Co-ordinated growth between aerial and root systems in young apple plants issued from in vitro culture

BACKGROUND AND AIMS: In several species exhibiting a rhythmic aerial growth, the existence of an alternation between root and shoot growth has been demonstrated. The present study aims to investigate the respective involvement of the emergence of new organs and their elongation in relation to this phenomenon and its possible genotypic variation in young apple plants. METHODS: Two apple varieties, X6407 (recently named 'Ariane') and X3305 ('Chantecler' x 'Baujade'), were compared. Five plants per variety, issued from in vitro culture, were observed in minirhizotrons over 4 months. For each plant, root emergence and growth were observed twice per week. Growth rates were calculated for all roots with more than two segments and the branching density was calculated on primary roots. On the aerial part, the number of leaves, leaf area and total shoot length were observed weekly. KEY RESULTS: No significant difference was observed between varieties in any of the final characteristics of aerial growth. Increase in leaf area and shoot length exhibited a 3-week rhythm in X3305 while a weaker signal was observed in Ariane. The primary root growth rate was homogeneous between the plants and likewise between the varieties, while their branching density differed significantly. Secondary roots emerged rhythmically, with a 3-week and a 2-week rhythm, respectively, in X3305 and 'Ariane'. Despite a high intra-variety variability, significant differences were observed between varieties in the secondary root life span and mean length. A synchronism between leaf emergence and primary root growth was highlighted in both varieties, while an opposition phase was observed between leaf area increments and secondary root emergence in X3305 only. CONCLUSION: A biological model of dynamics that summarizes the interactions between processes and includes the assumption of a feedback effect of lateral root emergence on leaf emergence is proposed.     

The Rieske protein: a case study on the pitfalls of multiple sequence alignments and phylogenetic reconstruction

Previously published phylogenetic trees reconstructed on "Rieske protein" sequences frequently are at odds with each other, with those of other subunits of the parent enzymes and with small-subunit rRNA trees. These differences are shown to be at least partially if not completely due to problems in the reconstruction procedures. A major source of erroneous Rieske protein trees lies in the presence of a large, poorly conserved domain prone to accommodate very long insertions in well-defined structural hot spots substantially hampering multiple alignments. The remaining smaller domain, in contrast, is too conserved to allow distant phylogenies to be deduced with sufficient confidence. Three-dimensional structures of representatives from this protein family are now available from phylogenetically distant species and from diverse enzymes. Multiple alignments can thus be refined on the basis of these structures. We show that structurally guided alignments of Rieske proteins from Rieske-cytochrome b complexes and arsenite oxidases strongly reduce conflicts between resulting trees and those obtained on their companion enzyme subunits. Further problems encountered during this work, mainly consisting in database errors such as wrong annotations and frameshifts, are described. The obtained results are discussed against the background of hypotheses stipulating pervasive lateral gene transfer in prokaryotes.     

Genetic diversity in the blackberry rust pathogen, Phragmidium violaceum, in Europe and Australasia as revealed by analysis of SAMPL

Indigenous to Europe, the blackberry rust fungus Phragmidium violaceum was introduced to Australia and subsequently appeared in New Zealand, with the most recent authorised introductions to Australia specifically for the biological control of European blackberry. Markers for ‘selective amplification of microsatellite polymorphic loci’ (SAMPL) were developed for studying the population genetics of P. violaceum. Modification of one of the two SAMPL primers with a HaeIII adapter (H) revealed significantly greater levels of genetic variation than primers used to generate AFLPs, the latter revealing little or no variation among 25 Australasian and 19 European isolates of P. violaceum. SAMPL was used to describe genetic variation among these 44 isolates of P. violaceum from 51 loci generated using primer pairs (GACA)₄ + H–G and R1 + H–G. The European isolates were more diverse than Australasian isolates, with 37 and 22 % polymorphic loci, respectively. Cluster analysis revealed geographic clades, with Australasian isolates forming one cluster separated from two clusters comprising the European isolates. However, low bootstrap support at these clades suggested that Australian isolates had not differentiated significantly from European isolates since the first record of P. violaceum in Australia in 1984. In general, the results support two hypotheses. First, that the population of P. violaceum in Australia was founded from a subset of individuals originating from Europe. Second, that P. violaceum in New Zealand originated from the Australian population of P. violaceum, probably by wind dispersal of urediniospores across the Tasman Sea. The application of SAMPL markers to the current biological control programme for European blackberry is discussed.     

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.     

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

Background

Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis.

Methology/Principal Findings

We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21–66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process.

Conclusions/Significance

Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis.     

Activation of Wnt/β-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

Background

Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity.

Methodology/Principal Findings

Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/β-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3β, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/β-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/β-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes.

Conclusions/Significance

We conclude that activation of Wnt/β-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/β-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations.     

Threading bis-intercalation of a macrocyclic bisacridine at abasic sites in DNA: nuclear magnetic resonance and molecular modeling study

The macrocyclic bisacridine (CBA) has been reported previously to specifically recognize single-stranded nucleic acid structures, especially DNA hairpins. The binding of the drug with an abasic site-containing oligonucleotide, was investigated by (1)H NMR and molecular modeling. We have used a DNA undecamer, the d(C(1)G(2)C(3)A(4)C(5)X(6)C(7)A(8)C(9)G(10)C(11)) x d(G(12)C(13)G(14)T(15)G(16)T(17)G(18)T(19)G(2)(0)C(21)G(22)) duplex in which the X residue is a stable analogue of the abasic site [3-hydroxy-2-(hydroxymethyl) tetrahydrofuran]. Analysis of the NMR data reveals that the bisacridine molecule forms two different intercalation complexes in a 80/20 (+/- 10) ratio. For the major complex, a molecular modeling study was performed guided by nineteen intermolecular drug-DNA restraints, determined from NOESY spectra. In this model, the ligand interacts in the threading binding mode with an acridine ring intercalated between the C(7)-A(8) and T(15)-G(16) base pairs, while the other acridine ring resides in the abasic pocket. The two linker chains are positioned in the minor and in the major groove, respectively. A comparable study was performed to evaluate the interaction of CBA with the parent unmodified duplex in which X(6) was replaced by an adenine residue. No complex formation was observed when operating in identical conditions. This shows the selective binding of CBA to the abasic site and its potential interest to target the abasic site lesion.