Electron microprobe analysis of the different epithelial cells of toad urinary bladder

Summary The electrolyte composition of toad urinary bladder epithelial cells has been measured using the technique of electron microprobe analysis. Portions of hemi-bladders, which had been mounted in chambers and bathed with a variety of media, were layered with albumin solution on their mucosal surfaces and immediately shock-frozen in liquid propane at –180°C. From the frozen material 1–2m thick cryosections were cut and promptly freeze-dried for 12 hr at –80°C and 10^–6 Torr. Electron microprobe analysis using a scanning electron microscope, an energy dispersive X-ray detector, and a computer programme, to distinguish between characteristic and uncharacteristic radiations, allowed quantification of cellular ionic concentrations per kg tissue wet wt by comparison of the intensities of the emitted radiations from the cells and from the albumin layer. Granular, mitochondrial-rich, and basal cells, and the basal portions of goblet cells, showed a similar composition, being high in K (about 110mm/kg wet wt) and low in Na (about 13mm/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K, presumably reflecting the composition of the mucus within them. With Na-Ringer's as the mucosal medium, cells gained Na and lost K, when their serosal surfaces were exposed to ouabain, 10^–2 m. Replacement of mucosal Na by choline virtually prevented these ouabain-induced changes. Cellular ion contents were unchanged when Na in the serosal medium was replaced by choline. No differences in Na and K concentrations were detected between nuclei and cytoplasm. These results provide independent support for the hypothesis that the cellular Na transport pool in toad bladder epithelial cells derives exclusively from the mucosal medium and that no important recycling of Na occurs from the serosal medium to the cells.     

Miconazole: An effective antifungal agent for plant tissue culture

Miconazole at concentrations between 5 and 20 mgl^-1 inhibited hyphal growth and sporulation in a wide range of fungi commonly associated with plants. These fungi included 4 Oomycetes, 11 Deuteromycetes, 4 Ascomycetes and 4 common airborne contaminants of plant tissue culture. The phytotoxicity of 20 mgl^-1 miconazole was also tested against a wide range of in vitro plant cultures. Shoot cultures from 15 species showed either no response or a slight growth reduction in the presence of miconazole. Although the growth of shoot cultures in 2 other species was significantly reduced by miconazole, a positive growth rate was maintained. Callus and hairy root cultures from 5 species were more sensitive to miconazole than shoot cultures, although they also retained a positive growth rate in the presence of the antifungal agent. Reconstruction experiments demonstrated the effectiveness miconazole had for the rescue of plant material from in vitro cultures contaminated from fungi.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - EBI ergosterol biosynthetic inhibitor - IAA indole-3-acetic acid - MZ miconazole - NAA -naphthaleneacetic acid     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

Be more specific! Laser-assisted microdissection of plant cells

Laser-assisted microdissection (LAM) is a powerful tool for isolating specific tissues, cell types and even organelles from sectioned biological specimen in a manner conducive to the extraction of RNA, DNA or protein. LAM, which is an established technique in many areas of biology, has now been successfully adapted for use with plant tissues. Here, we provide an overview of the processes involved in conducting a successful LAM study in plants and review recent developments that have made this technique even more desirable. We also discuss how the technology might be exploited to answer some pertinent questions in plant biology.     

Conservation of Arabidopsis flowering genes in model legumes

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.     

Developing a method for customized induction of flowering

Background  

The ability to induce flowering on demand is of significant biotechnological interest. FT protein has been recently identified as an important component of the mobile flowering hormone, florigen, whose function is conserved across the plant kingdom. We therefore focused on manipulation of both endogenous and heterologous FT genes to develop a floral induction system where flowering would be inhibited until it was induced on demand. The concept was tested in the model plant Arabidopsis thaliana (Arabidopsis).