Evolutionary constraints on the plastid tRNA set decoding methionine and isoleucine

The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons

Can infants develop meningitis in the absence of bacteremia in the first ninety days of life? A retrospective chart review

The overall incidence of meningitis in infants 0-90 days is low; however, it remains a serious cause of morbidity and mortality among affected patients. It is standard of care to perform lumbar punctures as part of the work-up of fever in the first four weeks of life and sick-looking babies up to the age of 90 days. This particular procedure is often refused by parents, and physicians are left to predict the possibility of meningitis based on blood culture results.The aim of this study is to determine whether it would be safe to rule out meningitis based on a negative blood culture in this age group

Nonessential Plastid-Encoded Ribosomal Proteins in Tobacco: A Developmental Role for Plastid Translation and Implications for Reductive Genome Evolution[W][OA]

Of seven plastid genome-encoded ribosomal proteins analyzed by reverse genetics in tobacco (Nicotiana tabacum), two were found to be nonessential: S15 and L36. Elimination of ribosomal protein S15 produced normal plants, but elimination of L36 resulted in mutants with reduced apical dominance and strikingly altered leaf morphology, uncovering a role for plastid translational activity in plant development

Phytochemical Composition, Antioxidant and Antiproliferative Activities of <i>Citrus hystrix</i>, <i>Citrus limon</i>, <i>Citrus pyriformis,</i> and <i>Citrus microcarpa</i> Leaf Essential Oils against Human Cervical Cancer Cell Line

The essential oil derived from Citrus plants has long been used for medicinal purposes, due to its broad spectrum of therapeutic characteristics. To date, approximately 162 Citrus species have been identified, and many investigational studies have been conducted to explore the pharmacological potential of Citrus spp. oils. This study investigated the volatile constituents of essential oil distilled from the leaves of C. hystrix, C. limon, C. pyriformis, and C. microcarpa, using gas chromatography–quadrupole mass spectrometry. A total of 80 secondary compounds were tentatively identified, representing 84.88–97.99% of the total ion count and mainly comprising monoterpene (5.20–76.15%) and sesquiterpene (1.36–27.14%) hydrocarbons, oxygenated monoterpenes (3.91–89.52%) and sesquiterpenes (0.21–38.87%), and other minor chemical classes (0.10–0.52%). In particular, 27 compounds (1.19–39.06%) were detected across all Citrus species. Principal component analysis of the identified phytoconstituents and their relative quantities enabled differentiation of the Citrus leaf oils according to their species, with the loading variables contributing to these metabolic differences being identified. The Citrus leaf oils were tested for their antioxidant and antiproliferative activities using 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results indicated that C. limon displayed the highest DPPH radical scavenging ability (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix exhibited the lowest activity (IC50 value of 279.03 ± 10.37 mg/mL). On the other hand, all the Citrus oils exhibit potent antiproliferative activities against the HeLa cervical cancer cell line, with IC50 values of 11.66 μg/mL (C. limon), 20.41 μg/mL (C. microcarpa), 25.91 μg/mL (C. hystrix), and 87.17 μg/mL (C. pyriformis)

The Contributions of Wobbling and Superwobbling to the Reading of the Genetic Code

<div><p>Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but—contrary to previous suggestions—it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus.</p> </div

Targeted deletion of the plastid <i>trnS-GGA</i> gene.

<p>(A) Physical map of the region in the tobacco plastid genome harboring the <i>trnS-GGA</i> gene. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. The bent arrows indicate the borders of the transformation plasmid. Restriction sites used for RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in Δ<i>trnS-GGA</i> transplastomic lines. The <i>aadA</i> cassette is shown as grey box. (C) RFLP analysis of Δ<i>trnS-GGA</i> plastid transformants. All lines are homoplasmic and show exclusively the 2.9 kb band diagnostic of the transplastome. Independently generated transplastomic lines are designated by Arabic numerals following the tRNA gene name, the following capital letter indicates an individual plant. Wt: wild type. (D) tRNA-Ser(GGA) accumulation in wild-type plants and Δ<i>trnS-GGA</i> transplastomic lines. Hybridization to a plastid <i>trnS-GGA</i> probe reveals weak signals in all transplastomic plants, which are presumably caused by cross-hybridization to the mitochondrial <i>trnS-GGA</i>. To control for RNA loading, part of the ethidium bromide-stained gel (showing the two largest 23S rRNA hidden break products) prior to blotting is also shown. (E) tRNA-Ser(GGA) accumulation in isolated chloroplasts of wild type plants and a Δ<i>trnS-GGA</i> knock-out line. Hybridization to the plastid <i>trnS-GGA</i> probe confirms complete absence of the tRNA from the transplastomic line. (F) Confirmation of homoplasmy of the Δ<i>trnS-GGA</i> lines by inheritance assays. Germination of seeds harvested from transplastomic plants on spectinomycin-containing medium results in a homogeneous population of green antibiotic-resistant seedlings. (G) Comparison with spectinomycin-sensitive wild-type seedlings. Antibiotic sensitivity is evidenced by the white phenotype of all seedlings.</p

Analysis of plastid protein synthesis and photosynthetic parameters in Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i>, Δ<i>trnT-GGU</i>, and Δ<i>trnV-GAC</i> plants.

<p>(A) Assessment of RbcL protein accumulation by western blotting using a specific anti-RbcL antibody. For semiquantitative analysis, a dilution series of wild-type protein was loaded. Consistent with the differences in the severity of the growth phenotypes (cf. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen-1003076-g005" target="_blank">Figure 5</a>), the Δ<i>trnV-GAC</i> and Δ<i>trnL-CAA</i> mutants show the smallest reduction in RbcL accumulation, whereas the Δ<i>trnS-GGA</i> mutant and especially the Δ<i>trnT-GGU</i> mutant are more strongly affected, with RbcL levels being in the range of the 25% dilution of the wild-type sample in the Δ<i>trnT-GGU</i> mutant. Reduced synthesis of chloroplast proteins is also apparent, when total plant protein samples are separated by gel electrophoresis and stained with Coomassie (lower panel). The two most abundant proteins (representing the large and small subunits of Rubisco, RbcL and RbcS) are indicated. Reduced abundance of chloroplast proteins in the Δ<i>trnS-GGA</i> and Δ<i>trnT-GGU</i> mutants also becomes evident by a stronger background staining (coming from a large number of lower abundant nuclear-encoded proteins). (B) Analysis of chlorophyll content, chlorophyll a∶b ratio and the maximum quantum efficiency of photosystem II (F_V/F_M) in wild-type plants and homoplasmic transplastomic tRNA knock-out mutants. Datasets are shown for plants grown under ∼80 µE m⁻² s⁻¹ light intensity. Young Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i> and Δ<i>trnV-GAC</i> plants were measured after 7 weeks of growth, Δ<i>trnT-GGU</i> plants after 30 weeks (when they had reached a similar size as the other lines after 7 weeks). Mature Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i> and Δ<i>trnV-GAC</i> plants were measured after 20 weeks of growth, Δ<i>trnT-GGU</i> plants were raised at ∼20 µE m⁻² s⁻¹ for 40 weeks and then grown for 4 weeks at ∼80 µE m⁻² s⁻¹. The fourth leaf from the top was analyzed. For each plant line, three different plants were measured. F_V/F_M represents the maximum quantum efficiency of PSII in the dark adapted state. The error bars indicate the standard deviation, statistically significant differences from the wild type (p<0.05; Student's t-test) are indicated by asterisks.</p

Targeted deletion of the plastid <i>trnL-CAA</i> gene.

<p>(A) Physical map of the region in the tobacco plastid genome containing the gene for <i>trnL-CAA</i>. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning and RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in Δ<i>trnL-CAA</i> transplastomic plants. The <i>aadA</i> cassette replacing <i>trnL-CAA</i> is shown as grey box. (C) RFLP analysis of Δ<i>trnL-CAA</i> plastid transformants. All lines are homoplasmic and show exclusively the 3.1-kb band diagnostic of the transplastome. Wt: wild type. (D) tRNA-Leu(CAA) accumulation in the wild type and Δ<i>trnL-CAA</i> lines assessed by northern blotting. Hybridization to a plastid <i>trnL-CAA</i> probe confirms complete absence of the tRNA from homoplasmic knock-out lines. The ethidium bromide-stained agarose gel prior to blotting is also shown. (E) Confirmation of the homoplasmic state of the Δ<i>trnL-CAA</i> lines by inheritance assays. Germination of seeds from transplastomic plants on spectinomycin-containing medium results in a homogeneous population of green antibiotic-resistant seedlings. (F) Wild-type seedlings are sensitive to spectinomycin and bleach out in the presence of the antibiotic.</p

Targeted inactivation of the two plastid <i>trnT</i> genes.

<p>(A) Physical map of the <i>trnT-UGU</i> containing region in the tobacco plastid genome (ptDNA; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Shinozaki1" target="_blank">[44]</a>). Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning or RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in <i>ΔtrnT-UGU</i> transplastomic plants. The selectable marker gene <i>aadA</i> (grey box) is inserted into the <i>trnT-UGU</i> gene in the same transcriptional orientation. (C) Physical map of the <i>trnT-GGU</i> containing region in the tobacco ptDNA. (D) Map of the transformed plastid genome in Δ<i>trnT-GGU</i> plants. (E) RFLP analysis of Δ<i>trnT-UGU</i> transplastomic lines. Independently generated transplastomic lines are designated by Arabic numerals following the tRNA gene name. All transplastomic lines remain heteroplasmic and show both the 1.9 kb wild type-specific hybridization band and the 3.1 kb band diagnostic of the transformed plastid genome. Wt: wild type. (F) RFLP analysis of Δ<i>trnT-GGU</i> transplastomic plants. All lines are homoplasmic and show exclusively the 3.7 kb band diagnostic of the transgenic ptDNA. (G) Seed assays to confirm heteroplasmy of Δ<i>trnT-UGU</i> plants and homoplasmy of Δ<i>trnT-GGU</i> plants. Seeds were germinated in the absence or in the presence of spectinomycin. Δ<i>trnT-UGU</i> plants produce mostly antibiotic-sensitive seedlings and a few antibiotic-resistant seedlings, as expected for a heteroplasmic situation. Moreover, many of the resistant seedlings are variegated indicating their composition of tissues possessing and tissues lacking the transgenic plastid genome. In contrast, the Δ<i>trnT-GGU</i> lines produce homogeneous antibiotic-resistant progeny, confirming their homoplasmic status. (H) Analysis of tRNA-Thr(GGU) accumulation in the wild type, a heteroplasmic Δ<i>trnT-UGU</i> line and a homoplasmic Δ<i>trnT-GGU</i> line by northern blotting. Hybridization of electrophoretically separated RNA isolated from purified chloroplasts to a plastid <i>trnT-GGU</i> probe confirms complete lack of mature tRNA-Thr(GGU) in the Δ<i>trnT-GGU</i> homoplasmic knock-out line, whereas its accumulation is unaltered in the heteroplasmic Δ<i>trnT-UGU</i> line. Note accumulation of a ∼1.5 kb hybridizing RNA species in the Δ<i>trnT-GGU</i> line, which corresponds to the tRNA-Thr(GGU) disrupted with the <i>aadA</i> cassette. To control for RNA loading, part of the ethidium bromide-stained gel (containing the two largest 23S rRNA hidden break products) prior to blotting is also shown.</p

Decoding of the 64 triplets of the genetic code in plastids.

<p>Codon recognition by standard Watson-Crick base pairing, wobbling and/or superwobbling is indicated by the nucleotide in the wobble position of the anticodon of the tRNA species that can decode the triplet. Essential tRNA species are indicated in bold, non-essential tRNAs in normal font. The codon usage in plastids of <i>Nicotiana tabacum</i> is shown on a greyscale. Superscript numbers and indices indicate nucleoside modifications in the wobble position (N₃₄) of the anticodon of the tRNA species. ¹: 2′-O-methyluridine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Pillay1" target="_blank">[26]</a>; ²: 2′-O-methylcytidine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Pillay1" target="_blank">[26]</a>; ³: unknown modification <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Pfitzinger1" target="_blank">[23]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Sprouse1" target="_blank">[45]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Francis1" target="_blank">[46]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Francis2" target="_blank">[47]</a>; ⁴: inosine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Karcher1" target="_blank">[28]</a>; ⁵: lysidine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Francis3" target="_blank">[48]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Muramatsu1" target="_blank">[49]</a>; ⁶: 5-carboxymethylaminomethyl uridine (cmnm⁵U; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Schn1" target="_blank">[50]</a>); ⁷: 5-methylaminomethyl-2-thiouridine (mam⁵s²U; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Schn2" target="_blank">[51]</a>; <a href="http://trnadb.bioinf.uni-leipzig.de/" target="_blank">http://trnadb.bioinf.uni-leipzig.de/</a>); ^*: modification status of the wobble uridine unknown (RNA sequence not determined); -: stop codon.</p