Chlorosis from lime-induced Fe deficiency limits grapevine (Vitis L.) growth and productivity (Bavaresco et al., 2003; Gruber and Kosegarten, 2002; Mengel et al., 1984a). As soil pH increases, Fe solubility decreases, and an increase in soil pH by one unit decreases Fe3+ activity 1000-fold (Lindsay and Schwab, 1982). ‘Concord’ grapevines (V. labruscana) are particularly susceptible to lime-induced chlorosis, and this situation can be made worse under conditions of high soil moisture and low soil temperature (Davenport and Stevens, 2006).
When Fe is limiting, dicotyledons can facilitate Fe uptake by acidifying the rhizosphere to increase Fe solubility and exuding organic acids and phenolics to chelate Fe (de Vos et al., 1986; Marschner et al., 1986; Römheld and Marschner, 1983). In addition, roots can increase the activity of plasma membrane-bound ferric chelate reductase (FCR), which uses cytosolic NAD(P)H to cleave Fe(III)-chelates and reduce Fe3+ to Fe2+ before transport across the membrane by inducible Fe2+ transporters (Chaney et al., 1972; Vert et al., 2002). Reduction of Fe(III)-chelates is proposed to be the rate-limiting step for Fe assimilation in dicotyledons (Grotz and Guerinot, 2002), and root FCR activity is often higher in Fe-efficient vs. Fe-inefficient species (Brancadoro et al., 1995; Römheld and Marschner, 1981).
Iron is transported to the leaves primarily as Fe(III)-citrate, although there is some evidence that nicotianamine and carboxylic acids such as malate may play a role in Fe transport (Rombolà et al., 2000; Tiffin, 1970; von Wiren et al., 1999). Foliar Fe assimilation is dependent on FCR activity to reduce Fe(III)-chelates and provide Fe2+ for transport across the mesophyll plasma membrane (Brüggemann et al., 1993). Unlike root FCR, foliar FCR does not appear to be induced by Fe deficiency; however, foliar FCR activity is regulated by light (Brüggemann et al., 1993; de la Guardia and Alcantara, 1996; Gonzalez-Vallejo et al., 2000; Larbi et al., 2001).
It has been proposed that bicarbonate uptake increases the pH of xylem sap and leaf apoplast and interferes with foliar Fe utilization (Mengel et al., 1984a, 1984b), but this is not well-agreed on. For instance, the xylem sap pH of corn (Zea mays L.) seedlings increased 0.6 and 1.1 units when supplied with 5 to 20 mm HCO3 − in the nutrient solution (Wegner and Zimmermann, 2004), and López-Millàn et al. (2001b) found that the pH of the apoplast in Fe-deficient pear (Pyrus communis L.) leaves was 6.5 to 6.6, whereas the apoplast pH of green leaves was lower, from 5.5 to 5.9. Yet on the other hand, Nikolic and Römheld (2002) found that the addition of 10 mm HCO3 − to the nutrient solution did not change the pH of the leaf apoplastic fluid in sunflower and that there was no difference in the leaf apoplast pH between chlorotic and field-grown Vitis vinifera L. In addition to bicarbonate, nitrate may also influence the pH of the xylem sap and leaf apoplast and impact Fe assimilation, but this is not fully supported either (Kosegarten et al., 2001; Lucena, 2000; Nikolic and Römheld, 2003).
The accumulation of organic acids, primarily malate and citrate, commonly occurs in iron-deficient roots and leaves (Abadía et al., 2002). For example, citrate and malate increased in the root tips of four different grapevine genotypes in response to Fe limitation, and chlorosis-resistant genotypes contained higher levels of organic acids (Ollat et al., 2003). An increase in malate and citrate in response to Fe deficiency has a number of potential benefits. Increased root exudation of citric and malic acid will improve soil Fe availability both through chelation and rhizosphere acidification (Jones, 1998; Landsberg, 1981). In addition, greater concentrations of citrate could also aid in transport of Fe in the xylem (Tiffin, 1970).
It is not clear, however, why organic acids accumulate in Fe-deficient tissues. Citrate accumulation in Fe-deficient plants was originally thought to result from a decreased conversion of citrate to isocitrate by aconitase, which requires Fe as a cofactor (Bacon et al., 1961). However, aconitase activity is not consistently decreased under Fe deficiency (de Vos et al., 1986; López-Millàn et al., 2001a). Alternatively, organic acid accumulation could result from increased carbon fixation by phosphoenolpyruvate (PEP) carboxylase when soil HCO3 − is high or Fe is limiting. According to this theory, high rates of H+ efflux under Fe deficiency would increase the pH of the cytoplasm and activate PEP carboxylase (Felle, 1988; Rabotti et al., 1995). Carboxylation of PEP through PEP carboxylase would result in oxaloacetate (OAA) and malate synthesis and a lowering of cytosolic pH (Davies, 1986).
Considering the importance of FCR and organic acids in Fe assimilation, there were two main objectives for our work with low pH-tolerant (Fe-inefficient) ‘Concord’ grapevines. The first objective was to characterize root and leaf ferric chelate reductase activity in response to lime-induced Fe limitation. The second objective was to quantify how lime-induced Fe limitation affects key enzymes and metabolites involved with glycolysis and the tricarboxylic acid cycle in leaves. We hypothesized that Fe-deficient leaves would have lower aconitase activity and that this would result in an accumulation of citrate. We also hypothesized that an increase in CaCO3 in the rooting medium would raise the xylem sap pH and negatively impact Fe utilization.
BavarescoL.FregoniM.FraschiniP.1991Investigations on iron uptake and reduction by excised roots of different grapevine rootstocks and a Vitis vinifera cultivarPlant Soil130109114
BavarescoL.GiachinoE.PezzuttoS.2003Grapevine rootstock effects on lime-induced chlorosis, nutrient uptake, and source-sink relationshipsJ. Plant Nutr.2614511465
BradfordM.M.1976Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye bindingAnal. Biochem.72248254
BrancadoroL.RabottiG.ScienzaA.ZocchiG.1995Mechanisms of Fe-efficiency in roots of Vitis spp. in response to iron-deficiency stressPlant Soil171229234
BrüggemannW.MaaskantelK.MoogP.R.1993Iron uptake by leaf mesophyll-cells—The role of the plasma membrane-bound ferric-chelate reductasePlanta190151155
ChenL.-S.SmithB.R.ChengL.2004CO2 assimilation, photosynthetic enzymes, and carbohydrates of ‘Concord’ grape leaves in response to iron supplyJ. Amer. Soc. Hort. Sci.129738744
ChenL.S.ChengL.2003Both xanthophyll cycle-dependent thermal dissipation and the antioxidant system are up-regulated in grape (Vitis labrusca L. Cv. Concord) leaves in response to N limitationJ. Expt. Bot.5421652175
DavenportJ.R.StevensR.G.2006High soil moisture and low soil temperature are associated with chlorosis occurrence in Concord grapeHortScience41418422
de la GuardiaM.D.AlcantaraE.1996Ferric chelate reduction by sunflower (Helianthus annuus L.) leaves: Influence of light, oxygen, iron-deficiency and leaf ageJ. Expt. Bot.47669675
de VosC.R.LubberdingH.J.BienfaitH.F.1986Rhizosphere acidification as a response to iron-deficiency in bean-plantsPlant Physiol.81842846
EspenL.Dell'OrtoM.De NisiP.ZocchiG.2000Metabolic responses in cucumber (Cucumis sativus L.) roots under Fe-deficiency: A 31P-nuclear magnetic resonance in-vivo studyPlanta210985992
FournierJ.M.AlcantaraE.de la GuardiaM.D.1992Organic-acid accumulation in roots of 2 sunflower lines with a different response to iron-deficiencyJ. Plant Nutr.1517471755
GentyB.BriantaisJ.M.BakerN.R.1989The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescenceBiochim. Biophys. Acta9908792
GezginS.ErF.2001Relationship between total and active iron contents of leaves and observed chlorosis in vineyards in Konya-Hadim-Alada region of turkeyCommun. Soil Sci. Plant Anal.3215131521
Gonzalez-VallejoE.B.MoralesF.CistueL.AbadíaA.AbadíaJ.2000Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplastsPlant Physiol.122337344
GruberB.KosegartenH.2002Depressed growth of non-chlorotic vine grown in calcareous soil is an iron deficiency symptom prior to leaf chlorosisJ. Plant Nutr. Soil Sci.165111117
JennerH.L.WinningB.M.MillarA.H.TomlinsonK.L.LeaverC.J.HillS.A.2001NAD malic enzyme and the control of carbohydrate metabolism in potato tubersPlant Physiol.12611391149
KosegartenH.HoffmannB.MengelK.2001The paramount influence of nitrate in increasing apoplastic pH of young sunflower leaves to induce Fe deficiency chlorosis, and the re-greening effect brought about by acidic foliar spraysJ. Plant Nutr. Soil Sci.164155163
KsouriR.M'rahS.GharsalliM.LachaâlM.2006Biochemical responses to true and bicarbonate-induced iron deficiency in grapevine genotypesJ. Plant Nutr.29305315
LandsbergE.C.1981Organic-acid synthesis and release of hydrogen ions in response to Fe deficiency stress of monocotyledonous and dicotyledonous plant speciesJ. Plant Nutr.3579591
LarbiA.MoralesF.López-MillànA.F.GogorcenaY.AbadíaA.MoogP.R.AbadíaJ.2001Technical advance: Reduction of Fe(III)-chelates by mesophyll leaf disks of sugar beet. Multi-component origin and effects of Fe-deficiencyPlant Cell Physiol.4294105
LeegoodR.C.1993Carbon metabolism247267HallD.O.ScurlockJ.M.O.Bolhar-NordenkampfH.R.LeegoodR.C.LongS.P.Photosynthesis and production in a changing environment: A field and laboratory manualChapman & HallLondon
López-MillànA.F.MoralesF.AbadíaA.AbadíaJ.2001aChanges induced by Fe deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris) leavesPhysiol. Plant.1123138
López-MillànA.F.MoralesF.AbadíaA.AbadíaJ.2001bIron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) treesJ. Expt. Bot.5214891498
LucenaJ.J.2000Effects of bicarbonate, nitrate and other environmental factors on iron deficiency chlorosis. A reviewJ. Plant Nutr.2315911606
McCluskeyJ.HerdmanL.SkeneK.R.2004Iron deficiency induces changes in metabolism of citrate in lateral roots and cluster roots of Lupinus albus. Physiol. Plant.121586594
MengelK.BreiningerM.T.BublW.1984aBicarbonate, the most important factor inducing iron chlorosis in vine grapes on calcareous soilPlant Soil81333344
MoralesF.AbadíaA.AbadíaJ.1998Photosynthesis, quenching of chlorophyll fluorescence and thermal energy dissipation in iron-deficient sugar beet leavesAust. J. Plant Physiol.25403412
OllatN.LabordeW.NeveuxM.Diakou-VerdinP.RenaudC.MoingA.2003Organic acid metabolism in roots of various grapevine (Vitis) rootstocks submitted to iron deficiency and bicarbonate nutritionJ. Plant Nutr.2621652176
PichA.ScholzG.1993The relationship between the activity of various iron-containing and iron-free enzymes and the presence of nicotianamine in tomato seedlingsPhysiol. Plant.88172178
PoonnachitU.DarnellR.2004Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium speciesAnn. Bot. (Lond.)93399405
RabottiG.DenisiP.ZocchiG.1995Metabolic implications in the biochemical responses to iron-deficiency in cucumber (Cucumis sativus L.) rootsPlant Physiol.10711951199
RombolàA.D.BrüggemannW.López-MillánA.F.TagliaviniM.AbadíaJ.MarangoniB.MoogP.R.2002Biochemical responses to iron deficiency in kiwifruit (Actinidia deliciosa)Tree Physiol.22869875
RombolàA.D.BrüggemannW.TagliaviniM.MarangoniB.MoogP.R.2000Iron source affects iron reduction and re-greening of kiwifruit (Actinidia deliciosa) leavesJ. Plant Nutr.2317511765
RömheldV.MarschnerH.1981Effect of Fe stress on utilization of Fe chelates by efficient and inefficient plant speciesJ. Plant Nutr.3551560
RömheldV.MarschnerH.1983Mechanism of iron uptake by peanut plants.1. FeIII reduction, chelate splitting, and release of phenolicsPlant Physiol.71949954
SiedowJ.N.DayD.A.2000Respiration and photorespiration676728BuchananB.B.GruissemW.JonesR.L.Biochemistry & molecular biology of plantsAmerican Society of Plant PhysiologistsRockville, MD
StittM.LilleyR.M.GerhardtR.HeldtH.W.1989Metabolite levels in specific cells and subcellular compartments of plant-leavesMethods Enzymol.174518552
TerryN.1983Limiting factors in photosynthesis. 4. Iron stress-mediated changes in light-harvesting and electron-transport capacity and its effects on photosynthesis in vivo. Plant Physiol.71855860
VertG.GrotzN.DedaldechampF.GaymardF.GuerinotM.L.BriatJ.F.CurieC.2002IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growthPlant Cell1412231233
von WirenN.KlairS.BansalS.BriatJ.F.KhodrH.ShioiriT.LeighR.A.HiderR.C.1999Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plantsPlant Physiol.11911071114
WegnerL.H.ZimmermannU.2004Bicarbonate-induced alkalinization of the xylem sap in intact maize seedlings as measured in situ with a novel xylem pH probePlant Physiol.13634693477