1 Introduction

During the last decade, the importance of grain legumes for human nutrition increased considerably in the Mediterranean basin. These legumes fix nitrogen by symbiosis with rhizobia, and supply the necessary of nitrogen for plant growth and development, and contribute to the improvement of the soil nitrogen status. Consequently, rhizobia–legume symbiosis, will allow farmers to economise the cost of chemical fertilizers and to avoid nitrate pollution. The low and unstable yields of common bean (Phaseolus vulgaris) production is, particularly, due to the effects of biotic and abiotic factors, especially when its growth depends on biological nitrogen fixation (BNF) (Jonas et al. 2010; Faghire et al. 2011; Mandri et al. 2012). Common bean cultivars are salt-sensitive species (Lauchli 1984; Soussi et al. 1999; Faghire et al. 2011) and the limitation of their productivity is associated with a low growth of the host plant, less symbiotic development of the root-nodule bacteria (Georgiev and Atkins 1993) and, consequently, a reduction capacity of nitrogen-fixation (Delgado et al. 1993). Under stress conditions, many physiological and biochemical mechanisms such as, the accumulation of compatible osmolytes like proline, glycine betaine, sucrose and mannitol (Zhu 2002) were switched on to protect major processes like cell respiration, photosynthetic activity, nutrient transport, nitrogen and carbon metabolism.

In salt conditions, the acid phosphatase (APase, EC 3.1.3.2) activity is known to contribute to salt tolerance by maintaining an inorganic phosphate level which can be co-transported with H+ along a gradient of proton motive force (Olmos and Hellin 1997). Protective roles of the antioxidant enzymes, under salt constraint, have been reported for a number of plants such as wheat (Almeselmani et al. 2006; Esfandiari et al. 2007), and Catharanthus roseus (Jaleel et al. 2007). Intracellular acid phosphatases (APase, EC 3.1.3.2) are ubiquitous in plant organs, they were implicated in providing P during seed germination from stored phytate, hydrolysing of orthophosphate monoesters into more mobile orthophosphate anions, synthesis glycolate from P-glycolate and glycerate from 3-PGA during photorespiration (Vance et al. 2003). Moreover, the characteristics of some acid phosphatases (APase, EC 3.1.3.2) purified from nodules of soybean (Glycine max L.) showed that these enzymes were involved in the conversion of purines to ureides during symbiotic N fixation (Penheiter et al. 1997).

Trehalose 6-phosphate phosphatase (TPP, EC 3.1.3.12) is an enzyme that catalyzes the chemical reaction (alpha, alpha-trehalose 6-phosphate + H2O ⇌ alpha,alpha-trehalose + phosphate) (Cabib and Leloir 1958). The trehalose (D-glucopyranosyl-1,1-D-glucopyranoside) is a non-reducing disaccharide that has been found in a wide variety of organisms such as yeasts, fungus, bacteria, plants, insects and other invertebrates (Crowe et al. 1984; Wiemken 1990). This molecule plays an important role in the protection from abiotic stress by stabilizing dehydrated enzymes and membranes as well as protecting biological structures from desiccation damage (Lopez et al. 2008; Lopez and Lluch 2012).

Therefore, the overall aim of this study was to use biochemical and molecular approaches to evaluate the effect of salt stress on (i) The activities of acid phosphatase (APase, EC 3.1.3.2) and trehalose 6-phosphate phosphatase (TPP, EC 3.1.3.12) and (ii) The abundance of APase gene transcripts in common bean-rhizobia root nodules.

2 Materials and methods

2.1 Plant material and experimental treatments

The experiments were conducted in a glasshouse under natural light with day/night temperatures of 28/20 °C and 16 h photoperiod consists of 16 h of light and 8 h of darkness with an additional illumination of 400 μmol photons m−2·S −1. The relative humidity during the day was 70 %. The study was carried out using four common bean inbred lines 147, 115, 104 and 83 originating from the International Centre of Tropical Agriculture. Seeds were surface sterilized with 3 % calcium hypochlorite and then washed thoroughly in ten successive baths of sterile distilled water. Thereafter, they were germinated for 4 days at 28 °C in paper roll germination soaked with sterile distilled water. Seedlings with uniform roots were inoculated with the reference Rhizobium strain, Rhizobium tropici CIAT899, or with the local Rhizobium strain, Rhizobium gallicum RhM11 (Faghire et al. 2011) grown in a liquid of yeast extract mannitol medium (YEM) at 28 °C for 2 days. Thereafter, seedlings were transferred to 40 l (40,000 cm3) plastic containers with a nutrient solution, where the seedlings were aerated intensely and arranged in a fully randomized block design. Roots of selected seedlings were passed through the hole of a rubber stopper on the vat cover. Wool cotton was fitted at the hypocotyl level to maintain root system suspended in the nutrient solution (Vadez et al. 1999). Urea was supplied at 2 mM per plant into the nutrient solution during the initial week of growth to avoid N deficiency during nodule development. Thereafter, the plants were grown in N-free nutrient solution with NaCl concentrations of 0 and 25 mM added to the nutrient solution at 7th day after their transplantation (appearance of the first true leaves). The pH nutrient solution was adjusted, every week, to around pH 7 by the addition of 0.2 g·l−1 of CaCO3 and the medium was aerated during the experiment by an ambient air flow of 400 ml·min−1.

Plants were harvested 42 days after their transplantation, which coincide with the flowering stage and the optimum of functional activities of nodules. At harvest, root systems were superficially washed with deionized water and plants were separated into shoots, roots and nodules. Nodule samples were frozen at −80 °C for the essay of the enzymes activities. Thereafter, each plant organs and nodules samples were oven-dried at 75 °C for 48 h to determine their dry weight (DW).

2.2 Preparation of extracts and enzyme assays

2.2.1 Acid phosphatase (APase) activity

The extracts were prepared by homogenizing 0.2 g of fresh nodules in a mortar with 33 % (w/w) polyvinyl-polypyrrolidone and 500 μl of acetate-buffer (0.1 M pH 5), and 5 μl β-mercapto-ethanol. The extracts were centrifuged at 13,000 g for 30 min at 4 °C, and three aliquots of 100 μl of the supernatant were used for enzyme assays. All the operations were carried out at 4 °C.

For the determination of APase activity, 100 μl of nodular extracts were incubated during 30 min at 37 °C with a mixture 200 μl of acetate-buffer and 200 μl of p-nitrophenyl phosphate (p-NPP). The reaction was stopped by addition of 1.0 ml NaOH 0.5 N. Then the optical density (OD) was measured by spectrophotometer at 410 nm. APase activity was defined as the amount of the protein hydrolysing 1 nmol of p-NPP per min per g of fresh nodules.

2.2.2 Trehalose 6-phosphate phosphatase (TPP, EC 3.1.3.12) activity

The extracts were prepared by homogenizing 0.2 g of fresh nodules in a mortar with 33 % (w/w) polyvinyl-polypyrrolidone and 2 ml of 50 mM Tris–HCl buffer (pH 7.5) containing 2.5 mM MgCl2, 100 mM NaCl and 10 mM β-mercaptoethanol. The extracts were centrifuged at 13,000 g for 20 min and the supernatants were used to determine the enzyme activities. All operations were carried out at 4 °C.

The TPP activity was assayed by monitoring the phosphate released from trehalose 6-phosphate (Padilla et al. 2004). The reaction was carried out in a supernatant volume of 0.25 ml that contained 25 mM Tris–HCl (pH 7.0), 10 mM MgCl2 and 1 mM trehalose-6-phosphate. Samples were assayed for phosphate activity by malachite green method (Ohno and Zibilske 1991).

2.2.3 Sample preparation and fixation for in situ RT-PCR

Sample preparation and fixation for in situ RT-PCR were carried out according to the protocol described by Molina et al. (2011) and Bargaz et al. (2012). The method involves in situ the amplification of specific nucleic acid sequences on nodules sections, followed by fluorescence detection (Van Aarle et al. 2007) of the localized PCR product using epifluorescence microscopy. In brief, 5 mm of the diameter of root nodules of the two combinations 115-CIAT899, 147-CIAT899 and their corresponding salt treatment, were immediately and separately harvested from plants 42 days after their transplantation, and thoroughly washed with diethyl pyrocarbonate (DEPC) treated water, and then fixed in 4 % (v/v) paraformaldehyde, 45 % (v/v) ethanol and 5 % (v/v) acetic acid, and kept for 2 h under vacuum, and then stored overnight at 4 °C. Fixed nodules were included in 9 % (m/v) low-melting-point-agarose and cut into 50 μm thick sections using a microtome (Micro-cut H1200 Vibrating Microtome; Bio-Rad, Marnes la Coquette, France). The obtained sections were collected into a small tubes containing 0.5 ml of DEPC-treated water and freed from residual agarose by three washes with DEPC-treated water heated to 60 °C.

For reverse transcription, the first cDNA strand was synthesized from the total of RNA of 50 μm of nodules slices using MMLV reverse transcriptase (following the manufacturer’s recommendations). Nodules slices were incubated in 40 μl reverse transcriptase mix containing the APase gene-specific reverse primer APrev. Negative controls were prepared by omitting the reverse transcriptase. Then the reverse transcriptase mix was removed and 40 μl of PCR mix was added including 0.25 μM of each constituent, APdir specific-gene (5′-CTGCTTACGAATGACGACGGC-3′) and APrev (5′-CCAGTTTCAAAGGCGTTACCG-3′) primers. The thermocycling was performed using 30 cycles of 95° for 30 s, 60 °C for 30 s, and 72 °C for 45 s, with an extension at 72 °C for 2 min.

Then amplified cDNA in the fixed tissues was detected after removing the PCR mix. The samples were washed and incubated in 100 μl blocking solution under gentle agitation in the dark at 37 °C. The blocking solution was replaced by 100 μl of phosphatase-conjugated anti-digoxigenin Fab fragment (Roche Diagnostics, Basel Switzerland) diluted at 1:1,000 in 2 % BSA. Afterwards, the samples were incubated at room temperature for 90 min and then washed three times to remove the excess of antibody. Detection of phosphatase was carried out using tELF-97® endogenous phosphatase detection kit (Molecular Probes, Leiden, The Netherlands). The observations were made with an Olympus BX61® microscope equipped with an epifluorescence condenser, Hoechst/DAPI long-pass filter set configured at an excitation filter of 360–370 nm, a dichroic mirror of 400 nm and an emission of 420 nm. The images were photographed with grey View II® camera (ORCA AG; Hamamastu) using Analysis® software (Soft Imaging System, Munster, Germany). The analysis image was performed using ImageJ software as an image analysis program.

2.3 Statistical analysis

The experimental layout was a randomized block design. The values representing dry biomass are means of seven replicates per treatment. Three replicates were performed for the other studied parameters. All results were subjected to a two-way analysis of variance, with a Least Significant Difference test (LSD) for the comparison of means using Costat software.

3 Results

In the plant experiment, one set of pots with different combinations of bean lines and Rhizobium strains that did not receive any salt treatments was used as controls. The symbiotic combinations 83-CIAT899, 104-CIAT899 and 104-RhM11, showed high SDW that reached 2.4 g per plant whereas 115-CIAT899 symbiotic combination presented the lowest SDW that was less than 2 g per plant (Fig. 1a). The addition of 25 mM NaCl to the nutrient solution reduced significantly the SDW in the combinations 83-CIAT899, 104-RhM11 and 147-RhM11 respectively to 57, 15 and 40 % comparison with their corresponding controls. The other symbiotic combinations did not show, under salinity treatment, any significant variation in the growth except for 83-RhM11 that showed a significant increase in SDW (Fig. 1a).

Fig. 1
figure 1

Effects of salinity on growth parameters: a shoot dry weight (SDW), b RDW, root dry weight c NDW, nodule dry weight. Means followed by the same letter within a column are not significantly different (P < 0.05) using the LSD test

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For the RDW (Fig. 1b) and NDW (Fig. 1c) measured, the effect of salinity treatment on these parameters did not show any regular or significant variation in all of the tested symbiotic combinations, except for 83-CIAT899 which caused a significant reduction on both RDW (47 %) and NDW (52 %), whereas this constraint caused a significant increase of these parameters for 104-RhM11 combination (Fig. 1b and c).

3.1 Effect of NaCl on nodule enzyme activities

In the plant experiment, one set of pots with different combinations of bean lines and Rhizobium strains that did not receive any salt treatments was used as controls. The nodules of the majority of common bean–rhizobia symbiotic combinations showed high APase activity that exceeded 1,000 nmol min−1 g−1 FM (Fig. 2a), while salinity treatment induced a significant decrease of APase activity in the nodules of plants inoculated with CIAT899 in all four inbred lines of common bean. This decrease was not significant when plants were inoculated with RhM11 except for nodules of 147-RhM11 symbiotic combination. Also, salinity affected nodules APase activity of 115-CIAT899 and 147-CIAT899 symbiotic combinations and reductions of 24 % and 34 % were respectively noted comparatively to their corresponding controls (Fig. 2a).

Fig. 2
figure 2

Effects of salinity on nodules enzymes activities: a Phosphatase acid (APase) and b Trehalose phosphate-6P phosphatase (TPP). Means followed by the same letter within a column are not significantly different (P < 0.05) using the LSD test

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For TPP activity and in absence of salt treatment, the nodules of 147-RhM11 presented the highest value followed by 83-CIAT899 or 83-RhM11, while 104-RhM11 symbiotic combination presented the lowest nodular TPP activity which did not exceed 100 nmol·min−1·g FM−1 (Fig. 2b). However, the presence of salt in rooting medium caused generally a decrease of TPP activity in nodules of the tested symbiotic combinations that was significant for the nodules of 83-CIAT899, 83-RhM11 and 147-RhM11 symbiotic combinations (Fig. 2b).

3.2 Localisation of acid phosphatase (APase) transcripts

To test the transferability of the information between Uni-Tag profiles and other platforms, selected nodule-metabolism-associated transcripts were localized in common bean nodules. For this purpose, PCRs of acid phosphatase-annotated Uni Tags, in situ, were performed in nodules slices. Main features of the anatomy of common bean nodules are depicted in Fig. 3.

Fig. 3
figure 3

Cross section through a common bean nodule. Abbreviations: (INF) Infected zone, (E) Endodermis, (VT) Vascular trace, (IC) Inner cortex, (OC) Outer cortex

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Under salt treatment, acid phosphatase gene transcripts were mostly detected in the outer cortex and less extended in the inner cortex. These transcripts were also detected in the infected zone for both symbiotic combinations 115-CIAT899 and 147-CIAT899 (Fig. 4b and c). We noted that negative controls did not show any fluorescent signal (Fig. 4a). In salinity conditions, APase transcripts showed less fluorescent signal for both combinations compared with their corresponding controls, also APase transcripts were more abundant in 147-CIAT899 nodules than in 115-CIAT899 nodules of the symbiotic combination (Fig. 4c). In contrast, nodules of the symbiotic combination 115-CIAT899 did not show any difference of fluorescent signal compared with the control of 147-CIAT899 symbiotic combination.

Fig. 4
figure 4

In situ localization of phosphatase acid transcript isoforms in common bean root nodules (115-CIAT899 and 147-CIAT899). a Control without reverse transcription b Nodules from non-stressed plants c Nodules from stressed plants. Bars, 500 μm (this figure is available in colour at Symbiosis online)

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4 Discussion

Many previous studies have shown that salt stress conditions induced a decrease of plant growth and its development processes (Sairam and Tyagi 2004; Faghire et al. 2011). Nevertheless, it is known that plants have many morphological and physiological strategies that allow them to survive under salt stress conditions. Because of the importance of legume–Rhizobium symbiosis in agriculture, we evaluated the symbiotic interaction involving the Moroccan native strain RhM11 and CIAT899 with four lines of common bean under salt stress conditions.

Our results showed that under salinity and symbiotic conditions, the reaction of host plant depends on the plant line and also on the rhizobial strain used for inoculation (RhM11 or CIAT 899). In this regard, salt conditions affected the nodulation of common bean except for the plants of the line 83 inoculated with RhM11. Under salinity treatment, the nodule dry weight of the line 104 plants, inoculated with RhM11, increased significantly compared to the control treatment. In our work, the mean biomass of nodules represented approximately 8 % of the whole plant biomass. These results are consistent with the findings of several researchers (Tang et al. 2001; Kouas et al. 2005; Faghire et al. 2011). We noted that SDW plant was not affected by salinity in the majority of the tested symbiosis combinations except for 83-CIAT899 and 147-RhM11 (Fig. 1a). Our results showed also that the proportion of the allocated mass decreased in shoot from 74 % (control assay) to 69 % (stressed assay) and it increased in roots from 19 % to 22 % (data not shown). Based on this observation, we noted that the shoot was more sensitive to salinity than the root (Fig. 1a, b). Similar results were also found in other grain legumes such as Cicerarietinum (Soussi et al. 1998) and P. vulgaris (Tejera et al. 2004; Lopez et al. 2006).

In order to understand the effect of salt stress and its relationship with nodulation, we evaluated the nodule enzyme activities under salt conditions. Indeed, this constraint implies many enzymatic processes including phosphatase (Tejera et al. 2004; Adelson et al. 2008; Mandri et al. 2012). Generally, our results under control treatment (0 mM NaCl), can be compared to similar values measured by Adelson et al. (2008); Bargaz et al. (2012) and Mandri et al. (2012) in nodules of common bean plants. The results of biochemical assays of APase activity are consistent with the results in situ of the Acid Phosphatase (APase) transcripts visualisation. Our study, as well as previous studies of Penheiter et al. (1997); Tejera et al. (2004); Adelson et al. (2008); and Mandri et al. (2012) did not allow to distinguish whether this enzyme activity, specified in crude extracts of ground nodules, derived from tissue fractions of nodules or rhizobia. The our results of nodules TPP activity have agreed with those reported by Lopez et al. (2006) in nodules of common bean plants grown with or without NaCl. Lopez et al. (2006) have also observed that TPP activity was less affected by salinity treatment in common bean nodules.

Nodules APase activity is known to contribute to resistance under salt stress, by maintaining a certain level of inorganic phosphate that can be co-transported with H+ along a gradient of proton motive force (Olmos and Hellin 1997). Our results showed a positive correlation (up to r = 0.6) between nodules APase activity and nodules TPP activity, which suggests high contribution of these enzymes in salt stress. This observation has been reported for a number of plants (Almeselmani et al. 2006; Esfandiari et al. 2007; Jaleel et al. 2007). Based on these observations, we conclude that the inoculation with CIAT899 strain can allow common bean to acquire stress tolerance, whereas inoculation with the RhM11 local strain had the most remarkable capacity to improve tolerance of bean genotypes under salt conditions.