Influence of abscisic acid and other plant growth regulators on citrus defense mechanisms to salt stress

El estres salino afecta notablemente el crecimiento y la produccion del cultivo de citricos. En el presente trabajo se ha evaluado la efectividad de distintos tratamientos con fitorreguladores: acido abscisico, acido jasmonico y 8' -metilen abscisato de metilo, como paliativos de los efectos nocivos producidos por una concentracion alta de sales. Para ello se cultivaron plantas de la variedad Salustiana, injertadas sobre el patron citrange Carrizo, y se sometieron a tratamientos con NaCi (100 mM). Como parametros de su comportamiento fisiologico se midieron abscision, produccion de etileno, acumulacion de iones cloruro y tasa fotosintetica neta en organos foliares. Los resultados mostraron que, en plantas no tratadas como fitorreguladores, el estres salino ocasiono una bajada drastica de la actividad fotosintetica y un aumento de la produccion de etileno Como resultado de la acumulacion masiva de iones cloruro y, por tanto, una abscision foliar generalizada. Los tratamientos con acido jasmonico u 8' -metilen abscisato de metilo no alteraron el patron de cambios fisiologicos inducidos por el estres salino. Por el contrario, las plantas tratadas con acido abscisico mostraron una clara reduccion en estos parametros. Por tanto, los datos indican que el acido abscisico modifica el comportamiento fisiologico de las plantas de citricos y puede ayudar en su aclimatacion a condiciones de estres salino.

The physiological response to salt stress in citrus, as well as in many other species, is modulated by plant hormones, mainly abscisic acid (ABA) and ethylene (Gómez-Cadenas et al., 1998). ABA is involved, apart from in many other physiological processes, in water use efficiency in plants under environmental stress. This hormone regulates the stomatal opening and, therefore, transpiration. One of the responses of citrus plants to water deficit is a rapid rise of the endogenous levels of ABA (Gómez-Cadenas et al., 1996. Ethylene, on the other hand, regulates leaf abscission in citrus plants under water stress (Tudela and Primo-Millo, 1992;Gómez-Cadenas et al. 1996), or salt stress (Gómez-Cadenas et al., 1998). The elevated levels of chloride ions in leaves of citrus under salt stress is linked to an increased concentration of 1-aminocyclopropano-1-carboxylic acid (ACC) followed by its oxidation to ethylene which, in turn, triggers leaf abscission (Gómez-Cadenas et al., 1998).
Finally, evidence exists for the involvement of jasmonic acid (JA) in plant responses to stress, probably interacting with ABA and ethylene (Lehmann et al., 1995;Wasternack and Parthier, 1997). However, data on the role of this hormone as a mediator of the responses of citrus to salt stress have not been reported.
Different combinations of phytohormones have been tested to alleviate salinization symptoms in cultivated plants. Pre-treatments with ABA effectively increased salt tolerance in crops such as tobacco and barley and forest species such as Pinus banksiana Lamb. (Larosa et al., 1987;Popova et al., 1995;Rajasekaran and Blake, 1999). In spite of this beneficial effect, ABA could have a limited effect since it could form conjugates and/or be degraded rapidly (Cutler and Krochko, 1999). JA has also been found to facilitate acclimation of crops such as barley and strawberry to saline conditions (Tsonev et al., 1998).
In this article, the effects of ABA, JA and a synthetic derivative of ABA, 8'-methylene methyl abscissate (which chemical structure appears to delay its catabolism and, therefore, enhance its action), on the physiological behaviour of citrus grown under severe salt stress are studied. ] Raf) obtained from a commercial nursery. Plants were placed in 2-l plastic pots filled with inert sand as a substrate and kept in a greenhouse under the following conditions: day temperature, 24-28ºC, night temperature 18-20ºC; 16 h light/8 h dark photoperiod and relative humidity between 60 and 95%. All plants were watered twice a week with 500 ml of Hoagland solution modif ied for citrus (Bañuls et al., 1997). Only intermediate leaves were used for the different determinations. Abscission was expressed as the percentage of leaves that shed with a gentle touch.

Chemical and salt treatments
Salt stress was imposed by adding sodium chloride to the irrigation solution to achieve a final concentration of 100 mM. The irrigation solution was supplemented with ABA, JA (both obtained from Sigma, Madrid, Spain) or 8-MAMe (Precision Biochemicals Inc., Vancouver, Canada) at a concentration of 10 µM each. Treatments with plant growth regulators were initiated ten days before the beginning of salinization and maintained during the whole experimental period.

Leaf water potential
Leaf water potential was determined by using a pressure chamber (model 3000, Soilmoisture Equipment, Santa Barbara, CA), as described in Gómez-Cadenas et al. (1996).

Measurement of photosynthetic rate
Net photosynthetic rate was measured with a LI-6200 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA) equipped with a 250 ml cuvette. Determinations were made in the morning (9 a.m.). For the measurements, the inside of the cuvette was illuminated with a 150 W lamp (Philips EFR A1/232) and cooled with external fans to prevent the sample from heating. All measurements were made at a photosynthetic photon flux (PPF, 400-700 nm) of 900-1000 µmol m -2 s -1 , which exceeds saturating PPF for citrus. At least, ten mature leaves per treatment were used for the measurements.

Chloride analysis
Total chloride content in leaf tissue was determined by AgNO 3 titration (Chapman and Pratt, 1961). One gram of lyophilized leaf tissue was extracted in a basic medium achieved by adding 250 mg of CaO (PRS, Panreac, Barcelona) and some drops of water to the tissue to compact the powdered tissue. Samples were calcined in a muffle oven for 90 min at 550ºC. Ashes were then resuspended in 15 ml of ultra pure water at approximately 70ºC. This suspension was vacuum filtered using a Whatman paper nº 1. The filtered residue was washed five times with 10 ml of ultra pure hot water to obtain a final volume of 65 ml. The pH of the filtrate was adjusted to a value between 6.0 and 7.0 with diluted acetic acid. The titration was performed with 0.05 N AgNO 3 using 5% KCrO 2 as an indicator.

Ethylene production
Ethylene production was measured from intermediate leaves by gas chromatography. Leaves were se-parated from the plant by making an incision at the base of the petiole with a scalpel. Then, leaves were placed individually in 12-ml tubes with the petiole submerged in 100 µl of distilled water in the bottom of the tube to prevent dehydration. The tubes were aerated for 30 min and then hermetically closed with silicon stoppers. Following a 4 h incubation period, one ml of the enclosed atmosphere was injected into a gas chromatograph (AGILENT 4890D, Agilent Technologies, Inc., Wilmington, DE, USA) equipped with an activated alumina column and a flame ionization detector.

Water potential
As a first approach to evaluate the effect of salinity on citrus plants, leaf water potential was measured (Fig. 1). Non-salinized plants showed leaf water potential values ranging between -8.42 and -12.50 bar throughout the period studied (data not shown). To facilitate the interpretation of data, a relative value of 100% was assigned to results obtained in control plants and those corresponding to the remaining treatments were expressed as a percentage of this value. All the plants exposed to salt stress showed a reduction in leaf water potential. After seven days of salinization, values began to drop and reached the lowest value on day 14 (-18.7 bar, 2.3 times smaller than the value for the non-salt treated control plants at that moment). After this, leaf water potential in plants under salt stress remained at low levels. Plants treated with plant growth regulators did not exhibit any differences in leaf water potential compared to non-treated salinized plants (Fig. 1).

Leaf abscission
Leaves in non-salinized plants did not abscise during the period studied (Fig. 2). In contrast, addition of 100 mM NaCl to the irrigation solution induced a massive drop of leaves. Therefore, after 20 days of saline treatment, leaf abscission was obvious and, in a short period of time (18 days), the salinized plants had lost nearly all their leaves. Treatment with 10 µM ABA resulted in an important reduction of the salt-induced abscission (56% abscission in plants treated with ABA compared to 98% in those not treated, 38 days after the onset of salinization). JA addition delayed leaf abscission induced by salt stress until day 30 although after this date leaf abscission rate increased and, 36 days after the beginning of salinization, 100% of leaves had abscised. Finally, 8-MAMe treatment did not delay the abscission process induced by salinity.

Leaf ethylene production
Leaves from non-salt treated control plants produced basal levels of ethylene, ranging between 0.30 and 0.68 nl g -1 h -1 (Fig. 3). Ethylene production in stressed plants remained low until day 18. However, after this date, leaves continuously increased the endogenous production of the plant hormone to reach 30 nl g -1 h -1 on day 38 (Fig. 3). Abscisic acid treatment suppressed ethylene production until 32 days after the onset of salt stress. Thereafter, leaf ethylene production in ABA-treated plants increased but levels on day 38 were still lower than those of non-treated plants.
On the other hand, neither treatment with JA nor with 8-MAMe significantly modified leaf ethylene production in response to salt stress. It is interesting to note that the pattern of ethylene production in leaves mimicked that of leaf abscission.

Photosynthesis net rate
To demonstrate the effect of salt stress on the photosynthetic activity, CO 2 net assimilation rate was measured. The data obtained were normalized as indicated above. Non-salt treated control plants presented photosynthetic rates ranging from 5.7 to 7.9 Each value is the mean of 3 independent measurements ± SE.
µmol m -2 s -1 during the whole experimental period (data excluded). In salt-treated plants, a progressive inhibition of photosynthetic activity was observed obtaining, 18 days after the onset of the salinization, values 90.5% lower than those obtained in control plants (Fig. 4). ABA treatment caused a reduction in the initial photosynthetic rate (Fig. 4, day 0) but it remained constant after adding NaCl. After the f ifth day, photosynthesis in ABA-treated plants substantially decreased although it still remained slightly higher that in non-treated plants. Treatments with JA or 8-MAMe did not have significant effects on the reduction of the photosynthetic rate induced by salt stress (Fig. 4).

Accumulation of chloride ions in leaf tissue
To determine the level of intoxication, the amount of chloride ions in leaf tissue was measured (Fig. 5). Leaf Clconcentration in all plants under salt stress, treated or not with plant growth regulators, progressively increased over the time studied (Fig. 5). However, the final accumulation was clearly lower in ABAtreated plants than in those not treated with hormones. Treatments with JA or 8-MAMe did not cause any reduction in the accumulation of chloride ions in the leaf tissue of plants under salt stress (Fig. 5).

Discussion
The data presented here indicate that the deleterious effects of salt stress on citrus plants can be reduced by treatment with 10 µM ABA. However, neither JA nor 8-MAMe, applied in the same conditions, had any apparent effect on the physiological response of citrus to salt stress.
It was previously demonstrated that one of the first responses of Carrizo citrange to severe salt stress conditions (200 mM NaCl) was the massive abscission of leaves (Gómez-Cadenas et al., 1998). This response has been directly related to the accumulation of Clions (Bañuls and Primo-Millo, 1995;Gómez-Cadenas et al., 1998) as well as other damage and physiological alterations (Walker and Douglas, 1983;Bañuls et al., 1997;Romero-Aranda et al., 1998). It has also been confirmed that this response is mediated by ethylene, the hormone that triggers leaf and fruit abscission in citrus under salt, water and nutritional stress (Tudela and Primo-Millo, 1992;Gómez-Cadenas et al., 1996. The data presented here reinforce these conclusions and extend the results to grafted citrus plants under less drastic conditions of salt stress. Hence, the massive and rapid leaf abscission observed 20 days after imposing the saline treatment (Fig. 2) was preceded by an important rise in leaf ethylene production (Fig. 3)

Plant growth regulators and salinity in citrus 63
Days and was also correlated with an important accumulation of Clions in these organs (Fig. 5).
Considering that the accumulation of chloride ions is responsible for the overproduction of ethylene and the subsequent leaf abscission, the reduction in Clconcentration observed in plants treated with ABA (Fig.  5) can account for the inhibition of ethylene production (Fig. 3) and, therefore, in leaf abscission (Fig. 2). The mechanisms involved in Cluptake and root to shoot transport are still unknown (Storey and Walker, 1999), although previous reports indicate that chloride uptake under salinization is primarily driven by passive forces, mainly water absorption (Moya et al., 1999) which depends on transpiration and consequently on the stomatal aperture regulated by ABA. Thus, treatment with this phytohormone can induce stomatal closure and, in term, decrease transpiration, radicular absorption and the amount of chloride ions that pass from the substrate to the plant.
The data indicate that net photosynthetic rate is also affected by the salt stress imposed (Fig. 4). Previous studies have demonstrated the relationship between reduced photosynthetic rate and high levels of Cland Na + (Walker et al., 1982;Bañuls and Primo-Millo, 1992;García-Legaz et al., 1993). Data shown in Fig. 4 are consistent with this proposal and show that the decrease in photosynthesis in salinized plants untreated with plant growth regulators was linear and coincident with the increase in leaf Clions (Fig. 5). On the other hand, treatment with ABA modifies the behaviour of CO 2 assimilation in saline conditions and appears to protect the photosynthetic machinery, as described in other species exposed to conditions of water deficit or high salt concentrations (Popova et al., 1995;Rajasekaran and Blake, 1999).
It has also been proposed that ABA can act protecting membranes from the damage induced by reactive oxygen species (Rajasekaran and Blake, 1999), improving xylematic nutrient transport (Gadallah, 1996), regulating gene expression and inducing antioxidant enzymes (Gómez-Cadenas et al., 1999;Bellaire et al., 2000), etc. It is, therefore, likely that ABA works at different levels to reduce the deleterious effect of salinity on citrus.
On the other hand, Tsonev et al. (1998) showed that JA treatment in barley plants improved their resistance to high salinity. The data presented here, which indicate that treatment with JA does not modify the physiological responses of citrus to salt stress, do not agree with this results. This could be due to a different effect of the phytohormone in cereal and citrus plants or to the fact that the concentration of JA used was below the threshold required for its action. However, the application of JA 10 µM appears to have some effect on plant metabolism since, for example, a 17% reduction in the photosynthetic rate is observed on day 0 (Fig.  4). Moreover, in previous experiments it was shown that the addition of 100 µM JA produces toxic effects in citrus plants (data not shown). It, therefore, seems that although JA was absorbed by the plant, it had no significant effects on leaf abscission nor did it prevent the harmful effects of salt stress.
Finally, treatment with 8-MAMe, the chemically modified abscisic acid methyl ester, did not have the desired effects on leaf abscission, ethylene production or chloride ion accumulation. Although this compound had not been tested previously on citrus, it could be hypothesized that the chemical modification delayed its breakdown, favouring the action of this compound similar to abscisic acid. However, 8-MAMe failed to modify the behaviour of citrus plants in the parameters studied. This failure could be due to the presence of the methylic ester or to the fact that the chemical modification in the 8' position affects its physiological activity.
As a conclusion, abscisic acid, regularly added to the irrigation solution can reduce the damaging effects of high NaCl concentrations on young citrus plants. Treatment with ABA reduces defoliation triggered by ethylene by reducing the amount of chloride ions in the leaf tissue as well as protecting the photosynthetic machinery under saline conditions. This work opens up new possibilities for future studies on the management of citrus crops grown in areas with salinized irrigation water.