Biochemical responses to iron deficiency in kiwifruit (Actinidia deliciosa)

Tree Physiology 22, 869–875 © 2002 Heron Publishing—Victoria, Canada

Biochemical responses to iron deficiency in kiwifruit (Actinidia deliciosa) A. D. ROMBOLÀ,1,2 W. BRÜGGEMANN,3 A. F. LÓPEZ-MILLÁN,4 M. TAGLIAVINI,1 J. ABADÍA,4 B. MARANGONI1 and P. R. MOOG3 1

Dipartimento di Colture Arboree, Università di Bologna, Via G. Fanin, 46-40127 Bologna, Italy

2

Author to whom correspondence should be addressed ([email protected])

3

Botanisches Institut, Johann-Wolfgang Goethe-Universität, Frankfurt, Germany

4

Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, CSIC, Apdo. 202, 50080 Zaragoza, Spain

Received September 26, 2001; accepted March 2, 2002; published online July 2, 2002

Keywords: ferric chelate reductase, iron nutrition, organic acids, phosphoenol pyruvate carboxylase.

Introduction Dicotyledonous plants reduce chelated Fe(III) to Fe(II) at the root surface, prior to uptake (Chaney et al. 1972, Römheld and Marschner 1986). This reduction is mediated by a plasma membrane-bound ferric chelate reductase (FCR) that transports electrons from cytosolic NADH to apoplastic Fe(III) (Brüggemann et al. 1990). Iron (Fe) translocation to the shoots occurs as Fe(III)-citrate (Tiffin 1970, López-Millán et al. 2000b), whereas Fe uptake by leaf mesophyll cells relies on a second reduction step mediated by another FCR (Brüggemann et al. 1993), similar to the enzyme occurring at the root surface. Iron-efficient dicotyledonous plants respond to Fe deficiency by enhancing root FCR activity (see Moog and Brüggemann 1994), but no induction of FCR activity seems to occur

in the leaves (Brüggemann et al. 1993, de la Guardia and Alcántara 1996, Rombolà et al. 2000, Larbi et al. 2001). Plants are classified as Fe-efficient or Fe-inefficient based on their ability to induce root FCR activity in response to Fe deficiency. Increased root FCR activity is not the only adaptive mechanism employed by plants to improve Fe uptake when Fe is scarce. Iron deficiency affects many physiological and biochemical features of plant metabolism, including increasing proton pump activity and increasing release of reductants into the rhizosphere (Römheld and Marschner 1986). Also, marked increases in root organic acid concentrations, particularly malate and citrate, have been observed in response to Fe shortage (De Vos et al. 1986, Landsberg 1986, Fournier et al. 1992, Rabotti et al. 1995, López-Millán et al. 2000a). It is unclear, however, how increases in organic acid pools enhance the ability of the plant to cope with Fe shortage (see Abadía et al. 2002). Plant responses to Fe deficiency are common in calcareous soils, where the soluble Fe concentration is extremely low and bicarbonate concentration is high (up to 10 mM; Boxma 1982). Although bicarbonate has long been considered the major causal factor of Fe-deficiency chlorosis (Mengel 1994), the mechanisms by which elevated bicarbonate concentrations in the soil impair plant Fe nutrition are not completely understood. Bicarbonate can be absorbed through the roots and utilized in the synthesis of organic acids, which may accumulate in the root (Lee and Woolhouse 1969, Gout et al. 1993). Root organic acid accumulation has been related to increased rates of CO2 root fixation and increased activity of phosphoenolpyruvate carboxylase (PEPC) (Landsberg 1986, Bialczyck and Lechowski 1992, Rabotti et al. 1995). The enzyme PEPC (EC 4.1.1.31) catalyzes the incorporation of bicarbonate into a C3 organic acid, phosphoenolpyruvate (PEP), generating oxalacetate, which is converted to malate by malate dehydrogenase (Lance and Rustin 1984). This process is an important component of the pH-stat mechanism (Davies 1973). We studied two kiwifruit (Actinidia deliciosa (A. Chev.)

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Summary A comparative study of two kiwifruit genotypes (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson var. deliciosa) with different tolerance to iron (Fe) deficiency was conducted to identify biochemical features associated with tolerance to Fe deficiency. After 14 days of growth in hydroponic culture under Fe-deficient and Fe-sufficient conditions, leaf chlorophyll concentration, activities of ferric chelate reductase (FCR), phosphoenolpyruvate carboxylase (PEPC) and citrate synthase in root extracts, concentrations of organic acids in roots, leaves and xylem sap, and xylem sap pH were measured. In response to Fe deficiency, the tolerant genotype D1 showed: (i) higher FCR activity associated with a longer lasting induction of FCR; (ii) higher PEPC activity; (iii) higher concentrations of citric acid in roots; and (iv) lower xylem sap pH compared with the susceptible genotype Hayward. These findings imply that induction of FCR and PEPC activities in roots in response to Fe deficiency are important physiological adaptations enabling Fe-efficient kiwifruit plants to tolerate Fe deficiency.

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C.F. Liang et A.R. Ferguson var. deliciosa) genotypes, Hayward and D1, with different tolerances to Fe deficiency. We assayed FCR activity in both genotypes grown with and without Fe supply. To understand the physiological significance of organic acids, we measured their concentrations in roots, leaves and xylem sap and the root activities of PEPC and citrate synthase under Fe-deficient and Fe-sufficient conditions.

Materials and methods Plant material and growth conditions

Ferric chelate reductase Ferric chelate reductase (FCR) activity was determined as the reduction of Fe(III)-EDTA by excised roots by a modification of the method described by Moog et al. (1995). Roots (2 g fresh weight (FW)) were excised and incubated in plastic tubes (covered with aluminum foil) for 20 min in 26 ml of assay medium containing 500 µM BPDS and 300 µM Fe(III)EDTA in half-strength Hoagland solution without micronutrients, buffered with Mes-KOH at pH 6.0. Assays were performed on Days 2, 9, 14 and 20 after imposing Fe deficiency. Phosphoenolpyruvate carboxylase and citrate synthase Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and citrate synthase (CS; EC 4.1.3.7) activities were measured in root extracts on Day 14 after imposing Fe deficiency according to Hostrup and Wiegleb (1991). Roots were excised and homogenized with a pre-cooled (4 °C) mortar and pestle in 50 mM Tris-HCl, pH 7.8, containing 5 mM EDTA, 2 mM dithiothreitol, 20% (v/v) ethylene glycol and 1% (w/w) insolu-

Xylem sap collection and analysis To collect xylem sap, the root system of each plant was placed in a Schölander pressure chamber. The stem was cut about 20 cm above the insertion of the main root laterals, a few cm of bark were removed at the cut end to avoid contamination with phloem sap, and a pressure of 0.3 MPa was then applied to the root system. Approximately 500–3000 µl of xylem sap per plant was collected in microcapillary tubes. The pH of the xylem samples was immediately determined with a microelectrode (Ingold, Steinbach, Germany). Samples were filtered (0.45 µm, Millipore, Schwalbach, Germany) and then frozen at –20 °C. Organic acids in the xylem sap were quantified by HPLC with a 300 × 7.8 mm Aminex ion-exchange column (HPX-87H, Bio-Rad, Hercules, CA) as described by López-Millán et al. (2000a). Organic acid concentrations in roots and leaves Roots and fully expanded apical leaves were oven-dried at 60 °C for 48 h, milled and extracted (100 mg dry weight) for 24 h at 25 °C in 8 ml of 80% (v/v) ethanol after the addition of 1 mg of β-phenyl-glucopyranoside as internal standard. Samples were centrifuged for 5 min at 5000 g and the supernatants were collected and dried at 50 °C in a continuous air stream (Bartolozzi et al. 1997). Samples and standards were then treated with 270 µl of pyridine, 90 µl of hexamethyldisilazane and 30 µl of trimethylchlorosilane, heated at about 60 °C for 2 h and then stored at 4 °C until analyzed. Separation of trimethylsilyl derivatives was carried out with a Carlo Erba HRCG (Milano, Italy) gas chromatograph equipped with a flame ionization detector and a MEGA (Legnano, Milano, Italy) capillary fused-silica column (25 m × 0.25 mm I.D., OV1 methyl polysiloxane stationary phase). Injector and detector temperatures were set at 310 and 300 °C, respectively. Column temperature was increased at 13 °C min –1, from 125 to 300 °C. Organic acids were identified by comparison of their retention times with those of standards and quantified by the internal standard calculation method. Statistics Comparison of means and analysis of variance for factorial experiments (genotype × Fe treatment) was performed with SAS software (SAS Institute, Cary, NC).

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Micropropagated, 2-year-old plants of two genotypes of kiwifruit, differing in tolerance to lime-induced Fe deficiency, were used. The Fe-deficiency-susceptible genotype Hayward is frequently used as a scion for fruit production, whereas the Fe-deficiency-tolerant clone D1 is commonly used as a rootstock (Viti et al. 1990, Vizzotto et al. 1998). Both genotypes are widely used for fruit production in kiwifruit orchards in the Mediterranean region. Plants were cultivated in a greenhouse, with additional light supplied by Phillips SOLT 400 W lamps to provide a 14-h photoperiod at a minimum photon flux density of 150 µmol m –2 s –1 at the plant level. Day/night temperature was 25/20 °C. Pruning ensured only one shoot per plant. The plants were grown in peat and regularly watered, and supplied with nutrients by adding half-strength Hoagland solution once a week. When shoots were 10–15 cm in length (3–4 leaves; approximately 5 months), plants were transferred from peat to an aerated half-strength Hoagland solution, pH 6.0, containing 25 µM Fe(III)-EDTA. After 10 days (Day 0), six randomly chosen plants of each genotype were transferred to nutrient solutions without Fe. The other set of plants was placed in fresh nutrient solution with 25 µM Fe(III)-EDTA. Nutrient solutions were renewed twice a week. On Days 2, 9, 14 and 20, the chlorophyll concentration in the youngest fully expanded leaves was determined according to Arnon (1949).

ble polyvinylpolypyrrolidone. The root fresh weight to buffer ratio was 1:7. The homogenate was filtered with Miracloth (Calbiochem, Bad Soden, Germany) and the filtrate was centrifuged for 10 min at 10,000 g and 4 °C. The supernatant was used to determine PEPC activity according to Hostrup and Wiegleb (1991), by following NADH consumption, and CS activity was assayed as described by Srere (1967) and Kurz and LaRue (1977), by following the reduction of acetyl CoA to Co-A. Protein concentrations in the extracts were determined according to Bradford (1976), with bovine serum albumin as a standard.

FE DEFICIENCY IN KIWIFRUIT

Results Leaf chlorophyll concentration Regardless of genotype, Fe deficiency caused progressive decreases in leaf chlorophyll concentrations (Table 1). Leaf chlorophyll concentrations leveled off, after Day 14, at about 12 and 9 µg cm –2 in D1 and Hayward, respectively. Leaf chlorophyll concentration was always higher in D1 than in Hayward (Table 1). Even when receiving a supply of Fe (+Fe), the Hayward plants were slightly chlorotic, suggesting that 25 µM Fe in the hydroponic solution was insufficient to meet plant Fe demand. Ferric chelate reductase Root FCR activity was always higher in D1 than in Hayward (Table 2). No significant effect of Fe supply on FCR was found at Day 2 or Day 20. Iron deficiency increased FCR activity at Day 9 in both genotypes, whereas at Day 14, Fe deficiency increased FCR activity in D1 but not in Hayward (Table 2).

Table 2. Time course of root Fe-chelate reductase activity (nmol FeII g–1 FW min–1) in two kiwifruit genotypes (D1 and Hayward) grown under Fe-sufficient (+Fe) and Fe-deficient (–Fe) conditions. Data are means of six replicates. Days of treatment 2

Organic acid concentrations in roots and leaves After Day 14 of the Fe-deficiency treatment, the major organic acids in root and leaf extracts were malic, citric, quinic and succinic (Tables 4 and 5). Root concentrations of citric and quinic acids were higher in D1 than in Hayward, whereas root

Table 1. Time course of leaf chlorophyll concentration (µg Chl cm –2) in two kiwifruit genotypes (D1 and Hayward) grown under Fe-sufficient (+Fe) and Fe-deficient (–Fe) conditions. Data are means of six replicates.

1.47 0.23 *1

6.42 0.52 ***

Treatment (T) +Fe –Fe Significance

1.34 0.36 ns

2.52 4.52 **

ns

ns

G × T Interaction SEM2 1

9

14

20

Genotype (G) D1 Hayward Significance

26.49 21.72 ns1

21.74 15.06 ***

18.18 14.42 *

18.13 15.02 *

Treatment (T) +Fe –Fe Significance

24.08 24.13 ns

21.40 15.03 ***

20.40 10.96 ***

21.85 11.03 ***

G × T Interaction

ns

ns

ns

ns

1

Abbreviation and symbols: ns, *, *** = not significant and significant at the 5 and 0.1% levels, respectively.

20

+Fe

–Fe

3.87 0.81

5.15 0.51

4.25 0.59 *** 2.88 2.15 ns

* 0.62

ns

Abbreviation and symbols: ns, *, **, *** = not significant and significant at 5, 1 and 0.1% levels, respectively. SEM = standard error of the interaction means.

concentrations of malic and succinic acids did not differ between genotypes (Table 4). Regardless of the genotype, the concentration of citric acid in root extracts increased significantly with Fe deficiency (P ≤ 0.05), whereas increases in the concentrations of malic, succinic and quinic acids were not significant. In leaf extracts, the concentrations of citric and succinic acids did not differ between genotypes and did not change significantly with Fe deficiency (Table 5). Leaf malate and quinate concentrations decreased significantly in response to Fe defi-

Table 3. Activities of phosphoenolpyruvate carboxylase (PEPC; nmol mg –1 protein min –1) and citrate synthase (CS; nmol mg –1 protein min –1) in root extracts of two kiwifruit genotypes (D1 and Hayward) after 14 days of growth under Fe-sufficient (+Fe) or Fe-deficient (–Fe) conditions. Data are means of six replicates. PEPC

Days of treatment 2

14

Genotype (G) D1 Hayward Significance

CS

+Fe

–Fe

8.6 10.0

19.7 12.6

Treatment (T) +Fe –Fe Significance G × T Interaction SEM2 1

2

6.6 6.4 ns 6.0 7.0 ns

* 2.7

ns

Abbreviation and symbol: ns, *, = not significant and significant at 5% level, respectively. SEM = standard error of the interaction means.

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Phosphoenolpyruvate carboxylase activity in root extracts increased significantly in response to Fe deficiency in D1 (by 130%) but not in Hayward (Table 3). In contrast, citrate synthase activity in root extracts did not increase significantly with Fe deficiency and was not influenced by genotype. Protein concentrations were significantly higher in root extracts of Fe-deficient plants than in root extracts of Fe-sufficient controls (1.7 versus 1.0 mg g FW –1 on average).

9

Genotype (G) D1 Hayward Significance

2

Phosphoenolpyruvate carboxylase and citrate synthase

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Table 4. Effects of genotype (D1 and Hayward) and Fe supply (with (+Fe) and without Fe (–Fe)) on the concentration (mg g –1 DW) of organic acids in the roots. Data are means of six replicates. Malic acid

Citric acid

Succinic acid

Quinic acid

Genotype (G) D1 Hayward Significance

0.88 0.63 ns

2.27 1.23 *

0.27 0.23 ns

0.50 0.22 *

Treatment (T) +Fe –Fe Significance

0.71 0.80 ns1

1.08 2.29 *

0.23 0.26 ns

0.27 0.44 ns

G × T Interaction

ns

ns

ns

ns

1

Abbreviation and symbol: ns, * = non significant and significant at 5% level, respectively.

Table 6. Effects of genotype (D1 and Hayward) and Fe supply ((+Fe) and without Fe (–Fe)) on the pH and the concentration (µM) of organic acids in xylem sap. Data are means of six replicates. Abbreviation: Asc. = Ascorbic. pH

Genotype (G) D1 Hayward Significance

+Fe

–Fe

+Fe

–Fe

6.64 6.17

6.28 6.39

210 300

380 360

Treatment (T) +Fe –Fe Significance G × T Interaction SEM2 1

2

ciency in D1 but not in Hayward.

Malic acid

**1 0.12

* 60

Citric acid Asc. acid

140 160 ns

49 105 **

90 180 *

72 78 ns

ns

ns

Abbreviation and symbols: ns, *, ** = non significant and significant at 5 and 1% levels, respectively. SEM = standard error of the interaction means.

The effect of Fe deficiency on xylem pH depended on the genotype, causing a pH decrease in D1 and a pH increase in Hayward (Table 6). In Fe-deficient and Fe-sufficient plants of both genotypes, the predominant organic acid in the xylem sap was malic acid, followed by citric and ascorbic acids (Table 6). Iron deficiency led to a 2-fold increase in the concentration of citric acid in the xylem sap of both genotypes. The concentration of malic acid increased significantly in D1 but not in Hayward (Table 6). We observed similar effects of genotype and Fe status on minor organic acids (cis-aconitic, 2-oxoglutaric, fumaric and oxalic). In D1 plants, the concentration of minor organic acids ranged from 2 to 6 µM and from 4 to 11 µM for

Table 5. Effects of genotype (D1 and Hayward) and Fe supply (with (+Fe) and without Fe (–Fe)) on the concentration (mg g –1 DW) of organic acids in the leaves. Data are means of six replicates. Acid Malic

Genotype (G) D1 Hayward Significance

Citric

+Fe

–Fe

3.45 2.99

2.28 3.22

Treatment (T) +Fe –Fe Significance G × T Interaction SEM2 1

2

*1 0.27

Succinic Quinic

1.99 2.36 ns

0.61 0.79 ns

1.86 2.33 ns

0.71 0.70 ns

ns

ns

+Fe

–Fe

8.25 6.46

5.56 7.68

* 0.85

Abbreviation and symbol: ns, * = non significant and significant at 5% level, respectively. SEM = standard error of the interaction means.

+Fe and –Fe plants, respectively (data not shown). Concentrations of ascorbic acid did not increase in response to Fe deficiency, but were higher in Hayward than in D1, indicating that the Hayward genotype was more susceptible to Fe deficiency, independently of Fe supply (Table 6).

Discussion In both genotypes, Fe deficiency (–Fe) caused a large decrease in leaf chlorophyll concentration. The treatment effect increased between Days 2 and 9 and then leveled off at Day 14 (Table 1). Although leaf chlorophyll concentrations of Hayward and D1 were similarly affected by Fe deficiency, at Day 14, the two genotypes showed a differential response with respect to the effects of Fe deficiency on root enzymatic activities (Tables 2 and 3). The tolerant genotype D1 developed a high and persistent induction of FCR activity (Table 2) and a marked stimulation of PEPC activity in response to Fe deficiency (Table 3), whereas these responses were less pronounced in the susceptible Hayward genotype. Root FCR activity, with and without Fe supply, was much higher in the tolerant genotype D1 than in the susceptible Hayward genotype, and probably represents the most important mechanism for providing tolerance to Fe deficiency in D1. Iron deficiency elicited a stimulation of root FCR activity in both genotypes between Days 2 and 9. Withdrawal of Fe from the nutrient solution also increased root FCR activity and the increase persisted until Day 14 in D1 (Table 2). A rapid decline in FCR activity following its induction in response to Fe deficiency has been described in Lycopersicon esculentum Mill. (Brown and Jolley 1988, Zouari et al. 2001), Ficus benjamina L. (Rosenfield et al. 1991), several Prunus spp rootstocks (Romera et al. 1991, Gogorcena et al. 2000), Phaseolus vulgaris L. (Schmidt 1993) and Arabidopsis thaliana L.

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Xylem sap analysis

FE DEFICIENCY IN KIWIFRUIT

(2.98 versus 1.45 mg g –1 DW), suggesting that tolerance to Fe deficiency in kiwifruit also depends on changes in root citrate concentrations. Increases in citrate concentrations may be a consequence of enhanced glycolysis leading to enhanced proton extrusion and FCR (Bienfait 1996). Iron deficiency also resulted in a decrease in xylem sap pH in the tolerant genotype D1 (Table 6), whereas it caused an increase in xylem sap pH in the sensitive genotype Hayward. This difference between genotypes may be associated with differing activities of proton pumps in the xylem parenchyma cells. A decrease in xylem sap pH in response to Fe deficiency has been found in Fe-efficient species Vicia faba L. (Kosegarten et al. 1998) and Beta vulgaris L. (López-Millán et al. 2000a). Kosegarten et al. (1999) reported that an increase in the pH of the leaf apoplast hampers Fe(III) reduction and consequently causes a substantial amount of Fe to be trapped outside the leaf cells. This might be the case with kiwifruit, because significant re-greening of chlorotic leaves occurs after leaves are sprayed with dilute acids (Tagliavini et al. 1995). We conclude that tolerance to Fe deficiency in kiwifruit is associated with (1) a high and persistent induction of FCR, (2) a stimulation of PEPC in the roots, and (3) more efficient use of malate and quinate in leaves. In plants growing in calcareous soils with high bicarbonate concentrations (Boxma 1982), a shift in the relative contribution of photosynthesis and dark fixation of carbon may occur. Fixation of C, which is normally negligible (Vapaavuori and Pelkonen 1985), may become relatively important in Fe-deficient plants (Rombolà 1998, López-Millán et al. 2000a, 2000b). The role of enhanced root PEPC activity as a mechanism to cope with Fe deficiency in calcareous soils deserves further research. Acknowledgments Authors gratefully acknowledge the help of Ms. Lucia Cabrini with the gas chromatographic analysis. The study was supported by grants AIR3-CT94-1973 to B.M., W.B. and J.A., MURST ex 40% (Roma, grant Cofin 1998) to B.M. and MURST-Ministerio del Ciencia y Tecnología “Azioni Integrate/Acción integrada” (HI-1999-0158) to J.A. and M.T. The Centro Nazionale delle Ricerche (Italy) provided financial support for travel to A.D. Rombolà. The authors thank Anunciación Abadía and Yolanda Gogorcena for reading the manuscript and providing helpful comments. Special thanks to Harald Kosegarten for final critical reading.

References Abadía, J., A.F. López-Millán, A. Rombolà and A. Abadía. 2002. Organic acids and Fe deficiency: a review. Plant Soil. In press. Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1–15. Bartolozzi, F., G. Bertazza, D. Bassi and G. Cristoferi. 1997. Simultaneous determination of soluble sugars and organic acids as their trimethylsilyl derivatives in apricot fruits by gas-liquid chromatography. J. Chromatogr. A 758:99–107. Bialczyck, J. and Z. Lechowski. 1992. Absorption of HCO−3 by roots and its effect on carbon metabolism of tomato. J. Plant Nutr. 15: 293–312.

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(Moog et al. 1995). Two mechanisms could be responsible for the decrease in FCR with time. First, the signal eliciting the FCR increase could be overruled by other physiological consequences of Fe deficiency (Grusak and Pezeshgi 1996). Second, the FCR increase could be induced by only a narrow range of Fe concentrations in the nutrient solution (Zouari et al. 2001). In both genotypes, FCR activity of +Fe plants slightly increased between Days 2 and 9 (Table 2), perhaps indicating that a temporary Fe-stress response also occurred in +Fe plants. The kiwifruit genotypes differed in root PEPC activity, which was stimulated by Fe deficiency in D1 only (Table 3). A similar response has previously been observed in Capsicum annuum L. (Landsberg 1986), Cucumis sativus L. (Rabotti et al. 1995) and Beta vulgaris L. (López-Millán et al. 2000b). Increased PEPC activity may enable roots (by the pH-stat mechanism; Davies 1973, 1979) to avoid increases in cytoplasmic pH caused by acidification of the rhizosphere, one of the common responses of Strategy I species to Fe deficiency. Rhizosphere acidification has been reported to occur in kiwifruit (Vizzotto et al. 1997). The composition of organic acids in the xylem sap did not differ significantly between genotypes (Table 6). The increase in root PEPC activity of Fe-deficient D1 plants (Table 3) was associated with increases in malate and citrate concentrations in the xylem. This suggests that a significant proportion of the organic acids produced by way of root PEPC were loaded into the xylem, as has been shown in studies with labeled C (Bialczyck and Lechowski 1992, 1995). In kiwifruit, it is likely that root malate concentrations are kept low as a result of loading into the xylem, thereby avoiding PEPC inhibition by malate (e.g., Guern et al. 1983, López-Millán 2000b). Because similarly high concentrations of malate and citrate were measured in the xylem sap of Hayward (Table 6), it is possible that the flow of organic acids from roots to leaves in the xylem helps prolong the survival of chlorotic shoots. The increases in concentrations of minor organic acids observed in roots of D1 presumably reflected changes in TCA cycle enzymatic activities caused by alterations in other side-reactions in response to Fe deficiency. We obtained circumstantial evidence that 25 µM Fe in the growing medium was sufficient to meet the Fe demands of D1 plants but might have caused some Fe deficiency stress in Hayward plants. If true, this would partly explain the relatively high concentration of malate in the xylem of +Fe Hayward plants. The concentrations of citrate in roots (Table 4) and xylem sap (Table 6) significantly increased in both genotypes in response to Fe deficiency. Increased citrate concentrations in different plant parts is a typical plant response to Fe deficiency (De Vos et al. 1986, Bienfait 1996). Citric acid could chelate Fe(III) in the xylem (Tiffin 1966, Clark et al. 1973, LópezMillán et al. 2000b). The CS activity in root extracts did not change significantly either between treatments or genotypes (Table 3). Nevertheless, in response to Fe deficiency, root citrate concentrations were twice as high in D1 as in Hayward

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