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ENVIRONMENTAL STRESS AND ADAPTATION

Nitrate Does Not Result in Iron Inactivation in the Apoplast of Sunflower Leaves¹

Institut für Pflanzenernährung (330), Universität Hohenheim, D–70593 Stuttgart, Germany

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
It has been hypothesized that nitrate (NO₃^–) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069–1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO₃^–, NH₄⁺ and NH₄NO₃), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 µM ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO₃^– supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted ⁵⁹Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO₃^– (6 mM) showed no effect on ⁵⁹Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased ⁵⁹Fe uptake, independently of apoplastic pH or of the presence of NO₃^– in the apoplastic solution. Finally, there are no indications in the present study that NO₃^– supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO₃^– nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.

It is well known that at high concentration in the soil solution, HCO₃^– penetrates the root apoplast, thereby neutralizing H⁺ ions and lowering Fe uptake by root cells as a consequence of inhibition of plasma membrane-bound Fe^III reductase at high apoplastic pH (Fleming et al., 1984; Römheld and Marschner, 1986; Toulon et al., 1992). There is no evidence, however, that free dissolved HCO₃^– can be transported from root to shoot via the xylem to a high enough extent to cause a substantial increase in the pH of the xylem sap (Bialczyk and Lechowski, 1995; Peiter et al., 2001). So far, there is good confirmation that most of the dissolved HCO₃^– taken up is utilized in the processes of organic carbon fixation (e.g. phosphoenolpyruvate carboxylase) by roots (López-Milán et al., 2000). The presence of high HCO₃^– in the nutrient solution was shown to affect neither the bulk pH of apoplastic fluid obtained by centrifugation of intact leaves (Nikolic and Römheld, 1999, 2002) nor the apoplastic pH measured by using a pH-sensitive fluorescent dye (fluorescein isothiocyanate-dextran) after infiltration of xylem sap into excised leaves (Kosegarten et al., 1999). Furthermore, HCO₃^– does not appear as a cause of physiological Fe inactivation in the leaf apoplast nor does it cause an inhibition of Fe uptake into the leaf symplast (Nikolic and Römheld, 2002), which is also in agreement with the recent conclusions of Kosegarten et al. (1999, 2001).

Although the effect of the form of nitrogen (N) supply (NO₃^– versus NH₄⁺) on pH changes in nutrient solutions and rhizosphere have been extensively studied over the past two decades, the effect of N form on the pH of leaf apoplast is still a matter of controversial debate (for review, see Sattelmacher, 2001). It has been argued that NO₃^– nutrition induces an alkalization of leaf apoplast (Mengel et al., 1994; Kosegarten et al., 1999, 2001), although there is no clear evidence that this occurs to any great extent (Mühling and Sattelmacher, 1995; Sattelmacher et al., 1998; Mühling and Läuchli, 2001). Also, there are other factors, including NO₃^– reductase activity in the roots (Andrews, 1986) and light-induced pH changes in the leaf apoplast (Mühling and Läuchli, 2001), which may modify any possible potential effect of NO₃^– supply in the nutrient solution on leaf apoplastic pH. According to Mengel's hypothesis, inactivation of Fe in the leaf is caused primarily by NO₃^– supplied from the roots (Mengel, 1994; Mengel et al., 1994), and it is postulated that Fe trapped in the leaf apoplast at high pH can be mobilized by lowering the apoplastic pH that favors Fe^III reduction and, thus, Fe uptake into the symplast (Kosegarten et al., 2001). The main support for this Fe inactivation concept comes from the findings that spraying chlorotic leaves with dilute organic or mineral acids is followed by leaf regreening of field-grown kiwifruit (Actinidia deliciosa) and peach (Prunus persica) trees (Tagliavini et al., 1995; 2000) or sunflower (Helianthus annuus) plants grown in nutrient solutions (Kosegarten et al., 2001). However, Nikolic and Römheld (1999) reported that although the pH of leaf apoplast is important in regulation of extracellular Fe^III reduction and Fe uptake into the symplast of leaf cells, this effect was of only marginal significance within the physiologically relevant pH range as measured in the leaf apoplastic fluid. In contrast to roots, there is little information in the literature on Fe distribution between apoplast and symplast in leaves (Pich and Scholz, 1991; Becker et al., 1992; Nikolic and Römheld, 2002), and there appears to be a particular lack of experimental data relating to Fe-binding forms in the leaf apoplast caused by various sources of N nutrition.

In this study, we tested Mengel's hypothesis of Fe inactivation in leaves caused by NO₃^– nutrition using the same model plant, sunflower, as has commonly been used in many experiments performed by Mengel et al. (Mengel et al., 1994; Hoffmann and Kosegarten, 1995; Kosegarten et al., 1998a, 1998b, 1999, 2001). The objectives of the work presented here were: (a) to clarify the effect of NO₃^– supply to roots on postulated Fe inactivation in the leaf apoplast as a primary cause of NO₃^–-induced Fe deficiency chlorosis; (b) to investigate the influence of form of N supply and pH of nutrient solution stabilized by pH buffers on concentration of Fe in the two-leaf compartments, apoplast, and symplast; and c) to characterize the leaf apoplast for various binding forms of Fe. Furthermore, we studied the direct effect of pH and the presence of NO₃^– in the leaf apoplast of excised leaves on Fe uptake by the symplast of leaf cells. In the present work, a method suitable for determination of different Fe-binding forms in the leaf apoplast, combining extraction of apoplastic washing fluid (AWF) with isolation of cell walls from sunflower leaves has been developed. In this new approach, Fe uptake by leaf symplast as affected by pH and NO₃^– is measured using various radioactive labeled ⁵⁹Fe solutions infiltrated into the apoplast of excised leaves via petiole by the transpiration stream.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Effects of N Form and Nutrient Solution pH on Chlorophyll, Growth, and Fe Utilization

The pH of unbuffered nutrient solution supplied with NO₃^– shifted from 5.6 in fresh nutrient solution to about 6.8 before the next solution change (data not shown). However, the pH of NH₄NO₃-supplied nutrient solution without pH buffering was continually low, alternating between 5.5 and 4.5. As shown in Figure 1 and Table I, Fe deficiency chlorosis on the leaves of sunflower plants only occurred in the treatments with a high pH in the nutrient solution (NO₃^–, unbuffered; NH₄⁺ and NH₄NO₃, buffered at pH 7.5) at low Fe supply (1 µM). Although severe chlorosis symptoms were found in either NH₄⁺ or NH₄NO₃ treatment with pH stabilization at 7.5 using HEPES buffer, NO₃^– treatment without pH control (about pH 6.8) showed only moderate chlorosis (Fig. 1; Table I). Maintaining low pH at 5.0 using MES buffer, even at an extremely high concentration of NO₃^– (40 mM), did not result in any decrease in leaf chlorophyll content compared with the unbuffered NH₄NO₃ treatment (about pH 4.5).

View larger version (69K): [in this window] [in a new window]   Figure 1. Effects of N form and nutrient solution pH on Fe deficiency chlorosis occurrence in sunflower plants after 2 weeks of growth in nutrient solution supplied with 1 µM Fe. Top (left to right): 2 mM NH4NO3 (pH 4.5, unbuffered) and NH4NO3 (pH 7.5, HEPES). Middle (left to right): 4 mM NH4+ (pH 5.0, MES) and 4 mM NH4+ (pH 7.5, HEPES). Bottom (left to right): 4 mM NO3– (pH 5.0, MES), 4 mM NO3– (pH 6.8, unbuffered), and 40 mM NO3– (pH 5.0, MES).

The growth of sunflower plants, estimated as dry weights of shoot and roots, were significantly reduced in all treatments with high pH (NO₃^–, unbuffered; NH₄⁺ and NH₄NO₃, pH 7.5, HEPES). Furthermore, this was closely related to chlorophyll content, especially in severely chlorotic plants (Table I). The NH₄NO₃ treatment with pH of the nutrient solution buffered to 7.5 showed strong inhibition of leaf expansion growth, whereas leaf area was only slightly decreased in NO₃^–-treated plants without pH control (Table I). However, plant growth was significantly reduced in green plants too but only when the NH₄⁺-N was the sole form supplied at low pH. Root Fe concentration and particularly Fe amount were significantly lower in chlorotic plants than in green ones (Table II). Both concentration and amount of leaf Fe also markedly decreased in chlorotic plants (2–3 times) grown in various N treatments with high pH in the nutrient solution. It is obvious that in all treatments where nutrient solution pH was high, NH₄⁺ and NH₄NO₃ treatments with pH of nutrient solution buffered to 7.5, and NO₃^– treatment without pH buffering, Fe uptake was strongly restricted (Table II). Also, as clearly shown in Figure 2, Fe concentration in xylem sap significantly decreased in both NO₃^–- and NH₄NO₃-fed plants grown at high external pH in the nutrient solution supplied with various N forms (NO₃^–, unbuffered; NH₄NO₃, pH 7.5, HEPES) as compared with plants grown at low external pH (NO₃^–, pH 5.0, MES; NH₄NO₃, unbuffered).

View larger version (11K): [in this window] [in a new window]   Figure 2. Effect of N form and pH of the nutrient solution on Fe concentration in xylem sap. Plants were grown for 2 weeks in nutrient solutions at 4 mM N supplied with either NO3– or NH4NO3. Black bars, Green (NO3–, pH 5.0; NH4NO3, unbuffered); white bars, chlorotic (NO3–, unbuffered; NH4NO3, pH 7.5). Data are means ± SD of four replications. Significant differences (P < 5%) between treatments are indicated by different letters.

Effects of N Form and pH of Nutrient Solution on pH and NO₃^– Concentration in Xylem Sap and Leaf Apoplastic Fluid

No significant pH differences were found in xylem sap among treatments with different forms of N supply (Table III). NO₃^–-treated plants always showed higher pH of bulk apoplastic fluid for 0.2 to 0.3 pH units than other N treatments (i.e. NH₄⁺ and NH₄NO₃), although these differences were not significant (Table III). However, no differences in the pH of apoplastic fluid were found between green and chlorotic leaves of plants solely fed with NO₃^– with or without pH control. Moreover, xylem sap and leaf apoplastic pH were not decreased even in the plants where Fe deficiency chlorosis was alleviated by adding 25% of N as NH₄⁺ (data not shown).

In all N treatments, the NO₃^– concentration in leaf apoplastic fluid was much lower than in xylem sap (Fig. 3). The NO₃^– concentration did not differ either in xylem sap or was the same in the apoplastic fluid of both green and chlorotic plants grown in either NO₃^– or NH₄NO₃ treatment supplied with 4 mM N. However, when plants were grown in nutrient solution with 40 mM NO₃^–, the NO₃^– concentration in both xylem and apoplastic sap increased 2 to 3 times (Fig. 3), but high apoplastic NO₃^– did not result in any decrease of chlorophyll content (Table I; Fig. 1). Almost no NO₃^– was detected in xylem sap (below 0.5 mM) and apoplastic fluid (below 0.1 mM) in NH₄⁺ treatments (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 3. Concentration of NO3– in xylem sap (A) and leaf apoplastic fluid (B) as affected by N forms and external pH. Plants were grown for 2 weeks in nutrient solutions supplied with NO3– (4 or 40 mM N) or NH4NO3 (4 mM N). Black bars, Green (NO3–, pH 5.0; NH4NO3, unbuffered); white bars, chlorotic (NO3–, unbuffered; NH4NO3, pH 7.5). Data are means ± SD of four replications. Significant differences between treatments (P < 5%) are indicated by different letters.

Effects of N Form and Nutrient Solution pH on Fe Compartmentation in the Leaves

The Fe concentrations in both leaf compartments, apoplast and symplast, significantly decreased in chlorotic leaves of sunflower plants grown at high external pH in nutrient solutions supplied with various N forms (NO₃^–, NH₄⁺, and NH₄NO₃) as compared with green leaves of plants grown at low external pH (Table IV). Although the absolute apoplastic Fe concentration was about 2-fold higher in green leaves as compared with chlorotic ones, the relative proportion of apoplastic Fe remained almost unchanged (22%–28%) in all treatments. Thus, there were only relatively small differences in distribution of symplastic Fe and various apoplastic Fe binding forms between green (low external pH) and chlorotic (high external pH) leaves, regardless of whether NH₄NO₃^– or sole NO₃^–-N form was supplied (Fig. 4). In contrast, the relative proportion of soluble Fe presents in AWF was about 2 times lower in chlorotic leaves (all N treatments with high external pH) as compared with green ones (all N treatments with low external pH), but this fraction only represents 0.4% to 1% of total leaf Fe. Total cell wall Fe concentration and concentrations of both Fe-binding forms in the cell walls (i.e. weakly and strongly bound) decreased about 2-fold in chlorotic leaves in all N treatments when nutrient solution pH was kept high (Table V).

View larger version (26K): [in this window] [in a new window]   Figure 4. Effects of N forms and external pH on relative amount of Fe fractions (symplastic and apoplastic) and apoplastic Fe binding forms of sunflower leaves. Light gray, Symplastic Fe; gray, apoplastic Fe, weakly bound (cell walls); dark gray, strongly bound (cell walls); black, soluble (AWF).

Maintaining a constant low pH (pH 5.0, MES), plants grown even at an extremely high NO₃^– concentration in the nutrient solution (40 mM) did not differ in Fe concentration in leaf cell wall from those grown in a solution at a 10 times lower NO₃^– concentration (4 mM; 3.9 and 4.2 µmol g^–1 dry weight, respectively) and, as a consequence, the leaves remained green (Fig. 1; Tables I and V). The concentration of leaf cell wall Fe, however, was significantly lower in plants grown in at high pH (about 6.8) in nutrient solution without pH regulation (1.7 µmol g^–1 dry weight). Concentration of Fe in leaf cell walls showed a close correlation with leaf chlorophyll content regardless of N form, whereas increase in apoplastic NO₃^– concentration did not affect Fe concentration in the cell walls (Fig. 5, A and B). This clearly indicates that high leaf apoplastic NO₃^– concentration (up to 12 mM) did not induce any excessive Fe trapping in the cell walls (Fig. 5B).

View larger version (15K): [in this window] [in a new window]   Figure 5. Cell wall Fe concentration in relation to chlorophyll content (A) and NO3– concentration in leaf apoplastic fluid (B) in young, fully expanded leaves of sunflower. Plants were grown for 2 weeks in nutrient solutions supplied with NO3– (4–40 mM N) or NH4NO3 (4 mM N).

Fe Uptake by the Symplast of Intact Leaves as Dependent on Apoplastic pH and NO₃^– Concentration

Uptake solutions with radiolabeled ⁵⁹Fe citrate were infiltrated into the leaf apoplast by the transpiration stream via the petioles of excised leaves. This approach was used to clarify using short-term experiments whether high leaf apoplastic pH and/or high apoplastic NO₃^– concentration can inhibit Fe uptake by leaf symplast. To maintain the apoplastic pH at a defined value, various pH-buffered ⁵⁹Fe-labeled solutions were perfused into the xylem apoplast for 2 h. As shown in Figure 6, unbuffered solution, representing the indigenous pH conditions in the apoplast, and solutions buffered at low pH (5.0 and 6.0, respectively) did not differ significantly, whereas at a high buffered pH (7.0), Fe uptake was substantially decreased. These differences were much more pronounced when leaves were exposed to high light intensity (500 µmol m^–2 s^–1) than in darkness (Fig. 6). Furthermore, regardless of the pH of the uptake solutions, light strongly affected Fe uptake by the symplast of leaf cells. It is interesting to note that Fe uptake rate was even higher at pH buffered to 7.0 under light as compared with the uptake rate in darkness at low pH.

View larger version (12K): [in this window] [in a new window]   Figure 6. Uptake of 59Fe by the leaf symplast of sunflower, as depending of apoplastic NO3–. –NO3–, Plants were precultured in NO3–-free nutrient solution for 48 h, and less than 0.5 mM NO3– was measured in the apoplastic fluid obtained by centrifugation of intact leaves; no NO3– was added in 59Fe-labeled solution. +NO3–, Plants were grown in NO3–-supplied nutrient solution (4 mM), resulting in up to 5 mM NO3– in the leaf apoplastic fluid; 6 mM NO3– was added to the 59Fe-labeled solution. After perfusion of radioactive labeled ± NO3– solution into the leaf apoplast of excised leaves via the petiole (2 h) by the transpiration stream, leaves were further incubated in darkness or under light (500 µmol m–2 s–1) for 2 h. Black bars, Dark; white bars, light. Data are means ± SD of four replications. Significant differences (P < 5%) between treatments are indicated by different letters.

To study the effect of apoplastic NO₃^– on Fe uptake, 6 mM NO₃^– was infiltrated together with ⁵⁹Fe citrate as described previously (Fig. 7; see "Materials and Methods"). To avoid any effect of NO₃^– present in the leaf apoplast, plants were precultured in NO₃^–-free nutrient solution 48 h before the experiment. The presence of NO₃^– in the apoplastic solution showed no effect on Fe uptake by leaf symplast, either in darkness or when leaves were exposed to high light intensity (Fig. 7). However, light strongly stimulated ⁵⁹Fe uptake, regardless of the presence of NO₃^– in the apoplastic solution.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of apoplastic pH on 59Fe uptake by the symplast of intact sunflower leaves. 59Fe-labeled solution (10 µM Fe citrate, 1:100 molar ratio) without or with various pH buffers (50 mM MES [pH 5.0 and 6.0] and 50 mM HEPES [pH 7.0]) was infiltrated into the leaf apoplast of excised leaves (2 h) via the petiole by the transpiration stream. Leaves were further incubated in darkness or under light (500 µmol m–2 s–1) for 2 h. Black bars, Dark; white bars, light. Data are means ± SD of four replications. Significant differences (P < 5%) between treatments are indicated by different letters.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
To clarify any possible influence of NO₃^– (and other N forms) on the availability of Fe in sunflower leaves, it is necessary to distinguish the effects of changes in pH at the root surface induced by the various N forms on the uptake and translocation of Fe by the plant from the comparative effects of pH per se. To achieve this, the pHs of the nutrient solutions supplied with various N forms were stabilized using organic pH buffers (see "Materials and Methods"). These pH buffers diffuse from the nutrient solution to penetrate the root apoplast and stabilize it at desired values, thus counteracting possible pH changes modulated by the plasma membrane proton pump (Logan et al., 1997) or by proton cotransport of NO₃^– (Meharg and Blatt, 1995). From the literature, the buffers used (MES, acid dissociation constant_a = 6.15; and HEPES, pK_a = 7.55) at 5 mM concentration are effective enough to maintain the desired pH in the root apoplast either at 5.0 or 7.5, taking into account the buffering capacity of the isolated cell walls (Sentenac and Grignon, 1981; Wang et al., 1992).

Fe deficiency chlorosis always occurred when the pH of the nutrient solution was high regardless of the form of N supply (Fig. 1; Table I). When the pH of the nutrient solution was maintained low (pH 5.0, MES), the growth of NH₄⁺-fed plants was strongly inhibited in comparison with plants supplied with NO₃^– (Fig. 1; Table I), an observation reported for other plant species (Kirkby and Hughes, 1970; Raab and Terry, 1994; Walch-Liu et al., 2000). Strong growth inhibition was also observed in plants cultivated in high pH-buffered nutrient solution (pH 7.5, HEPES) supplied with NH₄⁺ or NH₄NO₃ and was associated with severe Fe deficiency (Fig. 1; Table I). The inhibition of both chlorophyll synthesis and plant growth was more prominent in both NH₄⁺- and NH₄NO₃-fed plants grown in the nutrient solutions pH buffered at 7.5 as compared with NO₃^–-fed plants grown in the nutrient solution without pH control (Fig. 1; Table I). This can be explained by the fact that the pHs of the buffered NH₄NO₃- and NH₄⁺-supplied nutrient solutions were constantly high (7.5), whereas the NO₃^–-fed plants were subjected to an alternating pH from 5.6 (initial pH in the renewed solution) to about 6.8 as measured before the nutrient solution change (data not shown), probably because of H⁺ consumption by roots via H⁺/NO₃^– symport (Meharg and Blatt, 1995). Thus, this NO₃^– treatment could provide a few hours of unrestricted Fe uptake at low pH after renewal of the nutrient solution every 2 d (see "Materials and Methods"). However, when the pH of NO₃^–-supplied nutrient solution was maintained high (pH 7.5, HEPES), plants showed severe chlorosis and rapid inhibition in shoot and root growth as a consequence of restricted Fe uptake (data not shown).

The higher root apoplastic pH caused either by the unbuffered NO₃^– supply or maintained by a HEPES buffer in NH₄⁺- or NH₄NO₃-fed plants significantly decreased both the concentration and the amount of Fe in roots and shoots, compared with the low pH treatments (Table II). Thus, restricted Fe uptake due to inhibited Fe^III reduction at high root apoplastic pH (Römheld and Marschner, 1986; Toulon et al., 1992) is most likely the primary cause of Fe deficiency chlorosis in sunflower plants. This conclusion is supported by the significantly lower Fe concentration in xylem sap in both NO₃^–-fed plants grown in the nutrient solution without pH control or either NH₄⁺- or NH₄NO₃-fed plants with buffered pH at 7.5 as compared with plants grown at low external pH (Fig. 2).

In contrast to our findings, Kosegarten et al. (1998a, 1998b) found that at a low-nutrient solution pH (from 4.0–5.0), controlled by a pH-stat device, NO₃^–-fed sunflower plants had a significantly lower chlorophyll concentration or even reduced leaf growth compared with those grown with NH₄NO₃. It would seem that the limited capability of the pH stat system to maintain constant low pH in the root apoplast of NO₃^–-fed plants used in these studies could be the major reason for a latent Fe deficiency before a decrease in leaf Fe concentration. Furthermore, Kosegarten at al. (1998a) and Zou et al. (2001), respectively, reported a significantly higher root Fe concentration in chlorotic sunflower and maize plants grown in nutrient solutions supplied with NO₃^– as compared with green, NH₄⁺-, or NH₄NO₃-fed plants. In both these studies, Fe concentration may have been overestimated as a consequence of Fe precipitation because of low chelate stability of FeEDTA above pH 6.5 (Strasser et al., 1999). In the present study, precipitation of Fe was avoided by growing the plants in the nutrient solutions supplied with ferric ethylenediaminedi(O-hydroxyphenylacetic acid), which is much more stable at high pH (up to 9.0) in well-aerated hydroponic solutions (Halvorson and Lindsay, 1972). Moreover, washing the roots by the method of Bienfait et al. (1985) ensured greater removal of extraplasmatic Fe, thus minimizing the possibility of overestimation of root Fe concentration (Table II; see also Strasser et al., 1999).

The pH of apoplastic fluid collected by the centrifugation technique from intact leaves (Table III) represents the bulk pH of the whole leaf apoplast as an average of various cells (i.e. parenchyma, adaxial, and abaxial epidermal cells) with different ion concentrations (Karley et al., 2000) so that any possible spatial pH changes are not revealed. The pH of apoplastic fluid from leaves of NO₃^–-fed plants collected 3 h after light onset was always higher by about 0.1 to 0.3 units than in NH₄⁺- or NH₄NO₄-fed plants (Table III); however, these differences were not statistically significant. These findings are in accord with the results of Kosegarten et al. (1999) showing mean data of the apoplastic pH determined by the fluorescence microscope imaging technique. However, a clear increase in apoplastic pH above 6.5 found in their study was only in the small intervenial area of young leaves (about 10% of the total apoplastic space), which they called microsites. Kosegarten et al. (1999) supposed that such increases in apoplastic pH of growing but chlorotic leaf microsites were a consequence of high NO₃^– uptake by rapidly expanding cells, presumably caused by the removal of protons from the apoplast. Experimental evidence is still lacking, however, that these microsites of high apoplastic pH represent areas of growth.

As evident from Figure 3, the NO₃^– concentration either in xylem sap or in apoplastic fluid collected from young leaves was not significantly different between NO₃^–- and NH₄NO₃-fed plants. Measured NO₃^– concentration in both xylem and leaf apoplastic fluids was in the range reported for sunflower plants in various studies (Dannel et al., 1995; Kosegarten et al., 1999; Sattelmacher et al., 1998). Higher NO₃^– concentrations in the xylem sap than in the apoplastic fluid (Fig. 3; Dannel et al., 1995) could presumably be due to NO₃^– uptake into the vacuoles of xylem parenchyma cells or because the exudates were from decapitated plants and, hence, obtained in the absence of a transpiration stream (Mühling and Läuchli, 2001). Interestingly, although NO₃^– was supplied in the nutrient solution in an excessive amount (40 mM), leading to a 2- to 3-fold increase in apoplastic NO₃^– concentration, no Fe chlorosis was found in young leaves when the pH of nutrient solution was maintained low with MES buffer (Table I; Figs. 3 and 5A).

Because nearly all of the assimilated NH₄⁺ is translocated to the shoot in organically bound form (e.g. amino acids), predominantly via the xylem, no marked uptake of NH₄⁺ ions from the apoplast into the symplast of leaf cells is to be expected (Dannel et al., 1995); therefore, a pH fall in the leaf apoplast appears unlikely if NH₄⁺ is supplied to roots (Mühling and Läuchli, 2001). Apoplastic pH may even be raised as a consequence of the removal of protons, presumably via proton/amino acids cotransport (Williams et al., 1990; Logan et al., 1997).

Loading of 6 mM NO₃^– into the leaf apoplast via the petiole of excised leaves of sunflower plants precultured in NO₃^– free nutrient solution did not affect ⁵⁹Fe uptake into the leaf symplast (Fig. 6). Infiltration of various buffer solutions into the excised leaves was very efficient in controlling the apoplastic pH of xylem vessels, allowing only minor pH changes (Kosegarten et al., 1999). As shown in Figure 7, perfusion of high pH-buffered solution (50 mM HEPES, pH 7.0) into the leaf apoplast restricted ⁵⁹Fe uptake rate by about 35% to 40% of that found at low pH (unbuffered solution; 50 mM MES, pH 5.0 and 6.0, respectively). These results are in agreement with the results of Nikolic and Römheld (1999) showing the pH dependence of Fe^III citrate reduction and ⁵⁹Fe uptake by leaf discs infiltrated by the solutions buffered at various pHs (5.0–7.0) and incubated in darkness.

A decrease in reduction of Fe^III citrate occurring only at high apoplastic pH caused by the infiltration of HEPES buffer (e.g. pH 7.0) into the apoplast of sunflower leaves has been demonstrated by two different experimental approaches, i.e. in leaf discs by measuring the absorbance of the ferrous biphenyl-phenanthrolinedisulfonic acid complex (Nikolic and Römheld, 1999) or in the excised leaves determined by microscope image analysis as formation of the Fe^II-ferrozine complex (Kosegarten et al., 1999). Considering that such a high pH (7.0) accounts for less than 3% of the total intervenial leaf apoplast of NO₃^–-fed plants (Kosegarten et al., 1999), it seems difficult to explain how such a small space can affect Fe uptake in the range sufficient to induce leaf chlorosis. In the remainder, which makes up about 90% of total intervenial apoplastic space, Fe^III reduction reactions are not inhibited (see Kosegarten et al., 1999), presumably providing enough Fe for that required for metabolic processes. Thus, quite regardless of the presence of high concentrations of NO₃^– in the leaf apoplast, Fe chlorosis does not appear if sufficient Fe is taken up by roots (e.g. at low pH-buffered nutrient solution) or if it has been loaded into the leaf xylem apoplast (Tables I and II; Figs. 3 and 5A).

As clearly shown in the presented study, light strongly increased Fe uptake rates into the symplast of leaf cells apart from the effect of apoplastic pH. Hence, the Fe uptake rate at high apoplastic pH (7.0) under high light intensity was even greater than that found at low apoplastic pH (5.0 and 6.0, respectively) in the dark (Fig. 7). Temporal variations in H⁺ concentration resulting from changes in metabolic activity caused by a dark/light transition leads to a biphasic apoplastic pH response (Mühling and Sattelmacher, 1995; Sattelmacher, 2001). Increase in apoplastic pH under dark conditions probably might be caused by inhibition of light-induced H⁺ extrusion into the leaf apoplast (Mühling and Läuchli, 2001). Light might also directly affect Fe uptake into the symplast of leaf cells by nonenzymatic photoreduction of Fe^III citrate in the leaf apoplast (Brown et al., 1979; Schmidt, 1999). In vitro photoreduction of Fe^III citrate by high light intensity (500 µmol m^–2 s^–1) was much less pH regulated in the range from 5.0 to 7.5 as compared with reduction by leaf tissue in darkness (M. Nikolic, unpublished data; Nikolic and Römheld, 1999).

Once taken up by the roots, Fe is transported to the shoots via the xylem, mainly in the form of Fe^III citrate complexes (Tiffin, 1966), and through the petioles, the xylem stream enters the leaf via the veins and moves toward the sites of high evaporation such as leaf margins (Sattelmacher, 2001). In accordance with this accepted concept, Fe^III can be bound to cell wall components, presumably to free carboxyl groups (e.g. pectins) before being taken up by the symplast of mesophyll cells. Thus, the relative proportion of soluble Fe present in AWF as various complexes with organic anions (e.g. citrate and malate) is very low, and most of the apoplastic Fe is bound to the cell wall (over 95%) and held at different binding strengths (Fig. 4). Because of spatial limitations for cations in vivo, the cation exchange capacity of isolated cell wall material is much higher than the cell walls of intact plant tissue (Sattelmacher, 2001). Thus, during the isolation procedure, the cell walls are able to fix a certain portion of Fe of symplastic origin (e.g. soluble Fe-containing proteins and Fe in vacuoles), which leads to an overestimation of the proportion of Fe located in the cell walls.

In contrast to what would have been expected from Mengel's hypothesis, NO₃^– supply to the unbuffered nutrient solution resulted in significant decreases in concentration of leaf Fe in both compartments, symplast and apoplast, with a tendency to maintain of the relative proportion of apoplastic Fe unchanged (Table IV). These findings are in agreement with results of Nikolic and Römheld (2002) reported for both green and chlorotic grapevine (Vitis vinifera) plants grown on calcareous soil under field conditions. Moreover, the relative proportions of symplastic and the various apoplastic binding forms of leaf Fe did not differ greatly between green (low external pH) and chlorotic (high external pH) leaves in both NH₄NO₃- and NO₃^–-fed plants (Fig. 4).

Considering further Mengel's hypothesis that high apoplastic pH due to high NO₃ uptake in young developing leaves depresses Fe^III reduction and thereby Fe²⁺ uptake into symplast (Kosegarten et al., 2001), the question has to be raised as to the fate of this nonutilized Fe. Presumably, it must be accumulated outside the plasma membrane and most probably trapped in the cell walls, which implies a relative increase in the concentration of Fe. Our results, however, were unable to confirm this expected increase in the cell wall Fe isolated from leaves of NO₃^–-fed plants. They showed the contrary: Both binding forms of leaf cell wall Fe (weakly and strongly bound) were significantly lower in all chlorotic plants grown in the high-pH nutrient solutions regardless of N form (Table V) due to restricted Fe uptake caused by high pH in the root apoplast (Table II; Nikolic et al., 2000). The lack of correlation between leaf apoplastic NO₃^– concentration and Fe concentration in the cell walls (Fig. 5B) also provides further evidence against the postulated hypothesis of Fe inactivation. Therefore, we suggest that the reported regreening of chlorotic leaves after spraying with citric acid (Tagliavini et al., 2000; Kosegarten et al., 2001) may be explained by the effect of citric acid in strongly extracting Fe bound in the cell walls, thereby increasing soluble Fe^III citrate in the leaf apoplastic fluid, rather than by the postulated enhanced reduction of Fe^III and, thus, Fe uptake into the symplast due to lowering of apoplastic pH (Kosegarten et al., 2001).

Despite the fact that NO₃^– is a predominant N form in most crop soils (Römheld, 2000), Fe deficiency chlorosis does not appear in perennial woody plants (e.g. grapevine, pear [Pyrus communis], peach, etc.) grown on non calcareous soils and in the case of sunflower is rare even on calcareous soils. Thus, the recently reported NO₃^–-induced Fe deficiency chlorosis in sunflower grown in the nutrient solution culture at low Fe supply (Kosegarten et al., 1998b, 1999, 2001) is an interesting physiological phenomenon but of little relevance to Fe deficiency in field grown plants. Nevertheless, even in this case, Fe chlorosis is mainly a consequence of lower uptake and shoot translocation of Fe by NO₃^–-fed plants as compared with plants grown in the nutrient solutions with NH₄⁺-N supply (Table II; Mengel and Geurtzen, 1986; Kosegarten et al., 1998b). Furthermore, the results from Kosegarten's experiments with the controlled pH of nutrient solutions do not differ in principal whatsoever from ours because the uptake of Fe by roots was influenced by pH rather than by N form.

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
High-pH buffers (e.g. HCO₃^– and HEPES) restrict uptake and shoot translocation of Fe by nutrient solution grown plants regardless of whether N is supplied solely as NH₄⁺ or as NO₃^– or as both forms as NH₄NO₃. Increasing N supply to roots solely as NO₃^– (up to 40 mM) does not change the relative distribution of Fe between leaf apoplast and symplast, and there are no differences in either total Fe leaf concentrations or Fe distribution between various binding fractions in the cell walls if the external pH of the root medium is kept constantly low (5.0). In unbuffered nutrient solution, however, NO₃^– supply decreases Fe uptake due to an increase in root surface pH. Similarly, uptake of Fe is depressed if the nutrient solution is supplied with NH₄⁺ as the sole N source and buffered at high pH (7.5). We found no evidence that NO₃^– supply to roots inactivates Fe in the leaf apoplast as postulated by Mengel and coworkers. The results presented here clearly show that occurrence of NO₃^–-induced Fe deficiency chlorosis is exclusively caused by inhibited uptake and translocation of Fe from roots to shoots as a consequence of high pH at the root surface.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material and Growth Conditions

Sunflower (Helianthus annuus L. cv Frankasol) was grown under controlled environmental conditions in a growth chamber with light/dark regime of 16/8 h, temperature regime of 24°C/20°C, photon flux density of approximately 300 µmol^–2 s^–1 at plant height, and relative humidity of about 70%. After soaking in 1 mM CaSO₄ overnight, seeds were germinated in quartz sand moistened with saturated CaSO₄ solution in darkness for 3 d. The 5-d-old seedlings were then transferred to full-strength nutrient solutions (four plants per 2.5-L plastic pot) containing: 0.7 mM K₂SO₄, 0.1 mM KH₂PO₄, 0.1 mM KCl, 0.5 mM MgSO₄, 10 µM H₃BO₃, 0.5 µM MnSO₄, 0.2 µM CuSO₄, 0.1 µM ZnSO₄, and 0.01 µM (NH₄)₆Mo₇O₂₄. N was applied in the different forms as Ca(NO₃)₂, (NH₄)₂SO₄, and NH₄NO₃, at a concentration of 4 mM N for each treatment, unless mentioned separately in the text. For NH₄⁺ and NH₄NO₃ treatments, CaCl₂ was added at a final concentration of 2 mM to compensate for Ca supplied in the NO₃^– treatment. In all experimental sets, Fe was supplied as ferric ethylenediaminedi(O-hydroxyphenylacetic acid) at 1 µM. In addition, the pH of nutrient solutions (adjusted with NaOH) was kept constant by buffering either at 5.0 with 2-(N-morpholino)ethanesulfonic acid or 7.5 with HEPES, both at final concentration of 5 mM. If used at such low concentrations, these organic buffers appeared to be biologically inert without adverse effects on plant growth (Bugbee and Salisbury, 1985). The nutrient solutions were renewed completely every 2 d to avoid nitrification (Walch-Liu et al., 2001) and continuously aerated. Two weeks after transfer to nutrient solutions, plants were harvested.

Chlorophyll Determination

Leaf chlorophyll content was determined nondestructively using a portable Chlorophyll Meter SPAD-502 device (Minolta Camera Co., Osaka).

Determination of Total Fe in Plant Tissues

After 14 d of growth in the nutrient solutions, sunflower plants were harvested and separated to shoots (leaves + stems) and roots. To remove extraplasmatic Fe, roots were washed with bipyridyl and sodium dithionite as described by Bienfait et al. (1985). Roots and shoots were weighted after oven drying at 65°C for 48 h, ashed at 550°C for 8 h, and digested in 5 M HNO₃. Fe was determined by atomic absorption spectrometry (AAS; UNICAM 939, Unicam, Kassel, Germany) after dissolving the residues in 1% (w/v) HCl.

Determination of Apoplastic and Symplastic Fe in Leaves

Apoplastic Fe in intact leaf discs was determined according to the method of Nikolic and Römheld (2002). Samples containing 15 leaf discs (1.5-cm diameter) were vacuum infiltrated in solution containing 1.5 mM 2,2'-bipyridyl and 10 mM MES (pH 6.0) and than incubated for 20 min under reductive conditions by adding sodium dithionite at a final concentration of 5 mM under continuous N₂ bubbling through the solution. Apoplastic Fe was removed as Fe^II[bipyridyl]₃ complex, and Fe concentration was determined by measuring A₅₂₀ (spectrophotometer U-3300, Hitachi, Tokyo) and using extinction coefficient of 8.65 mM^–1 (Bienfait et al., 1985). After removal of apoplastic Fe, leaf discs were shortly washed several times in deionized water, and remaining Fe (symplastic) was determined as previously described by Nikolic and Römheld (2002).

Isolation of Cell Walls

The young, fully expanded leaves were excised, and major veins were removed. Leaf discs (0.5-cm diameter) were taken and plasmolyzed under vacuum (–40 kPa) in 20 mL of 0.4 M Suc solution for 20 min. The samples (about 1 g fresh weight) were homogenized in 5 mL of 0.4 mM Suc solution using precooled mortar and pestle. Leaf homogenate was brought to 15 mL, and the pellet obtained after centrifugation at 1,000g for 15 min was resuspended and successively pelleted in 15 mL of 0.4, 0.6, and 1 M Suc solutions, respectively (1,000g, 10 min each). The final pellet was washed three times with 10 mL of 0.5% (w/w) SDS and centrifuged at 1,000g for 10 min, followed by several washings (at least six times) with deionized water, until these cell wall materials became free of plasma membranes and other cytoplasmic fragments as observed by a light microscope. All procedures were carried out at 4°C except plasmolysis and washing with SDS, which were performed at room temperature.

Determinations of Different Fe-Binding Forms in the Leaf Apoplast

For determination of water-soluble Fe in the leaf apoplast, young expanded leaves were excised, and central veins were removed. Leaflets were briefly washed and incubated for 30 min in deionized water to prevent water uptake during further vacuum infiltration (Mühling and Sattelmacher, 1995). Leaves were infiltrated with ice-cold deionized water in a vacuum desiccator several times reducing the pressure to –45 kPa, followed with slow relaxation to atmospheric pressure within 2 to 3 min. Infiltrated leaves that become darker than noninfiltrated were carefully blotted dry, rolled over a plastic tube, orientated with the cut edge up and fixed into a plastic 50-mL syringe. The leaves-filled syringes were then centrifuged at 1,000g for 15 min at 4°C. Collected AWF samples were evaporated in a rotary evaporator, and dry residue was dissolved in 1% (w/v) HCl for determination of Fe by AAS. Leaves remaining after centrifugation were dried at 65°C overnight and weighted. Concentration of soluble Fe fraction was calculated per leaf dry weight basis. All samples were tested for possible cytoplasmic contamination using the malate dehydrogenase (MDH) activity assay (Husted and Schjoerring, 1995) as a cytosolic (i.e. mitochondria) marker enzyme.

Isolated cell wall materials were resuspended in 10 mL of solution containing 1.5 mM 2,2'-bipyridyl and 0.5 mM CaSO₄, and weakly bound Fe fraction was removed during reductive extraction in the presence of sodium dithionite at final concentration of 10 mM under continuous flow of N₂. After incubation for 30 min, the A₅₂₀ was measured in a 3-mL aliquot, and cell wall materials were washed several times with deionized water for determination of Fe remaining in the cell walls (strongly bound Fe fraction). Finally, the pellet was resuspended in deionized water and transferred to a glass beaker, evaporated, dried, weighted, and ashed. The residue was digested in 5 M HNO₃ and dissolved in 1% (w/v) HCl for Fe determination by AAS.

Collection and Analysis of Xylem Sap and Leaf Apoplastic Fluid

Xylem sap was obtained by exudation after sunflower plants were decapitated at the stem about 2 cm above the root base. Silicon tubes were fixed over the decapitated stem, and xylem sap was collected for 2 h after discarding the exudates obtained during the first few minutes. The apoplastic fluid from intact fully expanded young leaves was collected 3 h after light onset by the centrifugation method described previously by Dannel et al. (1995). The first fraction, obtained at low centrifugal speed of 1,500g to remove xylem sap, was discarded, and the apoplastic fluid was collected by the second centrifugation at 2,500g for 15 min at 4°C. Cytoplasmic contamination of all samples was quantified by comparing the MDH activity and K⁺ concentration in the apoplastic fluid with total leaf homogenate. The MDH activity of about 2% of the total leaf homogenate and measured K⁺ concentration up to 15 mM indicated that there was no symplastic contamination of the apoplastic fluid (Dannel et al., 1995; Nikolic and Römheld, 2002).

The pH measurements were carried out in the samples immediately after their collection using a glass microelectrode (InLab 423, Mettler Toledo, Giessen, Germany). After pH measurements, the samples of xylem sap and leaf apoplastic fluid were frozen in liquid N₂ and stored at –20°C for further analysis of NO₃^– and Fe. NO₃^– was determined using an RQflex reflectometer (Merck, Darmstadt, Germany). Fe was determined by flame AAS using a microinjection device (50 µL) after dilution of the samples at a ratio of 1:1 (v/v) with 5 M HCl.

Measurement of ⁵⁹Fe Uptake by Leaf Symplast in Intact Leaves

Radiolabeled ⁵⁹Fe citrate was prepared by mixing ⁵⁹FeCl₃ with citric acid with a molar ratio of 1:100. Young, fully expanded young leaves were excised, and the cut ends of petioles were placed in a 2-mL plastic tube containing ⁵⁹Fe citrate solution (10 µM Fe; specific activity of 10 µCi µmol^–1 Fe) in the presence of various buffers: 50 mM MES (pH 5.0 and 6.0) and 50 mM HEPES (pH 7.0). Unbuffered ⁵⁹Fe-labeled uptake solutions in the presence or absence of 6 mM NO₃^– were used in an alternative experiment. Radioactive labeled solution was perfused (2 h) via the petiole by transpiration stream into the leaf apoplast under light (500 µmol m^–2 s^–1). Thereafter, radioactive solutions were replaced with non-radiolabeled solutions, and leaves were incubated for a further 2 h under light or in darkness. To prevent photoreduction of Fe^III citrate under light exposure, plastic tubes were wrapped with aluminum foil and covering the leaf lamina with a light transparent plastic bag regulated water losses by transpiration. For determination of ⁵⁹Fe uptake by leaf symplast, leaf discs (1-cm diameter) were punched by a calibrated cork borer from the leaf area devoid of major veins and washed twice in ice-cold nonradioactive solution containing 10 mM MES (pH 6.0), 0.5 mM CaSO₄, and 10 µM FeCl₃ for 10 min each. Apoplastic ⁵⁹Fe was removed with bipyridyl and sodium dithionite during 30 min of reductive incubation as mentioned above. After incubation, leaf discs were finally washed twice in 0.5 mM CaSO₄ for 5 min each, oven dried, ashed, and residue was dissolved in 1% (w/v) HCl for measuring of ⁵⁹Fe radioactivity by a liquid scintillation counter (Wallac 1414 Win Spectral, Wallac Oy, Turku, Finland).

Statistics

Data were subjected to analysis of variance using the statistical software SigmaStat for Windows (version 2.0, 1997, SPSS Inc., Chicago). Means were compared by Duncan's test at P < 5%.

	ACKNOWLEDGMENTS

 
We thank Ernest Kirkby (University of Leeds, UK) and Dr. Leon Kochian (Cornell University, Ithaca, NY) for critical reading of the manuscript and Maria Ruckwied (University Hohenheim, Stuttgart, Germany) for excellent technical assistance during AAS analysis.

Received November 15, 2002; returned for revision January 20, 2003; accepted March 23, 2003.

	FOOTNOTES

 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.017889.

¹ This work was supported by the Deutsche Forschungsgemein-schaft as part of the special research project "The Apoplast of Higher Plants: Compartment for Storage, Transport, and Reactions."

² Present address: Centre for Multidisciplinary Studies, University of Belgrade, Kneza Viseslava 1, 11000 Belgrade, Yugoslavia.

^* Corresponding author; e-mail roemheld{at}uni-hohenheim.de; fax 49–711–459–3295.

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