Plant Physiology and Biochemistry 45 (2007) 315e322www.elsevier.com/locate/plaphy

Research article

Genotypic variability within Tunisian grapevine varieties(Vitis vinifera L.) facing bicarbonate-induced iron deficiency

Riadh Ksouri a, Ahmed Debez a,1, Henda Mahmoudi a, Zeineb Ouerghi b,Mohamed Gharsalli a, Mokhtar Lachaal b,*

a Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, Centre de Biotechnologies de laTechnopole de Borj-Cedria (CBBC), BP 901, 2050 Hammam-Lif, Tunisia

b Unite de Physiologie et de Biochimie de la Tolerance au Sel chez les Plantes, Faculte

des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia

Available online 15 March 2007

Abstract

Morpho-physiological responses to bicarbonate-induced Fe deficiency were investigated in five Vitis vinifera L. Tunisian varieties (Khamri,Blanc3, Arich Dresse, Beldi, and Balta4). One-month-old woody cuttings were cultivated for 85 days on a free calcareous soil irrigated with tapwater containing increasing bicarbonate levels (0, 4, 8, 12, and 16 mM NaHCO3). After this screening, a second experiment compared root bio-chemical responses of two contrasting genotypes (tolerant-sensitive) dealing with bicarbonate-induced iron deprivation (20 mM Fe � 10 mMHCO3

�) for 75 days. Using morpho-physiological criteria, grapevine tolerance to HCO3�-induced Fe shortage appeared to be genotype-depen-

dent: Balta4 and Beldi varieties showed the highest leaf-chlorosis score (especially at the extreme HCO3� levels), in contrast to Khamri variety.

Growth parameters (shoot height, total leaf area, leaf number, and biomass production) as well as juvenile leaf chlorophyll content were alsodifferently affected depending on both genotype and bicarbonate dose. At 16 mM HCO3

�, Khamri was the less sensitive variety, contrasting withBalta4. On the other hand, chlorophyll content correlated positively with HCl-extractible Fe content of the juvenile leaves, suggesting that thegrapevine response to iron deficiency may partly depend on to the plant ability to adequately supply young leaves with this element. Root bio-chemical responses revealed a relatively higher root acidification capacity in Khamri (tolerant) under Fe-deficiency while no significant changesoccurred in Balta4 (sensitive). In addition, Fe(III)-reductase and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities were stronglystimulated by Fe-deficiency in Khamri, while remaining constant in Balta4. These findings suggest that biochemical parameters may constitutereliable criteria for the selection of tolerant grapevine genotypes to iron chlorosis.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Acidification; Fe(III)-reductase; Iron chlorosis; Native grapevine; Phosphoenolpyruvate carboxylase; Variability

1. Introduction

Several native grapevine (Vitis vinifera L.) genotypes,highly appreciated for their organoleptic characteristics andcommercial potential, are widely cultivated in Tunisia, fromthe Kroumirie-Mogods mountains (North-West, humid cli-mate) to the desert region of Rjim-maatoug (South-West,

* Corresponding author. Tel./fax: þ216 71 872 600.

E-mail address: mokhtar.lachaal@fst.rnu.tn (M. Lachaal).1 Present address: Institut fur Botanik, Universitat Hannover, Herrenhauser

Str. 2, 30419, Hannover, Germany.

0981-9428/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserve

doi:10.1016/j.plaphy.2007.03.014

arid climate) [41]. Developing viticulture requires the conser-vation of autochthonous varieties that have evolved severalmechanisms enabling them to cope with the local bioclimaticand edaphic conditions [6]. However, the calcareous soils pre-dominating in Tunisia (North and Centre) and the irrigationwater containing high levels of bicarbonate (South), decreaseconsiderably iron availability and expose the plants to severerestrictions in iron acquisition [7,23]. Iron is a micronutrientof high importance for the plant, since mediating vital growthand development processes [14]. For instance, chlorophyllsynthesis and electron transport in mitochondria and the pho-tosynthetic chain are closely related to the plant iron status[16,17].

d.

316 R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322

Excessive concentration of bicarbonate in the soils is themain factor inducing Fe chlorosis for the plants [24], mainlythrough the HCO3

� ‘‘buffer’’ effect. The latter is known to neu-tralize the root Hþ-ATPase activity [33] and to decrease theFe(III)-reductase and iron reduction, hence restricting iron ab-sorption by roots and its transport towards shoots [20]. Yet,several data showed the presence of a genotypic-dependentvariability of the plant response to low soil iron availability,especially among grapevine genotypes [27,40]. Moreover,the grapevine resistance to iron chlorosis seems to be stronglycorrelated with the plant ability to acidify the external mediumand/or to improve Fe(III)-reductase activity. Therefore, it hasbeen suggested that these two parameters are reliable andcould be used for the screening of plants tolerant to iron defi-ciency [15,39]. Other biochemical criteria such as phospho-enolpyruvate carboxylase (PEPC) activity were stronglycorrelated with the genotype tolerance to iron deficiency ingrapevine [27] and in cucumber [32]. Exploring such a poly-morphism for selecting the most tolerant varieties may ulti-mately result into the improvement of the productivity in themost affected regions [11]. The valuable potential of localgrapevine varieties may be useful in this promising perspec-tive. The purpose of the present study was thus (i) to investi-gate the variability of morpho-physiological responses of fivelocal grapevine varieties to iron chlorosis, induced by increas-ing bicarbonate levels in the irrigation water, and (ii) tocompare the root ability to acidify root medium acidification,Fe(III)-reductase and PEPC activities of two contrastingvarieties (tolerant-sensitive).

2. Material and methods

2.1. Culture conditions

Woody cuttings of five autochthonous varieties (Khamri,Blanc3, Arich Dresse, Beldi and Balta4), previously character-ised by biochemical or molecular markers [41], and obtainedfrom 20-cm-long shoot cuts with two buds and IBA (indolebu-tyric acid)-treatment, were transferred to a heated greenhouse(25 �C). One month later, well rooted woody cuttings (five rep-licates) were cultivated in 2-L pots filled with a humid silt-sandysoil sampled from our Institute and weekly irrigated for 85 dayswith tap water containing increasing bicarbonate concentrations(0, 4, 8, 12, and 16 mM NaHCO3). Cultures were performed ina greenhouse (22e30 �C temperature, 75e85% relative humidity)

under natural light (PAR: 250 mmol m�2 s�1). Soil and tap waterchemical characteristics are presented in Table 1.

2.2. Second experiment for root-biochemical parametersdetermination

One-month-old woody cuttings of Khamri and Balta4 varie-ties were cultivated on a liquid medium (one plant per pot).The experiment was carried out in a greenhouse under controlledconditions of temperature (22e30 �C) and humidity (75e85%).Rooted woody cuttings of each variety were divided in two lotsand transferred to hydroponic medium, for 75 days, containinga nutrient solution [9]: KH2PO4 (0.5 mM), K2SO4 (0.75 mM),Ca(NO3)2,4H2O (2 mM), MgSO4 $ 7H2O (0.65 mM) H3B3

(0.5 mM), MnSO4 (0.5 mM), CuSO4 (0.045 mM), ZnSO4 (0.05mM), (NH4)6Mo7O24 (0.02 mM), containing either 20 mMFe(III)-Na-EDTA (control) or Fe-deficient. Iron shortage was in-directly (ID) achieved by adding bicarbonate (10 mM NaHCO3)to the control medium (with 20 mM Fe). The nutrient solutionwas replaced weekly.

2.3. Morpho-physiological parameters andHCl-extractible iron assay

Plant aspect was weekly monitored during the growth pe-riod. Chlorosis symptoms on the young leaves were evaluatedaccording to Pouget and Ottenwaelter’s [31] scale, rankingfrom 0 (no symptoms) to 5 (severe chlorosis with necrosis).At the end of the treatment, shoot length, leaf number andarea (using a planimeter, type LI-3050 A/4) were determined.

Harvested plants were separated in juvenile leaves (the 4thleaf from the shoot tip) [2], old leaves, stems plus petioles, androots. Fresh and dry weight (respectively FW and DW) ofthese organs, leaf chlorophyll concentration (mg g�1 FW)and HCl-extractible iron concentration (mg g�1 DW) of the ju-venile leaves were then determined. Extraction and assay oftotal chlorophyll were performed as described by Torrecillaset al. [38]. Iron was extracted by HCl (1 N), according tothe method of Oserkowsky [28] which was modified by Llor-ente et al. [18]: leaf material was dried at 70 �C for 72 hand grinded (using an agate mortar type ‘‘pulverisette6-FRISCH’’). After 10 ml HCl (1 N) were then added to400 mg of grounded leaf, the mixture was shaken for30 min, filtered through a Whatman filter paper and the filtratevolume brought to 25 ml with deionised water. Extractible Fe

Table 1

Chemical characteristics of the soil (A) and irrigation water (B) used for plant culture

A. INRST soil

N P Kþ Ca2þ Naþ Cl� AC EEI pH EC

0.45 0.24 4.1 � 10�3 13.0 � 10�3 1.7 � 10�3 0.5 � 10�3 2.0 37 6.6 0.05

B. Irrigation water

Kþ Ca2þ Naþ Cl� Mg2þ HCO3� SO4

2� DR pH EC

0.15 3.5 4.48 7.76 1.65 2.36 6.3 1.2 7.9 1.5

Soil ion contents, nitrogen and inorganic phosphorus are expressed as g per kg of dried soil. EC, soil electric conductivity, mmohs cm�1; AC, active calcareous,%;

EEI, easily extractible iron, ppm. Water ion contents are expressed as mM. DR, dry residue, g L�1; EC, water electric conductivity, mmohs cm�1.

317R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322

Fig. 1. Leaf chlorosis score of native grapevine genotypes irrigated for 85 days with tap water containing NaHCO3 (B4, 4 mM; B8, 8 mM; B12, 12 mM and B16,

16 mM). The B0 treatment (0 mM NaHCO3) was removed since B0-plants score was equal to 0 (no leaf chlorosis symptoms). Means (n ¼ 5, �SE) labelled by at

least one same letter are not statistically different at P < 0.05.

was assayed on the filtrate using an atomic absorption spectro-photometer (Model Perkin Elmer 4000).

2.4. Acidification and Fe(III)-reductase activity

Medium acidification by roots was assessed by monitoringthe evolution of the nutrient solution pH during the week pre-ceding the final harvest, using a digital pH meter (Metrohm663) (initial pH was 6.1 for the control and 7.9 for ID medium).Fe(III)-reductase activity was assayed after 1 hr on segmentsexcised from the root apical region, using Bathophenanthrolinedisulphonate, BPDS (0.3 mM) and Fe(III)-Na-EDTA (0.1 mM)[9]. The FeII(BPDS)3 complex absorbance was measured at535 nm, while its concentration was determined using theextinction coefficient 22.1 mM�1 cm�1 [10].

2.5. PEP carboxylase extraction and assay

The activity of phosphoenolpyruvate carboxylase (PEPC;EC 4.1.1.31) was assayed according to Ouerghi et al. [29].Fresh excised root samples (0.2 g) were ground in liquid nitro-gen, in 100 mM Trisebicine (pH 8.0) containing 1 mM ethyl-enediamine tetra-acetic acid (EDTA), 1% b-mercaptoethanol(v/v), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5%polyvinylpyrrolidone (PVP) (w/w of sample FW), and centri-fuged at 12,000 � g for 10 min at 4 �C. PEPC reaction mixturecontained 100 mM Trisebicine (pH 8.0), 5 mM MgCl2, 1 mMDTT, 5 mM NaHCO3, 0.2 mM NADH, 4 mM PEP, 5 enzymeunits of malate dehydrogenase (MDH) and 100 ml of the crudeextract. Enzyme activity (l ¼ 340 nm, at 30 �C) was expressedas mmol h�1 g FW (three replicates per treatment).

2.6. Statistical analysis

A two-way analysis of variance (ANOVA), with the geno-type (G) and bicarbonate treatment (T) as factors, was

achieved for the whole data, using the STATI-CF statisticalprogram. Means were compared using the NewmaneKeulstest at the P < 0.05 level, when significant differences werefound. Values are the means of 5 and 3 replicates for physio-logical and biochemical parameters, respectively.

3. Results

3.1. Leaf aspect and chlorophyll status

Chlorosis symptoms were observed on young leaves of thegrapevines grown with bicarbonate, their intensity howeverdepending on the genotype and the treatment (Fig. 1). Leafsymptoms appeared first in Balta4 and Beldi varieties, as com-pared to Khamri, Blanc3, and Arich Dresse. The two-way AN-OVA showed significant effects of bicarbonate treatment (T),genotype (G) and their interaction (T * G) on leaf chlorosisscore, which was estimated in parallel with visual observations(Table 2). Khamri displayed the lowest values (0.4 and 1.8, at4 mM HCO3

� and 16 mM HCO3�, respectively), while the

highest scores were found in Balta4 (1.2 and 3.6, respectivelyat 4 mM and 16 mM HCO3

�) (Fig. 1). Leaf chlorosis was asso-ciated with a significant decrease of leaf chlorophyll contents,

Table 2

Results of a two-way analysis of variance (ANOVA) of plant characteristics by

treatment (T) and genotype (G)

Dependent variables T G T * G

Leaf chlorosis score 253.71*** 17.77*** 2.31**

Leaf chlorophyll content 828.62*** 79.54*** 4.07***

Shoot length 555.55*** 158.2*** 12.87***

Plant biomass 515.81*** 41.02*** 7.51***

Total leaf area 1010.87*** 209.42*** 12.27***

Leaf number 187.48*** 91.94*** 6.83***

Ferrous iron 1218.7*** 61.79*** 5.55***

Numbers represent F values: *P < 0.01, **P < 0.001, ***P < 0.0001.

318 R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322

Fig. 2. Chlorophyll content in the leaf No. 4 of native grapevine genotypes irrigated for 85 days with tap water containing NaHCO3 (B0, 0 mM; B4, 4 mM; B8,

8 mM; B12, 12 mM and B16, 16 mM). Means (n ¼ 5, �SE) labelled by at least one same letter are not statistically different at P < 0.05.

Fig. 3. Shoot length (A) and biomass production (B) of native grapevine genotypes irrigated for 85 days with tap water containing NaHCO3 (B0, 0 mM; B4, 4 mM;

B8, 8 mM; B12, 12 mM and B16, 16 mM). Means (n ¼ 5, �SE) labelled by at least one same letter are not statistically different at P < 0.05.

319R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322

with significant effects of T, G, and T * G (Table 2). For in-stance, leaf chlorophyll content was 35% and 58% lower, re-spectively in Khamri and Balta4 varieties challenged with16 mM HCO3

� (Fig. 2).

3.2. Plant growth

The lowest bicarbonate levels reduced shoot length after3 weeks of treatment. As for leaf chlorosis status, a significanteffect of T, G, and T * G was found (Table 2). At the final har-vest, the reduction of shoot height ranged from 31% in Khamrito 60% in Balta4 irrigated with 16 mM HCO3

� (Fig. 3A). Beldi,Arich Dresse, and Blanc3 genotypes showed an intermediatebehaviour (�41% to �55%). T, G, and T * G had also a signif-icant effect on plant biomass production (Table 2). Growthrestriction was particularly pronounced at HCO3

� concentra-tions exceeding 4 mM. In addition, 16 mM HCO3

� clearly dis-tinguished between the genotypes, the biomass reductionvarying from 30% in Khamri to 60% in Balta4 (Fig. 3B).

Bicarbonate addition to the medium resulted in a significantdecrease of both total leaf area and leaf number, despite beinggenotype-dependent (Fig. 4A and B). The Two-way ANOVAindicated the occurrence of a significant effect of T, G, andT * G on both of these parameters (Table 2), but leaf numberappeared to be more sensitive than leaf expansion. For in-stance, the decrease of the latter was 23% in Khamri (tolerantvariety), while exceeding 40% in Balta4 at 16 mM HCO3

�. Atthis concentration, leaf number was 33% and 60% lower thanthat of the control in Khamri and Balta4, respectively.

3.3. HCl-extractible iron content of young leaves

Fe content of young leaves declined significantly in Fe-lacking grapevines. This was apparent at the lowest Bicarbonatedoses (4 mM) and was more pronounced with increasing HCO3

�

concentration (Fig. 5A). T, G, and T * G had a significant effecton this parameter (Table 2). At the highest bicarbonate concen-tration (16 mM), the young leaf iron content was less than 50%

Fig. 4. Total leaf area (A) and leaf number (B) of native grapevine genotypes irrigated for 85 days with tap water containing NaHCO3 (B0, 0 mM; B4, 4 mM; B8,

8 mM; B12, 12 mM and B16, 16 mM). Means (n ¼ 5, �SE) labelled by at least one same letter are not statistically different at P < 0.05.

320 R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322

y = 0.019x + 0.05

R = 0.978

0.0

0.4

0.8

1.2

1.6

20 40 60 80

Chl

orop

hyll

cont

ent

(mg.

g-1 F

W)

Fe content (µg.g-1 DW)

A

B

Fig. 5. (A) Iron content in the leaf NO. 4 of native grapevine genotypes irrigated for 85 days with tap water containing NaHCO3 (B0, 0 mM; B4, 4 mM; B8, 8 mM;

B12, 12 mM and B16, 16 mM). Means (n ¼ 5 � SE) labelled by at least one same letter are not statistically different at P < 0.05. (B) Relationship between leaf

chlorophyll and iron concentrations of young chlorotic leaves. Each point corresponds to a grapevine variety and represents the mean value of five replicates.

of the control in the sensitive genotype Balta4, while reaching65% in Khamri (tolerant).

3.4. Root biochemical responses to iron deficiency incontrasting behaviour variety

3.4.1. Root acidificationIncreasing medium pH occurred in the control plants of

both genotypes (Table 3). Under induced Fe-deficiency (ID),a slight acidification of the medium in the most tolerant geno-type (�0.5 pH units in Khamri) whereas Balta4 expressed noroot acidification capacity.

3.4.2. Iron reduction activityFe(III)-reductase in vivo activity of excised roots was low in

control plants of both genotypes (78 nmol h�1 g�1 FW to88 nmol h�1 g�1 FW) (Table 3). This activity was markedlystimulated in Khamri (tolerant) under indirect Fe-loweringavailability (up to ca. 4.1-fold higher than the control), whileno significant changes were recorded in Balta4 (sensitive)(Table 3).

3.4.3. PEPC activitySimilar values were found in control plants of both Khamri

and Balta4. At 10 mM NaHCO3, PEPC was ca. 4.7-fold higherin Khamri as compared to the control (Table 3), while nochanges occurred in the sensitive variety Balta4.

4. Discussion

HCO3�-induced Fe-shortage had a depressive effect on

physiological parameters (leaf chlorosis score, shoot length,whole plant biomass production, chlorophyll and iron con-tents), depending on both bicarbonate concentration andgrapevine genotype. Balta4, Beldi and at a lesser extent ArichDresse and Blanc3, manifested early chlorosis symptoms ontheir young leaves (notably at the highest HCO3

� levels), whileKhamri variety displayed the lowest chlorosis score. The var-iability of sensitivity to bicarbonate is seen as a major criterionallowing genotypic discrimination of fruit trees growing incalcareous soils [13,19,30]. A variable restriction (dependingon T, G, and T * G) occurred with respect to physiological pa-rameters (ponderal growth, shoot length, and leaf expansion)

321R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322

Table 3

Root biochemical parameters: acidification, Fe-reductase and PEPC activities in Khamri and Balta4 grown for 75 days on a nutrient solution containing either

20 mM Fe (control), or Fe-deficient (ID), by adding bicarbonate (10 mM HCO3�) to the control medium

Acidification Fe-reductase PEPC

pHi pHf nmol h�1 g�1 FW mmol h�1 g�1 FW

Balta4 C 6.05 � 0.0 6.98 � 0.16 77.7 � 12.50b 0.96 � 0.08b

ID 7.95 � 0.0 8.26 � 0.13 85.4 � 7.20b 1.50 � 0.38b

Khamri C 6.05 � 0.0 7.17 � 0.22 88.5 � 6.70b 1.02 � 0.34b

ID 7.95 � 0.0 7.48 � 0.19 359.7 � 36.2a 4.18 � 0.70a

Means (n ¼ 3, �SE) labelled by at least one same letter are not statistically different at P < 0.05. pHi, initial pH; pHf, final pH (after 7 days); C, control; ID,

induced deficiency.

too. Comparing the control to 16 mM HCO3�-treated plants

showed that Khamri was the less affected variety (�23% to�30% depending on the parameter), while Balta4 was themost sensitive (�47% to �60%). These findings are in agree-ment with previous reports relative to bicarbonate impacton contrasting genotypes of grapevine and other species[1,5,12,36].

Bicarbonate had a depressive effect on both chlorophyll andiron status of young leaves, (�50% in the 16 mM HCO3

�-treated sensitive varieties, Balta4 and Beldi). The strong rela-tionship between these both parameters (R ¼ 0.97) confirmsthat the response to iron shortage may be closely linked toleaf iron content (Fig. 5B). Moreover, as already documented[25,34,37], our findings suggest that tolerance to low iron avail-ability may rely on the plant aptitude to insure adequate ironabsorption and reduction. In dicotyledonous (among whichgrapevine) and monocotyledonous excepting Graminae, thisis mediated by several mechanisms known as Strategy I [22].In sensitive genotypes, the absence of such mechanisms maybe partly responsible for their growth restriction and the ap-pearance of typical intervein chlorosis at the leaf level [3,4].In addition, root growth reduction in calcareous soils may bedue to direct and/or indirect (lower chlorophyll in photosyn-thetic active young leaves) effects of bicarbonate ions [5,21].

Using root biochemical criteria, the present study estab-lished that the tolerance of Khamri was associated with an im-portant root activity of Fe(III)-reductase, as well as a relativecapacity of lowering the culture medium pH, a necessary pro-cess for the mobilisation of soluble ferric compounds presentin the soil [26]. Our data hence suggest that the above-men-tioned parameters clearly discriminated between the grapevinegenotypes coping with Fe-deficiency. Root acidification hasalready been proposed as a useful criterion for the evaluationof resistant varieties in cultivated species [15]. In contrastinggrapevine varieties, the presence of a close relationship be-tween root acidification and Fe(III)-reductase activities andtheir resistance to ferric chlorosis, suggested the utilisationof these parameters as criteria to select tolerant grapevinesin calcareous soils [25]. In the same way, an important rootPEPC ability was registered in the tolerant genotype (Khamri)treated with bicarbonate, in contrast to Balta4. Ollat et al. [27]indicated that this enzyme may control organic acid biosynthe-sis and accumulation in Fe-shortage. Bienfait [8] pointed outtoo that citrate metabolism was associated with both stimu-lated proton release and Fe-reductase activity.

As a whole, our findings confirm the variable response ofnative grapevines to bicarbonate-induced iron deficiency. Rel-ative tolerance of Khamri variety was correlated with higherleaf iron content, indicating that the observed genotypical dif-ferences in the response to Fe-induced deficiency may bemainly due to differences in Fe acquisition rather than Fe uti-lisation in leaves, as previously documented [30,35]. Consid-ering the biochemical aspects, the most tolerant genotypewas relatively able to decrease the medium pH while exhibit-ing higher Fe-reductase and PEPC activities. Finally, the fivegenotypes could be distinguished as: more tolerant (Khamri),intermediate (Blanc3 and Arich Dresse), and sensitive (Balta4and Beldi).

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