Attenuation of salt-induced changes in photosynthesis by exogenous nitric oxide in tomato ( Lycopersicon esculentum Mill . L . ) seedlings

Exogenous sodium nitroprusside (SNP), a NO donor, was applied in this study to investigate the potential role of NO in photosynthetic performance of tomato (Lycopersicon esculentum L. cv. Hufan2560) seedlings under salt-stressed conditions. Exogenous NO alleviated the decrease in dry mass of shoot and root caused by salt stress. In parallel, NO application in salt-stressed plants attenuated the decrease in the photosynthetic parameters such as leaf chlorophyll, net photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E), the ratio of variable to maximum fluorescence (Fv/Fm), electron transport rate (ETR), the efficiency of excitation energy capture by open photosystem II (PSII) reaction centers (Fv ́/Fm ́), and the photochemical quenching coefficient (qP), and counteracted the increase in on-photochemical quenching coefficient (qN). Furthermore, the changes as mentioned above reversed by NO treatment are specific to salt stress since application of NO alone to tomato seedlings without salt stress had slight effects on the tested parameters. The results obtained here demonstrated that the photosynthetic performance was improved by NO application in salt-stressed plants and such an improvement was associated with an enhancement of gas-exchange and the actual PSII efficiency, which revealed an important role of NO in enhancing resistance of plants to salt stress.


INTRODUCTION
It is estimated that about half of the irrigated land and 20% of the world's cultivated land are currently affected by salinity (Zhu, 2001).Especially, cultivated plants are threatened by NaCl, which becomes of great importance for agriculture production (Tester and Davenport, 2003).Currently, therefore, further steps in understanding molecular and physiological mechanisms for salt tolerance and finding ways to enhance salinity stress tolerance are crucially important in agriculture.
The decline in growth observed in many plants under salinity condition is generally associated with several *Corresponding author.E-mail: wmzhusaas@126.com.Tel/Fax: +86-21-62200977.physiological actions including ion toxicity, osmotic stress, reactive oxygen species (ROS), etc. (Parida and Das, 2005).Among the primary processes affected by salinity stress, photosynthesis is the most sensitive process (Munns et al., 2006).The salt-induced limitation of the photosynthetic performance may be due to stomatal limitation and/or non-stomatal limitation (Hichem et al., 2009).Salinity stress influenced net photosynthetic rate (P N ), stomatal conductance (gs) and intercellular CO 2 concentration (Ci) of plants (Parida and Das, 2005;Qu et al., 2009).Moreover, reduction in PSII efficiency caused by salt stress seems to be associated with the photosystem II (PSII) complex, primary charge separation in PSII and pigment-protein complexes of thylakoid membranes of chloroplasts, PSII activity, and the quantum yield of PSII electron transport (Zhang et al., 2009).Afr.J. Biotechnol.
However, the mechanisms of the inhibited photosynthetic capacity under salt stress are still largely unknown.Nitric oxide (NO), a relatively stable free radical gas, acts as a key signaling molecule with multiple biological functions in plants.NO was reported to be involved in seed germination, growth and cell proliferation, maturation and senescence, programmed cell death, stomatal movement and responses to abiotic and biotic stresses (Neill et al., 2003;Wendehenne et al., 2004;Lamotte et al., 2005;Besson-Bard et al., 2008).The involvement of NO in salinity tolerance has drawn much attention in the past few years.For instance, under salinity conditions, the exogenous NO can enhance salt tolerance by alleviating oxidative damage (Shi et al., 2007;Zheng et al., 2009), stimulating activities of protonpump and Na + / H + antiport in the tonoplast (Zhang et al., 2006), and increasing the K + /Na + ratio (Zhao et al., 2007).However, the effect of NO on phytosynthetic activity has so far been poorly addressed, with often conflicting results.Takahashi and Yamasaki (2002) showed that the NO donor, S-nitroso-N-acetyl penicillinamine (SNAP), does not modify the maximal quantum efficiency (Fv/Fm), but inhibits the linear electron transport rate and pH formation across the thylakoid membrane.Yang et al. (2004) and Wodala et al. (2008) found that treatment with sodium nitroprusside (SNP), a NO donor, decreased Fv/Fm values and photochemical quenching coefficient (qP).Contrarily, Zhang et al. (2006) reported that application of SNP increased Fv/Fm and the effective quantum yield of PSII ( II) in light-mediated greening of barley seedlings.Exogenous SNP was also reported to retard decrease in Fv/Fm caused by osmotic stress or copper toxicity (Singh et al., 2003;Tan et al., 2008).Nevertheless, little information is available about the influence of exogenous NO on photosynthetic performance under salinity condition.
Tomato is an important greenhouse crop worldwide, which is greatly affected by high salinity in soil (Cantore et al., 2008).Based on the above observation, the objective of the present study is to investigate whether exogenous SNP plays an important role in protecting photosynthetic performance against salt stress, as assessed by chlorophyll contents, photosynthetic gas exchange and chlorophyll fluorescence that are three sets of commonly used parameters.The physiological mechanism of exogenous NO on photosynthetic performance of saltstressed tomato seedlings is also discussed, which leads to improve understanding of the mechanisms regarding the alleviation of salt toxicity.

Plant culture and treatments
The experiment was carried out using plants grown in an environment-controlled greenhouse of Shanghai Academy of Agricultural Sciences.Tomato seeds (Lycopersicon esculentum L. cv.Hufan2560) were rinsed thoroughly with distilled water and germinated on two layers of moist filter paper in Petri dishes at 28°C.The germinated seeds were sown in quartz sands in the greenhouse at a mean temperature cycle of 30°C (day)/25°C (night) and relative humidity of 73/85%.After 15 days, tomato seedlings at the second-true leaf stage were watered with one quarter-strength Hoagland nutrient solution.On day 40, uniformly growing tomato seedlings at the third-true leaf stage were removed from plastic plates, and the roots were rinsed with distilled water.The tomato seedlings were then transferred to plastic vessels containing 10 L half-strength Hoagland nutrient solution.Nutrition solution was replaced with fresh solution every 5-days during the growth period, and the solution was aerated by an aquarium air pump.

Plant treatments
On day 60, when tomato seedlings were at the fourth or fifth-true leaf stage, salinity and NO treatments were performed by adding sodium chloride (NaCl) and SNP, a NO donor, to the nutrient solution.Experiment included four treatment groups designated as: (1) Control: 0 mM NaCl and 0 M SNP; (2) SNP: 0 mM NaCl and 100 M SNP; (3) NaCl: 100 mM NaCl and 0 M SNP, and (4) SNP + NaCl: 100 M SNP and 100 mM NaCl.The solutions as described above were renewed every 2 days.All plants were arranged in a randomized, complete block design with three replicates per treatment, giving a total of 12 containers for each experiment.The number of plants used per cultivar was four in each replicate.The fifth pairs of leaves at upper cotyledons were used for the determination of all experimental parameters.The plants were harvested after 8 days of treatment, divided into shoot and root, and dried in an oven at 65°C to constant weight.

Leaf gas-exchange parameters
PN, gs, transpiration rate (E) and Ci were measured in four plants per cultivar per treatment by a photosynthesis system (LI-6400, LICOR Inc., Lincoln, NE, USA).During the measurements, photosynthetic photon flux density (PPFD) was set to 800 µmol m -2 s -1 , the air relative humidity was about 80%, the leaf temperature was maintained at 25°C and the ambient CO2 concentration (Ca) was about 400 l L -1 .
PN-PPFD, PN-Ci response curves were generated on sunny days between 8:00 and 13:00.For PN-PPFD measurement, the air cuvette temperature and the air CO2 concentration were maintained at 25°C and 400 l L -1 , respectively.PPFD was gradually decreased from 1500 to 50 µmol m -2 s -1 1 (1500,1200,1000,800,600,400,300,200,180,150,100,80 and 50 µmol m -2 s -1 ).Assimilation was recorded at each light level following a 10 min acclimation period.Measurements were repeated to obtain at least three stable readings for each of the marked leaves.For PN-Ca measurement, the marked leaf was placed in the cuvette at 25°C and 1000 µmol m -2 s -1 PPFD, the CO2 concentration was increased from 50 to 1600 l L -1 (50, 80, 100, 150, 180, 200, 300, 400, 600, 800, 1000, 1200,  1500, 1600 l L -1 ).The air within the cuvette was maintained at 80% relative humidity to minimize stomatal conductance.Assimilation was recorded at each CO2 concentration following a 10 min acclimation period.Measurements were repeated to obtain at least three stable readings for each of the marked leaves.

Chlorophyll fluorescence
Chlorophyll fluorescence was measured with a PAM-2100 pulse modulated fluorometer (Walz, Effeltrich Germany).The minimal fluorescence (Fo) was determined by a weak modulated light which was low enough to induce significant variable fluorescence.A 0.8 s saturating light of 8000 mol m -2 s -1 was used on dark-adapted Wu et al. 7839 leaves to determine the maximal fluorescence (Fm).Then the leaf was illuminated by an actinic light of 600 mol m -2 s -1 .When the leaf reached steady-state photosynthesis, the steady-state fluorescence (Fs) was recorded and a second 0.8 s saturating light of 8000 mol m -2 s -1 was applied to determine the maximal fluorescence (Fm´) in the light adapted state.The actinic light was turned off; the minimal fluorescence in the light-adapted state (Fo´) was determined by the illumination with 3 s far-red light; the excitation capture efficiency of open centers (Fv´/Fm´), electron transport rate (ETR), photochemical quenching coefficient (qP), non-photochemical quenching coefficient (qN) were measured.The following fluorescence parameters were calculated based on the above measurements: (1) Maximal efficiency of PSII photochemistry Fv/Fm = (Fm-Fo)/Fm; (2) qP = (Fm´ -Fs)/ (Fm´-Fo); (3) qN = ( Fm -Fm´)/ (Fm -Fo).
Chlorophyll content measurements 0.5 g of leaf samples from each group were homogenized with 80% acetone (v/v) and then the homogenate was filtered through filter paper.Absorbance of the resulting solution was read by spectrophotometric measurements at 603, 647 and 664 nm.Specific chlorophyll content was calculated by using the equations of Moran and normalized to the total fresh weight for each sample (Mochizuki et al., 2001).

Effect of NO on growth
Salt-exposure caused a significant reduction in the dry mass of shoot and root of Hufan2560 on the 8 th day after treatment (Figure 1).Under salt stress condition, shoot and root dry mass decreased by 17.8 and 28.6%, respectively, compared to the control.However, application of exogenous NO dramatically alleviated the decrease in dry mass caused by salt stress (Figure 1).In the presence of 100 M SNP under salt stress, the reduction in shoot and root dry mass declined to 9.38 and 19.6%, respectively.NO alone had little effect on the dry mass, but no significant differences were found between NO treatment and normal condition.

Effect of NO on photosynthetic pigments
Changes of photosynthetic pigment contents were measured in response to salt stress and/or to the addition of NO.The contents of chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll (chl a+b) in the leaves of tomato seedlings are presented in Figure 2.There are significant declines in the concentration of chl a, chl b and chl a+b under salt stress compared with that of the control.However, treatment with exogenous NO increased photosynthetic pigment composition markedly under stress condition.For example, at the 8 th day after NaCl stress, the contents of chl a, chl b and chl a+b were decreased by 64.7, 59.1 and 67%, respectively, in non-NO applied plants, but decreased by 52.5, 42.4 and 55% in NO applied plants, respectively.NO alone caused smaller changes under normal conditions.under salt treatment, we determined the effects of SNP application on gas exchange parameters.Gas exchange and photosynthetic parameters, at photosynthetic photon flux density (800 µmol m -2 s -1

Effects of NO on gas exchange parameters
) and ambient CO 2 concentration (400 l L -1 ) were estimated to be modified by treatments with NaCl and/or NO described below.As shown in Figure 3, in comparison with the control, saltstressed tomato plants displayed significant reduction in net P N , gs and E after 2 -8 days of treatment (Figures 3A  to C).These parameters dropped to the lowest level on day 8.However, this reduction was alleviated by exogenous NO application.Compared to the control, P N , gs and E decreased by 56.3, 70.1 and 63.3%, respectively, under salt stress, whereas decreased by 44.8, 50.2 and 45.7%, respectively, under NO and salt stress.Furthermore, Ci was decreased within 4 days after salt treatment, and then increased from 6 to 8 days.Exposure to exogenous NO attenuated the decrease in Ci at the early stage (0 -4 days) and the increase at the later stages (6 -8 days) under salt stress (Figure 3D).The effects of plants without NaCl showed slight influence caused by NO application.

Effects of NO on P N -PPFD and P N -Ci curves
To further analyze the mechanism by which NO regulate CO 2 fixation, the effects of salt and/or NO stress on photosynthesis depend on PPFD and Ci as indicated in Figure 4.Both P N -PPFD and P N -Ci curves showed similar increases in the P N of tomato seedlings in response to increase in PPFD and Ci under different experimental conditions.However, the increases were much slower and reached lower levels when tomato seedlings were treated with salt, and addition of NO again partially reversed these changes caused by salt stress.

Effects of NO on chlorophyll fluorescence
As shown in Figure 5, in the longer-term of salinity (8 days), the ratio of Fv/Fm were significantly lower in saltstressed plants than in the control, in parallel to the increases in Fo and the decreases in Fm, especially on the 8th day after treatment.Treatment with NO significantly improved Fm, Fv/Fm and Fv/Fo values, and remarkably reduced Fo value under salt stress.Under normal conditions, however, no significant changes in all these parameters were observed after NO application.
The changes of ETR, the efficiency of excitation energy capture by open PSII reaction centers (Fv´/Fm´), qN, and the qP in non-salt-stress and salt-stress after NO application were also examined (Figure 6).The ETR significantly declined from day 2 after NaCl treatment and reached the maximum inhibition at day 8, compared with the control (Figure 6A).Both the Fv´/Fm´ and qP were strongly inhibited by salt stress (Figures 6B and C).These two parameters decreased before day 2, then increased between day 2 and 4, decreased again from day 4 to 8 relative to the control.A different pattern of change of qN in NaCl stressed plants was clearly shown by quenching analyses (Figure 6D).Application of NO significantly increased qP, ETR and Fv´/Fm´, but reduced qN dramatically under salt stress.NO treatment also caused small increases in the values of qP, ETR and Fv´/Fm´ and decreases in qN under the control conditions.

DISCUSSION
The use of NO donors is a general tool for investigating the biological roles of NO in plants surviving under adverse environmental conditions (Shi et al., 2007;Zhang et al., 2006;Zheng et al., 2009).In this research, SNP was used as NO donor to study the role of NO in photosynthetic performance of tomato seedlings under salt stress.Our results showed that SNP application enhanced the salt tolerance of tomato plants which was reflected in a greater increase in the dry mass of shoot and root in NO applied plants than in non-NO applied plants when subjected to salt stress (Figure 1), supporting that NO is actively involved in the regulation of plant growth.Previous studies have demonstrated that the exogenous NO mitigated decrease in plant growth caused by salinity is through increasing antioxidant system, alleviating oxidative damage (Shi et al., 2007;Zheng et al., 2009) and stimulating vacuolar H + -ATPase and H + -PPase activities (Zhang et al., 2006), etc.In the present study, it is suggested that the growth of tomato is associated with the effects of NO on photosynthetic performance under salt stress.The photosynthetic performance was estimated by the changes in pigment content photosynthetic rate and chlorophyll fluorescence parameters.The level of total chlorophyll, chlorophyll a and chlorophyll b concentrations reduced significantly by salt stress in tomato plants (Figure 2).However, treatment with exogenous NO increased leaf chlorophylls under salt stress, which suggested that NO treatment protected the photosynthetic apparatus in tomato.This protective effect of NO on the photosynthetic pigments was expected in view of earlier few studies showing increased photosynthetic pigments by NO treatment (Laxalt et al., 1997;Shi et al., 2005;Pahwa et al., 2009).Decrease in chlorophyll content induced by NaCl was probably due to the destruction of structure of chloroplasts, inhibited synthesis of new chlorophyll, and increased degradation of chlorophylls (Sakaki et al., 1983), which may be arrested by exogenous NO application in this study.Meanwhile, the changes of chlorophyll content were accompanied by the changes of photosynthesis in tomato plants.
The salt-induced limitation of the photosynthetic performance may be due to stomatal limitation and/or nonstomatal limitation (Debez et al., 2008).Salinity stress as well as other stress conditions can reduce CO 2 -assimilation rate due to stomatal closure and further decrease photosynthetic capacity.Salt stress resulted in a reduction of P N , gs and E during the whole experimental period (Figures 3A to C), which was alleviated by exogenous NO treatment.In parallel, however, the change of Ci was not associated with deceased P N , gs and E. Salt stress caused the decrease in CO 2 concentration at the first four days, which was alleviated by NO, and then the gradual increase afterwards, which was also reduced by NO.This observation suggested that the effect of NO on reductions in CO 2 -assimilation in salt-stressed tomato plants were largely dependent on stomatal limitation at earlier stages, and then this dependency switched to the non-stomatal limitation at later stages.
P N is an important exterior mark of photosynthetic metabolism level in plants; its improvement is a compre-7844 Afr.J. Biotechnol.
hensive outcome of the increase of light using efficiency, the acceleration of CO 2 transportation, and the enhancement of CO 2 fixation (Wang et al., 2010).The PN-PPFD and PN-Ci curve also showed that salt stress significantly affected the shape of the P N -PPFD and P N -Ci response in tomato leaves, especially under high PPFD and higher Ci.In contrast, attenuation in the decreases in P N was observed in plants co-treated with NO under salt stress.These results can be interpreted in two ways.One is that NO increase the level of chlorophyll pigments and results in changes of gs and E under salt stress.The other is that application of NO significantly improved light saturation point (LSP), light-saturated photosynthetic capacity (Amax), cerebrospinal fluid (CSP) and CO 2 -saturated photosynthetic capacity (RuBPmax), but reduced cerebral perfusion pressure (CCP) and intracranial pressure (ICP) under salt stress (Wu et al., 2007).The phenomenon suggests that NO strongly affect photosynthetic performance not only involving enhancement of regulation of stomatal closure or opening, but also increase in photochemical quenching.The reduction of photochemical activity is considered to be one of the non-stomatal factors that limit photosynthesis (Souza et al., 2004).Thus, the activity of PSII was investigated in the present study by using chlorophyll fluorescence technology.In particular, fluorescence provides information on the ability of a plant to tolerate environmental stresses and the extent to which those stresses have damaged the photosynthetic apparatus (Maxwell and Johnson, 2000).Results of this study showed that treatment with NaCl caused significant changes in PSII photochemistry, evidenced by the significant decrease in Fv/Fm and the damage to the donor side or the acceptor side of PSII in tomato leaves.Fv/Fm, the maximal quantum efficiency of PSII, is frequently used as an indicator of the photoinhibition or of stress damage to the PSII (Calatayud and Barreno, 2004).More importantly, NO application resulted in a less decrease in Fv/Fm ratio in salt-stressed tomato plants (Figure 5), suggesting that improved PSII efficiency by NO treatment in salt-stressed plants was associated with the decrease in inhibition of electron flow at oxidizing site of PSII.On the other hand, a significant increase in Fo and a decline in Fm (Figures 5A and B) in response to salt stress could be attributed to the separation of LHCII from the PSII complex, activation of PSII reaction centre, and decrease of electron flow within the PQ pool (Yamane et al., 1997).The ratio of variable to minimal fluorescence (Fv/Fo), is a well-known indicator of the changes in the rates of photosynthetic quantum conversion (Maxwell and Johnson, 2000).According to Pereira et al. (2000), as a conesquence of damage, the decrease in Fv/Fo is one of the probable reasons for the structural damage of thylakoids.In this study, Fv/Fo ratio was lower in plants with NaCl treatments than those in the controls (Figure 5C).Exogenous NO application significantly improved Fm, Fm/Fo and Fv/Fo and also reduced Fo, which implied that NO application showed positive effects on PSII efficiency and could preserve PSII efficiency from salt stress.
There is a general agreement that photoinhibition is primarily caused by an inactivation of the electron transport system in thylakoids.The dominant effect seems to be an alteration of the reaction centers of PSII leading to a decreased photochemical efficiency.The significant changes in PSII photochemistry in the light adapted leaves, such as the decreased ETR, Fv´/Fm´ and qP, as well as the increased qN, can be seen as the regulatory response to down-regulate PSII activity (Genty et al., 1989), in consistent with the high decrease of CO 2 assimilation rate.Since qP was expressed as an indicator of the proportion of closed PSII centers, attenuation in the decreases in qP was observed in plants co-treated with NO under salt stress (Figure 6C), suggesting that under salt stress, exogenous NO kept more PSII reaction centers in an open state so that more excitation energy was used for electron transport.In our study, decreases in Fv´/Fm´ and increases in qN observed under salt stress (Figure 6B and D), indicated the occurrence of a non-radiative energy dissipation mechanism in which a higher proportion of absorbed photons is lost as thermal energy instead of being used to drive photosynthesis (Shangguan et al., 2000).Indeed, there was a less decrease in Fv´/Fm´ and less increase in qN in NO applied plants than in non-NO applied plants under salt stress, which suggested that NO application resulted in a less dissipation of excitation energy as heat in the PSII antennae.The observed decreases in ETR and qP indicated an over-excitation of the photochemical system.When this is the case, accumulation of reduced electron acceptors may increase the probability of the generation of reactive radicals that render damages to PSII components (Barber and Andersson, 1992).Under these conditions, exposure to exogenous NO improved ETR, Fv´/Fm´ and qP, indicating that NO alleviated the adverse effect of salt stress by improvement of photochemical efficiency.
There were some conflicting evidences on the effects of NO on phytosynthetic performance.Yang et al. (2004) found that SNP treatment alone decreased photochemical activity of PSII of intact Solanum tuberosum leaves.However, in this study, NO alone applied to tomato seedlings under normal conditions had very slight effects on photosynthetic performance.The discrepancy may be due to different plant species or environmental conditions.The detail physiological mechanisms of NO on phytosynthetic performance remain to be resolved.
In summary, our results suggest that the photosynthetic performance improved by NO application may be explained by an improvement in photosynthetic gas-exchange and the actual PSII efficiency of tomato seedlings when subjected to salt stress.Taken together, these findings demonstrate the positive function of NO on phytosynthetic performance under salt stress, and offer promising perspectives for NO as a potential regulation of plant growth.

Figure 1 .
Figure 1.Effects of NaCl and/or NO treatment on shoot and root dry mass of tomato Hufan2560 cultured in nutrient solution.Tomato seedlings were stressed in nutrient solutions for 8 days.Different treatments represents: Control: 0 M SNP + 0 mM NaCl treatment; SNP: 100 M SNP + 0 mM NaCl treatment; NaCl: 0 M SNP + 100 mM NaCl treatment; Salt + NaCl: 100 M SNP +100 mM NaCl treatment.Values are represented as the mean ± S.E.(n = 10).

Figure 2 .
Figure 2. Effects of NaCl and/or NO treatment on chlorophyll content of tomato seedlings grown in nutrient solutions on 0,

Figure 4 .
Figure 4. Effects of NaCl and/or NO treatment on the response curves of photosynthetic rate (PN) to photosynthetic photon flux densities (PPFD) and intercellular CO2 concentrations (Ci) of tomato seedlings grown in nutrient solutions on 0, 2, 4, 6, and 8 days after treatment.Vertical bars represent the mean ± S.E.(n = 8).

Figure 5 .Figure 6 .
Figure 5. Effects of NaCl and/or NO treatment on minimal fluorescence level (Fo) (A), maximal fluorescence level (Fm) (B), maximum quantum yield of System II (Fv/Fm) (C), and ratio of variable to minimal fluorescence (Fv/Fo) (D) of tomato seedlings grown in nutrient solutions on 0, 2, 4, 6 and 8 days after treatment.Vertical bars