Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rale of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity.     

Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana , betacyanin accumulation was reduced when cell division was inhibited by treatment with various inhibitors of DNA synthesis or anti-microtubule drugs. Aphidicolin (APC), an inhibitor of DNA synthesis, reduced the incorporation of radioactivity from labeled tyrosine into betacyanin, but the incorporation of radioactivity from labeled 3,4-dihydroxyphenylalanine (DOPA) into betacyanin was not affected by similar treatments. Propyzamide, another anti-microtubule drug, reduced incorporation of radioactivity from tyrosine and DOPA into betacyanin. However, the rate of incorporation from DOPA was higher than that from tyrosine. The results suggest that inhibition of betacyanin accumulation in Phytolacca americana cells by APC and propyzamide is due to suppression of the reaction converting tyrosine to DOPA, which may be closely related to cell division.     

Phytohormonal changes in intact shoots of wheat and oilseed rape treated with the acylcyclohexanedione growth retardant prohexadione calcium

The effects of the acylcyclohexanedione-type growth retardant prohexadione calcium on seedling growth and endogenous levels of immunoreactive phytohormone-like substances in shoots of wheat ( Triticum aestivum L. cv. Kanzler) and oilseed rape ( Brassica napus L. ssp. napus cv. Lirajet) were studied. After treatment of seedlings with increasing retardant concentrations in hydroponics, plant height and fresh weight of shoots were reduced by up to 40%. Concomitantly, the amount of immunoreactive gibberellins decreased, on a fresh weight basis, when compared with levels in the shoots of control plants. In contrast, the levels of abscisic acid and dihydrozeatin riboside and isopentenyladenosine-type cytokinins were considerably elevated by the growth retardant. The content of 3-indoleacetic acid decreased slightly. These results suggest that, in addition to its effect on gibberellin content, prohexadione calcium also influences the levels of endogenous abscisic acid and cytokinins.     

Comparison of the chemical changes in the rhizosphere of the nickel hyperaccumulator Alyssum murale with the non-accumulator Raphanus sativus

Changes in pH and redox potential were studied in the rhizosphere soil of a nickel hyperaccumulator plant (Alyssum murale) and of a crop plant, radish (Raphanus sativus). Differences in rhizosphere pH and reducing activity were found between the lateral and the main roots of both species, but the pH changes in the rhizosphere were similar in both species. Changes in pH were associated with the relative uptakes of cations and anions; whether the concentrations of heavy metals in the growth medium did not have any effect on the rhizosphere pH. The source of nitrogen (ammonium or nitrate) was the major factor determining the pH of the rhizosphere of both species. The redox potential of the rhizosphere was influenced by both the N-source and the concentrations of heavy metals. When heavy metals were not present in the growth medium, and nitrate was the N-source, the reducing capacity of A. murale roots was enhanced. However, the reducing activity of A. murale was always smaller than that of radish. Therefore, the mechanism of metal solubilization by the hyperaccumulator plant does not involve either the reduction of pH in the rhizosphere or the release of reductants from roots. The acidification and reducing activity of the roots of A. murale was always smaller than that of R. sativus.     

Simultaneous extraction of soil phytoavailable cadmium,copper, and lead by chelating resin membrane

Cadmium, copper, and lead were extracted from suspensions of contaminated soils using metal chelating exchange resin membranes. Nine soils with widely varying properties and Cd, Cu and Pb levels were tested. Soil suspensions made up with 4 g in 40 mL deionized water were equilibrated with 5 cm² Ca-saturated Chelex exchange resin membrane which was retained inside a polypropylene bag and shaken at 150 rpm for 24 hrs. Resin membrane extractable Cd, Cu and Pb of the soils were correlated with Cd, Cu, and Pb uptake by young wheat seedlings grown in these soils and compared with soil Cd, Cu, and Pb extracted by 0.1 M HCl, 0.01 M CaCl₂, and 0.005 M Diethylenetriamine pentaacetic acid (DTPA). The amounts of Cd, Cu and Pb extracted by the Ca-saturated Chelex membrane from all tested soils correlated well with those absorbed by young wheat seedlings. The Ca-saturated Chelex membrane extractable Cd, Cu and Pb of the soil had the strongest correlation with plant uptake Cd, Cu and Pb among the extraction methods we tested. It was demonstrated that the Ca-saturated Chelex membrane extraction is an appropriate method in simultaneously estimating Cd, Cu and Pb phytoavailability of soil and is applicable to a wide range of soils.     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

Effect of snow removal on leaf water potential,soil moisture,leaf and soil nutrient status and leaf peroxidase activity of sugar maple

Effect of removal of snow cover in winter was investigated in an 80-year-old sugar maple (Acer saccharum Marsh.) stand in southern Quebec. We hypothesized that winter soil frost would induce some of the decline symptoms observed in sugar maple stands in southern Quebec in the early 1980's. Snow was continuously removed from around trees for a one week (partial removal) or for a four-month period (complete removal) during the 1990–1991 winter. Foliage and soils were sampled periodically during the summer of 1991. The complete snow removal treated trees showed decreased leaf water potential and increased peroxidase activity over most of the growing season. Foliar Ca was reduced in both snow removal treatments early in the growing season while foliar N was reduced in the complete snow removal trees late in the growing season. Soil NO ₃ ^– and K⁺ were elevated in both snow removal treatments at various times throughout the growing season. Prolonged soil frost in a sugar maple stand can induce lower leaf water potential, higher leaf peroxidase activity and early leaf senescence during the following growing season. Soil frost may have reduced nutrient uptake without affecting significantly the leaf nutrient status.     

The impact of wildfire on vesicular-arbuscular mycorrhizal fungi and their potential to influence the re-establishment of post-fire plant communities

Wildfires are a typical event in many Australian plant communities. Vesicular-arbuscular mycorrhizal (VAM) fungi are important for plant growth in many communities, especially on infertile soils, yet few studies have examined the impact of wildfire on the infectivity of VAM fungi. This study took the opportunity offered by a wildfire to compare the infectivity and abundance of spores of VAM fungi from: (i) pre-fire and post-fire sites, and (ii) post-fire burned and unburned sites. Pre-fire samples had been taken in May 1990 and mid-December 1990 as part of another study. A wildfire of moderate intensity burned the site in late December 1990. Post-fire samples were taken from burned and unburned areas immediately after the fire and 6 months after the fire. A bioassay was used to examine the infectivity of VAM fungi. The post-fire soil produced significantly less VAM infection than the pre-fire soil. However, no difference was observed between colonization of plant roots by VAM fungi in soil taken from post-fire burned and adjacent unburned plots. Soil samples taken 6 months after the fire produced significantly more VAM than corresponding soil samples taken one year earlier. Spore numbers were quantified be wet-sieving and decanting of 100-g, air-dried soil subsamples and microscopic examination. For the most abundant spore type, spore numbers were significantly lower immediately post-fire. However, no significant difference in spore numbers was observed between post-fire burned and unburned plots. Six months after the fire, spore numbers were the same as the corresponding samples taken 1 year earlier. All plants appearing in the burned site resprouted from underground organs. All post-fire plant species recorded to have mycorrhizal associations before the fire had the same associations after the fire, except for species of Conospermum (Proteaceae), which lacked internal vesicles in cortical cells in the post-fire samples.     

Vertical thin-layer photoreactor for controlled cultivation of cyanobacteria

Nostoc sp. was cultivated in an air-lift reactor with continuous recirculation of the head gas phase that aerated and agitated the cyanobacterial suspension at regulated flow rates. The supply of inorganic carbon for growth was coupled with pH control, in the range of 7.7 to 8.1, by intermittent sparging of CO₂-head gas mixtures. The formation of irregular bubbles with swirling motion at the photostage of the reactor promoted efficient CO₂ transference in dense populations of Nostoc sp. (1.1 g/l) when bubbling at flow rates of 10 l/min. Biomass productivity was almost six-fold higher in the photoreactor (16.4 mg/l.h) than in a conventional system (2.8 mg/l.h). The exponential growth phase of cultures in the photoreactor amounted to 60% of the total growth period.The authors are with the Laboratorio de Alimentos, Area Microbiologia, Facultad de Quimica Bioquimica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina