Salinity tolerance of Valencia orange trees on rootstocks with contrasting salt tolerance is not improved by moderate shade

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1 Journal of Experimental Botany Advance Access published September 15, 2006 Journal of Experimental Botany, Page 1 of 10 doi: /jxb/erl121 RESEARCH PAPER Salinity tolerance of Valencia orange trees on rootstocks with contrasting salt tolerance is not improved by moderate shade F. García-Sánchez 1,3, J. P. Syvertsen 1, *, V. Martínez 3 and J. C. Melgar 2 1 University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA 2 Universidad de Córdoba. Dpto. Agronomía, Apdo. 3048, E Córdoba, Spain 3 Centro de Edafologia y Biologia Aplicada del Segura, CSIC, Campus Universitario de Espinardo, Espinardo, E Murcia, Spain Received 17 February 2006; Accepted 14 July 2006 Abstract The effects of shading in combination with salinity treatments were studied in citrus trees on two rootstocks with contrasting salt tolerance to determine if shading could reduce the negative effects of salinity stress. Well-nourished 2-year-old Valencia orange trees grafted on Cleopatra mandarin (Cleo, relatively salt tolerant) or Carrizo citrange (Carr, relatively salt sensitive), were grown either under a 50% shade cloth or left unshaded in full sunlight. Half the trees received no salinity treatment and half were salinized with 50 mm Cl 2 during two 9 week salinity periods in the spring and autumn interrupted by an 11 week rainy period. The shade treatment reduced midday leaf temperature and leaf-to-air vapour pressure deficit regardless of salinity treatments. In non-salinized trees, shade increased midday CO 2 assimilation rate (A CO2 ) and stomatal conductance, but had no effect on leaf transpiration (E lf ). Shade also increased leaf chlorophyll and photosynthetic water use efficiency (A CO2 /E lf ) in leaves on both rootstocks and increased total plant dry weight in Cleo. The salinity treatment reduced leaf growth and leaf gas exchange parameters. Shade decreased Cl 2 concentrations in leaves of salinized Carr trees, but had no effect on leaf or root Cl 2 of trees on Cleo. There were no significant differences in leaf gas exchange parameters of shaded and unshaded salinized plants but the growth reduction from salinity stress was actually greater for shaded than for unshaded trees. Shaded trees on both rootstocks had higher leaf Na 1 than unshaded trees after the first salinity period, and this shade-induced elevated leaf Na 1 persisted after the second salinity period in trees on Carr. Thus, shading did not alleviate the negative effects of salinity on growth and Na 1 accumulation. Key words: Cl 2,CO 2 assimilation, Na 1, stomatal conductance, water use efficiency. Introduction Secondary salinization from irrigation water is a growing worldwide problem as more than 6% of agricultural land has become saline (Ghassemi et al., 1995). Since subtropical citrus is susceptible to freezing temperatures and poor soil drainage, citrus is grown in warm areas with high evaporative demand and where irrigation is required to produce maximum yield (Levy and Syvertsen, 1981). In these areas, many soils and sources of water contain high amounts of salts that can inhibit the growth and yield of salt-sensitive citrus (Murkute et al., 2005). All commercial citrus trees are grafted on rootstocks which can regulate the amount of Cl ÿ and/or Na + accumulated in foliage (Levy and Syvertsen, 2004). For example, the citrus rootstock Cleopatra mandarin is considered to be a Cl ÿ excluder, whereas Carrizo citrange is considered to be a Cl ÿ accumulator but a Na + excluder. Na + exclusion in rootstocks may depend on removing Na + from xylem by an exchange of Na + for K + (Walker, 1986). The Cl ÿ ion is considered to be a more important limitation than Na + * To whom correspondence should be addressed. JMSN@ufl.edu ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 2 of 10 García-Sánchez et al. on citrus growth and yield (Bañuls et al., 1997). The accumulation of Cl ÿ and, thus, relative salt tolerance, has been linked to plant growth, water use (Castle and Krezdorn, 1975; Syvertsen et al., 1989), and transpiration (Moya et al., 1999, 2003). The relationship between Cl ÿ accumulation and water use in citrus is not universal, however, because when water use was reduced during growth at elevated CO 2, leaf Cl ÿ was reduced only in relatively saltsensitive Carrizo seedlings but not in relatively salt-tolerant Cleopatra (García-Sánchez and Syvertsen, 2006). Reduced photosynthesis in well-watered but salt-stressed citrus leaves has been associated with the specific toxicity of Cl ÿ and/or Na + rather than with osmotic stress (Bañuls and Primo-Millo, 1992; Levy and Syvertsen, 2004). Reductions in CO 2 assimilation (A CO2 ) have been attributed to a direct biochemical inhibition of photosynthetic capacity (Lloyd et al., 1987b; García-Sánchez and Syvertsen, 2006) which was more important than reduced stomatal conductance (g s ) in limiting A CO2. Salinity stress often occurs in conjunction with flooding, drought, and/or high temperature stress. Shade can improve the physiological response of the plants to drought (Duan et al., 2005) or to excess boron stress (Sotiropoulos et al., 2004) compared with unshaded plants. In citrus, 50% shade screens reduced excessively high leaf temperatures and leaf-to-air vapour pressure differences (D) at midday such that A CO2, g s, and photosynthetic leaf water use efficiency were increased above that of unshaded leaves (Syvertsen et al., 2003). Thus, salt stress should be reduced by shade. Increases in midday g s by shade were accompanied by decreases in D, however, such that leaf transpiration and whole plant water use were unchanged (Jifon and Syvertsen, 2003a, b). If chloride uptake and transport in citrus are indeed linked to water use (Moya et al., 1999, 2003) and shade has little effect on water use, we hypothesized that growing trees under shade should have little affect on leaf Cl ÿ concentration under salinity stress. Valencia orange trees grafted on two rootstocks with contrasting salinity tolerance, Cleopatra mandarin (relatively salt tolerant) and Carrizo citrange (relatively salt sensitive), were tested to determine their physiological responses to salinity in sun and shade. These responses should yield insights into mechanisms of salinity tolerance in citrus. Since salinity problems in Florida citrus normally occur only in the relatively dry spring and autumn irrigation periods (Syvertsen et al., 1989), the salinity treatment was applied in spring and autumn with an intervening nonsaline summer rainy period that leached any accumulated salts from the soil. Materials and methods Plant culture and treatments The study was carried out at the University of Florida/IFAS Citrus Research and Education Center, Lake Alfred, FL ( N, W; 51 m a.s.l.). Two-year-old Valencia orange (Citrus sinensis L. Osbeck) trees grafted on Cleopatra mandarin (Cleo, C. reticulata Blanco) or Carrizo citrange (Carr, C. sinensis L. Osb3Poncirus trifoliata L.) were used in this experiment. Twenty uniform trees on each rootstock were grown outdoors in plastic containers (5.0 l) filled with native Candler fine soil sand. The trees were watered three times per week with 1.0 l of soluble fertilizer solution (9N 2P 9K), (NO 3 ) 2 Ca and iron-chelate (6%) with an N concentration of 66 mg l ÿ1. The 1.0 l volume of nutrient solution was enough to achieve leaching from the bottom of all containers. The shade treatment was applied from April to November 2003 by placing shade screens on top of 2.2-m-tall PVC frames constructed over the trees. Shade screens were Aluminet-50 (Polysack Plastic Industries, Nir Yitzhak, Israel), a spectrally neutral, aluminized polypropylene shade screen with a mesh size of 633 mm, which transmits about 50% of incident photosynthetically active radiation (PAR). Ten trees on each rootstock were placed under the shade and 10 trees served as an unshaded control. Two salinity treatments, 0 and 50 mm Cl ÿ (NaCl and CaCl 2 ; 3:1), were evaluated on five trees on each rootstock in both full sun under the shade. The salinity treatment was begun at the same time as the shade treatment but the salinity was applied during two 9 week dry periods, 23 April to 24 June and 18 September to 21 November. At the beginning of each period, the salinity treatment was added in increasing increments of 15 mm Cl ÿ per day during two consecutive days (to avoid osmotic shock) and on the third day, salinity was increased by 20 mm Cl ÿ to reach the final concentration of 50 mm Cl ÿ. Although the shade treatment was maintained during the intervening typical summer rainy period (25 June to 17 September), the previously salinized trees were irrigated only as necessary with the standard nutrient solution without salt. The experimental design was a two rootstocks (Cleo and Carr)3 two salt concentrations (0 and 50 mm Cl ÿ )3two light intensities (unshaded and 50% shade) factorial design with five replicate trees in each treatment. Gas exchange and water relations Gas exchange and water relations were measured on selected clear days near the end of each salinity period. Measurements were made on two leaves chosen from the mid-shoot area of each plant, giving 10 replicate leaves per treatment. Net assimilation of CO 2 (A CO2 ), stomatal conductance (g s ), and leaf transpiration (E lf ) were determined with a Li-Cor portable photosynthesis system (LI-6200; Li-Cor Inc., Lincoln, NB, USA) equipped with a well-stirred 0.25 l leaf chamber with constant-area inserts (9 cm 2 ). Leaf temperature (T lf ) was measured using the thermocouple inside the gas exchange cuvette. Intercellular CO 2 concentration (C i ) and photosynthetic water use efficiency (A CO2 /E lf ) were calculated based on the equations of von Caemmerer and Farquhar (1981). All gas exchange measurements were made in the morning from h to h. The measurement conditions within the cuvette are included in the Results section below. Leaf water potential was measured near midday (13.00 h to h) using a Scholander-type pressure chamber (PMS instrument, Corvallis, OR, USA; Scholander et al., 1965) on leaves similar to those used for net gas exchange. Chlorophyll analysis After gas exchange measurements, two leaf discs (0.45 cm 2 each) were sampled from the same leaves avoiding major veins. Chlorophyll was eluted from the discs by submerging them in 2 ml of N,N-dimethylformamide in the dark for at least 72 h. The amount of absorbance was read at 647 nm and 664 nm with a Shimadzu UV-vis spectrophotometer (Model UV2401PC, Shimadzu, Riverwood Drive, Columbia, MD, USA) and used to calculate leaf chlorophyll concentrations according to equations of Inskeep and Bloom (1985).

3 Leaf ion concentration and growth parameters At the end of each salinity period, five leaves per tree were used to analyse leaf Cl ÿ and Na + concentrations. Leaves were briefly rinsed with deionized water, oven-dried at 60 8C for at least 48 h, weighed, and ground to a fine powder. Samples were extracted with a 0.1 N solution of nitric acid and 10% acetic acid. Chloride concentration was measured using a silver ion titration chloridometer (HBI Chloridometer; Haake Buchler, Saddle Brook, NJ, USA). Leaf Na + concentration was determined by a commercial laboratory (Waters Agricultural Laboratory, Camilla, GA, USA). At the end of the experiment, root Cl ÿ and Na + concentrations were also analysed as above. Plants were harvested and total dry weights of leaves, stem, and roots were determined. Total leaf area was measured using a leaf area meter (Li-3000; Li-Cor). Statistical analysis Analysis of variance used two rootstocks3two shade levels3two salinity levels and five replicate plants per treatment. Treatment means were separated by Duncan s multiple range test at P <0.05 using the SPSS statistical package (SPSS, Chicago, IL, USA). Linear regression was used to describe relationships between selected variables and analysis of covariance was used to compare slopes of relationships. Results Tree growth and leaf chlorophyll In non-salinized Cleo, shading increased total plant dry weight (TPDW) by increasing leaf dry weight, but in nonsalinized Carr, TPDW was similar in shaded and unshaded trees (Table 1). Salinity reduced TPDW and leaf dry weight of trees on both rootstocks but reductions in leaf dry weight were greater in unshaded Carr (42%) than in unshaded Cleo trees (24%). In addition, salinity reduced root dry weight in shaded and unshaded Carrizo, and in shaded Cleo but Salinity tolerance of shaded citrus trees 3of10 not in unshaded Cleo. Leaf dry weight of shaded trees on Cleo, however, was reduced similarly (about 59%) by salinity to that of Carr. Root dry weight was reduced by salinity in all the trees except in unshaded Cleo trees. Leaf dry weight/area was decreased by shade but was only decreased by salinity in unshaded Carr trees. After the first 9 weeks of salinity treatment, chlorophyll concentrations in leaves were decreased by salinity (Table 2). Leaf chlorophyll a, b and total chlorophyll, followed similar patterns with respect to treatments. The shade treatment increased leaf chlorophyll in trees on both rootstocks although not significantly so for Carr (rootstock3shade significant at P <0.05). Measurement conditions and leaf gas exchange Shade reduced PAR about 58%, and, at the end of the first 9 weeks of salinity treatment (spring salinization), the mean leaf temperature at midday in unshaded trees was C compared with C in shaded trees (Table 3). Shade also lowered D compared with unshaded leaves. There were significant differences in the two-way interaction shade3salt on A CO2, g s, and A CO2 /E lf. In the non-saline treatment, A CO2, g s, and A CO2 /E lf were significantly increased by shade. Salinity reduced A CO2, g s, and A CO2 /E lf in both unshaded and shaded leaves, but reductions were greater in leaves on Cleo than Carr. In salinized trees, however, the already reduced gas exchange responses were not affected by shade. Leaf transpiration rate (E lf ) was not affected by rootstock or shading but, in shaded Cleo leaves, E lf was reduced by salinity. Salinity increased C i in leaves on both shaded and unshaded Cleo trees and on shaded Carr trees compared with the non-salinized treatment. Shade Table 1. Effects of 31 weeks of shade cover (unshaded versus shade) and two periods of salt treatment [control (0S) versus 50 mm Cl ÿ (+S)] on dry weight of leaves and roots, leaf area, leaf dry weight:area ratio, and shoot:root ratio of Valencia orange trees grafted on Cleopatra mandarin and Carrizo citrange rootstock (n=5 trees) Within each column and each rootstock, values with the same letter are not significantly different at 5%. Ns, *, **, and ***, non-significant differences or significant differences at P <0.05, 0.01, or 0.001, respectively. Rootstock Cover Salt Dry wt leaves (g) Dry wt roots (g) TPDW (g) Leaf area (m 2 ) LDW:area (g m ÿ2 ) Shoot:root ratio Cleopatra Unshaded 0S 52.7 b 31.5 ab b 0.48 c a 3.58 a +S 39.9 c 25.5 bc c 0.35 d a 3.00 a Shade 0S 67.1 a 39.7 a a 0.72 a 93.1 c 3.38 a +S 27.1 d 17.9 cd 75.9 d 0.28 de 94.7 c 3.16 a Carrizo Unshaded 0S 53.5 b 32.6 ab ab 0.52 bc b 3.53 a +S 31.2 cd 18.5 cd 81.1 cd 0.33 de 95.0 c 3.53 a Shade 0S 50.6 b 35.1 a b 0.59 b 85.6 d 2.91 a +S 20.6 d 14.3 d 64.4 d 0.22 e 90.9 cd 3.76 a Analysis of variance Rootstock ** Ns * Ns *** Ns Shade Ns Ns Ns Ns *** Ns Salt *** *** *** *** Ns Ns Rootstock3shade Ns Ns Ns * * Ns Rootstock3salt Ns Ns Ns Ns Ns Ns Shade3salt ** ** * *** Ns Ns Rootstock3shade3salt Ns Ns Ns Ns * Ns

4 4 of 10 García-Sánchez et al. increased C i in salinized trees on Carr, but not significantly so in trees on Cleo. At the end of the second salinity period (autumn salinization), shade again reduced midday leaf temperatures by about 2 8C from about 34.6 to C, and average D from about 3 kpa to 2.4 kpa (Table 4). Overall, stomatal conductance and E lf were lower during this autumn Table 2. Effects of cover (unshaded and shaded) and salt treatment, control (0S) and 50 mm Cl ÿ (+S), on mean (n=5) leaf chlorophyll concentration (g m ÿ2 ) of Valencia orange trees grafted on Cleopatra mandarin and Carrizo citrange rootstocks after 9 weeks of the shade and salt treatments Within each column and each rootstock, values with the same letter are not significantly different at 5%. Ns, *, **, and ***, non-significant differences or significant differences at P <0.05, 0.01, or 0.001, respectively. 9 weeks Cover Salt Chl a Chl b Chl total Cleopatra Unshaded 0S ab de cd +S c e e Shade 0S a a a +S a abc abc Carrizo Unshaded 0S ab bcd bcd +S b de de Shade 0S a ab ab +S ab cd cd Analysis of variance Rootstock Ns Ns Ns Shade *** *** *** Salt ** ** *** Rootstock3shade * * * Rootstock3salt Ns Ns Ns Shade3salt Ns Ns Ns Rootstock3shade3salt Ns Ns Ns sampling day than during the previous spring sampling day as environmental conditions changed and leaves aged. Again, shading tended to increase A CO2, g s, and A CO2 /E lf of non-saline trees on both rootstocks. The salt treatment reduced these net gas exchange characteristics for trees on both rootstocks except for the g s of unshaded Carr trees, which was not significantly reduced by salinity. Shading increased g s and A CO2 /E lf of salinized Carr trees. Leaf transpiration rate was not affected by shading, but salinity decreased E lf of shaded and unshaded Cleo trees and that of unshaded Carr. Salinity consistently increased C i for trees on both Cleo and Carr. Shade increased C i in salinized trees on Cleo, but not in trees on Carr. Pooling gas exchange measurements across all treatments revealed that high leaf temperature increased D and decreased g s (Fig. 1). The slope of g s versus T lf appeared greater for the non-saline than for the salinized trees but these slopes were not significantly different when tested by analysis of covariance at P >0.05. A CO2 and A CO2 /E lf both increased linearly with increasing g s and the slopes were greater (P <0.05) in the non-saline treatment than in the saline treatment (Fig. 2). Although C i was greater for salinized than for the non-saline treatment, the relationship between C i versus g s was not significant as C i was similar over a wide range of g s values. Chloride and sodium concentration in leaves and roots Leaves on Carr trees had higher leaf chloride concentrations than those on Cleo after both salinization periods, but these differences were not significant for shaded trees after Table 3. Effects of cover (unshaded and shaded) and salt treatment, control (0S) and 50 mm Cl ÿ (+S), on mean (n=10) photosynthetically active radiation (PAR), leaf temperature (T lf ), leaf-to-air vapour pressure deficits (D), CO 2 assimilation (A CO2 ), stomatal conductance (g s ), leaf transpiration rate (E lf ), photosynthetic water use efficiency (A CO2 /E lf ), and total internal CO 2 partial pressure (C i ) in leaves of Valencia orange trees grafted on Cleopatra mandarin and Carrizo citrange rootstock The measurements were done on a clear day (22 June 2003) after 9 weeks of shade and salinity treatment. Within each column and each rootstock, values with the same letter are not significantly different at 5%. Ns, *, **, and ***, non-significant differences or significant differences at P <0.05, 0.01, or 0.001, respectively. Rootstock Cover Salt PAR (lmol m ÿ2 s ÿ1 ) T lf (8C) D (kpa) A CO2 (lmol m ÿ2 s ÿ1 ) g s (mmol m ÿ2 ) E lf (mmol m ÿ2 ) A CO2 /E lf (mmol mol ÿ1 ) C i (ll l ÿ1 ) Cleopatra Unshaded 0S 1906 a 37.0 a 3.4 a 5.69 bc 233 bcd 6.21 ab 0.93 b 314 c +S 1786 a 37.6 a 3.6 a 2.70 d 187 e 5.91 ab 0.46 c 333 ab Shade 0S 788 b 34.4 b 2.5 b 9.07 a 365 a 6.81 a 1.34 a 308 c +S 735 b 35.3 b 3.0 b 2.69 d 213 cde 5.38 b 0.47 c 340 a Carrizo Unshaded 0S 2050 a 37.8 a 3.5 a 6.54 b 247 bc 6.49 a 1.01 b 309 c +S 1794 a 37.6 a 3.6 a 4.37 c 203 de 6.17 ab 0.69 c 320 bc Shade 0S 849 b 34.2 b 2.4 b 9.55 a 370 a 6.29 a 1.51 a 312 c +S 718 b 34.7 b 2.7 b 4.15 cd 272 b 5.95 ab 0.68 c 332 ab Analysis of variance Rootstock Ns Ns Ns ** * Ns ** Ns Shade *** *** *** *** *** Ns *** Ns Salt Ns Ns Ns *** *** ** *** *** Rootstock3shade Ns Ns Ns Ns Ns Ns Ns Ns Rootstock3salt Ns Ns Ns Ns Ns Ns Ns Ns Shade3salt Ns Ns Ns *** *** Ns ** Ns Rootstock3shade3salt Ns Ns Ns Ns Ns Ns Ns Ns

5 Salinity tolerance of shaded citrus trees 5of10 Table 4. Effects of cover (unshaded and shaded) and salt treatment, control (0S) and 50 mm Cl ÿ (+S), on mean (n=10) photosynthetically active radiation (PAR), leaf temperature (T lf ), leaf-to-air vapour pressure deficits (D), CO 2 assimilation (A CO2 ), stomatal conductance (g s ), leaf transpiration rate (E lf ), photosynthetic water use efficiency (A CO2 /E lf ), and total internal CO 2 partial pressure (C i ) in leaves of Valencia orange trees grafted on Cleopatra mandarin and Carrizo citrange rootstock The measurements were done on a clear day (7 November 2003) after 31 weeks of the shade treatment at the end of the autumn salinization. Within each column and each rootstock, values with the same letter are not significantly different at 5%. Ns, *, **, and ***, non-significant differences or significant differences at P <0.05, 0.01, or 0.001, respectively. Rootstock Cover Salt PAR (lmol m ÿ2 s ÿ1 ) T lf (8C) D (kpa) A CO2 (lmol m ÿ2 s ÿ1 ) g s (mmol m ÿ2 ) E lf (mmol m ÿ2 ) A CO2 /E lf (mmol mol ÿ1 ) C i (ll l ÿ1 ) Cleopatra Unshaded 0S 1254 a 34.8 a 3.1 a 5.03 ab 82 c 2.41 ab 1.95a 253 c +S 1255 a 34.9 a 3.1 a 2.76 cd 62 d 1.94 c 1.37 b 283 b Shade 0S 616 b 32.9 b 2.5 b 5.77 a 105 ab 2.46 ab 2.27 a 280 b +S 592 b 32.1 b 2.4 b 2.64 cd 80 cd 1.84 c 1.37 b 306 a Carrizo Unshaded 0S 1491 a 34.5 a 2.9 a 4.08 bc 75 cd 2.56 a 1.53 b 261 bc +S 1208 a 34.5 a 3.0 a 1.89 d 69 cd 2.04 bc 0.91 c 309 a Shade 0S 581 b 32.3 b 2.3 b 6.09 a 119 a 2.63 a 2.27 a 273 bc +S 666 b 32.0 b 2.3 b 3.30 bcd 100 b 2.19 abc 1.46 b 308 a Analysis of variance Rootstock Ns Ns Ns Ns * Ns Ns Ns Shade *** *** *** ** *** Ns *** * Salt Ns Ns Ns *** *** *** *** *** Rootstock3shade Ns Ns Ns Ns * Ns * Ns Rootstock3salt Ns Ns Ns Ns Ns Ns Ns Ns Shade3salt Ns Ns Ns Ns Ns Ns Ns Ns Rootstock3shade3salt Ns Ns Ns Ns Ns Ns Ns Ns Fig. 1. Relationship between leaf-to-air vapour pressure deficits (D) and stomatal conductance (g s ) versus leaf temperature (T lf ) in leaves of Valencia orange trees grafted on Cleopatra mandarin or Carrizo citrange for the salinity treatments combined. The linear regression (y=b1x+bo) for each treatment are indicated. ***, Significant slopes at P < Data are from 6 weeks and 9 weeks after the experiment started.

6 6 of 10 García-Sánchez et al. Fig. 2. Relationship between CO 2 assimlation rate (A CO2, lmol m ÿ2 s ÿ1 ), internal CO 2, and photosynthetic water use efficiency (A CO2 /E leaf, lmol mol ÿ1 ) versus stomatal conductance (g s, mmol m ÿ2 s ÿ1 ) in leaves of Valencia orange trees on Cleopatra mandarin or Carrizo citrange. The linear regression (y=b1x+bo) for each treatment is indicated. Ns and ***, Non-significant differences and significant slopes at P <0.001, respectively. Data are from 6 weeks and 9 weeks after the experiment started. the second salinization period (Fig. 3). At the end of the spring salinization, leaf Cl ÿ concentration was greater than at the end of the autumn salinization that followed the leaching rainy period. Shade decreased leaf Cl ÿ in salinized Carr at the end of both salinization periods. At the end of the autumn salinization, shaded Carr trees had significantly lower root Cl ÿ concentration than unshaded trees. Leaves of salinized shaded trees had higher leaf Na + concentration than those of unshaded trees on both rootstocks at the end of the spring salinization treatment (Fig. 3). Overall, leaf Na + and Cl ÿ were lower at the end of the autumn salinization than after the spring salinization. Leaves on Carr trees had a significantly lower leaf Na + concentration than those on Cleo at the end of the spring salinization under shade and at the end of the autumn salinization in full sun. Shade increased leaf Na + in salinized trees except in Cleo at the end of the autumn salinization. There was no rootstock or shade effect on root Na + concentration.

7 Salinity tolerance of shaded citrus trees 7of10 Fig. 3. Effects of soil NaCl (0S=0 mm; +S=50 mm Cl ÿ ) and growing in shade or unshaded conditions on mean (n=5) Cl ÿ and Na + concentration (mg g ÿ1 dry weight) in leaf and root tissue of Valencia orange trees grafted on Cleopatra mandarin or Carrizo citrange. Data are from 6 weeks and 9 weeks after the experiment started. Different letters indicate significant differences at P <0.05 (Duncan s test). Leaf water potential At the end of the both salinization periods, leaf water potential was consistently decreased by salinity in leaves on both rootstocks under both light treatments (Fig. 4). Shade had no significant effect on leaf water potential at the end of the spring salinity period but, at the end of the autumn salinity period, shade decreased leaf water potential in all trees except non-salinized trees on Cleo. Discussion Effect of shading on non-salinized trees Shading trees with 50% screen cloth reduced T lf and D and resulted in higher A CO2, g s, and A CO2 /E lf in non-salinized trees on both Cleo and Carr. Leaf transpiration, however, was affected little by shading. Although g s was higher in shaded leaves, the driving force for transpiration (D) was lower in shaded leaves and, thus, compensated for the higher g s such that E lf was not affected. Despite the significant correlation between g s and A CO2, the shadeinduced increases in A CO2 occurred with no change or a slight increase in C i (in the second set of gas exchange measurements), implying that g s was not the dominant limitation to A CO2 (Farquhar and Sharkey, 1982). High leaf temperatures were apparently more important than g s in limiting A CO2 because, if CO 2 diffusion at low g s in unshaded leaves had been the major limitation to A CO2,a decrease in C i would have occurred with the decrease in A CO2. Excessively high T lf at high PAR can also lead to

8 8 of 10 García-Sánchez et al. Fig. 4. Effects of soil NaCl (0S=0 mm; +S=50 mm Cl ÿ ) and growing in shade or unshaded conditions on mean (n=5) midday leaf water potential W w (MPa) in leaves of Valencia orange trees grafted on Cleopatra mandarin or Carrizo citrange. Data are from 6 weeks and 9 weeks after the experiment started. Different letters within each figure indicate significant differences at P <0.05 (Duncan s test). reductions of A CO2 in unshaded leaves due to an increase in photoinhibition (Jifon and Syvertsen, 2003b). Salinity also had a direct effect on the reduction of A CO2 rather than an indirect effect via lowered g s, as indicated by the consistent increases in C i in salinized trees compared with non-salinized trees at the end of both salinity periods. Similar responses of T lf, D, and gas exchange parameters to shade occurred during both measurement periods in nonsalinized trees on Carr. In trees on Cleo, however, there was no effect of shading on A CO2 at the end of the second period. Thus, after 31 weeks of shading, citrus leaves on Cleo may have acclimated to shade and reduced A CO2 (Syvertsen, 1984). In an experiment with young Murcott citrus trees, shading increased growth by increasing the leaf dry weight during a 3 month shade period (Medina et al., 2002). In the present study, TPDW increased in shaded trees on Cleopatra from increased leaf growth but shaded trees on Carrizo had a similar TPDW to those in full sun. Higher leaf gas exchange of shaded plants is typically only observed during the middle of the day, since PAR can be limiting in early morning and late afternoon (Medina et al., 2002; Syvertsen et al., 2003). Therefore, the positive effect of shading on A CO2 at midday may have been insufficient to increase the overall growth of trees on Carrizo. In addition, at the end of the experiment, leaves of trees on Carr had a lower leaf water potential in shade than in unshaded conditions. This decrease in leaf water status could have negated any effect of shade on growth of trees on Carr. Effect of shading on salinized trees At the end of both the spring and autumn salinization periods, A CO2 of salinized trees was not enhanced by shading, despite the fact that T lf and D were consistently decreased. Stomatal conductance and C i, however, were consistently increased by shade in trees on both rootstocks regardless of salinity. Thus, the salinity-induced decrease in A CO2 was not primarily due to stomatal constraints, but was more likely attributable to direct effects of Cl ÿ or/and Na + ion toxicity (Storey and Walker, 1999; García-Sánchez and Syvertsen, 2006). Leaf Cl ÿ in salinized Carr was reduced by shade, but apparently not enough to affect A CO2. Shaded leaves had higher leaf Na + concentration than unshaded leaves in trees on both rootstocks. Therefore, high Na + could have been responsible for negating any A CO2 response in shaded trees. In salinized Valencia orange trees grafted on Troyer citrange or Cleopatra mandarin, A CO2 inhibition by salinity was more readily attributable to Na + toxicity than to Cl ÿ toxicity (Lloyd et al., 1987a). In the present experiment, reduction of PAR by shading could be a limiting factor for photosynthesis in salinized citrus leaves, especially during the morning and afternoon. Non-optimal growth conditions can even decrease the light saturation point (Liaw and Chen, 1991) by as much as 50% in drought-stressed Plantago leaves (Mudrik et al., 2003). Since shade increased leaf chlorophyll in trees on both rootstocks (Table 2), expressing A CO2 on a leaf chlorophyll basis (A CO2 /chl) resulted in a non-significant effect of shade on A CO2 /chl (data not shown) that previously was significant when A CO2 was expressed on a leaf area basis (Table 3). All other treatment effects on A CO2 were similar, regardless of whether expressed on a leaf area basis or on a leaf chlorophyll basis. Unshaded salinized trees on Cleo had lower leaf Cl ÿ than trees on Carr (Fig. 3), supporting salinity-tolerance differences attributable to these rootstocks (Levy and Syvertsen, 2004). This well-known regulation of leaf Cl ÿ concentration in citrus leaves has been associated with leaf transpiration and total water absorbed per plant (Moya et al., 1999, 2003), shoot:root ratio (Storey and Walker, 1999), and efficiency of the root system for limiting Cl ÿ uptake

9 (Storey and Walker, 1999). In this experiment, the higher exclusion of Cl ÿ from shoots in trees on Cleo than on Carr was more likely related to the ability of roots to restrict the movement of Cl ÿ since their shoot:root ratio, leaf dry weight, and leaf transpiration were similar. In addition, shade decreased leaf Cl ÿ concentration in leaves on Carr without changing leaf transpiration. Thus, leaf Cl ÿ concentration was not necessarily closely linked to water use. The overall growth reduction by salinity in unshaded trees was greater for Carr than for Cleo trees (45% and 28%, respectively), and was related to their relative leaf Cl ÿ concentrations. Such differences in effects of salinity on growth between trees on Cleo and Carr were consistent with earlier findings (García-Sánchez et al., 2002; García- Sánchez and Syvertsen, 2006). Growth reductions by salinity under shaded conditions, however, were greater than those in unshaded conditions and were similar for trees on Cleo and Carr (55 59%). Negative effects of high salinity on strawberry fruit yield (Awang and Atherton, 1995), bean plants (Helal and Mengel, 1981), or melons (Meiri et al., 1982) were also greater under shaded than in unshaded conditions. In the present study, the lower amount of growth of salinized trees under shaded conditions than in full sun could have been due to the greater increase in leaf Na + concentration in shaded trees. This important effect of Na + occurred in spite of the high amount of Ca 2+ in the salinity treatment as high amounts of Ca 2+ can mitigate the negative effects of Na + (Gratten and Grieve, 1992). Leaf Na + concentration of salinized trees on Cleo tended to be higher than on Carr at the end of the spring salinization period, and this difference remained in unshaded trees at the end of the second period. Shade increased leaf Na + in salinized Carr trees by the end of the second salinity period. Shading affected the Cl ÿ and Na + concentration in different ways since leaf Na + concentration was higher for shaded trees on both Cleo and Carr, whereas leaf Cl ÿ concentration was lower for leaves and roots of shaded trees on Carr. So as not to damage roots in the middle of the experiment, root Na + was not sampled at the end of the first salinity period. The decrease in the leaf temperature by shade did not affect the final accumulation of Na + in roots but must have increased root uptake, since Na + transport to leaves was apparently increased. Since shade consistently increased root growth in non-salinized trees on both rootstocks, a greater uptake of Na in shaded trees could have occurred through a transient increase in root growth that was not measured at the end of the experiment. In conclusion, citrus leaves growing in full sun experience high temperatures that decreased midday A CO2, g s, and water use efficiency. Lowering leaf temperature by shading increased chlorophyll, midday A CO2, g s, and A CO2 /E lf but did not affect E lf. Shade did decrease Cl ÿ concentrations in leaves of salinized Carr trees but shade had no effect on Cl ÿ in Cleo. Salinity stress limited the positive effect of shading on net gas exchange of leaves on both rootstocks and negated any effects of shade on growth of Valencia orange trees on Carr. In salt-stressed trees, growth was reduced more under shade than in full sun and leaf Na + was increased more than 2-fold after the spring salinization period. Even though the overall amount of leaf Na + after the autumn salinization period was lower, Carr shade leaves still had twice the Na + as sun leaves. Root Na + in Cleo tended to be higher than in Carr roots and root Na + was higher than leaf Na + in both rootstocks. Although root Na + was not significantly affected by shade, salinized root Na + was consistently reduced by shade. Thus, the redistribution of Na + from roots to leaves under shade conditions may have been responsible for the increase in leaf Na +. This idea is supported by previous studies (García-Sánchez and Syvertsen, 2006) where patterns of changes in Na + and Cl ÿ occurred in opposite directions in roots and leaves. Acknowledgements We thank Jill Dunlop and Eva Ros for their skilled assistance. This research was supported by a fellowship from the Ministerio de Educacion, Cultura y Deportes of Spain (AGL CO4-02/ AGR) and the University of Florida Agricultural Experiment Station. References Salinity tolerance of shaded citrus trees 9of10 Awang YB, Atherton JG Growth and fruiting responses of strawberry plants grown on rockwool to shading and salinity. 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