Journal of Experimental Botany, Vol. 52, No. 360, pp. 1489-1498,
July 1, 2001
© 2001 Oxford University Press
Original Papers |
Iron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) trees
Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Apdo. 202, E-50080 Zaragoza, Spain
Received 24 October 2000; Accepted 6 March 2001
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Abstract |
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Experiments have been carried out with field-grown pear trees to investigate the effect of iron chlorosis on the composition of the leaf apoplast. Iron deficiency was associated with an increase in the leaf apoplastic pH from the control values of 5.5–5.9 to 6.5–6.6, as judged from direct pH measurements in apoplastic fluid obtained by centrifugation and fluorescence of leaves incubated with 5-CF. The major organic acids found in leaf apoplastic fluid of iron-deficient and iron-sufficient pear leaves were malate, citrate and ascorbate. The total concentration of organic acids was 2.9 mM in the controls and increased to 5.5 mM in Fe-deficient leaves. The total apoplastic concentration of inorganic cations (Ca, K and Mg) increased with Fe deficiency from 15 to 20 mM. The total apoplastic concentration of inorganic anions (Cl-, NO3-, SO42- and HPO42-) did not change with Fe deficiency. Iron concentrations decreased from 4 to 1.6 µM with Fe deficiency. The major Fe species predicted to exist in the apoplast was [FeCitOH]-1 in both Fe-sufficient and deficient leaves. Organic acids in whole leaf homogenates increased from 20 to 40 nmol m-2 with Fe deficiency. The accumulation of organic anions in the Fe-deficient leaves does not appear to be associated to an increased C fixation in leaves, but rather it seems to be a consequence of C transport via xylem.
Key words: Apoplast, iron deficiency, iron chlorosis, pear, organic acids.
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Introduction |
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Iron deficiency is one of the major abiotic stresses affecting fruit tree crops in the Mediterranean area. One of the most obvious characteristics of the plants affected by Fe deficiency is that their leaves become chlorotic (Terry and Abadía, 1986
; Abadía, 1992; Abadía and Abadía, 1993; Morales et al., 1994). In a geographical region of great agricultural importance in north-eastern Spain, such as the Ebro river basin, many fruit tree crops are affected by Fe chlorosis. Crops affected include peach, pear, apple, cherry, citrus, grape, and plum (Sanz et al., 1992). Pear is one of the fruit tree species most affected by Fe chlorosis, that causes decreases in yield and shortens the productive lifetime of the orchards by 5–6 years (Sanz et al., 1992). In the Ebro river basin, approximately 67% of pear orchards (over 13 200 ha) are currently being treated with Fe compounds to correct Fe chlorosis.
Iron chlorosis is caused by low Fe availability in calcareous, high pH soils (Lindsay and Schwab, 1982). Iron deficiency produces physiological and biochemical responses at the plant root level in dicotyledonous plants. The physiological responses include an enhanced proton extrusion which decreases the pH of the rhizosphere (Brown, 1978), a release of reducing and/or chelating substances such as phenolics and flavins (Susín et al., 1996) and a two-step mechanism for Fe uptake in which Fe(III) is first reduced by a plasma membrane (PM)-bound ferric-chelate reductase (FC-R) (Moog and Brüggemann, 1994; Robinson et al., 1999) and subsequently absorbed as Fe(II) (Chaney et al., 1972; Eide et al., 1996). Among the biochemical responses, dicotyledonous plants accumulate organic acids, mainly citrate and malate, both in leaves (Landsberg, 1981) and roots (de Vos et al., 1986; López-Millán et al., 2000b).
In the xylem, Fe is transported to the leaves as Fe(III), probably chelated by citrate (Schmidt, 1999; López-Millán et al., 2000a). The way Fe enters leaf cells has been much less studied than the corresponding processes in the roots. Once in the leaf apoplast, Fe(III) is reduced before uptake by leaf cells (Brüggemann et al., 1993; Nikolic and Römheld, 1999). Reduction of Fe-chelates are mediated by a PM-bound FC-R enzyme (González-Vallejo et al., 2000). Therefore, the composition of the apoplast is of crucial importance to understand the processes involved in the mechanism of Fe uptake by leaves. For instance, apoplastic pH could be important for the mobility of Fe and the activity of the leaf PM FC-R (Mengel, 1995). No report has been made so far, to our knowledge, on the effects of Fe deficiency on the composition of the leaf apoplast in field-grown plants, although two recent papers (Nikolic and Römheld, 1999; López-Millán et al., 2000a) and a communication to a Symposium (Biino et al., 1997) reported some data obtained in controlled conditions.
The leaf apoplast is involved in transmission of signals (Hartung et al., 1992), transport and storage of mineral nutrients (Zhang et al., 1991), trace gas exchange with the atmosphere (Gabriel et al., 1999) and different responses to plant environmental stresses (Dietz, 1997). Studies have been made on the composition of the apoplast under different environmental conditions (Grignon and Sentenac, 1991; Canny, 1995). The apoplast contains significant concentrations of inorganic and organic anions (Schurr and Schulze, 1995; Dietz, 1997).
The aim of this work was to investigate the effects of Fe deficiency on the composition of the leaf apoplast in pear trees grown in the field, in order to understand the role of this compartment in the transport and acquisition of Fe by leaf cells. The changes induced by Fe deficiency on the concentrations of different inorganic and organic ions in pear leaf apoplast have been investigated. Chemical speciation has been carried out with the MinteqA2 software. The activities of different enzymes, including PEPC and several TCA-cycle enzymes, and the concentrations of pyridine nucleotides and organic anions have been measured in leaf extracts of Fe-deficient and control leaves.
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Plant material
Leaves were sampled from pear trees (Pyrus communis L.) growing in calcareous soils and affected by iron chlorosis. The orchard was located in El Temple (Huesca) in the Ebro basin in north-eastern Spain. Chlorosis was not associated to soil patchiness since the soil in the orchard was fairly homogeneous. The soil has a clay-loamy texture, with 32% total calcium carbonate, 12.6% active lime, 1.89% organic matter, and pH in water of 8.4. The pear cultivar used was ‘Blanquilla’ grafted on quince ‘BA29’. The trees were 17 years old, trained as palmette, with a frame of 3x4 m. The orchard had not been treated with Fe-chelates in the last two or three years. Under these conditions chlorosis became more marked in the orchards studied every year. Chlorosis was only due to Fe deficiency, since the application of Fe(II) salts or Fe(III)-chelates to the chlorotic leaves produced complete leaf regreening. In the 1997 growing season there were trees with marked chlorosis symptoms (average leaf chlorophyll 200 µmol m-2) and other trees that still remained green (average leaf chlorophyll 600 µmol m-2). Samplings were conducted during the summer of 1997. Iron chlorosis in pear often leads to little or not interveinal chlorosis (Abadía et al., 1989
). Young, fully-expanded leaves, showing almost homogeneous colour throughout the leaf, were chosen for all measurements. Iron-deficient and control leaves had 230 and 220 g FW m-2, respectively. Other morphological characteristics of these control and Fe-deficient pear leaves have been reported (Morales et al., 1998). In particular, leaf thickness did not change by Fe deficiency. The leaf Fe concentrations in these trees have been also reported (Morales et al., 1998).
Chlorophyll determination
Chlorophyll concentration was estimated non-destructively with a SPAD-502 device (Minolta, Osaka, Japan). For calibration, leaf discs with different degrees of Fe deficiency were first measured with the SPAD, then extracted with 100% acetone in the presence of Na ascorbate and chlorophyll measured spectrophotometrically (Abadía and Abadía, 1993).
Apoplastic fluid collection
Apoplastic fluid was obtained from whole pear leaves by direct centrifugation (Dannel et al., 1995) with some modifications. Leaves were excised at the base of the petiole with a razor blade in the field and transported to the laboratory (within approximately 30 min) in dim light with the petiole immersed in de-ionized water. Once in the laboratory the petiole was excised under water. Five leaves were then rolled and placed into a plastic syringe barrel, with the petiole side at the narrow end of the syringe. Leaf-filled syringes were centrifuged at 4 °C. A first centrifugation was made at low speed (2500 g, 15 min) to remove the xylem sap of the main vein. Apoplastic fluid was obtained after a second centrifugation for 15 min at 4000 g and 4°C. A small volume of apoplastic fluid was obtained from the bottom of the centrifuge tube. The phosphate (0.1–0.3 mM) and K+ (5–12 mM) concentrations in the apoplastic fluid were considerably lower than the assumed cytoplasmic concentrations (5 and 100 mM, respectively) and, therefore, strongly support the conclusion that apoplastic fluid was devoid of significant cytoplasmic contaminants.
Malate dehydrogenase (c-mdh; EC 1.1.1.37; 67 kDa molecular weight) was also used as a cytosolic contamination marker for apoplastic fluid. The activity of c-mdh was determined using oxalacetate as substrate and measuring the decrease in A340 due to the enzymatic oxidation of NADH. The final reaction mixture (pH 9.5) was 46.5 mM Tris, 0.1 mM NADH and 0.4 mM oxalacetate (Dannel et al., 1995). The activity of the marker in the leaf apoplast was related to the total activity in whole leaf homogenates. To measure enzymatic activities in total leaf homogenates three leaf discs of 0.95 cm2 were homogenized with 2 ml of buffer (pH 8.0) containing 100 mM Hepes, 30 mM sorbitol, 2 mM DTT, 1 mM CaCl2, 1% BSA, and 1% PVP. The supernatant was collected and analysed immediately after 10 min centrifugation at 10 000 g.
pH measurements
The pH of the apoplastic fluid was measured directly in the fluid obtained by centrifugation with a microelectrode (Physitemp, USA). Apoplastic pH was also measured in vivo by fluorescence (Hoffman et al., 1992) with 5-carboxyfluorescein (5-CF) (López-Millán et al., 2000a). Leaves were excised and the cut end of the petiole was exposed to incubation medium containing 5 µM 5-CF, 1 mM KCl, 0.1 mM NaCl, and 0.1 mM CaCl2 at pH 5.5. The incubation was carried out for 5 h at room ambient light and temperature (15–25 µmol photons m-2 s-1, 20–25 °C). The level of autofluorescence was subtracted from total fluorescence. Three leaves per Chl level were taken and four measurements were carried out in different areas of each leaf.
Inorganic ion determination
Ca and Mg were determined by atomic absorption spectrophotometry and K by emission spectrophotometry. Fe was determined in the apoplastic fluid by atomic absorption spectrometry with a graphite furnace (Varian SpectrAA with Zeeman correction). Each sample was analysed in triplicate.
Inorganic anions (
were quantified by HPLC with a 4.6x75 mm IC-Pak A HR ion-exchange column as described previously (López-Millán et al., 2000a).
Organic ion analysis
Leaf samples (3 leaf discs of 0.95 cm2 taken with a calibrated cork borer) were frozen in liquid N2 and ground in a mortar with 8 mM sulphuric acid. Homogenates were boiled for 30 min, centrifuged 10 min at 10 000 g, filtered with a 0.2 µm PVDF filter (LIDA, Kenosha, WI, USA), taken to a final volume of 2 ml with 8 mM sulphuric acid and kept at -80 °C until analysis. Apoplastic fluid samples were filtered (0.45 µm PVDF, LIDA) before HPLC analysis.
Organic anions were analysed by HPLC with a 300x7.8 mm Aminex ion-exchange column (HPX-87H from Bio-Rad, Hercules, CA, USA) as described earlier (López-Millán et al., 2000a). Peaks corresponding to oxalate, citrate, 2-oxoglutarate, ascorbate, malate, shikimate, and fumarate were identified by comparison of their retention times with those of known standards from Bio-Rad and Sigma (Fig. 1). The peak with a retention time of 12 min was composed of succinate and an unidentified compound with maxima at 205 and 262 nm. Because of this reason, succinate was not quantified. Another peak with a retention time of approximately 14 min, with absorption maxima at 219 and 281 nm, was tentatively identified as p-coumaric acid. The identity of oxalate, citrate, malate, fumarate, and succinate was further confirmed by MS.
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Sugar analysis
Sugars (glucose, fructose and sucrose) were analysed by HPLC with a 300x4 mm Spherisorb-NH2 column (Waters) as described previously (López-Millán et al., 2000a). Peaks corresponding to glucose, fructose and sucrose were identified by comparison of their retention times with those of known standards from Sigma.
Enzyme assays
Extracts for measuring enzyme activities were made by grinding three leaf discs of 0.95 cm2 each in a mortar with 2 ml of extraction buffer containing 30 mM sorbitol, 1% BSA and 1% PVP in 100 mM HEPES-KOH, pH 8.0. The slurry was centrifuged for 15 min at 10 000 g and 4 °C, and the supernatant was collected and analysed immediately.
Malate dehydrogenase (MDH; EC 1.1.1.37), citrate synthase (CS; EC 4.1.3.7), aconitase (EC 4.2.1.3), isocitrate dehydrogenase (ICDH; EC 1.1.1.42), fumarase (EC 4.2.1.2), phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), lactate dehydrogenase (LDH; EC 1.1.1.27), and pyruvate decarboxylase (PDC; EC 4.1.1.1) were assayed as described earlier (López-Millán et al., 2000b).
Nucleotide analysis
Pyridine nucleotides were extracted from liquid N2-frozen leaf discs (0.95 cm2) in 1 ml of 100 mM NaOH (for NAD(P)H) or 5% TCA (for NAD(P)+). The extracts were boiled for 6 min, cooled on ice and centrifuged at 12 000 g for 6 min. Samples were adjusted to pH 8.0 with HCl or NaOH and 100 mM Bicine (pH 8.0). Nucleotides were quantified by the enzyme-cycling method (Matsumura and Miyachi, 1980).
Chemical speciation
Concentrations of the different Fe-chelate species were estimated with the software MinteqA2 (Allison et al., 1991) by using the ionic environment characteristics of the apoplastic fluid. Chelate formation constants used for citrate and malate were derived from those given by Holden et al. and Cline et al., respectively (Holden et al., 1991; Cline et al., 1982). At an ionic strength of 0 M, the log10 of the chelate formation constants used for the Fe-citrate species [FeCit]0, [FeCitH]+1, [FeCitOH]-1, [FeCit2]-3, and [Fe2Cit2(OH)2]-2 were 13.13, 14.43, 10.11, 20.13, and 24.51, respectively. The log10 of the chelate formation constants for the Fe-malate species [FeMal]+1, [Fe2Mal2(OH)2]0, [Fe2Mal3(OH)2]-2, and [Fe3Mal3(OH)4]-1 were 8.39, 15.32, 20.33, and 27.75, respectively.
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Results |
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Isolation of apoplastic fluid
The volume of apoplastic fluid obtained by centrifugation increased gradually when the centrifugal forces increased (Fig. 2A
). In routine experiments apoplastic fluid was collected at 4000 g, after carrying out a preliminary centrifugation of the leaves at 2500 g to discard fluid containing the xylem sap of the main vein. The activity of the cytosolic marker c-mdh was approximately 2 nmol NADH s-1 ml-1 both at 2500 and 4000 g. This value was less than 2% of the total activity found in leaf homogenates (Fig. 2A; approximately 100–120 nmol NADH s-1 ml-1), indicating a low degree of cytosolic contamination (Dannel et al., 1995). Compared to the controls, moderate and severe Fe deficiency caused 25% increases and 15% decreases, respectively, in the volume of apoplastic fluid recovered g-1 of leaf fresh weight (Fig. 2B).
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Apoplastic fluid pH
The pH of the apoplastic compartment of pear leaves was measured by two different methods, direct pH determination in the apoplastic fluid obtained by centrifugation and in vivo estimation by means of the fluorescent dye 5-CF (Hoffman et al., 1992
). When determined with a microelectrode the pH of the apoplastic fluid increased with Fe deficiency by one unit, from approximately 5.5 in control to 6.6 in Fe-deficient leaves (Fig. 3). This increase was statistically significant (P<0.01). The changes in pH values estimated in vivo using 5-CF follow a similar trend than those found by direct pH measurement in the apoplastic fluid, with values of approximately 5.9 in control and 6.5 in Fe-deficient leaves (Fig. 3). In this case, however, pH changes were not statistically significant at the P<0.01 level.
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Ionic composition of the apoplastic fluid
Calcium and Mg concentrations in apoplastic fluid increased in response to Fe deficiency (Fig. 4A). Increases were 1.13-fold for Ca and 4.3-fold for Mg. The K concentration increased from 9 mM in the controls to 11 mM in moderately deficient leaves (200–400 µmol Chl m-2) and decreased to 5.2 mM in severely deficient leaves (<200 µmol Chl m-2).
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Iron deficiency was associated to a 3-fold decrease in
concentration in apoplastic fluid of pear leaves (Fig. 4B
). Phosphate concentration increased 3.6-fold with Fe deficiency (Fig. 4B). Concentrations of Cl– increased 2-fold in moderately deficient leaves and decreased again to values slightly lower than the controls in severely deficient leaves (Fig. 4B). Iron deficiency did not cause significant changes in the 
concentrations in leaf apoplastic fluid (Fig. 4B).
The concentration of Fe in the apoplastic fluid of pear leaves was 4.0–4.4 µM in leaves with 300–700 µmol Chl m-2 (Fig. 5). Below this Chl concentration there was a linear decrease in Fe in apoplastic fluid with Fe deficiency, down to 1.6 µM in leaves with 30 µmol Chl m-2 (Fig. 5).
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Iron deficiency was associated with a general increase in organic anion concentrations in the apoplastic fluid, reaching maximal values in chlorotic leaves with Chl concentrations between 100–150 µmol m-2 (Fig. 6A
). The major organic anions found in pear leaf apoplastic fluid were citrate, ascorbate and malate, the latter being the major anion. Maximum increases in the concentrations of the three major anions in the apoplastic fluid were 2.2-fold for malate, 2-fold for citrate and 1.4-fold for ascorbate. Minor organic anions in the leaf apoplastic fluid were shikimate, 2-oxoglutarate and fumarate (Fig. 6B). Iron deficiency caused a 2-fold increase in fumarate concentration and a marked decrease in 2-oxoglutarate concentration (Fig. 6B). Shikimate concentrations in apoplastic fluid ranged between 250 and 150 µM (Fig. 6B).
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Sugar composition of the apoplastic fluid
The apoplastic fluid usually contains sugars (Tetlow and Farrar, 1993; López-Millán et al., 2000a). Iron deficiency caused changes in the sugar concentrations of the apoplastic fluid (Fig. 7). Moderate Fe deficiency caused 2.6- and 2-fold increases in concentrations of glucose and sucrose (Fig. 7). Severely deficient samples had glucose concentrations similar to the controls and sucrose concentrations 2-fold lower than the control values (Fig. 7). Fructose concentrations decreased only slightly with Fe deficiency (Fig. 7).
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Iron chemical speciation in the apoplastic fluid
Iron species predicted to exist in equilibrium in the apoplastic fluid were mainly complexes with citrate, the major being [FeCitOH]-1 (Table 1 ). Iron deficiency caused changes in the predicted distribution of total Fe. With Fe deficiency the amount of Fe predicted to exist as [FeCitOH]-1 increased from 62.2% to 84.3%, whereas the amount predicted to be present as [FeCit2]-3 decreased from 29.6% to 7.5%. The amount of the [Fe2Cit2(OH)2]-2 species accounted for 8% of the total Fe in both Fe-sufficient and deficient apoplastic fluid.
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Organic anion concentration in leaves
The total organic anion concentrations in pear leaves increased 1.6-fold with Fe deficiency. Major organic anions found in whole leaf homogenates were citrate, oxalate, malate and ascorbate. Leaves with 50 µmol Chl m-2 had 15- and 1.7-fold increases in citrate and ascorbate concentrations and 1.3-fold decreases in malate concentrations, respectively, when compared to control leaves (Fig. 8A). Oxalate decreased markedly with Fe deficiency (Fig. 8A). Minor organic anions in whole leaf homogenates were shikimate, 2-oxoglutarate and fumarate (Fig. 8B). Iron deficiency caused a 3-fold decrease in shikimate, whereas 2-oxoglutarate and fumarate increased 1.3- and 4.3- fold, respectively. The peak at approximately 14 min, tentatively identified as p-coumaric acid, decreased 3-fold with Fe deficiency.
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Enzyme activities in leaf extracts
The activities of five enzymes involved in the TCA cycle were measured in leaf homogenates. Enzymatic activities were increased by moderate Fe deficiency (Fig. 9A, B), and maximal activities were found with leaves of approximately 100 µmol Chl m-2. Increases were 2.4-fold for fumarase, 2.3-fold for ICDH, 1.4-fold for aconitase, and 3.4-fold for CS. Malate dehydrogenase did not show significant changes in activity with Fe deficiency.
The activities of PEPC, an enzyme related to C-fixation, and of LDH and PDC, two enzymes involved in anaerobic metabolism, were also measured in leaf homogenates. The activity of PEPC was approximately 5.5 nmol s-1 m-2 and did not show significant changes with Fe deficiency (Fig. 9C). The activity of LDH decreased with Fe deficiency 5.6-fold whereas the PDC activity decreased only slightly (Fig. 9C).
Leaf nucleotide concentrations
The pool of pyridine nucleotides increased 1.6-fold in the leaves of severely Fe-deficient plants (100–150 µmol Chl m-2) when compared to the controls. Iron deficiency increased the concentrations of both reduced and oxidized nucleotide forms. The largest relative increase was 5.5-fold for NADH (from 0.02 to 0.11 nmol m-2), followed by 1.7-fold for NADP+ (from 0.68 to 1.13 nmol m-2), 1.6-fold for NAD+ (from 0.41 to 0.67 nmol m-2) and 1.4-fold for NADPH (from 0.12 to 0.17 nmol m-2) (Fig. 10A). As a result of these changes, the NADH/NAD+ and NADPH/NADP+ ratios increased 4.2- and 2.2-fold in response to Fe deficiency (Fig. 10B).
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Discussion |
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For the first time the effects of Fe deficiency on the chemical composition of leaf apoplastic fluid in field-grown pear trees have been characterized. Several reports have investigated the ionic composition of the apoplast in leaves (for reviews see Grignon and Sentenac, 1991
; Canny, 1995; Sattelmacher et al., 1998), but little is known about the composition of this leaf compartment in field-grown trees. No report so far has been published on the changes induced by Fe deficiency in this compartment in field-grown plants.
Iron deficiency was associated with a marked increase in the apoplastic pH of pear leaves as judged from direct pH measurements in apoplastic fluid collected by centrifugation. Data obtained from fluorescence of leaves incubated with 5-CF followed a similar trend, although differences were not statistically significant. The increase in pH was almost one unit, from 5.5–5.9 to 6.5–6.6. These data support Mengel's view (Mengel, 1995) that Fe deficiency could indeed be associated with pH increases of the leaf apoplastic space in field-grown plant species. This pH increase, however, is not likely to cause major problems in Fe acquisition by mesophyll cells (see discussion below). The pH increase found in pear contrasts with the significant decrease (0.4 pH units) found recently with Fe deficiency in the apoplastic fluid of sugar beet (López-Millán et al., 2000a). The data from this study confirm that leaves from Fe-deficient trees are under a short supply of apoplastic Fe. Iron deficiency caused a marked (2-fold) decrease in the Fe concentration of the apoplastic fluid, similar to the decrease in total leaf Fe concentration (data expressed on a leaf volume basis) in the pear leaves in the same orchard (Morales et al., 1998).
The major Fe-chelate species predicted to exist in the apoplastic fluid of Fe-deficient and Fe-sufficient leaves were [FeCitOH]-1 and [FeCit2]-3. Iron deficiency caused changes in the chemical speciation of Fe-citrate in pear apoplast, with the monomeric [FeCitOH]-1 form increasing from 60% to 85% of the total Fe with Fe deficiency. This chemical species may favour Fe acquisition by the mesophyll plasma membrane FC-R, since it would experience less electrostatic repulsion with the negatively charged PM than [FeCit2]-3. The formation of citrate-Fe polymers (Spiro et al., 1967a) is frequently assumed to occur in plant shoots (Bienfait and Scheffers, 1992; Moog and Brüggemann, 1994; Schmidt, 1999). However, no experimental evidence is currently available in support of this theory. In fact, excess citrate would compete effectively with the formation of the citrate-Fe polymers, therefore inhibiting polymerization (Spiro et al., 1967b). Given the high citrate:Fe ratios, the formation of citrate-Fe polymers in the pear apoplast seems unlikely.
The marked increases in the organic anion:Fe ratios with Fe deficiency may constitute a major factor decreasing Fe availability by mesophyll cells. The malate : citrate : Fe molar ratios increased with Fe deficiency from 300 : 150 : 1 to 1750 : 900 : 1 in the apoplastic fluid. The large citrate:Fe molar ratios found in the leaf apoplastic fluid of Fe-deficient plants may impair significantly Fe uptake by mesophyll cells, since the activity of the FC-R leaf PM enzyme has been recently shown to decrease markedly when the citrate:Fe ratio increases. Plasma membrane FC-R activities decreased 5-fold when the citrate:Fe molar ratio increased from 100 to 500 (González-Vallejo et al., 1999). On the other hand, high malate:Fe ratios in apoplastic fluid may favour Fe acquisition by mesophyll cells, because the activity of the FC-R enzyme increases markedly with increases in the malate:Fe ratio (González-Vallejo et al., 1999). Chemical speciation, however, indicates that Fe-malate complexes are unlikely to be formed in significant amounts in the apoplast to be substrates for the FC-R. It should be also taken into account that Fe deficiency decreases largely by itself leaf FC-R activities, both in protoplasts and in mesophyll tissue (González-Vallejo et al., 2000; Larbi et al., 2001).
Several other factors that change with Fe deficiency may modulate Fe availability from the apoplastic space to a lower extent than the citrate:Fe ratios. It has been hypothesized that an increase in apoplastic pH could affect the FC-R of the plasma membrane of mesophyll cells, leading to Fe immobilization in the apoplastic space (Mengel, 1995). The pH dependence of the FC-R activity depends on the leaf materials used, being different for plasma membrane preparations (Brüggemann et al., 1993; González-Vallejo et al., 1999), protoplasts (González-Vallejo et al., 2000), and leaf discs (Larbi et al., 2001). The one-unit pH change found in pear apoplast with Fe deficiency would lead to 30–50% decreases in FC-R enzyme activity of sugar beet leaf protoplasts (González-Vallejo et al., 2000) and to approximately 20% increases in the FC-R enzyme activity of sugar beet leaf discs (Larbi et al., 2001). This suggests that pH is unlikely to be a major factor in controlling the FC-R activity, although it may modulate it to a certain extent. Another factor modulating FC-R activities could be changes in pyridine nucleotide redox state. The increases found in mesophyll NADPH/NADP+ and NADH/NAD+ ratios (2.2- and 4.2-fold, respectively) would also tend to increase PM FC-R activities.
A quantitative physicochemical approach to ion relations in biological solutions has been put forward recently (Gerendás and Schurr, 1999). This theory—the strong ion theory—allows for the analysis of dependent variables such as pH in relation to independent variables such as concentrations of strong and weak ions, dissociation constants and pCO2. According to this theory the changes in the difference between cations and anions could explain pH shifts. The difference between cations and anions was (in mM) 6.1 and 6.7 in apoplastic fluid of control and Fe-deficient leaves. This increase would theoretically lead to an increase in pH, as it occurs indeed experimentally. So, these data are in support of the strong ion theory.
The organic acid increases found in the apoplastic fluid and whole leaf homogenates are unlikely to be caused by an increase of the rate of C fixation in leaves. The leaf photosynthetic rate of fruit tree leaves (Hurley et al., 1986) and other plant species (Terry, 1980) is markedly decreased by Fe deficiency. Also, the activity of leaf PEPC, that might fix bicarbonate, does not increase with Fe deficiency. Instead, this study's data suggest that the increase in organic anion concentrations in the leaves could be due to C import via xylem. Iron-deficient roots have increased activities of PEPC and other enzymes, accompanied by metabolite changes suggesting the existence of a non-autotrophic, anaplerotic C fixation in roots (López-Millán et al., 2000b). This C can be exported via xylem to the leaves (Bialzyk and Lechowski, 1992; López-Millán et al., 2000a) thus contributing to the increases in organic anions found in apoplastic fluid and whole leaves.
The increase in leaf organic anion concentrations with Fe deficiency may be the cause underlying other leaf biochemical changes. This includes the slight increases in leaf TCA cycle enzymatic activities, the 60% increase in the leaf pyridine nucleotide pool and the increases in the leaf NADPH/NADP+ and NADH/NAD+ ratios. In general, these changes seem to improve the conditions for an enhancement of leaf PM FC-R activity.
Ionic concentrations of organic and inorganic ions in the apoplast of control pear leaves are in the same range as those reported in the literature for leaves of Quercus, sunflower, pea, spinach, and bean (Bowling, 1987; Speer and Kaiser, 1991; Dannel et al., 1995; Gabriel et al., 1999). Sulphate concentrations were, however, much higher, possibly due to agrochemical treatments made usually in the area. The total anion:cation ratios for Fe-sufficient and deficient apoplast fluid were 0.64 and 0.63. Such a large excess of cations compared to anions in apoplastic fluid is a common observation (Dietz, 1997).
In summary, iron deficiency in pear was associated with organic anion accumulation in the apoplast and also in whole leaf tissue. The major predicted Fe chemical species was [FeCitOH]-1 in apoplastic fluid of Fe-deficient and sufficient plants, although its relative weight increased with Fe deficiency. Leaf apoplastic pH increased by 1 unit with Fe deficiency. Citrate accumulation could potentially be deleterious for Fe availability through a decrease of the PM FC-R activity caused by the high citrate:Fe ratios. It is hypothesized that the accumulation of organic anions in the Fe-deficient plants is a consequence of C transport via xylem. The production and accumulation of organic acids may have also two significant advantages. First, it would improve organic anion release to the rhizosphere, thus improving Fe-chelation and microbial growth. Second, it would provide substrates for respiratory and other maintenance processes to leaves with very low photosynthetic activity.
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Acknowledgments |
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Supported by grants AGR97-1177 from the Comisión Interministerial de Ciencia y Tecnología to AA, and PB97-1176 from the Dirección General de Investigación Científica y Técnica and AIR3-CT94-1973 from the Commission of European Communities to JA. AFL-M was supported by a fellowship from the Spanish Ministry of Science and Education. FM was scientist on contract from the Spanish Ministry of Education and Culture.
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Notes |
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1 To whom correspondence should be addressed. Fax: +34 976 716145. E-mail: jabadia{at}eead.csic.es
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Abbreviations |
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CS, citrate synthase; DTNB, 5-5'dithio-bis-2-nitrobenzoic acid; FC-R, ferric-chelate reductase; FDH, formate dehydrogenase; ICDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; MS, mass spectroscopy; PDC, pyruvate decarboxylase; PEPC, phosphoenol pyruvate carboxylase; PM, plasma membrane; PVDF, polyvinyl fluoride; SPAD, portable Chl meter; 5-CF, 5-carboxy fluorescein.
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References |
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Abadía J. 1992. Leaf responses to Fe deficiency: a review. Journal of Plant Nutrition15, 1699–1713.
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