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Biochemical Responses toTrue and Bicarbonate-InducedIron Deficiency in GrapevineGenotypesRiadh Ksouri a , Sabah M'rah b , Mohamed Gharsalli a

& Mokhtar Lachaâl ba Laboratoire d'Adaptation des Plantes aux StressAbiotiques , Hammam-Lif, Tunisiab Département de Biologie, Faculté des Sciences deTunis , Campus Universitaire , Tunis, TunisiaPublished online: 14 Feb 2007.

To cite this article: Riadh Ksouri , Sabah M'rah , Mohamed Gharsalli & MokhtarLachaâl (2006) Biochemical Responses to True and Bicarbonate-Induced IronDeficiency in Grapevine Genotypes, Journal of Plant Nutrition, 29:2, 305-315, DOI:10.1080/01904160500476897

To link to this article: http://dx.doi.org/10.1080/01904160500476897

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Journal of Plant Nutrition, 29: 305–315, 2006

Copyright © Taylor & Francis Group, LLC

ISSN: 0190-4167 print / 1532-4087 online

DOI: 10.1080/01904160500476897

Biochemical Responses to Trueand Bicarbonate-Induced Iron Deficiency

in Grapevine Genotypes

Riadh Ksouri,1 Sabah M’rah,2 Mohamed Gharsalli,1

and Mokhtar Lachaal2

1Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, Hammam-Lif, Tunisia2Departement de Biologie, Faculte des Sciences de Tunis, Campus Universitaire,

Tunis, Tunisia

ABSTRACT

Biochemical responses to direct or bicarbonate-induced iron (Fe) deficiency were com-pared in two Tunisian native grapevine varieties, Khamri (tolerant) and Balta4 (sensi-tive), and a tolerant rootstock, 140Ru. Woody cuttings of each genotype were irrigatedwith a nutrient solution containing one of the following: 20 µM Fe (control), 1 µM Fe(direct Fe-deficiency), or 20 µM Fe + 10 mM HCO−

3 (indirect bicarbonate-induced Fe-deficiency). Under direct Fe-deficient conditions, lower leaf chlorosis score and higherchlorophyll and leaf Fe contents were found in Khamri and 140Ru compared with Balta4.Moreover, indirect Fe deficiency caused similar effects on these parameters, which weremore pronounced in Balta4. Both tolerant genotypes, Khamri and 140Ru, showed higherroots-acidification capacity and phenol release under the direct Fe deficiency comparedwith the bicarbonate-induced condition. In the sensitive variety, Balta4, no significantchanges were found between the control and Fe-deficient plants. Root Fe(III)-reductaseactivity was strongly stimulated by both types of Fe deficiency in Khamri and 140Ru,and displayed no significant changes in Balta4. In the three genotypes, root and leafactivities of two Fe-containing enzymes, catalase and guaiacol peroxidase, were signif-icantly affected under Fe deficiency (either direct or induced), though to a greater extentin the sensitive variety, Balta4. The latter also displayed higher leaf malonyldialdehyde(MDA) content, traducing an important membrane lipid peroxidation.

Keywords: acidification, Fe-containing enzymes, Fe(III)-reductase, iron deficiency,malonyldialdehyde, Tunisian grapevine

Received 28 May 2004; accepted 15 April 2005.Address correspondence to Mokhtar Lachaal, Departement de Biologie, Fac-

ulte des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia. E-mail:Mokhtar.Lachaal@fst.rnu.tn

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INTRODUCTION

Developing the viticulture requires the conservation of autochthonous varietiesof grape, which have evolved several mechanisms that enable them to adapt tolocal bioclimatic and edaphic conditions. Several native grapevine genotypesappreciated for their organoleptic characteristics are currently widely cultivatedin Tunisia (Ben Abdallah et al., 1998). However, the calcareous soils that pre-dominate in the country lead to low iron (Fe) availability, thus exposing theplants to severe deficiencies of this nutrient (Ksouri et al., 2001). The produc-tivity of this local grapevine could potentially be improved by selecting themost tolerant genotypes.

A previous study investigating the variability responses to Fe deficiency inseven local grapevine genotypes using morpho-physiological criteria showedthat the variety Khamri and rootstock 140Ru were tolerant to this nutrient stress,while Balta4 appeared to be sensitive to it (Ksouri et al., 2004 and 2005). Thegrapevine resistance to Fe chlorosis seems to be strongly correlated with (1) theplant’s ability to acidify the external medium and (2) the improvement of Fe(III)-reductase activity (Dell’Orto et al., 2000). It has been suggested that these twoparameters are reliable and could be used for the screening of plants tolerantto Fe deficiency (Ellsworth et al., 1997; Wie et al., 1997). On the other hand,other biochemical criteria such as APX and catalase activity were found to bestrongly correlated with the content of the Fe “active” fraction in, for instance,chickpea (Cicer grietinum) suggesting the role of these enzymes in the responseof plants is dealing with this abiotic constraint (Iturbe-Ormaetxe et al., 1995).

The present study investigated the biochemical responses of local Tunisiangrapevine varieties to Fe deficiency (direct or HCO−

3 -induced) by comparingtheir medium acidification and iron mobilization abilities and assessing theactivities of two Fe-containing enzymes (catalase and guaiacol peroxidase) indifferent organs. Varieties with contrasting behavior were used: Khamri (toler-ant) and Balta4 (sensitive) along with a tolerant rootstock (140Ru).

MATERIALS AND METHODS

Culture Conditions

One-month-old woody cuttings representing the rootstock 140Ru and the localgrapevine varieties (Khamri and Balta4), previously characterized by biochem-ical markers (Ben Abdallah et al., 1998), were cultivated on a liquid medium.The experiment was performed in a glass greenhouse under controlled condi-tions of temperature (22◦C–30◦C) and humidity (75%–85%). Rooted woodycuttings representing each variety were divided into three lots and irrigated for75d with a nutrient solution (Brancadoro et al., 1995) containing either 20 µMFe (control), or 1 µM Fe (deficient). Lowering Fe availability was done directly

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Fe-Deficiency Responses of Grapevine 307

(DD) (1 µM Fe) or indirectly (ID), by adding bicarbonate (10 mM HCO−3 ) to

the control medium (with 20 µM Fe). Nutrient solution (pH 6.1) was replacedevery week.

Leaf Chlorosis Parameters

At harvest, the young leaf chlorotic status was determined using two methods.The first was non-destructive, and used a score based on the visual appreciationof chlorosis symptoms (Pouget and Ottenwaelter, 1978), with values rangingfrom 0 (absence of chlorosis) to 5 (severe chlorosis). The second method con-sisted of measuring the chlorophyll and bivalent Fe contents of the expandingleaf no. 4 (the fourth leaf from the shoot tip). Chlorophyll extraction and assaywere achieved according to Torrecillas et al. (1984). Iron fraction was extractedon dry matter with HCl (1N) (Llorente et al., 1976) and assayed by atomicabsorption spectrophotometry (Perkin Elmer Model 4000).

Acidification and Iron Mobilization Capacity

Medium acidification by roots was assessed by monitoring the evolution of thenutrient solution pH during the week preceding the final harvest using a digi-tal pH meter (Metrohm 663). Initial pH was 6.1 for both control and DD and7.9 for ID medium. Iron(III)-reductase activity was measured on segments ex-cised from the root apical region using bathophenanthroline disulfonate (BPDS)(Brancadoro et al., 1995). The FeII (BPDS) complex absorbance was measuredat 535 nm, while its concentration was determined using the extinction coeffi-cient 22.1 mM−1cm−1 (Chaney et al., 1972). Phenol index allows an estimationof plant ability to release phenol compounds in the medium (Vivas et al., 2003).This parameter was spectrophotometrically (UV/visible PBU 2000) assessed at280 nm in the culture medium one week after the nutrient solution was replaced.

Enzyme Extractions and Assays

Root and young leaf samples (displaying chlorosis symptoms or not) were usedfor enzyme extraction. Two g of fresh matter were homogenized with a mortarcontaining sterile sand in 2 ml of ice-cold 50 mM tricine-KOH buffer withpH 8.0 (Ranieri et al., 2001) and polyvinylpyrrolidone (PVP) 10% (w/w). Thehomogenate was then centrifuged at 14000 g for 15 min. All the extractionoperations were carried out at 4◦C. The supernatant was used for the enzymeactivity assay. Guaiacol peroxidase (EC. 1.11.1.7) and catalase (EC 1.11.1.6)were assayed at 470 nm (Fielding and Hall, 1978) and 240 nm (Chance andMaehly, 1955), respectively. Lipid peroxidation was estimated by determiningthe malonyldialdehyde (MDA) contents (Hernandez and Almansa, 2002) in the

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organs mentioned above. Fresh samples (200 mg) were homogenized in 2 mL0.1% trichloroacetic acid (TCA). The homogenate was centrifuged at 15000g for 10 min at 4◦C. Then 0.5 ml of the supernatant was mixed with 1.5 mlof 0.5% thiobarbituric acid (TBA) prepared in 20% TCA, and incubated at90◦C for 20 min. After the reaction was stopped by placing materials in anice bath, samples were centrifuged at 10000 g for 5 min. Lipid peroxidationwas colorimetrically determined by reading the absorbance of the supernatantat 532 nm. After subtracting the nonspecific absorbance at 600 nm, MDAconcentration was determined using the extinction coefficient 155 mM−1cm−1.

Statistical Analysis

A two-way ANOVA statistical analysis at the significance level P < 0.05 wasperformed to determine the significance of each factor (genotype-treatment) andtheir interaction in affecting the different parameters. Newman-Keuls post-hoctest was used when significant differences were found among salt treatments.

RESULTS

Leaf Chlorosis and Iron Status

Iron-chlorosis symptoms appeared earlier and were more pronounced underinduced Fe deficiency (ID). Also, grapevine genotypes showed a contrasted be-havior using the morphological observations. Under both Fe-deficiency modes,significantly higher chlorosis scores were found in Balta4 than in Khamri androotstock 140Ru, which showed similar values (Table 1). Leaf chlorophyll

Table 1Variability of chlorosis score and total chlorophyll contents in young leaves of Tunisiangrapevine genotypes grown for 75 d in nutrient solutions differing in iron availability

Total chlorophyllChlorosis score (mg g−1FW)

Variety C DD ID C DD ID

140Ru 0.00e 1.29d 1.86c 1.21c 1.10d 0.98eKhamri 0.00e 1.43d 2.14c 1.45a 1.31b 1.17cdBalta4 0.00e 3.29b 3.71a 1.16cd 0.66f 0.60f

C: control (20 µM Fe); DD: direct deficiency (1 µM Fe); ID: indirect deficiency(20 µM Fe + 10 mM HCO−

3 ). Means of seven replicates ± SE. Values followed by oneor more of the same letters were not significantly different at P < 0.05, according to theNewan-Keuls post-hoc test.

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Table 2Variability of bivalent iron contents in young leaves of Tunisian grapevinegenotypes grown for 75 d in nutrient solutions differing in iron availability

Leaf No. 4 iron content (µg g−1DW)

Variety C DD ID

140Ru 55.7bc 48.0de 44.9eKhamri 63.4a 54.5c 51.2cdBalta4 59.4b 32.3f 28.1g

C: control (20 µM Fe); DD: direct deficiency (1 µM Fe); ID: indirect defi-ciency (20 µM Fe + 10 mM HCO−

3 ). Means of seven replicates ± SE. Valuesfollowed by one or more of the same letters were not significantly different atP < 0.05, according to the Newan-Keuls post-hoc test.

contents were significantly affected by lower Fe availability, though to a greaterextent in Balta4 (about 43%–48% lower than control values under DD and ID,respectively), while ranging from -10% (DD) to -19% (ID) in Khamri and140Ru, respectively. In the three genotypes, the depressive effect of Fe defi-ciency on this parameter was significantly higher under ID.

A significant decline of leaf HCl-extractible Fe contents was observed inplants exposed to Fe deficiency. However, it varied depending on the genotypeand the induction mode of Fe deficit (Table 2). Indeed, Balta4 was the most

Figure 1. Variability of root acidification capacity in Tunisian grapevine genotypesgrown for 75 d in nutrient solutions differing in iron availability. C: control (20 µMFe); DD: direct deficiency (1 µM Fe); ID: indirect deficiency (20 µM Fe + 10 mMHCO−

3 ). This parameter was estimated by measuring the nutrient solution pH. Means ofseven replicates ± SE. Means with one or more of the same letters were not significantlydifferent at P < 0.05, according to the Newan-Keuls post-hoc test.

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markedly affected variety, with reductions reaching approximately 53% (ID),compared with −20% (ID) of bivalent Fe contents in the control for Khamri and140Ru (both tolerant). As was found for the chlorosis parameters, the negativeimpact of indirect Fe deficiency (ID) was more pronounced than DD.

Root Acidification and Iron Mobilization Capacity

Increasing pH values were found in the control plants of the three genotypes(Figure 1). Under direct Fe deficiency (DD), an acidification of the mediumwas registered, and was stronger in the most tolerant genotypes (−1.4 pH unitsin Khamri compared with only −0.4 in Balta4). Under indirect Fe deficiency(ID), root acidification capacity was lower (−0.6 pH units in Khamri).

Iron(III)-reductase activity in vivo measured on excised roots showed lowvalues in control plants of the three genotypes (70 nmol.h−1 g−1 FW to 90 nmol

Figure 2. Variability of iron uptake efficiency in Tunisian grapevine genotypes grownfor 75 d in nutrient solutions differing in iron availability. Root Fe(III)-reductase activity(A) and phenol index (B). C: control (20 µM Fe); DD: direct deficiency (1 µM Fe);ID: indirect deficiency (20 µM Fe + 10 mM HCO−

3 ). Means of seven replicates ± SE.Means with one or more of the same letters were not significantly different at P < 0.05,according to the Newan-Keuls post-hoc test.

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Fe-Deficiency Responses of Grapevine 311

h−1 g−1 FW) (Figure 2A). This activity was strongly stimulated in the toler-ant grapevine, whether subjected to direct (DD) or indirect (ID) Fe deficiency(up to about four-fold higher than control values in Khamri and 140Ru, re-spectively), while no significant changes were recorded in the sensitive varietyBalta4 (Figure 2A).

On the other hand, DD led to a significant improvement of the phenol index(Figure 2B) for the tolerant genotypes (six-fold to nine-fold higher than controlvalues), contrasting with constant values in Balta4. Unexpectedly, indirect Fedeficiency (ID) had no effect on this parameter.

Enzyme Activity and Lipid Peroxidation

Whether measured in leaves or roots, guaiacol peroxidase activities significantlydecreased under direct (DD) or indirect (ID) Fe deficiency, though a genotypevariability was observed (Figure 3). Under DD, Balta4 displayed the strongestdrop (about 10% of control values), whereas the reduction rate ranged fromabout 35% to about 55% in both of the tolerant genotypes (Khamri and 140Ru).The same trend was found for plants submitted to indirect Fe deficiency (ID),

Figure 3. Variability of guaiacol peroxidase (A) and catalase (B) activities in youngleaves and roots of Tunisian grapevine genotypes grown for 75 d in nutrient solutionsdiffering in iron availability. C: control (20 µM Fe); DD: direct deficiency (1 µM Fe);ID: indirect deficiency (20 µM Fe + 10 mM HCO−

3 ). Means of seven replicates ± SE.Means with one or more of the same letters were not significantly different at P < 0.05,according to the Newan-Keuls post-hoc test.

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Figure 4. Variability of MDA contents in young leaves and roots of Tunisian grapevinegenotypes grown for 75 d in nutrient solutions differing in iron availability. C: control(20 µM Fe); DD: direct deficiency (1 µM Fe); ID: indirect deficiency (20 µM Fe +10 mM HCO−

3 ). Means of seven replicates ± SE. Means with one or more of the sameletters were not significantly different at P< 0.05, according to the Newan-Keuls post-hoctest.

with a severe drop seen in Balta4. Conversely, 140Ru especially maintainedhigher values (−34% and −16% for leaf and root guaiacol peroxidase activities,respectively).

Catalase activity also seemed to be more affected by DD or ID treatments inBalta4 than in the other genotypes (Figure 3). This was particularly true for leafactivity (35% and 70% of control values in Balta4 and Khamri, respectively).Root activity seemed to be more sensitive regardless of the genotype and theFe-deficiency type, as it declined sharply (up to −93% compared with thecontrol). However, it is notable that the activities of the tolerant genotypes weresignificantly higher than in the sensitive one.

Malonyldialdehyde (MDA) is a good indicator of the possible inductionof oxidative damage caused by Fe deficiency. Low MDA contents were foundin control leaves and roots, while they varied in the Fe deficient grapevines,depending on genotype (Figure 4). For instance, leaf MDA contents in thesensitive variety, Balta4, were eight-fold higher under DD or ID treatments thanin the control, whereas MDA was maintained at similar levels in the tolerantKhamri and 140Ru genotypes. Root MDA contents were slightly increased bylowering Fe availability in the culture medium (Figure 4).

DISCUSSION AND CONCLUSIONS

Using biochemical criteria, the present study confirmed previous investigationsshowing genotype-dependent variable responses to Fe deficiency in Tunisiangrapevines (Ksouri et al., 2004, 2005). Tolerance of Khamri and 140Ru wasassociated with higher leaf contents of chlorophyll and bivalent Fe. Also, thesegenotypes showed (1) an important root capacity for lowering the culture

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Fe-Deficiency Responses of Grapevine 313

medium pH, as well as (2) stimulated activity of Fe(III)-reductase. Compar-ing both Fe deficiency induction types revealed that direct deficiency (DD)was accompanied by a stronger root acidification, while no significant dif-ference was found concerning Fe(III)-reductase activity. This result is prob-ably related to the buffer role of HCO−

3 , which maintains a high pH in themedium, and neutralizes H+ ions excreted by the root proton pumps (Romeraet al., 1992). These protons are known to be necessary for the mobilizationof soluble ferric compounds present in the soil (Ohwaki and Sugahar, 1997).Thus, these data suggest that the parameters mentioned above clearly discrim-inate between the grapevine genotypes in terms of dealing with Fe deficiency,notably when it is induced directly. Root acidification has already been pro-posed as a useful criterion for the evaluation of resistant varieties in cultivatedspecies (Ellsworth et al., 1997). Moreover, Dell’Orto et al. (2000) investigatedthe variability of the response of 11 grapevine varieties and showed a closerelationship between these two root activities and their resistance to ferricchlorosis.

On the other hand, root-acidification capacity and phenolic-acid leach-ing (which was significantly stimulated in the tolerant genotypes, Khamri and140Ru) displayed similar trends under lower Fe availability. This result agreeswith a previous study on Parietaria diffusa exposed to the same nutrient con-straint (Dell’Orto et al., 2003).

Measuring the activities of two Fe-containing enzymes (guaiacol peroxi-dase and catalase) that contribute to the plant defense system against oxidativestress showed that they were affected differently by the Fe deficiency, depend-ing on genotype. Though affected, the most tolerant ones (Khamri and 140Ru)maintained higher guaiacol peroxidase or catalase activities, whether submittedto direct or indirect Fe deficiency, suggesting that these antioxidant enzymesare crucial.

Using MDA to assess the degree of lipid peroxidation confirmed the en-zyme activity results, with MDA contents found to be significantly higher inthe young leaves of the sensitive variety (Balta4). MDA was shown to be a re-liable indicator of the oxidative stress resulting from several abiotic constraints(Zhang and Kirkham, 1994; Hernandez et al., 2001). Such a variability in en-zyme response was also reported by Dasgan et al. (2003) in tomato (Lycopersi-con esculentum) exposed to Fe deficiency, with the tolerant variety, ‘Pakmor,’characterized by stronger guaiacol peroxidase and catalase activities than thesensitive one (‘Target’). According to the same authors, the better leaf status ofFe used in the plant metabolism explained the better behavior of ‘Pakmor.’

In conclusion, this study showed that the biochemical responses to Fedeficiency (direct or induced) of the autochthonous Tunisian grapevines aregenotype-related, though the impact of the indirect form was more pronounced.The most tolerant genotypes were able to acidify efficiently the external mediumand conserved higher antioxidant enzyme activities under these unfavorableconditions.

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ACKNOWLEDGMENTS

We are deeply grateful to Professor Chedly Abdelly (Laboratoire d’Adaptationdes Plantes aux Stress Abiotiques, INRST, Hammam-Lif) for kindly offeringlaboratory facilities and material support.

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