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TECHNICAL REPORTS

Plant and Environment Interactions

Responses to Iron Limitation in Hordeum vulgare L. as Affected by the Atmospheric CO₂ Concentration

^a Inst. of Soil Science, Univ. of Hohenheim, 70599 Stuttgart, Germany
^b Inst. of Plant Nutrition, Univ. of Hohenheim, 70593 Stuttgart, Germany
^c Creative Research Initiative ‘Sousei’ (CRIS), Hokkaido Univ., N21W10, Kita-ku, Sapporo 001-0021, Japan
^d Inst. of Plant Cultivation and Plant Nutrition, Humboldt Univ., Berlin, Germany

^* Corresponding author (gd.neumann{at}t-online.de).

Received for publication April 6, 2006.

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Material and Methods Results Discussion Concluding Remarks REFERENCES

 
Elevated atmospheric CO₂ treatments stimulated biomass production in Fe-sufficient and Fe-deficient barley plants, both in hydroponics and in soil culture. Root/shoot biomass ratio was increased in severely Fe-deficient plants grown in hydroponics but not under moderate Fe limitation in soil culture. Significantly increased biomass production in high CO₂ treatments, even under severe Fe deficiency in hydroponic culture, indicates an improved internal Fe utilization. Iron deficiency-induced secretion of PS in 0.5 to 2.5 cm sub-apical root zones was increased by 74% in response to elevated CO₂ treatments of barley plants in hydroponics but no PS were detectable in root exudates collected from soil-grown plants. This may be attributed to suppression of PS release by internal Fe concentrations above the critical level for Fe deficiency, determined at final harvest for soil-grown barley plants, even without additional Fe supply. However, extremely low concentrations of easily plant-available Fe in the investigated soil and low Fe seed reserves suggest a contribution of PS-mediated Fe mobilization from sparingly soluble Fe sources to Fe acquisition of the soil-grown barley plants during the preceding culture period. Higher Fe contents in shoots (+52%) of plants grown in soil culture without Fe supply under elevated atmospheric CO₂ concentrations may indicate an increased efficiency for Fe acquisition. No significant influence on diversity and function of rhizosphere-bacterial communities was detectable in the outer rhizosphere soil (0–3 mm distance from the root surface) by DGGE of 16S rRNA gene fragments and analysis of marker enzyme activities for C-, N-, and P-cycles.

Abbreviations: PS, phytosiderophores • MAs, mugineic acids • DAS, days after sowing • DGGE, denaturing gradient gel electrophoresis

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Material and Methods Results Discussion Concluding Remarks REFERENCES

 
ANTHROPOGENIC activities have caused the concentration of atmospheric CO₂ to rise from about 280 µmol mol^–1 at the beginning of the industrial revolution to currently over 380 µmol mol^–1 (Keeling and Whorf, 2003). CO₂ is one of the greenhouse gases and further increasing concentrations, predicted for the next decades, may contribute to global warming (IPCC, 2001). Moreover, numerous model investigations simulating elevation of atmospheric CO₂ concentration demonstrated an increase in biomass production of plants grown at sufficient nutrient supply (Hodge and Millard, 1998), which is caused by a higher photosynthetic CO₂ assimilation rate (Eamus, 2000). An increased belowground translocation of carbohydrates (Rogers et al., 1994) in response to elevation of CO₂ may improve spatial acquisition of nutrients by stimulation of fine root production (Zak et al., 2000), and also improve chemical availability of nutrients in the rhizosphere by increased root exudation of organic compounds (Norby, 1994; Hungate et al., 1997; Cheng and Johnson, 1998; Pendall et al., 2004). Elevated CO₂ concentrations may affect the concentrations as well as the qualitative composition of specific root exudates in the rhizosphere, which could have a direct impact on mobilization of sparingly soluble nutrients by modifications of the rhizosphere chemistry. Alternatively, higher overall root exudation at elevated CO₂ may be simply the consequence of a larger root system with unchanged exudation rates per unit root biomass or root length, and therefore unchanged rhizosphere concentrations (Wasaki et al., 2005). Increased root growth and altered rhizodeposition under elevated atmospheric CO₂ concentrations may also affect biomass, activity, and the structure of rhizosphere microbial communities (Rogers et al., 1994; Paterson et al., 1997; Sadowsky and Schortemeyer, 1997; Marilley et al., 1999; Wiemken et al., 2001; Klamer et al., 2002). Since microorganisms have a significant impact on dynamics of organic matter and on plant nutrient availability in soils, they may play a key role in the responses of ecosystems to global climate changes (Freeman et al., 2004).

Most of the studies conducted so far have focused on the role of elevated atmospheric CO₂ on carbon-, nitrogen-, and phosphorus cycling (Mayr et al., 1999; Kang et al., 2001; Larson et al., 2002; Kang et al., 2005). However, little attention has been paid to CO₂ effects on rhizosphere availability of micronutrients, such as iron (Fe), zinc (Zn), or manganese (Mn) despite their function as essential mineral nutrients (Marschner, 1995), as factors determining pathogen resistance (Graham and Webb, 1991) and plant-microbial competition (von Wirén et al., 1993, 1995; Yehuda et al., 1996; Hördt et al., 2000).

The aim of this study was to investigate the effects of elevated atmospheric CO₂ concentrations on Fe acquisition in plants, considering plant growth responses, root-induced changes in rhizosphere chemistry and related impacts on rhizosphere microbial communities. Barley (Hordeum vulgare L.) was selected as a model system, representing a plant species with efficient acquisition of Fe and Zn, mediated by high release rates of organic metal chelators (phytosiderophores) under conditions of Fe limitation (Takagi, 1976; Marschner and Römheld, 1994). Phytosiderophores (PS) are non-proteinogenic amino acids (mugineic acids), derived from methionine, and are synthesized in roots of graminaceous plant species (strategy II plants) from nicotianamine, which is a ubiquitous intracellular Fe-chelator in higher plants (Neumann and Römheld, 2000). Mugineic acids mobilize sparingly soluble inorganic forms of Fe^III and Zn by formation of stable complexes, even under conditions of alkaline soil pH as a major factor, limiting the solubility of micronutrients in soils (Marschner, 1995). Subsequent uptake of Fe^III–phytosiderophore complexes is mediated by a specific transporter in the plasma membrane of root cells (Marschner and Römheld, 1994; Curie et al., 2001). The biosynthesis of PS in the root tissue seems to be regulated by the intracellular Fe level. In barley, PS secretion follows a distinct diurnal rhythm (Ma and Nomoto, 1996), and is localized in 1 to 3 cm sub-apical root zones (Marschner et al., 1987; Walter et al., 1995a,b). Under non-axenic conditions PS are readily decomposed by certain microorganisms which use PS as a source of carbon (Mori et al., 1987; Takagi et al., 1988). The temporal and spatial concentration of PS secretion may be a strategy to counteract microbial degradation and unspecific adsorption of PS in the rhizosphere (Römheld, 1991; Neumann and Römheld, 2000). Moreover, there is evidence that graminaceous plants can use PS to acquire Fe by exchange chelation from complexes with native or partially degraded microbial siderophores (Yehuda et al., 1996; Hördt et al., 2000). Therefore, barley provides a well-characterized model system to assess effects of elevated atmospheric CO₂ concentrations on Fe acquisition and related plant microbial interactions in the rhizosphere.

In the present study, barley plants were grown in a hydroponic culture system to study CO₂ effects on Fe-deficiency-induced responses in plant growth and PS exudation under strictly controlled conditions. Rhizobox microcosms with an Fe-limited calcareous subsoil were employed to address the more complex situation in soil culture and to investigate the effects of Fe supply on PS release, Fe acquisition, and the structural and functional diversity of rhizosphere bacteria under ambient (400 µmol mol^–1) and elevated (800 µmol mol^–1) atmospheric CO₂ concentrations.

	Material and Methods

TOP NOTES ABSTRACT INTRODUCTION Material and Methods Results Discussion Concluding Remarks REFERENCES

 
Plant Culture
Barley (Hordeum vulgare L. cv. Europa) seeds were surface-sterilized by shaking in 30% H₂O₂ for 10 min, subsequently washed with deionized water, followed by 4 h imbibition in 10 mmol L^–1 CaSO₄. Germination was conducted in rolls of moist filter paper soaked with 2.5 mmol L^–1 CaSO₄ for 4 d in darkness and subsequent 3-d incubation with a 16 h light period. Thereafter, the seedlings were transferred to nutrient solution (10 groups of three plants each per 2.5 L pot) or to rhizobox microcosms (2 plants per box), respectively. In both the nutrient solution and in the rhizobox experiment each treatment was comprised of five replicates. The nutrient solution (pH 5.5) for hydroponic culture was composed of 2 mmol L^–1 Ca(NO₃)₂; 0.7 mmol L^–1 K₂SO₄; 0.5 mmol L^–1 MgSO₄; 0.25 mmol L^–1 KH₂PO₄; 0.1 mmol L^–1 KCl; 1 µmol L^–1 H₃BO₄; 0.5 µmol L^–1 MnSO₄; 0.5 µmol L^–1 ZnSO₄; 0.2 µmol L^–1 CuSO₄; 0.01 µmol L^–1 (NH₄)MO₇O₂₄ without (–Fe) or with (+Fe, 20 µmol L^–1 Fe-EDTA) Fe application in 2.5 L of continuously aerated nutrient solution.

Rhizobox microcosms contained 300 g of an air-dried calcareous loess subsoil low in plant-available Fe (CaCO₃ 215 g kg^–1; pH [CaCl₂] 7.6; C_org 1 g kg^–1; plant-available P [P_CAL] 5 mg kg^–1 [Schüller, 1969]; N_total 0.2 g kg^–1; Fe [DTPA extract]: 2.5 mg kg^–1 [Martens and Lindsay, 1990]; Fe [ammonium acetate extract] [Childs, 1981]: not detectable; Fe [water saturation extract; Rhoades, 1992]: not detectable) and were inoculated with 10% (w/w) of a fresh agricultural soil (Ap horizon) for microbial inoculation, and sieved to 2 mm mesh size. Fertilization was performed by soil application of 150 mg K kg^–1 as K₂SO₄; 100 mg N kg^–1 as Ca(NO₃)₂; 80 mg P kg^–1 as Ca(H₂PO₄)₂; 50 mg Mg kg^–1 as MgSO₄ without (–Fe) or with Fe application (+Fe) at 20 µmol Fe kg^–1 soil as Fe-EDTA. Soil moisture was adjusted daily to 20% (w/v) by gravimetric determination. The rhizoboxes were fixed with a horizontal angle of 50° to stimulate root growth along the transparent root observation window of the boxes. Plant cultivation was performed under controlled environmental conditions in growth chambers with a 16/8 h day/night cycle, light intensity of 300 µmol m^–2 s^–1, a 25/20°C day/night temperature regime with a relative humidity of 60%, and either 400 µmol mol^–1 (ambient) or 800 µmol mol^–1 (elevated) atmospheric CO₂ concentrations. Elevated CO₂ concentrations were adjusted to 800 µmol mol^–1 ± 5% by automatic injection of pure CO₂ using an automatic CO₂ controller (Siemens, Ditzingen, Germany) in one growth chamber. The ambient CO₂ concentration of approximately 400 µmol mol^–1, characteristic for the region of Stuttgart, was applied in the other growth chamber.

Collection of Root Exudates, Plant Harvest, and Soil Sampling
Collection of phytosiderophores exuded from apical root zones was performed by application of filter papers as sorption media onto the root surface (Neumann et al., 1999). At 16, 22, and 28 d after sowing (DAS), plants were removed from the nutrient solution at 2 h after onset of the light period. The roots were spread on a moist perspex plate, five first-order lateral roots of each plant selected for exudate collection were carefully separated from the root system, and the remaining roots were covered with filter paper and moistened with nutrient solution. Filter discs (5 mm diameter; filter paper: Schleicher & Schuell 2992, Dassel, Germany; previously washed with methanol and deionized water; water uptake capacity 63 µL cm^–2), moistened with distilled water, were placed onto the surface of the sub-apical root zone (at 0.5–2.5 cm distance from the root tip) for trapping of PS released from the roots. During incubation, the filters were occasionally re-moistened with 10 to 20 µL of distilled water. After 3 h, the filter discs from 5 root tips and the corresponding root segments were harvested separately into 1.5 mL reaction vials and stored at –20°C until further analysis. At each sampling date, plants were harvested and separated into shoots and roots for biomass determination.

The same method for exudate collection was performed at 16, 22, and 28 d after sowing (DAS) for roots of soil-grown barley plants, appearing at the root observation windows of the rhizobox microcosms, a technique which has been successfully employed for the collection of carboxylates in rhizosphere soil solutions of Lupinus albus (Wasaki et al., 2005). Short-term collection (3 h) was performed to minimize microbial degradation of PS and to recover a high proportion of root exudates (Neumann and Römheld, 2000). Rhizosphere soil samples were collected by use of forceps at 28 DAS in the same sub-apical root zones used for exudate collection at a distance of 0 to 3 mm from the root surface. All samples were frozen immediately and stored at –20°C until further analysis.

Determination of Chlorophyll, Plant Dry Matter, and Iron Analysis
Nondestructive determination of leaf chlorophyll content in soil-grown plants was conducted at 28 d after sowing by SPAD readings (Monje and Bugbee, 1992) using a portable chlorophyll meter (Minolta SPAD-502, Tokyo, Japan). Dry matter of shoot and roots was determined after oven-drying at 60°C for 2 d. An aliquot of 200 mg dry plant tissue, homogenized in a mill, was digested by dry ashing at 500°C for 4 h in a muffle furnace. After cooling, the samples were extracted twice with 2 mL of 3.4 mol L^–1 HNO₃ and evaporated to dryness on a heating plate to precipitate SiO₂. The ash was dissolved in 2 mL of 4 mol L^–1 HCl, subsequently diluted 10 times with hot deionized water, and boiled for 2 min. After addition of 0.1 mL Cs/La buffer to 4.9 mL ash solution, Fe concentration was measured by atomic absorption spectrometry (UNICAM 939, Offenbach/Main, Germany).

Analysis of Phytosiderophores in Root Exudates and Root Tissue
Extraction of root exudates from the filters was performed by addition of 500 µL 12.5% (w/w) NaOH solution with subsequent vigorous mixing and centrifugation. The supernatant was used for high performance liquid chromatography (HPLC) analysis according to Neumann et al. (1999). Extraction of PS from root tissue was performed with lateral roots of soil-grown barley plants modified after the method of Stephan and Rudolph (1984). Dried root material was homogenized to a fine powder with a mill. Hot distilled water (100°C) was added to aliquots of the powdered root tissue (500 µL 100 mg^–1 dry matter) and incubated for 10 min at 80°C in a thermo-mixer. Insoluble material was removed by 10 min centrifugation at 12000 rpm and the pellet was re-extracted with 500 µL of boiling water as described above. After a second centrifugation step, the supernatants were combined and subjected to HPLC analysis. Anion exchange HPLC analysis of PS in root exudates and root tissue extracts was performed on a Shimadzu liquid chromatograph (HPLC-LC 10 series) equipped with an automated sample injector (SIL– 10 A), a low pressure gradient mixer (FCV– 10 AL), a post column derivatization reactor (CRB-6A) with two reaction coils connected in series, a fluorescence detector (RF-10A), and a chromatography data system (LC Workstation Class LC10, Shimadzu, Duisburg, Germany). Separation of 20-µL samples was performed at 32°C on a Dionex AS11 column (250 x 4 mm i.d.), equipped with a Dionex AG11 guard column (50 x 4 mm I.D., Dionex, Idstein, Germany), by use of an aqueous NaOH gradient with a flow rate of 1 mL min^–1. Post-column fluorescence labeling of separated mugineic acids (MAs) was performed with orthophthaldialdehyde (OPA) according to Neumann et al. (1999) without addition of Brij-30. In a first reaction coil (Teflon capillary, 800 x 0.25 mm i.d.), MAs were oxidized with a sodium hypochlorite (NaOCl) solution (Neumann et al., 1999) to enable the reaction with OPA, which was performed in the second reaction coil (Peek capillary, 1900 x 0.25 mm i.d.). Derivatization reagents were mixed with the eluent stream at a flow rate of 0.8 mL min^–1 and the reaction temperature was adjusted to 50°C. Fluorescence detection was performed with a wavelength of 330 nm for excitation and 440 nm for fluorescence emission.

Soil Enzyme Activities and PCR-DGGE Analysis for 16S rRNA Genes
The functional diversity of rhizosphere microbial communities was characterized by activities of marker enzymes (Kandeler et al., 2002). A range of hydrolytic enzymes, involved in C, N, and P cycling in soils, were investigated using a fluorescence microplate assay according to Marx et al. (2001). The activities of β-glucosidase, xylosidase, cellobiosidase, N-acetyl-β-glucosaminidase, and acid phosphatase were measured using MUF (4-methylumbelliferyl)-, β-1,4-glucoside, β-xyloside, cellobioside, N-acetyl-β-1,4-glucosaminide, and phosphate as substrates, respectively. AMC (7-amino-4-methylcoumarin)-leucine and -tyrosine were used as substrates for leucine- and tyrosin-aminopeptidase analysis. TRIZMA buffer (0.05 mol L^–1, pH 7.8) was used for the analysis of peptidase activities and MES buffer (0.1 mol L^–1, pH 6.1) for the measurement of the other enzyme activities. The substrates (10 mmol L^–1) were dissolved in DMSO (dimethylsulfoxide) and diluted to 1 mmol L^–1 with the appropriate autoclaved buffer. Rhizosphere soil (0.5 g) was dispersed with 50 mL sterile, distilled water by an ultrasonic dis-aggregator (50 J s^–1 for 120 s). 50 µL of the soil suspension, 100 µL of the respective buffer, and 100 µL substrate solution were combined in 96 well microplates (OptiPlate 96F; Greiner Bio-one, Frickenhausen, Germany). Microplates were incubated at 30°C and fluorescence measurements were performed after 30, 60, 120, and 180 min using a microplate reader (Flx 800; Bio-Tec Instruments, Winooski, VT, USA; excitation wavelength 360 nm, emission wavelength 460 nm). Enzyme activities were expressed as the increasing release rates of MUF and AMC (µmol kg^–1 h^–1).

Changes in rhizosphere bacterial community structures were investigated by PCR-DGGE analysis for 16S rRNA genes according to Wasaki et al. (2005).

Statistical Analysis
Enzyme activities, shoot Fe content, shoot Fe concentration, and PS concentration of lateral root tissues were calculated on dry weight basis (105°C for soil, 60°C for plant material). Data of plant biomass, PS exudation, Fe nutritional status, leaf chlorophyll levels, PS concentration in lateral root tissue, and soil enzyme activities were analyzed by two-factorial analysis of variance (ANOVA). The factors were CO₂ (ambient and elevated CO₂ concentrations) and Fe (with and without Fe fertilization). Tukey's HSD test was used for comparison of means. A statistical probability P < 0.05 was considered as significant. The STATISTICA 6.0 software package was used for ANOVA.

	Results

TOP NOTES ABSTRACT INTRODUCTION Material and Methods Results Discussion Concluding Remarks REFERENCES

 
Plant Growth and Iron Nutritional Status
In hydroponic culture, elevation of atmospheric CO₂ concentrations significantly increased shoot and root biomass production, both in Fe-sufficient and Fe-deficient plants (Fig. 1 , Fig. 2a ). This effect was already detectable in early stages of plant growth in hydroponics at 16 DAS (Fig. 2a and Table 1 ). Chlorosis symptoms appeared under more severe Fe deficiency after 2 to 3 wk of the culture period (Fig. 1). Iron deficiency chlorosis was particularly expressed under elevated CO₂ treatments but was still associated with higher biomass production than in Fe-deficient plants cultivated at ambient CO₂ concentrations (Fig. 1). Severe Fe limitation at 28 d after sowing increased the root/shoot ratio and the appearance of this effect was accelerated in response to elevated atmospheric CO₂ concentration, while no CO₂ effects were detectable in Fe-sufficient plants grown in nutrient solution (Fig. 2b).

View larger version (99K): [in this window] [in a new window]   Fig. 1. Hordeum vulgare grown in nutrient solution 26 days after sowing (DAS). Appearance of strong iron deficiency chlorosis particularly expressed under elevated CO2.

View larger version (47K): [in this window] [in a new window]   Fig. 2. (a-d) Shoot and root biomass and root/shoot ratio of Hordeum vulgare grown in nutrient solution and in soil culture on a calcareous Loess subsoil, depending on plant age, Fe supply, and atmospheric CO2 concentration. Bars represent means and standard errors. Different letters indicate significant differences at the P < 0.05 level between treatments for each single harvest time

View larger version (24K): [in this window] [in a new window]   Fig. 3. (a-c) Fe-nutritional status and leaf chlorophyll levels (SPAD values) of soil-grown barley plants at 28 days after sowing (DAS), depending on Fe supply and atmospheric CO2 concentration. Bars represent means and standard errors. Different letters indicate significant differences at the P < 0.05 level. The dashed line symbolizes the average Fe concentration sufficient for average plant growth (Marschner, 1995).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Phytosiderophore exudation in 0.5 to 2.5 cm apical root zones of barley plants grown in nutrient solution, depending on plant age, Fe supply, and atmospheric CO2 concentration. Bars represent means and standard errors. Different letters indicate significant differences at the P < 0.05 level between treatments for each single harvest time.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Phytosiderophore concentrations in lateral root tissue of barley plants grown in rhizoboxes at 28 days after sowing (DAS). Bars represent means and standard errors. Different letters indicate significant differences at the P < 0.05 level.

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Material and Methods Results Discussion Concluding Remarks REFERENCES

Apart from the increased internal Fe use efficiency under elevated CO₂, there are also indications for an improved efficiency in Fe acquisition: Increased root biomass production of Fe-deficient barley plants in response to elevated CO₂ treatments (Fig. 2a, c) may reflect an improved capacity for spatial Fe acquisition, although other morphological root traits important for nutrient uptake, such as root length density or production of fine roots and root hairs (Neumann and Römheld, 2002) have not been investigated in this study.

Moreover, also the well-documented "Strategy-II" mechanism for chemical Fe acquisition in graminaceous plant species, based on release of phytosiderophores as metal chelators, with particularly intense expression in barley (Takagi et al., 1976; Neumann and Römheld, 2000) seems to be further stimulated by elevated atmospheric CO₂ concentrations. In hydroponic culture, PS release from lateral roots of Fe-deficient barley plants was increased by elevated CO₂ in the root zone of maximum release at a distance of 0.5 to 2.5 cm behind the root tip (Marschner et al., 1987, Walter et al., 1995a,b). This effect was particularly expressed under conditions of severe Fe deficiency at 28 d after sowing (Fig. 4) and may be explained by a higher vitality of CO₂–treated plants as a consequence of the increased internal Fe use efficiency, associated with an improved function of basic metabolic processes, such as photosynthesis. As an alternative explanation, the well-documented increased root/shoot partitioning of carbohydrates in response to elevated atmospheric CO₂ concentrations (Rogers et al., 1994) may provide an improved carbohydrate supply for the biosynthesis of PS. Accordingly, Erenoglu et al. (1996) found increased release rates of PS in barley when the photosynthetic carbohydrate supply for biosynthesis of PS in the root tissue was improved with increasing light intensities.

In barley, the highly localized exudation of large PS amounts in subapical root zones, concentrated to a distinct time interval during the few hours after onset of the light period (Marschner et al., 1987; Walter et al., 1995a,b), is assumed to be a strategy to compensate for unspecific adsorption and microbial degradation of PS in the rhizosphere (von Wirén et al., 1993, 1995). Increased PS exudation from roots of Fe-deficient barley plants under elevated atmospheric CO₂ concentrations may therefore improve Fe mobilization in the rhizosphere and increase the competitiveness for Fe mobilization with rhizosphere microorganisms (von Wirén et al., 1993, 1995; Yehuda et al., 1996; Hördt et al., 2000).

Unfortunately, detection of localized PS release by filter papers, applied as sorption media onto the root surface (Neumann et al., 1999; Wasaki et al., 2005), was not possible. In soil-grown plants, however, this technique has been successfully employed in previous studies to detect carboxylate release from plant roots in soil culture (Dinkelaker et al., 1997; Marschner et al., 2002; Wasaki et al., 2005). This may be attributed to limited rates of PS release, suppressed by the high Fe-nutritional status of the soil-grown plants (1.0–3.7 mmol Fe kg^–1 DM), which did not drop below the sufficiency levels of 0.6 to 2.7 mmol Fe kg^–1 DM reported in the literature for barley (Reuter and Robinson, 1997), even in the treatments without Fe supply (Fig. 3b). Recovery of PS can be further limited by adsorption to the soil matrix (Römheld, 1991) as a consequence of equilibrium formation between PS collected in the filter papers and the soil solution (Neumann and Römheld 2000), by intense microbial degradation, or by the well documented re-uptake of PS as metal chelates by the roots of barley plants (Neumann and Römheld, 2000; Curie et al., 2001).

However, the comparatively high Fe nutritional status of the soil-grown barley plants even without additional Fe application (Fig. 3b), does not reflect a high Fe availability in the calcareous loess sub-soil used for the experiments, which was selected to induce the expression of Fe deficiency responses as strongly as possible. Soil analysis data revealed no easily plant-available Fe fractions in the water saturation extract (Rhoades, 1982) or after 1 mol L^–1 ammonium acetate extraction (Childs, 1981). Even DTPA-(chelator)-extractable Fe (2.8 mg Fe kg^–1 soil) was below the critical Fe level of approximately 5 mg kg^–1 soil, reported in the literature (Martens and Lindsay, 1990). The seed reserves of 0.05 µmol Fe plant^–1could account only for 2.5 to 10% of the total Fe content in shoots of the test plants (0.5–2.0 µmol plant^–1; Fig. 3a) at final harvest. Therefore, the comparatively high Fe concentrations in shoot tissue of soil-grown barley plants without Fe supply can be only explained by intense mobilization of sparingly soluble Fe fractions in the soil, probably mediated by release of phytosiderophores. The resulting high intracellular Fe accumulation, detected at the end of the culture period, probably induced a temporal downregulation of PS exudation, leading to PS release rates below the detection limit of the exudate analysis. A contribution of PS to Fe acquisition of the investigated barley plants is further supported by the finding that PS accumulation in the root tissue was detectable even in the variants with additional Fe fertilization (Fig. 5) at a level which is comparable to the PS concentrations reported by Walter et al. (1995a,b) for the root tissue of Fe-deficient barley, grown under controlled conditions in hydroponic culture. Higher shoot contents of Fe in barley plants exposed to elevated atmospheric CO₂ concentrations in soil culture (although not in all cases significant, Fig. 3a), may reflect improved Fe acquisition by CO₂–induced stimulation of root growth (Fig. 2c), and higher release rates of PS as a consequence of a higher Fe demand for plant growth at elevated CO₂ concentrations (Fig. 2c). Also higher root to shoot translocation of Fe under conditions of elevated CO₂ may contribute to an increased Fe content in the shoot. However, in contrast to Fe contents, the concentrations of Fe in the shoot tissue declined in plants without Fe supply but there were no significant differences between CO₂ treatments (Fig. 3b). Obviously, the surplus of Fe, which accumulated in the shoot of CO₂–treated plants, was immediately used for new biomass production without increasing the Fe tissue concentrations. This was, however, not associated with symptoms of Fe-deficiency chlorosis in young leaves and accordingly, there were no significant differences in SPAD readings between treatments (Fig. 3c). The comparatively low expression of CO₂ effects in soil-grown barley plants may be attributed to the better Fe-nutritional status as a consequence of Fe mobilization in soil culture, compared with plants grown in hydroponics without any Fe supply, where CO₂ effects were most apparent under severe Fe deficiency at 28 DAS with strong expression of Fe deficiency chlorosis (Fig. 1, 4).

Rhizosphere Soil Bacterial Community
Various studies have suggested that elevated CO₂ may increase C input into soils through enhanced primary production by plants, which may stimulate microbial activities (Ebersberger et al., 2004; Freeman et al., 2004; Kang et al., 2005). As PS are a type of non-proteinogenic amino acids, released in high amounts into the rhizosphere of graminaceous plant species in response to Fe limitation, they may be easily degraded by rhizosphere microorganisms, providing an excellent source of both carbon and nitrogen (Watanabe and Wada, 1989; von Wirèn et al., 1993).

However, this study with barley plants revealed no indication for distinct effects of Fe supply or atmospheric CO₂ concentrations on structural and functional diversity of rhizosphere bacteria in 0.5 to 2 cm sub-apical root zones (Table 2), as the sites with the most intense Fe deficiency-induced PS exudation (Marschner et al., 1987; Walter et al., 1995a,b). In contrast, using the same methodology, earlier studies on the release of carboxylates in P-deficient Lupinus albus revealed significant effects of root exudation on rhizosphere-microbial communities in a distance of 1 to 3 mm from the root surface (Wasaki et al., 2005). Calculated release rates of PS in barley could easily reach millimolar levels of PS accumulation in the rhizosphere (Römheld, 1991), but in the current experiment, the expression of this effect may be much smaller due to suppression of PS release when the Fe nutritional status of the plants was high enough after a period of Fe mobilization. Moreover, the restriction of exudation pulses over a short time period of several hours in apical root zones with low densities of microbial colonization, continuously moving through the soil with root growth rates of 1 to 2 cm d^–1 (Neumann and Römheld, 2000), as well as efficient re-uptake of Fe-PS by a highly selective transport system (Curie et al., 2001), are probably much too fast to induce any detectable changes in rhizosphere soil bacterial diversity by DGGE analysis of 16S rRNA gene fragments or by activity testing for marker enzymes of C, N, and P cycles. This view is further supported by findings of von Wirén et al. (1993, 1995), demonstrating Fe-deficiency chlorosis in maize and Sorghum with slow continuous release rates of PS, grown in nutrient solution and on a calcareous substrate. Chlorosis symptoms were induced by microbial PS degradation and could be avoided under axenic culture conditions. In contrast, no comparable symptoms were observed in barley with highly localized pulses of intense PS exudation in sub-apical root zones.

In contrast to the present study, conducted with rhizosphere soil at a distance of up to 3 mm from the root surface, Marschner and Crowley (1998) and Crowley (2000) reported distinct root-induced changes, investigating structure and function of eubacterial communities directly at the root surface in apical root zones of barley, depending on the Fe-nutritional status. DGGE analysis of 16S rDNA profiles revealed the formation of characteristic communities in response to Fe limitation, and species diversity increased with increasing distance from the root apex (Crowley, 2000). Iron deficiency of Pseudomonas fluorescens, indicated by enhanced production of the bacterial siderophore pyroverdin, was increased at the rhizoplane in apical root zones of Fe-deficient barley plants when PS exudation was suppressed by foliar Fe application (Marschner and Crowley, 1998). According to radial diffusion gradients of root exudates (Neumann and Römheld, 2002), a stronger expression of effects on microbial communities can be expected directly at the root surface compared with the outer rhizosphere soil, investigated in the present study.

	Concluding Remarks

TOP NOTES ABSTRACT INTRODUCTION Material and Methods Results Discussion Concluding Remarks REFERENCES

 
Elevated atmospheric CO₂ concentrations have a strong impact on the expression of adaptations to Fe deficiency in barley as a representative Strategy II plant. The CO₂–induced modifications comprise: (i) increased internal Fe use efficiency; (ii) stimulation of root growth, and (iii) increased root exudation of Fe-mobilizing phytosiderophores in the sub-apical root zones, as demonstrated by experiments conducted under controlled environmental conditions in hydroponic culture. If this holds true also for graminaceous plant species grown under natural conditions, a further improvement of the efficient Strategy II mechanism for acquisition of Fe and Zn in response to elevated atmospheric CO₂ may have important consequences for interspecific competition in plant communities, particularly on calcareous soils with low availability of Fe and micronutrients, favoring the growth of plants with a high potential for PS exudation. The absence of detectable Fe, and CO₂ effects on diversity of eubacterial communities in the rhizosphere may reflect the comparatively low expression of Fe deficiency responses in soil-grown plants. This however, does not exclude an impact on plant-microbial interactions, which have not been considered in the present study. Improved acquisition of Fe and micronutrients such as Zn and Mn under elevated atmospheric CO₂ may improve plant pathogen resistance as co-factors for defense reactions or by direct competition for Fe with pathogens (Graham and Webb, 1991; Crowley, 2000). Further investigations should also consider (i) longer culture periods in pot experiments for a stronger expression of Fe deficiency responses in soil culture, (ii) other plant species, and (iii) long-term studies under field conditions with different soils to assess the ecological significance of the observed CO₂ effects on Fe efficiency in graminaceous plants.

	ACKNOWLEDGMENTS

 
This work was supported by the German Research Foundation.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Material and Methods Results Discussion Concluding Remarks REFERENCES

 
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