Genome size diversity in orchids: consequences and evolution

Background

The amount of DNA comprising the genome of an organism (its genome size) varies a remarkable 40 000-fold across eukaryotes, yet most groups are characterized by much narrower ranges (e.g. 14-fold in gymnosperms, 3- to 4-fold in mammals). Angiosperms stand out as one of the most variable groups with genome sizes varying nearly 2000-fold. Nevertheless within angiosperms the majority of families are characterized by genomes which are small and vary little. Species with large genomes are mostly restricted to a few monocots families including Orchidaceae.

Scope

A survey of the literature revealed that genome size data for Orchidaceae are comparatively rare representing just 327 species. Nevertheless they reveal that Orchidaceae are currently the most variable angiosperm family with genome sizes ranging 168-fold (1C = 0·33–55·4 pg). Analysing the data provided insights into the distribution, evolution and possible consequences to the plant of this genome size diversity.

Conclusions

Superimposing the data onto the increasingly robust phylogenetic tree of Orchidaceae revealed how different subfamilies were characterized by distinct genome size profiles. Epidendroideae possessed the greatest range of genome sizes, although the majority of species had small genomes. In contrast, the largest genomes were found in subfamilies Cypripedioideae and Vanilloideae. Genome size evolution within this subfamily was analysed as this is the only one with reasonable representation of data. This approach highlighted striking differences in genome size and karyotype evolution between the closely related Cypripedium, Paphiopedilum and Phragmipedium. As to the consequences of genome size diversity, various studies revealed that this has both practical (e.g. application of genetic fingerprinting techniques) and biological consequences (e.g. affecting where and when an orchid may grow) and emphasizes the importance of obtaining further genome size data given the considerable phylogenetic gaps which have been highlighted by the current study.Key words: AFLP, C-value, chromosome, evolution, genome size, guard cell size, Orchidaceae, Robertsonian fission, Robertsonian fusion     

Paclitaxel reduces regulatory T cell numbers and inhibitory function and enhances the anti-tumor effects of the TLR9 agonist PF-3512676 in the mouse

The anti-tumor properties of Toll-like receptor (TLR) 9 agonist CpG oligodeoxynucleotides (ODN) are enhanced by combinations with several cytotoxic chemotherapy regimens. The mechanisms of this added benefit, however, remain unclear. We now report that, similar to the depletion of regulatory T cells (Treg) using anti-CD25, paclitaxel increased the anti-tumor effect of the TLR9 agonist PF-3512676 in a CD8⁺ T cell-dependent fashion. Paclitaxel treatment decreased Treg numbers in a TLR4-independent fashion, and preferentially affected cycling Treg expressing high levels of FoxP3. The paclitaxel-induced reduction in Treg FoxP3 expression was associated with reduced inhibitory function. Adoptively transferred tumor-antigen specific CD8⁺ T cells proliferated better in mice treated with paclitaxel and their recruitment in the tumor was increased. However, the systemic frequency of PF-3512676-induced tumor-antigen specific effector CD8⁺ T cells decreased with paclitaxel, suggesting opposite effects of paclitaxel on the anti-tumor response. Finally, gene expression profiling and studies of tumor-associated immune cells revealed a complex modulation of the PF-3512676-induced immune response by paclitaxel, including a decrease of IL-10 expression and an increase in IL-17-secreting CD4⁺ T cells. Collectively, these data suggest that paclitaxel combined with PF-3512676 may not only promote a better anti-tumor CD8⁺ response though increased recruitment in the tumor, possibly through Treg depletion and suppression, but also exerts more complex immune modulatory effects.     

Native Plant and Microbial Contributions to a Negative Plant-Plant Interaction

A number of hypotheses have been suggested to explain why invasive exotic plants dramatically increase their abundance upon transport to a new range. The novel weapons hypothesis argues that phytotoxins secreted by roots of an exotic plant are more effective against naïve resident competitors in the range being invaded. The common reed Phragmites australis has a diverse population structure including invasive populations that are noxious weeds in North America. P. australis exudes the common phenolic gallic acid, which restricts the growth of native plants. However, the pathway for free gallic acid production in soils colonized by P. australis requires further elucidation. Here, we show that exotic, invasive P. australis contain elevated levels of polymeric gallotannin relative to native, noninvasive P. australis. We hypothesized that polymeric gallotannin can be attacked by tannase, an enzymatic activity produced by native plant and microbial community members, to release gallic acid in the rhizosphere and exacerbate the noxiousness of P. australis. Native plants and microbes were found to produce high levels of tannase while invasive P. australis produced very little tannase. These results suggest that both invasive and native species participate in signaling events that initiate the execution of allelopathy potentially linking native plant and microbial biochemistry to the invasive traits of an exotic species.Invasive weeds are a major source of agricultural costs due to reduced productivity and the labor expended for weed control. In addition, the extensive use of herbicides to control weed populations has undesirable environmental consequences. Therefore, understanding mechanisms that facilitate exotic plant dispersal and displacement of natives in new ranges is critical to predicting and controlling invasions and may yield insights into the ecological processes that govern homeostasis and perturbation in natural plant communities.Phragmites australis (Cav.) Trin ex. Steud. (common reed) has been present in the United States for at least 10,000 years as a major component of mixed tidal wetland plant communities (Saltonstall, 2002). However, over the past 200 years its distribution and abundance has expanded rapidly and it is now considered one of the most aggressive invasive species in marsh communities in North America. Chloroplast DNA analysis has shown that 13 native North American Phragmites haplotypes exist, while invasive populations possess a single chloroplast DNA haplotype (M) that is also widespread in Europe and Asia (Saltonstall, 2002). These data are supported by nuclear microsatellite DNA analysis (Saltonstall, 2003) and morphological differences that distinguish native, noninvasive from exotic, invasive Phragmites in North America (Saltonstall et al., 2004). When grown under the same conditions, exotic Phragmites has significantly higher aboveground and belowground biomass than native Phragmites (Vasquez et al., 2005; Saltonstall and Stevenson, 2007), and this pattern is typically observed under field conditions as well although exceptions exist (League et al., 2006; Meadows and Saltonstall, 2007). Unfortunately today, only remnant native P. australis populations remain along the Atlantic Coast of North America, indicating the near total displacement of native populations by exotic P. australis.Various hypotheses have been forwarded to explain the rapid invasion of P. australis, of which human activities, stress regimes, and hydrologic disturbances have received the greatest attention (Chambers et al., 1999). Compared to invasion in terrestrial ecosystems, invasiveness in marsh communities is less well documented and it is still not clear how environmental factors relate to the establishment of specific dominant marsh species. Although allelopathy has been superficially suggested as the main displacing mechanism in P. australis (Kaneta and Sugiyama, 1972; Drifmeyer and Zieman, 1979), there has been minimal success in characterizing the responsible allelochemical. Interestingly, three triterpenoids (β-amacin, taraxerol, and taraxerone) and a flavone (tricin) have been identified from aerial portions of P. australis (Kaneta and Sugiyama, 1972; Drifmeyer and Zieman, 1979). Regrettably, none of these identified chemicals were tested for possible allelopathic activity.Previously, we showed that a root exudate component of P. australis roots inhibits seedling growth, and that production of the exudates is higher in the invasive P. australis haplotype (Rudrappa et al., 2007). The active fraction of this exudate was found to be composed of gallic acid (3,4,5-trihydroxybenzoic acid). Gallic acid is toxic to a variety of weeds, crop plant species, and the model plant species Arabidopsis (Arabidopsis thaliana; Rudrappa et al., 2007; Rudrappa and Bais, 2008). Our published results also show the persistence of gallic acid in soil extracts from P. australis-invaded fields, which validates our in vitro results and strongly supports the idea that P. australis'' invasive behavior may partly be due to the exudation of gallic acid in the soil/marsh (Rudrappa et al., 2007). Our studies concur with the earlier established reports of phytotoxicity and persistence of gallic acid in soil (Weidenhamer and Romeo, 2004).Biochemically, the transition from simple galloylglucoses to complex gallotannins is marked by addition of further galloyl moieties to the pentagalloylglucose (Niemetz and Gross, 2005). It is now known that free gallic acid is released from complexed gallotannins by simple hydrolysis reactions, wherein a tannase activity breaks gallate ester to form free gallic acid, ellagic acid, and Glc (Mahoney and Molyneux, 2004). Treatment of fungal tannase from Aspergillus flavus results in hydrolysis of pellicle-localized gallotannin to form gallic acid, and ellagic acid as two phenolic components (Mahoney and Molyneux, 2004). As gallic acid is often complexed as gallotannins (Niemetz and Gross, 2005), we speculated that plant- or microbial-derived tannase may facilitate free gallic acid release in salt marsh soils.Aside from allelopathy, invasive plants may deleteriously affect interactions between rhizospheric microbial communities and native plant species (Klironomos, 2002; Wardle et al., 2004; Callaway et al., 2008) to promote their expansion in new ranges. One specific example is the disruption of interactions between native species and their arbuscular mycorhizae, upon which the native species rely for nutrient acquisition (Stinson et al., 2006). Another recent study suggests that the recruitment or establishment of an altered soil microbial community may negatively impact the ability of native species to survive in the same soils (Batten et al., 2008). Evidences suggest that soil biota have several effects on the success of invasive plants and the interactions are based in part on the biochemistry, i.e. novel biochemical weapons (Callaway and Ridenour, 2004). However, to our knowledge, no previous studies have directly tested whether P. australis or any other exotic plant may exploit the biochemical potential of native plant and microbial communities to release a phytotoxin (gallic acid) from a relatively benign precursor (gallotannin) in the rhizosphere. This report presents evidence that links native plant and microbial biochemistry to the invasive traits of an exotic species.     

The p14 FAST protein of reptilian reovirus increases vesicular stomatitis virus neuropathogenesis

The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor.     

FolX and FolM Are Essential for Tetrahydromonapterin Synthesis in Escherichia coli and Pseudomonas aeruginosa

Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The ΔtyrA strain was, as expected, prototrophic for tyrosine, whereas the ΔtyrA ΔfolX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis.Pterins contain the bicyclic pteridine ring with an amino group in the 2-position and an oxo group in the 4-position; they can be reduced through the dihydro forms to the tetrahydro forms, which are active as cofactors (Fig. ​(Fig.1A).1A). Tetrahydropterins are known to be the cofactors for phenylalanine hydroxylases from Pseudomonas and Chromatium species as well as for mammalian aromatic amino acid hydroxylases and other mammalian enzymes (13, 17, 38, 41) (Fig. ​(Fig.1B).1B). Although the identity of the mammalian tetrahydropterin cofactor, tetrahydrobiopterin (H₄-BPt), is firmly established (38), the same is not true for bacteria, and the biosynthesis of bacterial tetrahydropterins is not well understood.Open in a separate windowFIG. 1.Tetrahydropterin structure, cofactor role, and biosynthesis. (A) The pterin nucleus, its levels of reduction, and the structures of compounds relevant to this study. (B) The requirement for a tetrahydropterin (H₄-pterin) cofactor for phenylalanine hydroxylase (PAH) and the cofactor regeneration cycle involving pterin-4a-carbinolamine dehydratase (PCD) and quinonoid dihydropterin (q-H₂-pterin) reductase (q-DHPR; EC 1.5.1.34). (C) The established steps in tetrahydrobiopterin (H₄-BPt) biosynthesis and possible routes for tetrahydromonapterin (H₄-MPt) biosynthesis in relation to the folate pathway. H₄-BPt is formed by the sequential action of 6-pyruvoyltetrahydropterin (P-H₄-Pt) synthase (PTPS-II) and sepiapterin reductase (SR). H₄-MPt could originate via the FolX-catalyzed epimerization of dihydroneopterin triphosphate (H₂-NPt-P₃) to dihydromonapterin triphosphate (H₂-MPt-P₃), followed by dephosphorylation to dihydromonapterin (H₂-MPt) and reduction by a dihydropterin reductase (EC 1.5.1.33), putatively FolM. H₄-MPt also could come from the FolB-mediated epimerization of dihydroneopterin (H₂-NPt) followed by reduction. FolB also mediates the side chain cleavage of H₂-NPt or H₂-MPt to give 6-hydroxymethyldihydropterin (H₂-HMPt); the H₂-MPt cleavage is omitted for simplicity. Other abbreviations: P-ase, phosphatase; H₂-HMPt-P₂, 6-hydroxymethyldihydropterin diphosphate; pABA, p-aminobenzoate; H₂-pteroate, dihydropteroate; H₂-folate, dihydrofolate; H₄-folate, tetrahydrofolate.While a few bacterial taxa, such as Cyanobacteria and Chlorobia, produce H₄-BPt, most do not, as judged directly from pterin analysis and indirectly from the rarity of H₄-BPt biosynthesis genes 6-pyruvoyltetrahydropterin synthase II (PTPS-II) and sepiapterin reductase (SR) (Fig. ​(Fig.1C)1C) among sequenced genomes (12, 25). As bacteria lacking H₄-BPt include Pseudomonas and many others with phenylalanine hydroxylase genes, it is clear that bacterial phenylalanine hydroxylases generally must use a cofactor other than H₄-BPt. The most prominent candidate is tetrahydromonapterin (H₄-MPt), which occurs in Escherichia coli (21) and almost certainly also in Pseudomonas species (11, 17). H₄-MPt could be derived from the dihydropterin intermediates of folate biosynthesis via two different routes (Fig. ​(Fig.1C).1C). These are (i) the conversion of dihydroneopterin triphosphate (H₂-NPt-P₃) to dihydromonapterin triphosphate (H₂-MPt-P₃) by H₂-NPt-P₃ epimerase (FolX) followed by dephosphorylation and reduction to the tetrahydro level, and (ii) the conversion of dihydroneopterin (H₂-NPt) to dihydromonapterin (H₂-MPt) by the epimerase action of dihydroneopterin aldolase (FolB) and then reduction. FolB is a fairly well-understood enzyme of folate synthesis (9), but FolX has no known biological role and a folX deletant has no obvious phenotype (19). folX genes apparently are confined to Gammaproteobacteria (9).Although the epimerase activities of FolX and FolB have been demonstrated amply in vitro (1, 5, 19), no genetic evidence links either enzyme to H₄-MPt formation in vivo. The situation with the reduction of H₂-MPt to H₄-MPt is even less clear, because this activity has not been investigated experimentally. A candidate enzyme for this step nevertheless can be proposed on bioinformatic grounds: the somewhat mysterious FolM protein (9). FolM belongs to a subset of the short-chain dehydrogenase/reductase (SDR) family having the characteristic motif TGX₃RXG (in place of TGX₃GXG, which typifies other SDRs). The archetype of this subset is Leishmania pteridine reductase 1 (PTR1), which reduces various dihydropterins to the tetrahydro state (15). E. coli FolM has low dihydrofolate (H₂-folate) and dihydrobiopterin (H₂-BPt) reductase activities in vitro (14), but neither of these is likely to be its physiological function, since H₂-folate reduction normally is mediated by FolA and E. coli lacks H₄-BPt. folM genes occur in many Gram-negative organisms, including Chlamdiae, Chloroflexi, Cyanobacteria, Acidobacteria, Planctomycetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria (9).We report here comparative genomic and genetic evidence that FolX and FolM are required for H₄-MPt synthesis in E. coli and P. aeruginosa, the bacteria in which H₄-MPt has been most studied, and biochemical evidence that FolM has high H₂-MPt reductase activity. We also point out gaps in the understanding of pterin metabolism that our data bring sharply into focus.     

Combining oncolytic virotherapy and tumour vaccination

The interactions between the immune system, a malignant tumour and an oncolytic virus are complex and poorly understood. For oncolytic viruses to become successful therapeutics we need to better understand these interactions and identify strategies to take advantage of defects in the innate immune response within tumours and avoid cellular anti-viral responses while capitalizing on anti-tumoural immunity. In this review we will discuss the evidence for the induction of tumour-specific immune responses by oncolytic viruses as well as by cancer vaccines. We will then describe some of the barriers to successful cancer immunotherapy, and finally we will outline a strategy for enhancing anti-tumoural immunity while reducing anti-viral immunity by combining tumour vaccination with oncolytic viral therapy.     

Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.     

Clonal derivation and characterization of human embryonic stem cell lines

Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.