Protocol: optimised electrophyiological analysis of intact guard cells from arabidopsis

Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell. We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves. These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels of intact stomatal guard cells to that achieved previously in work with Vicia and tobacco guard cells

Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth

Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K+ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K+ conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation

Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation

Differential metabolism of deoxyribonucleosides by leukaemic T cells of immature and mature phenotype

Experimental evidence has indicated that T lymphoblasts are more sensitive to deoxynucleoside toxicity than are B lymphoblasts. These data have led to the use of purine enzyme inhibitors as selective chemotherapeutic drugs in the treatment of T cell malignancies ranging from T cell acute lymphoblastic leukaemia to cutaneous T cell lymphomas. We have compared the toxicities of 2′-deoxyadenosine, 2′-deoxyguanosine, and thymidine for T cell lines derived from patients with T cell acute lymphoblastic leukaemia with those for mature T cell lines derived from patients with cutaneous T cell leukaemia/lymphoma. We have found that both deoxynucleosides are far less toxic to the mature T cell lies than to T lymphoblasts and that the mature cells accumulate much lower amounts of dATP and dGTP when exposed to deoxyadenosine and deoxyguanosine, respectively. Similar studies performed on peripheral blood cells from patients with T cell leukaemias of mature phenotype and on peripheral blood T cells demonstrate similar low amounts of deoxynucleotide accumulation. Measurements of the activities of several purine metabolizing enzymes that participate in deoxynucleoside phosphorylation or degradation do not reveal differences which would explain the toxicity of deoxynucleosides for immature, as compared to mature, T cells. We conclude that deoxynucleoside metabolism in leukaemic T cells varies with their degree of differentiation. These observations may be relevant to the design of chemotherapeutic regimes for T cell malignancies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72479/1/j.1365-2141.1985.tb04067.x.pd

Effect of screening of the electron-phonon interaction on the temperature of Bose-Einstein condensation of intersite bipolarons

Here we consider an interacting electron-phonon system within the framework of extended Holstein-Hubbard model at strong enough electron-phonon interaction limit in which (bi)polarons are the essential quasiparticles of the system. It is assumed that the electron-phonon interaction is screened and its potential has Yukawa-type analytical form. An effect of screening of the electron-phonon interaction on the temperature of Bose-Einstein condensation of the intersite bipolarons is studied for the first time. It is revealed that the temperature of Bose-Einstein condensation of intersite bipolarons is higher in the system with the more screened electron-phonon interaction.Comment: 6 pages, 4 figure

Unitarity and Interfering Resonances in pipi Scattering and in Pion Production piN->pipiN

Additivity of Breit-Wigner phases has been proposed to describe interfering resonances in partial waves in $\pi\pi$ scattering. This assumption leads to an expression for partial wave amplitudes that involves products of Breit-Wigner amplitudes. We show that this expression is equivalent to a coherent sum of Breit-Wigner amplitudes with specific complex coefficients which depend on the resonance parameters of all contributing resonances. We use analyticity of $\pi\pi$ partial wave amplitudes to show that they must have the form of a coherent sum of Breit-Wigner amplitudes with complex coefficients and a complex coherent background. The assumption of additivity of Breit-Wigner phases restricts the partial waves to analytical functions with very specific form of residues of Breit-Wigner poles. We argue that the general form provided by the analyticity is more appropriate in fits to data to determine resonance parameters. The partial wave unitarity can be imposed using the modern methods of constrained optimization. We discuss unitarity and the production amplitudes in $\pi N\to\pi\pi N$ and use analyticity in the dipion mass variable to justify the common practice of writing the production amplitudes as a coherent sum of Breit-Wigner amplitudes with free complex coefficients and a complex coherent background in fits to mass spectra with interfering resonances.Comment: 31 page

Clustering Algorithms: Their Application to Gene Expression Data

Gene expression data hide vital information required to understand the biological process that takes place in a particular organism in relation to its environment. Deciphering the hidden patterns in gene expression data proffers a prodigious preference to strengthen the understanding of functional genomics. The complexity of biological networks and the volume of genes present increase the challenges of comprehending and interpretation of the resulting mass of data, which consists of millions of measurements; these data also inhibit vagueness, imprecision, and noise. Therefore, the use of clustering techniques is a first step toward addressing these challenges, which is essential in the data mining process to reveal natural structures and iden-tify interesting patterns in the underlying data. The clustering of gene expression data has been proven to be useful in making known the natural structure inherent in gene expression data, understanding gene functions, cellular processes, and subtypes of cells, mining useful information from noisy data, and understanding gene regulation. The other benefit of clustering gene expression data is the identification of homology, which is very important in vaccine design. This review examines the various clustering algorithms applicable to the gene expression data in order to discover and provide useful knowledge of the appropriate clustering technique that will guarantee stability and high degree of accuracy in its analysis procedure

SNAREs-molecular governors in signalling and development

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive membrane fusion and contribute to membrane and protein targeting and delivery in all eukaryotic cells. SNAREs are essential to the mechanics of cell growth and development, and they facilitate a number of homeostatic and evoked responses in plants, from hormone signalling to pathogen defence. Additionally, there is now unambiguous evidence that SNAREs play roles in anchoring other membrane proteins and in facilitating ion channel gating through direct, physical interaction with channel proteins. What is the physiological significance of these additional features of plant SNAREs? We explore possible interpretations and suggest functions as scaffolds for effective signal transmission between proteins and, by analogy with a mechanical device invented by James Watt, as molecular governors to coordinate solute transport with cell expansion and growt

A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC)

Gene expression and binary interaction techniques are vital tools that shape our understanding of protein complexes. An inherent flaw, however, with most current protein-protein interaction techniques is the variability in expression levels for fusion proteins when using several individual plasmids. Here, we describe a novel recombination-based cloning strategy called 2in1 that enables co-expression of fusion proteins on a cell-by-cell basis from a single plasmid. We demonstrate the utility of 2in1 through the development of a ratiometric bimolecular fluorescence complementation assay (rBiFC), in which both candidate genes are simultaneously cloned into a single vector backbone containing an internal fluorescent marker for expression control and ratiometric analysis. rBiFC significantly increases the credibility of protein-protein interaction results allowing ratiometric comparison between different protein pairs. In addition to its use in rBiFC, 2in1 can be introduced easily into other vector systems that rely on multiple gene expression and prove feasible in future synthetic biological approaches