Antje Reinecke

Disrupting Two Arabidopsis thaliana Xylosyltransferase Genes Results in Plants Deficient in Xyloglucan, a Major Primary Cell Wall Component

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.

Metal-Mediated Molecular Self-Healing in Histidine-Rich Mussel Peptides

Mussels withstand high-energy wave impacts in rocky seashore habitats by fastening tightly to surfaces with tough and self-healing proteinaceous fibers called byssal threads. Thread mechanical behavior is believed to arise from reversibly breakable metal coordination cross-links embedded in histidine-rich protein domains (HRDs) in the principle load-bearing proteins comprising the fibrous thread core. In order to investigate HRD behavior at the molecular level, we have synthesized a histidine-rich peptide derived from mussel proteins (His5-bys) and studied its reversible adhesive self-interaction in the presence and absence of metal ions using PEG-based soft-colloidal probes (SCPs). Adhesion energies of greater than 0.3 mJ/m(2) were measured in the presence of metal ions, and the stiffness of the modified SCPs exhibited a 3-fold increase, whereas no adhesion was observed in the absence of metals. Raman spectroscopy confirmed the presence of metal-coordination via histidine residues by the peptide-supporting the role of His-metal complexes in the mechanical behavior of the byssus.

Pectin May Hinder the Unfolding of Xyloglucan Chains during Cell Deformation: Implications of the Mechanical Performance of Arabidopsis Hypocotyls with Pectin Alterations

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on mur1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants (Col-0) and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels (approximately tensile strength) of the hypocotyls of the mutants with pectin alterations (mur1, qua2) were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for mur1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.

“Green” gold nanotriangles: synthesis, purification by polyelectrolyte/micelle depletion flocculation and performance in surface-enhanced Raman scattering

The aim of this study was to develop a one-step synthesis of gold nanotriangles (NTs) in the presence of mixed phospholipid vesicles followed by a separation process to isolate purified NTs. Negatively charged vesicles containing AOT and phospholipids, in the absence and presence of additional reducing agents (polyampholytes, polyanions or low molecular weight compounds), were used as a template phase to form anisotropic gold nanoparticles. Upon addition of the gold chloride solution, the nucleation process is initiated and both types of particles, i.e., isotropic spherical and anisotropic gold nanotriangles, are formed simultaneously. As it was not possible to produce monodisperse nanotriangles with such a one-step procedure, the anisotropic nanoparticles needed to be separated from the spherical ones. Therefore, a new type of separation procedure using combined polyelectrolyte/micelle depletion flocculation was successfully applied. As a result of the different purification steps, a green colored aqueous dispersion was obtained containing highly purified, well-defined negatively charged flat nanocrystals with a platelet thickness of 10 nm and an edge length of about 175 nm. The NTs produce promising results in surface-enhanced Raman scattering.

Cooperative behavior of a sacrificial bond network and elastic framework in providing self-healing capacity in mussel byssal threads.

The dissipative and self-healing properties of mussel byssal threads are critical for their function as anchoring fibers in wave-battered habitats and central to their emergence as an exciting model system for bio-inspired polymers. Much is now understood about the structure-function relationships defining this remarkable proteinaceous bio-fiber; however, the molecular mechanisms underlying the distinctive tough, viscoelastic and self-healing behavior are still unclear. Here, we investigate elastic and dissipative contributions from the primary load-bearing proteins in the distal region of byssal threads (the preCols) using X-ray diffraction (XRD) combined with in situ tensile testing. Specifically, we identified cross β-sheet structure in the preCol flanking domains that functions as an elastic framework, providing hidden length. Dissipative behavior was associated with a strain-rate dependent phase transition of a sacrificial network stabilized by strong, reversible cross-links. Based on these findings, we posit a new model for byssal thread deformation and self-healing.

Age effects on hypocotyl mechanics

Numerous studies deal with composition and molecular processes involved in primary cell wall formation and alteration in Arabidopsis. However, it still remains difficult to assess the relation between physiological properties and mechanical function at the cell wall level. The thin and fragile structure of primary cell walls and their large biological variability, partly related to structural changes during growth, make mechanical experiments challenging. Since, to the best of our knowledge, there is no reliable data in the literature about how the properties of the fully elongated zone of hypocotyls change with age. We studied in a series of experiments on two different seed batches the tensile properties the region below the growth zone of 4 to 7 day old etiolated Arabidopsis hypocotyls. Additionally, we analysed geometrical parameters, hypocotyl density and cellulose content as individual traits and their relation to tissue mechanics. No significant differences of the mechanical parameters of the non-growing region between 5–7 day old plants could be found whereas in 4 day old plants both tensile stiffness and ultimate tensile stress were significantly lower than in the older plants. Furthermore hypocotyl diameters and densities remain almost the same for 5, 6 and 7 day old seedlings. Naturally, hypocotyl lengths increase with age. The evaluation whether the choice–age or length—influences the mechanical properties showed that both are equally applicable sampling parameters. Additionally, our detailed study allows for the estimation of biological variability, connections between mechanics and hypocotyl age could be established and complement the knowledge on biochemistry and genetics affecting primary plant cell wall growth. The application of two different micromechanical devices for testing living Arabidopsis hypocotyls allows for emphasizing and discussing experimental limitations and for presenting a wide range of possibilities to address current and future questions related to plant cell wall mechanics, synthesis and growth in combination with molecular biology methodologies.

Enhancing soft matter mechanics via metal coordination: lessons from the mussel byssus

Tissue engineering holds the promise of producing functional biological replacements to repair damaged and diseased tissues in the body. The complex signals that are implicated in tissue morphogenesis, repair and homeostasis can be used as a guide for the development of innovative biomaterial systems for tissue regeneration. Through the precise temporal and spatial presentation of soluble bioactive factors, mechanical forces, and biomaterial physical and biochemical properties, we aspire to create biomaterials and microenvironments that regulate cell gene expression and new tissue formation. Today’s talk will explore a new biodegradable biomaterial system that has been engineered with the capacity for independent modulation of the soluble (e.g., growth factors) and insoluble (e.g., cell adhesion signals) biochemical signaling environment and biomaterial physical properties (e.g., the elastic moduli). The ability to independently control and spatially pattern these environmental parameters will then be demonstrated to be a powerful tool for elucidating their individual and combined effects on cell function for regenerative medicine applications. K62 Biomimetic polyurethanes scaffolds for cardiac tissue engineering M Boffito, S Sartori and G Ciardelli Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy Introduction: Cardiac tissue engineering (TE) focuses on creating functional cardiac muscle constructs. In this work polyurethane scaffolds for cardiac repair, were produced by thermally induced phase separation, in order to obtain oriented fibers texture, like the muscle tissues topography. These scaffolds were further surface functionalized with ECM proteins, to mimic the native tissue composition and encourage cell colonization. Materials and Methods: The polyurethane (PUR) used in this study was synthesized starting from poly(e-caprolactone) diol, 1,4-budandiisocyanate and L-lysine ethyl ester, as previously described[1]. PUR scaffolds were fabricated by thermally induced phase separation (TIPS). Briefly, the polyurethane was first dissolved at 60 °C in dimethyl sulfoxide (DMSO), the solution was poured in a stainless steel mould and cooled. The quenching was performed under application of a thermal cooling gradient. The scaffold was placed for seven days in a water/ ethanol solution and refreshed twice a day. The functionalization was performed by plasma treatment with acrylic acid, followed by the activation of carboxylic groups and the coupling with ECM proteins. The constructs were characterized by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), DSC and Tensile Test. The functionalization steps were studied by Contact Angle and X-ray photoelectron spectroscopy (XPS). Image J software analysis was used to evaluate porosity and pore size. Alamar tests were conducted to estimate cell viablity. Primary cultures of cardiac cells were prepared from neonatal Sprague-Dawley rats. Results: The PUR was successfully synthesized as confirmed by ATR-IR and Size Exclusion Chromatography (Mn=40.000Da). By changing fabrication parameters, scaffolds with different porosity were obtained. The porosity and pore size were respectively in the range of 85-90% and 100-150lm. The formation of a structure preferably oriented in one direction was observed in the scaffolds by SEM (Figure 1). TIPS allowed the fabrication of scaffolds with suitable mechanical properties for Cardiac TE (Young modulus of about 1/1.2 MPa in the dry state, and in the range of 0.3/0.5 MPa in wet conditions; strain at break values were higher than the typical deformation of the heart during cardiac cycle). Contact angle tests showed a value of about 133° for the non-functionalized scaffold, 84° for the scaffold functionalized with acrylic acid and about 100° after protein coupling. XPS analysis confirmed the surface immobilization of ECM proteins. Three-dimensional confocal laser scanning microscopy (CLSM) time-lapse imaging was performed on PUR scaffolds seeded with Neonatal rat cardiomyocytes scaffolds, showing high cell viability. The synchronized beating of cardiomyocytes was observed after 7 days and for over 40 days.