Annals of Botany 80 : 553–559, 1997

Differences in Iron Nutrition Strategies of Two Calcifuges,

Carex pilulifera L. and Veronica officinalis L.

ANGELIKA ZOHLEN* and GERMUND TYLER

Department of Ecology, Soil-Plant Research, Ecology Building, Lund Uni�ersity, S-223 62 Lund, Sweden

Received: 6 January 1997 Accepted: 3 July 1997

Veronica officinalis and Carex pilulifera, widespread calcifuge plants in Europe, were cultivated in acid and calcareoussoils to study differences in Fe aquisition strategies indicated in previous studies. The experiments were performed ina computer-controlled glasshouse at a soil solution moisture content of 50–60% water holding capacity ; additionallight was supplied at 70 W m−# if ambient light was ! 100 W m−# between 0600 and 1800h. Both species developedchlorosis when grown in the calcareous soil. C. pilulifera proved unable to translocate sufficient amounts of Fe to theleaves when cultivated in calcareous soil, but much Fe accumulated in, and especially as a precipitate on the surfaceof roots. In contrast, V. officinalis tended to increase Fe taken up into the leaves of plants grown on calcareous soil,but a much greater proportion of the leaf tissue Fe was accumulated as less active forms not extracted by Fecomplexing agents, e.g. 1,10-phenanthroline, than was the case in acid-soil grown plants. Considerably less Fe wasaccumulated in the root biomass of V. officinalis compared to C. pilulifera. It is concluded that chlorosis in C. piluliferais related to insufficient Fe uptake in the leaves, whereas leaf immobilization of Fe in physiologically less active formsis the problem in V. officinalis. # 1997 Annals of Botany Company

Key words : Iron, chlorosis, calcifuge, iron immobilization, leaf tissue, fractionation, Carex pilulifera, Veronicaofficinalis.

INTRODUCTION

The majority of plants in central and northern Europe areable to grow and compete successfully on calcareous soils.However, an appreciable minority of species, the so calledcalcifuges, are unable to grow in calcareous soils because oflimitations in their mineral nutrition. Often, the primarylimitation for these species is an inability to render theindigenous forms of soil phosphate available for uptake(Tyler, 1992), but there are also species that are limited bya deficiency of micronutrients, especially iron. The typicalsymptom of lime-induced chlorosis is a yellowing of leaves.The veins may remain more or less green because Fe ispoorly redistributed within the tissue (Chaney, 1984).

Several calcifuges, e.g. Veronica officinalis L., Carexpilulifera L. and Rumex acetosella L., develop chloroticsymptoms, probably due to Fe deficiency, when cultivatedin limestone soils (Tyler, 1996a). This is particularly evidentwhen the primary growth limitation by phosphate is reversedby phosphate addition to the soil. In C. pilulifera, but not inV. officinalis, chlorosis is related to a decreased uptake rateof Fe. Chlorosis caused by a physiological Fe deficiencymay be due to an immobilization of this element inmetabolically inactive forms in the tissues (Abadı!a et al.,1989). Interactions with pH, bicarbonate, phosphate, andlow-molecular organic acids may be involved (Kinzel,1982), but mechanisms are not well understood.

The aim of this study was to elucidate the difference in the

* E-mail Angelika.Zohlena!planteco.lu.se

Fe nutrition strategies of the two calcifuges C. pilulifera andV. officinalis using tissue fractionation methods to char-acterize the Fe solubility in leaves and roots. It washypothesized that ‘ lime’ chlorosis in C. pilulifera may bedue to insufficient uptake or transport of Fe to the shoots,whereas in V. officinalis it may be due to an immobilizationof tissue Fe in non-metabolic forms.

MATERIALS AND METHODS

Two cultivation experiments were performed in a glasshousein three different soils : an acid soil (the original substrate),a calcareous soil and a calcareous soil with added phosphateto prevent growth limitations caused by phosphorusdeficiency. The two soil types used were a Rendzic leptosol(pH-BaCl

#7±5) from the Ordovician limestone ‘alvar ’ of

O$ land (56°25« N, 16°30« E), approx. 10 cm deep, and thetop 10 cm (A

hhorizon) of a dystric cambisol (pH BaCl

#4±0),

developed from a silicate-rock moraine in central Scania(55°57« N, 13°50« E), south Sweden. We sieved the soils(through a 6 mm mesh) and stored them for 2–3 d at5 °C before further treatment. To half of the samples oflimestone soil we added 5 mmol dm−$ of water-solubleCaHPO

%[2 H

#O to provide additional phosphate.

For the first experiment we collected adult, well-developedplants of V. officinalis and C. pilulifera from the same areaas the acid soil. Equal-sized plants with roots and shoots cutdown to approx. 3 cm were planted in 3 l polythene vesselswith basal drainage, using five replicates per species andtreatment, and five plants per vessel.

0305-7364}97}100553­07 $25.00}0 bo970493 # 1997 Annals of Botany Company

554 Zohlen and Tyler—Iron Strategies in Calcifuges

The experiment began on 8 May 1996 and finishedbetween 5–8 July. The glasshouse was computer-controlled,with a mean minimum temperature at night of 16±5³2±2 °C(95% confidence limit) and mean maximum temperature byday of 26±4³3±5 °C. The soil moisture was maintained at50–60% of the water holding capacity (WHC) during theexperiment (which corresponded to 15±5–18±6% d.wt of theacid soil and 30±0–36±0% d.wt of the calcareous soil), byadding H

#O to the soil surface every 24–48 h, as necessary.

Additional light, 70 W m−#, was provided by high pressuresodium lamps between 0600h and 1800h if ambient lightwas less than 100 W m−#.

We continuously removed all flower buds produced bythe plants during the experiment. Before harvesting, weestimated the leaf colour of the plants, using the Swedishstandard colour atlas (sheet 2, SIS 1989; transformed into a15 degree scale from yellow to deep green).

At harvest, the above-ground biomass of plants was cutnear the soil surface with titanium scissors and washed byvigorous shaking in polythene vessels for 30 s with 100 ml0±1 HCl and H

#O, respectively. Excess water was removed

by blotting. Leaves produced during the experiment werefinely chopped with the same scissors. Within a few hours ofstorage at 7 °C, we extracted 0±30 g of the freshly choppedmaterial with 7 ml of a 1±5% 1,10-phenanthroline (Katyaland Sharma, 1980, 1984) and a 1 HCl solution (Takkarand Kaur, 1984), respectively, shaken continuously for 17 hin 36 ml plastic tubes. Before use, all equipment had beensoaked overnight in 0±1 HCl and rinsed in H

#O. 1,10-

phenanthroline is an Fe complexing agent considered tochelate mainly Fe#+ and used to characterize differences inmetabolically active Fe between chlorotic and non-chloroticplants. We solubilized 15 g 1,10-phenanthroline in 850 mlH

#O, adjusted the pH to 3±0 with 1 HCl and made the

volume to 1 l. The 1 HCl extractable fraction is consideredto reflect the Fe fraction that is active in chlorophyllformation (Oserkowsky, 1933).

To measure the pH of the plant tissue, 0±30 g freshchopped material was extracted with 7 ml 0±2 KCl, pH 7for 17 h. Before chopping, the plant material was preparedas described above, but washed by shaking for 2¬30 s withH

#O only.Subsamples of the chopped plant material were used for

dry weight determination at 85 °C and determination oftotal Fe by wet digestion (conc. HNO

$) for complete

destruction of organic matter. We evaporated excess acid to2 ml and added H

#O to 25 ml.

The concentrations of Ca, Fe, Al and Mg in theexperimental soils, exchangable with 0±2 BaCl

#(10 g soil

at field moisture and 100 ml extractant, 1 h), were de-termined by plasma emission (ICP-ES) and exchangable Kwas determined by flame AAS in 1000 mg l−" of Cs as CsCl.We also extracted soil Fe (10 g soil at field moisture plus50 ml extractant for 1 h) with two complexing agents,0±02 Na citrate (pH 8±5) and 0±02 Na EDTA (pH 4±7),respectively. The extraction by organic ligands shouldsimulate a possible influence of root exudates on Femobilization. For easily exchangable phosphate, the Sb}Moblue method was used with ascorbic acid reductant onfiltered and centrifuged extracts obtained by shaking 10 g

soil at field moisture for 20 min with 100 ml unbufferedneutral 0±05 Na

#SO

%plus 0±02 NaF (Olsen and

Sommers, 1982; Tyler 1992).We determined the pH of the soil solution obtained by

high-speed centrifugation (12000 rpm) of approx. 130 g soilat field moisture for 1 h, in a specially equipped coolingcentrifuge at 10 °C (Falkengren-Grerup and Tyler, 1993).

The organic matter content of the soil was estimated asloss on ignition (550 °C for 3 h) and calculated as apercentage of dry weight. Clay content was not determined,but did not exceed 5% in any of the soils according toprevious studies using the same soils (Tyler, 1996b).

A second growth experiment was performed in order toconfirm the results of the first one using the same soils andthe sameplant species but somedifferent extractionmethods.Eight replicate vessels of each species and treatment wereused in the second experiment which ran between 9 Aug.and 24 Oct. 1996, using the same growing conditions as inthe first experiment. The mean minimum temperature atnight was 15±5³0±4 °C (95% confidence limit) and the meanmaximum temperature by day was 24±6³0±1 °C.

For the extractions, we washed the plants in 0±1 HCl asabove, followed by three washes in H

#O. After removing

surplus water between blotting paper, we extracted 0±5 gfresh chopped sample with 5 ml 1,10-phenanthroline sol-ution for 17 h. We also used TPTZ (2,4,6-tris-(2-pyridyl)-1,3,5-triazine) as an alternative extractant, being consideredmore specific for Fe#+ than 1,10-phenanthroline (Piersonand Clark 1984a). A 0±02 TPTZ solution was prepared bydissolving 6±25 g in 700 ml H

#O, adding HCl until the TPTZ

was dissolved and making the volume to 1 l with dilutedHCl. The pH of this solution was 2±1. To 0±5 g freshlychopped plant material we added 5 ml TPTZ solution andthe sample was shaken continuously at room temperaturefor 17 h. We performed the total digestion as in the firstexperiment. Before use, we had rinsed all equipment in10−& TPTZ and H

#O.

We also measured the Fe contents of the roots in thesecond experiment. We distinguished between the Fe‘plaque’, i.e. the insoluble amorphous and crystalline Fe-oxides and oxyhydrates precipitated on the root surface(Crowder and St Cyr, 1991) using reducing extraction withDCB (dithionite-citrate-bicarbonate), and the remaining Feinside the root determined by wet digestion. The DCBsolution was prepared according to St Cyr and Campbell(1996) and Otte et al. (1989). It contained 17±8 ml 0±03 Nacitrate solution, 2±2 ml 0±125 Na bicarbonate solution and1±5 g Na dithionite per 20 ml. After shaking the roots withapprox. 300 ml H

#O in 500 ml flasks overnight, we cleaned

them in running H#O, washed them again with approx.

300 ml H#O, shaken continuously overnight and, before

extraction, removed surplus water between blotting paper.We then added 20 ml of DCB solution and extracted for30 min.

All extracts of samples were cleaned by micropore filtering(Gelman Acrodisc PF 0±8 µm prefilter}0±2 µm supor) andanalysed directly by flame atomic absorption spectrophoto-metry (AAS; Varian SpectraAA-400) against standardsprepared in the appropriate extractant.

Statistical treatment comprised the calculation of mean

Zohlen and Tyler—Iron Strategies in Calcifuges 555

values and 95% confidence limits, Pearson correlationcoefficientswith P-values, Tukey test and P-values accordingto one-way Anova.

RESULTS

The pH differed by more than 3 units between the soils(Table 1). The organic matter content was about 50%higher in the acid soil. Exchangeable Mg and K were not in

T 1. Chemical characteristics of the soils used in theculti�ation experiments

Element}extractant Acid Calc

pH}0±2 BaCl#

3±99 (0±02) 7±53 (0±05)pH, soil solution 4±97 (0±11) 8±02 (0±07)Organic matter (% d.wt) 9±3 (0±3) 6±1 (0±3)Iron}0±2 BaCl

#0±012 (0±002) 0±003 (0±001)

}0±02 Na EDTA 3±08 (0±10) 0±088 (0±006)}0±02 Na citrate 2±85 (0±06) 0±189 (0±027)

Calcium}0±2 BaCl#

8±9 (0±6) 68±9 (8±5)Magnesium}0±2 BaCl

#1±49 (0±05) 0±64 (0±02)

Potassium}0±2 BaCl#

1±12 (0±08) 1±91 (0±43)Aluminium}0±2 BaCl

#6±9 (0±3) ! 0±1

Phosphate}0±05 Na#SO

%­

0±02 NaF 0±010 (0±002) ! 0±001

Acid, Acid silicate soil ; Calc, calcareous soil. Data are means and(in parentheses) 95% confidence limits of the means (n¯ 5). Con-centrations of elements calculated as micromol g−" d.wt.

T 2. Leaf and root biomass produced (g d.wt per �essel),leaf colour, estimated by a 15 degree scale from greenishyellow to dark green, and the pH of leaf tissue extract, at the

end of the experiments

C. pilulifera V. officinalis

Leaf biomassExpt 1 Acid 4±81 (0±93) 1±94 (0±23)

Calc 0±97 (0±15) 0±42 (0±06)Calc­P 0±83 (0±23) 0±94 (0±19)

Expt 2 Acid 5±08 (0±48) 3±05 (0±08)Calc 1±32 (0±10) 1±17 (0±12)Calc­P 1±46 (0±08) 1±28 (0±10)

Root biomassExpt 2 Acid 0±97 (0±18) 0±45 (0±12)

Calc 0±15 (0±03) 0±15 (0±02)Calc­P 0±16 (0±03) 0±20 (0±05)

Leaf colourExpt 1 Acid 9±8 (1±0) 12±6 (0±6)

Calc 5±6 (0±6) 6±0 (1±2)Calc­P 1±6 (0±6) 3±4 (1±0)

Expt 2 Acid 9±2 (0±6) 15±0 (0±0)Calc 4±0 (0±6) 9±0 (0±6)Calc­P 3±5 (0±6) 7±9 (0±9)

Leaf pHExpt 1 Acid 5±18 (0±12) 5±12 (0±08)

Calc 4±93 (0±24) 5±24 (0±08)Calc­P 5±16 (0±35) 5±21 (0±06)

Means and (in parentheses) 95% confidence limits. (n¯ 5 in expt 1;n¯ 8 in expt 2). Acid, Acid silicate soil ; Calc, calcareous soil,Calc­P, calcareous soil supplied with CaHPO

%.

T 3. Expt 1. Total, 1,10-phenanthroline and 1 M HClsoluble Fe in freshly sampled leaf tissue of Carex piluliferaand Veronica officinalis, grown in acid silicate soil (Acid),calcareous soil (Calc), and calcareous soil supplied with

CaHPO%

(Calc­P)

Species Soil Total 1,10-phen. HCl

C. pilulifera Acid 0±781a 0±283a 0±691a

Calc 0±491b 0±163b 0±272b

Calc­P 0±360b 0±115b 0±202b

V. officinalis Acid 1±148a 0±689a 0±818a

Calc 1±588a 0±399b 0±654ab

Calc­P 1±311a 0±480b 0±593b

Mean concentrations (n¯ 5) calculated as micromol g−" d.wt. Sig-nificant (P! 0±05) differences between soils concerning the samespecies and fraction of Fe are indicated by different superscripts.

a critical range for plant uptake in either of the soils, buteasily exchangeable phosphate was below the detection limitin the calcareous soil. Exchangeable Al was only present inthe acid soil, but not at a level likely to be toxic, accordingto previous studies on these plants (Tyler, 1996a). Ex-changeable Ca was more than seven times higher in thecalcareous than in the acidic soil, whereas exchangeable Fewas much higher in the acidic soil. Fe differed greatlyamong the extractants : BaCl

#solubilized less Fe than Na

EDTA and Na citrate. The latter two extractants releasedsimilar amounts of Fe from the acidic soil, but Na citrateextracted more Fe from the calcareous soil than wasobtained with Na EDTA.

The plants grown on the calcareous soils exhibitedchlorosis and this was most severe in the phosphatetreatment, whereas leaf colour of the silicate soil grownplants did not differ visibly from plants in the field (Table 2).V. officinalis showed intercostal chlorosis, whereas leaves ofC. pilulifera were more uniformly chlorotic. Biomassproduction in both species was two- to nearly five-foldhigher on the acidic than on the calcareous soils, but only inV. officinalis did growth seem to be supported by phosphateaddition.

The two species had different Fe uptake rates (Table 3).C. pilulifera contained less Fe per unit leaf dry weight whencultivated on calcareous soils, whereas V. officinalis tendedto increase its Fe uptake compared to plants grown on theacid soil. However, the 1,10-phenanthroline and HClextractable Fe was lower in plants grown on the calcareoussoils in both species. The proportion of extractable Fedepended on the extractant. HCl extracted more Fe than1,10-phenanthroline and reached nearly 90% of the total Fein acid-soil-grown C. pilulifera (Table 4).

The most interesting finding was that the proportion of1,10-phenanthroline extractable compared to total Fe dif-fered considerably between species and treatments (Fig. 1).For C. pilulifera this ratio was similar in all treatments. Incontrast, the proportion of extractable to total Fe in V.officinalis was high on the acid, and low on the calcareoussoil. The P treatment had no significant influence on thetotal Fe status of the plants (Table 3), but seemed to

556 Zohlen and Tyler—Iron Strategies in Calcifuges

T 4. 1,10-phenanthroline, TPTZ and HCl soluble Fe in freshly sampled leaf tissue of Carex pilulfera and Veronicaofficinalis grown in acid silicate soil (Acid), calcareous soil (Calc) and calcareous soil supplied with CaHPO

%(Calc­P). All

�alues calculated as a percentage of total Fe

C. pilulifera V. officinalis

Acid Calc Calc­P Acid Calc Calc­P

1,10-phenanthroline,Expt 1 36±5a 33±5a 33±2a 60±0a 26±4b 40±4b

Expt 2 15±1a 13±2ab 11±4b 30±4a 14±4b 14±7b

TPTZ 13±7a 8±9b 8±6b 12±8a 6±6b 6±2b

Hydrochloric acid 88±7a 57±3b 55±1b 71±3a 41±5b 47±9b

Significant differences (P! 0±05) between soils concerning the same species and fraction of Fe are indicated by different superscripts.

30

0Acid

B

Per

cen

tage

of

tota

l Fe

5

25

20

15

10

Calc. Calc. + P Acid Calc. Calc. + P

aab

b

a

b b

C. pilulifera V. officinalis

60

0Acid

A

Per

cen

tage

of

tota

l Fe

10

50

40

30

20

Calc. Calc. + P Acid Calc. Calc. + P

aa a

a

b

cC. pilulifera

V. officinalis

F. 1. 1,10-phenanthroline extractable Fe, measured as a percentageof total Fe, in freshly sampled leaves of Carex pilulifera and Veronicaofficinalis grown in acid silicate soil (Acid), calcareous soil (Calc) andphosphate-amended calcareous soil (Calc­P). Means which differsignificantly (P! 0±05) among treatments are indicated by different

letters above the columns. A, expt 1; B, expt 2.

increase the extractable Fe fraction in V. officinalis (Fig. 1)compared to the untreated calcareous soil. The pH of theplant tissue, as reflected by the 0±2 KCl extraction, wassimilar in both species and showed no influence of soils ortreatment (Table 2).

The second experiment essentially confirmed the resultsof the first, with only minor differences. Though stillapparent, the chlorosis was not as severe as in the firstexperiment, and the leaf colour on the non-amendedcalcareous soil was nearly the same as on the P amendedsoil. On the other hand, the biomass production was higher

T 5. Expt 2. Total, 1,10-phenanthroline, and TPTZsoluble Fe in freshly sampled leaf tissue of Carex piluliferaand Veronica officinalis, grown in acid silicate soil (Acid),calcareous soil (Calc), and calcareous soil supplied with

CaHPO%

(Calc­P)

Species Soil Total 1,10-phen. TPTZ

C. pilulifera Acid 0±872a 0±130a 0±118a

Calc 0±596b 0±078b 0±051b

Calc­P 0±575b 0±066b 0±050b

V. officinalis Acid 1±143a 0±345a 0±142a

Calc 1±390a 0±203b 0±092b

Calc­P 1±352a 0±185b 0±077b

Mean concentrations (n¯ 8) calculated as micromol g−" d.wt. Sig-nificant (P! 0±05) differences between soils concerning the samespecies and fraction of Fe are indicated by different superscripts.

than in the first experiment, especially in V. officinalis, butthe relative differences among soils and treatments weresimilar (Table 2).

The total Fe values for C. pilulifera were slightly higherthan in expt 1, whereas for V. officinalis they were similar(Table 5). The 1,10-phenanthroline extractable Fe was alsohigher on the acidic than on the calcareous soils. The TPTZextractable Fe fraction, only analysed in expt 2, was alwayslower than 1,10-phenanthroline extractable Fe, but therelation between both extractants was the same for V.officinalis.

The much higher 1,10-phenanthroline extractable fractionof total leaf Fe in acid-soil-grown, compared to calcareous-soil-grown V. officinalis, demonstrated in expt 1, wasconfirmed in expt 2 (Fig. 1). There was no differencebetween untreated and P treated V. officinalis in this respect.Also for C. pilulifera the 1,10-phenanthroline fraction wassomewhat higher in acidic than in calcareous-soil-grownplants, though differences were much smaller than for V.officinalis. Compared with the first experiment, the 1,10-phenanthroline values were generally lower in the secondexperiment, probably due to different proportions of sampleand extractant volume, according to method tests performed(data not shown).

A correlation analysis between extractable and total Feand leaf colour confirmed the differences between the two

Zohlen and Tyler—Iron Strategies in Calcifuges 557

T 6. Linear correlation coefficients (r �alues) for leafcolour on tissue Fe �ariables

C. pilulifera V. officinalis

Expt 1Total Fe 0±880*** ®0±267Fe-phen. 0±882*** 0±707**Fe-1 HCl 0±904*** 0±592*

Expt 2Total Fe 0±891*** ®0±489*Fe-phen. 0±893*** 0±893***Fe-TPTZ 0±913*** 0±822***Total Fe—Fe-phen. 0±854*** ®0±657**Total Fe—Fe-TPTZ 0±836*** ®0±416

***P! 0±001, **P! 0±01, *P! 0±05.

T 7. Expt 2. DCB soluble ‘plaque ’ Fe, remaining rootFe, and ‘plaque ’ Fe as a percentage of total Fe in the roots ofCarex pilulifera and Veronica officinalis, grown in acidsilicate soil (Acid), calcareous soil (Calc) and calcareous

soil supplied with CaHPO%

(Calc­P)

Species Soil ‘Plaque’ Root ‘Plaque’ % total

C pilulifera Acid 10±27a 3±04a 77±3a

Calc 4±50b 3±03a 59±2b

Calc­P 3±03b 4±92a 40±6c

V. officinalis Acid 5±55a 1±02a 84±5a

Calc 1±38b 1±12a 55±1b

Calc­P 1±95b 1±19a 60±0b

Mean concentrations (n¯ 8) calculated as micromol g−" d.wt. Sig-nificant (P! 0±05) differences between soils concerning the samespecies and fraction of Fe are indicated by different superscripts.

species (Table 6). For C. pilulifera, both total and extractableFe were significantly positively correlated with leaf colour.For V. officinalis this was only true for the extractablefractions. Total Fe actually had a negative relation to leafcolour in this species, which was particularly valid for the Fefraction not extracted by 1,10-phenanthroline (Table 6).

The root biomass, only measured in expt 2, varied amongtreatments in a similar way to the leaf biomass and washigher on the acid soil (Table 2). An analysis of the DCBsoluble ‘plaque’ Fe on the roots demonstrated much highervalues in the acid soil in both species. However, the Feremaining in the roots after DCB extraction did not varyamong treatments, but differed considerably between thespecies (Table 7). In contrast to leaf Fe, the ‘plaque’ and theroot Fe were higher in C. pilulifera than in V. officinalis.However, the ratio of ‘plaque’ to total root Fe (‘plaque’Fe­root Fe) did not differ greatly between species exceptfor the phosphorus treatment.

DISCUSSION

The Fe complexing reagent, 1,10-phenanthroline is notperfectly selective for Fe#+, though it has higher affinity forFe#+ than for Fe$+ (Pierson and Clark 1984a, b). Moreover,if the extracted solution is measured spectrophotometrically,

the Fe values can easily be overestimated because of thebackground colour of other extracted compounds (Abadı!aet al., 1984, Katyal and Sharma, 1984; Takkar and Kaur,1984). Therefore the Fe determination was done by atomicabsorption spectrometry, though a discrimination betweenthe two oxidation states is not possible. On the other hand,even in the dark Fe$+ is rapidly reduced to Fe#+ by leafcompounds such as ascorbic acid, phenols or organic acids(Abadı!a et al., 1984, Mehrotra and Gupta 1990, 1991b) and,therefore, it is hardly possible to discriminate between Fe#+

and Fe$+ using the usual extraction methods. 2,4,6-tris-(2-pyridyl)-1,3,5-triazine (TPTZ) is quite selective for Fe#+,especially at low concentrations (Collins and Diehl, 1959;Pierson and Clark, 1984a). HCl is not selective for Fe#+ orFe$+ but Takkar and Kaur (1984) found a better correlationbetween HCl extractable Fe and chlorosis than with 1,10-phenanthroline.

In our experiment the correlation between leaf colour and1,10-phenanthroline or TPTZ extraction did not differgreatly, but both values were higher than for the HClextracts (Table 6). This is not in agreement with Takkar andKaur (1984) who did not find a strong relation to chlorosisusing the 1,10-phenanthroline extraction. The finding that1,10-phenanthroline accounted somewhat better than TPTZfor the variability in chlorosis in fresh plant material wasalso reported by Mehrothra and Gupta (1991a). However,Benton Jones (1991) could not distinguish between chloroticand non-chlorotic plants using 1,10-phenanthroline. Thedifferent extraction methods do not provide a direct measureof physiologically ‘active’ Fe (Chaney, 1984) but they aregenerally better related to chlorosis than total tissue Fe(Oserkowsky, 1933; Abadı!a et al., 1984; Mehrotra andGupta, 1991a). However, the origin and localization of theextracted Fe remains undetected (Abadı!a, 1992). Total Fe inchlorotic plants is often higher than in non-chlorotic plants(Oserkowsky, 1933; Mengel and Buebl, 1983; Takkar andKaur, 1984). This was supported by the results for V.officinalis in our study. There was no positive correlationbetween total Fe and the degree of chlorosis or betweentotal and extractable Fe in this species. In contrast, for C.pilulifera both total and extractable Fe were proportionallylower in calcareous-soil-grown plants and both fractionswere highly interrelated and well correlated with chlorosis.Ao et al. (1987) found decreased total Fe contents in plantssuffering from lime-induced chlorosis. Dicotyledonousspecies are more effective in Fe solubilization than mono-cotyledonous plants (Olsen and Brown, 1980a). This couldexplain the difference between C. pilulifera and V. officinalisforced to grow on calcareous soil.

Plants mainly take up Fe in the form of Fe#+ (Chaney,Brown and Tiffin, 1972; Olsen and Brown, 1980b), but inwell-drained soils most Fe is present as Fe$+ (Olsen andBrown, 1980b ; Jolley and Brown, 1994) and has to bereduced by the root before uptake (Chaney et al., 1972).During translocation in the plant, Fe#+ is reoxidized to Fe$+

and transported mainly as citrate or malate (Tiffin, 1970),and most of the Fe in plants is present as Fe$+ (Machold,Meisel and Schnorr, 1968; Jolley and Brown, 1994).

Dicotyledons and non-gramineous monocotyledons areregarded as ‘strategy I’ plants (Marschner, Roemheld and

558 Zohlen and Tyler—Iron Strategies in Calcifuges

Acid

Leaves

Roots

Root'plaque'

58%

17%

25%

Calc.

35%

24%

41%

Acid

39%

7%

54%

Calc.

10%9%

81%

Carex pilulifera Veronica officinalis

F. 2. Percentage distribution of Fe between leaves, roots and root ‘plaque’ (8) in Carex pilulifera and Veronica officinalis, grown in acid silicatesoil (Acid) and unamended calcareous soil (Calc). The area of each compartment is proportional to the percentage.

Kissel, 1986; Marschner, 1995). Under Fe deficiency theyexhibit several mechanisms to maintain an adequate level ofFe. Many plants release H+ ions, extruded by an enhancedactivity of a plasma membrane-bound reductase. Reduc-tants}chelators are often released as well, but this dependson the plant species (Jolley, Brown and Nugent, 1991). Thereduction of Fe$+ to Fe#+ at the root plasmalemma isenhanced, and citrate is accumulated in root tissues,accelerating the transport of solubilized Fe to the upperparts of the plant (De Vos, Lubberding and Bienfait, 1986;Jolley and Brown, 1994). That citrate is capable ofsolubilizing Fe in soils was shown in our experiment by theincreased Fe extraction, especially from the calcareous soil(Table 1).

A high HCO$

− content of the soil or plant tissue isregarded as the main cause of lime-induced chlorosis,whereas P is considered to be of little influence (Boxma,1972; Pissaloux, Morard and Bertoni, 1995; Mengel,Breininger and Buebl, 1984, Romera, Alca! ntara and de laGuardia, 1991). Due to HCO

$

−, a high pH inhibits rootsresponses, including neutralization of the H+ and reductionof Fe$+ at the plasmamembrane (Marschner and Ro$ mheld,1994). Our results might indicate some influence ofphosphorus on Fe dynamics (Fig. 1), but the differencesbetween non-amended and phosphate-amended calcareoussoil were not distinct enough for consistent conclusions tobe drawn. Leaf P concentrations were not determined in ourstudy, but previous work has demonstrated considerableincreases in P uptake by these species when grown inphosphate-amended limestone soil (Tyler, 1996b). A highCa content does probably not induce chlorosis, but canimpair plant growth (Pissaloux et al., 1995). Also severe Fe-deficiency may inhibit cell growth and thus leaf growth(Abbott, 1967). This was evident for both experimentalplants, especially for the root biomass, though phosphateaddition apparently promoted growth of calcareous-soil-grown V. officinalis. In contrast to Mengel (1994), we foundno correlation between leaf colour and tissue pH (Table 2)but this could be due to the rather coarse method for pHdetermination.

Shoot and root biomass exhibited a significant shift

towards shoot biomass on calcareous soils in C. pilulifera,whereas V. officinalis had a similar shoot:root ratio in alltreatments. Mengel (1994) reported increased levels of Fe inroots under Fe deficiency, but in our experiments the ratioof total shoot Fe and root Fe was similar for each species inall treatments. Comparing the Fe distribution among leaves,roots and root ‘plaque’, a relatively greater proportion ofthe Fe was located in the leaves of the calcareous-soil-grownplants, compared to the acid-soil-grown plants (Fig. 2). C.pilulifera accumulated more ‘plaque’ than V. officinalis. Thestrategy of V. officinalis was to translocate most of the totalbiomass Fe to the leaves, whereas most of the Fe in C.pilulifera was recovered as ‘plaque’ from the root surface.

These differences in Fe distribution strategy between thetwo species were particularly apparent in the calcareous-soil-grown plants. In C. pilulifera from the calcareous soilalmost 60% of the total plant Fe was located in or on theroot biomass, whereas this value was less than 20% for V.officinalis. This, together with the low total uptake of Fefrom calcareous soil in the leaves of C. pilulifera, and thehigh leaf uptake but immobilization of Fe in forms notextractable by 1,10-phenanthroline in V. officinalis, consti-tutes a major difference between these species in Fe nutritionstrategy and calcifuge behaviour.

ACKNOWLEGEMENT

We thank Maj-Britt Larsson for her skilful technicalassistance.

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