Water status of drought-resistant and drought-sensitive sorghum treated with ethephon

The effects of ethephon on stomatal resistance, water potential, osmotic potential, turgor potential, and ethylene production were determined on leaves of a drought-resistant (KS 65) and a drought-sensitive (IA 25) genotype of sorghum [Sorghum bicolor (L.) Moench] grown under wellwatered or drought-stressed conditions. With both sufficient and limited water supply, ethephon had no effect on the adaxial, abaxial, or total stomatal resistance of either genotype. For both water treatments, the adaxial stomatal resistance of the drought-sensitive genotype was higher than that of the drought-resistant genotype. Ethephon increased the amount of ethylene produced by the plants under both levels of water. For plants with sufficient water, water potentials of both genotypes were lowered by ethephon. Ethephon had no effect on the water potentials under drought or on the osmotic potentials under either water regime. With drought, the turgor potential of the drought-sensitive genotype, but not that of the drought-resistant, was increased by ethephon.     

Leaf water potential of differentially salinized plants

Water and osmotic potential energies were measured with thermocouple psychrometers, at intervals during a 4-week period, in growing leaves of bean (Phaseolus vulgaris, var. Blue Lake) and barley (Hordeum vulgare, var. Liberty) plants having roots equally split between 2 differentially salinized nutrient solutions. The osmotic potentials of plants with half their roots in saline solutions were about halfway between the osmotic potentials of plants grown in nonsaline solutions and those grown in saline solutions. By the end of the 4-week measurement period, the beans and barley were almost mature. The final dry weights of shoots of plants with half their roots in saline solutions were about halfway between the dry weights of the shoots of plants grown in nonsaline solutions and the dry weights of those in saline solutions. The results obtained showed that the degree of osmotic adjustment and the rate of growth were functions of the proportion of the root system exposed to saline conditions.     

Enhanced Growth and Activity of a Biocontrol Bacterium Genetically Engineered To Utilize Salicylate

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 μg/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 × 10⁵ CFU/g to approximately 3 × 10⁸ CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 × 10⁶ CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 μg of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10⁸ CFU/g of soil by 48 h of incubation, with the fastest increase at 100 μg/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 μg/g; at 1,000 μg/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 μg of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.     

Water potential gradients of tall and short cultivars of winter wheat

Summary Water and osmotic potentials were measured with thermocouple psychrometers, weekly after heading, two times each day at pre-dawn and at noon, in flag leaves and grain of tall and short cultivars of winter wheat grown in the field under rain-fed conditions.Water was held with less tension in the grain than in the leaf for both tall and short cultivars. The tall cultivars had lower leaf water potentials, but higher grain water potentials, than the short cultivars. The grain osmotic potential was lower in the short cultivars compared to the tall ones. Grain yield of short cultivars (1810 kg/ha) was more than that of tall cultivars (1730 kg/ha). Apparently higher leaf water potentials of short cultivars enabled more photosynthates to move into the grain.     

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.     

Patterns of liana community diversity and structure in a tropical rainforest reserve,Ghana: effects of human disturbance

Most studies have concluded that liana diversity and structure increase with disturbance. However, a contradictory pattern has emerged recently calling for more research in the area. Liana diversity and structure were investigated in three forest types that differ in disturbance intensity (nondisturbed, moderately disturbed and heavily disturbed forest: NDF, MDF and HDF, respectively) in the Atewa Range Forest Reserve, Ghana. In each forest type, 10 square plots of 0.25 ha were demarcated. Lianas with diameter ≥1 cm located on trees with diameter ≥10 cm were enumerated. A total of 429 individuals representing 40 species, 29 genera and seventeen families were identified in the study. Shannon diversity and species richness of lianas were significantly lower in the HDF (P < 0.05). Liana density and basal area differed significantly across all forest types (P < 0.0001). The importance value index (IVI) of most liana species varied greatly across the forest types. The current study has provided evidence to support the pattern of decreasing liana diversity and structure with disturbance in some tropical forests. Further studies are recommended to gain more understanding of the factors that are responsible for the divergent liana responses to disturbance in tropical forests.     

Targeting Photoreceptors via Intravitreal Delivery Using Novel,Capsid-Mutated AAV Vectors

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.