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.     

紫椴根际土壤肥力与外生菌根侵染关系

调查帽儿山老山生态站紫椴(Tilia amurensis Rupr.)外生菌根的侵染情况与根际土壤肥力指标,结合Pearson相关性分析与逐步回归分析的统计方法,分析紫椴外生菌根侵染率与根际土壤肥力指标的相关性。结果表明:紫椴外生菌根侵染率与碱性磷酸酶、碱解氮、有效磷、土壤水分含量呈极显著负相关,与脲酶呈极显著正相关,与速效钾呈显著正相关。逐步回归分析发现,影响紫椴外生菌根侵染率的主要土壤肥力因子是碱性磷酸酶和土壤水分含量。     

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.     

Effects of red pine (Pinus resinosa Ait.) mycorrhizoplane-associated actinomycetes onin vitro growth of ectomycorrhizal fungi

Mycorrhizoplane-associated actinomycetes were isolated using an enrichment technique from red pine (Pinus resinosa Ait.) roots of seedlings recently outplanted onto cleared northern hardwood sites in the Upper Peninsula of Michigan, USA. Interactions were assessedin vitro between actinomycete isolates and three commonly occurring ectomycorrhizal fungi (Laccaria bicolor (Maire) Orton,L. laccata (Scop.: Fr.) Berk. and Br., andThelephora terrestris Fr.). Most actinomycete isolates exerted a range of effects on the growth of the three fungus isolates during the four week test period, inhibiting some while stimulating others; several inhibited growth of all three fungus isolates. Mycorrhizoplane-associated actinomycetes show potential for use as coinoculants with selected ectomycorrhizal fungi to optimize the soil microflora for developing seedlings.     

Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity

Unlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine. Occasionally non-enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4. 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay. There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags. Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity. As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine. By using site-directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head-to-tail homodimer with two active sites acting in trans across the interface of the dimer. Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites. Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre-protein monomer.     

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 - JHB ₃ juvenile hormone III bisepoxide     

外生菌根菌对樟子松苗木生长的影响

以内蒙古大兴安林樟子松分布区较典型的6个乡土菌种与本实验室从法国引进的宿主范围较广的2个菌种及内蒙古地区分布广泛的土生空团菌为研究对象,采用固体和液体两种菌剂类型,对出苗30d左右的樟子松实生苗进行外生菌根菌接种试验,接种130d后观察菌根侵染情况及生长特性,同时对菌根化的樟子松苗木进行自然干旱胁迫,研究外生菌根菌接种对樟子松苗木生长及抗逆性的影响。结果显示:(1)5个乡土菌种和1个法国引进菌种均能够与樟子松实生苗共生菌根,菌根化苗木较对照的苗高增长10.5%～111.4%、地茎增长4.55%～92.8%、苗木干重增加6.8%～184.5%,且固体菌剂效果优于液体菌剂。(2)在干旱胁迫下,菌根化苗木叶绿素含量较对照增加5.82%～36.92%、净光合速率提高120.2%～217.03%,叶片推迟10.3～25h出现萎蔫,临界致死时间推迟13.8～38.2h。(3)接种菌种厚环乳牛肝菌和点柄乳牛肝菌的苗木菌根侵染率、苗木生长量、干旱胁迫下各项生理指标均显著优于其他菌种和对照。研究表明,樟子松实生苗菌根化对苗木生长有明显的促进作用,能够提高苗木对干旱胁迫的抵御能力;厚环乳牛肝菌和点柄乳牛肝菌是有潜在研究和应用价值的优良菌株,这两个菌株的固体菌剂可用于樟子松菌苗生产。     

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.     

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.     

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     

Rapid in vitro ectomycorrhizal infection onPinus densiflora roots byTricholoma matsutake

The root systems of 11-wk-oldPinus densiflora seedlings were inoculated with a hyphal suspension ofTricholoma matsutake and aseptically incubated for 4 wk in a forest soil without supplying exogenous carbohydrates. One week following inoculation, fungal hyphae had colonized the root surface and bound soil particles together establishing a root-substrate continuum. Fungal hyphae were visible within the main root cortex following clearing bleaching and staining. In the ensuing days, fungal colonization was observed within elongating lateral roots in which Hartig net formation was confirmed 4 wk after inoculation. This is the first report of rapid ectomycorrhizal infection ofP. densiflora seedings byT. matsutake.     

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.