Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P_f) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield P_f.     

Exome Sequencing Identifies a Founder Frameshift Mutation in an Alternative Exon of USH1C as the Cause of Autosomal Recessive Retinitis Pigmentosa with Late-Onset Hearing Loss

We used a combined approach of homozygosity mapping and whole exome sequencing (WES) to search for the genetic cause of autosomal recessive retinitis pigmentosa (arRP) in families of Yemenite Jewish origin. Homozygosity mapping of two arRP Yemenite Jewish families revealed a few homozygous regions. A subsequent WES analysis of the two index cases revealed a shared homozygous novel nucleotide deletion (c.1220delG) leading to a frameshift (p.Gly407Glufs*56) in an alternative exon (#15) of USH1C. Screening of additional Yemenite Jewish patients revealed a total of 16 homozygous RP patients (with a carrier frequency of 0.008 in controls). Funduscopic and electroretinography findings were within the spectrum of typical RP. While other USH1C mutations usually cause Usher type I (including RP, vestibular dysfunction and congenital deafness), audiometric screening of 10 patients who are homozygous for c.1220delG revealed that patients under 40 years of age had normal hearing while older patients showed mild to severe high tone sensorineural hearing loss. This is the first report of a mutation in a known USH1 gene that causes late onset rather than congenital sensorineural hearing loss. The c.1220delG mutation of USH1C accounts for 23% of RP among Yemenite Jewish patients in our cohort.     

Comparative morphology and cytology of the eye,with particular reference to the retina,in lizardfishes (Synodontidae,Teleostei)

Fishelson, L., Delarea, Y. and Goren, M. 2012. Comparative morphology and cytology of the eye, with particular reference to the retina, in lizardfishes (Synodontidae, Teleostei). —Acta Zoologica (Stockholm) 93 : 68–79. The retinas of nine species of lizardfishes (Synodontidae) are composed of double cones, single cones, and rods. The cones are 16–28 μm long, and their number in the fundus of adult Synodus variegatus reaches ca. 32,900 mm² (varying from ca. 300,000 to ca. 390,000 in a 10 mm² of the retina), while in Saurida spp., they number ca. 12,000–14,000/mm². The cone ellipsoids are with up to 600 mitochondria, 0.5–1.6 μm in diameter. The rods are 30–50 μm long; their outer segments 0.6–1.2 μm thick and 15–18 μm long; their inner segments elongated. Their number varies from 15 to 128 million/retina. In fish of similar dimensions but of different species, the number of visual cells in the retina differs. In all species, the eyes increase from 2.0 mm in diameter in the smallest fish studied to 12 mm in the largest one. With eye growth, the retina in the various species increases from ca. 3.8 mm² in the smallest fish to ca.160.0 mm² in the large Saurida macrolepis. The possible ecological aspects of the observed phenomena are discussed.     

Influence of Growth Phase on Adhesion Kinetics of Escherichia coli D21g

The influence of bacterial growth stage and the evolution of surface macromolecules on cell adhesion have been examined by using a mutant of Escherichia coli K-12. To better understand the adhesion kinetics of bacteria in the mid-exponential and stationary growth phases under flow conditions, deposition experiments were conducted in a well-controlled radial stagnation point flow (RSPF) system. Complementary cell characterization techniques were conducted in combination with the RSPF experiments to evaluate the hydrophobicity, electrophoretic mobility, size, and titratable surface charge of the cells in the two growth phases considered. It was observed that cells in stationary phase were notably more adhesive than those in mid-exponential phase. This behavior is attributed to the high degree of local charge heterogeneity on the outer membranes of stationary-phase cells, which results in decreased electrostatic repulsion between the cells and a quartz surface. The mid-exponential-phase cells, on the other hand, have a more uniform charge distribution on the outer membrane, resulting in greater electrostatic repulsion and, subsequently, less adhesion. Our results suggest that the macromolecules responsible for this phenomenon are outer membrane-bound proteins and lipopolysaccharide-associated functional groups.     

Imaging the assembly and disassembly kinetics of cis-SNARE complexes on native plasma membranes

Mild sonication of eukaryotic cells produces native plasma membrane sheets that retain their docked organelles, cytoskeleton structures and cytoplasmic complexes. While the delicate organization of membranous protein complexes remains undisturbed, their inner plasmalemmel leaflet can be rapidly exposed to bathing solutions, enabling specific biochemical manipulations. Here, we apply this system to track membrane-biochemistry kinetics. We monitor soluble NSF-attachment protein receptor (SNARE) complex assembly and disassembly on the plasma membrane at high time resolution. The results suggest two-phase kinetics for the assembly process and dependence of the disassembly kinetics on both N-ethyl maleimide-sensitive factor (NSF) and soluble NSF-attachment protein (alpha-SNAP) concentrations.     

A combination of stomata deregulation and a distinctive modulation of amino acid metabolism are associated with enhanced tolerance of wheat varieties to transient drought

Introduction

Mediterranean winter crops are commonly and increasingly exposed to irregular rainfall and high temperatures, which lead to transient drought events of different degrees, adversely affecting growth and yield. Hence, exploring the diverse degrees of tolerance to drought existing in the crop and the molecular strategies behind it is pivotal for the development of ad hoc breeding programs.

Objective

We investigated the physiological and metabolic response of six commercial wheat cultivars to transient water stress at the tillering and grain-filling stages.

Methods

Drought experiments in lysimeters were set up at two developmental stages including six wheat cultivars. Newly expanded youngest leaves and flag leaves were sampled during the drought and following recovery. Metabolite profiles were generated using a GC–MS based protocol. Data on transpiration were continually acquired by measuring the weight variation of pots using electronic temperature compensated load cells.

Results

The tillering stage in wheat is more sensitive to droughts than the grain filling stage. The former stage was characterized by pronounced metabolic alterations also during recovery from the drought, and plants exhibited reduced transpiration. Notably, cultivars varied considerably in their susceptibility to drought. Exceptionally only in cv Zahir was transpiration not reduced at tillering. During recovery, the transpiration rate of Yuval and Zahir was not significantly affected, while except Ruta the other varieties maintained lower values. At grain-filling, a moderate decrease in transpiration in response to drought was evident in Bar-Nir, Yuval and Zahir varieties as compared with the stronger response of Gedera, Galil and Ruta. The transpiration trend during recovery remained lower than the control plants, particularly in Gedera and Zahir, while it reached higher values than control plants in Yuval and Ruta varieties. Metabolite profiling of leaves across cultivars showed varietal specific trends of response. Particularly during tillering, amino acid metabolism was differentially regulated across cultivars. For instance, Ruta and Zahir exhibited major changes in central carbon nitrogen metabolism during stress response, accumulating large amounts of proline and threonine during tillering, while in Bar-Nir a general decrease in relative amino acid content was noted. Changes in stress related GABA were common to Galil, Ruta, Yuval and Zahir. Desiccation related raffinose family oligosaccharides were mostly associated with a later stage of grain-filling and recovery stages of response.

Conclusion

The results indicate the occurrence of stage-dependent metabolic diversification along with a physiological response during transient droughts among wheat cultivars. It can be concluded that the most tolerant cultivar was Zahir, where a combination of stomatal closure deregulation and a significant accumulation rate of stress-related metabolites were evident.

     

Ecosystem Management of Desertified Shrublands in Israel

The objectives of this study were to understand the ecological processes and possible management strategies in desertified shrublands. We hypothesized that biological production and diversity in desertified shrublands in the Negev in Israel are low due to water, soil, and nutrient leakage from the ecosystem. We designed a series of field experiments in order to examine (a) whether source–sink relationships exist between the crusted soil and the shrub patches, (b) whether resources (water, soil, and nutrients) leak from the system, and (c) whether management, which changes the landscape mosaic by introducing new sink patches that reduce leakage of resources, may increase productivity and diversity. The results indicate that the low number of shrub patches, which serve as sinks for resources, leads to water, soil, and nutrient leakage from the ecosystem. This leakage reduces ecosystem production and diversity. We found that artificially created pits, which act as sinks for resources, decrease leakage and increase biomass production and annual plant species diversity. Based on the experimental results, we developed conceptual models for shrubland desertification and ecosystem management. The models are based on a source–sink relationship between two patch types characteristic of shrublands. The models relate landscape productivity to the number of sink patches and suggest that, in cases where there are too few sinks, artificially created sink patches should be added. Management methods were developed to reduce resource leakage in the desertified shrubland of the Negev. Methods included construction of man-made pits in the landscape that add resource-enriched patches to the landscape. These patches are used to create parks consisting of clusters of trees integrated into a matrix of shrubs and herbaceous vegetation. The managed parks are used for recreational purposes and for rangeland. Received 8 July 1997; accepted 7 July 1998.