Identification of shared single copy nuclear genes in Arabidopsis, Populus, Vitis and Oryza and their phylogenetic utility across various taxonomic levels

Background

Although the overwhelming majority of genes found in angiosperms are members of gene families, and both gene- and genome-duplication are pervasive forces in plant genomes, some genes are sufficiently distinct from all other genes in a genome that they can be operationally defined as 'single copy'. Using the gene clustering algorithm MCL-tribe, we have identified a set of 959 single copy genes that are shared single copy genes in the genomes of Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa. To characterize these genes, we have performed a number of analyses examining GO annotations, coding sequence length, number of exons, number of domains, presence in distant lineages, such as Selaginella and Physcomitrella, and phylogenetic analysis to estimate copy number in other seed plants and to demonstrate their phylogenetic utility. We then provide examples of how these genes may be used in phylogenetic analyses to reconstruct organismal history, both by using extant coverage in EST databases for seed plants and de novo amplification via RT-PCR in the family Brassicaceae.

Results

There are 959 single copy nuclear genes shared in Arabidopsis, Populus, Vitis and Oryza ["APVO SSC genes"]. The majority of these genes are also present in the Selaginella and Physcomitrella genomes. Public EST sets for 197 species suggest that most of these genes are present across a diverse collection of seed plants, and appear to exist as single or very low copy genes, though exceptions are seen in recently polyploid taxa and in lineages where there is significant evidence for a shared large-scale duplication event. Genes encoding proteins localized in organelles are more commonly single copy than expected by chance, but the evolutionary forces responsible for this bias are unknown. Regardless of the evolutionary mechanisms responsible for the large number of shared single copy genes in diverse flowering plant lineages, these genes are valuable for phylogenetic and comparative analyses. Eighteen of the APVO SSC single copy genes were amplified in the Brassicaceae using RT-PCR and directly sequenced. Alignments of these sequences provide improved resolution of Brassicaceae phylogeny compared to recent studies using plastid and ITS sequences. An analysis of sequences from 13 APVO SSC genes from 69 species of seed plants, derived mainly from public EST databases, yielded a phylogeny that was largely congruent with prior hypotheses based on multiple plastid sequences. Whereas single gene phylogenies that rely on EST sequences have limited bootstrap support as the result of limited sequence information, concatenated alignments result in phylogenetic trees with strong bootstrap support for already established relationships. Overall, these single copy nuclear genes are promising markers for phylogenetics, and contain a greater proportion of phylogenetically-informative sites than commonly used protein-coding sequences from the plastid or mitochondrial genomes.

Conclusions

Putatively orthologous, shared single copy nuclear genes provide a vast source of new evidence for plant phylogenetics, genome mapping, and other applications, as well as a substantial class of genes for which functional characterization is needed. Preliminary evidence indicates that many of the shared single copy nuclear genes identified in this study may be well suited as markers for addressing phylogenetic hypotheses at a variety of taxonomic levels.     

Exposure to acute hypoxia induces a transient DNA damage response which includes Chk1 and TLK1

Severe hypoxia has been demonstrated to induce a replication arrest which is associated with decreased levels of nucleotides. Chk1 is rapidly phosphorylated in response to severe hypoxia and in turn deactivates TLK1 through phosphorylation. Loss of Chk1 has been shown to sensitize cells to hypoxia/reoxygenation. After short (acute) exposure to hypoxia this is due to an increased rate of reoxygenation-induced replication restart and subsequent p53-dependent apoptosis. After longer (chronic) exposure to hypoxia S phase cells do not undergo reoxygenation-induced replication restart. Cells exposed to these levels of hypoxia are however sensitive to loss of Chk1. This suggests a new role for Chk1 in the cell cycle response to reoxygenation.Key words: hypoxia, reoxygenation, replication restart, Chk1, TLK1     

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A2 homologue: an example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)s or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA(2) homologues and, together with additional strategies, supports the concept of the presence of others "bioactive sites" distinct from the catalytic site in snake venom myotoxic PLA(2)s.     

Biochemical and genomic analysis of sucrose metabolism during coffee (Coffea arabica) fruit development

Sucrose metabolism and the role of sucrose synthase were investigated in the fruit tissues (pericarp, perisperm, and endosperm) of Coffea arabica during development. Acid invertase, sucrose phosphate synthase, and sucrose synthase activities were monitored and compared with the levels of sucrose and reducing sugars. Among these enzymes, sucrose synthase showed the highest activities during the last stage of endosperm and pericarp development and this activity paralleled closely the accumulation of sucrose in these tissues at this stage. Carbon partitioning in fruits was studied by pulse-chase experiments with (14)C-sugars and revealed high rates of sucrose turnover in perisperm and endosperm tissues. Additional feeding experiments with (14)CO(2) showed that leaf photosynthesis contributed more to seed development than the pericarp in terms of photosynthate supply to the endosperm. Sugar analysis, feeding experiments, and histological studies indicated that the perisperm plays an important role in this downloading process. It was observed that the perisperm presents a transient accumulation of starch which is degraded as the seed develops. Two full-length cDNAs (CaSUS1 and CaSUS2) and the complete gene sequence of the latter were also isolated. They encode sucrose synthase isoforms that are phylogenetically distinct, indicating their involvement in different physiological functions during cherry development. Contrasting expression patterns were observed for CaSUS1 and CaSUS2 in perisperm, endosperm, and pericarp tissues: CaSUS1 mRNAs accumulated mainly during the early development of perisperm and endosperm, as well as during pericarp growing phases, whereas those of CaSUS2 paralleled sucrose synthase activity in the last weeks of pericarp and endosperm development. Taken together, these results indicate that sucrose synthase plays an important role in sugar metabolism during sucrose accumulation in the coffee fruit.     

Influence of toxic and non-toxic phytoplankton on feeding and survival of Dreissena polymorpha (Pallas) larvae

Grazing and survival of larvae of the zebra mussel, Dreissena polymorpha, on a green alga and cyanobacteria were studied in laboratory experiments. Clearance rates of the larvae were determined for Chlamydomonas reinhardtii (green alga), two non-toxic and two toxic Microcystis aeruginosa strains (Cyanobacteria). Clearance rates of larvae on non-toxic Microcystis were significantly higher than on toxic Microcystis. The clearance rate on Chlamydomonas reinhardtii was in between the clearance rates on toxic and non-toxic Microcystis strains and not significantly different from them. Effects of toxicity of Microcystis on the survival of zebra mussel larvae was investigated in a short-term experiment. Survival of larvae fed toxic Microcystis was lower than that of larvae fed non-toxic Microcystis, but higher than that of starved larvae. This may imply that, for survival of zebra mussel larvae, it is better to have bad quality (toxic) food than no food.     

Genetic diversity and population structure of Eugenia dysenterica DC. (``cagaiteira' – Myrtaceae) in Central Brazil: Spatial analysis and implications for conservation and management

Studies about the organization of the genetic variability and population structure in natural plant populations are used to support conservation and management programs. Among the Cerrado fruit tree species that possess potential economic importance in agriculture, the “Cagaiteira” (Eugenia dysenterica DC. – Myrtaceae), deserves an special position. We obtained information about allele and genotypic frequencies in 10 local populations, situated up to 250 km apart, from six isozymes that furnished a total of 8 loci. The average within-population fixation index (f) was 0.337, and the out crossing rate was 0.835, suggesting a mixed mating system for this species, which seems to be preferably alogamous. Based on genetic diversity and analysis of variance techniques, a high degree of population differentiation (θ_P = 0.154) was found, in comparison with other tropical tree species. Genetic divergence, analyzed by Nei's genetic distances, clustered with UPGMA and ordinated by non-metric multidimensional scaling, showed spatial patterns of clusters of local populations. Explicit spatial analyses, using Mantel tests and boundary tests, basically confirmed these patterns and revealed a complex pattern of genetic variation in geographic space. The intercept of the multivariate spatial correlograms was around 120 km, an indication of the minimum distance between samples needed to conserve genetic diversity among samples. This spatial scale can be used to define population genetics units for conservation programs or to establish sampling strategies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparing grazing by Dreissena polymorpha on phytoplankton in the presence of toxic and non-toxic cyanobacteria

SUMMARY 1. The feeding behaviour of the zebra mussel ( Dreissena polymorpha ) was studied in the laboratory on different combinations of food, including a green alga ( Chlamydomonas reinhardtii ) and toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa .
2. The highest clearance rate of phytoplankton by zebra mussels was found when the mussels were feeding on a mixture of Chlamydomonas and non-toxic Microcystis , the lowest on a mixture of Chlamydomonas and toxic Microcystis .
3. The differences found in the clearance rates between food combinations can be partly explained by the production of pseudofaeces containing live phytoplankton cells. Zebra mussels expelled significantly more live phytoplankton cells in the presence of toxic Microcystis than in the presence of non-toxic Microcystis . The pseudofaeces contained predominantly live Chlamydomonas cells. Proportionately much less live Microcystis cells were encountered in the pseudofaeces.
4. Consequently, grazing of zebra mussels on a combination of Chlamydomonas and Microcystis may finally result in a dominance of Chlamydomonas over Microcystis . The presence of toxic Microcystis may even strengthen this shift.     

Partitioning beta‐diversity through different pond hydroperiod lengths reveals predominance of nestedness in assemblages of immature odonates

Patterns of freshwater invertebrate assemblage structure in the transition from permanent to non‐permanent lentic habitats are well described in the literature. However, the effects of small changes in the hydroperiod of non‐permanent ponds on invertebrate assemblage structure remain less studied, especially on β‐diversity. Thus, we tested the effects of different pond hydroperiod lengths on the assemblage structure of immature odonates, in terms of both α‐ and β‐diversity. Small high‐altitude ponds with different hydroperiod lengths (assigned to ‘short’, ‘medium’ and ‘long’ hydroperiods) were sampled in southern Brazil between 2013 and 2014. Based on the hypothesis that shorter hydroperiods filter constituents of lentic fauna, i.e. that long‐living species cannot inhabit shorter‐hydroperiod ponds, we expected to find higher α‐ and β‐diversity in longer hydroperiods, as well as predominance of the nestedness component in β‐diversity. Restricted occurrence of some genera and higher α‐diversity of immature odonate assemblages was detected in long‐hydroperiod ponds. Within‐hydroperiod β‐diversity values did not vary among hydroperiods, because the occasional occurrence of some genera with high dispersal ability of adults in short‐hydroperiod ponds yielded similar values of the β‐diversity among hydroperiods. Partitioning of β‐diversity among hydroperiods revealed a significant higher contribution of the nestedness component rather than turnover. This pattern is explained by the occurrence of some generalist genera across the whole gradient of hydroperiod, as a subset of fauna in longer‐hydroperiod ponds. Thus, our results suggest that reduction in hydroperiod length, if occurring in the future climate change, would favor habitat‐generalist taxa in lentic ecosystems.     

Edaphically distinct habitats shape the crown architecture of <Emphasis Type="Italic">Lychnophora ericoides</Emphasis> Mart. (Asteraceae) on tropical mountaintops

Different architectural arrangements may represent contrasting morphological solutions to different environmental pressures. This work aims to elucidate whether the crown architecture of Lychnophora ericoides (Asteraceae) modifies in response to harsh soil conditions (nutrient poor and heavy metal rich) and how its crown architecture affects its reproduction. One hundred and sixty L. ericoides individuals were randomly sampled from eight populations, four on quartzite and four on iron canga rocky complexes in the Iron Quadrangle, southeastern Brazil. We performed soil analyses to characterize edaphic differences and used eight morphometric parameters to describe the crown architecture of the plants. We calculated the population density and reproductive potential to verify the relationship between habitat, architecture, and fitness. Canga soils were more nutrient rich than quartzite soils and plants were architecturally distinct in each habitat. Plants established on canga soils were shorter, had a thinner main branch, and a smaller leaf than those established on quartzite soils. Moreover, plants on canga soils had a larger crown diameter and a greater number of branches and inflorescences. There was no difference in population density but the reproductive potential varied among populations and habitats. The crown architecture of L. ericoides closely relates to reproductive potential and may favor the reproduction of more architectonically complex plants, regardless of habitat.