Different strategies for salt tolerance in determined

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Different strategies for salt tolerance in determined and indeterminate nodules of Lotus japonicus and Medicago truncatula a

a

a

Miguel López-Gómez , Noel A. Tejera , Carmen Iribarne , José a

A. Herrera-Cervera & Carmen Lluch

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a

Departamento de Fisiología Vegetal, Universidad de Granada, Granada, Spain Available online: 06 Jul 2011

To cite this article: Miguel López-Gómez, Noel A. Tejera, Carmen Iribarne, José A. Herrera-Cervera & Carmen Lluch (2011): Different strategies for salt tolerance in determined and indeterminate nodules of Lotus japonicus and Medicago truncatula , Archives of Agronomy and Soil Science, DOI:10.1080/03650340.2011.561836 To link to this article: http://dx.doi.org/10.1080/03650340.2011.561836

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Archives of Agronomy and Soil Science 2011, 1–13, iFirst article

Different strategies for salt tolerance in determined and indeterminate nodules of Lotus japonicus and Medicago truncatula Miguel Lo´pez-Go´mez*, Noel A. Tejera, Carmen Iribarne, Jose´ A. Herrera-Cervera and Carmen Lluch

Downloaded by [UGR-BTCA Gral Universitaria] at 00:49 18 October 2011

Departamento de Fisiologıá Vegetal, Universidad de Granada, Granada, Spain (Received 9 December 2010; final version received 5 February 2011) In this study, we examine how indeterminate and determined nitrogen-fixing root nodules of model legumes Lotus japonicus and Medicago truncatula adapt their non-structural carbohydrate pool during salt stress, with particular emphasis on trehalose, a compatible solute abundant in nodules of some legumes. M. truncatula and L. japonicus plants were inoculated with Sinorhizobium meliloti and Mesorhizobium loti, respectively, and the effect of 50 mM sodium chloride (NaCl) added to the nutrient solution was studied in a time-course experiment. Sucrose and pinitol were the predominant carbohydrates in nodules of both legumes, contributing to osmoprotection in nodules of L. japonicus under salt stress. Trehalose concentration increased under salt stress in L. japonicus nodules; however, compared with sucrose and pinitol, its concentration was too low to contribute efficiently to osmoregulation. By contrast, proline showed a dramatic increase in nodules and leaves of M. truncatula under salt stress, contributing to osmotic adjustment in this species. Results found in this study showed different mechanisms for salt tolerance in determined and indeterminate nodules of model legumes L. japonicus and M. truncatula that might be a general feature in the mentioned different types of nodules. Keywords: carbohydrate; Lotus japonicus; Medicago truncatula; salt stress; trehalose

Introduction Roots of legumes have the ability to establish a symbiosis with nitrogen-fixing rhizobial bacteria to form nodules, highly specialized organs, in which atmospheric dinitrogen is reduced to ammonia. Two types of nodules have been defined: determined and indeterminate nodules (Hadri and Bisseling 1998). Model legumes Lotus japonicus and Medicago truncatula develop determined and indeterminate nodules, respectively, providing convenient and powerful systems for the study of plant–rhizobium interaction (Stougaard 2001). Plant salt stress has become a major concern worldwide due to the salinization of agricultural land caused by irrigation and desertification progress. Salinity imposes at least two primary stresses on plants: hyperosmotic stress, caused by the reduction

*Corresponding author. Email: [email protected] ISSN 0365-0340 print/ISSN 1476-3567 online Ó 2011 Taylor & Francis DOI: 10.1080/03650340.2011.561836 http://www.informaworld.com


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M. Lo´pez-Go´mez et al.

of water potential and consequently reduced water availability; and hyperionic stress, related to the toxic effects of the accumulated ions (Munns and Tester 2008). Legumes are classified as salt-sensitive crop species (Laüchli 1984) and their productivity is particularly affected by salt stress because symbiotic nitrogen fixation markedly decreases upon exposure to mild saline conditions (Lo´pez et al. 2008). As sessile organisms, plants have evolved a number of strategies to acclimatize to various kinds of deleterious conditions and thus to increase competitiveness in various ecological niches. Among others, salinity acclimatization responses include accumulation of compatible osmolytes such as amino acids, sugars and polyols (Zhu 2002; Sanchez et al. 2008), to protect cell respiration, photosynthetic activity, nutrient transport and nitrogen metabolism. It has been assumed that the accumulation of organic solutes in response to salt stress is involved in restoration of the cell volume and turgor, reduction of the cell damage induced by free radicals, and protection and stabilization of enzymes and membrane structures (Bartels and Sunkar 2005). Salinity is known to increase the nodular carbohydrate accumulation and sucrose is the predominant carbohydrate in legume root nodules (Fouge`re et al. 1991; Gordon et al. 1997). Trehalose (a-D-glucopyranosyl-1,1-a-D-glucopyranoside) is a non-reducing disaccharide that has been found in a wide variety of organisms such as yeast, fungi, bacteria, plants, insects and other invertebrates (Elbein et al. 2003). This molecule plays an important role as an abiotic stress protectant in many organisms. However, in plants, its precise role remains unclear, although some data indicate that trehalose has a protective role during abiotic stresses (for review see Fernańdez et al. 2010). In higher vascular plants, trehalose is a rare sugar (Hoekstra et al. 1992) although they express genes encoding for trehalose biosynthesis, and some even contain trehalose in very small amounts (Leyman et al. 2001; Iordachescu and Imai 2008). However, a large amount of trehalose has been detected in the root nodules of soybean (Mu¨ller et al. 1994), being mainly localized in the bacteroids (Streeter 1987), which suggests a bacterial origin for this disaccharide. Synthesis of trehalose is a two-step process catalysed by the sequential action of trehalose 6-phosphate-synthase (TPS; E.C. 2.4.1.15) and trehalose 6-phosphate-phosphatase (TPP; E.C. 3.1.3.12) (Goddijn and van Dun 1999). It has been suggested that genetically engineered trehalose accumulation in crop plants could improve their tolerance to drought and salinity (Romero et al. 1997). However, limited amounts of trehalose were found to accumulate, probably because of the ubiquitous presence of the enzyme trehalase in plants (Mu¨ller et al. 2001). Trehalase (TRE; E.C. 3.2.1.28) activity, the only enzyme capable of hydrolysing trehalose to glucose, has been found to be downregulated at transcriptional level in root nodules of M. truncatula under salt-stress conditions, suggesting a regulatory role for this enzyme at the concentration of trehalose found in such conditions (Lo´pez et al. 2008). In addition to carbohydrates, accumulation of amino acids such as proline has been widely discussed as a mechanism of protection against salinity in plants (Szabados and Savoure´ 2009), although the role of this amino acid in osmotolerance remains controversial. Considering the importance of legumes in agriculture and human diet, together with their critical role in natural and agricultural ecosystems due to their ability to fix nitrogen, further knowledge of legume stress physiology is required to address current and future threats to food security. In this work, nodulated M. truncatula

Archives of Agronomy and Soil Science

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and L. japonicus plants were exposed to salinization with 50 mM NaCl; subsequently, several physiological and biochemical markers related to salt stress were measured to determine how the species mentioned above adapt their non-structural carbohydrate pool during salt stress. To achieve this, the levels of trehalose and the activities of three enzymes responsible for the trehalose metabolism in nodules (TPS, TPP and TRE) were examined. These and other results are discussed in terms of the differences found in the mechanisms of salt stress acclimatization in M. truncatula and L. japonicus.


Materials and methods Plant material and experimental treatments Lotus japonicus (cv. Gifu) and Medicago truncatula (var. Jemalong) seeds were scarified by immersion in concentrated H2SO4 for 5 min, washed with sterile water, surface sterilized by immersion in 5% NaClO plus Tween 20 for 20 min and germinated onto 0.8% water–agar plates at 288C in the dark. After 4 days, Lotus and Medicago seedlings were transferred to sterile vermiculite and watered with Hornum (Handberg and Stougaard 1992) and a modified Rigaud and Puppo (1975) nutrient solution, respectively. Two days later, L. japonicus seedlings were inoculated with 1 mL of a stationary culture of Mesorhizobium loti R7A strain and M. truncatula seedlings with Sinorhizobium meliloti GR4 strain (*109 cell mL71) grown in a tryptone–yeast extract (TY) medium. One plant per pot of *300 mL was grown in a controlled environmental chamber with a 16/8 h light/ dark cycle, 23/188C day/night temperature, relative humidity 55/65% and photosynthetic photon flux density (400–700 nm) of 450 mmol m72 s71 supplied by combined fluorescent and incandescent lamps. Once symbiosis was well established and plants were at the vegetative stage (5 weeks M. truncatula and 7 weeks L. japonicus after sowing), they were subjected to salt treatment by adding NaCl to the nutrient solution (50 mM). Control plants were watered with a NaCl-free nutrient solution. Plants were harvested at 0 (vegetative stage), 14 (flowering), 21 (early fructification) and 28 (advanced fructification) days after salt addition. Nodules and leaves were frozen at 7808C for further analyses. Samples of leaves, stems, roots and nodules were dried at 708C for 24 h and their dry weight was determined. Nitrogen fixation Nitrogenase activity (E.C. 1.7.9.92) was measured as the representative H2-evolution in an open-flow system (Witty and Minchin 1998) using an electrochemical H2 sensor (Qubit System Inc., Canada). H2 production was recorded on intact nodulated roots of plants at 0, 14, 21 and 28 days after salt addition, because both strains used to inoculate have no uptake of hydrogenase (hup-) (Brito et al. 2005). Apparent nitrogenase activity (ANA, rate of H2 production in air) was determined under N2/O2 (80%:20%) at a total flow of 0.4 L min71. After reaching steady-state conditions total nitrogenase activity (TNA) was determined under Ar:O2 (79%:21%). Nitrogen fixation rate (NFR) was calculated as: NFR ¼ ðTNA ANAÞ=3

ð1Þ

4


The electron allocation coefficient (EAC) of nitrogenase activity was calculated as: EAC ¼ 1 ðANA=TNAÞ

ð2Þ

Standards of high purity H2 were used to calibrate the detector.


Carbohydrate analysis Carbohydrates sucrose, glucose, fructose, maltose, trehalose and pinitol were separated and quantified by gas chromatography (Streeter and Strimbu 1998). Samples of nodules (200 mg) were ground in methanol (80% v/v) and incubated at 608C for 10 min followed by centrifugation at 13,000 rpm for 10 min. The pellet was re-extracted a further three times, and supernatants were collected and vacuumdried. Solids were dissolved in 125 mL pure pyridine plus 125 mL STOX reagent (Pierce Biotechnology, Inc.); this reagent contains hydroxylamine for conversion of anomeric forms to the oxime derivatives, and also contains an internal standard, b-phenylglucose. Samples were thoroughly mixed by vortex and were incubated in a 708C heating block for 30 min with occasional mixing by vortex. After cooling, 200 mL hexamethyldisilazane and 20 mL trifluoroacetic acid were added, mixed and allowed to react for 60 min before analysis. TMS-oxime derivatives were separated on a packed column of 3% OV-17 on Chromosorb WHP using a HewlettPackard 5890 Series II gas chromatograph and peak areas were quantified using a Hewlett-Packard 3396A integrator. Proline determination Proline in leaves and nodules were extracted using 250 mg of tissue and 3 mL of 95% (v/v) ethanol. The insoluble fraction of the extract was washed twice with 5 mL of 70% (v/v) ethanol. All soluble fractions were centrifuged at 3500 g for 10 min. The supernatants were collected and stored at 48C for proline determination. Proline content was measured according to the method described by Bates et al. (1973). As standard curve, L-proline was used to estimate concentration. Preparation of extracts and enzyme assays Protein extracts were prepared by homogenizing 0.2 g of nodules in a mortar with 33% (w/w) polyvinyl-polypyrrolidone and 2 mL of 100 mM MES buffer (pH 6.3) containing 2 mM EDTA and 2 mM phenylmethylsulfonyl fluoride (PMSF) for TRE activity; and 2 mL 50 mM Tris/HCl buffer (pH 7.5) containing 2.5 mM MgCl2, 100 mM NaCl and 10 mM b-mercaptoethanol for TPS and TPP activities. Extracts were centrifuged at 30,000 g for 20 min and supernatants were used to determine enzyme activities. All operations were carried out at 48C, and enzyme activities were monitored between 2 and 4 h. TRE activity (E.C. 3.2.1.28) was determined colorimetrically according to Mu¨ller et al. (1994) by measuring the glucose released. The reaction mixture contained 100 mM trehalose in 50 mM MES/KOH (pH 6.3). After incubation at 378C for 45 min, the reaction was stopped by heating at 1008C for 5 min. The glucose released was measured by the glucose oxidase-peroxidase method using a test kit (ATOM, BioSystems).



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The TPS assay (E.C. 2.4.1.15) was based on the method of Salminen and Streeter (1986) by determining the release of UDP from UDP-glucose in the presence of glucose-6-phosphate. The reaction mixture (0.2 mL) contained 100 mM Tris/HCl buffer (pH 7.5), 8 mM UDP-glucose, 30 mM glucose-6-phosphate, 100 mM MgCl2, 3 mM EDTA and 25 mM KCl. The reaction was started by the addition of the nodule enzyme extract (0.04 mL). After 60 min at 308C, reactions were stopped by heating at 1008C for 2 min. Samples were centrifuged at 2000 g for 10 min, and the amount of UDP in the supernatant measured in terms of oxidation of NADH in a linked assay with pyruvate kinase and lactic acid dehydrogenase. The assay mixture contained 50 mM Tris/HCl (pH 7.5), 5 mM phosphoenol pyruvate, 0.24 mM NADH, 10 mM MgCl2, 3.5 U pyruvate kinase and 5 U lactic acid dehydrogenase. The decrease in absorbance at 340 nm was measured continuously over a period of 20 min. TPP activity (E.C. 3.1.3.12) was assayed by monitoring phosphate released from trehalose-6-phosphate (Padilla et al. 2004). The reaction was carried out in a final volume of 0.25 mL containing 25 mM Tris/HCl (pH 7.0), 10 mM MgCl2 and 1 mM trehalose-6-phosphate. Samples were assayed for phosphate by the zinc acetate method (Bencini et al. 1983). Statistical analyses The experimental layout was a randomized complete block design. For growth and nitrogen fixation, the values are the mean of 12 replicates per treatment. Four replicates were performed for the other parameter studied per experiment. The experiment was performed twice. All results were subjected to two-way analysis of variance with a least significant difference (LSD) test between means using a Statgraphics 5.0 (Statistical Graphics Corp., Rockville, MD, USA). The standard errors (SEs) were also calculated. Results Plant growth and nitrogen fixation The effect of 50 mM NaCl treatment on the growth of M. truncatula and L. japonicus was followed at 14, 21 and 28 days after saline treatment (DAT). In both legumes, shoot dry weight (SDW) and root dry weight (RDW) were positively affected by salt treatment at 14 and 21 DAT (Table 1), especially in M. truncatula where both parameters showed significant differences. However, at 28 DAT in both legumes the SDW decreased by approx 13% and RDW decreased by 28% in M. truncatula, but was not significantly different in L. japonicus. Parameters related to the nitrogen fixation of both symbioses decreased in the presence of the saline stress (Table 1). Nodule dry weight (NDW) did not show statistically significant differences at 14 DAT, but decreased by 40% and 60% at 21 DAT in M. truncatula and L. japonicus, respectively; and by 40% in L. japonicus at 28 DAT. The nitrogen fixation rate (NFR) showed similar behaviour to the NDW in L. japonicus, meanwhile, in M. truncatula, the salinity effect was higher at 14 DAT (30% decrease) than at 28 DAT (no significant difference). The electron allocation coefficient (EAC) did not show significant differences by the salinity in either species, which means that the electron flux through nitrogenase used for proton reduction was not affected. On the other hand, an important difference in NFR was observed

6 Table 1.

M. Lo´pez-Go´mez et al. Effect of sodium chloride on growth and nitrogen fixation parameters.

Legume

DAT

NaCl (mM)

SDW

RDW

NDW

NFR

EAC

0 14

0 0 50 0 50 0 50 LSD (0.05)

121a 141a 203b 279c 306d 437f 383e 23

27a 45b 51b 78c 102d 159e 114d 13

4.44a 6.08b 5.28ab 12.08d 7.33c 12.15d 11.75d 1.21

41.11d 28.40c 19.53b 17.66ab 16.04a 15.69a 15.00a 3.27

0.67a 0.65a 0.61a 0.57b 0.58b 0.53b 0.59b 0.06

0 0 50 0 50 0 50 LSD (0.05)

77a 139b 159b 165cb 167cb 232c 202d 21

31a 35a 70c 37a 83d 46ab 51b 10

4.75a 10.38c 8.47bc 17.33d 7.28b 26.85e 16.92d 2.28

74.10e 59.09cd 50.57c 66.88de 32.32b 24.60b 13.49a 10.19

0.74c 0.85a 0.80a 0.79b 0.79b 0.80a 0.82a 0.05

Medicago truncatula

21 28


Lotus japonicus 0 14 21 28

Note: SDW, shoot dry weight (mg71 plant); RDW, root dry weight (mg71 plant); NDW, nodule dry weight (mg71 plant), NFR, nitrogen fixation rate (mmol N2 g71 NDW h71); EAC, electron allocation coefficient in Medicago truncatula and Lotus japonicus inoculated with Sinorhizobium meliloti GR4 and Mesorhizobium loti R7A strains, respectively. DAT, days after treatment. Means followed by the same letter within a column are not significantly different (P 0.05) using the LSD test.

between both types of nodules at all development stages, NFR being almost double in L. japonicus nodules (determined) than in M. truncatula nodules (indeterminate). Nodule carbohydrates Changes in carbohydrate content in nodules of M. truncatula and L. japonicus plants treated with NaCl are shown in Figure 1. Sucrose was the predominant sugar in nodules of both legumes and its concentration showed an extraordinary increase under salinity conditions in L. japonicus nodules, with 6.0- 3.0- and 1.1-fold higher concentrations at 14, 21 and 28 DAT, respectively, relative to control plants. By contrast, in M. truncatula a decrease in the sucrose content of 34% and 18% was detected under salinity conditions at 21 and 28 DAT, respectively. Glucose, fructose and maltose in plants of L. japonicus showed a similar pattern, with a double concentration under salinity at 14 and 21 DAT (except for fructose 21 DAT, which increased by 50%) and a 20% increase at 28 DAT (except for maltose which increased by 80%). In M. truncatula, the glucose concentration decreased by 10–30% at all harvest times with salinity, meanwhile fructose and maltose were augmented by 22% and 64%, respectively, at 14 DAT, and declined at 21 and 28 DAT relative to control plants. Trehalose concentration showed differences in both symbioses by the salinity treatment: in M. truncatula its concentration only increased 20% at 14 DAT showing a 20% and 70% decrease after 21 and 28 days of treatment. By contrast, in nodules of L. japonicus, the trehalose concentration increased at all harvest times reaching double concentrations at 14 DAT, and 60% and 40% higher concentrations at 21 and 28 DAT. Pinitol was the only carbohydrate augmented by



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Figure 1. Effect of NaCl treatment on carbohydrates (mmol g71 NFW) in nodules of Medicago truncatula and Lotus japonicus inoculated with Sinorhizobium meliloti GR4 and Mesorhizobium loti R7A strains, respectively. Vertical bars represent + SE (n ¼ 4).

the salinity in M. truncatula at all experimental times, with an increase of 50%, 44% and 13% at 14, 21 and 28 DAT, respectively. In L. japonicus, pinitol showed the greatest increase under salinity compared with the other carbohydrates analyzed, with concentrations 6.6-, 6.0- and 3.0-fold higher at 14, 21 and 28 DAT, respectively. Proline in leaves and nodules The proline content in nodules and leaves of both species increased with NaCl treatment (Table 2). In leaves of M. truncatula, this increase was more than double under salt stress after 21 and 28 days of treatment, and in nodules, the increase was more drastic at 28 DAT, with 3.4 times higher concentrations. In general, in

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Table 2. Effect of sodium chloride on proline content in Medicago truncatula and Lotus japonicus inoculated with Sinorhizobium meliloti GR4 and Mesorhizobium loti R7A strains, respectively. Proline content (mmol g71 FW) M. truncatula DAT 0 14


21 28

NaCl (mM) 0 0 50 0 50 0 50 LSD (0.05)

Leaf a

0.161 0.255a 0.404b 0.967c 3.287e 1.706d 4.420f 0.104

L. japonicus

Nodule a

0.530 0.712a 0.692a 1.185b 1.652c 1.161b 4.397d 0.252

Leaf a

0.109 0.122a 2.797e 0.278b 0.560c 0.620c 1.007d 0.069

Nodule 0.056a 0.264b 1.701g 0.459c 0.764d 0.864e 1.127f 0.035

Note: DAT, days after treatment. Means followed by the same letter within a column are not significantly different (P 0.05) using the LSD test.

L. japonicus, the proline concentration showed a greater increase by salinity at the first harvest (14 DAT), with stronger accumulation in leaves (23-fold) than in nodules (6.5-fold). An increase in the proline accumulation in leaves and nodules of both species was also detected over the treatment period with a 10-fold increase in M. truncatula leaves and a 15-fold increase in L. japonicus nodules. Enzyme activities involved in the trehalose metabolism in root nodules TPS, TPP and TRE activities were measured in nodules of both symbioses throughout the experiment (Figure 2). TPS and TPP showed inhibition of trehalose synthesis under salinity, although no significant differences were found in L. japonicus TPS activity, and the maximum inhibition was detected in M. truncatula at 21 DAT. TPP behaved similarly in both symbioses, showing a decrease of 40% and 25% of activity in M. truncatula and L. japonicus at 28 DAT, respectively. Trehalose catabolism by TRE activity was diminished under salinity conditions in L. japonicus throughout the studied period, meanwhile, in M. truncatula, no significant differences were found between unstressed and stressed nodules and an increase in activity was observed at flowering stage (14 DAT). Discussion In this study, the effect of 50 mM NaCl applied at the vegetative growth stage on nodulated M. truncatula and L. japonicus plants was investigated. Our results showed that salt stress decreased nitrogen fixation parameters NDW and NFR in both legumes, as previously reported by Lo´pez et al. (2008). It is noteworthy that the pattern of NFR inhibition over the course of the experiment was different between indeterminate and determined nodules. In the first case (M. truncatula), the salinity effect was higher after 14 days, showing recovery of the NFR after 28 days, probably because of the capacity for tissue regeneration in this type of nodule. By contrast, the effect of salinity was greater in the NFR of determined nodules (L. japonicus) after 4 weeks of salt treatment, despite the carbohydrate accumulation induced by salinity.



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Figure 2. Effect of NaCl treatment on nodule enzyme activities: trehalose 6P-synthase (TPS), trehalose 6P-phosphatase (TPP) and trehalase (TRE) on Medicago truncatula and Lotus japonicus inoculated with Sinorhizobium meliloti GR4 and Mesorhizobium loti R7A strains, respectively. Vertical bars represent + SE (n ¼ 4).

The reduction in NFR under salt-stress conditions has been attributed to a retardation in the initiation and growth of new nodules, reduction in the efficiency of fully formed nodules or a decrease in the proportion nodules able to fully differentiate into active N2-fixing nodules (Zahran 1999). However, the availability of carbohydrates in nodules during the fructification stage (21 and 28 DAT), did not seem to be the cause of the decline in nitrogen-fixing activity detected in L. japonicus over the experimental time, becase senescent nodules contained substantial quantities of all the carbohydrates analyzed. Growth parameters decreased with salinity at the last experimental stage, which correlated with the inhibition in NFR in L. japonicus. However, 50 mM NaCl promoted an increase in SDW and RDW in both legumes relative to control plants


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at 14 and 21 DAT, which has been reported previously in several L. japonicus genotypes (Quero et al. 2007). This root growth enhancement by salinity in L. japonicus has been considered profitable in other legumes because it might improve plant water status by increasing the root requirements to explore a higher soil volume (Khadri et al. 2006). The accumulation of organic solutes under saline conditions in plants has been well documented (Bartels and Sunkar 2005), particularly in legume root nodules, where salinity is known to increase the nodular carbohydrate accumulation. Sucrose is the predominant carbohydrate in root nodules of M. truncatula and L. japonicus (Figure 1), as previously reported by Lo´pez et al. (2008). A strong correlation between sugar accumulation and osmotic stress tolerance has been widely reported (Taji et al. 2002), suggesting a higher tolerance for determined nodules of L. japonicus to salt stress since they accumulated a higher concentration of all disaccharides and monosaccharides studied (Figure 1). Trehalose was not an exception and its concentration increased under salt stress at all experimental times in L. japonicus nodules, where the concentration detected was more than double that in M. truncatula. Nevertheless, compared with sucrose and pinitol, the trehalose concentration under salt-stress conditions was too low to contribute efficiently to osmoregulation, which is related to the findings of Fouge`re et al. (1991) and Mu¨ller et al. (1994) in M. sativa nodules. This result suggests a possible role for this disaccharide as cell protein stabilizer, membrane protector against damage provoked by salinity (Sampedro and Uribe 2004) or indirectly against reactive oxygen species (Luo et al. 2008). Pinitol proved to be another carbohydrate that accumulated when plants were submitted to salt stress, showing the higher increase in L. japonicus at all harvest times. This result, together with its slow turnover and limited reactivity, make pinitol a good candidate as an osmoprotectant. Pinitol has been previously described as a compatible solute in plants (Obendorf et al. 2008) and in legumes as a major carbohydrate (up to 50–60% of soluble sugars), especially under water-stress conditions (Streeter et al. 2001), and may act as an osmolyte (Reddy et al. 2004). In addition to the carbohydrates, proline accumulation was observed in both legumes with NaCl supply; with leaves showing a higher accumulation than nodules (Table 2). Zhu et al. (1992) and Kohl et al. (1994) reported that soybean bacteroids display enhanced proline dehydrogenase (ProDH) activity, the main proline catabolic enzyme, when subjected to salt stress, suggesting that proline could be used as an energy source for nitrogen fixation. Nevertheless, the role of this amino acid in osmotolerance in plants has been widely discussed and remains controversial. Transgenic plants that accumulate high levels of proline are reported to display increased tolerance to salt and osmotic stress, as well as to cold and frost (Kishor et al. 2005, Han and Hwang 2003). In this sense, several studies have attributed an antioxidant feature to proline, suggesting reactive oxygen species scavenging activity (Szabados and Savoure´ 2009). In addition, proline accumulation might serve as a mechanism to protect the protein turnover machinery against stress damage (Khedr et al. 2003). In general, the proline concentration in many salt-tolerant plants has been found to be higher than in salt-sensitive ones (Ashraf and Harris 2004), which indicates that proline is an interesting marker for salt tolerance. Regarding trehalose metabolism, trehalase activity decreased with salinity after 21 and 28 DAT, contributing to trehalose accumulation in L. japonicus nodules, as reported by Lo´pez et al. (2008), although, after 14 days of treatment, when trehalose


11

concentration doubled, other unknown mechanisms must be involved in the regulation of trehalose accumulation. By contrast, in M. truncatula nodules, TPS activity appears to be involved in the decrease of trehalose content detected at 28 days of salt treatment.


Conclusion The results of this study showed a different mechanism for salt tolerance in determined and indeterminate nodules of model legumes L. japonicus and M. truncatula that might be a general feature in the above-mentioned nodule types. The accumulation of carbohydrates such as pinitol and sucrose is the main strategy in determined nodules of L. japonicus, and proline is the major osmoprotector in indeterminate nodules of M. truncatula. Trehalose appears not to act as an osmoprotectant because of its low concentration, although it might be involved in cell protein stabilization and membrane protection against damage provoked by salinity in nodules of L. japonicus. Acknowledgements The authors are grateful to Dr J.G. Streeter for carbohydrate measurements. Financial support was obtained through the Andalusian Research Program (AGR-139) and the Spanish Ministry of Education and Culture Grant BOS2002-04182-C02-02.

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