This article was downloaded by: [The University of Manchester Library]On: 07 December 2014, At: 04:00Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Soil Science and Plant NutritionPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tssp20

Distribution and secondary effects of EDDHA in somevegetable speciesH. Frits Bienfait a , Josemaria Garcia-Mina b c & Angel Maria Zamareño ca Afdeling Ecofysiologie van Planter , Wentgebouw, Sorbonnelaan 16, 3984 CA, Utrechtb Department of Chemistry and Soil Chemistry , University of Navarra , Spainc R&D Department , Inabonos-Roullier Group , Poligono Arazuri-Orcoyen, C/C, No. 32,31160, Orcoyen, SpainPublished online: 14 Sep 2011.

To cite this article: H. Frits Bienfait , Josemaria Garcia-Mina & Angel Maria Zamareño (2004) Distribution andsecondary effects of EDDHA in some vegetable species, Soil Science and Plant Nutrition, 50:7, 1103-1110, DOI:10.1080/00380768.2004.10408581

To link to this article: http://dx.doi.org/10.1080/00380768.2004.10408581

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Soil Sci. Plant Nutr., 50 (7), 1103 - 1110, 2004

Distribution and Secondary Effects of EDDHAin Some Vegetable Species

H. Frits Bienfait, Josemaria Garcia-Mina*'**, and Angel Maria Zamarefio**

Afdeling Ecofvsiologie van Plamen, Wentgebouw, SorbonlZelaan 16,3984 CA Utrecht, The Netherlands; *Departmentof Chemistry and Soil Chemistry, University ofNavarra, Spain; and **R&D Department, 1nabonos-Roullier Group,

Poligono Arazuri-Orcoyen, C/C, No. 32. 31160 Orcoyen, Spain

Received April 12,2004; accepted in revised fOlm June 6, 2004

Tomato (Lycopersicum esculentum), cucumber (Cucumis sativus), pepper (Capsicumannuum), and lettuce (Lactuca sativa) were grown on rockwool or perlite substrate withnutrient solution. Fe was administered as the Fe complex of the chelator ethylenediaminedi-(o-hydroxyphenylacetic acid) (EDDHA) or Fe(NH4)2(S04)2 in the nutrient solution or asinorganic iron in the substrate. Roots and leaves of plants grown on Fe-EDDHA containedEDDHA in quantities up to 0.27 x the amount of Fe, which is interpreted as an indication ofthe contribution of passive chelate absorption to Fe uptake. Fruits of tomato and pepper,and leaves of lettuce contained only traces of EDDHA. Breakdown of the chelator in leavesof pepper and tomato is estimated to have been between 0.5 and 2% per day. In tomatofruits, lycopene content was lowered in plants growing on Fe-EDDHA. Cucumber growingon Fe-EDDHA suffered from serious infection by the mildew Sphaerotheca fuscaj theplants growing on an inorganic source of iron were resistant. These results exemplify physi­ological effects of EDDHA other than those directly associated with iron nutrition.

Key Words: cucumber, Fe-EDDHA, pepper, Sphaerotheca, tomato.

1103

Uptake of iron by dicotyledonolls plants is generallyassumed to follow the mechanism baptized Strategy I byRbmheld and Marschner (1986). Characteristic featuresof this mechanism are two enzyme activities in the plas­ma membrane of the root epidermis cells: a protonexcreting ATPase (Rbmheld et al. 1984), and an enzymewhich reduces outside ferric chelatcs (Chancy ct al.1972). Proton excretion stimulates, by acidification ofthe rhizosphere, the dissolution of ferric complexes inthe soil, and reduction of dissolved ferric chelatesresults in ferrous chelates, which then dissociate sincegood ferric chelators are, in general, bad ferrous chela­tors. Ferrous ions are easily taken up by the roots (Halland Williams 2003). The two enzymes are induced oractivated upon Fe deficiency, and it has been reportedseveral times that, in general, the better the plants canexpress these "Fe efficiency" reactions, the larger theresistance to iron deficiency in the more or less calcare­ous field.

Abbreviations: DTPA, diethylenetriamine pentaacetic acid; EDDHA,ethylenediamine di(o-hydroxyphenylacetic acid); HEDTA, N-(2­

hydroxyethyl)ethylenediaminetriacetic acid; BPDS, bathophenan­throline disulphonate.

Iron can also be taken up as a complex with syntheticchelators of the EDTA family, for which there is strongSupp0l1ing evidence (Holmes and Brown 1955; Hill­Cottingham and Lloyd-Jones 1965; Rbmheld 1979). Inrecent phytoremediation literature uptake of other biva­lent metal chelates such as Pb-EDTA has been shown(Epstein et al. 1999).

In the present day culture of vegetables and fruits onartificial substrates such as rockwool or perlite, iron isroutinely given in the nutrient solution as a chelate withDTPA or EDDHA. This is not the natural situation. TheEDTA family of chelators is purely synthetical; theirmembers were introduced in plant culture in the 1950's.Second, uptake of Fe as a chelate has other conse­quences for plant metabolism than uptake via Strategy I(see DISCUSSION) and might thus influence the composi­tion and the taste of fruits and vegetables. EDDHA mayalso have other effects, e.g. as on the flowering responseof Lemna (Pieterse et al. 1970).

A commercial grower may therefore have a number ofreasons to culture his products without the use of syn­thetic chelators. We undertook a study to see whethertomato, cucumber, pepper, and lettuce could be success­fully grown on substrates with iron in a non-chelated

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14

1104 H.E BIENFAIT, J. GARCIA-MINA, and A.M. ZAMARENO

form. In doing so, we observed resistance against mil­dew in cucumber grown without chelates.

MATERIALS AND METHODS

Growth of plants. Tomato (Lycopersicum escu­lentum cv. "Vision") and cucumber (Cucumis sativus cv."Aviance") were grown in Utrecht on rockwool or glass­wool slabs, 3 plants per slab, with a nutrient solutioncontaining: 5.4 mM Ca2+, 9.5 mM K+, 2.4 mM Mg2+,16 mM (N03)·, 1.2 mM (NH4t, 4.4 mM SO/-, 1.5 mMphosphate, 30 i-LM H3B03, 10 i-LM Mn2+, 5 i-LM Zn2+,0.75 i-LM Cu2+, 0.5 i-LM Mo04

2-, kept with HCI on pH 5.7

to 5.9 and when no iron was added, an Fe content of0.8 i-LM. Fresh nutrient solution was added to the slabs atintervals of 2 h, at a daily rate of 0.2 to 0.3 X the watervolume present in the slabs. In the first 6 weeks, (NH4)+was omitted from the nutrient solution in order to preventacidification by ammonia uptake.

Italian green pepper (Capsicum annuum var. Estilo),tomato (cv. Foumaise Hybrid FI) and lettuce (Lactucasativa var. Masaida) were grown in Pamplona on perlitein 6-L pots with two regimes: I) in winter ("hydropon­ics") the pots standing in dishes with 3 cm high nutrientsolution which was weekly refreshed; 2) in summer ("fer­tigation," a farmers practice) irrigated at 12 h with500 mL of water and at 24.00 h with 670 mL of nutrientsolution. The nutrient solution contained 1.5 mM Ca2+,

3mM K+, 2.5mM Mg2+, 5mM (NO))-, 5mM (NR4)+,3.5 mM SO/-, I mM phoshate, 2.5 mM CI-, 25 i-LMH)BO), 54 i-LM Mn-EDTA, 5 i-LM Zn-EDTA, 3 i-LM Cu­EDTA, and 5 i-LM MoO/-.

In the Utrecht solution Fe was added as 15 i-LM Fe­EDDHA (no fmther specification) from Duchefa, Haar­lem, the Netherlands, and in the Pamplona solution asFc(NH4)iS04)2 or Fe-%-EDDHA in concentrations asindicated. In both laboratOlies (Utrecht: May-September,Pamplona: September-February and May-October) theplants were grown in a greenhouse on a light regime ofabout 14 h L/l0 h D, at temperatures of about 26°C (light)and 18T (dark) and RH between 45 and 80%. In Utrecht,daytime temperatures could occasionally rise to 32T.

Preparation. of iron-enriched rockwooi and!.glasswool substrate. Rockwool and glasswool slabs(Cultilene) of 7 X 20 X 100 cm were taken out of theirplastic hulls and cut horizontally in 8-1mn slices. For thecontrol treatments A and D the slabs were simply recon­structed and put back in the hulls. For treatment B, Fewas added as finely divided steel wool between the slices,for treatment C as a colloidal precipitate of Fe(OR)); inboth cases 70 g Fe per slab. The slabs were then reassem­bled and put back in their hulls.

Determination of ortho-ortho isomers ofEDDBIA in plant material.

Storage: Harvested plant material which could not beused immediately for analysis, was stored at -20T(Utrecht) or -40T (Pamplona).

Extraction of EDDHA as the fen-ic complex: Five gpulverised frozen plant material and 25 mL water werehomogenized for 30 s at -40T with an Ultra-Tun-axT25 at 24,000 rpm. After I h shaking of the homogenatein a Heidolph Multi Reax at 2,000 rpm, the solution wasfiltered (8 i-Lm pore size). To 7 mL of filtrate were added35 i-LL of 0.12 M fen-ic nitrate and the pH was adjustedto 7, in order to reconstitute the Fe-EDDHA chelatefrom the possibly free EDDHA present in the plantmaterial. After I h the pH was measured and adjustedagain if needed and the solution was filtered using a0.45 i-Lm filter prior to HPLC analysis.

HPLC analysis: Fifty i-LL of the filtrate obtained fromthe extraction process was injected into a Waters HPLCsystem consisting of a 616 Waters pump, 717 Plusautosampler, 996 photodiode alTay detector, FractionCollector II, and Empower Login chromatographic soft­ware. The analytical chromatographic conditions were:Column Lichrospher 60 RP-seleet B (5 /lm) 250 X 4mm (Merck, ref. 1.50984.0001) flow 1 mL min-I, oventemperature 40T, detection wavelength 480 nm. Gradi­ent mobile phase: solvent A, methanol; solvent B,ammonium acetate 0.0 I M, pH 6.8. Time 0, 4% A, 96%B. Time 4 min: 4% A, 96% B. Time 8 min: 25% A, 75%B. Time 13 min: 25% A, 75% B. Time 17 min: 40% A,60% B. Time 19 min: 40% A, 60% B. Time 21 min: 4%A, 96% B. Time 25 min: 4% A, 96% B. Retention times(min): rac-FeEDDHA 5.2; meso-FeEDDHA 14.2 (seeFig. I).

The concentration of Fe-EDDHA was calculated usingan extemal standard method with a Fe-EDDHA standardpreviously prepared in the laboratory.

The recuperation degree was calculated by addingknown amounts of Fe-EDDHA to tissue samples fromnon-Fe-EDDHA treated plants. All measurements werecan-ied out in duplicate. The recuperation rate for addedFe-EDDHA was between 84 and 94% for all tested tis­sues of tomato, pepper and lettuce. The quantificationlimit of the technique used was 0.77 mg kg- 1 FW.

Determination of total Fe in plant material.Shoot, root, and fruit total Fe content was measured byatomic absorption spectrometry after acid digestion(HNOiHP2) of fresh plant material in a microwavedigester as described by Pinton et al. (1999).

Determination of apoplasmic Fe. ApoplasmicFe in roots was removed and measured according toBienfait et al. (1985).

Determination of acid-soluble iron in rock­wool or glasswool samples. A sample of about

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14

Growth on Chelated and Non-Chelated Iron 1105

a

0.00 -....~200~~~~~.'oo~----r--,8.tn"..--~,'doo~--,--,,2-00~~14'~OO~--,--,16.-00~~,'.OO~~ZJ.'OO'~~22,--100M",ffi

b

20.00 72.00

bJj·~-------------- --"~~---000 zoo ~rn MD ~ 1000 1200 1~OO 1~OO 1800

_ffi

~

'" 0

Fig. 1. HPLC chromatograms. a: PureFe EDDHA. b: Extract of leaves of tomatogrown on Fe-EDDHA. c: Extract of leavesof tomato grown on Fe(NH4MS04)2' AU,absorbance units. The peaks of EDDHAisomers in b are both shifted to a slightlyearlier elution time relative to the puresample. This is a nOlmal phenomenon withextracts from plant tissue.

c

18,00 :,!),OO 22.0016.0014.00-{lOOlt--e-~--,-~~~~"..--~-,---c~c--r-~-,-,~~~~-,--,~r-r~--,--,~~,--I

noo zoo ~oo ~ 8,00 1000 1200

M""ffi

0.2 g was incubated with 20 mL 6 N HCI, and softly shak­en during 1 h at room temperature. The suspension wasthen shortly centrifuged at 1,000 X g, 5 mL supernatantwas added to 12.5 mL 0.2 M MES pH 6, 1.2 mM bipy­ridyl, and the pH was adjusted to 5.5 with concentratedKGB. The absorption at 520 nm was determined beforeand after addition of sodium dithionite.

Physiologically available iron. Unless men­tioned otherwise, a piece of maximally 0.6 g glasswool orrockwool was softly shaken in 20 mL 0.5 mM HCI for 1 hat room temperature. Thereafter I mL was centrifuged for5 min at 9,000 X 9 and dissolved Fe(II) and Fe(III) weredetennined on the supernatant as follows: to the samplewere added bipyridyl or BPDS to 0.3 mM, MES orHEPES buffer to 50 mM, the pH was if necessary adjust­ed to a value between 6 and 8, and A520 (bipyridyl) or Am(BPDS) were determined. Fe(III), when present, showed

itself by an increase of absorption after addition of Na­dithionite. The mM extinction coefficient for Fe(II)bipy­ridyl was taken to be 8.65, for Fe(II)BPDS 22.14.

RESULTS

Growth with and without EDDHATomato. In the Utrecht experiment, tomato in the

control regime A on Fe-EDDHA grew well and gave nor­mal fruits (17.3 kg), but in the last two weeks of thegrowing period showed increasing chlorosis. Whether thischlorosis was due to Fe shortage could not be determined.During periods of high temperature the concentration ofFe-EDDHA in the nutrient solution in the outflow fromthe slabs could reach values of 60-80 J-LM, probably dueto fast transpiration by the plants.

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14

1106 H.P. BIENFAIT, J. GARCIA-MINA, andA.M. ZAMARENO

Table 1. Acid-soluble Iron In rockwool and glasswoolsubstrates.

Taste-related Fe(NH4MS04),. _ Fe-EDDHA

parameters 20 fLM 20 fLM 40 fLM----_ ..-._-_._-_.-

pH 4.03 4.04 4.06

Acidity (%) 0.53 0.56 0.55

Sucrose (%) ("Brix") 5.19 5.29 5.23

Fructose (%) 1.78 1.73 1.78

Glucose (%) 1.40 1.39 1.35

Bostwick (em) 11.25 12.25 11.00

Dry matter (%) 7.05 7.20 7.25

Colour-related parameters

Hunter L 49.32 53.16 57.08

Hunter a 27.42 b 27.23 b 22.52 a

Hunter 1J 20.05 b 20.49 ab 21.70 a

Hunter a/ Hunter 1J 1.37 b 1.33 ab 1.04 a

Lycopene (mg/ I00 g) 6.25 b 5.55 a 4.70 a

Presence of EDDHA in plant partsLeaves and roots of tomato and pepper, grown on Fe­

EDDHA, contained significant amounts of EDDHA(Table 3, Fig. I); the fruits of these plants contained only

Composition of the frilltsAnalysis of the tomato fruits, grown in Pamplona,

showed that the source of iron for the plants had no sig­nificant influence on several parameters, with the excep­tion of lycopene, which was significantly lower in plantsgrowing on Fe-EDDHA (Table 2). There was a cone­sponding effect on the color, the fruits grown with Fe­EDDHA being less intensely red.

Table 2. Effects of growth with Fe(NH4MS04)" or Fe­EDDHA on different properties of tomato fruits.

It appeared that in glasswool B the steelwool had conod­ed to brown and black spots in the glasswool. GlasswoolC showed practically only the same brown spots whichhad been applied to them at the outset (fenic hydroxide).The black and the brown spots in glasswool B were cutout and subjected to incubation with HCI at an initial pHof 3.3, in order to assess the in situ availability of ironupon local proton extrusion by roots.The results are sum­marized in Table 1. It shows that this treatment releasedsignificantly more Fe, especially in the ferrous formwhich is easily taken up by the roots, from the black spotsthan from the brown ones.

Values are averages of two values, each value resulting from twodetenninations on 3 tomatoes. Contents are on the basis of freshweight. Acidity, titrated acidity expressed as % of citric acid;Bostwick, degree of consistency or fiImness, Hunter L, degree ofluminosity; Hunter G, degree of redness; Hunter b, degree of yel­lowness. Significant differences indicated by different letters forJi < n.n5 (LSD test).

100

Dissolved Extracted

at th~~ld of------- a~e~ ~hthe incuba- Fe (II) Fe(III) HCI

tion (fLM) (fLM)(fLM)

------

19,000

Substrate

Rockwool, untreated 4.3 15 I4.3 15 2

Glasswool, untreated 3.3 0 0

Glasswool from regime 3.8 0 3B, with brown spot 3.7 0 9

Glasswool from regime 3.5 85 22B, with black spot 3.4 48 12_._. --- --_.----_.... - -----

Fragments of the substrates, about 0.6 g, were incubated for 55min in 20 mL 0.5 mM HCI at initial pH 3.3 or in 6 M HCI as indi­cated. After 55 min samples were taken and centrifuged for 5 minat 9,000 X g, and Fe was determined on the supernatants.

The plants growing on Fe-free nutrient solutionregimes B-D gave different results: On regime B (withsteelwool) growth and fruit development were the sameas in A, with now and then a tiny chlorosis, but no chloro­sis at the end of the growth period. The yield (17.1 kg)was slightly lower than in culture A, but we consider thedifference not to be significant.

On growing regime C (with Fe(OH)3) the plantsshowed a continually recuning chlorosis; this responsewas even stronger, as to be expected, on regime D (Fe­free), the strongest chlorosis appearing on glasswool.

In the Pamplona experiment, no differences wereobserved between plants grown on Fe-EDDHA or onFe(NH4MS04)2 both in relation to plant growth rate andto fruit production (data not shown).

Cucumber gave basically the same results astomato, with two exceptions.

a: In the control regime A (with Fe-EDDHA) theplants were struck after 8 weeks by a mildew infection(Sphaerotheca fusca) which soon covered all the leavesexcept the five youngest. The plants on nutrient solutionwithout Fe-EDDHA (B-D) were not infected. Attemptsto transfer the infection from the infected plants to thoseon EDDHA-free nutrient solution were in vain.

b: Plants which canied a large burden of fruits tendedto develop chlorosis in the youngest leaves. After removalof the largest fruits, the new leaves were green again.

With cucumber, as with tomato, the concentration ofFe-EDDHA in the outflow from the slabs of culture Acould reach values of 60-80 i-LM in periods of high tem­perature. Since the culture on the steelwool regime Bgave comparable results, especially with tomato, as withthe Fe-EDDHA regime A, and better than the ferrichydroxide regime C, samples of the used glasswool weretaken at the end of the culture period in order to detectpossible differences in Fe availability from the substrate.

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14

Growth on Chelated and Non-Chelated Iron 1107

Table 3. Fe and EDDHA in tissues of tomato, pepper, and lettuce.

Total Fe (j..lmol g-I DW) EDDHA (j..lmol g-I DW) EDDHA/FeRoot

apopl. RootLeaf Fruit

RootLeaf Fruit Root Leaf Fruit

plasm. Plasm ..__._-_....._---_ .._---- .... -.--_. __.-._-_.------.---_ .._---- .........._...._-_....__ ._---. .-..............................._._--

nd' 1.95 1.34 0.75nd 1.53 1.42 0.65 0.24 0.11 < 0.013 0.16 0.08 < 0.020nd 1.88 1.40 0.91 0.38 0.18 < 0.013 0.20 0.13 < 0.014

nd 1.68 1.78 0.35nd 1.21 1.89 0.41 0.09 0.10 < 0.014 0.D7 0.05 < 0.034nd 1.96 1.61 0.40 0.15 0.14 < 0.014 0.08 0.08 < 0.035

1.43 2.70 1.54 0.35 0.43 0.13 0.274.13 1.20 2.04 1.41 0.23 0.40 < 0.03 0.19 0.20 < 0.023

Treatment

Pepper under fertigationb

60 j..lM Fe EDDHA3 weeks after retum on

Fe(NH4MS04)2

Pepper20 j..lM Fe(NH4MS04)220 j..lM Fe EDDHA40 j..lM Fe EDDHA

Tomato20 j..lM Fe(NH4MS04)220 j..lM Fe EDDHA40 j..lM Fe EDDHA

nd 1.17 0.43 0.07'nd 1.57 0.48 0.25 < 0.01 0.16 < 0.02nd 1.43 OSl 0.48 <0.01 0.19 < 0.02

..__~.~ .._.-

Lettuce20 j..lM Fe(NH4MS04)220 j..lM Fe EDDHA40 j..lM Fe EDDHA

~~~~~~~~~~~__~~~~~~~~~~

Apopl, apoplasmic Fe; plasm, total Fe after removal of apoplasmic Fe. Apoplasmic Fe was removed and determined after Bien­fait et aI. (1985). a contamination, see RESULTS. b See MATERIAL AND METHODS. C It was not detemlined.

traces. For the interpretation of these results it is neces­sary to know the stability of the chelator molecule in theplants. Therefore,S pepper plants grown under fertigationwith Fe-EDDHA for 15 weeks were transfened to aregime with Fe(NH4MS04)2 as an iron source. After 3morc weeks these plants were harvested and tissue sam­ples were analysed. Table 3 shows a reduction of theEDDHA content in the roots by one-third during the threeweeks of EDDHA-free growth, which can be ascribed toexport to the sprout via the xylem, an increase of root tis­sue biomass, and to breakdown. In the leaves, the con­tents of both the racemic and the meso forms of 0/0­

EDDHA were only slightly (about 10%) lowered duringthis 21 d period, probably due to the combined and partlyopposite effects of a continued import from the roots afterthe change of regime, export via the phloem and degrada­tion. Dilution by growth could not have played an impor­tant role since the plants were at the end of their growthperiod. Whatever the precise contrihution of all these fac­tors to the still high content of chelator in the leaves afterthree weeks EDDHA-free growth, it can be reasonablyassumed that breakdown in that tissue could not havebeen more than roughly 2% per day for roots and 0.5%per day for leaves.

Table 3 also shows that leaf EDDHA contents in toma­to, pepper, and lettuce did not go parallel to those in totalFe. In pepper the culture conditions had a significanteffect on the contents of both Fe and EDDHA. Table 3also gives the conesponding differences in the ratios ofEDDHA and total Fe contents. Fruits of pepper and toma­to, grown on Fe-EDDHA, contained only traces of

EDDHA. (The EDDHA content of lettuce roots onFe(NH4MS04)2 may have been due to a contamination inthe greenhouse, or represent a remainder from pre-cultureat the provider of seedlings.)

Resistance to mildew in cucumberThe variety of cucumber we used was seriously

attacked by the mildew Sphaerotheca fusca, when grownon Fe-EDDHA. All the plants which grew without thechelate were insensitive. The infection must have come inthrough the open windows of the greenhouse where thecucumber plants were growing close together. Sincetransfer of the infection from plant to plant by insects wasapparently absent and by hand unsuccessful, and the cor­relations infection-EDDHA and no infection-no EDDHAwere 100%, we conclude that growth on Fe-EDDHA andsensitivity to Sphaerotheca fusca were causally related.

DISCUSSION

Uptake of iron and EDDHA. Our results showthat plants growing on perlite and a nutrient solution withFe in the fonn of the EDDHA complex take up both ironand the chelator (Table 3). In the tissues tested the ironcontent was always much higher than that of EDDHA.The difference could not be caused by a fast breakdownof the chelator in the plant tissue, since in plants that hadbeen growing on Fe-EDDHA and then transferred to achelator-free substrate, the EDDHA level in the leaveswas only reduced by about 0.5% per day, and in the roots

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14

1108 H.P. BIENFAIT, J. GARCIA-MINA, andA.M. ZAMARENO

Table 4. Changing ratio's of EDDHA isomers in planttissues.

[Racemic]/[Meso] in Decrease

Roots Leaves (%)... _-_._- .. _. __._.-

Tomato20 fLM Fe-EDDHA 0.93 0.72 2340 fLM Fe-EDDHA 0.95 0.77 19

Pepper20 fLM Fe-EDDHA 1.09 0.69 3740 fLM Fe-EDDHA 1.12 0.73 35

Pepper undcr fcrtigation60 fLM Fe-EDDHA 1.17 1.00 1521 d after return to 0.83 0.94

Fe(NH4MSO4)2

The ratio of Racemic / Meso isomers of 0-0 EDDHA are givenas isolated from roots and leaves of plants grown on two con­centrations of Fe-EDDHA.

by 1.5% per day (Table 3). This slow decrease could inroots easily be caused by export via the xylem, and inleaves via the phloem, combined with the increase ofplant dry mass in the period of growth without chelator.Apparently, breakdown of EDDHA in tomato or pepperleaf and root tissue, if at all, is very slow. Nevertheless,there are indications for some breakdown. The ratios ofracemic / meso in roots, 0.93-1.17 in the original chelatemixture, were systematically lower in leaves (0.7-1.00,Table 4). In tomato the ratio dropped with about 20%from roots to leaves, in pepper about 35%. This suggestsa difference in translocation or a difference in degrada­tion, or both. If breakdown is enzymatic, the enzyme mayhave different affinities for the three isomers. Hill-Cot­tingham and Lloyd-Jones (1965) observed, in bean plants,a difference in breakdown rates of the meso and racemicfOillIS of EDDHA (their Fig. 3). A possible non-enzymat­ic mechanism of breakdown, suggested by Hill-Cotting­ham and Lloyd-Jones (1961), is photoreduction of theferric chelate in the leaves, followed by decarboxylationof the chelator (cf. Bienfait and Scheffers 1992).

Since we did not find a tissue in which EDDHA levelswere higher than those of Fe, the difference in Fe andchelate contents of the tissues tested must have beencaused by a difference in uptake rates. The expected ratioof EDDHA/total Fe in the plant would be 0 if Fe was tak­en up by the Fe efficiency reaction only. The ratio wouldbe 1 if all Fe was taken up by passive chelate absorptionand with no breakdown of EDDHA in the plant. With amaximal breakdown rate of 1% per day and the usualgrowth rate during 15 weeks, the EDDHA/Fe ratio in theplant then would be at least 0.5. The EDDHA/Fe ratiosfound varied between 0.05 and 0.27 for tomato and pep­per roots and leaves (Table 3), and since these tissues con­tain the bulk of both the amount of iron taken up and thebiomass dry weight, we conclude that iron uptake in these

two species occurred both by chelate splitting after ferricreduction (via the Fe efficiency reaction or by otherreducing processes in the rhizosphere), and by passivechelate absorption.

The balance of these different processes was apparentlydifferent for the three species that we tested, and alsodepended on the experimental conditions. A higher Fe­EDDHA concentration resulted in a higher EDDHA/Feratio, indicating an increased contribution of passive che­late absorption; and in pepper cultured in the summerwith 60 IJvM under feltigation, the EDDHA/Fe ratio inleaves was more than three times higher as in the winteron 40 IJvM in hydroponic culture (Table 3).

A number of factors may influence the rates of the twoFe uptake mechanisms. Ferric chelate reduction by rootsdepends in the first place on the Fe status of the plant, andhas flllther been found to obey mixed Michaelis-Mentenand diffusion kinetics (e.g., Bienfait et al. 1983); the samemay hold for uptake of the resulting ferrous ion. Passivechelate absorption, on the other hand, will be proportionalto concentration. Only traces of EDDHA were found inthe leaves of lettuce and the fruits of pepper and tomato.

Both pepper and tomato fruits are mainly fed via thephloem, and the loading of phloem with EDDHA or oneof its complexes, or its unloading, may be slow. We haveno good explanation for the low EDDHA content of let­tuce leaves; EDDHA does enter the roots (Table 3). Per­haps the cause is to be found in the way of growth oflettuce, with only the outside leaves capable to transpirefreely. Breakdown in the leaves remains of course a possi­bility.

The most plausible mechanism of passive (Fe-)EDDHAentry into the root is by passage through openings in theendodennis, caused by the breakthrough of newly fonn­ing lateral roots (cf. Mahaffee et al. 1994). In that case thedriving force for entry is the transpiration stream. Bienfait(1988) noticed that the Fe status of the tomato fer mutant,which is unable to express the Fe efficiency reactions(Brown et al. 1971), upon Fe nutrition with Fe-HEDTAwas closely related to the product (transpirationrate) X (Fe-chelate concentration in the nutrient solution),as is to be expected on the basis of passive chelate absorp­tion.

Available forms of iron in rockwool. Rockwoolis produced from basaltic material, and the material fromCultilene or Grodan that we tested contained about 3-5%acid-soluble iron. Incubation with 0.5 mM HCl (pH 3.3,which is easily realized in nutrient solution by several Fe­deficient dicots) can release ferrous ions to concentrationsup to 15 IJvM within an hour (Table I). Plants growing onpure rockwool and iron free nutrient solution did not tumas chlorotic as in the same regime on glasswool, whichmeans that the plants were indeed capable to mobilizeiron from the rockwool substrate; but they were not con-

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14

Growth on Chelated and Non-Chelated Iron 1109

tinually as green as in the regimes A (with Fe-EDDHA)and B (with added iron in the substrate). The post harvestanalysis on the glasswool substrate of culture B showedthat the black oxidation products were most effective inyielding iron, up to 100 f.LM, mostly reduced and thusdirectly available for uptake by the plant, upon incubationfor 55 min with dilute acid (Table I). We assume that thisproduct is most probably magnetite, Fe10 4. Magnetite canbe fanned from metallic iron in water and under low orvariable oxygen levels, a condition which is easily real­ized in the rhizosphere of glasswool or rockwool culture.Chapman (1939) showed already that finely divided mag­netite could be a good source of iron for citrus on sandculture, especially when the plants had been given time toacidify their rhizosphere. The less effective brown oxida­tion product, most probably Fe(OH)3' yielded only smallamounts of ferric iron upon incubation with dilute acid(Table I), which explains the lesser results with culture C.

Growth on Fe-EDDHA or on inorganic iron made nodifference for the content of some taste- and colour-deter­mining compounds of tomato fruits, with the exception oflycopene (Table 2). The synthesis of lycopene requires, inits final steps, the activity of an iron-containing desaturaseenzyme complex (Carol and Kuntz 2001; Cunninghamand Gantt 1998). There are xylem vessels reaching theskin of the tomato fruit (Malone and Andrews 2001);EDDHA entering through these vessels might competefor iron with the enzyme and in this way interfere withlycopene synthesis.

The fact that no more effects of the iron source werefound on the other compounds tested does not mean thatthere are no effects relevant to the taste. Expression of theFe efficiency reactions, especially proton excretion, hasconsequences for metabolism, such as an increased pro­duction of citrate and malate in the root and export ofthese metabolites to the sprout (Landsberg 1981; de Vaset al. 1986). For grape it is known that soil characteristics,like its content of CaC03 have effects on the taste ofwine. In bean plants, Fe deficiency changes the malate/sugar ratio in phloem sap (de Vas et al. 1986).

Resistance to mildew in cucumber. There is anextensive literature on the relation between Fe nutritionand resistance against microbial infections, both in theroots and the sprout (e.g. van Peer et al. 1990; Liu et al.1995; van Loon et al. 1998; Expert 1999; Persello­Cartieux et al. 2003). We can conceive at least two mech­anisms to explain our results:

1) Growth on Fe-chelates implies a passive influx ofiron which therefore is not under the control of the plant'sFe sensing system. Thus, depending on the circumstances(Fe-chelate concentration in the nutrient solution, transpi­ration rate, etc.) the sprout may be confronted with anoverdose of soluble Fe via the transpiration stream. In thatcase an attacking microorganism may easily get hold of

sufficient iron for its growth. In a plant which depends onthe activation of its Fe efficiency reactions, the influx ofiron is well regulated and the infection has a problem ingetting enough iron. The bacterium Erv.!inia chrysan­themi, which cannot use the xylem iron carrier fen-ic cit­rate as an iron source, excretes the siderophorechrysobactin into the intercellular leaf space after infect­ing Saintpaulia (Neema et al. 1993); this illustrates theimportance of iron supply for microorganisms in order tosuccesfully invade plant tissue. Rahman and Punja (2003)applied Fe-EDTA to leaves of ginseng which led in theroots both to increased Fe levels and to larger lesions afterinfection by Cylindrocarpon destructans. Deak et al.(1999) found that transgenic tobacco ectopically express­ing alfalfa fenitin showed less necrotization in its leavesupon local infection with tobacco necrosis virus or thefungi Alternaria alternata and Botrytis cinerea than con­trols. They ascribed this effect to a lowering of Fe levelsin the leaves by the scavenging action of fen-itin, therebyinhibiting the local fonnation of reactive oxygen species.

2) EDDHA or one of its breakdown products inter­fered with the plants' natural resistance against microbialinfection. We do not know of any publication reporting onsuch an effect. The concentration of EDDHA in theleaves (Table 3), when expressed on the basis of totalwater in the leaves, varied between 14 and 60 f.LM. IfEDDHA was not taken up by all the leaf cells, the con­centration in the intercellular spaces must have beenaccordingly higher, and if the chelator had given off partof its iron to the cells, e.g. via the action of a reductase inthe plasma membranes, it could have bound other metalions, as already suggested by Haertl and Martell (1956).

Conclusions. EDDHA is not just a carrier of ironto the roots, but it is also taken up and can have effects onthe physiology of the plant. Properties of plants culturedon Fe-EDDHA are therefore not necessarily the same asthose of plants growing on natural Fe sources.

Acknov....tedgments. HFB wishes to thank Dr. Wim Voogt ofPractijkonderzoek Plant & Omgeving in Naaldwijk, and at UtrechtUniversity Dr. Peter Bakker for expel1 advice, Fred Siesling andthe Afdeling Ecofysiologie van Planten for their generous hospi­tality and support. JG-M thanks Fabrice Houdusse and Sara SanFrancisco for their valuable teclmical assistance.

REFERENCES

Bienfait HF 1988: Proteins under the control of the gene for Feefficiency in tomato. Plant Physiol., 88, 785-787

Bienfait HF, Bino RJ, van der Bliek AM, Duivenvoorden JF, andFontaine JM 1983: Characterization of ferric reducing activityin roots of Fe-deficient Phaseolus vulgaris. Physiol. Plan­tarum, 59, 196-202

Bienfait HF and SchetTers MR 1992: Some properties of felTic

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14

1110 H.E BIENFAIT, J. GARCIA-MINA, andA.M. ZAMARENO

citrate relevant to the iron nutrition of plants. Plant Soil, 143,141-144

Bienfait HF, van den Briel ML, and Mesland-Mul N 1985: Freespace iron pools in roots, generation and mobilization. PlantPhysiol., 81, 596-600

Brown IC, Chaney RL, and Ambler JE 1971: A new tomatomutant inefficient in the transpOlt of iron. Physiol. Plantarum,25,48-53

Carol P and Kuntz M 2001: A plastid tetminal oxidase comes tolight: Implications for carotenoid biosynthesis and chlorores­piration. Trends Plant Sci., 6, 31-36

Chaney RL, Brown JC, and Tiffin LO 1972: Obligatory reductionof ferric chelates in iron uptake by soybeans. Plant Physiol.,50,208-213

Chapman HD 1939: Absorption of iron from finely ground mag­netite by citrus seedlings. Soil Sci., 48, 309-315

Cunningham FX Jr and Gantt E 1998: Genes and enzymes of car­otenoid biosynthesis in plants. Annu. Rev. Plant Phys. PlantMol. BioI., 49, 557-583

Deak. M, Horvath GV, Davletova S, Torok K, Sass L, Vass I,Bama B, Kiraly Z, and Dudits D 1999: Plants extopicallyexpressing the iron-binding protein, ferritin, are tolerant tooxidative damage and pathogens. Nat. Biotechnol., 17, 192­196

de Vos CR, Lubberding HJ, and Bienfait HF 1986: Rhizosphereacidification as a response to iron deficiency in bean plants.Plant Physiol., 81, 842-846

Epstein AL, Gussman CD, Blaylock MJ, Yenniyahu U, HuangJW, Kapulnik Y, and Orser CS 1999: EDTA and Pb-EDTAacculation in Brassica juncea grown in Pb-amended soil.Plant Soil, 208, 87-94

Expert D 1999: Withholding and exchanging iron: Interactionsbetween Ervvinia spp. and their plant hosts. Annu. Rev. Phyto­pathol., 37, 307-334

Haertl EJ and Martell AE 1956: Metal chelates in plant and ani­mal nutrition. J. Agric. Food Chem., 4, 26-32

Hall JL and Williams LE 2003: Transition metal transporters inplants. J. Exp. Bot., 54, 2601-2613

Hill-Cottingham DG and Lloyd-Jones CP 1961: Absorption andbreakdown of iron-ethylene tetraacetic acid by tomato plants.Nature, 189, 312

Hill-Cottingham DG and Lloyd-Jones CP 1965: The behaviour ofiron chelating agents with plants. J. Exp. Bot., 16, 233-242

Holmes RS and Brown JC 1955: Chelates as correctives for chlo­rosis. Soil Sci., 80,167-179

Landsberg E-C 1981: Organic acid synthesis and release of hydro­gen ions in response to Fe deficiency stress of mono- anddicotyledonous plant species. J. Plant Nutl~, 3, 579-591

Liu L, Kloepper JW, and Tuzun S 1995: Induction of systemicresistance in cucumber by plant growth-promoting rhizobac­teria: Duration of protection and effect of host resistance onprotection and root coloniaztion. Phytopathology, 85, 1064­1068

Mahaffee WF, Kloepper JW, van Vuurde JWL, van der Wolf JM,and van den Blink M 1994: Endophytic colonization ofPhaseolus vulgaris by Pseudomonas jiuorescens strain 89B­27 and Enterobacter asburiae strain JM22. In ImprovingPlant Productivity with Rhizobactelia, Ed. MH Ryder, PMStevens, and GD Bowen, p. 180, CSIRO Division of Soils,Glen Osmond

Malone M and Andrews J 2001: The distribution of xylemhydraulic resistance in the fruiting truss of tomato. Plant CellEnviron., 24, 565-570

Neema C, Laulhere J-P, and Expert D 1993: Iron deficiencyinduced by chrysobactin in Saintpaulia leaves inoculated withElwinia chrysanthemi. Plant Physiol., 102,967-973

Persello-Cartieux F, Nussaume L, and Robaglia C 2003: Talesfrom the underground: Molecular plant-rhizobacteria interac­tions. Plant Cell Environ., 26, 189-199

Pieterse AH, Bhalla PR, and Sabharwal PS 1970: Induction offlowering in Lemna gibba G3 by Fe-EDDHA. Plant CellPhysiol., 11, 675-676

Pinton R, Cesco S, Santi S, Agnolon F, and Varanini Z 1999:Water-extractable humic substances enhance iron deficiencyresponses by Fe-deficient cucumber plants. Plant Soil, 210,145-157

Rahman M and Punja ZK 2003: Growth of Cylindrocarpondestructans and interactions with ginseng roots to cause rootrot. Can. J. Plant Pat/wI., 25, 112

Romheld V 1979: Mechanismus der Aufnahme und Verlagerungvon Eisenchelaten bei hoheren Pflanzen. PhD Thesis, TUBerlin

Romheld V and Marschner H 1986: Mobilization of iron in therhizosphere of different plant species. Adv. Plant Nutr., 2,155-204

Romheld V, MUller C, and Marschner H 1984: Localization andcapacity of proton pumps in roots of intact sunflower plants.Plant Physiol., 76, 603-606

van Loon LC, Bakker PAHM, and Pieterse CMJ 1998: Systemicresistance induced by rhizosphere bacteria. Annu. Rev. Phyto­pathol., 36, 453-483

van Peer R, van Kuik AJ, Rattink H, and Schippers B 1990: Con­trol of Fusarium wilt in carnation on rockwool by Pseudomo­nas sp. strain WCS417r and by Fe-EDDHA. Neth. J. PlantPat/wI., 96, I 19-132

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

4:00

07

Dec

embe

r 20

14