Large old trees increase growth under shifting climatic constraints: Aligning tree longevity and individual growth dynamics in primary mountain spruce forests

In a world of accelerating changes in environmental conditions driving tree growth, tradeoffs between tree growth rate and longevity could curtail the abundance of large old trees (LOTs), with potentially dire consequences for biodiversity and carbon storage. However, the influence of tree-level tradeoffs on forest structure at landscape scales will also depend on disturbances, which shape tree size and age distribution, and on whether LOTs can benefit from improved growing conditions due to climate warming. We analyzed temporal and spatial variation in radial growth patterns from ~5000 Norway spruce (Picea abies [L.] H. Karst) live and dead trees from the Western Carpathian primary spruce forest stands. We applied mixed-linear modeling to quantify the importance of LOT growth histories and stand dynamics (i.e., competition and disturbance factors) on lifespan. Finally, we assessed regional synchronization in radial growth variability over the 20th century, and modeled the effects of stand dynamics and climate on LOTs recent growth trends. Tree age varied considerably among forest stands, implying an important role of disturbance as an age constraint. Slow juvenile growth and longer period of suppressed growth prolonged tree lifespan, while increasing disturbance severity and shorter time since last disturbance decreased it. The highest age was not achieved only by trees with continuous slow growth, but those with slow juvenile growth followed by subsequent growth releases. Growth trend analysis demonstrated an increase in absolute growth rates in response to climate warming, with late summer temperatures driving the recent growth trend. Contrary to our expectation that LOTs would eventually exhibit declining growth rates, the oldest LOTs (>400 years) continuously increase growth throughout their lives, indicating a high phenotypic plasticity of LOTs for increasing biomass, and a strong carbon sink role of primary spruce forests under rising temperatures, intensifying droughts, and increasing bark beetle outbreaks.     

Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.     

Selector Screening for Enantioseparation of dl‐α‐Methyl Phenylglycine Amide by Liquid–Liquid Extraction

Enantioseparation through liquid extraction technology is an emerging field, e.g., enantioseparations of amino acids (and derivatives thereof), amino alcohols, amines, and carboxylic acids have been reported. Often, when a new selector is developed, the versatility of substrate scope is investigated. From an industrial point of view, the problem is typically approached the other way around, and for a target racemate, a selector needs to be found in order to accomplish the desired enantioseparation. This study presents such a screening approach for the separation of the enantiomers of dl ‐α‐methyl phenylglycine amide (dl ‐α‐MPGA), a model amide racemate with high industrial relevance. Chiral selectors that were reported for other classes of racemates were investigated, i.e., several macrocyclic selectors and Pd‐BINAP complexes. It appeared very challenging to obtain both high extraction yields and good enantioselectivity for most selectors, but Pd‐BINAP‐based selectors performed well, with enantioselectivities up to 7.4 with an extraction yield of the desired enantiomer of 95.8%. These high enantioselectivities were obtained using dichloromethane as solvent. Using less volatile chlorobenzene or 1‐chloropentane, reasonable selectivities of up to 1.7 were measured, making these the best alternative solvents for dichloromethane. Chirality 27:123–130, 2015. © 2014 Wiley Periodicals, Inc.     

Cytoskeletal Components Define Protein Location to Membrane Microdomains

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases.Living cells need borders and molecular compartments for biochemical reactions and storage of metabolites. The plasma membrane therefore is a prerequisite for the evolution of different life forms. It consists of a phospholipid bilayer into which proteins and special lipid species such as sterols, sphingolipids, and glycolipids are inserted. The first complex model of plasma membrane was proposed in 1972 by Jonathan Singer and Garth Nicolson (1), replacing the concept of the plasma membrane as a strict protein–lipid–protein sandwich that was generally accepted until then. In Singer and Nicolson''s model, the cell membrane is a two-dimensionally oriented viscous solution in which the membrane constituents are orientated in the most thermodynamically favorable manner, hiding hydrophobic hydrocarbon chains inside the lipid bilayer and exposing polar and ionic groups to the aqueous phase. This fluid mosaic model also implied that membrane proteins as well as lipid components are distributed in a homogeneous lipid bilayer at long range, but they can form specific aggregates and phases at short range, which were also termed “lipid rafts” or membrane microdomains.Over the past 30 years, it has become evident that the plasma membrane is not such a homogeneous structure as it was initially proposed. We now know that the lipid bilayer is asymmetric (2) and that the free diffusion of membrane proteins is restricted by their interactions with intracellular and extracellular components (3). More recently, Simons and Ikonen suggested that large ordered phases, enriched with cholesterol and sphingolipids, emerge within the plasma membrane and that they function as platforms for enrichment of certain proteins while excluding others (4). This current membrane model suggests that the mixture of sterols and polar lipids within the plasma membrane can appear in two distinct phases: liquid disordered (L_d) and liquid ordered (L_o) phase (5). In this view, the so-called membrane microdomains are considered to be part of the L_o phase. Based on work on model membranes, it is suggested that lateral segregation of components into L_d and L_o phases occurs spontaneously (6) with the self-associating properties between sterols and highly saturated hydrocarbon chains of phopsho- and sphingolipids as the main driving force (7). Additionally, it is suggested that also specific lipid-protein and protein-protein interactions are essential for the formations of membrane domains as well as for stabilization of smaller nanodomains which subsequently may cause formation of larger platforms. In contrast to the animal cells, in plants these membrane microdomains seem to be rather immobile (8), possibly due to their attachment to the outer cell wall. More recently, it became obvious that membrane microdomains within a single cell are highly diverse and of different compositions (9). Generally, in the plant model, organisms'' plasma membrane microdomains turned out to be important in plant defense (10, 11), cell polarity (12, 13), and general signaling properties of the plasma membrane (14, 15).The cytoskeleton was identified as an essential cellular component with important roles in membrane topography, bordering, trafficking, and organelle movement (16). Single particle tracking in mammalian cells revealed that the transferrin receptor and macroglobulin receptor demonstrate normal Brownian diffusion but only within a specific membrane compartment (17). Two hypothetical models were proposed in order to explain this phenomenon (supplemental Fig. 1). Direct interactions between transmembrane proteins and cytoskeleton are suggested to creates a barrier, called “fence,” where cytosolic parts of transmembrane proteins collides with cytoskeletal components, limiting their diffusion to certain areas. These molecules can jump over the “fence” to a neighboring compartment, possibly due to the dynamic nature of the interaction of membrane proteins and cytoskeleton, where they are again temporally trapped (17). This phenomenon was recently described also in A. thaliana where the interplay between membrane microdomains and microtubules plays a role in secondary cell wall formation (reviewed in (18)). The second model assumes, additionally, that particular transmembrane proteins are anchored to and lined up along cytoskeleton and act as “pickets” to arrest free diffusion of other membrane components, including nontransmembrane proteins, within the enclosed compartment (19).For plants, the composition of these sterol-rich membranes phases was analyzed in several biochemical studies (14, 20–22). Thereby, low-density preparations of plasma membrane fractions after treatment with nonionic detergents (DRM¹ fractions) were considered as a biochemical representation enriched in cellular membrane ordered phases or microdomains. Proteomic studies in mammalian cells consistently reported that the DRM fraction is highly enriched with several cytoskeletal proteins such as actin, tubulin, myosin, dynamin, actinin, and supervillin (23–25). Additionally, the level of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a lipid connecting the plasma membrane to actin filaments, was also significantly elevated in DRM preparations (26). Treatment with microtubule and actin depolymerizing agent results in drastic loss of many signaling proteins from these DRM fractions prepared from adult rat cardiac myocytes (27) or human embryonic retinal cells (28).Based on this knowledge, we propose two hypothetical models for the relationship between cytoskeleton and membrane microdomains for plant cells: (i) Actin filaments and microtubules could be important in the membrane phase separation or formation of the membrane microdomains themselves. In this case, disruption of the cytoskeleton would cause a lack of phase segregation in the plasma membrane. (ii) The cytoskeleton is only important for the incorporation of specific protein into the sterol-enriched regions but not for the general formation of these phase separations. This view implies that phase separations or membrane microdomains would still be present after cytoskeleton disruption but their protein composition can be different. Another possible scenario is (iii) that cytoskeletal elements serve as anchors for membrane microdomains at particular position in the plasma membrane, so the absence of these anchors would cause the increased mobility of microdomains (supplemental Fig. 1).The primary aim of this study was to characterize the interplay between cytoskeletal components and different membrane phases (microdomains) in A. thaliana suspension cell cultures. To reach this goal, biochemical and proteomic approaches were combined with confocal microscopy and activity assays measuring the influence of actin or tubulin disruption on the composition, localization, and biochemical properties of the sterol-enriched membrane microdomains. Thereby, for biochemical analyses, low-density detergent-resistant membrane fractions are analyzed as containing cellular sterol-rich membrane compartments.     

Glass‐Type Polyamorphism in Li‐Garnet Thin Film Solid State Battery Conductors

Ceramic Li₇La₃Zr₂O₁₂ garnet materials are promising candidates for the electrolytes in solid state batteries due to their high conductivity and structural stability. In this paper, the existence of “polyamorphism” leading to various glass‐type phases for Li‐garnet structure besides the known crystalline ceramic ones is demonstrated. A maximum in Li‐conductivity exists depending on a frozen thermodynamic glass state, as exemplified for thin film processing, for which the local near range order and bonding unit arrangement differ. Through processing temperature change, the crystallization and evolution through various amorphous and biphasic amorphous/crystalline phase states can be followed for constant Li‐total concentration up to fully crystalline nanostructures. These findings reveal that glass‐type thin film Li‐garnet conductors exist for which polyamorphism can be used to tune the Li‐conductivity being potential new solid state electrolyte phases to avoid Li‐dendrite formation (no grain boundaries) for future microbatteries and large‐scale solid state batteries.     

Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles

The comparisons of 170 sequences of kinetoplast DNA minicircle hypervariable region obtained from 19 stocks of Trypanosoma cruzi and 2 stocks of Trypanosoma cruzi marenkellei showed that only 56% exhibited a significant homology one with other sequences. These sequences could be grouped into homology classes showing no significant sequence similarity with any other homology group. The 44% remaining sequences thus corresponded to unique sequences in our data set. In the DTU I ("Discrete Typing Units") 51% of the sequences were unique. In contrast, in the DTU IId, 87.5% of sequences were distributed into three classes. The results obtained for T. cruzi marinkellei, showed that all sequences were unique, without any similarity between them and T. cruzi sequences. Analysis of palindromes in all sequence sets show high frequency of the EcoRI site. Analysis of repetitive sequences suggested a common ancestral origin of the kDNA. The editing mechanism that occurs in kinetoplastidae is discussed.