sodium transport and mechanism(s) of sodium tolerance in frankia strains

Journal of Basic Microbiology 2012, 52, 1–12 1

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Sodium transport and mechanism(s) of sodium tolerance in Frankia strains

Amrita Srivastava, Satya Shila Singha and Arun Kumar Mishra

Laboratory of Microbial genetics, Department of Botany, Banaras Hindu University, Varanasi, India

The mechanism(s) underlying differential salt sensitivity/tolerance were investigated in the terms of altered morphological and physiological responses against salinity such as growth, electrolyte leakage, Na+ uptake, efflux, accumulation and intracellular concentrations of macronutrients among the Frankia strains newly isolated from Hippöphae salicifolia D. Don. Growth was minimally reduced at 500 and 250 mM NaCl respectively in HsIi10 and rest of the strains (HsIi2, HsIi8, HsIi9) which proved that 500 and 250 mM NaCl are the critical concen-trations for the respective strains. The differences in the sodium influx/ efflux rate was responsible for the differential amount of remaining sodium among the frankial strains and might be one of the primary determinants for the reestablishment of macronutrients (Mg2+, Ca2+ and K+) during salinity. Secondly, the interactive effect of sodium influx/efflux rate, remaining sodium and intracellular macronutrients (Mg2+, Ca2+ and K+) concentration has been responsible for the extent of membrane damage and growth sustenance of the tolerant/sen-sitive frankial strains during salinity. HsIi10 showed better co-regulation of various factors and managed to tolerate salt stress up to considerable extent. Therefore, HsIi10 can serve as a potential biofertilizer in the saline soil.

Supporting Information for this article is available from the authors on the WWW under http://www.wiley-vch.de/contents/jc2248/2011/201100586_s.pdf

Keywords: Electrolyte leakage / Frankia / Na+ regulation / salinity stress / macronutrients / micronutrients

Received: November 22, 2011; accepted: February 16, 2012

DOI 10.1002/jobm.201100586

Introduction*

In agricultural soil, salt stress is the most prevalent among the various stresses that leads to the consider-able loss of cultivable lands. According to FAO report (FAO land and plant nutrition management service, 2008; http:/www.fao.org/ag/ag1/ag1I/spush), the total landscape in the world affected by salt is estimated to be more than 800 million hectares. Presence of high concentration of salt in the soil creates adverse effect on the organisms [1]. It changes the membrane integ-rity, disturbs the K+/Na+ ratios and ultimately creates aPresent address: Department of Botany, Guru Ghasidas Vishwavidya-laya, Bilaspur, Chhattisgarh Correspondence: Dr. Arun Kumar Mishra, Associate professor, Labora-tory of Microbial Genetics, Department of Botany, Banaras Hindu Uni-versity, Varanasi-221005, India E-mail: [email protected]; [email protected] Phone: +91-542-6701103(Office); +91-9335474142(Mobile) Fax: +91-542-2368174

ionic and osmotic stress into the cells of the organisms [2, 3]. Microorganisms have the capacity to counter salinity as well as other kinds of stress in an active or a passive manner [4–6]. Sodium is not considered as an essential nutrient even though certain plant and microbes have the capac-ity to grow very well in salt affected regions and show greater utilization of Na+ in their metabolism. The basic physiological and biochemical cellular interaction with sodium ions among different organisms are almost same. The sensitivity and tolerance of the strains de-pend on the fundamental mechanism of sodium accu-mulation and exclusion [7–10]. Salinity causes altera-tions in kinetic steady states of ion transport for Na+, Cl– as well as for other related cations such as K+ and Ca2+ in higher plants [11, 2]. Na+ is also known to affect intracellular content of cations including Ca2+ and K+ [12, 13].

2 A. Srivastava et al. Journal of Basic Microbiology 2012, 52, 1–12


Frankia is a microaerophilic, filamentous actinobacte-ria which establishes actinorhizal association with the root nodules of non leguminous and angiospermic plants including Hippöphae salicifolia D. Don [14]. It fixes atmospheric nitrogen in the root nodules of H. salicifolia D. Don which is widely distributed along the high alti-tude areas (above 1400 asl) of Eastern Himalayas, India. Due to actinorhizal association with H. salicifolia D. Don and tremendous nitrogen fixing capability, Frankia makes Hippöphae a pioneer species during early ecologi-cal succession and is also helpful in the improvement of soil fertility [15, 16]. Additionally, there are reports that the nitrogen fixers have better adaptability and survival towards salinity [1, 10]. We know from the earlier observations that the plants and Frankia have the ability to resist cold stress, metal stress as well as the nitrogen deficient conditions [17, 14]. Few reports are available which are only confined to the study of the growth, nitrogenase activity and sodium ion accre-tion among Frankia strains isolated from Casuarina, Eleagnus and Allocasuarina under salinity [18–20]. Till date, no work has been done on the mechanism of so-dium transport and its regulation in Frankia strains isolated from any host. Therefore, it is a first attempt to gain a detailed knowledge of the Na+ transport and its kinetics as well as the role of macro nutrients (added as a nutritional supplement) during salt stress condition among the different frankial strains isolated from H. salicifolia D. Don growing in eastern Himalayas (Sik-kim and adjoining areas). Additionally, we have also investigated the role of interaction of Na+ with macro-nutrients (Ca2+, K+ and Mg2+) in provision of better adaptability, growth and survival among the frankial strains towards salinity.

Materials and methods

Isolation of Frankia from root nodules Individual nodules from the roots of H. salicifolia were washed properly with water followed by 5.25% sodium hypochlorite and 0.1% tween 80 (1:1). After rewashing and soaking in 5% sodium bicarbonate solution for 10 minutes. The nodules were placed in sterilized petri dishes with wet filter paper placed at the bottom. The outer epidermis was peeled off in a dissecting micro-scope and the nodules were sterilized again in 30% H2O2. After washing the lobes with double distilled water, they were crushed in tissue homogenizer. The nodule homogenate was mixed with phosphate buffer saline and centrifuged at 2000 rpm for 5 min. The pel-let was centrifuged again with double distilled water at

10,000 rpm for 5 min and the suspension was inocu-lated in sterilized 250 ml Erlenmeyer flasks containing 100 ml BAP medium in a laminar flow for culture maintenance.

Culture conditions Frankia strains- HsIi2 (NAIMCC-B-00726; accession no. JQ480013), HsIi8 (NAIMCC-B-00730; accession no. JQ480011), HsIi9 (NAIMCC-B-00731; accession no. JQ480009) and HsIi10 (NAIMCC-B-00732; accession no. JQ480012) were isolated from root nodules of H. sali-cifolia D. Don growing in North Sikkim, India. Reference strain CpI2 was obtained from Dr. Johannes Pasi Haan-suu, Department of Biological and Environmental Sci-ences, FIN-00014, University of Helsinki. All the strains were grown in properly washed and autoclaved Erlen-meyer flasks containing liquid BAP medium, pH 7.4 [21] containing Mg2+ and Ca2+ as macronutrients and com-plimented with Hoagland’s micronutrients. In the cul-ture medium, sucrose (5 mM) and ammonium chloride (5 mM) were used as a sole carbon and nitrogen source respectively. Separately autoclaved phosphate buffer (10 mM) and Fe-EDTA were added to the cooled medium. Filter sterilized antibiotics cycloheximide (50 μg ml–1) and nalidixic acid (10 μg ml–1) were also added in the autoclaved medium for the prevention of the bacterial and fungal contamination. Strains had an absolute requirement of vitamin supplement and were cultivated in the appropriate maintenance medium containing 10 μg l–1 each of folic acid, calcium panto-thenate and riboflavin, 22.5 μg l–1 of biotin, 50 μg l–1 each of pyridoxine HCl and nicotinic acid and 100 μg l–1 of thiamine HCl (filter sterilized). To maintain the ho-mogeneity, cultures were maintained in B.O.D. (Bio-logical Oxygen Demand) incubator fitted with rotary shaker (120 rpm) under dark conditions at 29 ± 0.5 °C. The strains were subcultured once a week to obtain uniform inocula.

Protein determination Frankia cells were collected by centrifugation at 10,000 rpm for 10 minutes and the obtained pellets were homogenized with autoclaved double distilled water. 0.5 ml of the homogenized culture was taken and protein was estimated as per the protocol of Lowry et al. [22] with Bovine Serum albumin (BSA) as a stand-ard.

Growth measurement Frankia cells growing at a concentration of 35 μg pro-tein ml–1 were inoculated in sterilized Erlenmeyer flasks containing 150 ml sterilized BAP medium sup-

Journal of Basic Microbiology 2012, 52, 1–12 Sodium transport and tolerance in Frankia strains 3


plemented with different concentrations of NaCl (0, 50, 100, 250, 500, 750, 1000 mM). The cultures were main-tained at standard growth conditions. 3 ml cultures were withdrawn from the flask at every alternate day and growth was observed at 663 nm up to 30th day of incubation using Systronics Double Beam Spectropho-tometer 2203.

Estimation of electrolyte leakage Electrolyte leakage was measured as per the protocol of Dionisio-Sese and Tobita [23]. Inocula (1 ml) were trans-ferred from 12 d old Frankia cultures in 50 ml of steril-ized BAP medium supplemented with or without dif-ferent concentrations of NaCl (0, 50, 100, 250, 500, 750, 1000 mM) and incubated at standard growth condi-tions. Cultures were centrifuged at 10,000 rpm for 10 min after 12 d of incubation. Fresh pellet (0.5 ml) was taken and washed with deionized water to remove surface-adhered electrolytes. The washed pellet was placed in test tubes containing 10 ml distilled deionized water and incubated for 2 h in water bath at 32 °C. The initial electrical conductivity of the above medium (EC1) was measured. The same sample was autoclaved at 121 °C for 20 min and cooled. All the electrolytes re-leased in the medium were measured as the final elec-trical conductivity (EC2). The electrolyte leakage (EL) was calculated using the formula EL = (EC1/EC2) × 100.

Measurement of sodium uptake and intracellular sodium ion concentration The sterilized growth medium with or without sup-plementation of different concentration of NaCl (0, 50, 100, 250, 500, 750, 1000 mM) was prepared in the steril-ized 250 ml flasks. The exponentially grown cultures (1 ml) were transferred in sterilized flasks and the ex-periment was set at standard growth conditions. Cul-tures (1 ml) were taken from the different flasks at different time intervals (0, 1, 2, 3, 5, 10, 15, 30, 45 and 60 min) and centrifuged at 10,000 rpm for 10 minutes. The supernatant was transferred into another tube and its absorbance was determined at 689 nm using atomic absorption spectrophotometer (Perkin Elmer model 2380). Depletion of sodium in the medium represents the uptake of sodium by the Frankia cells. The uptake of Na+ increased linearly and proportionately with time but deviated after 5 min among all the strains. Therefore, the uptake rate for Na+ was calculated at 5 minutes and expressed in terms of μmol Na+ μg–1 protein min–1. For determination of intracellular sodium ion con-centration, inocula from 12 d old Frankia cultures (1 ml) were transferred in 50 ml of sterilized BAP medium

supplemented with or without different concentrations of NaCl (0, 50, 100, 250, 500, 750, 1000 mM) and incu-bated for 5 min at the standard growth conditions. Then, cultures were centrifuged at 10,000 rpm for 10 min and the obtained pellet was rinsed with aerated iso-osmotic solution of sorbitol to remove the adhering ions. Further, the pellet was digested with HNO3:HClO4 (10:1 v/v) in a boiling water bath for 30 min. Samples were cooled at room temperature and were centrifuged at 10,000 rpm for 10 min. The supernatant was taken for the determination of intracellular Na+ content in terms of μmol Na+ μg–1 protein and its absorbance was determined at 689 nm using atomic absorption spec-trophotometer (Perkin Elmer model 2380).

Measurement of sodium efflux and remaining sodium Different frankial strains were incubated in NaCl de-plete and replete (50, 100, 250, 500, 750, 1000 mM) BAP medium at standard growth conditions for 5 min. Then, the centrifugation was done at 10,000 rpm for 10 min and the pellets were rinsed with an aerated iso-osmotic solution of sorbitol for removal of adhering ions. The obtained pellets were again re-suspended in 5 ml sterilized deionized water for 5 min, the re-centrifugation was done at 10,000 rpm for 10 min and absorbance of the supernatant was taken for the de-termination of rate of Na+ efflux in terms of μmol Na+ μg–1 protein min–1 using atomic absorption spectropho-tometer (Perkin Elmer model 2380). Remaining sodium ion content (after efflux) of the frankial cells was calculated by deducting sodium ion effluxed from the intracellular sodium ion content of the cells of Frankia.

Measurement of intracellular cation concentration Frankia cultures were grown for 12 d at standard growth conditions in three different sets of BAP medium i.e. medium without Ca2+; medium without Mg2+ and medium without K+ and then after the above mentioned media were used for determination of the intracellular Ca2+, Mg2+ and K+ respectively. The intra-cellular cation content (Ca2+, Mg2+ and K+) at different concentrations of NaCl was measured after 5 min of incubation at standard growth conditions with the addition of the respective cation and different concen-trations of NaCl (0, 50, 100, 250, 500, 750 and 1000 mM) in the above medium deprived of such cation. The con-centration of Ca2+, Mg2+ and K+ in the experimental medium was same as used with standard growth con-ditions. Then after cultures were centrifuged at 10,000 rpm for 10 min and the obtained pellets were



treated similarly as for the estimation of intracellular Na+ content. Concentration of Ca2+, Mg2+ and K+ was measured in terms of μmol μg–1 protein by using atomic absorption spectrophotometer (Perkin Elmer model 2380).

Statistical analysis In all the graphs, bars indicate the standard error of the six replicates (n = 6). Results were subjected to two-way ANOVA in order to assess the significance of quantita-tive changes in the experimental parameters because of different NaCl treatments and strains. Growth re-sponses were subjected to three-way ANOVA to ap-praise the significance of change in growth due to dif-ferent NaCl treatments, strains and days. Duncan’s multiple range test was performed as post hoc on pa-rameters subjected to ANOVA (only if the ANOVA was significant). SPSS software (SPSS Inc., version 16.0) was used to perform all the statistical tests.

Results

Effect of salinity on growth Initially, growth (in terms of cell density) was compared among 18 different strains of Frankia isolated from the root nodules of H. salicifolia D. Don under standard (–NaCl) and salt replete (50, 100, 250, 500, 750, 1000 mM) conditions. All the strains showed dimin-

ished growth under salt grown conditions (data not shown). Finally, four frankial strains were selected on the basis of maximum (HsIi10), intermediate (HsIi2 and HsIi9) and minimum (HsIi8) growth (in terms of cell density) under different levels of salinity (Fig. 1) and the reference strain CpI2 was taken to understand the mechanism underlying salt tolerance/salt sensitivity in Frankia strains. At the standard growth conditions (–NaCl) growth was going on increased up to 24th day of incubation whereas the maximum growth was ob-served between 12th and 14th day of incubation at different concentrations of NaCl. Though the growth started from the initial day in the presence of NaCl but remained lesser throughout the entire growth period (up to 30 days) as compared to the control (–NaCl). Among the various NaCl concentrations that were tried, growth was maximum at 50 mM and decreased on further increment in NaCl concentration. Growth was observed up to 250 mM (500 mM for HsIi10) be-yond which there was sharp reduction in growth. At 250 mM NaCl, growth was inhibited by 23.53% and 27.03% as compared to control in HsIi2 and CpI2 re-spectively on 14th day of incubation whereas in HsIi8 and HsIi9 the growth was inhibited by 32.35% and 21.43% respectively as compared to the control on 12th day of incubation (Fig. 1A, B, C, E). In HsIi10, the growth response was slightly changed and the critical concentration for growth was 500 mM NaCl (an inhibi-

Figure 1. Effect of different concentrations of NaCl on the growth of Frankia strains (A) HsIi2, (B) HsIi8, (C) HsIi9, (D) HsIi10 and (E) CpI2.



Table 1. Results of analysis of variance (ANOVA) for repeated measures of NaCl concentrations (Treatments), strains, days and their interactions for growth.

Parameter Treatment Strain Days × Strain

Treatment × Days

Treatment × Days

Strain × Days

Treatment × Strain

Growth 0.00175*** 80.893*** 192.479*** 9.470*** 26.618*** 6.951*** 2.172***

Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant tion of 17.5% as compared to control on 12th day of incubation) (Fig. 1D). Then after the growth declined in all the NaCl concentrations and maximum inhibition was observed at the last day of incubation. At 750 mM NaCl, all the strains showed 45% to 55.56% inhibition in growth at 12th to 14th day of incubation with re-spect to control (–NaCl). Thus, 750 mM concentration of NaCl was considered to be inhibitory for the growth of the frankial strains. 1000 mM concentration of NaCl was found to be lethal and growth was suddenly de-creased from the very beginning and the strains further tried to maintain their survival throughout the ex-perimental phase. Three-way ANOVA revealed signifi-cant effect of treatment, strain, days, combinations of treatment and strains, treatment and days, strains and days and also due to overall combined effect of treat-ment, strains and days (Table 1).

Effect of salinity on membrane integrity Membrane integrity was determined in terms of elec-trolyte leakage (EC) among the five frankial strains. A considerable difference was observed in the electrolyte leakage at different NaCl concentrations. Percentage of electrolyte leakage was maximum in HsIi8 followed by HsIi2, CpI2, HsIi9 and HsIi10 at all the tried concentra-tions of NaCl (Fig. 2). Electrolyte leakage was maximum at 1000 mM (79.95 to 90.14%) followed by 750 mM (60.28 to 82.93%), 500 mM (25.79 to 57.36%) and 250 mM (19.69 to 42.97%) against their control. Two-way ANOVA showed that response of electrolyte leak-age varied significantly due to the effect of treatment (NaCl supplementation), strain and treatment × strain (Table 2).

Figure 2. Effect of NaCl on the electrolyte leakage from different frankial strains exposed to a gradient of NaCl.

Na+ uptake and kinetics The uptake of sodium ions among all the five strains was calculated at different time intervals (1, 2, 3, 5, 10, 15, 30, 45 and 60 min) (data shown only up to 15 min of incubation as supplementary figure). The rate of uptake increased linearly up to 5 min of incubation. After that, linearity deviated and saturation was observ-ed after 15 min of incubation and therefore the uptake rate was calculated at 5 min of incubation (Fig. 3). A biphasic pattern of Na+ uptake (in terms of μmol μg–1 protein min–1) was observed among all the frankial strains whereby the uptake rate increased sharply

Table 2. Results of analysis of variance (ANOVA) for repeated measures of NaCl concentrations (Treatments), strains and their interactions for electrolyte leakage, Na+ uptake, intracellular Na+ concentration, Na+ efflux and intracellular concentrations of Ca2+, K+ and Mg2+.

Parameter Treatment Strain Treatment × Strain

Electrolyte Leakage 0.0002814*** 0.0016*** 86.386*** Na+ Uptake 0.00246*** 191.005*** 17.459*** Intracellular Na+ 0.005532*** 331.139*** 40.984*** Na+ Efflux 0.009888*** 546.379*** 71.513*** Intracellular Ca2+ 3.379** 1.156ns 1.173ns Intracellular K+ 155.412*** 104.893*** 3.031*** Intracellular Mg2+ 88.051*** 19.473*** 1.063ns

Level of significance: *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant



Figure 3. Sodium uptake by different strains of Frankia (A) HsIi2, (B) HsIi8, (C) HsIi9, (D) HsIi10 and (E) CpI2 exposed to a gradient of NaCl concentrations.

and significantly from 50 to 250 mM NaCl in the first phase. Then linearity deviated and uptake rate was almost stabilized and slight increase was observed be-tween 250 and 500 mM. In second phase, a rapid in-crease was again started and linearity continued up to 1000 mM NaCl (lethal concentration for growth). Maxi-mum uptake rate was observed in HsIi8, followed by HsIi2, CpI2, HsIi9 and HsIi10 at all the tried NaCl con-centrations. Similarly, rate of Na+ uptake was also com-pared at 1000 mM NaCl. Na+ uptake was maximum in HsIi8 followed by HsIi2, HsIi9 and CpI2 while the minimum rate was reported in HsIi10. The first phase of Na+ uptake rate followed the Michaelis-Menten kinet-ics up to 500 mM NaCl and substrate dependent sodium uptake was observed. Two-way ANOVA showed that Na+ uptake response varied significantly due to the effect of treatment (NaCl supplementation), strain and treat-ment × strain (Table 2). While analyzing the kinetic behavior of the first phase of Na+ uptake in different frankial strains (Sup-plementary Table), the Michaelis constant (Km) was found to be minimum for HsIi8 (0.182 × 106 μM), almost similar for HsIi2 (0.24 × 106 μM), CpI2 (0.235 × 106 μM) and HsIi9 (0.22 × 106 μM) and maximum for HsIi10 (0.32 × 106 μM). Vmax followed a reciprocal relationship with respect to Km and was maximum for HsIi8 (0.99 μmol μg–1 protein min–1) followed by HsIi2 (0.81 μmol μg–1 protein min–1) and CpI2 (0.74 μmol μg–1 protein min–1). It was almost similar in HsIi9

(0.675 μmol μg–1 protein min–1) and HsIi10 (0.67 μmol μg–1 protein min–1)

Maintenance of cellular Na+ status (intracellular, efflux and remaining Na+) As the uptake of Na+ was increased at different concen-trations of NaCl, internal sodium concentration (in terms of μmol μg–1 protein) also increased in the cells of Frankia and maximum was observed in HsIi8 (4.399 μmol μg–1 protein) followed by HsIi2 (3.66 μmol μg–1 protein), CpI2 (3.4452 μmol μg–1 protein), HsIi9 (3.2506 μmol μg–1 protein) and HsIi10 (2.4517 μmol μg–1 protein) at 1000 mM NaCl (Fig. 4). Two-way ANOVA show-ed that the response of intracellular Na+ content varied significantly due to the effect of treatment (NaCl sup-plementation), strain and treatment × strain (Table 2). The increase in Na+ efflux rate was observed in two phases (Fig. 5). The first phase lasted up to 250 mM NaCl, second phase extended from 500 to 1000 mM NaCl as observed in the case of Na+ uptake of the same strains. There was a very insignificant increase in efflux rate from 250 to 500 mM NaCl. Among the five strains, efflux rate was maximum for HsIi8 (0.883 μmol μg–1 protein min–1) followed by HsIi2 (0.7339 μmol μg–1 pro-tein min–1), CpI2 (0.6888 μg–1 protein min–1), HsIi9 (0.6541 μmol μg–1 protein min–1) and minimum in HsIi10 (0.4943 μmol μg–1 protein min–1) at 1000 mM NaCl. Two-way ANOVA showed that the response of Na+ efflux varied significantly due to the effect of treatment (NaCl supplementation), strain and treatment × strain (Table 2).

Figure 4. Effect of NaCl on intracellular sodium ion content in different strains of Frankia.



Figure 5. Effect of NaCl on the rate of sodium efflux in different frankial strains. Remaining Na+ concentration (intracellular Na+ con-centration after efflux) showed a particular trend where it remained within a narrow range from 50 to 250 mM (50 to 500 mM in HsIi10) for an individual strain and then dropped steeply as the concentration of NaCl reached 1000 mM (Fig. 6).

Intracellular macronutrient status during salinity When the intracellular macronutrients (K+, Mg2+ and Ca2+) were compared at standard growth conditions

Figure 6. Remaining sodium ion content of different Frankia strains exposed to a gradient of NaCl concentrations. (–NaCl) among different strains, Ca2+ was highest in amount (0.61 μmol μg–1 protein) followed by K+ (0.055 μmol μg–1 protein) and Mg2+ (0.026 μmol μg–1 protein) (Fig. 7). Increasing concentration of Na+ in the external medium decreased the intracellular concentra-tion of the macronutrients among all the strains. There was rapid decline in the intracellular macronutrient concentrations at lower supplements of NaCl (up to 100 mM); exception was only in the case of HsIi10 in which a gradual decrease in the intracellular Ca2+ con-

Figure 7. Effect of NaCl on the intracellular macronutrient concentration in Frankia strains (A) HsIi2, (B) HsIi8, (C) HsIi9, (D) HsIi10 and (E) CpI2.



centration was observed. Intracellular K+ content de-clined rapidly at 50 mM NaCl and maximum decline was observed in HsIi8 (68.42%) followed by HsIi2 (50.98%), CpI2 (48.15%), HsIi9 (45.45% ) and HsIi10 (10.71% ). At different concentrations of NaCl, intracel-lular Ca2+ content was always higher than that of K+ whereas intracellular Mg2+ was lower or equal to that of K+. An exception to the above trend was observed in HsIi8, where the intracellular K+ content was always lesser than that of Mg2+ content at all the tried NaCl concentrations. Two-way ANOVA revealed that Intracel-lular Ca2+ concentration varied only due to the signifi-cant effect of treatment, intracellular K+ content varied significantly due to the effect of treatment, strain and treatment×strain and intracellular Mg2+ content varied significantly mainly due to the effect of treatment and species (Table 2).

Discussion

It is well known that salinity imposes ionic stress in which Na+ inclusion is one of the major contributors. Each frankial strain showed a different level of salt tolerance as in other organisms [24, 25]. Considerable growth was observed only up to 500 and 250 mM NaCl respectively in HsIi10 and rest of the strains which proved that 500 and 250 mM NaCl are the critical con-centrations for their respective strains. Differential salt sensitivity might be the result of genotypic diversity towards salinity in frankial strains [26, 27]. Various stresses including salt cause first and major impact on the cell membrane [28] which is observed in the form of leakage of essential solutes from the internal organelles [29–35]. The increment in electrolyte leakage on in-creasing the concentration of NaCl was a measure of the extent of stress on the membrane stability of Frankia strains as reported in other organisms [36, 37]. Lower degree of membrane damage might be one of the probable reasons for the considerable growth upto 250 mM NaCl. In HsIi10, the extent of membrane dam-age was not as severe at 500 mM in comparison to the other strains including reference strain CpI2 at the same concentration validating its better survival against salin-ity. As compared to other strains, highest percentage of electrolyte leakage may be one of the probable reasons for poor growth and salt sensitivity in HsIi8 (Fig. 2). Loss of membrane integrity leads to solute leakage which was evident in the form of increase in electrolyte leakage from lower to higher NaCl concentrations. On provision of NaCl in the frankial medium, a ratio among instant and rapid uptake of Na+, its accumula-

tion and its efflux was established from the very begin-ning to 1000 mM supplement of NaCl in all the five strains. The uptake of Na+ was rapid and increased line-arly up to 5 min which represents rapid saturation of the uptake machinery. This is in accordance with the results reported for the other nitrogen fixing organisms in which a plateau was achieved very rapidly [38, 10]. Probably, elevated level of NaCl establishes an electro-chemical gradient which might be the most probable reason for increased Na+ uptake among frankial strains. Simultaneously, a quick rise in the intracellular status of Na+ among all the strains supports the fact that up-take of Na+ is a passive process mediated by electro-chemically motored carriers [8, 39] and it is stopped when the external and internal electrochemical poten-tial gets equalized, or is maintained at a particular level by parallel occurring Na+ exclusion. A comparison of intake of sodium, its accumulation, efflux and residual sodium ion (after efflux) among all the frankial strains provides a clear picture about the mechanisms which effectively participated in the transportation and regu-lation of sodium i.e. influx and efflux. Differences in the influx or efflux rate of the different strains might be responsible for the varied level of remaining sodium which would be really the required sodium concentra-tion for the sustenance and survival of the respective strains. Results suggested that beyond critical NaCl concentration most of the accumulated Na+ was ex-pelled out on provision of appropriate conditions and the expulsion was maximum at 1000 mM NaCl. It means above the critical level, accumulated sodium is deleterious to frankial physiology and that’s why efflux rate might be increased in the second phase (500 to 1000 mM NaCl) of sodium regulation at higher concen-trations only to flush out maximum sodium from the cells. High Na+ concentration (or Na+: K+ ratio) is in-volved in disruption of several cytoplasmic enzymatic pathways as well as for inducing osmotic effects [40, 41]. As compared to other strains, poorer growth and survival in HsIi8 at different sodium levels might be the result of higher uptake and extrusion of Na+ because it is well known that the extrusion of Na+ demands more cellular energy. Simultaneously, some more energy requiring physiological and biochemical salt adaptive mechanisms (reestablishment of ion concentration and synthesis of osmoprotectants) would also have been operated for overall protection of HsIi8 against salinity. Possibly, there would have been an extreme energy crisis during salt stress conditions. In addition to these; highest percentage of accumulated sodium through fast uptake in HsIi8 might be one of the possible de-



terminants of salt sensitivity because it minimizes the nutrient and metabolite transport along with protein synthesis, energy and lipid metabolism. Higher salinity is the major cause for the disturbed ionic and osmotic homeostasis, reduction in many of the enzymatic ac-tivities and molecular damages [42, 43]. It has been earlier reported that the enzymes (H+-ATPase, H+-PPase, V-ATPase and V-PPase) of the plasma membrane and tonoplast are actively involved in the salt tolerance as well as in nutrient and metabolite transport [44]. Extru-sion of Na+ is also an active mechanism for regulating salt tolerance in cyanobacteria operating via Na+/H+ antiporter [8]. In contrast to this, lowest intracellular Na+ profile in HsIi10 via slowest rate of sodium uptake might be the probable reason for better growth and adaptation of HsIi10 towards salinity. Due to slowest influx and efflux (energy requiring process), there would be spared energy which might be utilized for all the energy requiring salt adaptive machinery to work at full strength. The kinetics of Na+ uptake was deduced by applying Michaelis-Menten relationship to the first phase of the biphasic pathway (from 50 to 500 mM NaCl; Supple-mentary Table). The saturation at 250 mM NaCl indi-cated that all the carriers are fully engaged and there was crisis of carriers for further uptake at 500 mM NaCl [45]. A Michaelis-Menten curve might have been ob-tained on considering NaCl concentrations above 1000 mM NaCl but since growth of Frankia was barely supported at the above mentioned NaCl concentration, consideration of higher concentration of NaCl was not taken into account. High value of Km in HsIi10 is an indication of high demand of substrate for saturation of absorption system and this coupled with low Vmax suggests a system that is bound to restrict entry of Na+

inside the cell. Similarly, low Km and high Vmax in HsIi8 suggests heavy and less restricted inflow of Na+ into the cellular system. Deviation in the linear rate of uptake at 250 mM NaCl (Fig. 3) indicates saturation and further non-availability of carriers for Na+ uptake. Thus, no addi-tional carriers are vacant for surplus Na+ made avail-able at 500 mM NaCl. The biphasic pattern might be a manifestation of two different categories of carriers [46]. One possibility is that a particular class of carriers gets activated only on saturation of the initial ones. The other probability is that at lower concentration of NaCl, certain carriers might be functional while higher con-centration of NaCl might trigger the remaining carri-ers. Similar biphasic pattern of uptake has been ob-served for various substrates in a number of symbiotic organisms [47–52].

Some of the deleterious effects of salt stress are at-tributed to the deficiency of other nutrients [53]. Elec-tric potential might be disturbed in the presence of NaCl that governs the activity of ions at the membrane surface [54]. Changes in the above potential might be the reason for electrolyte leakage resulting in loss of essential solutes. Ca2+ attachment stabilizes the plasma membrane and its exchange with Na+ during salt stress is also known to exacerbate membrane disintegrity [55, 56]. Overall presence of salt might have reduced the surface charge density leading to a diminished cation activity at the interface which ultimately causes an interference with the rates of transport of other ions. Also, as the intracellular content of Na+ increased, there would have been a higher imbalance of charge density in the cell interior. In order to ameliorate the standard ion density of the cell, a higher efflux of certain cations would have occurred. The initial intracellular content of Mg2+ in salt de-plete cells and the drop in its concentration under salt replete conditions was less in comparison to that of Ca2+ and K+, emphasizing the least requirement of Mg2+

during salt toxicity and also proves that Na+ plays mi-nor role (exerts minimal interference) in Mg2+ trans-port. It is well known that an increasing trend in external Na+ concentration always tends to decrease uptake and intracellular concentration of Ca2+ [57]. An initial rapid decrease in the intracellular content of Ca2+ in the pres-ence of NaCl might be due to repulsion of other cations from the cell surface in presence of abundant Na+

which ultimately reduced Ca2+ uptake. Probably, due to high amount of cytosolic Na+, extrusion of Ca2+ would have occurred so as to maintain a constant charge den-sity of the cell interior. Ca2+ is known to have consider-able role in salt tolerance and counteract NaCl effect in several instances [17, 58, 59]. Contrary to its protective capability, the movement of Ca2+ is known to slow down when Na+ is present on the same side of the membrane [12, 60]. Similar decrease in cytosolic Ca2+ content has been reported in plasma membrane vesi-cles and protoplasts of Capsicum annuum, roots of maize and Arabidopsis on being subjected to salinity [61–63]. Reduced K+ content due to Na+ interference made it evident that there is high probability of similar trans-porters being used for Na+ and K+. In salt deplete condi-tions, high cytosolic K+ and low Na+ is maintained by H+-ATPase motored primary transport and channel/co-transporter mediated secondary transport while in salt replete condition, most of the transporters might have been engaged in movement of Na+ which outnumbered K+ [3]. Similarity in ionic radius and ion hydration ener-



gies of Na+ and K+ provides an overwhelming advantage to Na+ for an easy mode of transportation. In wheat, TaHKT1 is known to act as a Na+/K+ symporter at mi-cromolar and as Na+ uniporter at milimolar Na+ con-centration [64, 65]. At low concentrations of NaCl, a single locus KNA1 was found responsible for differential uptake of Na+ and K+ in plants [66, 67, 13]. Fungal K+ transporters known as TRK are also capable of adjusting Na+/K+ selectivity according to the cellular requirement and external environment [68]. HKT1 transporter in Xenopus oocytes [65] has two binding sites, one for Na+ alone and the other for K+ as well as Na+. At high NaCl concentration, all the binding sites of HKT1 become saturated with Na+ and it acts solely as a Na+ trans-porter [39]. Such selective favor of Na+ would obviously lead to deficit of intracellular K+. Several symporters and co-transporters have been reported in plant system such as KUP or HAK1- of Arabidopsis and barley that show homology in microbial systems [69, 70]. There-fore, the mechanism of inter-relation of Na+ and K+ observed in plants can be extrapolated to frankial sys-tem. Plasma membrane depolarization because of in-ward movement of Na+ might have activated the depo-larization activated channels (DAPCs) and/or non-selec-tive cation channels (NSCCs) which result in an elevated K+ efflux [55]. During salt stress, disruption of NHX1 gene causes heavy loss to intracellular K+ content [71]. Thus, the disruption of K+ homeostasis can be attrib-uted to reduced uptake, increased efflux and reduced cytosolic pools. K+ plays important role in many meta-bolic functions such as protein synthesis – for binding tRNA to ribosomes, starch synthesis, activation of more than 50 enzymes and salt toxicity [2, 72, 73]. In spite of various similarities and univalency, Na+ cannot substi-tute K+ in its varied functions [74] and becomes a major cause of nutrient deficiency. Thus, with an increasing membrane destabilization, increased uptake and intra-cellular content of Na+ during salt stress, difference in transport regulation and distribution of vital solutes among the strains decides the success of salt tolerance machinery. As indicated by two-way ANOVA, response of all the experimental parameters (except that of in-tracellular Ca2+ in response to strain and treatment × strain and intracellular Mg2+ in response to treat-ment × strain) varied significantly both due to strains’ individual performance and treatment effect (Tables 1, 2). Both the factors i.e. treatment and strain in combi-nation also affected the response of different traits significantly. Conclusively, strain HsIi8 showed maxi-mum electrolyte leakage and membrane disintegration along with highest uptake and remaining Na+ content, suffered highest deficit of macronutrients and was the

most salt sensitive strain. On the other hand, HsIi10 showed better co-regulation of various factors and managed to tolerate salt stress up to considerable ex-tent. Therefore, HsIi10 can be further utilized for test-ing other parameters which might be useful in exploit-ing its utility as a potential biofertilizer in the saline soils.

Acknowledgements

We are thankful to DBT, DST and CSIR, New Delhi for their financial support. The Head, Department of Bot-any, Banaras Hindu University, Varanasi, India is grate-fully acknowledged for providing laboratory facilities.

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sodium transport and mechanism(s) of sodium tolerance in frankia strains

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