Nodule carbohydrate metabolism and polyols involvement in the response of Medicago sativa to salt stress

Environmental and Experimental Botany 85 (2013) 43–49

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Nodule carbohydrate metabolism and polyols involvement in the response of Medicago sativa to salt stress Francisco Palma ∗ , Noel A. Tejera, Carmen Lluch Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain

a r t i c l e

i n f o

Article history: Received 13 April 2012 Received in revised form 7 August 2012 Accepted 19 August 2012 Keywords: Legumes Medicago sativa Poliols Salt stress, Symbiosis

a b s t r a c t Alterations of plant growth, chlorophyll fluorescence parameters, nodule carbon metabolism and polyols concentration as result of salt stress were examined in alfalfa (Medicago sativa). Plants, in symbiosis with Sinorhizobium meliloti GR4 strain, were grown under controlled conditions for 35 days (DAS) and subjected to 150 mM of NaCl stress. Plant biomass (PDW) and nitrogen fixation rate (NFR) were markedly affected by salt stress conditions; the highest reductions of PDW (50%) and NFR (40%) were registered at 84 DAS and 56 DAS, respectively. In addition, salinity affected the chlorophyll fluorescence parameters, decreased initial chlorophyll fluorescence (F0 ) and increased the optimum quantum yield of PSII (Fv /Fm ratio). The enzyme activities sucrose synthase activity and phosphoenolpyruvate carboxylase, responsible for the carbon supply to the bacteroids by the formation of dicarboxylates, were drastically inhibited by salinity, mainly at 56 DAS with the beginning of flowering. The content of total soluble sugars and proline increased under salt stress, and these concentrations were higher in nodule than in leaf. This last result suggests that the nodule is an organ specially protected in order to maintain its functioning, even under stress conditions. Besides, the content of myoinositol and pinitol in leaves and nodules changed with the plant growth stage and the saline treatment. Under salinity stress, the concentrations of pinitol in nodule were higher than in leaf, which supports the central function of this molecule in the adaptive response of nodules to salt stress. The increase of pinitol synthesis in nodule of M. sativa under salt stress could be one of the adaptive features used by the plant. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Salinity is one of the major abiotic factors limiting global agricultural productivity, and it is estimated that one-third of the world’s irrigated land are unsuitable for crops (Frommer et al., 1999). Salt stress drastically affects the photosynthesis (Soussi et al., 1998), the nitrogen metabolism (Cordovilla et al., 1994), the carbon metabolism (Delgado et al., 1993; Balibrea et al., 2003) and the plant nutrition (Mengel and Kirkby, 2001). Legumes are classified as salt-sensitive crop species (Läuchli, 1984) and their production is particularly affected by salt stress because these plants depend on symbiotic N2 fixation for their nitrogen requirement (Elsheikh and Wood, 1995). The limitation of productivity is associated with a lower growth of the host plant, poor development of the root nodules (Georgiev and Atkins, 1993) and consequently with a reduction of the nitrogen-fixation capacity (Ben Salah et al., 2011).

∗ Corresponding author at: Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain. Tel.: +34 958 243382; fax: +34 958 248995. E-mail address: [email protected] (F. Palma). 0098-8472/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.envexpbot.2012.08.009

Under the variation of saline environments, plants develop different adaptive mechanisms (Rhodes et al., 2002; Sairam et al., 2006), some of which include the synthesis and accumulation of low-molecular weight organic compounds in the cytosol and organelles (Ashraf and Harris, 2004; Sairam et al., 2006). These compounds, collectively called compatible osmolytes, are simple sugars, disaccharides, sugar alcohols or polyols, amino acids and sulfonium compounds (Ashraf and Harris, 2004; Bartels and Sunkar, 2005; Ashraf and Foolad, 2007). A major function of the accumulation of the compatible osmolytes is the osmotic adjustment to counteract the high concentrations of inorganic salts in the vacuole and in the root medium (Zhu, 2001; Rhodes et al., 2002). Another function of the compatible osmolytes is the osmoprotection which may occur at lower salt concentrations. This role involves the protection of thylakoids and the plasma membrane, as well as the stabilization of proteins. Under salinity stress these compounds also act as a sink of energy or reducing power, as a source of carbon and nitrogen, or scavenging reactive oxygen species (Bartels and Sunkar, 2005; Sairam et al., 2006). Soluble carbohydrates and their polyol derivatives are the most common osmolytes accumulating in plants in response to low water potentials. Cyclic polyols involve myoinositol, ononitol and pinitol. Myoinositol is derived from glucose-6-phosphate and can

44

F. Palma et al. / Environmental and Experimental Botany 85 (2013) 43–49

be further methylated to sequoyitol or ononitol, which are epimerized to d-pinitol (Loewus and Loewus, 1980). An increase in pinitol content has been shown to occur in plants subjected to a water deficit (Streeter et al., 2001; Matos et al., 2010) as well as to a high salinity (Sengupta et al., 2008). Salinity induces the expression of genes, one of which has been shown to be involved in pinitol synthesis (Vernon and Bohnert, 1992). Thus, the introduction of genes involved in the synthesis of proline (P5CF127A), betaines (betA) and polyols (mt1D, imt1) into plants contributes to abiotic stress tolerance (Rathinasabapathi, 2000; Chen and Murata, 2002). It has been also suggested that exogenous application of compatible solutes is an alternative approach to improve crop productivity under saline conditions (Makela et al., 1999; Chen and Murata, 2002). The objective of the present work was to investigate changes induced by salinity on the content of some compatible osmolytes and enzyme activities of carbon metabolism in Medicago sativa grown under symbiotic conditions. In addition, the content of the polyols myoinositol and pinitol, as well as their relation with the adaptation of the symbiosis to salt stress, was also evaluated. 2. Materials and methods 2.1. Biological material and growth conditions Seeds of M. sativa (var. Aragon) were surface sterilized by immersion in 5% NaClO for 3 min and germinated in 0.8% wateragar plates at 28 ◦ C in darkness. Two days after, seedlings were transferred to individual pots of about 200 ml containing a vermiculite–perlite mixture (3:1) and watered with N-free nutrient solution (Rigaud and Puppo, 1975). Each seedling, inoculated with 1 ml of a stationary culture of Sinorhizobium meliloti GR4 strain (ca. 109 cell ml−1 ), was grown in a controlled environmental chamber with a 16/8 h light–dark cycle, 23/18 ◦ C day–night temperature, relative humidity 55/65% and photosynthetic photon flux density (400–700 nm) of 450 ␮mol m−2 s−1 supplied by combined fluorescent and incandescent lamps. 2.2. Treatments and harvest When plants were 35 days old (symbiosis established) they were treated with sodium chloride (150 mM) by adding it to the growth medium. Ten plants were harvested from both treated and control group on days 42, 56, 70 and 84 respectively. A fresh sample of the root, containing nodules of each plant, was used for the nitrogenase assay, and afterward nodules were removed, weighed, and dried at 70 ◦ C for 48 h to calculate dry weight. Other samples of nodules from each plant, leaves, and roots were pooled and stored at −80 ◦ C for the enzyme assays and analytical determinations. The fresh weight (FW) of roots (including the portion used for the nitrogenase assay), stems, and leaves were recorded, whereupon all the organs were dried at 70 ◦ C for 48 h and their dry weight calculated. 2.3. Chlorophyll fluorescence measurement Chlorophyll fluorescence, of the first full expanded leaf of each plant, was measured on each plant using a portable pulsemodulated fluorometer (OS5-FL, Opti-Sciences). After the leaves were dark-adapted for 30 min, the fluorescence parameters initial fluorescence (F0 ), maximal fluorescence (Fmax ) and variable fluorescence (Fv ) were analyzed (Schreiber et al., 1994). The F0 was obtained with modulated low intensity light (<0.1 ␮mol m−2 s−1 ), Fmax was determined by a 0.8 s long saturating light pulse (180 ␮mol m−2 s−1 ) and Fv was estimated by the difference between F0 and Fmax . The ratio of variable fluorescence to maximal fluorescence (Fv /Fmax ), represents the relative state of PSII and

was used to assess the functional damage to the plants (Schreiber et al., 1994). 2.4. Nitrogen fixation assay Nitrogenase activity (EC 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). For nitrogenase measurements, pots maintained in a controlled environmental chamber (as described above) were sealed and H2 production was recorded. Apparent nitrogenase activity (ANA, rate of H2 production in air) was determined under N2 :O2 (80%:20%) with a total flow of 0.4 l min−1 . After reaching steadystate conditions, total nitrogenase activity (TNA) was determined under Ar:O2 (79%:21%). The nitrogen fixation rate (NFR) was calculated as (TNA − ANA)/3. Standards of high-purity H2 were used to calibrate the detector. 2.5. Preparation of extracts and enzyme assays Extracts were prepared homogenizing 0.2 g of nodules in a mortar with 33% (w/w) polyvinyl–polypyrrolidone and 1.5 ml of 50 mM phosphate K buffer (pH 8) containing 1 mM EDTA and 20% (v/v) ethylene glycol for sucrose synthase activity and 100 mM maleic–KOH buffer (pH 6.8) containing 100 mM sucrose, 2% (v/v) ␤-mercaptoethanol and 20% (v/v) ethylene glycol for phosphoenolpyruvate carboxylase activity. Extracts were centrifuged at 30 000 × g for 20 min and desalted supernatants were used to determine enzyme activities. All operations were carried out at 4 ◦ C. Sucrose synthase activity (E.C.2.4.1.13) was measured according to (Morell and Copeland, 1985). The production of UDP–glucose was coupled to the reduction of NAD+ in the presence of excess UDP–glucose dehydrogenase. Reaction mixtures were contained in a volume of 1 ml, 100 mM bicine KOH buffer (pH 8.5), 100 mM sucrose, 2 mM UDP, 0.025 U UDP–glucose dehydrogenase and 1.5 mM NAD+ . The assay was started by the addition of the enzyme extract. The activity was spectrophotometrically measured by following the NAD+ reduction at 340 nm. Phosphoenolpyruvate carboxylase activity (E.C.4.1.1.31) assays were optimized from the (Soussi et al., 1998) method. The reaction mixtures contained 100 mM bicine KOH (pH 8.5), 5.0 mM MgCl2 , 0.2 mM NADH, 10 mM NaHCO3 and 2.0 mM PEP. The activity was followed by the oxidation of NADH at 340 nm. 2.6. Proline, total soluble sugars, malate and protein analysis Proline, soluble sugars and malate were extracted using 1 g of sample (leaves and nodules) and 12 ml of extraction medium. Both soluble sugars and proline were quantified by means of a colorimetric reaction with anthrone and ninhydrin reagents, respectively (Irigoyen et al., 1992). Standard curves, prepared with l-proline and glucose, were used to estimate concentrations. Malate content was determined enzymatically monitoring the production of NADH at 340 nm (Delhaize et al., 1993). The sample was incubated with buffer (0.5 M glycine, 0.4 M hydrazine, pH 9.0), NAD (40 mM) and malate dehydrogenase (5 mg/ml). Protein concentration was measured at 660 nm by the method of (Lowry et al., 1951) using the Folin–Ciocalteau reagent with bovine-serum albumin as a standard. 2.7. Myoinositol, pinitol and trehalose analysis Sugar alcohols and trehalose were separated and quantified by isocratic ion chromatography with pulsed amperometric detection according to Cataldi et al. (2000) with modifications, conducted on a Dionex ICS-3000 chromatograph (Dionex Corp., Sunnyvale, CA). Leaf and nodule samples (0.2 g) were homogenizing in mortars with

F. Palma et al. / Environmental and Experimental Botany 85 (2013) 43–49

3 ml ethanol:chloroform:water (12:5:1, v/v/v) and centrifuged at 3800 × g for 10 min (Irigoyen et al., 1992). The supernatant was separated into aqueous and chloroform phases by addition of chloroform and water. Finally, dry residues were resolubilized in 0.5 ml of Milli-Q water at 40 ◦ C, centrifuged (10 000 × g 15 min 4 ◦ C) and the supernatant was suitably diluted. The separation was performed on a CarboPac MA1 column plus precolumn (Dionex Corp., Sunnyvale, CA) kept at 30 ◦ C, using 0.5 M NaOH as eluent and a flow rate of 1 ml min−1 . Mixtures containing myoinositol, pinitol and trehalose (1 mM each) were used as standards. 2.8. Statistical design and analyses The experimental layout was a randomized complete block design. The growth values and parameters related to nitrogen fixation were means of ten replicates per treatment. Four replicates were performed for all biochemical determinations. Data were subjected to an analysis of variance (ANOVA) using the StatgraphicsPlus 5.1 software (Statistical Graphics Corp., Rockville, MD, USA). Means were compared by Fisher’s least significant difference test (LSD) and differences at p ≤ 0.05 were considered significant. 3. Results Plant biomass and nitrogen fixation rate were markedly affected by salt stress conditions (Fig. 1). In control (0 mM NaCl) and treated plants (150 mM NaCl), the plant dry weight (PDW) increased with plant age, although plants grown without salt showed values of PDW higher than salinized plants in all harvests. At fructification

stage (84 days after sowing, DAS) PDW decreased about 50% with the saline treatment. The NFR was maximal at the beginning of flowering stage (56 days after sowing, DAS) in both control and salinized plants; however, in this stage this parameter decreased about 40% under salt stress. Regarding fluorescence parameters (Fig. 1), F0 (initial chlorophyll fluorescence) increased under NaCl treatment, but optimum quantum yield of PSII, represented by the Fv /Fm ratio, decreased with salinity. At the last harvest (84 DAS), plants exposed to salt stress increased F0 about 60% and decreased Fv /Fm about 23% as compared with non-salinized plants. The enzyme activities of nodule carbon metabolism sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) are shown in Fig. 2. In control plants, the SS activity (involved in sucrose cleavage) showed the maximum value at the beginning of flowering (56 DAS), coinciding with the NFR behavior. On the other hand, the maximum PEPC activity was recorded at flowering stage (70 DAS) where the value was two fold higher as compared with the one obtained in the vegetative stage (42 DAS). Both nodule enzyme activities were drastically affected by salinity, mainly in the second harvest, where SS and PEPC activities decreased about 85% and 80% respectively. The nodule malate content was higher in control than salttreated plants (Fig. 2). In absence of salt, nodules increased the malate content at the beginning of flowering (56 DAS), reaching values ten fold higher than those found in stressed plants at this grow stage. Fig. 3 shows the contents of total soluble sugars (TSS), trehalose and proline in leaves and root nodules. In general, the concentration

150 mM NaCl

0 mM NaCl 2.4

a

LSD (P≤0.05) PDW

g plant -1

1.8

b

d f f 42

c

e

μmol N2 g-1 NDW h-1

F0

b

a

c d

80

de

de

e

f

42

56

70

84

a

a

b

b

70

84

0

56

70

84 2.0

400 LSD (P≤0.05)

a

NFR

LSD (P≤0.05) Fv/Fm

a

1.5 1.0

200

b

100 0

LSD (P≤0.05)

160

b

0.6

300

320 240

1.2

0.0

45

c c

d

d 42

d

56

70

Days after sowing

84

0.5

a

a

a

a

0.0 42

56

Days after sowing

Fig. 1. Plant dry weight (PDW), nitrogen fixation rate (NFR), initial chlorophyll fluorescence (F0 ) and photochemical efficiency of Photosystem II (Fv /Fm ) of M. sativa treated with NaCl at 35 days after sowing. Values are mean ± S.E. (n = 10) and differences between means were compared using LSD (P ≤ 0.05). The same letter represents no significant difference.

46

F. Palma et al. / Environmental and Experimental Botany 85 (2013) 43–49

150 mM NaCl

μmol NAD+ mg-1 prot min-1

0 mM NaCl 0.0060 0.0045 0.0030

a b

b cd

0.0015

c d

cd

e

0.0000 42

μmol NADH mg-1 prot min-1

SS

LSD (P≤0.05)

56

4. Discussion

70

84

0.4 LSD (P≤0.05) PEPC

0.3

a

0.2

c

0.1

de

b

c cd

e

f

0.0 42

56

70

84

20

mg g-1 FW

LSD (P≤0.05) Malate

15

a 10

c

c 5

d

b e

f

f

56

70

0 42

treatment (Fig. 4). The concentrations of myoinositol decreased significantly in leaf under salt effect, however, was higher in the nodules, especially two weeks after treatment. In nodules, the highest value was recorded 7 days after NaCl application (42 DAS); reaching a six folds higher concentration than non-salinized plants. On the other hand, pinitol content increased with salt stress and plant age in both organs (leaf and nodule). Under salt stress, the increases of pinitol content were more important in nodules, being 1.5-fold higher at 42 DAS and about 2–3-fold higher at the rest of harvests. In leaf, the increase of pinitol resulted from the salt stress was about 20–25% along the whole experiment.

84

Days after sowing Fig. 2. Sucrose synthase activity (SS), phosphoenolpyruvate carboxylase activity (PEPC), and content of malate in root nodules of M. sativa treated with NaCl at 35 days after sowing. Values are mean ± S.E. (n = 10) and differences between means were compared using LSD (P ≤ 0.05). The same letter represents no significant difference.

of TSS and proline was higher in nodules than in leaves; besides, the values were increased in both organs by the saline conditions. Under the effect of salinity, TSS increased two fold in leaf and about 42% in nodule, in the last harvest. Proline content also increased significantly by the salinity in both organs; at 84 DAS, the values enhanced 93% and 45% in leaf and nodule, respectively. Regarding trehalose content in leaves, the behavior was different in the control treatment and stressed plants along the whole experiment. At the stages vegetative and beginning of the flowering, trehalose concentrations were higher in leaves of unstressed plants, but at flowering and fructification stages, the values were higher in NaCltreated plants. Salinity also induced an accumulation of trehalose in nodules, which was found in all harvests. In general, the content of myoinositol and pinitol in leaves and nodules changed with the plant growth stage and the saline

Several papers have been written regarding the effect of salt stress on nitrogen fixation and on the enzymes of the carbon metabolism in nodules (Lopez et al., 2008; Lopez and Lluch, 2008) as well as on the accumulation of sugars and other compatible solutes (Bartels and Sunkar, 2005; Khadri et al., 2007), but the mechanisms implicated in these processes remain unclear. In the present work, we examined the changes in the content of polyols (myoinositol and pinitol), the presence of soluble carbohydrates and the carbon metabolism in nodules of M. sativa plants under salt stress. Our study showed a significant decrease of PDW and NFR of M. sativa under salt stress (Fig. 1). The highest reductions of PDW (50%) and NFR (40%) were registered at 84 DAS and 56 DAS, respectively. These results are in agreement with those reported in grain legumes, such as Phaseolus vulgaris (Tejera et al., 2005) and Cicer arietinum (Soussi et al., 1998), and forage legumes as Lotus japonicus and Medicago truncatula (Lopez et al., 2008). In addition, salinity affected the chlorophyll fluorescence parameters, decreased initial chlorophyll fluorescence (F0 ) and increased the optimum quantum yield of PSII (Fv /Fm ratio). This last result might be due to the lesions in the reaction center of PSII, as it was previously pointed out in sorghum (Masojidek and Hall, 1992), or indirectly by an accelerated senescence as it happens in rice (Moradi and Ismail, 2007). The enzyme activities SS and PEPC, responsible of the carbon supply to the bacteroids by the formation of dicarboxylates, were drastically inhibited by salinity, mainly at 56 DAS in the beginning of flowering (Fig. 2). These results agree with the experiments of Ben Salah et al. (2009), who reported a decrease of SS activity in Medicago ciliaris subjected to salt stress. The sucrose synthase activity of legumes ensures the supply of hexoses for the demands of energy and reducing power of nodules during the active stage of nitrogen fixation and this enzyme may regulate carbon metabolism and nitrogen fixation (Lopez and Lluch, 2008). In this regard, nodules of non-stressed plants showed the maximum value of SS activity at the beginning of flowering stage, coinciding with the peak of nitrogenase activity (Fig. 1). It has been previously hypothesized that there is a close relation between nitrogen fixation and the capacity of the nodules to metabolize sucrose (Lopez and Lluch, 2008). Our results showed that malate content in nodules of non stressed plants (control) was higher than in salt treated plants; besides, in absence of NaCl the malate concentration was more elevated at the beginning of flowering stage (Fig. 2). The decrease in SS activity by salt stress could lead to a reduction of malate content in nodules, and this decline could result a shortage of substrates for bacteroid respiration and probably a disturbance in the regulation of the oxygen permeability (Gálvez et al., 2000). Consequently, it may explain the observed decline in the nitrogen fixation rate and also could provide additional evidences on the central role of SS in the regulation of nodule carbon metabolism and nitrogen fixation. In general, ours results showed that NaCl treatment increased the content of total soluble sugars, trehalose and proline in leaves

F. Palma et al. / Environmental and Experimental Botany 85 (2013) 43–49

150 mM NaCl

0 mM NaCl 100 LSD (P≤0.05)

TSS

27

a b

18

bc c

e

c

d

e

Nodule (mg g-1 FW)

Leaf (mg g-1 FW)

36

9

42

56

70

a

50

b d

25

c

f g 42

f

e

56

70

84

0.60 LSD (P≤0.05) Trehalose

a

1.8

b

b

1.2

d d

0.6

e

c

de

Nodule (mg g-1 FW)

Leaf (mg g-1 FW)

TSS

75

84

2.4

LSD (P≤0.05) Trehalose

0.45

a

0.30

e 0.15

c f

cd

b

de

g 0.00

0.0 42

56

70

42

84

56

70

84

6 LSD (P≤0.05)

Proline

a

2

1

b

bc c 42

d d 56

c 70

d

Nodule (mg g-1 FW)

3

Leaf (mg g-1 FW)

LSD (P≤0.05)

0

0

0

47

LSD (P≤0.05)

Days after sowing

a

4

2

0 84

Proline

c d de 42

e

de 56

b

c

70

84

Days after sowing

Fig. 3. Content of total soluble sugar (TSS), trehalose and proline in leaves and root nodules of M. sativa treated with NaCl at 35 days after sowing. Values are mean ± S.E. (n = 10) and differences between means were compared using LSD (P ≤ 0.05). The same letter represents no significant difference.

and root nodules (Fig. 3). A strong correlation between sugar accumulation and osmotic stress tolerance has been widely reported, including transgenic experiments (Gilmour et al., 2000; Streeter et al., 2001; Taji et al., 2002; Bartels and Sunkar, 2005). It was also thought that the role of the compatible solutes was the osmotic adjustment but there is an increased discussion about other roles of these molecules (Serraj and Sinclair, 2002). In our experiment, the concentrations of soluble sugars and proline were higher in nodule than in leaf; besides, values were increased in both organs by the saline conditions. According with these results, we suggest that the nodule is an organ specially protected in order to maintain its crucial function, even under stress conditions. In addition, trehalose content in nodules increased with salt stress in all harvests (Fig. 3). This last result is in accordance with Lopez et al. (2006), who reported higher trehalose content in L. japonicus nodules under salt stress than in control nodules, indicating the possible osmoprotectant role of trehalose in legume nodules.

Accumulation of cyclic polyols such as myoinositol or pinitol has frequently been reported in response to drought and salinity (Vernon and Bohnert, 1992; Streeter et al., 2001). Using transgenic tobacco plants, Sheveleva et al. (1997) related the accumulation of ononitol with the tolerance to these stresses. In general our results show that the content of myoinositol and pinitol in leaves and nodules changed with the plant growth stage and the saline treatment (Fig. 4). Although the fundamental biological functions of myoinositol are still far from be clear in plants, a recent study of molecular biology in Arabidopsis informs that myoinositol serves as the main substrate for synthesizing phosphatidylinositides, which are essential for the endomembrane structure and the substances traffic and thus for auxins-regulated embryogenesis (Luo et al., 2011). In addition, under stress conditions, myoinositol is methylated and isomerized to O-methyl inositols (ononitol and pinitol), which have roles in plant stress responses (Ahn et al., 2011).

48

F. Palma et al. / Environmental and Experimental Botany 85 (2013) 43–49

150 mM NaCl

0 mM NaCl 4

Leaf (mg g-1 FW)

LSD (P≤0.05) Myoinositol

4

a a

b

ab

c c

2

d e

Nodule (mg g-1 FW)

6

0 56

70

a

2

b 1

84

c c

c

c

42

56

70

c d 84

36 LSD (P≤0.05)

Pinitol

27

a 18

d

d

e

e

c

b c

0

Nodule (mg g-1 FW)

36

Leaf (mg g-1 FW)

3

0 42

9

LSD (P≤0.05) Myoinositol

LSD (P≤0.05) Pinitol

b

56

70

b

18 9 0

42

a

27

84

e f 42

Days after sowing

c d ef 56

70

84

Days after sowing

Fig. 4. Content of myoinositol and pinitol in leaves and root nodules of M. sativa treated with NaCl at 35 days after sowing. Values are mean ± S.E. (n = 10) and differences between means were compared using LSD (P ≤ 0.05). The same letter represents no significant difference.

The pinitol is the major component of the soluble carbohydrates present in plants subjected to osmotic stress (McManus et al., 2000). Under salinity stress, the concentrations of pinitol in nodule were higher than in leaf and this response was similar to the observed in sugars and proline contents (Fig. 3). However, the molar concentration of pinitol in stressed plants was greater than the concentration of proline, as it has been also observed in soybean plants exposed to drought (Streeter et al., 2001). According with our data, we suggest that the pinitol have a central function in the adaptive response of the nodule to salt stress. Interestingly, under saline condition, nodules showed the highest myoinositol content at the vegetative stage, decreasing at the following harvests, whereas the response of the pinitol to salt stress was the opposite, showed the highest values at the fructification stage. In this regard, Sengupta et al. (2008) found that salt stress increased the inositol methyl transferase activity, which is involved in the biosynthesis of pinitol through conversion of myoinositol. This response could explain our results previously commented, and in this way the increase of pinitol synthesis in nodule of M. sativa under salt stress could be one of the adaptive features used by the plant.

Acknowledgments Financial support was obtained through the Andalusian Research Program (AGR-139) and the Spanish Ministry of Education and Science AGL2006-01279. The authors are grateful to anonymous reviewers for making valuable suggestions to earlier drafts of this study.

References Ahn, C., Park, U., Park, P.B., 2011. Increased salt and drought tolerance by d-ononitol production in transgenic Arabidopsis thaliana. Biochemical and Biophysical Research Communications 415, 669–674. Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59, 206–216. Ashraf, M., Harris, P.J.C., 2004. Potential biochemical indicators of salinity tolerance in plants. Plant Science 166, 3–16. Balibrea, M.E., Cuartero, J., Bolarín, M.C., Pérez-Alfocea, F., 2003. Sucrolytic activities during fruit development of Lycopersicon genotypes differing in tolerance to salinity. Physiologia Plantarum 118, 38–46. Bartels, D., Sunkar, R., 2005. Drought and salt tolerance in plants. Critical Reviews in Plant Sciences 24, 23–58. Ben Salah, I., Albacete, A., Martínez Andújar, C., Haouala, R., Labidi, N., Zribi, F., Martinez, V., Pérez-Alfocea, F., Abdelly, C., 2009. Response of nitrogen fixation in relation to nodule carbohydrate metabolism in Medicago ciliaris lines subjected to salt stress. Journal of Plant Physiology 166, 477–488. Ben Salah, I., Slatni, T., Gruber, M., Mahmoudi, H., Zribi, K., Abdelly, C., 2011. Variability in the response of six genotypes of N2 -fixing Medicago ciliaris to NaCl. Symbiosis 53, 139–147. Cataldi, T.R.I., Margiotta, G., Iasi, L., Di Chio, B., Xiloyannis, C., Bufo, S.A., 2000. Determination of sugar compounds in olive plant extracts by anion-exchange chromatography with pulsed amperometric detection. Analytical Chemistry 72, 3902–3907. Chen, T.H.H., Murata, N., 2002. Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Current Opinion in Plant Biology 5, 250–257. Cordovilla, M.P., Ligero, F., Lluch, C., 1994. The effect of salinity on N fixation and assimilation in Vicia faba. Journal of Experimental Botany 45, 1483–1488. Delgado, M.J., Garrido, J.M., Ligero, F., Lluch, C., 1993. Nitrogen fixation and carbon metabolism by nodules and bacteroids of pea plants under sodium chloride stress. Physiologia Plantarum 89, 824–829. Delhaize, E., Ryan, P.R., Randall, P.J., 1993. Aluminum tolerance in wheat (Triticum aestivum L.). II. Aluminum stimulated excretion of malic acid from root apices. Plant Physiology 103, 695–702.

F. Palma et al. / Environmental and Experimental Botany 85 (2013) 43–49 Elsheikh, E.A.E., Wood, M., 1995. Nodulation and N2 fixation by soybean inoculated with salt-tolerant rhizobia or salt-sensitive bradyrhizobia in saline soil. Soil Biology and Biochemistry 27, 657–661. Frommer, W.B., Ludewig, U., Rentsch, D., 1999. Taking transgenic plants with a pinch of salt. Science 285, 1222–1223. Gálvez, S., Hirsch, A.M., Wycoff, K.L., Hunt, S., Layzell, D.B., Kondorosi, A., Crespi, M., 2000. Oxygen regulation of a nodule located carbonic anhydrase in alfalfa. Plant Physiology 124, 1059–1068. Georgiev, G.I., Atkins, C.A., 1993. Effects of salinity on N2 fixation, nitrogen metabolism and export and diffusive conductance of cowpea root nodules. Symbiosis 15, 239–255. Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D., Thomashow, M.F., 2000. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology 124, 1854–1865. Irigoyen, J.J., Emerich, D.W., Sánchez-Díaz, M., 1992. Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants. Physiologia Plantarum 84, 55–60. Khadri, M., Tejera, N.A., Lluch, C., 2007. Sodium chloride–ABA interaction in two common bean (Phaseolus vulgaris) cultivars differing in salinity tolerance. Environmental and Experimental Botany 60, 211–218. Läuchli, A., 1984. Salt exclusion: an adaptation of legumes for crops and pastures under saline conditions. In: Staples, R.C. (Ed.), Salinity Tolerance in Plants. WileyInterscience, New York, USA, pp. 171–188. Loewus, F.A., Loewus, M.W., 1980. Myoinosytol: biosynthesis and metabolism. In: Preiss, J. (Ed.), The Biochemistry of Plants. Carbohydrates: Structure and Function. Academic Press, New York, USA, pp. 43–76. Lopez, M., Herrera-Cervera, J.A., Iribarne, C., Tejera, N.A., Lluch, C., 2008. Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism. Journal of Plant Physiology 165, 641–650. Lopez, M., Herrera-Cervera, J.A., Lluch, C., Tejera, N.A., 2006. Trehalose metabolism in root nodules of the model legume Lotus japonicus in response to salt stress. Physiologia Plantarum 128, 701–709. Lopez, M., Lluch, C., 2008. Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress. Journal of Plant Interactions 3, 137–144. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275. Luo, Y., Qin, G., Zhang, J., Liang, Y., Song, Y., Zhao, M., Tsuge, T., Aoyama, T., Liu, J., Gu, H., Qu, L.J., 2011. d-myo-Inositol-3-phosphate affects phosphatidylinositolmediated endomembrane function in Arabidopsis and is essential for auxin-regulated embryogenesis. Plant Cell 23, 1352–1372. Makela, P., Kontturi, M., Pehu, E., Somersalo, S., 1999. Photosynthetic response of drought and salt stressed tomato and turnip rape plants to foliar applied glycinebetaine. Physiologia Plantarum 105, 45–50. Masojidek, J., Hall, D.O., 1992. Salinity and drought stresses are amplified by high irradiance in sorghum. Photosynthetica 27, 159–171. Matos, M.C., Campos, P.S., Passarinho, J.A., Semedo, J.N., Marques, N.M., Ramalho, J.C., Ricardo, C.P., 2010. Drought effect on photosynthetic activity, osmolyte accumulation and membrane integrity of two Cicer arietinum genotypes. Photosynthetica 48, 303–312.

49

McManus, M.T., Bieleski, R.L., Caradus, J.R., Barker, D.J., 2000. Pinitol accumulation in mature leaves of white clover in response to a water deficit. Environmental and Experimental Botany 43, 11–18. Mengel, K., Kirkby, E.A., 2001. Principles of plant nutrition. Annals of Botany 93, 479–480. Moradi, F., Ismail, A.M., 2007. Responses of photosynthesis, chlorophyll fluorescence and ROS-Scavenging systems to salt stress during seedling and reproductive stages in rice. Annals of botany 99, 1161–1173. Morell, M., Copeland, L., 1985. Sucrose synthase of soybean nodules. Plant Physiology 78, 149–154. Rathinasabapathi, B., 2000. Metabolic engineering for stress tolerance: installing osmoprotectant synthesis pathways. Annals of botany 86, 709–716. Rhodes, D., Nadolska-Orczyk, A., Rich, P.J., 2002. Salinity, osmolytes and compatible solutes. In: Läuchli, A., Lüttge, U. (Eds.), Salinity: Environment-Plants-Molecules. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 181–204. Rigaud, J., Puppo, A., 1975. Indole-3-acetic-acid catabolism by soybean bacteroids. Journal of General Microbiology 88, 223–228. Sairam, R.K., Tyagi, A., Chinnusamy, V., 2006. Salinity tolerance: cellular mechanisms and gene regulation. In: Huang, B. (Ed.), Plant–Environment Interactions. , third edition. CRC Press, Boca Raton, FL, pp. 121–175. Schreiber, U., Bilger, W., Newbauer, C., 1994. Chlrophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In: Schulze, E.D., Caldwell, M.M. (Eds.), Ecophysiology and Photosynthesis. Springer Verlag, Heidelberg, pp. 49–70. Sengupta, S., Patra, B., Ray, S., Majumder, A.L., 2008. Inositol methyl tranferase from a halophytic wild rice, Porteresia coarctata Roxb. (Tateoka): regulation of pinitol synthesis under abiotic stress. Plant, Cell & Environment 31, 1442–1459. Serraj, R., Sinclair, T.R., 2002. Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant, Cell & Environment 25, 333–341. Sheveleva, E., Chmara, W., Bohnert, H.J., Jensen, R.G., 1997. Increased salt and drought tolerance by d-ononitol production in transgenic Nicotiana tabacum L. Plant Physiology 115, 1211–1219. Soussi, M., Ocana, A., Lluch, C., 1998. Effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.). Journal of Experimental Botany 49, 1329–1337. Streeter, J.G., Lohnes, D.G., Fioritto, R.J., 2001. Patterns of pinitol accumulation in soybean plants and relationships to drought tolerance. Plant, Cell & Environment 24, 429–438. Taji, T., Ohsumi, C., Seki, M., Iuchi, S., Yamaguchi-Shinozaki, K., Shinozaki, K., 2002. Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant and Cell Physiology 43, S233–S233. Tejera, N.A., Campos, R., Sanjuan, J., Lluch, C., 2005. Effect of sodium chloride on growth, nutrient accumulation, and nitrogen fixation of common bean plants in symbiosis with isogenic strains. Journal of Plant Nutrition 28, 1907–1921. Vernon, D.M., Bohnert, H.J., 1992. A novel methyl transferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum. EMBO Journal 11, 2077–2085. Witty, J.F., Minchin, F.R., 1998. Methods for the continuous measurement of O2 consumption and H2 production by nodulated legume root systems. Journal of Experimental Botany 49, 1041–1047. Zhu, J.K., 2001. Plant salt tolerance. Trends in Plant Science 6, 66–71.