BackgroundThe complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays.ResultsWe established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)25 bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts.ConclusionsThe described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0142-6) contains supplementary material, which is available to authorized users.

We analyze in detail how the interplay between electronic structure and cluster geometry determines the stability and the fragmentation channels of single Pd-doped cationic Au clusters, PdAu N −1 + (N = 2−20). For this purpose, a combination of photofragmentation experiments and density functional theory calculations was employed. A remarkable agreement between the experiment and the calculations is obtained. Pd doping is found to modify the structure of the Au clusters, in particular altering the two-dimensional to three-dimensional transition size, with direct consequences on the stability of the clusters. Analysis of the electronic density of states of the clusters shows that depending on cluster size, Pd delocalizes one 4d electron, giving an enhanced stability to PdAu 6 + , or remains with all 4d 10 electrons localized, closing an electronic shell in PdAu 9 +. Furthermore, it is observed that for most clusters, Au evaporation is the lowest-energy decay channel, although for some sizes Pd evaporation competes. In particular, PdAu 7 + and PdAu 9 + decay by Pd evaporation due to the high stability of the Au 7 + and Au 9 + fragmentation products.

The reactivity of small metallic clusters, nanoparticles composed of a countable number of atoms (typically up to ~100 atoms), has attracted much attention due to the fascinating properties these objects possess toward a variety of molecules. Cluster reactivity often is significantly different from the homologous bulk, with gold as prototypical example. Bulk gold is the noblest of all metals, whereas small gold clusters react with carbon monoxide, molecular oxygen, and hydrocarbons, among others. Furthermore, cluster reactivity is strongly size and composition dependent, allowing a wide range of tuning possibilities.The study of cluster reactivity usually follows two routes of investigation. In the first, research aims for fundamental understanding of mechanisms, mainly driven by curiosity. One consequence of the inherent small size of a cluster is that atoms can arrange themselves very differently from the crystallographic structure of the homologous bulk. In addition, quantum confinement effects dominate the electronic structure of a cluster with atom-like electronic shells instead of the Flanders (FWO).

Far-infrared multiple photon dissociation spectroscopy is used in combination with density functional theory calculations to determine the structures of isolated Aun+ (n ≤ 9) clusters.

The interaction of hydrogen with singly rhodium doped aluminum clusters AlnRh + (n = 1−12) is investigated experimentally by a combination of time-of-flight mass spectrometry and infrared multiple photon dissociation (IRMPD) spectroscopy. Density functional theory (DFT) is employed to optimize the geometric and electronic structures of bare and hydrogenated AlnRh + clusters and the obtained infrared spectra of hydrogenated clusters are compared with the corresponding IRMPD spectra. The reactivity of the AlnRh + clusters towards H2 is found to be strongly size-dependent, with n = 1−3, and 7 being the most reactive. Furthermore, it is favorable for H2 to adsorb molecularly on Al2Rh + and Al3Rh + , while it prefers dissociative adsorption on other sizes. The initial molecular adsorption of H2 is identified as the determining step for hydrogen interaction with the AlnRh + clusters, because the calculated molecular adsorption energies of H2 correlate well with the experimental abundances of the hydrogenated clusters. Natural charge populations and properties of the AlnRh + clusters are analyzed to interpret the observed size-dependent reactivity.

The effect of vanadium doping on the hydrogen adsorption capacity of aluminum clusters (Al , n=2-18) is studied experimentally by mass spectrometry and infrared multiple photon dissociation (IRMPD) spectroscopy. We find that vanadium doping enhances the reactivity of the clusters towards hydrogen, albeit in a size-dependent way. IRMPD spectra, which provide a fingerprint of the hydrogen binding geometry, show that H dissociates upon adsorption. Density functional theory (DFT) calculations for the smaller Al V (n=2-8,10) clusters are in good agreement with the observed reactivity pattern and underline the importance of activation barriers in the chemisorption process. Orbital analysis shows that the activation barriers are due to an unfavorable overlap between cluster and hydrogen orbitals.

Small positively charged gold clusters have been found to emit thermal radiation at a very high rate, with time constants ranging from one to 35 μs for Au n + (n = 6-13,15). For sizes n = 14,16-20 the radiation occurs on much longer time scales. Strong thermal suppression of the population of higher-lying states puts constraints on the possible energies of excited states that can contribute to the radiation. Taking that into account, an evaluation of the experimentally determined rate constants shows that the strong radiation originates from thermally excited low-lying electronic states hitherto not observed. The origin of these states is discussed and two possibilities are suggested: one is related to electron correlation and electron pairing, and the other results from thermal shape fluctuations.

The linear and nonlinear optical properties of metal nanoparticles are highly tunable by variation of parameters such as particle size, shape, composition, and environment. To fully exploit this tunability, however, quantitative information on nonlinear absorption cross sections is required, as well as a sufficient understanding of the physical mechanism underlying these nonlinearities. In this work, we present a detailed and systematic investigation of the wavelength-dependent nonlinear optical properties of Ag nanoparticles embedded in a glass host, in which the most important parameters determining the nonlinear behavior of the system are characterized. This allows a proper quantification of absorption cross sections and elucidation of the excitation mechanism. Based on small-angle X-ray scattering measurements average particle diameters of 3 and 17 nm are estimated for the studied samples. The nonlinear optical properties of the nanoparticle−glass composite are studied in an extended wavelength range with the open aperture z-scan technique. The experiments reveal a strong dependence of the nonlinear optical response on the excitation wavelength. Based on the wavelengthdependent response, excited-state absorption is determined as the excitation mechanism of the nanoparticles. Electromagnetic simulations demonstrate that the contributions from electric field enhancement and plasmonic coupling between the particles in the diluted glasses are limited, which implies that the very high two-photon absorption cross section at 460 nm ((6.9 ± 1.6) × 10 6 GM for the 3 nm particles and (19.5 ± 2.2) × 10 9 GM for the 17 nm particles) is an intrinsic property. In addition, irradiance-dependent measurements elucidate the role of saturation of the excited-state absorption process on the observed nonlinearities.