Improving salt stress responses of the symbiosis in alfalfa using salt-tolerant cultivar and rhizobial strain

Applied Soil Ecology 87 (2015) 108–117

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Improving salt stress responses of the symbiosis in alfalfa using salt-tolerant cultivar and rhizobial strain Annick Bertrand a, *, Catherine Dhont b , Marie Bipfubusa b , François-P. Chalifour b , Pascal Drouin c,1, Chantal J. Beauchamp b a b c

Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, Québec, QC G1V 2J3, Canada Departement de phytologie, 2425 rue de l’agriculture, Université Laval, Québec, QC G1V 0A6, Canada Université du Québec en Abitibi-Témiscamingue (UQAT), Rouyn-Noranda, QC J9X5E4, Canada

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 July 2014 Received in revised form 29 October 2014 Accepted 20 November 2014

Salt stress can affect alfalfa growth directly by adversely affecting metabolism, or indirectly by its effect on Rhizobium capacity for symbiotic N2 fixation. Growth and carbohydrate metabolism in leaves, roots and nodules of two alfalfa cultivars (Medicago sativa cv Apica and salt-tolerant cv Halo) in association with two rhizobial strains (A2 and salt-tolerant Rm1521) exposed to different levels of NaCl (0, 20, 40, 80 or 160 mM NaCl) were assessed under controlled conditions. For both cultivars, shoot and root biomasses and shoot to root ratio significantly declined with increasing NaCl concentrations. Under 80 mM NaCl, Halo plants yielded 20% more fresh shoot biomass than Apica while plants inoculated with Rm1521 allocated more biomass to the roots than to the shoots compared to A2. Halo plants maintained a steady shoot water content (about 80%) under the entire range of NaCl concentrations. Shoot water content was more variable in Apica. Apica in association with salt-tolerant strain Rm1521 maintained a better water status than with strain A2, as indicated by the higher shoot water content at 80 mM NaCl. Under salt stress, two major compatible sugars involved in plant osmoregulation, sucrose and pinitol, increased in leaves while a large accumulation of starch was observed in roots. In nodules, pinitol, sucrose and starch increased under salt stress and were much more abundant with strain Rm1521 than with A2. This suggests that there could be an active transport from the shoot to the nodules to help maintain nodule activity under NaCl stress and that strain Rm1521 increases the sink strength toward nodules. Our results show that combining cultivars and rhizobial strains with superior salt tolerance is an effective strategy to improve alfalfa productivity in salinity affected areas. Crown Copyright ã 2014 Published by Elsevier B.V. All rights reserved.

Keywords: Medicago sativa L. Salt stress Osmoregulation Pinitol Nodule Salt-tolerant cultivar

1. Introduction Salt stress is one of the most important abiotic factors limiting plant growth and productivity especially in arid and semi-arid regions. In Canada, this occurs extensively in the Prairie Provinces, where 79% of Canada’s agricultural lands are located. The total extent of moderate-to-severe salinity in the Prairies, resulting in a 50% reduction in productivity, was estimated to be around 1.4 million hectares (Eilers et al., 1997). Plants tend to cope with salt stress by different adaptation strategies including physiological, biochemical, and molecular mechanisms (Bohnert et al., 1995; Munns and Tester, 2008) that

* Corresponding author. E-mail address: [email protected] (A. Bertrand). 1 Current address: Lallemand Animal Nutrition, 586 Ridge Road, Chazy, NY 12921, USA. http://dx.doi.org/10.1016/j.apsoil.2014.11.008 0929-1393/ Crown Copyright ã 2014 Published by Elsevier B.V. All rights reserved.

facilitate retention and/or acquisition of water, protect chloroplast functions and maintain ion homeostasis (Parida and Das, 2005). Biochemical mechanisms include accumulation of compatible solutes such as amino acids, sugars and polyols (Ford, 1984). These organic solutes are able to accumulate at high concentration in the cytoplasm, contributing to turgor maintenance and protecting enzymes and other cellular structures against damage by ions or dehydration (Bartels and Sunkar, 2005). Plant species or cultivars greatly vary in their tolerance to salt (Noble et al., 1984; Al-Khatib et al., 1994; Munns, 2002) and thus, differ in their capacity to remain productive as soil salinity increases. Alfalfa is the most important forage crop species in Canada where it is the third largest crop by area. It is cultivated over 4.5 million hectares in the Canadian Prairies accounting for 76% of the total national production area of pure alfalfa or alfalfa mixtures (Statistics Canada, 2012). Alfalfa is considered as moderately tolerant to salt and can tolerate an equivalent of 20 mM NaCl. Alfalfa with improved salt tolerance has been developed using conventional

A. Bertrand et al. / Applied Soil Ecology 87 (2015) 108–117

breeding and genetic engineering approaches (Bohnert and Jensen, 1996; Flowers, 2004). High salinity may also affect alfalfa performance by reducing the efficiency of the Rhizobium–legume symbiotic N2 fixation (Bernstein and Ogata, 1966; Delgado et al., 1993; Zahran, 1999). It has been established that Rhizobia are, in general, more tolerant to salt stress than the host plant, but rhizobial strains greatly vary in growth and survival under salt stress (Soussi et al., 1998; Swaraj and Bishnoi, 1999). It has been suggested that a combination of stress-tolerant cultivars and stress-tolerant Rhizobia may result in synergistic advantages in the ability of legumes to grow and survive under saline conditions (Hashem et al., 1998; Nogales et al., 2002). This approach has been successfully used for improving biological nitrogen fixation under salt stress of soybean (Glycine max) (Elsheikh and Wood, 1995), Acacia ampliceps (Zou et al., 1995) and Phaseolus vulgaris (Nogales et al., 2002). Mohammad et al. (1989) showed that salt stress-tolerant Medicago sativa plants exhibited higher drought tolerance when inoculated with a salt stress-tolerant strain of Sinorhizobium meliloti. In the present study, we investigated the mechanisms of alfalfa tolerance to salt stress. Our hypothesis was that tolerant symbiotic partners would act in synergy to alleviate salt stress in alfalfa. To reach this goal, we measured the concentration of carbohydrate compounds typically associated with stress tolerance in leaves, roots and nodules of alfalfa under five levels of NaCl. We compared the response of one salt-sensitive and one salt-tolerant cultivar in association with one salt-sensitive and one salt-tolerant rhizobial strain. 2. Materials and methods 2.1. Plant material This study was carried out on two cultivars of alfalfa (M. sativa subsp. sativa), Apica and Halo. Apica was developed at the Agriculture and Agri-Food Canada Research Station in Québec City (Québec, Canada) and was tested across Eastern Canada for its yield, persistence and winter survival (Michaud et al., 1983). Halo is a synthetic variety with 192 parent plants selected sequentially for germination, seedling growth, and mature plant re-growth, as well as salinity tolerance (Steppuhn et al., 2012). 2.2. Inoculum production Two strains of S. meliloti were used, A2 (Balsac) isolated from Eastern Canada (Bordeleau et al., 1977) and Rm1521, a salt-tolerant strain isolated from the Ottawa vicinity (Bromfield et al., 1994). Rhizobial strains were grown on yeast mannitol agar plates (Vincent, 1970). Each strain was resuspended in yeast extract mannitol broth, and then placed in a shaking incubator (120 rpm, Lab-Line Orbit Environ-shaker, Melrose Park, IL) at 28
C for 1 week. Viability counts were then performed and the inoculum was adjusted to 109 cells mL1. 2.3. Plant growth conditions Alfalfa seeds were surface-sterilized by immersion in ethanol 95% for 30 s, and in 5% NaClO for 10 min, and then washed five times with sterile water. Sowing was performed in 20 cm diameter, 20 cm deep plastic pots filled with turface (Profile Products LLC, Buffalo Grove, IL). For each cultivar, twenty seeds were sown per pot: two seeds were placed in each of ten small cavities in which 200 mL of inoculum was added. Half of the pots were inoculated with A2 and the other half with Rm1521 inoculum. Plants were grown in a large growth room under a 21/17
C day/night temperatures regime, a 16 h photoperiod and a

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photosynthetic photon flux density of 600–800 mmol photons m2 s1 provided by a mixture of high pressure sodium and metal halide 400 W lamps (PL light Systems, Beamsville, ON, Canada). After one week, seedlings were thinned to ten plants per pot and a second inoculation was done using 200 mL of inoculant per seedling. Plants were fertigated daily with 0.25X N-free nutrient solution during the first week and with 0.5X N-free nutrient solution during the second week (1X nutrient solution contained 111.8 mg P L1, 141.1 mg K L1, 2.100 mg Fe L1, 0.600 mg Mn L1, 0.120 mg Zn L1, 0.030 mg Cu L1, 0.390 mg B L1, 0.018 mg Mo L1, 48.62 mg Mg L1, and 0.952 mg Co L1). Two weeks after sowing, plants were fertilized once with N (2 mM N + 0.5 X nutrient solution). Salt stress treatments consisted of 0, 20, 40, 80, or 160 mM NaCl which were applied on 3 week-old plants. To avoid osmotic shock, NaCl concentrations were gradually increased during one transition-week starting on day 1 with 20 mM NaCl for all NaCl-treated pots. NaCl concentration was then doubled every two days until final concentrations were reached. Once the targeted salt stress levels were reached, plants were fertigated daily with 0.5X nutrient solution containing the appropriate NaCl concentration. Salt stress was applied for six weeks and then plants were sampled. 2.4. Plant biomass measurements and tissue sampling At sampling, plants were carefully removed from substrate and gently washed under tap water. Excess water was removed by gently pressing roots in absorbent paper towels. Roots were cut from shoots, and root and shoot fresh weights (FW) were recorded. To reduce within-pot variation, pooled subsamples of shoots and roots from three plants were dried at 55
C to a constant weight for total dry weight (DW) and water content assessments. For the remaining plants, shoots were separated into leaves and stems while nodules were detached from roots. Subsamples of leaves, roots and nodules were lyophilized prior to grinding for biochemical assessments. Nodule samples were ground using a Tissue Lyser (Tissue Lyser II, Retsch, QiaGen) while other tissues were ground using a mixer mill (Mixer Mill 301, Retsch Inc.). The relative biomass curve response to salt stress was established for shoot and root biomass using the following equation: Relative biomassð%Þ ¼

biomass yield under NaCl stress  100 biomass yield at0mM NaCl

2.5. Extractions and analyses of carbohydrates Lyophilized samples of leaves and roots (0.2 g) were extracted in 7 mL and nodules (0.1 g) were extracted in 4 mL of deionized water. Tubes were heated 20 min at 65
C to stop enzymatic activity. Tubes were kept overnight at 4
C for optimal extraction and then homogenized on a vortex mixer and centrifuged 10 min at 3000 rpm at 4
C (Bertrand et al., 2003). 1 mL subsample of supernatant was transferred to 1.5 mL microtubes and centrifuged for 3 min at 13,000  g prior to HPLC analyses. The non-soluble residues left after extraction were washed twice with 10 mL of methanol and used for starch determination. Mono-, di-, tri- and tetra-saccharides were separated and quantified on a Waters analytic system controlled by the Empower II software (Waters, Milford, MA, USA). Sugars were separated on a HPX-87P column (Bio-Rad) at 80
C with a flow rate of 0.5 mL min1 with water. Peak identity and quantity were determined for sucrose, glucose, fructose and pinitol by

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Table 1 Analysis of variance of NaCl, cultivars and rhizobial strain effects and interactions on plant growth traits (p-value). Total biomass

Shoot biomass

Root biomass

Shoot/root ratio

Effects

FW

DW

FW

DW

FW

DW

FW

DW

NaCl Cultivars NaCl  cultivars Rhizobial strains NaCl  rhizobial strains Cultivars  rhizobial strains NaCl  cultivars  rhizobial strains

<0.0001*** 0.086 0.241 0.206 0.110 0.131 0.736

<0.0001*** 0.406 0.633 0.150 0.103 0.201 0.407

<0.0001*** 0.049* 0.117 0.495 0.110 0.179 0.955

<0.0001*** 0.463 0.446 0.522 0.092 0.261 0.546

<0.0001*** 0.427 0.853 0.107 0.318 0.221 0.211

0.0010** 0.571 0.842 0.096 0.301 0.312 0.446

<0.0001*** 0.494 0.770 0.120 0.769 0.952 0.216

<0.0001*** 0.884 0.651 0.030* 0.581 0.931 0.373

Shoot water content

0.203 0.165 0.983 0.763 0.392 0.770 0.040*

*

P < 0.05. P < 0.01. *** P < 0.001. **

comparison to standards (Sigma–Aldrich, Oakville, ON, Canada). Total water soluble carbohydrates (WSC) were calculated as the sum of sucrose, glucose, fructose, glucose and pinitol. Starch was quantified in non-soluble residues as glucose equivalents with the p-hydroxybenzoic acid hydrazide method of Blakeney and Mutton (1980) after gelatinization at 100
C and digestion for 90 min with amyloglucosidase (Sigma A7255, Sigma Chemical Co., St. Louis, MO). Starch amounts were determined spectrophotometrically by reference to a glucose standard curve. 2.6. Statistical analysis An analysis of variance model for a split plot design with four blocks was adjusted to study the effect of NaCl treatments, strains and cultivars on biomass and carbohydrates characteristics. The MIXED procedure of the SAS program was used (SAS, 2011). When there was some heterogeneity in the data it was corrected with the group option in the REPEATED statement. Residual analysis was performed to verify the assumptions underlying the model. When the normality assumption was not met, a log transformation was used. Pairwise comparisons were made using protected Fisher LSD (least significant difference) at P = 0.05. 3. Results 3.1. Plant biomass Total biomass yield was significantly affected by salt stress (Table 1): increasing NaCl concentration reduced gradually plant biomass expressed either on a fresh weight (FW) or dry weight (DW) basis (Table 2). At 160 mM NaCl, yield was reduced by 67% on a FW basis and by 62% on a DW basis as compared to unstressed plants (0 mM NaCl, Table 2). However, these negative effects were more pronounced on shoot (72% and 74% of fresh and dry shoot biomass, respectively) than on root (55% fresh root biomass and 44% dry root biomass; Table 2). We observed a significant difference in shoot FW biomass response to salt stress between the two cultivars (Table 1); Halo plants averaged 20% more fresh

weight than Apica under a moderate salt stress of 80 mM NaCl treatments (Fig. 1A). On the basis of the relative biomass curves, a 50% reduction of biomass occurred for shoot with 60 mM NaCl in Apica and with 80 mM in Halo plants (Fig. 1B), and for roots with 140 mM NaCl in both cultivars (Fig. 1C). Rhizobial strains did not significantly affect root or shoot biomass per se (Table 1) but shoot/root ratio was higher for plants inoculated with A2 than with Rm1521 (Fig. 1D). For both cultivars, the shoot/root ratio decreased significantly with salt stress intensity (Fig. 1D). Interaction between salt stress, cultivar and rhizobial strain was significant for the shoot water content (Table 1). In Apica treated with 40 mM NaCl, shoot water content was higher when plants were inoculated with A2 than with Rm1521 (Fig. 1E). In the same cultivar treated with 80 mM NaCl, shoot water content was higher in plants inoculated with Rm1521 (81% of water) as compared to the ones inoculated with A2 (76% of water). In Halo, shoot water content showed less variation in response to salt stress and was slightly higher in plants inoculated with A2 than with Rm1521 at 20 mM NaCl (Fig. 1E). 3.2. Carbohydrate concentrations in leaves Interaction between NaCl treatment and cultivars was significant for total WSC and glucose concentrations in leaves (Table 3). In both cultivars, total WSC concentrations increased from 80 mg g1 DM without salt stress to 104 mg g1 DM with the 20 mM NaCl treatment (Fig. 2A). A significant difference was observed between the two cultivars under the 40 mM NaCl stress, total WSC concentration being 45% higher in Halo than in Apica. In Apica submitted to the 40 mM NaCl stress, total WSC concentration was similar to values of unstressed plants (78 mg g1 DM) while in Halo, the total WSC concentrations reached up to 112 mg g1 DM under the same treatment (Fig. 2A). At 80 and 160 mM NaCl treatments, no difference was detected between cultivars for total concentration of WSC, but total WSC concentrations were higher than those measured in unstressed plants. Changes in glucose concentrations were very similar to those observed for

Table 2 Effects of salt stress on total biomass, shoot and root biomass and shoot/root ratio for the two cultivars tested. NaCl (mM)

0 20 40 80 160

Total biomass

Shoot biomass

Root biomass

Shoot/root ratio

FW (g)

DW (g)

FW (g)

DW (g)

FW (g)

DW (g)

FW

78.09a 61.50b 52.46b 41.16c 25.66d

19.30a 15.55b 14.13b 11.13c 7.25d

54.30a 40.91b 33.08c 25.11d 14.99e

12.00a 9.01a 7.46c 5.42d 3.07e

23.79a 20.61b 19.38b 16.06c 10.67d

7.30a 6.5ab 6.67a 5.72b 4.15c

2.36a 2.01b 1.72c 1.60d 1.41e

For each parameter, means in the same column followed by the same letter are not significantly different, as determined by the Fisher’s least significant difference (LSD) test at P = 0.05. FW: fresh weight; DW: dry weight. For each value, n = 8.

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[(Fig._1)TD$IG] A

B

C

D

111

E

Fig. 1. Shoot biomass (A), relative shoot biomass (B), relative root biomass (C), shoot/root ratio (D), and shoot water content (E) of two alfalfa cultivars (Apica and Halo) in combination with two rhizobial strains (A2 or Rm1521) and exposed to different levels of NaCl (0, 20, 40, 80 or 160 mM NaCl). Each value represents the mean of four replicates. For each cultivar, means followed by a same letter are not significantly different, as determined by the Fisher’s least significant difference (LSD) test at P = 0.05. When triple interactions were significant, graphics representing the effects of NaCl and rhizobial strains on each cultivar were presented separately, and * indicated significant difference between Halo and Apica cultivars while letters indicated significant differences between strains A2 and Rm1521 at the corresponding NaCl level. Dotted lines in (1B) and (1C) indicates the 50% reduction of relative shoot and root biomass respectively.

total WSC in response to salt stress except for the fact that the glucose concentration under 80 and 160 mM NaCl was similar to the values measured in unstressed plants (Fig. S1A). Leaf sucrose concentrations increased slightly but significantly with salinity stress, from 21 mg g1 DM in unstressed plants to 30 mg g1 DM in plants stressed at 160 mM NaCl (Table 3; Fig. 2B). Interaction between NaCl treatment, cultivars and rhizobial strains was significant for leaf pinitol concentrations (P = 0.05; Table 3). For both cultivars, pinitol concentrations increased markedly under a 20 mM NaCl stress and were higher in NaCl stressed than in unstressed plants (Fig. S1B). Apica treated with 40 mM NaCl accumulated less pinitol in leaves than those treated with higher NaCl levels. Unstressed Apica showed higher pinitol concentrations when inoculated with Rm1521 than with A2 strain. Fructose concentration in leaves was significantly affected by salt stress (Table 3). Fructose concentration gradually decreased with salt stress intensity from 13 mg g1 DM in unstressed plants to 4 mg g1 DM in 160 mM NaCl-treated plants (data not shown). Leaf starch concentrations remained unchanged, at around 80 mg g1 DM, regardless of salt stress, cultivar or rhizobial strain (Table 3). 3.3. Carbohydrate concentrations in roots Interaction between salt stress, cultivars and rhizobial strains was significant for total WSC and sucrose concentrations in roots

(Table 3). Total WSC concentrations in unstressed plants were much higher in Halo than in Apica (71 vs 56 mg g1 DM; Fig. 3A). The 20 mM NaCl treatment induced a major decrease in root WSC concentrations in Apica and Halo as compared to unstressed plants (Fig. 3A). The rhizobial strain had a major impact on root WSC concentration of Apica. This cultivar accumulated more total WSC in roots when inoculated with Rm1521 than with A2 under the 40 mM NaCl treatment, and reversely for the 80 mM NaCl stress. In cultivar Halo, root WSC concentrations were reduced by 33% by salt stress regardless the NaCl concentration. No difference was detectable between strains for this cultivar. Changes in root sucrose concentrations were similar to those observed for total WSC in response to salt stress and symbiotic association for each cultivar (Fig. 3B). Root pinitol concentrations were significantly affected by salt stress in interaction with alfalfa cultivar (Table 3). In both cultivars, pinitol concentrations significantly increased with salt stress intensity (Fig. S1C). In unstressed and 20 mM NaCl stressed plants, pinitol concentrations were higher in Halo than in Apica, and reversely at 160 mM NaCl, pinitol concentrations were higher in Apica (Fig. S1C). Triple interaction was significant for root glucose concentration (Table 3). In both cultivars, glucose concentrations decreased in response to salt stress (Fig. S1D). The lowest concentration of root glucose was measured under 20 mM NaCl in Apica and 40 mM NaCl

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Table 3 Analysis of variance of NaCl, cultivars and rhizobial strain effects and interactions on carbohydrate concentrations in leaves, roots and nodules (p-value). Effects

WSC

Sucrose

Pinitol

Glucose

Fructose

NaCl Cultivars NaCl  cultivars Rhizobial strains NaCl  rhizobial strains Cultivars  rhizobial strains NaCl  cultivars  rhizobial strains

Leaves <0.0001*** 0.002** 0.001*** 0.609 0.875 0.945 0.163

Starch

0.017* 0.524 0.658 0.121 0.467 0.138 0.080

<0.0001*** 0.015* 0.280 0.040* 0.062 0.905 0.052

<0.0001*** 0.001** <0.0001*** 0.955 0.706 0.170 0.274

<0.0001*** 0.322 0.095 0.344 0.665 0.804 0.123

NaCl Cultivars NaCl  cultivars Rhizobial strains NaCl  rhizobial strains Cultivars  rhizobial strains NaCl  cultivars  rhizobial strains

Roots 0.001*** 0.029** 0.039* 0.743 0.001** 0.554 0.009**

0.002** 0.035* 0.337 0.639 0.005** 0.625 0.001***

<0.0001*** 0.902 0.001** 0.293 0.379 0.692 0.349

<0.0001*** 0.889 <0.0001*** <0.0001*** 0.080 0.405 0.013*

ND ND ND ND ND ND ND

<0.0001*** 0.118 0.894 0.002** 0.014* 0.713 0.353

NaCl Cultivars NaCl  cultivars Rhizobial strains NaCl  rhizobial strains Cultivars  rhizobial strains NaCl  cultivars  rhizobial strains

Nodules <0.0001*** 0.020* 0.348 0.016* 0.204 0.237 0.029*

0.002** 0.665 0.635 0.498 0.221 0.115 0.040*

<0.0001*** <0.0001*** 0.014* <0.0001*** 0.001*** 0.171 0.066

0.171 0.002** 0.153 0.075 0.460 0.308 0.468

0.017* 0.456 0.097 0.079 0.320 0.809 0.849

<0.0001*** 0.218 0.488 <0.0001*** 0.005** 0.656 0.064

0.097 0.064 0.414 0.446 0.929 0.437 0.434

WSC: water soluble carbohydrates (sum of sucrose, pinitol, glucose and fructose). ND: non-detectable level of fructose. * P < 0.05. ** P < 0.01. *** P < 0.001.

in Halo. In unstressed Apica roots, glucose concentrations were higher upon the symbiotic association with strain A2 as compared to strain Rm1521. No significant effect of the strain was detected for Halo (Fig. S1D).

[(Fig._2)TD$IG]

Starch concentration in roots was significantly affected by salt stress in interaction with rhizobial strains (Table 3). Starch concentration was higher in unstressed plants and in 20 mM- and 40 mM-NaCl stressed plants inoculated with

Fig. 2. Total water soluble sugars (WSC) (A) and sucrose (B) concentrations in leaves of two alfalfa cultivars (Apica and Halo) in combination with two rhizobial strains (A2 or Rm1521) and exposed to different levels of NaCl (0, 20, 40, 80 or 160 mM NaCl). Each value represents the mean of four replicates. For each cultivar, means followed by a same letter are not significantly different, as determined by the Fisher’s least significant difference (LSD) test at P = 0.05. When triple interactions were significant, graphics representing the effects of NaCl and rhizobial strains on each cultivar were presented separately, and * indicated significant difference between Halo and Apica cultivars while letters indicated significant differences between strains A2 and Rm1521 at the corresponding NaCl level.

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[(Fig._3)TD$IG]

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Fig. 3. Total water soluble sugars (WSC) (A) and sucrose (B) concentrations in roots of two alfalfa cultivars (Apica and Halo) in combination with two rhizobial strains (A2 or Rm1521) and exposed to different levels of NaCl (0, 20, 40, 80 or 160 mM NaCl). Each value represents the mean of four replicates. For each cultivar, means followed by a same letter are not significantly different, as determined by the Fisher’s least significant difference (LSD) test at P = 0.05. When triple interactions were significant, graphics representing the effects of NaCl and rhizobial strains on each cultivar were presented separately, and * indicated significant difference between Halo and Apica cultivars while letters indicated significant differences between strains A2 and Rm1521 at the corresponding NaCl level.

Rm1521 than with strain A2. Starch concentration increased with salt stress intensity (Fig. S1E). Starch concentration was higher in Apica than in Halo under 160 mM NaCl. Fructose concentration in roots was undetectable in response to salt stress. 3.4. Carbohydrate concentrations in nodules Interaction between NaCl treatments, cultivars and rhizobial strains was significant for the total WSC and sucrose concentrations in nodules (Table 3), and changes in WSC and sucrose concentrations were similar (Fig. 4A and B). In Apica, rhizobial strain had a major impact on the accumulation of WSC and sucrose in response to NaCl treatment WSC and sucrose concentrations were higher in nodules of plants inoculated Rm1521 in unstressed and in 40 mM NaCl stressed plants while they were higher with strain A2 under a 80 mM NaCl stress (Fig. 4A and B). In Halo, WSC and sucrose concentrations increased in nodules in response to salt stress as compared to unstressed plants (Fig. 4A and B). However, no major differences were detected in response to NaCl treatments. Even though WSC and sucrose concentration in Halo tended to be higher in Rm1521 than in A2 nodules, the differences were not significant between the rhizobial strains. Interaction between salt stress and cultivars, and between salt stress and rhizobial strains were significant for pinitol concentration in nodules (Table 3). In both cultivars, nodule pinitol concentration increased with salt stress intensity, the highest concentration being observed under 40 mM NaCl with strain Rm1521, and under 160 mM NaCl with strain A2 (Fig. 4C). Pinitol concentration was higher in plants inoculated with Rm1521, especially when submitted to 20 and 40 mM NaCl in both cultivars. Pinitol concentration under salt stress was slightly higher in Apica than in Halo (Fig. 4C).

Nodule starch concentration was significantly affected by NaCl stress in interaction with rhizobial strain (Table 3). For both cultivars, the 20, 40 and 80 mM NaCl treatment induced an increase in starch concentration compared to unstressed plants. Rm1521 nodules accumulated up to 70% more starch than A2 nodules (Fig. 4D). Starch concentrations in nodules stressed with 160 mM NaCl were similar to values measured in unstressed nodules, except for Apica inoculated with Rm1521 where it remained higher. Glucose and fructose concentrations were low in nodules. Glucose concentration differed between the two cultivars (Table 3) and was 42% higher in nodules of Apica than in the ones of Halo (6.3 vs 4.4 mg g1 DM). Fructose concentration in nodules was significantly affected by salt stress (Table 3) and was higher in plants submitted to 80 mM NaCl (3 mg g1 DM) than for the other treatments (2.2 mg g1 DM). 4. Discussion 4.1. Biomass response to salt stress Our results show that alfalfa biomass production is adversely affected by increasing soil salinity. The primary effect of salt stress is to reduce plant growth and salt tolerant plants typically show less growth reduction under salt stress (Munns, 2002). Plants of cultivar Halo were more NaCl tolerant than those of cv Apica as indicated by the difference in NaCl concentration that caused 50% reduction in fresh shoot biomass (80 mM NaCl for Halo vs 60 mM NaCl for Apica, Fig. 1B). This result supports the observation of Steppuhn et al. (2012) that among nine alfalfa populations selected for their salinity tolerance, Halo plants showed greater relative shoot biomass yield than any other alfalfa population under a salt stress of 80 mM NaCl (8 dS m1).

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[(Fig._4)TD$IG]

A

B

C

D

Fig. 4. Total water soluble sugars (WSC) (A), sucrose (B), pinitol (C) and starch (D) concentrations in nodules of two alfalfa cultivars (Apica and Halo) in combination with two rhizobial strains (A2 or Rm1521) and exposed to different levels of NaCl (0, 20, 40, 80 or 160 mM NaCl). Each value represents the mean of four replicates. For each cultivar, means followed by a same letter are not significantly different, as determined by the Fisher’s least significant difference (LSD) test at P = 0.05. When triple interactions were significant, graphics representing the effects of NaCl and rhizobial strains on each cultivar were presented separately, and * indicated significant difference between Halo and Apica cultivars while letters indicated significant differences between strains A2 and Rm1521 at the corresponding NaCl level.

The shoot to root ratio has been suggested as a reliable indicator to screen for drought or salt tolerance (Kramer, 1983; Munns, 2002). Although roots are directly exposed to salt stress, our data show that the shoot/root ratios of both cultivars progressively decreased with increasing NaCl stress level, showing that root growth was less adversely affected by NaCl than shoot growth. Interestingly, we found a significant difference in the shoot/root ratio according to the rhizobial strain in symbiosis with alfalfa. At the threshold of 80 mM NaCl, the shoot/root ratio was lower for alfalfa in association with Rm1521 than with A2. It seems that alfalfa in association with Rm1521 possesses an advantage against NaCl stress because more biomass is allocated to the roots as compared to the shoots under the full range of salt concentrations. Decrease in shoot/root ratio is considered an adaptive mechanism (Gorham et al., 1985; Munns, 2002) as it leads both to an increase surface for water extraction through the roots and a lower water loss through reduced transpiration area. The importance of the root system in providing a better water supply to the plant has recently been highlighted by Campanelli et al. (2013) who showed that arbuscular mycorrhiza symbiosis alleviates salt stress in alfalfa, likely by increasing root density. It should however be noticed that rhizospheric microorganisms could also compete with each other and mycorrhiza could decrease the positive effects of rhizobia as it was observed with soybean (Juge et al., 2012). It is noticeable that strain Rm1521 has been shown to be salt tolerant per se when growing in agar media with the addition of

NaCl (Bromfield et al., 1994). Thus, in addition to favor a lower shoot/root ratio, Rm1521 is more adapted to salt stresses and could likely help maintain a higher N2 fixing capacity under NaCl stress. Biological nitrogen fixation was not measured in this study but it is well known that any factor that limits plant growth, such as drought or salinity, will also limit symbiotic nitrogen fixation (Athar and Johnson, 1996). Furthermore, stress-tolerant rhizobium strains could help legumes to maintain a higher level of nitrogen fixation as well as higher biomass yield (Athar and Johnson, 1996; Bertrand et al., 2011). Plant growth responds to salinity in two phases, a rapid osmotic phase during which growth inhibition is mainly due to the difficulty for the plant to absorb soil water, and a slower ionic phase in which growth inhibition is due to the toxic effect of the salt within the plant (Munns and Tester, 2008). NaCl stress lasted six weeks in our study, thus both effects have likely contributed to growth reduction. For instance, in response to the osmotic stress, we found that root biomass reduction was more pronounced for fresh than for dry weight suggesting that NaCl stress negatively affected the water retention and/or uptake ability of plant roots due to the osmotic effect of the salt in the rooting medium. A similar response was observed for shoot fresh weight that was higher for cultivar Halo than Apica while shoot dry weight was similar for both cultivars. This indicates a better water retention for cultivar Halo under salt stress. In addition, we observed a significant difference in shoot water content in response to NaCl stress according to the cultivar and the

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rhizobial strain. NaCl-tolerant cultivar Halo maintained shoot water content around 80% showing a regulation of turgescence under the full range of NaCl stress. The shoot water content of Apica was much more variable and, at the threshold of 80 mM NaCl, we observed a sharp drop of water content when in symbiotic association with A2 and a large increase when in symbiotic association with Rm1521. Under this level of NaCl stress, a difference of 6% in shoot water content was recorded between the two strains. The capacity of a plant to maintain turgor under NaCl stress is a feature linked with NaCl tolerance as it helps maintain cell growth and stomatal opening to allow photosynthesis. The ability of a plant to maintain turgor under salt stress has also been linked to the mitigation of salt toxicity by a dilution effect (Cuartero et al., 1992; Asmare, 2013). According to our results, it seems that one of the mechanisms of salt tolerance of cultivar Halo is through a better osmotic adjustment under NaCl stress. Interestingly, we observed that under a damaging stress of 80 mM NaCl, the salt-sensitive cultivar Apica maintained a higher water status when in association with salt-tolerant strain Rm1521 than with A2. A similar beneficial association has been recently reported between a drought-sensitive cultivar of common bean and Rhizobium gallicum (Sassi-Aydi et al., 2012). R. gallicum was then shown to maintain an adequate water status in common bean by stimulating the accumulation of compatible solutes. It has been suggested that salt stress-tolerant M. sativa plants exhibited higher drought tolerance when inoculated with a salt stress-tolerant strain of S. meliloti (Mohammad et al., 1989). One of the modes of action of Rm1521 strain could be linked to its sink strength for compatible solutes as shown by the higher accumulation of carbohydrates in nodules of plants inoculated with strain Rm1521 as compared to A2 and discussed below. 4.2. Accumulation of compatible carbohydrates 4.2.1. Leaves and roots Osmotic adjustment is mainly due to the accumulation of compatible solutes such as soluble sugars, amino acids and proteins, which is an effective mechanism for the protection and survival of plants under salt stress (Zhu, 2002). For instance, soluble sugars are known to play a role in membrane and enzyme protection from stress damage or in the reduction of cell damage induced by free radicals (Hare et al., 1998; Kaplan and Guy, 2004). Within a species, salt tolerant populations have been shown to accumulate larger amounts of protective metabolites to help maintain cell turgor by osmotic adjustment, compared to sensitive populations (Ashraf and Tufail, 1995; Ashraf and Harris, 2004; Krasensky and Jonak, 2012). We assessed the concentration of different carbohydrates known as compatible solutes to better understand the mechanisms of osmotic adjustment of alfalfa in response to salt stress. We found an accumulation in total WSC in alfalfa shoot in response to salt stress and, interestingly, total WSC accumulation was larger in plants of salt-tolerant cultivar Halo than in those of Apica. It is possible that higher levels of total WSC may have helped maintain a steadier shoot water-content in that cultivar than in Apica throughout the entire range of salt stress. Our results support the observation of Majid et al. (2012) of a higher content of soluble sugars in salt-tolerant than in salt-sensitive alfalfa varieties. This accumulation of sugars was probably induced by a lower utilization in the actively growing tissues (Munns, 1993), which could also explain the lower biomass production under salinity. We observed different response in WSC accumulation between source and sink organs. For instance, between 0 and 20 mM NaCl stress, we observed a large accumulation of total WSC in leaves along with a reduction in roots. When looking at individual sugar, we observed an increase in sucrose concentration in leaves in response to increasing salt

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stress intensity. Sucrose accumulation in leaves is considered a physiological adaptation mechanism to NaCl stress through its role of compatible solute for osmoregulation and of its interaction with membrane surfaces, possibly replacing water in the maintenance of membrane structure (Koster and Leopold, 1988). A positive contribution of sucrose in osmotic adjustment of alfalfa in our experiment is supported by the similarity between the evolution of sucrose accumulation and variations in shoot water content in plants exposed to increasing salinity (Figs. 1E and 2B). Among other sugars, we observed an increase in pinitol concentration under salt stress. Such an increase in pinitol concentrations has been observed in roots of alfalfa submitted to salt stress (Fougère et al., 1991) as well as in leaves of alfalfa (Aranjuelo et al., 2011), white clover (Trifolium repens L.) (McManus et al., 2000), and soybean (Streeter et al., 2001) under drought stress. Pinitol is considered a compatible solute and has been involved in osmotic adjustment in plant tissues under salt stress (Fougère et al., 1991; Adams et al., 1992) or water deficit (McManus et al., 2000; Reddy et al., 2004). In addition, polyols may help prevent oxidative damage, keep membrane integrity and maintain enzyme activities due to their ability to stabilize macromolecules and to scavenge hydroxyl radicals (Smirnoff and Cumbes, 1989). Our data show salt-induced accumulation of pinitol which may be involved in the regulation of water balance to prevent water loss from the roots to the soil (Ruiz-Lozano et al., 2012). Unlike in leaves, root sucrose concentrations declined in response to salt stress. Torabi and Halim (2013) also found that sucrose concentrations in alfalfa roots declined with increasing salinity. In our study, the decline in sucrose contents in roots under salt stress was concomitant with a decrease in glucose concentrations and an increase in starch concentrations. This suggests a partitioning of carbohydrate toward starch through salt stress-induced sucrose synthase (Fernandes et al., 2004; Krasensky and Jonak, 2012) or invertase activity (Pattanagul and Thitisaksakul, 2008). According to previous studies (Balibrea et al., 2000; Pattanagul and Thitisaksakul, 2008), decrease in root sucrose concentration toward starch synthesis is an adaptive mechanism since the ability of plants to partition sugars into starch help maintain the root sink strength. An excess amount of sucrose in roots causes a feedback inhibition and limits the capacity of roots to import sucrose. The sucrolytic activity, which could be used as an indicator for the ability of sink organs to grow and to import sucrose (Doehlert and Chourey, 1991; Sung et al., 1994), was enhanced by salinity in alfalfa roots in our experiment. This activity is considered as a good marker for the ability of sink organs to attract assimilates and could explain the greater allocation of biomass to the roots instead of the shoot. 4.2.2. Nodules We observed an increased concentration of starch in nodules with increasing salinity. As for roots, the partitioning of carbohydrates into starch increases the sink strength of nodules. The accumulation of starch in nodules was concomitant with the accumulation of major soluble sugars, suggesting that this accumulation could result from the translocation of sugars from other plant organs such as leaves and roots. This is consistent with Palma et al. (2013) who stated that the nodule is an organ specially protected in order to maintain its crucial function, even under stress conditions. We observed increases in sucrose and pinitol concentrations in alfalfa nodules as previously observed by Fougère et al. (1991) and Palma et al. (2013), and in Medicago truncatula and Ligustrum japonicum (López-Gómez et al., 2012) exposed to salt stress. Sucrose accumulation under salt stress was also observed in nodules of soybean (Gordon et al., 1997) and white lupin (Lupinus albus L.) (Fernandez-Pascual et al., 1996). Salt-induced

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accumulation of sucrose and pinitol in nodules can alleviate the negative effect of salt stress on nitrogen fixation. This is in accordance with numerous studies indicating that sucrose accumulation in nodules could boost nitrogen fixation by increasing the carbon flux for bacteroid respiration (Gordon et al., 1997; López et al. 2008). The compatible sugars such as sucrose and pinitol could also be involved in nodule osmoregulation (Aranjuelo et al., 2011; Ashraf and Bashir, 2003; López-Gómez et al., 2012). Interestingly, starch and pinitol concentrations were significantly higher in nodules of plants in symbiotic association with salt-tolerant strain Rm1521 than with A2 suggesting that the accumulation of sugars in nodules contributes both to the salt tolerance of the bacteroids and of the host plant. It has been suggested that many species of bacteria adapt to saline conditions by the intracellular accumulation of osmolytes (Csonka and Hanson, 1991). Sugar accumulation may improve the salt tolerance of bacteroids by acting as energy sources or osmolytes (Hunt and Layzell, 1993). Alfalfa in association with strain Rm1521 could also take advantage of the larger sink strength of this strain as compared to A2 by maintaining an active symbiosis under stress. In the present study, accumulation of starch and soluble sugars (sucrose and pinitol) in nodules in response to salt stress were stimulated by Rm1521 compared to A2. These findings support the relative salt tolerance of Rm1521compared to A2. 5. Conclusions Our study shows that alfalfa cultivar Halo has a better salt tolerance through various mechanisms resulting in a better biomass distribution and more stable shoot water content under the full range of NaCl concentrations studied as compared to Apica which has not been selected for salt tolerance. The rhizobial strains in symbiosis with alfalfa also affect the plant capacity to withstand salinity. For instance, the salt-tolerant strain Rm1521 provided major advantages to alfalfa cultivars to withstand severe NaCl stress by maintaining nodule sink strength for compatible solutes as well as nodule metabolism under stress. Our results also showed that combining salt-tolerant cultivar and rhizobial strain could improve the salt tolerance of alfalfa through various mechanisms. Acknowledgments The authors sincerely thank Sandra Delaney and Josée Bourassa for their technical assistance. This work was supported by the Centre SÈVE, Université de Sherbrooke, QC, Canada. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. apsoil.2014.11.008. References Adams, P., Thomas, J.C., Vernon, D.M., Bohnert, H.J., Jensen, R.G., 1992. Distinct cellular and organismic responses to salt stress. Plant Cell Physiol. 33, 1215–1223. Al-Khatib, M.M., McNeilly, T., Collins, J.C., 1994. Between and within cultivar variability in salt tolerance in lucerne: (Medicago sativa L.). Genet. Resour. Crop Evol. 41, 159–164. Aranjuelo, I., Molero, G., Erice, G., Avice, J.C., Nogués, S., 2011. Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.). J. Exp. Bot. 62, 111–123. Ashraf, M., Bashir, A., 2003. Salt stress induced changes in some organic metabolites and ionic relations in nodules and other plant parts of two crop legumes differing in salt tolerance. Flora 198, 486–498.

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