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. 2007 Jan-Feb;2(1):50–57. doi: 10.4161/psb.2.1.3862

A Tolerant Behavior in Salt-Sensitive Tomato Plants can be Mimicked by Chemical Stimuli

Víctor Flors 1, Mercedes Paradís 1, Javier García-Andrade 1, Miguel Cerezo 1, Carmen González-Bosch 2, Pilar García-Agustín 1,✉
PMCID: PMC2633898  PMID: 19516968

Abstract

Lycopersicon esculentum plants exhibit increased salt stress tolerance following treatment with adipic acid monoethylester and 1,3-diaminepropane (DAAME), known as an inducer of resistance against biotic stress in tomato and pepper. For an efficient water and nutrient uptake, plants should adapt their water potential to compensate a decrease in water soil potential produced by salt stress. DAAME-treated plants showed a faster and stronger water potential reduction and an enhanced proline accumulation. Salinity-induced oxidative stress was also ameliorated by DAAME treatments. Oxidative membrane damage and ethylene emission were both reduced in DAAME-treated plants. This effect is probably a consequence of an increase of both non-enzymatic antioxidant activity as well as peroxidase activity. DAAME-mediated tolerance resulted in an unaltered photosynthetic rate and a stimulation of the decrease in transpiration under stress conditions without a cost in growth due to salt stress. The reduction in transpiration rate was concomitant with a reduction in phytotoxic Na+ and Cl− accumulation under saline stress. Interestingly, the ABA deficient tomato mutant sitiens was insensitive to DAAME-induced tolerance following NaCl stress exposure. Additionally, DAAME treatments increased the ABA content of leaves, therefore, an intact ABA signalling pathway seems to be important to express DAAME-induced salt tolerance. Here, we show a possibility of enhance tomato stress tolerance by chemical induction of the major plant defences against salt stress. DAAME-induced tolerance against salt stress could be complementary to or share elements with induced resistance against biotic stress. This might be the reason for the observed wide spectrum of effectiveness of this compound.

Key Words: adipic acid monoethyl ester; 1,3-diaminepropane; Lycopersicon esculentum; salt stress; oxidative stress; ethylene; chemical induced tolerance

Introduction

Tomato is an important greenhouse crop in semi-arid regions of Mediterranean countries. Its growth is greatly affected by environmental stresses such as low temperatures, drought or high salinity. From an agricultural point of view, these types of stress together with biotic challenges are the most limiting factors in crop production. To counter such environmental stress plants generally respond to by activating defence mechanisms and adjusting their cellular metabolism.1,2

Salt in soil water inhibits plant growth for two reasons. First, it reduces the plant's ability to absorb water and second, it affects the transpiration stream by injuring cells in transpirating leaves. This is the so-called salt-specific or ion-excess effect of salinity.3 Salinity is one of the major environmental factors limiting plant growth and productivity because plant processes such as photosynthesis, protein synthesis and energy and lipid metabolism are all affected.

Soils with high salt concentrations are virtually dry because the availability of water is trapped by the ions. To cope with this problem, plants respond by overproducing compatible osmolites such as proline and absorbing ions from the substrate which act by decreasing the water potential in leaf tissues and thus creating a negative pump effect to maintain the water flux through xylem sap.4,5 This process is known as osmoregulation.6

In addition to the hyperosmotic shock, a subsequent oxidative stress is also generated.7–9 This oxidative damage may be caused by the imbalance between the generation of reactive oxygen species (ROS) and their detoxification by the antioxydative system of the plant.7,8 Alleviation of such oxidative damage and increased resistance to environmental stresses, including salt stress, is often correlated with a more efficient antioxydative system.10–12

Plants had to develop physiological and biochemical mechanisms to respond and adapt to these stresses and thus acquire tolerance. Adaptation to stress has been suggested to be mediated by both pre-existing and induced defences.10,12–14

Many molecules, for example, abscisic acid, calcium, jasmonic acid, ethylene and salicylic acid (SA) have been suggested as signal transducers or messengers after environmental stresses.15 Salicylic acid and carboxylic acid derivatives have received attention after the discovery of their ability to induce resistance to pathogens.16–22 We have shown that 1,2,3,4-Tetra-O-acetyl-6-ethyladipate-β-D-glucopyranose (a glycoside derivative of adipic acid monoethyl ester, AAME) and 1,3-diaminepropane treatment activates resistance against Botrytis cinerea and Alternaria solani and improves the protective effect previously shown by the FGA mixture (AAME, 1,2,3,4-O-acetyl-β-D-glucopyranose and furfurylamine).21,20 The amide 5-(3-aminopropylcarbamoil) ethyl pentanoate derived from 1,3-diaminepropane and AAME also provides high protection for pepper plants against the pathogen Alternaria solani.22 Recently, AAME has been shown to completely suppress the grey mould disease of tomato fruit, providing evidence for its efficacy in a biological context.23 Although most studies with AAME derivatives have been dealing with the induction of resistance against biotic stress,20,23,24 emerging evidence of its ability to induce tolerance against salt stress has appeared. Concretely, in the salt-susceptible citrus rootstock, citrange Carrizo, AAME derivatives can ameliorate the deleterious effects induced by 60 and 120 mM of NaCl.25 Therefore, there is a possible link between plant resistance mechanisms and biotic and abiotic stress.

Muthukumarasamy et al., 2000,26 in radish, and Senaratna et al., 2000,27 in bean and tomato, have shown that chemicals such as triadimefon and SA ameliorate the effect of multiple environmental stress. Recently, β-aminobutyric acid (BABA), a well known resistance-inducer against many pathogens, has been described as a compound capable of inducing priming in resistance mechanisms against osmotic stress.28 In this study, numerous SA-impaired mutants have been tested for their induced tolerance phenotype under salt stress conditions and all of them showed wild type phenotype. Therefore, it was concluded that SA production was not essential for expression of salt-induced tolerance. However, abscisic acid (ABA) accumulation or ABA-related gene expression is important to express chemical induced tolerance, thus all ABA mutants tested so far failed to express BABA-induced tolerance.28 This large body of evidence prompted us to study the possibility to induce tolerance against salt stress by treating plants with AAME derivatives.

In this communication, we demonstrate that both adipic acid monoethylester and 1,3-diaminepropane (DAAME) are able to protect a salt-sensitive tomato cultivar against abiotic stress such as high salinity. This protection could be based on the enhancement of the expression of stress tolerance, although the exact mechanisms of action still remains to be clarified.

Materials and Methods

Plant material.

Seeds of the salt-sensitive Lycopersicon esculentum cv. Ailsa craig, Money Maker (M.M.) and the ABA-deficient mutant sitiens in the background of Money Maker (a kind gift from M. Korneef) were germinated under greenhouse conditions. Two week-old seedlings were then transplanted and grown in a culture chamber in 10 L trays filled with modified Hoagland solution.29 Plants were maintained in the nutrient solution for one week before the experiment. Temperatures ranged between 18–20°C (night) and 25–27°C (day). Relative humidity was maintained at approximately 80%. The nutritive solution was renewed every seven days. Salt treatment started at the stage of four true leaves.

Chemicals and treatments.

To determine the response of Lycopersicon esculentum to salt treatment, 90 mM of NaCl was added to Hoagland's solution. To test whether the chemicals could induce resistance to saline stress, different treatments were carried out (chemicals were dissolved in nutrient solution): control plants; control plants supplied with 90 mM of NaCl; DAAME plants treated with 2.18 µM of adipic acid monoethylester and 1.75 µM of 1,3-diaminepropane (DAP); DAAME plants treated with adipic acid monoethylester and 1,3-diaminepropane supplied with 90 mM of NaCl. Salt and DAAME were added at the same time in the nutrient solution and renewed every week. The plants were maintained for 28 days and groups of five plants were harvested after 0, 7, 14, 21 and 28 days. Gas exchange, ethylene and water potential were measured in vivo and excised leaves from control and treated plants were washed with distilled water, frozen in liquid N2, lyophilized and stored at −80°C for posterior analysis.

Leaf water potential.

Leaf water potential was determined with a SUBDIV model pressure chamber, Santa Barbara, CA. USA.30

Leaf mineral concentration.

Sodium was determined by atomic absorption spectrometry31 and chloride by potentiometric titration with 0.01 N AgNO3.

Gas exchange.

Net photosynthetic and transpiration rates were measured in plants at different times during the experiment and water use efficiency (wue) was estimated. A closed gas-exchange portable photosynthesis system (model LCA4) was used. Leaf laminae were totally enclosed within a fan-stirred cuvette and maintained under controlled conditions (leaf temperature between 25–27°C, and irradiance of 1200 µmol m−2.s−1). Ten consecutive measurements were taken at 3–5 min intervals.

Peroxidase (POx) activity (E.C. 1.1.1.1.7).

Lyophilized tissue was ground to a powder with a mortar and pestil and homogenized in extraction buffer (100 mM borate buffer pH 8.5, 0.5 g PVP, 0.5 mM DTT, 20 mM βME) at a ratio of 100 mg of tissue per mL. The crude extract was obtained after centrifugation at 8000xg, 4°C for 30 min. Total soluble protein was estimated by the Bradford method.32

Total peroxidase activity was determined in the crude extract according to the method developed by Hemeda and Klein (1990).33 For peroxidase determination, 75 µL of the crude extract were added to a 3 mL of assay medium. The assay medium contained 100 mL of 50 mM phosphate buffer (pH = 6.6), 1% guayacol, 0.3% H2O2 and crude enzyme extract (25 mL/mL). The formation of the oxidized tetraguayacol was used for calculating activity rates. A medium containing all components except H2O2 was used as a control. The peroxidase activity was calculated using the extinction coefficient of 2.66 × 104 M−1 cm−1 at 470 nm and stoichometry of 4. One unit of activity was defined as the calculated consumption of 1 µmol of H2O2/min. The level of peroxidase activity is reported as cat un. per mg of protein.

Hormone measurements.

Ethylene was quantified using an Agilent 4890 D gas chromatograph model equipped with an activated alumina column and a flame ionisation detector. The procedures for ethylene handling and determination have previously been reported.34

ABA analysis was performed according to Gómez-Cadenas and coworkers (2002).4 ABA, extracted from 50 mg of lyophilized tissue, was dissolved in 1 mL of MeOH: H2O (10:90; v/v), filtered and injected into the HPLC. Analyses were carried out using a Waters (Milford, MA, USA) Alliance 2690 HPLC system with a Nucleosil ODS reversed-phase column (100 × 2 mm i.d.; 5 µm). The chromatographic system was interfaced to a Quatro LC (quadrupole-hexapole-quadrupole) mass spectrometer (Micromass, Manchester, UK). The Masslynx NT version 3.4 (Micromass) software was used to process the quantitative data from calibration standards and the plant samples. As internal standard, 100 ppb deuterated ABA (dABA, Sigma) was added to the tissue before the homogenization.

Lipid peroxidation.

Lipid peroxidation was determined by measuring the amount of malonildialdehyde (MDA) formation using the thiobarbituric acid method described by Stewart and Bewley (1980).35 Lyophilized tissue was ground to a powder with a mortar and homogenized with 0.5% (w/v) thiobarbituric acid solution containing 20% (w/v) trichloroacetic acid at a ratio of 200 mg of fw per mL. The mixture was heated at 95°C for 30 min and the reaction was stopped by immediately placing it in an ice-bath. The cooled mixture was centrifuged at 10000xg for 10 min, and the OD at 532 nm, and the MDA concentration was determined by its extinction coefficient of 156 mM−1 cm−1.

Proline determination.

Lyophilized tissue was ground to a powder with a mortar and homogenized with sulfosalicylic acid (3%; w/v) at a ratio of 10 mg of fw per mL. The proline extract was obtained after centrifugation at 5000xg, 4°C for 10 min. Proline was determined in the supernatant by the method developed by Bates et al. (1973).36

Antioxidant activity.

Frozen tissue was ground to a powder in liquid nitrogen with a mortar and pestle and homogenized in 80% ethanol (0.25 mg FW/mL). After centrifugation at 6000 g, 4°C for 10 min, 5 mL of supernatant were evaporated at 40°C. Sep-pack C18 cartridges were preconditioned with 5 mL of methanol and 10 mL of water. After sample loading (5 mL) Sep-Pak cartridges were washed with 4 mL of methanol. Aliquots (40 µL) of the methanol fraction were assayed for antioxidant activity.

The evaluation of antioxidant activity based on coupled oxidation of β-carotene and linoleic acid was carried out as previously described.37 β-carotene (2 mg) was dissolved in 20 mL of chloroform solution. A 3 mL aliquot of this solution was added to a conical flask containing 40 mg linoleic acid and 400 mg Tween-40. Chloroform was removed using a rotary evaporator at 40°C. Oxygenated distilled water (100 mL) was added to the β-carotene emulsion and mixed well. Aliquots (3 mL) of oxygenated β-carotene emulsion and 40 µL of tomato extract were placed in test tubes and mixed well. The tubes were immediately placed in a water bath and incubated at 50°C. Oxidation of β-carotene emulsion was monitored spectrophotometrically by measuring optical density at 470 nm. Absorbance was measured 10, 20 and 30 min after the addition of oxygenated water. A control sample consisted of 40 µl methanol, instead of tomato extract, and 3 mL of β-carotene emulsion. The degradation rate of β-carotene was calculated by first order kinetics. Antioxidant activity was expressed as % inhibition relative to the control using the equation: (dr control- dr sample)/ dr control) × 100.

Statistical analysis.

Statistical analysis was carried out using the Statgraphics software support. Means were expressed with their SE (n = 6). They were compared by an LSD test. Differences were taken into account only when they were significant at the 5% level. All experiments were repeated at least three times.

Results

DAAME induces faster and more efficient osmoregulation under salt stress conditions.

A decrease in water soil potential induced by salt must be followed by an adaptation in plant water potential for an efficient water and nutrient uptake. Salt stress induced a reduction in the water potential during the first two weeks (Fig. 1A), followed by a recovery in the potential probably due to a transient adaptation of plants to salt, however at late time-points, salt stress induced a strong reduction in the water potential. In stressed tomato plants treated with DAAME a significant decrease in water potential was observed within 28 days after stress application compared to stressed but non-treated plants (Fig. 1A).

Figure 1.

Figure 1

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Effect of salinity on water potential (A) and proline content (B). (•) Control, (▪) treated with 90 mM of NaCl, (•) treated with DAAME, (□) treated with DAAME and 90 mM of NaCl. Water potential was expressed as percentage of water potential in non-stressed control plants. Each point is the average of at least six independent measurements ± SE.

Proline is one of the the major metabolites that contributes to plant osmoregulation.38 Salt stress induced an increase of this organic osmolyte after 21 days of stress exposure (Fig. 1B). Proline content in control and DAAME-treated plants remained at low levels throughout the period of time studied, ranging from 1.89 ± 0.04 to 1.69 ± 0.18 (βmol g−1 f.w.) (Fig. 1B). Once the stress was present, DAAME potentiated the osmotic adaptation by increasing the Pro content 2.8 fold as opposed to the stressed non-treated plants at the end of the experiment (Fig. 1B).

DAAME treatment protected tomato plants from the oxidative damage induced by salinity.

Salinity causes strong electrolyte leakage and as a consequence a strong production of malondialdehyde (MDA), which is a secondary end product of the oxidation of polyunsaturated fatty acids and it is considered a useful index of general lipid peroxidation.39 The time course of membrane lipid peroxidation in tomato leaves, measured as the content of MDA, is given in the (Fig. 2A). Salt treatments induced an increase in oxidative damage, however, this deleterious effect was not observed in DAAME-treated plants after stress induction (Fig. 2). DAAME treatment, in the absence of stress, also reduced the MDA content by 50%. This suggests an increasing protection against oxidative damage in treated plants.

Figure 2.

Figure 2

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MDA (A) and ethylene emision (B) in Lycopersicon esculentum leaves. (•) Control, (▪) treated with 90 mM of NaCl, (•) treated with DAAME, (□) treated with DAAME and 90 mM of NaCl. Each point is the average of at least six independent measurements ± SE.

Ethylene is a common signal hormone synthesized under stress conditions.4,12 To assess the possibility that ethylene was acting as a marker of stress induced by salt application, we determined the levels of this hormone in our experimental system. Stressed plants showed a six fold increase in ethylene emission at the end of the experiment with respect to control plants (Fig. 2B). However, ethylene emission after stress application and DAAME treatment was reduced by 42% with respect to non-treated plants (Fig. 2B).

Among the antioxidant defences in plants, two major systems can be found, one is mainly enzymatically mediated by radical scavenging enzymes. The second is mediated by non-enzymatic pathways based on chemical species able to react with free radicals.12 A faster and stronger reaction of the enzymatic scavenging system lead by peroxidases was observed in DAAME-treated plants after stress application (Fig. 3A). DAAME-treated plants responded to the imposed stress 7 days after salt treatment, but non-treated plants reacted later at 21 days showing lower levels of peroxidase activity (Fig. 3A).

Figure 3.

Figure 3

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Total POx activity (A). (•) Control, (▪) treated with 90 mM of NaCl, (•) treated with DAAME, (□) treated with DAAME and 90 mM of NaCl. Non-enzymatic antioxidant activity in Lycopersicon esculentum leaves (B). Each bar is the average of at least six independent measurements ± SE.

We have also tested the potential of DAAME to stimulate non-enzymatic antioxidant defences (antioxidant activity), measured as flavonoids, lipophilic phenolics and other non-polar compounds able to scavenge oxidant radicals.37 Antioxidant activity significantly increased in DAAME-treated plants after stress induction (Fig. 3B). It is noteworthy that DAAME-treated plants already showed an increase in the non-enzymatic antioxidant defence in the absence of salt stress (Fig. 3B).

Preservation of photosynthesis and reduction of transpiration induced by DAAME can lead to a growth enhancement under saline conditions and a reduction in toxic ion uptake.

Among the abiotic stresses, NaCl has a strong influence on the photosynthetic activity. When salt stress was induced, transpiration was reduced as a reaction to the osmotic shock (Fig. 4A). DAAME treatment induces a decrease in transpiration independently of the stress imposed. The photosynthetic rate remained approximately constant in non-stressed control plants throughout the experiment (Fig. 4B). However, in the absence of stress DAAME treatments resulted in a strong increase in the photosynthetic rate (Fig. 4B). The photosynthetic rate was indeed reduced by salt stress. However, DAAME-treated plants did not show a decrease in photosynthetic activity after stress induction and the levels remained similar to those found in non-stressed control plants (Fig. 4B).

Figure 4.

Figure 4

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Effect of salinity on transpiration (A) and photosynthetic rate (B) and relative growth rate (RGR = 1/W × dW/dt) (C) in Lycopersicon esculentum leaves. (•) Control, (▪) treated with 90 mM of NaCl, (•) treated with DAAME, (□) treated with DAAME and 90 mM of NaCl. Values were expressed as a percentage of transpiration and photosynthetic rate in non-stressed control plants. Each point is the average of at least six independent measurements ± SE.

During the experimental period the dry weight of control plants increased by 40% (Fig. 4C). Differences in growth rate between stressed and non-stressed plants were evident after 28 days. Salt stress reduced strongly the growth rate down to 4% compared to the dry weigh at the beginning of the experiment. DAAME-treated plants did not show a delay in growth after stress induction (Fig. 4C), although a certain increase in dry weight has been observed during the experiment, after 28 days there were no differences between DAAME stressed and non-stressed plants.

Cl− uptake seemed to be enhanced during the first week after salt stress exposure in DAAME-treated plants. However, the Cl− content in DAAME-stressed plants remained constant during the rest of the experiment and was reduced by 80% in respect to the stressed control plants after 28 days (Fig. 5A). DAAME-treatment reduced strongly the Na+ leaf content compared to the non-treated plants (Fig. 5B).

Figure 5.

Figure 5

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Effect of salinity on Cl− (A) and Na+ (B) concentration in Lycopersicon esculentum leaves. (•) Control, (▪) treated with 90 mM of NaCl, (•) treated DAAME, (□) treated DAAME and 90 mM of NaCl. Each point is the average of at least six independent measurements ± SE.

Functional ABA-signalling is required for DAAME-induced tolerance.

Tomato sitiens mutants are not able to withstand high salt doses due to their defective ABA production. Dunlap and Binzel40 reported that sitiens plants died within 48 h after 150 mM of NaCl exposition. In our experimental conditions a range of NaCl concentrations was tested and 60 mM was determined as an appropriate dose of NaCl in order to extend sitiens survival within 21 days after stress application, to be comparable with results obtained with Ailsa wild type plants. Remarkably, at 90 mM sitiens plants suffered severe damage at seven days after stress application and died three days later (data not shown). DAAME had little effect on dry weight of M.M. wild type plants grown under normal conditions, however DAAME induced a growth enhancement of 32% after 21 days of culture in 60 mM of NaCl (Fig. 6A). Accordingly, DAAME induced an increase in ABA leave content in stressed and unstressed plants (Fig. 7A). Surprisingly, ABA did not increase in M.M. grown under NaCl. This might be due to the low salt concentration employed and an osmotic adjustment after 21 days of experiment. sitiens mutants presented reduced ABA production and growth that was further reduced by 50% when they were grown in NaCl (Figs. 6B and 7B). DAAME had no effect on stress amelioration in sitiens plants although DAAME still produced a residual increase in ABA leaf content that probably was too low to protect sitiens against NaCl.

Figure 6.

Figure 6

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Effect of salinity on dry weigh of Money Maker (A) and sitiens (B) 21 days after stress induction. Water and DAAME treated plants were stressed with 60 mM of NaCl. Each bar is the average of at least five independent measurements ± SE.

Figure 7.

Figure 7

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Effect of salinity on ABA concentration in Money Maker (A) and sitiens (B) 21 days after stress induction. Water and DAAME treated plants were stressed with 60 mM of NaCl. ABA was measured in freeze dried leaves by HPLC-MS. Each bar is the average of at least five independent measurements ± SE.

Discussion

We have reported that L. esculentum cv. Ailsa craig exhibits increased salt stress tolerance following treatment with adipic acid monoethylester and 1,3-diaminepropane (called DAAME). Although the mechanism of action is still unknown, DAAME confers tolerance to salt-induced stress by a faster water potential reduction, increasing proline content, reducing the MDA and ethylene emission. Treatment also augmented the antioxidant defences increasing the peroxidase activity and the non-enzymatic antioxidant activity. The induced tolerance resulted in a preservation of the photosynthetic activity and a reduction in the Na+ and Cl− uptake. The results also showed that an intact ABA signalling is required to express DAAME-induced tolerance.

In our previous research we showed that 1,2,3,4-Tetra-O-acetyl-6-ethyladipate-β-D-glucopyranose (a glycoside derivative AAME) and 1,3-diaminepropane, and amides of AAME could protect plants against different necrotrophs.22 AAME has been shown to completely suppress the grey mould disease of tomato fruit.23 Few reports exist about the effectiveness of these derivatives to induce tolerance against salt stress. Concretely, we showed in citrange Carrizo that AAME derivatives can ameliorate the deleterious effects by NaCl.25 This study done in citrus provides evidence that mechanisms controlling responses to biotic and abiotic stress share common components, or at least, are activated by the same chemicals.

The increase of salt in the root medium, mediated by high salinity of soil, could lead to an osmotic adjustment (lowering of leaf water potential) that is generally accepted as an important adaptation to salinity.41 A common plant response is an accumulation of ions and proline, which is the main compatible osmolite in most cultivars,42 hence this organic osmolyte is known as osmoprotectant and its concentration correlates with osmotic stress tolerance.43 Proline overproduction using transgenic plants can confer salt tolerance in tobacco, rice and Arabidopsis.44–46 Additionally, proline serves as a storage sink for carbon and nitrogen and free-radical scavengers,38 therefore, it contributes to ameliorate the deleterious effect of salt stress in different ways. We found that DAAME-induced tolerance could be mediated by a faster osmoregulation process. After DAAME treatment, a reduction of transpiration and an increase in proline accumulation are activated. DAAME induces tolerance to salt-stress by enhancing reduction in water potential and increasing proline levels, thereby DAAME improves plant adaptation to the imposed osmotic stress. Similar results have been obtained using the non-protein amino acid β-aminobutyric (BABA), a well known inducer of resistance against pathogens. BABA can induce tolerance to water and salt stress by enhancing osmoregulation by priming ABA accumulation and leading a faster stomata closure that contributes to an osmotic readjustment. However, proline is not implicated in BABA priming against water or salt stress.28 Therefore, this is another evidence that osmotic and pathogenic stresses share signalling mechanisms.

Proline, furthermore, can also provide protection by scavenging free radicals and regulating the redox potential by reestablishment of the NADP+ supply and, therefore, stabilizing membranes and proteins.47–49 Although in our experiments there is not a total correlation between proline content and water potential reduction in DAAME stressed and non-stressed plants (Fig. 1A and B), it must be considered that other metabolites can contribute to osmoprotection together with ions such as Na+ and Cl−, strongly accumulated under salt stress conditions.

Apart of a faster osmoregulation, prevention of oxidative damage to cells during stress has been suggested as another mechanism of stress tolerance.12,50,51 In our experimental conditions we have observed an increase in the proline content which could also have some influence in the reduction of oxidative damage and the improvement of salt stress tolerance after DAAME treatments. Accumulation of proline by over expressing proline synthase genes improves seedlings tolerance and low free radical levels.38 The increased ABA leaf content obtained after DAAME treatments lead us to hypothesize that ABA could be mediating the DAME-induced proline accumulation, it is noteworthy that under osmotic stress ABA can regulate P5CS gene involved in proline biosynthesis.52 Our data indicate that the increase in total antioxidant activity, measured as a pool of compounds capable of scavenging oxidant species,37 together with enhanced peroxidase activity after DAAME application, helped to avoid high levels of oxidative cellular damage (measured as MDA content). This suggests that DAAME can induce the capacity to cope with oxidative stress in Lycopersicon esculentum seedlings. This capacity deals not only with enzyme activities but also with other compounds in which synthesis could be induced. Similar results have been described in pea seedlings after SA treatment.53

Our data show a reduction of Na+ and Cl− leaf content after DAAME treatments, furthermore, the transpiration rate is significantly reduced by DAAME after stress application. A reduction in transpiration could interrupt the xylematic flux to minimize Na+ and Cl− entry in the plant4,54 and could definitely contribute to reduce the toxicity of these ions in the plant.

As consequence of salt injury, an increase in ethylene released in leaves is observed as a response to oxidative-derived damage, however, results support a decrease in this hormone in DAAME stressed plants. This fact could be due to the reduced accumulation of Na+ and Cl− ions in roots (data not shown) and leaves, and an increase of ROS scavenging mechanisms. The amelioration in oxidative stress could cause a reduction in the conversion of ACC to ethylene as it has been previously been shown.4,55

It is well known that ABA controls the stomatic closure producing a reduction of leaf transpiration.56 Previously, we have shown that adipic acid and other carboxylic acid derivatives have an anti-transpirant effect,20,21 therefore it is possible that the influence of these compounds on transpiration is mediated by ABA. This led us to study the implication of this hormone in DAAME signalling. DAAME treatments increased ABA leaf content under normal or salt stress conditions, additionally sitiens mutants did not display DAAME-induced tolerance and accordingly, they did not accumulate significant amounts of ABA after DAAME treatments. Therefore, the obtained evidence points to ABA playing an important role in DAAME signalling mechanisms.

Although the physiological and molecular basis are not clearly understood, probably, DAAME-induced salt tolerance is based on an enhancement of the mechanisms that function to minimize osmotic stress or ion disequilibrium, by decreasing the accumulation of Na+ and Cl−, as well as to alleviate the consequent secondary effects caused by salt stress. We propose that adipic acid monoethylester and 1,3-diaminepropane (DAAME) provide salt-stress tolerance in tomato seedlings, being more consistent with a signalling role for the expression of tolerance rather than a direct effect. Further physiological and molecular research has to be done in order to clearly define the signalling pathways involved in AAME derivatives-induced resistance or tolerance.

Taking together the results presented in this manuscript and previous research, the induced-resistance based on treatment with AAME derivatives has been proved to be effective in several crops against different fungi and also salt stress. Although the signalling mechanisms could be stress-dependent, we hypothesize that the final steps in AAME derivative based-protection could be complementary or share elements, thus these compounds can be effective in inducing protection against such different types of stress.

Thus, considering the use of non-transgenic organisms and the possible agronomical benefits against abiotic stress, these compounds could serve as an important research tool to investigate the biology and the mode of action of tolerance induced by chemicals. The use of such a family of organic compounds, non-aggressive to the environment, could help in the future to develop new strategies to fight against the progressive salinization of the groundwater resources and the loss of water quality.

Acknowledgements

We thank to the SCIC of the Universitat Jaume I its technical support on hormone measurements. This work was financially supported by the Ministerio de Ciencia y Tecnología though the CYCIT project BFI2003-06948 and by the Plan de Promoción de la Investigación Caixa de Castelló-UJI number P11B2204-35.

Footnotes

Previously published online as a Plant Signaling & Behavior E-publication: http://www.landesbioscience.com/journals/psb/abstract.php?id=3862

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