Nitrate improves growth in salt-stressed citrus seedlings through effects on photosynthetic activity and chloride accumulation

Tree Physiology 24, 1027–1034 © 2004 Heron Publishing—Victoria, Canada Nitrate improves growth in salt-stressed citrus seedlings through effects on p...
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Tree Physiology 24, 1027–1034 © 2004 Heron Publishing—Victoria, Canada

Nitrate improves growth in salt-stressed citrus seedlings through effects on photosynthetic activity and chloride accumulation DOMINGO J. IGLESIAS,1 YOSEPH LEVY,2 AURELIO GÓMEZ-CADENAS,3 FRANCISCO R. TADEO,1 EDUARDO PRIMO-MILLO1 and MANUEL TALON1,4 1

Departamento de Citricultura y Otros Frutales, Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial, E-46113 Moncada, Valencia, Spain

2

Gilat Experiment Station, Mobile Post Negev 85-280, Israel

3

Departamento de Ciencias Experimentales, Universitat Jaume I, Campus Riu Sec., E-12071 Castelló, Spain

4

Corresponding author ([email protected])

Received January 5, 2004; accepted January 28, 2004; published online July 1, 2004

Summary We analyzed the effects of nitrate availability on growth of Navelina (Citrus sinensis (L.) Osbeck) scions grafted on three citrus rootstocks differing in salt tolerance: Carrizo citrange (Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.), Citrus macrophylla Wester and Cleopatra mandarin (Citrus reshni Hort. ex Tanaka). Salt stress reduced total plant biomass by 27–38%, whereas potassium nitrate supplementation partially counteracted this effect by increasing dry matter and new leaf area. Salinized Carrizo citrange had the greatest response to nitrate supplementation, whereas the effects on salinized Cleopatra mandarin and C. macrophylla were less apparent. Nitrogen and chlorophyll contents and photosynthetic activity also increased in leaves of the nitrate-supplemented salinized plants. In salinized plants, nitrate supplementation reduced leaf abscission, stimulated photosynthetic activity and increased growth of new leaves. The nitrate treatment did not modify chloride concentration in leaves, but it reduced chloride concentrations in Carrizo and Macrophylla roots. Therefore, in both rootstocks, chloride content was similar in mature leaves, higher in immature leaves and lower in roots of the nitrate-supplemented salinized plants compared with salinized plants unsupplemented with nitrate. We suggest that the nitrate-induced stimulation of growth reduced chloride concentration in roots through the reallocation of chloride to new leaves. Keywords: Carrizo citrange, Citrus macrophylla, Cleopatra mandarin, gas exchange, Navelina, nitrogen, rootstocks, salinity.

Introduction In citrus, salt stress decreases photosynthetic activity, transpiration, stomatal conductance and root hydraulic conductivity, resulting in growth reduction (Romero-Aranda et al. 1998,

Moya et al. 1999). When photoassimilate production of citrus plants becomes limiting under saline conditions, a wide range of plant nutritional deficiencies are apparent (Alva and Syvertsen 1991). Leaf abscission may also contribute to the salt-induced inhibition of growth (Gómez-Cadenas et al. 1998). Current evidence indicates that chloride accumulation is the principal cause of salt toxicity in citrus (Moya et al. 2003). It has been suggested that salt tolerance is associated mainly with a reduction in chloride uptake or transport (Storey and Walker 1987, Storey 1995, Moya et al. 1999). Because chloride uptake depends on the rootstock’s ability to exclude chloride, salt tolerance in citrus is intimately linked to rootstock characteristics (Levy et al. 1999, Levy and Syvertsen 2003, Moya et al. 2003). It is well known that the mineral composition of soil modifies the response of citrus trees to salinity (Zekri and Parsons 1990, Romero-Aranda et al. 1998, Moya et al. 1999). Nitrate and other nitrogen-derived compounds like urea or ammonium have marked beneficial effects on the growth response of different tree species to salt stress, including apple (e.g., El Siddig and Ludders 1994a, 1994b) and citrus (Bañuls et al. 1990, Romero-Aranda and Syvertsen 1996). Such beneficial effects may be independent of chloride accumulation in the tree, but it has been suggested that nitrate mitigates the detrimental effects of salinity by reducing chloride uptake (Romero-Aranda and Syvertsen 1996, Cerezo et al. 1999, Tyerman and Skerrett 1999). However, the physiological role that nitrate plays in reducing detrimental effects of salinization in citrus remains controversial. Two mechanisms have been postulated: a chloride dilution effect as a result of growth stimulation; and an anionic antagonism between nitrate and chloride uptake. We evaluated these hypotheses by studying the responses of Navelina citrus seedlings grafted on three citrus rootstocks, Carrizo citrange (salt sensitive), C. macrophylla (intermediate salt sensitivity) and Cleopatra mandarin (salt tolerant) and treated with salt and nitrate.

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Materials and methods Plant material and treatments The experiments were performed with 1-year-old potted plants of Navelina orange scion (Citrus sinensis (L.) Osbeck) grafted onto either Carrizo citrange (Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.), Citrus macrophylla Wester or Cleopatra mandarin (Citrus reshni Hort. ex Tanaka) rootstock. The salt tolerance ranking of the rootstocks were: Cleopatra mandarin > C. macrophylla > Carrizo citrange (Chapman 1968, Bañuls et al. 1990, Levy et al. 1999). Uniformly sized scions (30 per rootstock) were selected, transplanted from commercial soil to a mixture of sand and turf (85:15), and placed in a greenhouse providing a day/night temperature of 24–29/16–18 °C. Trees were irrigated three times per week for 30 days with half-strength Hoagland’s solution (Bañuls et al. 1997). Ten trees per rootstock were then assigned to control, salinity and salinity + nitrate supplementation treatments. Control trees were irrigated three times per week with water. Trees in the salinity treatment were irrigated three times per week with water containing 25 mM NaCl:CaCl2 (15:1). Trees in the salinity + nitrate supplementation treatment were irrigated three times per week with water containing 25 mM NaCl:CaCl2 plus 10 mM KNO3. Nitrogen was supplied as a potassium salt to equilibrate the ionic imbalances induced by excess sodium and calcium in the soil (Liu and Liu 1992, Bañuls et al. 1997). After 60 and 90 days, seven plants per treatment and rootstock were randomly selected for analyses. Because the responses were similar for both the 60- and 90-day treatments, data are presented for the 60-day period only. Growth measurements At the end of the experiments, leaves from all plants were separated into mature (present before the salt treatment) and immature (produced during the experimental period and corresponding to the summer flush) leaves. Leaves were counted, weighed and washed with distilled water. Leaf area was measured with a Li-Cor 3100 (Li-Cor, Lincoln, NE) leaf area meter. Fibrous roots were collected and washed. Washed roots and leaves were lyophilized and then weighed. Dry material was ground to a powder for analyses. Photosynthesis measurements A week before the end of the experiments (52 days after the onset of treatments), net CO2 assimilation rates were determined with a Li-Cor LI-6400 portable photosynthetic system equipped with an 18 cm3 prismatic leaf chamber that included a Gallium Arsenide Phosphide (GaAsP) photosynthetic photon flux (PPF) sensor. All measurements were performed at a constant airflow rate of 500 µmol s–1, and at ambient humidity and CO2 concentration. Photosynthetic photon flux was adjusted to 750 µmol m –2 s –1, the minimum saturating value as in preliminary experiments (Iglesias et al. 2002). Within the cuvette, mean temperature was 23.0 ± 0.5 °C and the leaf-to-

air vapor pressure difference was 1.6 ± 0.2 kPa. Determinations were performed in the greenhouse during three consecutive days. Each photosynthetic determination consisted of a minimum of 25 measurements of different leaves per treatment. Measurements were always carried out on mature leaves (present before the onset of the treatments) because these leaves had higher photosynthetic rates than immature leaves (Iglesias et al. 2002). The LI-6400 was programmed to perform continuous determinations on a single leaf, until the stored data showed a coefficient of variation of less than 1%. Once this value was reached, the mean of the whole dataset was taken as the best estimate of the photosynthetic rate of each leaf. Within a treatment, there were no statistical differences in net CO2 assimilation rates among the 3 days. Chloride analyses Chloride content was estimated according to Gilliam (1971). Chloride was extracted from 500 mg dry mass of leaf or root tissue with 0.1 mol dm –3 HNO3 in 10% (v/v) glacial acetic acid. Samples were incubated overnight at room temperature and then filtered. Chloride concentration was determined by silver ion titration (Moya et al. 1999). At least three independent extractions were performed per treatment and rootstock. Nitrate and nitrogen determinations Nitrate was estimated according to Sempere et al. (1993). In general, nitrate was extracted from 100 mg dry mass of leaf or root tissue in 20 ml of aqueous solution saturated with calcium sulfate, and quantified through the maximum value of the second derivative of the absorbance spectrum of the extract. At least three independent extractions were performed per treatment and rootstock. Total nitrogen was determined as described by GómezCadenas et al. (2000). Samples were extracted according to the semi-micro Kjeldahl method (Bremner 1965), using sulfate-selenium catalysis and steam distillation. Chlorophyll determinations After completion of the gas exchange measurements, the same leaves were used for each chlorophyll extraction (n = 10). Three 8-mm diameter discs per leaf were extracted with N,N-dimethylformamide for 72 h in the dark at 4 °C. Chlorophyll absorbance was determined with a Shimadzu UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan) at 647 and 664 nm. Total chlorophyll concentration was calculated based on the absorbance values following Moran (1982). Statistical analyses Treatment effects were evaluated by analyses of variance, and comparisons of means were made by the Least Significant Differences (LSD) method (P < 0.05). Statistical analyses were performed using StatGraphics Plus for Windows, Version 2.1. (Statistical Graphics, Englewood Cliffs, NJ).

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Table 1. Effect of nitrate supplementation on growth (measured as dry mass) and leaf abscission (%) in Navelina seedlings grafted on Carrizo citrange, C. macrophylla or Cleopatra mandarin left untreated (CT = control), or treated for 60 days with 25 mM NaCl:CaCl2 (SL) or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (SL + NI). Values are means of at least three independent determinations. For each rootstock, means within each column followed by the same letter do not differ significantly at P ≤ 0.05. Treatment

Carrizo citrange

Citrus macrophylla

Cleopatra mandarin

Total dry mass per plant (g) CT SL SL + NI

73.4 a 45.6 c 61.8 b

69.7 a 48.3 c 58.9 b

56.0 a 41.1 c 50.7 b

Leaf abscission (%) CT SL SL + NI

0.0 c 32.4 a 20.2 b

0.0 c 21.0 a 15.1 b

0.0 c 28.2 a 12.6 b

Results Nitrate supplementation increased growth and reduced leaf abscission in salinized citrus seedlings Salinity reduced growth in Navelina orange scions grafted on three rootstocks differing in salinity tolerance (Table 1). The smallest reduction in whole plant dry biomass (27%) was in grafts with Cleopatra mandarin. The largest reduction (38%) was in grafts with Carrizo citrange. Nitrate supplementation always increased total plant biomass of salinity-treated seedlings (22–36%). During the experiment, only mature adult leaves abscised as a result of salt stress (Table 1). After 60 days of treatment, the proportion of leaves remaining was higher on seedlings with supplemental nitrogen than those without (12–16% in grafts with Carrizo and Cleopatra, and 6% in grafts with C. macrophylla). Figure 1 shows the mean dry mass of leaves and fibrous roots produced per plant during the 60-day experiment. Dry mass of mature leaves (i.e., leaves present before the beginning of the experiments) in salt treated seedlings was unaffected by nitrate supplementation. The dry mass of immature leaves produced by salinity-treated Navelina orange grafted on Carrizo citrange during the salt treatment was only 0.2 g, an amount that was increased more than 30-fold by nitrate supplementation. This effect was mostly the result of increased growth of individual shoots rather than an increase in the number of shoots produced. Nitrate supplementation increased the dry mass of immature leaves in salt treated Navelina on C. macrophylla and Cleopatra mandarin by 35 and 40%, respectively. Fibrous root production of salinized plants was unaffected by nitrate supplementation. Treatment effects on leaf area Nitrate supplementation did not modify total leaf area of mature leaves in salt treated Navelina orange on any rootstock, but it increased immature leaf area (Figure 2) by more than 150- 2.5- and 4-fold, in grafts with Carrizo, C. macrophylla and Cleopatra mandarin, respectively.

Figure 1. Dry mass per plant (g) of leaves and roots from Navelina seedlings grafted on Carrizo citrange (CA), C. macrophylla (MA) or Cleopatra mandarin (CL) treated for 60 days with 25 mM NaCl:CaCl2 (solid bars), or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (hatched bars) (empty bars = control plants). Data are means of 10 plants and bars show standard errors. For each rootstock, different letters above the error bars indicate significant differences (P ≤ 0.05).

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Thus, nitrate supplementation resulted in differences among root stocks in leaf dry mass per unit of area, the effect being confined to young leaves (Figure 3). Increases were from 200 to 1000 mg cm –2 in grafts with Carrizo citrange, from 215 to 375 mg cm –2 in grafts with C. macrophylla and from 180 to 530 mg cm –2 in grafts with Cleopatra mandarin. Photosynthetic activity and leaf chlorophyll concentration Salt-stressed plants had lower photosynthetic rates than controls (Figure 4A) (cf. Romero-Aranda et al. 1997, 1998). Nitrate supplementation increased photosynthetic rates of mature leaves of salt-treated Navelina orange by 25–30% in scions grafted on Carrizo citrange or C. macrophylla, and by 66% in scions grafted on Cleopatra mandarin (Figure 4A). Chlorophyll concentration of mature leaves of salt-treated Navelina orange always increased significantly in response to nitrate supplementation (15–25%, Figure 4B). Leaf chloride content in immature leaves and fibrous roots

Figure 2. Total leaf area per plant (cm 2 ) of Navelina seedlings grafted on Carrizo citrange (CA), C. macrophylla (MA) or Cleopatra mandarin (CL) treated for 60 days with 25 mM NaCl:CaCl2 (solid bars), or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (hatched bars) (open bars = control plants). Data are means of 10 plants and error bars show standard errors. For each rootstock, different letters above the bars indicate significant differences (P ≤ 0.05).

Figure 3. Dry mass per unit leaf area of Navelina seedlings grafted on Carrizo citrange (CA), C. macrophylla (MA) or Cleopatra mandarin (CL) treated for 60 days with 25 mM NaCl:CaCl2 (solid bars), or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (hatched bars) (open bars = control plants). Data are means of 10 plants and error bars show standard errors. For each rootstock, different letters above the bars indicate significant differences (P ≤ 0.05).

Chloride was undetectable in control grafts. Salt treatment caused chloride to accumulate in both mature and immature leaves of all grafts (Figure 5) in amounts that were not significantly affected by nitrate supplementation. In contrast, nitrate

Figure 4. Net assimilation rates (A) and total chlorophyll concentration (B) in mature leaves from Navelina seedlings grafted on Carrizo citrange (CA), C. macrophylla (MA) or Cleopatra mandarin (CL) treated for 60 days with 25 mM NaCl:CaCl2 (solid bars), or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (hatched bars) (open bars = control plants). Each photosynthetic determination is the mean of n = 25 measurements per treatment and rootstock. Chlorophyll extractions were performed on randomly selected leaves after photosynthetic measurements. Values are means of at least three independent extractions. Error bars show standard errors. For each rootstock, different letters above the bars indicate significant differences (P ≤ 0.05).

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supplementation significantly inhibited chloride accumulation in fibrous roots of salt-treated Navelina orange grafted on Carrizo citrange and C. macrophylla, but not Cleopatra mandarin. Nitrate supplementation had no effect on the total chloride content of mature leaves, but it increased the chloride content of immature leaves (Figure 6), reflecting the increase in biomass of young leaves in nitrate-supplemented plants (Figure 1). Nitrate supplementation had no effect on fibrous root biomass (Figure 1), so that the effect of nitrate supplementation on total chloride content of fibrous roots was comparable to its effects on chloride concentration (Figure 6). Nitrate concentration increased in roots of all nitrate-supplemented salt-treated plants (Table 2). In leaves, nitrate was undetectable (data not shown), although nitrogen concentrations increased in response to nitrate supplementation (Table 3). Nitrate supplementation induced nitrogen accumula-

Figure 5. Leaf and root chloride concentrations in Navelina seedlings grafted on Carrizo citrange (CA), C. macrophylla (MA) or Cleopatra mandarin (CL) treated for 60 days with 25 mM NaCl:CaCl2 (solid bars), or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (hatched bars). In control plants, chloride was undetectable. Chloride determinations were made at the end of the treatments and values are means of at least three independent extractions. Error bars show standard errors. For each rootstock, different letters above the bars indicate significant differences (P ≤ 0.05).

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tion in roots of all three rootstocks and in mature and immature leaves of Navelina on Carrizo citrange and Cleopatra mandarin. Although a similar pattern was observed in leaves of Navelina on C. macrophylla, the differences were not statistically significant. Total nitrogen content per plant was significantly higher in all nitrate-supplemented plants, with salttreated plants on Carrizo citrange showing the greatest increase. Discussion Salt stress produces a wide variety of detrimental effects in tree species including reductions in growth and gas exchange parameters, foliar damage and, eventually, leaf abscission (Levy and Syvertsen 2003). Various aspects of reproduction including flowering, fruit set and yield are affected by salt stress (Howie and Lloyd 1989). In citrus, detrimental effects of

Figure 6. Leaf and root chloride content in Navelina seedlings grafted onto Carrizo citrange (CA), C. macrophylla (MA) or Cleopatra mandarin (CL) treated for 60 days with 25 mM NaCl:CaCl2 (solid bars), or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (hatched bars). In control plants, chloride was undetectable. Chloride contents were calculated from data presented in Figures 1 and 5. Error bars show standard errors. For each rootstock, different letters above the bars indicate significant differences (P ≤ 0.05).

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Table 2. Nitrate concentrations in fibrous roots from Navelina seedlings grafted on Carrizo citrange, C. macrophylla or Cleopatra mandarin left untreated (control = CT), or treated for 60 days with 25 mM NaCl:CaCl2 (SL) or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (SL + NI). Values are means of at least three independent extractions. For each rootstock, means within each column followed by the same letter do not differ significantly at P ≤ 0.05. Treatment

CT SL SL + NI

−1 Nitrate concentration (mg g DW )

Carrizo citrange

Citrus macrophylla

Cleopatra mandarin

< 0.1 b < 0.1 b 8.3 a

< 0.1 b 0.2 b 7.1 a

< 0.1 b < 0.1 b 5.4 a

salt have been related to chloride accumulation (Moya et al. 1999). However, there is evidence that the severity of the effect of chloride accumulation on growth depends on the quantities of other ions (Bañuls et al. 1992, 1997). We subjected 1-year-old citrus trees to 25 mM NaCl:CaCl2 (15:1), a treatment that has been shown to produce typical salt-stress effects on citrus, especially in plants grafted on salt-sensitive rootstocks (Bañuls et al. 1990, Ruiz et al. 1997, Moya et al. 1999). This treatment combines sodium chloride with calcium chloride in proportions that decrease the detrimental effects of sodium (Bañuls et al. 1992, Ebert et al. 2002), without reducing chloride uptake (Moya et al. 1999). We also supplemented salt-treated plants with 10 mM KNO3, a much higher concentration than that at which nitrate is reported to act as a chloride antagonist (Heuer and Feigin 1993, El Siddig and Ludders 1994a, 1994b, Cerezo et al. 1999). At the applied concentrations, potassium equilibrates the imbalances resulting from the excess of sodium and calcium without affecting plant physiology or reducing chloride uptake

(Bañuls et al. 1997, Moya et al. 1999). Nitrate supplementation improved the performance of salinized plants based on the increase in total plant biomass and the significant reduction in leaf abscission (Table 1). Nitrate-supplementation increased leaf nitrogen (Table 3) and chlorophyll concentrations (Figure 4B) and photosynthetic rates (Figure 4A) of salt-treated plants. Heuer and Feigin (1993) suggested that, under saline conditions, decreases in photosynthetic activity and chlorophyll concentration are a consequence of leaf chloride accumulation. However, nitrate supplementation had no effect on chloride content in mature leaves (Figure 6), although it increased photosynthetic activity (Figure 4A), indicating that its effect on photosynthesis was not mediated by a reduction in leaf chloride concentration (Figure 5). Likewise, the reduction in leaf abscission (Table 1) in response to nitrate supplementation was unrelated to leaf chloride concentration (Figure 6). In citrus, high carbohydrate concentrations are reported to reduce abscission (Gómez-Cadenas et al. 2000). It is possible, therefore, that the increase in photosynthetic rates of salttreated plants supplemented with nitrate reduced leaf abscission by increasing leaf carbohydrate content (Iglesias et al. 2003). However, saline conditions can promote leaf abscission through the induction of ethylene production (Bar et al. 1998, Gómez-Cadenas et al. 1998), whereas high nitrogen availability can inhibit ethylene production (Bar et al. 1998). Nitrate supplementation increased leaf production in salt-treated Navelina orange grafted onto all three rootstocks (Figures 1 and 2). In addition to the direct effect of new leaves on plant growth, the additional leaf biomass will, all other things being equal, have reduced chloride concentration by dilution. The data presented in Figures 5 and 6 are compatible with the assumption that, in salinized plants on Carrizo citrange and in C. macrophylla, the decrease in root chloride concentration that was observed in response to nitrate supple-

Table 3. Nitrogen concentrations of leaves and roots and total nitrogen content per plant in Navelina seedlings grafted onto Carrizo citrange, C. macrophylla or Cleopatra mandarin left untreated (control = CT), or treated for 60 days with 25 mM NaCl:CaCl2 (SL), or 25 mM NaCl:CaCl2 supplemented with 10 mM KNO3 (SL + NI). Data are means of at least three independent extractions. For each rootstock, means within each column followed by the same letter do not differ significantly at P ≤ 0.05. Treatment

Nitrogen concentration (%)

Nitrogen content (mg plant–1 )

Mature leaves

Immature leaves

Roots

Carrizo citrange CT SL SL + NI

2.83 a 2.31 c 2.50 b

2.72 a 2.17 c 2.34 b

2.70 a 2.12 c 2.37 b

52.43 a 16.25 c 35.25 b

Citrus macrophylla CT SL SL + NI

2.77 a 2.24 b 2.37 b

2.75 a 2.18 b 2.30 b

2.71 a 2.04 c 2.43 b

43.25 a 17.36 c 21.76 b

Cleopatra mandarin CT SL SL + NI

2.81 a 2.21 c 2.39 b

2.74 a 2.16 c 2.40 b

2.72 a 2.10 c 2.46 b

54.55 a 21.00 c 25.76 b

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mentation resulted from the re-allocation of root chloride into new leaves. Chloride present in leaves and roots of salt-treated plants was similar whether or not they were nitrate-supplemented, despite greater growth of the nitrate-supplemented trees. This may indicate that chloride was reduced in the nitrate-supplemented trees through an antagonism between chloride and nitrate uptake at the fibrous root–soil interface, as suggested by Cerezo et al. (1999) and Tyerman and Skerrett (1999). In conclusion, nitrate showed two related effects that probably improved the performance of citrus growing under saline conditions. First, nitrate supplementation stimulated photosynthesis and growth and reduced leaf abscission. Second, the nitrogen-induced increase in leaf biomass resulted in chloride dilution, leading to a reduction in chloride concentration, the critical parameter for salt damage. Acknowledgments The authors thank A. Almenar, A. Boix, R. Ibáñez, H. Montón and I. López for help in both field and lab work. We also thank Dr. Carlos Ramos for assistance on nitrate extraction and quantification and F. Legaz, M.C. Prieto and P. Giner for the nitrogen determinations.

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TREE PHYSIOLOGY VOLUME 24, 2004

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