Comparison of Sugars,Iridoid Glycosides and Amino Acids in Nectar and Phloem Sap of Maurandya barclayana,Lophospermum erubescens,and Brassica napus

Background

Floral nectar contains sugars and amino acids to attract pollinators. In addition, nectar also contains different secondary compounds, but little is understood about their origin or function. Does nectar composition reflect phloem composition, or is nectar synthesized and/or modified in nectaries? Studies where both, the nectar as well as the phloem sap taken from the same plant species were analyzed in parallel are rare. Therefore, phloem sap and nectar from different plant species (Maurandya barclayana, Lophospermum erubescens, and Brassica napus) were compared.

Methodology and Principal Findings

Nectar was collected with microcapillary tubes and phloem sap with the laser-aphid-stylet technique. The nectar of all three plant species contained high amounts of sugars with different percentages of glucose, fructose, and sucrose, whereas phloem sap sugars consisted almost exclusively of sucrose. One possible reason for this could be the activity of invertases in the nectaries. The total concentration of amino acids was much lower in nectars than in phloem sap, indicating selective retention of nitrogenous solutes during nectar formation. Nectar amino acid concentrations were negatively correlated with the nectar volumes per flower of the different plant species. Both members of the tribe Antirrhineae (Plantaginaceae) M. barclayana and L. erubescens synthesized the iridoid glycoside antirrhinoside. High amounts of antirrhinoside were found in the phloem sap and lower amounts in the nectar of both plant species.

Conclusions/Significance

The parallel analyses of nectar and phloem sap have shown that all metabolites which were found in nectar were also detectable in phloem sap with the exception of hexoses. Otherwise, the composition of both aqueous solutions was not the same. The concentration of several metabolites was lower in nectar than in phloem sap indicating selective retention of some metabolites. Furthermore, the existence of antirrhinoside in nectar could be based on passive secretion from the phloem.     

An evolutionary relationship between Stearoyl-CoA Desaturase (SCD) protein sequences involved in fatty acid metabolism

Background:

Stearoyl-CoA desaturase (SCD) is a key enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs) in fat biosynthesis. Despite being crucial for interpreting SCDs’ roles across species, the evolutionary relationship of SCD proteins across species has yet to be elucidated. This study aims to present this evolutionary relationship based on amino acid sequences.

Methods:

Using Multiple Sequence Alignment (MSA) and phylogenetic construction methods, a hypothetical evolutionary relationship was generated between the stearoyl-CoA desaturase (SCD) protein sequences between 18 different species.

Results:

SCD protein sequences from Homo sapiens, Pan troglodytes (chimpanzee), and Pongo abelii (orangutan) have the lowest genetic distances of 0.006 of the 18 species studied. Capra hircus (goat) and Ovis aries (Sheep) had the next lowest genetic distance of 0.023. These farm animals are 99.987% identical at the amino acid level.

Conclusions:

The SCD proteins are conserved in these 18 species, and their evolutionary relationships are similar. Key Words: Phylogenetic analysis, Stearoyl-CoA desaturase (SCD) proteins, Multiple sequence alignment     

Whole genome comparative analysis of transposable elements provides new insight into mechanisms of their inactivation in fungal genomes

Background

Transposable Elements (TEs) are key components that shape the organization and evolution of genomes. Fungi have developed defense mechanisms against TE invasion such as RIP (Repeat-Induced Point mutation), MIP (Methylation Induced Premeiotically) and Quelling (RNA interference). RIP inactivates repeated sequences by promoting Cytosine to Thymine mutations, whereas MIP only methylates TEs at C residues. Both mechanisms require specific cytosine DNA Methyltransferases (RID1/Masc1) of the Dnmt1 superfamily.

Results

We annotated TE sequences from 10 fungal genomes with different TE content (1-70%). We then used these TE sequences to carry out a genome-wide analysis of C to T mutations biases. Genomes from either Ascomycota or Basidiomycota that were massively invaded by TEs (Blumeria, Melampsora, Puccinia) were characterized by a low frequency of C to T mutation bias (10-20%), whereas other genomes displayed intermediate to high frequencies (25-75%). We identified several dinucleotide signatures at these C to T mutation sites (CpA, CpT, and CpG). Phylogenomic analysis of fungal Dnmt1 MTases revealed a previously unreported association between these dinucleotide signatures and the presence/absence of sub-classes of Dnmt1.

Conclusions

We identified fungal genomes containing large numbers of TEs with many C to T mutations associated with species-specific dinucleotide signatures. This bias suggests that a basic defense mechanism against TE invasion similar to RIP is widespread in fungi, although the efficiency and specificity of this mechanism differs between species. Our analysis revealed that dinucleotide signatures are associated with the presence/absence of specific Dnmt1 subfamilies. In particular, an RID1-dependent RIP mechanism was found only in Ascomycota.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1347-1) contains supplementary material, which is available to authorized users.     

Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data

Background

The intra- and inter-species genetic diversity of bacteria and the absence of ‘reference’, or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia.

Methods

A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM) of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization.

Results

The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52%) corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as ‘centroids’ in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578.

Conclusion

The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra-species variability.     

Mistletoes and mutant albino shoots on woody plants as mineral nutrient traps

Background and Aims

Potassium, sulphur and zinc contents of mistletoe leaves are generally higher than in their hosts. This is attributed to the fact that chemical elements which are cycled between xylem and phloem in the process of phloem loading of sugars are trapped in the mistletoe, because these parasites do not feed their hosts. Here it is hypothesized that mutant albino shoots on otherwise green plants should behave similarly, because they lack photosynthesis and thus cannot recycle elements involved in sugar loading.

Methods

The mineral nutrition of the mistletoe Scurrula elata was compared with that of albino shoots on Citrus sinensis and Nerium oleander. The potential for selective nutrient uptake by the mistletoe was studied by comparing element contents of host leaves on infected and uninfected branches and by manipulation of the haustorium–shoot ratio in mistletoes. Phloem anatomy of albino leaves was compared with that of green leaves.

Key Results

Both mistletoes and albino leaves had higher contents of potassium, sulphur and zinc than hosts or green leaves, respectively. Hypothetical discrimination of nutrient elements during the uptake by the haustorium is not supported by our data. Anatomical studies of albino leaves showed characteristics of release phloem.

Conclusions

Both albino shoots and mistletoes are traps for elements normally recycled between xylem and phloem, because retranslocation of phloem mobile elements into the mother plant or the host is low or absent. It can be assumed that the lack of photosynthetic activity in albino shoots and thus of sugars needed in phloem loading is responsible for the accumulation of elements. The absence of phloem loading is reflected in phloem anatomy of these abnormal shoots. In mistletoes the evolution of a parasitic lifestyle has obviously eliminated substantial feeding of the host with photosynthates produced by the mistletoe.     

Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae)

Background and Aims

Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data.

Methods

Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals.

Key Results

Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation.

Conclusions

The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile.     

Hemolymph proteome changes during worker brood development match the biological divergences between western honey bees (Apis mellifera) and eastern honey bees (Apis cerana)

Background

Hemolymph plays key roles in honey bee molecule transport, immune defense, and in monitoring the physiological condition. There is a lack of knowledge regarding how the proteome achieves these biological missions for both the western and eastern honey bees (Apis mellifera and Apis cerana). A time-resolved proteome was compared using two-dimensional electrophoresis-based proteomics to reveal the mechanistic differences by analysis of hemolymph proteome changes between the worker bees of two bee species during the larval to pupal stages.

Results

The brood body weight of Apis mellifera was significantly heavier than that of Apis cerana at each developmental stage. Significantly, different protein expression patterns and metabolic pathways were observed in 74 proteins (166 spots) that were differentially abundant between the two bee species. The function of hemolymph in energy storage, odor communication, and antioxidation is of equal importance for the western and eastern bees, indicated by the enhanced expression of different protein species. However, stronger expression of protein folding, cytoskeletal and developmental proteins, and more highly activated energy producing pathways in western bees suggests that the different bee species have developed unique strategies to match their specific physiology using hemolymph to deliver nutrients and in immune defense.

Conclusions

Our disparate findings constitute a proof-of-concept of molecular details that the ecologically shaped different physiological conditions of different bee species match with the hemolymph proteome during the brood stage. This also provides a starting point for future research on the specific hemolymph proteins or pathways related to the differential phenotypes or physiology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-563) contains supplementary material, which is available to authorized users.     

A complete analysis of HA and NA genes of influenza A viruses

Background

More and more nucleotide sequences of type A influenza virus are available in public databases. Although these sequences have been the focus of many molecular epidemiological and phylogenetic analyses, most studies only deal with a few representative sequences. In this paper, we present a complete analysis of all Haemagglutinin (HA) and Neuraminidase (NA) gene sequences available to allow large scale analyses of the evolution and epidemiology of type A influenza.

Methodology/Principal Findings

This paper describes an analysis and complete classification of all HA and NA gene sequences available in public databases using multivariate and phylogenetic methods.

Conclusions/Significance

We analyzed 18975 HA sequences and divided them into 280 subgroups according to multivariate and phylogenetic analyses. Similarly, we divided 11362 NA sequences into 202 subgroups. Compared to previous analyses, this work is more detailed and comprehensive, especially for the bigger datasets. Therefore, it can be used to show the full and complex phylogenetic diversity and provides a framework for studying the molecular evolution and epidemiology of type A influenza virus. For more than 85% of type A influenza HA and NA sequences into GenBank, they are categorized in one unambiguous and unique group. Therefore, our results are a kind of genetic and phylogenetic annotation for influenza HA and NA sequences. In addition, sequences of swine influenza viruses come from 56 HA and 45 NA subgroups. Most of these subgroups also include viruses from other hosts indicating cross species transmission of the viruses between pigs and other hosts. Furthermore, the phylogenetic diversity of swine influenza viruses from Eurasia is greater than that of North American strains and both of them are becoming more diverse. Apart from viruses from human, pigs, birds and horses, viruses from other species show very low phylogenetic diversity. This might indicate that viruses have not become established in these species. Based on current evidence, there is no simple pattern of inter-hemisphere transmission of avian influenza viruses and it appears to happen sporadically. However, for H6 subtype avian influenza viruses, such transmissions might have happened very frequently and multiple and bidirectional transmission events might exist.     

Molecular phylogeny and habitat diversification of the genus Farfugium (Asteraceae) based on nuclear rDNA and plastid DNA

Background and Aims

Farfugium (Asteraceae) is a small genus that contains the two species F. japonicum and F. hiberniflorum and is distributed along a long archipelago in east Asia. The common taxon, F. japonicum, includes three varieties associated with a wide range of habitats, including forest understorey (sciophytes), coastal crag (heliophytes) and riverbed (rheophytes). Leaf shape is an important taxonomic character within this genus and is associated with the habitat.

Methods

Twenty populations that included all Farfugium taxa were collected throughout its range. Leaf morphology was measured to determine differences amongst the taxa. Phylogenetic analyses based on sequences of the internal transcribed spacer of nuclear rDNA and four plastid DNA regions (matK, trnL-trnF, trnH-psbA and rpl20-rps12) were conducted separately.

Key Results

Leaf morphology was significantly different amongst taxa, but morphological variations were partly explained by adaptation to certain environmental conditions that each population inhabited. Molecular phylogenies for the nDNA internal transcribed spacer and cpDNA were consistent in classifying F. hiberniflorum and the Taiwanese var. formosanum, whilst suggesting polyphyletic origins for the rheophyte, sciophyte and heliophyte taxa. All samples from the southern Ryukyus (Japan) and Taiwan clustered into a monophyletic group, which corroborates the land configuration theory involving Quaternary land-bridge formation and subsequent fragmentation into islands. The incongruence between the two DNA datasets may imply traces of introgressive hybridization and/or incomplete lineage sorting.

Conclusions

The occurrence of rheophyte, sciophyte and heliophyte plants within Farfugium may be attributable to their isolation on islands and subsequent adaptation to the riparian, coastal crag and forest understorey environments, following their migration over the Quaternary land-bridge formation along their distribution range. Nearly identical DNA sequences coupled with highly divergent morphologies amongst these taxa suggest that diversification was rapid.     

Geographically widespread swordfish barcode stock identification: a case study of its application

Background

The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species.

Methodology

We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5′ hyper-variable fragment of the control region (5′dloop), were also used to validate the efficacy of COI as a stock-specific marker.

Case Report

This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products.

Conclusions

The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5′dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.     

Evolutionary history and phylodynamics of influenza A and B neuraminidase (NA) genes inferred from large-scale sequence analyses

Background

Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking.

Methodology/Principal findings

Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The d _N /d _S ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding.

Conclusions/Significance

The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance.     

Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop

Background and Aims

Floral development depends on photosynthetic products delivered by the phloem. Previous work suggested the path to the flower involved either the apoplast or the symplast. The objective of the present work was to determine the path and its mechanism of operation.

Methods

Maize (Zea mays) plants were grown until pollination. For simplicity, florets were harvested before fertilization to ensure that all tissues were of maternal origin. Because sucrose from phloem is hydrolysed to glucose on its way to the floret, the tissues were imaged and analysed for glucose using an enzyme-based assay. Also, carboxyfluorescein diacetate was fed to the stems and similarly imaged and analysed.

Key Results

The images of live sections revealed that phloem contents were released to the pedicel apoplast below the nucellus of the florets. Glucose or carboxyfluorescein were detected and could be washed out. For carboxyfluorescein, the plasma membranes of the phloem parenchyma appeared to control the release. After release, the nucellus absorbed apoplast glucose selectively, rejecting carboxyfluorescein.

Conclusions

Despite the absence of an embryo, the apoplast below the nucellus was a depot for phloem contents, and the strictly symplast path is rejected. Because glucose and carboxyfluorescein were released non-selectively, the path to the floret resembled the one later when an embryo is present. The non-selective release indicates that turgor at phloem termini cannot balance the full osmotic potential of the phloem contents and would create a downward pressure gradient driving bulk flow toward the sink. Such a gradient was previously measured by Fisher and Cash-Clark in wheat. At the same time, selective absorption from the apoplast by the nucellar membranes would support full turgor in this tissue, isolating the embryo sac from the maternal plant. The isolation should continue later when an embryo develops.