Effect of rootstock on salinity tolerance of sweet almond

South African Journal of Botany 102 (2016) 50–59

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Effect of rootstock on salinity tolerance of sweet almond (cv. Mazzetto) A. Zrig a, H. Ben Mohamed b, T. Tounekti a,d, H. Khemira a,d,⁎, M. Serrano c, D. Valero c, A.M. Vadel a a

Unité de Recherche Biodiversité et Valorisation des Bioresources en Zones Arides, Faculté des Sciences de Gabès, University of Gabes, Zrig, Gabes 6072, Tunisia Laboratory of Horticulture, Tunisian National Agricultural Research Institute (INRAT), Rue Hédi Karray, 2049 Ariana, Tunisia c Department of Food Technology, University Miguel Hernández, Ctra. Beniel km. 3, 2, 03312 Orihuela, Alicante, Spain d Centre for Environmental Research & Studies (CERS), Jazan University, Jazan, Saudi Arabia b

a r t i c l e

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Article history: Received 20 November 2014 Received in revised form 3 June 2015 Accepted 6 September 2015 Available online xxxx Edited by A.K. Cowan Keywords: Anthocyanins Carotenoids Garnem GF677 Rootstock Sweet almond Polyamines

a b s t r a c t The current work aims to assess whether the rootstock can improve the salinity tolerance of an almond cultivar. The study included a description of the physiological and biochemical adaptations of one-year old scions of Mazzetto grafted onto either GF677 or Garnem rootstocks and exposed to increasing concentrations of NaCl in the growing medium. The plants were watered with a nutrient solution containing 0 mM (control), 25 mM, 50 mM or 75 mM NaCl. There was a significant reduction in shoot extension in response to increasing NaCl concentration in the growing medium especially in the plants grafted onto GF677. The better shoot growth of Mazzetto/Garnem plants appears to be due to their ability to limit the loss of photosynthetic activity by maintaining stomatal conductance and protecting chlorophyll and cytosolic assimilatory enzymes from toxic ions. Leaves of Mazzeto/Garnem had higher gs rates, carotenoids/chlorophyll and anthocyanins/chlorophyll ratios and a better nutritional status (higher K+ and Ca2+ and lower Na+) compared to Mazzeto/GF677. Furthermore, the former maintained higher proline and soluble sugar concentrations and lower leaf water potential. Our results suggest that Garnem offers a degree of protection against salinity which can be exploited in breeding programmes. © 2015 SAAB. Published by Elsevier B.V.

1. Introduction Salinity represents one of the most limiting factors to plant growth and agricultural productivity (Schwarz et al., 2010). Fruit tree species are particularly sensitive to soil salinity. For instance, shoot growth of peach (Tattini, 1990) and almond (Zrig et al., 2011) is suppressed by relatively low concentrations (25 mM NaCl) of salt in the soil solution. Therefore, the development of stress-tolerant crops through the selection and breeding of cultivars able to produce economic yields under saline or drought conditions is vital to minimising the detrimental effects of these stresses (Cuartero et al., 2006). Unfortunately, the genetic complexity of abiotic stress tolerance renders this task extremely difficult (Ashraf and Foolad, 2007). Accordingly, other alternatives such as grafting were considered. For instance, grafting has been used for more than 50 years for vegetable production in many parts of the world whereby elite commercial cultivars were grafted onto selected vigorous rootstocks to improve plant tolerance of adverse environmental conditions (Oztekin et al., 2007; Flores et al., 2010; Lee et al., 2010; Schwarz et al., 2010). Similarly, rootstocks are widely used to improve

Abbreviations: Ψw, water potential; RWC, relative water content; A, photosynthetic assimilation; gs, stomatal conductance; E, transpiration; TSS, total soluble sugar; ROS, reactive oxygen species; TAA, total antioxidant activity. ⁎ Corresponding author. Tel.: +966 537135188. E-mail address: [email protected] (H. Khemira).

http://dx.doi.org/10.1016/j.sajb.2015.09.001 0254-6299/© 2015 SAAB. Published by Elsevier B.V.

the tolerance of woody fruit crops to soil related stresses (Massai et al., 2004; Colla et al., 2008). The ability of woody plants to tolerate salinity varies among species but largely depends on the nature of their root systems (Moya et al., 1999). Another consideration is the rootstock/scion combination, since the response of the grafted plant depends on the functional relationships between the scion, the rootstock and the graft union (Don et al., 1997; Gyu et al., 1997). Rootstocks were shown to improve salt tolerance of plants by reducing Na+ toxicity. Several studies suggest that lower accumulation of Na+ and/or Cl− in the plant's shoots is the main reason for higher salt tolerance of grafted plants such as citrus (Moya et al., 1999) and prunus cultivars (Massai et al., 2004; Zrig et al., 2011). The influence of the rootstock on the concentration of certain minerals in the aerial parts of the plant was attributed to physical characteristics of the root system, such as lateral and vertical development, which results in enhanced or reduced uptake of water and minerals (Heo, 1991; Jang, 1992); this is one of the reasons for the widespread use of rootstocks to overcome salinity (Lee et al., 2010). Others studies demonstrated that higher salt tolerance of grafted plants is closely associated with increased translocation of K+, Ca2+ or Mg2+ to the leaves (Moya et al., 1999; Zhu et al., 2008). High salt concentration in the medium causes ion imbalances, ion toxicity and hyperosmotic stress in plants. As a consequence of these primary effects, secondary stresses such as oxidative damage often

A. Zrig et al. / South African Journal of Botany 102 (2016) 50–59

occur (Zhu, 2001). The resulting reactive oxygen species (ROS) such as superoxide radicals (O− 2 ) and hydrogen peroxide (H2O2) can seriously disrupt metabolism through oxidative damage to lipids, proteins, and nucleic acids (Apel and Hirt, 2004). Plants deploy various physiological and biochemical mechanisms to cope with salt stress. These strategies include accumulation of compatible solutes and osmolytes in the cytosol and activation of antioxidant defence systems (Liu et al., 2007; He et al., 2009). Plants need to maintain their internal water potential below that of the soil in order to maintain turgor and water uptake for growth. This requires an increase in osmotica concentration in the cells either through uptake of inorganic ions or synthesis of metabolically compatible solutes such as sucrose and proline (Munns and Tester, 2008). Although compatible solutes have a high energy construction cost, they are often the best defence at the disposition of the cell against toxic ions. The accumulation of Na+ in the cell can damage membranes and enzymes often leading to premature leaf senescence or even plan death; compatible solutes can delays these detrimental effects (Tester and Davenport, 2003). Plants do have the capability to scavenge or detoxify ROS by producing different types of antioxidants. Such antioxidants were used as markers for salinity tolerance in grafted vegetables. Thus, an efficient antioxidant system is an important factor for the enhanced salt tolerance of grafted plants. This is achieved by obtaining higher activities of anti-oxidative enzymes and higher contents of non-enzymatic antioxidants. The activity of non-enzymatic antioxidant in the leaves of grafted eggplant seedlings were found to be significantly higher than those in self-rooted seedlings under NaCl stress (Liu et al., 2007). Compared with those on the leaves, there have been fewer studies on the antioxidant system in the roots of grafted vegetables under salt stress. Cucumber grafted onto salt tolerant rootstocks has lower root H2O2 content, and higher root SOD, POD, and CAT activities than plants grafted onto salt sensitive rootstocks under NaCl stress (Zhen et al., 2010). In most glycophytic plants like fruit trees, the degree of salinity tolerance depends on the roots' ability to exclude or retain potentially toxic ions. Therefore, the role of the rootstock is crucial in determining the tree's performance under saline conditions (Grattan and Grieve, 1999). The present study investigated the physiological and biochemical mechanisms involved in the reaction of the sweet almond cv. Mazzetto grafted onto two different rootstocks (GF677 and Garnem) to increasing NaCl concentrations in the growing medium. 2. Materials and methods 2.1. Plant material and salt treatments The present study was performed on one year old sweet almond cv Mazzetto grafted onto two rootstocks: GF677 (Prunus amygdalus × Prunus persica) and Garnem GN15 (P. amygdalus cv. Garfi × P. persica cv. Nemared). The plants were cultivated in perforated 4-L plastic pots containing desert-dune sand under controlled conditions (temperature: 25 ± 2 °C; light intensity (PAR): 500 to 700 μmol m−2 s− 1). They were irrigated every 4 days with a complete nutrient solution (N, 1.8 mM; P, 0.35 mM; K, 0.64 mM; Ca, 1.0 mM; Mg, 0.35 mM; S, 0.35 mM; Fe, 0.03 mM; Zn, 0.4 μM; Mn, 5.0 μM; Cu, 0.1 μM and B, 0.02 mM). The salinity stress was obtained by adding NaCl to the nutrient solution to obtain 25, 50 and 75 mM total ion concentrations. Control treatment consisted of no NaCl added. To avoid osmotic shock, the concentration of the nutrient solution was increased by 25 mM per day until the final salinity level for each treatment was reached. From the moment when the final concentration of NaCl was obtained for the most severe stress level, the concentrations of nutrient solutions were kept constant for the following four weeks. Four fully expanded leaves from each plant were harvested in the morning (between 9 and 11 a.m. local time), weighed and divided into two batches; one was frozen in liquid nitrogen and stored at − 80 °C for biochemical analyses; the other was washed in de-ionized water, dried at 80 °C in

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a forced-air oven for 48 h and ground into a fine powder to pass through a 30-mesh screen for ion analyses. 2.2. Growth and leaf water Before the treatments were imposed, the tip of the main shoot of each plant was marked to be able later to assess shoot extension during the period of the experiment. After four weeks of applying saline water, the plants were harvested and divided into stems, leaves and roots. The latter were washed free of soil particles. The material was then dried for 48 h at 80 °C and total dry weights were determined. Percent leaf relative water content (% RWC) was measured according the method described by Kramer and Brix (1965) and calculated according the following equation: RWC ¼ 100 ½ð FW–DWÞ=ðTW−DWÞ; where FW is the fresh weight, DW is the dry weight and TW is the turgid weight. Turgid weight was determined after soaking the leaf samples in distilled water for 24 h at 4 °C in a refrigerator; dry weight was measured after oven drying the samples for 48 h at 80 °C. Leaf water potential (Ψw) was measured on mid-shoot leaves with a pressure chamber using standard methodology (Gucci et al., 1997). Leaf osmotic potential (Ψπ) was measured on expressed sap from frozen and thawed leaves with a vapour pressure osmometer (Wescor 5520, Logan, UT, USA). The osmolality of the sap expressed from leaves was converted to osmotic potential (Ψπ) by the van't Hoff equation: Ψπ = −ciRT (Nobel, 1992). 2.3. Ion analyses For ion analyses, 1 g of dry ground leaves from each plant was extracted with 20 mL of 0.1 M HNO3. After filtration, Na+, Ca2+ , Mg2+ and K+ contents were determined with an atomic absorption spectrometer (Avanta, GBC, Australia). 2.4. Gas exchange measurements Gas exchange measurements were carried out after 30 days of salt treatment. Net photosynthetic rate (A), transpiration rate (E) and stomatal conductance (gs) of upper mature leaves were measured with a portable photosynthesis measurement system (Lcp pro+, ADC Systems Ltd., UK) under ambient conditions (PAR was 500–700 μmol m−2 s−1 and air temperature of 25 ± 3 °C). 2.5. Proline content Frozen leaves or roots (0.2 g) were ground to a fine powder in a precooled mortar with liquid nitrogen then homogenized with 5 mL of 3% aqueous sulfosalicylic acid and centrifuged at 8000 × g for 15 min. Two millilitres of acid-ninhydrin and 2 mL of glacial acetic acid were added to 2 mL of the homogenate in a test tube. The mixture was then incubated at 100 °C for 1 h, after which the reaction was stopped by placing the test tube in an ice bath. Four millilitres of toluene were added to each test tube and vortexed for 20 s. The absorbance at 520 nm of the organic toluene phase containing the chromophore was used to quantify the amount of proline as described by Bates et al. (1973). 2.6. Soluble sugar concentrations Total soluble sugars in the leaves and roots were determined in each replicate according to the method of Robyt and White (1987). Frozen leaves or roots (0.2 g) were extracted in 80% methanol solution. The extract was incubated for 30 min at 70 °C. This extract was used for the estimation of total soluble sugar concentration. Five millilitres of sulphuric

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acid was added to the plant extract and mixed vigorously. Soluble sugar concentrations were estimated at 645 nm with a spectrophotometer (Unicam Helio, Cambridge, UK). 2.7. Determination of chlorophylls and carotenoids Leaf chlorophyll and carotenoid contents were determined by using the method of Arno (1949). In short, 0.5 g of fresh leaves was ground to a fine powder in liquid nitrogen in a pre-cooled mortar and homogenized for 30 s in 5 mL of 95.5% acetone. The pigments' concentrations were estimated from absorbance at 647 nm and 664 nm. A solution of 95.5% acetone was used as a blank. Pigment concentrations were calculated as follows: Chl aðmg=g FWÞ ¼ ½12:7 ðA664Þ 2:69 ðA647Þ Chl bðmg=g FWÞ ¼ ½22:9 ðA647Þ 4:69 ðA664Þ: Total carotenoids were extracted in duplicates according to Mınguez-Mosquera and Hornero-Mendez (1993). One gramme of frozen leaf tissue was briefly extracted with acetone and shaken with diethyl ether and 10% NaCl. Two phases were obtained; the lipophilic phase was washed with Na2SO4 (2%), saponified with 10% KOH in MeOH, and the pigments were subsequently extracted with diethyl ether, evaporated and then made up to 25 mL with acetone. Total carotenoids were estimated by reading the absorbance at 450 nm with a spectrophotometer (UNICAM Helios, Cambridge, UK), and expressed as μg of β-carotene equivalent per 100 mg FW, taking into account the molar absorption coefficient (ε1% cm) of 2560. The results are presented as means ± SE.

for 30 min at 20,000 ×g. A 2-ml aliquot of the supernatant was used to determine free polyamines by benzoylation, and the derivatives were analysed by HPLC according to Serrano et al. (2003). The elution system consisted of MeOH/H2O (64:36) solvent, running isocratically with a flow rate of 0.8 ml min−1. The benzoylpolyamines were eluted through a reverse-phase column (LiChro Cart 250 — 4.5 mm) and detected by absorbance at 254 nm. A relative calibration procedure was used to determine the polyamines in the samples, using 1.6 hexanediamine as the internal standard and standard curves covered the range 1–320 nM. 2.11. Antioxidant activity The total antioxidant activity (TAA) was also quantified in duplicate for each subsample according to the protocol described by Serrano et al. (2009) which enables the determination of TAA due to hydrophilic compounds (H-TAA) in the same extraction. Briefly, 1 g of fresh leaves or roots was homogenized in 5 mL of 50 mM phosphate buffer (pH 7.8) and 3 mL of ethyl acetate, and then centrifuged at 15.000 rpm for 15 min at 4 °C. Two fractions were obtained, with the lower fraction being used for H-TAA quantification. TAA was determined using the enzymatic system composed of the chromophore 2.2azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), the horseradish peroxidase enzyme (HRP) and its oxidant substrate (hydrogen peroxide), in which ABTS•+ radicals are generated and monitored at 730 nm. The decrease in absorbance after adding the extracts was proportional to TAA of the sample. A calibration curve (0–20 nM) was performed with Trolox (R)-(+)-6-hydroxy-2.5.7.8tetramethylcroman-2-carboxylic acid (Sigma, Madrid, Spain) in aqueous media for H-TAA. The results are expressed as the mean ± SE in mg of Trolox equivalent per kg FW.

2.8. Anthocyanin content 2.12. Statistical analyses Leaf anthocyanins were determined according to Serrano et al. (2005). Total anthocyanins' content was calculated using cyaniding-3glucoside (molar absorption coefficient of 23.900 L cm−1 mol−1 and molecular weight of 449.2 g mol− 1) as a standard. The results were expressed as mg of anthocyanin per 100 g FW and presented as the mean ± SE of duplicate determinations made on each of four samples. Leaf individual anthocyanins, cyanidin-3.5-glucoside and petunidin3-glucoside, were assayed by high performance liquid chromatography using a diode array detector (HPLC-DAD) (Serrano et al., 2005). One millilitre of extract obtained for total anthocyanin quantification was filtered through a 0.45-μm Millipore filter and injected into a HewlettPackard HPLC series 1100 equipped with a C18 Supelco column (Supelcogel C-610H, 30 cm × 7.8 mm, Supelco Park, Bellefonte, USA) and detected by absorbance at 510 nm. The peaks were eluted by a gradient using the following mobile phases: 95% water + 5% MeOH (A); 88% water + 12% MeOH (B); 20% water + 80% MeOH (C); and MeOH (D) at a rate of 1 ml min−1. Peaks were identified using authentic standards by comparison of the retention times and peak spectral analysis. The anthocyanin standards were provided by Dr. C. García-Viguera of the Dpto. Ciencia y Tecnología de Alimentos, CEBAS-CSIC, Murcia, Spain. 2.9. Phenolic compound contents Phenolic compounds were extracted from 0.5 g of fresh leaves according to Tomás-Barberán et al. (2001) using water:MeOH (2:8) containing 2 mM NaF and quantified using the Folin–Ciocalteu reagent. The results were expressed as mg gallic acid equivalent per kg FW of duplicate determinations made on each subsample. 2.10. Free polyamine content For each replicate, 1 g fresh leaves were extracted with 10 mL of 5% cold perchloric acid, with 1.6 hexanediamine (100 nmol g− 1 tissue) added as an internal standard. The homogenate was then centrifuged

Variance of the data was analysed with GLM procedure of SAS software for a Randomized Complete Block design with four replicates (SAS, 1996). Both main effects (salinity level and rootstock) and interactions were considered in the analyses. Where applicable, means were separated by Duncan's Multiple Range Test (P ≤ 0.05). 3. Results 3.1. Growth and leaf water relations Plant scion growth is significantly influenced by salinity and rootstock. Reduction in leaf DW was generally more acute in the case of Mazzetto/GF677 than in the case of Mazzetto/Garnem (Table 1). Compared with the plants grown under unstressed conditions, 75 mM NaCl treatment decreased leaf DW of Mazzetto/GF677 and Mazzetto/ Garnem plants by 30% and 23%, respectively. There was also a significant reduction in shoot extension in response to increasing medium salinity especially in the plants grafted onto GF677. Mazzetto/Garnem maintained a larger shoot extension than Mazzetto/GF677 at all salinity levels. By contrast, the RWC was not significantly affected by salinity. The Ψw of the scion became increasingly more negative with increasing NaCl concentration for both rootstocks but more so for GF677. The Ψπ decreased also as medium salinity increased; however, the rootstock had no effect on this parameter. 3.2. Mineral content Variability in leaf Na+ concentration was mainly due to changes in NaCl concentration in the nutrient solution. Nevertheless, untreated Mazzetto/Garnem plants had a lower Na concentration than Mazzetto/GF677 (Table 2). The concentrations of Ca2 +, K + and Mg2 + were significantly influenced by salinity and rootstock. The reduction in the concentration of these was acute in Mazzetto/GF677


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Table 1 Growth parameters and water relations of sweet almond cv. Mazzetto grafted on two different almond rootstocks (GF677 and Garnem) fed with increasing concentrations of NaCl. NaCl (mM)

0 25 50 75

Ψπ (MPa)

Ψw (MPa)

Shoot extension (cm)

Leaf FW (g)

M/GF677

M/Garnem

M/GF677

M/Garnem

M/GF677

RWC (%) M/Garnem

M/GF677

M/Garnem

M/GF677

M/Garnem

15.5 ± 0.60a 12.3 ± 0.71a 6.5 ± 1.01b 6 ± 1.44b

32.3 ± 1.73a 30 ± 0.00a 18.5 ± 0.31ab 9.5 ± 2.42a

24.2 ± 1.50a 18.8 ± 0.91b 20.7 ± 0.72b 17.1 ± 0.44b

18.8 ± 0.00a 17.4 ± 0.80a 16 ± 0.51ab 14.5 ± 0.82b

89.86 ± 2.30a 88.61 ± 0.81a 87.11 ± 4.11a 75.82 ± 3.70a

85.39 ± 2.70a 85.13 ± 3.62a 81.75 ± 4.18a 72.95 ± 3.50a

−0.85 ± 0.01a −1.45 ± 0.01b −1.5 ± 0.01b −1.82 ± 0.01b

−0.65 ± 0.02a −0.87 ± 0.03a −1.05 ± 0.01a −1.7 ± 0.05b

−1.48 ± 0.11a −1.67 ± 0.09a −1.67 ± 0.04a −2.15 ± 0.06b

−1.48 ± 0.11a −1.67 ± 0.02b −1.67 ± 0.05b −1.99 ± 0.05c

Values are the means ± SE of four replicates. Different letters indicate significant differences between treatments (Duncan test, P ≤ 0.05).

than in Mazzetto/Garnem. The Ca2 +/Na+, K+/Na+ and Mg2 +/Na+ ratios were generally higher in the leaves of Mazzetto/Garnem than in Mazzetto/GF677 except for 50 mM NaCl (Fig. 1). 3.3. Gas exchange measurements After four weeks of NaCl treatments, A, gs and E were reduced in all graft combinations compared to control. The largest reductions were consistently found in Mazzetto/GF677 (Fig. 2). The reduction in gs was 57% and 32%, respectively, for in Mazzetto/GF677 and Mazzetto/ Garnem at the 75 mM level.

Similarly, leaf Chl(a + b) and Chla/Chlb of Mazzetto/Garnem did not vary with medium salinity. In contrast, both parameters tended to decrease in Mazzetto/GF677. . The addition of NaCl to the watering solution reduced leaf carotenoid content in Mazzetto/GF877 but increased it in Mazzetto/Garnem (the highest concentration was 20 μg/100 mg reached with 25 mM NaCl treatment) (Fig. 3). In Mazzetto/GF677, no significant differences among salinity levels existed for Car/Chl ratio,

3.4. Chlorophyll and carotenoid content The leaves of Mazzetto grafted onto the green-leaved GF677 rootstock had higher Chla, Chlb and Chl(a + b) concentrations than those of Mazzetto grafted onto the red-leaved Garnem (Fig. 3). Chla was not affected by salinity in either scion/rootstock combinations while Chlb concentration increased in the leaves of Mazzetto/GF677 but not in Mazzetto/Garnem as the medium became more saline (Fig. 3). Table 2 Ion concentrations in the leaves of sweet almond cv. Mazzetto grafted on two different almond rootstocks (GF677 and Garnem) fed with increasing concentrations of NaCl. Values are the means ± SE of four replicates. Different letters indicate significant differences between treatments (Duncan test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Na (μEq·g−1 DW)

Analysis of variance (F) Salinity Rootstocks Salinity × rootstocks Ca (μEq·g−1DW)

Analysis of variance (F) Salinity Rootstocks Salinity × rootstocks K (μEq·g−1 DW)

Analysis of variance (F) Salinity Rootstocks Salinity × rootstocks Mg (μEq·g−1 DW)

Analysis of variance (F) Salinity Rootstocks Salinity × rootstocks

NaCl (mM)

M/GF677

M/Garnem

0 25 50 75

879.9 ± 7.7c 997.8 ± 23.1c 1547.3 ± 81.4b 2207.4 ± 47.8a

467.2 ± 26.4d 884.2 ± 7.7c 1757.6 ± 53.6b 1849.3 ± 4.6a

658.8 ± 2.7a 613.5 ± 6.4a 558.9 ± 9.6a 406.2 ± 40.5b

647.3 ± 27.9a 603.9 ± 9.4a 607.52 ± 14.6a 557.9 ± 22.6a

330.80*** 26.82*** 13.75*** 0 25 50 75 13.2*** 10.28** 3 ns 0 25 50 75

608.1 ± 43.8a 593.1 ± 16.7a 483.3 ± 56.2ab 378.3 ± 35.4b

694.8 ± 40.7a 668.9 ± 7.6a 640.0 ± 34.6a 469.8 ± 33.7b

669.5 ± 57.9a 620.3 ± 83.0a 606.0 ± 23.1a 290.7 ± 67.6b

806.5 ± 54.6a 598.3 ± 20.3b 583.8 ± 9.8b 559.8 ± 9.3b

12.81*** 7.05** 0.21 ns 0 25 50 75 11.21*** 6.54** 2.92 ns

Fig. 1. Effect of NaCl on Ca2+/Na+ , K+/Na+ and Mg2+/Na+ ratios in leaves of almond cv. Mazzetto grafted on two different almond rootstocks GF677 and Garnem. Values are the means ± SE of four replicates.

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Fig. 2. Effect of NaCl on gas exchange in almond cv. Mazzetto grafted on two different almond rootstocks GF677 and Garnem. Values are the means ± SE of four replicates.

whereas in Mazzetto/Garnem the Car/Chl ratio was significantly higher at the 75 mM NaCl level compared to the control.. 3.5. Anthocyanin content It is remarkable that among control plants, the leaves of Mazzetto grafted onto the red-leaved Garnem had higher concentrations of total anthocyanins, cyanidin-3.5-glucoside and petunidin-3-glucoside and a higher anthocyanin/Chl ratio than those of Mazzetto grafted onto the green-leaved GF677 rootstock (Fig. 4). The level of total anthocyanins and petunidin-3-glucoside tended to decrease as salinity of the growing medium increased in the case of Mazzetto/Garnem. The effect of salinity on leaf anthocyanins and anthocyanin to Chl ratio was inconsistent in the case of Mazzetto/GF677. The anthocyanin/Chl ratio increased slightly with 25 mM in Mazzetto/GF677 trees whereas in the Mazzetto/ Garnem trees, this ratio increased strongly at 75 mM NaCl. Averaged across all salinity treatments, anthocyanin/Chl ratio of Mazzetto/ Garnem was nearly four times higher than that of Mazzetto/GF677. 3.6. Polyphenol content The level of polyphenols increased slightly above control levels in Mazzetto/Garnem with the 25 mM NaCl treatment (Fig. 4). Polyphenols then tended to decrease as salinity level increased. With Mazzetto/ GF677 plants, polyphenol content was significantly higher at the highest NaCl dose (75 mM) as compared with control plants. Generally, the level of polyphenols is higher in the leaves of Mazzetto/GF677. 3.7. Proline content Leaves of Mazzetto/GN15 had approximately five times more proline than those of Mazzetto/GF677, regardless of NaCl concentration in the culture medium (Fig. 5). These proline levels tended to increase

slightly with the highest of NaCl concentrations in Mazzetto/Garnem but not in Mazzetto/GF677.

3.8. Total soluble sugars The addition of NaCl in the culture medium caused generally an increase in leaf total soluble sugars (TSS) (Fig. 5). The change depended on rootstock and NaCl concentration. In Mazzetto/GF677, the concentration of TSS doubled with the 25 mM NaCl treatment then fell back to control level with the 50 and 75 mM NaCl treatments. In Mazzetto/ Garnem, TSS concentration increased over control levels by 66% and 80% with 25 mM and 75 mM NaCl, respectively; plants which were treated with 50 mM NaCl had an expectedly low concentration of TSS.

3.9. Free polyamines Free polyamines' concentration in the leaves varied with NaCl level in the culture medium (Fig. 6). Generally, salinity depressed their concentration in both scion/rootstock combinations. The exception was the 25-mM NaCl treatment which tended to boost putrescine and spermidine concentrations in Mazzetto/GF677 leaves. This was not the case with Mazzetto/Garnem.

3.10. Total antioxidant activity Regardless of NaCl level, the leaves of Mazzetto/GF677 had higher total antioxidant activity (TAA) than those of Mazzetto/Garnem (Fig. 7). The addition of 75 mM NaCl to Mazzetto/GF677 increased leaf TAA by 30% over the control. TAA in Mazzetto/Garnem leaves was almost doubled by the 25 and 75 mM NaCl treatments but was unchanged by the 50 mM treatment.


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Fig. 3. Effect of NaCl on total chlorophyll content and carotenoids in almond cv. Mazzetto grafted on two different almond rootstocks GF677 and Garnem. Values are the means ± SE of four replicates. For each scion/rootstock combination, different letters indicate significant differences between treatments according to Duncan's Multiple Range Test at P ≤ 0.05.

4. Discussion The present study suggests that Mazzetto growth was significantly influenced by both rootstock and salt stress. The reduction in shoot extension and leaf dry weight of Mazzetto subjected to increasing concentrations of NaCl was more acute when the rootstock was GF677 than when it was Garnem. It appears, therefore, that grafting on certain rootstocks can effectively limit the deleterious effects of salinity on the scion; this is consistent with previous reports dealing with various plant species (Martinez-Rodriguez et al., 2008; He et al., 2009; Zhen et al., 2010; Zrig et al., 2011). It is generally accepted that the rootstock effect on shoot growth is related to its ability to minimise toxic ions' absorption and transport over time (Martinez-Rodriguez et al., 2008). In the present study, Na+ concentration in the aerial parts was lower in Mazzetto/Garnem compared to Mazzetto/GF677. Such an exclusion mechanism can explain, at least partly, the larger shoot extension and leaf biomass of Mazzetto/Garnem observed in our study. Similar results were obtained in melon plants grafted onto pumpkin rootstocks

subjected to salt stress (Edelstein et al., 2005). A higher Na+ concentration in the soil or in the irrigation water can reduce essential nutrients' availability and uptake and depress Ca2+/Na+, K+/Na+ and Mg2+/Na+ ratios in the plant (Grattan and Grieve, 1999). As a result, the plant becomes susceptible to specific ion injury as well as to nutritional disorders which may affect growth and yield (Grattan and Grieve, 1999). Water relations in the rootstock–scion system were studied with emphasis on improving plant adaptation to environmental conditions. In this study, the plant's RWC did not differ between rootstocks and the reduction due to increasing NaCl concentration was small to be statistically significant. Similar results were obtained in tomato and cucumber under stress conditions (Estan et al., 2005; Huang et al., 2010). Nevertheless, Ψw decreased appreciably even at the lowest NaCl concentrations (25 mM NaCl) especially in Mazzetto/GF677. The plants grafted onto Garnem maintained consistently higher leaf Ψw compared to those on GF677. The Ψπ too decreased in response to soil salinity but there was no difference between the two rootstocks which indicates that Mazzetto/Garnem maintained a higher turgor potential

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Fig. 4. Effect of NaCl on total anthocyanin content, individual anthocyanin and total polyphenols content in leaves of almond cv. Mazzetto grafted on two different almond rootstocks GF677 and Garnem. Values are the means ± SE of four replicates. For each scion/rootstock combination, different letters indicate significant differences between treatments according to Duncan's Multiple Range Test at P ≤ 0.05.

(since Ψw of Mazzetto/Garnem was consistently less negative than that of Mazzetto/GF677) which apparently contributed to its higher shoot extension rate. The better water-relations of Mazzetto/Garnem plants appear to be the cause of higher gas exchange rates of their leaves over those of Mazzetto/GF677. Leaves of Mazzetto/Garnem plants were able to limit the loss of photosynthetic activity and transpiration by maintaining stomatal conductance. In addition to maintaining cell turgidity needed for growth, Mazzetto/Garnem plants appear to be better protected against toxic ions by partitioning less Na and more K, Ca and Mg to their leaves compared to Mazzetto/GF677. In a previous study, we suggested that the reduction in leaf gas-exchange of almond trees in response to salinity is due mainly to an increase in leaf Na+ content (Zrig et al., 2011). Furthermore, salinity interferes with several physiological and biochemical aspects, including photosynthesis, nutrient absorption and pigments and antioxidant biosynthesis and function (Colla et al., 2008). In this respect, the ability of grafted plants to counteract the effects induced by salt stress depends largely on the

rootstock. Indeed, Mazzetto/Garnem plants appear to have a better osmoprotection against toxic ions. First, their leaves had higher carotenoids/Chl and anthocyanin/Chl ratios and higher concentrations of proline and TSS than those of Mazzetto/GF677. Second, these parameters tended to increase as soil salinity increased. The protection of Chl and assimilatory enzymes against oxidation and loss of conformation due to the presence of Na+ and Cl− should contribute to maintaining photosynthetic assimilation rates. According to Dejampour et al. (2012), proline is a reliable indicator of salt tolerance for almond trees. Cytosolic compatible solutes play also an important role in osmoregulation (Hare et al., 1998; Parida and Das, 2005); they slow water efflux to the apoplast and the vacuole. In this regard, Mazzetto/Garnem is better protected than Mazzetto/GF677. Photosynthesis can be limited by a reduction in photochemical activity independently of stomatal conductance (Souza et al., 2004). It has been shown that several biotic and abiotic stresses reduce Chla + b content (Liu et al., 2007; Rouphael et al., 2008) and the


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Fig. 5. Effect of NaCl on proline and sugar content in leaves of almond cv. Mazzetto grafted on two different almond rootstocks GF677 and Garnem. Values are the means ± SE of four replicates. For each scion/rootstock combination, different letters indicate significant differences between treatments according to Duncan's Multiple Range Test at P ≤ 0.05.

photochemical efficiency of photosystem II (PSII), but these effects can be improved by grafting (Ahn et al., 1999; Zheng et al., 2009; He et al., 2009). In the present study, Chla + b concentration in the leaves decreased at the highest NaCl concentration in Mazzetto/GF677, whereas it remained unchanged in plants grafted onto Garnem. Hence, it appears that the rootstock affected the rate of Chl turnover or biosynthesis. This result agrees with one of our previous studies (Zrig et al., 2011). Under salinity stress, the leaves of Mazzetto/Garnem had higher carotenoids/ Chl and anthocyanins/Chl ratios and maintained higher proline and soluble sugar concentrations than those of Mazzetto/GF677. These are indications of more efficient antioxidant mechanisms protecting the light-harvesting antenna and the photosystems. Besides their role as light-harvesting pigments that contribute to photosynthesis, carotenoids protect chlorophylls against oxidative destruction by scavenging two of the reactive oxygen species, singlet molecular oxygen and peroxyl radicals (Salisbury and Ross, 1992; Stahl and Sies, 2005). These results suggest that the rootstock Garnem enhanced the antioxidation capacity of Mazzetto cells. Furthermore, even in control plants, Mazzetto/Garnem leaves had higher anthocyanin concentrations and anthocyanins/Chl ratios than those of Mazzetto/GF677. The leaves of the scion seem to acquire some of the characters of the rootstock; the red-leafed Garnem has more anthocyanins in its tissues than the green-leafed GF677 (Zrig et al., 2011). The mechanism of this influence is not well understood but growth regulators such as auxin may be involved. Several reports documented the effect of the rootstock on the

biochemical composition, in particular in terms of enzymes and phenols, of the scion of various species, such as almond (Zrig et al, 2011), grape (Satisha et al., 2007) and melon (Schmutz and Lüdders, 1999). Zrig et al. (2015) emphasised the photo-protective role of anthocyanins against photo-oxidation during salinity stress in Garnem rootstock total antioxidant activity was positively correlated with carotenoids and anthocyanins concentrations more than with polyamines. It appeared that Mazzetto/Garnem plants have a more effective mechanism of photoprotection which apparently involves carotenoids and anthocyanins and more osmo-protectants in the form of proline and soluble sugars. In the case of Mazzetto/GF677, anti-oxidation activity appears to be achieved mainly by polyphenols and spermidine which were abundant in its leaves. 5. Conclusion In conclusion, tree tolerance to soil salinity can be improved by grafting onto tolerant rootstocks. Mazzetto/Garnem plants maintained a more active shoot growth than Mazzetto/GF677 at all levels salinity considered. This appears to be due to their ability to limit the loss of photosynthetic assimilation and cell turgor. Photosynthetic activity of Mazzetto/Garnem leaves may have benefited from a higher Ψw and stomatal conductance. Furthermore, more proline and higher carotenoids/ Chl and anthocyanins/c\Chl ratios in Mazzetto/Garnem leaves suggest a better protection for Chl and cytosolic assimilatory enzymes against

Fig. 6. Effect of NaCl on free polyamine content in leaves of almond cv. Mazzetto grafted on two different almond rootstocks GF677 and Garnem. Values are the means ± SE of four replicates. For each scion/rootstock combination, different letters indicate significant differences between treatments according to Duncan's Multiple Range Test at P ≤ 0.05.

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Fig. 7. Effect of NaCl on total antioxidant activity in leaves of almond cv. Mazzetto grafted on two different almond rootstocks GF677 and Garnem. Values are the means ± SE of four replicates. For each scion/rootstock combination, different letters indicate significant differences between treatments according to Duncan's Multiple Range Test at P ≤ 0.05.

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