JXB Advance Access originally published online on January 17, 2007
Journal of Experimental Botany 2007 58(5):993-1000; doi:10.1093/jxb/erl259
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RESEARCH PAPER |
Iron deficiency differently affects metabolic responses in soybean roots*
Dipartimento di Produzione Vegetale, University of Milano, Via Celoria 2, I-20133 Milano, Italy
To whom correspondence should be addressed. E-mail: graziano.zocchi{at}unimi.it
Received 18 March 2006; Revised 17 September 2006 Accepted 26 October 2006
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Abstract |
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Iron deficiency responses were investigated in roots of soybean, a Strategy I plant species. Soybean responds to iron deficiency by decreasing growth, both at the root and shoot level. Chlorotic symptoms in younger leaves were evident after a few days of iron deficiency, with chlorophyll content being dramatically decreased. Moreover, several important differences were found as compared with other species belonging to the same Strategy I. The main differences are (i) a lower capacity to acidify the hydroponic culture medium, that was also reflected by a lower H+-ATPase activity as determined in a plasma membrane-enriched fraction isolated from the roots; (ii) a drastically reduced activity of the phosphoenolpyruvate carboxylase enzyme; (iii) a decrease in both cytosolic and vacuolar pHs; (iv) an increase in the vacuolar phosphate concentration, and (v) an increased exudation of organic carbon, particularly citrate, phenolics, and amino acids. Apparently, in soybean roots, some of the responses to iron deficiency, such as the acidification of the rhizosphere and other related processes, do not occur or occur only at a lower degree. These results suggest that the biochemical mechanisms induced by this nutritional disorder are differently regulated in this plant. A possible role of inorganic phosphate in the balance of intracellular pHs is also discussed.
Key words: Fe(III)-chelate reductase, Glycine max L., intracellular pH, iron deficiency, PM H+-ATPase
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Introduction |
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Iron is an essential oligo-element for all living organisms, including plants, since, as a transition element, it takes part in fundamental biological redox processes, such as respiration and photosynthesis, and in chlorophyll biosynthesis (Marschner, 1995). Soils are normally well furnished with iron, which is the fourth element in the Earth's crust, but in well-aerated and in calcareous soils it is found in oxide and hydroxide compounds with a very low solubility (Lindsay and Schwab, 1982), so that, in these conditions, plants often have to face an iron availability that is limited. Plants respond to shortage of iron by inducing responses directed towards the acquisition of the element from the rhizosphere, and, according to the way they respond, they have been divided in Strategy I (dicots and non-Graminaceous monocots) and Strategy II (Graminaceae) (Marschner and Römheld, 1994) plants.
The most complex responses are induced in Strategy I plants, that base their iron supply on a reduction-based mechanism (Schmidt, 1999), while Strategy II plants base their iron acquisition on the release of phytosiderophores and the subsequent uptake of the Fe3+-phytosiderophore complex (Curie and Briat, 2003). The reduction mechanism implies the activation of a Fe(III)-chelate reductase (FC-R), that is the ‘conditio sine qua non’ for the acquisition of the ion, since only the ferrous form is transported into the root (Chaney et al., 1972; Yi and Guerinot, 1996). The putative redox chain has not yet been identified in plasma membrane, but two sequences coding FC-R activities, named FRO2 and FRO1 have recently been cloned from A. thaliana, pea, and tomato, respectively (Robinson et al., 1999; Waters et al., 2002; Li et al., 2004).
However, the whole response to Fe-deficiency is not only limited to this mechanism, since there are other important events that come along with it. First of all, the induction of a specific transporter belonging to the ZIP family, named IRT1 (Iron-Regulated Transporter) (Guerinot, 2000) localized on the root plasma membrane, has been shown to be co-regulated with FRO2 in A. thaliana (Vert et al., 2003). The gene for IRT1 has been cloned from A. thaliana (Eide et al., 1996; Vert et al., 2002) and its activity identified through a functional expression in the yeast double mutant fet3fet4 (Eide et al., 1996). IRT1 isologues have also been found in pea (Cohen et al., 1998) and tomato (Eckhardt et al., 2001).
As stated before, the major problem plants have to cope with regarding iron acquisition is its scarce availability largely due to its low solubility. In fact, in well-aerated soils the predominant form is the ferric form and its solubility is very scarce in the physiological pH range (Marschner, 1995). To solve this problem, plants have developed, under low-iron conditions, the capacity to decrease the rhizospheric pH by increasing proton extrusion. It has been shown that this process is linked to the activation of a specific plasma membrane H+-ATPase of the root epidermal cells (Zocchi and Cocucci, 1990; Rabotti and Zocchi, 1994; Dell'Orto et al., 2000), with the aim of increasing the solubility of the sparingly insoluble iron forms by decreasing the pH of the rhizosphere, in order to generate a driving force for the uptake of the ion (Zocchi and Cocucci, 1990) and to facilitate the access of Fe-chelates to the site of reduction by neutralizing the negative charges, thereby decreasing the repulsion effect. As well as these activities, which are all located on the root plasma membrane, it has been found that the metabolism is strongly involved in order to sustain the production of reducing equivalents [NAD(P)H] and ATP (Rabotti et al., 1995; Espen et al., 2000; Zocchi, 2006). In particular, it has been shown that the activity of the PEPC is increased up to several fold (De Nisi and Zocchi, 2000; Lopéz-Millán et al., 2000). This increase could be linked with the production of substrates for the FC-R and H+-ATPase activities and generation of H+ for the cytosolic pH-stat (Espen et al., 2000). The activation of these processes makes a plant more or less efficient in the acquisition of iron. In this work it is shown that, in soybean, the mechanisms supporting the Fe-deficiency response are differently activated with respect to other Strategy I plants already studied.
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Materials and methods |
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Plant material and growth conditions
Seeds of soybean (Glycine max L. cv. A2012 from Asgrow, Italy) were used in this work. According to the company this cultivar ranks excellent for the tolerance to the Fe-deficiency chlorosis. Seeds were surface-sterilized, sown in agriperlite, watered with 0.1 mM CaSO4, allowed to germinate in the dark at 26 °C for 6 d, and then transferred to a nutrient solution with the following composition: 2 mM Ca(NO)3, 0.75 mM K2SO4, 0.65 mM MgSO4, 0.5 mM KH2PO4, 10 µM H3BO3, 1 µM MnSO4, 0.5 µM CuSO4, 0.5 µM ZnSO4, 0.05 µM (NH4)Mo7O24, and 0.1 mM Fe-EDTA (when added). The pH was adjusted to 6.0–6.2 with NaOH. Aerated hydroponic cultures were maintained in a growth chamber with a day/night regime of 16/8 h and a PPFD of 200 µmol m–2 s–1 at the plant level. The temperature was 18 °C in the dark and 24 °C in the light. Culture medium was changed weekly. Plants showed chlorotic symptoms after approximately 10 d of culture in the absence of Fe. Previous experiments carried out with agar-embedded roots in the presence of a pH indicator (bromocresol purple) and of BPDS had shown that the responses (acidification and Fe(III) reduction, respectively) were localized in the first 3–4 cm of the root apices (not shown). For this reason all the in vivo and in vitro analyses were carried out on 4-cm-long root apical segments.
Leaf chlorophyll determination
Leaf chlorophyll was extracted in 80% (v/v) acetone from fully expanded youngest leaves; solutions were centrifuged at 10 000 g for 10 min prior to measuring the absorbance in a spectrophotometer (model V550, Jasco). Chlorophyll content was determined according to Lichtenthaler (1987).
Measurement of the acidification capacity of the nutrient solution
Acidification of the medium was determined directly in the nutrient solution by measuring the pH every day with a pHM64 (Radiometer, Copenhagen) pH-meter.
In vivo FC-R activity
Fe(III)-reductase activity was measured in excised roots by using bathophenanthrolinedisulphonate (BPDS) (Chaney et al., 1972). Ten apical root segments, about 4 cm long, were incubated in 5 ml of a solution with the following composition: 0.5 mM CaSO4, 0.1 mM Fe(III)-EDTA, 0.25 mM BPDS, 10 mM MES-NaOH pH 5.5 in the dark at 25 °C. After 3 h 2 ml of the solution were withdrawn and the absorbance at 535 nm was determined spectrophotometrically. The results are linear over the experimental period. The amount of reduced Fe was calculated by the concentration of the Fe(II)- (BPDS)3 complex formed (
of BPDS is 22.1 mM–1 cm–1). Contribution of the material released by the roots on the reduction of Fe3+ was less than 5% of the total.
Isolation of plasma membrane vesicles
Plasma membrane-enriched vesicles from 14-d-old plant root apical segments grown in the presence or absence of Fe were prepared by the two-phase partitioning procedure as previously described (Rabotti and Zocchi, 1994). Final pellet collected at 80 000 g for 30 min was resuspended in a medium containing 250 mM sucrose, 2 mM MES-BTP (pH 7.0) and 4 mM PMSF.
Determination of H+-ATPase activity
The H+-ATPase activity of plasma membrane (PM) vesicles was determined with a spectrophotometric method (Palmgren et al., 1990), coupling ATP hydrolysis to NADH oxidation at 25 °C in 1.5 ml final volume as already reported (Rabotti and Zocchi, 1994). Reaction was started by addition of 20–50 µl of PM preparation. NADH oxidation was followed at 340 nm and the absorbance changes monitored over a 5 min period.
Determination of FC-R activity
The NADH-dependent FC-R activity of PM vesicles was assayed in a medium containing 250 mM sucrose, 15 mM MOPS-BTP (pH 6.0), 0.25 mM K3Fe(CN)6, 0.25 mM NADH, and 0.01% Lubrol (Sigma-Aldrich). Reaction was started by the addition of 20–50 µl of PM preparation. NADH oxidation was followed at 340 nm and the absorbance changes monitored over a 5 min period.
Enzyme assays
Cytosolic soluble enzymes were extracted from 14-d-old plant root apical segments grown in the presence or absence of Fe as already reported (Rabotti et al., 1995).
Hexokinase (HK) (EC 2.7.1.1 [EC] ) activity was determined in a buffer containing 82.5 mM triethanolamine-NaOH (pH 7.6), 2 mM MgSO4, 1 mM EDTA, 0.5 mM PEP, 0.2 mM NADH, 1 mM ATP, 5 mM glucose, 30 µg ml–1 PK (EC 2.7.1.40 [EC] ), and 15 µg ml–1 LDH (EC 1.1.1.27 [EC] ).
Phosphofructokinase 1 (PFK-1) (EC 2.7.1.11 [EC] ) and pyruvate kinase (PK) were determined as already reported by Espen et al. (2000) while phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31 [EC] ) was determined as reported by De Nisi and Zocchi (2000).
Reaction was started by adding aliquots of protein extracts and the enzymatic assay was performed at 25 °C in 1.5 ml final volume. Oxidation of NADH was followed spectrophotometrically at 340 nm.
Proteins were determined by the Bradford method using 
-globulin as the standard (Bradford, 1976).
Collection of root exudates
After 14 d of hydroponic culture with or without Fe, plants were rinsed and transferred to vessels containing 300 ml of distilled water. Root exudates were collected for a period of 4 h. After the collection, Micropur® was added to the exudates to prevent microbial degradation of organic solutes. The samples were then cooled to 0 °C and filtered through filter paper. Solutions containing root exudates were then concentrated by evaporation and again filtered on a 0.22 µm cellulose acetate filters. This final solution was used for quantification of citrate, amino acids, phenols, and total organic carbon.
Quantification of citrate, amino acids, phenols and total soluble carbon in root exudates
Citrate was quantified enzymatically, using a specific kit from Boehringer-Mannheim; the assay was performed according to the manufacturer's instructions.
Total amino acids were quantified by the ninhydrin method according to Hirs et al. (1954).
Total phenolics in the root exudates were estimated by Folin–Ciocalteau method, as described by Singleton and Rossi (1965), measuring the absorbance at 750 nm. Phenolics concentration was calculated from the calibration curve using caffeic acid as a standard.
Finally, soluble organic carbon was determined by using a modified procedure according to Von Wirén et al. (1995) through oxidation with K2Cr2O7, and the Cr3+ formation was determined spectrophotometrically at 578 nm.
Nuclear magnetic resonance spectrometry
The 31P-NMR spectra were recorded on a standard broad-band 10 mm probe on a Bruker AMX 600 spectrometer (Bruker Analytische Messtechnik, Rheinstetten-Forchheim, Germany) equipped with an X32 data system, running UXNMR software, version 920801. The 31P-NMR spectra were recorded at 242.9 MHz without lock, with a Waltz-based broad-band proton decoupling and a spectral window of 16 kHz. The spectra were acquired using a 90 °C pulse angle and a 6 s recycle time to give fully relaxed resonance (except for vacuolar phosphate). In vivo 31P-NMR experiments were carried out by packing about 35 root segments (up to 4 cm, excised from the tip) obtained from 14-d-old plants grown in the presence or absence of Fe, into a 10-mm-diameter NMR tube equipped with a perfusion system connected to a peristaltic pump in which the aerated, thermoregulated (26 °C) medium [0.5 mM CaSO4, 1 mM MDP, 1 mM MES-BTP (pH 6.1)] flowed at 10 ml min–1, as described by Espen et al. (2000). Cytoplasmic and vacuolar pHs were estimated from the chemical shift of Pi resonance after construction of a standard titration curve (Roberts et al., 1980, 1981). Pi content was estimated measuring the area of the 31Pi resonances by Lorential line-shape analysis and the values obtained were referred to the percentage volume of the tissue in the NMR tube (Spickett et al., 1992; Espen et al., 2000).
Measurement of Pi levels
Roots from plants grown in the presence or in the absence of Fe were homogenized in 4 vols of 10% (v/v) ice-cold trichloroacetic acid and centrifuged at 13 000 g for 15 min. Inorganic phosphate was determined in the supernatant using the Fiske and Subbarow (1925) method.
Statistical analyses
Values are the means ±SE of three independent experiments in triplicate.
One-way analysis of variance (ANOVA) was used for all the tested parameters and the means were compared by Student t test at P 
0.05. Data reported in Table 8 were treated with a two-way analysis of variance (ANOVA) by using the Tukey test at P 0.05.
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Results |
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Soybean plants responded to Fe shortage by greatly decreasing growth both at the root and shoot level (Table 1). Interveinal chlorosis of the younger leaves is a classical symptom of iron deficiency and in these leaves the chlorophyll content was dramatically decreased after 14 d of Fe deficiency (Table 2).
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The time-course of the medium acidification by the soybean plant roots grown in hydroponic culture in the presence and in the absence of iron, along with their capacity to reduce the iron chelate Fe(III)-EDTA in vivo, are shown in Fig. 1. The acidification of the medium occurs, but at a very low degree, less than 1.0 pH unit, even after 16 d of Fe deficiency. When compared with other plants like cucumber, for instance, where the acidification reaches a difference of about 2.5 pH units in a few h (Zocchi and Cocucci, 1990; Rabotti and Zocchi, 1994), pH changes occurring with soybean are very poor. Conversely, the activity of the Fe3+ reduction in vivo is efficient and reaches a maximum after 12 d of hydroponic culture. This is the reason why the choice was made to use 14-d-old plants to perform all the measurements both in vivo and in vitro. The assay of the enzymatic activities involved in the iron reduction and in the acidification of the medium was also determined on a plasma membrane-enriched fraction isolated from roots. As determined in vivo, the activities of the two enzymes involved were enhanced under Fe deficiency also in vitro, with the increase in FC-R being roughly around 100%. The increase in H+-ATPase activity was small, only around 24% (Table 3), confirming data obtained in vivo. The lower H+-ATPase activity under Fe deficiency is also coherent with a very low transmembrane electrical potential difference determined between +Fe and –Fe roots, less than 10 mV (not shown), as compared to values found in cucumber which were around 40 mV (Zocchi and Cocucci, 1990).
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Other metabolic activities that are usually increased with Fe deficiency, and it has been assumed to be important for the whole response to Fe deficiency (Espen et al., 2000; Zocchi, 2005). These include phosphoenolpyruvate carboxylase, that has been shown to increase in cucumber and sugar beet roots (De Nisi and Zocchi, 2000; Lopéz-Millán et al., 2000). The determination of some glycolytic enzyme activities also confirms such an increase for the soybean plants, while, on the contrary, it was quite surprising to find that the PEPC activity was decreased under the same condition (Table 4).
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The total organic carbon in exudates from soybean roots under Fe-deficiency was almost doubled with respect to the control; the analysis of the specific compounds in exudates (citrate, amino acids, and phenolics) shows that their increase follows more or less what occurs for the total organic carbon (Table 5). On a per cent basis, these compounds account for 6.0, 7.5, and 4.8% of the total exuded carbon, respectively, for the –Fe condition. These values are of the same order of magnitude of those found by Römheld and Marschner (1983) for Fe-deficient peanut plants.
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More information on the biochemical changes that occur in soybean roots grown in the presence and in the absence of iron were obtained by a 31P-NMR analysis. The in vivo spectra obtained with roots of 14-d-old plants are shown in Fig. 2. The spectra are characteristic of a well oxygenated plant tissue and were stable over several hours of data acquisition. The well-defined peaks of resonance allowed for the definition of the intracellular pHs and a quantitative determination of the Pi. Iron starvation induced several changes and the most evident was the increase in the vacuolar phosphate content and the increase in the sugar phosphate and NTP (more likely ATP) peaks (Fig. 2). A quantification of the intracellular Pi concentration is shown in Table 6, where an increase in the vacuolar Pi content (+83%) is evident. Values of the pHc and pHv are reported in Table 7. Iron-deficiency induces a slight, but still significant decrease, both in the pHc and to a greater extent in the pHv. These data are consistently different from those determined in a similar study conducted with cucumber roots, where it was found that the vacuolar phosphate was almost completely depleted under Fe-deficiency and the intracellular pHs were slightly increased (Espen et al., 2000). The concentration of Pi in TCA extracts from roots at different growing times is reported in Table 8. The results show that the Pi level in the tissue was in agreement with the increase seen in the in vivo 31P-NMR experiments.
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-phosphate of NTP and NDP, UDP-Glc and NAD(P)H; 8, UDP-Glc; 9, ß-phosphate of NTP. In three independent experiments the resonance intensities differed by 12% at the most. The nucleotide region is shown on an expanded (x6) scale.
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Discussion |
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Strategy I plants have been classified on the basis of their biochemical responses to Fe deficiency (Marschner and Römheld, 1994), the main process being a reduction-based mechanism that reduces apoplastic Fe3+ to Fe2+, the form necessary for uptake. In the strategy adopted by dicots and non-graminaceous monocots, every species can modulate the activation of biochemical mechanisms as a function of specific environmental conditions or according to intrinsic characteristics, such as the capacity to modify the metabolism in order to sustain the energetic effort or to extrude inorganic (ions, protons) or organic (amino acids, organic acids, phenolics, flavins, etc.) compounds (Zocchi, 2006, and references therein). To understand the whole response to Fe deficiency it is important to know not only which mechanisms are induced by the Fe-deficient plants but, also, how plants may regulate those mechanisms. Alkaline and calcareous soils represent a serious concern for iron acquisition by plants, since in these conditions the range of inorganic iron availability is around 0.1–10% of the normal requirement for optimal plant growth (Römheld and Marschner, 1986). One-third of the world's soil are below the level of iron bioavailability, limiting plant growth and productivity. All plant species belonging to the Strategy I growing on calcareous soils suffer from Fe deficiency, but there is an inter- and an intraspecific variability in their susceptibility to this nutritional disorder, despite a similar demand for iron. These genotypic differences are strongly related to the ability of plants to mobilize iron in the rizosphere and to their capacity to take up the ion. Soybean plants are particularly sensitive to the presence of bicarbonate and/or to elevated soil pH, since even tolerant genotypes greatly suffer under these conditions (M Dell'Orto, personal communication). If the results obtained with other plant species, such as cucumber, are compared, distinctive differences in the responses to Fe deficiency can be noticed. Rizosphere acidification has been recognized for many years as an important response to iron deficiency for Strategy I plants (Römheld and Marschner, 1986; Schmidt, 1999), and this response has been associated with the activity of a plasmalemma-localized H+-ATPase (Zocchi and Cocucci, 1990; Dell'Orto et al., 2000). However, unlike the FC-R, increased H+-ATPase activity has not always been observed, and the extent of such stimulation seemed to differ considerably among plant species and genotypes, ranging from very low values to about 100% (Schmidt, 1999, and references therein). For instance, in cucumber roots, one of the most active acidifying species, it was shown that a higher activity under iron deficiency was associated with an enhancement in the level of the enzyme (Dell'Orto et al., 2000) and of its transcript (Dell'Orto et al., 2002). However, for many other dicots, including soybean, this capacity is strongly limited even in the efficient genotypes (Brown and Jones, 1976; Landsberg, 1982), although there is evidence for localized acidification of the rhizosphere (Römheld and Marschner, 1984). In the literature only one report (Yi and Guerinot, 1996) has clearly shown acidification under iron deficiency by Arabidopsis roots. This could explain why in Arabidopsis, Thimm et al. (2001), using microarray analysis, did not find any change in the expression of the gene encoding the root H+-ATPase. A similar scenario seems to occur in soybean, where the increase in the H+-ATPase activity is quite low both in vivo and in vitro (Fig. 1; Table 3). Another striking difference is the development of transfer cells in the apical root portion. Some authors correlated the increase in the medium acidification with the development of transfer cells. However, these morphological modifications do not occur in soybean roots (Landsberg, 1982; this work, not shown). Therefore it could be hypothesized that, beyond the fundamental step of iron reduction, the efficiency responses rely on other reactions that could be differently regulated among different species.
The 31P-NMR study has shown that both pHc and pHv, as determined by the chemical shift of Pi, are decreased in soybean plant roots grown under Fe deficiency. This was quite surprising, since, working with cucumber, it had previously been found that both pHc and pHv had increased (Espen et al., 2000). From Fig. 2 and from Table 6 further important information is apparent. When comparing the results obtained with those found in cucumber plants (Espen et al., 2000), a major difference exists with regard to the relative amount of vacuolar Pi, which is enhanced in soybean whereas it is almost completely depleted in cucumber (Espen et al, 2000). How could this different compartmentalization of Pi be explained? It was claimed (Davies, 1973) that the plasmalemma H+-ATPase and the PEPC activities are part of the plant pH-stat mechanism. Plants might have developed different extra-help systems to balance the pHc, one of these could involve the Pi movements in the cell. As suggested by Kurkdjian and Guern (1989) the vacuole could actively participate in the physiological responses and to the poise of pHc and, as hypothesized by Espen et al. (2000), Pi could also be involved in a mechanism of pH-stat. Two different scenarios might be predicted. In the first one, suitable for species active in the acidification of the rhizosphere, increased PM-H+-ATPase activity corresponds with the alkalinization of the pHc which, in turn, activates PEPC. The major need of H+ in the cytosol to keep the pH at its physiological level might come from increasing the rate of glycolysis (Sakano, 1998) and/or from a de-protonation of 
released from the vacuole (Espen et al., 2000).
In the case of soybean a second picture might be hypothesized. The lower activity of the PM H+-ATPase (Fig. 1; Table 3) is associated with a decrease in the pHc (Table 7) also as the result of a more active glycolytic pathway producing H+ (Table 4). Decrease in the pHc would, as a consequence, decrease the activity of the PEPC (Table 4).
In soybean, H+ could be pumped into the vacuole to re-equilibrate the pHc to its physiological value (in fact the pHv decreases, Table 7) by a tonoplast-associated H+-ATPase and/or H+-translocating PPase, which would facilitate the Pi transport into the vacuole (Table 6). There is evidence for ATP-dependent Pi transport across the tonoplast (Sakano et al., 1995). The last two mechanisms could be stimulated by the increased amount of the cytosolic ATP (Fig. 2) and/or PPi as the consequence, from either a lower utilization by the PM-H+-ATPase, or from active catabolism. A simple model to explain the movement of Pi across the tonoplast and its possible involvement in the pH-stat mechanisms are depicted in Fig. 3. At present, no biochemical or molecular information are available for the Pi transporters nor on the activity of the H+ translocating mechanisms at the tonoplast, and further work will be necessary to elucidate these mechanisms and their implication in the mechanism of intracellular pH poise. In conclusion, from this work it emerges that the efficient response to iron starvation comes from many different responses; the more a plant is able to activate such activities the more efficient it will be in iron acquisition. Expression and regulation of these responses could be different among species or genotypes providing them with a different level of efficiency.
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Acknowledgements |
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This work was supported by grants from the Italian Ministry of Education (Cofin 2004) and FIRST 2004 to GZ. We thank Dr Silvia Donnini for her help in the statistical analysis.
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Footnotes |
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* This paper is dedicated to the memory of Professor John B Hanson, a great scientist, who passed away on 23 October 2006.
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Abbreviations |
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BPDS, bathophenanthrolinedisulphonate; BTP, 1, 3-bis[tris(hydroxymethyl)-methylamino]-propane; FC-R, Fe(III)-chelate reductase; MDP, methylenediphosphonate; MOPS, 4-morpholinopropanesulphonic acid; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; pHc, cytosolic pH; pHv, vacuolar pH; PM, plasmalemma; PMSF, phenylmethylsulphonyl fluoride; PPFD, photosynthetic photon flux density.
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