Effect of nickel and grafting combination on yield, fruit quality, antioxidative enzyme activities, lipid peroxidation, and mineral composition of tomato

2.1 Plant material, growth conditions and experimental design

Tomato (Solanum lycopersicum L. cv. Ikram, Syngenta, Milan, Italy) plants were either self-grafted or grafted onto the following commercial rootstocks: Maxifort and Unifort (S. lycopersicum × S. habrochaites, De Ruiter/Monsanto, Bergschenhoek, The Netherlands) and eggplant (Solanum melongena L.) Heirloom cv. Black Beauty. Maxifort and Unifort were selected as being the most representative commercial rootstocks used in Italy. Eggplant cv. Black Beauty was included as rootstock due to its tolerance against abiotic stresses (Abdelmageed and Gruda, 2009).

The experiment was carried out in a greenhouse situated on the experimental farm of Tuscia University, Central Italy (42°25′ N; 12°08′ E) from April 23 to August 8, 2013. The air temperature and the day/night relative humidity inside the greenhouse were maintained between 18/33°C and 55/85%, respectively. Seedlings were produced by a commercial company (Centro SEIA, Sicily, Italy) using the slant cut (splice) graft technique. At three-four true leaf stage tomato seedlings were transferred into pots (6 L) containing quartziferous sand. Pots were arranged in double rows in separate plastic covered channel, leaving the pot head open, on a fixed platform providing sufficient slope to enable drainage collection into tanks fixed at the lateral end of each channel. The space between plants within a row was 0.45 m and the distance between the centers of double rows was 1.2 m, giving a plant density of 3.7 plants m⁻². Pollination was facilitated by bumble bees. Plants were grown as vertical cordons at which all laterals branches were pruned and later the terminal bud was also removed after the 5^th fruit cluster.

The experiment was designed as a factorial combination of three Ni concentrations [0 (control), 25, or 50 µM] and four grafting combinations (self-grafted Ikram/Ikram, Ikram/Black Beauty, Ikram/Unifort, or Ikram/Maxifort). The treatments were arranged in a randomized complete-block design with three replicates per treatment. The Ni treatments were initialized on May 20 (28 days after transplanting, DAT).

The basic (control) nutrient solution used in this experiment was a modified Hoagland and Arnon formulation. All chemicals used were of analytical grade, and composition of the basic nutrient solution was: 14.0 mM N-NO₃, 1.6 mM S, 1.5 mM P, 6.0 mM K, 4.5 mM Ca, 1.5 mM Mg, 0.5 mM Cl, 20 μM Fe, 9 μM Mn, 0.3 μM Cu, 1.6 μM Zn, 20 μM B, and 0.3 μM Mo. The enriched-Ni treatments had the same nutrient composition plus 25 or 50 µM Ni supplied as NiCl₂. The pH of the nutrient solution for all treatments was 5.8 ± 0.3. Deionized water was used for the preparation of all nutrient solutions. Nutrient solution was pumped from independent supply tanks of 478 L through a drip irrigation system with one emitter per plant of 2 L h⁻¹ flow rate. Irrigation scheduling was performed using electronic low-tension tensiometers (LT-Irrometer, Riverside, CA, USA) that controlled irrigation based on substrate matric potential (Norrie et al., 1994). The duration of each irrigation event was tuned to provide at least 35% of the nutrient solution draining from the pots (Rouphael et al., 2004; Rouphael and Colla, 2005). The drainage solutions were collected in tanks placed at the lateral end of each channel. The drainage solutions were not reused in the system.

2.2 Yield and biomass measurements

Fully ripe fruits were harvested from June 26 and continued until the termination of the experiment (August 8). The fruit yield, number of fruits, and mean fruit weight were recorded on all plants. At the end of the experiment (108 days after transplanting, DAT), each plant was separated into different plant organs (leaf, stem, and root) and their tissues were dried in a forced-air oven at 80°C until constant weight for biomass determination. Shoot biomass was equal to the sum of aerial vegetative plant parts (leaves + stems). Leaf area (LA) was measured with an electronic area meter (Delta-T Devices Ltd., Cambridge, UK).

2.3 Fruit quality analysis

At peak harvesting period, nine full red-ripe fruits were selected per plot (3 from each plant) to determine the fruit quality. Fruit shape index (SI), defined as the ratio of width to length, was measured. Fruit firmness (N cm⁻²) was determined by using a penetrometer (Bertuzzi FT 011; Brugherio, Milan, Italy), fitted with an 8 mm-diameter round-head probe. Hunter color values parameters: L* (brightness), a* (redness), and b* (yellowness) were measured on the surface of the external pulp with a Minolta colorimeter (Model CR-300; Minolta, Osaka, Japan). Total soluble solids (TSS, °Brix) content of the fruit juice was measured by an Atago N1 refractometer (Atago Co. Ltd., Japan), and titraTable acidity was determined by potentiometric titration with 0.1 M NaOH up to pH 8.1 and the results were expressed as percentage of citric acid in the juice. Fruit dry matter (DM) was also determined.

2.4 Leaf pigment content

At the termination of the experiment (August 6, 106 DAT), the leaf pigments (total chlorophyll and carotenoids) were extracted by homogenization of fresh leaf tissues (0.5 g) in acetone (80%) adding a small amount of MgCO₃. The resulting extracts were centrifuged at 4,800 × g for 20 min. The total chlorophyll and carotenoid contents were determined by taking absorbance of the supernatant at 470, 647, and 664 nm by a UV-Vis spectrophotometer (Perkin Elmer, Norwalk, CT, USA). Formula and extinction coefficients used for the determination of leaf pigments were described by Lichtenhaler and Wellburn (1983) and the content of chlorophyllous pigments was expressed in mg g⁻¹ of fresh weight.

2.5 SPAD index and fluorescence measurements

Both the SPAD index and the fluorescence measurements were recorded 106 DAT. A portable chlorophyll meter (SPAD-502, Minolta corporation, Ltd., Osaka, Japan) was used to measure the relative leaf chlorophyll concentration as a rational unit. Measurements were made at a central point on the leaflet between the midrib and the leaf margin of the 3^rd leaf from the top. Twelve random measurements per plot were taken and averaged to a single SPAD value for each treatment.

The chlorophyll fluorescence was recorded on 15 min dark-adapted leaves by means of a chlorophyll fluorimeter Handy PEA (Hansatech Instruments Ltd,King's Lynn, UK) with an excitation source intensity higher than 3,000 μmol m⁻² s⁻¹ at the sample surface. The minimal fluorescence intensity (F₀) in a dark-adapted state was measured in the presence of a background weak light signal (about 2–3 µmol photons m⁻² s⁻¹). The maximal fluorescence level in the dark-adapted state (F_m) was induced by 0.8 s saturating light pulse (3,000 μmol photons m⁻² s⁻¹). The maximum quantum yield of open photosystem II (PSII) (F_v/F_m) was calculated as (F_m − F₀)/F_m, as described by Maxwell and Johnson (2000). Six measurements per experimental unit were performed in the 3^rd leaf from the top as were used for SPAD.

2.6 Assay of antioxidant enzyme activity

At the same date of the SPAD and fluorescence measurements, fresh leaf samples from the 3^rd leaves from top were harvested from each plant and immediately frozen in liquid nitrogen and stored at –80°C for later antioxidant enzyme activity, proline, and lipid peroxidation analysis. Enzyme extractions were performed using a pre-chilled mortar and pestle with two volumes of an ice-cold extraction buffer (0.05 M potassium phosphate buffer, pH 7.0) containing 0.1% (w/v) ascorbic acid, 1% (w/v) polyvinilpolypirrolidone, 1 mM Na₂-EDTA, and 0.1% ((v/v)) Triton X-100. After centrifugation (15,000 × g, 4°C, 30 min) the supernatant was set aside for the determination of the enzyme activity and protein content by a spectrophotometer (Perkin Elmer, Norwalk, CT, USA). Catalase (CAT, EC 1.11.1.6) activity was measured according to Havir and McHale (1989). The assay mixture (1 ml) contained 0.1 ml of 125 mM H₂O₂, and 20 µl of the crude extract in 0.05 M potassium phosphate buffer (pH 7.0). Enzyme activity was evaluated by following the decomposition of H₂O₂ at 240 nm for 1 min and calculated using the extinction coefficient (0.036 mM⁻¹ cm⁻¹). Activity of ascorbate peroxidase (APX, EC 1.11.1.11) was measured following the decrease of absorbance at 290 nm for 1 min (Nakano and Asada, 1981) corresponding to the oxidation of ascorbic acid. APX activity was calculated using its extinction coefficient (2.8 mM⁻¹ cm⁻¹). Activity of guaiacol peroxidase (GPX, EC 1.11.1.7) was measured according to Chance and Maehly (1955). The assay mixture (1 ml) contained 0.1 ml of 90 mM guaiacol, 0.1 ml of 125 mM H₂O₂, and 50 µl of the crude extract in 0.05 M potassium phosphate buffer (pH 7.0). Enzyme activity was evaluated following the increase of absorbance at 470 nm for 40 s due to guaiacol oxidation and calculated using the extinction coefficient (26.6 mM⁻¹ cm⁻¹). The specific enzyme activity for all enzymes was expressed as mmol mg⁻¹ protein min⁻¹.

2.7 Proline determination

Free proline content was determined according to the method of Bates et al. (1973) Around 0.5 g of leaf material was homogenized in 10 mL of 30 g L⁻¹ sulfosalicylic acid (Sigma Aldrich), and the homogenate was filtered through Whatman No. 2 filter paper. Then 2 mL of filtrate was reacted with 2 mL of acid-ninhydrin (1.25 g of ninhydrin in 30 mL of glacial acetic acid and 20 mL of 6 mol L⁻¹ phosphoric acid) and 2 mL of glacial acetic acid in a test tube at 100°C for 1 h. The reaction was terminated in an ice bath, then 4 mL of toluene was added, and the product of the reaction was extracted by vortex mixing. The absorption of the upper phase was read at 520 nm using toluene as a blank. Proline concentration was calculated on a fresh weight (FW) basis using L-proline for the standard curve.

2.8 Determination of lipid peroxidation

The Ni-induced oxidative damage (membrane lipid peroxidation) in fresh leaf tissue was estimated by measuring the malondialdehyde (MDA) concentrations. Fresh leaf tissues were homogenized in 0.1% (w/v) trichloroacetic acid (TCA) solution in 1:3 ratio. After centrifugation (15 min, 12,000 × g at 4°C), an aliquot of the supernatant was added to 0.5% thiobarbituric acid (TBA) made in 20% TCA and heated at 95°C for 30 min. After rapid cooling on ice, the mixture was centrifuged at 10,000 × g for 10 min. The absorbance was recorded at 532 and 600 nm using an UV-Vis spectrophotometer. The MDA concentration was calculated from the difference between the absorbance values at 532 and 600 nm (Amor et al., 2005).

2.9 Mineral analysis

Dried plant tissues (leaf, fruit and root) were ground separately in a Wiley mill to pass through a 20-mesh screen, then 0.5 g of the dried plant tissues were analyzed for the following elements: N, P, K, Ca, Mg, Fe, Mn, Zn, Cu, and Ni. Nitrogen concentration in the plant tissues was determined after mineralization with sulfuric acid by “Kjeldahl method” (Bremner, 1965). P, K, Ca, Mg, Fe, Mn, Zn, Cu, and Ni concentrations were determined by dry ashing at 400°C for 24 h, dissolving the ash in 1:20 HNO₃, and assaying the solution obtained using an inductively coupled plasma emission spectrophotometer (ICP Iris; ThermoOptek, Milano, Italy; Kalra, 1998).

2.10 Statistical analysis

All data were statistically analyzed by ANOVA using the SPSS software package (SPSS 10 for Windows, 2001). To separate treatment means within each measured parameter, Duncan's multiple range test was performed at P = 0.05.

3.1 Fruit yield components, biomass production and partitioning

In grafted and self-grafted plants, fruit yield decreased in response to an increase of Ni concentration in the nutrient solution (Table 1). The lowest yield observed under Ni enriched solutions (25 and 50 µM Ni) in comparison to the control was due to a reduction in both the number of fruits per plant and the fruit mean weight (Table 1). Moreover, irrespective of Ni level, the yield of the different grafting combinations was in the following order: Ikram/Maxifort > Ikram/Unifort > Ikram/Ikram > Ikram/Black Beauty (Table 1). Similarly, the final LA significantly decreased with increasing the Ni concentration in the nutrient solution with no significant difference between 25 and 50 µM Ni treatments (Table 1). Shoot and root dry biomass were highly affected by the interaction Ni × grafting combination (data not shown). At 0 µM Ni, shoot dry weight was highest in Ikram/Maxifort plants (291.4 g plant⁻¹), while at 25 µM Ni the highest shoot dry weight was recorded in Ikram/Maxifort and Ikram/Unifort grafting combinations (average 248.8 g plant⁻¹). The Ikram/Unifort combination gave the highest shoot dry weight biomass at 50 µM Ni (241.8 g plant⁻¹). At 0 µM Ni, the root dry weight was highest in Ikram/Maxifort and Ikram/Unifort grafting combinations (average 17.3 g plant⁻¹), while at 25 µM Ni no significant differences were recorded among grafting combinations (average 12.7 g plant⁻¹). Finally, Ikram/Black Beauty plants gave the highest root dry weight at 50 µM Ni (13.0 g plant⁻¹).

3.2 Fruit quality

When averaged over grafting combinations, increasing the concentration of Ni from 0 to 50 µM in the nutrient solution significantly enhanced the fruit firmness, fruit dry matter (DM), and total soluble solids (TSS) contents with the highest values recorded under severe Ni conditions, whereas an opposite trend was observed for titratable acidity (TA). Moreover, the brighter color (i.e., higher yellowness, b*) with higher lightness (L*) was observed under severe Ni stress (Table 2). Irrespective of Ni level, the lowest fruit shape index (SI) was recorded in Ikram/Black Beauty, whereas the higher fruit DM and TSS contents were observed in Ikram/Unifort and Ikram/Balck Beauty, respectively. Finally, the tomato fruit of Ikram/Black Beauty was characterized by lower redness (a*) and yellowness when compared to the other three grafting combinations (Table 2).

3.3 Leaf pigments, SPAD index, chlorophyll fluorescence and electrolyte leakage

Increasing the Ni from 0 to 50 µM in the nutrient solution decreased the leaf pigments in both self-grafted and grafted plants. Similarly, the SPAD index and the maximum quantum use efficiency of PSII measured as F_v/F_m ratio decreased with increasing Ni level with the lowest values recorded under severe Ni stress conditions, whereas an opposite trend was observed for the electrolyte leakage (EL, Table 3). Irrespective of Ni concentration, the highest SPAD index and F_v/F_m ratio values were recorded using both tomato rootstocks (Unifort and Maxifort), whereas an opposite trend was observed for the Chl a/b ratio. The highest EL was observed in both self-grafted and Ikram/Black Beauty combinations (Table 3).

3.4 Activity of antioxidative enzymes, proline and MDA content

The changes in the antioxidative enzyme activities were mainly due to the Ni stress treatment and to a lesser degree to the effect of grafting combination (Table 4). CAT activity in leaf crude extract declined gradually with the increase in Ni concentration from 0 to 50 µM. By contrast, the APX and GPX activities in the leaves of severe Ni-exposed tomato plants were significantly higher by 14.9% and 26.8%, respectively, than in the control (Table 4). Among the grafting combinations, the lowest APX and GPX activities were recorded in Ikram/Black Beauty combination. Analysis of proline in leaves revealed significant differences between the Ni treatments with the highest values recorded in the control (Table 4). The proline content in leaves of Ikram/Black Beauty was significantly higher by 30.8% in comparison to the Ikram/Maxifort combination (Table 4). The severe Ni treatment produced a consistent increase in the level of lipid peroxidation in leaves determined by measuring the amount of MDA. The MDA content recorded on plants receiving 50 µM Ni was significantly higher by 20% and 6.7% in comparison to tomato plants treated with 0 and 25 µM Ni, respectively. When averaged over Ni level, the MDA production reached a maximum value with Ikram/Black Beauty followed by Ikram/Ikram and Ikram/Unifort, whereas the lowest MDA content was recorded when Ikram was grafted onto the Maxifort rootstock (Table 4).

3.5 Mineral composition and partitioning

The concentration of N in leaves and fruits was significantly affected by Ni level where the lowest value of leaf concentration was at 50 µM Ni. Moreover, the concentration of N in leaves was higher in Ikram/Black Beauty than in the other grafting combinations, whereas an opposite trend was recorded for the concentration of N in root (Table 5). The concentration of P in leaves decreased under Ni-exposed treatments. Concerning the effect of grafting combination, the lowest concentration of P in leaves, fruits and roots were recorded when the cultivar Ikram was grafted onto the eggplant rootstock Black Beauty (Table 5).

In leaves, K concentration decreased as solution Ni concentration increased from 0 to 50 µM. The concentration of K in fruit and root tissues was significantly influenced by grafting combination, with the lowest values recorded on grafting combination Ikram/Black Beauty. At 50 µM Ni level, the leaves and roots had the lowest Ca concentration in comparison to those recorded in the control treatment. The highest Ca concentration in leaves, fruits and roots were observed in Ikram/Unifort and Ikram/Maxifort combinations. Similar to N and K, increasing Ni concentration in the nutrient solution significantly decreased the Mg concentration in leaves especially under severe Ni stress conditions. Moreover, the concentration of Mg in leaves and roots was lower in Ikram/Black Beauty in comparison to those recorded in plants self-grafted and grafted onto both tomato rootstocks (Table 5).

The effect of Ni-supply level on tissue micronutrient concentrations Fe, Zn, Mn and Cu was highly significant. The leaf and fruit tissue concentrations of Fe and Cu decreased as the external Ni concentration increased from 0 to 50 µM, whereas an opposite trend was observed for the root tissue (Table 6). The fruit tissue of Zn decreased with increasing Ni level, whereas an opposite trend was recorded for Mn in fruits (Table 6). Overall, the accumulation of micronutrient was significantly lower in Ikram/Black Beauty in comparison to the Ikram/Ikram, Ikram/Unifort, and Ikram/Maxifort combinations.

Irrespective of treatments, most of the Ni absorbed by tomato plants was recorded in root tissue. Ni concentration in leaves, fruits and roots was significantly raised by increased Ni levels from 0 to 50 µM (Table 6). Finally, the highest accumulation of Ni in leaf tissue was observed in both Ikram/Ikram and Ikram/Black Beauty combinations (Table 6), whereas in root tissue the highest values were recorded on self-grafted tomato plants treated with 50 µM Ni (data not shown).

4.1 Crop productivity and growth responses

The requirement of Ni in higher plants is quite low, whereas toxicity occurs at concentrations in the range of 10 µg g⁻¹ in sensitive and 50 µg g⁻¹ in moderately tolerant species (Seregin and Kozhevnikova, 2006; Marschner, 2011;Yusuf et al., 2011). In the present study, significant depression in fruit yield, leaf area, shoot and root biomass production was recorded (Table 1), and that effect varied as a function of Ni concentration in the nutrient solution. Root growth was inhibited more severely (reduction of 23–28%) than the growth of the shoot (reduction of 14%). These results are consistent with the findings of Pandey and Sharma (2002) and Seregin et al. (2003), who reported that under Ni stress conditions the root growth of cabbage and maize was more severely affected than that of the aerial parts. Overall, the decrease in crop performance due to Ni agrees with the findings of Balaguer et al. (1998) who observed that increasing the Ni concentration from 0 to 20 mg L⁻¹ in the nutrient solution negatively affected tomato growth and yield. Other reports show that the accumulation of Ni adversely affects the growth and yield of eggplant (Pandey and Sharma, 2002), wheat (Gajewska et al., 2006), cowpea (Kopittke et al., 2007), and mustard (Gopal and Nautiyal, 2012).

In general, abiotic stress (i.e., drought, salinity, heavy metals) reduces vegetable crop productivity, but can improve some quality attributes of the product as observed in plants grown in soilless and soil conditions (Rouphael et al., 2010; 2012b). In the current experiment, plants receiving 50 µM Ni enhanced some fruit quality aspects of tomatoes, which are particularly important for marketing and consumer satisfaction, in particular visual appearance (firmness and brightness) and taste (DM and TSS contents) (Table 2). This result is in agreement with findings of Gad et al. (2007) who observed that increasing Ni up to 30 mg kg⁻¹ soil improved the fruit DM, vitamin C and TSS concentration in tomato. However, excessive applications of Ni in the root zone lead to an increase of Ni content in fruits, which represents a serious risk for human health.

Recent studies have indicated that some rootstocks of Solanaceae and Cucurbitaceae species may restrict the uptake of heavy metals and toxic micronutrients (Edelstein et al., 2007; Rouphael et al., 2008a; Mori et al., 2009). In fact, our study showed that grafting Ikram onto tomato rootstocks, in particular Maxifort, improved the yield and growth parameters in comparison to self-grafted plants or Ikram/Black Beauty combination (Table 1). Ni-tolerance of plants grafted onto tomato rootstocks, in particular Maxifort, may be attributed to a reduction of Ni concentration in leaf tissue and the better uptake and translocation of mineral elements, in particular iron, to the shoots. Edelstein and Ben-Hur (2006) and Rouphael et al. (2008a) demonstrated that grafting fruit vegetables, particularly melon and cucumber, onto appropriate rootstocks (i.e., pumpkin) may limit the heavy metals (Cr and Ni) and trace elements (Cu) concentration in the aerial parts, thus, mitigating their adverse effects on crop performance and human health (Savvas et al., 2010).

4.2 Physiological and biochemical responses

Considering that the response to Ni relies on the plant ability to counteract the negative effects caused by Ni toxicity, the different changes induced by the eggplant (Black Beauty) and tomato (Unifort and Maxifort) rootstocks on crop performance (i.e., yield and growth) should be also reflected at physiological and biochemical levels. In fact, the higher yield and biomass production in grafted plants, in particular those grafted onto Unifort and Maxifort rootstocks, could be attributed to the capacity of maintaining higher SPAD index and maximum quantum efficiency of PSII (F_v/F_m) (Table 3). This result suggests that plants grafted onto appropriate rootstocks can delay photoinhibition under heavy metal stress especially that at biochemical level Ni interferes with the photosynthetic electron transport chain and the light harvesting complex II (Singh et al., 1989; Chen et al., 2009). Moreover, the lower Chl a/b ratio recorded in both tomato rootstocks demonstrated less damage to chlorophyll b, indicating better protection of PSII under metal stress conditions (Ünyayar et al., 2005).

The electrolyte leakage method for assessing cell membrane stability has been widely adopted as a benchmark to differentiate stress susceptible and tolerant species/genotypes, and in some cases higher membrane stability could be associated with abiotic stress tolerance in vegetable crops (Rouphael et al., 2008a; Colla et al., 2010a). The present study showed that the grafting combinations Ikram/Unifort and Ikram/Maxifort reduced the amount of ion leakage (Table 3), indicating that grafting Ikram onto the selected tomato rootstocks has facilitated the maintenance of membrane functions (i.e., semipermeability). Calcium increases the structural stability of the cell membrane because of electrostatic interactions with membrane phospholipids and proteins and due to its role as fundamental component of the cell wall (Borer et al., 2005). Ni application to the nutrient solution reduced leaf content of Ca leading to a reduction of cell membrane stability of leaf tissues. However, Unifort and Maxifort rootstocks were able to mitigate the detrimental effect of Ni on membrane stability by improving the Ca uptake and translocation in leaf tissues of tomato plants.

The production and accumulation of ROS in plant cells is another common damage under heavy metal stress conditions (Seregin and Ivanov, 2001). In the present study, the activities of antioxidant enzymes like APX and GPX were enhanced especially under severe Ni stress conditions, and more considerably with plants grafted onto Maxifort than onto Black Beauty (Table 4). The above findings indicate that certain rootstocks mitigated the Ni toxicity through a better detoxification process in plant cells. The increase in the leaf APX and GPX activities were also observed in cucumber, rice, and Luffa cylindrica under Ni stress conditions (Gajewska and Skłodowska, 2007; Maheshwari and Dubey, 2009; Wang et al., 2010). Contrary to APX and GPX, CAT activity in the tomato leaves decreased in response to Ni concentration with no significant differences among grafting combinations. Since CATs are metalloenzymes containing Fe, Cu or Zn (Chen et al., 2009), the decrease in CAT activity could be expected as excess Ni decreased micronutrients (Fe, Cu and Zn) in the leaf tissues. The decrease in CAT activity, recorded in the current study, is in line with the findings of Pandey and Sharma (2002) and Gajewska and Skłodowska (2008) on cabbage and wheat, respectively.

Another system of protection against Ni toxicity includes the synthesis of osmolites such as proline, which contribute to stabilization of protein molecules and membranes (Seregin and Kozhevnikova, 2006). The proline content decreased with increasing Ni concentration from 0 to 50 µM in disagreement with previous studies, in which proline enhancement was observed in response to heavy metal toxicity (Gajewska et al., 2006; Pandey and Sharma, 2002). The decrease in proline contents may be related to the oxidative stress resulting in hydrolysis of protein (Azmat and Khan, 2011). Finally, leaves of Ikram/Maxifort combination in Ni-stressed plants accumulate less proline than those of Ikram/Ikram, Ikram/Unifort, and Ikram/Black Beauty.

The changes in the membrane lipid composition were also observed in the current study, since malondialdehyde (MDA, a lipid peroxidation product) content, which is an important indicator of oxidative stress, increased linearly when tomato plants were treated with 50 µM Ni as observed earlier on rice, pigeonpea, and cucumber (Ros et al., 1992; Rao and Sresty, 2000; Khoshgoftarmanesh et al., 2014). The lowest MDA accumulation recorded in the Ikram/Maxifort combination confirmed that Maxifort rootstock plays an important role in mitigating the oxidative damage caused by Ni by enhancing the antioxidant capacity and reducing the proline and MDA content in leaf tissues.

4.3 Mineral nutrition, translocation and assimilation

The uptake and distribution of Ni in plants is still a debatable issue for plant scientists. Seregin and Kozhevnikova (2006) observed that more than 50% of the Ni absorbed by plants is accumulated in the roots, and they attributed the accumulation of this heavy metal in the underground organ to sequestration in the cation exchange sites of the walls of xylem parenchyma cells. Roots of grafted and self-grafted tomato plants accumulated higher amounts of Ni than in the aboveground parts (leaves, fruits). Our results are in agreement with those reported by Brune and Dietz (1995) and Baccouch et al. (2001) who demonstrated more Ni accumulation in roots than in shoots of barely and maize, respectively. The Ni concentration in fruit was not affected by grafting combination, and the concentration (average 5.2 mg kg⁻¹ DW) was within the recommended international standards (Poulik, 1999). Moreover, the Ni content in leaf tissue of tomato plants grafted onto Unifort and especially Maxifort had less Ni when compared to self-grafted plants and plants grafted onto eggplant rootstock. This suggests that some rootstocks of tomato more efficiently reduce the Ni uptake and the transport to leaves. The reduction in accumulation of Ni in leaves of Ikram/Maxifort combination was the principal mechanism that decreases the detrimental effect of Ni toxicity on crop yield and growth. The current results were supported by the findings of Savvas et al. (2013) who demonstrated the ability of four commercial Cucurbita maxima × Cucurbita moschata rootstocks to reduce the Ni concentration in leaves of cucumber when compared to self-grafted and ungrafted plants.

Ni above a threshold level (5–10 µg g⁻¹ DW) may inhibit the absorption, uptake, and accumulation of essential macro- and microelements such as K, Ca, Mg, Fe, Cu, Zn, and Mn, leading to their deficiency in plants (Chen et al., 2009; Yusuf et al., 2011). The decrease in the uptake may also be attributed to Ni-induced metabolic disorders that affect root function (Seregin and Kozhevnikova, 2006). This was the case in the current experiment since a significant reduction of macro- (N, K, Ca, and Mg) and microelements (Fe, Mn, and Cu) in leaf tissue of tomato was observed under severe Ni stress (Tables 5 and 6). These results are consistent with the findings of Palacios et al. (1998) who reported that high concentrations of Ni significantly decreased the uptake of several divalent cations (Mg²⁺, Fe²⁺, Mn²⁺, Cu²⁺, and Zn²⁺). Many rootstocks used in vegetable grafting were able of enhancing the uptake rates of some nutrients even under abiotic stress conditions (e.g., heavy metals) because they are characterized by more vigorous root systems (Savvas et al., 2010).