Technopole de Borj-Cedria (CBBC), BP 901, 2050 Hammam-Lif, Tunisiab 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
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 184.108.40.206) 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
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,
arid climate) . Developing viticulture requires the conser-vation of autochthonous varieties that have evolved severalmechanisms enabling them to cope with the local bioclimaticand edaphic conditions . 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 growthResearch
Genotypic variability within(Vitis vinifera L.) facing bicarb
Riadh Ksouri a, Ahmed Debez a,1, HeMohamed Gharsalli a,
a Laboratoire dAdaptation des Plantes aux Stre
Plant Physiology and Biochem* Corresponding author. Tel./fax: 216 71 872 600.E-mail address: email@example.com (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 reservedoi:10.1016/j.plaphy.2007.03.014article
Tunisian grapevine varietiesonate-induced iron deficiency
nda Mahmoudi a, Zeineb Ouerghi b,Mokhtar Lachaal b,*
ss Abiotiques, Centre de Biotechnologies de la
istry 45 (2007) 315e322www.elsevier.com/locate/plaphyand development processes . For instance, chlorophyllsynthesis and electron transport in mitochondria and the pho-tosynthetic chain are closely related to the plant iron status[16,17].
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  and in cucumber . 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 . 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 , 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 tapwater containing increasing bicarbonate concentrations(0, 4, 8, 12, and 16 mM NaHCO3). Cultures were performed ina greenhouse (22e30 C temperature, 75e85% relative humidity)
Chemical characteristics of the soil (A) and irrigation water (B) used for plant c
A. INRST soil
N P K Ca2 Na
0.45 0.24 4.1 103 13.0 103 1.7 10B. Irrigation water
K Ca2 Na Cl Mg2
0.15 3.5 4.48 7.76 1.65Soil ion contents, nitrogen and inorganic phosphorus are expressed as g per kg of
EEI, easily extractible iron, ppm. Water ion contents are expressed as mM. DRThe experimentwas 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 : 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 shortagewas in-directly (ID) achieved by adding bicarbonate (10 mMNaHCO3)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 Ottenwaelters  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) , old leaves, stems plus petioles, androots. Fresh and dry weight (respectively FW and DW) ofthese organs, leaf chlorophyll concentration (mg g1 FW)and HCl-extractible iron concentration (mg g1 DW) of the ju-venile leaves were then determined. Extraction and assay oftotal chlorophyll were performed as described by Torrecillaset al. . Iron was extracted by HCl (1 N), according tothe method of Oserkowsky  which was modified by Llor-ente et al. : 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
Cl AC EEI pH EC3 0.5 103 2.0 37 6.6 0.05
2 DR pH EC2.36 6.3 1.2 7.9 1.5
1Excessive concentration of bicarbonate in the soils is themain factor inducing Fe chlorosis for the plants , mainlythrough the HCO3
buffer effect. The latter is known to neu-tralize the root H-ATPase activity  and to decrease theFe(III)-reductase and iron reduction, hence restricting iron ab-sorption by roots and its transport towards shoots . Yet,several data showed the presence of a genotypic-dependentvariability of the plant response to low soil iron availability,
under natural light (PAR: 250 mmol m2 s1). 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).
316 R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322dried soil. EC, soil electric conductivity, mmohs cm ; AC, active calcareous,%;
, dry residue, g L1; EC, water electric conductivity, mmohs cm1.
nextinction coefficient 22.1 mM1 cm1 .
2.5. PEP carboxylase extraction and assay
The activity of phosphoenolpyruvate carboxylase (PEPC;EC 220.127.116.11) w