Slow and temperature-compensated autonomous disassembly of KaiB-KaiC complex.

KaiC is the central pacemaker of the circadian clock system in cyanobacteria and forms the core in the hetero-multimeric complexes, such as KaiB-KaiC and KaiA-KaiB-KaiC. Although the formation process and structure of the binary and ternary complexes have been studied extensively, their disassembly dynamics have remained elusive. In this study, we constructed an experimental system to directly measure the autonomous disassembly of the KaiB-KaiC complex under the condition where the dissociated KaiB cannot reassociate with KaiC. At 30°C, the dephosphorylated KaiB-KaiC complex disassembled with an apparent rate of 2.1±0.3 d-1, which was approximately twice the circadian frequency. Our present analysis using a series of KaiC mutants revealed that the apparent disassembly rate correlates with the frequency of the KaiC phosphorylation cycle in the presence of KaiA and KaiB and is robustly temperature-compensated with a Q 10 value of 1.05±0.20. The autonomous cancellation of the interactions stabilizing the KaiB-KaiC interface is one of the important phenomena that provide a link between the molecular-scale and system-scale properties.

Regulation mechanisms of the dual ATPase in KaiC.

KaiC is a dual adenosine triphosphatase (ATPase), with one active site in its N-terminal domain and another in its C-terminal domain, that drives the circadian clock system of cyanobacteria through sophisticated coordination of the two sites. To elucidate the coordination mechanism, we studied the contribution of the dual-ATPase activities in the ring-shaped KaiC hexamer and these structural bases for activation and inactivation. At the N-terminal active site, a lytic water molecule is sequestered between the N-terminal domains, and its reactivity to adenosine triphosphate (ATP) is controlled by the quaternary structure of the N-terminal ring. The C-terminal ATPase activity is regulated mostly by water-incorporating voids between the C-terminal domains, and the size of these voids is sensitive to phosphoryl modification of S431. The up-regulatory effect on the N-terminal ATPase activity inversely correlates with the affinity of KaiC for KaiB, a clock protein constitutes the circadian oscillator together with KaiC and KaiA, and the complete dissociation of KaiB from KaiC requires KaiA-assisted activation of the dual ATPase. Delicate interactions between the N-terminal and C-terminal rings make it possible for the components of the dual ATPase to work together, thereby driving the assembly and disassembly cycle of KaiA and KaiB.

Elucidation of master allostery essential for circadian clock oscillation in cyanobacteria.

Spatiotemporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor-transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and adenosine 5'-diphosphate release from its N-terminal domain during adenosine triphosphatase cycle. An engineered KaiC protein oscillator consisting of a minimal set of the identified master allosteric events exhibited a monophosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple posttranslational biochemical circadian oscillators.

Tree xylem water isotope analysis by Isotope Ratio Mass Spectrometry and laser spectrometry: A dataset to explore tree response to drought.

Water isotopes from plant xylem and surrounding environment are increasingly used in eco-hydrological studies. Carrière et al. [1] analyzed a dataset of water isotopes in (i) the xylem of three different tree species, (ii) the surrounding soil and drainage water and (iii) the underlying karst groundwater, to understand tree water uptake during drought in two different Mediterranean forests on karst setting. The xylem and soil water were extracted by cryogenic distillation. The full dataset was obtained with Isotope Ratio Mass Spectrometry (IRMS) and Isotope Ratio Infrared Spectrometer (IRIS), and included 219 measurements of δ2H and δ18O. Prompted by unexpected isotopic data characterized by a very negative deuterium excess, a subsample of 46 xylem samples and 9 soil water samples were double checked with both analytical techniques. IRMS and IRIS analyses yielded similar data. Therefore, the results reveal that laser spectrometry allows an accurate estimation of xylem and soil water isotopes. The dataset highlights a strong 2H depletion in xylem water for all species. Deuterium does not seem adequate to interpret ecological processes in this dataset given the strong fractionation.

The role of deep vadose zone water in tree transpiration during drought periods in karst settings - Insights from isotopic tracing and leaf water potential.

Karst environments are unusual because their dry, stony and shallow soils seem to be unfavorable to vegetation, and yet they are often covered with forests. How can trees survive in these environments? Where do they find the water that allows them to survive? This study uses midday and predawn water potentials and xylem water isotopes of branches to assess tree water status and the origin of transpired water. Monitoring was conducted during the summers of 2014 and 2015 in two dissimilar plots of Mediterranean forest located in karst environments. The results show that the three monitored tree species (Abies alba Mill, Fagus sylvatica L, and Quercus ilex L.) use deep water resources present in the karst vadose zone (unsaturated zone) more intensively during drier years. Quercus ilex, a species well- adapted to water stress, which grows at the drier site, uses the deep water resource very early in the summer season. Conversely, the two other species exploit the deep water resource only during severe drought. These results open up new perspectives to a better understanding of ecohydrological equilibrium and to improved water balance modeling in karst forest settings.

Identification of genes involved in the ACC-mediated control of root cell elongation in Arabidopsis thaliana.

BACKGROUND: Along the root axis of Arabidopsis thaliana, cells pass through different developmental stages. In the apical meristem repeated cycles of division increase the numbers of cells. Upon leaving the meristem, these cells pass the transition zone where they are physiologically and mechanically prepared to undergo subsequent rapid elongation. During the process of elongation epidermal cells increase their length by 300% in a couple of hours. When elongation ceases, the cells acquire their final size, shape and functions (in the differentiation zone). Ethylene administered as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is capable of inhibiting elongation in a concentration-dependent way. Using a microarray analysis, genes and/or processes involved in this elongation arrest are identified. RESULTS: Using a CATMA-microarray analysis performed on control and 3h ACC-treated roots, 240 differentially expressed genes were identified. Quantitative Real-Time RT-PCR analysis of the 10 most up and down regulated genes combined with literature search confirmed the accurateness of the analysis. This revealed that inhibition of cell elongation is, at least partly, caused by restricting the events that under normal growth conditions initiate elongation and by increasing the processes that normally stop cellular elongation at the end of the elongation/onset of differentiation zone. CONCLUSIONS: ACC interferes with cell elongation in the Arabidopsis thaliana roots by inhibiting cells from entering the elongation process and by immediately stimulating the formation of cross-links in cell wall components, diminishing the remaining elongation capacity. From the analysis of the differentially expressed genes, it becomes clear that many genes identified in this response, are also involved in several other kind of stress responses. This suggests that many responses originate from individual elicitors, but that somewhere in the downstream signaling cascade, these are converged to a 'common pathway'. Furthermore, several potential keyplayers, such as transcription factors and auxin-responsive genes, were identified by the microarray analysis. They await further analysis to reveal their exact role in the control of cell elongation.

Apoplastic alkalinization is instrumental for the inhibition of cell elongation in the Arabidopsis root by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid.

In Arabidopsis (Arabidopsis thaliana; Columbia-0) roots, the so-called zone of cell elongation comprises two clearly different domains: the transition zone, a postmeristematic region (approximately 200-450 µm proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the adjacent proximal region (450 µm away from the root tip up to the first root hair) with a high rate of elongation. In this study, the surface pH was measured in both zones using the microelectrode ion flux estimation technique. The surface pH is highest in the apical part of the transition zone and is lowest at the basal part of the fast elongation zone. Fast cell elongation is inhibited within minutes by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid; concomitantly, apoplastic alkalinization occurs in the affected root zone. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, can partially rescue this inhibition of cell elongation, whereas the inhibitor N,N'-dicyclohexylcarbodiimide does not further reduce the maximal cell length. Microelectrode ion flux estimation experiments with auxin mutants lead to the final conclusion that control of the activity state of plasma membrane H(+)-ATPases is one of the mechanisms by which ethylene, via auxin, affects the final cell length in the root.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Universal tree structures in directed polymers and models of evolving populations.

By measuring or calculating coalescence times for several models of coalescence or evolution, with and without selection, we show that the ratios of these coalescence times become universal in the large size limit and we identify a few universality classes.

Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia.

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.