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Environmental and Experimental Botany 66 (2009) 270–278

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

rafting increases the salt tolerance of tomato by improvement of photosynthesisnd enhancement of antioxidant enzymes activity

ong Hea, Zhujun Zhua,b,∗, Jing Yanga, Xiaolei Nia, Biao Zhua

Department of Horticulture, Zhejiang University, Hangzhou 310029, PR ChinaDepartment of Horticulture, School of Agricultural and Food Science, Zhejiang Forestry University, Lin’an, Hangzhou 311300, PR China

r t i c l e i n f o

rticle history:eceived 31 July 2008eceived in revised form 21 January 2009ccepted 18 February 2009

eywords:omatorafting

a b s t r a c t

Plant growth, K and Na concentration, gas exchange, chlorophyll fluorescence parameters and antioxidantenzymes activities were investigated in non-grafted, self-grafted and rootstock grafted plants, exposed to0, 50, 100 and 150 mM NaCl concentration, over a 2-week period. 100 and 150 mM NaCl significantlydecreased the net CO2 assimilation rate (A) and stomatal conductance (Gs), as well as photochemi-cal quenching coefficient (qP), efficiency of the excitation energy capture by open PSII reaction centers(F ′

v/F ′m) and actual quantum yield of photosynthesis (˚PSII). However, only slight reversible photoinhibi-

tion occurred at 150 mM NaCl in non-grafted and self-grafted plants. However, the inhibition induced bysalinity was significantly alleviated in rootstock-grafted plants. Lipid peroxidation was largely increased

ntioxidant enzymes

hotosynthesishlorophyll fluorescence parameters

by 100 and 150 mM NaCl treatment in non-grafted and self-grafted tomato plants, and was less influencedin rootstock-grafted plants. This alleviation of oxidative damage was due to the increase in activities ofcatalase (CAT) and enzymes involved in ascorbate–glutathione cycle such as ascorbate peroxidase (APX),dehydroascorbate reductase (DHAR) and glutathione reductase (GR). The alleviation of growth inhibi-tion of rootstock-grafted plants by salt stress may be related to improvement of photosynthesis and

ant e

enhancement of antioxid

. Introduction

Salinity is one of the major environmental stresses affecting theerformance of many crop plants. About 7% of the world’s land areand 20% of the world’s irrigated land are affected by soil salin-ty (Munns, 2002; Yamaguchi and Blumwald, 2005). The growthnhibition observed in many plants subjected to salinity is oftenssociated with a decrease in their photosynthetic capacity. Theecrease in net photosynthesis rate induced by salinity differs in dif-erent genotypes. In general, net photosynthesis rate and stomatalonductance of salt tolerant genotypes were less affected by salin-ty than salt-sensitive genotypes (Naumann et al., 2007; Zhao et al.,007; Lopez-Climent et al., 2008). The salt-induced limitation ofhe photosynthetic performance may be due to stomatal limitationnd/or non-stomatal limitation (Debez et al., 2008). Since salinitytrongly limits the photosynthetic CO2 fixation, the light energy

bsorption rate by photosynthetic pigments exceeds the rate of itsonsumption in chloroplasts (Foyer and Noctor, 2005). The excessnergy may accelerate the photodamage to PSII via the generationf the reactive oxygen species (ROS) (Melis, 1999; Yang et al., 2007).

∗ Corresponding author at: Department of Horticulture, Zhejiang University,angzhou 310029, PR China. Tel.: +86 571 86971354; fax: +86 571 86971354.

E-mail addresses: zhjzhu@zju.edu.cn, zhuzj@zjfc.edu.cn (Z. Zhu).

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.02.007

nzymes activity.© 2009 Elsevier B.V. All rights reserved.

A number of pathways are proposed to cooperate in protectingthe photosynthetic apparatus from photo-oxidative stress. Theseinclude photorespiration, xanthophyll cycle-dependent energy dis-sipation, the cyclic electron flows through either PSI or PSII andantioxidant system (Asada, 2006). Besides chloroplast, the mito-chondria and peroxisome are important intracellular generatorsof ROS (Dat et al., 2000; Navrot et al., 2007). ROS includes super-oxide (O2

•−), hydroxyl radicals (OH•), hydrogen peroxide (H2O2)and singlet oxygen (1O2). These ROS are highly reactive and theycan seriously disrupt normal metabolism through oxidative dam-age to lipids, protein and nucleic acids in the absence of anyprotective mechanism (Mittler, 2002). There is evidence that salin-ity may cause oxidative stress by inducing an imbalance in cellcompartment in the production of ROS and antioxidant defense(Mittler, 2002; Apel and Hirt, 2004). The antioxidative system inplants includes low-molecular mass antioxidants as well as antiox-idant enzymes, such as superoxide dismutase (SOD), catalase (CAT),ascorbate peroxidase (APX), guaiacol peroxidase (GPOD), dehy-droascorbate reductase (DHAR) and glutathione reductase (GR)(Noctor and Foyer, 1998).

The application of grafting began towards the end of 1920,initially to limit the effects of soil pathogens such as Fusarium oxyso-rum (Ke and Saltveit, 1988). Recently the technique has been widelyused to enhance nutrient uptake (Ahmedi et al., 2007), to induceresistance against drought (Erismann et al., 2008), to increase

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esistance to low (Zhou et al., 2007) and high temperatures (Riverot al., 2003), to induce resistance against heavy metal toxicityRouphael et al., 2008) and to increase synthesis of endogenousormones (Dong et al., 2008). In relation to salt tolerance, manytudies have been carried out to determine the response of graftedlants to salinity. These results suggested that grafting providedn alternative way to improve salt tolerance by reducing the ionictress (Romero et al., 1997; Santa-Cruz et al., 2002; Chen et al., 2003;stan et al., 2005; Martinez-Rodriguez et al., 2008). Romero et al.1997) observed that root characteristics are of primary importancen determining salt tolerance in melon plants, whereas Chen et al.2003) showed the significance of shoot tolerance rather than thatf the root in tomato. Santa-Cruz et al. (2002) suggested that bothhoot and root characteristics played important roles in salt toler-nce. However, few studies are concerned about the photosynthesiserformance and antioxidant system of grafted plants under salineonditions.

In the present study, we exposed non-grafted, self-grafted andootstock-grafted tomato plants to different concentration of salttresses and investigated whether grafted plants could improve tol-rance to salinity, and whether the induced tolerance to salt stressas associated with the protection of the photosynthetic apparatus

nd the enhancement of the plant antioxidative system. Accord-ngly, gas exchange, chlorophyll fluorescence, antioxidant enzymesnd K and Na concentrations were determined in non-grafted, self-rafted and rootstock-grafted tomato plants.

. Materials and methods

.1. Materials

The commercial tomato hybrid Lycopersicon esculentum Mill. cv.ezuo903 was used as scion and Zhezhen No. 1 (a widely usedommercial rootstock variety in Zhejiang Province, China, providedy Zhejiang Academy of Agricultural Science) was selected as root-tock. Tomato seeds were germinated on moisture filter paper inhe dark at 28 ◦C for 3 days, then grown in vermiculite for about0 days. Grafting was performed when seedlings have developed–4 true leaves using the procedure described by Santa-Cruz et al.2002). Non-grafted and self-grafted plants were included as con-rols. In order to maintain a high humidity level and facilitate graftormation, seedlings were covered with a transparent plastic lid andere left in the shade for 24 h. The plastic was opened slightly everyay to allow reduction in relative humidity and it was removed 6ays after grafting.

Then the seedlings were transplanted into 10 l plastic con-ainers containing aerated full nutrient solution: 5 mM Ca(NO3)2,mM KNO3, 2.5 mM KH2PO4, 2 mM MgSO4, 29.6 �M H3BO3, 10 �MnSO4, 50 �M Fe-EDTA, 1.0 �M ZnSO4, 0.05 �M H2MoO4, 0.95 �M

uSO4, four seedlings per container. The pH of the nutrient solutionas maintained close to 6.5 by adding H2SO4 or KOH. The seedlingsere cultivated under control growth conditions (70% RH, 28/20 ◦C

day/night) temperature, a light intensity of 800 �mol m−2 s−1 and14 h photoperiod). 7 days after transfer to the hydroponic medium,

he salt treatments were applied by adding 50 mM NaCl, 100 and50 mM NaCl to the nutrient solution and maintained for 14 days.our trays (16 plants) were included in each treatment and in theontrols.

.2. Gas exchange measurements

After 14 days of salt treatment, four plants were randomlyelected from each treatment for leaf gas exchange measurements.he most recently fully expanded leaf was used for measurementith an open gas exchange system (Li-6400, Li-Cor, Inc., Lincoln,E, USA). The system was calibrated prior to measurement. Net

ental Botany 66 (2009) 270–278 271

photosynthetic rate (A), stomatal conductance (Gs), intercellularCO2 concentration (Ci) and transpiration rate (E) were determinedbetween 9:00 and 12:00 at 800 �mol m−2 s−1 photosynthetic pho-ton flux density (PPFD). During measurement, relative humiditywas maintained at 70% and leaf temperature was set at 28 ± 0.5 ◦Cin the leaf chamber. Water use efficiency (WUE) was calculated asA/E.

2.3. Fluorescence measurements

Light-adapted and dark-adapted measurements of chlorophyllfluorescence were conducted on the same leaf for gas exchangemeasurements using a pulse amplitude modulated leaf chamberfluorometer (Li-6400, Li-Cor, Inc., Lincoln, NE, USA). Minimal flu-orescence values in the dark-adapted state (Fo) were obtained byapplication of a low-intensity red measuring light source (630 nm),whereas maximal fluorescence values (Fm) were measured afterapplying a saturating light pulse of 8000 �mol m−2 s−1, and thusmaximum quantum use efficiency of PSII in the dark-adapted statewas calculated, Fv/Fm = (Fm − Fo)/Fm. The leaf area assayed was darkadapted for at least 15 min prior to Fv/Fm measurements. Minimum(F ′

o) and maximum (F ′m) values of fluorescence in the light-adapted

state at 800 �mol m−2 s−1 were also obtained in this manner.After leaves were continuously illuminated with actinic light for6 min, the steady-state fluorescence (Fs) was recorded. Using theseparameters, the following ratios were calculated: effective quan-tum use efficiency of PSII in the light-adapted state, F ′

v/F ′m = (F ′

m −F ′

o)/F ′m, effective quantum yield, ˚PSII = (F ′

m − Fs)/F ′m, photochem-

ical quenching, qP = (F ′m − Fs)/F ′

m − F ′o and non- photochemical

quenching, NPQ = (Fm − F ′m)/F ′

m.

2.4. Determination of plant growth and cation concentrations

After 14 days of treatment, the plants were harvested anddivided into shoots and roots. The shoots and roots were dried at70 ◦C to constant mass and their dry mass was measured.

For determination of cation concentrations, the mixtures ofdried samples were digested in a mixture of H2SO4–H2O2 (38.1%H2SO4, w/v; 3% H2O2, w/v), and the concentrations of K and Na weredetermined with atomic absorption spectrometry (SHIMADZU AA-6300, Japan).

2.5. Determination of lipid peroxidation

Lipid peroxidation was determined in terms of concentrationof thiobarbituric acid-reactive substances (TBARS) according tothe method of Cakmak and Marschner (1992). 0.3 g fresh tissuehomogenized in 3 ml of 0.1% (w/v) trichloroacetic acid (TCA). Thehomogenate was centrifuged at 10,000 × g for 10 min at 4 ◦C. Thereaction mixture in a total volume of 4 ml containing 1 ml ofextracts, 3 ml of 2% (w/v) TBA (2-thiobarbituric acid) made in 20%TCA. The reaction mixture was heated at 95 ◦C for 30 min and thenstopped by quickly placing it in an ice-bath. After centrifugation at10,000 × g for 10 min, the absorbance of the supernatant at 532 and600 nm was read. After subtracting the non-specific absorbance at600 nm, the TBARS concentration was determined by its extinctioncoefficient of 155 mM−1 cm−1.

2.6. Determination of enzymatic activities

For the enzyme assays, 0.3 g leaves were ground with 2 ml ice-

cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mMascorbate and 2% PVP. The homogenates were centrifuged at 4 ◦C for20 min at 12,000 × g and the resulting supernatants were used forthe determination of enzymatic activity and protein content assays(Zhu et al., 2000). All steps in the preparation of the enzyme extract

272 Y. He et al. / Environmental and Experimental Botany 66 (2009) 270–278

F ion ont test (

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ig. 1. Effects of NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solutomato plants. Different letters indicate significant differences according to the LSD

ere carried out at 4 ◦C. All spectrophotometric analyses wereonducted on a SHIMADZU UV-2410PC spectrophotometer. Eacheasurement of protein concentration or antioxidant enzymes wasade four replicates in four plants.Protein concentration was determined using a Coomassie bril-

iant blue with bovine serum albumin as the standard (Bradford,976).

The activity of superoxide dismutase (SOD) was assayed by mea-uring its ability to inhibit the photochemical reduction of nitroblueetrazolium (NBT) (Rao and Sresty, 2000). The reaction mixture3 ml) contained 50 mM phosphate buffer (pH 7.8), 13 mM methio-ine, 75 �M NBT nitroblue tetrazolium, 2 �M riboflavin, 0.1 mMDTA and 0.05 ml of enzyme extract. The reaction was started bydding 2 �M riboflavin and placing the tubes under 15 W fluo-escent lamps for 15 min. A complete reaction mixture withoutnzyme, which gave the maximal color, served as control. The reac-ion was stopped by switching off the light, and then the tubes wereovered with a black cloth. A non-irradiated complete reaction mix-ure served as a blank. The absorbance was recorded at 560 nm, andne unit of SOD was defined as being presented in the volume ofxtract that caused inhibition of the photo-reduction of NBT by 50%.

Catalase (CAT) activity was measured as the decline inbsorbance at 240 nm due to the decomposition decline of extinc-

ion of H2O2. The reaction mixture contained 25 mM potassiumhosphate buffer (pH 7.0), 10 mM H2O2 and 0.1 ml enzyme extracts.he reaction was started by adding H2O2 (Cakmak and Marschner,992).

able 1ffects of NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solution contentootstock-grafted tomato plants. Different letters indicate significant differences accordin

reatment (mM) Na concentration (mg g−1 DW) K concen

Leaf Root Leaf

on-grafted0 5.05 ± 0.07d 5.48 ± 0.17d 39.05 ±50 17.79 ± 1.26c 21.67 ± 0.85c 21.41 ±100 56.68 ± 5.56b 62.36 ± 2.57b 15.49 ±150 75.13 ± 4.25a 78.45 ± 6.93a 9.61 ±

elf-grafted0 4.88 ± 0.21d 5.54 ± 0.59d 39.16 ±50 21.31 ± 0.31c 23.42 ± 2.94c 22.50 ±100 58.59 ± 1.29b 65.15 ± 3.69b 17.49 ±150 80.22 ± 4.16a 79.95 ± 1.98a 12.10 ±

ootstock-grafted0 5.13 ± 0.10d 5.68 ± 0.37d 42.43 ±50 22.37 ± 3.26c 21.61 ± 0.38c 25.88 ±100 59.84 ± 1.97b 62.95 ± 0.69b 14.34 ±150 76.12 ± 1.36a 77.47 ± 2.48a 11.00 ±

shoot (A) and root dry mass (B) of non-grafted, self-grafted and rootstock-graftedP < 0.05). Values are the means of four replicate samples.

Guaiacol peroxidase (GPOD) activity was measured using mod-ification of the procedure of Egley et al. (1983). The reactionmixture in a total volume of 2 ml contained 25 mM potassiumphosphate buffer (pH 7.0), 0.1 mM EDTA, 0.05% guaiacol (2-ethoxyphenol), 1.0 mM H2O2 and 0.1 ml enzymes extract. Theincrease of absorbance due to polymerization of guaiacol totetraguaiacol was measured at 470 nm (E = 26.6 mM−1 cm−1) over30 s.

Ascorbate peroxidase (APX) activity was measured according toNakano and Asada (1981) as the decrease in absorbance at 290 nmdue to ascorbate oxidation. The reaction mixture (2 ml) contained25 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate,0.1 mM H2O2, 0.1 mM EDTA and 0.1 ml enzyme extract. The reactionwas started by adding H2O2.

The assay of dehydroascorbate reductase (DHAR) activity wascarried out by measuring the increase in absorbance at 265 nmdue to ASA formation (Nakano and Asada, 1981). The reaction mix-ture contained 25 mM potassium phosphate buffer (pH 7.0), 2.5 mMreduced glutathione (GSH), 0.4 mM dehydroascorbate (DHA) and0.1 ml enzyme extract.

Glutathione reductase (GR) activity was determined by themethod of Foyer and Halliwell (1976), using a reaction mixtureof 25 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA,

0.5 mM oxidized glutathione (GSSG), 0.12 mM NADPH and 0.1 mlenzyme extract in a total volume of 2 ml. The GR activity wasmeasured by following the decrease in absorbance at 340 nm(E = 6.2 mM−1 cm−1) due to NADPH oxidation over 30 s.

s of Na and K and K/Na ratio in leaves and roots of non-grafted, self-grafted andg to LSD test (P < 0.05). Values are the means of four replicate samples.

tration (mg g−1 DW) K/Na

Root Leaf Root

2.26a 48.06 ± 2.14a 7.74 ± 0.46a 8.77 ± 0.13a2.45b 30.25 ± 0.82b 1.21 ± 0.22b 1.33 ± 0.11b1.06c 20.53 ± 1.10c 0.27 ± 0.01c 0.33 ± 0.03c0.29d 10.44 ± 1.96d 0.13 ± 0.01d 0.14 ± 0.03d

1.38a 43.85 ± 2.16a 8.04 ± 0.35a 9.77 ± 1.45a3.16b 29.89 ± 0.90b 1.28 ± 0.31b 1.28 ± 0.07b2.27c 20.55 ± 0.66c 0.24 ± 0.03c 0.32 ± 0.04c0.91d 10.32 ± 0.56d 0.15 ± 0.01d 0.13 ± 0.02d

2.30a 46.68 ± 2.90a 8.27 ± 0.57a 10.03 ± 1.14a0.75b 30.65 ± 2.70b 1.63 ± 0.27b 1.35 ± 0.13b1.08c 22.75 ± 0.62c 0.26 ± 0.02c 0.36 ± 0.01c0.72d 17.82 ± 0.83d 0.14 ± 0.01d 0.23 ± 0.02d

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Y. He et al. / Environmental and E

.7. Statistical analysis

Data presented are the mean values of four replicates. Statisticalssays were carried out by analysis of variance (ANOVA) test usingAS software (SAS Institute, Cary, NC) and means were comparedy the least significant difference (LSD) test. Comparisons with Palues < 0.05 were considered significantly different.

. Results

.1. Plant growth

Shoot dry mass of tomato plants was not affected at 50 mMaCl treatment, but decreased significantly at 100 and 150 mMaCl treatment (P < 0.05). Shoot dry mass was decreased by 51.8%,1.7% and 34.8% over control at 100 mM NaCl treatment in non-

ig. 2. Effects of NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solution ononcentration Ci (C), transpiration rate E (D) and water use efficiency WUE (E) of non-gignificant differences according to LSD test (P < 0.05). Values are the means of four replic

ental Botany 66 (2009) 270–278 273

grafted, self-grafted and rootstock-grafted plants, respectively, andfurther decreased by 80.3%, 79.0% and 58.3% at 150 mM NaCltreatment (Fig. 1A). Similarly, root dry mass declined at 100 and150 mM NaCl, however, less significantly in rootstock-grafted plants(Fig. 1B).

3.2. K and Na concentrations

As shown in Table 1, leaf and root Na concentrationincreased dramatically with increasing NaCl concentration in

solution (P < 0.05). The highest leaf and root Na concentra-tions were observed in plants supplied with 150 mM NaCl.Non-grafted, self-grafted and rootstock-grafted plants showedsimilar leaf and root Na concentrations under the same saltlevels.

leaf net CO2 assimilation rate A (A), stomatal conductance Gs (B), intercellular CO2

rafted, self-grafted and rootstock-grafted tomato plants. Different letters indicateate samples.

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74 Y. He et al. / Environmental and E

Leaf and root K concentration decreased significantly inesponse to increase of NaCl concentration in tomato plantsP < 0.05). The lowest K concentration was observed in 150 mM NaClreatment plants. Non-grafted, self-grafted and rootstock-graftedlants showed similar leaf or root K concentrations under the samealt levels except for root K concentration under 150 mM NaClreatment, at which rootstock-grafted plants had higher root K con-entration than non-grafted and self-grafted plants (Table 1).

Non-grafted, self-grafted and rootstock-grafted plants had sim-lar leaf or root K/Na ratio under the same salt levels exceptor root K/Na ratio under 150 mM NaCl treatment, at whichootstock-grafted plants had higher K/Na ratio than non-grafted andelf-grafted plants (Table 1).

ig. 3. Effects of NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solution ohotochemical quenching (qP) (B), effective quantum use efficiency of PSII in the light-adauenching (NPQ) (E) of non-grafted, self-grafted and rootstock-grafted tomato plants. Difre the means of four replicate samples.

ental Botany 66 (2009) 270–278

3.3. Leaf gas exchange

As shown in Fig. 2, 50 mM NaCl did not affect net CO2assimilation rate (A), transpiration rate (E), intercellular CO2 con-centration (Ci) and water use efficiency (WUE), but considerablyreduced stomatal conductance (Gs). A, Gs and E (Fig. 2A, B andD) were decreased dramatically by 100 and 150 mM NaCl treat-ment (P < 0.05), especially under 150 mM NaCl treatment. A (Fig. 2A)

was decreased by 82.7% and 79.1% in non-grafted and self-graftedplants at 150 mM NaCl treatment compared with control, respec-tively, while decreased by 45.6% in rootstock-grafted plants. Gs

and E (Fig. 2B and D) in rootstock-grafted plants were significantlyhigher than that in non-grafted and self-grafted plants under 100

n maximum quantum use efficiency of PSII in the dark-adapted state (Fv/Fm) (A),pted state (F ′

v/F ′m) (C), effective quantum yield (˚PSII) (D) and non-photochemical

ferent letters indicate significant differences according to LSD test (P < 0.05). Values

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Y. He et al. / Environmental and E

nd 150 mM NaCl treatment (P < 0.05). Ci (Fig. 2C) was significantlyecreased at 100 mM treatment (P < 0.05), and further decreased inootstock-grafted plants at 150 mM NaCl treatment, while increasedignificantly (P < 0.05) in non-grafted and self-grafted plants at50 mM NaCl treatment. WUE (Fig. 2D) was enhanced in non-rafted and self-grafted plants at 100 mM NaCl treatment, and theneduced at 150 mM NaCl treatment, while WUE in rootstock-graftedlants increased at both 100 and 150 mM NaCl treatments.

.4. Chlorophyll fluorescence parameters

Compared to controls, the maximal quantum yield of PSII (Fv/Fm)as not significantly affected by 50 and 100 mM NaCl. However,

v/Fm was significantly decreased at 150 mM NaCl treatment in non-rafted and self-grafted plants. But the decrease by 150 mM NaCl inootstock-grafted plants was not significant (P > 0.05) (Fig. 3A). Thehotochemical quenching coefficient (qP), efficiency of excitationaptured by open PSII (F ′

v/F ′m) and actual quantum yield of photo-

ynthesis (˚PSII) was not significantly influenced by 50 mM NaClreatment (Fig. 3B–D). qP, F ′

v/F ′m and ˚PSII decreased significantly

n non-grafted and self-grafted plants at 100 and 150 mM NaClreatment (P < 0.05), especially at 150 mM NaCl. However, thesearameters were less affected in rootstock-grafted plants. Only′v/F ′

m and ˚PSII at 150 mM NaCl treatment were significantly influ-nced (P < 0.05). As shown in Fig. 3E, non-photochemical quenchingNPQ) increased significantly (P < 0.05) in grafted or non-graftedlants at different salt concentration. The similar NPQ was observed

n non-grafted plants, self-grafted and rootstock-grafted plants at00 mM NaCl and 150 mM NaCl treatment.

.5. Lipid peroxidation

TBARS concentration, a measure of lipid peroxidation, is givenn Fig. 4. Compared to controls, leaf TBARS concentration wasot significantly influenced at 50 mM NaCl treatment, however,

ncreased greatly in non-grafted and self-grafted plants at 100 and50 mM NaCl treatments, especially at 150 mM NaCl treatment. TheBARS concentration was not changed in rootstock-grafted plants

t 100 mM NaCl with respect to control, increased significantly at50 mM NaCl treatment (P < 0.05). TBARS concentration increasedy 336.4%, 340.2% and 33.1% compared with control in non-grafted,elf-grafted plants and rootstock-grafted plants at 150 mM NaClreatment, respectively.

ig. 4. Effects of NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solu-ion on leaf TBARS concentration of non-grafted, self-grafted and rootstock-graftedomato plants. Different letters indicate significant differences according to LSD testP < 0.05). Values are the means of four replicate samples.

ental Botany 66 (2009) 270–278 275

3.6. Antioxidant enzymes

The activities of SOD and GPOD (Fig. 5A and C) were significantlyincreased in non-grafted and self-grafted tomato leaves exposedto 100 and 150 mM NaCl treatments, while the increase of SODwas only observed in rootstock-grafted plants at 150 mM NaCl. Thehighest SOD and GPOD activities were observed in non-grafted andself-grafted plants under 150 mM NaCl treatment (Fig. 5A and C).Unlike the activities of SOD and GPOD, CAT and DHAR activitiesdecreased considerably in salt stressed non-grafted and self-graftedplants, while increased significantly in rootstock-grafted plants(Fig. 5B and E). 50 mM NaCl treatment did not affect the APXactivity in non-grafted and self-grafted plants, while considerablyincreased the APX activity in rootstock-grafted plants. 100 and150 mM NaCl treatments increased APX activity, and there wereno significant difference in APX activity among non-grafted, self-grafted and rootstock-grafted plants (Fig. 5D). GR activity increasedin non-grafted and grafted tomato plants at 50, 100 and 150 mMNaCl. Rootstock-grafted plants showed much higher GR activitythan non-grafted and self-grafted plants in both control conditionsand salt stress conditions (Fig. 5F).

4. Discussion

In the present experiment, grafting did not affect shoot and rootdry mass under non-salinity condition. These results confirm pre-vious data obtained with shoots (Santa-Cruz et al., 2002; Estan etal., 2005) and roots (Martinez-Rodriguez et al., 2008) of differenttomato genotypes. Mild salt stress (50 mM) had no effects on shootand root dry mass of tomato plants in this experiment, while somereports indicated that 50 mM NaCl considerably reduced tomatofruit yield (Estan et al., 2005; Martinez-Rodriguez et al., 2008). Thismay be due to the difference of genotype and/or exposure time tosalinity (Ashraf and Harris, 2004). Moderate (100 mM) and severe(150 mM) salt stress dramatically decreased the shoot and root drymass, and rootstock-grafted plants were less affected (Fig. 1). This isin agreement with earlier findings that grafting a commercial culti-var on salt tolerant cultivar improves the salt tolerance (Santa-Cruzet al., 2002; Estan et al., 2005; Martinez-Rodriguez et al., 2008).

Net CO2 assimilation rate (A) declined with increasing level ofsalt stress. In rootstock-grafted plants, the decrease was accom-panied by a significant decrease in stomatal conductance (Gs)and intercellular CO2 concentration (Ci), implying that stomatallimitations were responsible for the reduction in A by salt treat-ment (Farquhar and Sharkey, 1982). In non-grafted and self-graftedplants, Ci decreased under moderate stress conditions, but did notchange under severe conditions, while A and Gs decreased dra-matically, implying the occurring of non-stomatal limitations. Thismight indicate that when exposing to 150 mM NaCl the non-graftedand self-grafted plants were suffered from severe stress (Flexasand Medrano, 2002). Under moderate and severe salt stresses, therootstock-grafted plants showed higher A than non-grafted andself-grafted plants (Fig. 2A). This is in agreement with previousfindings that rootstock-grafting improves the photosynthesis per-formance of plants under salinity (Moya et al., 2002; Massai et al.,2004). Unlike A and Ci, Gs significantly decreased under mild saltstress (Fig. 2B), which corroborates previous findings that Gs is verysensitive to salt stress (Netondo et al., 2004; Jiang et al., 2006a).However, there were some conflicting results that Gs was resistantto salt stress (Lu et al., 2003; Naumann et al., 2007). This may be dueto different plant species or environmental conditions. Water use

efficiency (WUE), calculated by the ratio of A to E, increased in mod-erate salt stressed tomato plants (Fig. 2E), owing to the fast decreaseof transpiration rate. Compared with the non-grafted and self-grafted plants, rootstock-grafted plants had a higher WUE undersevere saline conditions, which might result from the improve-

276 Y. He et al. / Environmental and Experimental Botany 66 (2009) 270–278

F n on an ificans

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o2sioatado

ig. 5. Effects of NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solutioon-grafted, self-grafted and rootstock-grafted plants. Different letters indicate signamples.

ent of photosynthesis performance of rootstock-grafted plants.he higher WUE is of importance for salt tolerance, since high WUEay reduce the uptake of salt and alleviate the water deficiency

nduced by salinity (Moya et al., 1999; Karaba et al., 2007).The reduction of photochemical activity is considered to be one

f the non-stomatal factors that limit photosynthesis (Souza et al.,004). Thus, the activity of PSII was investigated in the presenttudy by using chlorophyll fluorescence technology. Fv/Fm, the max-mal quantum efficiency of PSII, is frequently used as an indicatorf the photoinhibition or of stress damage to the PSII (Calatayud

nd Barreno, 2004). In the present study, Fv/Fm was not affected inomato plants under mild and moderate salt stress. In non-graftednd self-grafted plants under severe salinity conditions, Fv/Fm

ecreased to 0.76 and 0.77, respectively (Fig. 3A), suggesting theccurrence of photoinhibition, and this could be a consequence of

ctivities of SOD (A), CAT (B), GPOD (C), APX (D), DHAR (E) and GR (F) in the leaves oft differences according to LSD test (P < 0.05). Values are the means of four replicate

damage to PSII (Demmig-Adams and Adams, 1992). Similar resultswere observed in barley (Maxwell and Johnson, 2000). The constantFv/Fm in rootstock-grafted plants might show that photoinhibitionof PSII was not triggered. There were also some conflicting resultsthat reduction in Fv/Fm was much less marked under high salin-ity, which was attributed to the increase in chlorophyll a content(Redondo-Gómez et al., 2007). Compared with Fv/Fm, actual quan-tum yield of photosynthesis (˚PSII) was more sensitive to salt stress.Moderate and severe salt stress considerably decreased ˚PSII innon-grafted and self-grafted plants (Fig. 3D), which was attributed

to both the decrease of open PSII reaction centers (qP) and theefficiency of the excitation energy capture by open PSII reactioncenters (F ′

v/F ′m) (Fig. 3B and C), since ˚PSII is a product of qP and

F ′v/F ′

m (Genty et al., 1989). Decreased ˚PSII implies lower electrontransport to carbon fixation, and thus decreases the net CO2 assim-

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lation rate (Maxwell and Johnson, 2000). In the present study, theigher ˚PSII in rootstock-grafted plants was well correlated to theetter performance of photosynthesis under moderate and severealt stress. Under moderate and severe salt stresses, ˚PSII was con-iderably decreased with a slight or no decrease in Fv/Fm, might beue to an enhancement in NPQ, which suggested that more energyas dissipated through thermal in PSII (Fig. 3E).

Another common damage under saline conditions is the accu-ulation of excessive ROS (Asada, 2006), which occurs when the

eduction in photosynthesis is much higher than the extent ofhe reduction in ˚PSII, suggesting electrons flowing to oxygen

olecules rather than being used for carbon assimilation. Multi-le antioxidant enzymes systems are believed to play an importantole in the scavenging of ROS, and thus protect cells from oxidativeamage. The antioxidant enzymes respond differently under salineonditions. As observed by Zhu et al. (2004), the activities of CAT andHAR were decreased under salinity, while activities of SOD, APX,POD and GR were enhanced. In the present study, SOD activity

ncreased under salt stress, more considerably in non-grafted andelf-grafted plants (Fig. 5A). Similar results were observed in graftedlants under thermal stress (Rivero et al., 2003), and this might beue to the production of ROS under stress conditions remaining

ower in rootstock-grafted plants than in non-grafted or self-graftedlants. CAT activities, together with SOD which is thought to be theost effective antioxidant enzymes in preventing cellular damage

Scandalios, 1993), increased with salinity level in rootstock-graftedlants, while declined in non-grafted and self-grafted plants. Theigher CAT activities observed in rootstock-grafted plants might beeneficial to scavenge the ROS. APX, DHAR and GR are importantntioxidant enzymes involved in ascorbate–glutathione cycle. Inhis cycle, APX reduces H2O2 to H2O and O2, using ASA as a reduc-ng substrate. DHAR and GR are required for the regeneration of ASAnd GSH, respectively, which were of two important antioxidantscavenging ROS directly or indirectly (Noctor and Foyer, 1998). Theigher DHAR and GR activities in rootstock-grafted plants especiallynder salinity may contribute to higher ASA and GSH contents.ecause of the higher activity of antioxidant system in rootstock-rafted plants, the TBARS concentration was much lower comparedith the non-grafted and self-grafted plants (Fig. 5), suggesting less

xidative damage occurring.It is reported that grafting might mitigate salt stress by reduc-

ng the Na uptake, enhancing K uptake and keeping a higher K/Naatio in leaves and roots (Romero et al., 1997; Santa-Cruz et al.,002; Chen et al., 2003; Estan et al., 2005; Martinez-Rodriguez et al.,008). However, in the present study, except for a higher K concen-ration and K/Na ratio in rootstock-grafted plant roots under severealine conditions, there were no differences in the K, Na content and/Na ratio among non-grafted, self-grafted and rootstock-graftedlants at the same salt levels (Table 1). We assumed that this mighte due to different genotypes of tomato plants.

. Conclusion

In conclusion, this study showed that grafting with a salt tol-rant rootstock improved the photosynthesis performance withigher Gs and WUE under saline conditions, kept higher pho-ochemical activity of PSII, increased the capacity of antioxidantystem by enhancing the activities of CAT and enzymes involved inscorbate–glutathione cycle and decreased the level of lipid perox-dation, as a result, the growth of tomato was promoted.

cknowledgement

This work was supported by the National Science Foundation ofhina (No. 30471183). In addition, the authors thank the reviewers

or their constructive comments and detailed corrections.

ental Botany 66 (2009) 270–278 277

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