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

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<ul><li><p>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</p><p>des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia</p><p>Available online 15 March 2007</p><p>Abstract</p><p>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</p><p>) for 75 days. Using morpho-physiological criteria, grapevine tolerance to HCO3-induced Fe shortage appeared to be genotype-depen-</p><p>dent: Balta4 and Beldi varieties showed the highest leaf-chlorosis score (especially at the extreme HCO3 levels), in contrast to Khamri variety.</p><p>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</p><p>, 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.</p><p>Keywords: Acidification; Fe(III)-reductase; Iron chlorosis; Native grapevine; Phosphoenolpyruvate carboxylase; Variability</p><p>1. Introduction</p><p>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,</p><p>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 growthResearch</p><p>Genotypic variability within(Vitis vinifera L.) facing bicarb</p><p>Riadh Ksouri a, Ahmed Debez a,1, HeMohamed Gharsalli a,</p><p>a Laboratoire dAdaptation des Plantes aux Stre</p><p>Plant Physiology and Biochem* Corresponding author. Tel./fax: 216 71 872 600.E-mail address: mokhtar.lachaal@fst.rnu.tn (M. Lachaal).</p><p>1 Present address: Institut fur Botanik, Universitat Hannover, Herrenhauser</p><p>Str. 2, 30419, Hannover, Germany.</p><p>0981-9428/$ - see front matter 2007 Elsevier Masson SAS. All rights reservedoi:10.1016/j.plaphy.2007.03.014article</p><p>Tunisian grapevine varietiesonate-induced iron deficiency</p><p>nda Mahmoudi a, Zeineb Ouerghi b,Mokhtar Lachaal b,*</p><p>ss Abiotiques, Centre de Biotechnologies de la</p><p>istry 45 (2007) 315e322www.elsevier.com/locate/plaphyand 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].</p><p>d.</p></li><li><p>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).</p><p>2. Material and methods</p><p>2.1. Culture conditions</p><p>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 tapwater containing increasing bicarbonate concentrations(0, 4, 8, 12, and 16 mM NaHCO3). Cultures were performed ina greenhouse (22e30 C temperature, 75e85% relative humidity)</p><p>Table 1</p><p>Chemical characteristics of the soil (A) and irrigation water (B) used for plant c</p><p>A. INRST soil</p><p>N P K Ca2 Na</p><p>0.45 0.24 4.1 103 13.0 103 1.7 10B. Irrigation water</p><p>K Ca2 Na Cl Mg2</p><p>0.15 3.5 4.48 7.76 1.65Soil ion contents, nitrogen and inorganic phosphorus are expressed as g per kg of</p><p>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 [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 shortagewas in-directly (ID) achieved by adding bicarbonate (10 mMNaHCO3)to the control medium (with 20 mM Fe). The nutrient solutionwas replaced weekly.</p><p>2.3. Morpho-physiological parameters andHCl-extractible iron assay</p><p>Plant aspect was weekly monitored during the growth pe-riod. Chlorosis symptoms on the young leaves were evaluatedaccording to Pouget and Ottenwaelters [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.</p><p>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 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. [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</p><p>ulture</p><p>Cl AC EEI pH EC3 0.5 103 2.0 37 6.6 0.05</p><p>HCO3 SO4</p><p>2 DR pH EC2.36 6.3 1.2 7.9 1.5</p><p>1Excessive concentration of bicarbonate in the soils is themain factor inducing Fe chlorosis for the plants [24], mainlythrough the HCO3</p><p> buffer effect. The latter is known to neu-tralize the root H-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,</p><p>under natural light (PAR: 250 mmol m2 s1). Soil and tap waterchemical characteristics are presented in Table 1.</p><p>2.2. Second experiment for root-biochemical parametersdetermination</p><p>One-month-old woody cuttings of Khamri and Balta4 varie-ties were cultivated on a liquid medium (one plant per pot).</p><p>316 R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322dried soil. EC, soil electric conductivity, mmohs cm ; AC, active calcareous,%;</p><p>, dry residue, g L1; EC, water electric conductivity, mmohs cm1.</p></li><li><p>nextinction coefficient 22.1 mM1 cm1 [10].</p><p>2.5. PEP carboxylase extraction and assay</p><p>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 340 nm, at 30 C) was expressedas mmol h1 g FW (three replicates per treatment).</p><p>2.6. Statistical analysis</p><p>symptoms 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</p><p> and 16 mM HCO3, respectively), while the</p><p>highest scores were found in Balta4 (1.2 and 3.6, respectivelyat 4 mM and 16 mM HCO3</p><p>) (Fig. 1). Leaf chlorosis was asso-ciated with a significant decrease of leaf chlorophyll contents,</p><p>Table 2</p><p>Results of a two-way analysis of variance (ANOVA) of plant characteristics by</p><p>treatment (T) and genotype (G)</p><p>Dependent variables T G T * G</p><p>Leaf chlorosis score 253.71*** 17.77*** 2.31**</p><p>Leaf chlorophyll content 828.62*** 79.54*** 4.07***</p><p>Shoot length 555.55*** 158.2*** 12.87***</p><p>Plant biomass 515.81*** 41.02*** 7.51***</p><p>Total leaf area 1010.87*** 209.42*** 12.27***</p><p>Leaf number 187.48*** 91.94*** 6.83***</p><p>Ferrous iron 1218.7*** 61.79*** 5.55***was assayed on the filtrate using an atomic absorption spectro-photometer (Model Perkin Elmer 4000).</p><p>2.4. Acidification and Fe(III)-reductase activity</p><p>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 the</p><p>Fig. 1. Leaf chlorosis score of native grapevine genotypes irrigated for 85 days w</p><p>16 mM). The B0 treatment (0 mM NaHCO3) was removed since B0-plants score</p><p>least one same letter are not statistically different at P &lt; 0.05.</p><p>R. Ksouri et al. / Plant Physiology aA two-way analysis of variance (ANOVA), with the geno-type (G) and bicarbonate treatment (T) as factors, wasachieved for the whole data, using the STATI-CF statisticalprogram. Means were compared using the NewmaneKeulstest at the P &lt; 0.05 level, when significant differences werefound. Values are the means of 5 and 3 replicates for physio-logical and biochemical parameters, respectively.</p><p>3. Results</p><p>3.1. Leaf aspect and chlorophyll status</p><p>Chlorosis symptoms were observed on young leaves of thegrapevines grown with bicarbonate, their intensity howeverdepending on the genotype and the treatment (Fig. 1). Leaf</p><p>ith tap water containing NaHCO3 (B4, 4 mM; B8, 8 mM; B12, 12 mM and B16,</p><p>was equal to 0 (no leaf chlorosis symptoms). Means (n 5, SE) labelled by at</p><p>317d Biochemistry 45 (2007) 315e322Numbers represent F values: *P &lt; 0.01, **P &lt; 0.001, ***P &lt; 0.0001.</p></li><li><p>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,</p><p>8 mM; B12, 12 mM and B16, 16 mM). Means (n 5, SE) labelled by at least one same letter are not statistically different at P &lt; 0.05.</p><p>318 R. Ksouri et al. / Plant Physiology and Biochemistry 45 (2007) 315e322Fig. 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;</p><p>B8, 8 mM; B12, 12 mM and B16, 16 mM). Means (n 5, SE) labelled by at least one same letter are not statistically different at P &lt; 0.05.</p></li><li><p>Fig. 4. Total leaf area (A) and leaf number (B) of native grapevine genotypes irri</p><p>8 mM; B12, 12 mM and B16, 16 mM). Means (n 5, SE) labelled by at leaswith 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</p><p> (Fig. 2).</p><p>3.2. Plant growth</p><p>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</p><p> (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</p><p> concentra-tions exceeding 4 mM. In addition, 16 mM HCO3</p><p> clearly dis-tinguished between the genotypes, the biomass reductionvarying from 30% in Khamri to 60% in Balta4 (Fig. 3B).</p><p>Bicarbonate addition to the medium resulted in a signif...</p></li></ul>

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