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Journal of soil science and plant nutrition

versión On-line ISSN 0718-9516

J. Soil Sci. Plant Nutr. vol.15 no.4 Temuco dic. 2015  Epub 18-Oct-2015

http://dx.doi.org/10.4067/S0718-95162015005000070 

 

Crucial variations in growth and ion homeostasis of Glycine gracilis seedlings under two types of salt stresses

  

L. Shi1*, S. Ma1, Y. Fang1, J. Xu1

1School of Life Sciences, Northeast Normal University, Changchun, 130024, China. *Corresponding author: lianxuanshi@nenu.edu.cn

 


Abstract

Based on Glycine gracilis growth and ion homeostasis testing, neutral salt (NS)and alkaline salt (AS) stress were characterized and the responses of G. gracilis were investigated.The injurious effects of AS on G. gracilis were obviously stronger than those of NS.The effects of both stresses on the Na+ content and Na+/K+ ratio were similar at low concentrations, but as the stress increased, the effects of a greater Na+ content and Na+/K+ratio increased slowly under NS conditions, but sharply under AS. The roots of G. gracilis accumulated NO3and H2PO4, while the stems and leaves accumulated C2O42− and H2PO4to maintain thein tracellular ion balance. The dominant intracellular anions in the stipes were NO3- and C2O42- under control conditions, and NO3- and H2PO4- under salt stress. With the increasing AS, the Cl, NO3 and H2PO4 concentrations decreased, and G.gracilis might have increased SO42− and C2O42−levels to compensate for the shortage of inorganic anions. Under NS, the NO3 and C2O42− concentrations decreased, and G. gracilis might have increased Cl, H2PO4 and SO42−levels to compensate for the shortage of inorganic anions. G. gracilis seedling showed a special nutritional metabolism and some growth adaptability under salt stress.

Keywords: Glycinegracilis, growth, ion homeostasis, saline stress, pH stress


1. Introduction

Land salinization, as a widespread environmental problem, is an important factor limiting agricultural productivity (Läuchli and Lüttge, 2002; Mekawy et al., 2015). Soil salinity and alkalinity seriously affect ~932 million hectares of land globally (Rao et al., 2008). In nort heastern China, alkalinized grassland covers more than 70% of the total land area. This alkalinized grassland area, where only a few alkali-tolerant halophytes can survive, is still expanding (Huang, J.C. et al, 2013). Along with over-exploitation of the earth, population growth and global climate changes will affect land usage. Therefore, the study of salt damage in plants is of growing importance.

Natural salt-alkalinized soils are very complicated, with Na+, Ca2+, Mg2+, K+, Cl, SO42−, HCO3, CO32−, and NO3 as the main ions (Läuchli and Lüttge, 2002). NaCl, Na2SO4, NaHCO3, and Na2CO3 are the main harmful salts in many inland areas, such as in northeastern China, where the soil became alkaline as a result of the hydrolysis of two carbonates (NaHCO3 and Na2CO3) (Ge and Li, 1990; Zubair et al., 2012).Two neutral salts (NaCl and Na2SO4) are the main salt components in saline soil, while two alkaline salts (NaHCO3 and Na2CO3) are the main salt components in alkali soil. In previous reports, we suggested that salt stress be defined as the stress caused by neutral salts (NS), and alkali stress as the stress caused by alkaline salts (AS) (Shi and Sheng, 2005). The existence of alkali stress has been demonstrated clearly by a number of reports, which have shown it to be more severe than salt stress in various plant species (Campbell and Nishio, 2000; Hartung et al., 2002). However, to date, research on salt stress has emphasized NaCl as the main contributing factor (Munns and Tester, 2008; Liu et al., 2013) and little attention has been paid to alkali stress (Wang et al., 2007; Yang et al., 2008; Gao et al., 2008). Even so, there are some reports on high-pH calcareous soils (Brand et al. 2002; Nuttall et al. 2003), alkaline soil (Hartung et al., 2002), AS (El-Samad and Shaddad, 1996; Campbell and Nishio, 2000; Yang et al., 2007) and salt-alkaline mixed stress (Shi and Sheng, 2005). These reports demonstrate the existence of AS stress. Therefore, the problem of alkali stress should be recognized and investigated as thoroughly as salt stress (Wang et al., 2012).

Soil salinization and alkalization usually occur together. Stress due to soil salinity generally involves osmotic stress and ion-induced injury (Munns and Tester, 2008). Comparisons of alkali and salt stress could reveal an additional effect of alkali stress due to its high pH. A high-pH environment surrounding the roots can cause metal ions and phosphorus to precipitate (Shi and Wang, 2005; Zhang et al., 2014). With the loss of the normal physiological root functions and destruction of the root cell structure (Li et al., 2009), absorption of inorganic anions, such as Na+, K+, Cl, NO3 and H2PO4, would be greatly affected, and thus disrupt the ionic balance (Yang et al., 2007, 2008; Chen et al., 2009). Thus, plants in alkaline soil must cope with both physiological drought and ion toxicity, and also maintain the intracellular ionic balance (Wang et al., 2011). A systematic analysis of ion contents and ratios in different plant organs are important parameters for plant growth assessment, and approaches to the study of the stress physiology. In the post-genome era, ionome research has developed into ionomics and become an important omics study, along with proteomics and metabolomics.

G. gracilis, commonly known as semi-wild soybean belongs to the most important legume genus. Glycine are generally divided into the wild (Glycine soja), semi-wild (G. gracilis) and cultivated soybeans (Glycine max), representing three kinds of genetic relationships in evolution (Wu et al., 2001). There are many studies on the physiological responses to salt stress in wild and cultivated soybean, but seldom reports in the semi-wild soybean (Wu et al., 2014). Having the high yield of the cultivated soybean and the high resistance of the wild soybean, the semi-wild soybean is the transition type. Studies on the physiological characteristics of stress in the semi-wild soybean reveal how these two dominant characteristics can be embodied in the same species. In this study, G. gracili seedlings were treated with different concentrations and types of salt stress. We compared the effects of NS and AS on the growth and ion balance of G. gracilis seedlings to elucidate the mechanisms of NS and AS damage to plants, and the physiological adaptive mechanism of plants to NS and AS. Consequently, by the study of ionomics, we provide a theoretical basis for the application in semi-wild soybean. Additionally, our data could provide a reference for the study of soybean evolution.

2. Materials and Methods

2.1. Plant materials

Seeds of G. gracili, provided by Jilin Academy of Agriculture Science, were sown in 25-cm-diameter plastic pots containing washed sand. Two seedlings in each pot were sufficiently watered with Hoagland,s nutrient solution daily. All pots were placed outdoors and sheltered from rain. During this experiment, the humidity was 60% and temperatures were 24–28 °C in the daytime and 17–20 °C at night.

2.2. Design of simulated salt conditions and stress treatments

Two NS were mixed in a 1:1 molar ratio (NaCl: Na2SO4) and applied to the NS stress group. For the AS stress group, two AS were mixed in a 1:1 molar ratio (NaHCO3: Na2CO3). Within each group, the total Na+ concentrations were applied at 30, 60, 90 and 120 mmol·L-1 and CDM-II electrical conductivity detector, mobile phase: Na2CO3/NaHCO4 = 1.7/1.8 mmol·L-1). The different salt stress groups were labeled at N1-N4 and A1-A4, respectively. The stress treatment fluid was made in Hoagland,s nutrient solution. In the NS stress and AS stress groups, the pH levels were 6.74–6.75 and 9.50–9.88, respectively.

The stress treatments were applied when the seedlings were six weeks old. Thirty pots of uniformly growing seedlings were chosen and randomly divided into ten sets (three pots per set). One set was used as an untreated control, and another one was used for the growth index determination at the beginning of the treatments. The remaining eight sets were watered thoroughly every day at 17:00–18:00 with nutrient solution containing the appropriate salts for the stress treatment. Control plants were maintained by watering with nutrient solution. The entire duration of treatment was 5 days.

2.3. Measurement of physiological indices

Plants were harvested in the evening after the final treatment, washed by tap water and then by distilled water. The growth indices, including shoot height and root length, were measured. Then the roots, stems, leaves and stipes were separately oven-dried at 100 °C for 10 min and vacuum-dried at 75 °C to a constant weight, after which, the dry weights were recorded.

The relative growth rate (RGR) was determined according to Kingsbury et al. (1984).Dry samples of plant material (100 mg) were treated with 10 mL of deionized water at 100 °C for 1 h, and the resulting extract was used to determine the contents of free inorganic ions. Cl, SO42−, NO3, H2PO4 and oxalic acid concentrations in the tissue sap were determined using ion chromatography (DX-300 ion chromatographic system, AS4A-SC chromatographic column, CDM-II electrical conductivity detector, mobile phase: Na2CO3/NaHCO3 = 1.7/1.8 mM; DIONEX, Sunnyvale, CA, USA). An atomic absorption spectrophotometer (TAS-990, Purkinje General, Beijing) was used to determine the concentrations of Na, K, Ca, Mg, P, Fe, Mn, B and Mo atoms. ST and SA were determined according to Suomin Wang (2004) as follows:

ST = (Na+root/K+root)/(Na+leave/K+leave) and

SA = (Na+soil/K+soil)/(Na+plant/K+plant)

2.4. Statistical analyses

All data were expressed as means ± SE, and each mean value was calculated from three replicates. Data were analyzed by one-way analysis of variance (ANOVA) using the statistical software SPSS 17.0 (SPSS Inc., Chicago, USA). The treatment values were compared using an F-test. The term significant indicates differences for which P≤0.05.

3. Results

3.1. Growth

Along with the increase in Na+ concentrations under the two types of salt stresses, the fresh weights of shoots and roots, the dry weights of shoots and roots, the shoot heights and the root lengths of G. gracilis seedlings all significant decreased (Figure 1, P < 0.01) and the decreases under the AS treatment were more obvious than under the NS treatment. The RGR and WC of G. gracilis seedlings are shown in Figure 1. The RGR of G. gracilis decreased with the increasing salt stress, and the degree of the decrease was greater under AS stress than under NS stress. The RGR of the aboveground portion of G.gracilis was more significant than that of the underground. With the increase in the Na+ concentrations, the WC of roots, stems and petioles decreased, but they did not reach significant levels under either salt stress and there was no significant difference between the two salt stresses (P>0.05). The WC of leaves was significantly decreased, especially under the AS stress (P < 0.01). However, under AS, when the Na+ concentrations was 30 mmol·L-1, the fresh weights of shoots, the dry weights of shoots and roots, the shoot heights and the root lengths of G. gracilis seedlings were significantly higher than in the control. This indicated a special compensatory effect.

Figure l. Effects of different type salts stresses on the fresh weight of shoot (A1), the fresh weight of root (B1), the dry weight of shoot (A2), the dry weight of root (B2), the shoot height (A3), the root length (B3) of G. Gracilis seedlings. The values are the means of three replicates. Means followed by different letters in the samestress type are significantly different at P<0.05 according to Duncan's method. Neutral salts stress: NaCl: Na2SO4=1:1; Alkaline salts stress: NaHCO3:Na2CO3=1:1

3.2. Cations

For both types of salt stress, the Na+ contents of roots, stems, petioles and leaves were all higher than those of controls. The Na+ contents in stems, leaves and petioles also increased with the increase in stress intensity. However, under the same concentrations, the Na+ content was higher in the AS than that in the NS treatment group and the increasing trend in the AS were greater than that in the NS. In the root system of G. gracilis, the Na+ content presented an increasing trend as the stress intensity increased under NS conditions, but presented a decreasing trend under AS conditions (Figure 2, A1, B1; P < 0.01). Under the NS treatment, mainly the underground organs accumulated Na+, but under the AS treatment, mainly the aboveground organs accumulated Na+.

Figure 2. Effects of different type salts stress on contents of Na+ (A1, B1), K+ (A2, B2), Ca+ (A3, B3) Mg+, (A4, B4), and P (A5, B5) of G. gracilis seedlings; The values are the means of three replicates. Means followed by different letters in the same stress type are significantly different at P<0.05 according to Duncan's method. Saline stress: Neutral salts stress: NaCl:Na2SO4=1:1; Alkaline salts stress: NaHCO3-:Na2CO3=1:1.

Under both types of salt stress, the Ca2+ and K+ contents of G. gracilis seedlings, roots and stems showed declining trends and were lower than those of the controls. However, the degree of decrease under the AS treatment was significantly greater than that under the NS treatment (Figure 2, A2, B2, A3, B3; P < 0.01). There were no obvious differences in the Ca2+ and K+ contents in leaves under the different Na+ concentrations of NS, but there were significant decreasing trends under AS (P < 0.05). The K + contents in petioles were significantly higher than that of the control under both types of salt stress (P < 0.01). However, there were not significant differences between the different Na+ concentrations (P >0.05). The Ca2+ contents, trends in petioles were similar to those in roots and stems under the different salt stresses. The main organ for K+ accumulation changed from underground to aboveground with the increased Na+ concentrations under both types of salt stress. For NS, petioles and leaves were the main organs for K+ accumulation, but K+ could be only accumulated in petioles under AS. Ca2+ showed no significant accumulation with the increase of Na+ concentrations. Under the same concentration stress, the Ca2+ and K+ contents of each organ were all lower under AS than under NS treatments.

The Mg2+content in roots, stems and leaves of G. gracilis seedlings showed decreasing trends under both kinds of salt stress, and especially under AS, where it decreased more significantly. With the same Na+ concentration stress, the Mg2+ content in roots was higher under NS, but in stems and leaves it was higher under AS. The Mg2+ content in petioles showed no significant change under higher concentrations of NS, but significantly decreased under high concentrations of AS (Figure 2, A4, B4; P < 0.01). The P3+ contents in roots, stems, petioles and leaves showed significantly increasing trends under NS, and were all higher than that of control. However, the P3+content in roots showed a decreasing trend and were significantly lower than controls. In stems and leaves the P3+ contents showed increasing trends, and in petioles there were no significant differences between different Na+ concentration under AS. However, the P3+contents in each organ were all lower than those in the controls (Figure 2, A5, B5; P< 0.01). With an increase in the two types of salt stress intensity, the Fe3+ contents in the roots showed no significant differences, but in stems, petioles and leaves it showed a significant increasing trend (Figure 3, the A1, B1; P < 0.01).The P3+, Mg2+ and Fe3+ contents in G. gracilis seedlings moved from the underground organs to the aboveground organs under both kinds of salt stress, but under AS, the change was more significant.

Figure 3. Effects of different type salts stress on contents of Fe (A1, B1), Mn (A2, B2), Mo (A3, B3) and B (A4, B4) of G. gracilis seedlings; The values are the means of three replicates. Means followed by different letters in the same stress type aresignificantly different at P<0.05 according to Duncan's method. Neutral salts stress: NaCl: Na2SO2=1:1; Alkaline salts stress:NaHCO3: Na2CO3=1:1

The Mn2+ contents in roots, stems, petioles and leaves of G. gracilis seedlings showed increasing trends along as the AS stress intensity increased, and they were significantly higher than those of the NS and control. However, the Mn2+contentsin stems, petioles and leaves showed decreasing trends under NS, which were also greater than those of the control, and in the roots no regular changes were detected (Figure 3, A2, B2; P < 0.01).The Mo2+ contents in roots, stems, petioles and leaves of G. gracilis seedlings showed decreasing trends as both types of salt stress intensities increased. Under the same Na+ concentration stress, the Mo2+ contents under AS were higher than those of the NS and control, except in roots (Figures 3, A3, B3; P < 0.01). The changes in the B3+ contents were similar to those of Mo2+; however, with the same Na+ concentrations, the B3+ contents under NS were significantly higher than those under AS and the controls (Figure 3, A4, B4; P < 0.01).

3.3. Anions

The Cl contents in stems, petioles and leaves of G. gracilis seedlings were higher under the two salt stresses than in the controls, and the Clcontent in each organ increased as the Na+ concentration increased. Cl contents were significantly higher under NS than under AS at the same Na+ concentrations. Additionally, the Cl concentrations, changing trend in the roots was the same as in other organs under NS. However, the Cl concentrations were significantly lower under AS than under control conditions, and showed a gradually decreasing trend as the Na+ concentration increased (Figure 4, A1, B1; P < 0.01).The experimental results showed that Cl contents were mostly accumulated in the roots and were significantly higher than in the stems, petioles and leaves under NS. Along with the increasing of stress strength, the distribution of Clchanged under AS, from the underground to the above ground. Cl in stems, petioles and leaves accumulated only under a high Na+ concentration of AS. The H2PO4 content, s accumulation behavior was consistent with Clin different organs under both types of salt treatments (Figure 4, A3, B3; P < 0.01).

Figure 4. Effects of different type salts stress on contents of Cl- (A1,B1), NO3- (A2, B2), H2PO4- (A3, B3), SO42- (A4, B4) and C2O42- (A5, B5) of G. gracilis seedlings; The values are the means of three replicates. Means followed by different letters in the same stress type aresignificantly different at P<0.05 according to Duncan's method. Neutral salts stress: NaCl:Na2SO4=1:1; Alkaline salts stress: NaHCO3: Na2CO3=1:1

As the Na+ concentration of both types of salt stress increased, the changes in the NO3contentsin roots, stems, petioles and leaves of the G. gracilis seedlings were similar. The NO3content decreased in roots, was not significantly different in stems, and increased in petioles and leaves. At the same time, the NO3content was lower under stress treatments than in controls. The NO3contents under AS were lower than under NS and the range of changes under AS were greater than under NS (Figure 4, A2, B2; P < 0.01).

The SO42–contents in roots, stems, petioles and leaves increased with increasing Na+ concentrations of NS, showing accumulation specificity along with the increased stress. The SO42–contents in roots were higher than in stems, petioles and leaves. The SO42– contents in roots decreased with the increasing Na+ concentrations of AS, but increased in stems, petioles and leaves. With the AS intensity increased, the distribution of SO42–changed from the underground to the above ground organs (Figure 4, A4, B4; P < 0.01).

C2O42–accumulated mostly in stems in the whole plant under both types of salt stress. Under NS, the C2O42–contents in roots decreased linearly with the increasing salinity, but in the stems, petioles and leaves there was an initial decrease, followed by increases under different treatments. This showed that the roots could accumulateC2O42–only in low concentrations, but stems, petioles and leaves could accumulate it at both low and high concentrations. The C2O42–in roots decreased with the increasing salinity under AS. The C2O42– in stems, petioles and leaves showed trends of increasing first, and then decreasing, with the increasing salinity. With the increase of AS intensity, the distribution of C2O42– changed. Under high concentrations of AS, the C2O42–in stems, petioles and leaves specifically accumulated (Figure 4, A5, B5; P < 0.01).

3.4. Ion balance

3.4.1. Na+/K+

With the increasing levels of NS and AS, the Na+/K+ in different organs of G. gracilis seedlings rose, but the extent under AS was more significant than under NS. Under the same stress intensity, Na+/K+ under AS was higher than under NS. Under both types of salt stresses, Na+/K+ gradually moved from underground organs to aboveground organs, and the reduction range under NS was greater than under AS. Na+/K+values in roots were the highest under AS at all treatment levels (Table 1). The damage under AS was greater than under NS, and the damage to the underground parts of G. gracilis seedlings was greater than to the above ground part under both kinds of salt stress. The roots suffered the most serious damage under AS.

Table 1. The ratio of Na+/K+inroot, stem, stipe, leaf and SA, ST of G. gracili seedlings under different type salts stresses. Neutral salts stress: NaCl: Na2SO4=1:1; Alkaline salts stress:NaHCO3: Na2CO3=1:1

3.4.2. Selective absorption of Na+ and K+

SA and ST were used to measure the selective absorption of Na+ and K+. ST shows the capability of roots to transport Na+ upward and K+ down ward. SA shows the roots selective absorption of K+ and blockage of Na+ absorption. A higher ST value indicated that the root controlled Na+ uptake and capability of K+ transport to the leaves were stronger, indicating that the selective transportation ability of the root was stronger. Meanwhile, a higher SA value indicated that the root refused Na+ absorption and its selective absorption of K+ was stronger, indicating thatthe selective absorption capability of the root was stronger (Wang et al., 2004).

The experimental result showed that, along with the increase in the two types of salt stress, the ST values of G. gracilis seedlings first increased and then decreased, and the proportion of treatments were greater than the control. However, there also were some differences between NS and AS. Under NS, ST rose when the concentration of Na+ was 15–60 mmol·L-1 then decreased at 60–120 mmol·L-1. While under AS, ST rose significantly when the concentration of Na+ was 15–30 mmol·L-1. The increase dintensity under AS was greater than that under NS, which were 834% and 301%, respectively. When the stress intensity was higher than 30 mmol·L-1, ST significantly decreased, to 0.10%, 0.07% and 0.12% of NS, respectively.

Under the two different types of salt stress, the SA values of G. gracilis seedlings were all higher than those of the control, and there were no significant changes among the different Na+ concentration treatments. However, SA value sunder NS were greater than those under AS. At the same Na+concentrations, the SA of NS were 343%, 248%, 375%, 366% and 425% of AS, respectively.

3.4.3. Ion percentage

Under control conditions, G. gracilis seedling roots the accumulated cations were mainly P3+ and K+, and the accumulated anions were mainly NO3 and H2PO4. The accumulated ions in stems and leaves were mainly cationic P3+ and K+, and anionic H2PO4 and C2O42–. Petioles mainly accumulated in the cations of P3+and K+ and the anions of NO3 and C2O42 –(Table 2-3).

Table 2. The percentages of different cations in root, stem, stipe and leaf of G. gracili seedlings under different type salts stresses. Neutral salts stress: NaCl: Na2SO4=1:1; Alkaline salts stress: NaHCO3: Na2CO3=1:1.

Table 3. The percentages of different anions in root, stem, stipe and leaf of G. gracili seedlings under different type salts stresses. Neutral salts stress: NaCl: Na2SO4=1:1; Alkaline salts stress: NaHCO3: Na2CO3=1:1.

Under NS, when the stress concentration was low, the accumulated cations in roots were mainly P3+ and K+. But when the stress concentration became higher, Na+ became the major cation instead of P3+ and K+. The accumulated anions for roots were mainly NO3–and H2PO4. The accumulated cations of stem and leaf were mainly P3+ and K+, and anions were mainly NO3 and H2PO4. Petioles mostly accumulated cationic P3+ and K+, and anionic H2PO4 and NO3 (Table 2-3).

With the increase of NS, the percentages of Na+, P3+, SO42–, Cland H2PO4in roots and petioles of G.gracilis seedlings increased and percentages of K+, Mg2+, Mo2+, NO3 and C2O42–decreased. Percentages of Na+, P3+, SO42– and Cl in stems increased and percentages of K+, Mg2+, Mo2+, NO3 and C2O42–decreased. In blades, the percentages of Na+, P3+, Mo2+, SO42– and Clincreased, but percentages of K+, Mg2+, NO3 and C2O42–decreased. Compared with other cations, percentages of Fe2+, Mn2+, B3+ and Mo2+ were very small, less than 1% of the total cations. The percentage of Fe2+ was the smallest and its percentage content had no obvious difference with the control group (Table 2-3).

Under AS, the accumulated cations in roots were mainly Na+ and K+ atlow concentrations and Na+ athigh stress concentrations, and the accumulated anions were mainly NO3and H2PO4. The main accumulated cations in stems and petioles were P3+ and K+ at low concentrations and Na+ at high stress concentrations, and the accumulated anions were NO3 and H2PO4-.The accumulated cations in leaves were mainly Mg2+ and K+ at low concentrationsand Na+ at high stress concentrations, and the accumulated anions were NO3 and H2PO4. Under AS, the percentage of Na+, C2O42–, Cland SO42– in roots and stem sincreased, but the percentage of K+, Mg2+, Ca2+, P3+, Mo2+, NO3 and H2PO4decreased. The percentages of Na+, SO42-, C2O42–, Cland H2PO4 in petioles increased, and the percentages of K+, Mg2+, Ca2+, P3+, Mo2+ and NO3 decreased. The percentage of Na+, Mo2+, SO42–, C2O42– in leaves increased, and the percentage of K+, Mg2+, Ca2+, P3+, NO3 and H2PO4decreased. Compared with other cations,the percentages of Fe2+, Mn2+, B3+and Mo2+were very small, less than 1% of the total cations. The percentage of Fe2+ content was the smallest, and its percentage content had no obvious difference with the control group (Table 2-3).

4. Discussion

High salt-stress generally leads to growth arrest and even plant death (Cuartero and Fernández-Muñoz, 1998; Maggio et al., 2007). However, in the present study, there was no decrease in RGR of G. gracilis under NS and AS stress. The RGR value reflects the life-sustaining activities of a plant, and is considered as an optimum index for the degree of stress and plant responses to stresses (Yang et al., 2008). Shoot height and root length showed the direct performance of in vitro plants based on the salinity-alkalinity stress influence degree of physiology. The roots conta in the first perceptible stress information and influence on growth. The decrease of RGR with increasing stress was also supported by the change in shoot height and root length. However, the fact that the RGR decrease under AS was greater than under NS, implies not only that NS and AS stresses are distinct, but also the resistance of G. gracilis to NS stress is stronger than to SA stress. The injurious effect caused by AS was greater than that of NS at the same salinity concentration, consistent with previous reports (Shi and Sheng, 2005; Yang et al., 2007; Wang et al., 2011).

The different injurious effects of the two stresses may be related to different mechanisms. The injurious effects of salinity are commonly thought to be a result of low water potentials and ion toxicities (Munns and Tester, 2008). The AS exerts the same stress factors as NS but with the added influence of high-pH stress.

The high-pH environment surrounding the roots not only can directly cause some ions, such as Ca2+, Mg2+, HPO42–and H2PO4,to precipitate (Shi and Wang, 2005), but also may create some microelement toxicity, which can be detrimental to plants, especially for root growth. Plant survival under alkali stress, therefore, depends on not only its capability to cope with water stress and ion toxicity, but also its resistance to high pH levels. Therefore, to adapt to the AS stress environment, plants need to expend more material and energy than to adapt to NS stress, and this might bee reason for the lower RGR value under AS stress than under NS stress, as observed in this research.

Data show high pH levels as an important factor in limiting plant growth and development under alkaline conditions (Yang et al., 2007, 2008). High pH clearly affects plant growth differently at various developmental stages.

Plants in saline conditions usually accumulate inorganic ions in vacuoles to decrease their cell water potential, because the energy consumption for absorbing inorganic ions is far less than for synthesizing organic compounds. If excessive amounts of ions enter the plant, they rise to toxic levels, inhibit photosynthesis and thus reduce the growth rate (Munns and Tester, 2008). Na+ is the main poisonous ion in salinized soil. (Zhang et al. 2014) Low Na+ and high K+ levels in the cytoplasm are essential to maintain a number of enzymatic processes (James et al., 2006; Hussain et al., 2013).

Ionic imbalance in plants is mainly caused by the influx of superfluous Na+ (Munns and Tester, 2008; Blumwald, 2000). Plants in saline conditions usually accumulate inorganic anions, such as Cl (Santa-Cruz et al., 2002), NO3, and SO42–, or synthesize organic anions to neutralize the high concentrations of cations and maintain ionic balance (Yang et al., 2007).

A stable internal environment, as a result of intracellular ion balance, is necessary for plants to maintain a normal metabolism (Yang et al., 2007). In a living plant, as long as the plant can adapt to the environment, the proportion of ions in its tissue should be stable regardless of how the environmental pH value changes.

The dominant intracellular cations under control, in this study, were P+ and K+, contributing>70% of the total positive charge. However, while the Na+ concentrations increased with increasing stress, K+ concentrations decreased. The Na+ and K+ were the dominant intracellular cations under both NA and SA stresses. The contribution of Fe2+, Mn2+, B3+and Mo2+to the total positive charge was minimal (<1%). This is in contrast to that observed in K. sieversiana (Yang et al., 2007) where the contribution of K+ to the total positive charge was dramatically greater than that in G. glauca.

At lower stress intensities, the effects of both salts on the Na+ content and Na+/ K+ of G. gracilis were similar. But when the salinity was higher than 60 mM, as the salinity increased, the Na+ contents and Na+/ K+ values increased slowly under NS, but sharply under AS. This implied that the high pH level of AS might interfere with control of Na+ uptake in the shoots and increase the intracellular Na+ content to a toxic level. This could explain some of the damage that emerged in higher AS environments. James et al. (2006) also reported that the photosynthetic capacity was related to the cellular and subcellular partitioning of Na+, K+ and Cl. Moreover, the high pH level led to the H+ deficit outside the roots and may limit the Na+ extrusion from the root cytosol to the external environment. This may be why the injurious effects caused by AS were greater than those of NS. However, the behavior of G. gracilis was significantly different from that of Kochia sieversiana, a naturally alkali-resistant halophyte (Yang et al., 2007). However, the effects of both stresses on Na+-K+ selective absorption and other K. sieversiana responses were similar. This indicates that K. sieversiana root cells may be resistant to the highpH surrounding the roots, and prevented from invading the intracellular environment. Therefore, we propose that the high-pH environment surrounding the roots is an important physiological mechanism for plant resistance to AS. The process of pH adjustment may occur outside the roots, in the roots or in both simultaneously. However, the mechanisms governing the ionic balance under both stresses were different.

That the dominant intracellular anion in G. gracilis roots, stems and leaves under both stresses was similar to in the control and that the stress intensity did not have an effect on the anions, proportions suggested that G. gracilis was able to maintain the ionic balance in cells, not only under NS, but also under AS, even at pH >9.88. In addition, the dominant anion adjustment differed among roots, stems and leaves. The present results indicated that the roots of G. gracilis accumulated NO3and H2PO4to maintain the intracellular ionic balance under both saline and alkaline conditions. While in stems and leaves, C2O42–and H2PO4were accumulated to maintain the intracellular ionic balance. The dominant intracellular anions in G. gracilis stipes were NO3 and C2O42–under the control treatment, while NO3and H2PO4 accumulated under both stress.

While the SO42–concentrations increased with the increasing stress, the NO3concentrations decreased. However, under AS stress, the Cl and H2PO4concentrations decreased, and G. gracilis might have enhanced the C2O42– concentration to compensate for the shortage of inorganic anions. The accumulation of C2O42– in G. gracilis may be a response to an inorganic anion deficit. Under NS stress, the C2O42–concentrations decreased, and G. gracilis might have enhanced Cl and H2PO4concentrations to compensate for the shortage of inorganic anions. The accumulation of Cl and H2PO4in G. gracilis may be a response to an inorganic anion deficit.Therefore, Cl, H2PO4 and C2O42–accumulationsmay result from a negative charge deficit, and the C2O42–metabolic regulation may play an important role in maintaining the ionic balance.

5. Conclusion

In summary, the effects of different types of salt stress onthe growth of G. gracilis seedlings were significantly different. Under alkali salts stress, the growth of G. gracilis seedlings was more intensely inhibited than under neutral salts stress, which related to specific ion accumulations under different types of salt stress. The accumulation of Na+ and Clwere significant under neutral salts stress. However, under alkali salts stress, Na+ accumulated but K+ declined, and the Na+/K+ increased significantly, which showed that the damage mechanism of both types of salt stresses on plants were different. Due to the high pH under alkali salts treatments, the accumulation of anions in plants was breached. Under the different types of salt stress, G. gracilis seedlings had obviously different ionic balances and specific ion, such as Mn2+ and Mo2+, accumulation capabilities, which were associated with the relief of the high pH stress. Due to specific ion levels slowly dropping and accumulation specificity, G. gracilis seedlings also showed some adaptability in growth under both types of salt stress, though the adaptability of G. gracilis to the neutral salts stress was better than to the alkali salts stress.

Abbreviations: NS - neutral salts stress; AS - alkali salts stress; DM - dry mass; WC - water content; RGR - relative growth rate.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 31270366).

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