Physiological and Biochemical Responses of Jerusalem Artichoke Seedlings to Mixed Salt-Alkali Stress Conditions

Soil salinization and alkalization frequently co-occur in the grassland, but little information exists concerning the mixed effects of salt-alkaline stress on plant. Jerusalem artichoke is an economically and ecologically important energy crop and also considered as a salt-tolerant species. In this study, we investigated the effects of 12 mixed salt-alkaline conditions on the seedling growth and responses of Jerusalem artichoke to such conditions. The results showed that the seedling growth decreased with the increasing salinity and pH, and the destructive effects were more markedly under the interactions of highest salinity and pH. The Na, Mg and Ca concentrations were all increased with the increasing salinity and pH, but the K kept stable. The Cl concentration increased when the treatment without alkali salts, and the NO3 and H2PO4 concentrations were decreased with the increasing salinity. Jerusalem artichoke seedlings enhanced organic acids and proline to supply the shortage of inorganic anions and cope with osmotic stress from the high Na concentration. Above results show that the toxicity effects of the interactions of salt stress and alkali stress on plant is much greater than that only salt or alkali stress. A better understanding of the seedlings of Jerusalem artichoke under mixed salt-alkali stress conditions should facilitate the effective utilization of this species under such complex environment in Northeast China.


Introduction
Jerusalem artichoke (Helianthus tuberosus L.) is a tuberous plant belongs to the sunflower family.It was introduced into Europe in the late 1500s from North America, and then cultivated across Europe and Asia over the centuries, both for human consumption and as a feed for grazing livestock (Baldini et al., 2004).Currently, Jerusalem artichoke is cultivated for many application areas such as source of sugars, green forage, crude material for production of ethanol, and pharmaceuticals applications (Baldini et al., 2004;Saengthongpinit and Sajjaanantakul, 2005).In addition, it grows very rapidly and can reach a height of 2-4 m.It is also has highly tolerant to salt soil due to the deep root system (Zhao et al., 2008).In conclusion, Jerusalem artichoke is an economically and ecologically important energy crop species for humans.
Soil salinization and alkalization is an important environmental problem all over the world, especially in China.Salinity-alkalinity soil decreases the plant growth and production and also lead to large area of land degradation.For example, arable land acreage of the world is 1.5×10 9 ha, but 23% (0.34×10 9 ha) of the area is saline, and 37% (0.56×10 9 ha) is sodic (Tanji, 1990;Lin et al., 2014).In the Northeast of China, almost 70% of the grassland has been serious degraded due to salt-alkali soil, and the area is expanding all the time (Deng et al., 2006).
In addition, soil salinization and alkalization are co-occur, and the stress effect on the plant is always much greater than that only salt or alkali stress.Previous studies have been proved that salt stress and alkali stress are great differed due to the high pH, and the alkali stress is more severe than salt stress (Shi and Yin, 1993;Guo et al., 2009;Zhang and Mu, 2009).Under salt stress, plants always maintain high K + and Ca 2+ levels and exclude Na + from the shoots, synthesize many organic solutes such as proline in the cytoplasm to resist the osmotic stress, and also accumulate inorganic ions such as Cl − in order to keep ion balance (Kerepesi and Galiba, 2000;Ashraf et al., 2007).While under alkali stress, accumulation of organic acids is the main way to resist the high pH stress.However, the conditions of plant surviving in the saltalkali soil are very complex, contain both the salt and alkali stresses (Shi and Yin, 1993;Shi and Wang, 2005;Li et al., 2010).Thus, the effect of mixed salt-alkali stress should be distinguished from salt stress or alkali stress, and the physiological and biochemical responses to such conditions maybe also unique.

Stress treatment and seedling harvest
The Jerusalem artichoke seedlings were subjected to stress treatments after 40 d, and 52 pots with uniform seedlings were divided randomly into 13 sets, four pots per set.Each set contained four replicates.The pots were watered daily with nutrient solution containing the appropriate stress salts as above stated.The control seedlings were watered with only the nutrient solution.The total treatment duration was 8 d.Harvest seedlings were washed with distilled water twice, and then the shoots were retained, the fresh weight was determined for each plant.The shoots were oven-dried at 80 °C for 15 min and then at 65 °C to a constant weight.The water content (WC) was calculated using the formula (FW-DW)/DW.FW is short for fresh weight, DW is short for dry weight.

Determination of inorganic ions and organic solutes
Dry samples of plant material (50 mg) were treated with 10 mL deionized water at 100 °C for 1 h and the extract was taken to determine free inorganic ions, organic acids.An atomic absorption spectrophotometer (TAS-990, Purkinje General, Beijing) was used to determine the Na + , K + , Mg 2+ and Ca 2+ contents.The NO3 -, Cl - and H2PO4 -contents were determined by ion chromatography (DX-300 ion chromatographic system, USA), and the studies described how the plant changed under such condition.To our knowledge, the physiological adaptation mechanism of plant seedlings especially the Jerusalem artichoke is still unclear.A better understanding of the seedlings of Jerusalem artichoke under mixed salt-alkali stress conditions should facilitate the effective utilization of this species under such complex environment in the Northeast of China.
In this study, mixtures of two neutral salts (NaCl and Na2SO4), two alkaline salts (NaHCO3 and Na2CO3) were used in different proportions to simulate 12 treatments of mixed saltalkaline conditions.The aims were (1) to test the effects of mixed salt-alkali stresses on the seedling growth of Jerusalem artichoke, (2) to explore the physiological adaptation mechanism of Jerusalem artichoke under such conditions.

Design of the mixed salt-alkali stress conditions
In order to simulate the mixed salt-alkali conditions, NaCl, Na2SO4, NaHCO3 and Na2CO3 were chose based on the salt composition of the salt-alkali soil in the Northeast of China (Peng et al., 2008).Four salts were mixed in different proportions according to the salinity and pH in the Songnen grassland.Four treatment groups (A-D) were set with the increasing alkalinity.Salt composition and molar ratio of various treatments are shown in Table 1.Within each group, three concentrations were used (20, 40 and 60 mM).Total 12 mixed stress treatments (A1-D5, pH 7.02-10.18,and salinity 20-60 mM) are shown in Table 2. measurement conditions were as follows: AS4A-SCion exchange column, CD M-II electric conductivity detector, mobile phase was Na2CO3/ NaHCO3 = 1.7/1.8mM.For the analysis of organic acids, the measurements were also undertaken using ion chromatography (DX-300 ion chromatographic system, USA), and the measurement conditions were as follows: ICE-AS6 analytical column, CDM-II electric conductivity detector, AMMS-ICE II interference suppressor, mobile phase was 0.4 mM perfluorobutyric acid, flow speed was 1.0 mL/min, column temperature was 20 °C, sample size was 50 μL.The content of proline was measured using ninhydrin, according to Zhu et al. (1983).

Data analysis
All data were analyzed using SPSS 13.0.Tukey's tests were performed for multiple comparisons to determine significant (P＜0.05)differences between individual treatments.A two-way ANOVA was used to test the effects of the factors (pH and salinity) and their interactions on seedling growth and physiological change index.The data were expressed as mean ± S.E.

Effects on the seedling growth and water content
The fresh and dry weights of Jerusalem artichoke seedlings were significantly affected by pH and salinity (P<0.001,Table 3), but not significantly by the interaction of the two factors.Maximum seedling weight (fresh and dry) were obtained both in the distilled water, which were 43.6 g and 7.2 g, respectively.Seedling weight decreased with the increasing salinity in all the treatment groups, and more markedly in group D (Fig. 1).At the highest salinity (60 mM), the dry weight in group A was 4.6 g , while 4.3 g, 3.4 g and 2.8 g in the other 3 groups, respectively.The values are the means of four replicates.Means followed by different letters are significantly different at P < 0.05 according to a least significant difference test Water content in the shoot was significantly affected by pH, salinity and the interaction of the two factors (P<0.001,Table 3).Maximum water content was also obtained in distilled water.In group A, the water content significantly decreased only when the salinity reached 60 mM.In group D, when the salinity reached highest (60 mM), the water content decreased of 25% compared to the control (Fig. 1).

Effects on inorganic ions contents
The Na + , Mg 2+ and Ca 2+ contents were significantly affected by the pH, salinity and the interaction of the two factors (P<0.001,Table 3).However, the K + content was not significantly affected.Within each group, the Na + contents were increased with the increasing salinity, and reached the highest value at 60 mM.In addition, the ratio of the alkali salts were also affected the Na + content.The Na + content in group D is 16.3 mg/g but only 8.4 mg/g in the group A at 60 mM salinity (Fig. 2A).
The K + content changed unregularity in all the treatments, but compared with the control, the K + content did not change significantly (Fig. 2B).In addition, due to the Na + content increased sharply with the increasing alkali salts, the Na + /K + had the similar change trends with the Na + content.The maximum value (1.2) was appeared at 60 mM in the group D (Fig. 2E).In addition, the Mg 2+ and Ca 2+ contents were also showed a increasing trends with the increasing salinity and the ratio of alkali salts.The maximum values were both 60 mM in the group D (Fig. 2CD).
The NO3 -, Cl -and H2PO4 -contents were all significantly affected by the pH, salinity and the interaction of the two factors (P<0.001,Table 3).The Cl -content significantly increased with the increasing salinity at A group (P<0.05), and was 3.7 times than that in the control group at the highest salinity.However, the Cl - content significantly increased when the salinity reached 20 mM, and kept unchanged when the salinity was much higher.The changing trends were similar between NO3 -and H2PO4 -contents, both of the values were decreased with the increasing salinity and reached the lowest at 60 mM salinity in the final (Fig. 3).

Effects on organic solutes contents
Maliate acid, citric acid and oxalate acid were detected in the shoot of Jerusalem artichoke under mixed salt-alkali stress conditions.The three organic acids were significantly affected by the pH, salinity and the interaction of the two factors (P<0.001,Table 3).In addition, the three organic acids had the similar change trends in each stress group, they were all increased when the alkali salt ratio was higher.There was no significant difference in the maliate acid and citric acid contents compared with the control in group A except oxalate acid.At the highest salinity stress, the organic acids contents were 1.8 times, 3.6 times and 2.2 times than that of the control, respectively (Fig. 4).
The proline content was significantly affected by the pH, salinity and the interaction of the two factors (P<0.001,Table 3).Although the proline content increased with the salinity in the group A and B, the rangeability was not large.However, in the group D, when the salinity reached 60 mM, the proline content was 13.5 times than that of the control (Fig. 5).

Discussion
In this study, the 12 treatments covered various salt-alkali stress conditions in a range of pH from 7.02 to 10.18, which is approximate to the Songnen grassland in the Northeast of China.In the salt-alkali soil of Northern China, the stress factors are very difficult to simulate.However, the mixed stress treatments used in our research made the reasearch of complex natural environment conditions possible.Most privious studies  absorptions in this species.However, due to the great number of the Na + , the Na + / K + increased sharply at higher salinity in group D. High Na + /K + ratio can affect ions regionalization, lead to ions imbalance, and also reduce enzyme activities.This is why the fresh and dry weights were lowest in this treatment.
Under the circumstance of higher Na + concentration, most plants accumulate the inorganic anions, such as Cl -and NO3 -in order to keep the ionic balance.In our study, when the treatment was without alkali salts, Jerusalem artichoke accumulated much Cl -to balance the influx of Na + (Fig. 3A).However, once the treatment contained the alkali salts, all the anion contents decreased, indicating that high pH inhibited absorptions of Cl -.Similar results were also reported for other plants, like Chloris virgata (Yang et al., 2010).Seedlings accumulated much organic acids to compensate the anions deficit caused by high pH.Accumulation of organic acids not only played an important osmotic role but also buffered excess toxic cations and kept ionic balance, such as maliate acid, citric acid and oxalate acid in this species.Although a great number of inorganic ions accumulate in vacuoles, plants can also synthesize low molecular mass organic solutes in the cytoplasm, such as proline to prevent dehydration and protect biomacromolecules (Parida andDas, 2005, Long et al., 2010).In the present study, proline was accumulated under stress conditions, especially at higher salinity in group D, indicating that changes of proline content in seedlings may be a special physiological response to high intensity pH stress.only concerned salt stress of alkali stress (Munns, 2002;Debez et al., 2004;Ashraf and Foolad, 2007;Yang et al., 2007Yang et al., , 2009Yang et al., , 2010)).However, little information exsit concerning the mixed salt-alkali stress conditions.
Seedling establishment is the most important stage for plant surviving in the salt-alkali conditions.This stage is also very sensitive to salinity and alkalinity in the soil.Understanding the physiological responses of Jerusalem artichoke to mixed salt-alkali stress conditions during seedling stage has an important exploring significance.Our results showed that the more ratio of the alkali salts in the treatment, the lower of the fresh weight and dry weight.This indicated that the high pH played important roles in seedling growth, especially at higher salt concentration.The negative effects of salt stress on the plant are mainly contains osmotic stress and toxic ion, but the alkali stress exerts the high pH impacts.Once the salt stress and alkali stress are mixed, the destructive effects on the plant is much greater due to the mixed effects of salinity, pH and toxic ions.Losing water is also a quick way to osmotic adjustment in response to the osmotic stress for most plants (Lissner et al., 1999).With the increasing salt stress, the water content in the shoots of Jerusalem artichoke were decreased.The reasons maybe that the root was damaged under stress condions and then the ability of water absorption was decreased.In addition, many organic solutes were synthesis under such conditons and the water content in the shoots were reduced.
In general, the Na + entered the plant cells through the highaffinity K + transporter (HKT) and non-selective cation channels (Zhu, 2003).Most plants in the salt environment accumulated a large number of Na + and simultaneously inhibited the K + absorption (Munns, 2002).In this research, although the Na + concentration increased, the K + concentration was not affected.Similar results were also found in some other halophytes in Songnen grassland in China, such as Kochia sieversiana and Suaeda glauca (Yang et al., 2007(Yang et al., , 2008)).This showed that the increased Na + concentrations did not induce the decreased K + concentrations in shoot of Jerusalem artichoke, indicating that a specific ion transport mechanism may exit between Na + and K + The values are the means of four replicates.Means followed by different letters are significantly different at P < 0.05 according to a least significant difference test

Fig. 1 .
Fig. 1.Effects of mixed salt-alkaline stresses on fresh weight (A), dry weight (B) and water content (C) of Jerusalem artichoke

Fig. 2 .
Fig. 2. Effects of mixed salt-alkaline stresses on Na + content (A), K + content (B)，Mg 2+ content (C) Ca 2+ content (D) and Na + / K + (E) of Jerusalem artichokeThe values are the means of four replicates.Means followed by different letters are significantly different at P < 0.05 according to a least significant difference test

Fig. 4 .
Fig. 4. Effects of mixed salt-alkaline stresses on oxalate acid content (A), citric acid (B) and maliate acid (C) of Jerusalem artichokeThe values are the means of four replicates.Means followed by different letters are significantly different at P < 0.05 according to a least significant difference test

Table 1 .
Experiments were conducted in Northeast Forestry University (126°38'E, 45°45'N, Harbin, China) during May and June.Jerusalem artichoke (Helianthus tuberosus L.) tubers were 474 Salt composition and molar ratio of various treatments

Table 2 .
Stress factors of various treatments

Table 3 .
Two-way ANOVA of effects of salinity, pH and their interactions on the growth and physiological index of Jerusalem artichoke