New players in the interaction between beetle polygalacturonases and plant polygalacturonase-inhibiting proteins: Insights from proteomics and gene expression analyses

Plants possess various defense strategies to counter attacks from microorganisms or herbivores. For example, plants reduce the cell-wall-macerating activity of pathogen- or insect-derived polygalacturonases (PGs) by expressing PG-inhibiting proteins (PGIPs). PGs and PGIPs belong to multi-gene families believed to have been shaped by an evolutionary arms race. The mustard leaf beetle Phaedon cochleariae expresses both active PGs and catalytically inactive PG pseudoenzymes. Previous studies demonstrated that (i) PGIPs target beetle PGs and (ii) the role of PG pseudoenzymes remains elusive, despite having been linked to the pectin degradation pathway. For further insight into the interaction between plant PGIPs and beetle PG family members, we combined affinity purification with proteomics and gene expression analyses, and identified novel inhibitors of beetle PGs from Chinese cabbage (Brassica rapa ssp. pekinensis). A beetle PG pseudoenzyme was not targeted by PGIPs, but instead interacted with PGIP-like proteins. Phylogenetic analysis revealed that PGIP-like proteins clustered apart from “classical” PGIPs but together with proteins, which have been involved in developmental processes. Our results indicate that PGIP-like proteins represent not only interesting novel PG inhibitor candidates in addition to “classical” PGIPs, but also fascinating new players in the arms race between herbivorous beetles and plant defenses

Hijacking the mustard-oil bomb: How a glucosinolate-sequestering flea beetle copes with plant myrosinases

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the β-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle’s energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants

An integrated—omics/chemistry approach unravels enzymatic and spontaneous steps to form flavoalkaloidal nudicaulin pigments in flowers of Papaver nudicaule L.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions

Density functional calculations of nanoscale conductance

Density functional calculations for the electronic conductance of single molecules are now common. We examine the methodology from a rigorous point of view, discussing where it can be expected to work, and where it should fail. When molecules are weakly coupled to leads, local and gradient-corrected approximations fail, as the Kohn-Sham levels are misaligned. In the weak bias regime, XC corrections to the current are missed by the standard methodology. For finite bias, a new methodology for performing calculations can be rigorously derived using an extension of time-dependent current density functional theory from the Schroedinger equation to a Master equation.Comment: topical review, 28 pages, updated version with some revision

ButterflyBase: a platform for lepidopteran genomics

With over 100 000 species and a large community of evolutionary biologists, population ecologists, pest biologists and genome researchers, the Lepidoptera are an important insect group. Genomic resources [expressed sequence tags (ESTs), genome sequence, genetic and physical maps, proteomic and microarray datasets] are growing, but there has up to now been no single access and analysis portal for this group. Here we present ButterflyBase (http://www.butterflybase.org), a unified resource for lepidopteran genomics. A total of 273 077 ESTs from more than 30 different species have been clustered to generate stable unigene sets, and robust protein translations derived from each unigene cluster. Clusters and their protein translations are annotated with BLAST-based similarity, gene ontology (GO), enzyme classification (EC) and Kyoto encyclopaedia of genes and genomes (KEGG) terms, and are also searchable using similarity tools such as BLAST and MS-BLAST. The database supports many needs of the lepidopteran research community, including molecular marker development, orthologue prediction for deep phylogenetics, and detection of rapidly evolving proteins likely involved in host–pathogen or other evolutionary processes. ButterflyBase is expanding to include additional genomic sequence, ecological and mapping data for key species

Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-α, IL-1β) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells

Differential (2+1) Jet Event Rates and Determination of alpha_s in Deep Inelastic Scattering at HERA

Events with a (2+1) jet topology in deep-inelastic scattering at HERA are studied in the kinematic range 200 < Q^2< 10,000 GeV^2. The rate of (2+1) jet events has been determined with the modified JADE jet algorithm as a function of the jet resolution parameter and is compared with the predictions of Monte Carlo models. In addition, the event rate is corrected for both hadronization and detector effects and is compared with next-to-leading order QCD calculations. A value of the strong coupling constant of alpha_s(M_Z^2)= 0.118+- 0.002 (stat.)^(+0.007)_(-0.008) (syst.)^(+0.007)_(-0.006) (theory) is extracted. The systematic error includes uncertainties in the calorimeter energy calibration, in the description of the data by current Monte Carlo models, and in the knowledge of the parton densities. The theoretical error is dominated by the renormalization scale ambiguity.Comment: 25 pages, 6 figures, 3 tables, submitted to Eur. Phys.

Multiplicity Structure of the Hadronic Final State in Diffractive Deep-Inelastic Scattering at HERA

The multiplicity structure of the hadronic system X produced in deep-inelastic processes at HERA of the type ep -> eXY, where Y is a hadronic system with mass M_Y< 1.6 GeV and where the squared momentum transfer at the pY vertex, t, is limited to |t|<1 GeV^2, is studied as a function of the invariant mass M_X of the system X. Results are presented on multiplicity distributions and multiplicity moments, rapidity spectra and forward-backward correlations in the centre-of-mass system of X. The data are compared to results in e+e- annihilation, fixed-target lepton-nucleon collisions, hadro-produced diffractive final states and to non-diffractive hadron-hadron collisions. The comparison suggests a production mechanism of virtual photon dissociation which involves a mixture of partonic states and a significant gluon content. The data are well described by a model, based on a QCD-Regge analysis of the diffractive structure function, which assumes a large hard gluonic component of the colourless exchange at low Q^2. A model with soft colour interactions is also successful.Comment: 22 pages, 4 figures, submitted to Eur. Phys. J., error in first submission - omitted bibliograph

Low Q^2 Jet Production at HERA and Virtual Photon Structure

The transition between photoproduction and deep-inelastic scattering is investigated in jet production at the HERA ep collider, using data collected by the H1 experiment. Measurements of the differential inclusive jet cross-sections dsigep/dEt* and dsigmep/deta*, where Et* and eta* are the transverse energy and the pseudorapidity of the jets in the virtual photon-proton centre of mass frame, are presented for 0 < Q2 < 49 GeV2 and 0.3 < y < 0.6. The interpretation of the results in terms of the structure of the virtual photon is discussed. The data are best described by QCD calculations which include a partonic structure of the virtual photon that evolves with Q2.Comment: 20 pages, 5 Figure

Hadron Production in Diffractive Deep-Inelastic Scattering

Characteristics of hadron production in diffractive deep-inelastic positron-proton scattering are studied using data collected in 1994 by the H1 experiment at HERA. The following distributions are measured in the centre-of-mass frame of the photon dissociation system: the hadronic energy flow, the Feynman-x (x_F) variable for charged particles, the squared transverse momentum of charged particles (p_T^{*2}), and the mean p_T^{*2} as a function of x_F. These distributions are compared with results in the gamma^* p centre-of-mass frame from inclusive deep-inelastic scattering in the fixed-target experiment EMC, and also with the predictions of several Monte Carlo calculations. The data are consistent with a picture in which the partonic structure of the diffractive exchange is dominated at low Q^2 by hard gluons.Comment: 16 pages, 6 figures, submitted to Phys. Lett.