Changes in root growth patterns of (Picea abies) spruce roots by inoculation with an ectomycorrhizal fungusPisolithus tinctorius and jasmonic acid treatment

Symbiosis between fungi and plant roots forming a mycorrhiza involves extensive interactions at the molecular level between both partners. The role of plant hormones in the regulation of mycorrhizal infection is not known to involve jasmonates. Their endogenous levels increase during pathogen attack; however, little has been done on their involvement in mycorrhizae. In our recent work, root growth patterns of 2-month-old spruce seedlings after inoculation withPisolithus tinctorius and/or jasmonic acid (JA) treatment were studied using a paper-sandwich technique. Changes in root length, the degree of branching, presence and length of root hairs, and infection parameters were followed using a stereomicroscope. The first mycorrhizal contact of hyphae with roots was significantly accelerated upon treatment with 0.5 M JA. Interactions between root hairs and fungal hyphae were seen by scanning electron microscopy. The multiplication of root hairs of non-mycorrhized seedlings treated with 5.0 M JA and changes of the root surface were observed by the same technique.     

Sunflower root growth regulation: the role of jasmonic acid and its relation with auxins

Jasmonates are lipid-derived hormones that act as signal molecules in abiotic and biotic stresses and influence several aspects of plant growth and development. In this work we have investigated the effect of jasmonic acid (JA) on the root architecture of Helianthus annuus seedlings and if JA and auxins interact to modulate the growth of the primary root (PR) and lateral roots (LR). The addition of μM concentrations of JA to the growing medium of sunflower seedlings decreased the growth of the PR and LR, and also reduced the number of LR. Moreover, treatment with ibuprofen, an inhibitor of JA synthesis, increased PR and LR root length causing a deep effect on root architecture. Hence, not only exogenous but also the endogenous JA regulates sunflower root growth. Microscopic analysis showed that the application of JA reduces the cortex cell length and the estimated cell production rate in root meristem while ibuprofen only affects the cell elongation. A possible interaction between JA and auxins to regulate root growth was further analyzed. We show that JA produced its phenotype even in the presence of reduced levels of auxin generated by treatment with an auxin transport inhibitor. Besides, the auxin produced its phenotype even when ibuprofen was applied. In conclusion, JA may induce primary and lateral root growth inhibition in sunflower by an auxin-independent pathway.     

Stimulatory effects of jasmonic acid on potato stem node and protoplast culture

The effect of jasmonic acid (JA), in a wide range of concentrations (0.01–100 M), on the development of potato plantlets (Solanum tuberosum L.cv. Vesna) was investigated in order to discriminate between physiological and supraoptimal effects of this growth regulator in vitro. Concentrations from 0.1–1 M significantly increased the length of developed plants. Application of JA in these concentrations resulted in a very well differentiated root system with many lateral branches. With increasing JA concentrations, the main roots shortened and thickened. Concentrations higher than 10 M led to the compaction of the stem, roots, and root hairs, giving a stunted appearance to the plants. The effect of JA on cell wall regeneration and callus formation was observed in potato leaf protoplast culture. JA at concentrations from 0.01–1 M stimulated cell division and microcalli development.     

Effect of copper excess in environment on soybean root viability and morphology

The effects of increase copper concentrations in medium (10–150 μM CuSO₄) on growth and viability of the roots of two-week-old soybean seedlings (Glycine max L., cv. Dorintsa) were studied. Copper excess suppressed biomass accumulation and linear plant growth; copper affected root growth much stronger than shoot growth. The presence of 10 μM CuSO₄ in medium suppressed accumulation of plant biomass by 40% and the root length by 70%; in the presence of 25 μM CuSO₄, these indices were equal to 80 and 90%, respectively. In the presence of 50 μM CuSO₄, roots ceased to grow but biomass and shoot length still increased slightly. 150 μM CuSO₄ was lethal for plants. The earliest sign of excessive copper toxicity was the accumulation of MDA, indicating activation of membrane lipid peroxidation. A significant increase in MDA content was observed at plant incubation in medium with 10 μM CuSO₄ for 1 h; in this case, the content of copper in the roots increased from 36 ±1.8 (in control) to 48 ± 2.4 μg/g dry wt. The number of dead cells (permeable for the dye Evans Blue) was doubled in the presence of 200 μg/g dry wt within the root; this occurred in 72 h of growth in medium with 10 μM CuSO₄, in 6 h at 25 μM CuSO₄, in 3 h at 50 μM CuSO₄, and 1 h at 150 μM CuSO₄. Toxicity of copper excess was manifested stronger in dividing and elongation cells of the root apex (root meristem and the zone of elongation) than in more basal root regions. Copper excess resulted in the formation of breaks in the surface cell layers of the root tips and affect root morphology. When plant grew in medium with 10 μM CuSO₄, a distance of lateral root formation zone from the root tip decreased markedly, and spherical swellings were formed on the tips of lateral roots. The higher copper concentrations (50 and 150 μM) suppressed completely the development of lateral roots.     

Jasmonic acid induced changes in protein pattern, antioxidative enzyme activities and peroxidase isozymes in peanut seedlings

Protein pattern, ammonia content, glutamine synthetase activity, lipid peroxidation, superoxide dismutase, catalase, peroxidase and peroxidase isoforms were studied in the leaves and roots of 7-d-old peanut (Arachis hypogaea L. cv. JL-24) seedlings treated by 25, 100 and 250 μM jasmonic acid (JA). SDS-PAGE protein profile of leaves and roots after JA application showed a significant increase in 18, 21, 30, 45, 47 and 97.4 kDa proteins and significant decrease in 22 and 36 kDa proteins. Pathogenesis related PR-18 was specific in leaves at 250 μM JA and PR-21 have cross reacted differently with 21 and 30 kDa proteins in leaves and roots treated by all JA concentrations. Further, the immunoblot analysis with glutamine synthetase, GS-45 antibodies revealed a specific cross reaction with 45 and 47 kDa proteins of both control and JA treated leaves, however, higher at 100 and 250 μM JA treated leaves than control ones. Further, the malondialdehyde (MDA) content significantly increased in leaves and roots treated with JA, indicated membrane damage with JA treatments that led to the generation of peroxidation products. The peroxidase isozymic pattern showed two specific isoforms. Besides, the activities of SOD and catalase were significantly elevated in JA treated leaves.     

Growth response and phosphorus uptake of rye with long and short root hairs: Interactions with mycorrhizal infection

Plant growth and phosphorus (P) uptake of two selections of rye (Secale cereale L.) differing in length of root hairs, in response to mycorrhizal infection were investigated. Rye plants with short root hairs (SRH) had a greater length of root infected by Glomus intraradices (up to 32 m pot^–1) than those with long root hairs (LRH) (up to 10 m pot^–1). Application of P decreased the percentage of root length infected in both selections. In low-P soil, mycorrhizal infection increased shoot and root P concentration, especially in LRH plants. Generally, LRH had higher shoot dry weight than SRH plants. P uptake was increased both by LRH and by mycorrhizal infection. Differences in specific P uptake and P utilization efficiency between SRH and LRH plants were observed in non-mycorrhizal plants. With low P supply, P utilization efficiency (dry matter yield per unit of P taken up) of LRH plants increased with time. However, mycorrhizal infection reduced P utilization efficiency, particularly of SRH plants. SRH plants, which were agronomically less efficient (i.e. low dry matter yield at low P supply) were more responsive to either mycorrhizal infection or P addition than the LRH plants. No interaction was observed between mycorrhizal infection and root hair length.     

Changes in the Growth, Chemical Composition, and Antioxidant Activity in the Aquatic Plant Wolffia arrhiza (L.) Wimm. (Lemnaceae) Exposed to Jasmonic Acid

The present study was undertaken to test the influence of exogenously applied jasmonic acid (JA) at concentrations of 0.01–100 μM upon the growth and metabolism of the aquatic plant Wolffia arrhiza (Lemnaceae). JA acted in a concentration-dependent manner. JA at 0.1 μM stimulated plant growth and accumulation of cellular components (proteins, monosaccharides, chlorophylls, phaeophytins, and carotenoids). Treatment with JA at 0.1 μM enhanced W. arrhiza viability by the induction of biomass production and increased the level of photosynthetic pigments, monosaccharides, and soluble proteins. Moreover, JA at 0.1 μM activated the enzymatic (catalase, ascorbate peroxidase, NADH peroxidase) and nonenzymatic antioxidant (ascorbate, glutathione) system in W. arrhiza and, therefore, suppressed lipid peroxidation. In contrast, decreases in fresh weight, major photosynthetic pigments, monosaccharides, and soluble protein content were observed in W. arrhiza exposed to 100 μM JA. JA applied at 100 μM also stimulated the formation of lipid peroxides which are responsible for membrane damage. In the presence of 100 μM JA, antioxidant enzyme (catalase, ascorbate peroxidase, NADH peroxidase) activity and ascorbate as well as glutathione content were inhibited. The data support the hypothesis that JA plays an important role in W. arrhiza growth and metabolism, regulating oxidative status by direct influence on the enzymatic as well as nonenzymatic antioxidant machinery.     

Temporal and positional relationships between Mn uptake and low-pH-induced root hair formation in Lactuca sativa cv. Grand Rapids seedlings

We investigated whether low-pH-induced manganese (Mn) deficiency causes low-pH-induced root hair formation in lettuce seedlings. Both the number and length of root hairs increased in 0 μM Mn (Mn-free) at pH 6 and decreased in 3 mM Mn (excess Mn) at pH 4 compared with the values in 10 μM Mn (normal Mn). These results indicate an inhibitory effect of Mn on both root hair initiation and elongation. The time dependency of root hair induction caused by Mn deficiency corresponded to that caused by low pH. Within 1 h after the pH of the culture medium was reduced from pH 6 to pH 4, the Mn uptake by roots decreased to 43% of that at pH 6. These results suggest that low-pH-induced Mn deficiency promotes root hair formation. At low pH, the rate of Mn uptake was reduced in areas >2 mm from the root tip. Roots with low-pH-induced root hairs still showed low Mn uptake during 3 h of incubation at pH 6. Therefore, the additional root hairs induced by low pH did not compensate for the low-pH-induced decrease in Mn uptake     

Effect of zinc and cadmium on physiological and production characteristics in <Emphasis Type="Italic">Matricaria recutita</Emphasis>

Effects of zinc (12–180 μM) alone and in mixtures with 12 μM Cd on metal accumulation, dry masses of roots and shoots, root respiration rate, variable to maximum fluorescence ratio (F_V/F_M), and content of photosynthetic pigments were studied in hydroponically cultivated chamomile (Matricaria recutita) plants. The content of Zn in roots and shoots increased with the increasing external Zn concentration and its accumulation in the roots was higher than that in the shoots. While at lower Zn concentrations (12 and 60 μM) the presence of 12 μM Cd decreased Zn accumulation in the roots, treatment with 120 and 180 μM Zn together with 12 μM Cd caused enhancement of Zn content in the root. Presence of Zn (12–120 μM) decreased Cd accumulation in roots. On the other hand, Cd content in the shoots of plants treated with Zn + Cd exceeded that in the plants treated only with 12 μM Cd. Only higher Zn concentrations (120 and 180 μM) and Zn + Cd mixtures negatively influenced dry mass, chlorophyll (Chl) and carotenoid content, F_V/F_M and root respiration rate. Chl b was reduced to a higher extent than Chl a.     

Influence of nitrate concentration at the root surface on yield and nitrate uptake of kohlrabi (Brassica oleracea gongyloides L.) and spinach (Spinacia oleracea L.)

The objective of this study was to determine if plant roots have to take up nitrate at their maximum rate for achieving maximum yield. This was investigated in a flowing-solution system which kept nutrient concentrations at constant levels. Nitrate concentrations were maintained in the range 20 to 1000 μM. Maximum uptake rate for both species was obtained at 100 μM. Concentrations below 100 μM resulted in decreases in uptake rate per cm root (inflow) for both spinach and kohlrabi by 1/3 and 2/3, respectively. However, only with kohlrabi this caused a reduction in N uptake and yield. Thus indicating that this crop has to take up nitrate at the maximum inflow. Spinach, however, compensated for lower inflows by enhancing its root absorbing surface with more and longer roots hairs. Both species increased their root length by 1/3 at low nitrate concentrations.     

Root hair elongation is inhibited by hypaphorine, the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by indole-3-acetic acid

Hypaphorine, the major indolic compound isolated from the ectomycorrhizal fungus Pisolithus tinctorius, controls the elongation rate of root hairs. At inhibitory concentrations (100 μM), hypaphorine induced a transitory swelling of root hair tips of Eucalyptus globulus Labill. ssp. bicostata. When the polar tip growth resumed, a characteristic deformation was still visible on elongating hairs. At higher hypaphorine concentrations (500 μM and greater), root hair elongation stopped, only 15 min after application. However, root hair initiation from trichoblasts was not affected by hypaphorine. Hypaphorine activity could not be mimicked by related molecules such as indole-3-acetic acid (IAA) or tryptophan. While IAA had no activity on root hair elongation, IAA was able to restore the tip growth of root hairs following inhibition by hypaphorine. These results suggest that hypaphorine and endogenous IAA counteract in controlling root hair elongation. During ectomycorrhiza development, the absence of root hairs might be due in part to fungal release of molecules, such as hypaphorine, that inhibit the elongation of root hairs. Received: 27 October 1999 / Accepted: 14 March 2000     

Aluminum uptake and aluminum-induced rapid root growth inhibition of rice seedlings

Aluminum (Al) inhibits root growth in acidic soil, but the site of action of Al remains unclear. We investigated whether the rate of Al accumulation correlates to Al-indeced rapid root growth inhibition in rice seedlings (Oryza sativa L. cv. Youngnam). Growth of roots was significantly inhibited by 100 μM AICI₃, as early as 1 h after the treatment. The inhibition of root growth was strongly dependent on Al concentration (l₅₀ = 20 (μM) and Al-exposure time (l₅₀ = 23 min at 25 μM Al) in a solution of 10 mM KCI and 1 mM CaCl₂ buffered by 10 mM Mes/KOH (pH 4.5). Using ICPES, massive uptake of Al by roots was observed even at 15 min treatment of 25 μM Al. The kinetics of Al uptake by the roots closely corresponded to the inhibitory effects of Al on root growth. When the roots of seedlings were exposed to 50 (μM Al for 1 h, then sectioned and stained with hematoxylin, all cell types of the roots showed the presence of Al in the cytoplasm. These results indicate that Al was rapidly taken up into the root cells and thereby reduced root growth.     

Transport of [2-14C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris

Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-¹⁴C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-¹⁴C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-¹⁴C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-¹⁴C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA. Received: 14 April 1997 / Accepted: 9 June 1997     

Cytoarchitecture and pattern of cytoplasmic streaming in root hairs ofMedicago truncatula during development and deformation by nodulation factors

Summary The cytoarchitecture and the pattern of cytoplasmic streaming change during the development of root hairs ofMedicago truncatula and after a challenge with nodulation (Nod) factors. We measured the speed and orientation of movement of 1–2 μm long organelles. The speed of organelle movement in cytoplasmic strands in the basal part of growing root hairs is 8–14 μm/s and is of the circulation type like in trichoblasts, bulges before tip-growth initiation, and full-grown hairs. In the subapical area of growing hairs, reverse-fountain streaming occurs discontinuously at a slower net speed. The reason for the slower speed is the fact that organelles often stop and jump. Reverse-fountain streaming is a pattern in which the main direction of organelle transport reverses 180 degrees before the cell tip is reached. Within minutes after their application to roots,Rhizobium leguminosarum-derived Nod factors, cause an increase and divergence in the subapical cytoplasmic strands. This phenomenon can best be observed in the growth-terminating hairs, since in hairs of this developmental stage, subapical cytoplasmic strands are transvacuolar. First, the tips of these hairs swell. The organelle movement in the swelling tip increases up to the level normal for circulation streaming, and the number of strands with moving organelles increases. When a new polar outgrowth emerges, reverse-fountain streaming is set up again, with all its characteristics like those seen in growing hairs. This outgrowth may obtain a new full root hair length, by which these hairs may become twice as long as nonchallenged hairs. Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Root respiratory quotient and nitrate uptake in hydroponically grown non-mycorrhizal and mycorrhizal wheat

 Oxygen and CO₂ fluxes were measured in hydroponically grown mycorrhizal and non-mycorrhizal Triticum aestivum L. cv. Hano roots. The NO₃ ^– uptake of the plants was used to estimate the amount of root respiration attributable to ion uptake. Plants were grown at 4 mM N and 10 μM P, where a total and viable mycorrhizal root colonisation of 48% and 18%, respectively, by Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) was observed. The O₂ consumption and NO₃ ^– uptake rates were similar and the CO₂ release was higher in mycorrhizal than in non-mycorrhizal wheat. This resulted in a significantly higher respiratory quotient (RQ, mol CO₂ mol^–1 O₂) in mycorrhizal (1.27±0.13) than in non-mycorrhizal (0.79±0.05) wheat. As the biomass and N and P concentrations in mycorrhizal and non-mycorrhizal wheat were the same, the higher RQ resulted from the mycorrhizal colonisation and not differences in nutrition per se. Accepted: 26 March 1999     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

INTERACTION BETWEEN THE ECTOMYCORRHIZAL FUNGUS PISOLITHUS TINCTORIUS AND ROOT HAIRS OF PICEA MARIANA (PINACEAE)

The ectomycorrhizal fungus Pisolithus tinctorius interacts with roots of Picea mariana to form a typical mantle and Hartig net. Hyphae alter their growth pattern when in contact with susceptible root hairs in the mycorrhizal infection zone and grow acropetally, gradually covering the length of the hair to form a mantlelike structure. Initial contact with the hair may be influenced by a fibrillar material on the root hair surface. Although many root hairs become surrounded by fungal hyphae, they are not penetrated, and therefore are not entry points for this symbiotic fungus.     

Induction and cultures of mountain ginseng adventitious roots and AFLP analysis for identifying mountain ginseng

Among the various plant growth regulators (PGR), 9.0 μM 2,4-D(2,4-dichlorophenoxyacetic acid) turned out to be the best for callogenesis and 4.5 μM 2,4-D was revealed to be more suitable for callus proliferation. Of the four basal media, the highest number of 8.2 adventitious roots was induced in Woody plant medium (WPM) containing 14.8 μM IBA(indole 3-butyic acid). F tests on each auxin treatment in the ANOVA revealed highly significant difference at α=0.01. The number of multiple adventitious roots (MARs) formed in each adventitious roots highly depended on the types and levels of auxin tested. At both 24.6 μM and 34.4 μM IBA, more than 25 MARs were induced after 4 weeks of culture. When different medium were applied, immersion type showed the highest fresh weight of MARs at the time of harvest. With 160 g fresh weight of inoculum, about 14.2 times of biomass was obtained after 6 weeks of culture. Identical fractionation pattern of the amplified fragment length polymorphism (AFLP) analysis was observed between the adventitious root and donor of a mountain ginseng. This observation suggested that any rearrangements of the genomic DNA had not occurred in the adventitious roots after 1 year of cultures. The cultivated ginseng and the mountain ginseng groups could be distinguished by 2 AFLP markers.     

Improvement of in vitro propagation of apricot cultivar ‘Bebecou’

Factors affecting successful establishment in vitro, rapid proliferation and rooting of apricot cultivar ‘Bebecou’ were studied. Ethanol and NaOCl were applied in several combinations for disinfection; chilling, plant growth regulators BA, IAA and GA₃, antibiotics, different culture vessels and systems of subculture were evaluated for the optimization of shoot proliferation and the auxins NAA and IBA were assessed for root induction. The highest number of new microshoots/explant (18.7) was obtained in a culture medium supplemented with 2.2 μM BA+0.57 μM IAA after 300 h of chilling. The effect of GA₃ (11.4 μM) on shoot proliferation was positive in combination with 4.4 or 8.9 μM BA. Shoot length and productivity were highest at 2.2 μM BA+11.4 μM GA₃+0.57 μM IAA and at 2.2 μM BA+0.57 μM IAA, respectively and decreased as cytokinin concentration increased. The antibiotic ‘Na-cefotaxime’ had a minimal impact on shoot growth when used at the lowest concentration (250 mg l⁻¹). Subculture every 2 weeks in a medium supplemented with 2.2 μM BA and 0.57 μM IAA was more efficient for shoot induction than alternation of 20 days culture in a propagation medium supplemented with 2.2 μM BA and 10 days culture in an elongation medium supplemented with 1.1 μM BA and 5.71 μM IAA. The highest number of roots/shoot (8.1) was recorded at 19.6 μM IBA.     

Jasmonates induce over-accumulation of methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures

 Jasmonic acid (JA) and its methyl ester (MeJA) at concentrations ranging from 0.001 to 10 μM provoked large increases in methylputrescine levels in normal and hairy roots of Hyoscyamus muticus L.; generally, levels of free putrescine and perchloric acid-soluble conjugated putrescine, spermidine and spermine also increased dramatically. More ¹⁴C-putrescine was formed when hairy roots were incubated with labelled ornithine than with arginine; conjugated ¹⁴C-putrescine was also rapidly formed. In accord with these results, ornithine decarboxylase (EC 4.1.1.17) activity was higher than that of arginine decarboxylase (EC 4.1.1.19), and MeJA enhanced these activities about two- and fourfold, respectively. Although treatment of root cultures with jasmonates enhanced precursor (putrescine, methylputrescine) levels and accumulation of secondary metabolites such as acid-soluble conjugated di-/polyamines, it provoked only modest increases in tropane alkaloid tissue levels. Received: 24 March 1999 / Revision received: 5 October 1999 / Accepted: 26 October 1999