BACKGROUND
the accuracy of the translational machinery depends on the fidelity of codon recognition by the anticodons of transfer rnas .
trnas are short rna molecules of 70– <dig> nucleotides that form defined secondary structures.
over one hundred different modifications of rnas have been described, which are likely to influence their chemistry, metabolism, and stability
<cit> .
in trnas, such modifications can influence the codon-anticodon complex formation.
the uridine at the wobble base  of trnas for lys, glu, and gln is universally modified to 5-methyl-2-thiouridine derivatives which enhance codon reading accuracy
<cit> .
the process of u <dig> thiolation has been well studied in a variety of organisms and involves a number of proteins that activate and eventually transfer the sulfur onto the u <dig>  in yeast, the e <dig> ligase-like protein uba4p  activates and thiolates urm1p , a ubiquitin-related modifier  protein, resulting in a thiocarboxylate at the c-terminal glycine.
the sulfur is then further transferred via the activity of two cytoplasmic thiouridylase proteins , ncs6p and ncs2p  to the uridine residue of the target trnas
<cit> ).
the two ctu proteins interact with each other and share common motifs, but only ctu <dig> is able to bind trnas.
the atp-binding motif pp-loop in these proteins suggests that the sulfur transfer is an energy-consuming reaction
<cit> .
this process is reminiscent of the sulfur transfer reaction in prokaryotes
<cit>  but also shares similarities with the eukaryotic ubiquitylation system.
in yeast and human cell lines, the urm proteins have also been shown to be involved in protein conjugation, particularly under oxidative stress conditions.
the biological significance of this process, however, remains to be determined
<cit> .
the apparent conservation of the machinery involved in sulfur transfer and protein conjugation suggests an evolutionary relationship between these systems
<cit> .
an additional, interesting aspect of loci involved in trna thiolation is that they frequently have been identified as modifiers of the tor  network
<cit> , a major controller of cell growth in eukaryotes
<cit> .
since the regulation of translational activity is a target of the tor pathway, it is quite possible that the lack of trna thiolation, which affects translational accuracy
<cit> , has an impact on the tor pathway via a feedback mechanism.
in plants, the investigation of the sulfur transfer reaction including thiolation of trnas is best investigated in arabidopsis thaliana.
the e <dig> ligase-like protein cnx5/sir <dig> was shown to be important for the biosynthesis of molybdopterin
<cit> .
cnx5/sir <dig> also transfers the sulfur to urm <dig> and urm <dig>  the arabidopsis homologs of urm proteins of yeast and humans
<cit> .
rol <dig> represents the ctu <dig> homolog of arabidopsis which binds to urm <dig> and urm <dig> and transfers the sulfur group from the c-terminal thiocarboxylate of the urm proteins to the trnas.
rol <dig>  urm <dig> and urm <dig> can complement corresponding yeast mutants and mutations in the arabidopsis rol <dig>  urm <dig>  and urm <dig> genes interfere with trna thiolation.
these data show that urm <dig>  urm <dig>  and rol <dig> code for the orthologs of the respective yeast proteins and that the sulfur transfer process is conserved in arabidopsis
<cit> .
the ctu <dig> ortholog, however, has so far not been identified.
in arabidopsis, mutations in cnx5/sir <dig> severely impact plant growth, reflecting the position of this protein in several sulfur-dependent processes such as molybdopterin biosynthesis and trna thiolation
<cit> .
in contrast, urm <dig> urm <dig> double and rol <dig> single mutants are mainly affected in root growth
<cit> .
rol <dig> was initially identified as a suppressor of the root hair cell wall formation mutant lrx <dig>
<cit> .
correspondingly, mutating urm <dig> and urm <dig> also results in suppression of lrx <dig> <cit> .
a likely explanation for this observation is the impact of defective trna thiolation on the tor network in arabidopsis.
interfering with tor signaling by rnai constructs targeting the tor kinase or using specific inhibitors of the tor protein activity have similar effects on root development as found for the rol <dig> or urm <dig> urm <dig> mutants
<cit> .
this work experimentally identifies the second cytoplasmic thiouridylase ctu <dig> of arabidopsis.
ctu <dig> is ubiquitously expressed in the plant and is essential for trna thiolation.
analysis of protein-protein interaction shows binding of ctu <dig> to rol <dig>  the ctu <dig> homolog of arabidopsis.
the analysis of a ctu <dig> knock-out mutant reveals an effect on root development, suggesting that the modification of trnas is particularly important for root developmental processes.
RESULTS
ctu <dig> is poorly conserved among different species
based on protein homology, a potential ctu <dig> homolog of arabidopsis was identified .
an alignment of the proposed arabidopsis ctu <dig> with those of distantly related plants such as potato  and rice  reveals 43% and 55% identity, respectively, while the human protein shows around 20% identical residues .
dewez et al.
<cit>  identified a number of amino acid motifs that are conserved in ctu2-like proteins of a number of species and can thus be considered relevant for protein function.
these sequences are not fully conserved in the plant ctu <dig> homologs and blocks of well conserved sequences among the plant proteins go beyond those identified as being important .
the pp-loop motif  involved in atp binding
<cit>  is also found in ctu2-type proteins, but with the less conserved consensus sequence sggxxs.
to analyze a possible effect of the ctu2- <dig> mutation on trna thiolation, trna was isolated from wild-type and ctu2- <dig> mutant seedlings and separated on a polyacrylamide gel supplemented with n-acryloylamino phenyl mercuric chloride  which binds thiolated trnas, resulting in a higher molecular weight complex with slower migration in the gel.
while trna isolated from the wild type showed the expected retarded band corresponding to thiolated trnas, these retarded trna species were absent in the ctu2- <dig> mutant .
complementation of the ctu2- <dig> mutant with a wild-type ctu <dig> clone resulted in transgenic plants with reconstituted trna thiolation .
this suggests that the gene under investigation is indeed involved in trna thiolation.
ctu <dig> undergoes interaction with the ctu1-homolog rol5
different experimental evidence performed in several organisms established that the process of trna thiolation involves the interaction of ctu1-type and ctu2-type proteins
<cit> .
the ctu1-type protein is essential for trna thiolation and is encoded in arabidopsis by rol <dig> <cit> .
to provide further evidence that the identified arabidopsis protein is indeed ctu <dig>  the interaction with rol <dig> was tested in a yeast-two-hybrid experiment.
transformation of yeast cells with rol <dig> and ctu <dig> cdnas in bait and prey vectors, respectively, revealed growth of yeast cells on appropriate selective media indicative of the interaction of the two proteins.
in addition, strong β-galactosidase activity was observed, confirming the activation of the reporter system in yeast.
in control experiments with the vectors containing ctu <dig> or rol <dig> and the second empty plasmid did not show any growth of yeast under selective conditions, excluding auto-activation by ctu <dig> or rol <dig> alone .
hence, rol <dig> and the proposed ctu <dig> undergo protein-protein interaction, as is expected for the ctu1- and ctu2-homologs of arabidopsis.
ctu <dig> is ubiquitously expressed and influences root architecture
to investigate the expression pattern of ctu <dig>  a promoter:gus fusion construct was produced and transformed into wild-type arabidopsis.
gus activity was monitored in several independent transgenic lines.
at the seedlings stage, gus activity leading to blue staining was observed in all tissues, i.e.
roots, hypocotyl, rosette leaves, and cotyledons.
also in adult plants, gus staining was found in all tissues .
hence, ctu <dig> seems expressed evenly in all tissues, which is in agreement with microarray data
<cit> .
to investigate the importance of ctu <dig> in plant development, the ctu2- <dig> mutant was analyzed for aberrant morphological phenotypes.
seedlings grown for  <dig> days on ms agar plates in a vertical orientation revealed a reduction in lateral root formation and, hence, lateral root density in the mutant compared to the wild type .
mutant lines complemented with the wild-type ctu <dig> construct showed reversion to wild type-like lateral root formation, confirming that the ctu2- <dig> mutation is causing this developmental defect.
since the same effect on lateral root development was observed in the trna thiolation-deficient urm <dig> urm <dig> double mutant
<cit> , the trna thiolation-deficient rol <dig> mutant was also analyzed.
the quantification of lateral root formation revealed a reduction in the lateral root density in the rol <dig> mutant that was even stronger than in the ctu2- <dig> line .
the similar effect of the different mutations blocking trna thiolation suggests that root development is particularly sensitive to changes in this type of trna modification.
<dig>  the trna thiolation-defective rol <dig> mutant also shows a reduction, which is significantly stronger than in ctu2- <dig> .
error bars represent the standard error of the mean.
b) in contrast to wild-type columbia, 7 days-old lrx <dig> mutant seedlings develop aberrant root hairs.
this phenotype is suppressed by ctu2- <dig>  resulting in wild type-like root hair formation in the lrx <dig> ctu2- <dig> double mutant.
the rol <dig> mutant was initially identified as a suppressor of the root hair formation mutant lrx <dig> <cit> .
to test whether ctu2- <dig> would have the same effect on lrx <dig>  an lrx <dig> ctu2- <dig> double mutant was produced.
while wild-type seedlings developed normal root hairs, those of lrx <dig> mutant seedlings showed the previously reported deformation phenotype
<cit> .
the lrx <dig> ctu2- <dig> double mutant seedlings, however, developed wild type-like root hairs .
the phenotypic penetrance in the different lines was very high and consistent.
hence, also the ctu2- <dig> mutation acts as a suppressor of lrx <dig>
discussion
trnas of bacteria and eukaryotes are altered by a variety of modifications
<cit> .
the uridine in the wobble position of trnas recognizing the split-codon boxes of lys, glu, and gln codons are universally modified to 5-methyl-2-thiouridine derivatives ; the cytosolic trnas of eukaryotes containing 5-methoxycarbonylmethyl-2-thiouridine
<cit> .
the thiolation process requires a number of proteins that were identified in diverse organisms such as yeast, c. elegans, and humans.
among these proteins are an e1-like ligase, a ubiquitin-related modifier and two cytoplasmic thiouridylase  proteins
<cit> .
this work describes the experimental identification of the second cytoplasmic thiouridylase, ctu <dig>  while other plant homologs of the proteins involved could be readily identified based on amino acid sequence homology,
<cit> , the level of homology of ctu2-type proteins across eukaryotes is rather low, making experimental evidence for the correct annotation of the arabidopsis homolog necessary.
the proteins of arabidopsis and yeast or humans only share 20% identity, which is drastically lower than between ctu1-type proteins.
these proteins in yeast and arabidopsis  share 54% identical residues
<cit> .
this difference in conservation of the protein sequence suggests that ctu2-type proteins have undergone a stronger divergent evolution than ctu1-type proteins.
a low sequence homology can indicate that initial substitutions of amino acids had to be compensated for by secondary mutations to maintain protein function, resulting in constant conformational characteristics despite low sequence homology
<cit> .
alternatively, the function of ctu2-type proteins might tolerate more changes in the protein without affecting its intrinsic activity.
despite the limited sequence homology, the identified protein is highly likely to be ctu <dig> since it is essential for trna thiolation and interacts with the ctu1-type protein rol <dig>  the interaction of ctu1- and ctu2-type proteins has been well-established in several species
<cit> .
in in vitro experiments, the ctu1-ctu <dig> complex has been shown to be sufficient for trna thiolation, even though at a very low level
<cit> .
in vivo, however, the accumulation of biologically significant amounts of thiolated trnas requires the other protein components of this pathway.
ctu <dig> appears to be expressed in most if not all tissues.
this is supported by the staining of transgenic plants containing an ctu2:gus fusion construct and by gene expression profile analysis platforms such as genevestigator
<cit>  or at genexpress visualization tool
<cit>  that both indicate a moderate expression in all tissues.
despite the ubiquitous occurrence of trna thiolation, an obvious mutant phenotype of ctu2- <dig> is limited to root tissue.
this might be related to the laboratory conditions under which the plants were grown, or the increased susceptibility of this tissue to changes in trna modification.
the impact of trna thiolation on developmental processes
thiolation of the wobble position of specific trnas is important for effective translation by restricting base-pairing capability, preventing misreading of other near-cognate codons, and influencing the thermostability of the codon-anticodon interaction
<cit> .
yeast and worm mutants defective in this trna modification exhibit a temperature-sensitive growth phenotype
<cit> .
by contrast, increased ctu <dig> activity parallels increased cell growth, as observed in certain types of human cancer
<cit> .
hence, the level of trna thiolation can have a substantial effect on cell viability.
in yeast, blocking trna thiolation by mutating ncs <dig>  ncs <dig>  or urm <dig> causes a change in growth properties, resulting in the failure of yeast cells to induce pseudohyphal growth, an otherwise common response to nutrient-limited conditions in order to improve nutrient uptake efficiency
<cit> .
in plants, the trna thiolation mutants ctu2- <dig>  urm <dig> urm <dig>  and rol <dig> exhibit a reduction in lateral root density and are also affected in root hair development, exemplified by the suppression of the lrx <dig> root hair phenotype in lrx <dig> ctu2- <dig>  lrx <dig> urm <dig> urm <dig>  and lrx <dig> rol <dig> mutants .
similar to pseudohyphae in yeast, the plant root system is important for nutrient uptake, indicating that changes in trna thiolation affect functionally related differentiation processes in very distinct organisms.
rather than being a coincident, this similarity might have a common basis in the tor pathway, which senses nutrient availability and growth factors and adapts cell growth to prevailing conditions via influencing e.g.
translation, mitochondrial activity, or cytoskeleton dynamics
<cit> .
the lack of trna thiolation affects translational activity, which is likely to feedback to the tor network
<cit> , resulting in developmental alterations.
indeed, inhibiting tor signaling by different means in arabidopsis results in reduced root development and suppression of the lrx <dig> root hair phenotype as found for rol <dig>  urm <dig> urm <dig>  and ctu2- <dig> <cit> .
CONCLUSIONS
this work describes the experimental validation of the ctu <dig> protein of arabidopsis thaliana, the second cytoplasmic thiouridylase.
the finding that protein-protein interaction between ctu1- and ctu2-type proteins of arabidopsis takes place and that the ctu <dig> protein is essential for trna thiolation provides further support that the protein machinery involved in trna thiolation is well conserved among eukaryotes.
the thiolation of trnas is not only physiologically important but is also connected to cell growth control.
hence, in future experiments it will be interesting to assess the potential of modifying plant growth properties and stress responses via modulating expression levels of genes involved in trna modification.

 the accuracy of the translational machinery depends on the fidelity of codon recognition by the anticodons of transfer rnas. trnas are short rna molecules of 70– nucleotides that form defined secondary structures. over one hundred different modifications of rnas have been described, which are likely to influence their chemistry, metabolism, and stability. in trnas, such modifications can influence the codon-anticodon complex formation. the uridine at the wobble base of trnas for lys, glu, and gln is universally modified to 5-methyl-2-thiouridine derivatives which enhance codon reading accuracy. the process of u thiolation has been well studied in a variety of organisms and involves a number of proteins that activate and eventually transfer the sulfur onto the u in yeast, the e ligase-like protein uba4p activates and thiolates urm1p, a ubiquitin-related modifier protein, resulting in a thiocarboxylate at the c-terminal glycine. the sulfur is then further transferred via the activity of two cytoplasmic thiouridylase proteins, ncs6p and ncs2p to the uridine residue of the target trnas ). the two ctu proteins interact with each other and share common motifs, but only ctu is able to bind trnas. the atp-binding motif pp-loop in these proteins suggests that the sulfur transfer is an energy-consuming reaction. this process is reminiscent of the sulfur transfer reaction in prokaryotes but also shares similarities with the eukaryotic ubiquitylation system. in yeast and human cell lines, the urm proteins have also been shown to be involved in protein conjugation, particularly under oxidative stress conditions. the biological significance of this process, however, remains to be determined. the apparent conservation of the machinery involved in sulfur transfer and protein conjugation suggests an evolutionary relationship between these systems. an additional, interesting aspect of loci involved in trna thiolation is that they frequently have been identified as modifiers of the tor network, a major controller of cell growth in eukaryotes. since the regulation of translational activity is a target of the tor pathway, it is quite possible that the lack of trna thiolation, which affects translational accuracy, has an impact on the tor pathway via a feedback mechanism. in plants, the investigation of the sulfur transfer reaction including thiolation of trnas is best investigated in arabidopsis thaliana. the e ligase-like protein cnx5/sir was shown to be important for the biosynthesis of molybdopterin. cnx5/sir also transfers the sulfur to urm and urm the arabidopsis homologs of urm proteins of yeast and humans. rol represents the ctu homolog of arabidopsis which binds to urm and urm and transfers the sulfur group from the c-terminal thiocarboxylate of the urm proteins to the trnas. rol urm and urm can complement corresponding yeast mutants and mutations in the arabidopsis rol urm and urm genes interfere with trna thiolation. these data show that urm urm and rol code for the orthologs of the respective yeast proteins and that the sulfur transfer process is conserved in arabidopsis. the ctu ortholog, however, has so far not been identified. in arabidopsis, mutations in cnx5/sir severely impact plant growth, reflecting the position of this protein in several sulfur-dependent processes such as molybdopterin biosynthesis and trna thiolation. in contrast, urm urm double and rol single mutants are mainly affected in root growth. rol was initially identified as a suppressor of the root hair cell wall formation mutant lrx. correspondingly, mutating urm and urm also results in suppression of lrx. a likely explanation for this observation is the impact of defective trna thiolation on the tor network in arabidopsis. interfering with tor signaling by rnai constructs targeting the tor kinase or using specific inhibitors of the tor protein activity have similar effects on root development as found for the rol or urm urm mutants. this work experimentally identifies the second cytoplasmic thiouridylase ctu of arabidopsis. ctu is ubiquitously expressed in the plant and is essential for trna thiolation. analysis of protein-protein interaction shows binding of ctu to rol the ctu homolog of arabidopsis. the analysis of a ctu knock-out mutant reveals an effect on root development, suggesting that the modification of trnas is particularly important for root developmental processes.  ctu is poorly conserved among different species based on protein homology, a potential ctu homolog of arabidopsis was identified. an alignment of the proposed arabidopsis ctu with those of distantly related plants such as potato and rice reveals 43% and 55% identity, respectively, while the human protein shows around 20% identical residues. dewez et al. identified a number of amino acid motifs that are conserved in ctu2-like proteins of a number of species and can thus be considered relevant for protein function. these sequences are not fully conserved in the plant ctu homologs and blocks of well conserved sequences among the plant proteins go beyond those identified as being important. the pp-loop motif involved in atp binding is also found in ctu2-type proteins, but with the less conserved consensus sequence sggxxs. to analyze a possible effect of the ctu2- mutation on trna thiolation, trna was isolated from wild-type and ctu2- mutant seedlings and separated on a polyacrylamide gel supplemented with n-acryloylamino phenyl mercuric chloride which binds thiolated trnas, resulting in a higher molecular weight complex with slower migration in the gel. while trna isolated from the wild type showed the expected retarded band corresponding to thiolated trnas, these retarded trna species were absent in the ctu2- mutant. complementation of the ctu2- mutant with a wild-type ctu clone resulted in transgenic plants with reconstituted trna thiolation. this suggests that the gene under investigation is indeed involved in trna thiolation. ctu undergoes interaction with the ctu1-homolog rol5 different experimental evidence performed in several organisms established that the process of trna thiolation involves the interaction of ctu1-type and ctu2-type proteins. the ctu1-type protein is essential for trna thiolation and is encoded in arabidopsis by rol. to provide further evidence that the identified arabidopsis protein is indeed ctu the interaction with rol was tested in a yeast-two-hybrid experiment. transformation of yeast cells with rol and ctu cdnas in bait and prey vectors, respectively, revealed growth of yeast cells on appropriate selective media indicative of the interaction of the two proteins. in addition, strong β-galactosidase activity was observed, confirming the activation of the reporter system in yeast. in control experiments with the vectors containing ctu or rol and the second empty plasmid did not show any growth of yeast under selective conditions, excluding auto-activation by ctu or rol alone. hence, rol and the proposed ctu undergo protein-protein interaction, as is expected for the ctu1- and ctu2-homologs of arabidopsis. ctu is ubiquitously expressed and influences root architecture to investigate the expression pattern of ctu a promoter:gus fusion construct was produced and transformed into wild-type arabidopsis. gus activity was monitored in several independent transgenic lines. at the seedlings stage, gus activity leading to blue staining was observed in all tissues, i.e. roots, hypocotyl, rosette leaves, and cotyledons. also in adult plants, gus staining was found in all tissues. hence, ctu seems expressed evenly in all tissues, which is in agreement with microarray data. to investigate the importance of ctu in plant development, the ctu2- mutant was analyzed for aberrant morphological phenotypes. seedlings grown for days on ms agar plates in a vertical orientation revealed a reduction in lateral root formation and, hence, lateral root density in the mutant compared to the wild type. mutant lines complemented with the wild-type ctu construct showed reversion to wild type-like lateral root formation, confirming that the ctu2- mutation is causing this developmental defect. since the same effect on lateral root development was observed in the trna thiolation-deficient urm urm double mutant, the trna thiolation-deficient rol mutant was also analyzed. the quantification of lateral root formation revealed a reduction in the lateral root density in the rol mutant that was even stronger than in the ctu2- line. the similar effect of the different mutations blocking trna thiolation suggests that root development is particularly sensitive to changes in this type of trna modification. the trna thiolation-defective rol mutant also shows a reduction, which is significantly stronger than in ctu2-. error bars represent the standard error of the mean. b) in contrast to wild-type columbia, 7 days-old lrx mutant seedlings develop aberrant root hairs. this phenotype is suppressed by ctu2- resulting in wild type-like root hair formation in the lrx ctu2- double mutant. the rol mutant was initially identified as a suppressor of the root hair formation mutant lrx. to test whether ctu2- would have the same effect on lrx an lrx ctu2- double mutant was produced. while wild-type seedlings developed normal root hairs, those of lrx mutant seedlings showed the previously reported deformation phenotype. the lrx ctu2- double mutant seedlings, however, developed wild type-like root hairs. the phenotypic penetrance in the different lines was very high and consistent. hence, also the ctu2- mutation acts as a suppressor of lrx discussion trnas of bacteria and eukaryotes are altered by a variety of modifications. the uridine in the wobble position of trnas recognizing the split-codon boxes of lys, glu, and gln codons are universally modified to 5-methyl-2-thiouridine derivatives; the cytosolic trnas of eukaryotes containing 5-methoxycarbonylmethyl-2-thiouridine. the thiolation process requires a number of proteins that were identified in diverse organisms such as yeast, c. elegans, and humans. among these proteins are an e1-like ligase, a ubiquitin-related modifier and two cytoplasmic thiouridylase proteins. this work describes the experimental identification of the second cytoplasmic thiouridylase, ctu while other plant homologs of the proteins involved could be readily identified based on amino acid sequence homology,, the level of homology of ctu2-type proteins across eukaryotes is rather low, making experimental evidence for the correct annotation of the arabidopsis homolog necessary. the proteins of arabidopsis and yeast or humans only share 20% identity, which is drastically lower than between ctu1-type proteins. these proteins in yeast and arabidopsis share 54% identical residues. this difference in conservation of the protein sequence suggests that ctu2-type proteins have undergone a stronger divergent evolution than ctu1-type proteins. a low sequence homology can indicate that initial substitutions of amino acids had to be compensated for by secondary mutations to maintain protein function, resulting in constant conformational characteristics despite low sequence homology. alternatively, the function of ctu2-type proteins might tolerate more changes in the protein without affecting its intrinsic activity. despite the limited sequence homology, the identified protein is highly likely to be ctu since it is essential for trna thiolation and interacts with the ctu1-type protein rol the interaction of ctu1- and ctu2-type proteins has been well-established in several species. in in vitro experiments, the ctu1-ctu complex has been shown to be sufficient for trna thiolation, even though at a very low level. in vivo, however, the accumulation of biologically significant amounts of thiolated trnas requires the other protein components of this pathway. ctu appears to be expressed in most if not all tissues. this is supported by the staining of transgenic plants containing an ctu2:gus fusion construct and by gene expression profile analysis platforms such as genevestigator or at genexpress visualization tool that both indicate a moderate expression in all tissues. despite the ubiquitous occurrence of trna thiolation, an obvious mutant phenotype of ctu2- is limited to root tissue. this might be related to the laboratory conditions under which the plants were grown, or the increased susceptibility of this tissue to changes in trna modification. the impact of trna thiolation on developmental processes thiolation of the wobble position of specific trnas is important for effective translation by restricting base-pairing capability, preventing misreading of other near-cognate codons, and influencing the thermostability of the codon-anticodon interaction. yeast and worm mutants defective in this trna modification exhibit a temperature-sensitive growth phenotype. by contrast, increased ctu activity parallels increased cell growth, as observed in certain types of human cancer. hence, the level of trna thiolation can have a substantial effect on cell viability. in yeast, blocking trna thiolation by mutating ncs ncs or urm causes a change in growth properties, resulting in the failure of yeast cells to induce pseudohyphal growth, an otherwise common response to nutrient-limited conditions in order to improve nutrient uptake efficiency. in plants, the trna thiolation mutants ctu2- urm urm and rol exhibit a reduction in lateral root density and are also affected in root hair development, exemplified by the suppression of the lrx root hair phenotype in lrx ctu2- lrx urm urm and lrx rol mutants. similar to pseudohyphae in yeast, the plant root system is important for nutrient uptake, indicating that changes in trna thiolation affect functionally related differentiation processes in very distinct organisms. rather than being a coincident, this similarity might have a common basis in the tor pathway, which senses nutrient availability and growth factors and adapts cell growth to prevailing conditions via influencing e.g. translation, mitochondrial activity, or cytoskeleton dynamics. the lack of trna thiolation affects translational activity, which is likely to feedback to the tor network, resulting in developmental alterations. indeed, inhibiting tor signaling by different means in arabidopsis results in reduced root development and suppression of the lrx root hair phenotype as found for rol urm urm and ctu2-.  this work describes the experimental validation of the ctu protein of arabidopsis thaliana, the second cytoplasmic thiouridylase. the finding that protein-protein interaction between ctu1- and ctu2-type proteins of arabidopsis takes place and that the ctu protein is essential for trna thiolation provides further support that the protein machinery involved in trna thiolation is well conserved among eukaryotes. the thiolation of trnas is not only physiologically important but is also connected to cell growth control. hence, in future experiments it will be interesting to assess the potential of modifying plant growth properties and stress responses via modulating expression levels of genes involved in trna modification. 
a specific subset of trnas contains a thiolated uridine residue at the wobble position to improve the codon-anticodon interaction and translational accuracy. based on the arabidopsis protein sequence, ctu2-type proteins of other plant species can now be readily identified. ctu is ubiquitously expressed, yet its activity seems to be particularly important in root tissue. ctu is essential for trna thiolation as a ctu mutant fails to perform this trna modification. the proteins involved in trna thiolation are reminiscent of prokaryotic sulfur transfer reactions and of the ubiquitylation process in eukaryotes. ctu is essential for trna thiolation and interacts with rol the previously identified ctu homolog of arabidopsis. the analysis of ctu adds a new component to the so far characterized protein network involved in trna thiolation in arabidopsis. a ctu knock-out mutant shows an alteration in root development. this manuscript describes the identification of ctu the second cytoplasmic thiouridylase protein of arabidopsis thaliana. for other proteins, the identification of the plant homologs is much less clear, due to the low conservation in protein sequence.
 a large number of post-transcriptional modifications of transfer rnas have been described in prokaryotes and eukaryotes. they are known to influence their stability, turnover, and chemical/physical properties. a specific subset of trnas contains a thiolated uridine residue at the wobble position to improve the codon-anticodon interaction and translational accuracy. the proteins involved in trna thiolation are reminiscent of prokaryotic sulfur transfer reactions and of the ubiquitylation process in eukaryotes. in plants, some of the proteins involved in this process have been identified and show a high degree of homology to their non-plant equivalents. for other proteins, the identification of the plant homologs is much less clear, due to the low conservation in protein sequence.  this manuscript describes the identification of ctu the second cytoplasmic thiouridylase protein of arabidopsis thaliana. ctu is essential for trna thiolation and interacts with rol the previously identified ctu homolog of arabidopsis. ctu is ubiquitously expressed, yet its activity seems to be particularly important in root tissue. a ctu knock-out mutant shows an alteration in root development.  the analysis of ctu adds a new component to the so far characterized protein network involved in trna thiolation in arabidopsis. ctu is essential for trna thiolation as a ctu mutant fails to perform this trna modification. the identified arabidopsis ctu is the first ctu2-type protein from plants to be experimentally verified, which is important considering the limited conservation of these proteins between plant and non-plant species. based on the arabidopsis protein sequence, ctu2-type proteins of other plant species can now be readily identified. trnathiolationcytoplasmic thiouridylasectu2rootlateral rootsroot hairslrx1rol5 