears different. In C. reinhardtii, of the 256 tRNA genes detected in the genome, 161 have an intron (Merchant et al., 2007). The intron size is more variable compared to higher plants, and ranges from 9 to 56 nucleotides. Alignment of these intronic sequences does not reveal any obvious conservation at the nucleotide level, and nothing is known about the pre-tRNA structure in C. reinhardtii at present. The tRNAMete and tRNATyr genes also contain an intron in C. reinhardtii (Figure S11). Mature sequences of these two tRNA families present in the anticodon stem and the variable loop, the characteristics found in higher plants. Although the tRNATyr intron shows similarities in size and sequence to those of higher plants (T at position 1 and a G residue 2–4 nucleotides before the intron’s 3¢ end), tRNAMete introns do not share the GCT and GAGT motifs and are much longer in size compared to tRNAMete introns in higher plants. In the green alga O. tauri, of 48 tRNA genes identified, 16 contain an intron, whereas of the 432 tDNAs identified in the moss P. patens, only tRNAMete and tRNATyr genes contain an intron. As a whole, flowering plants and bryophytes appear to have evolved differently from the green alga kingdom with respect to the presence of introns in tRNA genes. TRNA-RELATED SINES Short interspersed nuclear elements (SINEs) are mobile non-autonomous elements that are found in a wide variety of eukaryotic genomes and are transcribed by PolIII. The best known family of SINEs is the Alu family (Kramerov and Vassetzky, 2005). SINE retroelements use the enzymatic machinery of autonomous long interspersed elements (LINEs) for propagation by retrotransposition. The SINE/ LINE partnership is based on common 3¢ regions or poly(A) tails, and a general model of SINE and LINE retroposition has been proposed by Sun et al. (2007). Although most SINEs are ancestrally derived from tRNA genes (or in rare cases from 7SL or 5S RNAs), the typical tRNA cloverleaf structure is not apparent. SINEs derived from tRNAs have a composite structure comprising a tRNA-related 5¢ portion followed by a tRNA-unrelated region. In all cases, the A and B boxes corresponding to the internal promoter are present in the tRNA-related portion. A eukaryotic phylogenetic tree of SINE structures has been tentatively constructed (Sun et al., 2007). The authors proposed that SINE RNAs started as simple structures with a single tRNA-related segment. Then either this segment is amplified, or short or long tRNAunrelated segments are added to the original molecule. The first SINE described in plants, p-SINE1, was found in the O. sativa Waxy gene (Umeda et al., 1991). In A. thaliana and Brassica species, various families of tRNA-related SINEs, called RathE1, RathE2, RathE3, S1 and S2 have been identified (Pelissier et al., 2004). SINEs are very short (<500 bp) and have no conserved sequence, making research into them very difficult. Despite these difficulties, SINEs have now been identified in many plant species, and are usually dispersed randomly in nuclear genomes (for review, see Deragon and Zhang, 2006). SINEs are usually not present in micro-organisms, and Chlamydomonas is the first unicellular organism in which such elements have been identified (Merchant et al., 2007). Remarkably, the three SINE tRNArelated families identified in this green alga have retained an authentic tRNA structure, resembling tRNAArg(CCG), tRNAAsp(AUC) and tRNAAsp(GUC), as well as an intron between positions 37 and 38 of the tRNA sequences. However, in vivo, no mature tRNA from the three types of SINE RNAs was detected, demonstrating that the correct cloverleaf folding required by tRNA-processing enzymes is not present, and that Chlamydomonas tRNA-related SINE RNAs do not fold like their ancestral molecule, as already demonstrated for other SINEs (Pelissier et al., 2004; Cognat et al., 2008). Completion of the poplar whole-genome sequencing project enabled us to identify a new tRNA-related SINE family of 35 members in P. trichocarpa (Figure 6 and Figure S1). Surprisingly, as for Chlamydomonas, the tRNA-related portion can be folded into a mostly perfect cloverleaf tRNA structure with an anticodon AAG (theoretically specifying leucine). The tRNA-unrelated portion is very well conserved among the family, and is terminated by a poly(A)-rich region followed by a poly(AT)-rich domain. The 35 members of the family are randomly dispersed on the various scaffolds (Figure S1). Identification of this new family of SINEs represents an additional example of the complexity of these elements, and their probable importance in both the evolution and organization of plant nuclear genomes. In most cases, their function remains unknown and needs to be elucidated. ORGANELLAR TRNA GENES In plants, translation occurs in the cytosol but also in mitochondria and plastids. These two organelles originate from endosymbiotic a-proteobacteria or cyanobacteria, respectively. Both organelles have a genome, but many genes have been lost or transferred to the nucleus during ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 tRNA genes in plants 89 (a) tRNA-related A Internal region Poly AT B Poly A 75 110 15 40 (b) Figure 6. tRNA-related SINEs in P. trichocarpa. (a) Schematic representation of the structure of the putative tRNALeu-related SINE members. The A and B boxes (dark grey) within the tRNA (black) required for transcription, and the poly(A)-rich (pale grey) and poly(AT)-rich (medium grey) regions are indicated. The size (in nucleotides) of each domain is given. (b) Cloverleaf structure of a representative tRNA-like region of the putative tRNALeu-related SINE transcripts. The location of the intron-like sequence is indicated. The three black dots correspond to the anticodon. Blue and red dots correspond to U–A or U.G and C–G pairings respectively. Thirty-five tRNA-related SINE sequences were identified (Figure S1). evolution. The few remaining genes encode proteins and structural RNAs (all rRNAs and some tRNAs). In A. thaliana, O. sativa and P. trichocarpa chloroplast genomes, 37, 38 and 39 tDNAs have been annotated, respectively. These genes correspond to 30 tRNA isoacceptor species (Hiratsuka et al., 1989; Sato et al., 1999; http:// genome.ornl.gov/poplar_chloroplast/). In Chlamydomonas, only 30 tDNAs are present on the plastidial genome, corresponding to 25 tRNA isoacceptor species (Maul et al., 2002). In all cases, the various tRNAs are sufficient to allow protein synthesis, and it is generally assumed that the chloroplast genomes encode all tRNAs required for translation. In agreement with this hypothesis, knockout of tRNAAsn(GUU) or tRNACys(GCA) tobacco plastidial genes showed that these two genes are essential for plant development, and that the absence of plastidial tRNAAsn or tRNACys cannot be compensated for by import of their cytosolic counterparts into chloroplasts (Legen et al., 2007). However, it appears that some tRNA genes have been lost on the plastidial genome of a few organisms, such as the parasitic non-photosynthetic plants Epifagus virginiana and Oronbranche minor (Wolfe et al., 1992; Lohan and Wolfe, 1998) and some dinoflagellates (Howe et al., 2008). Although it has not been shown, it is tempting to speculate that these missing tRNAs are imported from the cytosol into plastids. Overall, 17–29 tDNAs have been identified in plant mitochondrial genomes, with the notable exception of Chlamydomonas mitochondria, which contain only three tDNAs (Vinogradova et al., 2009). Transfer RNA genes corresponding to Ala, Arg, Leu, Thr and Val are missing in all sequenced angiosperm mitochondrial genomes. Nuclear-encoded tRNA import has been shown to compensate for the deficiency of mitochondrial tDNAs (Schneider and Maréchal-Drouard, 2000; Duchêne et al., 2009) (see below). Another characteristic of mitochondrial genomes is acquisition of tDNAs of another origin, particularly plastidial origin. As an example, in Arabidopsis, 11 tRNA species called ‘native’ tRNAs are encoded by authentic mitochondrial genes, and four others are chloroplast-like tRNAs. The remaining tRNAs, corresponding to seven amino acids, are imported from the cytosol (Duchêne and Maréchal-Drouard, 2001). Similar data have been obtained in numerous plant species (Rainaldi et al., 2003). Expression of organellar tDNAs Plastids have two types of RNA polymerases: plastidencoded eubacterial-type RNA polymerase (PEP), and nuclear-encoded phage-type RNA polymerases (NEPs). Standard PEP promoters resemble E. coli promoters, with consensus sequences at positions )35 (TTGACA) and )10 (TATAAT). NEP-dependent promoters are of two types. Some (type I) have a YRTA motif, and others (type II) have no identified consensus. Some tRNAs are co-transcribed with other RNAs. For example, the four rRNA molecules are co-transcribed with tRNAIle, tRNAAla and tRNAArg from an NEP and/or a PEP promoter in higher plants (LealKlevezas et al., 2000; Shiina et al., 2005). Transcription of tRNAGlu(UUC) and tRNAVal(UAC) genes is controlled by a PEP promoter in A. thaliana (Hanaoka et al., 2003). Mitochondrial genes are transcribed by two NEPs, and many promoter regions have a CRTA consensus sequence (Fey et al., 1999; Kühn et al., 2005). Numerous promoters are scattered along the genome, and multiple promoters for a single gene are also common. Indeed transcription of protein, rRNA or tRNA genes in mitochondria appears to be a relaxed process with little control or modulation (Holec et al., 2006). Import of nuclear-encoded tRNAs into mitochondria To provide an active mitochondrial translation system and to compensate for the lack of mitochondrial-encoded tRNAs, some nuclear-encoded tRNAs used in the cytosolic transla- ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 90 Morgane Michaud et al. tion machinery are imported into mitochondria. Gene loss and tRNA import acquisition occurred very early during plant evolution (Akashi et al., 1996; Vinogradova et al., 2009). The number and identity of imported tRNAs vary from one plant to another. Import of tRNAs corresponding to Ala, Arg, Leu, Thr and Val occurs in all known angiosperms, but gene loss and import of the corresponding tRNA can also occur in individual species such as tRNATrp in Arabidopsis (Salinas et al., 2008, Duchêne et al., 2009) or tRNASer in Helianthus annuus (sunflower; Ceci et al., 1996). The most detailed surveys in higher plants have been performed for potato (Solanum tuberosum; Maréchal-Drouard et al., 1990; Brubacher-Kauffmann et al., 1999), wheat (Triticum aestivum; Glover et al., 2001) and Chlamydomonas mitochondria (Vinogradova et al., 2009). Various tRNA isoacceptors are usually found in mitochondria, although the extent of their steady-state levels in the organelle may vary (Glover et al., 2001; Vinogradova et al., 2009). Comparing tRNA isoacceptors in C. reinhardtii, the extent of mitochondrial localization was shown to be highly variable. For instance, tRNAGly(CCC), tRNAVal(CAC), tRNALeu(CAG) are mainly cytosol-specific, tRNAGly(GCC), tRNAVal(AAC) are equally present in both compartments, whereas tRNAVal(UAC) and tRNALeu(CAA) are mainly localized in mitochondria. As discussed below, the observed differences might reflect the need for mitochondria to optimize translation. Many studies have been and are still being performed to understand the tRNA import process, in particular to determine the specificity and targeting determinants, and to identify mitochondrial membrane channels. The anticodon, D and T loops have been identified as import determinants in plant mitochondria. At the level of the mitochondrial outer membrane, the voltage-dependent anion channel and some components of the protein import machinery (TOM complex) have been shown to be essential for tRNA import (Salinas et al., 2008; Duchêne et al., 2009). However, elucidating the tRNA mitochondrial import process is still a huge challenge. Organellar tRNAs and codon usage In many organisms, there is a good correlation between codon usage and the level of corresponding tRNAs. Adjustment of the tRNA population to codon usage has been demonstrated in chloroplasts (Pfitzinger et al., 1987), and the concentration of various tRNAs is not controlled by gene dosage (Maréchal-Drouard et al., 1993). Plastidial genomes of photosynthetic plants usually encode 30 tRNA species and do not import tRNAs from the cytosol. According to standard rules, 32 tRNAs are required to read all codons of the universal genetic code (Crick, 1966). However, the existence of relaxed wobble rules has been proposed (Percudani, 2001). In particular, the presence of a modified uridine at the first position of the anticodon enables a single tRNA to decode the four codons of a codon box. For example, Pfitzinger et al. (1990) showed that, due to a modified uridine at position 34, plastidial tRNAAla or tRNAPro are able to decode the four-codon family. In addition, by producing knockout mutants for the plastidial tRNAGly(UCC) or tRNAGly(GCC) genes, Rogalski et al. (2008) demonstrated that tRNAGly(UCC) containing an unmodified U in the anticodon can read all four glycine codons due to ‘superwobbling’, whereby the U can pair with the four nucleotides. Similarly, tobacco plants from which the tRNAVal(GAC) gene had been deleted from the plastidial genome showed normal development (Corneille et al., 2001). As no import of cytosolic tRNAVal occurs in these transplastomic plants, the GUN codon family is probably recognized by the only remaining plastidial tRNAVal(UAC) (P. Maliga, S. Corneille, Waksman Institute of Microbiology Rutgers, The State University of New Jersey, USA, and L. Maréchal-Drouard, unpublished data). In the plant mitochondrial translation system, wobble pairing rules for codon–anticodon recognition have not been established, and it is difficult to draw precise conclusions on the minimal set of mitochondrial tRNAs required for an active translation machinery. However, using classical wobble rules, it is worth noting that, in Chlamydomonas mitochondria, a good correlation was found between the steady-state levels of imported tRNAs and the frequency of the cognate codon, not only in mitochondria but also in the cytosol (Vinogradova et al., 2009). Furthermore, it is possible to estimate the steady-state levels of potato mitochondrial tRNAs fractionated using 2D-PAGE (Maréchal-Drouard et al., 1990) and to compare their level to the plant mitochondrial codon usage (L. Maréchal-Drouard, unpublished data). Leucine codons are the most frequently used mitochondrial codons (approximately 12.5%) and imported tRNAsLeu are the most abundant mitochondrial tRNAs (approximately 15%). Codons corresponding to cysteine and tryptophan are the least frequently used (approximately 1.7% for each amino acid), and are recognized by mitochondrial-encoded tRNAs that are present in low amounts in the organelle (approximately 1.5% for each tRNA). There are also tRNAs corresponding either to mitochondrial-encoded (e.g. tRNAPhe) or nuclear-encoded (e.g. tRNAVal, tRNAArg) species that are present in equal amounts and recognize codons similarly used in the mitochondria (5–7%). In addition, it should be noted that, although the plant mitochondrial codon usage does not fluctuate greatly, the origin of the mitochondrial tRNA population varies significantly from one plant species to another. Thus, it appears likely that no correlation exists between the origin (mitochondrial or nuclear) of a mitochondrial tRNA and the frequency of the cognate codons. CONCLUSION Analysis of tDNAs among various plant genomes highlights many common characteristics, but also some differences ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93 tRNA genes in plants 91 within angiosperms or between angiosperms and green alga. A good correlation was observed between the number of tDNAs and genome size. The number of isoacceptors is also nearly identical in all species and correlates with codon usage. However, Arabidopsis breaks these rules due to the very high numbers of three tRNA isoacceptor genes. The various genomes are not homogenous with regard to the number of pseudogenes, organellar-like tDNAs or SINEs. Whether these elements are a source of genetic instability in plant nuclear genomes will need to be assessed in the future. Flowering plants share a common transcriptional and post-transcriptional organization (single transcription units, upstream and downstream signals, types of introns) and are clearly different from the green alga Chlamydomonas. The great flexibility in tDNA organization and transcription generates the ubiquitous, highly structured tRNA molecules. However, the presence of a large number of tRNA isodecoder genes raises a question regarding their potential function: are they simply the result of neutral mutations during evolution, or do they play an important role within the cell? This comprehensive analysis provides a basis for further studies, and the next challenges will be to explore the influence of this flexibility on genome maintenance and expression. ACKNOWLEDGEMENTS We thank Lee Tuddenham (Architecture et Réactivité de l’ARN, UPR 9002 CNRS, Strasbourg, France) for thorough assessment of our manuscript. M.M. holds a fellowship from the French Ministère Délégué à l’Enseignement Supérieur et à la Recherche. This work was supported by the Centre National de la Recherche Scientifique and the University of Strasbourg. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Sequence alignments of the tRNA-related SINEs found in P. trichocarpa. Figure S2. Distribution of tDNAs on B. distachyon and A. thaliana chromosomes. Figure S3. Number of tRNA isoacceptors and tRNA isodecoders per nuclear genome, and ratio between the number of tRNA isodecoders and the number of tRNA genes. Figure S4. Correlation between the number of tDNA copies specific for each amino acid and the frequency of occurrence of the same amino acid in M. truncatula, P. trichocarpa and C. reinhardtii. Figure S5. Correlation between the number of tRNA gene copies specific for each amino acid and the frequency of occurrence of the same amino acid in A. thaliana. Figure S6. Number of tRNAPhe(GAA) or tRNAGln(CTG) isodecoders per number of tDNAs. Figure S7. A and B box sequences in A. thaliana tDNAs. Figure S8. tRNAGly–snoRNA co-transcripts in A. thaliana, M. truncatula and P. trichocarpa. Figure S9. tRNAMete–snoRNA co-transcripts in O. sativa and B. distachyon. Figure S10. Cloverleaf structure of the two orphan tDNAs found in O. sativa and B. distachyon. Figure S11. Alignment of pre-tRNATyr and pre-tRNAMete from C. reinhardtii. Table S1. Accession numbers or websites from which tDNAs were retrieved for plant genomes. Table S2. Number of mitochondrial or plastidial-like tDNAs per chromosome. Table S3. Nuclear tRNA gene content and codon frequency in A. thaliana and O. sativa. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. 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