Kinetics of arsenite removal by halobacteria from a highland Andean Chilean Salar

Díaz-Palma Paula, Alfaro Gleny, Hengst Martha, Pozo Patricia, Stegen Susana, Queirolo Fabrizio,Rojo Gonzalo, Silva Pedro, Arias Diana, Gallardo Karem and Contreras-Ortega Carlos

Abstract
Background: The purpose of this study was to identify arsenite-oxidizing halobacteria in samples obtained from Salar de Punta Negra, II Region of Chile. Seven bacterial isolates, numbered as isolates I to VII, grown in a culture medium with 100 ppm as NaAsO2 (As (III)) were tested. Bacterial growth kinetics and the percent of arsenite removal (PAR) were performed simultaneously with the detection of an arsenite oxidase enzyme through Dot Blot analysis.

Results: An arsenite oxidase enzyme was detected in all isolates, expressed constitutively after 10 generations grown in the absence of As (III). Bacterial growth kinetics and corresponding PAR values showed significant fluctuations over time. PARs close to 100% were shown by isolates V, VI, and VII, at different times of the bacterial growth phase; while isolate II showed PAR values around 40%, remaining constant over time.

Conclusion: Halobacteria from Salar de Punta Negra showed promising properties as arsenite removers under control conditions, incubation time being a critical parameter.

Keywords: Arsenic, Removal, Halobacteria, Arsenite-Oxidase

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Molecular identification of microorganisms associated with the brine shrimp Artemia franciscana

Misty R Riddle, Bonnie K Baxter and Brian J Avery

Abstract
Background: Prior research on the microorganisms associated with the brine shrimp, Artemia franciscana, has mainly been limited to culture-based identification techniques or feeding studies for aquaculture. Our objective was to identify bacteria and archaea associated with Artemia adults and encysted embryos to understand the role of microbes in the Artemia life cycle and, therefore, their importance in a hypersaline food chain.

Results: We used small subunit (SSU) 16S ribosomal RNA gene sequencing to identify bacteria and archaea associated with adults and encysted Artemia embryos from one of their natural environments – Great Salt Lake (GSL), Utah, USA. We found that bacterial sequences most closely related to the genera Halomonas and Vibrio were commonly extracted from GSL adult Artemia, while bacterial sequences most similar to the genera Halomonas, Psychroflexus and Alkalilimnicola dominate in GSL water. Encysted embryos (cysts) yielded bacterial sequences from the genera Idiomarina and Salinivibrio, which were absent from adults and water. Common archaeal sequences in adults were most closely related to the genera Haloterrigena and Haloarcula, while all of the archaeal sequences from GSL water were most similar to the genus Halogeometricum. Cyst derived archaeal sequences were most closely related to the genera Halorubrum and Haloarcula.

Conclusions: In addition to identifying microbial rRNA sequences that are specific to different stages of the Artemia life cycle, we observed striking differences in the sequences associated with the adult Artemia population in samples collected from GSL at different times and locations. While our study was limited in scope and the sample was small, our findings provide a foundation for future research into how the bacteria and archaea associated with Artemia influence the Artemia life cycle, and GSL food web

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Reduction of photosynthetic sensitivity in response to abiotic stress in tomato is mediated by a new generation plant activator

Jason J Wargent, Douglas A Pickup, Nigel D Paul and Michael R Roberts

 

Abstract
Background: Yield losses as a result of abiotic stress factors present a significant challenge for the future of global food production. While breeding technologies provide potential to combat negative stress-mediated outcomes over time, interventions which act to prime plant tolerance to stress, via the use of phytohormone-based elicitors for example, could act as a valuable tool for crop protection. However, the translation of fundamental biology into functioning solution is often constrained by knowledge-gaps.

Results: Photosynthetic and transcriptomic responses were characterised in young tomato (Solanum lycopersicumL.) seedlings in response to pre-treatment with a new plant health activator technology, ‘Alethea’, followed by a subsequent 100 mM salinity stress. Alethea is a novel proprietary technology composed of three key constituent compounds; the hitherto unexplored compound potassium dihydrojasmonate, an analogue of jasmonic acid; sodium benzoate, a carboxylic acid precursor to salicylic acid, and the α-amino acid L-arginine. Salinity treatment led to a maximal 47% reduction in net photosynthetic rate 8 d following NaCl treatment, yet in Alethea pre-treated seedlings, sensitivity to salinity stress was markedly reduced during the experimental period. Microarray analysis of leaf transcriptional responses showed that while salinity stress and Alethea individually impacted on largely nonoverlapping, distinct groups of genes, Alethea pre-treatment substantially modified the response to salinity. Alethea affected the expression of genes related to biotic stress, ethylene signalling, cell wall synthesis, redox signalling and photosynthetic processes. Since Alethea had clear effects on photosynthesis/chloroplastic function at the physiological and molecular levels, we also investigated the ability of Alethea to protect various crop species against methyl viologen, a potent generator of oxidative stress in chloroplasts. Alethea pre-treatment produced dramatic reductions in visible foliar necrosis caused by methyl viologen compared with non-primed controls.

Conclusions: ‘Alethea’ technology mediates positive recovery of abiotic stress-induced photosynthetic and foliar Loss of performance, which is accompanied by altered transcriptional responses to stress.

Keywords: Photosynthesis, Abiotic stress, Priming, Tomato, Transcriptomics, Potassium dihydrojasmonate, Sodium
benzoate, L-arginine

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Sequencing-based variant detection in the polyploid crop oilseed rape

Rachel Wells, Martin Trick, Fiona Fraser, Eleni Soumpourou, Leah Clissold, Colin Morgan,Jérôme Pauquet and Ian Bancroft

Abstract
Background: The detection and exploitation of genetic variation underpins crop improvement. However, the polyploid nature of the genomes of many of our most important crops represents a barrier, particularly for the analysis of variation within genes. To overcome this, we aimed to develop methodologies based on amplicon sequencing that involve the incorporation of barcoded amplification tags (BATs) into PCR products.

Results: A protocol was developed to tag PCR products with 5’ 6-base oligonucleotide barcode extensions before pooling for sequencing library production using standard Illumina adapters. A computational method was developed for the de-convolution of products and the robust detection and scoring of sequence variants. Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome.Furthermore, using one-dimensional 8-fold pooling, 4608 lines of a B. napus mutation population were screened for induced mutations in a locus-specific amplicon (an orthologue of GL2.b) and mixed product of three co-amplified loci (orthologues of FAD2), identifying 10 and 41 mutants respectively.

Conclusions: The utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species. Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.

Keywords: SNP, Mutation, Polyploid, Crop

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Characterization of temperature and light effects on the defense response phenotypes associated with the maize Rp1-D21 autoactive resistance gene

Adisu Negeri, Guan-Feng Wang, Larissa Benavente, Cromwell M Kibiti, Vijay Chaikam, Guri Johal and Peter Balint-Kurt

 

Abstract
Background: Rp1 is a complex locus of maize, which carries a set of genes controlling race-specific resistance to the common rust fungus, Puccinia sorghi. The resistance response includes the “Hypersensitive response” (HR), a rapid response triggered by a pathogen recognition event that includes localized cell death at the point of pathogen penetration and the induction of pathogenesis associated genes. The Rp1-D21gene is an autoactive allelic variant at the Rp1 locus, causing spontaneous activation of the HR response, in the absence of pathogenesis.Previously we have shown that the severity of the phenotype conferred by Rp1-D21 is highly dependent on genetic background.

Results: In this study we show that the phenotype conferred by Rp1-D21 is highly dependent on temperature, with lower temperatures favoring the expression of the HR lesion phenotype. This temperature effect was observed in all the 14 genetic backgrounds tested. Significant interactions between the temperature effects and genetic background were observed. When plants were grown at temperatures above 30°C, the spontaneous HR phenotype conferred by Rp1-D21 was entirely suppressed. Furthermore, this phenotype could be restored or suppressed by alternately reducing and increasing the temperature appropriately. Light was also required for the expression of this phenotype. By examining the expression of genes associated with the defense response we showed that, at temperatures above 30°C, the Rp1-D21 phenotype was suppressed at both the phenotypic and molecular level.

Conclusions: We have shown that the lesion phenotype conferred by maize autoactive resistance gene Rp1-D21 is temperature sensitive in a reversible manner, that the temperature-sensitivity phenotype interacts with genetic background and that the phenotype is light sensitive. This is the first detailed demonstration of this phenomenon in monocots and also the first demonstration of the interaction of this effect with genetic background. The use of temperature shifts to induce a massive and synchronous HR in plants carrying the Rp1-D21 genes will be valuable in identifying components of the defense response pathway.

Keywords: Maize, Hypersensitive response, Disease resistance, Temperature sensitive, Light dependent, Rp1,Autoactive R gene

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Dynamic compartment specific changes in glutathione and ascorbate levels in Arabidopsis plants exposed to different light intensities

Elmien Heyneke, Nora Luschin-Ebengreuth, Iztok Krajcer, Volker Wolkinger, Maria Müller and Bernd Zechmann

 

Abstract
Background: Excess light conditions induce the generation of reactive oxygen species (ROS) directly in the chloroplasts but also cause an accumulation and production of ROS in peroxisomes, cytosol and vacuoles. Antioxidants such as ascorbate and glutathione occur in all cell compartments where they detoxify ROS. In this study compartment specific changes in antioxidant levels and related enzymes were monitored among Arabidopsis wildtype plants and ascorbate and glutathione deficient mutants (vtc2-1 and pad2-1, respectively) exposed to different light intensities (50, 150 which was considered as control condition, 300, 700 and 1,500 μmol m-2 s -1) for 4 h and 14 d.

Results: The results revealed that wildtype plants reacted to short term exposure to excess light conditions with the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol and an increased activity of catalase in the leaves. Long term exposure led to an accumulation of ascorbate and glutathione mainly in chloroplasts. In wildtype plants an accumulation of ascorbate and hydrogen peroxide (H2 O2 ) could be observed in vacuoles when exposed to high light conditions. The pad2-1 mutant reacted to long term excess light exposure with an accumulation of ascorbate in peroxisomes whereas the vtc2-1 mutant reacted with an accumulation of glutathione in the chloroplasts (relative to the wildtype) and nuclei during long term high light conditions indicating an important role of these antioxidants in these cell compartments for the protection of the mutants against high light stress.

Conclusion: The results obtained in this study demonstrate that the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol is an important reaction of plants to short term high light stress. The accumulation of ascorbate and H2 O2 along the tonoplast and in vacuoles during these conditions indicates an important route for H2 O2 detoxification under these conditions.

Keywords: Arabidopsis, Ascorbate, Chloroplast, Glutathione, High light, Reactive oxygen species

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Cadmium uptake and partitioning in durum wheat during grain filling

Neil S Harris and Gregory J Taylor

Abstract
Background: Concentrations of cadmium (Cd) in the grain of many durum wheats (Triticum turgidum subsp.durum) grown in North American prairie soils often exceed international trade standards. Genotypic differences in root-to-shoot translocation of Cd are a major determinant of intraspecific variation in the accumulation of Cd in grain. We tested the extent to which changes in whole-plant Cd accumulation and the distribution of Cd between tissues influences Cd accumulation in grain by measuring Cd accumulation throughout the grain filling period in two near-isogenic lines (NILs) of durum wheat that differ in grain Cd accumulation.

Results: Roots absorbed Cd and transported it to the shoots throughout the grain filling period, but the low- and high-Cd NILs did not differ in whole-plant Cd uptake. Although the majority of Cd accumulation was retained in the roots, the low- and high-Cd NILs differed substantively in root-to-shoot translocation of Cd. At grain maturity, accumulation of Cd in the shoots was 13% (low-Cd NIL) or 37% (high-Cd NIL) of whole-plant Cd accumulation. Accumulation of Cd in all shoot tissue, including grain, was at least 2-fold greater in the high-Cd NIL at all harvests. There was no net remobilization of shoot Cd pools during grain filling. The timing of Cd accumulation in grain was positively correlated with grain biomass accumulation, and the rate of grain filling peaked between 14 and 28 days
post-anthesis, when both NILs accumulated 60% of total grain biomass and 61-66% of total grain Cd content.

Conclusions: These results show that genotypic variation in root-to-shoot translocation of Cd controls accumulation of Cd in durum wheat grain. Continued uptake of Cd by roots and the absence of net remobilization of Cd from leaves during grain filling support a direct pathway of Cd transport from roots to grain via xylem-tophloem transfer in the stem.

Keywords: Cadmium (Cd), Durum wheat, Near-isogenic lines (NIL), Grain filling, Uptake, Translocation,Remobilization

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Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains

Christopher T DeFraia, Yongsheng Wang, Jiqiang Yao and Zhonglin Mou

Abstract
Background: Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other .Elongator subunits in plant immunity has not been characterized.

Results: In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine
domains of AtELP3 are essential for its function in plant immunity.

Conclusion: Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not In SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin.

Keywords: Arabidopsis, Elongator, Plant immunity, AtELP3, Transcription

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Natural variation in floral nectar proteins of two Nicotiana attenuata accessions

Pil Joon Seo, Natalie Wielsch, Danny Kessler, Ales Svatos, Chung-Mo Park, Ian T Baldwin and Sang-Gyu Kim

Abstract
Background: Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata.

Results: We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LCMS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary.

Conclusions: Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions.

Keywords: LC-MS/MS, Nectar protein, Nectarin, Nicotiana attenuata

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The dual targeting ability of type II NAD(P)H dehydrogenases arose early in land plant evolution

Lin Xu, Simon R Law, Monika W Murcha, James Whelan and Chris Carrie

Abstract
Background: Type II NAD(PH) dehydrogenases are located on the inner mitochondrial membrane of plants, fungi, protists and some primitive animals. However, recent observations have been made which identify several Arabidopsis type II dehydrogenases as dual targeted proteins. Targeting either mitochondria and peroxisomes or mitochondria and chloroplasts.

Results: Members of the ND protein family were identified in various plant species. Phylogenetic analyses and subcellular targeting predictions were carried out for all proteins. All ND proteins from three model plant species Arabidopsis, rice and Physcomitrella were cloned as N- and C-terminal GFP fusions and subcellular localisations were determined. Dual targeting of plant type II dehydrogenases was observed to have evolved early in plant evolution and to be widespread throughout different plant species. In all three species tested dual targeting to both mitochondria and peroxisomes was found for at least one NDA and NDB type protein. In addition two NDB type proteins from Physcomitrella were also found to target chloroplasts. The dual targeting of NDC type proteins was found to have evolved later in plant evolution.

Conclusions: The functions of type II dehydrogenases within plant cells will have to be re-evaluated in light of this newly identified subcellular targeting information.

Keywords: Type II NAD(P)H dehydrogenases, Dual targeting, Mitochondria, Peroxisomes, Plastids

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Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants

Arne Weinhold, Mario Kallenbach and Ian Thomas Baldwin

Abstract
Background: Genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear.

Results: Transgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants.

Conclusions: The unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits.

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Different evolutionary histories of two cation/ proton exchanger gene families in plants

Inês S Pires, Sónia Negrão, Melissa M Pentony, Isabel A Abreu, Margarida M Oliveira and Michael D Purugganan

Abstract
Background: Gene duplication events have been proposed to be involved in the adaptation of plants to stress conditions; precisely how is unclear. To address this question, we studied the evolution of two families of antiporters. Cation/proton exchangers are important for normal cell function and in plants, Na+,K+/H+
antiporters have also been implicated in salt tolerance. Two well-known plant cation/proton antiporters are NHX1 and SOS1,which perform Na+and K+
compartmentalization into the vacuole and Na+efflux from the cell, respectively.However, our knowledge about the evolution of NHX and SOS1 stress responsive gene families is still limited.

Results: In this study we performed a comprehensive molecular evolutionary analysis of the NHX and SOS1 families. Using available sequences from a total of 33 plant species, we estimated gene family phylogenies and gene duplication histories, as well as examined heterogeneous selection pressure on amino acid sites. Our results show that, while the NHX family expanded and specialized, the SOS1 family remained a low copy gene family that appears to have undergone neofunctionalization during its evolutionary history. Additionally, we found that both families are under purifying selection although SOS1 is less constrained.

Conclusions: We propose that the different evolution histories are related with the proteins’ function and localization, and that the NHX and SOS1 families are examples of two different evolutionary paths through which duplication events may result in adaptive evolution of stress tolerance.

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Lr67 and Lr34 rust resistance genes have much in common – they confer broad spectrum resistance to multiple pathogens in wheat

Wolfgang Spielmeyer, Rohit Mago, Colin Wellings and Michael Ayliffe

Abstract
Background: Adult plant rust resistance genes Lr67 and Lr34 confer race non-specific resistance to multiple fungal pathogens of wheat. Induced, susceptible mutants were characterised for both genes.

Results: Three categories of Lr34 mutants were identified that were either partial susceptible, fully susceptible or hyper-susceptible to stripe rust and leaf rust. The likely impact of the mutational change on the predicted Lr34 protein correlated with differences in response to rust infection. Four independent Lr67 mutants were recovered that were susceptible to stripe rust, leaf rust and stem rust pathogens, including one possible hyper-susceptible Lr67 mutant.

Conclusions: Detailed study of Lr34 mutants revealed that subtle changes in resistance response to multiple pathogens were correlated with mutational changes in the predicted protein. Recovery of independent Lr67 mutants indicates that as for Lr34, a single gene at the Lr67 locus is likely to confer resistance to multiple
pathogens. The infection phenotypes of Lr67 mutants closely resembled that of Lr34 mutants.

Keywords: Lr34, Lr67, Rust resistance, Mutants

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Soil water stress affects both cuticular wax content and cuticle-related gene expression in young saplings of maritime pine (Pinus pinaster Ait)

Grégoire Le Provost, Frédéric Domergue, Céline Lalanne, Patricio Ramos Campos, Antoine Grosbois,Didier Bert, Céline Meredieu, Frédéric Danjon, Christophe Plomion and Jean-Marc Gion

Abstract
Background: The cuticle is a hydrophobic barrier located at the aerial surface of all terrestrial plants. Recent studies performed on model plants, such as Arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. Although forest trees will face more intense drought stresses (in duration and intensity) with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported.

Results: This aspect was investigated in a conifer, maritime pine (Pinus pinaster Ait.), in a factorial design with two genetic units (two half-sib families with different growth rates) and two treatments (irrigated vs non-irrigated), in field conditions. Saplings were grown in an open-sided greenhouse and half were irrigated three times per week for two growing seasons. Needles were sampled three times per year for cuticular wax (composition and content) and transcriptome (of 11 genes involved in cuticle biosynthesis) analysis. Non-irrigated saplings (i) had a higher cuticular wax content than irrigated saplings and (ii) overexpressed most of the genes studied. Both these trends were more marked in the faster growing family.

Conclusions: The higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. This study provides also a set of promising candidate genes for future forward genetic studies in conifers.

Keywords: Cuticle biosynthesis, Drought, Edaphic stress, Field experiment, Gene expression, Maritime pine

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The Arabidopsis LRR-RLK, PXC1, is a regulator of secondary wall formation correlated with the TDIF-PXY/TDR-WOX4 signaling pathway

Jiehua Wang, Melis Kucukoglu, Linbin Zhang, Peng Chen, Daniel Decker, Ove Nilsson, Brian Jones,Göran Sandbergand Bo Zheng

Abstract
Background: Although a number of leucine-rich repeat receptor-like kinase-encoding genes (LRR-RLKs) have been identified in plants, a functional role has been determined for only a few. Recent studies have demonstrated that an LRR-RLK, PXY/TDR, is important for the process of secondary vascular development. Other studies have indicated that PXY/TDR is unlikely to be the sole LRR-RLK involved in this complex process.

Results: In this study, in silico analyses led to the identification of three Arabidopsis LRR-RLK genes (PXY-correlated; PXC1, 2, 3) with transcript accumulation profiles that correlated strongly with several key regulators of vascular development, including PXY/TDR, HB-8, REV, and CLE41. Expression profiling using qPCR and promoter:reporter lines indicated that all three PXC genes are associated with the vasculature. One in particular, PXC1 (At2g36570), had a strong correlation with PXY/TDR. Shifting pxc1 mutants from long-days to short-days showed that loss of the gene led to a dramatic reduction in secondary wall formation in xylem fibers. Transcript analysis of mutants for a variety of secondary cell wall-associated genes, including PXY/TDR indicated that the pathways mediated by PXC1 connect with those mediated by the TDIF-PXY/TDR-WOX4 system.

Conclusions: The data indicate that the LRR-RLK, PXC1 is involved in secondary cell wall formation in xylem fibers. Whereas further study is needed to identify the ligands and mode of action of the PXC1 protein, it is clear from this work that similarly to the shoot apical meristem (SAM), secondary vascular development requires contributions from a number of LRR-RLKs.

Keywords: LRR-RLK, Arabidopsis, Secondary Wall Formation, TDIF-PXY/TDR-WOX4 Signaling

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A fungal endophyte induces transcription of genes encoding a redundant fungicide pathway in its host plant

Sameh SM Soliman, Christopher P Trobacher, Rong Tsao, John S Greenwood and Manish N Raizada

Abstract
Background: Taxol is an anti-cancer drug harvested from Taxus trees, proposed ecologically to act as a fungicide. Taxus is host to fungal endophytes, defined as organisms that inhabit plants without causing disease. The Taxus endophytes have been shown to synthesize Taxol in vitro, providing Taxus with a second potential biosynthetic route for this protective metabolite. Taxol levels in plants vary 125-fold between individual trees, but the underlying reason has remained unknown.

Results: Comparing Taxus trees or branches within a tree, correlations were observed between Taxol content, and quantity of its resident Taxol-producing endophyte, Paraconiothyrium SSM001. Depletion of fungal endophyte in planta by fungicide reduced plant Taxol accumulation. Fungicide treatment of intact plants caused concomitant decreases in transcript and/or protein levels corresponding to two critical genes required for plant Taxol biosynthesis. Taxol showed fungicidal activity against fungal pathogens of conifer wood, the natural habitat of the Taxol-producing endophyte. Consistent with other Taxol-producing endophytes, SSM001 was resistant to Taxol.

Conclusions: These results suggest that the variation in Taxol content between intact Taxus plants and/or tissues is at least in part caused by varying degrees of transcriptional elicitation of plant Taxol biosynthetic genes by its Taxol-producing endophyte. As Taxol is a fungicide, and the endophyte is resistant to Taxol, we discuss how this endophyte strategy may be to prevent colonization by its fungal competitors but at minimal metabolic cost to itself.

Keywords: Taxus, Paraconiothyrium, Fungus, Endophyte, Taxol, Biosynthesis, Fungicide, DXR, Taxadiene synthase

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Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa

Kelly Vining, Kyle R Pomraning, Larry J Wilhelm, Cathleen Ma, Matteo Pellegrini, Yanming Di,Todd C Mockler, Michael Freitag and Steven H Strauss

Abstract
Background: Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation.

Results: We investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue,
then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes.

Conclusions: DNA methylation varies in a highly gene- and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements.

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Genetic variation in four maturity genes affects photoperiod insensitivity and PHYA-regulated post-flowering responses of soybean

Meilan Xu, Zeheng Xu, Baohui Liu, Fanjiang Kong, Yasutaka Tsubokura, Satoshi Watanabe, Zhengjun Xia ,Kyuya Harada, Akira Kanazawa, Testuya Yamada and Jun Abe

 

Abstract

Background: Absence of or low sensitivity to photoperiod is necessary for short-day crops, such as rice and soybean,to adapt to high latitudes. Photoperiod insensitivity in soybeans is controlled by two genetic systems and involves three important maturity genes: E1, a repressor for two soybean orthologs of Arabidopsis FLOWERING LOCUS T (GmFT2a and GmFT5a), and E3 and E4, which are phytochrome A genes. To elucidate the diverse mechanisms underlying
photoperiod insensitivity in soybean, we assessed the genotypes of four maturity genes (E1 through E4) in early-flowering photoperiod-insensitive cultivars and their association with post-flowering responses.

Results: We found two novel dysfunctional alleles in accessions originally considered to have a dominant E3 allele according to known DNA markers. The E3 locus, together with E1 and E4, contained multiple dysfunctional alleles. We identified 15 multi-locus genotypes, which we subdivided into 6 genotypic groups by
classifying their alleles by function. Of these, the e1-as/e3/E4 genotypic group required an additional novel gene (different from E1, E3, and E4) to condition photoperiod insensitivity. Despite their common pre-flowering photoperiod insensitivity, accessions with different multi-locus genotypes responded differently to the post-flowering photoperiod. Cultivars carrying E3 or E4 were sensitive to photoperiod for post-flowering characteristics, such as reproductive period
and stem growth after flowering. The phytochrome A–regulated expression of the determinate growth habit gene Dt1, an ortholog of Arabidopsis TERMINAL FLOWER1, was involved in the persistence of the vegetative activity at the stem apical meristem of flower-induced plants under long-day conditions.

Conclusions: Diverse genetic mechanisms underlie photoperiod insensitivity in soybean. At least three multi-locus genotypes consisting of various allelic combinations at E1, E3, and E4 conferred pre-flowering photoperiod insensitivity to soybean cultivars but led to different responses to photoperiod during post-flowering vegetative and reproductive development. The phyA genes E3 and E4 are major controllers underlying not only pre-flowering but also post-flowering
photoperiod responses. The current findings improve our understanding of genetic diversity in pre-flowering photoperiod insensitivity and mechanisms of post-flowering photoperiod responses in soybean.

Keywords: Photoperiod, Soybean, Flowering, Determinate habit, Post-flowering, Genetic variation

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A non-synonymous SNP within the isopentenyl transferase 2 locus is associated with kernel weight in Chinese maize inbreds (Zea mays L.)

Jianfeng Weng, Bo Li, Changlin Liu, Xiaoyan Yang, Hongwei Wang, Zhuanfang Hao, Mingshun Li,Degui Zhang, Xiaoke Ci, Xinhai Li and Shihuang Zhang

Abstract
Background: Kernel weight, controlled by quantitative trait loci (QTL), is an important component of grain yield in maize. Cytokinins (CKs) participate in determining grain morphology and final grain yield in crops. ZmIPT2, which is expressed mainly in the basal transfer cell layer, endosperm, and embryo during maize kernel development,encodes an isopentenyl transferase (IPT) that is involved in CK biosynthesis.

Results: The coding region of ZmIPT2 was sequenced across a panel of 175 maize inbred lines that are currently used in Chinese maize breeding programs. Only 16 single nucleotide polymorphisms (SNPs) and seven haplotypes were detected among these inbred lines. Nucleotide diversity (π) within the ZmIPT2 window and coding region were 0.347 and 0.0047, respectively, and they were significantly lower than the mean nucleotide diversity value of 0.372 for maize Chromosome 2 (P< 0.01). Association mapping revealed that a single nucleotide change from cytosine (C) to thymine (T) in the ZmIPT2 coding region, which converted a proline residue into a serine residue, was significantly associated with hundred kernel weight (HKW) in three environments (P <0.05), and explained 4.76% of the total phenotypic variation. In vitro characterization suggests that the dimethylallyl diphospate (DMAPP) IPT activity of ZmIPT2-T is higher than that of ZmIPT2-C, as the amounts of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) consumed by ZmIPT2-T were 5.48-, 2.70-, and 1.87-fold, respectively, greater than those consumed by ZmIPT2-C. The effects of artificial selection on the ZmIPT2 coding region were evaluated using Tajima’s D tests across six subgroups of Chinese maize germplasm, with the most frequent favorable allele identified in subgroup PB (Partner B).

Conclusions: These results showed that ZmIPT2, which is associated with kernel weight, was subjected to artificial selection during the maize breeding process. ZmIPT2-T had higher IPT activity than ZmIPT2-C, and this favorable allele for kernel weight could be used in molecular marker-assisted selection for improvement of grain yield components in Chinese maize breeding programs.

Keywords: Maize, Isopentenyl transferase 2, Association mapping, Artificial selection

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Mosquitoes established in Lhasa city, Tibet, China

Qiyong Liu, Xiaobo Liu, Cirendunzhu, Alistair Woodward, Pengcuociren, Li Bai, Baimaciwang,Shaowei Sang, Dazhen, Fangjun Wan, Lin Zhou, Yuhong Guo, Haixia Wu, Guichang Li,Liang Lu, Jun Wang, Dawa, Cordia Chu and Xiraoruodeng

Abstract
Background: In 2009, residents of Lhasa city, Tibet Autonomous Region (TAR), China reported large numbers of mosquitoes and bites from these insects. It is unclear whether this was a new phenomenon, which species were involved, and whether these mosquitoes had established themselves in the local circumstances.
Methods: The present study was undertaken in six urban sites of Chengguan district Lhasa city, Tibet. Adult mosquitoes were collected by bed net trap, labor hour method and light trap in August 2009 and August 2012.The trapped adult mosquitoes were initially counted and identified according to morphological criteria, and a
proportion of mosquitoes were examined more closely using a multiplex PCR assay.

Results: 907 mosquitoes of the Culex pipiens complex were collected in this study. Among them, 595 were females and 312 were males. There was no significant difference in mosquito density monitored by bed net trap and labor hour method in 2009 and 2012. Of 105 mosquitoes identified by multiplex PCR, 36 were pure mosquitoes (34.29%) while 69 were hybrids (65.71%). The same subspecies of Culex pipiens complex were observed by bed net trap,
labor hour method and light trap in 2009 and 2012.

Conclusion: The local Culex pipiens complex comprises the subspecies Cx. pipiens pipiens, Cx. pipiens pallens, Cx. pipiens quinquefasciatus and its hybrids. Mosquitoes in the Cx. pipiens complex, known to be, potentially, vectors of periodic filariasis and encephalitis, are now present from one season to the next, and appear to be established in Lhasa City, TAR.

Keywords: Mosquitoes, Culex pipiens complex, Multiplex PCR, Established, Lhasa

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Specific structure and unique function define the hemicentin

Xuehong Xu, MengMeng Xu, Xin Zhou, Odell B Jones, Edward Moharomd, Yuexin Pan, Guifang Yan,Donald D Anthony and Williams B Isaac

 

Abstract
Hemicentin has come a long way from when it was first identified in C. elegans as him-4 (High incidence of males).The protein is now a recognized player in maintaining the architectural integrity of vertebrate tissues and organs.Highly conserved hemicentin sequences across species indicate this gene’s ancient evolutionary roots and functional importance. In mouse, hemicentin is liberally distributed on the cell surface of many cell types, including epithelial cells, endothelial cells of the eye, lung, and uterus, and trophectodermal cells of blastocyst. Recent discoveries have uncovered yet another vital purpose of hemicentin 1. The protein also serves a unique function in mitotic cytokinesis, during which this extracellular matrix protein plays a key role in cleavage furrow maturation.Though understanding of hemicentin function has improved through new discoveries, much about this protein remains mysterious.

Keywords: Extracellular matrix (ECM), Fibulin, Hemicentin, Embryogenesis, Tissue/Organ architecture, Cell division,Mitosis

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Genetic structure of the gentle Africanized honey bee population (gAHB) in Puerto Rico

Alberto Galindo-Cardona, Jenny P Acevedo-Gonzalez, Bert Rivera-Marchand and Tugrul Giray

Abstract
Background: The Africanized honey bee is one of the most spectacular invasions in the Americas. African bees escaped from apiaries in Brazil in 1956, spread over Americas and by 1994 they were reported in Puerto Rico. In contrast to other places, the oceanic island conditions in Puerto Rico may mean a single introduction and different dynamics of the resident European and new-coming Africanized bees. To examine the genetic variation of honey bee feral populations and colonies from different locations in Puerto Rico, we used eight known polymorphic microsatellite loci.

Results: In Puerto Rico, gAHB population does not show any genetic structure (Fst= 0.0783), and is best described as one honey bee population, product of hybridization of AHB and EHB. The genetic variability in this Africanized population was similar to that reported in studies from Texas. We observed that European private allele frequencies are high in all but one locus. This contrasts with mainland Africanized populations, where European allele frequencies are diminished. Two loci with European private alleles, one on Linkage Group 7, known to carry two known defensiveness Quantitative Trait Loci (QTLs), and the other on Linkage Group 1, known to carry three functionally studied genes and 11 candidate genes associated with Varroa resistance mechanisms were respectively, significantly greater or lower in European allele frequency than the other loci with European private alleles.

Conclusions: Genetic structure of Puerto Rico gAHB differs from mainland AHB populations, probably representing evolutionary processes on the island.

Keywords: Apis mellifera, Honeybee population, Hybridization, Africanized, European bees

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Linking the potato genome to the conserved ortholog set (COS) markers

Hannele Lindqvist-Kreuze, Kwangsoo Cho, Leticia Portal, Flor Rodríguez, Reinhard Simon, Lukas A Mueller,David M Spooner and Merideth Bonierbale

Abstract
Background: Conserved ortholog set (COS) markers are an important functional genomics resource that has greatly improved orthology detection in Asterid species. A comprehensive list of these markers is available at SolGenomics Network (http://solgenomics.net/) and many of these have been placed on the genetic maps of anumber of solanaceous species.
Results: We amplified over 300 COS markers from eight potato accessions involving two diploid landraces of Solanum tuberosum Andigenum group (formerly classified as S. goniocalyx, S. phureja), and a dihaploid clone derived from a modern tetraploid cultivar of S. tuberosum and the wild species S. berthaultii, S. chomatophilum, andS. paucissectum. By BLASTn (Basic Local Alignment Search Tool of the NCBI, National Center for Biotechnology Information) algorithm we mapped the DNA sequences of these markers into the potato genome sequence.Additionally, we mapped a subset of these markers genetically in potato and present a comparison between the physical and genetic locations of these markers in potato and in comparison with the genetic location in tomato.
We found that most of the COS markers are single-copy in the reference genome of potato and that the genetic location in tomato and physical location in potato sequence are mostly in agreement. However, we did find some COS markers that are present in multiple copies and those that map in unexpected locations. Sequencecomparisons between species show that some of these markers may be paralogs.
Conclusions: The sequence-based physical map becomes helpful in identification of markers for traits of interest thereby reducing the number of markers to be tested for applications like marker assisted selection, diversity, and phylogenetic studies.

Keywords: Conserved ortholog set (COS), Genetic mapping, Potato genome, Solanum

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Analysis of Main Proteins Associated with Lipid Droplets from PeriAdrenal Adipose Tissue of Patients with Cushing’s Syndrome

Névéna Christeff, Cedric Broussard, Jérôme Bertherat, Françoise Hotellier, Luc Camoin and Tarik Issad

Abstract:

The Cushing’s syndrome results from chronic exposure to excess glucocorticoids produced by the adrenal cortex. The most common feature of patients with Cushing’s syndrome is central obesity associated with dyslipidaemia and insulin resistance leading to type 2 diabetes and cardiovascular diseases. In the adipocytes, triacylglycerol and cholesterol esters are stored within an intracellular lipid droplet covered by a monolayer of phospholipids and surrounded by proteins involved in the regulation of lipolysis. Since the protein composition of adipocyte lipid droplets from patients with Cushing’s syndrome has not yet been reported, we used two-dimensional gel electrophoresis followed by mass spectrometry to identify the main lipid droplet-associated proteins from peri-adrenal adipose cell of patients with this syndrome.
The lipid droplet proteome of peri-adrenal adipose cell of patients with Cushing’s syndrome is highly complex and contains more than 500 proteins. MALDI-TOF MS analysis of silver-stained protein spots revealed the identity of 27 proteins clustered into 7 groups according to their known function. Interestingly, proteins involved in response to oxidative stress and endoplasmic reticulum stress were found on the lipid droplets of adipose cell from patients with
Cushing’s syndrome.

Keywords: Adipocyte, Cushing’s syndrome, endoplasmic reticulum, lipid droplet, two-dimensional gel electrophoresis.

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Impact of p21 Knockout on Topotecan-Induced Stress Responses in Human Colon Carcinoma Cells: A Proteomic Analysis

Kyunghee Lee and Sayed S. Daoud

Abstract:

There are few reports describing the role of p21-dependent protein repression in cell death. To identify such cell death-associated proteins and to shed the light into the molecular mechanisms by which p21 is responding to pharmacological stress, we used a subcellular proteomic approach for the analysis of protein expression profiles of fractionated nuclei, mitochondria, and cytosols of isogenic p21 null (p21-/-) and wild-type human HCT-116 cells following treatment with sublethal doses (1μM) of the topoisomerase I inhibitor, topotecan (TPT). In total, 174 unique deregulated proteins were identified in HCT-116 cells following treatment with TPT, whereas only 146 proteins were identified in p21-/- cells. They contributed to multiple functional activities of stress signaling pathways, and that p21-/- cells are accelerated to be more responsive to topotecan-induced cell death due to the following: 1) down regulation of proteins involved in the transcriptional and replication machinery of cells like DNA (cytosine-5)-methyltransferase 1, Matrin 3, DNA replication licensing factor MCM4, heterogeneous nuclear ribonucleoprotein Q, poly(Rc)-binding protein 1 and splicing factor arginine serine rich 7; 2) the activation of a caspase-independent apoptosis by the upregulation of the Bcl2 inhibitor of transcription (Bit1) protein; and 3) the activation of TNF signaling by the upregulation of macrophage immigration inhibitory factor (MIF), and 26S proteosome non-ATPase regulatory subunit 2 (TRAP2) proteins. We suggest that the upregulation of these proteins are contributing factors to the molecular mechanisms of topotecan-induced cell death in p21-/- cells; and that the data present an opportunity for developing new therapeutic approaches for selective targeting of p21-
signaling pathways.

Key Words: Proteomics, topotecan, p21, colon cancer, mass spectrometry, Bit1

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Analysis of Proteins Associated with Chinch Bug (Blissus leucopterus leucopterus Say)-Infested Corn (Zea mays L.) Seedlings

Sita R. Ghimire, Sonya M. Baird, Gerald T. Baker and Peter W.K. Ma

Abstract:

A proteomics approach was used to study the proteins associated with chinch bug infested corn seedlings. Examination of two-dimensional gels revealed the presence of more than 600 high quality protein spots each from chinch bug-infested and healthy corn seedlings. A total of 31 protein spots was selected for matrix-assisted laser desorption and ionization time-of-flightmass spectrometric analysis. Among the protein spots selected, 13 were from infested plants, 10 from healthy plants, and four each from healthy and infested plants having differential expressions. Peptide mass fingerprinting revealed that each spot analyzed represents a different protein. Thirty-nine percent of the proteins had confirmed identity and the rest were tentatively identified. Among 13 proteins analyzed from infested seedlings most were related to defense, cell rescue, virulence and metabolism. Some of these proteins related to metabolism and protein synthesis were
down-regulated in the infested seedlings. All proteins except one from infested corn seedlings seem to be activated in the plant system because of the chinch bug-induced stresses including osmotic, oxidative and acid stresses, and wounding.

Key Words: Goose-necking, peptide mass fingerprinting, post-translational modification, protein extraction.

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Effect of Growth Temperature and Culture Medium on the Cryotolerance of Permafrost Exiguobacterium Sibiricum 255-15 by Proteome-Wide Mass Mapping

Yinghua Qiu, Tatiana A. Vishnivetskaya, Weilian Qiu and David M. Lubman

Abstract:

Exiguobacterium sibiricum 255-15 has shown significantly improved cryotolerance after liquid broth growth at 4oC and agar surface growth at both 4oC and 25oC compared with liquid broth growth at 25oC. The ability to survive freeze-thaw stress is expected to depend on the physiological state and protein composition of cells prior to freezing. Using 2-D liquid separation and an ESI-TOF MS-based mass mapping technique, we examined the differences in the proteomic profiles of the permafrost bacterium E. sibiricum 255-15 grown at two temperatures (4oC and 25oC) and two media (liquid broth and agar surface) before freeze-thawing treatments. In this study, a total of 330 proteins were identified. The cells cultured under the growth conditions associated with the improved cryotolerance have revealed a general downregulation of enzymes involved in major metabolic processes (glycolysis, anaerobic respiration, ATP synthesis, fermentation, electron transport, and sugar metabolism) as well as in the metabolism of lipids, amino acids, nucleotides and nucleic acids. In addition, eight proteins (2’-5’ RNA ligase, hypoxanthine phosphoribosyl transferase, FeS assembly ATPase SufC, thioredoxin reductase and four hypothetical proteins) were observed to be up-regulated. This suggests these eight proteins might have a potential role to induce the improved cryotolerance.

Key Words: Bacterial cryotolerance, Exiguobacterium sibiricum, 2-D mass mapping, ESI-TOF MS, MALDI-TOF MS, MALDI-QIT-TOF MS

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Isolation and Partial Characterization of an Antiviral Proteolytic Fraction from the Venom of Echis Carinatus Sochureki

G. Borkow, D. Marco and M. Ovadia

Abstract:

The venom of the viper Echis carinatus sochureki suppresses the hemolytic activity of Sendai virus on human erythrocytes, when pre-incubated with the virions prior to their binding to cells. A fraction (C1), with an IC50 of 1.25 g/ml, was isolated from the venom. Fraction C1 possesses strong azocollase, azocaseinase and gelatinase activity. The proteolytic and anti-hemolytic potency of C1 depends on the period and temperature of incubation. Its antiviral activity is inhibited by Sodium-EDTA but not by PMSF. SDS PAGE of Sendai virus incubated with fraction C1 shows disappearance of several of the virion high molecular weight bands. We suggest that inhibition of the hemolytic activity of the virions is probably a result of the cleavage of viral surface proteins, such as the hemagglutinin-neuraminidase glycoprotein found on the virion envelope that mediates the absorption of the virus to cells.

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DNA Barcoding in a Crop Genebank: The Capsicum annuum Species Complex

Robert L. Jarret

Abstract:

Variability within eight cpDNA introns including trnS-trnfM, trnL-trnT, trnH-psbA, trnF-trnL, trnD-trnT, trnCrpoB, rps16 and matK, and the nuclear waxy introns was examined in seven species of Capsicum (C. annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens, C. chacoense and C. rhomboideum) in order to evaluate the feasibility of utilizing these loci for DNA barcoding within the C. annuum complex. Numerous insertions/deletions (indels) and substitutions were detected in all cpDNA introns. However, none was sufficient to differentiate the individual members of the C. annuum complex (C. annuum, C. chinense and C. frutescens). Variation within trnL-trnT, trnF-trnLand trnH-psbA enabled the differentiation of the complex from the other taxa examined. In contrast, single base indels and substitutions within the waxy introns permitted the differentiation of all taxa within the plant materials examined. The use of trnH-psbA or trnL-trnT, and the waxy introns is proposed for barcoding members of the C. annuum complex

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Does 'Relationship Intelligence' Make Big Brains in Birds?

Isabella B.R. Scheiber, Brigitte M. Weiß, Katharina Hirschenhauser, Claudia A.F. Wascher, Iulia T. Nedelcu and Kurt Kotrschal

 

Abstract:

Lately, Emery et al. developed a bird-specific modification of the “social brain hypothesis”, termed “relationship intelligence hypothesis”. Although the idea may be valuable, we doubt that it is supported by sufficient evidence and critically discuss some of the arguments raised by the authors in favour of their new idea.

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Absence of Dystrophin Does Not Affect Myogenesis

Maziar Assadi, Thomas Schindler, John D. Porter and Hanno Langen

Abstract:

Duchenne muscular dystrophy (DMD) is caused by the absence of the protein dystrophin in the muscle cells. The function of dystrophin is still not clear. For enabling study of the molecular function of dystrophin, we used small inhibitory RNA (siRNA) for suppressing the expression of the protein in two muscle cell lines and achieved a quantitative knockdown. We applied two-dimensional differential gel electrophoresis (2-D DIGE) in this new in vitro model for DMD to investigate if the absence of dystrophin during myogenesis causes any changes in protein expression. We did not observe statistical relevant changes. The result of our study suggests that the absence of dystrophin does not have any effect on myogenesis.

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Widespread Occurrence of Non-Enzymatic Deamidations of Asparagine Residues in Yersinia pestis Proteins Resulting from Alkaline pH Membrane Extraction Conditions

Moo-Jin Suh, Hamid Alami, David J. Clark, Prashanth P. Parmar, Jeffrey M. Robinson, Shih-Ting Huang, Robert D. Fleischmann, Scott N. Peterson and Rembert Pieper

Abstract:

Extraction of crude membrane fractions with alkaline solutions, such as 100-200 mM Na2 CO3 (pH ~11), is often used to solubilize peripheral membrane proteins. Integral membrane proteins are largely retained in membrane pellets. We applied this method to the fractionation of membrane proteins of the plague bacterium Yersinia pestis. Extensive horizontal spot trains were observed in 2-DE gels. The pI values of the most basic spots part of such protein spot trains usually
matched the computationally predicted pI values. Regular patterns of decreasing spot pI values and in silico analysis with the software ProMoST suggested ‘n-1’ deamidations of asparagine (N) and/or glutamine (Q) side chains for ‘n’ observed spots of a protein in a given spot train. MALDI-MS analysis confirmed the occurrence of deamidations, particularly in N side chains part of NG dipeptide motifs. In more than ten cases, tandem MS data for tryptic peptides provided strong evidence for deamidations, with y- and b-ion series increased by 1 Da following N-to-D substitutions. Horizontal spot trains in 2-DE gels were rare when alkaline extraction was omitted during membrane protein sample preparation. This study strongly supports the notion that exposure to alkaline pH solutions is a dominant cause of extensive N and Q side chain deamidations in proteins during sample preparation of membrane extracts. The modifications are of non-enzymatic nature
and not physiologically relevant. Therefore, quantitative spot differences within spot trains in differential protein display experiments following the aforementioned sample preparation steps need to be interpreted cautiously.

Key Words: Alkaline membrane extraction, deamidation, membrane proteome, spot train, two-dimensional gel electrophoresis

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Analysis of the Human Salivary Peptidome by Differential Peptide Display and LC-MS/MS Overview Sequencing

N. Le Yondre, H. Tammen, R. Hess, P. Budde and M. Jürgens

Abstract:

In recent years interest in the characterization of the human salivary proteome has increased in order to explore its diagnostic potential. Major constituents of human saliva are highly polymorphic proteins that may have biological roles in oral lubrication and protection, e.g. proline-rich proteins (PRPs), statherins, histatins and cystatins. Interestingly, manyof theses proteins are rapidly degraded in the oral cavity by host- and bacteria- or viral-derived proteases. Thus, comprehensive analysis of peptides up to 15 kDa (peptidomics) of whole saliva may yield basic information on proteolytic patterns. By employing a LC-ESI-MS/MS approach a total of 107 native peptides from 17 distinct protein precursors was identified from whole saliva. Subsequently the catalog of peptides was used to analyze inter-individual differences in saliva samples from four donors by differential peptide display technology. Genetic polymorphisms were found in peptides from the PRB4M_HUMAN and PROL4_HUMAN precursors. Analysis of the proposed N- and C-termini of the peptides revealed frequent cleavage after Lys or Arg which is characteristic for salivary kallikrein enzymes. Furthermore, we highlight the cleavage motif Gln/Gly in the PRP-C precursor, which suggests a new proteolytic pattern in saliva.

Key Words: Differential peptide display, peptidomics, proteomics, saliva, mass spectrometry.

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Caveolin-1 and Flotillin-1 Differential Expression in Clinical Samples of Renal Cell Carcinoma

Francesca Raimondo, Davide Ticozzi Valerio, Fulvio Magni, Roberto Perego, Cristina Bianchi, Cecilia Sarto, Stefano Casellato, Ester Fasoli, Stefano Ferrero, Ingrid Cifola, Francesco Rocco, Marzia Galli Kienle, Paolo Mocarelli, Paolo Brambilla and Marina Pitto

Abstract:

Caveolin-1 and flotillin-1 belong to plasma membrane microdomains. They are characterized by peculiar lipid and protein composition and are involved in fundamental cellular events such as: signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 30 human renal cell carcinoma (RCC) and adjacent normal kidney (ANK) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 over-expression was found in RCC tissues compared to ANK, and was confirmed by immunohistochemistry. Caveolin-1 and flotillin-1 protein levels were found by 1-D, 2-DE, and MS to be increased also in RCC microdomain-enriched subcellular fractions purified from paired RCC and ANK samples.

Key Words: Caveolin, Flotillin, mass spectrometry, renal cell carcinoma, subcellular proteomics, two-dimensional electrophoresis.

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Yeast Two-Hybrid Assay Reveals Several Interactors of EphB6 Receptor

Brian P. Fox and Raj P. Kandpal

Abstract:

EphB6, a kinase-deficient receptor, belongs to the Eph family of receptor tyrosine kinases. It can participate in active cell-to-cell signaling pathways following dimerization with and phosphorylation by EphB1 and possibly other members of the EphB family. EphB6 is a positive prognostic marker in neuroblastoma. The loss of EphB6 expression in melanomas, non-small cell lung carcinomas and breast cancer appears to be associated with a more advanced stage of these cancers. Despite the apparent role for EphB6 in preventing the progression of these cancers, little is known about EphB6-mediated signaling in cancer. We have identified 16 unique proteins that are capable of interacting with the cytoplasmic domain of EphB6 in the yeast two-hybrid assay. Furthermore, the interaction of a subset of these proteins (aldolaseA, dynactin, clusterin, TMEM25 and plekstrin homology domain-containing family B member 1) with EphB6 in mammalian cells was confirmed by employing a co-immunoprecipitation strategy. The identification of these interacting proteins suggests pathways mediated by EphB6.

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Quantification of Serum Proteins of Metastatic Oral Cancer Patients Using LC-MS/MS and iTRAQ Labeling

Lifeng Zhang, Jiang Jiang, Martha Arellano, Lei Zhang, Xinmin Yan, David T. Wong and Shen Hu

Abstract:

Metastasis is a critical event in oral squamous cell carcinoma (OSCC) progression. In this study, we have performed quantitative analysis of serum proteins from non-metastatic (lymph-node metastasis free) and metastatic OSCC patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with iTRAQ labeling (isobaric tagging for relative and absolute quantitation). To eliminate highly abundant proteins, the serum samples were initially separated by
SDS-PAGE and only low abundant protein bands were excised for subsequent in-gel tryptic digestion. The resulting peptides were then extracted from each sample gels and labeled with iTRAQ reagent 114 (control), 116 (non-metastatic) and 117 (metastatic), respectively. Afterwards, the labeled samples were combined and subjected to LC-MS/MS analysis using linear ion trap (LIT) MS with pulsed Q collision induced dissociation (PQD). A total of 64 proteins were identified and quantified by this approach. Our study showed that iTRAQ labeling and LIT-MS with PQD is a valuable approach to quantification of serum proteins. We also demonstrated the presence of differentially expressed serum proteins between non-metastatic and metastatic OSCCs that may be further validated as biomarkers for metastatic OSCC. However, in order to comprehensively quantify low abundant serum proteins, a more efficient approach is needed to deplete highly abundant proteins prior to quantitative serum proteome analysis of OSCC.

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Proteomic Analysis of Responses to Drought Stress in Sunflower (Helianthus annuus) Leaves by 2DE Gel Electrophoresis and Mass Spectrometry

Mª Ángeles Castillejo, Ana Mª Maldonado, Samuel Ogueta and Jesús V. Jorrín

Abstract:

By using a differential proteomic approach, responses to drought stress in sunflower have been studied. Two sunflower genotypes, showing different levels of tolerance to drought have been utilized. Following TCA-acetone protein extraction, the 2-DE leaf protein profile of well watered and drought stressed plants have been compared. Coomassie staining of the gels allowed visualization of around 350 well resolved spots within the 5-8 pH and 10–100 kDa ranges.
Image analysis revealed the presence of both, qualitative and quantitative changes between genotypes and treatments. Differential spots were subjected to trypsin digestion and peptides were analyzed by MALDI-TOF mass spectrometry. After database search using peptide mass fingerprinting, 2 genotype-dependent and 23 (susceptible genotype) and 5 (tolerant genotype) stress-responsive protein spots were identified. The two proteins spots differentiating sunflower genotypes corresponded to phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. In response to drought conditions a general decrease in protein spots corresponding to enzymes of the photosynthesis and carbohydrate metabolism was observed in the more susceptible genotype, suggesting inhibition of the energetic metabolism. Such changes have not been observed in the tolerant genotype, indicating a normal metabolism under drought stress.

Key Words: Differential expression proteomics, drought stress, MALDI-TOF, sunflower proteomics, two-dimensional gel electrophoresis

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Targeted Mass Spectrometric Immunoassay for Detection of Cystatin C Isoforms in Cerebrospinal Fluid

Dobrin Nedelkov, Shainag Shaik, Olgica Trenchevska, Vasko Aleksovski, Angel Mitrevski and Kiro Stojanoski

Abstract:

Targeted cerebrospinal fluid (CSF) Mass Spectrometric Immunoassay for cystatin C was developed and applied to a small number of CSF and serum samples obtained from healthy and individuals diagnosed with multiple sclerosis. Cystatin C and its variants were retrieved from the CSF samples via affinity micro columns derivatized with cystatin C antibodies, and were eluted onto MALDI mass spectrometer targets. The resulting mass spectra revealed various levels of cystatin C isoforms in the samples. Truncated Cystatin C isoforms missing 3, 4, 7, 8, 9, 10, 11, 14, 15, and 17 Nterminal amino acid residues were detected. Correlation in the level of truncation between the matched CSF and serum samples was also observed. The mass spectrometric immunoassay provided rapid, high-throughput assessment of cystatin C and its isoforms present in biological fluids.

Key Words: Cystatin C, CSF, mass spectrometry, immunoassay, serum.

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Assessing Conservation of Disordered Regions in Proteins

Ágnes Tóth-Petróczy, Bálint Mészáros, István Simon, A. Keith DunkerVladimir N. Uversky and Monika Fuxreiter

Abstract:

Intrinsically disordered regions (IDRs) are highly populated in eukaryotic proteomes and serve pivotal, mostly regulatory functions. Many IDRs appear to be functionally conserved and analysis of protein domains indicates high propensity of conserved regions predicted to be disordered. Nevertheless, it is difficult to assess conservation of IDRs in general due to their fast evolution and low sequence similarity. We propose three measures to evaluate conservation of IDRs:
i) similarities of the disorder profiles using different prediction conditions; ii) the conservation of amino acids with propensities for promoting either disorder or order; and iii) the overlap between disordered/ordered regions. These measures are computed on multiple sequence alignments that also include low-complexity regions of proteins. Using three subunits of the Mediator complex of transcription regulation from Homo sapiens and Drosophila melanogaster as an example we
show that despite of their sequence dissimilarity IDRs can be conserved and likely carry out the same function in different organisms.

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Quantitative Proteomic Analysis of Formalin Fixed Paraffin Embedded Oral HPV Lesions from HIV Patients

Mohit Raja Jain, Tong Liu, Jun Hu, Marlene Darfler, Valerie Fitzhugh, Joseph Rinaggio and Hong Li

Abstract:

Human immunodeficiency virus (HIV) infection is associated with dysplastic changes in oral human papilloma virus (HPV) lesions, suggesting changes in keratinocytes. In the present study, we seek to identify proteomic changes in oral HPV lesions between HIV(+) and HIV(-) patients. While fresh tissues represent the most desirable samples for proteomic investigations, they are often difficult to obtain in large numbers under clinical settings. We therefore have developed a new method to identify protein changes in formalin fixed and paraffin-embedded (FFPE) oral HPV lesions utilizing iTRAQ™ technology in conjunction with Liquid Tissue® sample preparation method. Using this method, we identified nine proteins that were differentially expressed in oral HPV lesions as a result of HIV infection. The quantitative proteomic method presented here will be valuable for others who plan to analyze FFPE tissues.

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Quantitative Serum Proteomics of Tryptophan Nutrition Using Bi-directional Heavy Oxygen Labeling with a RuBisCO Internal Standard

Amanda M. Cooksey, Alejandro Corzo, Marek D. Koter and Shane C. Burgess

Abstract:

Tryptophan plays an important role in vertebrate metabolism as not only a building block of proteins, but also as a precursor of serotonin, melatonin, niacin and kynurenines, which influence immune tolerance. Here we use an animal paradigm and quantitative serum proteomics to model tryptophan deficiency. We applied bidirectional H216/18O labeling to serum proteins from chickens fed either a tryptophan-deficient or- adequate diet and used the plant protein RuBisCO as an
internal standard. The proteins were trypsin digested and processed by 2-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (2D LC ESI MS2). The resulting mass spectra were analyzed using the SEQUEST algorithm and the ProteinMapper program to identify proteins that had increased or decreased expression. We identified 4161 proteins labeled bidirectionally, of which 46 were increased and 90 decreased (~3%). Using Ingenuity Pathways
Analysis (IPA) software, we found that a tryptophan nutritional deficiency may affect not only the immune and neurological systems, but our modeling also suggests that it may be important in cancer, optic atrophy and cardiomyopathy.

Key Words: Isotope labeling, Ingenuity, modeling, systems biology, quantitation, diet, deficiency, biomarkers.

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Proteomics and Pathway Mapping Analyses Reveal Phase-Dependent Over-Expression of Proteins Associated with Carbohydrate Metabolic Pathways in Candida albicans Biofilms

Ali Abdul Lattif, Jyotsna Chandra, Jinsook Chang, Shuqing Liu, Guangyin Zhou, Mark R.Chance, Mahmoud A. Ghannoum and Pranab K. Mukherjee

Abstract:

Candida albicans is the most commonly isolated fungus associated with biofilms, which are extracellular matrix (ECM)-encased, drug-resistant microbial communities formed on indwelling medical devices. Protein profiles of fungal biofilms have not been investigated in detail, although such profiles are believed to play critical roles in fungal biofilm formation. In this study, we used two-dimensional difference-in-gel electrophoresis (DIGE)-based proteomics to identify
differentially expressed proteins in C. albicans biofilms grown to early and mature phases, compared to planktonic cells. The resulting proteomic data set was subjected to pathway mapping to reveal phase-specific pathways that were differentially expressed in biofilm cell walls and extracellular matrix (ECM). Our analyses showed 107 proteins to be differentially expressed in ECM, while 44 were differentially expressed in cell walls during biofilm formation, compared to
planktonic controls. Furthermore, 95% (102/107) and 68% (30/44) of these differentially expressed proteins were upregulated in ECM and cell walls of biofilms, respectively. These proteins were mapped to cellular pathways, which revealed that these differentially expressed proteins were associated with several metabolic pathways, in a phase-dependent manner. For example, among ECM-associated proteins, proteins within 18 pathways were differentially expressed, with two
pathways (glutamate and nitrogen metabolism) unique to early phase, and four pathways (purine, Gly/Ser/Thr, and inositol metabolism, and carbon fixation) unique to mature phase biofilms. Such differences were also observed in cell wallassociated proteins, where proteins associated with 14 specific pathways were differentially regulated. We also found glycolytic enzymes including the key enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were overexpressed in
biofilms at both early and mature phases, compared to planktonic controls. Iodoacetate-mediated inhibition of this enzyme completely abrogated the ability of C. albicans to form biofilms, indicating the role of glycolysis/gluconeogenesis pathways in biofilm formation. Taken together, we demonstrate that ECM and cell walls of C. albicans biofilms express increased levels of specific proteins within pathways in a phase-dependent manner, suggesting that these pathways, especially glycolysis/gluconeogenesis, play critical roles in fungal biofilm formation and maintenance.

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Most of the Abundant Protein Fractions of Embryonic Cerebrospinal Fluid are Produced Out of the Brain Anlagen

M. Parvas, M. Rius and D. Bueno

Abstract:

The microenvironment of the central nervous system is important for neuronal function and development. During the early stages of embryo development the cephalic vesicles are filled by embryonic cerebrospinal fluid, a complex fluid containing different protein fractions, which contributes to the regulation of the survival, proliferation and neurogenesis of neuroectodermal stem cells. The protein content of embryonic cerebrospinal fluid from chick and rat embryos at the start of neurogenesis has already been determined. Most of the identified gene products are thought to be involved in the regulation of developmental processes during embryogenesis. However, due to the crucial roles played by embryonic cerebrospinal fluid during brain development, the embryological origin of the gene products it contains remains an intriguing question. According to the literature most of these products are synthesised in embryonic tissues other than the
neuroepithelium. In this study we examined the embryological origin of the most abundant embryonic cerebrospinal fluid protein fractions by means of slot-blot analysis and by using several different embryonic and extraembryonic protein extracts, immunodetected with polyclonal antibodies. This first attempt to elucidate their origin is not based on the proteins identified by proteomic methods, but rather on crude protein fractions detected by SDS-PAGE analysis and to which
polyclonal antibodies were specifically generated. Despite some of the limitations of this study, i.e. that one protein fraction may contain more than one gene product, and that a specific gene product may be contained in different protein fractions depending on post-translational modifications, our results show that most of the analysed protein fractions are not produced by the cephalic neuroectoderm but are rather stored in the egg reservoir; furthermore, few are produced by embryo tissues, thus indicating that they must be transported from their production or storage sites to the cephalic cavities, most probably via embryonic serum. These results raise the question as to whether the transfer of proteins from these two embryo compartments is regulated at this early developmental stage.

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Environmental Factors Affecting the Accumulation of Rosmarinic Acid in Spearmint (Mentha spicata L.) and Peppermint (Mentha piperita L.)

Ronald S. Fletcher, Tannis Slimmon, and Laima S. Kott

Abstract:

Four spearmint, and two peppermint clonal lines, selected for enhanced rosmarinic acid content (50-120 mg g-1rosmarinic acid DW), where up to 80% of the antioxidant activity was correlated to rosmarinic acid content, were examined to determine the effects of environmental and physiological conditions on the accumulation of rosmarinic acid in leaf tissues. Exposure to a short photoperiod of 12 hours in comparison to 16 hours reduced rosmarinic acid accumulation in two mint lines, but no significant difference was found between photoperiods of 14 and 16 hours. The physiological age of the plant strongly influenced the accumulation of rosmarinic acid with the highest levels recorded in the vegetative state, and a significant reduction in the concentration of rosmarinic acid in the leaves in both the bud initiation and flowering stages in the mint lines. Cold stress, impacted over a six week period had no effect on rosmarinic acid production. A field study of the commercial chemotype 700B indicated that soil type plays an essential role in the accumulation of rosmarinic acid in the leaf tissue, probably due to retention of moisture which favours rosmarinic acid production. For producers and extractors, taking these factors into account would significantly increase rosmarinic acid
accumulation in commercially high rosmarinic acid mint and increase the quality control of plant extracts for the natural products industry.

Keywords: Rosmarinic acid, spearmint, peppermint, environmental and physiological conditions.

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