Chloroplast tRNA Research Articles

C2-methyladenosine in tRNA promotes protein translation by facilitating the decoding of tandem m2A-tRNA-dependent codons

RNA modification C2-methyladenosine (m2A) exists in both rRNA and tRNA of Escherichia coli (E. coli), installed by the methyltransferase RlmN using a radical-S-adenosylmethionine (SAM) mechanism. However, the precise function of m2A in tRNA and its ubiquity in plants have remained unclear. Here we discover the presence of m2A in chloroplast rRNA and tRNA, as well as cytosolic tRNA, in multiple plant species. We identify six m2A-modified chloroplast tRNAs and two m2A-modified cytosolic tRNAs across different plants. Furthermore, we characterize three Arabidopsis m2A methyltransferases—RLMNL1, RLMNL2, and RLMNL3—which methylate chloroplast rRNA, chloroplast tRNA, and cytosolic tRNA, respectively. Our findings demonstrate that m2A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency in chloroplast and cytosol by facilitating decoding of tandem m2A-tRNA-dependent codons. This study provides insights into the molecular function and biological significance of m2A, uncovering a layer of translation regulation in plants.

Novel structures and evolution of tRNA genes: insight into the chloroplast tRNAs of family Sapindaceae

Novel structures and evolution of tRNA genes: insight into the chloroplast tRNAs of family Sapindaceae

Anticodon table of the chloroplast genome and identification of putative quadruplet anticodons in chloroplast tRNAs

The chloroplast genome of 5959 species was analyzed to construct the anticodon table of the chloroplast genome. Analysis of the chloroplast transfer ribonucleic acid (tRNA) revealed the presence of a putative quadruplet anticodon containing tRNAs in the chloroplast genome. The tRNAs with putative quadruplet anticodons were UAUG, UGGG, AUAA, GCUA, and GUUA, where the GUUA anticodon putatively encoded tRNAAsn. The study also revealed the complete absence of tRNA genes containing ACU, CUG, GCG, CUC, CCC, and CGG anticodons in the chloroplast genome from the species studied so far. The chloroplast genome was also found to encode tRNAs encoding N-formylmethionine (fMet), Ile2, selenocysteine, and pyrrolysine. The chloroplast genomes of mycoparasitic and heterotrophic plants have had heavy losses of tRNA genes. Furthermore, the chloroplast genome was also found to encode putative spacer tRNA, tRNA fragments (tRFs), tRNA-derived, stress-induced RNA (tiRNAs), and the group I introns. An evolutionary analysis revealed that chloroplast tRNAs had evolved via multiple common ancestors and the GC% had more influence toward encoding the tRNA number in the chloroplast genome than the genome size.

Evolutionary characteristics and phylogeny of cotton chloroplast tRNAs.

The novel structural variations were identified in cotton chloroplast tRNAs and gene loss events were more obvious than duplications in chloroplast tRNAs. Transfer RNAs (tRNA) have long been believed an evolutionary-conserved molecular family, which play the key roles in the process of protein biosynthesis in plant life activities. In this study, we detected the evolutionary characteristics and phylogeny of chloroplast tRNAs in cotton plants, an economic and fibered important taxon in the world. We firstly annotated the chloroplast tRNAs of 27 Gossypium species to analyze their genetic composition, structural characteristics and evolution. Compared with the traditional view of evolutionary conservation of tRNA, some novel tRNA structural variations were identified in cotton plants. I.g., tRNAVal-UAC and tRNAIle-GAU only contained one intron in the anti-condon loop region of tRNA secondary structure, respectively. In the variable region, some tRNAs contained a circle structure with a few nucleotides. Interestingly, the calculation result of free energy indicated that the variation of novel tRNAs contributed to the stability of tRNA structure. Phylogenetic analysis suggested that chloroplast tRNAs have evolved from multiple common ancestors, and the tRNAMet seemed to be an ancestral tRNA, which can be duplicated and diversified to produce other tRNAs. The chloroplast tRNAs contained a group I intron in cotton plants, and the evolutionary analysis of introns indicated that group I intron of chloroplast tRNA originated from cyanobacteria. Analysis of gene duplication and loss events showed that gene loss events were more obvious than duplications in Gossypium chloroplast tRNAs. Additionally, we found that the rate of transition was higher than the ones of transversion in cotton chloroplast tRNAs. This study provided new insights into the structural characteristics and evolution of chloroplast tRNAs in cotton plants.

Evolution and structural variations in chloroplast tRNAs in gymnosperms

BackgroundChloroplast transfer RNAs (tRNAs) can participate in various vital processes. Gymnosperms have important ecological and economic value, and they are the dominant species in forest ecosystems in the Northern Hemisphere. However, the evolution and structural changes in chloroplast tRNAs in gymnosperms remain largely unclear.ResultsIn this study, we determined the nucleotide evolution, phylogenetic relationships, and structural variations in 1779 chloroplast tRNAs in gymnosperms. The numbers and types of tRNA genes present in the chloroplast genomes of different gymnosperms did not differ greatly, where the average number of tRNAs was 33 and the frequencies of occurrence for various types of tRNAs were generally consistent. Nearly half of the anticodons were absent. Molecular sequence variation analysis identified the conserved secondary structures of tRNAs. About a quarter of the tRNA genes were found to contain precoded 3′ CCA tails. A few tRNAs have undergone novel structural changes that are closely related to their minimum free energy, and these structural changes affect the stability of the tRNAs. Phylogenetic analysis showed that tRNAs have evolved from multiple common ancestors. The transition rate was higher than the transversion rate in gymnosperm chloroplast tRNAs. More loss events than duplication events have occurred in gymnosperm chloroplast tRNAs during their evolutionary process.ConclusionsThese findings provide novel insights into the molecular evolution and biological characteristics of chloroplast tRNAs in gymnosperms.

Structural variation and evolution of chloroplast tRNAs in green algae.

As one of the important groups of the core Chlorophyta (Green algae), Chlorophyceae plays an important role in the evolution of plants. As a carrier of amino acids, tRNA plays an indispensable role in life activities. However, the structural variation of chloroplast tRNA and its evolutionary characteristics in Chlorophyta species have not been well studied. In this study, we analyzed the chloroplast genome tRNAs of 14 species in five categories in the green algae. We found that the number of chloroplasts tRNAs of Chlorophyceae is maintained between 28–32, and the length of the gene sequence ranges from 71 nt to 91 nt. There are 23–27 anticodon types of tRNAs, and some tRNAs have missing anticodons that are compensated for by other types of anticodons of that tRNA. In addition, three tRNAs were found to contain introns in the anti-codon loop of the tRNA, but the analysis scored poorly and it is presumed that these introns are not functional. After multiple sequence alignment, the Ψ-loop is the most conserved structural unit in the tRNA secondary structure, containing mostly U-U-C-x-A-x-U conserved sequences. The number of transitions in tRNA is higher than the number of transversions. In the replication loss analysis, it was found that green algal chloroplast tRNAs may have undergone substantial gene loss during the course of evolution. Based on the constructed phylogenetic tree, mutations were found to accompany the evolution of the Green algae chloroplast tRNA. Moreover, chloroplast tRNAs of Chlorophyceae are consistent with those of monocotyledons and gymnosperms in terms of evolutionary patterns, sharing a common multi-phylogenetic pattern and rooted in a rich common ancestor. Sequence alignment and systematic analysis of tRNA in chloroplast genome of Chlorophyceae, clarified the characteristics and rules of tRNA changes, which will promote the evolutionary relationship of tRNA and the origin and evolution of chloroplast.

Novel Structural Variation and Evolutionary Characteristics of Chloroplast tRNA in Gossypium Plants.

Cotton is one of the most important fiber and oil crops in the world. Chloroplast genomes harbor their own genetic materials and are considered to be highly conserved. Transfer RNAs (tRNAs) act as “bridges” in protein synthesis by carrying amino acids. Currently, the variation and evolutionary characteristics of tRNAs in the cotton chloroplast genome are poorly understood. Here, we analyzed the structural variation and evolution of chloroplast tRNA (cp tRNA) based on eight diploid and two allotetraploid cotton species. We also investigated the nucleotide evolution of chloroplast genomes in cotton species. We found that cp tRNAs in cotton encoded 36 or 37 tRNAs, and 28 or 29 anti-codon types with lengths ranging from 60 to 93 nucleotides. Cotton chloroplast tRNA sequences possessed specific conservation and, in particular, the Ψ-loop contained the conserved U-U-C-X3-U. The cp tRNAs of Gossypium L. contained introns, and cp tRNAIle contained the anti-codon (C-A-U), which was generally the anti-codon of tRNAMet. The transition and transversion analyses showed that cp tRNAs in cotton species were iso-acceptor specific and had undergone unequal rates of evolution. The intergenic region was more variable than coding regions, and non-synonymous mutations have been fixed in cotton cp genomes. On the other hand, phylogeny analyses indicated that cp tRNAs of cotton were derived from several inferred ancestors with greater gene duplications. This study provides new insights into the structural variation and evolution of chloroplast tRNAs in cotton plants. Our findings could contribute to understanding the detailed characteristics and evolutionary variation of the tRNA family.

Phylogeny and evolution of chloroplast tRNAs in Adoxaceae.

Chloroplasts are semiautonomous organelles found in photosynthetic plants. The major functions of chloroplasts include photosynthesis and carbon fixation, which are mainly regulated by its circular genomes. In the highly conserved chloroplast genome, the chloroplast transfer RNA genes (cp tRNA) play important roles in protein translation within chloroplasts. However, the evolution of cp tRNAs remains unclear. Thus, in the present study, we investigated the evolutionary characteristics of chloroplast tRNAs in five Adoxaceae species using 185 tRNA gene sequences. In total, 37 tRNAs encoding 28 anticodons are found in the chloroplast genome in Adoxaceae species. Some consensus sequences are found within the Ψ‐stem and anticodon loop of the tRNAs. Some putative novel structures were also identified, including a new stem located in the variable region of tRNATyr in a similar manner to the anticodon stem. Furthermore, phylogenetic and evolutionary analyses indicated that synonymous tRNAs may have evolved from multiple ancestors and frequent tRNA duplications during the evolutionary process may have been primarily caused by positive selection and adaptive evolution. The transition and transversion rates are uneven among different tRNA isotypes. For all tRNAs, the transition rate is greater with a transition/transversion bias of 3.13. Phylogenetic analysis of cp tRNA suggested that the type I introns in different taxa (including eukaryote organisms and cyanobacteria) share the conserved sequences “U‐U‐x2‐C” and “U‐x‐G‐x2‐T,” thereby indicating the diverse cyanobacterial origins of organelles. This detailed study of cp tRNAs in Adoxaceae may facilitate further investigations of the evolution, phylogeny, structure, and related functions of chloroplast tRNAs.

CFM1, a member of the CRM-domain protein family, functions in chloroplast group II intron splicing in Setaria viridis.

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.

Evolutionary analysis of chloroplast tRNA of Gymnosperm revealed the novel structural variation and evolutionary aspect.

Gymnosperms such as ginkgo, conifers, cycads, and gnetophytes are vital components of land ecosystems, and they have significant economic and ecologic value, as well as important roles as forest vegetation. In this study, we investigated the structural variation and evolution of chloroplast transfer RNAs (tRNAs) in gymnosperms. Chloroplasts are important organelles in photosynthetic plants. tRNAs are key participants in translation where they act as adapter molecules between the information level of nucleic acids and functional level of proteins. The basic structures of gymnosperm chloroplast tRNAs were found to have family-specific conserved sequences. The tRNAΨ -loop was observed to contain a conforming sequence, i.e., U-U-C-N-A-N2. In gymnosperms, tRNAIle was found to encode a “CAU” anticodon, which is usually encoded by tRNAMet. Phylogenetic analysis suggested that plastid tRNAs have a common polyphyletic evolutionary pattern, i.e., rooted in abundant common ancestors. Analyses of duplication and loss events in chloroplast tRNAs showed that gymnosperm tRNAs have experienced little more gene loss than gene duplication. Transition and transversion analysis showed that the tRNAs are iso-acceptor specific and they have experienced unequal evolutionary rates. These results provide new insights into the structural variation and evolution of gymnosperm chloroplast tRNAs, which may improve our comprehensive understanding of the biological characteristics of the tRNA family.

A Natural Variation in PLEIOTROPIC DEVELOPMENTAL DEFECTS Uncovers a Crucial Role for Chloroplast tRNA Modification in Translation and Plant Development.

The modification of tRNA is important for accurate, efficient protein translation. A number of tRNA-modifying enzymes were found to influence various developmental processes in distinct organisms. However, few genetic or molecular studies have focused on genes encoding tRNA-modifying enzymes in green plant organelles. Here, we discovered that PDD OL , a natural variation allele of PLEIOTROPIC DEVELOPMENTAL DEFECTS (PDD), leads to pleiotropic developmental defects in a near-isogenic line (NIL) generated by introgressing the wild rice Oryza longistaminata into the rice (Oryza sativa) cv 187R. Map-based cloning revealed that PDD encodes an evolutionarily conserved tRNA-modifying GTPase belonging to the tRNA modification E family. The function of PDD was further confirmed by genetic complementation experiments and mutant analysis. PDD mRNA is primarily expressed in leaves, and PDD is localized to chloroplasts. Biochemical analyses indicated that PDD187R forms homodimers and has strong GTPase activity, whereas PDDOL fails to form homodimers and has weak GTPase activity. Liquid chromatography-coupled tandem quadrupole mass spectrometry revealed that PDD is associated with the 5-methylaminomethyl-2-thiouridine modification of chloroplast tRNA. Furthermore, compared to 187R, NIL-PDD OL has severely reduced levels of proteins involved in photosynthesis and ribosome biogenesis but increased levels of plastid-encoded RNA polymerase subunits. Finally, we demonstrate that the defect due to PDD OL alters chloroplast gene expression, thereby affecting communication between the chloroplast and the nucleus.

Intergenomic gene transfer in diploid and allopolyploid Gossypium

BackgroundIntergenomic gene transfer (IGT) between nuclear and organellar genomes is a common phenomenon during plant evolution. Gossypium is a useful model to evaluate the genomic consequences of IGT for both diploid and polyploid species. Here, we explore IGT among nuclear, mitochondrial, and plastid genomes of four cotton species, including two allopolyploids and their model diploid progenitors (genome donors, G. arboreum: A2 and G. raimondii: D5).ResultsExtensive IGT events exist for both diploid and allotetraploid cotton (Gossypium) species, with the nuclear genome being the predominant recipient of transferred DNA followed by the mitochondrial genome. The nuclear genome has integrated 100 times more foreign sequences than the mitochondrial genome has in total length. In the nucleus, the integrated length of chloroplast DNA (cpDNA) was between 1.87 times (in diploids) to nearly four times (in allopolyploids) greater than that of mitochondrial DNA (mtDNA). In the mitochondrion, the length of nuclear DNA (nuDNA) was typically three times than that of cpDNA. Gossypium mitochondrial genomes integrated three nuclear retrotransposons and eight chloroplast tRNA genes, and incorporated chloroplast DNA prior to divergence between the diploids and allopolyploid formation. For mitochondrial chloroplast-tRNA genes, there were 2-6 bp conserved microhomologies flanking their insertion sites across distantly related genera, which increased to 10 bp microhomologies for the four cotton species studied. For organellar DNA sequences, there are source hotspots, e.g., the atp6-trnW intergenic region in the mitochondrion and the inverted repeat region in the chloroplast. Organellar DNAs in the nucleus were rarely expressed, and at low levels. Surprisingly, there was asymmetry in the survivorship of ancestral insertions following allopolyploidy, with most numts (nuclear mitochondrial insertions) decaying or being lost whereas most nupts (nuclear plastidial insertions) were retained.ConclusionsThis study characterized and compared intracellular transfer among nuclear and organellar genomes within two cultivated allopolyploids and their ancestral diploid cotton species. A striking asymmetry in the fate of IGTs in allopolyploid cotton was discovered, with numts being preferentially lost relative to nupts. Our results connect intergenomic gene transfer with allotetraploidy and provide new insight into intracellular genome evolution.

Genomic and evolutionary aspects of chloroplast tRNA in monocot plants

BackgroundChloroplasts are one of the most indispensable organelles that make life forms on the earth possible by their capacity to photosynthesize. These organelles possess a circular genome with a number of coding genes responsible for self-regulation. tRNAs are an important evolutionary-conserved gene family that are responsible for protein translation. However, within the chloroplast genome, tRNA machinery are poorly understood.ResultsIn the present study, the chloroplast genome of six monocot plants, Oryza nivara (NC_005973), Oryza sativa (NC_001320), Sachharum officinarum (NC_006084), Sorghum bicolor (NC_008602), Triticum aestivum (NC_002762), and Zea mays (NC_001666) were downloaded and analyzed to identify tRNA sequences. Further analysis of the tRNA sequences in the chloroplast genomes of the monocot plants resulted in the identification of several novel features. The length of tRNAs in the chloroplast genome of the monocot plants ranged from 59 to 155 nucleotides. Pair-wise sequence alignment revealed the presence of a conserved A-C-x-U-A-x-U-A-x-U-x5-U-A-A nucleotide consensus sequence. In addition, the tRNAs in chloroplast genomes of the monocot plants also contain 21–28 anti-codons against 61 sense codons in the genome. They also contain a group I intron and a C-A-U anti-codon for tRNAIle, which is a common anti-codon of tRNAMet. Evolutionary analysis indicates that tRNAs in the chloroplast genome have evolved from multiple common ancestors, and tRNAMet appears to be the ancestral tRNA that underwent duplication and diversification to give rise to other tRNAs.ConclusionThe results obtained from the study of chloroplast tRNA will greatly help to increase our understanding of tRNA biology at a new level. Functional studies of the reported novel aspects of the chloroplast tRNA of the monocot plants will greatly help to decipher their roles in diverse cellular processes.

The Roles of Mitochondrion in Intergenomic Gene Transfer in Plants: A Source and a Pool.

Intergenomic gene transfer (IGT) is continuous in the evolutionary history of plants. In this field, most studies concentrate on a few related species. Here, we look at IGT from a broader evolutionary perspective, using 24 plants. We discover many IGT events by assessing the data from nuclear, mitochondrial and chloroplast genomes. Thus, we summarize the two roles of the mitochondrion: a source and a pool. That is, the mitochondrion gives massive sequences and integrates nuclear transposons and chloroplast tRNA genes. Though the directions are opposite, lots of likenesses emerge. First, mitochondrial gene transfer is pervasive in all 24 plants. Second, gene transfer is a single event of certain shared ancestors during evolutionary divergence. Third, sequence features of homologies vary for different purposes in the donor and recipient genomes. Finally, small repeats (or micro-homologies) contribute to gene transfer by mediating recombination in the recipient genome.

APPLE PROLIFERATION DISEASE IN CROATIAN ORCHARDS: A MOLECULAR CHARACTERIZATION OF ‘CANDIDATUS PHYTOPLASMA MALI’

Apple proliferation (AP) causes major economic losses to the European apple industry. The causal agent associated with AP is ‘Candidatus Phytoplasma mali’ which belongs to the same phylogenetic cluster as two other fruit tree phytoplasmas: ‘Ca. P. pyri’ and ‘Ca. P. prunorum’. ‘Ca. P. pyri’ and ‘Ca. P. solani’ have sporadically been associated with symptomatic apples in Croatia in previous surveys. There was no molecular evidence of ‘Ca. P. mali’ presence until the start of this study, when it was confirmed for the first time. In this four-year survey (2011-2014), the detection and characterization of ‘Ca. P. mali’ from apples and psyllid vectors was carried out. Detection was based on PCR-RFLP analysis of the 16S rRNA gene. Real-time PCR was also used for amplifying ‘Ca. P. mali’ 16S rDNA and Malus domestica chloroplast tRNA leucine gene. Phylogenetic analysis of 16S rDNA and aceF gene sequences confirmed the identification of selected phytoplasma strains. Considerable aceF variability among the strains was detected, and a new, yet unrecorded aceF genotype was found. ‘Ca. P. mali’ has consistently been identified with high incidence in apple samples from Croatian continental region orchards, whereas it occurred sporadically in the coastal areas. This is the first molecular detection and characterization of ‘Ca. P. mali’ strains from both apple (Malus domestica Burkh.) and psyllid vector Cacopsylla picta (Forster) in Croatia.

Abiotic stresses affect differently the intron splicing and expression of chloroplast genes in coffee plants (Coffea arabica) and rice (Oryza sativa)

Despite the increasing understanding of the regulation of chloroplast gene expression in plants, the importance of intron splicing and processing of chloroplast RNA transcripts under stress conditions is largely unknown. Here, to understand how abiotic stresses affect the intron splicing and expression patterns of chloroplast genes in dicots and monocots, we carried out a comprehensive analysis of the intron splicing and expression patterns of chloroplast genes in the coffee plant (Coffea arabica) as a dicot and rice (Oryza sativa) as a monocot under abiotic stresses, including drought, cold, or combined drought and heat stresses. The photosynthetic activity of both coffee plants and rice seedlings was significantly reduced under all stress conditions tested. Analysis of the transcript levels of chloroplast genes revealed that the splicing of tRNAs and mRNAs in coffee plants and rice seedlings were significantly affected by abiotic stresses. Notably, abiotic stresses affected differently the splicing of chloroplast tRNAs and mRNAs in coffee plants and rice seedlings. The transcript levels of most chloroplast genes were markedly downregulated in both coffee plants and rice seedlings upon stress treatment. Taken together, these results suggest that coffee and rice plants respond to abiotic stresses via regulating the intron splicing and expression of different sets of chloroplast genes.

DNA barcoding for efficient identification of Ixiolirion species (Ixioliriaceae).

Ixiolirion is a genus of unresolved taxonomy. DNA barcoding is a technique that allows species identification using standardized DNA sequences. In this study, a total of 23 individuals, representing 2 Chinese Ixiolirion species, were sampled to test the effectiveness of 3 DNA barcodes [internal transcribed spacer (ITS), chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase] for species identification. Of the 3 DNA barcodes, ITS displayed the maximum level of polymerase chain reaction and sequencing success as well as the highest sequence variation. Intra-specific sequence distances of ITS, chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase were 0, 0, and 0-0.1%, respectively, with 8.3, 0.6, and 0.5% as mean inter-specific distances, respectively. All individuals of each species formed a monophyletic group (clade) in the neighbor-joining trees constructed using the 3 single-DNA barcodes. Our results demonstrated that ITS, chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase DNA markers could be used to identify Ixiolirion species. Our results indicate that DNA barcoding provides a reliable and effective means for discriminating Ixiolirion species, and is a robust tool for resolving taxonomic controversies of Ixiolirion in combination with morphology-based taxonomy.

Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis

Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2'-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed.

Ice Core Genetics Reveals Palaeo- Ecosystems in Greenland and Antarctica

Although a significant proportion of plant tissue is located in roots and other below-ground parts of plants, little is known on the dietary choices of root-feeding insects. This is caused by a lack of adequate methodology which would allow tracking below-ground trophic interactions between insects and plants. Here, we present a DNA-based approach to examine this relationship. Feeding experiments were established where either wheat (Triticum aestivum) or maize (Zea mays) was fed to Agriotes larvae (Coleoptera: Elateridae), allowing them to digest for up to 72 h. Due to the very small amount of plant tissue ingested (max = 6.76 mg), DNA extraction procedures and the sensitivity of polymerase chain reaction (PCR) had to be optimized. Whole-body DNA extracts of larvae were tested for the presence of both rbcL and trnL plastid DNA using universal primers. Moreover, based on cpDNA sequences encoding chloroplast tRNA for leucine (trnL), specific primers for maize and wheat were developed. With both, general and specific primers, plant DNA was detectable in the guts of Agriotes larvae for up to 72 h post-feeding, the maximum time of digestion in these experiments. No significant effect of time since feeding on plant DNA detection success was observed, except for the specific primers in maize-fed larvae. Here, plant DNA detection was negatively correlated with the duration of digestion. Both, meal size and initial mass of the individual larvae did not affect the rate of larvae testing positive for plant DNA. The outcomes of this study represent a first step towards a specific analysis of the dietary choices of soil-living herbivores to further increase our understanding of animal–plant feeding interactions in the soil.

A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2

BackgroundtRNase Z removes the 3'-trailer sequences from precursor tRNAs, which is an essential step preceding the addition of the CCA sequence. tRNase Z exists in the short (tRNase ZS) and long (tRNase ZL) forms. Based on the sequence characteristics, they can be divided into two major types: bacterial-type tRNase ZS and eukaryotic-type tRNase ZL, and one minor type, Thermotoga maritima (TM)-type tRNase ZS. The number of tRNase Zs is highly variable, with the largest number being identified experimentally in the flowering plant Arabidopsis thaliana. It is unknown whether multiple tRNase Zs found in A. thaliana is common to the plant kingdom. Also unknown is the extent of sequence and structural conservation among tRNase Zs from the plant kingdom.ResultsWe report the identification and analysis of candidate tRNase Zs in 27 fully sequenced genomes of green plants, the great majority of which are flowering plants. It appears that green plants contain multiple distinct tRNase Zs predicted to reside in different subcellular compartments. Furthermore, while the bacterial-type tRNase ZSs are present only in basal land plants and green algae, the TM-type tRNase ZSs are widespread in green plants. The protein sequences of the TM-type tRNase ZSs identified in green plants are similar to those of the bacterial-type tRNase ZSs but have distinct features, including the TM-type flexible arm, the variant catalytic HEAT and HST motifs, and a lack of the PxKxRN motif involved in CCA anti-determination (inhibition of tRNase Z activity by CCA), which prevents tRNase Z cleavage of mature tRNAs. Examination of flowering plant chloroplast tRNA genes reveals that many of these genes encode partial CCA sequences. Based on our results and previous studies, we predict that the plant TM-type tRNase ZSs may not recognize the CCA sequence as an anti-determinant.ConclusionsOur findings substantially expand the current repertoire of the TM-type tRNase ZSs and hint at the possibility that these proteins may have been selected for their ability to process chloroplast pre-tRNAs with whole or partial CCA sequences. Our results also support the coevolution of tRNase Zs and tRNA 3'-trailer sequences in plants.