Management of polyamine pools and the regulation of ornithine decarboxylase

The management of polyamine synthesis and polyamine pools differs fundamentally from that of most other small molecular-weight endproducts. The polyamines are vital to growth and important cellular functions, but they are toxic in excess. I argue here that their multivalent cationic character, leading to binding to cell constituents, precludes fluent feedback inhibition of synthesis. This has led to the development of elaborate alternative regulatory mechanisms controlling ornithine decarboxylase, the key initial enzyme of the pathway. Poorly regulated polyamine synthesis and the toxicity of polyamines impose upon cells a need to control uptake and to dispose of excess polyamines. Recent data on polyamine transport suggest unorthodox mechanisms of accomplishing these functions.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Effect of nitrogen level on acid phosphatase activity of eight isolates of ectomycorrhizal fungus Paxillus involutus cultured in vitro

The higher levels of nitrogen in ammonium form stimulated the growth of mycelia and increased the accessible as well as the total acid phosphatase activity of Paxillus involutus (Batsch) Fr. isolates grown in pure culture. Rates of mycelia growth and acid phosphatase activities varied widely from one isolate to another. Scots pine (Pinus sylvestris L.) seedlings were inoculated with different P. involutus isolates in axenic conditions. Shoots of pine seedlings with mycorrhizae contained more phosphorus than shoots of non-mycorrhizal seedlings. The relations between growth and phosphatase activity of P. involutus isolates and their efficiency in supplying the host plant with phosphate are discussed.     

Nitrogen nutrition,photosynthesis and carbon allocation in ectomycorrhizal pine

Summary Studies examined net photosynthesis (Pn) and dry matter production of mycorrhizal and nonmycorrhizalPinus taeda at 6 intervals over a 10-month period. Pn rates of mycorrhizal plants were consistently greater than nonmycorrhizal plants, and at 10 months were 2.1-fold greater. Partitioning of current photosynthate was examined by pulse-labelling with¹⁴CO₂ at each of the six time intervals. Mycorrhizal plants assimilated more¹⁴CO₂, allocated a greater percentage of assimilated¹⁴C to the root systems, and lost a greater percentage of¹⁴C by root respiration than did nonmycorrhizal plants. At 10 months, the quantity of¹⁴CO₂ respired by roots per unit root weight was 3.6-fold greater by mycorrhizal than nonmycorrhizal plants. Although the stimulation of photosynthesis and translocation of current photosynthate to the root system by mycorrhiza formation was consistent with the source-sink concept of sink demand, foliar N and P concentrations were also greater in mycorrhizal plants.Further studies examined Pn and dry matter production ofPinus contorta in response to various combinations of N fertilization (3, 62, 248 ppm), irradiance and mycorrhizal fungi inoculation. At 16 weeks of age, 6 weeks following inoculation with eitherPisolithus tinctorius orSuillus granulatus, Pn rates and biomass were significantly greater in mycorrhizal than nonmycorrhizal plants. Mycorrhizal plants had significantly greater foliar %P, but not %N, than did nonmycorrhizal plants. Fertilization with 62 ppm N resulted in greater mycorrhiza formation than either 3 or 248 ppm. Increased irradiance resulted in increased mycorrhiza formation.     

Androgen and estrogen stimulation of ornithine decarboxylase activity in mouse kidney

In the present work, the activity of mouse renal ornithine decarboxylase (ODC) from CBA female mice was used as a biological marker to detect (anti)androgenic activity of different groups of endocrine disruptors and steroids. Daily injections of testosterone or dihydrotestosterone (DHT) into 60 day old female mice for 4 days increased renal ODC activity in a dose-dependent manner that reached up to 100-fold (testosterone) or 250-fold (DHT) above the baseline when the highest dose, 200 microg/mouse, was used. Administration of flutamide concurrently with testosterone (75 microg/mouse) caused a potent decrease of ODC induction in a dose-dependent manner, suppressing the enzyme activity at the doses of 0.1 and 0.5 mg/mouse by about 88 and 95%, respectively. In contrast, estradiol at the doses of 0.5 and 1 mg/mouse induced a significant stimulation of renal ODC activity in a dose-dependent manner when it was given alone or in combination with testosterone. Using a sensitive increase in ODC activity in response to androgens as an end point, we did not detect an antiandrogenic effect of several antiandrogens, such as cyproterone acetate, spironolactone, p,p'DDE and vinclozolin. Also, none of these antiandrogens were able to change the basal level of renal ODC activity, with the exception of cyproterone acetate that at a dose of 0.1 mg/mouse stimulated ODC activity. The data obtained suggest that mouse renal ODC from CBA females is not strictly androgen-specific and cannot be used for estimation of antiandrogenic effects of compounds having an affinity to different types of receptors.     

Juvenile hormone stimulation of ornithine decarboxylase activity during vitellogenesis in Drosophila melanogaster

Summary The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, becomes elevated in intact female Drosophila melanogaster shortly after adult eclosion. This activity reaches a peak at 24 h following eclosion, and then drops to lower levels by 48 h. This pattern is not observed in males, consistent with the hypothesis that polyamine synthesis is involved in ovarian maturation in Drosophila. Abdomens isolated within 2 h of adult eclosion do not display elevated ODC activity or ovarian maturation. However, a 250-ng dose of the juvenile hormone analog methoprene (ZR-515) applied in acetone to these abdomens, recovers ovarian maturation and causes a 5–10 fold increase in enzyme activity over controls treated with acetone alone. The same dose of the inactive precursor methyl farnesoate caused no such increase, whereas a 500-ng dose of the newly discovered natural Drosophila JHB₃ stimulated a four-fold response. The response to methoprene was dose-dependent, showing stimulatory activity at a dose as low as 10 ng. This stimulation by JHA is rapid, occurring between 1 and 3 h following hormone treatment, reminiscent of JH induction of fat body vitellogenin synthesis in Drosophila. Elevated ODC activity appeared to be localized in the adult fat body. During embryogenesis, ODC activity remained undetectable until just prior to hatching, when a large increase was detected. We postulate that JH may, either directly or indirectly, regulate polyamine biosynthesis in vivo, and that this synthesis may be required for the production of macromolecules during Drosophila vitellogenesis or embryogenesis.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - ODC ornithine decarboxylase - SAMDC S-adenosyl-methionine decarboxylase - JHB₃ juvenile hormone III bisepoxide     

Recovery from winter depression of photosynthesis in pine and spruce

Summary Recovery from winter depression of photosynthesis was studied in Pinus sylvestris, Pinus conforta and Picea abies by means of chlorophyll fluorescence and gas exchange measurements. During the winter 1986–1987 the fluorescence yield was low and no variable fluorescence was detectable before the end of March. In the field recovery of variable fluorescence/maximum fluorescence (F_v/F_m) during spring was slow for all three species studied. The temperature dependence of recovery was confirmed from measurements of the potential rate of recovery of F_v/F_m at different temperatures in the laboratory. At 20° C, F_v/F_m increased from 0.1 to 0.8 within 3 days. Recovery of F_v/F_m was paralleled by an increase in apparent photon yield. No significant differences could be demonstrated between the studied tree species in potential rate of recovery in the laboratory or in actual recovery in the field.     

Zinc toxicity in ectomycorrhizal Pinus sylvestris

Six strains of ectomycorrhizal fungi were compared for their ability to increase zinc tolerance in Pinus sylvestris L. seedlings. Pioneer and late-stage fungi as well as one strain collected from a Zn-polluted site were included in the study. The accumulation of zinc in the host plants was determined at two different sublethal substrate Zn concentrations. The mycobionts varied considerably in their protection of the autobionts against zinc toxicity. Several fungal species reduced zinc accumulation in the pine seedlings. A Thelephora terrestris (Ehrh.) Fr. Strain, however, increased the Zn concentration in its host plants. Specific zinc-retaining capacity of the mycelium and density of the extramatrical mycelium were important features determining the effectiveness of the zinc retention in the fungal symbiont.The experiments were carried out in a growth chamber where plants were cultivated in root observation chambers. The determination of the extent of substrate colonization showed that an elevated zinc concentration in a substrate might be an important stress factor for the growth of an ectomycorrhizal fungus. The growth of the extramatrical mycelium of some species was inhibited at the highest zinc concentration supplied. A determination of the in vitro zinc tolerance of the fungi could not always predict their tolerance as symbionts, where the latter characteristic did not determine the Zn retention capacity of their mycelium.In this paper we also discuss the possibility that on Zn-polluted soils ectomycorrhizal species and strains are selected that are Zn-tolerant and, in addition, are able to protect their own energy source, the autobiont, from toxicity. These mycorrhizal fungi act as a safety net, that can immobilize large amounts of zinc, thus preventing transport to the host plant.     

Inhibition of polyamine biosynthesis in Acanthamoeba castellanii and 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by chitosanoligosaccharide

Growth of Acanthamoeba castellaniiwas inhibited by chitosanoligosaccharide (up to 20 mg ml^–1) from the shells of crabs but was reversed by the polyamines, putrescine or spermidine, at 0.8 mM. Chitosanoligosaccharide strongly inhibited the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a key enzyme of polyamine biosynthesis, which is enhanced in tumour promotion.     

Motile tubular vacuoles in extramatrical mycelium and sheath hyphae of ectomycorrhizal systems

Summary Extramatrical mycelium and outer hyphae of the sheath ofEucalyptus pilularis-Pisolithus tinctorius mycorrhizas contain abundant motile tubular vacuoles which accumulate the carboxyfluorescein analogue Oregon Green 488 carboxylic acid. The fluorochrome accumulates in a system of small vacuoles, tubules, and larger vacuoles, which are interlinked, motile, and pleiomorphic, in external hyphae, cords, and hyphae of the outer sheath. There is often a difference in fluorescence between two neighbouring cells, indicating that the dolipore septum exercises control on the movement of material between cells. Generally the motile tubular vacuole system in mycorrhizas resembles that previously found in isolated mycelium. The majority of fungal cells in the sheath contain no fluorochrome even after long exposure of the mycorrhiza to the solution, but with differential interference optics the cells are clearly seen to be alive and to contain vacuoles resembling those in the outer hyphae. In translocation experiments, long-distance transport of the fluorochrome is slow and slight, or even nonexistent in some cases.Abbreviations carboxy-DFF Oregon Green 488 carboxylic acid - carboxy-DFFDA Oregon Green 488 carboxylic acid diacetate - DIC differential interference contrast Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Hyphal mats formed by two ectomycorrhizal fungi and their association with Douglas-fir seedlings: A case study

The ectomycorrhizal fungi Gautieria monticola and Hysterangium setchellii both form dense hyphal mats in coniferous forest soils of the Pacific Northwest. We recently observed that all Douglas-fir seedlings found under the canopy of a maturing 60–75 year stand were associated with mats formed by ectomycorrhizal fungi. The significance of these mat communities in relation to seedling establishment and survival is discussed.     

Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Putrescine carbamoyltransferase activity in pea is associated with the ornithine carbamoyltransferase polypeptide and is not involved in putrescine synthesis under physiological conditions

Putrescine carbamoyltransferase (PutCT) has been postulated to function in the synthesis of putrescine (Put) from an N-carbamoylputrescine (NCPut) intermediate in plants. In pea, PutCT activity was associated entirely with ornithine carbamoyltransferase (OCT) protein, which was purified to homogeneity using an immobilized transition-state analog inhibitor ( N-(phosphonacetyl)-L-ornithine). No evidence for a separate PutCT enzyme, similar to that in Streptococcus [15], or PutCT activity associated with a putrescine synthase-type multifunctional enzyme [13] was found. OCT carried out the carbamoylation of Put and other diamine and polyamine substrates inefficiently and at non-physiological pH (Put carbamoylation: pH 10.8 optimum, V_max 0.11 kat/mg protein, K_m=6.7 mM for Put and 1.0 mM for carbamoyl-P), when compared with ornithine carbamoylation (pH 8.5 optimum, V_max=313.9 kat/mg protein, K_m=4.4 mM for ornithine and 0.6 mM for carbamoyl-P). Different subcellular compartmentation of PutCT activity (chloroplast) and the NCPut substrate (cytosol), coupled with a thermodynamically-unfavorable reverse reaction (i.e., Put synthesis from NCPut), suggest that the OCT-associated PutCT activity does not significantly contribute to in vivo Put synthesis in plants.Abbreviations ADC Arginine Decarboxylase - Agm Agmatine - AIH Agmatine Iminohydrolase - Cad Cadaverine - Cit Citrulline - CP Carbamoyl-P - Dap 1,3-Diaminopropane - Dns- Dansyl- - NCPut N-Carbamoylputrescine - OCT Ornithine Carbamoyltransferase - Put Putrescine - PutCT Putrescine Carbamoyltransferase - Spd Spermidine - Spm Spermine     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

Multilocus structure in Pinus contorta Dougl.

We studied isozyme variation at 21 loci in 66 populations from three subspecies of Pinus contorta Dougl.; 35 in spp. latifolia, 20 in spp contorta and 11 in spp. murrayana. The objectives were to assess gametic disequilibria and multilocus structure. There was considerable differentiation of allele frequencies at 19 polymorphic loci across the 66 populations and within the subspecies. Allele frequencies at many loci correlated with geographic variables. Genetic variability varied considerably among populations within subspecies but the subspecies means were similar. The mean number of polymorphic loci and the mean heterozygosity over 19 polymorphic loci were, respectively, 13 and 0.194 in latifolia, 12 and 0.196 in murrayana, and 12 and 0.180 in contorta. The mean heterozygosity correlated with longitude and altitude across the 66 populations and with latitude in latifolia. Gametic disequilibria were evident in 40 populations; 29 in latifolia, eight in murrayana and three in contorta. Gametic disequilibria correlated with latitude across the 66 populations and with longitude in latifolia. The single-locus F _ST averaged 0.0339 in latifolia, 0.0567 in murrayana, and 0.0764 in contorta. The multilocus F _STM was 0.1227 in latifolia, 0.2926 in murrayana, and 0.3328 in contorta. Multilocus Wahlund and founder effects, migration patterns, and natural selection, probably played significant roles in generating and maintaining the multilocus genetic structure in P. contorta in general and the subspecies latifolia in particular.     

Effect of a synthetic pyrethroid on the growth of ectomycorrhizal fungi and mycorrhiza formation in Pinus patula

Th effect of fenvalerate 20% EC (a synthetic pyrethroid) on the growth of two ectomycorrhizal fungi, Thelephora terrestris and Laccaria laccata, was studied in vitro. In the case of T. terrestris, mycelial growth was promoted at lower concentrations of the insecticide but inhibited at higher concentrations; mycelial growth of L. laccata decreased with increase in concentration. The effect of the insecticide at three concentrations (0.25, 0.5, 1.5 ppm) was also studied on ectomycorrhizal formation in 4-month-old seedlings of Pinus patula in nursery trials. At lower concentrations the insecticide had no significant effect on mycorrhizal formation.     

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.     

Response of ectomycorrhizalPinus banksiana andPicea glauca to heavy metals in soil

Pinus banksiana andPicea glauca inoculated or not with the ectomycorrhizal fungusSuillus luteus were grown in a sandy loam soil containing a range of Cd, Cu, Ni, Pb and Zn concentrations. Ectomycorrhizal colonization rates were significantly reduced on Pinus and Picea seedlings by the heavy metals, particularly Cd and Ni. Needle tissue metal concentrations were lower in ectomycorrhizal seedlings at low soil metal concentrations. However, at higher soil concentrations, heavy metal concentrations of needle tissue were similar in ectomycorrhizal and nonmycorrhizal plants. The growth of nonmycorrhizal seedlings exposed to heavy metals was reduced compared to those inoculated withSuillus luteus. Apparently ectomycorrhizal colonization can protect Pinus and Picea seedlings from heavy metal toxicity at low or intermediate soil concentrations of Cd, Cu, Ni, Pb and Zn.     

Growth and survival of container-grownPinus caribaea infected with various ectomycorrhizal fungi

Summary Young pine seedlings were inoculated with mycelial cultures of 8 fungi, and basidiospore cultures of 4 fungi. Four months later distinctive mycorrhizas had been formed by all 4 spore cultures and by 5 of the mycelial cultures.At 10 months one further mycelial culture had given rise to distinctive mycorrhizas. However, by that time contamination between treatments, or from outside, was at a higher level.Survival of outplanted seedlings after 6 months was generally poor, but was slightly greater in some treatments.