Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.     

Modeling yeast spoilage in cold-filled ready-to-drink beverages with Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica

Mathematical models were developed to predict the probability of yeast spoilage of cold-filled ready-to-drink beverages as a function of beverage formulation. A Box-Behnken experimental design included five variables, each at three levels: pH (2.8, 3.3, and 3.8), titratable acidity (0.20, 0.40, and 0.60%), sugar content (8.0, 12.0, and 16.0 degrees Brix), sodium benzoate concentration (100, 225, and 350 ppm), and potassium sorbate concentration (100, 225, and 350 ppm). Duplicate samples were inoculated with a yeast cocktail (100 microl/50 ml) consisting of equal proportions of Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica (approximately 5.0 x 10(4) CFU/ml each). The inoculated samples were plated on malt extract agar after 0, 1, 2, 4, 6, and 8 weeks. Logistic regression was used to create the predictive models. The pH and sodium benzoate and potassium sorbate concentrations were found to be significant factors controlling the probability of yeast growth. Interaction terms for pH and each preservative were also significant in the predictive model. Neither the titratable acidity nor the sugar content of the model beverages was a significant predictor of yeast growth in the ranges tested.     

Exocytosis in plants

Exocytosis is the final event in the secretory pathway and requires the fusion of the secretory vesicle membrane with the plasma membrane. It results in the release to the outside of vesicle cargo from the cell interior and also the delivery of vesicle membrane and proteins to the plasma membrane. An electrophysiological assay that measures changes in membrane capacitance has recently been used to monitor exocytosis in plants. This complements information derived from earlier light and electron microscope studies, and allows both transient and irreversible fusion of single exocytotic vesicles to be followed with high resolution in protoplasts. It also provides a tool to investigate bulk exocytotic activity in single protoplasts under the influence of cytoplasmic modulators. This research highlights the role of intracellular Ca²⁺, GTP and pressure in the control of exocytosis in plants.In parallel to these functional studies, plant proteins with the potential to regulate exocytosis are being identified by molecular analysis. In this review we describe these electrophysiological and molecular advances, and emphasise the need for parallel biochemical work to provide a complete picture of the mechanisms controlling vesicle fusion at the plasma membrane of plant cells.     

The development of ISSR-derived SCAR markers around the<Emphasis Type="Italic"> SEASONAL FLOWERING LOCUS</Emphasis> (<Emphasis Type="Italic">SFL</Emphasis>) in<Emphasis Type="Italic"> Fragaria vesca</Emphasis>

Fragaria vesca is a short-lived perennial with a seasonal-flowering habit. Seasonality of flowering is widespread in the Rosaceae and is also found in the majority of temperate polycarpic perennials. Genetic analysis has shown that seasonal flowering is controlled by a single gene in F. vesca, the SEASONAL FLOWERING LOCUS (SFL). Here, we report progress towards the marker-assisted selection and positional cloning of SFL, in which three ISSR markers linked to SFL were converted to locus-specific sequence-characterized amplified region (SCAR1–SCAR3) markers to allow large-scale screening of mapping progenies. We believe this is the first study describing the development of SCAR markers from ISSR profiles. The work also provides useful insight into the nature of polymorphisms generated by the ISSR marker system. Our results indicate that the ISSR polymorphisms originally detected were probably caused by point mutations in the positions targeted by primer anchors (causing differential PCR failure), by indels within the amplicon (leading to variation in amplicon size) and by internal sequence differences (leading to variation in DNA folding and so in band mobility). The cause of the original ISSR polymorphism was important in the selection of appropriate strategies for SCAR-marker development. The SCAR markers produced were mapped using a F. vesca f. vesca × F. vesca f. semperflorens testcross population. Marker SCAR2 was inseparable from the SFL, whereas SCAR1 mapped 3.0 cM to the north of the gene and SCAR3 1.7 cM to its south.Communicated by H. Nybom     

Issues in high-throughput comparative modelling: a case study using the ubiquitin E2 conjugating enzymes

Sequences of the ubiquitin-conjugating enzyme (UBC or E2) family were used as a test set to investigate issues associated with the high-throughput comparative modelling of protein structures. A semi-automatic method was initially developed with particular emphasis on producing models of a quality suitable for structural comparison. Structural and sequence features of the E2 family were used to improve the sequence alignment and the quality of the structural templates. Initially, failure to correct for subtle structural inconsistencies between templates lead to problems in the comparative analysis of the UBC electrostatic potentials. Modelling of known UBC structures using Modeller 4.0 showed that multiple templates produced, on average, no better models than the use of just one template, as judged by the root-mean-squared deviation between the comparative model and crystal structure backbones. Using four different quality-checking methods, for a given target sequence, it was not possible to distinguish the model most similar to the experimental structure. The UBC models were thus finally modelled using only the crystal structure template with the highest sequence identity to the target to be modelled, and producing only one model solution. Quality checking was used to reject models with obvious structural anomalies (e.g., bad side-chain packing). The resulting models have been used for a comparison of UBC structural features and of their electrostatic potentials. The work was extended through the development of a fully automated pipeline that identifies E2 sequences in the sequence databases, aligns and models them, and calculates the associated electrostatic potential.     

Contribution of gastrin-releasing peptide and its receptor to villus development in the murine and human gastrointestinal tract

Recent studies have shown that aberrantly expressed gastrin-releasing peptide (GRP) and its receptor (GRP-R) critically regulate tumor cell differentiation in colon cancers developing in humans and mice. This finding suggested that the ability of GRP/GRP-R to promote a well-differentiated phenotype in colon cancer might reflect a re-capitulation of a normal role in regulating intestinal organogenesis. To determine if this was the case, we compared and contrasted intestinal development in GRPR-/- mice with their wild type littermates. GRP/GRP-R co-expression in wild type mice was only observed in villous enterocytes between N-1 and N-12. During this time frame villous growth was completely attenuated in GRPR-/- mice. The contribution of GRP/GRP-R to villous growth was due to their act in increasing enterocyte proliferation prior to N-8 but increasing enterocyte size thereafter. From N-12 onwards, small intestinal villous growth in GRPR-/- mice resumed such that no difference in this structure could be detected at adulthood between mice of either genotype. We next studied GRP/GRP-R expression in human abortuses. These proteins were co-expressed by villous enterocytes only between weeks 14 and 20 post-conception, a time frame analogous to when they are expressed in the murine intestine. Thus, this study shows for the first time that GRP/GRP-R play a transient and non-critical role in intestinal development, yet provides a rationale for their re-appearance in colon cancer.