Journal of Experimental Botany, Vol. 58, No. 5, pp. 993–1000, 2007 doi:10.1093/jxb/erl259 Advance Access publication 17 January, 2007
RESEARCH PAPER
Iron deficiency differently affects metabolic responses in soybean roots* Graziano Zocchi†, Patrizia De Nisi, Marta Dell’Orto, Luca Espen and Pietro Marino Gallina Dipartimento di Produzione Vegetale, University of Milano, Via Celoria 2, I-20133 Milano, Italy Received 18 March 2006; Revised 17 September 2006; Accepted 26 October 2006
Introduction
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.
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 Ro¨mheld, 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).
Key words: Fe(III)-chelate reductase, Glycine max L., intracellular pH, iron deficiency, PM H+-ATPase.
* This paper is dedicated to the memory of Professor John B Hanson, a great scientist, who passed away on 23 October 2006. y To whom correspondence should be addressed. E-mail:
[email protected] Abbreviations: 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. ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail:
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Abstract
994 Zocchi et al.
Materials and methods 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 lM H3BO3, 1 lM MnSO4, 0.5 lM CuSO4, 0.5 lM ZnSO4, 0.05 lM (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 lmol 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 agarembedded 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 (e 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 ll of PM preparation. NADH oxidation was followed at 340 nm and the absorbance changes monitored over a 5 min period.
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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; Lope´z-Milla´n 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.
Iron deficiency in soybean roots 995 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 ll of PM preparation. NADH oxidation was followed at 340 nm and the absorbance changes monitored over a 5 min period.
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 lm 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 Wire´n 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 31 P-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
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 6SE 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
