Genetic transformation of willows (Salix spp.) byAgrobacterium tumefaciens

Anin vitro transformation method has been developed for stem explants of fast-growing willow clones (Salix spp.) usingAgrobacterium tumefaciens as a vector. Transformants obtained with the strains C58 and GV3101 (pGV3851::pLD1) were selected on hormone-free medium and on medium containing kanamycin, respectively. Transformation was confirmed by Southern blot analysis and nopaline assay. Inoculation of green-house grown plants with nopaline and octopine wildtype strains and shoot or root inducing mutant strains caused undifferentiated tumors at a frequency of 0 to 80%, depending on theSalix genotype and the bacterial strain used.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - NPT neomycin phosphotransferase     

Computational modeling of progressive damage and rupture in fibrous biological tissues: application to aortic dissection

This study analyzes the lethal clinical condition of aortic dissections from a numerical point of view. On the basis of previous contributions by Gültekin et al. (Comput Methods Appl Mech Eng 312:542–566, 2016 and 331:23–52, 2018), we apply a holistic geometrical approach to fracture, namely the crack phase-field, which inherits the intrinsic features of gradient damage and variational fracture mechanics. The continuum framework captures anisotropy, is thermodynamically consistent and is based on finite strains. The balance of linear momentum and the crack evolution equation govern the coupled mechanical and phase-field problem. The solution scheme features the robust one-pass operator-splitting algorithm upon temporal and spatial discretizations. Based on experimental data of diseased human thoracic aortic samples, the elastic material parameters are identified followed by a sensitivity analysis of the anisotropic phase-field model. Finally, we simulate an incipient propagation of an aortic dissection within a multi-layered segment of a thoracic aorta that involves a prescribed initial tear. The finite element results demonstrate a severe damage zone around the initial tear and exhibit a rather helical crack pattern, which aligns with the fiber orientation. It is hoped that the current contribution can provide some directions for further investigations of this disease.

     

Noncompaction of the ventricular myocardium is associated with a de novo mutation in the beta-myosin heavy chain gene

Noncompaction of the ventricular myocardium (NVM) is the morphological hallmark of a rare familial or sporadic unclassified heart disease of heterogeneous origin. NVM results presumably from a congenital developmental error and has been traced back to single point mutations in various genes. The objective of this study was to determine the underlying genetic defect in a large German family suffering from NVM. Twenty four family members were clinically assessed using advanced imaging techniques. For molecular characterization, a genome-wide linkage analysis was undertaken and the disease locus was mapped to chromosome 14ptel-14q12. Subsequently, two genes of the disease interval, MYH6 and MYH7 (encoding the alpha- and beta-myosin heavy chain, respectively) were sequenced, leading to the identification of a previously unknown de novo missense mutation, c.842G>C, in the gene MYH7. The mutation affects a highly conserved amino acid in the myosin subfragment-1 (R281T). In silico simulations suggest that the mutation R281T prevents the formation of a salt bridge between residues R281 and D325, thereby destabilizing the myosin head. The mutation was exclusively present in morphologically affected family members. A few members of the family displayed NVM in combination with other heart defects, such as dislocation of the tricuspid valve (Ebstein's anomaly, EA) and atrial septal defect (ASD). A high degree of clinical variability was observed, ranging from the absence of symptoms in childhood to cardiac death in the third decade of life. The data presented in this report provide first evidence that a mutation in a sarcomeric protein can cause noncompaction of the ventricular myocardium.     

Studying global change through investigation of the plastic responses of xylem anatomy in tree rings

Variability in xylem anatomy is of interest to plant scientists because of the role water transport plays in plant performance and survival. Insights into plant adjustments to changing environmental conditions have mainly been obtained through structural and functional comparative studies between taxa or within taxa on contrasting sites or along environmental gradients. Yet, a gap exists regarding the study of hydraulic adjustments in response to environmental changes over the lifetimes of plants. In trees, dated tree-ring series are often exploited to reconstruct dynamics in ecological conditions, and recent work in which wood-anatomical variables have been used in dendrochronology has produced promising results. Environmental signals identified in water-conducting cells carry novel information reflecting changes in regional conditions and are mostly related to short, sub-annual intervals. Although the idea of investigating environmental signals through wood anatomical time series goes back to the 1960s, it is only recently that low-cost computerized image-analysis systems have enabled increased scientific output in this field. We believe that the study of tree-ring anatomy is emerging as a promising approach in tree biology and climate change research, particularly if complemented by physiological and ecological studies. This contribution presents the rationale, the potential, and the methodological challenges of this innovative approach.     

Metabolic capabilities and systems fluctuations in Haloarcula marismortui revealed by integrative genomics and proteomics analyses

The 1310 Haloarcula marismortui proteins identified from mid-log and late-log phase soluble and membrane proteomes were analyzed in metabolic and cellular process networks to predict the available systems and systems fluctuations upon environmental stresses. When the connected metabolic reactions with identified proteins were examined, the availability of a number of metabolic pathways and a highly connected amino acid metabolic network were revealed. Quantitative spectral count analyses suggested 300 or more proteins might have expression changes in late-log phase. Among these, integrative network analyses indicated approximately 106 were metabolic proteins that might have growth-phase dependent changes. Interestingly, a large proportion of proteins in affected biomodules had the same trend of changes in spectral counts. Disregard the magnitude of changes, we had successfully predicted and validated the expression changes of nine genes including the rimK, gltCP, rrnAC0132, and argC in lysine biosynthesis pathway which were downregulated in late-log phase. This study had not only revealed the expressed proteins but also the availability of biological systems in two growth phases, systems level changes in response to the stresses in late-log phase, cellular locations of identified proteins, and the likely regulated genes to facilitate further analyses in the postgenomic era.     

Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions

COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.     

From ATP as substrate to ADP as coenzyme: functional evolution of the nucleotide binding subunit of dihydroxyacetone kinases

Dihydroxyacetone kinases are a family of sequence-related enzymes that utilize either ATP or a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as a source of high energy phosphate. The PTS is a multicomponent system involved in carbohydrate uptake and control of carbon metabolism in bacteria. Phylogenetic analysis suggests that the PTS-dependent dihydroxyacetone kinases evolved from an ATP-dependent ancestor. Their nucleotide binding subunit, an eight-helix barrel of regular up-down topology, retains ADP as phosphorylation site for the double displacement of phosphate from a phospho-histidine of the PTS protein to dihydroxyacetone. ADP is bound essentially irreversibly with a t((1/2)) of 100 min. Complexation with ADP increases the thermal unfolding temperature of dihydroxyacetone L from 40 (apo-form) to 65 degrees C (holoenzyme). ADP assumes the same role as histidines, cysteines, and aspartic acids in histidine kinases and PTS proteins. This conversion of a substrate binding site into a cofactor binding site reflects a remarkable instance of parsimonious evolution.     

Long-term functional plasticity in plant hydraulic architecture in response to supplemental moisture

Background and Aims

Plasticity in structural and functional traits related to water balance may determine plant performance and survival in ecosystems characterized by water limitation or high levels of rainfall variability, particularly in perennial herbaceous species with long generation cycles. This paper addresses whether and the extent to which several such seasonal to long-term traits respond to changes in moisture availability.

Methods

Using a novel approach that integrates ecology, physiology and anatomy, a comparison was made of lifetime functional traits in the root xylem of a long-lived perennial herb (Potentilla diversifolia, Rosaceae) growing in dry habitats with those of nearby individuals growing where soil moisture had been supplemented for 14 years. Traditional parameters such as specific leaf area (SLA) and above-ground growth were also assessed.

Key Results

Individuals from the site receiving supplemental moisture consistently showed significant responses in all considered traits related to water balance: SLA was greater by 24 %; roots developed 19 % less starch storing tissue, an indicator for drought-stress tolerance; and vessel size distributions shifted towards wider elements that collectively conducted water 54 % more efficiently – but only during the years for which moisture was supplemented. In contrast, above-ground growth parameters showed insignificant or inconsistent responses.

Conclusions

The phenotypic changes documented represent consistent, dynamic responses to increased moisture availability that should increase plant competitive ability. The functional plasticity of xylem anatomy quantified in this study constitutes a mechanistic basis for anticipating the differential success of plant species in response to climate variability and change, particularly where water limitation occurs.