Synthetic iron chelates as substrates of root ferric chelate reductase in green stressed cucumber...

Journal of Plant Nutrition, 29: 423–439, 2006

Copyright © Taylor & Francis Group, LLC

ISSN: 0190-4167 print / 1532-4087 online

DOI: 10.1080/01904160500524886

Synthetic Iron Chelates as Substrates of RootFerric Chelate Reductase in Green Stressed

Cucumber Plants

Juan J. Lucena1 and Rufus L. Chaney2

1Seccion Quımica Agrıcola, Universidad Autonoma de Madrid, Madrid, Spain2Environmental Chemistry Lab. USDA-Agricultural Research Service,

Beltsville, MD, USA

ABSTRACT

This work studied the behavior of different iron (Fe)-chelates as substrates of ferricchelate reductase (FCR) and their ability as Fe suppliers for mildly chlorotic plants. FCRactivity and Fe concentration in xylem sap were determined in green stressed cucumberplants with different stress levels using different synthetic chelates as substrates. Bothreduction and Fe concentration in the xylem sap were higher for the less-stable Fechelates, except for Fe-EDTA, which presented a relatively low Fe concentration in sap.It was concluded that a high stability of the chelate in the nutrient solution reduces theFe reduction, but other factors, such as the complexation of the Fe(II) by the chelatingagents, should be considered when the complete process of Fe uptake is studied. Theuse of both indexes together, i.e., FCR determination and xylem sap concentration, isuseful for understanding the Fe uptake from different Fe chelates.

Keywords: iron, chelate, chlorosis, reductase, Cucumis sativus

INTRODUCTION

Iron (Fe)(III)-chelate reductase (FCR) is an enzymatic system that isup-regulated by strategy I plants to control the amount of Fe taken from themedium (Robinson et al., 1999). The substrates of the FCR are highly stable,Fe(III)-selective chelates that are reduced by the enzyme at the root surface.The effect of the Fe(III) chelate concentration on the rate of the reduction

Received 21 December 2004; accepted 20 July 2005.Address correspondence to Juan J. Lucena, Seccion de Quımica Agrıcola, Facultad

de Ciencias, Universidad Autonoma de Madrid, Madrid 28049, Spain. E-mail: [email protected]

423

424 J. J. Lucena and R. L. Chaney

was studied by Chaney (1989) using 10 µM solutions of the Fe chelates ofethylene-diamine-diacetic acid (EDDA), 2-hydroxy-ethyl-ethylene-diamine-triacetic acid (HEDTA), ethylene-diamine-tetraacetic acid (EDTA), anddiethylene-triamine-pentaacetic acid (DTPA). The study did not find any clearrelationship between the kinetic parameters and the stability of the chelates,moreover, the most stable Fe chelates ethylene diamine di-(ortho-hidroxyphenyl acetic) acid (EDDHA) and ethylene diamine di-(ortho-hidroxy p-methylphenyl acetic) acid (EDDHMA) were not used in these experiments. FCR ismore active at pH below 6.5 (Moog and Bruggemann, 1995; Susın et al., 1996;Romera et al., 1998). Strategy I requires lowering the pH in the immediatevicinity of the root (the rhizosphere or apoplast) (Romheld and Marschner,1986; Alcantara et al., 1991; Wei et al., 1997), this must occur despite the highbuffering effect of bicarbonate (Lucena, 2000). Most authors measure FCRactivity at bulk solution pH below 6.5, and very few attempts have been madeat pH 7.5 or higher due to experimental difficulties. Another problem in studieswith FCR is the reoxidation that may occur once the reduction takes place.This process may be catalyzed by the chelating agent and is favored at highpH values (Kurimura et al., 1968). It also depends on the Fe(II) trapping agentused in the experiment (Chaney, 1989). The reoxidation processes explain thelarge differences between the amount of Fe(II) formed in the reduction assay(RA) and the Fe that can be taken in by the plants under the same conditions.The importance of reoxidation in the effectiveness of the different chelatesunder natural conditions is not well known.

In this work, the study of Fe-EDTA is described and a series of highly stableFe(III) chelates, namely Fe-EDDHA, racemicFe-EDDHA, mesoFe-EDDHA,Fe-EDDHMA, Fe(III)-ethylene diamine di-(2-hydroxy-5-sulphophenyl ac-etate) (Fe-EDDHSA), Fe(III)-propilene diamine di-(ortho-hidroxy phenylacetate)(Fe-PDDHA), and Fe(III)-N,N′-di(2-hydroxy benzyl) ethylene di-amine N,N′-diacetate (Fe-HBED) were used as substrates of the FCR in mildlyFe-stressed cucumber plants. The objective was to determine the factors thatcan affect the chelates’ efficacy as FCR substrates. Since plants do not use allthe reduced Fe, the amount of Fe taken under the conditions of the reductionassay (RA) would be estimated from the Fe concentration in the xylem sap.

MATERIALS AND METHODS

Plant Culture

Two plant experiments were developed. For both, cucumber seeds(Cucumis sativus L. cv. ‘Burpless’) were germinated in standard seed ger-mination papers. The base of the rolled papers was placed in macronutrientsolution in diffuse light in the growth chamber for 5 d. Seed coats were re-moved. Uniform seedlings were selected. The stems of two individual plants

Iron Chelates as Substrates for FCR 425

were wrapped together with polyurethane foam and placed in a hole in a blackPlexiglas lid over a 9 L polyethylene bucket (12 holes per lid, 24 plants perbucket) for the first and second experiment and 2.5 L polyethylene vessels(three holes per lid, six plants per pot) for the third experiment. The bucketscontained 8 L of continuously aerated nutrient solution (2 L in the third experi-ment) of the following composition: macros: 1 mM Ca(NO3)2, 0.9 mM KNO3,0.3 mM MgSO4, 0.1 mM KH2PO4; cationic micronutrients: 1.0 µM MnSO4,0.5 µM CuSO4, 0.5 µM ZnSO4, 0.1 µM CoSO4, 0.1 µM NiSO4; anionic mi-cronutrients: 35 µM NaCl, 10 µM H3BO3, and 0.05 µM Na2MoO4. The pH wasbuffered at 7.25–7.5 with the addition of 0.1 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 5 g of CaCO3(2 g in the third experiment)per bucket. Water was added every 2 d.

Plants were grown in a growth chamber with cool-white fluorescent andincandescent lamps. The lights provided 200 µE/m2 at plant height during 16 hper day. The air temperature was 30◦C/25◦C (day/night).

During the growth period, Fe was added as 20 µM Fe-HBED. When plantsshowed the first fully expanded leaf (at 4 d in the first experiment, 3 d in thesecond), solutions were changed to include different Fe-stress-level treatmentsin EDTA-buffered nutrient solutions with the following composition: no metalmicronutrients as before, macronutrients and metal micronutrients: manganese(Mn) 2.5 µM, copper (Cu) 1.0 µM, zinc (Zn) 10 µM, cobalt (Co) 1.0 µM, andnickel (Ni) 1.0 µM. In the first experiment, Fe was added at rates of 0, 5.0, 20,and 100 µM (pretreatment solutions T0, T1, T2, and T3 respectively). In thesecond experiment, only 5.0 µM Fe was used. EDTA was added to complex allthe metal micronutrients plus an “excess” of 100 µM (Parker et al., 1995). ThepH was buffered at 7.5 with 0.1 mM HEPES and 5g/8L CaCO3. Plants weregrown for 6 d on the treatment solutions and then used for the RA.

Root Fe(III)-Chelate Reduction with Intact Plants

One-hundred-twenty-five mL disposable steriline beakers inserted in darkenedboxes with black lids were placed in the growth chamber. Each beaker contained100 mL RA solutions consisting of: macronutrient solution as in the growthperiod; 100µM Fe chelate (see later for treatments) used as substrate of the FCR;2 mM 2-morpholinoethanesulfonic acid (MES) or 2 mM to buffer the pH at 6.0or 7.5, respectively; and 300 µM bathophenanthroline disulfonic acid (BPDS)as an Fe2+ trapping and coloring reagent. The lids had one hole for a plastic gas-dispersion tube, another for a pipette, and a third to hold the pair of cucumberplants held by polyurethane foam. Each solution was continuously aeratedand allowed to reach temperature equilibrium before plants were transferred.Experiments began after 3 h into the daylight period. Initial 3 mL sampleswere obtained for each beaker. The roots of plants were washed three times inmacronutrient solution containing 37.5 µM Na2BPDS, then transferred to the


RA solutions. Three-milliliter samples were withdrawn at 5, 10, 20, 60, and120 min.

Iron chelates used as substrates were Fe-EDTA and Fe-EDDHA in the firstexperiment and Fe-EDTA, Fe-EDDHA, Fe-mesoEDDHA, Fe-racEDDHA, Fe-EDDHMA, Fe-EDDHSA, Fe-PDDHA, and Fe-HBED in the second (see belowfor preparation).

Three replicates were prepared for each treatment, with Fe(III)-chelateused as substrate of the FCR and solution pH. For each Fe(III)-chelate used assubstrate and pH, two beakers without plants (one in the second experiment)were included in order to correct reduction rates for slow photoreduction. TheFe(II) produced by root reduction was measured spectrophotometrically at 535nm the same day, using a Gilford Stasar III spectrophotometer (Nova Biotech,San Diego, CA, USA). Since all iron chelates used as substrates, except Fe-EDTA, presented a significant absorption at 535 nm, measurements were alsomade at 480 nm in order to calculate both the Fe(II)(BPDS)3 concentration andthe remaining Fe-chelate concentration. Previously, standard solutions of bothchelates were prepared and the molar absorption coefficients at both wave-lengths were determined. The concentration of each chelate was calculatedsolving the two-equation system (e.g., for Fe-EDDHA):

A535 = aFeBPDS535 × [Fe(BPDS)3] + aFeEDDHA535 × [Fe-EDDHA]

A480 = aFeBPDS480 × [Fe(BPDS)3] + aFeEDDHA480 × [Fe-EDDHA]

where A535 and A480 are the absorbencies measured for each sample at 535 and480 nm, respectively; aFeBPDS535, aFeBPDS480), aFeEDDHA535, and aFeEDDHA480 arethe molar absorption coefficients in our experimental conditions (see Table 1)and [Fe(BPDS)3] and [Fe-EDDHA] are the concentrations of the chelates. The

Table 1Absorptivity coefficients at 535 nm (a535) and at 480 nm (a480)of the Fe(III)-chelates used

L mol−1 cm−1

Iron chelate a535 a480

Fe(BPDS)4−3 2.23 · 104 1.77 · 104

Fe-EDDHA− 3.28 · 103 4.76 · 103

Fe-mesoEDDHA− 3.44 · 103 4.64 · 103

Fe-racEDDHA− 3.07 · 103 4.93 · 103

Fe-EDDHMA− 3.39 · 103 4.47 · 103

Fe-EDDHSA3− 2.87 · 103 4.85 · 103

Fe-PDDHA− 3.11 · 103 4.80 · 103

Fe-HBED− 2.63 · 103 3.54 · 103


addition of both is the total Fe in solution, useful for considering the concen-tration effect due to evapotranspiration (less than 5.1% in 1 h).

The slope of the plots of Fe(II) produced versus time were used as the rateof Fe(II) reduction for each plant.

Iron Concentration in Xylem Sap

When the RA were finished, nutrient solutions were replaced with 100 mLsolutions with the same composition as the RA solutions, but without BPDS.Plant roots were washed twice in macronutrient solution and once in the finalsolution before being placed again in the beakers, which were aerated continu-ously during the experiment. After 1 h, shoots were cut and stems were placedin Pasteur pipettes connected to a 1.5 mL eppendorf tube. Xylem sap from bothplants in the bunch was collected together for 2 h.

The fresh weight of blotted roots was then obtained at the end of thexylem-sap collection assay. Shoot dry weight and sap weight were alsomeasured.

Iron Chelates

EDTA was obtained from Sigma-Aldrich, Alcobendas, Spain and used as re-ceived (titrimetric purity >99%).

EDDHA was also obtained from Sigma-Aldrich, Alcobendas, Spain. Chro-matographic characterization (Lucena et al., 1996) revealed that the ratio be-tween racemic and meso isomers was close to 1.0. EDDHA was recrystallizedas described by Bell et al. (1991). Spectrophotometric titration with an Fe(III)standard was performed in the presence of 2 mM HEPES, with pH close toneutral, and was found to be 96.2% pure (based on complexing capacity byphotometric titration).

The meso-EDDHA and rac-EDDHA are the spatial isomers of the ED-DHA and were obtained by Prof. Augustin Garate of Universidad Autonomade Madrid (UAM) using the method of Bailey et al. (1981) as modified by us.Their chromatographic purity was 99% for the meso isomer and 100% for theracemic isomer. Their complexing capacity was 94.2% and 98.5% by photo-metric titration, respectively.

EDDHMA was provided by Prof. Miguel A. Sierra cf Universidad Com-plutense of Madrid (UCM), prepared as in Sierra et al., 2002. Its chromatogramshows only two peaks corresponding to the racemic and meso isomers, and thespectrophotometric titration after recrystallization showed a 99.0% complexingcapacity by photometric titration.

EDDHSAK4 was provided by NAC Quımica (Reus, Spain). The chro-matogram showed the presence of 4-hydroxybenzenesulfonic acid. Its com-plexing capacity for Fe was 65.3%. Attempts to purify it (Bell et al., 1991) bydissolving it in water instead of in HCl did not yield any greater purity.


PDDHA was synthesized by Prof. Sierra (Sierra et al., 2002) and, afterrecrystallization, showed a 93.6% complexing capacity.

HBED·HCl·H2O had a 103.2% complexing capacity as measured byphotometric titration.

Two hundred fifty µmol of each chelating agent, based on the measuredcomplexing capacity, were dissolved in 10 mL water containing 750 µmolKOH. After complete dissolution, an aliquot of a Fe(NO3)3 solution containing250 µmol of Fe was added, and pH was immediately adjusted to neutral. Volumewas brought to 25 mL in order to obtain 10 mM Fe-chelate stock solutions tobe used for spectrophotometric calibrations, RA, and xylem sap collection.

Stability of the Chelates: Ligand Competition with DTPA

In order to compare the stability of the chelates under these experimental con-ditions of pH and ionic strength, all of them were treated with excess DTPAand the changes in absorbance at 480 nm were measured for several days.Since the rate of the Fe transfer from the chelate to the DTPA is normallyslow, the experiment was performed in two different series (A and B), withthe same composition but different order of addition of the reactants. In seriesA, Fe-DTPA was formed first, and the chelating agents were then added todisplace the Fe from the Fe-DTPA. In series B, the chelates were formed firstand DTPA was then used as the competing ligand. The following reagents werethen consecutively added to 25 mL volumetric flasks:

– Series A: 10 mL H2O, 1 mL 0.25 M buffer (MES pH 6.0 or HEPES pH 7.5),1 mL 0.25 M Ca-DTPA, 1 mL 2.5 mM Fe and, after thorough agitation, 0.5mL of 5.0 mM chelating agents.

– Series B: 10 mL H2O, 1 mL 0.25 M buffer (MES pH 6.0 or HEPES pH 7.5),0.5 mL of 5.0 mM chelating agents, 1 mL 2.5 mM Fe and, after thoroughagitation, 1 mL 0.25 M Ca-DTPA.

In both series, pH was then adjusted to 6.0 or 7.5 and made to volume. Allthe solutions were prepared in triplicate. To speed the process, solutions werekept at 40◦C in the dark until 2 h before measurement, when they were placedat room temperature.

Ca-DTPA was prepared from Ca(NO3)2 and H5DTPA after the H5DTPAwas dissolved with 3 mol of KOH per mol of DTPA and the pH was then adjustedto 6.0 or 7.5, depending on the final pH desired. The relative concentrationsof DTPA and the other chelating agents were established after modeling withMINTEQA2 (Allison et al, 1990). Concentrations used would displace 87.5%of the Fe at pH 6.0 and 7.2% at pH 7.5 from Fe-EDDHA using the Lindsaythermodynamic database (Lindsay, 1979).

Iron solution was freshly prepared from a 1000 mg L−1 standard for atomicabsorption spectrophotometry.


Statistics

All the results were subjected to statistical analysis using the SAS program.Statistical comparison of means was conducted to reveal the differences amongdoses (first experiment), chelates used as substrates (first and second experi-ment), and pH of the RA solutions. Duncan’s multiple range test (α = 0.05)was used.

RESULTS

Stability of the Chelates: Ligand Competition with DTPA

The ability of EDDHSA and PDDHA to chelate Fe(III) in the presence ofCa(II) and H+ is not well known, so the study of their interaction with DTPAwas made initially. The results are shown in Figure 1. Clearly, for HBED, noconvergence was obtained between the two series. Although HBED has beenfound to form very stable Fe(III) chelates (Motekaitis et al., 1990; Chaney,1988) the kinetics of the displacement reactions involving HBED are so slowthat during the period of this study (20 d) almost no displacement was observedin either series (A or B) at pH 6.0 or 7.5. At pH 6.0, Fe-PDDHA was im-mediately displaced by DTPA in both series, precipitating a coalescent brownsubstance. At pH 7.5, the convergence was very slow, as some precipitationwas observed in Series A. The data obtained for the chelates studied are col-lected in Table 2. Although equilibrium had not been reached, the ability of thecompounds studied to bind Fe(III) in the presence of other cations followedthe order: HBED � EDDHSA > racEDDHA > EDDHA > EDDHMA >

mesoEDDHA > PDDHA at pH 6.0 and HBED > racEDDHA > EDDHSA> EDDHA > EDDHMA > mesoEDDHA > PDDHA at pH 7.5. The experi-mental conditions used are similar to those of the RA and xylem sap collection.The results obtained are in agreement with the stabilities predicted by Yunta etal. (2003) from thermodynamic data. The small differences between EDDHAand EDDHMA have already been reported (Ahrland et al., 1990), but the highstability of the Fe-EDDHSA that we found is somewhat surprising. In fact, thiscomplex is generally considered less stable than Fe-EDDHA and for this reasonits use as a soil fertilizer has decreased in recent years. Finally, the increase ofthe length of the chain linking the two nitrogen (N) atoms is responsible for thelower stability of the Fe-PDDHA complex compared with that of Fe-EDDHA(Bernauer, 1976; Lucena et al., 1996).

Plant Response with Different Iron Chlorosis Status

In the first experiment, a different Fe nutrition status was expected. Plantspretreated with no Fe showed intervenial chlorosis in the leaves and typical

Fig

ure

1.T

ime

cour

seof

Fech

elat

edin

com

petit

ion

with

Ca-

DT

PA.S

erie

sA

:se

quen

ceof

addi

tion:

100

µM

Fe,0

.01

MD

TPA

,and

100

µM

chel

atin

gag

ent.

Seri

esB

:seq

uenc

eof

addi

tion:

100

µM

Fe,1

00µ

Mch

elat

ing

agen

t,an

d0.

01M

DT

PA.

430


Table 2Percentage of Fe that is chelated after 20 d of competition

%

pH = 6.0 pH = 7.5

Chelating agent A B Avg A B Avg

EDDHA 36.4 38.3 37.3 83.3 85.6 84.5meso EDDHA 24.7 25.9 25.3 73.8 75.6 74.7racEDDHA 57.5 59.9 58.7 96.0 100.0 98.0EDDHMA 28.0 33.4 30.7 76.6 79.9 78.3EDDHSA 64.1 69.7 66.9 89.5 95.3 92.4PDDHA 1.9 3.4 2.7 11.6 14.4 13.0

Series A: sequence of addition, 100 µM Fe, 0.01 M DTPA, and 100 µM chelatingagent. Series B: sequence of addition, 100 µM Fe, 100 µM chelating agent, and 0.01M DTPA. The average value between series (Avg) is also included.

Fe- deficiency symptoms in the roots, but the three other pretreatments didnot present evident symptoms of chlorosis. However, growth parameters weredifferent, and as is shown in Table 3, both shoot and root development increasedas the Fe(III)-EDTA concentration in the nutrient solution was increased up to20 µM.

In Figure 2, the reduction rates, relative to the amount of Fe in the pretreat-ment solution, pH, and substrate in the RA, are presented. Reduction rates weremeasured for 2 h, although in the second hour, the rates decreased slightly formost of the plants. The decrease was more significant in the plants with a higherreduction capacity, surely due to the lower Fe(III)-chelate remaining after thefirst hour of reduction. Additionally, some of the plants had reduced almost50% of the initial Fe(III) after this time; hence, only the reduction rate duringthe first hour has been considered. Statistical comparison of the means usingthe Duncan test (α = 0.05) revealed significant differences among treatments

Table 3Growth parameters for plants in the first experiment

Iron pretreatment (Fe µM)

0 5 20 100

Roots fresh weight (g) 2.52 ± 0.26 3.48 ± 0.49 4.85 ± 0.72 3.69 ± 0.42Shoot dry weight (g) 0.55 ± 0.05 0.67 ± 0.07 0.71 ± 0.08 0.75 ± 0.08

Confident limits at α = 0.05.


Figure 2. Rate of Fe reduction from Fe-EDTA (TA) or Fe-EDDHA (HA) at two pHlevels for cucumber plants grown with different Fe-EDTA treatments (T0: 0 µM, T1:5.0 µM, T2: 20 µM, and T3: 100 µM). Error bars represent standard deviations.

(T0:a, T1:b, T2:c T3:d), pH (pH = 6:a, pH = 7.5:b), and substrates (Fe-EDTA:a,Fe-EDDHA:b). The determinations made at pH 6.0 showed more reduction thanthose made at pH 7.5. This result is in agreement with the findings of severalother authors (Moog and Bruggemann, 1995; Susın et al., 1996), includingthose who focussed on cucumber plants (Romera et al., 1998), but in our casethe differences found were not so large. In general, they were larger for pre-treatment T1 than for the others and were also larger for Fe-EDDHA than forFe-EDTA as substrate. As the Fe-nutritional status of the plants improved, thereduction decreased, and hence in pretreatment T3 no reduction was observed.These plants had an adequate amount of Fe for normal growth, while thoseunder treatments T1 and T2 (which did not show visual symptoms of chlorosis)had a measurable reduction capacity. T0 plants were treated under conditionsfar from those usually used, for Fe chelates, so in order to select the best exper-imental conditions for the study of different chelates as FCR substrates (secondexperiment), T1 conditions were selected.

Fe-EDTA was a better substrate than Fe-EDDHA. The largest differencesbetween these substrates were observed for pretreatments T2 and T1 and at pH7.5. These results encouraged us to use more Fe chelates as substrates, as wasdone in the second experiment.

The concentration of Fe in the xylem sap is presented in Figure 3. Trendswere quite similar to those of the Fe(III)-chelate reduction. There were statistical


Figure 3. Fe concentration in xylem sap from Fe-EDTA (TA) or Fe-EDDHA (HA) attwo pH levels for cucumber plants grown with different Fe-EDTA treatments (T0: 0 µM,T1: 5.0 µM, T2: 20 µM, and T3: 100 µM). Error bars represent standard deviations.

differences among pretreatments (T0:a, T1:b, T2:c T3:d), and between pHs (pH6.0:a, pH 7.5:b), but in this case Fe concentration was significantly higher forFe-EDDHA than for Fe-EDTA.

Iron Reduction and Uptake by Mildly Chlorotic Plants Using DifferentChelates as Substrates

In the second experiment, all of the plants were grown to be green, albeit Fe-stressed, using the 5 µM Fe-EDTA solution (as T1 in the first experiment). Thefresh weight of the roots was 3.82 ± 0.23 g and the dry weight of the shoots0.64 ± 0.03 g. Plants did not show typical chlorosis symptoms (Helianthusannuus).

Figure 4 presents the rate of Fe reduction during the first hour whendifferent chelates were used as substrates for reduction, at two pH lev-els. Statistical comparison of the means using the Duncan test (α = 0.05)revealed significant differences between pH (pH 6.0:a, pH 7.5:b) and amongsubstrates (Fe-EDTA:a, Fe-mesoEDDHA:b, Fe-EDDHA:b, Fe-PDDHA:c, Fe-EDDHMA:cd, Fe-HBED:de, Fe-racEDDHA:de, Fe-EDDHSA:e). For allchelates (except Fe-EDDHSA), plants exhibited more reduction at pH 6.0 thanat pH 7.5. The best substrate was Fe-EDTA, at both pHs. Fe-EDDHA behaved


Figure 4. Rate of Fe reduction from different Fe-chelates at two pH levels for Fe-stressed cucumber plants grown with 5.0 µM Fe-EDTA buffered with EDTA. Error barsrepresent standard deviations.

similar to Fe-mesoEDDHA, while the Fe-rac EDDHA (the most stable of bothisomers) showed lower reduction rates. As during the RA, not even 50% of theFe-EDDHA was used, so it is likely that only the Fe-meso isomer was beingreduced when Fe-EDDHA was used as substrate. This result is in agreementwith the recent findings of Alvarez-Fernandez et al (2005), which showed thatonly the meso-isomer was depleted from a nutrient solution when Fe-EDDHAwas used as Fe(III)-chelate for sunflower plants (Helianthus annuus).

Fe-EDDHMA also exists in two isomeric forms. According to Ahrlandet al. (1990) the racemic isomer is less stable than the meso form, surely dueto the steric hindrance caused by the two spatially close methyl groups. Theyalso stated that both Fe-EDDHMA isomers are more stable than any of theFe-EDDHA isomers, which does not agree with our findings using the ligandcompetition method. In fact, Fe-EDDHMA seems to be a better substrate ofthe FCR than Fe-rac EDDHA but worse than Fe-meso EDDHA under our testconditions.

Fe-EDDHSA has three negative charges. The experimental results showthat the reduction rate for this substrate is very low, although due to thelow purity of the chelate it is difficult to draw conclusions in this case. Asdiscussed earlier, Fe-PDDHA is less stable Fe-EDDHA. It also exists as a


mixture of isomers and behaves almost like Fe-EDDHMA at pH 6. Fe-HBEDis more stable than Fe-EDDHA or Fe-EDDHMA. In this case, the observedreduction rate was not significantly different from that of rac Fe-EDDHA andFe-EDDHMA.

Figure 5 presents the Fe concentration in xylem sap collected for var-ious treatments. Results for Fe-mesoEDDHA are similar to those found inthe first experiment for Fe-EDDHA (Figure 3, using Fe-EDTA as substrate),which are in agreement with the results from the FCR assay. Statistical com-parison of the means using the Duncan test (α = 0.05) revealed significantdifferences between pH (pH = 6.0:a, pH = 7.5:b) and among substrates (Fe-mesoEDDHA:a, Fe-racEDDHA:b, Fe-EDDHMA:b and Fe-EDDHSA:b). Ingeneral, Fe concentration in xylem sap (Figure 5) followed the same trends asthe Fe reduction.

DISCUSSION

The first experiment tested the plant response to different Fe sources, findingresults similar to those previously reported for other authors (Susın et al., 1996).The most Fe-stressed cucumber plants showed higher FCR activity than thoseplants that had been given a more adequate Fe supply. The use of green, Fe-stressed plants to characterize responses to Fe stress (as has been done in our

Figure 5. Fe concentration in xylem sap from different substrates at two pH levels forFe-stressed cucumber plants grown with 5.0 µM Fe-EDTA buffered with EDTA. Errorbars represent standard deviations. Where marked with “a,” collection of xylem sap fromthese substrates was done with less agitation and O2 in the medium.


experiments) seems more realistic than the use of severely chlorotic plantsgrown without Fe. In this later case, the plants often show responses that aremore like artifacts than actual up-regulated Fe-acquisition mechanisms (Chaneyet al., 1992). In fact, under field conditions, chlorosis is due not to the lackof total Fe, but to the low phytoavailability of the Fe in soil. Moreover, inagronomic practices, Fe chelates are normally used to control Fe chlorosis inplants, but they are added to prevent the occurrence of yellowing, or when thefirst symptoms of chlorosis are visible. The first experiment demonstrated thatgreen, stressed cucumber plants present a measurable FCR activity (Figure 2)even at a buffered pH of 7.5, a pH value similar to that of calcareous soils wherechlorosis problems occur. Then, green, stressed plants seem to be the preferredmodel with which to test Fe-chelates as Fe suppliers.

The different Fe(III)-chelates may act as substrates for the FCR activity ina different way. Judging from these results, the more stable chelates are thosethat produce lower reduction rates. Chaney’s results for FCR activity (Chaney,1989) indicate that not only the stability but also the charge of the chelate isimportant. He found that Fe-EDDA0 was less effective as a Fe substrate thanFe-EDTA−, despite its being a less stable chelate. Fe-EDDHSA3− was the onlychelate used in our experiments with a charge other than −1, but due to its lowpurity, no conclusions regarding the charge effect can been drawn.

The measurement of the amount of Fe reduced by the plants gives infor-mation only about a part of the uptake process. The result of the completeprocess could be better evaluated according to the Fe absorption by the plant,here estimated from the amount of Fe present in the xylem sap. The valuesobtained in all the experiments show that only a small fraction (around 1%) ofthe Fe that has been reduced in the RA reaches the xylem sap. Chaney and Bell(1987) presented data on reduction from Fe-DTPA and 59Fe uptake in stressedpeanut plants. In that case, the fraction of supplied Fe reaching the shoots waslarger than that found here (even reaching about 100%), but the pH values of theuptake assay solutions were considerably lower (around 4.0) than those usedin our experiments. They observed separate regulation of reduction and uptakerate; acidification was found to be one of the factors related to these differencesin regulation. In that case, the fact that in our experiments the Fe uptake ratewas lower than the reduction rate should be due to the higher pH (6.0 or 7.5)used. Moreover, Chaney and Bell (1987) did not use pH buffer in the uptakesolutions, so acidification by the roots most likely increased both the reductionand the uptake processes. In this study, however, the final pH was close to theinitial value, preventing the effect of acidification in both uptake and reductionprocesses. The uptake and other previous steps, such as reoxidation, shouldbe more sensitive to the pH of the solution than to the Fe-chelate reductionrate.

The differences in the percentages of Fe uptake with respect to the Fereduced were important in the first experiment. The average values were 0.65%for Fe-EDTA and 1.31% for Fe-EDDHA for treatments T0, T1, and T2. This


different behavior should not be due to differences in reoxidation, since theFe-EDDHA is a more stable chelate and should produce more reoxidation. Abetter explanation considers the differences in the complexation of Fe(II). WhileFe(II)-EDTA is quite stable, the stability of Fe(II)-EDDHA is considerablylower. Thus, the EDTA may serve as an Fe(II) trapping agent, lowering theamount of Fe that can be taken up by the plant compared to the amount reduced.Since the Fe(III)-EDTA is the weaker chelate among those used in this work, itshould be the best substrate for the FCR. However, EDTA can complex Fe(II),thus inhibiting Fe(II) uptake and thereby acting similarly to BPDS to impair Feuptake by the plant.

The Fe concentration in xylem sap of plants transferred to highly availableFe test solution has been revealed to be a good index of the Fe uptake fromdifferent FCR substrates. In our methodology, sap was collected starting 1 hafter transfer to the test solutions and the collection period was only 2 h because,according to White et al. (1981) longer periods may produce significant changesin solute composition.

CONCLUSIONS

These data suggest that the best substrates for the FCR activity in green Fe-stressed cucumber roots are the weaker Fe(III) chelates. However, using the Feconcentration in the xylem sap as an index of the Fe uptake, we can conclude thatthis process is also dependent on the ability of the chelating agent to complexFe(II).

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of Carrie E. Greenand Francisca Blas for their help in conducting the experiments, and thank Dr.M. Gomez-Gallego for her helpful review of the manuscript. This work wasperformed during a research visit by J. J. Lucena to the USDA, which wassupported by a MEC-CICyT grant. Part of the work was supported by projectsAGL2001-1142 from the Spanish Ministry of Sciences and Technology.

REFERENCES

Ahrland, S., A. Dahlgren, and I. Persson. 1990. Stabilities and hydrolysis ofsome iron(III) and manganese(III) complexes with chelating ligands. ActaAgriculturae Scandinavica 40: 101–111.

Alcantara, E., M. D. De la Guardia, and F. J. Romera. 1991. Plasmalemmaredox activity and H+ extrusion in roots of Fe-deficient cucumber plants.Plant Physiology 96: 1034–1037.


Allison, J. D., D. S. Brown, and K. J. Novo-Gradac. 1990. Minteqa2/Prodefa2:Geochemical assessment model for environmental systems, Version 3.0.User’s manual EPA 600/3-91-0221. Athens, GA: USEPA.

Alvarez-Fernandez, A., S. Garcıa-Marco, and J. J. Lucena. 2005. Evalua-tion of synthetic iron(III)-chelates (EDDHA/Fe3+, EDDHMA/Fe3+ andEDDHSA/Fe3+) to correct iron chlorosis. European Journal of Agronomy22: 119–130.

Bailey, N. A., D. Cummins, E. D. McKenzie, and J. M. Worthington. 1981.Iron(III) compounds of phenolic ligands: The crystal and molecular struc-ture of iron(III) compounds of the sexadentate ligand N,N′-ethylene-bis-(o-hydroxyphenylglycine). Inorganica Chimica Acta 50: 111–20.

Bell, P. F., R. L. Chaney, and J. S. Angle. 1991. Free metal activity and total metalconcentrations as indices of micronutrient availability to barley [Hordeumvulgare (L.) cv. ‘Klages’]. Plant and Soil 130: 51–62.

Bernauer, K. 1976. Diasteroisomerims and disateroselectivity in metal com-plexes. Topics in Current Chemistry 65: 1–35.

Chaney, R. L. 1988. Plant can utilize iron from iron N,N’-di (2-hydroxybenzoyl)ethylene diamine-N,N′-diacetic acid, a ferric chelate with 106greater for-mation constant than iron EDDHA. Journal of Plant Nutrition 11: 1033–1050.

Chaney, R. L. 1989. Kinetics of ferric chelate reduction by roots of iron-deficientpeanut (Arachis Hypogea). Acta Bototanica Neerlandica 38: 155–163.

Chaney, R. L. and P. F. Bell. 1987. The complexity of iron nutrition: Lessonsfor plant soil interaction research. Journal of Plant Nutrition 10: 963–994.

Chaney, R. L., Y. Chen, C. E. Green, M. J. Holden, P. F. Bell, D. G. Luster,and J. S. Angle. 1992. Root hairs on chlorotic tomatoes are an effect ofchlorosis rather that part of the adaptive Fe-stress response. Journal ofPlant Nutrition 15: 1857–1875.

Kurimura, Y., R. Ochai, and N. Matsuura. 1968. Oxygen oxidation of ferrousions induced by chelation. Bulletin of the Chemical Society of Japan 41:2234–2239.

Lindsay, W. L. 1979. Chemical Equilibria in Soils. New York: John Wiley.Lucena, J. J. 2000. Effect of bicarbonate, nitrate and other environmental factors

on iron deficiency chlorosis: A review. Journal of Plant Nutrition 23: 1591–1606.

Lucena, J. J., P. Barak, and L. Hernandez-Apaolaza. 1996. Isocratic ion-pair high-performance liquid chromatographic method for the deter-mination of various iron(III) chelates. Journal of Chomatography A727: 253–264.

Moog, P. R., and W. Bruggemann. 1995. Iron reductase systems on the plasmamembrane: A review. In Iron nutrition in soils and plants, ed. J. Abadıa,343–362. Dordrecht, Netherlands: Kluwer Academic Pubs.

Motekaitis, R. J., A. E. Martell, and M. J. Welch. 1990. Stabilities of trivalentmetal complexes of phenolic ligands related to N,N′-bis(2-hydroxybenzyl)


ethylene diamine-N,N’-diacetic acid (HBED). Inorganic Chemistry29: 1463–467.

Parker, D. R., R. L. Chaney, and W. A. Norvell. 1995. Chemical equilibriamodels: Applications to plant nutrition research. In Chemical Equilibriumand Reaction Models, eds. R. M. Loeppert, A. P. Schwab, and S. Goldberg,163–200. Soil Sci. Soc. of America, Madison, WI.

Robinson, N. J., C. M. Procter, E. L. Connolly, and M. L. Guerinot. 1999. Aferric-chelate reductase for iron uptake from soils. Nature 397: 695–697.

Romera, F. J., E. Alcantara, and M. D. De la Guardia. 1998. The inductionof the “turbo reductase” is inhibited by cycloheximide, cordycepin andethylene inhibitors in Fe-deficient cucumber (Cucumis sativus L.) plants.Protoplasma 205: 156–162.

Romheld, V., and H. Marschner. 1986. Mobilization of iron in the rizosphereof different plant species. Advances in Plant Nutrition 2: 155–204.

Sierra, M. A., M. Gomez-Gallego, R. Alcazar, J. J. Lucena, A. Alvarez-Fernandez, and F. Yunta. 2002. Novel method for preparing bis(2-hydroxyarylaminoacetic) acids using cyanide transfer agents. World PatentWO 02/00604 A1, PCT7ES-01/00243, issued January 3, 2002.

Susın, S., A. Abadıa, J. A. Gonzalez-Reyes, J. J. Lucena, and J. Abadıa. 1996.The pH requirement for in vivo activity of the iron-deficiency-induced“Turbo” ferric chelate reductase. Plant Physiology 110: 111–123.

Wei, L. C., R. H. Loeppert, and W. R. Ocumpaugh. 1997. Fe-deficiency stressresponse in Fe-deficiency resistant and susceptibility subterranean clover:Importance of induced H+ release. Journal of Experimental Botany 48:239–246.

White, M. C., A. M. Decker, and R. L. Chaney. 1981. Metal complexation inxylem fluid. I. Chemical composition of tomato and soybean stem exudate.Plant Physiology 67: 292–300.

Yunta, F., S. Garcıa-Marco, J. J. Lucena, M. Gomez-Gallego, R. Alcazar,and M. A. Sierra. 2003 Chelating agents related to ethylenediaminebis(2-hydroxyphenyl)acetic acid (EDDHA): Synthesis, characterizationand equilibrium studies of the free ligands and their Mg2+, Ca2+, Cu2+

and Fe3+ chelates. Inorganic Chemistry 42: 5412–5421.

Synthetic iron chelates as substrates of root ferric chelate reductase in green stressed cucumber...

Documents

Transcript of Synthetic iron chelates as substrates of root ferric chelate reductase in green stressed cucumber...