CH3Cl emissions by freshly harvested tubers of 61 potato cultivars ranged from less than 4 to 650 ng g−1 f. wt d−1. In experiments with Anna, the cultivar displaying the highest release rate, maximum emission of CH3Cl occurred at 20°C but the rate diminished after 48 h, a decrease due in part to inhibition of CH3Cl release by the accumulation of CH3Cl in the headspace. In 1995 and 1996, CH3Cl emission by tubers was first detectable 6 wk before the normal harvest date. If tubers were stored at 20°C immediately following harvest, release attained a maximum of 350 ng g−1 f. wt d−1 about 4 d after harvest, falling to less than 4 ng g−1 f. wt d−1 within 3 wk of harvest. On storage at 6°C post-harvest, maximum release of CH3Cl (c. 600 ng g−1) was delayed until 5 wk after harvest and release fell below 4 ng g−1 f. wt d−1 after 200 d. Release of CH3Cl was not significantly affected by cutting or bruising tuber tissue, and CH3Cl biosynthesis occurred in both core and superficial tissues of the tuber. The S-methyl group of either L- or D-methionine acted as a precursor of CH3Cl in sliced tubers, but the methylating system was specific for Cl−. An isotope dilution technique using C2H3Cl demonstrated that the amount of CH3Cl metabolized by the intact tuber was approximately 36% of the net release. However, investigations with 14CH3Cl showed that only a small proportion (7%) of the CH3Cl metabolized was fixed in involatile form in the tuber. In both skin and core tissues, fixed 14C was located primarily (80%) in the fraction soluble in phosphate buffer, but the specific activity of skin tissue was about twice that of core tissue. Autoradiography demonstrated that 14C fixation in the tuber was greatest within the lenticels, possibly indicating a microflora adapted to use the locally high concentrations of CH3Cl. Significant 14C fixation was also associated with the periderm but was not attributable to labelling of the O-methyl groups of the phenolic components of suberin. Within the core of the tuber, 14C fixation was located primarily in the phloem, pith and medullary rays.

Numerous biochemical and physiological studies have demonstrated the importance of ascorbate (ASC) as a reducing agent and antioxidant in higher plant metabolism. Of special note is the capacity of ASC to eliminate damaging activated oxygen species (AOS) including O2−· and H2O2. N2-fixing legume nodules are especially vulnerable to oxidative damage because they contain large amounts of leghaemoglobin which produces AOS through spontaneous autoxidation; thus, ASC and other components of the ascorbate–reduced glutathione (ASC–GSH) pathway are critical antioxidants in nodules. In order to establish a meaningful correlation between concentrations of ASC and capacity for N2 fixation in legume root nodules, soybean (Glycine max) plants were treated with excess ASC via exogenous irrigation or continuous intravascular infusion through needles inserted directly into plant stems. Treatment with ASC led to striking increases in nitrogenase activity (acetylene reduction), nodule leghaemoglobin content, and activity of ASC peroxidase, a key antioxidant enzyme. The concentration of lipid peroxides, which are indicators of oxidative damage and onset of senescence, was decreased in ASC-treated nodules. These results support the conclusion that ASC is critical for N2 fixation and that elevated ASC allows nodules to maintain a greater capacity to fix N2 over longer periods.

An improved method of cell fractionation allowed the extraction of soluble (sNR) and membrane-associated (mNR) forms of nitrate reductase (NR) from a dinoflagellate, even though in previous studies only mNR had been found in these algae. Both activities were assayed in cell-free extracts of Peridinium gatunense from Lake Kinneret, Israel, after disruption of the cells and differential centrifugation. In the cultures used, sNR showed much higher NO3−-reducing activity. Only a low proportion, 2.5–3% of NR activity, was found to be associated with mNR. Moreover, mNR comprised two forms as indicated by protein solubilization: a tightly membrane-bound and a more weakly attached NR. Ascorbate inhibited all NR activities, but that of mNR recovered after its removal. Polyvinyl pyrrolidone (PVP) and DTT also diminished sNR and mNR activities. For both enzymes, pH optima (7.65) and temperature optima (13–25°C) were similar, and agreed with those for optimum growth of P. gatunense both in culture and in the lake. The most efficient electron donor was NADH, though NADPH sustained low NR activities. Carboxylic anions such as succinate and malate did not support any reduction of NO3−, nor did they cause any stimulation of sNR or mNR activities. Both forms of NR showed a high affinity for their substrates: Km was c. 10 μM for NO3− and c. 5 μM for NADH. The high efficiency of NO3− assimilation by Peridinium seems to be limited mainly by energy under otherwise optimal nutritional conditions and, at low nitrate concentrations, the low Km may be one of the main reasons for the high competitivity of this alga in Lake Kinneret.

Studies on the rhizomatous clonal sedge Carex bigelowii, at a fellfield site in subarctic Swedish Lapland examined the ecological potential of rhizomes as nitrogen uptake systems. Direct application of a solution of 15NH415NO3 to the rhizomes of C. bigelowii significantly enriched the 15N content of the plant tissue. The pattern of enrichment indicated movement of labelled nitrogen into rhizomes and adjoining tissue (including roots and shoots), showing both uptake and translocation via the rhizome system. There was a gradient of decreasing 15N enrichment with increasing distance from the point of labelling, and estimation of the total mass of nitrogen taken up via labelled rhizomes showed low levels and rates of uptake. Quantification of the size of rhizome and root systems of C. bigelowii at the study site indicates that 80% of the living biomass may be below ground, and that the surface area of the rhizome system of C. bigelowii is approx. two-thirds that of the roots. The rhizome system of C. bigelowii can therefore act as a route for nitrogen uptake, with the potential to exploit almost as great a volume of soil as the root system. This mechanism of nitrogen uptake may play an important role in the Arctic, where many species have a clonal, rhizomatous growth form. In addition, plant growth in many arctic ecosystems is limited by low soil nutrient availability, a result of low temperatures (leading to slow soil decomposition rates) and patchy resource distribution. Nitrogen uptake via rhizomes may provide plants with the capacity to take advantage of transient nutrient supplies, and may partly compensate for the cost of developing and maintaining persistent rhizome networks in ecosystems where nutrient resources are in short supply.

Many soil fungi colonize the roots of pines to form symbiotic organs known as ectomycorrhizas. Dichotomous branching of short lateral roots and the formation of coralloid organs are diagnostic of ectomycorrhizas in many pine species, although the regulation of these changes in root morphology is not well understood. We used axenic root cultures of six pine species to examine the role of auxin, cytokinin, ethylene and nutrients in the regulation of root architecture. Surprisingly, extensive dichotomous and coralloid branching of lateral roots occurred spontaneously in Pinus taeda, P. halepensis and P. muricata. In P. sylvestris, P. ponderosa and P. nigra, treatment with auxin transport inhibitors (ATIs), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the ethylene-releasing compound 2-chloroethylphosphonic acid (CEPA or ethephon) induced extensive dichotomous branching and coralloid organ formation. Formation of both spontaneous and ATI-induced coralloid structures was blocked by treatment with an ethylene synthesis inhibitor L-α-(2-aminoethoxyvinyl)glycine; this inhibition was reversed by either ACC or CEPA. In addition, the induction of this unique morphogenetic pattern in pine root cultures was regulated by nutrient levels. The morphology and anatomical organization of the chemically induced dichotomous and coralloid structures, as well as the regulation of their formation by nutrient levels, show a striking similarity to those of ectomycorrhizas.

Gravitropic responses of oat coleoptiles were measured in different growth media; humid air, natural soil and artificial soil (glass beads). The oat coleoptiles in soil and glass beads were monitored by NMR imaging, while those in humid air were imaged in darkness with an infrared-sensitive charge-coupled device (CCD) camera. The present study shows for the first time that gravitropic experiments can be performed in artificial soil using NMR imaging as a convenient and suitable recording method. Not only was it possible to follow the gravitropic curvatures in natural soil, but the artificial soil allowed plant images of sufficient spatial and temporal resolution to be recorded. The advantages of using artificial soil in magnetic resonance imaging studies are that the iron content of glass beads is very low compared with natural soil, and that the artificial soil matrix can easily be standardized with regard to particle size distribution and nutrient content. Two types of glass beads were used, the diameter of the small and the large beads being 300–400 and 420–840 μm, respectively. The growth rate of the coleoptiles in soil and in big beads was roughly the same and only slightly lower than in humid air, whereas small beads reduced the growth rate by approx. 16%. The bending rate of the coleoptiles during the gravitropic response was reduced by c. 65% in soil and 75% in bead mixtures relative to bending in air. It should be noted, however, that the maximum curvature of the coleoptile tip was of the same order in all cases, about 35°. This value may represent the largest possible curvature of the organ. The potential of NMR imaging to study how plant organs penetrate the soil under the influence of gravitropism, mechanical impedance and thigmotropism is also discussed.

The effects of three conditions likely to affect soluble carbohydrate pools, namely drought, expression of barley sucrose: fructan 6-fructosyl transferase (6-SFT, EC 2.4.1.10) and the establishment of the arbuscular mycorrhizal symbiosis with Glomus mosseae were studied in a multifactorial experiment using tobacco (Nicotiana tabacum). Tobacco, a plant naturally unable to form fructan, accumulated fructan in leaves, and to a larger extent in the roots, when transformed with 6-SFT. Under drought conditions, growth was considerably reduced, but neither expression of 6-SFT nor mycorrhiza formation had an effect on growth rate. However, in response to drought, carbon partitioning was significantly altered towards accumulation of soluble sugars. In plants exposed to drought, pools of sucrose were greater than those of unstressed plants, particularly in their roots. In the transgenic plants expressing 6-SFT, there were also increased contents of the products of 6-SFT, namely fructan, most probably because of the increased availability of the substrate, sucrose. These effects were the same in the presence or absence of mycorrhiza. Hexoses (glucose and fructose) also increased in response to drought, primarily in the leaves. This effect of drought was little affected by the expression of 6-SFT, except that it slightly enhanced drought-induced glucose accumulation in roots. However, the presence of mycorrhiza led to a considerable reduction in drought-induced accumulation of hexoses in the leaves. The content of the fungal disaccharide trehalose was greatly increased in the roots of all mycorrhizal plants upon exposure to drought, particularly in some of the transgenic plants expressing 6-SFT.

A fructan polymerase activity was partially purified and concentrated by sequential acid and salt precipitation from extracts of excised, illuminated leaves of timothy grass (Phleum pratense). The polymerase catalysed the de novo synthesis of oligo- and polyfructan from sucrose as sole substrate at near-physiological rates (0.5–0.9 mg g−1 fresh mass h−1; 0.9–1.5 nkat g−1). Rates of in vitro polymerisation were high, at up to 4.1 mg cm−3 h−1 (7.1 nkat cm−3) of total products of degree of polymerization greater than 2 (DP>2). The trisaccharides 1-kestose and 6-kestose together with oligosaccharides of up to DP[les ]c. 10 were synthesized in under 2 h at 30°C. In longer incubations, ethanol-precipitable polymers of DP=c. 10–35 (1.6–5.7 kDa) were detected by anion-exchange chromatography and pulsed amperometry. When this polymeric product was used as a primer and re-incubated with fresh enzyme and sucrose, abundant polymers of up to DP=50 (8.1 kDa) were formed. The structure of the polymeric enzyme product was compared with native fructan from timothy leaves and with standard inulin, using glycosyl-linkage analysis followed by identification of partially methylated alditol acetate derivatives by GC–MS. The deduced structure was a linear (unbranched) 2,6-linked fructose chain terminated with glucose and fructose. The linkage structures of the native and enzyme-generated polymers were identical, increasing confidence in the physiological relevance of the activity. After ultracentrifugation of tissue homogenates at 265 000 gav, the polymerase remained in the supernatant, demonstrating no tight association with particulate components. The polymerizing reaction was dependent on enzyme concentration, requiring at least 3 g fresh mass equivalent cm−3 (c. 2.7 nkat cm−3) for the efficient in vitro generation of fructans of DP>3. In common with other trisaccharide-synthesizing and oligofructan-glycosylating enzymes from grasses, the polymerase reaction exhibited both a maximal velocity at pH 5.0–5.5 and a low affinity for sucrose. The polymerization reaction did not saturate fully even at 1.5 M sucrose, and the concentration causing half maximal velocity (apparent Km) was c. 560 mM. The preparation contained substantial invertase activity (1.8 mg sucrose g−1 fresh mass h−1=1.5 nkat g−1 fresh mass) with a Km for sucrose hydrolysis of 5 mM. A single peak of polymerase activity with an Mr of 51 kDa was recovered from size-exclusion chromatography (SEC). Invertases of Mr 51 and 110 kDa were identified in the preparation. The 110-kDa invertase isoform exhibited no polymerase activity, but synthesized trisaccharide (mainly 1-kestose) from sucrose. The 51-kDa isoform co-eluted with the polymerase. The trisaccharide fraction produced by this isoform contained abundant 1- and 6-kestose. After SEC, the purification of the polymerase was 41-fold relative to the original tissue homogenate. The properties of enzymatic polymerization of fructan are discussed with respect to the physiology of accumulation in grass leaves and other systems.

Natural abundances of 15N and N concentrations of 34 fruit bodies from 24 species of ectomycorrhizal and saprophytic fungi were measured in a temperate Central European mixed forest stand. The fungi of the two life forms are known to be capable of utilizing different types of N sources (organic N compounds from the humus, inorganic N from the soil and N from litter or wood) differing by their 15N natural abundance values. Based on the two life forms and the three different N sources, four functional groups of fungi were distinguished: (1) ectomycorrhizal fungi capable of utilizing organic N from the humus; (2) ectomycorrhizal fungi known to depend on inorganic N compounds in the soil; (3) saprophytes capable of utilizing organic N from the humus; and (4) saprophytes utilizing N from dead wood or litter. Large differences were found between species in the δ15N values (−3.0 to 3.3‰) and in the N concentrations (0.84 to 6.61 mmol eq N g dw−1) of the fruit bodies. In most cases fungi were more enriched in 15N than their respective bulk N source was. Fungi living in humus, and presumably having access to organic N compounds (groups 1 and 3), were significantly more enriched in 15N than fungi which are known to depend on inorganic N (e.g. Laccaria, group 2), or fungi living on litter or wood (group 4), irrespective of whether they were ectomycorrhizal or saprophytic species. Fungi living in humus had significantly higher N concentrations than fungi living on litter or wood.

In an experiment to determine the influence of mycorrhizal colonization on root development, fertilized seedlings of cotton were grown in long tubular pots. Inoculation of soil with an isolate of Glomus mosseae at 10, 25 or 40 cm below the soil surface resulted in spread of arbuscular mycorrhizas up and down the root system, and localized elongation of roots following colonization. Specific root length was not influenced by colonization. Increases in local root density at the point of inoculation were observed, though total root mass in relation to shoot mass declined following initiation of mycorrhizas.

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F1 hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn5-luxAB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn5[ratio ][ratio ]luxAB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux-tagged strain DF57-40E7, which carries a constitutively expressed luxAB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.

The extent of arbuscular mycorrhizal colonization was assessed in 10 field-collected plant species, representing three annual forbs, three perennial forbs, three perennial grasses and one annual grass. Each root system of each plant was split into four portions, and for each portion, mycorrhizal structures were revealed with epifluorescence microscopy (under which only arbuscules are generally visible) and three commonly used stains (Chlorazol Black E, Acid Fuchsin and Trypan Blue). The aim of the study was not to evaluate the efficacy of each method, but to compare results obtained by each under standard laboratory conditions. The recorded colonization levels of arbuscules, total arbuscular mycorrhizal fungal material and total fungal (arbuscular mycorrhizal+non-arbuscular mycorrhizal) material differed significantly between visualization methods in a number of species. However, there were also interactions between stain and plant species, indicating that the performance of a stain is dependent on the plant species being examined. In some cases (e.g. Plantago lanceolata), each visualization method produced the same colonization level, while in others (e.g. Dactylis glomerata), each method gave a different result. These data therefore suggest that the level of mycorrhizal colonization recorded in any particular plant species at a particular time is dependent on the technique employed.

Interactive effects of elevated atmospheric CO2 and arbuscular mycorrhizal (AM) fungi on biomass production and N2 fixation were investigated using black locust (Robinia pseudoacacia). Seedlings were grown in growth chambers maintained at either 350 μmol mol−1 or 710 μmol mol−1 CO2. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The 15N isotope dilution method was used to determine N source partitioning between N2 fixation and inorganic fertilizer uptake. Elevated atmospheric CO2 significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO2 had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO2 levels enhanced nodule and root mass production, as well as N2 fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO2 enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N2-fixing tree species is affected by enriched atmospheric CO2.

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.

Seedlings and old trees of Scots pine in self-regenerating, old, virgin boreal forest in northern Sweden were compared in terms of the species composition of their associated ectomycorrhizal (EM) fungi. The natural regeneration of seedlings was mimicked by annual seeding for 10 yr (1986–1995) in intact field vegetation at three sites. In 1995, all seedlings were harvested, and 171 were examined for mycorrhizas. Twenty-five soil cores were taken in order to study the mycorrhizas on the established trees. Most short roots were mycorrhizal. Using mycorrhizal morphology and restriction fragment length polymorphisms (RFLP) analysis, we were able to distinguish 43 ectomycorrhizal taxa. Fourteen of these taxa were identified using a sporocarp and mycelial culture based internal transcribed spacer (ITS)-RFLP reference database, and another three were identified by mycorrhizal morphology. Cenococcum geophilum, Piloderma croceum and Suillus variegatus were present on the seedlings, irrespective of age, as well as on the old trees. At one forest, 23 ectomycorrhizal taxa were found as mycorrhizas, of which 10 occurred on both seedlings and old trees. These 10 species accounted for 92 and 73% of the mycorrhizas on the seedlings and old trees, respectively. A 3-yr survey of ectomycorrhizal sporocarps at the same site revealed 62 EM taxa. The degree of similarity between the composition of EM species reflected by sporocarps and by mycorrhizas, was low. Ectomycorrhizal species present in our ITS-RFLP database constituted 7.5–19% of the mycorrhizas on seedlings and old trees, whereas they constituted 80–95% of the total production of epigeous sporocarps. The unidentified taxa comprised 70–87% of the mycorrhizas. Our findings support the view that the species composition of mycorrhizas colonizing naturally regenerated seedlings in forests is similar to that of mycorrhizas colonizing surrounding trees. We suggest that the concept of the mycelial network be expanded in order to embrace both the significance of interconnections between different trees as well as the continuity or perpetuation of EM fungal communities.

Previous work in the Colorado alpine ecosystem has shown that amino acids are a potentially important N source for the sedge, Kobresia myosuroides. This plant is the only known sedge to harbour associations with ectomycorrhizal fungi. The aim of the present work was to test the hypothesis that these ectomycorrhizas transfer N from amino acids in the soil solution to the host plant, and thereby have an important role in the N nutrition of this species. We used a two-chamber system (rhizoboxes) in which K. myosuroides plants were separated from a soil chamber by nylon mesh that allowed fungal hyphae, but not plant roots, to cross it. Injections of [15N, 2-13C]glycine were made into the soil chamber. The hyphal crossings on half of the rhizoboxes were regularly disrupted to control for leakage of label across the barrier. Plants in the intact rhizoboxes showed significantly higher 15N enrichment than those in controls, and mycorrhizal root tips were significantly more enriched than bulk roots. The mycorrhizas transferred an average of 1.3% of the added 15N label to plants, a figure comparable to those obtained in previous studies in which plant roots were directly exposed to label. We conclude that fungal associations have an important role in the N nutrition of K. myosuroides by transferring N from amino acids to their hosts.