Journal of Experimental Botany, Vol. 51, No. 345, pp. 695-701,
April 2000
© 2000 Oxford University Press
Development of Fe-deficiency responses in cucumber (Cucumis sativus L.) roots: involvement of plasma membrane H+-ATPase activity
1 Dipartimento di Produzione Vegetale, University of Milan, Via Celoria 2, I-20133 Milano, Italy
2 Dipartimento di Produzione Vegetale e Tecnologie Agrarie, University of Udine, Via Delle Scienze 208, I-33100 Udine, Italy
Received 27 September 1999; Accepted 25 November 1999
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Abstract |
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One of the mechanisms through which some strategy I plants respond to Fe-deficiency is an enhanced acidification of the rhizosphere due to proton extrusion. It was previously demonstrated that under Fe-deficiency, a strong increase in the Hplus;-ATPase activity of plasma membrane (PM) vesicles isolated from cucumber roots occurred. This result was confirmed in the present work and supported by measurement of ATP-dependent proton pumping in inside-out plasma membrane vesicles. There was also an attempt to clarify the regulatory mechanism(s) which lead to the activation of the H+-ATPase under Fe-deficiency conditions. Plasma membrane proteins from Fe-deficient roots submitted to immunoblotting using polyclonal antibodies showed an increased level in the 100 kDa polypeptide. When the plasma membrane proteins were treated with trypsin a 90 kDa band appeared. This effect was accompanied by an increase in the enzyme activity, both in the Fe-deficient and in the Fe-sufficient extracts. These results suggest that the increase in the plasma membrane H+-ATPase activity seen under Fe-deficiency is due, at least in part, to an increased steady-state level of the 100 kDa polypeptide.
Key words: Fe-deficiency, H+-ATPase, plasma membrane, proton extrusion, rhizosphere.
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Introduction |
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Increased acidification of the rhizosphere is acknowledged as a part of the mechanism through which dicotyledon plants (Strategy I plants) respond to Fe starvation. It has been proposed that this process involves the activation of root plasma membrane (PM) H+-ATPase. By pumping protons outside the cell this enzyme contributes to enhance the solubility of Fe oxides and generates the proton motive force for ion uptake. In addition, its activity would help to maintain an adequate environment for the activity of the Fe-deficiency-induced PM Fe(III)-chelate reductase (low apoplastic pH and membrane potential homeostasis).
Unlike Fe(III)-chelate reductase (Moog and Brüggemann, 1994
) increased H+-ATPase activity was not frequently observed in isolated plasma membrane vesicles; moreover, the extent of stimulation of the enzyme activity seems to differ considerably between plant species and genotypes (Bienfait, 1988; Buckhout et al., 1989; Chosack et al., 1991; Rabotti and Zocchi, 1994; Schmidt et al., 1997). This could be due to the relative importance of the Fe-deficiency-induced reactions (increased net proton efflux and enhanced root PM Fe(III)-chelate reductase activity) among the different species (Wei et al., 1997). The stage of plant development and/or severity of Fe-deficiency might also contribute to the observed differences, since these parameters are often associated with root morphological changes and localization of the response reactions (Römheld and Marschner, 1986). It has also been suggested that Fe(III) reduction and proton extrusion activities may be regulated independently (Yi and Guerinot, 1996).
Recently, considerable progress has been made in elucidating the molecular mechanisms of regulation of PM H+-ATPase (for review see Palmgren, 1998), which involve de novo synthesis of the enzyme (Hager et al., 1991; Niu et al., 1993; Santi et al., 1995), the presence of an autoinhibitory domain at the C-terminus (Palmgren et al., 1990), phosphorylation/dephosphorylation cycle and the occurrence of different isoforms (Sussman, 1994; De Nisi et al., 1999).
In a previous work (Rabotti and Zocchi, 1994), it was demonstrated that Fe-deficiency caused a significant increase in H+-ATPase activity of plasma membrane vesicles isolated from cucumber roots. The present study was undertaken to cast some light on the regulatory aspects of the increased PM H+-ATPase activity in roots of Fe-deficient plants. Plasma membrane vesicles were isolated from roots of cucumber plants during the development of the physiological response to Fe stress. Scalar (ATP hydrolysis) and vectorial (H+ pumping) components of the PM H+-ATPase activity were assayed. Furthermore, polyclonal antibodies were used to assess quantitative and qualitative changes of the enzyme. It was possible to show that an increase in the steady-state level of the enzyme is, at least in part, responsible for the enhanced activity of the plasma membrane proton pump in Fe-deprived cucumber roots.
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Materials and methods |
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Plant material
Cucumber seeds (Cucumis sativus L., cv. Marketmore 76, F.lliIngegnoli, Italy) were germinated on filter paper moistened with 0.1 mM CaSO4. After 5 d, seedlings were transferred to an aerated nutrient solution (iron contamination not higher than 10 ppb) with the following composition (mM): Ca(NO3)2 2.0, K2SO4 0.7, MgSO4 0.5, KCl 0.1, KH2PO4 0.1; (µM), H3BO3 10, MnSO4 0.5, ZnSO4 0.5, CuSO4 0.2, (NH4)6Mo7O24 0.01. The nutrient solution was changed every 4 d and adjusted to pH 6.3 with 1 N NaOH. Iron, when added, was supplied as Fe(III)-EDTA at a final concentration of 80 µM. Plants were grown under 16/8 h light/dark regime at 25 °C, relative humidity 65–75%, light intensity 200 µE m-2 s-1. Plant age was operationally defined as the time span since transfer of seedlings to the nutrient solution.
Fe(III)-EDTA reduction by intact roots
Fe(III)-EDTA reduction by roots of intact cucumber plants was measured by using the bathophenanthrolinedisulphonate (BPDS) reagent (Chaney et al., 1972). Roots of a single plant were incubated in 5 ml of an aerated solution containing CaSO4 0.5 mM, Fe(III)-EDTA 0.25 mM, BPDS 0.6 mM, MES-NaOH 10 mM (pH 5.5) in the dark at 25 °C. After 30–60 min, the absorbance of the solution was read at 535 nm, and the amount of Fe(III) reduced was calculated by the concentration of the Fe(II)-BPDS3 complex formed, using an extinction coefficient of 22.1 mM-1 cm-1.
Isolation of plasma membrane vesicles
Plasma membrane vesicles were purified from root homogenates using the sucrose gradient (Pinton et al., 1993) or two-phase partitioning (Rabotti and Zocchi, 1994) procedure as previously described. Final pellets were resuspended in a medium containing 2 mM MES (2-[N-morpholino]ethanesulphonic acid)-BTP (1,3-bis[tris(hydroxymethyl)-methylamino]-propane), pH 7.0, 1 mM DTT (omitted for redox activity assays and trypsin treatment), 1 mM phenylmethylsulphonyl fluoride (PMSF, omitted for trypsin treatment) and 10% (v/v) glycerol (sucrose gradient) or 250 mM sucrose (two-phase-partition).
Enzyme assays
ATPase activity of inside-out plasma membrane vesicles prepared by sucrose gradient was assayed by incubating membrane vesicles (2–5 µg protein) at 25 °C in 0.6 ml of 60 mM MES-BTP (pH 6.5), 6 mM MgSO4, 6 mM ATP-BTP (pH 6.5), 0.7 mM Na-molybdate, 1 mM NaN3, and 100 mM KNO3. The released inorganic phosphate was determined by the method of Forbusch (Forbusch, 1983).
ATP-dependent proton accumulation into membrane vesicles was assayed determining the decrease in the absorbance of acridine orange (AO) in 1.5 ml of standard assay medium including 60 mM MES-BTP (pH 6.5), 6 mM MgSO4, 6 mM ATP-BTP (pH 6.5), 10 µM AO, 0.5 mM NaN3, 100 mM KNO3, and 15–20 µg membrane protein. Absorbance changes were spectrophotometrically recorded at 492 nm, at 25 °C. Initial rates were calculated to determine proton pumping activity.
The enrichment of plasma membrane relative to other endomembrane vesicles was estimated by assaying, at 25 °C, the vanadate-sentitive (pH 6.5), nitrate-sensitive and azide-sensitive (pH 8.0) ATPase activity in the presence or absence of 0.1 mM vanadate, 100 mM KNO3 or 1 mM NaN3, respectively. In these assays 0.015% (w/v) Brij 58 was added.
ATPase activity of two-phase-partitioned vesicles was assayed as reported above at 25 °C, except that 0.015% (w/v) Brij 58 was added to the reaction mixture. Alternatively, it was measured with a spectrophotometric method (as described by Palmgren et al., 1990), coupling ATP hydrolysis to NADH oxidation, at 25 °C.
Plasma membrane vesicles obtained by the two-phase partition method were routinely checked for purity by marker enzyme analysis as previously described (Rabotti and Zocchi, 1994).
The NADH-dependent Fe(III)-EDTA-reductase activity of plasma membrane vesicles isolated from cucumber roots by two-phase partition was determined in the dark at 26 °C in 1 ml volume containing 250 mM sucrose, 40 mM MES-BTP (pH 7), 0.25 mM Fe(III)-EDTA, 0.5 mM BPDS, 5 µg of plasma membrane protein and, when added, 0.015% (w/v) Brij 58. The reaction was started by the addition of 0.25 mM NADH (final concentration) and the absorbance changes at 535 nm were monitored over a 20 min period. The rate of Fe(III) reduction was calculated as reported above for intact roots.
Protein content of membrane vesicles preparations was determined, using BSA as a standard (Bradford, 1976).
Trypsin treatment of PM proteins
Aliquots of two-phase-partitioned PM vesicles were mixed with an equal volume of 50 mM MES-BTP pH 7.5, 250 mM sucrose, 10 mM EDTA, 4 mM ATP, and 0.08% (w/v) Brij 58 containing trypsin (0.04 µg µg-1PM protein) to give a final PM protein concentration of about 1.5 µg µl-1. Samples were incubated 10 min at 20 °C. Proteolysis was stopped by adding trypsin inhibitor (10 µg µg-1 trypsin). In controls, trypsin and trypsin inhibitor were omitted or trypsin inhibitor was added at the end of the experiment, but no difference was found between these two treatments.
SDS-PAGE and immunoblotting
Membrane vesicles were solubilized in 1 vol of SDS-loading buffer containing 0.125 M TRIS-HCl, pH 7.4, 10% (w/v) SDS, 0.2 M DTT, 10% (w/v) glycerol, 0.002% bromophenol blue, 500 µg ml-1 chymostatin, and 5 mM PMSF (Gallagher and Leonard, 1987). After a 30 min incubation at 37 °C, samples were loaded on a discontinuous SDS-polyacrylamide gel (3.75% [w/v] acrylamide stacking gel, and typically 9% [w/v] acrylamide separating gel) (Laemmli, 1970). The resulting gels were stained with 0.045% (w/v) Coomassie Brilliant Blue R-250 in 17% (v/v) ethanol, 5.3% (w/v) trichloracetic acid and 6% (v/v) acetic acid, and then destained with a 10% ethanol and 7.5% acetic acid solution.
After SDS-PAGE PM samples were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane filters (Sigma) using a semi-dry blotting system with a buffer containing 10 mM 3-cyclohexylamino-1-propane sulphonic acid (pH 11.0 with NaOH) and 10% (v/v) methanol for 1.5 h at room temperature at a intensity current of 0.8 mA cm-2. Two different antisera have been used; one raised against the PM H+-ATPase of maize roots (a kind gift from Dr RT Leonard, University of Arizona, Tucson) (Fig. 2) and a second raised against the central domain of Arabidopsis thaliana PM H+-ATPase (a kind gift from Dr R Serrano, Universidad Politécnica, Valencia, Spain) (Fig. 3). Antiserum was diluted 1 : 3000 in TBS-T buffer [20 mM TRIS-HCl (pH 7.5, 200 mM NaCl, 0.05% (w/v) Tween 20] and incubation was carried out overnight at 4 °C. After rinsing with TBS-T, PVDF membranes were incubated at room temperature for 2 h with a 1 : 25 000 diluted secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG, Sigma). After rinsing in TBS-T the filters were incubated in 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (FAST BCIP/NBT, Sigma). The blots were scanned and the H+-ATPase immunoreactive bands were quantified by using a Panasonic videocamera linked to a personal computer. Images were processed with a CREAM sofware (KEM-EN-TEC, Copenhagen, Denmark).
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Results |
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Development of Fe-deficiency response in intact plants: Fe(III)-reduction and H+release
Higher Fe(III)-EDTA reductase activity of intact roots was observed as soon as 2 d after transferring cucumber seedlings to the Fe-free nutrient solution. Preliminary time-course experiments showed that a maximum rate was reached at day 5. This pattern resulted in a 12-fold increase in the reductase activity measured in Fe-deficient plants at day 5, as compared to Fe-sufficient plants (Table 1
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Plants grown in the presence of nitrate and the absence of Fe started to decrease the pH of the nutrient solution after 3 d. An average value of 5.6 was recorded at day 4. After renewal of the nutrient solution the pH dropped from the initial value of 6.3 to 4.4 in 1 d and reached values around 4.0 at day 8. This pattern was repeatedly observed up to 14 d in hydroponic culture. A raise in the pH of the nutrient solution was routinely found when plants were adequately supplied with Fe (Table 1
). Deficient plants supplied with 80 µM Fe-EDTA at day 4 showed a considerably lower capacity to decrease the pH of the nutrient solution, in comparison with plants maintained in the Fe-free status (an average value of 5.5 was measured at day 5 instead of 4.4). On the other hand, this treatment caused a considerable increase in the reductase activity measurable at day 5 (1160 versus 780 nmol Fe(III) g-1 fw h-1).
No significant difference in root and shoot dry weight accumulation between 5-d-old Fe-deficient and Fe-sufficient plants was observed; however, at this stage of development, in plants grown in Fe-free nutrient solution primary leaves were slightly chlorotic, production of lateral roots occurred and proliferation of root hairs all along the main root axis, with the exception of the apical zone, and at the base of laterals was visible (data not shown). Visual symptoms of Fe-deficiency and morphological modification became more pronounced with increasing plant age. In order to minimize secondary effects of Fe-deficiency, 5-d-old plants were used for subsequent membrane isolation.
H+-ATPase and Fe(III)-chelate reductase of plasma membrane vesicles
Plasma membrane vesicles were isolated from 5-d-old cucumber roots by using a simplified sucrose density gradient and/or the two-phase partition method. The first procedure allows the recovery of a large amount of inside-out plasma membrane vesicles suitable for studies on proton and ion fluxes, while the latter provides high purity plasma membrane vesicles in which the activity of Fe(III)-chelate reductase can be monitored, thus minimizing the interference of other intracellular membrane-bound reductases.
The assay of marker enzyme activities (vanadate-sensitive, nitrate-sensitive and azide-sensitive ATPases) indicated that the 30/40% sucrose interface represents a plasma membrane-enriched fraction (Table 2). Membrane vesicles displayed ATP-dependent proton pumping activity, which was largely insensitive to nitrate, azide and oligomycin (less than 10% inhibition), while it was almost completely inhibited by 0.1 mM vanadate (c. 90%), indicating the presence of tightly sealed inside-out plasma membrane vesicles (data not shown). The composition of the membrane vesicles was similar both for Fe-sufficient and Fe-deficient plants. Latency of the vanadate-sensitive ATPase activity, calculated on the basis of the stimulation in the presence of Brij 58, was about 40% for both membrane vesicle preparations from Fe-deficient and Fe-sufficient roots. However, both the ATP hydrolytic activity and, to a greater extent, ATP-dependent H+ pumping were enhanced in plasma membrane-enriched vesicles isolated from roots of Fe-deficient plants (Table 3).
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A492 mg-1 protein min-1) and Fe(III)-EDTA reduction determined as NADH oxidation (nmol mg-1 protein min-1) of plasma membrane vesicles isolated from roots of 5-d-old Fe-sufficient (+Fe) and Fe-deficient (-Fe) cucumber plants by sucrose gradient (SG) or two phase-partition (TPP) procedures. Data are means ±SE (n=4). Per cent values are given in brackets.Plasma membrane vesicles prepared by two-phase partitioning were minimally contaminated by endomembranes, confirming previous observations (Rabotti and Zocchi, 1994
). In these vesicles an increase in the rate of ATP hydrolysis was monitored when cucumber plants were grown for 5 d in the absence of Fe (Table 3). Moreover, in the same plant roots PM Fe(III)-EDTA reductase activity was nearly 3-fold enhanced when compared to Fe-sufficient plants.
Immunoblotting and trypsin treatment of plasma membrane H+-ATPase
Vesicles isolated from 5-d-old Fe-deficient and Fe-sufficient cucumber roots were compared with respect to their content in PM H+-ATPase. Proteins from plasma membrane vesicles were analysed using polyclonal antibodies raised against the PM H+-ATPase from Zea mays (Fig. 1) and the central domain of Arabidopsis thaliana PM H+-ATPase (Fig. 2). Western blotting analysis (Fig. 1) revealed an increased steady-state level of the PM H+-ATPase in plants grown for 5 d in Fe-free nutrient solution, as compared with plants adequately supplied with Fe. The increase estimated by densitometric analysis was around +40% with a SE never higher than 10% of the means.
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Immunoblotting of plasma membrane proteins typically showed the 100 kDa H+-ATPase band while no 90 kDa band was observed. When membrane vesicles were treated with trypsin a 90 kDa polypeptide band was evident (Fig. 2
). This effect was accompanied by an increase in the enzyme hydrolytic activity, which was more evident when assayed at pH 7.4 (Table 4) in agreement with other reports (Palmgren et al., 1990; Rasi-Caldogno et al., 1993). The effect of the trypsin treatment on the hydrolytic activity and on the electrophoretic behaviour of PM H+-ATPase was similar in membrane vesicles isolated from either Fe-sufficient or Fe-deficient plants. The present per cent stimulation of the H+-ATPase activity caused by Fe-deficiency remained essentially the same both when measured at pH 6.5 and pH 7.4 and in treated and non-treated samples (Table 4).
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Discussion |
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Activation of the plasma membrane proton pump (PM H+-ATPase) of root cortical cells is acknowledged as one of the biochemical mechanisms by which strategy I plants increase their capacity to mobilize and acquire iron from the sparingly soluble forms present in the soil (Marschner and Römheld, 1994
; Schmidt, 1999). It was previously demonstrated that the ATP-hydrolytic activity of the enzyme was enhanced by Fe-deficiency in plasma membrane vesicles isolated from cucumber roots (Zocchi and Cocucci, 1990; Rabotti and Zocchi, 1994). This result was confirmed in the present work and supported by the measurement of the ATP-dependent H+ accumulation in inside-out plasma membrane-enriched vesicles. As far as is known, an intensified ATP-driven H+ transport in isolated plasma membrane-enriched vesicles has only been shown for cotton roots (Chosack et al., 1991), following the accumulation of the lipophilic weak base 14C-methylamine. A higher increase in H+ pumping compared to ATP hydrolysis in PM vesicles from Fe-deficient plants was repeatedly found. This discrepancy may be due to the fact that the acridine orange signal is not linearly related to the ATPase activity (Palmgren, 1991). Passive membrane permeability to protons was apparently not involved since it was unaffected or even higher, in some cases, in vesicles from Fe-deficient plants as compared to those isolated from Fe-sufficient plants (not shown). Alternatively, a change in the coupling between the two components of the H+-ATPase activity might be hypothesized (Palmgren et al., 1991; Johansson et al., 1993).
Using polyclonal antibodies, it was also demonstrated for the first time, that the development of a higher PM H+-ATPase activity in Fe-deficient roots was associated with an increase in the steady-state level of the enzyme. Immunoblotting analysis revealed the presence of a 100 kDa PM H+-ATPase polypeptide while no band at 90 kDa was observed (Fig. 1), suggesting that the proteolytic removal of the C-terminal autoinhibitory domain (Palmgren et al., 1991) might not be responsible for the increase in the H+-ATPase activity induced by the Fe-deficiency. Furthermore, treating membrane vesicles with trypsin led to the appearance of a 90 kDa band in addition to the 100 kDa band (Fig. 2) and to the activation of the enzyme with a shift in the pH optimum either in PM vesicles isolated from Fe-deficient or in PM vesicles from Fe-sufficient plants (Table 4), suggesting, at least within the limit of the immunodetection technique, that a displacement of the C-terminal domain of the proton pump might be not involved in the activation which occurs under Fe-deficient conditions. Nevertheless, it is still possible that a subtle modification of the C-terminal domain and, consequently, the abolition of its autoinhibitory function might occur under Fe-deficiency in vivo, leading to an increase in PM H+-ATPase activity. Jahn et al., using detergent-solubilized plasma membrane isolated from fusicoccin-treated maize shoots, suggested that the interaction between the C-terminal domain of the H+-ATPase and the 14-3-3 proteins could have a regulatory role on the enzyme activity in vivo (Jahn et al., 1997), although stabilization of the labile ATPase/14-3-3 protein complex (Oecking et al., 1997) and the activation of the enzyme by different isoforms of 14-3-3 proteins (Baunsgaard et al., 1998) could be achieved only in the presence of the toxin. The use of two antibodies raised against the PM H+-ATPase of Z. mays and A. thaliana did not show any significant difference revealing the PM H+-ATPase from cucumber roots. These results leave open the question about the participation of different isoforms of the enzyme to the response to Fe-deficiency in cucumber roots, also considering the fact that only a small portion of the total immunoreactive H+-ATPase band was cleaved by trypsin (Fig 2). Interestingly, in Arabidopsis it has been reported that the AHA2 isoform is upregulated under Fe-deficiency (Fox and Guerinot, 1998).
Fe(III)-EDTA reductase activity of cucumber root plasma membrane vesicles was also increased by Fe-deprivation, as was Fe(III)-EDTA reduction in intact plants; the ratio of the activity in Fe-deficient plants against the activity of Fe-sufficient plants was about 12 times more with intact plants, while it was reduced to three times with isolated PM vesicles. Discrepancies between in vivo and in vitro results have been tentatively explained in terms of the localization of the Fe(III)-chelate reductase to a limited surface area of the roots. In addition, it has been suggested that the loss of a cofactor necessary for the Fe(III)-chelate reductase might occur during isolation of plasma membrane vesicles (Susin et al., 1996).
In a number of species it was reported that Fe(III)-EDTA reductase activity of root plasma membranes was 1.3- to 2.6-fold increased by Fe-deficiency (Moog and Brüggemann, 1994). On the other hand, H+-ATPase activity was not correspondingly affected by the Fe-status, for example, in sugar beet (Susin et al., 1996) or tomato (Buckhout et al., 1989; Valenti et al., 1991). A considerable increase was observed only in a limited number of plants, for example, cotton (Chosack et al., 1991) and cucumber (Zocchi and Cocucci, 1990; Rabotti and Zocchi, 1994).
Response to Fe-deficiency involves biochemical, physiological and morphological modifications; however, the relative importance of each individual stress-response reaction may vary depending on the plant species. Spatial and temporal separation in the expression of Fe reduction and proton extrusion has also been observed (Grusak et al., 1989; Grusak and Pezesghi, 1996), which support the view of independent regulation of the two responses (Yi and Guerinot, 1996). These aspects may be related to the differences in the levels of activity and stimulation of Fe(III)-chelate reductase and H+-ATPase observed in plasma membrane vesicles isolated from various Fe-deficient dicotyledonous plants.
In cucumber plants grown for 5 d in Fe-free nutrient solution (present work), proliferation of root hairs and lateral root formation started to be clearly evident. At this stage of development, Fe(III)-EDTA reduction by intact roots was maximal and supply of Fe(III)-EDTA to deficient plants caused a further increase in the reductase activity. It is likely that these morphological and physiological changes all need to be supported by a higher H+-ATPase activity (Serrano, 1989).
At day 5, a sharp decrease in the pH of the nutrient solution bathing Fe-deficient plants was also observed; however, the supply of Fe(III)-EDTA to these plants caused an increase in the root external pH. This result supports the view that acidification of the nutrient solution, which is often used as a parameter of the H+-ATPase-based Fe-deficiency stress response, reflects not only the activity of the plasma membrane proton pump, but other mechanisms, like cation/anion imbalance could be also responsible for the shift in the pH of the root external medium (Marschner et al., 1986; Kochian, 1991).
In conclusion these results show that the increase in root PM H+-ATPase occurring during the onset of the physiological and biochemical response to Fe deprivation in cucumber plants is, at least in part, dependent on a higher amount of the enzyme present at the plasma membrane of root cells suggesting that the control of this enzyme (isoform) is under transcriptional regulation.
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Acknowledgments |
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This work was supported by grants from MURST and CNR to GZ and RP. We thank Dr RT Leonard (University of Arizona, Tucson, Arizona, USA) and Dr R Serrano (Universidad Politécnica, CSIC, Valencia, Spain) for the kind gift of the H+-ATPase antibodies from maize roots and A. thaliana, respectively.
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3 To whom correspondence should be addressed. Fax: +39 02 2663057. E-mail:graziano.zocchi{at}unimi.it
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