Membrane flow in plants: preparation and kinetics of labelling of plasma membranes from growing pollen tubes of tobacco

Summary Growing pollen tubes of tobacco germinated in suspension culture, were labelled with [³H]leucine and after varying times of chase with unlabelled leucine at 23, 16, or 4°C, were separated into plasma membrane-enriched and plasma membrane-depleted fractions by aqueous two-phase partition. At 23°C, the specific radioactivity of the plasma membrane increased with time to a maximum at 60 min. At 16°C and 4°C, labelling of the plasma membrane was respectively 40% and 10% that at 23°C. However, if labelling was at 23°C and subsequent transfer was at 4°C, plasma membrane labelling was much less affected and labelling of the plasma membrane was 60% that at 23°C. Additionally, quantitation of various morphological parameters revealed no accumulations of 50–70 nm transition vesicles in the space between endoplasmic reticulum and cis Golgi apparatus that might suggest formation of a low temperature compartment similar to those described for mammalian cells and tissues. Similarly, growth of pollen tubes was reduced but not blocked even at temperatures of 12°C. The results suggest that tube elongation is accompanied by a steady state flow of membranes to the cell surface that is relatively insensitive to interruption by low temperatures. Whereas leucine incorporation is reduced by low temperature even at 16°C, the flow pathway to the cell surface, including the endoplasmic reticulum to Golgi apparatus transfer step, as well as elongation growth does not exhibit a pronounced low temperature block in this tip growing system.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Thermographic visualization of cell death in tobacco and Arabidopsis

Pending cell death was visualized by thermographic imaging in bacterio‐opsin transgenic tobacco plants. Cell death in these plants was characterized by a complex lesion phenotype. Isolated cell death lesions were preceded by a colocalized thermal effect, as previously observed at sites infected by tobacco mosaic virus (TMV) ( Chaerle et al. 1999 Nature Biotechnology 17, 813–816). However, in most cases, a coherent front of higher temperature, trailed by cell death, initiated at the leaf base and expanded over the leaf lamina. In contrast to the homogenous thermal front, cell death was first visible close to the veins, and subsequently appeared as discrete spots on the interveinal tissue, as cell death spread along the veins. Regions with visible cell death had a lower temperature because of water evaporation from damaged cells. In analogy with previous observations on the localized tobacco–TMV interaction ( Chaerle et al. 1999 ), the kinetics of thermographic and continuous gas exchange measurements indicated that stomatal closure preceded tissue collapse. Localized spontaneous cell death could also be presymptomatically visualized in the Arabidopsis lsd2 mutant.     

Focus: Addiction: Obese Smokers as a Potential Subpopulation of Risk in Tobacco Reduction Policy

Smoking and obesity represent the largest challenges to public health. There is an established inverse relationship between body mass index (BMI) and smoking, but this relationship becomes more complicated among obese smokers. Smokers with higher BMI consume more cigarettes per day and may be more nicotine-dependent than lean smokers. Rates of obesity are lower among smokers than non-smokers, indicating that chronic exposure to tobacco smoke may prevent excess weight gain in people who would otherwise become obese. Furthermore, obese smokers may be more sensitive to the weight-suppressive and reinforcing effects of nicotine. Consequently, obese smokers may respond differently to reduction in the nicotine content of cigarettes, a tobacco control policy being considered both in the Unites States and abroad. Here, we review the interrelationship between nicotine and obesity in the context of a potential nicotine reduction policy. We discuss the implications of nicotine-induced body weight suppression in obese smokers, as well as the possibility that obesity might increase susceptibility to smoking and nicotine dependence.     

Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

Summary A protoplast fusion experiment was carried out aiming to obtain somatic hybrid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S CaMV promotors. Hybrid selection was based on dual bialaphos— kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolio-like hybrids had nuclear DNA contents near that of N. tabacum (9.40±0.24pg) or N. rotundifolia (5.29±0.36 pg), respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels—N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8.90±0.30 to 19.57±0.33 pg. The data from the morphological and cytological analysis provided vidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.     

Hatch and Reproduction of Globodera tabacum tabacum in Response to Tobacco,Tomato, or Black Nightshade

The effects of broadleaf tobacco, tomato, and black nightshade on juvenile hatch and reproduction of Globodera tabacum tabacum were determined in laboratory and greenhouse experiments. Root exudates from nightshade stimulated greater egg hatch than those from either ''Rutgers'' tomato or ''86-4'' tobacco. Hatch was greater at higher proportions of root exudates for all three plant species. Root exudates from plants greater than 3 weeks old stimulated more hatch than younger plants. No regression relationships existed between plant age and nematode batch. In other experiments, hatch from eggs in cysts was higher for tomato and nightshade after 10 weeks in greenhouse pots compared to tobacco and bare soil. Numbers of second-stage juveniles in eggs in cysts produced from a previous generation on the same host were highest on nightshade and less on tomato and tobacco. Cysts of variable age recovered from field soil had increased hatch in both root exudates or water compared to recently produced cysts from plants in growth chambers. Globodera t. tabacum may be subject to both host and environmentally mediated diapause.     

Spin-lattice Relaxation in the Proton NMR of Hydrated Tobacco Cut-fillers

The spin-lattice relaxation time was measured by proton NMR of hydrated tobacco cut-fillers. The relaxation decays of adsorbed water were expressed by a single phase system below 70% relative humidity, while a two-phase system was applicable to water adsorbed at more than 80% relative humidity. From the two-phase model, it was considered that 0.12–0.13 kg water/kg dry tobacco is bound water.     

Evaluation of the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium cations by monoamine oxidase (MAO) enzymes and its use to search for new MAO inhibitors and protective agents

Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of amines and neurotransmitters and inhibitors of MAO are useful as neuroprotectants. This work evaluates the human MAO-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin, to the directly-acting neurotoxic metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenylpyridinium (MPP⁺) measured by High-Performance Liquid Chromatography (HPLC), and this approach is subsequently used as a new method for screening of MAO inhibitors and protective agents. Oxidation of MPTP by human MAO-B was more efficient than by MAO-A. R-Deprenyl, a known neuroprotectant, norharman (β-carboline), 5-nitroindazole and menadione (vitamin K3) inhibited MAO-B and reduced the formation of toxic pyridinium cations. Clorgyline and the β-carbolines, harman and norharman, inhibited the oxidation of MPTP by MAO-A. Cigarette smoke, as well as the naturally occurring β-carbolines (norharman and harman) isolated from smoke and coffee inhibited the oxidation of MPTP by MAO-B and/or MAO-A, suggesting protective effects against MPTP. The results show the suitability of the approach used to search for new MAO inhibitors with eventual neuroprotective activity.     

Ribonucleases in Nicotiana tabacum leaf extracts treated with phenol

When tobacco leaf extracts are treated with phenol, ca 20% of the ribonuclease (RNase) activity survives and can be measured when the phenol is removed. After purification, the resistant RNase is inactivated by phenol; this suggests that tobacco leaves contain material that protects the RNase. Phenol-resistant RNase may be one of the TMV-RNA inactivating systems present in phenol extracts of tobacco leaves.