Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR⁻) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH₄¹⁵NO₃ medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas ¹⁵N enrichment values indicated that 13–27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.     

Simultaneous measurement of ammonium,nitrate and proton fluxes along the length of eucalypt roots

Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH₄ ⁺, NO₃ ^– and H⁺ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 m NH₄NO₃. The pattern of fluxes was such that there was a large influx of NH₄ ⁺, a smaller influx of NO₃ ^– and large H⁺ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:–6 (NH₄ ⁺:NO₃ ^–:H⁺). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH₄ ⁺ and H⁺, and NH₄ ⁺ and NO₃ ^–. Overall, these results are consistent with NH₄ ⁺ being the preferred source N for E. nitens.     

Cotton root and shoot response to localized supply of nitrate,phosphate and potassium: Split-pot studies with nutrient solution and vermiculitic soil

Vertical stratification of plant-available K in vermiculitic soil profiles contributes to a late-season K deficiency that limits cotton (Gossypium hirsutum L.) yields on affected soils. Split-root solution culture and split-pot soil experiments were conducted to determine whether root distribution and cultivar differences in root extension in these stratified profiles result from a compensatory response to localized enrichment with NO₃-N, PO₄-P, and/or K in the root zone. Compensatory root growth was greatest in response to localized NO₃-N enrichment. For two cultivars examined in solution culture, 74% of new root development occurred in the half-pot providing 90% of the total NO₃-N supply. Only 60% of cultivar root development occurred in the half-pot providing 90% of the PO₄-P. No compensatory root growth was observed in response to localized K enrichment. In the split-pot system, the proportion of total root surface area developing in a half-pot was highly correlated with localized soil NO₃-N levels (r²=0.81), while increased K availability in one half of the root zone did not affect root distribution. Mean soil NO₃-N supply to the whole root system determined shoot N accumulation (r²=0.97). Shoot K accumulation was not related to soil K availability but was strongly correlated with mean root surface area density (r²=0.86). Cultivar Acala GC510, known to be less sensitive to K deficiency than Acala SJ-2, had significantly larger root diameter in all nutrient-supply environments. Under conditions of K stress, Acala GC510 had increased root branching and allocated greater dry matter to roots relative to shoots than Acala SJ-2. The results demonstrate that K acquisition by cotton is strongly influenced by the quantity and distribution of NO₃-N in the root zone through its effects on root proliferation, and that distinct cultivar differences associated with crop performance on low K soils can be detected in short-term, solution culture growth systems.     

A comparison of NH4+ and NO3– net fluxes along roots of rice and maize

Net fluxes of NH₄⁺ and NO₃^– along adventitious roots of rice ( Oryza sativa L.) and the primary seminal root of maize ( Zea mays L.) were investigated under nonperturbing conditions using ion-selective microelectrodes. The roots of rice contained a layer of sclerenchymatous fibres on the external side of the cortex, whereas this structure was absent in maize. Net uptake of NH₄⁺ was faster than that of NO₃^– at 1 mm behind the apex of both rice and maize roots when these ions were supplied together, each at 0·1 mol m^–3. In rice, NH₄⁺ net uptake declined in the more basal regions, whereas NO₃^– net uptake increased to a maximum at 21 mm behind the apex and then it also declined. Similar patterns of net uptake were observed when NH₄⁺ or NO₃^– was the sole nitrogen source, although the rates of NO₃^– net uptake were faster in the absence of NH₄⁺. In contrast to rice, rates of NH₄⁺ and NO₃^– net uptake in the more basal regions of maize roots were similar to those near the root apex. Hence, the layer of sclerenchymatous fibres may have limited ion absorption in the older regions of rice roots.     

Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.     

Localization and quantification of net fluxes of H+ along maize roots by combined use of pH-indicator dye videodensitometry and H+-selective microelectrodes

Two methods for measuring proton fluxes along intact maize roots grown with NH ₄ ⁺ or NO ₃ ⁻ at pH 6.5 were compared. Videodensitometric measurement of changes in a pH-indicator dye by video camera was used to map pH around roots and determine the amounts of protons released by various root regions. This method was compared with potentiometric determination of the concentration of H⁺ in the unstirred layer at the root surface using ion-selective microelectrodes. With NH ₄ ⁺ the roots released large amounts of H⁺ in preferential regions where the rate of flux can reach 1.4 or even 2.5 nmol m⁻¹ s⁻¹. Videodensitometry indicated a first region of root acidification in the subapical zone, but this was more difficult to localize with microelectrodes. With NO₃ ⁻ both methods showed that the roots released small amounts of H⁺ and that the apical region took up H⁺ in the first 10 mm then sometimes released H⁺ over the following 10 mm of root. The H⁺ flux profiles obtained by both methods were in good agreement in terms of both order of magnitude of the fluxes and spatial differences along the root. These results suggest that videodensitometry, which is easier to use than potentiometry, can be used to screen different plant species or cultivars under various experimental conditions. The microelectrode technique is indispensable, however, for studying the underlying mechanisms of net H⁺ fluxes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Subcellular compartmentation of elements in non-mycorrhizal and mycorrhizal roots of Pinus sylvestris: an X-ray microanalytical study. II. The distribution of calcium, potassium and sodium

The inter- and intracellular distribution of the elements calcium, potassium and sodium in non-mycorrhizal and mycorrhizal roots of Pinus sylvestris dependent on different external nutrient supply conditions was detected by energy-dispersive X-ray microanalysis after cryofixation, freeze-drying and pressure infiltration of the material. In non-mycorrhizal and mycorrhizal roots, calcium was mainly detectable in the apoplastic regions. The levels in vacuoles and cytoplasm were near the limits of detection by X-ray microanalysis. Incubation with high concentrations of potassium and sodium, or mycorrhizal infection with Suillus bovinus and Pisolithus tinctorius reduced the amounts of calcium detectable in the roots, especially in the apoplast of cortical cells. The studies revealed that: potassium is mainly localized in cytoplasm and cell walls; the cytoplasmic content is regulated over a wide range of external potassium concentrations; potassium levels in the inner parts of roots are higher than in the outer parts. Mycorrhizal infection with Suillus bovinus had no effect on the inter- and intracellular distribution of potassium in roots but, if the external supply was low, the potassium content in shoots was reduced. In non-mycorrhizal pine roots and those infected with Paxillus involutus an increase in the sodium content of all cell compartments was observed after treatment with high external concentrations of NaH₂PO₄. However, an increase in sodium content in mycorrhizas of S. bovinus was not detected. The X-ray microanalytical results are discussed in relation to the apoplastic movement of nutrients in non-mycorrhizal and mycorrhizal fine roots of pine and to the demand for these nutrients in different intracellular compartments.     

Effects of defoliation and atmospheric CO2 depletion on nitrate acquisition,and exudation of organic compounds by roots of Festuca rubra

The aim of this study was to investigate the physiological basis of increased root exudation from Festuca rubra, in response to defoliation. The hypothesis, that assimilate supply to roots is a key determinant of the response of root exudation to defoliation was tested by imposing CO₂-deplete (< 50 mol mol^–1) atmospheres to F. rubra. This was done as a non-destructive means of preventing supply of new assimilate to roots of intact and defoliated plants. F. rubra was grown in axenic sand systems, with defoliation and CO₂-depletion treatments applied to plants at 14 and 35 days after planting. Root exudation and NO₃ ^– uptake were quantified throughout, and post-treatment uptake and allocation of N were determined from the distribution of ¹⁵N label, supplied as ¹⁵NO₃ ^–. Defoliation of F. rubra resulted in significantly (P <0.01) increased root exudation, CO₂-depletion did not result in increased exudation from plants of either age. When treatments were applied to F. rubra after 14 days, defoliation and CO₂-depletion each reduced NO₃ ^– uptake significantly (P <0.05). However, in older plants, uptake of NO₃ ^– was less sensitive to defoliation and CO₂-depletion. The results indicate that increased root exudation following defoliation is not related directly to reduced assimilate supply to roots. This was evident from the lack of effect of CO₂-depletion on root exudation, and the absence of correlation between root-C efflux and the rate of NO₃ ^– uptake. The physiological basis of increased exudation following defoliation remains uncertain, but may be dependent on physical damage, either directly or as a consequence of systemic responses to wounding.