Physiological mechanism of enhancing salinity tolerance of Gleditsia sinensis Lam. by arbuscular mycorrhizal fungi

The protective effects of arbuscular mycorrhizal fungi (AMF) on salt-stressed crop plants had been well studied. However, the physiological mechanism of AMF in mitigating adverse impact caused by salinity stress in different tissues of woody plants is not clear. Gleditsia sinensis Lam. is a valuable tree species with various phamaceutical uses; however, high soil NaCl concentration limits its growth in saline soil including coastal areas. This study aimed to investigate the effects of AMF on G. sinensis salinity tolerance and reveal its underlying physiological mechanism. Methods A greenhouse experiment was performed. G. sinensis seedlings with and without AMF inoculation were subjected to four salinity levels (0, 50, 100, and 150 mM NaCl). After 2 months, the seedlings were harvested and analyzed for growth and biochemical parameters. High AMF colonization rates (over 95%) and high mycorrhizal dependency (over 75%) were observed across all NaCl levels, and AMF-inoculated plants presented significantly higher aboveground and below ground growth than non-inoculated plants. AMF effectively enhanced the salinity tolerance of G. sinensis seedlings by enhancing leaf gas exchanges inducing higher leaf net photosynthetic rates; improving peroxidase, catalase, and superoxide dismutase activities resulting in higher membrane stability indexes and lower malondialdehyde contents in leaves and roots; increasing P uptake and P/N ratio to mitigate P-limited biomass products; selectively absorbing less Na + and more Ca 2+ in their tissues to alleviate ion toxicity and maintain more favorable ion balances (e.g., K + /Na + ) in their tissues. The results suggested the feasibility of using AMF to improve salinity tolerance as well as afforestation and rehabilitation of G. sinensis in coastal areas.


Introduction
Salinization of soil is a severe and common environmental problem, particularly in arid and semiarid regions or low-lying coastal areas around the world (Porcel et al. 2012). One of the significant natural factors contributing to salinization of soils is oceanic salt deposition. Globally, salinization of soil, especially in the coastal areas, is increasing owing to the rising sea level and climate change, significantly affecting the multifunction, conservation, and rehabilitation of coastal ecosystems (Wilson 1999). Salinity stress restricts plant growth and development by inducing osmotic stress, oxidative stress, and ion toxicity, ultimately resulting in biomass production losses (Evelin et al. 2009). In the short term, accumulation of salt in the root zone causes decrease in osmotic potential, which leads to a decrease in water and nutrient availability. In the long-term, excessive uptake of Na + and Cl − cause nutrient imbalances and ion toxicity, which disrupt cell organelles, plasma membrane, Cl − , creating a dilution effect on the toxicity of these ions to plants (Augé 2001). Moreover, AMF regulate the physiological, biochemical, and molecular processes of plants, such as photosynthesis pathway, ion balance, antioxidant system, osmoregulators, sodium compartmentalization, hormones, and aquaporins, ultimately helping plants to better cope with salinity stress (Porcel et al. 2012).
The positive effects and underlying mechanisms of AMF involving in crop plant salt tolerance had been widely studied (Sharifi et al. 2007; Daei et al. 2009 Especially, Gleditsia sinensis Lam. is a leguminous plant whose rhizobia symbiosis (nitrogen fixation) is more favorable for AMF functioning, but no researches had been made on the influences of AMF in salt tolerance of G. sinensis currently. Moreover, G. sinensis is a previous economically important tree species with multiple phamaceutical values (Zhang et al. 2016). It is widely distributed in China and well adapted to many soil types. However, G. sinensis has very low salinity tolerance, withstanding only 0.3% sanility under greenhouse condition (Lei et al. 2008). As a result, its suitability as an afforestation and rehabilitation tree species in coastal areas is severely limited.
To investigate the effects of AMF on improving salinity tolerance of woody plant species and reveal the underlying physiological mechanism, we performed a greenhouse experiment with G. sinensis.
The membrane stability index, malondialdehyde contents, growth parameters including height growth, diameter growth, leaf area, dry biomass, and root morphology were measured to ascertain whether AMF could effectively enhance the salinity tolerance of G. sinensis seedlings. Besides, the chlorophyll contents, photosynthetic parameters, osmoregulators, antioxidant system, N, P contents and ion balance in different tissues were determined to analyze the physiological mechanism of AMF in alleviating salt-induced adverse effects on G. sinensis seedlings.

Plant material and soil
The seeds of G. sinensis were provided by Jiangsu forestry station. All the seeds were soaked in concentrated H 2 SO 4 for 10 min until the color of the seeds turned crimson, and then washed with sterile distilled water until the pH of residual water on the surface of the seeds turned to about 7.0.
After that, the seeds were soaked in warm water for 2 days. The inflated seeds were embedded in wet yellow sand (which was previously sterilized in an autoclave for 2 h at 0.14 MPa and 121 °C) and incubated in plant incubator under dark condition at 25 °C.
Inoculation treatment F. mosseae (isolate number: BGC G201C) was obtained from the Beijing Academy of Agriculture and Forestry Science, China. The inoculum was bulked in an open-pot sterilized yellow sand culture together with maize and clover as trap plants. After 3 months, the aboveground was cleared, and the roots were chopped into small pieces and mixed with the sand of the culture pot. This sand-based inoculum, consisting of yellow sand, infected root fragments, and mycorrhizal spores (> 7 g − 1 ), were collected and used in this study. The uniform seedlings (5 cm in length) were transported to the pots (1 seedling per pot). Before transportation, the pots were soaked in 0.3% KMnO 4 solution for 3 h and washed with tap water. About 2.5 kg of the autoclaved nursery substrates were dispensed into each pot and 80 g of sand-based inoculum were added 5 cm below the surface of the nursery substrates.
The non-inoculated control pots contained the fungal inoculums filtration and the same dosage of sterilized inoculum to provide the same microbial community (except for AMF) with inoculated treatment.

Growth conditions
The seedlings were grown in the glasshouse under the following conditions: 18 °C night/30 °C day temperature, 50-80% relative humidity, and 14 h/10 h diurnal light/dark cycles with a photosynthetic photon flux density of about 700-1,000 µmol m − 2 ·s − 1 . Water was supplied adequately during the entire period of the experiment to avoid any drought effects, and modified Hoagland's nutrient solution containing only 25% P concentration (300 mL per pot every time) was irrigated every month.
The seedlings were cultivated for about 4 months prior to salinization to allow adequate plant growth and symbiotic establishment. Subsequently, the four groups of non-mycorrhizal control and mycorrhizal treatments were respectively gradually supplemented with aqueous NaCl solution (300 mL per pot) at the concentrations of 0, 50, 100, and 150 mM NaCl every week for 2 months. In order to avoid salt shock, all three salt treatments (50, 100, and 150 mM NaCl treatments) were treated with 50 mM NaCl for the first week; salt treatment (50 mM NaCl treatment) were treated with 50 mM NaCl, and salt treatments (100 and 150 mM NaCl treatments) were treated with 100 mM NaCl for the second week; salt treatments (50, 100, and 150 mM NaCl treatments) were treated with 50, 100, and 150 mM NaCl, respectively, from week 3 onwards, the seedlings were harvested and analyzed for growth and biochemical parameters.

Plant harvest and chemical analyses
Before and after salt stress, the seedling height was measured using a steel ruler, and basal diameter was measured using calipers. After harvesting, the plants were rinsed with tap water, and separated into leaf, stem, and root. The leaf area and root system characteristics (root length, root surface area, and root tip number) were determined using a LA2400 Scanner (Expression 12000XL, EPSON, Long Beach, CA, USA). The dry weights of plant tissues (leaf, stem, and root) were recorded after drying the plant tissues in an oven at 70 °C to a constant weight. The mycorrhizal dependency was calculated using the formula (Wang et al. 2018): mycorrhizal dependency (%) = (dry weight biomass of inoculated seedlings -mean of dry weight biomass of non-inoculated seedlings) / dry weight biomass of inoculated seedlings × 100%.
The dried plant tissues were ground separately, sieved through a 0.5-mm sieve. 50 mg of each sample was weighed to determine the concentrations of N using an elemental analyzer (Vario MACRO cube, Elementar Trading Shanghai, Shanghai, China). 0.2 g of each sample was digested in 10 mL of acid mixture (HClO 4 :HNO 3, 1:5), and diluted with double-distilled water. The concentrations of P were ascertained spectrophotometrically using ammonium molybdate blue method, the concentrations of K + , Ca 2+ , Mg 2+ , and Na + were ascertained with an atomic absorption spectrophotometer (AA900T, Perkin Elmer, Norwalk, CA, USA) (Allen 1989), and the K + /Na + , Ca 2+ /Na + , and Mg 2+ /Na + ratios in the tissues were calculated using K + , Ca 2+ , Mg 2+ , and Na + data.

Estimation of root mycorrhizal colonization
For the quantification of mycorrhizal colonization, the washed fine roots were cut into 1-cm-long segments. The root segments were clarified with 10% (w/v) KOH at 90 °C for 1 h, stained with basic

Determination of chlorophyll contents and photosynthetic parameters
The chlorophyll contents (Chl) in leaves were determined according to Lichtenthaler (1987) with minor modification. Fresh mature leaves (0.1 g) of each plant were cut into small pieces and completely submerged in acetone solution (0.5 mL of pure acetone and 15 mL of 80% acetone). The samples were incubated at 35 °C under dark condition. After the leaf turned white in color, the samples were diluted with 80% acetone to 25 mL. The absorbance of the extracts was determined using an ultraviolet spectrophotometer (UV 2700, Shimadzu) at 663, 645, and 470 nm, respectively.

Measurement of relative water content and membrane stability
The leaf relative water content (RWC) was measured according to the previous method described by Wang  was heated in a water bath at 95 °C for 30 min. Then, the boiled reaction mixture was immediately cooled in an ice bath and centrifuged at 3,000 rpm for 10 min. The absorbance of the supernatant was measured at 532, 600, and 450 nm, respectively. The concentration of MDA was calculated by using the formula given by Hodges et al. (1999). The concentration of proline (Pro) generated was ascertained via ninhydrin reaction as described by Bates et al. (1973). The leaves and roots were cut into small pieces, completely submerged in 3% (w/v) sulfosalicylic acid solution, and heated in a water bath at 100 °C for 15 min. Then, 2 mL of the extract were added to 2 mL of glacial acetic acid and 2 mL of 2.5% ninhydrin solution, and heated in a water bath at 100 °C for 15 min. Subsequently, the reaction mixture was cooled down and 5 mL of methylbenzene were added to it and placed under dark condition. After the mixture completely separated into different layers, the absorbance of methylbenzene layer was measured at 520 nm. Superoxide dismutase (SOD) activity was assayed using nitro blue tetrazolium (NBT) reduction test by measuring the ability of SOD to inhibit photochemical reduction of NBT (Giannopolitis and Ries 1977).

Soluble proteins and antioxidant enzymes assay
A 50% inhibition of NBT reduction was considered as one unit of SOD activity at 560 nm. Peroxidase (POD) activity was assayed using guaiacol test and spectrophotometrically determined at 470 nm

Statistical analysis
The data obtained were analyzed using SPSS19.0 (SPSS Inc., Chicago, IL, USA). Two-way ANOVA was used to determine the effects of NaCl levels, AMF inoculation, and their interactions. Multiple comparisons of means were performed by Tukey's test (P ≤ 0.05). All the figures were derived using Origin 8.5 (Origin Lab, Northampton, USA), and all data are presented as mean ± standard deviation of at least three plants.

Mycorrhizal colonization, dependency and plant growth
No AMF structure was found in the roots of non-inoculated seedlings at all NaCl levels, whereas arbuscules, vesicles, and hyphae were observed in AMF-inoculated seedlings. The percentages of AMF colonization were very high (above 95%) across all NaCl levels (Fig. 1a). Mycorrhizal dependency was significantly influenced by salinity (Table S1), the values of mycorrhizal dependency were also very high (above 75%) across all NaCl levels, and significantly increased under high salinity conditions (100 and 150 mM NaCl) ( Fig. 1b and Table S1).
Salinity and AMF inoculation had significant interactions on all growth parameters (height growth, basal diameter growth, leaf area, root length, root surface, root tip number, leaf biomass, stem biomass, and root biomass), except for leaf area and stem biomass (Table S1). In general, the growth parameters decreased with the increasing NaCl levels, and AMF inoculation significantly and positively influenced plant growth parameters, except leaf area, across all NaCl levels (Table 1 and Fig. 2).

Leaf chlorophyll contents and photosynthetic parameters
Salinity significantly decreased the chlorophyll contents (Table S1), with the reduced values reaching a significant level at 150 mM NaCl (Table S2). In contrast, AMF inoculation enhanced the photosynthesis pigments under salinity condition, presented significant increase at 150 mM NaCl, when compared with those in non-inoculated plants. Photosynthetic parameters (P n , G s , T r and Ls) significantly decreased by salinity stress, among the photosynthetic parameters, P n , G s and T r were significantly enhanced by AMF under salinity conditions (Table S2).
Relative water contents, membrane stability and lipid peroxidation Salinity significantly decreased the leaf RWC at 150 mM NaCl (Fig. 3a), whereas AMF inoculation had no significant positive effect on leaf RWC (Table S1 and Fig. 3a). Salinity had significant effects on leaf MSI and MDA contents in leaves and roots (Table S1). Salinity decreased the leaf MSI, with the reduced value reaching a significant level at 100 and 150 mM NaCl (Fig. 3b). However, AMF inoculation improved leaf MSI, especially at high NaCl levels, with increased values reaching 10.33% (P < 0.05) and 9.49% at 100 and 150 mM NaCl, respectively. The MDA contents in leaves and roots increased as the NaCl levels increased, and were significantly higher at high NaCl levels (100 and 150 mM NaCl), when compared with no-salinity treatments ( Fig. 3c and d). AMF inoculation decreased the MDA contents across all the NaCl levels, with decreased values reaching 11.69%, 16.00%, 18.44% (P < 0.05), and 28.44% in leaves and 12.83%, 16.15% (P < 0.05), 12.97% (P < 0.05), and 23.48% in roots at 0, 50, 100, and 150 mM NaCl, respectively, when compared with those in non-inoculated seedlings.
Proline and soluble protein Salinity, AMF inoculation, and their interaction had significant effects on the Pro content in leaves, whereas only salinity significantly affected the Pro content in roots (Table S1). The Pro content in the leaves and roots of non-inoculated plants increased with the increasing NaCl levels, reaching significant values in leaves and roots at 100 and 150 mM NaCl, respectively ( Table 2). In contrast, under salinity condition, AMF inoculation significantly reduced the Pro content in leaves, but produced insignificant increase in Pro content in roots (Table 2). Furthermore, salinity significantly increased the SP content in leaves (Table S1). However, AMF inoculation decreased the SP content in leaves and increased it in roots, especially at 150 mM NaCl (P < 0.05), when compared with non-inoculated plants (Table 2).

Antioxidant enzymes activities
Salinity significantly affected the antioxidant enzymes (POD, SOD, CAT, and APX) activities, whereas AMF inoculation only significantly affected POD, SOD, and CAT, and their interaction showed significant effects only on POD, SOD, and APX in roots (Table S1). The activities of POD and CAT reached the highest values at 100 mM NaCl, while the highest SOD activity was noted in the leaves, but not in roots ( Table 2). The APX activities in leaves and roots increased with the increase in NaCl levels in non-inoculated plants, whereas such trend was not observed in AMF-inoculated plants. AMF inoculation significantly enhanced the activities of POD in leaves and roots under salinity conditions. AMF inoculation enhanced the activities of CAT, the increased values were 101.51% (P < 0.05), 124.36% (P < 0.05), 86.48%, and 88.38% (P < 0.05) in leaves and 82.08% (P < 0.05), 67.39% (P < 0.05), 15.32%, and 87.96% in roots at 0, 50, 100, and 150 mM NaCl, respectively. AMF inoculation enhanced the activities of SOD mainly in roots, and the enhanced values reached significant levels at 0, 50, 100 mM NaCl. The activities of APX were not significantly enhanced by AMF both in leaves and roots. N, P concentration and N/P ratio Salinity, AMF inoculation, and their interaction had significant effects on N, P concentrations and N/P ratios of plants (Table S1). While AMF inoculation did not increase the N concentrations in the tissues of plants, it significantly enhanced the P concentrations in stems and roots at 100 and 150 mM NaCl, when compared with those in non-inoculated plants (Fig. 4a-f). AMF decreased the N/P ratios in the tissues of plants under salinity condition, the decreased values of stems and roots reached significant levels across all NaCl levels ( Fig. 4g-i).

Ion concentration and ion balance
Salinity, AMF inoculation, and their interaction had significant effects on the Na + and Ca 2+ content in the three plant tissues (leaf, stem, and root), K + content in leaves, and Mg 2+ content in stems (Table   S1). While salinity significantly enhanced the concentrations of Na + , AMF inoculation significantly decreased the concentrations of Na + in the three tissues, when compared with those in noninoculated plants (Fig. 5a-c). The concentrations of K + were not significantly influenced by AMF inoculation across all NaCl levels except for 0 mM in leaves (Fig. 5d-f). AMF inoculation increased the concentrations of Ca 2+ , especially at high NaCl levels (100 and 150 mM), and the increase was significantly higher in roots, when compared with that in non-inoculated plants (Fig. 5g-i). However, the concentrations of Mg 2+ were lower, especially in stem, following AMF inoculation, when compared with those in non-inoculated plants (Fig. 5k).
Besides, AMF inoculation also had positive effects on Ca 2+ /Mg 2+ ratios, especially in stems and roots.

Effects of AMF inoculation on plant growth and root morphology under salinity stress
The results of the present study showed that the AMF, F. mosseae, had more than 95% colonization rate on the roots of G. sinensis across all NaCl levels (Fig. 1a), confirming previous reports indicating that F. mosseae had high tolerance to various stress, including salinity stress, and produced positive branched roots and few root hairs generally present higher mycorrhizal dependency (Yang et al. 2015). In the present study, G. sinensis seedlings with coarse root architecture were found to grow very slowly without AMF colonization, but grew much faster in the presence of AMF. Furthermore, AMF-inoculated seedlings showed considerably higher height growth, diameter growth, and biomass accumulation (Table 1 and Fig. 2), indicating that the growth of G. sinensis highly depended on AMF, the values of mycorrhizal dependency (above 75%) confirmed it (Fig. 1b) (Table S2)

Effects of AMF inoculation on osmotic adjustment under salinity stress
There are some physiological mechanisms related to the protective effects of AMF on plants under salt stress condition. First, the present study showed that AMF-inoculated G. sinensis seedlings exhibited higher Pro content in the roots, but significant lower Pro content in leaves (Table 2), which is consistent with that noted in mycorrhizal soybean (Sharifi et al. 2007). Similar results of higher accumulation of Pro in AMF-inoculated plants have also been reported in previous studies (Evelin et al. 2013;Talaat and Shawky 2014). In particular, higher accumulation of Pro in roots has been found to be beneficial for maintaining osmotic balance between water-absorbing root cells and external media (Evelin et al. 2009), whereas low accumulation of Pro in the leaves of AMF-inoculated plants might suggest less injury because Pro is also considered as an indicator of salt-induced damage (Evelin et al. 2013). The increase in the Pro content in roots could be attributed to the reduction in oxidation of Pro to glutamate or induction of Pro biosynthesis enzymes (Stewart 1981). Besides, higher SP concentration especially at 150 mM NaCl was also noted in the roots of AMF-inoculated plants ( Table 2) showed increased antioxidant enzymes activities to resist oxidative stress at a certain NaCl level (< 100 mM). However, these higher activities were not adequate to scavenge ROS, especially when the NaCl level reached 100 mM; hence, the MDA content in the leaves of plants at 100 mM NaCl was significantly higher than that in plants under no salinity treatment (Fig. 3c) In the present study, AMF inoculation mainly enhanced the P concentration, but not the N concentration of plants at high NaCl level. In addition, lower N/P ratios in the tissues especially in stems and roots of mycorrhizal plants (Fig. 4g-i) provided support for that the enhancement of P uptake as one of the underlying mechanism by AMF which alleviate salt damage to plants. N/P ratio in the shoot biomass could be used to evaluate whether N or P is the limiting factor for plant biomass product, generally N/P ratios < 10 and > 20 correspond to N-and Plimited biomass production (Güsewell 2004). The N/P ratios in leaves and stems of no-inoculation plants were > 20 under salinity conditions indicated that G. sinensis was P-limited biomass product when exposed to salinity, the lower N/P ratios in mycorrhizal plants clearly showed that AMF inoculation could effectively allviate P-limited biomass product caused by salinity stress.

Effects of AMF inoculation on ion contents and ion balances under salinity stress
High Na + concentration in soil has been reported to inhibit the uptake of other nutrients such as K + , Ca 2+ , Mg 2+ , etc., resulting in nutrient imbalance and thus plant growth restriction (Parida and Das 2005). K + plays a key role in plant metabolism, including stomatal movement, protein synthesis, and enzymes activation (Khalil et al. 2011). In the present study, the K + content significantly decreased when the Na + content increased (Fig. 5a-f), because Na + ions compete with K + ions for binding sites essential for various cellular activities. It has been revealed that mycorrhizal colonization can enhance K + absorption under salinity condition (Giri et al. 2007;Evelin et al. 2012). The results of the present study showed no obvious difference in the K + content between AMF-inoculated and non-inoculated plants, but indicated obviously higher K + /Na + ratios in the tissues of mycorrhizal plants at all NaCl levels (Table S3), which can be attributed to the lower Na + content, when compared with that in nonmycorrhizal plants.
The lower levels of Na + in AMF-inocualted plants have also been reported in many previous studies (Evelin et al. 2012;Lu et al. 2014;Pollastri et al. 2018). However, Allen and Cunningham (1983) indicated that AMF can occassionally enhance Na + uptake, suggesting that AMF could induce a buffering effect on the uptake of Na + causing higher Na + concentration in mycorrhizal plants at low salinity and lower Na + concentration at higher salinity. The strong enhancement of plant growth following AMF inoculation can also contribute to the decrease in Na + concentration in the tissues   Chancellors' postdoc fellowship.

Availability of data and materials
All data generated or analyzed during this study are in this article (and its supplementary information files) or are available from the corresponding author on reasonable request. GW and BZ revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate
Not applicable.

Competing interests
The authors declare that they have no competing interests. NM represents the groups without F. mosseae inoculation; AM represents the group with F. mosseae inoculation. Different capital letters indicate significant differences (P < 0.05) among inoculation treatments (NM and AM) within the same NaCl level. Different lowercase letters indicate significant differences (P < 0.05) among NaCl levels within the same inoculation treatment.  Figure 2 Effects of F. mosseae on biomass of G. sinensis seedling at different NaCl levels. NM represents the groups without F. mosseae inoculation; AM represents the group with F. mosseae inoculation. In the same tissues, different capital letters indicate significant differences (P < 0.05) among inoculation treatments (NM and AM) within the same NaCl level, different lowercase letters indicate significant differences (P < 0.05) among NaCl levels within the same inoculation treatment.  Effects of F. mosseae on N, P concentrations, and N/P ratios of G. sinensis seedling at different NaCl levels. (a, b c) N concentration in leaf, stem, and root, (d, e, f) P concentration in leaf, stem, and root, (g, h, i) N/P ratio in leaf, stem, and root; NM represents the groups without F. mosseae inoculation; AM represents the group with F. mosseae inoculation.
Different capital letters indicate significant differences (P < 0.05) among inoculation treatments (NM and AM) within the same NaCl level. Different lowercase letters indicate significant differences (P < 0.05) among NaCl levels within the same inoculation treatment.

Figure 5
Effects of F. mosseae on the ion concentrations of G. sinensis seedling at different NaCl levels. (a, b, c) Na+ concentration in leaf, stem, and root, (d, e, f) K+ concentration in leaf, stem, and root, (g, h, i) Ca2+ concentration in leaf, stem, and root, (j, k, l) Mg2+ concentration in leaf, stem, and root; NM represents the groups without F. mosseae inoculation; AM represents the group with F. mosseae inoculation. Different capital letters indicate significant differences (P < 0.05) among inoculation treatments (NM and AM) within the same NaCl level. Different lowercase letters indicate significant differences (P < 0.05) among NaCl levels within the same inoculation treatment.

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