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.
Bacon, J.S., DeKock, P.C. & Palmer, M.J. 1961 Aconitase levels in leaves of iron-deficient mustard plants (Sinapis alba) Biochem. J. 80 64 70
Bavaresco, L., Fregoni, M. & Fraschini, P. 1991 Investigations on iron uptake and reduction by excised roots of different grapevine rootstocks and a Vitis vinifera cultivar Plant Soil 130 109 114
Bavaresco, L., Giachino, E. & Pezzutto, S. 2003 Grapevine rootstock effects on lime-induced chlorosis, nutrient uptake, and source-sink relationships J. Plant Nutr. 26 1451 1465
Bradford, M.M. 1976 Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding Anal. Biochem. 72 248 254
Brancadoro, L., Rabotti, G., Scienza, A. & Zocchi, G. 1995 Mechanisms of Fe-efficiency in roots of Vitis spp. in response to iron-deficiency stress Plant Soil 171 229 234
Brüggemann, W., Maaskantel, K. & Moog, P.R. 1993 Iron uptake by leaf mesophyll-cells—The role of the plasma membrane-bound ferric-chelate reductase Planta 190 151 155
Chen, L.-S., Smith, B.R. & Cheng, L. 2004 CO2 assimilation, photosynthetic enzymes, and carbohydrates of ‘Concord’ grape leaves in response to iron supply J. Amer. Soc. Hort. Sci. 129 738 744
Chen, L.S. & Cheng, L. 2003 Both xanthophyll cycle-dependent thermal dissipation and the antioxidant system are up-regulated in grape (Vitis labrusca L. Cv. Concord) leaves in response to N limitation J. Expt. Bot. 54 2165 2175
Davenport, J.R. & Stevens, R.G. 2006 High soil moisture and low soil temperature are associated with chlorosis occurrence in Concord grape HortScience 41 418 422
de la Guardia, M.D. & Alcantara, E. 1996 Ferric chelate reduction by sunflower (Helianthus annuus L.) leaves: Influence of light, oxygen, iron-deficiency and leaf age J. Expt. Bot. 47 669 675
de Vos, C.R., Lubberding, H.J. & Bienfait, H.F. 1986 Rhizosphere acidification as a response to iron-deficiency in bean-plants Plant Physiol. 81 842 846
Espen, L., Dell'Orto, M., De Nisi, P. & Zocchi, G. 2000 Metabolic responses in cucumber (Cucumis sativus L.) roots under Fe-deficiency: A 31P-nuclear magnetic resonance in-vivo study Planta 210 985 992
Fournier, J.M., Alcantara, E. & de la Guardia, M.D. 1992 Organic-acid accumulation in roots of 2 sunflower lines with a different response to iron-deficiency J. Plant Nutr. 15 1747 1755
Genty, B., Briantais, J.M. & Baker, N.R. 1989 The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence Biochim. Biophys. Acta 990 87 92
Gezgin, S. & Er, F. 2001 Relationship between total and active iron contents of leaves and observed chlorosis in vineyards in Konya-Hadim-Alada region of turkey Commun. Soil Sci. Plant Anal. 32 1513 1521
Gonzalez-Vallejo, E.B., Morales, F., Cistue, L., Abadía, A. & Abadía, J. 2000 Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts Plant Physiol. 122 337 344
Gruber, B. & Kosegarten, H. 2002 Depressed growth of non-chlorotic vine grown in calcareous soil is an iron deficiency symptom prior to leaf chlorosis J. Plant Nutr. Soil Sci. 165 111 117
Jenner, H.L., Winning, B.M., Millar, A.H., Tomlinson, K.L., Leaver, C.J. & Hill, S.A. 2001 NAD malic enzyme and the control of carbohydrate metabolism in potato tubers Plant Physiol. 126 1139 1149
Kosegarten, H., Hoffmann, B. & Mengel, K. 2001 The 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 sprays J. Plant Nutr. Soil Sci. 164 155 163
Ksouri, R., M'rah, S., Gharsalli, M. & Lachaâl, M. 2006 Biochemical responses to true and bicarbonate-induced iron deficiency in grapevine genotypes J. Plant Nutr. 29 305 315
Landsberg, E.C. 1981 Organic-acid synthesis and release of hydrogen ions in response to Fe deficiency stress of monocotyledonous and dicotyledonous plant species J. Plant Nutr. 3 579 591
Larbi, A., Morales, F., López-Millàn, A.F., Gogorcena, Y., Abadía, A., Moog, P.R. & Abadía, J. 2001 Technical advance: Reduction of Fe(III)-chelates by mesophyll leaf disks of sugar beet. Multi-component origin and effects of Fe-deficiency Plant Cell Physiol. 42 94 105
Leegood, R.C. 1993 Carbon metabolism 247 267 Hall D.O., Scurlock J.M.O., Bolhar-Nordenkampf H.R., Leegood R.C. & Long S.P. Photosynthesis and production in a changing environment: A field and laboratory manual Chapman & Hall London
López-Millàn, A.F., Morales, F., Abadía, A. & Abadía, J. 2001a Changes induced by Fe deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris) leaves Physiol. Plant. 112 31 38
López-Millàn, A.F., Morales, F., Abadía, A. & Abadía, J. 2001b Iron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) trees J. Expt. Bot. 52 1489 1498
Lucena, J.J. 2000 Effects of bicarbonate, nitrate and other environmental factors on iron deficiency chlorosis. A review J. Plant Nutr. 23 1591 1606
Marschner, H., Römheld, V. & Kissel, M. 1986 Different strategies in higher-plants in mobilization and uptake of iron J. Plant Nutr. 9 695 713
McCluskey, J., Herdman, L. & Skene, K.R. 2004 Iron deficiency induces changes in metabolism of citrate in lateral roots and cluster roots of Lupinus albus. Physiol. Plant. 121 586 594
Mengel, K., Breininger, M.T. & Bubl, W. 1984a Bicarbonate, the most important factor inducing iron chlorosis in vine grapes on calcareous soil Plant Soil 81 333 344
Moore, A.L., Albury, M.S., Crichton, P.G. & Affourtit, C. 2002 Function of the alternative oxidase: Is it still a scavenger? Trends Plant Sci. 7 478 481
Morales, F., Abadía, A. & Abadía, J. 1998 Photosynthesis, quenching of chlorophyll fluorescence and thermal energy dissipation in iron-deficient sugar beet leaves Aust. J. Plant Physiol. 25 403 412
Nikolic, M. & Römheld, V. 2002 Does high bicarbonate supply to roots change availability of iron in the leaf apoplast? Plant Soil 241 67 74
Ollat, N., Laborde, W., Neveux, M., Diakou-Verdin, P., Renaud, C. & Moing, A. 2003 Organic acid metabolism in roots of various grapevine (Vitis) rootstocks submitted to iron deficiency and bicarbonate nutrition J. Plant Nutr. 26 2165 2176
Pascal, N. & Douce, R. 1993 Effect of iron-deficiency on the respiration of sycamore (Acer pseudoplatanus L.) cells Plant Physiol. 103 1329 1338
Pich, A. & Scholz, G. 1993 The relationship between the activity of various iron-containing and iron-free enzymes and the presence of nicotianamine in tomato seedlings Physiol. Plant. 88 172 178
Poonnachit, U. & Darnell, R. 2004 Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species Ann. Bot. (Lond.) 93 399 405
Pushnik, J.C. & Miller, G.W. 1989 Iron regulation of chloroplast photosynthetic function—mediation of PSI development J. Plant Nutr. 12 407 421
Rabotti, G., Denisi, P. & Zocchi, G. 1995 Metabolic implications in the biochemical responses to iron-deficiency in cucumber (Cucumis sativus L.) roots Plant Physiol. 107 1195 1199
Rombolà, A.D., Brüggemann, W., López-Millán, A.F., Tagliavini, M., Abadía, J., Marangoni, B. & Moog, P.R. 2002 Biochemical responses to iron deficiency in kiwifruit (Actinidia deliciosa) Tree Physiol. 22 869 875
Rombolà, A.D., Brüggemann, W., Tagliavini, M., Marangoni, B. & Moog, P.R. 2000 Iron source affects iron reduction and re-greening of kiwifruit (Actinidia deliciosa) leaves J. Plant Nutr. 23 1751 1765
Römheld, V. & Marschner, H. 1981 Effect of Fe stress on utilization of Fe chelates by efficient and inefficient plant species J. Plant Nutr. 3 551 560
Römheld, V. & Marschner, H. 1983 Mechanism of iron uptake by peanut plants.1. FeIII reduction, chelate splitting, and release of phenolics Plant Physiol. 71 949 954
Siedow, J.N. & Day, D.A. 2000 Respiration and photorespiration 676 728 Buchanan B.B., Gruissem W. & Jones R.L. Biochemistry & molecular biology of plants American Society of Plant Physiologists Rockville, MD
Stitt, M., Lilley, R.M., Gerhardt, R. & Heldt, H.W. 1989 Metabolite levels in specific cells and subcellular compartments of plant-leaves Methods Enzymol. 174 518 552
Terry, N. 1983 Limiting factors in photosynthesis. 4. Iron stress-mediated changes in light-harvesting and electron-transport capacity and its effects on photosynthesis in vivo. Plant Physiol. 71 855 860
Vert, G., Grotz, N., Dedaldechamp, F., Gaymard, F., Guerinot, M.L., Briat, J.F. & Curie, C. 2002 IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth Plant Cell 14 1223 1233
von Wiren, N., Klair, S., Bansal, S., Briat, J.F., Khodr, H., Shioiri, T., Leigh, R.A. & Hider, R.C. 1999 Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants Plant Physiol. 119 1107 1114
Wegner, L.H. & Zimmermann, U. 2004 Bicarbonate-induced alkalinization of the xylem sap in intact maize seedlings as measured in situ with a novel xylem pH probe Plant Physiol. 136 3469 3477